Discovery of S64315, a Potent and Selective Mcl-1 Inhibitor

pubs.acs.org/jmc

Article

Discovery of S64315, a Potent and Selective Mcl‑1 Inhibitor

Zoltan Szlavik, Marton Csekei, Attila Paczal, Zoltan B. Szabo, Szabolcs Sipos, Gabor Radics,

Agnes Proszenyak, Balazs Balint, James Murray, James Davidson, Ijen Chen, Pawel Dokurno,

̈

Allan E Surgenor, Zoe Marie Daniels, Roderick E. Hubbard, Gaetane Le Toumelin-Braizat,

̈

Audrey Claperon, Gaelle Lysiak-Auvity, Anne-Marie Girard, Alain Bruno, Maia Chanrion,

́

Frederic Colland, Ana-Leticia Maragno, Didier Demarles, Olivier Geneste, and Andras Kotschy*

Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c01234

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: Myeloid cell leukemia 1 (Mcl-1) has emerged as an

attractive target for cancer therapy. It is an antiapoptotic member

of the Bcl-2 family of proteins, whose upregulation in human

cancers is associated with high tumor grade, poor survival, and

resistance to chemotherapy. Here we report the discovery of our

clinical candidate S64315, a selective small molecule inhibitor of

Mcl-1. Starting from a fragment derived lead compound, we have

conducted structure guided optimization that has led to a

signiﬁcant (3 log) improvement of target aﬃnity as well as cellular

potency. The presence of hindered rotation along a biaryl axis has

conferred high selectivity to the compounds against other

members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and

achieved both eﬃcient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclinical candidate has

drug-like properties that have enabled its development and entry into clinical trials.

therapy.¹⁰Until recently, only compounds showing weak

INTRODUCTION

■

cellular potency on Mcl-1 (high μM range) were available and

Apoptosis, an evolutionary highly conserved form of

therefore useful only as in vitro chemical tools.¹¹Starting in

programmed cell death, is an essential process for the

late 2016, a series of potent and selective Mcl-1 inhibitors were

elimination of no longer needed and dangerous cells.¹Evasion

disclosed (Figure 1) some of which have also recently entered

of apoptosis is recognized as a critical element of the

clinical development.¹²The long-standing interest in Mcl-1 as

development as well as sustained expansion of tumors and

a target and the late emergence of Mcl-1 targeting drug

also underlies resistance to diverse anticancer treatments.²

candidates suggest that drugging Mcl-1 is highly desirable but

Mcl-1 is a member of the Bcl-2 family, critical regulatory

also very challenging. We have recently reported our successful

proteins of the mitochondrial apoptotic pathway, and is

eﬀorts to establish a drug discovery platform for antiapoptotic

frequently upregulated in cancer.³Moreover increased

targets¹³and its use in the identiﬁcation of fragment hits for

expression of the MCL1 gene through transcriptional or

Mcl-1 and their development into a lead compound targeting

Mcl-1.¹⁴The present manuscript describes our eﬀorts to

post-transcriptional mechanisms was observed as a down-

stream consequence of several key oncogenic pathways.⁴Mcl-1

optimize this lead into a clinical candidate and details the

is needed to sustain the growth of diverse tumors, including

unexpected events encountered in this process.

acute myeloid leukemia (AML),⁵MYC-⁶or BCR-ABL-driven

pre-B/B lymphomas,⁷certain breast cancers as well as non-

RESULTS AND DISCUSSION

■

small-cell lung carcinoma (NSCLC) derived cell lines that

Our starting point (1a) exhibited aﬃnity on Mcl-1 (28 nM

carry MCL1 gene ampliﬁcations.⁸Some compounds that

K_i)¹⁴and remarkable selectivity against other Bcl-2 family

broadly inhibit gene transcription or protein translation exert

their cytotoxic eﬀects in tumor cells (at least in part) by

downregulating Mcl-1.⁹

Received: July 27, 2020

In clinic, the highly promising activity of the Bcl-2-selective

inhibitor venetoclax (Venclexta), which led to its approval in

relapsed/refractory chronic lymphocytic leukemia (CLL)

patients with 17p deletion and in AML in combination, has

validated the concept of direct apoptosis activation in cancer

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

A

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 1. Structures of some recently disclosed potent and selective Mcl-1 inhibitors with reported in vivo eﬃcacy.

members with an impact on the viability of Mcl-1 dependent

multiple myeloma cell line H929^12aat the single digit

micromolar level. The primary objective of our lead

optimization eﬀort was to further enhance the aﬃnity of our

inhibitor. We determined the X-ray structure of 1a bound to

Mcl-1 (Figure 2), which suggested two main areas of

moiety in this region. Further exploring the limits of this

pocket, we investigated some monosubstituted aromatics.

Introducing a methyl group next to the biaryl link of thiophene

(1j) was detrimental, due probably to the fact that this change

does not allow the two aromatic rings to adopt a beneﬁcial

coplanar conformation. If the substituent was shifted into the

3- or 4-position of the monocycle, then it was well tolerated on

both the ﬁve (1k) and six membered (1l) substituent. On the

other hand, the replacement of the phenyl ring of 1h by 4-

pyridyl (1m) resulted in a decrease of both the aﬃnity and

activity in H929 cells, due probably to the polarity of the latter

leading to increased desolvation and decreased cell penetrance.

The next set of experiments explored the structure−activity

relationship with analogues carrying a methoxy substituent on

the lactic acid part (Table 1, 2a−l). The reference compound

2a exhibited an aﬃnity for Mcl-1 of 20 nM, while its cellular

activity was in the low micromolar range. The change of the 6-

ethyl substituent to propynyl (2b) led to a similar change as

observed for 1a. The aﬃnity was slightly decreased while the

cellular activity improved considerably. Next we tested the

ﬂuorophenyl analogues 2c−f. Neither the position of the

ﬂuorine on the benzene ring nor the number of ﬂuorine

substituents had a major eﬀect. All four compounds had similar

aﬃnity for Mcl-1 (9−20 nM) and showed good cellular activity

varying in a narrow range between 190 nM for 2c and 230 nM

for 2e. Changing the ﬂuorine to chlorine (2g) did not lead to

signiﬁcant changes. Replacement of the 3-chlorophenyl

substituent by 3-pyridyl (2h) was well tolerated for target

aﬃnity but was detrimental for the cellular activity. Finally, we

introduced a series of furane derivatives into the 6-position

(2i−l). The parent compound 2j showed a signiﬁcant

improvement both in aﬃnity (4.0 nM vs 20 nM for 2a) and

in cellular activity (110 nM). The halogenated furane

derivatives were equally potent. Their aﬃnity for Mcl-1 ranged

between 3 and 4 nM and achieved sub-100 nM cellular

activities for the ﬁrst time. The crystal structure of 2g bound to

Mcl-1 was obtained, and overlaying the bound structures of 1a

and 2g (Figure 3) nicely demonstrated the conformational

changes of Mcl-1 necessary to accommodate the latter

compound. The superimposition revealed the movement of

helix 4 as its backbone peels away from the binding site along

with some side chain reorientation to give a more capacious

pocket, which is necessary to hold a bigger group such as

chlorophenyl from 2g.

Figure 2. Structure of our Mcl-1 lead compound 1a and the X-ray

structure of its complex with Mcl-1 (PDB code 6QYO). S2 is

highlighted in cyan, S4 and S5 are in pink. Residues of interest are as

labeled.

exploitation: the more occluded hydrophobic S2 pocket that

the 6-ethyl occupies and the more solvent exposed S4−S5 area

extending from the benzyl across Thr266. The 6-ethyl moiety

occupies the S2 pocket surrounded by hydrophobic residues

(Val 249, Val253, Met231, and Met250) from helixes 3 and 4.

Our ﬁrst attempts were directed at optimizing the ﬁlling of

the S2 pocket. Since the X-ray structure of 1a suggested that

we only have limited space, a set of analogues (1b−d) carrying

a small apolar substituent were synthesized and tested. While

these modiﬁcations had only a minor eﬀect on the target

aﬃnity, their inﬂuence on the cellular activity was more

marked. The biggest improvement was achieved by the rigid 1-

propynyl substituted analogue (1d), which improved activity

10-fold, probably through improved cell penetration. We also

synthesized the cyclopropylated analogue 1e that should not ﬁt

into the S2 pocket but surprisingly we observed no detrimental

eﬀect on the on-target aﬃnity, suggesting that Mcl-1 was able

to accommodate bigger substituents in this region through

conformational rearrangement. In the next round we tested

some sterically more demanding aromatic groups. The

monocyclic furyl (1f), thienyl (1g), and phenyl (1h)

substituents were equally accommodated by the protein

resulting in aﬃnities like 1a, while their increased lipophilicity

led to a marked improvement of their cellular activity. Further

increasing the substituent to the bicyclic benzofurane (1i)

resulted in a considerable drop in aﬃnity marking the inability

of Mcl-1 to rearrange suﬃciently to accommodate a bicyclic

We also assessed the in vitro ADME properties of selected

compounds (Table 1 in Supporting Information). In general,

the modiﬁcations in the 6-position had only a minor eﬀect on

the predicted absorption (low to moderate) and liver

B

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

observed after 2i treatment, with 80 and 120 fold increase (at

25 mg/kg and 75 mg/kg, respectively) compared to untreated

tumors, showing in vivo apoptosis induction in the AMO-1

tumors after 2i treatment.

Having optimized the substituent in the S2 pocket, we

continued with the variation of the phenyllactic acid moiety for

compounds bearing the 4-ﬂuorofuryl (3) or the 4-ﬂuorophenyl

(4) moiety in position-6 in parallel (Table 2). We switched to

a new assay format more suitable to assess the aﬃnity of very

potent binders toward Mcl-1, a static quenching assay. We

introduced a set of ﬁve diﬀerent substituents onto the ortho-

position of the benzene ring, which was suggested to be the

preferred vector toward the S4−S5 pockets based on the X-ray

structures (Figure 2). The triﬂuoroethyl derivatives (3a, 4a)

showed a marked diﬀerence in aﬃnity with 3a being 10 times

more potent, and its cellular activity was also markedly better

(31 nM vs 114 nM, Table 2). A very similar behavior was

observed for the tetrahydrofurylmethyl (3b, 4b) and

pyridylmethyl (3c, 4c) molecule pairs. The ﬂuorofuryl

analogues (3b, 3c) showed 4- to 5-fold higher aﬃnity and

3−4 times higher cellular activity than the corresponding

ﬂuorophenyl derivatives 4b and 4c. Interestingly, replacing the

pyridine by pyrazine (3d and 4d) maintained the diﬀerence in

the aﬃnities (cf. 0.90 nM vs 5.0 nM), but the cellular activities

became equipotent (36 nM vs 42 nM). Finally, replacing the

pyrazine by N-methylpyrazole (3e, 4e) broadened the gap in

aﬃnity (0.86 nM vs 21 nM). In cellular activity we observed a

marked but smaller diﬀerence with 3e registering at 44 nM and

4e at 114 nM.

Figure 3. Comparison of the X-ray structures of the Mcl-1 inhibitors

1a (orange, PDB code 6QYO) and 2g (blue, 6YBG) bound to Mcl-1.

The backbone and side chain movements of Helix 4 improve the

binding of inhibitor 2g.

microsomal stability (high in rodents, intermediate to low in

human). Running the experiments with human hepatocytes for

selected compounds in the presence of plasma resulted in high

stability due probably to the very high plasma protein binding

of these molecules (unbound fraction (fu) below 1%). The in

vitro properties of 2i (good cellular activity and high metabolic

stability) suggested that this compound could also exhibit

some activity in vivo. Mice bearing the Mcl-1 dependent

human multiple myeloma AMO1^12atumors, as sensitive to

Mcl-1 inhibition as H929, were treated intravenously with 2i at

25 mg/kg and 75 mg/kg, respectively. The tumors were

harvested 2 h after treatment, and the amount of cleaved

PARP, a well-established apoptosis marker, was measured

(Figure 4a). Marked cleaved PARP dose dependent eﬀect was

We selected a potent compound from each series, 3e and 4d,

with similar ADME properties (Table 1 in Supporting

Information) and assessed their activity in the in vivo PD

study. Groups of xenografted mice bearing AMO1 tumors were

Figure 4. (A) Dose dependent apoptosis induction of 2i in AMO1 xenografted mice evidenced through PARP cleavage induction 2 h after

treatment (n = 3). (B) Time and dose dependent apoptosis induction by the Mcl-1 inhibitors 3e and 4d in AMO1 xenografted mice evidenced

through PARP cleavage induction (n = 3). (C) Dose dependent antitumor activity of 3e and 4d in AMO1 xenografted mice following iv

administration, for 5 consecutive days (n = 8).

C

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 1

Table 2

1

2

K_imeasured in Mcl-1 quench assay. Measured in H929 cell line

with 10% CFS, 48 h.

treated with 25 mg/kg or 50 mg/kg doses, and the fold

changes in the quantity of cleaved PARP were determined at

diﬀerent time points after iv bolus treatment. The results are

shown in Figure 4b. In each case we observed a robust onset of

apoptosis as evidenced by PARP cleavage registered after 6 h.

For both compounds we observed a dose eﬀect at the tested

doses at time points 6 and 16 h. Interestingly, data suggest that

in the PD study 3e might be about twice as potent as 4d,

showing similar fold activation at the diﬀerent time points at

half the dose. In the case of 3e we see a stronger dose

dependence of PARP activation at 16 h than at 6 h.

Irrespective of the compound or dose, the level of cleaved

PARP after 30 h was close to the baseline in all samples. In

order to test whether those PD results were predictive of

antitumor activity, we evaluated 4d and 3e eﬃcacy on AMO-1

grafted mice. Mice were treated intravenously (iv) with 25 mg/

kg or 50 mg/kg of the respective compound for 5 consecutive

days. In the case of 4d we observed moderate dose dependent

tumor growth inhibition (TGI_max= 96.2% and 125.6% at 25

mg/kg and 50 mg/kg, respectively) (Figure 4c and Table 4).

The antitumor activity of 3e was greater and led to tumor

regression on treatment at the tested doses. At 25 mg/kg, the

tumor regrew at the end of the treatment period, while at 50

mg/kg the eﬀect was lasting, and the regrowth of the tumors

started only 5 weeks later. The marked diﬀerence between

antitumor activity of 3e and 4d suggests that evaluation of

PARP cleavage 16 h after treatment might be used as a PD

marker for compound screening.

To further explore the chemistry in these two subseries, we

obtained the Mcl-1 bound X-ray structures of 3e and 4d

(Figure 5). Both compounds bind in a similar manner to

previous compounds of the series, and the heteroaromatic

rings sit on top of the ridge between the so-called S2 and S4

pockets providing suitable vectors to grow into the latter

region. The lasting tumor regression on 3e treatment

1

2

K_imeasured in Mcl-1 FP assay.¹³Measured in H929 cell line with

10% FCS, 48 h.

D

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 3

Table 4. Tumor Growth Inhibition Data for Compounds

Tested in the AMO1 Eﬃcacy Model

a

dose

compd (mpk) % TGI (day) % TGI_max(day) (median of the group)

time to reach 500 mm³

3e

50

25

50

25

12.5

129.9 (d7)

120.5 (d7)

95 (d7)

63.6 (d7)

111.9 (d7)

118.9 (d7)

113.8 (d7)

120 (d7)

154.9 (d2)

147.5 (d2)

125.6 (d2)

96.2 (d4)

173.1 (d2)

152.8 (d2)

120.5 (d2)

134.6 (d4)

132.4 (d2)

189.5 (d4)

130.8 (d2)

132.6 (d2)

174.3 (d2)

53

23

9

4d

5b

5e

8b

9b

9i

9m

9n

10b

11

7

18

32

31

28

14

38

21

28

32

Mcl-1

MTT IC

b⁵⁰

a

c

compd

R

X

(nM)

PARP 16 h

6.25

106 (d7)

5a

6a

5b

6b

5c

6c

5d

6d

5e

6e

5f

Et

O

0.077

0.34

0.12

21

44

13

31

4.8

16

7.8

11

6.0

41

47

18

6.25

189.5 (d4)

121.2 (d4)

116.8 (d4)

142.5 (d4)

O

NH

O

i-Pr

Bu

32

58

0.57

0.014

0.088

0.046

0.099

0.163

0.76

a

QD5, iv bolus treatment in each case, n = 8.

t-Bu

118

184

309

151

125

107

Changing the linker oxygen in 5c to nitrogen (5f) led to a

signiﬁcant decrease of on-target aﬃnity (0.42 nM vs 0.014

nM) as well as cellular activity (47 nM vs 4.8 nM). In contrast

the replacement of the 4-ﬂuorofuryl moiety by 1-propynyl (7)

led to the highest aﬃnity registered so far at 23 pM

accompanied by a slightly disappointing 18 nM cellular

activity.

Following the identiﬁcation of several compounds that

showed high cellular potency, a selection of them was assessed

in the in vivo PD study in AMO1 xenografted mice with iv

bolus treatment at 12.5 mg/kg. The fold activation results in

the tumors harvested 16 h after treatment are listed in Table 3.

Compounds 5b and 6b gave the weakest response with PARP

increases at 32-fold and 58-fold, respectively. For compounds

5d, 6d, 5e, and 6e we observed a very strong apoptosis

induction after 16 h with PARP increases between 118- and

309-fold, highlighting 5e in particular. The amino acid

analogue 5f was also active in the PD study registering a

125-fold PARP increase. Replacing the aromatic moiety in the

6-position by an acetylene analogue (7) was well tolerated and

gave strong apoptosis induction. In general, we can state that

all studied compounds led to apoptosis induction and most of

them showed a considerable pharmacodynamic eﬀect.

To probe the predictive power of the PD data, two

compounds were selected, 5b as the least active and 5e as the

most active, and tested in the AMO1 eﬃcacy model. Mice

were treated with 12.5 mg/kg of the respective compound on 5

consecutive days using iv bolus administration. The measured

eﬃcacy data are listed in Table 4. Gratifyingly, both

compounds induced rapid tumor regression evidenced by the

respective TGI_maxvalues of 173% and 153% observed 2 days

after beginning of the treatment. This eﬀect was persistent at

day 7 corresponding to the last day of remaining mice in the

CF₃-CH₂-

Bu

0.42

0.023

7

a

b

K_imeasured in Mcl-1 quench assay. Measured in H929 cell line

c

with 10% CFS, 48 h. Fold increase of cleaved PARP measured in

AMO1 xenografted mice 16 h after iv bolus treatment at 12.5 mg/kg

dose (n = 3).

prompted us to explore ﬁrst the ﬁlling of the shallow S4 pocket

through the variation of the substituent on the pyrazole ring.

The methyl substituent in 3e as shown in the X-ray does not

directly interact with Mcl-1 We expected that the conforma-

tional ﬂexibility of the two-atom linker should allow the

pyrazole ring to ﬂip and orient its substituent toward S4 to

interact with Mcl-1 more productively. A collection of alkyl-

substituted analogues was prepared and tested both in the 4-

ﬂuorofuryl (5a−e) and 4-ﬂuorophenyl (6a−e) series. Increas-

ing the length of the alkyl chain led to considerable

improvement in both subseries. Growing from methyl (3e,

4e) through ethyl (5a, 6a) to butyl (5c, 6c) resulted in a

signiﬁcant improvement of both aﬃnity and cellular activity.

Increasing the branching to isopropyl (5b, 6b) and tert-butyl

(5d, 6d) was equally tolerated reaching sub-100 pM aﬃnities

of 10 nM or better cellular activity for both 5d and 6d. The

triﬂuoroethyl-substituted molecules were also synthesized. In

the ﬂuorofuryl subseries with 5e we reached 6 nM activity in

the viability assay, while the ﬂuorophenyl analogue 6e was less

active at 41 nM. At this stage we also wanted to see if the

replacement of the hydroxy acid by an amino acid or the

propynyl substitution in the 6-position that was beneﬁcial for

less decorated molecules (e.g., 1d and 2b) is tolerated in 5c.

Figure 5. X-ray structures of the Mcl-1 inhibitors 3e (PDB code 6YBJ), 4d (6YBK), and 9m (6YBL) bound to Mcl-1.

E

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 5

a

b

c

compd

R

X

Mcl-1 (nM)

MTT IC₅₀(nM)

PARP 16 h

8a

9a

8b

9b

8c

9c

8d

9d

9e

9f

9g

9h

9i

9j

9k

9l

-OMe

O

N

O

N

0.12

0.36

0.13

0.64

0.043

0.72

0.030

0.036

0.052

0.050

0.081

0.055

0.73

0.034

0.048

0.028

0.029

0.220

0.011

0.014

15

24

NA

133

129

131

78

115

NA

32

138

141

151

237

120

25

morpholino

3.1

14.4

3.9

5.7

4.0

3.7

12

5.8

8.1

4.7

1.7

3.5

5.7

3.5

1.7

14

2-methoxyethyl

4-pyridyl

2-pyridyl

3-pyridyl

2-furyl

2-tolyl

4-methylpyrid-3-yl

3-methylpyrid-4-yl

5-methoxy-2-methylpyrid-4-yl

2-hydroxymethylphenyl

2-methoxyphenyl

68

285

60

83

59

9m

9n

10a

10b

11

3.9

0.9

4.7

0.17

251

a

b

c

K_imeasured in Mcl-1 quench assay. Measured in H929 cell line with 10% CFS, 48 h. Fold increase of cleaved PARP measured in AMO1

xenografted mice 16 h after iv bolus treatment at 12.5 mg/kg dose (n = 3).

Scheme 1. Synthesis Strategies Followed in the Preparation of our Mcl-1 Inhibitors

F

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

control group (TGI = 112% and 119% with 5b and 5e,

respectively). Diﬀerentiation between the two compounds was

observed on the time to relapse, as shown by the time to reach

500 mm³. Indeed, while time to reach 500 mm³occurred 18

days after treatment for 5b, this same size was reached 32 days

after 5e treatment showing a superiority of the latter

compound.

equipotent registering at 34 pM and 48 pM, respectively.

Interestingly the cellular activity of 9i was very high showing a

1.7 nM IC₅₀, while 9j and 9k were in the expected activity

range at 3.5 nM and 5.7 nM. Finally, we added an oxygen atom

to the ortho-tolyl moiety of 9h resulting in the hydrox-

ymethylphenyl (9l) and methoxyphenyl (9m) substituted

compounds. Compared to the tolyl analogue, both 9l and 9m

showed some improvement in aﬃnity (28 pM and 29 pM vs

55 pM) as well as in cellular activity (3.5 nM and 1.7 nM vs 4.7

nM), 9m being the more potent compound.

Out of this diverse set of very potent Mcl-1 inhibitors we

tested 9e−m in our PD assay at 12.5 mg/kg. All of the tested

compounds induced apoptosis, and the highest activities were

observed for 9i (237-fold) and 9m (285-fold). Although 9i and

9m were the most active both in the cellular and in vivo PD

assays, when we compared the ranking of the other compounds

in the cellular and PD assays, it was diﬃcult to see any

correlation. We progressed 9i and 9m and tested their in vivo

eﬃcacy treating tumor bearing mice on 5 consecutive days

with a dose of 6.25 mg/kg. Both compounds induced tumor

growth inhibition, and 9m showed the stronger eﬃcacy

considering both the magnitude and the duration of tumor

growth inhibition (189.5% of TGI_maxand 38 days to reach 500

mm³).

Having identiﬁed 9m as a very potent compound, we

prepared some of its analogues including the amino acid 9n,

the hydroxy (10a) and amino acids (10b) bearing a propynyl

substituent in the 6-position of the thienopyrimidine core

instead of ﬂuorophenyl, and the dimethylamino analogue 11.

Besides determining their aﬃnity and cellular activity, these

compounds were also characterized in the in vivo PD assay. By

comparison of the hydroxy acid−amino acid pairs (9m−9n,

10a−10b), 9m showed higher aﬃnity for the target and better

cellular activity (1.7 nM vs 14 nM) than 9n, while the aﬃnity

diﬀerence between 10a and 10b was marginal, and the

propynyl compounds 10b showed a remarkable 0.9 nM activity

surpassing its hydroxy acid analogue 10a (3.9 nM). Although

the aﬃnity of 11 toward Mcl-1 was only 170 pM, its cellular

activity was quite good at 4.7 nM. In the in vivo setting 11

showed a remarkable PD response (251-fold PARP cleavage

induction) as compared to the other compounds (9n, 10a−b)

(59- to 83-fold). As expected, 11 also showed the strongest

antitumor activity at 6.25 mg/kg, although not reaching the in

vivo eﬃcacy of 9m.

Although 5e showed very robust tumor regression, some of

its other characteristics (e.g., light sensitivity) were suboptimal;

therefore, we have also explored the further optimization of 4d.

As its Mcl-1 bound X-ray structure suggests (Figure 5), the

vector for growing into S4 is the position meta to the

oxymethyl moiety. We have established (data not shown) that

replacing the pyrazine by pyrimidine is beneﬁcial in terms of

both aﬃnity and compound behavior. For the ﬁrst set of

compounds bearing diverse substituents on the pyrimidine

ring, we prepared both the ﬂuorofuryl (8a−d) and

ﬂuorophenyl (9a−d) analogues. The small polar methoxy

substituent (8a, 9a) was tolerated in both series showing

aﬃnities in the several hundred picomolar range and 15 and 24

nM cellular activities, respectively (Table 5). Replacing

methoxy by morpholine (8b, 9b) had little eﬀect on the

aﬃnity but improved the cellular activity of the compounds.

The 2-methoxyethyl analogues showed an increase in aﬃnity

for 8c registering at 43 pM while 9c remained at 720 pM,

which was accompanied by single digit nanomolar cellular

potencies (3.9 and 5.7 nM, respectively). The introduction of

an aromatic substituent, the 4-pyridyl moiety, was also

beneﬁcial. At this point the so far apparent diﬀerence in

aﬃnity between the ﬂuorofuryl and ﬂuorophenyl subseries

disappeared, 8d and 9d having alike values of 30 pM and 36

pM, respectively, and their cellular activities were also very

close (4.0 nM vs 3.7 nM).

To assess the predictivity of the in vitro assays, compounds

8b−c and 9b−c were tested in our PD model. They were

injected at a dose of 12.5 mg/kg in AMO-1 grafted mice, and

the tumors were harvested 16 h after the treatment. The fold

increase values of cleaved PARP are described in Table 5. All

four compounds showed strong apoptosis induction, slightly

less pronounced for 9c as compared to 8b, 9b, and 8c, and it

was diﬃcult to correlate the PD response with their aﬃnity or

cellular activity. Interestingly, activity of those ﬂuorophenyl

derivatives matched the antitumor activity of the ﬂuorofuryl

compound 5e.

It was reassuring to see that despite its lesser activity in the

in vitro models, a ﬂuorophenyl analogue could match the in

vivo eﬃcacy of the ﬂuorofuryl derivatives, so at this point we

decided to focus our eﬀorts on the ﬂuorophenyl subseries.

First, we assessed the position of the nitrogen atom in the

pyridyl substituent. The 2-pyridyl derivative (9e) showed a

slight decrease of aﬃnity (52 pM vs 36 pM for 9d) and a more

marked drop of cellular activity (12 nM vs 3.7 nM), while the

3-pyridyl analogue (9f) lay in between with an aﬃnity of 50

pM and activity of 5.8 nM. Replacing 2-pyridyl by the 2-furyl

moiety (9g) led to a modest drop in aﬃnity (81 pM vs 52 pM)

that was accompanied by an improvement of cellular activity.

On the other hand, forcing the aromatic ring out of coplanarity

with the o-tolyl substituent (9h) did not break the aﬃnity (55

pM) with activity retained in the cellular assay at 4.7 nM. We

also assessed the eﬀect of methyl substitution on the pyridine

substituent. Of the 4-methylpyrid-3-yl (9i), 3-methylpyrid-4-yl

(9j), and 2-methyl-5-methoxypyrid-4-yl (9k) analogues, 9i

showed a marked drop in aﬃnity while 9j and 9k were nearly

The synthesis strategies furnishing our Mcl-1 inhibitors are

depicted in Scheme 1. All of the developed inhibitors could be

constructed from four key building blocks: a halogenated

thienopyrimidine core, a lactic acid derivative attached to the

4-position of the thienopyrimidine, a decorated ortho-

tolylboronic acid or ester coupled to the 5-position of the

core, and a smaller substituent in the 6-position connected to

the core through a C−C bond. In the early stages of our work

the late stage diversiﬁcation of the 6-position was achieved in

two ways. The synthesis of the 1 series started from 6-bromo-

4-chloro-5-iodothieno[2,3-d]pyrimidine and followed a nucle-

ophilic substitution (4-position), Suzuki coupling (5-position),

Suzuki-coupling (6-position) sequence that was concluded by

Mitsunobu coupling and ester hydrolysis to establish the

correct substitution pattern. The formed diastereoisomers were

separated after the ﬁrst Suzuki coupling. A setback of this

approach was the formation of 5,6-disubstituted byproducts in

the ﬁrst Suzuki coupling. The alternative route explored in the

synthesis of the 2 series started from 4-chloro-5-iodothieno-

G

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 6. (A) Coimmunoprecipitation assay of HeLa cells expressing Flag-tagged Mcl-1, BCL-2 or BCL-X_Las indicated. Cells were treated with

diﬀerent concentrations of 9m for 2 h before lysis. Cleared lysates were used for immunoprecipitation using anti-Flag antibody. The cell lysates

(input) and immunoprecipitates (IPs) were analyzed by immunoblotting with anti-Bax, Bak, or Flag antibodies as indicated. ∗ indicates nonspeciﬁc

bands. (B) Western blot analysis of endogenous Mcl-1 protein levels in HCT-116 cells treated with 9m for 16 h at the indicated concentrations.

Actin level is used as loading control.

Figure 7. (A) H929 cells were treated for 6 h with increasing concentration of 9m. Cleaved PARP was measured using MesoScale Discovery

Apoptosis panel. Mean data of two independent biological experiments are represented. (B) Apoptosis induction in WT or BAX/BAK KO THP1

cells treated with 9m at the indicated concentration for 2 h. Cells were analyzed by ﬂow cytometry for PI and annexin V-FITC labeling. Mean and

individual points from two biological replicates are shown. CT indicates that cells were treated with DMSO only.

[2,3-d]pyrimidine. On this compound the Suzuki coupling in

the 5-position was selective, and subsequent iodination of the

6-position yielded R9a. Introduction of the homochiral lactic

acid R2b by nucleophilic substitution was followed by the

separation of the diastereoisomers (1:1 mixture), derivatization

of the 6-position in a second Suzuki coupling, and the

aforementioned concluding steps.

For the diversiﬁcation of the phenyl lactate moiety in

position 4 (3−11 series) we used a diﬀerent strategy.

Exploiting the enhanced reactivity of the thienopyrimidine’s

6-position in cross-coupling reactions, we started from the 6-

iodo-4-chloro-thieno[2,3-d]pyrimidine bearing a bromine

(R1a) or iodine (R1b) in the 6-position. Selective and high

yielding Suzuki or Sonogashira coupling in the 6-position was

followed by nucleophilic substitution in the 4-position using

the THP-protected enantiopure lactate derivative R2a. We

could exploit the stereochemical directing eﬀect of the lactate

in the following Suzuki coupling in the 5-position to obtain

atropoisomer ratios up to 4:1 in our favor, which could be

separated by ﬂash column chromatography. Introduction of

the solubilizer (mostly 2-(4-methylpiperazin-1-yl)ethanol) in

Mitsunobu coupling and removal of the THP protecting group

gave key intermediates R5a−c. Diversiﬁcation of the 4-position

followed by the hydrolysis of the ester gave the desired

products. We carefully assessed that the stereochemical

integrity of our compounds remained intact during the applied

reaction conditions. The diastereomeric compounds that

would arise from epimerization of one of the chirality elements

are easy to detect due to their distinct chromatographic and

NMR spectroscopic behavior. In the case of our most

advanced compounds (i.e., 9m), we also developed a chiral

analytical method that was able to distinguish all four

stereoisomers and that conﬁrmed the stereochemical integrity

of our compounds.

On the basis of the in vitro and in vivo data, 9m was

identiﬁed as a potential preclinical candidate and was further

characterized. We obtained the X-ray structure of 9m in

complex with Mcl-1 (Figure 5). The thienopyridimine part of

the 9m binds exactly the same as in 1a, 2g, 3e, and 4d. The

oxymethyl linker from the benzyl crosses the Thr266 ridge and

positions the pyrimidine-methoxyphenyl in S4. While methox-

yphenyl sits in a small hydrophobic cavity surrounded by

H

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Val220, Val265, and Phe319, the pyrimidine engages with

Asn260 via two water molecules, one of which is further

coordinated by the methoxy of the methoxyphenyl group.

The fact that 9m inhibits the interaction of two proteins at a

hydrophobic surface predicted a signiﬁcant plasma protein

binding that was in line with the measured free fractions of

0.05% and 0.04% in human and mice plasma, respectively. As a

consequence, 9m showed good PK properties in mice, in

agreement with the very low in vitro hepatocytes clearance

measured in the presence of plasma. Administered at 6.25 or

12.5 mg/kg in mice, the observed clearance was 5.3 and 6.0

apoptosis readout, was monitored. As shown in Figure 7A, 9m

induced PARP cleavage in a dose-dependent manner

registering as early as 1 nM and with a maximal eﬀect

observed from 30 nM. Finally, to prove that the observed

apoptosis was Bax/Bak dependent, THP1 cell line expressing

Bax and Bak (WT) and also its BAX/BAK KO analogue were

treated with diﬀerent doses of 9m for 2 h. Cells analysis by

ﬂow cytometry revealed a dose-dependent onset of apoptosis

in the Bax/Bak WT cell line, while the cells devoid of Bax and

Bak showed no sign of apoptosis (Figure 7B).

We have also analyzed the activity of 9m in a panel of cell

lines from diﬀerent types of hematological malignancies (Table

4 in Supporting Information). We have considered the cell

lines to be highly sensitive to 9m when the IC₅₀was below 0.1

μM, moderately sensitive when the IC₅₀was between 0.1 μM

and 1 μM, and insensitive when the IC₅₀was higher than 1

μM. Interestingly, all the AML and DLBCL cell lines tested

and the majority of the multiple myeloma and other lymphoma

cell lines were highly sensitive to 9m. From the three ALL cell

lines tested, one was highly sensitive, and two were insensitive,

and all ﬁve CML cell lines were insensitive. These results are in

line with previous data published by our team with the S63845

Mcl-1 inhibitor compound.^12a

We next assessed the 9m compound in AMO1 xenografted

mice by treating the animals at diﬀerent doses (3.125 mg/kg,

6.25 mg/kg, and 12.5 mg/kg) by iv for 5 consecutive days

(Figure 8). A dose-dependent antitumor activity was observed

with TGI_maxof 89.1%, 115.8%, and 162.8% at 3.125 mg/kg,

6.25 mg/kg, and 12.5 mg/kg, respectively, with an outstanding

complete regression for all treated animals lasting for 35 days

observed at 12.5 mg/kg. Promisingly from a clinical

perspective, animals treated at 12.5 mg/kg weekly for 4

weeks experienced similar tumor growth inhibition as

compared to animals treated daily (TGI at D10 = 111.6% vs

108.4% after daily or weekly treatment, respectively). Taken

together, those results highlight the potential of S64315 as an

Mcl-1 inhibitor to be used weekly in hematological

malignancies in clinic.

A preliminary assessment of the safety pharmacology proﬁle

(hERG and oﬀ target activity assays) was performed with 9m.

The ﬁrst results showed no signiﬁcant alerts: the hERG

inhibition indicated IC₅₀> 3 μM (maximal solubility of 9m in

this assay) and the selectivity against all oﬀ targets was over

400-fold. The potential drug−drug interaction in human was

assessed with a high throughput assay using human CYP450

transfected cells: 9m was found mainly metabolized by

CYP2C8 and exhibited an inhibitory potential toward

CYP3A4 (IC₅₀= 1.8 μM, Table 3 in Supporting Information).

Figure 8. Antitumor activity of 9m on AMO-1 grafted mice (n = 10).

Mice were treated at diﬀerent doses of 9m by iv for 5 consecutive

days (treatment QD) or weekly for 4 weeks (treatment Q7D4). Data

are represented as the mean SEM.

mL min⁻¹kg⁻¹leading to high systemic exposures and a

terminal half-life of around 5 h at both doses.

To conﬁrm that 9m acts in cells through selectively

displacing proapoptotic BH3 domain containing proteins

from Mcl-1, HeLa cells expressing Flag tagged Mcl-1, Bcl-2,

and Bcl-x_Lwere treated with diﬀerent doses of 9m. Following

immunoprecipitation using anti-Flag antibody, the endogenous

Bak and Bax proteins complexed with Mcl-1, Bcl-2, and Bcl-x_L

were monitored (Figure 6A). As expected on the basis of its

low aﬃnity toward Bcl-2 and Bcl-x_L(58 μM and 237 μM,

respectively, in the FP assay¹³), 9m displaced Bak and Bax

proteins from Mcl-1 but not from Bcl-2 or Bcl-x_Lcomplexes.

The shift between the cytotoxic potency and the doses

required to visualize Bax and Bak displacement from Bcl-2

family members is well documented^12a,15,16and could be

explained by two reasons. First an experimental bias coming

from the fact that once the cell lysates are prepared, part of the

compound can dissociate from Mcl-1 allowing Bax and Bak to

reassociate, which would result in an apparent loss of potency

in this Co-Ip setting. Second, it is not known how much Bax

and/or Bak should be freed from Mcl-1 in order to induce

cytotoxicity, and this amount might be very little and not easily

detectable in a Western blot assay. Selective Mcl-1 inhibitors

were also reported to stabilize Mcl-1 protein in a dose-

dependent manner.^11a,12a,bWhen the HCT116 cell line (not

sensitive to Mcl-1 inhibition^12a) was treated with diﬀerent

doses of 9m, we observed a signiﬁcant dose-dependent

increase of the endogenous Mcl-1 protein. As reported earlier,

this assay was very sensitive registering activity at doses as low

as 0.3 nM. Having established Mcl-1 target hitting, we next

assessed whether cell killing observed following 9m treatment

is the consequence of apoptosis induction. To this end, H929

multiple myeloma cell line was treated for 6 h with diﬀerent

doses of 9m, and PARP cleavage, a commonly accepted

CONCLUSION

■

The systematic optimization of our lead Mcl-1 inhibitor that

possessed nanomolar aﬃnity and micromolar cellular activity

led to the identiﬁcation of the preclinical candidate S 64315/

MIK665 (9m). The structure guided optimization revealed the

plasticity of Mcl-1, enabling it to accommodate substituents

that were not expected to be tolerated on the basis of prior X-

ray structures. The ﬁlling of the S2 pocket resulted in a

signiﬁcant increase of activity. A signiﬁcant part of our eﬀorts

was directed at growing our molecule into the S4−S5 region of

Mcl-1. This part of the BH3 groove consists of more shallow

pockets so the structural guidance was complemented by

systematic modiﬁcations of our inhibitors. Our activities led to

the identiﬁcation of a series of selective and highly potent Mcl-

I

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

0.4% Triton X-100) and centrifuged for dissociation at 6500 rpm for

30 s using Precellys.

1 inhibitors that showed strong apoptosis induction in in vivo

models, which was also translated into eﬃcient tumor growth

inhibition. Of the compounds synthesized and tested, 9m

stood out and was selected as a preclinical candidate. Its mode

of action was conﬁrmed, and its other properties were also

favorable for progressing it into preclinical development. This

compound, also known as S64315/MIK665, is currently in

clinical development against diﬀerent hematological malig-

nancies.

Lysates (5 μg proteins) were assayed for immunodetection of

cleaved PARP with MSD apoptosis panel whole cell lysate kit (MSD

K15102D). Lysates were assayed in a MSD 96 well plate according to

the manufacturer’s instructions and analyzed on the Sector Image

2400. Data are expressed as relative light unit (RLU).

Bax/Bak Dependency. THP1 Bax/bak−/− generation was

described in Casara et al.¹⁷Brieﬂy, THP-1 (ATCC CCL-2) cells

were ﬁrst transduced with EF1a-Cas9- 2A neomycin (Sigma-Aldrich)

and selected under neomycin for 10 days before being sorted as

single-cells for the isolation of clones. One clone was then further

transduced with pLV-U6g-EGFP-gRNA BAK1, and cells were sorted

as single-cells based on the expression of the GFP reporter gene. Cells

were then further transduced with pLV-U6g-Puromycin gRNA BAX

and put under puromycin selection (1 μg/mL) for 9 days before being

sorted as single-cells for the isolation of clones. BAX and BAK

knockout eﬃciency on diﬀerent clones was veriﬁed by Western blot.

THP-1 cells were treated with the indicated compounds for 2 h,

centrifuged, and washed with binding buﬀer (10 mM Hepes, 140 mM

NaCl, 2.5 mM CaCl₂). Cells were incubated with 200 μL of binding

buﬀer containing annexin V-FITC (Invitrogen) and propidium iodide

(PI, Sigma) during 15 min at 20 °C in the dark. THP-1 cells were

incubated with 200 μL of binding buﬀer containing annexin V-APC

(BD Biosciences) and DAPI (Sigma). 400 μL of binding buﬀer was

added, and samples were kept at 4 °C before cytometric analysis. For

each sample, 10⁴cells were analyzed by ﬂow cytometry on Epics XL/

MCL ﬂow cytometer (Beckman Coulter, France). Fluorescence was

collected at 520 nm (FITC), 630 nm (PI), 660 nm (APC), and 470

nm (DAPI). The number of apoptotic cells (addition of primary

apoptosis, secondary apoptosis, and necrosis) was normalized to the

total number of cells per tube.

Animals and Treatment Groups. Healthy female CB-17 SCID

mice, 6−7 weeks old, were obtained from Charles River. Mice were

kept either at Oncodesign or in the Servier Institute-speciﬁed

pathogen-free animal area for mouse experimental purpose (facility

license numbers B78-100-2 and A21231011EA). The care and use of

animals used in this facility are strictly applying to European and

national regulation for the protection of vertebrate animals used for

experimental and other scientiﬁc purposes (Directives 86/609 and

2003/65).

SCID mice were inoculated with 0.1 mL volume containing 5 ×

10⁶cells subcutaneously in the right ﬂank. AMO-1 cells were

resuspended in a 50:50 mixture of growth media and Matrigel (BD

Biosciences). Width and length of the tumor were measured 2−3

times a week using an electronic caliper. Tumor volume was

calculated using the formula length × width²/2. When tumor volume

reached approximately 200 mm³, mice were randomized in diﬀerent

groups before treatment (n = 3 for pharmacodynamic studies and n =

8 to 10 for eﬃcacy studies). Compounds were formulated in HPbCD

20% (Fisher Scientiﬁcs)−HCl (25 mM) and administrated with the

doses and schedules described in the ﬁgure.

EXPERIMENTAL SECTION

■

MTT Cell Viability Assay of H929 Cell Line. H929 cells

(purchased from ATCC) were cultured in RPMI 1640 medium

supplemented with 10% heat inactivated FBS, 2 mM ^L-glutamine, 100

U/mL penicillin, 100 μg/mL streptomycin, and 10 mM Hepes, pH =

7.4 at 37 °C, in 5% CO2/95% air. Cells were grown at 37 °C in a

humidiﬁed atmosphere with 5% CO₂. Cell viability was measured

using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide) colorimetric assay. Cells were seeded in 96-well microplates

at a density to maintain control (untreated) cells in the exponential

phase of growth during the entire experiment. Cells were incubated

with compounds for 48 h followed by incubation with 1 mg/mL

MTT for 4 h at 37 °C. Lysis buﬀer (20% SDS) was added, and

absorbance was measured at 540 nm 18 h later. All experiments were

repeated at least 2 times in triplicate. The percentage of viable cells

was calculated and averaged for each well: % growth = (OD treated

cells/OD control cells) × 100, and the IC₅₀, concentration reducing

by 50% the optical density, was calculated by a linear regression

performed on the linear zone of the dose−response curve.

MCL-1 Stabilization. HCT116 cells (purchased from ATCC)

were seeded at 0.25× 10⁶cells in 35 mm dishes in RPMI 1640

medium supplemented with 10% decomplemented fetal bovine serum

(FBS), 2 mM ^L-glutamine, 100 U/mL penicillin, 100 μg/mL

streptomycin, and 10 mM Hepes, pH = 7.4. Cells were grown at

37 °C in 5% CO₂/95% air. 48 h later, cells were treated with S64315

at the indicated doses for 16 h and harvested in lysis buﬀer (10 mM

Hepes, pH7.4, 142.5 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1%

NP40, and phosphatase and protease inhibitors). Lysates (20 μg)

were analyzed by immunoblot using the following antibodies: anti-

Mcl-1 (Santa Cruz sc-819) and anti-actin (Millipore MAB1501R).

Coimmunoprecipitation. HeLa cells (purchased from ATCC)

were plated at 2.5 × 10⁵cells in 60 mm dishes 24 h before

transfection in DMEM medium (containing 10% FCS, 1 mM Hepes,

100 U/mL penicillin, 100 μg/mL streptomycin) in 5% CO₂

incubator. HeLa cells were transiently transfected, using Eﬀecten

reagent (Qiagen), with Flag-tagged Mcl-1, Bcl-x_Lor Bcl-2 expression

vectors. 24 h later, cells were treated with S64315 during 2 h and

harvested in lysis buﬀer (10 mM Hepes, pH 7.5, 150 mM KCl, 5 mM

MgCl₂, 1 mM EDTA, 0.4% TritonX100, protease inhibitors cocktail

(Calbiochem 539134) and phosphatase inhibitors cocktail (Calbio-

chem 524625)). The cleared lysates were then subjected to

immunoprecipitation with anti-Flag M2 agarose beads (Sigma). The

immunoprecipitates and inputs were analyzed by immunoblot using

the following antibodies: anti-Bax (Santa Cruz sc-493), anti-Bak (BD

556996) and anti-Flag M2 (Sigma).

Tumor growth inhibition TGI was calculated and at the last day of

control group and at the greatest response (TGI_max) using the

following equation:

Apoptosis Induction. H929 cells (purchased from ATCC) were

seeded at 2 × 10⁶cells in 35 mm dishes in RPMI 1640 medium

supplemented with 10% decomplemented fetal bovine serum (FBS),

2 mM ^L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin,

and 10 mM Hepes, pH = 7.4. Cells were grown at 37 °C in 5% CO₂/

95% air. 24 h after, cells were treated with S64315 for 6 h and

harvested in lysis buﬀer (10 mM Hepes, pH 7.4, 142.5 mM KCl, 5

mM MgCl₂, 1 mM EDTA, 1% NP40, phosphatases and proteases

inhibitors).

median of treated at day x − median of treated at day 0

× 100

median of control at day x − median of control at day 0

(1)

where day x is the day maximum where the number of animals per

group in the control group is suﬃcient to calculate the TGI (%).

The tumor growth delay (TGD) was expressed as a percentage by

which the treated group is delayed in attaining an arbitrary median

volume of 500 mm³relative to the control group. It was determined

using the following formula:

AMO-1 tumors collected after treatment at indicated time in the

ﬁgures were frozen. Piece of tumor was homogenized in lysis buﬀer

(10 mM Hepes, pH 7.5, 150 mM KCl, 5 mM MgCl₂, 1 mM EGTA,

J

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

[(median times of treated to reach 1000 mm³− median times of

control to reach 1000 mm³)/(median times of control to reach

1000 mm³)] × 100

MHz, DMSO-d₆) δ ppm: 12.57 (br s, 1H), 8.09 (s, 1H), 7.65 (s, 1H).

¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 168.3, 155.9, 146.1, 130.8,

126.7, 76.4. HRMS calculated for C₆H₃IN₂OS: 277.9011; found

278.9088 (M + H).

Step B: 4-Chloro-6-iodothieno[2,3-d]pyrimidine. A 1 L round

bottomed ﬂask equipped with mechanical stirrer, thermometer, reﬂux

condenser, and a tube ﬁlled with anhydrous CaCl₂was charged with

113 mL POCl₃and 35 mL N,N-dimethylaniline (0.29 mol). 75.5 g 6-

iodo-3H-thieno[2,3-d]pyrimidin-4-one (0.271 mol) was added

portionwise in 5 min. The reaction mixture was stirred at 105 °C

for 1 h. The resulting suspension was cooled to 10 °C, ﬁltered, and

washed with hexane. The ﬁltered solid was added to icy water and

stirred for 10 min, ﬁltered oﬀ, washed with cold water, Et₂O, and air-

dried to give 75.1 g 4-chloro-6-iodothieno[2,3-d]pyrimidine as a beige

crystalline solid (0.253 mol, 93%). ¹H NMR (500 MHz, DMSO-d₆) δ

ppm: 8.90 (s, 1H), 7.98 (s, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ

ppm: 172.3, 152.8, 151.8, 131.0, 128.9, 86.5. MS (EI, 70 eV) m/z (%

relative intensity, [ion]): 57 (9), 107 (27), 134 (13), 142 (15), 261

(48), 296 (100, [M⁺]).

A complete tumor regression response was considered for the

population with tumors of 25 mm³for at least three consecutive

measurements.

General Synthetic Remarks. All reagents obtained from

commercial sources were used without further puriﬁcation. Anhy-

drous solvents were obtained from commercial sources and used

without further drying.

The reactions were monitored using LCMS and GCMS instru-

ments. Analytical LCMS: Agilent HP1200 LC with Agilent 6140

quadrupole MS, operating in positive or negative ion electrospray

ionization mode. Molecular weight scan range was 100 to 1350 m/z.

Parallel UV detection was done at 210 and 254 nm. Samples were

supplied as a 1 mM solution in MeCN or in THF/water (1:1) with 5

μL loop injection. LCMS analyses were performed on two

instruments, one of which was operated with basic and the other

with acidic eluents.

Step C: 5-Bromo-4-chloro-6-iodothieno[2,3-d]pyrimidine. A 2 L

round bottomed ﬂask equipped with mechanical stirrer, thermometer,

and a bubbler was charged with 600 mL MeCN. 84.9 g 4-chloro-6-

iodothieno[2,3-d]pyrimidine (0.286 mol), 50.9 g NBS (0.286 mol),

and 8.5 mL HBF₄·Et₂O (62.5 mmol) were added. The reaction

mixture was stirred at rt for 16 h. Additional 22.9 g NBS (0.129 mol)

was added to the mixture in three portions in order to reach complete

conversion. After cooling the suspension to 0 °C and stirring for 1 h,

the precipitate was ﬁltered oﬀ, washed with MeCN, and air-dried to

Basic LCMS: Gemini-NX, 3 μm, C18, 50 mm × 3.00 mm i.d.

column at 23 °C, at a ﬂow rate of 1 mL min⁻¹using 5 mM aq

NH₄HCO₃solution and MeCN as eluents.

Acidic LCMS: ZORBAX Eclipse XDB-C18, 1.8 μm, 50 mm × 4.6

mm i.d. column at 40 °C, at a ﬂow rate of 1 mL min⁻¹using water and

MeCN as eluents, both containing 0.02 v/v % formic acid.

Combination gas chromatography and low-resolution mass

spectrometry were performed on Agilent 6850 gas chromatograph

and Agilent 5975C mass spectrometer using 15 m × 0.25 mm column

with 0.25 μm HP-5MS coating and helium as carrier gas. Ion source:

EI⁺, 70 eV, 230 °C. Quadrupole: 150 °C. Interface: 300 °C.

Flash chromatography was performed on ISCO CombiFlash Rf

200i with prepacked silica-gel cartridges (RediSepR_fGold High

Performance).

1

give 86.9 g R1a as a beige crystalline solid (0.231 mol, 81%). H

NMR (400 MHz, DMSO-d₆) δ ppm: 8.94 (s, 1H). ¹³C NMR (100

MHz, DMSO-d₆) δ ppm: 171.3, 152.9, 152.3, 126.0, 112.4, 92.9.

HRMS calculated for C₆HBrClIN₂S: 373.7777; found 374.7853 (M +

H).

R1b: 4-Chloro-5,6-diiodothieno[2,3-d]pyrimidine. Step A: 5,6-

Diiodo-3H-thieno[2,3-d]pyrimidin-4-one. To a well stirred slurry of

61.3 g 3H-thieno[2,3-d]pyrimidin-4-one (396 mmol), 92.4 g H₅IO₆

(405 mmol), 1 L AcOH, 200 mL water, 6 mL cc. H₂SO₄, and 203 g I₂

(799 mmol) were added. The reaction mixture was heated to 110 °C

and stirred for 3 h. The suspension was cooled to rt and then 940 mL

Et₂O was added and stirred further at 10 °C for 30 min. The

precipitate was ﬁltered oﬀ, washed with a 2:1 mixture of Et₂O and

EtOH, ﬁnally with Et₂O, and air-dried to give 94.4 g 5,6-diiodo-3H-

Preparative HPLC puriﬁcations were performed on an Armen Spot

liquid chromatography system with a Gemini-NX 10 μm C18, 250

mm × 50 mm i.d. column running at a ﬂow rate of 118 mL min⁻¹

with UV diode array detection (210−400 nm).

¹H NMR and proton-decoupled ¹³C NMR measurements were

performed on Bruker Avance III 500 MHz spectrometer and Bruker

Avance III 400 MHz spectrometer, using DMSO-d₆or CDCl₃as

solvent. ¹H and ¹³C NMR data are in the form of delta values, given in

parts per million (ppm), using the residual peak of the solvent as

internal standard (DMSO-d₆, 2.50 ppm (¹H)/39.5 ppm (¹³C);

CDCl₃, 7.26 ppm (¹H)/77.0 ppm (¹³C)). Splitting patterns are

designated as s (singlet), d (doublet), t (triplet), q (quartet), sp

(septet), m (multiplet), br s (broad singlet), dd (doublet of doublets),

td (triplet of doublets), qd (quartet of doublets). In some cases two

sets of signals appear in the spectra due to hindered rotation.

HRMS was determined on a Shimadzu IT-TOF, ion source

temperature 200 °C, ESI , ionization voltage ( )4.5 kV. Mass

resolution min 10000.

1

thieno[2,3-d]pyrimidin-4-one (234 mmol, 59%) as a tan powder. H

NMR (500 MHz, DMSO-d₆) δ ppm: 12.60 (br s, 1H), 8.13 (s, 1H).

¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 168.0, 155.0, 146.4, 124.6,

89.4, 88.4.

Step B: 4-Chloro-5,6-diiodothieno[2,3-d]pyrimidine. To a well

stirred slurry of 180 g 5,6-diiodo-3H-thieno[2,3-d]pyrimidin-4-one

(445 mmol) in 2.5 L POCl₃was added 64 mL N,N-dimethylaniline

(507 mmol). The reaction mixture was heated to 105 °C and stirred

for 1.5 h. The resulting suspension was cooled to rt, and 1.5 L hexane

was added, and it was stirred for further 20 min. The precipitate was

ﬁltered oﬀ, washed with hexane and water, then air-dried to give 159.8

1

g R1b (378 mmol, 85%) as a gray crystalline solid. H NMR (400

All obtained products had an LC purity above 96% that was

corroborated by their ¹H NMR spectrum unless speciﬁcally

mentioned otherwise.

All reactions and workup procedures, where 5-ﬂuoro-2-furyl moiety

was present, were performed in the dark.

Reagent Synthesis and General Procedures. R1a: 5-Bromo-

4-chloro-6-iodothieno[2,3-d]pyrimidine. Step A: 6-Iodo-3H-thieno-

[2,3-d]pyrimidin-4-one. A 2 L round bottomed ﬂask equipped with

mechanical stirrer, thermometer, and reﬂux condenser was charged

with the solution of 433 mL AcOH, 13 mL cc. H₂SO₄, and 87 mL

water. 69.3 g 3H-thieno[2,3-d]pyrimidin-4-one (0.455 mol), 51.9 g

H₅IO₆(0.228 mol) and 104 g I₂(0.410 mol) were added to the

stirred solution, and the mixture was heated to 60 °C for 1 h. The

resulting suspension was cooled to rt, ﬁltered oﬀ, washed with a

mixture of AcOH and water (5:1) and then with Et₂O. The resulting

beige crystalline solid was air-dried to give 95.6 g 6-iodo-3H-

thieno[2,3-d]pyrimidin-4-one (0.344 mol, 76%). ¹H NMR (500

MHz, DMSO-d₆): 8.91 (s, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ

ppm: 172.5, 152.5, 152.3, 129.1, 99.7, 89.9. MS (EI, 70 eV) m/z (%

relative intensity, [ion]): 106 (43), 141 (38), 233 (14), 268 (19), 387

(25), 422 (100, [M⁺]), 424 (37, [M⁺]).

R1c: 5-Bromo-4-chloro-6-(4-ﬂuorophenyl)thieno[2,3-d]-

pyrimidine. 75.08 g R1a (200 mmol), 53.63 g 2-(4-ﬂuorophenyl)-

4,4,5,5-tetramethyl-1,3,2-dioxaborolane (240 mmol), 130 g Cs₂CO₃

t

(400 mmol), 2.245 g Pd(OAc)₂(10 mmol), and 8.50 g BuX-Phos

(20 mmol) were placed in a 2 L ﬂask. 600 mL THF and 200 mL

water were added and then stirred overnight at 70 °C under Ar

atmosphere. The volatiles were evaporated under reduced pressure,

and then it was ﬁltered. The solid was sonicated in 250 mL MeCN

and ﬁltered again. Finally it was crystallized from EtOH/THF (2:1)

to give 52.57 g R1c as an oﬀ white solid (153 mmol, 76%). ¹H NMR

(400 MHz, DMSO-d₆) δ ppm: 9.02 (s, 1H), 7.80−7.77 (m, 2H),

7.47−7.43 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ ppm: 166.7,

K

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

162.8, 153.9, 153.0, 139.3, 132.3, 127.6, 126.5, 116.2, 100.8. HRMS

calculated for C₁₂H₅BrClFN₂S: 341.9029, found 342.9091 (M + H).

R1d: 4-Chloro-5-iodo-6-(prop-1-ynyl)thieno[2,3-d]pyrimidine.

42.24 g R1b (100 mmol), 3.509 g Pd(PPh₃)₂Cl (5.00 mmol), and

1.904 g CuI (10.0 mmol) were dissolved in 400 mL DIPA, then

propyne was bubbled through the reaction mixture, which was stirred

for 6 h at rt to reach complete conversion. Then the volatiles were

evaporated under reduced pressure and the crude product was

puriﬁed via ﬂash chromatography using heptane and EtOAc as eluents

to obtain 28.3 g R1d (84.6 mmol, 85%). ¹H NMR (500 MHz,

DMSO-d₆) δ ppm: 8.92 (s, 1H), 2.25 (s, 3H). ¹³C NMR (125 MHz,

DMSO-d₆) δ ppm: 166.9, 153.9, 153.1, 127.9, 126.9, 101.3, 82.1, 74.6,

4.8. MS (EI, 70 eV) m/z (% relative intensity, [ion]): 75 (13), 86

(13), 93 (11), 100 (12), 106 (13), 127 (15), 144 (13), 145 (17), 180

(32), 207 (23), 334 (100, [M⁺]), 336 (40, [M⁺]).

R1e: 4-Chloro-5-iodothieno[2,3-d]pyrimidine. 52.8 g R1b (125

mmol) was dissolved in 400 mL dry THF and cooled to 0 °C. 100 mL

tBuMgCl (200 mmol, 2 M in Et₂O) was added over 15 min. Then 50

mL water was added and stirred for 10 min. The solution was

decanted and concentrated under reduced pressure. The crude

product was sonicated in a mixture of 200 mL MeCN and water (3:1)

and then collected by ﬁltration to yield 30.4 g R1e (102 mmol, 82%).

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.95 (s, 1H), 8.45 (s, 1H).

¹³C NMR (100 MHz, DMSO-d₆) δ ppm: 167.6, 154.2, 152.6, 134.4,

migration from the phenolic oxygen to the alkyl oxygen was complete.

Then 30 mL AcOH was added and the volatiles were evaporated

under reduced pressure. Then 350 mL water was added and the

mixture was extracted with EtOAc. The combined organic layer was

washed with saturated aq NaHCO₃solution and with brine and then

dried over MgSO₄, ﬁltered, and the ﬁltrate was concentrated under

reduced pressure. Then 100 mL hexane was added, and it was stirred

for 30 min at 0 °C. The formed white crystals were collected by

ﬁltration and washed with hexane yielding 23.2 g ethyl 2-acetoxy-3-(2-

hydroxyphenyl)propanoate as a racemate (92.0 mmol, 23% for two

steps). The enantiomers were separated via chiral chromatography.

Column: OD. Eluents: heptane and EtOH. The enantiomer eluting

later was collected as ethyl (2R)-2-acetoxy-3-(2-hydroxyphenyl)-

propanoate with 99.9% ee. Retention times (Daicel Chiralcel OD-H

250 mm × 4.6 mm, 5 μm, eluent: ⁱPrOH/heptane 50:50; ﬂow rate 1.0

mL/min, detection wavelength: 210 nm, 276 nm; ambient temper-

1

ature): 4.7 min (S enantiomer), 6.3 min (R enantiomer). H NMR

(500 MHz, DMSO-d₆) δ ppm: 9.53 (s, 1H), 7.08−7.03 (m, 2H), 6.79

(dd, J = 7.9, 0.8 Hz, 1H), 6.71 (td, J = 7.4, 1.1 Hz, 1H), 5.10 (dd, J =

8.3, 6.0 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 3.06 (dd, J = 13.7, 6.0 Hz,

1H), 2.94 (dd, J = 13.7, 8.3 Hz, 1H), 2.00 (s, 3H), 1.09 (t, J = 7.1 Hz,

3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 170.0, 169.6, 155.5,

131.1, 128.1, 121.8, 118.8, 114.8, 71.4, 60.7, 31.8, 20.3, 13.9. HRMS

calculated for C₂₅H₂₆N₂O₆: 252.0998; found 275.0881 (M + Na).

Step C: Ethyl (2R)-2-Hydroxy-3-(2-tetrahydropyran-2-

yloxyphenyl)propanoate. 103.3 g ethyl (2R)-2-acetoxy-3-(2-

hydroxyphenyl)propanoate (409.4 mmol) was dissolved in 280 mL

DHP. 300 mg PTSA·H₂O (1.58 mmol, 0.39 mol %) was added, and

the mixture was stirred until no further conversion was observed.

Then it was diluted with 1 L EtOAc, washed with 200 mL saturated

aq NaHCO₃solution, then with 200 mL water. The organic layer was

dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under

reduced pressure. Then it was puriﬁed via ﬂash chromatography using

heptane and EtOAc as eluents to give 139.7 g ethyl (2R)-2-acetoxy-3-

(2-tetrahydropyran-2-yloxyphenyl)propanoate, as a 1:1 mixture of

diastereoisomers (contaminated with DHP oligomers).

127.3, 72.6.

R1f: Methyl (2R)-2-[(5,6-diiodothieno[2,3-d]pyrimidin-4-yl)oxy]-

3-phenylpropanoate. 8.45 g R1b (20.0 mmol), 5.41 g methyl (2R)-

2-hydroxy-3-phenylpropanoate (30.0 mmol), and 13.03 g Cs₂CO₃

(40.0 mmol) were placed in a ﬂask. 20 mL DMSO was added, and the

mixture was stirred at 60 °C for 35 min to reach complete conversion.

Then the reaction mixture was diluted with water, the pH was set to 5

with 2 M aq HCl solution, and then it was extracted with DCM. The

combined organic layer was dried over Na₂SO₄, ﬁltered, and the

ﬁltrate was concentrated under reduced pressure. The crude product

was puriﬁed via ﬂash chromatography using heptane and EtOAc as

1

eluents to obtain 10.13 g R1f (17.9 mmol, 89%). H NMR (400

This intermediate was dissolved in 600 mL EtOH, then 20 mL

NaOEt solution (20 mmol, 1.0 M in EtOH) was added, and it was

stirred at rt until no further conversion was observed. The mixture

was concentrated under reduced pressure to half of its initial volume,

then 300 mL water and 300 mL brine were added. It was extracted

twice with EtOAc. The combined organic layer was washed with

brine, then dried over Na₂SO₄, ﬁltered and the ﬁltrate was

concentrated to give 110.2 g ethyl (2R)-2-hydroxy-3-(2-tetrahydro-

pyran-2-yloxyphenyl)propanoate as a 1:1 mixture of diastereoisomers

(R2a, 374.4 mmol, 91% for two steps). The optical purity of the

MHz, CDCl₃) δ ppm: 8.49 (s, 1H), 7.45−7.39 (m, 2H), 7.34−7.21

(m, 3H), 5.78 (dd, J = 8.5, 4.8 Hz, 1H), 3.75 (s, 3H), 3.50−3.35 (m,

2H). ¹³C NMR (100 MHz, CDCl₃) δ ppm: 172.6, 170.2, 159.8,

152.8, 136.2, 129.5, 128.5, 127.1, 120.8, 90.4, 85.0, 76.0, 52.4, 37.6.

HRMS calculated for C₁₆H₁₂I₂N₂O₃S: 565.8658; found 566.8734 (M

+ H).

R2a: Ethyl (2R)-2-Hydroxy-3-(2-tetrahydropyran-2-yloxyphenyl)-

propanoate. Step A: [2-(Bromomethyl)phenyl]acetate. 60.07 g 2-

methylphenyl acetate (400 mmol), 106.8 g NBS (600 mmol), and

500 mL cyclohexane were placed in a 1 L ﬂask. Then 3.284 g AIBN

(20 mmol) was added under vigorous stirring over 30 min. The

mixture was stirred at 80 °C until no further conversion was observed,

then it was cooled to rt. The precipitate was ﬁltered oﬀ and washed

with cyclohexane. The ﬁltrate was concentrated under reduced

pressure, and the overweight crude product (100 g) was used in step

1

starting material was conserved. H NMR (500 MHz, DMSO-d₆) δ

ppm: 7.18−7.11 (m, 2H), 7.04 (d, J = 8.5 Hz, 1H), 6.89−6.84 (m,

1H), 5.52/5.50 (t, J = 3.0 Hz, 1H), 5.49−5.45 (m, 1H), 4.30−4.24

(m, 1H), 4.05/4.02 (q, J = 7.1 Hz, 2H), 3.77−3.69 (m, 1H), 3.59−

3.51 (m, 1H), 3.06/3.04 (dd, J = 13.1, 5.2 Hz, 1H), 2.74/2.71 (dd, J =

13.1, 8.8 Hz, 1H), 2.00−1.89 (m, 1H), 1.85−1.74 (m, 2H), 1.68−

1.50 (m, 3H), 1.12/1.10 (t, J = 7.1 Hz, 3H). ¹³C NMR (125 MHz,

DMSO-d₆) δ ppm: 173.87/173.86, 154.43/154.41, 131.35/131.30,

127.6, 126.4/126.3, 120.8/120.7, 114.0/113.9, 95.2/95.0, 70.04/

69.99, 61.2/61.1, 59.90/59.85, 35.7/35.6, 30.0/29.9, 24.8, 18.27/

18.25, 13.98/13.96. HRMS calculated for C₁₆H₂₂O₅: 294.1467; found

317.1355 and 317.1356 (M + Na).

R2b: Ethyl (2R)-2-Hydroxy-3-(2-methoxyphenyl)propanoate. To

a stirred mixture of 1.362 g 2-methoxybenzaldehyde (10.0 mmol) and

1.34 mL ethyl chloroacetate (12.5 mmol) in 10 mL dry THF at −78

°C, 12.5 mL NaHMDS solution (1.0 M in THF) was added dropwise.

It was allowed to reach rt and stirred for 30 min. The mixture was

quenched with sat. aq NH₄Cl solution, the layers were separated, the

aq layer was extracted with EtOAc. Then the combined organic layer

was dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated. The

obtained intermediate was dissolved in 20 mL EtOAc, then 0.2 g Pd/

C (10%) was added, and the mixture was stirred at rt under 4.5 bar H₂

atmosphere. In the case of low conversion glacial AcOH and

Pd(OH)₂were added to the mixture, and hydrogenation was

1

B without further puriﬁcation. H NMR (500 MHz, DMSO-d₆) δ

ppm: 7.42 (dd, J = 7.8, 1.7 Hz, 1H), 7.35 (td, J = 7.8, 1.7 Hz, 1H),

7.22 (td, J = 7.5, 1.2 Hz, 1H), 7.14 (dd, J = 7.5, 1.2 Hz, 1H), 4.43 (s,

2H), 2.37 (s, 3H).

Step B: Ethyl (2R)-2-Acetoxy-3-(2-hydroxyphenyl)propanoate.

23.10 g anhydrous LiCl (545 mmol) and 65.36 g anhydrous ZnCl₂

(479.6 mmol) were placed in a 2 L ﬂask, then dried at 160 °C under

0.1 mmHg for 1 h. After cooling to rt under Ar atmosphere, 26.49 g

Mg turnings (1090 mmol) and 1 L dry, precooled (0 °C) THF were

added. The resulting mixture was immersed into an ice-bath and then

stirred for 30 min. Then 100 g [2-(bromomethyl)phenyl] acetate was

dissolved in 120 mL dry THF and was added to the precooled

inorganics over 15 min. After addition of the reagent the resulting

mixture was stirred for 45 min while the temperature was kept

between 0 and 5 °C. Then 64.82 mL ethyl 2-oxoacetate (654 mmol,

50% in toluene) was added over 5 min, and the resulting mixture was

stirred for another 15 min. Then the insoluble inorganics were

removed by ﬁltration, and the ﬁltrate was diluted with 500 mL

MeOH. The mixture was stirred until the intramolecular acetyl group

L

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

continued. After an overnight stirring under H₂atmosphere the

mixture was ﬁltered through a pad of Celite, the Celite pad was

successively washed with EtOAc, then the ﬁltrate was concentrated

under reduced pressure and puriﬁed via ﬂash chromatography using

heptane and EtOAc as eluents to obtain 1.04 g ethyl 2-hydroxy-3-(2-

methoxyphenyl)propanoate (4.6 mmol, 46%) as a racemate. The

enantiomers were separated via chiral chromatography. Column: AD.

borolane impurity was removed by distillation (80 °C at 0.01 mmHg).

The crude product was triturated in MeOH aﬀording 160 g R3aa as a

white solid (0.377 mol, 80%). H NMR (500 MHz, DMSO-d₆) δ

ppm: 7.49 (d, J = 8.2 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 2.52 (s, 3H),

1.32 (sp, J = 7.4 Hz, 3H), 1.29 (s, 12H), 1.07 (d, J = 7.4 Hz, 18H).

¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 153.5, 143.6, 135.1, 125.3,

116.5, 83.4, 24.6, 19.2, 17.7, 12.3. MS (EI, 70 eV) m/z (% relative

intensity, [ion]): 83 (53), 211 (15), 381 (100), 383 (42).

1

i

Eluent: PrOH. The enantiomer eluting earlier was collected as R2b

with 99.8% ee. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 7.19 (dt, J =

8.1, 1.7 Hz, 1H), 7.12 (dd, J = 7.3, 1.4 Hz, 1H), 6.94 (d, J = 8.1 Hz,

1H), 6.84 (dt, J = 7.3, 0.8 Hz, 1H), 5.45 (d, J = 6.5 Hz, 1H), 4.21 (dq,

J = 6.5, 1.6 Hz, 1H), 4.01 (dq, J = 7.1, 1.6 Hz, 1H), 3.77 (s, 3H), 2.94

(dd, J = 13.3, 6.0 Hz, 1H), 2.77 (dd, J = 13.3, 8.0 Hz, 1H), 1.09 (t, J =

7.2 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 173.7, 157.3,

131.1, 127.8, 125.2, 120.0, 110.4, 69.7, 69.6, 59.9, 55.3, 35.2, 14.0.

R3aa: [2-Chloro-3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxabor-

olan-2-yl)phenoxy]triisopropylsilane. Step A: (4-Bromo-2-

chlorophenoxy)triisopropylsilane. 200 g 4-bromo-2-chlorophenol

(0.964 mol) and 126 mL TIPSCl (1.18 mol) were dissolved in 1.6 L

DCM. 167 g imidazole (2.45 mol) was added, and the mixture was

stirred at rt for 2 h. Then the volatiles were evaporated under reduced

pressure, and the residue was dissolved in 1.5 L EtOAc. The mixture

was washed with brine, dried over Na₂SO₄, ﬁltered, and the ﬁltrate

was concentrated under reduced pressure. The formed TIPSOH

impurity was removed by distillation (120 °C at 0.01 mmHg). The

residue was ﬁltered through a short pad of silica using hexane as

eluent and the ﬁltrate was concentrated under reduced pressure to

give 350 g (4-bromo-2-chlorophenoxy)triisopropylsilane as a colorless

oil (0.962 mol, 99.8%). ¹H NMR (400 MHz, CDCl₃) δ ppm: 7.49 (d,

J = 2.5 Hz, 1H), 7.21 (dd, J = 8.7, 2.5 Hz, 1H), 6.78 (d, J = 8.7 Hz,

1H), 1.31 (sp, J = 7.3 Hz, 3H), 1.14 (d, J = 7.3 Hz, 18H). ¹³C NMR

(100 MHz, CDCl₃) δ ppm: 151.4, 132.7, 130.4, 126.5, 121.2, 112.7,

17.9, 12.9. MS (EI, 70 eV) m/z (% relative intensity, [ion]): 63 (30),

79 (24), 93 (41), 170 (17), 235 (19), 251 (16), 265 (24), 293 (23),

319 (77), 321 (100), 323 (28), 362 (1, [M⁺]).

R3a: 2-Chloro-3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-

lan-2-yl)phenol. 2.23 g R3aa (5.25 mmol) was dissolved in 10 mL

dry THF, then 5.50 mL TBAF solution (5.50 mmol, 1 M in THF)

was added, and the mixture was stirred at rt for 10 min. Then the

solvent was evaporated under reduced pressure; the residue was

dissolved in EtOAc, washed with saturated aq NH₄Cl solution and

brine, dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated

under reduced pressure. The residue was puriﬁed via ﬂash

chromatography using heptane and EtOAc as eluents to give 1.07 g

R3a as a white solid (3.99 mmol, 76%). ¹H NMR (500 MHz, DMSO-

d₆) δ ppm: 10.40 (s, 1H), 7.42 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 8.2

Hz, 1H), 2.49 (s, 3H), 1.27 (s, 12H). ¹³C NMR (125 MHz, DMSO-

d₆) δ ppm: 156.0, 143.8, 135.6, 121.5, 113.7, 83.6, 25.1, 19.4. HRMS

calculated for C₁₃H₁₈BClO₃: 268.1037; found 267.0972 (M − H).

R3b: 1-[2-[2-Chloro-3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxa-

borolan-2-yl)phenoxy]ethyl]-4-methylpiperazine. 5.77 g R3a (21.5

mmol), 9.29 g 2-(4-methylpiperazin-1-yl)ethanol (64.4 mmol) and

43.0 g polymer bound PPh₃(1.5 mmol/g, 64.4 mmol) were stirred in

170 mL dry toluene, and then 14.79 g DTBAD (64.4 mmol) was

added. The mixture was stirred at 50 °C under Ar atmosphere for 3 h.

The crude reaction mixture was ﬁltered, then the ﬁltrate was

concentrated under reduced pressure. The residue was puriﬁed via

ﬂash chromatography using DCM and MeOH as eluents. The

resulting light brown oil was crystallized from hexane to give 5.58 g

1

R3b (14.2 mmol, 66%) as an oﬀ-white solid. H NMR (500 MHz,

DMSO-d₆) δ ppm: 7.56 (d, J = 8.5 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H),

4.15 (t, J = 5.8 Hz, 2H), 2.72 (t, J = 5.8 Hz, 2H), 2.51 (s, 3H), 2.50

(br s, 4H), 2.29 (br s, 4H), 2.13 (s, 3H), 1.29 (s, 12H). ¹³C NMR

(125 MHz, DMSO-d₆) δ ppm: 156.0, 143.0, 135.3, 110.2, 83.4, 67.1,

56.2, 54.8, 53.1, 45.8, 24.6, 19.0. HRMS calculated for

C₂₀H₃₂BClN₂O₃: 394.2195; found 395.2257 (M + H).

Step B: (4-Bromo-2-chloro-3-methylphenoxy)triisopropylsilane.

76.0 mL dry DIPA (0.54 mol) was dissolved in 1.2 L dry THF under

n

Ar atmosphere, and 51.2 mL BuLi solution (0.512 mol, 10 M in

hexanes) was added dropwise at −78 °C. The mixture was stirred for

45 min at the same temperature. Then 178 g (4-bromo-2-

chlorophenoxy)triisopropylsilane (0.489 mol) was added dropwise

at −78 °C, and the white suspension was stirred until the

regioselective deprotonation of the benzene ring was complete.

Then 36.5 mL MeI (0.586 mol) was added at this temperature and

the reaction mixture was stirred overnight without further cooling.

The volatiles were evaporated under reduced pressure. The residue

was dissolved in 1.5 L EtOAc, then washed with brine. The organic

phase was dried over Na₂SO₄, ﬁltered, and the ﬁltrate was

concentrated under reduced pressure. The crude product was ﬁltered

through a short pad of silica using hexane as eluent and then

concentrated under reduced pressure to obtain 172 g (4-bromo-2-

chloro-3-methylphenoxy)triisopropylsilane as a pale yellow oil (0.455

R4: Ethyl (2R)-2-[(5S_a)-5-(3-Chloro-4-hydroxy-2-methylphenyl)-

6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-tetrahydro-

pyran-2-yloxyphenyl)propanoate. Step A: Ethyl (2R)-2-[5-Bromo-6-

(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-tetra-hydro-

pyran-2-yloxyphenyl)propanoate. 48.45 g R1c (141 mmol), 45.63 g

R2a (155 mmol), and 137.8 g Cs₂CO₃(423 mmol) were placed in a 2

L ﬂask. 1.4 L tBuOH was added and the mixture was stirred at 70 °C

under N₂atmosphere for 4.5 h to reach 98% conversion.

Approximately 1 L solvent was evaporated under reduced pressure,

then it was diluted with water, the pH was set to 8 with 2 M HCl, and

then it was extracted with DCM. The combined organic layer was

dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under

reduced pressure. The crude product was puriﬁed via ﬂash

chromatography using heptane and EtOAc as eluents to obtain

62.19 g ethyl (2R)-2-[5-bromo-6-(4-ﬂuorophenyl)thieno[2,3-d]-

pyrimidin-4-yl]oxy-3-(2-tetrahydropyran-2-yloxyphenyl)propanoate

diastereoisomer mixture as a white solid (103 mmol, 73%). ¹H NMR

(500 MHz, DMSO-d₆) δ ppm: 8.67/8.66 (s, 1H), 7.76−7.72 (m,

2H), 7.43−7.39 (m, 3H), 7.20−7.16 (m, 1H), 7.08−7.06 (m, 1H),

6.90−6.87 (m, 1H), 5.81/5.69 (dd, J = 9.8, 3.8 Hz, 1H), 5.60−5.54

(m, 1H), 4.23−4.08 (m, 2H), 3.80−3.48 (m, 2H), 3.51/3.49 (dd, J =

13.8, 3.8 Hz, 1H), 3.19/3.17 (dd, J = 10.0, 1.8 Hz, 1H), 2.09−1.49

(m, 6H), 1.15/1.10 (t, J = 7.0 Hz, 3H). ¹³C NMR (125 MHz,

DMSO-d₆) δ ppm: 169.5, 165.84/165.82, 162.7, 162.0/161.9, 154.5/

154.4, 153.2, 135.67/135.66, 132.1, 132.0, 128.34/128.35, 127.8,

124.6, 120.98/120.95, 117.5, 116.1, 114.00/113.97, 99.8, 95.3/94.9,

74.5/74.4, 61.21/61.17, 61.1/61.0, 32.95/32.89, 29.95/29.88, 24.8,

18.24/18.21, 13.89/13.86. HRMS calculated for C₂₈H₂₆BrFN₂O₅S:

600.0729; found 601.0809 and 601.0798 (M + H).

1

mol, 93%). H NMR (500 MHz, DMSO-d₆) δ ppm: 7.45 (d, J = 8.8

Hz, 1H), 6.81 (d, J = 8.8 Hz, 1H), 2.44 (s, 3H), 1.30 (sp, J = 7.5 Hz,

3H), 1.06 (d, J = 7.5 Hz, 18H). ¹³C NMR (125 MHz, DMSO-d₆) δ

ppm: 150.9, 136.3, 130.9, 125.6, 118.4, 115.5, 20.9, 17.7, 12.2. MS

(EI, 70 eV) m/z (% relative intensity, [ion]): 93 (31), 265 (17), 307

(15), 333 (81), 335 (100), 337 (31), 376 (1, [M⁺]), 378 (1, [M⁺]).

Step C: [2-Chloro-3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxa-

borolan-2-yl)phenoxy]triisopropylsilane. 178 g (4-bromo-2-chloro-

3-methylphenoxy)triisopropylsilane (0.471 mol) was dissolved in 1.4

n

L dry THF under Ar atmosphere, and 52 mL BuLi solution (0.520

mol, 10 M in hexanes) was added dropwise at −78 °C. The mixture

was stirred for 5 min at this temperature. Then 116 mL 2-isopropoxy-

4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.569 mol) was added and

the mixture was allowed to warm up to rt. Then the volatiles were

evaporated under reduced pressure, and the residue was dissolved in

1.5 L EtOAc. It was washed with brine, then the organic phase was

dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under

reduced pressure. The 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxa-

Step B: Ethyl (2R)-2-[(5S_a)-5-(3-Chloro-4-hydroxy-2-methylphen-

yl)-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-tetrahy-

dropyran-2-yloxyphenyl)propanoate. 186.6 g ethyl (2R)-2-[5-

M

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

bromo-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-tetra-

hydropyran-2-yloxyphenyl)propanoate (310.3 mmol) and 99.99 g

R3a (372.3 mmol) were dissolved in 1.2 L THF, then 202.2 g

Cs₂CO₃(620.6 mmol) dissolved in 300 mL water was added. Then

11.0 g AtaPhos (15.51 mmol) was added, and the mixture was stirred

under N₂atmosphere at 80 °C for 2 h to reach 98% conversion. Most

of the volatiles were evaporated under reduced pressure, then it was

diluted with DCM and brine. The pH of the aq phase was set to 8

with 2 M aq HCl solution. After phase separation the aq phase was

extracted with DCM. The combined organic layer was dried over

Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. The atropoisomers were puriﬁed and separated via ﬂash

chromatography using heptane and EtOAc as eluents. The

diastereoisomer pair eluting later was collected as R4 (132.3 g,

4.16 g tBu-XPhos (9.80 mmol), and 120 mL water were added, and

the mixture was stirred for 3 h at 70 °C under N₂atmosphere. Most of

the THF was removed under reduced pressure, EtOAc and water

were added to the residue, and the resulting mixture was ﬁltered

through Celite. After separation of the phases, the aqueous phase was

extracted with EtOAc. The combined organic phase was dried over

Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. The residue was puriﬁed via ﬂash chromatography using

heptane and EtOAc as eluents to give a crude product which was

ﬁnally crystallized from a mixture of heptane and Et₂O to obtain 10.5

g 5-bromo-4-chloro-6-(5-ﬂuoro-2-furyl)thieno[2,3-d]pyrimidine as a

1

white solid (31.5 mmol, 45%). H NMR (400 MHz, DMSO-d₆) δ

ppm: 8.95 (s, 1H), 7.55 (t, J = 3.7 Hz, 1H), 6.23 (dd, J = 6.8, 3.7 Hz,

1H). ¹³C NMR (100 MHz, DMSO-d₆) δ ppm: 165.6, 158.7, 154.6 (d,

J = 268 Hz), 153.0, 136.8 (d, J = 1.5 Hz), 128.4, 126.7, 115.7, 97.6,

86.0 (d, J = 12.1 Hz). MS (EI, 70 eV) m/z (% relative intensity,

[ion]): 106 (29), 198 (42), 225 (100), 253 (73), 332 (62, [M⁺]), 334

(81, [M⁺]), 336 (24, [M⁺]).

1

199.5 mmol, 64%). H NMR (500 MHz, DMSO-d₆, 1:1 mixture of

diastereomers) δ ppm: 10.27 (br s, 1H), 8.60 (s, 1H), 7.32−7.28 (m,

2H), 7.24−7.19 (m, 2H), 7.14 (dd, J = 8.4, 6.6 Hz, 1H), 7.14−7.10

(m, 1H), 7.00 (dd, J = 8.4, 2.6 Hz, 1H), 6.96 (dd, J = 8.4, 2.6 Hz,

1H), 6.75−6.71 (m, 1H), 6.34/6.39 (dd, J = 7.4, 1.6 Hz, 1H), 5.55−

5.51 (m, 1H), 5.41/5.39 (d, J = 4.5 Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H),

3.70−3.65 (m, 2H), 3.10−3.05 (m, 1H), 2.44 (dd, J = 13.4, 9.2 Hz,

1H), 1.98/1.90 (br s, 1H), 1.85/1.83 (s, 3H), 1.79 (br s, 2H), 1.64

(br s, 1H), 1.59 (br s, 1H), 1.54 (br s, 1H), 1.09/1.08 (t, J = 7.0 Hz,

3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 169.3, 166.4, 162.54/

162.50, 162.1, 154.14/154.13, 153.1, 152.7, 136.7, 135.7/135.6,

131.1/131.0, 131.02/131.00, 130.06/130.00, 129.1/128.9, 128.26/

128.25, 125.9, 124.3, 120.7, 120.56/120.54, 118.83/118.82, 116.0,

113.9, 113.4, 95.1/94.8, 73.7/73.4, 61.2/61.0, 60.9/60.8, 32.7/32.6,

29.9/29.8, 24.8/24.7, 18.15/18.11, 17.6, 13.8. HRMS calculated for

C₃₅H₃₂ClFN₂O₆S: 662.1653; found 663.1728 and 663.1717 (M + H).

R5a: Ethyl (2R)-2-[(5S_a)-5-[3-Chloro-2-methyl-4-[2-(4-methylpi-

perazin-1-yl)ethoxy]phenyl]-6-(4-ﬂuorophenyl)thieno[2,3-d]-

pyrimidin-4-yl]oxy-3-(2-hydroxyphenyl)propanoate. 132.3 g R4

(199.5 mmol), 43.17 g 2-(4-methylpiperazin-1-yl)ethanol (299.3

mmol), and 94.20 g PPh₃(359.1 mmol) were dissolved in 1 L dry

toluene, then 78.09 g DTBAD (339.2 mmol) was added. The mixture

was stirred at 50 °C under N₂atmosphere for 1 h to reach >97%

conversion. 980 mL toluene was evaporated under reduced pressure,

then 500 mL Et₂O was added, and the mixture was stirred and

sonicated. The precipitated white crystals (PPh₃O) were ﬁltered,

washed with Et₂O. The ﬁltrate was concentrated under reduced

pressure and puriﬁed via ﬂash chromatography using EtOAc and

MeOH. The obtained intermediate was dissolved in 1 L EtOH, then 1

L 1.25 M HCl solution in EtOH was added and the mixture was

stirred at rt for 1 h to reach >99% conversion. Most of the EtOH was

evaporated, then 2 L Et₂O was added and the precipitated HCl salt

(white solid) was ﬁltered, washed with Et₂O. The HCl salt was

carefully treated with 2 L sat. aq NaHCO₃solution, then extracted

with DCM. The combined organic layer was dried over Na₂SO₄,

ﬁltered and the ﬁltrate was concentrated under reduced pressure to

give 128 g R5a (181.5 mmol, 91% for two steps).¹H NMR (500 MHz,

DMSO-d₆) δ ppm: 9.53 (br s, 1H), 8.60 (s, 1H), 7.32−7.28 (m, 2H),

7.28 (d, J = 8.8 Hz, 1H), 7.24−7.19 (m, 2H), 7.16 (d, J = 8.8 Hz,

1H), 6.97 (td, J = 7.8, 1.6 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H), 6.53 (td,

J = 7.4, 0.8 Hz, 1H), 6.18 (dd, J = 7.4, 1.6 Hz, 1H), 5.46 (dd, J = 9.0,

4.6 Hz, 1H), 4.25−4.18 (m, 2H), 4.07−4.00 (m, 2H), 2.92 (dd, J =

13.4, 4.8 Hz, 1H), 2.75 (t, J = 4.8 Hz, 2H), 2.53 (br s, 4H), 2.44 (dd, J

= 13.6, 9.2 Hz, 1H), 2.36 (br s, 4H), 2.17 (s, 3H), 1.88 (s, 3H), 1.06

(t, J = 7.0 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 169.4,

166.5, 163.1, 162.6, 161.1, 155.2, 153.8, 152.8, 136.9, 136.0, 131.1,

130.3, 129.0, 128.5, 128.0, 127.6, 122.1, 121.7, 118.8, 118.5, 116.0,

114.7, 110.7, 73.3, 67.3, 60.8, 56.2, 32.3, 17.7, 13.9. HRMS calculated

for C₃₇H₃₈ClFN₄O₅S: 704.2235; found 705.2288 (M + H).

Step B: Ethyl (2R)-2-[5-Bromo-6-(5-ﬂuoro-2-furyl)thieno[2,3-d]-

pyrimidin-4-yl]oxy-3-(2-tetrahydropyran-2-yloxyphenyl)-

propanoate. 12.0 g 5-bromo-4-chloro-6-(5-ﬂuoro-2-furyl)thieno[2,3-

d]pyrimidine (36.0 mmol), 11.8 g R2a (40.0 mmol), and 16.3 g

Cs₂CO₃(50 mmol) were mixed with 350 mL tBuOH, and the

mixture was stirred at 35 °C under N₂atmosphere for 4 h. Then the

mixture was cooled to rt and diluted with brine, extracted with DCM,

dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under

reduced pressure. The crude product was puriﬁed via ﬂash

chromatography using heptane and EtOAc as eluents to obtain 17.1

g ethyl (2R)-2-[5-bromo-6-(5-ﬂuoro-2-furyl)thieno[2,3-d]pyrimidin-

4-yl]oxy-3-(2-tetrahydropyran-2-yloxyphenyl)propanoate as a mixture

of diastereoisomers (28.9 mmol, 80%). ¹H NMR (500 MHz, DMSO-

d₆): 8.63/8.62 (s/s, 1H), 7.46−7.40 (m, 2H), 7.22−7.16 (m, 1H),

7.09−7.02 (m, 1H), 6.93−6.88 (m, 1H), 6.20−6.25 (m, 1H), 5.83−

5.77/5.71−5.66 (m, 1H), 5.61/5.55 (t/t, J = 3.0/3.1 Hz, 1H), 4.20−

4.07 (m, 2H), 3.78−3.40 (m, 3H), 3.23−3.14 (m, 1H), 2.06−1.33

(m, 6H), 1.17−1.09 (m, 3H). HRMS calculated for

C₂₆H₂₄BrFN₂O₆S: 590.0522; found 591.0599 (M + H).

Step C: Ethyl (2R)-2-[(5S_a)-5-(3-Chloro-4-hydroxy-2-methylphen-

yl)-6-(5-ﬂuoro-2-furyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-tetrahy-

dropyran-2-yloxyphenyl)propanoate. 14.0 g ethyl (2R)-2-[5-bromo-

6-(5-ﬂuoro-2-furyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-tetrahydro-

pyran-2-yloxyphenyl)propanoate (23.7 mmol) and 7.61 g R3a (28.3

mmol) were dissolved in 150 mL THF, then 13.0 g Cs₂CO₃(40.0

mmol), 50 mL water, and 668 mg AtaPhos (0.94 mmol) were added,

and the mixture was stirred under N₂atmosphere at 80 °C for 1.5 h.

Then the mixture was cooled to rt and diluted with brine. It was

neutralized using 1 M aq HCl solution, then extracted with DCM.

The combined organic layer was dried over Na₂SO₄, ﬁltered, and the

ﬁltrate was concentrated under reduced pressure. The atropisomers

were puriﬁed and separated via ﬂash chromatography using heptane

and EtOAc as eluents. The diastereoisomer pair eluting later was

isolated as ethyl (2R)-2-[(5S_a)-5-(3-chloro-4-hydroxy-2-methylphen-

yl)-6-(5-ﬂuoro-2-furyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-tetrahy-

dropyran-2-yloxyphenyl)propanoate (1:1 mixture of diastereoisomers,

1

11.6 g, 17.8 mmol, 75%, white solid). H NMR (500 MHz, DMSO-

d₆) δ ppm: 10.40 (s, 1H), 8.58/8.57 (s, 1H), 7.17−7.12 (m, 1H), 7.10

(d, J = 8.4 Hz, 1H), 7.04 (dm, J = 8.4 Hz, 1H), 7.01 (dm, J = 8.4 Hz,

1H), 6.83−6.78 (m, 1H), 6.38/6.36 (dd, J = 7.5, 1.7 Hz, 1H), 5.89

(dd, J = 6.9, 3.7 Hz, 1H), 5.69 (t, J = 3.7 Hz, 1H), 5.56/5.52 (m/t, J =

2.9 Hz, 1H), 5.56/5.43 (m/dd, J = 9.3, 4.0 Hz, 1H), 4.10−4.01 (m,

2H), 3.71−3.65 (m, 1H), 3.57−3.51 (m, 1H), 3.14/3.10 (dd, J =

13.6, 3.9/13.7, 3.6 Hz, 1H), 2.40−2.33 (m, 1H), 2.01−1.87 (m, 1H),

1.95/1.94 (s, 3H), 1.82−1.74 (m, 2H), 1.68−1.48 (m, 3H), 1.088/

1.086 (t, J = 7.1 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm:

169.24/169.23, 165.93, 162.49/162.46, 156.5 (d, J = 278 Hz), 154.1,

153.5, 152.9, 138.0, 135.6, 131.1/130.9, 128.72/128.70, 128.3,

127.20/127.18, 126.0/125.8, 124.30/124.28, 120.83, 120.77,

118.67/118.65, 113.97, 113.95/113.88, 111.1, 95.1/94.8, 85.3 (d, J

= 12.0 Hz), 73.7/73.4, 61.2, 61.0, 60.90/60.87, 32.8/32.7, 29.9/29.8,

R5b: Ethyl (2R)-2-[(5S_a)-5-[3-Chloro-2-methyl-4-[2-(4-methylpi-

perazin-1-yl)ethoxy]phenyl]-6-(5-ﬂuoro-2-furyl)thieno[2,3-d]-

pyrimidin-4-yl]oxy-3-(2-hydroxyphenyl)propanoate. Step A: 5-

Bromo-4-chloro-6-(5-ﬂuoro-2-furyl)thieno[2,3-d]pyrimidine. 26.3 g

R1a (70.0 mmol) and 61.5 g 2-(5-ﬂuoro-2-furyl)-4,4,5,5-tetramethyl-

1,3,2-dioxaborolane¹⁷(290 mmol) were dissolved in 350 mL THF,

then 29.3 g Cs₂CO₃(90.0 mmol), 1.10 g Pd(OAc)₂(4.90 mmol),

N

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

24.8/24.7, 18.12/18.07, 17.2, 13.8. HRMS calculated for

C₃₃H₃₀ClFN₂O₇S: 652.1446; found 653.1485 and 653.1492 (M + H).

Step D: Ethyl (2R)-2-[(5S_a)-5-[3-Chloro-2-methyl-4-[2-(4-methyl-

piperazin-1-yl)ethoxy]phenyl]-6-(5-ﬂuoro-2-furyl)thieno[2,3-d]-

pyrimidin-4-yl]oxy-3-(2-hydroxyphenyl)propanoate. 11.6 g ethyl

(2R)-2-[(5S_a)-5-(3-chloro-4-hydroxy-2-methylphenyl)-6-(5-ﬂuoro-2-

furyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-tetrahydropyran-2-

yloxyphenyl)propanoate (17.7 mmol), 5.77 g 2-(4-methylpiperazin-1-

yl)ethanol (40 mmol), and 10.5 g PPh₃(40 mmol) were dissolved in

100 mL dry toluene, then 9.21 g DTBAD (40 mmol) was added and

the mixture was stirred at 50 °C under N₂atmosphere for 1 h. Then

the mixture was concentrated under reduced pressure, and the residue

was puriﬁed via ﬂash chromatography using heptane, EtOAc, and

MeOH as eluents. The obtained intermediate was dissolved in 100

mL EtOH, then 100 mL HCl solution (1.25 M in EtOH) was added,

and the mixture was stirred at rt for 2 h. Then most of the solvent was

evaporated under reduced pressure, and then it was carefully

neutralized with saturated aq NaHCO₃solution and extracted with

DCM. The combined organic phase was dried over Na₂SO₄, ﬁltered,

and the ﬁltrate was concentrated under reduced pressure. The crude

product was puriﬁed via ﬂash chromatography using DCM and

MeOH as eluents to give 10.9 g R5b as a white solid (15.7 mmol, 89%

with brine and extracted with DCM. The organic layer was dried over

Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. The atropoisomers were puriﬁed and separated via ﬂash

chromatography using heptane and EtOAc as eluents. The

diastereoisomer pair eluting later was collected as 6.60 g ethyl

(2R)-2-[(5S_a)-(3-chloro-4-hydroxy-2-methylphenyl)-6-prop-1-

ynylthieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-tetrahydropyran-2-

yloxyphenyl)propanoate (10.9 mmol, 61%). ¹H NMR (500 MHz,

DMSO-d₆) δ ppm: 10.31 (s, 1H), 8.62/8.61 (s, 1H), 7.15−7.11 (m,

1H), 7.10 (d, J = 8.5 Hz, 1H), 7.00−6.97 (m, 1H), 6.98 (d, J = 8.5

Hz, 1H), 6.76−6.70 (m, 1H), 6.25−6.22/6.20−6.17 (m, 1H), 5.54/

5.51 (t, J = 2.6 Hz, 1H), 5.49/5.37 (dd/dd, J = 9.4, 4.1 Hz, 1H), 4.06

(q, J = 7.1 Hz, 2H), 3.71−3.61/3.56−3.49 (m, 2H), 3.14/3.11 (dd, J

= 13.3 Hz, 4.1 Hz, 1H), 2.43 (dd, J = 13.3 Hz, 9.4 Hz, 1H), 2.09 (s,

3H), 2.03 (s, 3H), 2.02−1.48 (m, 6H), 1.10/1.09 (t, J = 7.1 Hz, 3H).

¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 169.26/169.25, 166.20/

166.18, 162.15/162.11, 154.12, 154.09, 153.7/153.3, 135.91/135.87,

131.3/131.1, 129.62/129.56, 128.33/128.29, 128.26, 127.24/127.19,

125.63/125.63, 124.2, 120.6, 120.45/120.43, 119.36/119.35, 117.2,

113.9/113.0, 97.30/97.28, 95.0/94.8, 73.8/73.6, 71.9, 61.2/61.1,

60.93/60.90, 32.7/32.6, 29.86/29.82, 24.8/24.7, 18.1, 17.8, 14.0/13.8,

4.4. HRMS calculated for C₃₂H₃₁ClN₂O₆S: 606.1591; found 607.1685

and 607.1671 (M + H).

1

for 2 steps). H NMR (500 MHz, DMSO-d₆) δ ppm: 9.56 (s, 1H),

8.58 (s, 1H), 7.25 (s, 2H), 7.01−6.96 (m, 1H), 6.72 (dd, J = 8.1, 0.7

Hz, 1H), 6.61−6.57 (m, 1H), 6.23 (dd, J = 7.5, 1.5 Hz, 1H), 5.88 (dd,

J = 6.8, 3.5 Hz, 1H), 5.71 (t, J = 3.5 Hz, 1H), 5.48 (dd, J = 9.4, 4.4 Hz,

1H), 4.27 (t, J = 5.7 Hz, 2H), 4.09−3.99 (m, 2H), 2.95 (dd, J = 13.6,

4.4 Hz, 1H), 2.77 (t, J = 5.7 Hz, 2H), 2.53 (br s, 4H), 2.35 (dd, J =

13.6, 9.4 Hz, 1H), 2.29 (br s, 4H), 2.12 (s, 3H), 1.97 (s, 3H), 1.06 (t,

J = 7.1 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 169.3,

165.9, 162.5, 156.6 (d, J = 278 Hz), 155.1, 154.1, 152.9, 137.9, 135.8,

131.0, 129.0, 128.0, 127.4, 126.8 (d, J = 3.6 Hz), 126.1, 122.4, 121.7,

118.63, 118.56, 114.7, 111.23, 111.21, 85.3 (d, J = 12.4 Hz), 73.3,

67.4, 60.8, 56.3, 54.7, 53.0, 45.6, 32.4, 17.3, 13.8. HRMS calculated for

C₃₅H₃₆ClFN₄O₆S: 694.2028; found 695.2106 (M + H).

Step C: Ethyl (2R)-2-[(5S_a)-5-[3-cChloro-2-methyl-4-[2-(4-meth-

ylpiperazin-1-yl)ethoxy]phenyl]-6-prop-1-ynylthieno[2,3-d]-

pyrimidin-4-yl]oxy-3-(2-hydroxyphenyl)propanoate. 6.60 g ethyl

(2R)-2-[(5S_a)-(3-chloro-4-hydroxy-2-methylphenyl)-6-prop-1-

ynylthieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-tetrahydropyran-2-

yloxyphenyl)propanoate (10.9 mmol), 2.88 g 2-(4-methylpiperazin-1-

yl)ethanol (20.0 mmol), and 5.25 g PPh₃(20.0 mmol) were dissolved

in 50 mL dry toluene, then 4.61 g DTBAD (20.0 mmol) was added.

The mixture was stirred at 50 °C under N₂atmosphere for 1.5 h to

reach complete conversion. Then the mixture was concentrated under

reduced pressure, and the residue was puriﬁed via ﬂash chromatog-

raphy using EtOAc and MeOH as eluents. The obtained intermediate

was dissolved in 100 mL EtOH, then 40 mL 1.25 M HCl solution in

EtOH was added and the mixture was stirred at rt for 30 min to reach

complete conversion. Then most of the solvent was evaporated under

reduced pressure, and then it was carefully neutralized with saturated

aq NaHCO₃solution and extracted with DCM. The combined

organic phase was dried over Na₂SO₄, ﬁltered, and the ﬁltrate was

concentrated under reduced pressure. The crude product was puriﬁed

via ﬂash chromatography using DCM and MeOH as eluents to give

R5c: Ethyl (2R)-2-[(5S_a)-5-[3-Chloro-2-methyl-4-[2-(4-methylpi-

perazin-1-yl)ethoxy]phenyl]-6-prop-1-ynylthieno[2,3-d]pyrimidin-

4-yl]oxy-3-(2-hydroxyphenyl)propanoate. Step A: Ethyl (2R)-2-(5-

Iodo-6-prop-1-ynylthieno[2,3-d]pyrimidin-4-yl)oxy-3-(2-tetrahy-

dropyran-2-yloxyphenyl)propanoate. 8.92 g R1d (26.7 mmol), 8.83

g R2a (30.0 mmol), and 29.3 g Cs₂CO₃(90.0 mmol) were placed in a

500 mL ﬂask. 300 mL tBuOH was added, and the mixture was stirred

at 65 °C under N₂atmosphere for 4 h to reach >95% conversion.

Brine was added, then it was extracted with DCM. The combined

organic layer was dried over Na₂SO₄, ﬁltered, and the ﬁltrate was

concentrated under reduced pressure. The crude product was puriﬁed

via ﬂash chromatography using heptane and EtOAc as eluents to

obtain 10.59 g ethyl (2R)-2-(5-iodo-6-prop-1-ynylthieno[2,3-d]-

pyrimidin-4-yl)oxy-3-(2-tetrahydropyran-2-yloxyphenyl)propanoate

1

5.90 g R5c (9.00 mmol, 84% for 2 steps). H NMR (500 MHz,

DMSO-d₆) δ ppm: 9.53 (s, 1H), 8.62 (s, 1H), 7.24 (d, J = 8.6 Hz,

1H), 7.19 (d, J = 8.6 Hz, 1H), 6.99−6.95 (m, 1H), 6.71−6.68 (m,

1H), 6.54−6.50 (m, 1H), 6.07−6.04 (m, 1H), 5.41 (dd, J = 9.4, 4.3

Hz, 1H), 4.25 (t, J = 5.8 Hz, 2H), 4.11−4.00 (m, 2H), 2.97 (dd, J =

13.5, 4.3 Hz, 1H), 2.78−2.72 (m, 2H), 2.60−2.40 (m, 4H), 2.42 (dd,

J = 13.5, 9.3 Hz, 1H), 2.36−2.17 (m, 4H), 2.11 (s, 3H), 2.10 (s, 3H),

2.03 (s, 3H), 1.08 (t, J = 7.1 Hz, 3H). ¹³C NMR (125 MHz, DMSO-

d₆) δ ppm: 169.4, 166.2, 162.2, 155.1, 153.9, 153.7, 136.1, 135.5,

131.1, 129.8, 128.0, 127.2, 122.0, 121.6, 119.5, 118.4, 117.2, 114.7,

110.3, 97.5, 73.4, 71.8, 67.4, 60.8, 56.3, 54.7, 53.1, 45.8, 32.2, 17.8,

13.9, 4.4. HRMS calculated for C₃₄H₃₇ClN₄O₅S: 648.2173; found

649.2275 (M + H).

R6: (E)-4-(Dimethylamino)-1,1-dimethoxybut-3-en-2-one. 502.1

g 1,1-dimethoxypropan-2-one (4.25 mol) and 506.4 g 1,1-dimethoxy-

N,N-dimethylmethanamine (4.25 mol) were mixed in a 2 L ﬂask and

stirred at 105 °C for 3 h. The formed MeOH was removed

continuously via distillation. When MeOH formation stopped (at 65

°C head temperature), the reaction mixture was vacuum distilled

(decreasing the pressure slowly to 30 mbar) to remove side products

and unreacted starting materials. The crude product was distilled at

0.1 mbar. Fractions were collected between 107 and 118 °C head

temperature (bath temperature 160−165 °C) to give 488 g R6 as a

yellow oil (2.82 mol, 66%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm:

7.59 (d, J = 12.5 Hz, 1H), 5.17 (d, J = 12.5 Hz, 1H), 4.42 (s, 1H),

3.25 (s, 6H), 3.09 (s, 3H), 2.78 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆) δ ppm: 189.1, 153.8, 103.9, 53.6, 44.4, 36.8. MS (EI, 70

1

(17.9 mmol, 67%) as a mixture of diastereoisomers. H NMR (500

MHz, DMSO-d₆) δ ppm: 8.64/8.63 (s, 1H), 7.52−7.49 (m, 1H),

7.22−7.17 (m, 1H), 7.09−7.05 (m, 1H), 6.91−6.87 (m, 1H), 5.82/

5.71 (dd, J = 9.5, 4.1 Hz, 1H), 5.61/5.56 (t, J = 2.9 Hz, 1H), 4.18−

4.08 (m, 2H), 3.77−3.65 (m, 1H), 3.59−3.46 (m, 2H), 3.21 (dd, J =

13.9, 9.5 Hz, 1H), 2.21 (s, 3H), 2.05−1.92 (m, 1H), 1.86−1.76 (m,

2H), 1.68−1.50 (m, 3H), 1.13/1.11 (t, J = 7.1 Hz, 3H). ¹³C NMR

(125 MHz, DMSO-d₆) δ ppm: 169.4, 166.53/166.51, 161.4/161.3,

154.5/154.4, 153.69/153.67, 132.13/132.10, 128.43/128.40, 124.51/

124.50, 123.68/123.66, 120.91/120.88, 119.1, 114.01/114.97, 98.94/

98.93, 95.3/94.9, 79.6/79.5, 74.6/74.4, 74.3, 61.2/61.1, 61.04/61.01,

32.9, 29.9/29.8, 24.8, 18.21/18.19, 13.84/13.82, 4.7. HRMS

calculated for C₂₅H₂₅IN₂O₅S: 592.0529; found 593.0606 (M + H).

Step B: Ethyl (2R)-2-[(5S_a)-5-(3-Chloro-4-hydroxy-2-methylphen-

yl)-6-prop-1-ynylthieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-tetrahydro-

pyran-2-yloxyphenyl)propanoate. 10.59 g ethyl (2R)-2-(5-iodo-6-

prop-1-ynylthieno[2,3-d]pyrimidin-4-yl)oxy-3-(2-tetrahydropyran-2-

yloxyphenyl)propanoate (17.87 mmol) and 5.76 g R3a (21.45 mmol)

were dissolved in 100 mL THF, then 11.64 g Cs₂CO₃(35.74 mmol)

dissolved in 30 mL water was added. Then 1.26 g AtaPhos (1.79

mmol) was added, and the mixture was stirred at 60 °C under N₂

atmosphere for 3 h to reach complete conversion. Then it was diluted

O

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

eV) m/z (% relative intensity, [ion]): 55 (9), 75 (14), 98 (100), 114

(22), 143 (14), 173 (1, [M⁺]).

(11.9 mmol, 81%) was obtained. H NMR (400 MHz, DMSO-d₆) δ

ppm: 8.35 (d, J = 5.0 Hz, 1H), 6.75 (d, J = 5.0 Hz, 1H), 5.42 (t, J =

6.0 Hz, 1H), 4.36 (d, J = 6.0 Hz, 2H), 3.69−3.61 (m, 8H). ¹³C NMR

(100 MHz, DMSO-d₆) δ ppm: 171.6, 160.8, 158.2, 106.4, 66.0, 63.6,

43.8.

General Procedure 1: Acetal Deprotection and Reduction

of the Formed Aldehyde. The appropriate acetal (1.0 equiv) was

stirred with 2 M aq HCl solution (3 mL/mmol) at 60 °C until no

further conversion was observed. Then the mixture was cooled to 0

°C, then NaOH (5.7 equiv) was added portionwise. The pH was

adjusted to 8 using 10% aq K₂CO₃solution, then NaBH₄(2.0 equiv)

was added portionwise while keeping the temperature under 5 °C and

the mixture was stirred for 30 min at 0 °C. Then it was extracted with

EtOAc, dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated

under reduced pressure. The crude product was puriﬁed via ﬂash

chromatography using heptane and EtOAc as eluents.

General Procedure 2: Conversion of Amidines to Pyrimi-

dines. To the mixture of the appropriate amidine hydrochloride (1.2

equiv) and R6 (1.0 equiv) in dry MeOH (0.5 mL/mmol), NaOMe

(1.2 equiv) was added portionwise, and the mixture was stirred at 75

°C for 2 h. The reaction mixture was cooled and concentrated under

reduced pressure. To the residue water was added, and it was

extracted with DCM. The combined organic layer was dried over

MgSO₄, ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. The crude product was puriﬁed via ﬂash chromatography

using heptane and EtOAc as eluents.

R6c: [2-(2-Methoxyethyl)pyrimidin-4-yl]methanol. Step A: 4-

(Dimethoxymethyl)-2-(2-methoxyethyl)pyrimidine. Using general

procedure 2 and 25.0 g 3-methoxypropanamidine hydrochloride

(180 mmol) as the appropriate amidine hydrochloride, 13.8 g 4-

(dimethoxymethyl)-2-(2-methoxyethyl)pyrimidine (64.9 mmol, 36%)

R6aa: 4-(Dimethoxymethyl)-2-methylsulfanylpyrimidine. 198 g

NaOMe (3.67 mol) was dissolved in 3 L MeOH and cooled to 0 °C.

322 g thiocarbamide (4.23 mol) was added portionwise, and the

mixture was stirred for 1 h. Then 488 g R6 (2.82 mol) was added

dropwise at 0 °C, then it was heated to 70 °C for 4 h. It was cooled to

rt, 237 mL MeI (3.81 mol) was added dropwise, keeping the

temperature below 28 °C, and the resulting mixture was stirred

overnight at rt. It was ﬁltered, and the ﬁltrate was concentrated under

reduced pressure, diluted with EtOAc, washed with water and brine.

The combined aq layer was extracted with EtOAc. The combined

organic layer was dried over Na₂SO₄, ﬁltered, and the ﬁltrate was

concentrated under reduced pressure. The residue was dissolved in

500 mL Et₂O, ﬁltered through a pad of silica, using Et₂O as eluent.

The ﬁltrate was concentrated under reduced pressure to give 429 g

1

was obtained. H NMR (400 MHz, DMSO-d₆) δ ppm: 8.78 (d, J =

5.1 Hz, 1H), 7.38 (d, J = 5.1 Hz, 1H), 5.25 (s, 1H), 3.80 (t, J = 6.6

Hz, 2H), 3.32 (s, 6H), 3.22 (s, 3H), 3.11 (t, J = 6.6 Hz, 2H). ¹³C

NMR (100 MHz, DMSO-d₆) δ ppm: 167.9, 164.8, 158.1, 115.7,

102.6, 70.2, 57.9, 53.7, 38.8. MS (EI, 70 eV) m/z (% relative intensity,

[ion]): 75 (100), 135 (46), 165 (46), 167 (57), 182 (65), 197 (15).

Step B: [2-(2-Methoxyethyl)pyrimidin-4-yl]methanol. Using

general procedure 1 and 13.8 g 4-(dimethoxymethyl)-2-(2-

methoxyethyl)pyrimidine (64.9 mmol) as the appropriate acetal,

1

R6aa (2.14 mol, 76%) as a light brown oil. H NMR (400 MHz,

DMSO-d₆) δ ppm: 8.69 (d, J = 5.1 Hz, 1H), 7.23 (d, J = 5.1 Hz, 1H),

5.22 (s, 1H), 3.33 (s, 6H), 2.52 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆) δ ppm: 171.3, 165.3, 158.5, 113.6, 102.3, 53.7, 13.5. MS

(EI, 70 eV) m/z (% relative intensity, [ion]): 110 (12), 125 (100),

152 (29), 200 (20, [M⁺]).

R6a: (2-Methylsulfanylpyrimidin-4-yl)methanol. Using general

procedure 1 and 1202 mg R6aa (6.00 mmol) as the appropriate

acetal, 540 mg R6a (3.46 mmol, 58%) was obtained as white crystals.

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.61 (d, J = 5.1 Hz, 1H),

7.24 (d, J = 5.1 Hz, 1H), 5.63 (t, J = 6.0 Hz, 1H), 4.48 (d, J = 6.0 Hz,

2H), 2.49 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ ppm: 172.1,

170.9, 158.3, 113.4, 63.7, 13.9. MS (EI, 70 eV) m/z (% relative

intensity, [ion]): 81 (24), 92 (18), 110 (19), 127 (15), 138 (100),

156 (55, [M⁺]).

1

7.92 g R6c (47.1 mmol, 73%) was obtained. H NMR (400 MHz,

DMSO-d₆) δ ppm: 8.69 (d, J = 5.2 Hz, 1H), 7.38 (d, J = 5.2 Hz, 1H),

5.60 (t, J = 5.8 Hz, 1H), 4.52 (d, J = 5.8 Hz, 2H), 3.78 (t, J = 6.5 Hz,

2H), 3.22 (s, 3H), 3.05 (t, J = 6.5 Hz, 2H). ¹³C NMR (100 MHz,

DMSO-d₆) δ ppm: 170.9, 167.1, 157.5, 114.9, 70.3, 63.4, 57.8, 38.8.

LRMS calculated for C₈H₁₂N₂O2: 168.2; found 169.2 (M + H).

R6d: [2-(2-Methoxyphenyl)pyrimidin-4-yl]methanol. Step A: 4-

(Dimethoxymethyl)-2-(2-methoxyphenyl)pyrimidine. Using general

procedure 2 and 4.435 g 2-methoxybenzamidine AcOH salt (21.1

mmol) as the appropriate amidine hydrochloride, 4.20 g 4-

(dimethoxymethyl)-2-(2-methoxyphenyl)pyrimidine (19.42 mmol,

1

92%) was obtained. H NMR (400 MHz, DMSO-d₆) δ ppm: 8.93

(d, J = 5.1 Hz, 1H), 7.55−7.43 (m, 3H), 7.16 (d, J = 8.3 Hz, 1H),

7.08−7.02 (m, 1H), 5.31 (s, 1H), 3.76 (s, 3H), 3.37 (s, 6H). ¹³C

NMR (100 MHz, DMSO-d₆) δ ppm: 164.9, 164.8, 158.1, 157.3,

131.0, 130.9, 128.4, 120.2, 115.8, 112.3, 102.7, 55.7, 53.7. LRMS

calculated for C₁₄H₁₆N₂O₃: 260.3; found 261.2 (M + H).

Step B: [2-(2-Methoxyphenyl)pyrimidin-4-yl]methanol. Using

general procedure 1 and 2.36 g 4-(dimethoxymethyl)-2-(2-

methoxyphenyl)pyrimidine (9.10 mmol) as the appropriate acetal,

1.71 g [2-(2-methoxyphenyl)pyrimidin-4-yl]methanol (R6d, 7.91

mmol, 87%) was obtained. ¹H NMR (400 MHz, DMSO-d₆) δ

ppm: 8.84 (d, J = 5.2 Hz, 1H), 7.50−7.39 (m, 3H), 7.13 (d, J = 8.3

Hz, 1H), 7.06−6.99 (m, 1H), 5.65 (t, J = 5.9 Hz, 1H), 4.58 (d, J = 5.9

Hz, 2H), 3.74 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ ppm:

170.9, 164.4, 157.4, 157.1, 130.9, 130.6, 128.8, 120.1, 115.1, 112.2,

63.5, 55.7. LRMS calculated for C₁₂H₁₂N₂O₂: 216.2; found 217.2 (M

+ H).

R6b: (2-(Morpholin-4-yl)pyrimidin-4-yl)methanol. Step A: 4-

(Dimethoxymethyl)-2-methylsulfonylpyrimidine. 180 g R6aa (940

mmol) was dissolved in 1.5 L MeOH and 1.5 L water, then 752 g

Oxone (1220 mmol) was added portionwise at −5 °C, then stirred at

0 °C overnight. The reaction mixture was concentrated under reduced

pressure to half volume using a 30 °C bath and then the mixture was

ﬁltered, and the precipitate was washed with DCM. The ﬁltrate was

extracted with DCM. The combined organic layer was dried over

MgSO₄, ﬁltered and the ﬁltrate was concentrated under reduced

pressure to give 205 g 4-(dimethoxymethyl)-2-methylsulfonyl-

1

pyrimidine (868 mmol, 92%) as a light brown oil. H NMR (400

MHz, DMSO-d₆) δ ppm: 9.14 (d, J = 5.1 Hz, 1H), 7.86 (d, J = 5.1

Hz, 1H), 5.45 (s, 1H), 3.43 (s, 6H), 3.38 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆) δ ppm: 167.0, 165.4, 160.2, 121.3, 101.7, 54.0, 39.0.

LRMS calculated for C₈H₁₂N₂O₄: 232.3; found 233.0 (M + H).

Step B: 4-[4-(Dimethoxymethyl)pyrimidin-2-yl]morpholine. 3.50

g 4-(dimethoxymethyl)-2-methylsulfonylpyrimidine (15.1 mmol) was

stirred in 23 mL morpholine at rt for 2 h. The reaction mixture was

concentrated under reduced pressure and the residue was puriﬁed via

ﬂash chromatography using heptane and EtOAc as eluents to give

3.53 g 4-[4-(dimethoxymethyl)pyrimidin-2-yl]morpholine (14.8

R6e: [2-(4-Pyridyl)pyrimidin-4-yl]methanol. Using general proce-

dure 2 and 2.50 g pyridine-4-carboxamidine hydrochloride (15.9

mmol) as the appropriate amidine hydrochloride, 1.74 g 4-

(dimethoxymethyl)-2-(4-pyridyl)pyrimidine (7.53 mmol) was ob-

tained which was treated as described in general procedure 1 to yield

1

1.23 g R6e (6.60 mmol, 50%). H NMR (400 MHz, DMSO-d₆) δ

1

ppm: 8.97 (d, J = 5.1 Hz, 1H), 8.78−8.74 (m, 2H), 8.26−8−23 (m,

2H), 7.62 (d, J = 5.1 Hz, 1H), 5.76 (t, J = 5.8 Hz, 1H), 4.68 (d, J = 5.8

Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ ppm: 172.4, 161.2,

158.8, 151.0, 144.7, 122.0, 117.7, 63.9. LRMS calculated for

C₁₀H₉N₃O: 187.2; found 188.2 (M + H).

General Procedure 3: Hydroxymethylation of Pyrazoles. To

the solution of the appropriate alkyl pyrazole (1.0 equiv) in dry THF

mmol, 98%). H NMR (400 MHz, DMSO-d₆) δ ppm: 8.42 (d, J =

4.9 Hz, 1H), 6.71 (d, J = 4.9 Hz, 1H), 5.06 (s, 1H), 3.71−3.62 (m,

8H), 3.31 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ ppm: 165.6,

161.2, 158.8, 106.9, 103.0, 65.9, 53.6, 43.8.

Step C: (2-(Morpholin-4-yl)pyrimidin-4-yl)methanol. Using gen-

eral procedure 1 and 3.53 g 4-[4-(dimethoxymethyl)pyrimidin-2-

yl]morpholine (14.8 mmol) as the appropriate acetal, 2.33 g R6b

P

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(1.5 mL/mmol) n-BuLi solution (1.10 equiv) was added dropwise at

−70 °C. The mixture was stirred at −70 °C for 30 min, then allowed

to warm up to 0 °C in 30 min, then cooled again to −70 °C. Then

DMF (1.10 equiv) was added dropwise at −70 °C, then the reaction

mixture was stirred at rt overnight. Then the mixture was cooled to 15

°C, then sat. aq NH₄Cl solution was added dropwise, and then the

mixture was poured into sat. aq NH₄Cl solution. The phases were

separated, and the aq layer was extracted with EtOAc. The combined

organic layer was dried over Na₂SO₄, ﬁltered, and the ﬁltrate was

concentrated under reduced pressure. Then it was dissolved in EtOH

(0.5 mL/mmol), NaBH₄(1.30 equiv) was added portionwise at −15

°C, and then the mixture was stirred at rt for 1 h. Then it was poured

onto crushed ice and stirred for 16 h. The precipitate was ﬁltered oﬀ,

and the ﬁltrate was concentrated under reduced pressure. The oily

phase was separated, and the aq layer was extracted with EtOAc. The

combined organic layer was dried over Na₂SO₄, ﬁltered, and the

ﬁltrate was concentrated. The crude product was puriﬁed via ﬂash

chromatography, using heptane and EtOAc as eluents.

R6f: (1-Ethyl-1H-pyrazol-5-yl)methanol. Using general procedure

3 and 1.93 g 1-ethylpyrazole (20.0 mmol) as the appropriate alkyl

pyrazole, 1.67 g R6f (13.2 mmol, 66%) was obtained. ¹H NMR (400

MHz, DMSO-d₆) δ ppm: 7.31 (d, J = 1.8 Hz, 1H), 6.12 (d, J = 1.8

Hz, 1H), 5.25 (t, J = 5.5 Hz, 1H) 4.55 (d, J = 5.5 Hz, 2H), 4.10 (q, J =

7.2 Hz, 2H), 2.99 (br s, 1H), 1.31 (t, J = 7.2 Hz, 3H). ¹³C NMR (100

MHz, DMSO-d₆) δ ppm: 141.7, 137.2, 104.9, 53.7, 43.6, 15.6. LRMS

calculated for C₆H₁₀N₂O: 126.2; found 127.2 (M + H).

keeping the temperature under 5 °C, then it was stirred for 30 min at

0 °C. Then it was extracted with EtOAc, dried over Na₂SO₄, ﬁltered,

and the ﬁltrate was concentrated under reduced pressure. The crude

product was puriﬁed via ﬂash chromatography using heptane and

1

EtOAc as eluents to give 830 mg R6i (5.38 mmol, 88%). H NMR

(400 MHz, DMSO-d₆) δ ppm: 7.26 (d, J = 1.6 Hz, 1H), 6.19 (d, J =

1.6 Hz, 1H), 5.31 (t, J = 5.5 Hz, 1H), 4.61 (d, J = 5.5 Hz, 2H), 1.56

(s, 9H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 142.7, 135.6, 107.5,

59.8, 55.7, 29.8. MS (EI, 70 eV) m/z (% relative intensity, [ion]): 69

(40), 81 (100), 97 (35), 121 (37), 139 (11), 154 (20, [M⁺]).

R7: Ethyl (2R)-2-[(5S_a)-5-[3-Chloro-2-methyl-4-[2-(4-methylpi-

perazin-1-yl)ethoxy]phenyl]-6-(4-ﬂuorophenyl)thieno[2,3-d]-

pyrimidin-4-yl]oxy-3-[2-[(2-methylsulfanylpyrimidin-4-yl)-

methoxy]phenyl]propanoate. 1.39 g R5a (2.00 mmol), 0.94 g R6a

(6.00 mmol) and 1.57 g PPh₃(6.00 mmol) were dissolved in 40 mL

dry toluene, then 1.38 g DTBAD (6.00 mmol) was added. The

mixture was stirred at 50 °C under N₂atmosphere for 1 h. The

volatiles were evaporated under reduced pressure and the residue was

puriﬁed via ﬂash chromatography using DCM and MeOH as eluents

to give 1.24 g R7 (1.47 mmol, 74%). ¹H NMR (500 MHz, DMSO-d₆)

δ ppm: 8.70 (d, J = 5.2 Hz, 1H), 8.60 (s, 1H), 7.34 (d, J = 5.2 Hz,

1H), 7.33−7.28 (m, 3H), 7.25−7.14 (m, 4H), 6.98 (d, J = 8.2 Hz,

1H), 6.74 (t, J = 7.5 Hz, 1H), 6.31 (dd, J = 7.5, 1.3 Hz, 1H), 5.47 (dd,

J = 9.3, 4.5 Hz, 1H), 5.17 (d, J = 14.9 Hz, 1H), 5.11 (d, J = 14.9 Hz,

1H), 4.25−4.12 (m, 2H), 4.10−4.00 (m, 2H), 3.12 (dd, J = 13.9, 4.2

Hz, 1H), 2.75−2.65 (m, 2H), 2.56 (dd, J = 13.7, 9.4 Hz, 1H), 2.50 (s,

3H), 2.46 (br s, 4H), 2.24 (br s, 4H), 2.10 (s, 3H), 1.86 (s, 3H), 1.06

(t, J = 7.2 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 171.2,

169.3, 166.5, 166.3, 163.1, 161.7, 158.2, 155.3, 153.8, 152.7, 136.9,

135.9, 133.3, 131.0, 131.0, 130.2, 129.0, 128.8, 128.8, 128.5, 128.4,

127.6, 123.7, 122.1, 120.6, 118.8, 116.0, 113.3, 111.8, 110.7, 73.5,

68.7, 67.3, 60.9, 56.2, 54.6, 53.0, 45.6, 32.1, 17.6, 13.8, 13.5. HRMS

calculated for C₄₃H₄₄ClFN₆O₅S₂: 842.2487; found 843.2660 (M +

H).

R8: Methyl (2R)-2-[6-Bromo-(5S_a)-5-(3-chloro-4-hydroxy-2-

methylphenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-phenylpropa-

noate. Step A: 6-Bromothieno[2,3-d]pyrimidin-4(3H)-one. 10.0 g

3H-thieno[2,3-d]pyrimidin-4-one (65.7 mmol) was dissolved in 100

mL AcOH, then 4.04 mL Br₂(78.8 mmol, dissolved in 20 mL AcOH)

was added dropwise and the mixture was stirred at rt. Additional 3 mL

Br₂was added, and that was repeated until all starting material was

consumed. Then it was poured onto icy water. The formed precipitate

was ﬁltered oﬀ, washed with water, AcOH, and Et₂O and then air-

dried to give 14.9 g 6-bromothieno[2,3-d]pyrimidin-4(3H)-one (64.8

mmol, 98%) as an oﬀ-white solid. ¹H NMR (500 MHz, DMSO-d₆) δ

ppm: 12.63 (br s, 1H), 8.14 (d, J = 2.8 Hz, 1H), 7.55 (s, 1H). ¹³C

NMR (125 MHz, DMSO-d₆) δ ppm: 164.9, 156.0, 146.3, 125.6,

124.5, 110.3.

R6g: [1-(Propan-2-yl)-1H-pyrazol-5-yl]methanol. Using general

procedure 3 and 2.20 g 1-isopropylpyrazole (20.0 mmol) as the

appropriate alkyl pyrazole, 1.70 g R6g (12.1 mmol, 61%) was

1

obtained. H NMR (400 MHz, DMSO-d₆) δ ppm: 7.32 (d, J = 1.7

Hz, 1H), 6.10 (d, J = 1.7 Hz, 1H), 5.23 (t, J = 4.6 Hz, 1H), 4.60 (h, J

= 6.6 Hz, 1H), 4.49 (d, J = 4.6 Hz, 2H), 1.36 (d, J = 6.6 Hz, 6H). ¹³C

NMR (100 MHz, DMSO-d₆) δ ppm: 141.1, 137.0, 104.5, 53.6, 49.3,

22.7. LRMS calculated for C₇H₁₂N₂O: 140.2; found 141.2 (M + H).

R6h: (1-Butyl-1H-pyrazol-5-yl)methanol. Using general procedure

3 and 2.48 g 1-butylpyrazole (20.0 mmol) as the appropriate alkyl

pyrazole, 1.76 g R6h (11.4 mmol, 57%) was obtained. ¹H NMR (400

MHz, DMSO-d₆) δ ppm: 7.30 (d, J = 1.7 Hz, 1H), 6.12 (d, J = 1.7

Hz, 1H), 5.24 (t, J = 5.5 Hz, 1H), 4.48 (d, J = 5.5 Hz, 2H), 4.05 (t, J =

7.2 Hz, 2H), 1.76−1.67 (m, 2H), 1.31−1.21 (m, 2H), 0.88 (t, J = 7.4

Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ ppm: 142.0, 137.2,

104.8, 53.8, 48.3, 32.0, 19.4, 13.6. LRMS calculated for C₈H₁₄N₂O:

154.2; found 155.2 (M + H).

R6i: (1-tert-Butyl-1H-pyrazol-5-yl)methanol. Step A: 1-tert-Butyl-

5-(dimethoxymethyl)pyrazole. 3.50 g tert-butylhydrazine hydro-

chloride (28.1 mmol) and 4.06 g R6 (23.4 mmol) were dissolved

in 15 mL dry MeOH, then 1.52 g NaOMe (28.1 mmol) was added

portionwise and the mixture was stirred at 75 °C for 2 h. The reaction

mixture was cooled to rt and concentrated under reduced pressure.

Then water was added, and it was extracted with DCM. The

combined organic phase was dried over MgSO₄, ﬁltered, and the

ﬁltrate was concentrated under reduced pressure. The crude product

was puriﬁed via ﬂash chromatography using heptane and EtOAc as

eluents to give 1.21 g 1-tert-butyl-5-(dimethoxymethyl)pyrazole (6.10

mmol, 26%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 7.34 (d, J = 1.7

Hz, 1H), 6.33 (d, J = 1.7 Hz, 1H), 5.74 (s, 1H), 3.23 (s, 6H), 1.56 (s,

9H). ¹³C NMR (100 MHz, DMSO-d₆) δ ppm: 139.2, 135.6, 107.4,

96.9, 60.6, 52.4, 29.9. MS (EI, 70 eV) m/z (% relative intensity,

[ion]): 111 (100), 167 (28), 198 (4, [M⁺]).

Step B: 6-Bromo-4-chloro-5-iodothieno[2,3-d]pyrimidine. 1 L cc.

H₂SO₄was cooled with ice−water bath, and 72.0 g KI (0.434 mol)

was added in portions during 15 min and then 32.4 g NaIO₄(0.151

mol) during a 10 min period. The resulting mixture was stirred at rt

for 30 min, then 80.0 g 6-bromothieno[2,3-d]pyrimidin-4(3H)-one

(0.346 mol) was added to the mixture in portions in 30 min while the

internal temperature was kept between −25 °C and −19 °C. The

reaction mixture was stirred at −20 °C for 1.5 h. Ice was added to the

suspension, then the precipitate was ﬁltered oﬀ, washed with water,

ﬁnally with Et₂O, and air-dried to give 116 g 6-bromo-5-iodothieno-

[2,3-d]pyrimidin-4(3H)-one (0.325 mol, 94%). Then it was added to

910 mL POCl₃, then 41 mL N,N-dimethylaniline was added. The

mixture was stirred at 100 °C for 1.5 h. The resulting suspension was

cooled to rt, hexane was added, and it was stirred for further 20 min.

The precipitate was ﬁltered oﬀ, washed with hexane, water, and iPr₂O,

ﬁnally air-dried to give 101.6 g 6-bromo-4-chloro-5-iodothieno[2,3-

Note: 2.42 g 1-tert-butyl-3-(dimethoxymethyl)pyrazole (12.2 mmol,

1

52%) was also obtained. H NMR (400 MHz, DMSO-d₆) δ ppm:

7.75 (d, J = 2.3 Hz, 1H), 6.18 (d, J = 2.3 Hz, 1H), 5.34 (s, 1H), 3.23

(s, 6H), 1.49 (s, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ ppm: 148.4,

126.9, 102.9, 99.6, 57.9, 52.5, 29.5.

Step B: (1-tert-Butyl-1H-pyrazol-5-yl)methanol. 1.21 g 1-tert-

butyl-5-(dimethoxymethyl)pyrazole (6.10 mmol) was stirred with 18

mL 1 M aq HCl solution at 50 °C for 45 min. Then the mixture was

cooled to 0 °C, then 700 mg NaOH (17.5 mmol) was added

portionwise. The pH was adjusted to 8 using 10% aq K₂CO₃solution,

then 461 mg NaBH₄(12.2 mmol) was added portionwise, while

1

d]pyrimidine as a green shaded powder (0.270 mol, 83%). H NMR

(400 MHz, DMSO-d₆) δ ppm: 8.94 (s, 1H). ¹³C NMR (100 MHz,

DMSO-d₆) δ ppm: 168.9, 153.0, 152.5, 129.0, 123.4, 83.8.

Step C: Methyl (2R)-2-(6-Bromo-5-iodothieno[2,3-d]pyrimidin-4-

yl)oxy-3-phenylpropanoate. 15.4 g 6-bromo-4-chloro-5-iodothieno-

[2,3-d]pyrimidine (41.0 mmol), 11.1 g methyl (2R)-2-hydroxy-3-

Q

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

phenylpropanoate (61.6 mmol), and 26.7 g Cs₂CO₃(82 mmol) were

placed in a ﬂask. 40 mL dry DMSO was added, and the mixture was

stirred at 70 °C under Ar atmosphere for 30 min to reach complete

conversion. The reaction mixture was poured onto 200 mL icy water,

and then the pH was set to 5 with 2 M aq HCl solution Then it was

extracted with DCM, dried over Na₂SO₄, ﬁltered, and the ﬁltrate was

concentrated under reduced pressure. The crude product was puriﬁed

via ﬂash chromatography using heptane and EtOAc as eluents to

obtain 15.4 g methyl (2R)-2-(6-bromo-5-iodothieno[2,3-d]pyrimidin-

4-yl)oxy-3-phenylpropanoate (29.7 mmol, 73%). ¹H NMR (500

MHz, DMSO-d₆) δ ppm: 8.64 (s, 1H), 7.49−7.45 (m, 2H), 7.33−

7.28 (m, 2H), 7.26−7.21 (m, 1H), 5.71 (dd, J = 8.4, 4.8 Hz, 1H), 3.65

(s, 3H), 3.37−3.33 (m, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ

ppm: 169.6, 168.4, 160.1, 153.0, 136.0, 129.6, 128.4, 126.9, 119.9,

119.0, 80.7, 75.9, 52.3, 37.0. LRMS calculated for C₁₆H₁₂BrIN₂O₃S:

517.9; found 518.9 (M + H).

Step D: Methyl (2R)-2-[6-Bromo-(5S_a)-5-(3-chloro-4-hydroxy-2-

methylphenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-phenylpropa-

noate. 1.56 g methyl (2R)-2-(6-bromo-5-iodothieno[2,3-d]-

pyrimidin-4-yl)oxy-3-phenylpropanoate (3.00 mmol) and 1.29 g

R3a (4.80 mmol) were dissolved in dioxane, then 2.93 g Cs₂CO₃

(9.00 mmol) dissolved in 6 mL water and 219 mg Pd(ddpf)Cl₂(0.30

mmol) were added and the mixture was stirred at 120 °C in a

microwave reactor under N₂atmosphere for 30 min to reach

complete conversion. Then it was diluted with brine and the pH was

set to 5 with 2 M aq HCl solution. Then it was extracted with DCM.

The combined organic phase was dried over Na₂SO₄, ﬁltered, and the

ﬁltrate was concentrated under reduced pressure. The diaster-

eoisomers were puriﬁed and separated via ﬂash chromatography

using heptane and EtOAc as eluents. The diastereoisomer eluting later

was collected as R8 (311 mg, 0.58 mmol, 19%). ¹H NMR (500 MHz,

DMSO-d₆) δ ppm: 10.38 (s, 1H), 8.61 (s, 1H), 7.17−7.13 (m, 3H),

7.09 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.67−6.63 (m,

2H), 5.47 (dd, J = 9.1, 3.1 Hz, 1H), 3.58 (s, 3H), 2.97 (dd, J = 14.2,

3.1 Hz, 1H), 2.63 (dd, J = 14.2, 9.2 Hz, 1H), 2.02 (s, 3H). ¹³C NMR

(125 MHz, DMSO-d₆) δ ppm: 169.3, 167.5, 161.2, 153.6, 153.0,

135.9, 135.7, 133.4, 129.4, 129.0, 128.1, 126.7, 125.2, 120.6, 117.6,

114.1, 113.4, 75.1, 52.1, 37.0, 17.6. LRMS calculated for

C₂₃H₁₈BrClN₂O₄S: 531.0; found 532.0 (M + H).

ﬁltration to give 35.0 g R9a (159.3 mmol, 73%). ¹H NMR (500 MHz,

DMSO-d₆) δ ppm: 8.93 (s, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.98 (d, J =

8.4 Hz, 1H), 1.99 (s, 3H), 1.36 (sept, J = 7.5 Hz, 3H), 1.10 (dd, J =

7.5, 5.4 Hz, 18H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 172.3,

152.8, 152.4, 151.9, 138.6, 136.8, 129.7, 129.7, 127.2, 125.0, 117.4,

91.4, 17.8, 12.3.

R9: Ethyl (2R)-2-[(5S_a)-5-(3-Chloro-4-hydroxy-2-methylphenyl)-

6-iodothieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-methoxyphenyl)-

propanoate. 6.39 g R9a (10.8 mmol), 2.90 g R2b (12.9 mmol), and

10.5 g Cs₂CO₃(32.3 mmol) were placed in a 250 mL ﬂask. 50 mL

tBuOH was added, and the mixture was stirred at 70 °C for 3 h to

reach complete conversion. It was diluted with icy water, the pH was

set to 6 with 2 M aq HCl solution, and then it was extracted with

EtOAc. The combined organic layer was dried over Na₂SO₄, ﬁltered,

and the ﬁltrate was concentrated under reduced pressure. The residue

was dissolved in 150 mL THF, 13.6 mL TBAF (1 M solution in

THF) was added, and the mixture was stirred at rt for 3 h.

Approximately 100 mL solvent was evaporated under reduced

pressure, then it was diluted with EtOAc, washed with water and

brine. The organic layer was dried over Na₂SO₄, ﬁltered, and the

ﬁltrate was concentrated under reduced pressure. The crude product

was puriﬁed via ﬂash chromatography using heptane and EtOAc as

eluents. The diastereoisomer eluting later was collected as R9 (1.60 g,

1

2.5 mmol, 23%). H NMR (500 MHz, DMSO-d₆) δ ppm: 10.33 (s,

1H), 8.55 (s, 1H), 7.18 (dt, J = 7.9, 1.8 Hz, 1H), 7.02−6.96 (m, 2H),

6.90 (dd, J = 8.5, 0.8 Hz, 1H), 6.75 (dt, J = 7.4, 0.8 Hz, 1H), 6.29 (dd,

J = 7.4, 1.6 Hz, 1H), 5.36 (dd, J = 9.2, 4.3 Hz, 1H), 4.09−3.97 (m,

2H), 3.76 (s, 3H), 2.99 (dd, J = 13.8, 4.2 Hz, 1H), 2.42 (dd, J = 13.8,

9.2 Hz, 1H), 1.97 (s, 3H), 1.06 (t, J = 7.1 Hz, 3H). ¹³C NMR (125

MHz, DMSO-d₆) δ ppm: 171.3, 169.2, 160.8, 156.9, 153.3, 152.8,

138.7, 135.7, 130.9, 129.2, 128.4, 127.9, 123.2, 120.5, 119.9, 117.5,

113.3, 110.5, 85.9, 73.4, 60.8, 55.3, 32.2, 17.8, 13.8. HRMS calculated

for C₂₅H₂₂ClIN₂O₅S: 623.9983; found 625.0055 (M + H).

General Hydrolysis Procedure. The obtained intermediate was

dissolved in dioxane−water 1:1 (10 mL/mmol), and 10 equiv LiOH·

H₂O was added. The mixture was stirred at rt until no further

conversion was observed. Then it was diluted with brine, neutralized

with 2 M aq HCl solution, extracted with DCM. The combined

organic layer was dried over Na₂SO₄, ﬁltered, and the ﬁltrate was

concentrated under reduced pressure. The crude product was puriﬁed

via preparative reversed phase chromatography using 25 mM aq

NH₄HCO₃solution and MeCN as eluents.

General Procedure 4: Mitsunobu Reaction and Hydrolysis. 1

equiv of the appropriate phenol, 2 equiv of the appropriate alcohol,

and 2 equiv PPh₃were dissolved in dry toluene (0.2 M for the

phenol), then 2 equiv DTBAD was added. The mixture was stirred at

50 °C under N₂atmosphere until no further conversion was observed.

The volatiles were evaporated under reduced pressure, and the crude

intermediate was puriﬁed via ﬂash chromatography using EtOAc and

MeOH as eluents. The obtained intermediate was submitted to the

general hydrolysis procedure to give the desired product.

General Procedure 5: Liebskind−Schrogl Reaction. 1 equiv

R7, 3.0 equiv of the appropriate boronic acid derivative, and 3.0 equiv

copper(I) thiophenecarboxylate were dissolved in dry THF (0.1 M

for R7), then 0.15 equiv Pd(PPh₃)₄was added. The mixture was

stirred at 70 °C under N₂atmosphere until no further conversion was

observed. Then it was concentrated under reduced pressure, and the

crude intermediate was puriﬁed via ﬂash chromatography using DCM

and MeOH as eluents. The obtained intermediate was submitted to

the general hydrolysis procedure to give the desired product.

General Procedure 6: Suzuki Coupling−Mitsunobu Reac-

tion−Hydrolysis Sequence. Step A: Suzuki Coupling. 1 equiv R8,

2.5 equiv of the appropriate boronic ester or boronic acid, and 2.5

equiv Cs₂CO₃were dissolved in THF−water (4:1) (12.5 mL/mmol

of R8), then 0.1 equiv Pd(dppf)Cl₂was added. The mixture was

stirred at 110 °C under N₂atmosphere in a microwave reactor until

no further conversion was observed. Then it was diluted with brine,

neutralized with 2 M aq HCl solution, and extracted with DCM. The

combined organic layer was dried over Na₂SO₄, ﬁltered, and the

ﬁltrate was concentrated under reduced pressure. The crude product

R9a: [2-Chloro-4-(4-chloro-6-iodothieno[2,3-d]pyrimidin-5-yl)-3-

methylphenoxy]triisopropylsilane. Step A: [2-Chloro-4-(4-

chlorothieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy]-

triisopropylsilane. 34.50 g R1e (116.3 mmol), 59.32 g R3aa (139.6

n

mmol), 653 mg Pd(OAc)₂(2.908 mmol), 2.085 g BuPAd₂(5.817

mmol), and 74.09 g K₃PO₄(349.0 mmol) were placed in a 1 L ﬂask.

After addition of 450 mL DME and 150 mL water the reaction was

stirred at 60 °C under N₂atmosphere for 1 h. Then sat. aq NH₄Cl

solution was added and it was extracted with EtOAc. The combined

organic layer was dried over MgSO₄, ﬁltered, and the ﬁltrate was

concentrated under reduced pressure. The obtained solid was

sonicated in MeCN/water (3:1) and collected by ﬁltration to give

38.41 g [2-chloro-4-(4-chlorothieno[2,3-d]pyrimidin-5-yl)-3-

1

methylphenoxy]triisopropylsilane (82.6 mmol, 71%). H NMR (500

MHz, DMSO-d₆) δ ppm: 8.97 (s, 1H), 8.00 (s, 1H), 7.15 (d, J = 8.4

Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 2.06 (s, 3H), 1.35 (sept, J = 7.4 Hz,

3H), 1.10 (dd, J = 7.4, 3.3 Hz, 18H). ¹³C NMR (125 MHz, DMSO-

d₆) δ ppm: 169.1, 153.9, 152.8, 151.6, 136.9, 132.9, 129.5, 128.7,

128.2, 126.9, 124.7, 116.6, 17.8, 12.3.

Step B: [2-Chloro-4-(4-chloro-6-iodothieno[2,3-d]pyrimidin-5-

yl)-3-methylphenoxy]triisopropylsilane. 38.00 g [2-chloro-4-(4-

chlorothieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy]-

triisopropylsilane (81.27 mmol) was dissolved in 1 L dry THF, then

cooled to −78 °C under Ar atmosphere. 48.76 mL LDA (97.53 mmol,

2 M in THF, EtPh, hexanes) was added, and the mixture was stirred

at −78 °C for 1 h. Then 24.75 g I₂(97.53 mmol) was added and the

mixture was allowed to warm up to rt. Sat. aq NH₄Cl solution was

added, and it was extracted with EtOAc. The combined organic layer

was washed with aq Na₂S₂O₃solution, then dried over Na₂SO₄,

ﬁltered, and the ﬁltrate was concentrated under reduced pressure. The

obtained solid was sonicated in MeCN/water (3:1) and collected by

R

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

was puriﬁed via ﬂash chromatography using heptane and EtOAc as

eluents.

Step B: Mitsunobu Coupling−Hydrolysis. The product of step A

was transformed following general procedure 4 using 2-(4-

methylpiperazin-1-yl)ethanol as the appropriate alcohol.

the mixture was set to 4 with 2 M aq HCl solution, and then it was

extracted with DCM. The combined organic layer was dried over

Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. The crude intermediate was puriﬁed via ﬂash chromatog-

raphy using heptane and EtOAc as eluents to obtain a mixture of

diastereoisomers. It was dissolved in 10 mL THF, 0.6 mL TBAF

solution (0.6 mmol, 1 M in THF) was added, and the mixture was

stirred at rt until no further conversion was observed. Then it was

diluted with EtOAc, washed with water and brine. The organic layer

was dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated

under reduced pressure and puriﬁed via ﬂash chromatography using

heptane and EtOAc as eluents. The diastereoisomer eluting later was

collected as isopropyl (2R)-2-[6-acetyl-(5S_a)-5-(3-chloro-4-hydroxy-

2-methylphenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-phenylpropanoate

General Procedure 7: Suzuki Coupling−Mitsunobu Reac-

tion−Hydrolysis Sequence. Step A: Suzuki Coupling. 2.5 equiv of

the appropriate boronic acid was dissolved in dry dioxane (5 mL/

mmol R9), then 2.5 equiv pinacol and dry acidic Amberlyst (100 mg/

mmol boronic acid) were added and the mixture was stirred at rt

overnight, then it was ﬁltered (if the appropriate boronic ester was

available, then it was dissolved in dioxane (5 mL/mmol R9) and this

solution was used instead of the ﬁltrate). 1 equiv R9, 0.1 equiv PdCl₂·

dppf, 2.5 equiv Cs₂CO₃, and water (2.5 mL/mmol) were added to the

boronic ester solution, and the mixture was stirred at 110 °C under N₂

atmosphere in a microwave reactor until no further conversion was

observed. Then it was diluted with brine, neutralized with 2 M aq HCl

solution, and extracted with DCM. The combined organic layer was

dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under

reduced pressure. The crude product was puriﬁed via ﬂash

chromatography using heptane and EtOAc as eluents.

1

(73 mg, 0.14 mmol, 32%). H NMR (500 MHz, DMSO-d₆) δ ppm:

10.43 (br s, 1H), 8.71 (s, 1H), 7.22−7.17 (m, 3H), 7.15 (d, J = 8.4

Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 8.0 Hz, 2H), 5.46 (dd,

J = 8.4, 4.2 Hz, 1H), 4.78−4.71 (m, 1H), 2.86 (dd, J = 14.2, 4.2 Hz,

1H), 2.64 (dd, J = 14.2, 8.4 Hz, 1H), 2.03 (s, 3H), 1.94 (s, 3H), 1.07

(d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.2 Hz, 3H). ¹³C NMR (125 MHz,

DMSO-d₆) δ ppm: 192.6, 168.1, 167.9, 164.4, 155.2, 153.9, 139.2,

137.9, 135.5, 129.0, 128.6, 128.2, 126.7, 125.7, 120.8, 119.1, 113.5,

75.4, 68.7, 68.5, 36.8, 28.8, 21.3, 21.1, 17.8. HRMS calculated for

C₂₇H₂₅ClN₂O₅S: 524.1173; found 525.1244 (M + H).

Step B: Mitsunobu Reaction and Hydrolysis. The product of step

A was transformed following general procedure 4 using 2-(4-

methylpiperazin-1-yl)ethanol as the appropriate alcohol.

Examples in the Manuscript. (2R)-2-[((5S_a)-5-{3-cChloro-2-

methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-ethylthieno-

[2,3-d]pyrimidin-4-yl)oxy]-3-phenylpropanoic Acid (1a). 1a was

prepared according to the published procedure.¹⁴

Step C: (2R)-2-[(6-Acetyl-(5S_a)-5-{3-chloro-2-methyl-4-[2-(4-

methylpiperazin-1-yl)ethoxy]phenyl}thieno[2,3-d]pyrimidin-4-yl)-

oxy]-3-phenylpropanoic Acid (1c). Using general procedure 4 and 63

mg isopropyl (2R)-2-[6-acetyl-(5S_a)-5-(3-chloro-4-hydroxy-2-

methylphenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-phenylpropanoate

(0.12 mmol) as the appropriate phenol and 2-(4-methylpiperazin-1-

yl)ethanol as the appropriate alcohol, 15 mg 1c (0.024 mmol, 20%)

was obtained. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.69 (s, 1 H),

7.32 (d, J = 8.5 Hz, 1H), 7.22 (d, J = 8.5 Hz, 1H), 7.18−7.12 (m,

3H), 6.79 (d, J = 6.8 Hz, 1H), 5.39 (dd, J = 9.6, 3.0 Hz, 1H), 4.26 (t, J

= 5.4 Hz, 2H), 3.01 (dd, J = 14.4, 3.0 Hz, 1H), 2.82−2.74 (m, 2H),

2.62−2.37 (m, 9H), 2.22 (br s, 3H), 2.00 (s, 3H), 1.93 (s, 3H). ¹³C

NMR (125 MHz, DMSO-d₆) δ ppm: 192.4, 170.1, 167.8, 165.3,

155.5, 154.3, 138.8, 137.7, 137.1, 135.6, 128.9, 128.8, 128.2, 127.7,

126.4, 122.3, 119.3, 110.8, 76.6, 67.2, 56.1, 54.2, 52.3, 45.0, 37.1, 28.9,

17.8. HRMS calculated for C₃₁H₃₃ClN₄O₅S: 608.186; found 609.194

(M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(prop-1-yn-1-yl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-phenylpropanoic Acid (1d). Step A: Methyl (2R)-2-{[5-Iodo-

6-(prop-1-yn-1-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-phenylpropa-

noate. 1.13 g R1f (2.00 mmol), 70 mg Pd(PPh₃)₂Cl₂(5 mol %, 0.10

mmol), and 38 mg CuI (10 mol %, 0.20 mmol) were dissolved in 10

mL DIPA, then propyne gas was bubbled through the reaction

mixture, which was stirred at 45 °C for 20 min to reach complete

conversion. It was concentrated under reduced pressure and puriﬁed

via ﬂash chromatography using heptane and EtOAc as eluents to

obtain 760 mg methyl (2R)-2-{[5-iodo-6-(prop-1-yn-1-yl)thieno[2,3-

d]pyrimidin-4-yl]oxy}-3-phenylpropanoate (1.59 mmol, 79%). LRMS

calculated for C₁₉H₁₅IN₂O₃S: 478; found 479 (M + H).

Step B: Methyl (2R)-2-{[(5S_a)-5-(3-Chloro-4-hydroxy-2-methyl-

phenyl)-6-(prop-1-yn-1-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-phe-

nylpropanoate. 469 mg methyl (2R)-2-{[5-iodo-6-(prop-1-yn-1-

yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-phenylpropanoate (0.98

mmol) and 537 mg R3a (2.00 mmol) were dissolved in 10 mL 1,4-

dioxane, then 815 mg Cs₂CO₃(2.50 mmol) dissolved in 2 mL water

was added followed by 71 mg AtaPhos (0.10 mmol) and the mixture

was heated under N₂atmosphere at 110 °C in a microwave reactor for

10 min to reach complete conversion. After dilution with DCM and

brine the pH was set to 5 with 2 M aq HCl solution and the aq phase

was extracted with DCM. The combined organic layer was dried over

Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. The diastereoisomers were puriﬁed and separated via ﬂash

chromatography using heptane and EtOAc as eluents. The

diastereoisomer eluting later was collected as methyl (2R)-2-

{[(5S_a)-5-(3-chloro-4-hydroxy-2-methylphenyl)-6-(prop-1-yn-1-yl)-

thieno[2,3-d]pyrimidin-4-yl]oxy}-3-phenylpropanoate (225 mg, 0.46

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(2-methylprop-1-en-1-yl)thieno[2,3-d]-

pyrimidin-4-yl]oxy}-3-phenylpropanoic Acid (1b). Using general

procedure 6, 202 mg R8 (0.38 mmol) and 232 μL 4,4,5,5-tetramethyl-

2-(2-methylprop-1-enyl)-1,3,2-dioxaborolane (1.13 mmol) as the

appropriate boronic ester, 49 mg 1b (0.079 mmol, 21%) was

obtained. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.53 (s, 1H), 7.23

(d, J = 8.6 Hz, 1H), 7.17 (d, J = 8.6 Hz, 1H), 7.15−7.08 (m, 3H),

6.68−6.60 (m, 2H), 5.95 (br s, 1H), 5.30 (dd, J = 9.8 Hz, 2.8 Hz,

1H), 4.30−4.17 (m, 2H), 3.00 (dd, J = 14.1 Hz, 2.8 Hz, 1H), 2.83−

2.70 (m, 2H), 2.70−2.32 (br, 8H), 2.53 (dd, J = 14.1 Hz, 9.8 Hz,

1H), 2.23 (s, 3H), 2.21 (s, 3H), 1.98 (s, 3H), 1.82 (s, 3H). ¹³C NMR

(125 MHz, DMSO-d₆) δ ppm: 171.0, 166.7, 166.0, 154.1, 152.8,

140.3, 137.5, 136.6, 135.3, 130.7, 129.5, 129.3, 128.4, 128.3 126.8,

122.4, 118.1, 116.9, 110.8, 76.4, 67.5, 56.6, 54.5, 52.7, 45.3, 37.6, 27.9,

20.7, 18.2. HRMS calculated for C₃₃H₃₇ClN₄O₄S: 620.2224; found

621.2287 (M + H).

(2R)-2-[(6-Acetyl-(5S_a)-5-{3-chloro-2-methyl-4-[2-(4-methylpiper-

azin-1-yl)ethoxy]phenyl}thieno[2,3-d]pyrimidin-4-yl)oxy]-3-phenyl-

propanoic Acid (1c). Step A: 1-[4-Chloro-5-(3-chloro-2-methyl-4-

triisopropylsilyloxyphenyl)thieno[2,3-d]pyrimidin-6-yl]ethanone.

935 mg R9a (2.0 mmol) was dissolved in 20 mL dry THF and then

cooled to −78 °C under Ar atmosphere. 1.2 mL LDA solution (2.4

mmol, 2 M in THF, EtPh, hexanes) was added, and the mixture was

stirred at −78 °C for 1 h. Then 265 mg AcOAc (2.6 mmol) was

added and the mixture was allowed to warm up to rt. Then sat. aq

NH₄Cl solution was added and the mixture was extracted with

EtOAc. The combined organic layer was dried over Na₂SO₄, ﬁltered,

and the ﬁltrate was concentrated under reduced pressure. The crude

product was puriﬁed via ﬂash chromatography, using heptane and

EtOAc as eluents to obtain 289 mg 1-[4-chloro-5-(3-chloro-2-methyl-

4-triisopropylsilyloxyphenyl)thieno[2,3-d]pyrimidin-6-yl]ethanone

1

(0.57 mmol, 28%). H NMR (400 MHz, CDCl₃) δ ppm: 8.94 (s,

1H), 6.98 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 2.17 (s, 3H),

2.03 (s, 3H), 1.44−1.32 (m, 3H), 1.17 (d, J = 7.4 Hz, 18H).

Step B: Isopropyl (2R)-2-[6-Acetyl-(5S_a)-5-(3-chloro-4-hydroxy-2-

methylphenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-phenylpropa-

noate. 278 mg 1-[4-chloro-5-(3-chloro-2-methyl-4-

triisopropylsilyloxyphenyl)thieno[2,3-d]pyrimidin-6-yl]ethanone

i

(0.55 mmol) was dissolved in 5 mL PrOH, 118 mg methyl (2R)-2-

hydroxy-3-phenyl-propanoate (0.65 mmol) and 538 mg Cs₂CO₃

(1.65 mmol) were added, and the mixture was stirred at rt until no

further conversion was observed. It was diluted with water, the pH of

S

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

mmol, 47%). H NMR (500 MHz, DMSO-d₆) δ ppm: 10.35 (br s,

pyrimidin-4-yl]oxy}-3-phenylpropanoic Acid (1e). Using general

procedure 4 and 156 mg methyl (2R)-2-{[(5S_a)-5-(3-chloro-4-

hydroxy-2-methylphenyl)-6-(cyclopropylethynyl)thieno[2,3-d]-

pyrimidin-4-yl]oxy}-3-phenylpropanoate (0.30 mmol) as the appro-

priate phenol and 2-(4-methylpiperazin-1-yl)ethanol as the appro-

priate alcohol, 128 mg 1e (0.20 mmol, 68%) was obtained. ¹H NMR

(500 MHz, DMSO-d₆) δ ppm: 12.20 (br s, 1H), 8.61 (s, 1H), 7.31 (d,

J = 8.5 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H), 7.13−7.05 (m, 3H), 6.57−

6.53 (m, 2H), 5.28 (dd, J = 10.1, 2.5 Hz, 1H), 4.32−4.20 (m, 2H),

3.03 (dd, J = 14.1, 2.5 Hz, 1H), 2.87−2.74 (m, 2H), 2.80−2.50 (br m,

8H), 2.58 (dd, J = 14.1, 10.1 Hz, 1H), 2.42 (s, 3H), 2.11 (s, 3H),

1.55−1.48 (m, 1H), 0.92−0.84 (m, 2H), 0.67−0.54 (m, 2H). ¹³C

NMR (125 MHz, DMSO-d₆) δ ppm: 170.6, 166.0, 162.6, 153.80,

153.79, 136.9, 136.2, 136.1, 130.1, 128.9, 127.9, 127.4, 126.3, 121.8,

119.1, 117.1, 110.2, 104.1, 76.1, 67.7, 67.1, 55.8, 53.3, 51.2, 43.6, 37.1,

17.8, 9.01, 9.00, 0.0. HRMS calculated for C₃₄H₃₅ClN₄O₄S: 630.2068;

found 631.2096 (M + H).

1H), 8.63 (s, 1H), 7.16−7.10 (m, 4H), 7.00 (d, J = 8.4 Hz, 1H),

6.63−6.59 (m, 2H), 5.45 (dd, J = 8.9, 3.3 Hz, 1H), 3.57 (s, 3H), 2.97

(dd, J = 14.2, 3.3 Hz, 1H), 2.68 (dd, J = 14.2, 8.9 Hz, 1H), 2.09 (s,

3H), 2.03 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 169.3,

166.2, 162.1, 155.0, 153.6, 136.0, 135.9, 135.7, 129.6, 129.1, 128.1,

126.7, 120.5, 119.4, 117.2, 113.0, 97.3, 75.1, 71.9, 52.1, 37.0, 17.8, 4.4.

HRMS calculated for C₂₆H₂₁ClN₂O₄S: 492.0911; found 493.0973 (M

+ H).

Step C: (2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiper-

azin-1-yl)ethoxy]phenyl}-6-(prop-1-yn-1-yl)thieno[2,3-d]pyrimidin-

4-yl]oxy}-3-phenylpropanoic Acid (1d). Using general procedure 4

and 360 mg methyl (2R)-2-{[(5S_a)-5-(3-chloro-4-hydroxy-2-methyl-

phenyl)-6-(prop-1-yn-1-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-phe-

nylpropanoate (0.73 mmol) as the appropriate phenol and 2-(4-

methylpiperazin-1-yl)ethanol as the appropriate alcohol, 338 mg 1d

1

(0.56 mmol, 77%) was obtained. H NMR (500 MHz, DMSO-d₆) δ

ppm: 12.38 (br s, 1H), 8.62 (s, 1H), 7.32 (d, J = 8.7 Hz, 1H), 7.18 (d,

J = 8.7 Hz, 1H), 7.12−7.07 (m, 3H), 6.60−6.56 (m, 2H), 5.30 (dd, J

= 10.0, 2.7 Hz, 1H), 4.30−4.19 (m, 2H), 3.02 (dd, J = 14.2, 2.7 Hz,

1H), 2.85−2.74 (m, 2H), 2.80−2.50 (br m, 8H), 2.57 (dd, J = 14.2,

10.0 Hz, 1H), 2.38 (s, 3H), 2.11 (s, 3H), 2.02 (s, 3H). ¹³C NMR

(125 MHz, DMSO-d₆) δ ppm: 170.5, 166.0, 162.7, 153.9, 153.8,

136.9, 136.1, 135.6, 130.1, 128.9, 127.9, 127.4, 126.3, 121.9, 119.2,

117.2, 110.1, 97.2, 76.1, 71.9, 67.0, 55.9, 53.4, 51.4, 43.9, 37.1, 17.8,

4.4. HRMS calculated for C₃₂H₃₃ClN₄O₄S: 604.1911; found 605.2000

(M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(cyclopropylethynyl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-phenylpropanoic Acid (1e). Step A: Methyl (2R)-2-{[6-

(Cyclopropylethynyl)-5-iodothieno[2,3-d]pyrimidin-4-yl]oxy}-3-

phenylpropanoate. 1.13 g R1f (2.00 mmol), 152 mg ethynylcyclo-

propane (2.30 mmol), 70 mg Pd(PPh₃)₂Cl₂(5 mol %, 0.10 mmol),

and 38 mg CuI (10 mol %, 0.20 mmol) were dissolved in 4 mL DIPA,

and the mixture was stirred under N₂atmosphere at 40 °C for 30 min

to reach complete conversion. Then it was concentrated under

reduced pressure and puriﬁed via ﬂash chromatography using heptane

and EtOAc as eluents to obtain 968 mg methyl (2R)-2-{[6-

(cyclopropylethynyl)-5-iodothieno[2,3-d]pyrimidin-4-yl]oxy}-3-phe-

nylpropanoate (1.92 mmol, 96%). LRMS calculated for

C₂₁H₁₇IN₂O₃S: 504.0; found 505.0 (M + H).

Step B: Methyl (2R)-2-{[(5S_a)-5-(3-Chloro-4-hydroxy-2-methyl-

phenyl)-6-(cyclopropylethynyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-

phenylpropanoate. 968 mg methyl (2R)-2-{[6-(cyclopropylethynyl)-

5-iodothieno[2,3-d]pyrimidin-4-yl]oxy}-3-phenylpropanoate (1.92

mmol) and 670 mg R3a (2.50 mmol) were dissolved in 8 mL 2-

Me-THF, then 2.5 mL TBAOH (1 M solution in water, 2.50 mmol)

was added followed by 68 mg AtaPhos (5 mol %, 0.096 mmol). The

mixture was heated under N₂atmosphere at 110 °C in a microwave

reactor for 10 min to reach complete conversion. Then it was diluted

with DCM and brine, pH was set to 5 with 2 M aq HCl solution, and

the aq phase was extracted with DCM. The combined organic layer

was dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated

under reduced pressure. The diastereoisomers were puriﬁed and

separated via ﬂash chromatography using heptane and EtOAc as

eluents. The diastereoisomer eluting later was collected as methyl

(2R)-2-{[(5S_a)-5-(3-chloro-4-hydroxy-2-methylphenyl)-6-

(cyclopropylethynyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-phenylpro-

panoate (297 mg, 0.57 mmol, 30%) with a purity of 95.0%. ¹H NMR

(500 MHz, CDCl₃) δ ppm: 8.51 (s, 1H), 7.26 (d, J = 8.4 Hz, 1H),

7.16−7.12 (m, 3H), 7.04 (d, J = 8.4 Hz, 1H), 6.65−6.60 (m, 2H),

5.76 (br s, 1H), 5.41 (dd, J = 10.4, 2.8 Hz, 1H), 3.71 (s, 3H), 3.03

(dd, J = 14.2, 2.8 Hz, 1H), 2.71 (dd, J = 14.2, 10.4 Hz, 1H), 2.18 (s,

3H), 1.40−1.34 (m, 1H), 0.90−0.82 (m, 2H), 0.73−0.63 (m, 2H).

¹³C NMR (125 MHz, CDCl₃) δ ppm: 170.4, 167.1, 162.3, 153.3,

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(furan-2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-

phenylpropanoic Acid (1f). Using general procedure 6 and 133 mg

R8 (0.25 mmol) and 2-(2-furyl)-4,4,5,5-tetramethyl-1,3,2-dioxabor-

olane as the appropriate boronic ester, 35 mg 1f (0.055 mmol, 22%)

was obtained. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.57 (s, 1H),

7.80−7.77 (m, 1H), 7.28 (d, J = 8.6 Hz, 1H), 7.25 (d, J = 8.6 Hz,

1H), 7.19−7.10 (m, 3H), 6.74−6.68 (m, 2H), 6.51 (dd, J = 3.5, 1.8

Hz, 1H), 5.64 (d, J = 3.5 Hz, 1H), 5.34 (dd, J = 10.0, 2.5 Hz, 1H),

4.32−4.22 (m, 2H), 3.02 (dd, J = 14.2, 2.5 Hz, 1H), 2.86−2.73 (m,

2H), 2.70−2.34 (m, 8H), 2.53−2.45 (m, 1H), 2.25 (s, 3H), 1.94 (s,

3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 170.9, 166.4, 163.6,

154.5, 153.4, 147.4, 144.3, 137.6, 136.3, 129.7, 129.3, 128.5, 128.4,

127.7, 127.2, 126.8, 122.7, 119.3, 113.2, 111.6, 109.5, 76.5, 67.6, 56.6,

54.5, 52.7, 45.3, 37.7, 17.7. HRMS calculated for C₃₃H₃₃ClN₄O₅S:

632.186; found 633.1939 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(thiophen-2-yl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-phenylpropanoic Acid (1g). Using general procedure 6 and

133 mg R8 (0.25 mmol) and 2-thienylboronic acid as the appropriate

1

boronic acid, 21 mg 1g (0.032 mmol, 13%) was obtained. H NMR

(500 MHz, DMSO-d₆) δ ppm: 8.56 (s, 1H), 7.55 (d, J = 5.0 Hz, 1H),

7.32 (d, J = 3.8 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H), 7.24 (d, J = 8.6 Hz,

1H), 7.19−7.10 (m, 3H), 7.05 (dd, J = 5.0, 3.8 Hz, 1H), 6.75−6.70

(m, 2H), 5.32 (dd, J = 10.1, 2.4 Hz, 1H), 4.31−4.22 (m, 2H), 3.02

(dd, J = 14.2, 2.4 Hz, 1H), 2.84−2.72 (m, 2H), 2.70−2.30 (m, 8H),

2.50−2.43 (m, 1H), 2.20 (s, 3H), 1.91 (s, 3H). ¹³C NMR (125 MHz,

DMSO-d₆) δ ppm: 166.2, 163.4, 154.8, 153.4, 137.6, 137.0, 134.5,

130.5, 129.7, 129.2, 128.5, 128.5, 127.9, 127.7, 126.8, 122.8, 111.6,

76.5, 67.6, 56.5, 54.7, 52.8, 45.5, 37.7, 17.8. HRMS calculated for

C₃₃H₃₃ClN₄O₄S₂: 648.1632; found 649.172 (M + H).

(2R)-2-[((5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-phenylthieno[2,3-d]pyrimidin-4-yl)oxy]-3-phe-

nylpropanoic Acid (1h). Using general procedure 6 and 133 mg R8

(0.25 mmol) and 4,4,5,5-tetramethyl-2-phenyl-1,3,2-dioxaborolane as

the appropriate boronic ester, 20 mg 1h (0.031 mmol, 12%) was

obtained. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.60 (s, 1H),

7.39−7.29 (m, 4H), 7.27−7.20 (m, 2H), 7.16 (d, J = 8.5 Hz, 1H),

7.13−7.07 (m, 3H), 6.68−6.61 (m, 2H), 5.34 (dd, J = 10.0, 2.6 Hz,

1H), 4.30−4.14 (m, 2H), 3.02 (dd, J = 14.0, 2.6 Hz, 1H), 2.82−2.68

(m, 2H), 2.55 (dd, J = 14.0, 10.0 Hz, 1H), 2.70−2.30 (m, 8H), 2.24

(s, 3H), 1.84 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 170.9,

166.8, 163.6, 154.1, 153.2, 137.5, 136.3, 131.0, 128.9, 128.4, 122.4,

119.4, 111.0, 76.5, 67.5, 56.5, 54.5, 52.6, 45.2, 37.6, 18.0. HRMS

calculated for C₃₅H₃₅ClN₄O₄S: 642.2068; found 643.2135 (M + H).

(2R)-2-{[6-(1-Benzofuran-2-yl)-(5S_a)-5-{3-chloro-2-methyl-4-[2-

(4-methylpiperazin-1-yl)ethoxy]phenyl}thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-phenylpropanoic Acid (1i). Using general procedure 6 and

160 mg R8 (0.300 mmol) and 2-(benzofuran-2-yl)-4,4,5,5-tetrameth-

yl-1,3,2-dioxaborolane as the appropriate boronic ester, 23.5 mg 1i

(0.034 mmol, 11%) was obtained. ¹H NMR (500 MHz, DMSO-d₆) δ

ppm: 8.62 (s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H),

7.36 (d, J = 8.6 Hz, 1H), 7.37−7.32 (m, 1H), 7.29 (d, J = 8.6 Hz,

1H), 7.25−7.21 (m, 1H), 7.19−7.11 (m, 3H), 6.74 (d, J = 7.0 Hz,

151.2, 136.02, 135.98, 135.8, 130.6, 129.0, 128.2, 127.9, 126.7, 120.9,

120.3, 117.8, 112.5, 104.0, 75.3, 68.0, 52.4, 37.6, 18.0, 9.2, 9.1, 0.5.

HRMS calculated for C₂₈H₂₃ClN₂O₄S: 518.1067; found 519.1176 (M

+ H).

Step C: (2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiper-

azin-1-yl)ethoxy]phenyl}-6-(cyclopropylethynyl)thieno[2,3-d]-

T

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

2H), 6.10 (s, 1H), 5.36 (dd, J = 9.9, 2.6 Hz, 1H), 4.36−4.25 (m, 2H),

3.04 (dd, J = 14.4, 2.6 Hz, 1H), 2.86−2.76 (m, 2H), 2.49 (d, J = 14.4,

9.9 Hz, 1H), 2.63−2.39 (m, 8H), 2.24 (s, 3H), 1.98 (s, 3H). ¹³C

NMR (125 MHz, DMSO-d₆) δ ppm: 170.3, 166.5, 163.5, 154.2,

153.53, 153.45, 148.8, 135.7, 129.9, 129.2, 128.8, 128.1, 127.7, 126.6,

126.3, 125.9, 123.8, 122.5, 121.9, 119.0, 111.4, 111.1, 104.9, 76.4,

67.2, 56.1, 54.2, 52.4, 45.0, 37.2, 17.3. HRMS calculated for

C₃₇H₃₅ClN₄O₅S: 682.2017; found 683.2084 (M + H)

(2R)-2-[((5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-ethylthieno[2,3-d]pyrimidin-4-yl)oxy]-3-(2-

methoxyphenyl)propanoic Acid (2a). Step A: Ethyl (2R)-2-(6-Ethyl-

5-iodothieno[2,3-d]pyrimidin-4-yl)oxy-3-(2-methoxyphenyl)-

propanoate. 100 mg 4-chloro-6-ethyl-5-iodothieno[2,3-d]-

pyrimidine²(0.308 mmol), 104 mg R2b (0.462 mmol), and 502

mg Cs₂CO₃(1.54 mmol) were placed in a 10 mL ﬂask. 3.5 mL

tBuOH was added, and the mixture was stirred at 65 °C under N₂

atmosphere for 3 h. Then the mixture was cooled to rt and

concentrated under reduced pressure. It was diluted with water,

extracted with DCM. The combined organic layer was dried over

MgSO4, ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. The crude product was puriﬁed via ﬂash chromatography

using heptane and EtOAc as eluents to obtain 110 mg ethyl (2R)-2-

(6-ethyl-5-iodothieno[2,3-d]pyrimidin-4-yl)oxy-3-(2-

methoxyphenyl)propanoate (0.215 mmol, 70%). ¹H NMR (500

MHz, DMSO-d₆) δ ppm: 8.56 (s, 1H), 7.45−7.42 (m, 1H), 7.26−

7.21 (m, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.89−6.85 (m, 1H), 5.63 (dd,

J = 8.8, 5.5 Hz, 1H), 4.08 (q, J = 7.1 Hz, 2H), 3.80 (s, 3H), 3.38 (dd, J

= 13.8, 5.5 Hz, 1H), 3.24 (dd, J = 13.8, 8.8 Hz, 1H), 2.91 (q, J = 7.5

Hz, 2H), 1.26 (t, J = 7.5 Hz, 3H), 1.07 (t, J = 7.1 Hz, 3H). ¹³C NMR

(100 MHz, CDCl₃) δ ppm: 169.5, 166.6, 160.9, 157.3, 151.9, 144.8,

131.8, 128.5, 123.5, 120.1, 119.3, 110.6, 74.1, 71.4, 60.8, 55.4, 32.4,

26.5, 14.6, 13.9. HRMS calculated for C₂₀H₂₁IN₂O₄S: 512.0267;

found 513.0340 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(3-methylthiophen-2-yl)thieno[2,3-d]-

pyrimidin-4-yl]oxy}-3-phenylpropanoic Acid (1j). Using general

procedure 6 and 133 mg R8 (0.249 mmol) and 4,4,5,5-tetramethyl-

2-(3-methyl-2-thienyl)-1,3,2-dioxaborolane as the appropriate boronic

1

ester, 54.7 mg 1j (0.082 mmol, 34%) was obtained. H NMR (500

MHz, DMSO-d₆) δ ppm: 8.58 (s, 1H), 7.51 (d, J = 5.1 Hz, 1H), 7.31

(d, J = 8.7 Hz, 1H), 7.13 (d, J = 8.7 Hz, 1H), 7.11−7.07 (m, 3H),

6.90 (d, J = 5.1 Hz, 1H), 6.65−6.61 (m, 2H), 5.32 (dd, J = 10.2, 1.6

Hz, 1H), 4.27−4.11 (m, 2H), 3.03 (dd, J = 13.4, 1.6 Hz, 1H), 2.80−

2.67 (m, 2H), 2.59−2.51 (m, 9H), 2.28 (s, 3H), 2.14 (s, 3H), 1.90 (s,

3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 170.6, 166.8, 163.1,

153.8, 152.9, 137.4, 137.2, 136.3, 130.9, 130.6, 130.5, 130.4, 128.8,

127.9, 127.7, 127.1, 126.2, 121.8, 118.2, 110.3, 76.2, 67.0, 56.0, 53.8,

52.0, 44.5, 37.2, 17.6, 14.9. HRMS calculated for C₃₄H₃₅ClN₄O₄S₂:

662.1788; found 663.1882 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(2-methyl-1,3-thiazol-4-yl)thieno[2,3-d]-

pyrimidin-4-yl]oxy}-3-phenylpropanoic Acid (1k). Using general

procedure 6 and 133 mg R8 (0.25 mmol) and 2-methyl-4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-thiazole as the appropriate

Step B: (2R)-2-[((5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiper-

azin-1-yl)ethoxy] phenyl}-6-ethylthieno[2,3-d]pyrimidin-4-yl)oxy]-

3-(2-methoxyphenyl)propanoic Acid (2a). 105 mg ethyl (2R)-2-

(6-ethyl-5-iodothieno[2,3-d]pyrimidin-4-yl)oxy-3-(2-

methoxyphenyl)propanoate (0.205 mmol) and 97 mg R3b (0.246

mmol) and 134 mg Cs₂CO₃(0.410 mmol) were dissolved in 2 mL

1,4-dioxane and 0.4 mL water. Then 18 mg AtaPhos (0.025 mmol)

was added under N₂atmosphere. The mixture was stirred at 110 °C

under N₂atmosphere in a microwave reactor for 20 min to reach

complete conversion. Then it was diluted with brine and extracted

with DCM. The combined organic layer was dried over Na₂SO₄,

ﬁltered, and the ﬁltrate was concentrated under reduced pressure.

Then it was hydrolyzed according to the general hydrolysis procedure.

The diastereoisomer eluting later was collected as 2a (41 mg, 0.066

1

boronic ester, 21 mg 1k (0.032 mmol, 13%) was obtained. H NMR

(500 MHz, DMSO-d₆) δ ppm: 8.57 (s, 1H), 7.29 (d, J = 8.6 Hz, 1H),

7.26 (d, J = 8.6 Hz, 1H), 7.20−7.10 (m, 3H), 6.76−6.70 (m, 2H),

6.38 (s, 1H), 5.34 (dd, J = 10.0, 2.7 Hz, 1H), 4.30−4.23 (m, 2H),

3.02 (dd, J = 14.0, 2.7 Hz, 1H), 2.83−2.73 (m, 2H), 2.67 (s, 3H),

2.65−2.28 (m, 8H), 2.52−2.44 (m, 1H), 2.20 (s, 3H), 1.93 (s, 3H).

¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 170.8, 166.9, 166.3, 163.6,

154.6, 153.4, 146.6, 137.6, 136.1, 133.2, 129.6, 129.2, 128.7, 128.5,

128.4, 126.8, 123.0, 119.5, 116.2, 111.9, 76.4, 67.7, 56.6, 54.7, 52.9,

45.6, 37.6, 19.0, 17.7. HRMS calculated for C₃₃H₃₄ClN₅O₄S₂:

663.1741; found 664.1823 (M + H).

1

mmol, 32%). H NMR (500 MHz, DMSO-d₆) δ ppm: 8.51 (s, 1H),

7.10−7.24 (m, 3H), 6.89 (d, J = 8.2 Hz, 1H), 6.60−6.73 (m, 1H),

6.19 (d, J = 7.2 Hz, 1H), 5.31 (dd, J = 10.0, 3.1 Hz, 1H), 4.24 (t, J =

5.4 Hz, 2H), 3.76 (s, 3H), 3.09 (dd, J = 13.8, 3.1 Hz, 1H), 2.71−2.80

(m, 2H), 2.47−2.68 (m, 6H), 2.28−2.42 (m, 5H), 2.17 (s, 3H), 1.96

(s, 3H), 1.14 (t, J = 7.5 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ

ppm: 170.94, 165.9, 162.1, 156.9, 153.6, 152.1, 142.6, 135.9, 130.6,

129.7, 128.04, 127.96, 127.9, 124.3, 122.1, 119.7, 118.2, 110.6, 110.5,

73.9, 67.2, 56.2, 55.3, 54.3, 52.5, 45.2, 32.2, 21.6, 17.6, 15.5. HRMS

calculated for C₃₂H₃₇ClN₄O₅S: 624.2173; found 625.2259 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(prop-1-yn-1-yl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-methoxyphenyl)propanoic Acid (2b). Step A: Ethyl (2R)-

2-{[5-Iodo-6-(prop-1-yn-1-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-

methoxyphenyl)propanoate. 4.00 g R1d (12.0 mmol), 4.00 g R2b

(17.8 mmol), and 11.7 g Cs₂CO₃(36.0 mmol) were placed in a ﬂask.

40 mL tBuOH was added, and the mixture was stirred at 50 °C under

N₂atmosphere for 2 h to reach complete conversion. Then it was

diluted with water and the formed precipitate was ﬁltered, washed

with water, then dried in vacuo to obtain 6.16 g ethyl (2R)-2-{[5-iodo-

6-(prop-1-yn-1-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-

methoxyphenyl)propanoate (11.8 mmol, 98%). ¹H NMR (400 MHz,

CDCl₃) δ ppm: 8.52 (s, 1H), 7.38−7.35 (m, 1H), 7.26−7.20 (m,

1H), 6.90−6.83 (m, 2H), 5.79 (dd, J = 8.6, 5.7 Hz, 1H), 4.24−4.11

(m, 2H), 3.84 (s, 3H), 3.49 (dd, J = 13.8, 5.7 Hz, 1H), 3.39 (dd, J =

13.8, 8.6 Hz, 1H), 2.19 (s, 3H), 1.18 (t, J = 7.2 Hz, 3H). ¹³C NMR

(100 MHz, CDCl₃) δ ppm: 170.3, 167.1, 161.7, 157.8, 153.3, 131.8,

128.5, 125.0, 124.2, 120.3, 119.7, 110.1, 98.0, 76.0, 74.6, 74.6, 61.2,

55.3, 32.9, 14.0, 5.1. LRMS calculated for C₂₁H₁₉IN₂O₄S: 522.0;

found 523.0 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-phenylpropanoic Acid (1l). Using general procedure 6 and

133 mg R8 (0.25 mmol) and 2-(4-ﬂuorophenyl)-4,4,5,5-tetramethyl-

1,3,2-dioxaborolane as the appropriate boronic ester, 33 mg 1l (0.05

1

mmol, 20%) was obtained. H NMR (500 MHz, DMSO-d₆) δ ppm:

8.58 (s, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.28−7.24 (m, 2H), 7.21−7.17

(m, 2H), 7.16 (d, J = 8.5 Hz, 1H), 7.10−7.07 (m, 1H), 6.87−6.84 (m,

1H), 6.65−6.61 (m, 1H), 5.33 (dd, J = 10.1, 2.7 Hz, 1H), 4.27−4.17

(m, 2H), 3.03 (dd, J = 14.2, 2.7 Hz, 1H), 2.79−2.70 (m, 2H), 2.63−

236 (m, 8H), 2.54 (dd, J = 14.2, 10.1 Hz, 1H), 2.23 (s, 3H), 1.84 (s,

3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 170.7, 166.3, 163.1,

161.0, 153.7, 152.8, 137.2, 136.5, 135.8, 131.0, 130.6, 129.1, 128.9,

128.6, 127.9, 127.7, 126.3, 122.0, 118.8, 115.9, 110.6, 76.3, 67.1, 56.0,

54.0, 52.2, 44.8, 37.2, 17.5. HRMS calculated for C₃₅H₃₄ClFN₄O₄S:

660.1973; found 661.2042 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(pyridin-4-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-

3-phenylpropanoic Acid (1m). Using general procedure 6 and 177

mg R8 (0.30 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)pyridine as the appropriate boronic ester, 21 mg 1m (0.03 mmol,

11%) was obtained. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.64 (s,

1H), 8.54−8.50 (m, 2H), 7.35 (d, J = 8.8 Hz, 1H), 7.21−7.16 (m,

3H), 7.13−7.08 (m, 3H), 6.69−6.64 (m, 2H), 5.35 (dd, J = 9.8, 2.8

Hz, 1H), 4.29−4.18 (m, 2H), 3.03 (dd, J = 14.3, 2.8 Hz, 1H), 2.81−

2.71 (m, 2H), 2.66−2.31 (br m, 9H), 2.23 (s, 3H), 1.86 (s, 3H). ¹³C

NMR (125 MHz, DMSO-d₆) δ ppm: 170.4, 166.7, 163.7, 153.9,

153.5, 150.2, 140.2, 137.2, 135.7, 134.4, 131.0, 130.2, 128.8, 128.0,

127.3, 126.3, 122.8, 122.2, 118.9, 110.7, 76.4, 67.1, 56.0, 54.0, 52.2,

44.8, 37.2, 17.5. HRMS calculated for C₃₄H₃₄ClN₅O₄S: 643.2020;

found 644.2089 (M + H).

Step B: Ethyl (2R)-2-{[(5S_a)-5-(3-Chloro-4-hydroxy-2-methyl-

phenyl)-6-(prop-1-yn-1-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-

U

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

methoxyphenyl)propanoate. 472 mg ethyl (2R)-2-{[5-iodo-6-(prop-

1-yn-1-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-methoxyphenyl)-

propanoate (0.90 mmol) and 403 mg R3a (1.50 mmol) were

dissolved in 10 mL 1,4-dioxane, then 652 mg Cs₂CO₃(2.00 mmol)

dissolved in 2 mL water was added. Then 64 mg AtaPhos (10 mol %,

0.09 mmol) was added. The mixture was stirred at 110 °C under N₂

atmosphere in a microwave reactor for 10 min to reach complete

conversion. Then it was diluted with DCM and brine, pH was set to 5

with 2 M aq HCl solution, and the aq phase was extracted with DCM.

The combined organic layer was dried over Na₂SO₄, ﬁltered, and the

ﬁltrate was concentrated under reduced pressure. The diaster-

eoisomers were puriﬁed and separated via ﬂash chromatography

using heptane and EtOAc as eluents. The diastereoisomer eluting later

was collected as ethyl (2R)-2-[(5S_a)-5-(3-chloro-4-hydroxy-2-methyl-

phenyl)-6-prop-1-ynylthieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-

methoxyphenyl)propanoate (248 mg, 0.46 mmol, 51%). ¹H NMR

(500 MHz, DMSO-d₆): 10.34 (br s, 1H), 8.61 (s, 1H), 7.18−7.14 (m,

1H), 7.09 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.90−6.87

(m, 1H), 6.71−6.67 (m, 1H), 6.21−6.18 (m, 1H), 5.34 (dd, J = 9.3,

4.4 Hz, 1H), 4.11−3.99 (m, 2H), 3.75 (s, 3H), 3.00 (dd, J = 13.7, 4.4

Hz, 1H), 2.52−2.46 (m, 1H), 2.08 (s, 3H), 2.03 (s, 3H), 1.07 (t, J =

7.0 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 169.2, 166.2,

162.1, 156.9, 153.6, 153.2, 135.92, 135.89, 131.0, 129.6, 128.4, 125.6,

123.2, 120.4, 119.8, 119.3, 117.2, 113.0, 110.5, 97.3, 73.5, 71.9, 60.8,

55.3, 32.1, 17.8, 13.8, 4.4. HRMS calculated for C₂₈H₂₅N₂O₅SCl:

536.1173; found 537.1284 (M + H).

Step C: (2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiper-

azin-1-yl)ethoxy]phenyl}-6-(prop-1-yn-1-yl)thieno[2,3-d]pyrimidin-

4-yl]oxy}-3-(2-methoxyphenyl)propanoic Acid (2b). Using general

procedure 4 and 245 mg ethyl (2R)-2-[(5S_a)-5-(3-chloro-4-hydroxy-

2-methylphenyl)-6-prop-1-ynylthieno[2,3-d]pyrimidin-4-yl]oxy-3-(2-

methoxyphenyl)propanoate (0.46 mmol) as the appropriate phenol

and 2-(4-methylpiperazin-1-yl)ethanol as the appropriate alcohol, 188

mg 2b (0.30 mmol, 65%) was obtained. ¹H NMR (500 MHz, DMSO-

d₆) δ ppm: 12.00 (br s, 1H), 8.62 (s, 1H), 7.27 (d, J = 8.6 Hz, 1H),

7.18 (d, J = 8.6 Hz, 1H), 7.15−7.10 (m, 1H), 6.87 (d, J = 8.0 Hz,

1H), 6.65−6.61 (m, 1H), 6.00 (dd, J = 7.4, 1.4 Hz, 1H), 5.30 (dd, J =

10.3, 3.2 Hz, 1H), 4.30−4.23 (m, 2H), 3.75 (s, 3H), 3.13 (dd, J =

13.8, 3.2 Hz, 1H), 2.89−2.77 (m, 2H), 2.84−2.55 (m, 8H), 2.43 (s,

3H), 2.38 (dd, J = 13.8, 10.3 Hz, 1H), 2.10 (s, 3H), 2.02 (s, 3H). ¹³C

NMR (125 MHz, DMSO-d₆) δ ppm: 170.9, 166.1, 162.6, 156.9,

153.83, 153.79, 136.1, 135.5, 130.8, 130.0, 128.1, 127.3, 124.1, 121.8,

119.6, 119.2, 117.2, 110.5, 110.1, 97.3, 73.9, 71.9, 67.1, 55.8, 55.3,

53.3, 51.3, 43.7, 32.1, 17.8, 4.4. HRMS calculated for C₃₃H₃₅ClN₄O₅S:

634.2017; found 635.2082 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy] phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-methoxyphenyl)propanoic Acid (2c). Using general

procedure 4 and 176 mg R5a (0.25 mmol) as the appropriate phenol

and 16 mg MeOH (0.50 mmol) as the appropriate alcohol, 63 mg 2c

(0.088 mmol, 35%) was obtained. ¹H NMR (500 MHz, DMSO-d₆) δ

ppm: 8.58 (s, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.26−7.23 (m, 2H),

7.20−7.16 (m, 2H), 7.19 (d, J = 8.5 Hz, 1H), 7.10 (t, J = 8.0 Hz, 1H),

6.86 (d, J = 8.2 Hz, 1H), 6.61 (t, J = 7.6 Hz, 1H), 6.08 (d, J = 7.6 Hz,

1H), 5.33 (dd, J = 10.2, 3.2 Hz, 1H), 4.29−4.23 (m, 2H), 3.75 (s,

3H), 3.13 (dd, J = 13.8, 3.2 Hz, 1H), 2.81−2.71 (m, 2H), 2.63−2.41

(m, 8H), 2.34 (dd, J = 13.8, 10.2 Hz, 1H), 2.25 (s, 3H), 1.78 (s, 3H).

¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 171.1, 166.3, 163.3, 163.0,

161.0, 156.9, 153.6, 152.9, 136.5, 135.7, 131.0, 130.6, 129.1, 128.6,

128.0, 127.7, 124.5, 122.0, 119.6, 118.8, 116.0, 110.6, 110.5, 74.1,

67.2, 56.0, 55.3, 53.9, 52.2, 44.7, 32.3, 28.1, 21.1, 17.5. HRMS

calculated for C₃₆H₃₆ClFN₄O₅S: 690.2079; found 691.2147 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(3-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-methoxyphenyl)propanoic Acid (2d). Using general

procedure 7 and 144 mg R9 (0.249 mmol) and 2-(3-ﬂuorophenyl)-

4,4,5,5-tetramethyl-1,3,2-dioxaborolane as the appropriate boronic

acid derivative, 63 mg 2d (0.091 mmol, 36%) was obtained. ¹H NMR

(500 MHz, DMSO-d₆) δ ppm: 8.60 (s, 1H), 7.42−7.36 (m, 2H),

7.21−7.16 (m, 2H), 7.17−7.13 (m, 2H), 7.01−6.97 (m, 1H), 6.86 (d,

J = 8.0 Hz, 1H), 6.65−6.59 (m, 1H), 6.09 (d, J = 7.5 Hz, 1H), 5.34

(dd, J = 10.4, 3.2 Hz, 1H), 4.32−4.17 (m, 2H), 3.75 (s, 3H), 3.14

(dd, J = 13.8, 3.2 Hz, 1H), 2.81−2.70 (m, 2H), 2.67−2.40 (m, 8H),

2.32 (dd, J = 13.8, 10.4 Hz, 1H), 2.24 (s, 3H), 1.79 (s, 3H). ¹³C NMR

(125 MHz, DMSO-d₆) δ ppm: 171.1, 166.4, 163.5, 161.8, 156.9,

153.7, 153.1, 135.9, 135.7, 135.0, 131.0, 130.6, 130.5, 129.4, 127.9,

127.6, 125.0, 124.6, 122.0, 119.6, 118.8, 115.6, 115.4, 110.6, 110.4,

74.3, 67.1, 56.1, 55.3, 53.9, 52.2, 44.7, 32.3, 17.5. HRMS calculated for

C₃₆H₃₆ClFN₄O₅S: 690.2079; found 691.2152 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(2-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-methoxyphenyl)propanoic Acid (2e). Using general

procedure 7 and 144 mg R9 (2.49 mmol) and 2-(2-ﬂuorophenyl)-

4,4,5,5-tetramethyl-1,3,2-dioxaborolane as the appropriate boronic

acid derivative, 46 mg 2e (0.067 mmol, 27%) was obtained. ¹H NMR

(500 MHz, DMSO-d₆) δ ppm: 8.61 (s, 1H), 7.43−7.38 (m 1H), 7.32

(d, J = 8.6 Hz, 1H), 7.28−7.24 (m, 1H), 7.25−7.21 (m, 1H), 7.18−

7.14 (m, 1H), 7.12−7.06 (m, 2H), 6.86 (d, J = 8.2 Hz, 1H), 6.63−

6.58 (m, 1H), 6.09 (d, J = 7.5 Hz, 1H), 5.35 (dd, J = 10.0, 3.3 Hz,

1H), 4.26−4.21 (m, 1H), 4.17−4.10 (m, 1H), 3.75 (s, 3H), 3.12 (dd,

J = 14.0, 3.3 Hz, 1H), 2.79−2.72 (m, 1H), 2.72−2.66 (m, 1H), 2.62−

2.39 (m, 8H), 2.37 (dd, J = 14.0, 10.0 Hz, 1H), 2.25 (s, 3H), 1.82 (s,

3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 171.2, 167.2, 163.4,

159.0, 156.9, 153.5, 153.1, 135.7, 132.3, 131.5, 131.4, 130.9, 130.6,

130.5, 127.9, 127.5, 124.7, 124.6, 121.6, 120.1, 119.6, 118.1, 116.1,

110.4, 110.1, 74.5, 66.9, 56.1, 55.3, 53.8, 52.1, 44.5, 32.2, 17.5. HRMS

calculated for C₃₆H₃₆ClFN₄O₅S: 690.2079; found 691.2169 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(2,3,4-triﬂuorophenyl)thieno[2,3-d]pyrimidin-

4-yl]oxy}-3-(2-methoxyphenyl)propanoic Acid (2f). Using general

procedure 7 and 144 mg R9 (2.49 mmol) and 4,4,5,5-tetramethyl-2-

(2,3,4-triﬂuorophenyl)-1,3,2-dioxaborolane as the appropriate boronic

acid derivative, 87 mg 2f (0.120 mmol, 49%) was obtained. ¹H NMR

(500 MHz, DMSO-d₆) δ ppm: 8.63 (s, 1H), 7.36−7.28 (m, 2H),

7.17−7.07 (m, 3H), 6.86 (d, J = 8.3 Hz, 1H), 6.64−6.58 (m, 1H),

6.10 (d, J = 7.4 Hz, 1H), 5.36 (dd, J = 9.9, 3.2 Hz, 1H), 4.29−4.22

(m, 1H), 4.19−4.12 (m, 1H), 3.75 (s, 3H), 3.12 (dd, J = 13.9, 3.2 Hz,

1H), 2.80−2.73 (m, 1H), 2.73−2.66 (m, 1H), 2.65−2.40 (m, 8H),

2.37 (dd, J = 13.9, 9.9 Hz, 1H), 2.26 (s, 3H), 1.84 (s, 3H). ¹³C NMR

(125 MHz, DMSO-d₆) δ ppm: 171.2, 167.3, 163.6, 156.9, 153.6,

153.5, 150.5, 148.0, 139.0, 135.7, 132.4, 130.6, 130.4, 128.5, 127.9,

127.0, 126.8, 124.7, 121.7, 119.6, 118.2, 118.0, 113.1, 110.4, 110.2,

74.6, 67.0, 56.0, 55.3, 53.7, 52.0, 44.5, 32.2, 17.6. HRMS calculated for

C₃₆H₃₄ClF₃N₄O₅S: 726.1891; found 727.1963 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(3-chlorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-methoxyphenyl)propanoic Acid (2g). Using general

procedure 7 and 144 mg R9 (2.49 mmol) and 2-(3-chlorophenyl)-

4,4,5,5-tetramethyl-1,3,2-dioxaborolane as the appropriate boronic

acid derivative, 57 mg 2g (0.0805 mmol, 33%) was obtained. ¹H

NMR (500 MHz, DMSO-d₆) δ ppm: 8.59 (s, 1H), 7.42 (d, J = 8.6

Hz, 1H), 7.38−7.31(m, 2H), 7.21−7.14 (m, 3H), 7.11−7.07 (m,

1H), 6.85 (d, J = 8.1 Hz, 1H), 6.62−6.57 (m, 1H), 6.04 (d, J = 7.3

Hz, 1H), 5.33 (dd, J = 10.4, 2.9 Hz, 1H), 4.35−4.28 (m, 1H), 4.26−

4.18 (m, 1H), 3.75 (s, 3H), 3.17 (dd, J = 13.7, 2.9 Hz, 1H), 2.83−

2.71 (m, 2H), 2.67−2.40 (m, 8H), 2.30 (dd, J = 13.7, 10.4 Hz, 1H),

2.27 (s, 3H), 1.76 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm:

171.4, 166.4, 163.6, 156.9, 153.6, 153.1, 135.6, 134.7, 133.3, 130.66,

130.65, 130.6, 129.4, 128.5, 128.2, 127.9, 127.6, 127.4, 124.8, 122.1,

119.6, 118.8, 110.6, 110.4, 74.6, 67.2, 55.9, 55.3, 53.7, 52.0, 44.4, 32.4,

17.5. HRMS calculated for C₃₆H₃₆Cl₂N₄O₅S: 706.1783; found

707.1860 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(pyridin-3-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-

3-(2-methoxyphenyl)propanoic Acid (2h). Using general procedure

7 and 144 mg R9 (0.25 mmol) and 3-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)pyridine as the appropriate boronic ester, 70 mg 2h

1

(0.10 mmol, 42%) was obtained. H NMR (500 MHz, DMSO-d₆) δ

ppm: 13.00 (br s, 1H), 8.61 (s, 1H), 8.49 (dd, J = 4.8, 1.5 Hz, 1H),

8.37 (dd, J = 2.4, 0.7 Hz, 1H),7.67−7.63 (m, 1H), 7.41 (d, J = 8.7 Hz,

1H), 7.40−7.36 (m, 1H), 7.18 (d, J = 8.7 Hz, 1H), 7.12−7.08 (m,

V

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1H), 6.86 (d, J = 8.0 Hz, 1H), 6.61 (td, J = 7.5, 0.7 Hz, 1H), 6.09 (dd,

J = 7.5, 1.3 Hz, 1H), 5.35 (dd, J = 10.5, 3.2 Hz, 1H), 4.31−4.17 (m,

2H), 3.75 (s, 3H), 3.15 (dd, J = 13.9, 3.2 Hz, 1H), 2.82−2.69 (m,

2H), 2.67−2.39 (m, 8H), 2.33 (dd, J = 13.9, 10.5 Hz, 1H), 2.25 (s,

3H), 1.78 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 171.1,

166.6, 163.5, 156.9, 153.8, 153.2, 149.4, 148.9, 136.2, 135.6, 133.9,

130.7, 130.6, 129.9, 129.0, 127.9, 127.3, 124.6, 123.8, 122.0, 119.6,

118.7, 110.5, 110.4, 74.4, 67.0, 56.1, 55.3, 53.8, 52.1, 44.6, 32.3, 17.5.

HRMS calculated for C₃₅H₃₆ClN₅O₅S: 673.2126; found 674.2205 (M

+ H).

further conversion was observed. Then the mixture was diluted with

water and the pH was adjusted to 6 by the addition of 2 M aq HCl

solution. The mixture was extracted with DCM, the combined organic

layer was dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated

under reduced pressure. The crude product was puriﬁed via reversed

phase chromatography using 25 mM aq NH₄HCO₃solution and

1

MeCN as eluents to obtain 1.37 g 2l (1.90 mmol, 95%). H NMR

(500 MHz, DMSO-d₆) δ ppm: 8.58 (s, 1H), 7.27−7.21 (m, 2H),

7.17−7.10 (m, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.74−6.68 (m, 1H),

6.63 (d, J = 3.6 Hz, 1H), 6.19 (br d, J = 7.5 Hz, 1H), 5.65 (d, J = 3.6

Hz, 1H), 5.35 (dd, J = 10.4, 3.0 Hz, 1H), 4.32−4.22 (m, 2H), 3.76 (s,

3H), 3.13 (dd, J = 13.8, 3.0 Hz, 1H), 2.83−2.72 (m, 2H), 2.67−2.22

(br m, 8H), 2.28 (dd, J = 13.8, 10.4 Hz, 1H), 2.17 (s, 3H), 1.90 (s,

3H). ¹³C NMR (125 MHz, DMSO-d6) δ ppm: 171.2, 166.5, 163.8,

157.4, 154.5, 153.6, 149.7, 136.2, 131.0, 129.5, 128.4, 128.0, 127.9,

126.1, 125.0, 123.3, 122.8, 120.2, 119.1, 115.2, 112.0, 111.7, 111.0,

74.7, 67.8, 56.7, 55.8, 54.8, 53.1, 45.7, 32.8, 17.7. HRMS calculated for

C₃₄H₃₄BrClN₄O₆S: 740.1071; found 741.1165 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(furan-2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-

(2-methoxyphenyl)propanoic Acid (2i). Using general procedure 7

and 205 mg R9 (0.36 mmol) and 2-(2-furyl)-4,4,5,5-tetramethyl-

1,3,2-dioxaborolane as the appropriate boronic ester, 127 mg 2i (0.19

1

mmol, 54%) was obtained. H NMR (500 MHz, DMSO-d₆) δ ppm:

8.56 (s, 1H), 7.78 (br s, 1H), 7.27 (d, J = 8.6 Hz, 1H), 7.23 (d, J = 8.6

Hz, 1H), 7.17−7.10 (m, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.74−6.68 (m,

1H), 6.53−6.49 (m, 1H), 6.20 (br d, J = 7.4 Hz, 1H), 5.66−5.62 (m,

1H), 5.36 (dd, J = 10.5, 2.6 Hz, 1H), 4.34−4.20 (m, 2H), 3.76 (s,

3H), 3.14 (dd, J = 14.1, 2.6 Hz, 1H), 2.87−2.72 (m, 2H), 2.72−2.35

(br m, 8H), 2.32−2.20 (m, 1H), 2.27 (s, 3H), 1.91 (s, 3H). ¹³C NMR

(125 MHz, DMSO-d₆) δ ppm: 171.6, 166.4, 163.8, 157.4, 154.4,

153.4, 147.4, 144.3, 136.2, 131.0, 129.7, 128.4, 127.6, 127.2, 125.1,

122.7, 119.6, 119.3, 113.2, 111.6, 110.9, 109.4, 74.6, 67.6, 56.6, 55.8,

54.3, 52.5, 45.0, 32.8, 17.7. HRMS calculated for C₃₄H₃₅ClN₄O₆S:

662.1966; found 663.2028 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-(2-methoxyphenyl)propanoic Acid (2j). Using general

procedure 4 and 556 mg R5b (0.8 mmol) as the appropriate phenol

and MeOH as the appropriate alcohol, 237 mg 2j (0.35 mmol, 43%)

was obtained. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.56 (s, 1H),

7.27 (d, J = 8.8 Hz, 1H), 7.23 (d, J = 8.8 Hz, 1H), 7.17−7.11 (m,

1H), 6.87 (d, J = 8.2 Hz, 1H), 6.74−6.69 (m, 1H), 6.19 (d, J = 7.6

Hz, 1H), 5.86 (dd, J = 7.0, 3.6 Hz, 1H), 5.67 (t, J = 3.6 Hz, 1H), 5.33

(dd, J = 10.3, 3.2 Hz, 1H), 4.29−4.24 (m, 2H), 3.76 (s, 3H), 3.15

(dd, J = 14.2, 3.2 Hz, 1H), 2.79−2.76 (m, 2H), 2.63−2.29 (m, 8H),

2.25 (dd, J = 14.2, 10.3 Hz, 1H), 2.19 (s, 3H), 1.92 (s, 3H). ¹³C NMR

(125 MHz, DMSO-d₆) δ ppm: 170.9, 165.7, 163.4, 156.5, 154.0,

153.1, 138.1, 135.7, 130.5, 129.3, 127.9, 127.6, 127.1, 125.5, 124.8,

122.3, 119.8, 118.8, 111.1, 110.9, 110.5, 85.2, 74.5, 67.2, 56.2, 55.3,

54.2, 52.5, 45.1, 32.4, 17.2. HRMS calculated for C₃₄H₃₄ClFN₄O₆S:

680.1872; found 681.1947 (M + H).

(2R)-2-{[6-(5-Chlorofuran-2-yl)-(5S_a)-5-{3-chloro-2-methyl-4-[2-

(4-methylpiperazin-1-yl)ethoxy]phenyl}thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-(2-methoxyphenyl)propanoic Acid (2k). 99.5 mg 2i (0.15

mmol) was dissolved in 4 mL CHCl₃, then 300 mg NCS (2.25 mmol)

was added and the mixture was stirred at rt in the dark for 2 h to reach

complete conversion. Then it was diluted with DCM, washed with

water, dried over Na₂SO₄, ﬁltered and the ﬁltrate was concentrated

under reduced pressure. The crude product was puriﬁed via

preparative reversed phase chromatography using 25 mM aq

NH₄HCO₃solution and MeCN as eluents to obtain 20 mg 2k

(0.029 mmol, 19%). ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.57 (s,

1H), 7.26 (d, J = 8.6 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H), 7.16−7.11

(m, 1H), 6.88 (d, J = 8.1 Hz, 1H), 6.71 (d, J = 7.4 Hz, 1H), 6.54 (d, J

= 3.6 Hz, 1H), 6.21 (dd, J = 7.4, 1.3 Hz, 1H), 5.70 (d, J = 3.6 Hz,

1H), 5.37 (dd, J = 10.4, 3.0 Hz, 1H), 4.31−4.23 (m, 2H), 3.76 (s,

3H), 3.13 (dd, J = 14.0, 3.0 Hz, 1H), 2.84−2.73 (m, 2H), 2.66−2.50

(br m, 4H), 2.50−2.34 (br m, 4H), 2.29 (dd, J = 14.0, 10.4 Hz, 1H),

2.21 (s, 3H), 1.91 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm:

170.9, 166.0, 163.3, 156.9, 154.0, 153.2, 147.0, 135.8, 135.7, 130.5,

129.1, 128.0, 127.5, 125.6, 124.4, 122.3, 119.8, 118.7, 111.24, 111.18,

110.5, 109.9, 74.1, 67.2, 56.1, 55.3, 54.0, 52.2, 44.8, 32.3, 17.2. HRMS

calculated for C₃₄H₃₄Cl₂N₄O₆S: 696.1576; found 697.1656 (M + H).

(2R)-2-{[6-(5-Bromofuran-2-yl)-(5S_a)-5-{3-chloro-2-methyl-4-[2-

(4-methylpiperazin-1-yl)ethoxy]phenyl}thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-(2-methoxyphenyl)propanoic Acid (2l). 1.33 g 2i (2.00

mmol) was dissolved in 20 mL CHCl₃, then 534 mg NBS (3.00

mmol) was added. The resulting mixture was stirred at 0 °C until no

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-[2-(2,2,2-triﬂuoroethoxy)phenyl]propanoic Acid (3a). 209

mg R5b (0.30 mmol) and 138 mg K₂CO₃(1.00 mmol) were

dissolved in 2 mL DMF, then 232 mg 2,2,2-triﬂuoroethyl

triﬂuoromethanesulfonate (1.00 mmol) was added. The mixture was

stirred at rt under N₂atmosphere until no further conversion was

observed. Then it was diluted with brine, neutralized with 2 M aq HCl

solution, extracted with DCM. The combined organic layer was dried

over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. Then it was hydrolyzed according to the general hydrolysis

1

procedure to obtain 92 mg 3a (0.12 mmol, 40%). H NMR (500

MHz, DMSO-d₆) δ ppm: 8.58 (s, 1H), 7.26 (d, J = 8.6 Hz, 1H), 7.22

(d, J = 8.6 Hz, 1H), 7.20−7.15 (m, 1H), 7.00 (d, J = 8.1 Hz, 1H),

6.84−6.78 (m, 1H), 6.27 (br d, J = 7.7 Hz, 1H), 5.87 (dd, J = 6.8, 3.6

Hz, 1H), 5.66 (t, J = 3.6 Hz, 1H), 5.37 (dd, J = 10.1, 3.3 Hz, 1H),

4.82−4.64 (m, 2H), 4.32−4.20 (m, 2H), 3.12 (dd, J = 14.1, 3.3 Hz,

1H), 2.84−2.71 (m, 2H), 2.70−2.25 (br m, 8H), 2.36 (dd, J = 14.1,

10.1 Hz, 1H), 2.20 (s, 3H), 1.91 (s, 3H). ¹³C NMR (125 MHz,

DMSO-d₆) δ ppm: 171.2, 166.2, 163.6, 157.0, 155.2, 154.5, 153.5,

138.5, 136.2, 131.3, 129.6, 128.5, 128.0, 127.5, 125.5, 124.4, 122.7,

121.9, 119.1, 112.7, 111.6, 111.5, 85.7, 74.4, 67.7, 65.3, 56.6, 54.6,

52.9, 45.5, 32.5, 17.6. HRMS calculated for C₃₅H₃₃ClF₄N₄O₆S:

748.1745; found 749.1819 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-{2-[(2R)-tetrahydrofuran-2-ylmethoxy]phenyl}propanoic

acid (3b). Using general procedure 4 and 348 mg R5b (0.50 mmol)

as the appropriate phenol and [(2R)-tetrahydrofuran-2-yl]methanol

as the appropriate alcohol, 119 mg 3b (0.16 mmol, 32%) was

obtained. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.58 (s, 1H), 7.25

(d, J = 8.7 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 7.14−7.10 (m, 1H),

6.89 (d, J = 8.2 Hz, 1H), 6.73−6.69 (m, 1H), 6.23−6.20 (m, 1H),

5.87 (dd, J = 6.8, 3.7 Hz, 1H), 5.67 (t, J = 3.7 Hz, 1H), 5.39 (dd, J =

10.2, 3.4 Hz, 1H), 4.26 (t, J = 5.9 Hz, 2H), 4.17−4.11 (m, 1H), 3.96

(dd, J = 10.2, 4.5 Hz, 1H), 3.92 (dd, J = 10.2, 4.5 Hz, 1H), 3.78−3.74

(m, 1H), 3.69−3.65 (m, 1H), 3.11 (dd, J = 13.9, 3.4 Hz, 1H), 2.79−

2.74 (m, 2H), 2.63−2.29 (m, 8H), 2.32 (dd, J = 13.9, 10.2 Hz, 1H),

2.17 (s, 3H), 2.03−1.96 (m, 1H), 1.93 (s, 3H), 1.92−1.87 (m, 1H),

1.86−1.77 (m, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 170.8,

165.8, 163.1, 156.5, 156.3, 154.0, 153.1, 138.0, 135.7, 130.6, 129.1,

128.0, 127.5, 127.0, 125.7, 124.6, 122.3, 119.9, 118.7, 111.5, 111.2,

111.0, 85.2, 76.4, 74.0, 69.9, 67.7, 67.3, 56.2, 54.4, 52.6, 45.2, 32.4,

27.5, 25.5, 17.2. HRMS calculated for C₃₈H₄₀ClFN₄O₇S: 750.2290;

found 751.2375 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-{2-[pyridin-2-ylmethoxy]phenyl}propanoic acid (3c).

Using general procedure 4 and 104 mg R5b (0.15 mmol) as the

appropriate phenol and pyridin-2-yl methanol as the appropriate

alcohol, 36 mg 3c (0.05 mmol, 30%) was obtained with a purity of

94.3%. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.57 (s, 1H), 8.55 (d,

W

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

J = 4.4 Hz, 1H), 7.78 (td, J = 7.7, 1.6 Hz, 1H), 7.66 (d, J = 7.8 Hz,

1H), 7.34−7.30 (m, 1H), 7.27 (d, J = 8.6 Hz, 1H), 7.23 (d, J = 8.6

Hz, 1H), 7.16−7.10 (m, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.75 (t, J = 7.4

Hz, 1H), 6.27 (d, J = 7.3 Hz, 1H), 5.87 (dd, J = 6.8, 3.6 Hz, 1H), 5.67

(t, J = 3.5 Hz, 1H), 5.48 (dd, J = 10.2, 3.2 Hz, 1H), 5.21 (d, J = 13.6

Hz, 1H), 5.16 (d, J = 13.6 Hz, 1H), 4.29−4.19 (m, 2H), 3.27 (dd, J =

13.9, 3.0 Hz, 1H), 2.81−2.69 (m, 2H), 2.64−2.30 (m, 9H), 2.20 (s,

3H), 1.92 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 170.9,

165.7, 163.2, 157.0, 156.5, 155.6, 154.0, 153.1, 148.9, 138.0, 136.9,

135.7, 130.7, 129.1, 128.0, 127.6, 127.0, 125.7, 124.9, 122.8, 122.3,

120.9, 120.3, 118.7, 111.8, 111.2, 111.0, 85.2, 74.2, 70.0, 67.2, 56.1,

54.1, 52.4, 45.0, 32.5, 17.2. HRMS calculated for C₃₉H₃₇ClFN₅O₆S:

757.2137; found 379.6159 (M + 2H).

161.0, 154.7, 153.6, 152.9, 136.5, 135.8, 131.0, 130.5, 129.1, 128.6,

128.0, 127.7, 125.1, 122.0, 121.3, 118.8, 115.9, 112.2, 110.6, 74.1,

67.2, 64.9, 64.7, 56.0, 53.9, 52.2, 44.7, 32.0, 17.5. HRMS calculated for

C₃₇H₃₅ClF₄N₄O₅S: 758.1953; found 759.1999 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy] phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-{2-[(2R)-tetrahydrofuran-2-ylmethoxy]phenyl}propanoic

Acid (4b). Using general procedure 4 and 705 mg R5a (1.00 mmol)

as the appropriate phenol and [(2R)-tetrahydrofuran-2-yl]methanol

as the appropriate alcohol, 234 mg 4b (0.296 mmol, 30%) was

obtained. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.60 (s, 1H), 7.34

(d, J = 8.7 Hz, 1H), 7.22−7.29 (m, 2H), 7.17−7.22 (m, 2H), 7.16 (d,

J = 8.7 Hz, 1H), 7.06−7.11 (m 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.60−

6.64 (m, 1H), 6.12 (d, J = 7.4 Hz, 1H), 5.38 (dd, J = 10.0, 3.6 Hz,

1H), 4.17−4.28 (m, 2H), 4.12−4.17 (m, 1H), 3.89−3.98 (m, 2H),

3.73−3.79 (m, 1H), 3.64−3.69 (m, 1H), 3.11 (dd, J = 13.7, 6.6 Hz,

1H), 2.69−2.79 (m, 2H), 2.41−2.61 (m, 8H), 2.38 (dd, J = 13.7, 10.0

Hz, 1H), 2.19 (s, 3H), 1.95−2.03 (m, 1H), 1.87−1.95 (m, 1H), 1.80

(s, 3H), 1.75−1.87 (m, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ

ppm: 171.0, 166.3, 163.2, 162.0, 156.3, 153.6, 152.9, 136.5, 135.7,

131.0, 130.8, 130.5, 129.1, 128.6, 128.0, 127.7, 124.7, 122.0, 119.8,

118.8, 116.0, 111.4, 110.6, 76.4, 74.1, 69.9, 67.7, 67.2, 56.1, 54.2, 52.5,

45.0, 32.3, 27.5, 25.6, 17.5. HRMS calculated for C₄₀H₄₂ClFN₄O₆S:

760.2498; found 761.2550 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-[2-(pyrazin-2-ylmethoxy)phenyl]propanoic acid (3d).

Using general procedure 4 and 280 mg R5b (0.4 mmol) as the

appropriate phenol and pyrazin-2-ylmethanol as the appropriate

1

alcohol, 98 mg 3d (0.13 mmol, 32%) was obtained. H NMR (500

MHz, DMSO-d₆) δ ppm: 8.94 (d, J = 1.4 Hz, 1H), 8.64 (dd, J = 2.6,

1.4 Hz, 1H), 8.62 (d, J = 2.6 Hz, 1H), 8.55 (s, 1H), 7.27 (d, J = 8.6

Hz, 1H), 7.24 (d, J = 8.6 Hz, 1H), 7.17−7.14 (m, 1H), 7.04−7.01 (m,

1H), 6.78−6.76 (m, 1H), 6.29−6.25 (m, 1H), 5.87 (dd, J = 6.9, 3.6

Hz, 1H), 5.67 (t, J = 3.6 Hz, 1H), 5.46 (dd, J = 10.3, 3.0 Hz, 1H),

5.30 (d, J = 13.7 Hz, 1H), 5.24 (d, J = 13.7 Hz, 1H), 4.28−4.22 (m,

2H), 3.27 (dd, J = 13.9, 3.0 Hz, 1H), 2.80−2.71 (m, 2H), 2.67−2.33

(m, 8H), 2.36 (dd, J = 13.9, 10.3 Hz, 1H), 2.20 (s, 3H), 1.91 (s, 3H).

¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 170.9, 165.7, 163.2, 156.5,

155.5, 154.0, 153.0, 152.4, 144.04, 143.97, 143.0, 138.0, 135.7, 130.8,

129.2, 128.4, 128.1, 127.6, 127.0, 125.7, 125.1, 122.3, 120.5, 118.7,

111.7, 111.2, 111.0, 85.2, 74.3, 68.4, 67.2, 56.1, 54.1, 52.3, 44.9, 32.6,

17.2. HRMS calculated for C₃₈H₃₆ClFN₆O₆S: 758.2090; found

759.2159 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-[2-(pyridin-2-ylmethoxy)phenyl]propanoic Acid (4c). Using

general procedure 4 and 494 mg R5a (0.700 mmol) as the

appropriate phenol and 2-pyridylmethanol as the appropriate alcohol,

1

159 mg 4c (0.207 mmol, 30%) was obtained. H NMR (500 MHz,

DMSO-d₆) δ ppm: 8.59 (s, 1H), 8.55 (d, J = 4.9 Hz, 1H), 7.81−7.76

(m, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.37 (d, J = 8.7 Hz, 1H), 7.34−

7.30 (m, 1H), 7.29−7.24 (m, 2H), 7.22−7.15 (m, 2H), 7.18−7.14

(m, 1H), 7.12−7.07 (m, 1H), 6.95 (d, J = 8.2 Hz, 1H), 6.69−6.63 (m,

1H), 6.17 (d, J = 7.4 Hz, 1H), 5.47 (dd, J = 10.0, 3.4 Hz, 1H), 5.22−

5.13 (m, 2H), 4.28−4.22 (m, 1H), 4.21−4.14 (m, 1H), 3.27 (dd, J =

13.9, 3.4 Hz, 1H), 2.78−2.66 (m, 2H), 2.60−2.44 (m, 8H), 2.43 (dd,

J = 13.9, 10.0 Hz, 1H), 2.21 (s, 3H), 1.78 (s, 3H). ¹³C NMR (125

MHz, DMSO-d₆) δ ppm: 171.1, 166.3, 163.2, 162.1, 157.0, 155.6,

153.6, 152.9, 149.0, 136.9, 136.5, 135.8, 131.0, 130.8, 130.5, 129.1,

128.6, 128.0, 127.7, 124.9, 122.8, 122.0, 120.9, 120.1, 118.9, 116.0,

111.7, 110.6, 74.2, 70.0, 67.2, 56.1, 54.1, 52.3, 44.9, 32.5, 17.5. HRMS

calculated for C₄₁H₃₉ClFN₅O₅S: 767.2344; found 384.6257 (M +

2H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-{2-[(1-methyl-1H-pyrazol-5-yl)methoxy]phenyl}propanoic

Acid (3e). Using general procedure 4 and 1.74 g R5b (2.50 mmol) as

the appropriate phenol and (1-methyl-1H-pyrazol-5-yl)methanol as

the appropriate alcohol, 1.22 g 3e (1.61 mmol, 64%) was obtained. ¹H

NMR (500 MHz, DMSO-d₆) δ ppm: 8.55 (s, 1H), 7.36 (d, J = 1.7

Hz, 1H), 7.28−7.21 (m, 2H), 7.19−7.13 (m, 1H), 7.08 (d, J = 8.2 Hz,

1H), 6.78−6.72 (m, 1H), 6.41 (d, J = 1.7 Hz, 1H), 6.21 (br d, J = 7.7

Hz, 1H), 5.87 (dd, J = 6.7, 3.6 Hz, 1H), 5.68−5.65 (m, 1H), 5.38 (dd,

J = 10.0, 3.3 Hz, 1H), 5.22−5.12 (m, 2H), 4.31−4.21 (m, 2H), 3.88

(s, 3H), 3.15 (dd, J = 14.0, 3.3 Hz, 1H), 2.82−2.71 (m, 2H), 2.64−

2.35 (m, 8H), 2.28 (dd, J = 14.0, 10.0 Hz, 1H), 2.20 (s, 3H), 1.92 (s,

3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 170.8, 165.8, 163.2,

155.4, 154.0, 153.1, 138.1, 137.6, 137.5, 137.4, 130.9, 129.2, 128.0,

127.6, 127.0, 125.7, 124.9, 120.4, 118.7, 111.8, 111.2, 111.0, 106.7,

85.3, 74.2, 67.2, 60.2, 56.2, 54.1, 52.4, 44.9, 36.5, 32.5, 17.2. HRMS

calculated for C₃₈H₃₈ClFN₆O₆S: 760.2246; found 761.2343 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-[2-(2,2,2-triﬂuoroethoxy)phenyl]propanoic Acid (4a). 211

mg R5a (0.30 mmol) and 138 mg K₂CO₃(1.00 mmol) were

dissolved in 2 mL DMF, then 232 mg 2,2,2-triﬂuoroethyl

triﬂuoromethanesulfonate (1.00 mmol) was added. The mixture was

stirred at rt under N₂atmosphere until no further conversion was

observed. Then it was diluted with brine, neutralized with 2 M aq HCl

solution, extracted with DCM, dried over Na₂SO₄, ﬁltered and the

ﬁltrate was concentrated under reduced pressure. Then it was

hydrolyzed according to the general hydrolysis procedure to obtain 57

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-[2-(pyrazin-2-ylmethoxy)phenyl]propanoic Acid (4d). Using

general procedure 4 and 176 mg R5a (0.25 mmol) as the appropriate

phenol and 55 mg pyrazin-2-ylmethanol (0.5 mmol) as the

1

appropriate alcohol, 77 mg 4d was obtained (0.10 mmol, 40%). H

NMR (500 MHz, DMSO-d₆) δ ppm: 8.94 (d, J = 1.2 Hz, 1H), 8.63

(dd, J = 2.5, 1.5 Hz, 1H), 8.61 (d, J = 2.5 Hz, 1H), 8.57 (s, 1H), 7.38

(d, J = 8.6 Hz, 1H), 7.27−7.23 (m, 1H), 7.24 (d, J = 5.4 Hz, 1H),

7.20−7.15 (m, 2H), 8.63 (td, J = 8.0, 1.5 Hz, 1H), 7.00 (dm, J = 8.0

Hz, 1H), 6.67 (td, J = 7.4, 0.8 Hz, 1H), 6.16 (dd, J = 7.4, 1.5 Hz, 1H),

5.44 (dd, J = 10.0, 3.2 Hz, 1H), 5.29 (d, J = 13.5 Hz, 1H), 5.23 (d, J =

13.5 Hz, 1H), 4.28−4.16 (m, 2H), 3.28 (dd, J = 13.8, 3.2 Hz, 1H),

2.78−2.69 (m, 2H), 2.62−2.38 (m, 8H), 2.42 (dd, J = 13.8, 10.0 Hz,

1H), 2.23 (s, 3H), 1.77 (s, 3H). ¹³C NMR (500 MHz, DMSO-d₆) δ

ppm: 171.2, 166.2, 163.0, 162.2, 155.5, 153.6, 152.9, 152.4, 144.0,

144.0, 143.1, 136.5, 135.7, 130.9, 130.9, 130.6, 129.1, 128.6, 128.0,

127.7, 125.1, 122.0, 120.4, 118.8, 115.9, 111.7, 110.6, 74.4, 74.4, 68.4,

67.1, 56.0, 53.9, 52.1, 44.7, 32.5, 17.5. HRMS calculated for

C₄₀H₃₈ClFN₆O₅S: 768.2297; found 769.2422 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-{2-[(1-methyl-1H-pyrazol-5-yl)methoxy]phenyl}propanoic

Acid (4e). Using general procedure 4 and 494 mg R5a (0.700 mmol)

as the appropriate phenol and (1-methyl-1H-imidazol-5-yl)methanol

as the appropriate alcohol, 229 mg 4e (0.297 mmol, 42%) was

obtained. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.56 (s, 1H), 7.38

1

mg 4a (0.076 mmol, 25%). H NMR (500 MHz, DMSO-d₆) δ ppm:

8.59 (s, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.27−7.24 (m, 2H), 7.23−7.17

(m, 2H), 7.16 (d, J = 8.5 Hz, 1H), 7.16−7.12 (m, 1H), 7.00−6.95 (m,

1H), 6.75−6.71 (m, 1H), 6.20−6.16 (m, 1H) 5.33 (dd, J = 10.0, 3.5

Hz, 1H), 4.79−4.66 (m, 2H), 4.28−4.17 (m, 2H), 3.13 (dd, J = 14.1,

3.5 Hz, 1H), 2.79−2.70 (m, 2H), 2.56 (br s, 4H), 2.46 (br s, 4H),

2.42 (dd, J = 14.1, 10.0 Hz, 1H), 2.23 (s, 3H), 1.78 (s, 3H). ¹³C NMR

(125 MHz, DMSO-d₆) δ ppm: 171.1, 166.3, 163.4, 163.0, 162.2,

X

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(d, J = 8.6 Hz, 1H), 7.36 (d, J = 1.7 Hz, 1H), 7.26−7.22 (m, 2H),

7.20−7.14 (m, 3H), 7.14−7.10 (m, 1H), 7.05 (d, J = 8.5 Hz, 1H),

6.67−6.62 (m, 1H), 6.40 (d, J = 1.8 Hz, 1H), 6.08 (d, J = 7.7 Hz,

1H), 5.36 (dd, J = 10.2, 3.2 Hz, 1H), 5.20−5.12 (m, 2H), 4.30−4.24

(m, 1H), 4.22−4.15 (m, 1H), 3.88 (s, 3H), 3.16 (dd, J = 13.7, 3.2 Hz,

1H), 2.81−2.68 (m, 2H), 2.47−2.37 (m, 8H), 2.33 (dd, J = 13.7, 10.2

Hz, 1H), 2.24 (s, 3H), 1.76 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆)

δ ppm: 171.2, 166.3, 163.3, 162.1, 155.4, 153.6, 152.8, 137.6, 137.4,

136.5, 135.7, 131.0, 130.9, 130.6, 129.1, 128.5, 127.9, 127.7, 125.0,

122.0, 120.2, 118.8, 115.9, 111.8, 110.5, 106.7, 74.3, 67.1, 60.2, 56.0,

53.8, 52.1, 44.6, 36.5, 32.4, 17.5. HRMS calculated for

C₄₀H₄₀ClFN₆O₅S: 770.2453; found 771.2527 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-{2-[(1-ethyl-1H-pyrazol-5-yl)methoxy]phenyl}propanoic

Acid (5a). Using general procedure 4 and 348 mg R5b (0.50 mmol)

as the appropriate phenol and R6f as the appropriate alcohol, 112 mg

5a (0.14 mmol, 29%) was obtained. ¹H NMR (500 MHz, DMSO-d₆)

δ ppm: 8.55 (s, 1H), 7.39 (d, J = 1.7 Hz, 1H), 7.26 (d, J = 8.6 Hz,

1H), 7.22 (d, J = 8.6 Hz, 1H), 7.18−7.13 (m, 1H), 7.08 (d, J = 8.1

Hz, 1H), 6.77−6.73 (m, 1H), 6.41 (d, J = 1.7 Hz, 1H), 6.22 (dd, J =

7.4, 1.2 Hz, 1H), 5.87 (dd, J = 6.8, 3.7 Hz, 1H), 5.67 (t, J = 3.5 Hz,

1H), 5.38 (dd, J = 10.3, 3.2 Hz, 1H), 5.19 (d, J = 12.7 Hz, 1H), 5.14

(d, J = 12.7 Hz, 1H), 4.30−4.12 (m, 4H), 3.14 (dd, J = 13.9, 3.1 Hz,

1H), 2.83−2.71 (m, 2H), 2.66−2.35 (m, 8H), 2.30 (dd, J = 13.9, 10.3

Hz, 1H), 2.22 (s, 3H), 1.91 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H). ¹³C

NMR (125 MHz, DMSO-d₆) δ ppm: 170.9, 165.7, 163.2, 156.5,

155.5, 154.0, 153.0, 138.0, 137.7, 136.9, 135.7, 130.8, 129.2, 127.9,

127.5, 127.0, 125.7, 124.8, 122.3, 120.3, 118.7, 111.8, 111.1, 111.0,

106.6, 85.2, 74.1, 67.1, 60.1, 56.1, 54.0, 52.3, 44.8, 43.8, 32.4, 17.2,

15.4. HRMS calculated for C₃₉H₄₀ClFN₆O₆S: 774.2403; found

388.1265 (M + 2H).

calculated for C₄₁H₄₄ClFN₆O₆S: 802.2716; found 402.1447 (M +

2H).

(2R)-3-{2-[(1-tert-Butyl-1H-pyrazol-5-yl)methoxy]phenyl}-2-

{[(5S_a)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]-

phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-

propanoic Acid (5d). Using general procedure 4 and 417 mg R5b

(0.60 mmol) as the appropriate phenol and R6i as the appropriate

1

alcohol, 213 mg 5d (0.27 mmol, 44%) was obtained. H NMR (500

MHz, DMSO-d₆) δ ppm: 8.54 (s, 1H), 7.36 (d, J = 1.7 Hz, 1H), 7.26

(d, J = 8.6 Hz, 1H), 7.22 (d, J = 8.6 Hz, 1H), 7.18−7.13 (m, 1H),

7.06 (d, J = 8.1 Hz, 1H), 6.77−6.72(m, 1H), 6.48 (d, J = 1.7 Hz, 1H),

6.22 (dd, J = 7.5, 1.4 Hz, 1H), 5.87 (dd, J = 6.8, 3.7 Hz, 1H), 5.67 (t, J

= 3.5 Hz, 1H), 5.40 (dd, J = 10.2, 3.3 Hz, 1H), 5.25 (d, J = 12.7 Hz,

1H), 5.19 (d, J = 12.7 Hz, 1H), 4.30−4.20 (m, 2H), 3.17 (dd, J =

13.8, 3.2 Hz, 1H), 2.82−2.70 (m, 2H), 2.64−2.34 (m, 8H), 2.33 (dd,

J = 13.8, 10.3 Hz, 1H), 2.20 (s, 3H), 1.92 (s, 3H), 1.58 (s, 9H). ¹³C

NMR (125 MHz, DMSO-d₆) δ ppm: 170.8, 165.7, 163.2, 156.5,

155.5, 154.0, 153.0, 138.0, 137.3, 136.2, 135.7, 130.9, 129.2, 128.0,

127.5, 127.0, 125.7, 124.6, 122.3, 120.3, 118.7, 111.7, 111.1, 111.0,

108.9, 85.2, 74.1, 67.1, 61.9, 60.2, 56.1, 54.1, 52.3, 44.9, 32.4, 29.8,

17.2. HRMS calculated for C₄₁H₄₄ClFN₆O₆S: 802.2716; found

402.1422 (M + 2H).

(2R)-2-[(5S_a)-5-[3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl]-6-(5-ﬂuoro-2-furyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy-3-[2-[[2-(2,2,2-triﬂuoroethyl)pyrazol-3-yl]methoxy]phenyl]-

propanoic Acid (5e). 5e was prepared according to the published

procedure.⁵

2-[(1-Butyl-1H-pyrazol-5-yl)methoxy]-N-[(5S_a)-5-{3-chloro-2-

methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(5-ﬂuorofur-

an-2-yl)thieno[2,3-d]pyrimidin-4-yl]-^D-phenylalanine (5f). Step A:

4-Chloro-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)-

ethoxy]phenyl]thieno[2,3-d]pyrimidine. 48 g R1e (160 mmol) and

76 g R3b (191 mmol) were dissolved in 2.4 L DME, then 101 g

K₃PO₄(478 mmol), 3.60 g Pd(OAc)₂(16.0 mmol), 11.5 g nBuPAd₂

(32.0 mmol), and 800 mL water were added and the mixture was

stirred at 60 °C until no further conversion was observed. Then the

volatiles were evaporated under reduced pressure, ﬁltered, washed

with water and MeCN to give 60.0 g 4-chloro-5-[3-chloro-2-methyl-4-

[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]thieno[2,3-d]pyrimidine

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-(2-{[1-(propan-2-yl)-1H-pyrazol-5-yl]methoxy}phenyl)-

propanoic Acid (5b). Using general procedure 4 and 229 mg R5b

(0.33 mmol) as the appropriate phenol and R6g as the appropriate

1

alcohol, 88 mg 5a (0.11 mmol, 33%) was obtained. H NMR (500

1

MHz, DMSO-d₆) δ ppm: 8.54 (s, 1H), 7.42 (d, J = 1.6 Hz, 1H), 7.26

(d, J = 8.6 Hz, 1H), 7.22 (d, J = 8.6 Hz, 1H), 7.19−7.13 (m, 1H),

7.09 (d, J = 7.9 Hz, 1H), 6.78−6.71 (m, 1H), 6.39 (d, J = 1.6 Hz,

1H), 6.20 (br d, J = 7.3 Hz, 1H), 5.87 (dd, J = 6.8, 3.6 Hz, 1H), 5.66

(t, J = 3.6 Hz, 1H), 5.37 (dd, J = 10.3, 3.1 Hz, 1H), 5.18 (d, J = 12.5

Hz, 1H), 5.13 (d, J = 12.5 Hz, 1H), 4.67−4.57 (m, 1H), 4.30−4.21

(m, 2H), 3.14 (dd, J = 13.6, 3.1 Hz, 1H), 2.82−2.71 (m, 2H), 2.66−

2.46 (br m, 4H), 2.46−2.26 (br m, 4H), 2.31 (dd, J = 13.6, 10.3 Hz,

1H), 2.18 (s, 3H), 1.91 (s, 3H), 1.40 (d, J = 6.5 Hz, 3H), 1.37 (d, J =

6.5 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 153.5, 138.0,

136.7, 131.4, 129.7, 128.4, 120.8, 112.2, 111.7, 111.5, 106.8, 85.7,

74.6, 67.7, 60.4, 56.7, 54.7, 53.0, 50.2, 45.6, 32.8, 23.3, 17.7. HRMS

calculated for C₄₀H₄₂ClFN₆O₆S: 788.2559; found 789.2663 (M + H).

(2R)-3-{2-[(1-Butyl-1H-pyrazol-5-yl)methoxy]phenyl}-2-{[(5S_a)-5-

{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-

(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}propanoic Acid

(5c). Using general procedure 4 and 521 mg R5b (0.75 mmol) as the

appropriate phenol and R6h as the appropriate alcohol, 308 mg 5c

(139 mmol, 87%). H NMR (400 MHz, DMSO-d₆) δ ppm: 8.98 (s,

1H), 7.97 (s, 1H), 7.22 (d, J = 8.2 Hz, 1H), 7.09 (d, J = 8.2 Hz, 1H),

4.25−4.16 (m, 2H), 2.76 (t, J = 5.8 Hz, 2H), 2.54 (br s, 4H), 2.32 (br

s, 4H), 2.14 (s, 3H), 2.06 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6)

δ ppm: 178.2, 154.7, 151.9, 152.3, 137.0, 133.5, 130.0, 128.9, 126.8,

110.8, 91.9, 67.7, 56.9, 55.3, 53.6, 46.3, 18.5. HRMS calculated for

C₂₀H₂₂Cl₂N₄OS: 436.0891; found 437.0980 (M + H).

Step B: 4-Chloro-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-

1-yl)ethoxy]phenyl]-6-iodothieno[2,3-d]pyrimidine. 10.94 g 4-

chloro-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]-

phenyl]thieno[2,3-d]pyrimidine (25.0 mmol) was dissolved in 250

mL dry THF and cooled to −78 °C. 25 mL LDA solution (50 mmol,

2 M in THF, heptane, EtPh) was added dropwise under Ar

atmosphere, and the mixture was stirred for 15 min. Then 12.69 g I₂

(50 mmol) was added at −78 °C, and the mixture was allowed to

warm up to rt. Then the mixture was diluted with EtOAc and was

washed with sat. aq NH₄Cl solution, then with aq Na₂S₂O₃solution,

dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under

reduced pressure. The crude product was puriﬁed via ﬂash

chromatography using DCM and MeOH as eluents to obtain 11.8

g 4-chloro-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)-

1

(0.38 mmol, 51%) was obtained. H NMR (500 MHz, DMSO-d₆) δ

ppm: 8.53 (s, 1H), 7.39 (d, J = 1.8 Hz, 1H), 7.25 (d, J = 8.7 Hz, 1H),

7.23 (d, J = 8.7 Hz, 1H), 7.18−7.14 (m, 1H), 7.08 (dm, J = 8.0 Hz,

1H), 6.77−6.72 (m, 1H), 6.41 (d, J = 1.8 Hz, 1H), 6.22−6.19 (m,

1H), 5.88 (dd, J = 6.9, 3.6 Hz, 1H), 5.66 (t, J = 3.6 Hz, 1H), 5.38 (dd,

J = 10.5, 3.0 Hz, 1H), 5.18 (d, J = 12.7 Hz, 1H), 5.14 (d, J = 12.7 Hz,

1H), 4.29−4.22 (m, 2H), 4.17−4.07 (m, 2H), 3.14 (dd, J = 13.8, 3.0

Hz, 1H), 2.81−2.71 (m, 2H), 2.65−2.31 (m, 8H), 2.30 (dd, J = 13.8,

10.5 Hz, 1H), 2.18 (s, 3H), 1.91 (s, 3H), 1.77−1.71 (m, 2H), 1.30−

1.23 (m, 2H), 0.85 (t, J = 7.4 Hz, 3H). ¹³C NMR (125 MHz, DMSO-

d₆) δ ppm: 170.8, 165.8, 163.2, 156.5, 155.5, 154.0, 153.0, 138.0,

137.6, 137.2, 135.7, 130.9, 129.2, 128.0, 127.5, 127.0, 125.7, 124.8,

122.3, 120.4, 118.7, 111.8, 111.2, 111.0, 106.5, 85.2, 74.1, 67.2, 60.2,

56.2, 54.2, 52.5, 48.6, 45.1, 32.3, 31.9, 19.4, 17.2, 13.5. HRMS

1

ethoxy]phenyl]-6-iodothieno[2,3-d]pyrimidine (21 mmol, 84%). H

NMR (500 MHz, DMSO-d₆) δ ppm: 8.93 (s, 1H), 7.15 (d, J = 8.6

Hz, 1H), 7.13 (d, J = 8.6 Hz, 1H), 4.22 (t, J = 5.8 Hz, 2H), 2.77 (t, J =

5.8 Hz, 2H), 2.56 (br s, 4H), 2.34 (br s, 4H), 2.16 (s, 3H), 2.00 (s,

3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 172.3, 154.4, 152.8,

152.6, 138.7, 136.4, 129.8, 128.9, 127.2, 121.8, 110.7, 91.6, 67.2, 56.5,

54.8, 53.1, 45.8, 17.6. HRMS calculated for C₂₀H₂₁Cl₂IN₄OS:

561.9857; found 562.9935 (M + H).

Step C: 4-Chloro-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-

1-yl)ethoxy]phenyl]-6-(5-ﬂuoro-2-furyl)thieno[2,3-d]pyrimidine.

5.61 g 4-chloro-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)-

Y

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

ethoxy]phenyl]-6-iodothieno[2,3-d]pyrimidine (10.0 mmol) and 8.60

g 2-(5-ﬂuoro-2-furyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (40.0

mmol) were dissolved in 80 mL 1,4-dioxane and 20 mL water, then

7.12 g Cs₂CO₃(22.0 mmol) and 730 mg Pd(dppf)Cl₂(1.00 mmol)

were added and the mixture was stirred at 40 °C until no further

conversion was observed. The mixture was then diluted with water

and extracted with DCM. The combined organic layer was washed

with water, dried over MgSO₄, ﬁltered, and the ﬁltrate was

concentrated under reduced pressure. The crude product was puriﬁed

by preparative reversed phase chromatography using 25 mM aq

NH₄HCO₃solution and MeCN as eluents to give 2.60 g 4-chloro-5-

[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-

(5-ﬂuoro-2-furyl)thieno[2,3-d]pyrimidine (5.00 mmol, 50%). ¹H

NMR (500 MHz, DMSO-d₆) δ ppm: 8.93 (s, 1H), 7.24 (d, J = 8.4

Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 5.92 (dd, J = 6.8, 3.6 Hz, 1H), 5.68

(t, J = 3.5 Hz, 1H), 4.23 (t, J = 5.6 Hz, 2H), 2.79 (t, J = 5.6 Hz, 2H),

2.67−2.47 (m, 4H), 2.44−2.26 (m, 4H), 2.19 (s, 3H), 2.05 (s, 3H).

HRMS calculated for C₂₄H₂₃N₄O₂FSCl₂: 520.0903; found 521.0972

(M + H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 167.0, 158.0,

156.9, 155.8, 154.6, 153.4, 152.8, 137.6, 136.4, 129.4, 129.2, 128.3,

126.5, 126.4, 122.2, 112.9, 111.5, 85.8, 67.2, 56.4, 54.7, 53.0, 45.6,

30.4, 17.3. HRMS calculated for C₂₄H₂₃Cl₂FN₄O₂S: 520.0903; found

521.0972 (M + H).

14.4, 14.0. HRMS calculated for C₂₄H₃₅N₃O₅: 445.2577; found

446.2654 (M + H).

Step F: (2R)-2-Amino-3-[2-[(2-butylpyrazol-3-yl)methoxy]-

phenyl]propanoic Acid. 891 mg ethyl (2R)-2-(tert-butoxycarbonyla-

mino)-3-[2-[(2-butylpyrazol-3-yl)methoxy]phenyl]propanoate (2.00

mmol) was dissolved in 5 mL dry DCM, then 5 mL triﬂuoroacetic

acid was added in one portion. The mixture was stirred at rt for 2 h to

reach complete conversion. It was concentrated under reduced

pressure, then dissolved in 5 mL THF and 5 mL water. Then 840 mg

LiOH·H₂O (20.0 mmol) was added, and it was stirred at 50 °C for 3

h to reach complete conversion. Then it was neutralized with 2 M aq

HCl solution and puriﬁed via preparative reversed phase chromatog-

raphy using 25 mM aq NH₄HCO₃solution and MeCN as eluents to

obtain 400 mg (2R)-2-amino-3-[2-[(2-butylpyrazol-3-yl)methoxy]-

phenyl]propanoic acid (1.26 mmol, 63%). LRMS calculated for

C₁₇H₂₃N₃O₃: 317.4; found 318.4 (M + H).

Step G: 2-[(1-Butyl-1H-pyrazol-5-yl)methoxy]-N-[(5S_a)-5-{3-

chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(5-

ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-yl]-^D-phenylalanine (5f).

520 mg 4-chloro-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)-

ethoxy]phenyl]-6-(5-ﬂuoro-2-furyl)thieno[2,3-d]pyrimidine (1.00

mmol), 380 mg (2R)-2-amino-3-[2-[(2-butylpyrazol-3-yl)methoxy]-

phenyl]propanoic acid (1.20 mmol), and 400 mg K₂CO₃(3.00

mmol) were mixed in 10 mL dry DMSO and stirred at rt until no

further conversion was observed. The mixture was then diluted with

brine, acidiﬁed to pH 2 with 1 M aq HCl solution, and it was

extracted with DCM. The combined organic layer was washed with

brine and dried over Na₂SO₄, ﬁltered, and the ﬁltrate was

concentrated under reduced pressure, then puriﬁed via preparative

reversed phase chromatography using 25 mM aq NH₄HCO₃solution

and MeCN as eluents. The diastereoisomer eluting later was collected

Step D: Ethyl (2R)-2-(tert-Butoxycarbonylamino)-3-(2-

hydroxyphenyl)propanoate. 20.22

g (2R)-2-amino-3-(2-

hydroxyphenyl)propanoic acid hydrochloride (93.0 mmol) was

dissolved in 120 mL EtOH, then 18.2 mL SOCl₂(224 mmol) was

added slowly while vigorous stirring. Then it was heated at reﬂux

temperature for 4 h. Then it was cooled to rt and the volatiles were

removed under reduced pressure. Then it was dissolved in a biphasic

mixture of 100 mL DCM and 100 mL water. The pH of the aq layer

was set to 8 with NaHCO₃, then 20.31 g Boc₂O (93.0 mmol) was

added and the mixture was stirred overnight at rt. The phases were

separated, and the aq layer was extracted with DCM. The combined

organic layer was washed with brine and dried over Na₂SO₄, ﬁltered,

and the ﬁltrate was concentrated under reduced pressure. Then it was

sonicated with iPr₂O, when beige crystals were formed, which were

collected by ﬁltration to give 17.40 g ethyl (2R)-2-(tert-butoxycarbo-

nylamino)-3-(2-hydroxyphenyl)propanoate (56.2 mmol, 60%). ¹H

NMR (500 MHz, DMSO-d₆) δ ppm: 9.50 (s, 1H), 7.13 (d, J = 7.9

Hz, 1H), 7.07−7.00 (m, 2H), 6.80 (d, J = 7.9 Hz, 1H), 6.68 (t, J = 7.3

Hz, 1H), 4.26−4.19 (m, 1H), 4.07−3.95 (m, 1H), 4.01 (dd, J = 9.2,

7.2 Hz, 1H), 2.97 (dd, J = 13.2, 5.8 Hz), 2.73 (dd, J = 13.5, 9.6 Hz,

1H), 1.33 (s, 9H), 1.08 (t, J = 7.2 Hz, 3H). ¹³C NMR (125 MHz,

DMSO-d₆) δ ppm: 172.5, 155.4, 131.1, 127.7, 123.5, 118.7, 114.7,

78.1, 60.1, 53.5, 31.9, 28.1, 14.0. HRMS calculated for C₁₆H₂₃NO₅:

309.1576; found 332.1438 (M + Na).

1

as 5f (104 mg, 0.13 mmol, 13%). H NMR (500 MHz, DMSO-d₆) δ

ppm: 8.21 (s, 1H), 7.37 (d, J = 1.7 Hz, 1H), 7.24 (d, J = 1.0 Hz, 2H),

7.14−7.11 (m, 1H), 7.03 (d, J = 8.2 Hz, 1H), 6.83−6.79 (m, 2H),

6.28 (d, J = 1.8 Hz, 1H), 5.8 (dd, J = 6.8, 3.6 Hz, 2H), 5.61 (t, J = 3.6

Hz, 1H), 5.04 (d, J = 12.5 Hz, 1H), 4.96 (d, J = 12.5 Hz, 1H), 4.54−

4.47 (m, 2H), 4.18−4.15 (m, 2H), 4.06 (t, J = 7.2 Hz, 2H), 3.27 (dd,

J = 14.5, 5.6 Hz, 1H), 3.12−3.07 (m, 1H), 2.99−2.95 (m, 1H), 2.82−

2.54 (m, 8H), 2.75 (dd, J = 14.2, 6.0 Hz, 1H), 2.32 (s, 3H), 1.87 (s,

3H), 1.73−1.67 (m, 2H), 1.25−1.18 (m, 2H), 0.80 (t, J = 7.2 Hz,

3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 173.3, 172.8, 172.2,

172.2, 169.9, 164.0, 163.6, 160.7, 160.3, 159.3, 158.2, 157.3, 157.2,

156.0, 155.9, 155.1, 154.9, 153.9, 138.4, 137.6, 137.2, 135.7, 129.7,

128.7, 127.1, 126.4, 126.1, 123.3, 122.3, 120.4, 112.4, 111.8, 109.7,

106.6, 84.9, 84.8, 66.0, 60.0, 55.7, 54.1, 53.6, 49.9, 48.5, 43.9, 42.4,

31.9, 30.8, 19.3, 17.0, 13.5. HRMS calculated for C₄₁H₄₅ClFN₇O₅S:

801.2875; found 401.6505 (M + 2H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-{2-[(1-ethyl-1H-pyrazol-5-yl)methoxy]phenyl}propanoic

Acid (6a). Using general procedure 4 and 3.526 g R5a (5.00 mmol) as

the appropriate phenol and R6f as the appropriate alcohol, 3.02 g 6a

Step E: Ethyl (2R)-2-(tert-Butoxycarbonylamino)-3-[2-[(2-butyl-

pyrazol-3-yl)methoxy]phenyl]propanoate. 928 mg ethyl (2R)-2-

(tert-butoxycarbonylamino)-3-(2-hydroxyphenyl)propanoate (3.00

mmol), 925 mg R6h (6.00 mmol), and 1.57 g PPh₃(6.00 mmol)

were dissolved in 15 mL dry toluene under N₂atmosphere. It was

stirred at rt for 5 min, then 1.18 mL DIAD (1.21 g, 6.00 mmol) was

added dropwise over a period of 2 min (exothermic reaction). Then

the reaction mixture was stirred at 50 °C for 30 min to reach complete

conversion. The reaction mixture was directly injected onto a

preconditioned silica gel column, then it was puriﬁed via ﬂash

chromatography using heptane and EtOAc as eluents to give 890 mg

ethyl (2R)-2-(tert-butoxycarbonylamino)-3-[2-[(2-butylpyrazol-3-yl)-

1

(3.85 mmol, 77%) was obtained. H NMR (500 MHz, DMSO-d₆) δ

ppm: 8.56 (s, 1H), 7.39 (d, J = 1.7 Hz, 1H), 7.39−7−7.36 (m, 1H),

7.26−7.22 (m, 2H), 7.20−7.10 (m, 4H), 7.07−7.02 (m, 1H), 6.66−

6.62 (m, 1H), 6.40 (d, J = 1.7 Hz, 1H), 6.11−6.07 (m, 1H), 5.37 (dd,

J = 10.3, 3.3 Hz, 1H), 5.18 (d, J = 12.6 Hz, 1H), 5.13 (d, J = 12.6 Hz,

1H), 4.30−4.24 (m, 1H), 4.22−4.11 (m, 3H), 3.15 (dd, J = 13.9, 3.3

Hz, 1H), 2.81−2.69 (m, 2H), 2.55 (br s, 4H), 2.47 (br s, 4H), 2.35

(dd, J = 13.9, 10.3 Hz, 1H), 2.24 (s, 3H), 1.76 (s, 3H), 1.35 (t, J = 7.2

Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 171.2, 166.2,

163.3, 162.0, 155.5, 153.5, 152.8, 137.7, 136.9, 136.4, 135.7, 131.0,

130.9, 130.6, 129.1, 128.5, 127.9, 127.7, 124.9, 122.0, 120.2, 118.8,

115.9, 111.8, 110.5, 106.6, 74.2, 67.1, 60.1, 56.0, 53.8, 52.1, 44.6, 43.8,

32.3, 17.5, 15.5. HRMS calculated for C₄₁H₄₂ClFN₆O₅S: 784.2610;

found 785.2679 (M + H).

1

methoxy]phenyl]propanoate (2.00 mmol, 66%) as a colorless oil. H

NMR (400 MHz, DMSO-d₆) δ ppm: 8.88 (s, 1H), 7.41 (d, J = 1.8

Hz, 1H), 7.25−7.13 (m, 4H), 6.85 (t, J = 7.6 Hz, 1H), 6.41 (d, J = 1.6

Hz, 1H), 5.20 (d, J = 12.6 Hz, 1H) 5.17 (d, J = 12.6 Hz, 1H), 4.81−

4.72 (m, 1H), 4.21−4.16 (m, 1H), 4.13 (t, J = 7.4 Hz, 2H), 4.01−

3.94 (m, 1H), 3.98 (dd, J = 7.2, 1.8 Hz, 1H), 3.04 (dd, J = 13.4, 5.2

Hz, 1H), 2.72 (dd, J = 13.2, 10.0 Hz, 1H), 1.79−1.72 (m, 2H), 1.31

(s, 9H), 1.28−1.13 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H), 0.87 (t, J = 7.2

Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ ppm: 138.1, 131.6,

128.5, 121.0, 112.3, 106.9, 60.7, 60.7, 53.9, 49.0, 32.4, 32.2, 28.6, 19.8,

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[1-(propan-2-yl)-1H-pyrazol-5-yl]methoxy}phenyl)-

propanoic Acid (6b). Using general procedure 4 and 353 mg R5a

(0.50 mmol) as the appropriate phenol and R6g as the appropriate

Z

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

alcohol, 222 mg 6b (0.28 mmol, 56%) was obtained. H NMR (500

MHz, DMSO-d₆) δ ppm: 8.56 (s, 1H), 7.42 (d, J = 1.6 Hz, 1H), 7.35

(d, J = 8.6 Hz, 1H), 7.29−7.23 (m, 2H), 7.23−7.16 (m, 2H), 7.16 (d,

J = 8.6 Hz, 1H), 7.16−7.10 (m, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.68−

6.63 (m, 1H), 6.38 (d, J = 1.6 Hz, 1H), 6.11 (br d, J = 7.6 Hz, 1H),

5.36 (dd, J = 10.2, 3.5 Hz, 1H), 5.17 (d, J = 12.5 Hz, 1H), 5.13 (d, J =

12.5 Hz, 1H), 4.67−4.57 (m, 1H), 4.30−4.16 (m, 2H), 3.13 (dd, J =

13.8, 3.5 Hz, 1H), 2.80−2.68 (m, 2H), 2.65−2.25 (br m, 8H), 2.38

(dd, J = 13.8, 10.2 Hz, 1H), 2.18 (s, 3H), 1.77 (s, 3H), 1.40 (d, J = 6.5

Hz, 3H), 1.37 (d, J = 6.5 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ

ppm: 153.2, 138.0, 136.7, 131.5, 131.4, 131.0, 128.4, 120.6, 116.4,

111.0, 106.8, 74.4, 67.6, 60.4, 56.6, 54.7, 52.9, 50.2, 45.5, 32.7, 23.3,

18.0. HRMS calculated for C₄₂H₄₄ClFN₆O₅S: 798.2766; found

400.1469 (M + 2H).

(2R)-3-{2-[(1-Butyl-1H-pyrazol-5-yl)methoxy]phenyl}-2-{[(5S_a)-5-

{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-

(prop-1-yn-1-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}propanoic Acid

(7). Using general procedure 4 and 389 mg R5c (0.6 mmol) as the

appropriate phenol and R6h as the appropriate alcohol, 248 mg 7

1

(0.33 mmol, 55%) was obtained. H NMR (500 MHz, DMSO-d₆) δ

ppm: 8.57 (s, 1H), 7.38 (d, J = 1.7 Hz, 1H), 7.28 (d, J = 8.7 Hz, 1H),

7.15 (d, J = 8.7 Hz, 1H), 7.14−7.10 (m, 1H), 7.08−7.04 (m, 1H),

6.68−6.63 (m, 1H), 6.40 (d, J = 1.7 Hz, 1H), 6.01−5.97 (m, 1H),

5.31 (dd, J = 10.5, 3.0 Hz, 1H), 5.16 (d, J = 12.7 Hz, 1H), 5.12 (d, J =

12.7 Hz, 1H), 4.28−4.19 (m, 2H), 4.16−4.06 (m, 2H), 3.17 (dd, J =

13.8, 3.0 Hz, 1H), 2.81−2.69 (m, 2H), 2.65−2.32 (m, 8H), 2.35 (dd,

J = 13.8, 10.5 Hz, 1H), 2.21 (s, 3H), 2.08 (s, 3H), 2.02 (s, 3H), 1.76−

1.70 (m, 2H), 1.29−1.21 (m, 2H), 0.84 (t, J = 7.3 Hz, 3H). ¹³C NMR

(125 MHz, DMSO-d₆) δ ppm: 170.9, 166.0, 162.9, 155.5, 153.8,

137.6, 137.2, 136.0, 135.6, 131.1, 130.1, 127.9, 127.3, 121.8, 120.2,

119.1, 117.2, 111.8, 110.1, 106.5, 97.2, 74.3, 71.9, 67.1, 60.2, 56.2,

54.1, 52.3, 48.6, 44.9, 32.3, 31.9, 19.4, 17.8, 13.6, 4.4. HRMS

calculated for C₄₀H₄₅ClN₆O₅S: 756.2861; found 757.2953 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-{2-[(2-methoxypyrimidin-4-yl)methoxy]phenyl}propanoic

Acid (8a). Using general procedure 4 and 348 mg R5b (0.50 mmol)

as the appropriate phenol and (2-methoxypyrimidin-4-yl)methanol as

the appropriate alcohol, 95 mg 8a (0.12 mmol, 24%) was obtained.

¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.61 (d, J = 5.1 Hz, 1H),

8.55 (s, 1H), 7.43 (d, J = 5.1 Hz, 1H), 7.28 (d, J = 8.7 Hz, 1H), 7.25

(d, J = 8.7 Hz, 1H), 7.17−7.11 (m, 1H), 6.98−6.93 (m, 1H), 6.80−

6.74 (m, 1H), 6.30−6.25 (m, 1H), 5.87 (dd, J = 6.7, 3.6 Hz, 1H), 5.67

(t, J = 3.6 Hz, 1H), 5.46 (dd, J = 10.3, 3.0 Hz, 1H), 5.16 (d, J = 15.3

Hz, 1H), 5.09 (d, J = 15.3 Hz, 1H), 4.30−4.18 (m, 2H), 3.91 (s, 3H),

3.31 (dd, J = 13.9, 3.0 Hz, 1H), 2.80−2.69 (m, 2H), 2.65−2.52 (br s,

4H), 2.47−2.30 (br s, 4H), 2.37 (dd, J = 13.9, 10.3 Hz, 1H), 2.20 (s,

3H), 1.92 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 170.9,

168.7, 165.7, 164.7, 163.2, 160.2, 157.6, 155.4, 155.2, 154.0, 153.1,

138.0, 135.7, 130.7, 129.2, 128.1, 127.6, 127.0, 125.7, 125.0, 122.3,

120.5, 118.7, 111.7, 111.0, 85.3, 85.2, 74.3, 68.6, 67.2, 56.1, 54.5, 54.2,

52.4, 45.0, 32.6, 17.2. HRMS calculated for C₃₉H₃₈N₆O₇FSCl:

788.2195; found 789.2289 (M + H).

(2R)-3-{2-[(1-Butyl-1H-pyrazol-5-yl)methoxy]phenyl}-2-{[(5S_a)-5-

{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-

(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}propanoic Acid

(6c). Using general procedure 4 and 529 mg R5a (0.75 mmol) as

the appropriate phenol and R6h as the appropriate alcohol, 369 mg 6c

1

(0.45 mmol, 61%) was obtained. H NMR (500 MHz, DMSO-d₆) δ

ppm: 8.55 (s, 1H), 7.39 (d, J = 1.7 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H),

7.27−7.23 (m, 2H), 7.21−7.10 (m, 4H), 7.08−7.05 (m, 1H), 6.67−

6.63 (m, 1H), 6.40 (d, J = 1.7 Hz, 1H), 6.11−6.08 (m, 1H), 5.36 (dd,

J = 10.1, 3.3 Hz, 1H), 5.17 (d, J = 12.6 Hz, 1H), 5.13 (d, J = 12.6 Hz,

1H), 4.27−4.07 (m, 4H), 3.14 (dd, J = 13.7, 3.3 Hz, 1H), 2.78−2.68

(m, 2H), 2.59−2.30 (m, 8H), 2.36 (dd, J = 13.7, 10.1 Hz, 1H), 2.18

(s, 3H), 1.77 (s, 3H), 1.76−1.71 (m, 2H), 1.31−1.23 (m, 2H), 0.85

(t, J = 7.4 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 170.9,

166.3, 163.2, 162.0, 155.5, 153.6, 152.8, 137.6, 137.2, 136.5, 135.7,

131.0, 130.9, 130.6, 129.1, 128.5, 127.9, 127.6, 124.8, 122.0, 120.2,

118.8, 115.9, 111.8, 110.6, 105.5, 74.1, 67.2, 60.2, 56.1, 54.2, 52.5,

48.6, 45.0, 32.2, 31.9, 19.4, 17.5, 13.5. HRMS calculated for

C₄₃H₄₆ClFN₆O₅S: 812.2923; found 407.1551 (M + 2H).

(2R)-3-{2-[(1-tert-Butyl-1H-pyrazol-5-yl)methoxy]phenyl}-2-

{[(5S_a)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]-

phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-

propanoic Acid (6d). Using general procedure 4 and 176 mg R5a

(0.25 mmol) as the appropriate phenol and R6i as the appropriate

1

alcohol, 35 mg 6d was obtained (0.043 mmol, 17%). H NMR (500

MHz, DMSO-d₆) δ ppm: 8.58 (s, 1H), 7.36−7.34 (m, 2H), 7.28−

7.25 (m, 2H), 7.22−7.12 (m, 4H), 7.05 (d, J = 8.2 Hz, 1H), 6.66 (t, J

= 7.5 Hz, 1H), 6.47 (d, J = 1.5 Hz, 1H), 6.14 (dd, J = 7.6, 1.0 Hz,

1H), 5.39 (dd, J = 10.0, 3.5 Hz, 1H), 5.25 (d, J = 12.5 Hz, 1H), 5.19

(d, J = 12.5 Hz, 1H), 4.26−4.18 (m, 2H), 3.15 (dd, J = 13.8, 3.5 Hz,

1H), 2.78−2.69 (m, 2H), 2.62−2.26 (m, 8H), 2.41 (dd, J = 13.8, 10.0

Hz, 1H), 2.17 (s, 3H), 1.78 (s, 3H), 1.59 (s, 9H). ¹³C NMR (125

MHz, DMSO-d₆) δ ppm: 170.8, 166.3, 163.0, 162.1, 155.5, 153.6,

152.8, 137.3, 136.5, 136.2, 135.7, 131.0, 130.5, 129.1, 128.6, 128.0,

127.6, 124.5, 122.0, 120.2, 118.8, 116.0, 111.7, 110.6, 109.0, 74.0,

67.2, 61.9, 60.3, 56.1, 54.2, 52.5, 45.1, 32.2, 29.9, 17.5. HRMS

calculated for C₄₃H₄₆ClFN₆O₅S: 812.2923; found 813.3030 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[1-(2,2,2-triﬂuoroethyl)-1H-pyrazol-5-yl]methoxy}-

phenyl)propanoic Acid (6e). Using general procedure 4 and 423 mg

R5a (0.60 mmol) as the appropriate phenol and [1-(2,2,2-

triﬂuoroethyl)-1H-pyrazol-5-yl]methanol as the appropriate alcohol,

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-(2-{[2-(morpholin-4-yl)pyrimidin-4-yl]methoxy}phenyl)-

propanoic Acid (8b). Using general procedure 4 and 486 mg R5b

(0.7 mmol) as the appropriate phenol and R6b as the appropriate

1

alcohol, 130 mg 8b (0.15 mmol, 22%) was obtained. H NMR (500

MHz, DMSO-d₆) δ ppm: 8.54 (s, 1H), 8.38 (d, J = 4.8 Hz, 1H), 7.28

(d, J = 8.5 Hz, 1H), 7.22 (d, J = 8.5 Hz, 1H), 7.17−7.05 (m, 1H),

6.97 (d, J = 4.8 Hz, 1H), 6.94−6.85 (m, 1H), 6.80−6.69 (m, 1H),

6.29−6.18 (m, 1H), 5.91−5.80 (m, 1H), 5.71−5.62 (m, 1H), 5.52−

5.39 (m, 1H), 5.03 (d, J = 15.0 Hz, 1H), 4.96 (d, J = 15.0 Hz, 1H),

4.32−4.13 (m, 2H), 3.68 (br s, 4H), 3.62 (br s, 4H), 3.36−3.24 (m,

1H), 2.82−2.67 (m, 2H), 2.66−2.37 (m, 8H), 2.37−2.28 (m, 1H),

2.25 (s, 3H), 1.91 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm:

171.2, 166.6, 165.7, 163.5, 160.9, 158.7, 157.6, 155.4, 154.0, 153.1,

138.1, 135.7, 130.6, 129.3, 127.9, 127.7, 127.1, 125.6, 125.2, 122.3,

120.3, 118.8, 111.6, 111.0, 106.8, 85.2, 74.6, 68.9, 67.1, 66.0, 56.1,

53.8, 52.0, 44.5, 43.8, 32.7, 17.2. HRMS calculated for

C₄₂H₄₃ClFN₇O₇S: 843.2617; found 422.6360 (M + 2H).

1

311 mg 6e (0.37 mmol, 62%) was obtained. H NMR (500 MHz,

DMSO-d₆) δ ppm: 8.56 (s, 1H), 7.57 (d, J = 1.7 Hz, 1H), 7.37 (d, J =

8.5 Hz, 1H), 7.27−7.23 (m, 2H), 7.21−7.11 (m, 4H), 7.06−7.02 (m,

1H), 6.69−6.65 (m, 1H), 6.57 (d, J = 1.7 Hz, 1H), 6.14−6.10 (m,

1H), 5.37 (dd, J = 10.3, 3.0 Hz, 1H), 5.23 (d, J = 13.3 Hz, 1H), 5.20

(d, J = 13.3 Hz, 1H), 5.18 (q, J = 9.0 Hz, 2H), 4.26−4.17 (m, 2H),

3.16 (dd, J = 13.6, 3.0 Hz, 1H), 2.79−2.69 (m, 2H), 2.65−2.32 (m,

8H), 2.36 (dd, J = 13.6, 10.3 Hz, 1H), 2.22 (s, 3H), 1.78 (s, 3H). ¹³C

NMR (125 MHz, DMSO-d₆) δ ppm: 171.1, 166.3, 163.2, 162.0,

155.4, 153.6, 152.8, 140.03, 139.98, 139.9, 136.5, 135.7, 131.0, 130.6,

129.1, 128.5, 127.8, 127.9, 125.0, 123.7, 122.0, 120.4, 118.8, 116.0,

111.8, 110.6, 107.2, 74.2, 67.1, 60.3, 56.0, 54.0, 52.3, 49.5, 44.8, 32.4,

17.5. HRMS calculated for C₄₁H₃₉ClF₄N₆O₅S: 838.2327; found

839.2389 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-(2-{[2-(2-methoxyethyl)pyrimidin-4-yl]methoxy}phenyl)-

propanoic Acid (8c). Using general procedure 4 and 417 mg R5b

(0.60 mmol) as the appropriate phenol and R6c as the appropriate

1

alcohol, 249 mg 8c (0.3 mmol, 50%) was obtained. H NMR (500

MHz, DMSO-d₆) δ ppm: 8.74 (d, J = 5.2 Hz, 1H), 8.54 (s, 1H), 7.69

(d, J = 5.2 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 7.25 (d, J = 8.6 Hz, 1H),

7.16−7.12 (m, 1H), 6.98−6.94 (m, 1H), 6.78−6.75 (m, 1H), 6.26−

6.23 (m, 1H), 5.87 (dd, J = 6.8, 3.6 Hz, 1H), 5.67 (t, J = 3.6 Hz, 1H),

5.46 (dd, J = 10.6, 2.5 Hz, 1H), 5.19 (d, J = 15.2 Hz, 1H), 5.13 (d, J =

15.2 Hz, 1H), 4.30−4.20 (m, 2H), 3.79 (t, J = 6.7 Hz, 2H), 3.34 (dd,

J = 13.8, 2.5 Hz, 1H), 3.21 (s, 3H), 3.10 (t, J = 6.7 Hz, 2H), 2.81−

AA

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

2.70 (m, 2H), 2.69−2.37 (m, 8H), 2.33 (dd, J = 13.8, 10.6 Hz, 1H),

2.24 (s, 3H), 1.93 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm:

171.2, 167.6, 166.0, 165.7, 163.3, 157.9, 157.6, 155.4, 155.3, 154.0,

153.1, 138.1, 135.7, 130.7, 129.2, 128.1, 127.7, 127.1, 127.0, 125.6,

125.2, 122.3, 120.5, 118.8, 115.5, 111.7, 111.2, 110.9, 85.3, 85.2, 74.6,

70.2, 68.7, 67.2, 57.9, 56.1, 53.9, 52.1, 32.8, 17.2. HRMS calculated for

C₄₁H₄₂ClFN₆O₇S: 816.2508; found 409.1335 (M + 2H).

(0.25 mmol) as the appropriate phenol and R6c as the appropriate

alcohol, 72 mg 9c (0.088 mmol, 35%) was obtained. H NMR (500

1

MHz, DMSO-d₆) δ ppm: 8.74 (d, J = 5.2 Hz, 1H), 8.56 (s, 1H), 7.64

(d, J = 5.2 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 7.28−7.24 (m, 2H),

7.21−7.16 (m, 3H), 7.10 (td, J = 7.8, 1.5 Hz, 1H), 6.94 (d, J = 7.8 Hz,

1H), 6.68 (t, J = 7.4 Hz, 1H), 6.16 (d, J = 7.4 Hz, 1H), 5.45 (dd, J =

10.0, 3.0 Hz, 1H), 5.20 (d, J = 15.0 Hz, 1H), 5.12 (d, J = 15.0 Hz,

1H), 4.27−4.16 (m, 2H), 3.78 (t, J = 6.6 Hz, 2H), 3.34−3.29 (m,

1H), 3.20 (s, 3H), 3.09 (t, J = 6.6 Hz, 1H), 2.77−2.68 (m, 2H),

2.61−2.25 (m, 8H), 2.41 (dd, J = 13.8, 10.0 Hz, 1H), 2.22 (s, 3H),

1.79 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 171.2, 167.6,

166.3, 165.9, 163.0, 162.1, 157.9, 155.3, 153.6, 152.9, 136.5, 135.7,

131.0, 130.8, 130.6, 129.1, 128.6, 128.1, 127.7, 125.1, 122.0, 120.4,

118.9, 116.0, 115.5, 111.7, 110.6, 74.4, 70.2, 68.7, 67.2, 57.9, 56.0,

54.0, 52.2, 44.8, 32.6, 17.5. HRMS calculated for C₄₃H₄₄ClFN₆O₆S:

826.2716; found 414.1439 (M + 2H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[2-(pyridin-4-yl)pyrimidin-4-yl]methoxy}phenyl)-

propanoic Acid (9d). Using general procedure 4 and 176 mg R5a

(0.25 mmol) as the appropriate phenol and R6e as the appropriate

alcohol, 93 mg 9d (0.11, 44%) was obtained with a purity of 94.7%.

¹H NMR (500 MHz, DMSO-d₆) δ ppm: 9.02 (d, J = 5.0 Hz, 1H),

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(5-ﬂuorofuran-2-yl)thieno[2,3-d]pyrimidin-4-

yl]oxy}-3-(2-{[2-(pyridin-4-yl)pyrimidin-4-yl]methoxy}phenyl)-

propanoic Acid (8d). Using general procedure 4 and 348 mg R5b

(0.50 mmol) as the appropriate phenol and R6e as the appropriate

1

alcohol, 217 mg 8d (0.26 mmol 52%) was obtained. H NMR (500

MHz, DMSO-d₆) δ ppm: 9.02 (d, J = 5.1 Hz, 1H), 8.76−8.74 (m,

2H), 8.52 (s, 1H), 8.25−8.23 (m, 2H), 7.92 (d, J = 5.1 Hz, 1H), 7.29

(d, J = 8.6 Hz, 1H), 7.25 (d, J = 8.6 Hz, 1H), 7.18−7.14 (m, 1H),

7.05−7.02 (m, 1H), 6.81−6.77 (m, 1H), 6.30−6.27 (m, 1H), 5.87

(dd, J = 6.8, 3.6 Hz, 1H), 5.67 (t, J = 3.6 Hz, 1H), 5.48 (dd, J = 10.5,

2.6 Hz, 1H), 5.37 (d, J = 15.3 Hz, 1H), 5.29 (d, J = 15.3 Hz, 1H),

4.29−4.18 (m, 2H), 3.37 (dd, J = 14.1, 2.6 Hz, 1H), 2.79−2.68 (m,

2H), 2.62−2.34 (m, 8H), 2.37 (dd, J = 14.1, 10.5 Hz, 1H), 2.21 (s,

3H), 1.93 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 171.1,

167.0, 165.7, 163.3, 161.1, 158.8, 156.5, 155.3, 154.0, 153.1, 150.5,

144.0, 138.1, 135.7, 130.7, 129.2, 128.1, 127.7, 127.1, 125.7, 125.3,

122.3, 121.6, 120.6, 118.8, 117.8, 111.8, 111.2, 110.9, 85.2, 74.6, 68.7,

67.2, 56.1, 54.0, 52.2, 44.8, 32.7, 17.2. HRMS calculated for

C₄₃H₃₉ClFN₇O₆S: 835.2355; found 418.6246 (M + 2H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-{2-[(2-methoxypyrimidin-4-yl)methoxy]phenyl}propanoic

Acid (9a). Using general procedure 4 and 353 mg R5a (0.50 mmol)

as the appropriate phenol and (2-methoxypyrimidin-4-yl)methanol as

the appropriate alcohol, 150 mg 9a (0.19 mmol, 37%) was obtained.

¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.61 (d, J = 5.0 Hz, 1H),

8.57 (s, 1H), 7.43 (d, J = 5.0 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.30−

7.24 (m, 2H), 7.22−7.16 (m, 3H), 7.14−7.08 (m, 1H), 6.95−6.90

(m, 1H), 6.71−6.65 (m, 1H), 6.19−6.15 (m, 1H), 5.45 (dd, J = 10.0,

3.0 Hz, 1H), 5.15 (d, J = 15.2 Hz, 1H), 5.09 (d, J = 15.2 Hz, 1H),

4.28−4.21 (m, 1H), 4.21−4.14 (m, 1H), 3.91 (s, 3H), 3.30 (dd, J =

13.5, 3.0 Hz, 1H), 2.78−2.66 (m, 2H), 2.61−2.52 (br s, 4H), 2.47−

2.28 (br s, 4H), 2.42 (dd, J = 13.5, 10.0 Hz, 1H), 2.20 (s, 3H), 1.79

(s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 171.1, 168.7, 166.2,

164.7, 163.3, 161.0, 160.2, 155.2, 153.6, 152.9, 136.5, 135.7, 131.0,

130.9, 130.8, 130.6, 129.1, 128.6, 128.0, 127.7, 125.8, 125.0, 122.0,

120.4, 118.9, 115.9, 111.7, 111.6, 110.6, 74.4, 68.6, 67.2, 56.0, 54.5,

54.1, 52.3, 44.9, 32.5, 17.5. HRMS calculated for C₄₁H₄₀ClFN₆O₆S:

798.2403; found 400.1284 (M + 2H).

8.75 (dd, J = 4.4, 1.6 Hz, 1H), 8.54 (s, 1H), 8.24 (dd, J = 4.4, 1.6 Hz,

1H), 7.88 (d, J = 5.0 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.28−7.24

(m, 2H), 7.21−7.16 (m, 3H), 7.13 (td, J = 7.8, 1.6 Hz, 1H), 7.01 (d, J

= 8.2 Hz, 1H), 6.70 (t, J = 7.4 Hz, 1H), 6.19 (d, J = 7.8 Hz, 1H), 5.47

(dd, J = 10.2, 3.0 Hz, 1H), 5.36 (d, J = 15.2 Hz, 1H), 5.28 (d, J = 15.2

Hz, 1H), 4.26−4.14 (m, 2H), 3.36 (dd, J = 13.8, 2.5 Hz, 1H), 2.76−

2.66 (m, 2H), 2.61−2.32 (m, 8H), 2.44 (dd, J = 13.8, 10.2 Hz, 1H),

1

2.21 (s, 3H), 1.79 (s, 3H). H NMR (125 MHz, DMSO-d₆) δ ppm:

171.2, 167.0, 166.2, 162.2, 161.1, 158.8, 155.3, 153.6, 152.9, 150.6,

144.0, 136.5, 135.7, 131.0, 130.8, 130.6, 129.1, 128.6, 128.1, 127.8,

122.0, 121.6, 120.5, 118.9, 117.8, 116.0, 111.8, 110.6, 74.5, 68.8, 67.3,

67.1, 56.0, 54.0, 52.2, 44.8, 32.6, 17.5. HRMS calculated for

C₄₅H₄₁ClFN₇O₅S: 845.2562; found 423.6358 (M + 2H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[2-(pyridin-2-yl)pyrimidin-4-yl]methoxy}phenyl)-

propanoic Acid (9e). Step A: [2-(2-Pyridyl)pyrimidin-4-yl]methanol.

Using general procedure 2 and 2.50 g pyridine-2-carboxamidine

hydrochloride (15.9 mmol) as the appropriate amidine hydrochloride,

2.18 g 4-(dimethoxymethyl)-2-(2-pyridyl)pyrimidine (9.43 mmol)

was obtained which was treated as described in general procedure 1 to

yield 1.06 g [2-(2-pyridyl)pyrimidin-4-yl]methanol (5.66 mmol,

43%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.94 (d, J = 5.1

Hz, 1H), 8.76−8.71 (m, 1H), 8.37 (d, J = 7.9 Hz, 1H), 8.00−7.93 (m,

1H), 7.60 (d, J = 5.1 Hz, 1H), 7.55−7.50 (m, 1H), 5.74 (t, J = 5.8 Hz,

1H), 4.67 (d, J = 5.8 Hz, 2H). LRMS calculated for C₁₀H₉N₃O:

187.2; found 188.2 (M + H).

Step B: (2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiper-

azin-1-yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-

4-yl]oxy}-3-(2-{[2-(pyridin-2-yl)pyrimidin-4-yl]methoxy}phenyl)-

propanoic Acid (9e). Using general procedure 4 and 176 mg R5a

(0.25 mmol) as the appropriate phenol and [2-(2-pyridyl)pyrimidin-

4-yl]methanol as the appropriate alcohol, 61 mg 9e (0.073 mmol,

29%) was obtained. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.99 (d,

J = 5.0 Hz, 1H), 8.76−8.74 (m, 1H), 8.39−8.36 (m, 1H), 7.96 (td, J =

7.6, J = 1.8 Hz), 7.86 (d, J = 4.8 Hz, 1H), 7.55−7.52 (m, 1H), 7.39 (d,

J = 8.5 Hz, 1H), 7.28−7.24 (m, 2H), 7.21−7.16 (m, 3H), 7.13 (td, J =

7.8, 1.5 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 6.70 (t, J = 7.4 Hz, 1H),

6.19 (d, J = 7.4 Hz, 1H), 5.47 (dd, J = 10.2, 3.0 Hz, 1H), 5.34 (d, J =

15.2 Hz, 1H), 5.27 (d, J = 15.2 Hz, 1H), 4.27−4.15 (m, 2H), 3.37

(dd, J = 13.8, 2.5 Hz, 1H), 2.76−2.66 (m, 2H), 2.61−2.36 (m, 8H),

2.41 (dd, J = 13.8, 10.2 Hz, 1H), 2.21 (s, 3H), 1.79 (s, 3H). ¹³C NMR

(125 MHz, DMSO-d₆) δ ppm: 171.2, 166.8, 166.3, 166.2, 162.6,

162.2, 158.5, 155.3, 154.4, 153.6, 152.9, 149.7, 137.1, 136.5, 135.7,

131.0, 130.8, 130.6, 129.1, 128.6, 128.1, 127.8, 125.2, 123.5, 122.0,

120.5, 118.9, 117.1, 116.0, 111.8, 110.6, 74.6, 68.9, 67.1, 56.0, 54.0,

52.2, 52.2, 44.8, 32.6, 17.5. HRMS calculated for C₄₅H₄₁ClFN₇O₅S:

845.2562; found 423.6373 (M + 2H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[2-(morpholin-4-yl)pyrimidin-4-yl]methoxy}phenyl)-

propanoic Acid (9b). Using general procedure 4 and 1.06 g R5a (1.50

mmol) as the appropriate phenol and R6b as the appropriate alcohol,

1

335 mg 9b (0.39 mmol, 26%) was obtained. H NMR (500 MHz,

DMSO-d₆) δ ppm: 8.57 (s, 1H), 8.37 (d, J = 5.0 Hz, 1H), 7.37 (d, J =

8.4 Hz, 1H), 7.30−7.23 (m, 2H), 7.22−7.14 (m, 3H), 7.12−7.06 (m,

1H), 6.92 (d, J = 5.0 Hz, 1H), 6.91−6.86 (m, 1H), 6.70−6.63 (m,

1H), 6.18−6.12 (m, 1H), 5.45 (dd, J = 10.2, 3.2 Hz, 1H), 5.03 (d, J =

14.9 Hz, 1H), 4.97 (d, J = 14.9 Hz, 1H), 4.28−4.21 (m, 1H), 4.21−

4.13 (m, 1H), 3.72−3.66 (m, 4H), 3.65−3.60 (m, 4H), 3.28 (dd, J =

14.0, 3.2 Hz, 1H), 2.79−2.65 (m, 2H), 2.61−2.29 (m, 8H), 2.42 (dd,

J = 10.2, 3.2 Hz, 1H), 2.20 (s, 3H), 1.78 (s, 3H). ¹³C NMR (125

MHz, DMSO-d₆) δ ppm: 171.1, 166.5, 166.2, 163.3, 161.0, 160.9,

158.6, 155.4, 153.6, 152.9, 136.4, 135.7, 131.0, 130.8, 130.5, 129.1,

129.1, 128.6, 128.0, 127.7, 122.0, 120.2, 118.9, 116.0, 111.6, 110.5,

106.7, 74.3, 68.9, 67.2, 65.9, 63.7, 56.0, 54.0, 52.3, 44.9, 43.8, 32.4,

17.5. HRMS calculated for C₄₄H₄₅ClFN₇O₆S: 853.2825; found

427.6484 (M + 2H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[2-(2-methoxyethyl)pyrimidin-4-yl]methoxy}phenyl)-

propanoic Acid (9c). Using general procedure 4 and 176 mg R5a

AB

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[2-(pyridin-3-yl)pyrimidin-4-yl]methoxy}phenyl)-

propanoic Acid (9f). Step A: [2-(3-Pyridyl)pyrimidin-4-yl]methanol.

Using general procedure 2 and 2.50 g pyridine-3-carboxamidine

hydrochloride (15.9 mmol) as the appropriate amidine hydrochloride,

1.67 g 4-(dimethoxymethyl)-2-(3-pyridyl)pyrimidine (7.23 mmol)

was obtained which was treated as described in general procedure 1 to

yield 1.19 g [2-(3-pyridyl)pyrimidin-4-yl]methanol (6.37 mmol,

48%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 9.51−9.49 (m,

1H), 8.93 (d, J = 5.1 Hz, 1H), 8.73−8.70 (m, 1H), 8.68−8.63 (m,

1H), 7.58−7.53 (m, 2H), 5.73 (t, J = 5.8 Hz, 1H), 4.67 (d, J = 5.8 Hz,

2H). ¹³C NMR (100 MHz, DMSO-d₆) δ ppm: 172.2, 161.4, 158.7,

151.9, 149.4, 135.5, 133.1, 124.3, 116.8, 64.0. LRMS calculated for

C₁₀H₉N₃O: 187.2; found 188.2 (M + H).

Step B: (2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiper-

azin-1-yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-

4-yl]oxy}-3-(2-{[2-(pyridin-3-yl)pyrimidin-4-yl]methoxy}phenyl)-

propanoic Acid (9f). Using general procedure 4 and 176 mg R5a

(0.25 mmol) as the appropriate phenol and [2-(3-pyridyl)pyrimidin-

4-yl]methanol as the appropriate alcohol, 110 mg 9f (0.13 mmol,

52%) was obtained. ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 9.50 (d,

J = 2.2 Hz, 1H), 8.98 (d, J = 5.0 Hz, 1H), 8.71 (dd, J = 4.8, 1.7 Hz,

1H), 8.65 (dt, J = 8.0, 2.0 Hz, 1H), 8.54 (s, 1H), 7.82 (d, J = 5.0 Hz,

1H), 7.56−7.52 (m, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.28−7.24 (m,

2H), 7.21−7.16 (m, 3H), 7.13 (td, J = 7.6, 1.5 Hz, 1H), 7.00 (d, J =

8.2 Hz, 1H), 6.69 (t, J = 7.4 Hz, 1H), 6.18 (d, J = 7.8 Hz, 1H), 5.47

(dd, J = 10.0, 3.0 Hz, 1H), 5.34 (d, J = 15.2 Hz, 1H), 5.27 (d, J = 15.2

Hz, 1H), 4.27−4.15 (m, 2H), 3.36 (dd, J = 13.8, 2.5 Hz, 1H), 2.76−

2.66 (m, 2H), 2.61−2.36 (m, 8H), 2.41 (dd, J = 13.8, 10.0 Hz, 1H),

2.21 (s, 3H), 1.79 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm:

171.2, 166.8, 166.2, 163.0, 162.1, 161.4, 158.6, 155.3, 153.6, 152.9,

151.6, 149.0, 136.4, 135.7, 135.1, 132.4, 131.0, 130.8, 130.6, 129.1,

128.6, 128.1, 127.8, 125.2, 123.8, 122.0, 120.5, 118.9, 116.9, 116.0,

111.8, 110.6, 74.5, 68.8, 67.1, 56.0, 54.0, 52.2, 44.8, 32.6, 17.5. HRMS

calculated for C₄₅H₄₁ClFN₇O₅S: 845.2562; found 423.6337 (M +

2H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[2-(furan-2-yl)pyrimidin-4-yl]methoxy}phenyl)propanoic

Acid (9g). Using general procedure 5 and 1.10 g R7 (1.30 mmol) and

0.58 g 2-furylboronic acid (5.2 mmol) as the appropriate boronic acid

derivative, 358 mg 9g (0.43 mmol, 33%) was obtained. ¹H NMR (500

MHz, DMSO-d₆) δ ppm: 8.84 (d, J = 5.1 Hz, 1H), 8.57 (s, 1H), 7.93

(dd, J = 1.7, 0.8 Hz, 1H), 7.67 (d, J = 5.0 Hz, 1H), 7.39 (d, J = 8.9 Hz,

1H), 7.31 (dd, J = 3.4, 0.8 Hz, 1H), 7.30−7.24 (m, 2H), 7.23−7.16

(m, 3H), 7.15−7.10 (m, 1H), 7.00−6.95 (m, 1H), 6.70 (dd, J = 3.4,

1.7 Hz, 1H), 6.73−6.67 (m, 1H), 6.22−6.16 (m, 1H), 5.46 (dd, J =

10.2, 3.0 Hz, 1H), 5.26 (d, J = 15.3 Hz, 1H), 5.19 (d, J = 15.3 Hz,

1H), 4.28−4.21 (m, 1H), 4.21−4.13 (m, 1H), 3.34 (dd, J = 13.7, 3.0

Hz, 1H), 2.78−2.65 (m, 2H), 2.61−2.27 (m, 8H), 2.43 (dd, J = 13.7,

10.2 Hz, 1H), 2.20 (s, 3H), 1.80 (s, 3H). ¹³C NMR (125 MHz,

DMSO-d₆) δ ppm: 171.1, 166.5, 166.2, 163.0, 161.0, 158.3, 156.4,

155.2, 153.6, 152.9, 151.5, 145.9, 136.4, 135.7, 131.0, 130.8, 130.6,

129.1, 128.6, 128.0, 127.7, 125.2, 122.0, 120.4, 118.9, 116.0, 115.6,

113.6, 112.5, 111.7, 110.6, 74.5, 68.7, 67.1, 56.0, 54.0, 52.3, 44.9, 32.5,

17.5. HRMS calculated for C₄₄H₄₀ClFN₆O₆S: 834.2403; found

418.1278 (M + 2H).

3H), 2.16 (s, 3H), 1.80 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ

ppm: 171.0, 166.2, 166.0, 161.0, 157.9, 155.3, 153.6, 152.9, 137.6,

136.8, 136.4, 135.7, 131.1, 131.0, 130.8, 130.6, 130.4, 129.4, 129.1,

128.6, 128.0, 127.7, 125.7, 122.0, 120.4, 118.9, 116.0, 115.5, 111.7,

110.6, 68.8, 67.2, 56.1, 54.2, 52.5, 45.1, 32.5, 20.9, 17.5. HRMS

calculated for C₄₇H₄₄ClFN₆O₅S: 858.2766; found 430.1464 (M +

2H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[2-(4-methylpyridin-3-yl)pyrimidin-4-yl]methoxy}-

phenyl)propanoic Acid (9i). Using general procedure 5 and 548 mg

R7 (0.65 mmol) and (4-methyl-3-pyridyl)boronic acid as the

appropriate boronic acid derivative, 31 mg 9i (0.036 mmol, 5.5%)

1

was obtained. H NMR (500 MHz, DMSO-d₆) δ ppm: 8.99 (d, J =

5.4 Hz, 1H), 8.92 (br s, 1H), 8.53 (s, 1H), 8.49 (br s, 1H), 7.84 (d, J

= 4.6 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.32 (d, J = 4.2 Hz, 1H),

7.30−7.24 (m, 2H), 7.23−7.16 (m, 3H), 7.12 (td, J = 8.0, 1.4 Hz,

1H), 6.99 (d, J = 8.3 Hz, 1H), 6.70 (t, J = 7.5 Hz, 1H), 6.18 (d, J = 7.1

Hz, 1H), 5.46 (dd, J = 10.1, 2.8 Hz, 1H), 5.34 (d, J = 15.2 Hz, 1H),

5.27 (d, J = 15.2 Hz, 1H), 4.28−4.14 (m, 2H), 3.36 (dd, J = 13.7, 2.8

Hz, 1H), 2.77−2.65 (m, 2H), 2.60−2.24 (m, 9H), 2.47 (s, 3H), 2.16

(s, 3H), 1.80 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 166.3,

158.2, 155.3, 153.6, 152.8, 149.9, 136.4, 135.7, 131.0, 130.8, 130.6,

129.1, 128.6, 128.0, 122.0, 120.4, 118.9, 116.0, 115.9, 111.7, 110.6,

68.7, 67.2, 56.1, 54.2, 52.5, 45.1, 35.8, 32.6, 31.8, 20.2, 17.5. HRMS

calculated for C₄₆H₄₃ClFN₇O₅S: 859.2719; found 430.6434 (M +

2H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[2-(3-methylpyridin-4-yl)pyrimidin-4-yl]methoxy}-

phenyl)propanoic Acid (9j). Using general procedure 5 and 250 mg

R7 (0.30 mmol) and (3-methyl-4-pyridyl)boronic acid as the

appropriate boronic acid derivative, 52 mg 9j (0.06 mmol, 20%)

1

was obtained. H NMR (500 MHz, DMSO-d₆) δ ppm: 9.01 (d, J =

5.1 Hz, 1H), 8.58−8.48 (m, 3H), 7.91 (d, J = 4.8 Hz, 1H), 7.76 (d, J

= 5.1 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.30−7.23 (m, 2H), 7.23−

7.15 (m, 3H), 7.12 (td, J = 8.0, 1.6 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H),

6.69 (t, J = 7.3 Hz, 1H), 6.16 (d, J = 7.3 Hz, 1H), 5.46 (dd, J = 10.3,

2.6 Hz, 1H), 5.34 (d, J = 15.4 Hz, 1H), 5.26 (d, J = 15.4 Hz, 1H),

4.29−4.14 (m, 2H), 3.38 (m, 1H), 2.78−2.66 (m, 2H), 2.65−2.29

(m, 9H), 2.48 (s, 3H), 2.20 (s, 3H), 1.79 (s, 3H). ¹³C NMR (125

MHz, DMSO-d₆) δ ppm: 171.3, 166.5, 166.2, 163.8, 163.0, 161.0,

158.3, 155.2, 153.6, 152.9, 152.1, 147.5, 143.9, 136.4, 135.7, 131.6,

130.9, 130.8, 130.7, 129.2, 128.6, 128.0, 127.8, 123.6, 122.0, 120.4,

118.9, 116.7, 115.9, 111.7, 110.6, 74.8, 68.7, 67.2, 56.0, 54.0, 52.3,

44.8, 32.7, 17.7, 17.5. HRMS calculated for C₄₆H₄₃ClFN₇O₅S:

859.2719; found 860.2808 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[2-(5-methoxy-2-methylpyridin-4-yl)pyrimidin-4-yl]-

methoxy}phenyl)propanoic Acid (9k). Step A: Ethyl (2R)-2-[(5S_a)-5-

[3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-

(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyr-

imidin-4-yl)methoxy]phenyl]propanoate. Using step A of general

procedure 4 and 235 mg R5a (0.33 mmol) as the appropriate phenol

and (2-chloropyrimidin-4-yl)methanol as the appropriate alcohol, 197

mg ethyl (2R)-2-[(5S_a)-5-[3-chloro-2-methyl-4-[2-(4-methylpipera-

zin-1-yl)ethoxy]phenyl]-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-

yl]oxy-3-[2-[(2-chloropyrimidin-4-yl)methoxy]phenyl]propanoate

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[2-(2-methylphenyl)pyrimidin-4-yl]methoxy}phenyl)-

propanoic Acid (9h). Using general procedure 5 and 253 mg R7

(0.30 mmol) and o-tolylboronic acid as the appropriate boronic acid

1

(0.24 mmol, 71%) was obtained. H NMR (500 MHz, DMSO-d₆) δ

ppm: 8.89−8.85 (m, 1H), 8.60 (s, 1H), 7.71−7.66 (m, 1H), 7.36−

7.09 (m, 6H), 7.03−6.86 (m, 2H), 6.79−6.68 (m, 1H), 6.33−6.24

(m, 1H), 5.47 (dd, J = 9.0, 4.2 Hz, 1H), 5.26 (d, J = 15.6 Hz, 1H),

5.20 (d, J = 15.6 Hz, 1H), 4.29−4.15 (m, 2H), 4.11−4.00 (m, 2H),

3.29−2.15 (m, 12H), 2.09 (s, 3H), 1.86 (s, 3H), 1.06 (t, J = 7.0 Hz,

3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 169.9, 169.5, 166.5,

162.3, 162.2, 161.1, 155.0, 153.8, 153.7, 152.7, 137.0, 136.0, 131.1,

131.0, 130.2, 129.5, 128.9, 128.8, 128.5, 128.0, 123.7, 122.0, 120.8,

118.8, 116.0, 111.9, 110.7, 73.8, 68.4, 67.3, 61.0, 56.3, 54.6, 52.9, 45.7,

32.3, 18.0, 13.9. HRMS calculated for C₄₂H₄₁Cl₂FN₆O₅S: 830.2220;

found 831.2275 (M + H).

1

derivative, 71 mg 9h (0.083 mmol, 28%) was obtained. H NMR

(500 MHz, DMSO-d₆) δ ppm: 8.94 (d, J = 5.1 Hz, 1H), 8.55 (s, 1H),

7.81−7.77 (m, 1H), 7.76 (d, J = 5.1 Hz, 1H), 7.39 (d, J = 8.4 Hz,

1H), 7.39−7.34 (m, 1H), 7.33−7.25 (m, 4H), 7.23−7.16 (m, 3H),

7.15−7.09 (m, 1H), 7.01−6.96 (m, 1H), 6.73−6.66 (m, 1H), 6.23−

6.16 (m, 1H), 5.46 (dd, J = 10.1, 3.0 Hz, 1H), 5.30 (d, J = 15.5 Hz,

1H), 5.24 (d, J = 15.5 Hz, 1H), 4.28−4.14 (m, 2H), 3.35 (dd, J =

13.8, 3.0 Hz, 1H), 2.76−2.65 (m, 2H), 2.60−2.24 (m, 9H), 2.47 (s,

AC

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Step B: (2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiper-

azin-1-yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-

4-yl]oxy}-3-(2-{[2-(5-methoxy-2-methylpyridin-4-yl)pyrimidin-4-yl]-

methoxy}phenyl)propanoic Acid (9k). 192 mg ethyl (2R)-2-[(5S_a)-

5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-

(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyri-

midin-4-yl)methoxy]phenyl]propanoate (0.23 mmol), 115 mg 5-

methoxy-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

pyridine (0.46 mmol), 16 mg Pd(PPh₃)₂Cl₂(0.023 mmol), 0.6 mL 2

M aq Na₂CO₃solution, and 0.6 mL dioxane were stirred at 90 °C

under N₂atmosphere for 2 h. Then 97 mg LiOH·H₂O (2.30 mmol)

was added and the mixture was stirred at rt for 2 h. Then it was

diluted with brine, acidiﬁed with 2 M aq HCl solution, and extracted

with DCM. The combined organic layer was dried over Na₂SO₄,

ﬁltered, and the ﬁltrate was concentrated under reduced pressure and

puriﬁed via preparative reversed phase chromatography using 25 mM

aq NH₄HCO₃solution and MeCN as eluents to obtain 78 mg 9k

(0.088 mmol, 38%). ¹H NMR (500 MHz, DMSO-d₆) δ ppm: 8.94 (d,

J = 5.1 Hz, 1H), 8.56 (s, 1H), 8.38 (s, 1H), 7.83 (d, J = 5.1 Hz, 1H),

7.39 (s, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.29−7.25 (m, 2H), 7.20 (t, J =

8.7 Hz, 2H), 7.20 (d, J = 8.4 Hz, 1H), 7.15−7.11 (m, 1H), 7.00−6.97

(m, 1H), 6.72−6.68 (m, 1H), 6.20−6.18 (m, 1H), 5.45 (dd, J = 10.1,

3.0 Hz, 1H), 5.28 (d, J = 15.2 Hz, 1H), 5.22 (d, J = 15.2 Hz, 1H),

4.27−4.16 (m, 2H), 3.84 (s, 3H), 3.34 (dd, J = 14.2, 3.0 Hz, 1H),

2.76−2.66 (m, 2H), 2.61−2.27 (m, 8H), 2.46 (s, 3H), 2.45−2.41 (m,

1H), 2.18 (s, 3H), 1.80 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ

ppm: 166.4, 166.3, 163.3, 162.8, 162.1, 158.1, 153.7, 152.9, 150.9,

150.2, 136.5, 135.1, 134.9, 131.0, 130.9, 130.6, 128.1, 127.8, 123.6,

120.5, 118.9, 116.6, 116.0, 111.8, 110.7, 74.5, 68.8, 67.2, 56.7, 56.1,

54.2, 52.5, 45.1, 32.6, 23.1, 17.5. HRMS calculated for

C₄₇H₄₅ClFN₇O₆S: 889.2825; found 445.6481 (M + 2H).

calculated for C₄₇H₄₄ClFN₆O₆S: 874.2716; found 438.1415 (M +

2H).

N-[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)-

ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-2-

{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}-^D-phenylalanine

(9n). Step A: (2R)-2-[[5-Bromo-6-(4-ﬂuorophenyl)thieno[2,3-d]-

pyrimidin-4-yl]amino]-3-(2-hydroxyphenyl)propanoic Acid. 2.899

g R1c (8.44 mmol), 2.755 g (2R)-2-amino-3-(2-hydroxyphenyl)-

propanoic acid hydrochloride (12.66 mmol), and 3.500 g K₂CO₃

(25.32 mmol) were mixed in 84 mL DMSO and stirred at rt

overnight. The mixture was then diluted with brine, acidiﬁed with 1 M

aq HCl solution to pH 2. The formed precipitate was ﬁltered to give

2.839 g (2R)-2-[[5-bromo-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-

4-yl]amino]-3-(2-hydroxyphenyl)propanoic acid (5.80 mmol, 69%).

¹H NMR (500 MHz, DMSO-d₆) δ ppm: 11.20 (br s, 1H), 8.41 (s,

1H), 7.87 (d, J = 5.2 Hz, 1H), 7.73−7.66 (m, 2H), 7.45−7.34 (m,

2H), 7.10 (dd, J = 7.4, 1.6 Hz, 1H), 7.01 (td, J = 7.4, 1.6 Hz, 1H),

6.75 (dd, J = 8.0, 0.9 Hz, 1H), 6.68 (td, J = 7.4, 1.1 Hz, 1H), 4.72−

4.60 (m, 1H), 3.27 (dd, J = 13.8, 5.1 Hz, 1H), 3.06 (dd, J = 13.7, 6.5

Hz, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 173.4, 163.7,

161.2, 156.0, 155.6, 154.0, 131.9, 131.5, 128.1, 127.7, 124.5, 118.7,

116.0, 115.8, 114.0, 99.5, 55.6, 32.2. LRMS calculated for

C₂₁H₁₅BrFN₃O₃S: 487.0; found 488.0 (M + H).

Step B: Ethyl (2R)-2-[[(5S_a)-5-[3-Chloro-2-methyl-4-[2-(4-methyl-

piperazin-1-yl)ethoxy]phenyl]-6-(4-ﬂuorophenyl)thieno[2,3-d]-

pyrimidin-4-yl]amino]-3-(2-hydroxyphenyl)propanoate. 244 mg

(2R)-2-[[5-bromo-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

amino]-3-(2-hydroxyphenyl)propanoic acid (0.50 mmol) and 237 mg

R3b (0.60 mmol) were dissolved in 2.5 mL dioxane:water 2:1

mixture, then 489 mg Cs₂CO₃(1.50 mmol) and 35.4 mg AtaPhos

(0.05 mmol) were added and the mixture was stirred at 60 °C

overnight. Then the mixture was neutralized with 1 M aq HCl

solution and extracted with DCM. The combined organic layer was

dried over Na₂SO₄, ﬁltered, and the ﬁltrate was concentrated under

reduced pressure to yield a mixture of diastereoisomers, which were

separated via ﬂash chromatography using HILIC eluents. The

diastereoisomer eluting later was collected, then dissolved in 5 mL

1.25 M HCl solution in EtOH and stirred at 40 °C overnight. Then it

was carefully diluted with aq NaHCO₃solution and extracted with

DCM. The combined organic layer was dried over Na₂SO₄, ﬁltered,

and the ﬁltrate was concentrated under reduced pressure. The crude

product was puriﬁed via ﬂash chromatography using DCM and

MeOH as eluents to obtain 122 mg ethyl (2R)-2-[[(5S_a)-5-[3-chloro-

2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-

ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]amino]-3-(2-

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-[2-({2-[2-(hydroxymethyl)phenyl]pyrimidin-4-yl}methoxy)-

phenyl]propanoic Acid (9l). Using general procedure 5 and 53 mg

R7 (0.063 mmol) and 2-(hydroxymethyl)phenylboronic acid as the

appropriate boronic acid derivative, 24 mg 9l (0.027 mmol, 43%) was

1

obtained. H NMR (500 MHz, DMSO-d₆) δ ppm: 8.96 (d, J = 5.2

Hz, 1H), 8.54 (s, 1H), 7.94 (dd, J = 7.7, 1.2 Hz, 1H), 7.84 (d, J = 5.0

Hz, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.50 (td, J = 7.7, 1.2 Hz, 1H), 7.43

(d, J = 8.4 Hz, 1H), 7.38 (td, J = 6.6, 1.2 Hz, 1H), 7.29−7.23 (m,

2H), 7.22−7.15 (m, 3H), 7.11 (td, J = 7.7, 1.2 Hz, 1H), 6.99 (d, J =

8.4 Hz, 1H), 6.68 (t, J = 7.4 Hz, 1H), 6.16 (d, J = 7.4 Hz, 1H), 5.45

(dd, J = 10.5, 2.7 Hz, 1H), 5.31 (d, J = 15.2 Hz, 1H), 5.22 (d, J = 15.2

Hz, 1H), 4.81 (s, 2H), 4.28−4.21 (m, 1H), 4.21−4.14 (m, 1H),

3.44−3.35 (m, 1H), 2.78−2.65 (m, 2H), 2.61−2.24 (m, 9H), 2.19 (s,

3H), 1.78 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ ppm: 171.3,

166.2, 166.1, 165.0, 163.6, 163.0, 161.0, 158.0, 155.3, 153.6, 152.9,

142.0, 136.3, 135.9, 135.7, 130.9, 130.8, 129.8, 129.2, 128.7, 127.9,

127.8, 127.7, 126.6, 125.6, 122.0, 120.4, 118.9, 115.9, 115.6, 111.7,

110.6, 75.0, 68.8, 67.2, 61.7, 56.0, 54.0, 52.3, 44.9, 32.7, 17.5. HRMS

calculated for C₄₇H₄₄ClFN₆O₆S: 874.2716; found 875.2804 (M + H).

(2R)-2-{[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-1-

yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]-

oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)-

propanoic Acid (9m). Using general procedure 4 and 176 mg R5a

(0.25 mmol) as the appropriate phenol and R6d as the appropriate

alcohol, 91 mg 9m (0.105 mmol, 42%) was obtained. ¹H NMR (500

MHz, DMSO-d6) δ ppm: 8.88 (d, J = 5.2 Hz, 1H), 8.57 (s, 1H), 7.72

(d, J = 5.2 Hz, 1H), 7.52 (dd, J = 7.5, 1.8 Hz, 1H), 7.47−7.43 (m,

1H), 7.38 (d, J = 8.5 Hz, 1H), 7.29−7.25 (m, 2H), 7.22−7.17 (m,

3H), 7.15−7.10 (m, 2H), 7.03 (td, J = 7.4, 1.0 Hz, 1H), 7.98 (d, J =

8.5 Hz, 1H), 6.70 (t, J = 7.4 Hz, 1H), 6.19 (d, J = 7.4 Hz, 1H), 5.46

(dd, J = 10.0, 3.2 Hz, 1H), 5.25 (d, J = 15.0 Hz, 1H), 5.19 (d, J = 15.0

Hz, 1H), 4.26−4.16 (m, 2H), 3.75 (s, 3H), 3.33 (dd, J = 14.0, 3.0 Hz,

1H), 2.77−2.68 (m, 2H), 2.58−2.35 (m, 8H), 2.41 (dd, J = 14.0, 10.0

Hz, 1H), 2.20 (s, 3H), 1.80 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆)

δ ppm: 171.1, 166.3, 166.0, 164.7, 162.1, 157.8, 157.2, 155.3, 153.6,

152.9, 136.5, 135.8, 131.0, 130.8, 130.6, 129.1, 128.6, 128.4, 128.1,

127.7, 122.0, 120.4, 120.1, 118.9, 116.0, 115.6, 112.2, 111.8, 110.6,

74.3, 68.9, 67.2, 56.0, 55.7, 54.1, 52.3, 44.9, 32.5, 17.5. HRMS

1

hydroxyphenyl)propanoate (0.17 mmol, 34%). H NMR (500 MHz,

DMSO-d₆) δ ppm: 9.49 (s, 1H), 8.40 (s, 1H), 7.34 (d, J = 8.5 Hz,

1H), 7.26−7.20 (m, 3H), 7.20−7.15 (m, 2H), 7.00 (td, J = 7.8, 1.6

Hz, 1H), 6.71 (dd, J = 7.8, 0.9 Hz, 1H), 6.60 (td, J = 7.8, 0.9 Hz, 1H),

6.39 (dd, J = 7.8, 1.6 Hz, 1H), 5.03 (d, J = 7.8 Hz, 1H), 4.96−4.89

(m, 1H), 4.26 (t, J = 5.5 Hz, 2H), 4.09−3.96 (m, 2H), 3.03 (dd, J =

13.7, 5.5 Hz, 1H), 2.78 (t, J = 5.5 Hz, 2H), 2.60−2.47 (m, 4H), 2.36

(dd, J = 13.7, 9.4 Hz, 1H), 2.39−2.19 (m, 4H), 2.12 (s, 3H), 1.83 (s,

3H), 1.10 (t, J = 7.1 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ

ppm: 171.4, 164.3, 162.9, 160.9, 156.4, 155.4, 154.6, 153.4, 136.6,

134.4, 130.7, 130.2, 129.6, 129.0, 128.0, 126.4, 123.2, 122.0, 118.6,

115.9, 115.6, 114.9, 111.7, 67.4, 60.7, 56.2, 54.7, 53.0, 52.4, 45.7, 32.8,

17.4, 13.9. HRMS calculated for C₃₇H₃₉ClFN₅O₄S: 703.2395; found

704.2450 (M + H).

Step C: N-[(5S_a)-5-{3-Chloro-2-methyl-4-[2-(4-methylpiperazin-

1-yl)ethoxy]phenyl}-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-

yl]-2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}-^D-phenylala-

nine (9n). Using general procedure 4 and 63.4 mg ethyl (2R)-2-

[[(5S_a)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]-

phenyl]-6-(4-ﬂuorophenyl)thieno[2,3-d]pyrimidin-4-yl]amino]-3-(2-

hydroxyphenyl)propanoate (0.094 mmol) as the appropriate phenol

and R6d as the appropriate alcohol, 32.4 mg 9n (0.037 mmol, 39%)

1

was obtained. H NMR (500 MHz, DMSO-d₆) δ ppm: 8.85 (d, J =

5.2 Hz, 1H), 8.32 (s, 1H), 7.54 (dd, J = 7.5, 1.8 Hz, 1H), 7.49 (d, J =

5.2 Hz, 1H), 7.48−7.44 (m, 1H), 7.29−7.23 (m, 2H), 7.21−7.08 (m,

6H), 7.05 (td, J = 7.5, 0.9 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 6.91 (d, J

AD

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of S64315, a Potent and Selective Mcl-1 Inhibitor

DOI: 10.1021/acs.jmedchem.0c01234

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.0c01234

Full text of DOI:10.1021/acs.jmedchem.0c01234

Products guided by the article

R&D Labs maybe for 100305-93-9

Relevant to this article

Hot Product