Projected Dose Optimization of Amino- And Hydroxypyrrolidine Purine PI3KδImmunomodulators

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Projected Dose Optimization of Amino- and Hydroxypyrrolidine

Purine PI3K δ Immunomodulators

Joey L. Methot,* Hua Zhou, Meredeth A. McGowan, Neville John Anthony, Matthew Christopher,

Yudith Garcia, Abdelghani Achab, Kathryn Lipford, Benjamin Wesley Trotter, Michael D. Altman,

Xavier Fradera, Charles A. Lesburg, Chaomin Li, Stephen Alves, Craig P. Chappell, Renu Jain,

Ruban Mangado, Elaine Pinheiro, Sybill M. G. Williams, Peter Goldenblatt, Armetta Hill, Lynsey Sha ﬀ er,

Dapeng Chen, Vincent Tong, Robbie L. McLeod, Hyun-Hee Lee, Hongshi Yu, Sanjiv Shah,

and Jason D. Katz

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sı Supporting Information

ABSTRACT: The approvals of idelalisib and duvelisib have validated

PI3Kδ inhibitors for the treatment for hematological malignancies driven by

the PI3K/AKT pathway. Our program led to the identiﬁcation of

structurally distinct heterocycloalkyl purine inhibitors with excellent

isoform and kinome selectivity; however, they had high projected human

doses. Improved ligand contacts gave potency enhancements, while

replacement of metabolic liabilities led to extended half-lives in preclinical

species, aﬀording PI3Kδ inhibitors with low once-daily predicted human

doses. Treatment of C57BL/6-Foxp3-GDL reporter mice with 30 and 100

mg/kg/day of 3c (MSD-496486311) led to a 70% reduction in Foxp3-

expressing regulatory T cells as observed through bioluminescence imaging

with luciferin, consistent with the role of PI3K/AKT signaling in Treg cell proliferation. As a model for allergic rhinitis and asthma,

treatment of ovalbumin-challenged Brown Norway rats with 0.3 to 30 mg/kg/day of 3c gave a dose-dependent reduction in

pulmonary bronchoalveolar lavage inﬂammation eosinophil cell count.

INTRODUCTION

■

Phosphoinositide 3-kinase delta (PI3Kδ) is expressed primarily

in leukocytes where it regulates a broad range of cellular

activities such as cell survival, proliferation, diﬀerentiation, and

1

traﬃcking. The lipid kinase converts PIP2 (phosphatidylino-

sitol 4,5-bisphosphate) to PIP3 (phosphati-dylinositol 3,4,5-

trisphosphate), which in turn leads to the activation of the key

regulatory kinase AKT. The role of PI3Kδ in B-cell activation

has been investigated in hematologic malignancies driven by

the AKT pathway, culminating in the 2014 approval of the

Figure 1. Approved inhibitors of PI3Kδ.

selective PI3Kδ inhibitor idelalisib (Figure 1) for the treatment

of follicular B-cell non-Hodgkin lymphoma, chronic lympho-

cytic leukemia, and small lymphocytic lymphoma. This was

followed by the approval of the structurally related PI3Kδ

2

mice is consistent with involvement of PI3Kδ activity in

dysregulated inﬂammatory responses. Taken together with the

restricted expression and viability of PI3Kδ knock-out mice,

PI3Kδ inhibitors have been considered for the treatment of

inhibitor duvelisib in 2019 for the treatment of hematological

3

tumors. Interestingly, Ali and colleagues have reported that

inhibition of PI3Kδ in murine immunocompetent syngeneic

Received: February 6, 2021

Published: April 2, 2021

models protects against a broad spectrum of cancers, including

4

solid tumors with low expression of PI3Kδ. This unexpected

result may be due to an adaptive immune-mediated tumor

surveillance response. Evaluation of PI3Kδ inhibition in non-

oncology indications has been extensively studied as well.

Resistance to inﬂammatory disease in genetically modiﬁed

©

2021 American Chemical Society

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Figure 2. Potency, PK proﬁle, and crystal structure of 1 with PI3Kδ (3.05 Å). The purine makes hydrogen bond contacts to the kinase hinge

region, while the pyrrolidine amide gears the tetrahydropyran toward the Trp760 residue. The purine N(9)-methyl is directed toward a cavity

formed by gatekeeper Ile825 above and Tyr813 below. Unlike idelalisib and duvelisib, which induce a large hydrophobic pocket, the structure of

a

b

c

bound 1 is similar to the apo form of the kinase. PDB ID: 6MUM. LLC-PK1 cell line; 0.5 mg/kg iv, 1 mg/kg po; 0.25 mg/kg iv, 0.5 mg/kg po in

DMSO/PEG400/H O-20/60/20; n = 2.

2

inﬂammatory conditions such as severe asthma, allergic

both species the volume of distributions were very low (V_d_u=

5

6

rhinitis, COPD, or activated PI3Kδ syndrome. Preliminary

studies with idelalisib in patients with allergic rhinitis

demonstrated that PI3Kδ inhibition diminished total nasal

symptom scores, nasal airﬂow, nasal secretion weights, and

3.2 and 1.1 L/kg). While we did not detect signiﬁcant turnover

in microsome or hepatocyte incubations (Cl_i_n_tof <110 and

<40 mL/min/kg, respectively), a rat bile duct cannulation of 1

indicated tetrahydropyran oxidation, pyrazole N-dealkylation,

and cleavage of the ether bond. In addition, 17% of parent

compound 1 was recovered in urine out to 24 h post-dose,

with another 2% found in bile, suggesting a minor excretion

contribution to the observed clearance.

7

congestion. Consequently, numerous organizations have

reported PI3Kδ inhibitors to treat asthma and other

8

,9

inﬂammatory diseases, as well as liquid and solid tumors.

In a previous communication, we described SAR studies that

^B^a^s^e^d^oⁿ^m^aⁱⁿ^t^aⁱⁿⁱⁿ^g^a^Ctrough concentration of 335 nM, the

^C^D⁶⁹^w^h^o^l^e^b^l^o^o^d^I^C50 value, and the human PPB of only

led to potent and selective pyrrolidine PI3Kδ inhibitor 1 by

1

0

reconstruction of inhibitor XL-499. As an early lead

molecule, pyrrolidine 1 is structurally distinct from approved

inhibitors idelalisib and duvelisib and has promising potency

1

3

⁷^%^,^w^e^p^r^e^dⁱ^c^t^e^d^a^h^u^m^aⁿ^d^o^s^e^o^f³⁵⁰^m^g^bⁱ^d⁽^h^u^m^aⁿ^t1/2

=

h, peak/trough = 9) using the allometry method for

compound 1. Such a high human dose could present a safety

risk and prevent reaching higher levels of target engagement, if

needed. We sought to lower the predicted human dose by

improving both the potency and achieving a longer half-life in

preclinical species. The generally low intrinsic clearance and

unbound volume suggested that we could extend the half-life

by addressing the metabolic liabilities identiﬁed in the rat bile

duct cannulation study and by increasing the lipophilicity. We

also needed to maintain the excellent kinome selectivity as well

as selectivity over nonkinase oﬀ-targets such as adenosine

(

PI3Kδ IC₅₀= 3.9 nM; LBE 0.36) and good to excellent

selectivity versus PI3Kα (69x), PI3Kβ (1400x), and PI3Kγ

970x), as well as >1000× selectivity versus a 270-kinase panel.

(

Kinases PI3Kα and PI3Kβ are ubiquitously expressed with

roles in cell proliferation and metabolism, and genetic

knockouts are embryonically lethal. Hence, we sought high

selectivity versus PI3Kα and PI3Kβ, which have conserved

ATP-binding sites.

The phosphorylation status of serine 473 of AKT in the

Ramos Burkitt’s lymphoma-derived B cell line is driven by

PI3Kδ and is a measure of cellular activity of PI3Kδ inhibition

13

uptake activity that plagued earlier inhibitors in this series.

(

compound 1 AKT-pSer473 IC₅₀= 62 nM). Potency

LEAD OPTIMIZATION

evaluation in human whole blood was used for clinical

human dose prediction, in which we targeted the B-cell

surface biomarker CD69 IC at trough concentration. CD69 is

expressed in several hemopoietic cells as an early activation

marker in chronic lymphocytic leukemia and is correlated with

poor clinical prognosis. In human whole blood, compound 1

inhibited anti-human CD79b-induced expression of the B-cell

biomarker CD69 with an IC₅₀value of 335 nM.

The rat and dog PK proﬁles for compound 1 are

summarized in Figure 2. In rat, we observed a relatively

short half-life (0.8 h), low intrinsic clearance (126 mL/min/

kg), and good bioavailability (55%). In dogs, the half-life was

also short (1.5 h); however, the intrinsic clearance was low (10

mL/min/kg) and the bioavailability was excellent (100%). In

■

While the N(9)-methyl pyrazolopurine hinge-binding core

served as a consistent platform to identify novel selectivity

motifs leading to compound 1, we understood little of the

impact of purine C(8) and N(9) substitution on the potency,

selectivity, and physical properties of this series. Furthermore,

the pyrazole metabolism observed in the rat bile duct

cannulation experiment with 1 suggested an alternative to

the pyrazole that may lead to an improved half-life.

A portion of our SAR survey of this region of the molecule is

shown in Table 1. Using the (S)-aminopyrrolidine ethyl amide

selectivity motif, we ﬁrst deleted the N(9)-methyl group;

however, the corresponding inhibitor 2a bearing an N-H lost

5× in potency versus the N(9)-methyl analog 2b reference

50

11

12

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Table 1. Exploratory C(8)-Aryl and C(9)-Alkyl SAR with the (S)-Aminopyrrolidine Ethyl Amide Selectivity Motif

analog (PI3Kδ IC = 1.6 nM). We next studied the impact of

PI3Kα was lower than other isoforms; hence, PI3Kα was

chosen as a high-throughput indicator of selectivity.

50

larger substituents at N(9) given the space available in the

hydrophobic pocket lined by the gatekeepers Ile825 and

Tyr813. The positioning of the gatekeeper residue above the

plane of the hinge is unique to lipid kinases, creating a pocket

between it and Tyr813 below the plane that can be exploited

for potency and selectivity. Growing to an N-ethyl group (see

With the exceptional potency exhibited by inhibitor 2c, we

proﬁled the inhibitor in rats and dogs. In rats, the clearance

^w^a^s^m^o^d^e^r^a^t^e⁽^C^l^/^C^lint = 26/270 mL/min/kg) and the half-

life was short (V = 0.9 L/kg, t = 1.0 h; F = 52%). In dogs,

d

1/2

the clearance was again moderate (Cl/Cl_i_n_t= 12/37 mL/min/

kg) and the half-life was short (V = 1.3 L/kg, t = 1.2 h; F =

d

1/2

2

c) gave a nearly 10× boost in biochemical (PI3Kδ IC = 0.2

50

100%). Compound 2c, like 1, was stable to liver microsomes.

nM) and cellular potency relative to 2b, while an N-propyl

group (see 2d) lost 10× in potency. This remarkable 100×

span in potency from N-methyl to N-ethyl to N-propyl

revealed not only the potency achievable with improved

interaction with the gatekeeper Ile825 and Tyr813 but also the

limited space available for larger substituents in the pocket. It is

also noteworthy that the selectivity versus PI3Kα was

consistent through the methyl−ethyl−propyl progression,

which is not surprising given the high ATP site homology

among the class I PI3Ks. For this series, selectivity versus

Furthermore, compound 2c was a modest inhibitor of

adenosine uptake in HeLa cells (IC₅₀= 2280 nM), while O-

linked analog 1 was inactive in this assay (IC₅₀> 10,000 nM).

We continued to probe the SAR at C(9). Diﬂuoroethyl 2e

was equipotent to the parent N-methyl-substituted 2b;

although less ligand-eﬃcient, it may oﬀer other advantages

versus the N-methyl one. The larger triﬂuoromethyl group (2f)

was less potent. Importantly, ﬂuorinated analogs 2e and 2f had

rat PK proﬁles very similar to 2c, with comparable half-lives in

rat and again stable to rat liver microsomes. The cyclopropyl

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Table 2. Pyrrolidine Amide Survey with the 2-Methylpyrimidine Purine

a

b

c

d

LLC-PK1 cell line. 0.5 mg/kg iv, 1 mg/kg po. 0.25 mg/kg iv, 0.5 mg/kg po. 0.05 mg/kg in DMSO/PEG400/H O-20/60/20; n = 2.

2

analogs 2g and 2h were likely too large for the space available

and subsequently less potent than 2b. Based on the crystal

structures of 1 with PI3Kδ, we suspected a direct or water-

mediated contact between the pyrazole nitrogen and Tyr813.

Hence, we prepared pyrrole 2i and indeed observed a 16× loss

in potency versus pyrazole 2b, as expected. This one log unit

potency shift is consistent with the loss of a hydrogen bond

contact.

Given the N-dealkylation of the N-ethyl pyrazole ring of

compound 1 observed in vivo, we explored alternative

heterocycles that did not have an N-alkyl substituent. Potent

thiazole 2j (PI3Kδ IC₅₀= 0.7 nM) bearing a tert-butyl

substituent demonstrated not only the space available in the

aﬃnity pocket but also the potency gain achievable with a

hydrophobic interaction. Not unexpectedly, thiazole 2j had

high clearance in rats (Cl > Q_h_e_p). We turned to six-membered

heterocycles commonly found in the literature as potency-

enhancing motifs in the aﬃnity pocket. 2-Methylpyrimidine 2k

was found to be 3× less potent than pyrazole 2b; however,

selectivity versus PI3Kα improved from 60× to 375× with 2k.

With this improved selectivity, we explored the N(9)

substituent, ﬁnding that N(9)-ethyl 2l recovered the potency

loss with this pyrimidine (PI3Kδ IC₅₀= 1.9 nM) and

maintained promising selectivity versus PI3Kα (95×). As

expected from our observations with 2b−2d, N(9)-propyl-

substituted 2m was signiﬁcantly less potent. Importantly,

pyrimidine 2l had superior rat and dog PK proﬁles versus the

pyrazole analog 2c, with intrinsic clearances 3× lower in both

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Table 3. Pyrrolidine Amide Survey with Triﬂuoromethyl Pyridine Purine

a

b

c

d

LLC-PK1. 0.5 mg/kg iv, 1 mg/kg po. 0.25 mg/kg iv, 0.5 mg/kg po. 0.05 mg/kg in DMSO/PEG400/H O-20/60/20; n = 2.

2

rats and dogs (Cl_i_n_t= 100, 9.0 mL/min/kg for 2l), and an

improved half-life (1.5−2.7 h for 2l; note that 2l is numbered

potent in the Ramos pAKT cell assay. Unfortunately, both 2q

and 2r had high clearance in rats (Cl 69−71 mL/min/kg) and

a short half-life (t = 0.1 h), possibly due to metabolism of

3

a in Table 2 where PK parameters are displayed). The low

1

/2

unbound volumes in rats and dogs for 2l (V_d_u= 3.6, 1.9 L/kg)

were comparable to the pyrazole series, suggesting that the

improvements in the half-life were derived from lower intrinsic

clearances.

To provide additional options for lead optimization, we

identiﬁed 2-methylpyridine 2n and 2-triﬂuoromethylpyridine

the 2-methoxypyridine ring. Furthermore, analogs bearing the

2-methoxypyridine motif was also more active in the HeLa cell

adenosine transport assay (e.g., HeLa AdU IC₅₀= 0.3 μM for

2r).

Based on the metabolism and N-dealkylation of the ethyl

pyrazole ring observed in rat bile duct cannulation with

inhibitor 1 and improvements in intrinsic clearance and the

half-life observed when comparing 2c with 2l, we elected to

continue lead optimization eﬀorts with the 2-methylpyrimidine

at C(8) of the purine instead of the pyrazole group.

Furthermore, the boost in potency observed with the N(9)-

ethyl group directed our eﬀorts to this substituent versus the

N(9)-methyl group.

The metabolism observed on the tetrahydropyran ring of

compound 1 in a rat bile-duct cannulation study led us to

explore alternative amides to improve the half-life in preclinical

species. A small portion of the pyrrolidine amide cap SAR

surveyed is outlined in Table 2, with both the NH linker as

well as the O linker. Compound 2l is listed ﬁrst as a reference;

compound 2l is also 3a. For reasons still unknown, the NH-

linked pyrrolidine amides were frequently 4× more potent than

the corresponding O-linked pyrrolidine amides. For example,

O-linked ethyl amide 3b is 6× less potent than the

corresponding NH-linked analog, whereas with larger amides,

this diﬀerence tends to be smaller. However, the O-linked

analogs often oﬀered improved selectivity versus PI3Kα kinase

2o at C(8), which can impact the physical properties of the

series with one fewer aromatic nitrogen atom. A 2-

methylpyridine ring has a cLogP of about 0.4 units above

that of a 2-methylpyrimidine ring, while a 2-triﬂuoromethyl-

pyridine ring has a cLogP of 1.0 unit above that of a 2-

methylpyrimidine ring. This eﬀect was readily apparent in

HPLC-determined Log D values for pyrimidine 2k (0.4),

pyridines 2n (0.8), and 2o (1.6), demonstrating an

opportunity to tune polarity and introduce nonmetabolizable

lipophilicity as needed. For comparison, the 5-methyl-1-

ethylpyrazole analog 2b had a Log D value of 0.8. The

added lipophilicity did render 2o a more potent inhibitor of

adenosine transport in HeLa cells (AdU IC = 0.7 μM for 2o).

50

The 2-methoxypyridine moiety is frequently used to gain

potency in the aﬃnity pocket of kinases, often through

interactions with the Lys−Asp salt bridge. In our series, 2-

methoxypyridine 2p was no more potent than 2-methylpyr-

idine 2n. 3-Fluoro-2-methyoxypyridine 2q and 3-methyl-2-

methoxypyridine 2r were slightly more potent than 2-

methylpyridine 2n in the enzyme assay but 8−25× more

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compound 3b selectivity is 220×; twice the selectivity of the

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(

Table 4. Preliminary Human Dose Predictions Based on

Maintaining B-Cell Activation Biomarker CD69 IC₅₀

NH-linked 3a) and reduced adenosine uptake activity in HeLa

cells. Furthermore, O-linked analogs occasionally had better

half-lives in preclinical species. Hence, most pyrrolidine

capping groups were explored with both the NH-linked and

O-linked pyrrolidine series. Generally, amides bearing linear or

branched alkyl chains (e.g., 3a/3b) gave higher rat plasma

clearances than small cycloalkyl amides. We identiﬁed

numerous amide, urea, carbamate, and sulfonamide-linked

capping groups on the pyrrolidine that maintained good to

excellent enzymatic potency, with small aliphatic or hetero-

cyclic groups being generally preferred over polar residues such

as charged amines. The PI3Kδ potency SAR can be reviewed

b

human dose prediction

unb’d

human

PPB

target

a

target

compound C_t_r_o_u_g_h(nM)

C

t

P/

T

trough

(nM)

1/2

c

(%)

dose (mg) (h)

1

3

335

65

17

28

280

47

430

350, bid

35, qd

260, qd

3

9

4

c

590 (CD69

IC90

target)

(

76, bid)

3

3g

d

270

26

38

31

41

36

62

64

52

79

100

8

31

3

45

44

48

68

285, qd

40, qd

13, qd

180, qd

90, qd

130, qd

8, qd

5

14

5

10

5

6

4

13

4

27

10

43

4

in the patent literature. These diverse options for potency

gave us an opportunity to broadly evaluate PK in rats and dogs

to optimize for half-life in preclinical species. As noted earlier,

in vitro models for metabolism (microsome, hepatocyte) were

not predictive of the observed PK in rats or dogs, hence the

need for in vivo screening. Generally, promising compounds

with a rat half-life of 1.5 h or longer were further proﬁled. The

volume of distribution was consistently low for this series (e.g.,

3h

3k

4c

4d

4e

4f

75

9

73

68

100

380

10

20, qd

4

a

^H^u^m^aⁿ^w^h^o^l^e^b^l^o^o^d^C^D⁶⁹^I^C50 value unless otherwise noted.

b

Allometry approach using Tang’s method, based on rat and dog PK

^Vdu ⁼³^.⁶^,¹^.⁹^f^o^r²^l^/³^a⁾^,ⁿ^e^c^e^s^sⁱ^t^a^tⁱⁿ^g^a^l^o^wⁱⁿ^t^rⁱⁿ^sⁱ^c^c^l^e^a^r^aⁿ^c^e

to achieve an acceptable half-life of >1.5 h in rats.

15

c

data. Ratio of predicted C_m_a_x/C_t_r_o_u_g_h

.

Early in our amide screening campaign, we identiﬁed

cyclopropyl amides 3c and 3d. The NH-linked 3c was 4×

more potent than O-linked 3d; furthermore, 3c oﬀered better

PK proﬁles. The pharmacokinetic properties of compound 3c

in rats and dogs were characterized by low plasma clearance

suﬀered from increased intrinsic clearances in both rats and

dogs and consequently lower half-lives as well in both species,

presumably due to increased amide metabolism since the

unbound volumes were only slightly higher than 3c. As

expected, the HPLC Log D values increased by 0.3−0.4 units

in expanding from the cyclopropyl amide to a cyclobutyl

amide. To counter the eﬀect of increased lipophilicity, we

turned to substituents about the cyclobutane that may

rebalance polarity with a lower Log D. For example, a cis-3-

methoxy substituent brings the Log D closer to the original

cyclopropyl amide. cis-3-Methoxycyclobutyl amides 3g and 3h

were slightly less potent than cyclopropyl amides 3c and 3d;

however, the low intrinsic clearances and excellent half-lives in

rats and dogs were maintained (t₁_/₂= 1.8−2.3 h in rats and

3.6−4.3 h in dogs). Once again, the improved half-lives were

achieved by decreasing intrinsic clearance and not increasing

unbound volume. Taken together, with the excellent human

whole blood potency for these compounds (CD69 IC₅₀= 26

and 75 nM for 3g and 3h), human dose predictions based on

the allometry approach were 40 mg qd with a P/T of 13 for 3g,

slightly inferior to 3c, and 13 mg qd with a P/T of 4 for 3h,

slightly improved versus 3c (Table 4). The strategy to

rebalance the increased lipophilicity of a cyclobutane versus

a cyclopropane with the polarity of an ether was successful in

improving half-lives in rats and dogs. We also attempted to

deactivate the cyclobutyl amide toward oxidative metabolism

as well as increase the unbound volume of distribution with

ﬂuorine substitution, such as 3,3-diﬂuorocyclobutyl amides 3i

and 3j. The HPLC Log D values increased from 1.3 to 1.5;

however, any gains in the unbound volume of distribution were

nulled by increased intrinsic clearance, giving no improvement

in rat or dog half-lives.

(

6

^C^l^o^f⁸^aⁿ^d⁴^m^L^/^mⁱⁿ^/^k^g^,^r^e^s^p^e^c^tⁱ^v^e^l^y^;^wⁱ^t^h^C^lint of 64 and

.2 mL/min/kg), good terminal half-life (2.7 and 4.3 h,

respectively), and good bioavailability (55 and 95%,

respectively). The unbound volume of distribution for 3c

(

V_d_u= 6.3 and 1.8 L/kg in rats and dogs) is three-fold higher in

rats than for ethyl amide 3a but is unchanged in dogs versus

a, suggesting that the improved half-life in rats was at least

3

partially driven by a larger unbound volume of distribution.

Based on the allometry scaling method, the human

pharmacokinetic properties of compound 3c was predicted

to be Cl of 3.8 mL/min/kg, V of 3.1 L/kg (V of 4.4 L/kg),

d

du

t₁_/₂of 9.0 h, and a bioavailability of 72%. To achieve a

minimum blood concentration of 65 nM (the human whole

blood CD69 IC ), a dose of 35 mg once-daily will be required

5

0

(

5

see Table 4). To achieve a minimum blood concentration of

90 nM (the human whole blood CD69 IC ), a dose of 260

90

mg once-daily or 76 mg twice-daily will be required. Given the

high solubility, a dose number of <1 is expected.

Following the identiﬁcation of cyclopropyl amide 3c, we

continued our SAR campaign to identify further optimized

inhibitors, with longer predicted human half-lives. We placed

simple methyl and ﬂuoro substituents at various locations

about the pyrrolidine and cyclopropane in an attempt to

increase the unbound volume of distribution, identifying

numerous equipotent analogs. However, the PK proﬁles of

such close analogs were generally inferior to 3c , with higher

intrinsic clearances (see Supporting Information). We also

attempted to increase the volume of distribution via the

incorporation of basic amine substituents; however, half-lives

were not improved. Finally, we sought to increase structural

diversity in the series. For example, ring expansion of the

cyclopropane to cyclobutyl amides 3e/3f gave a 5× boost in

potency versus the cyclopropyl analogs, presumably through

improved van der Waals contacts with lipophilic residues Trp

Encouraged by the promising human dose predictions for 3-

methoxycyclobutyl amides 3g and 3h, we explored urea

analogs 3k and 3l. Ureas were generally as potent as amides in

biochemical, cell, and whole blood assays, and ureas 3k and 3l

were no exception. However, the PK proﬁles had eroded

signiﬁcantly for ureas relative to the optimized amides above,

with the best being urea 3k (t₁_/₂= 4, 1.7 h; bioavailability of 16

7

60 and Met752. Unfortunately, the cyclobutyl amides also

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a

b

Figure 3. Potency, PK proﬁle, and crystal structure of 3c (MSD-496486311) with PI3Kδ (2.8 Å). PDB ID: 7LM2. LLC-PK1 cell line; MDCKII

c

d

e

cell line; 0.5 mg/kg iv, 1 mg/kg po; 0.25 mg/kg iv, 0.5 mg/kg po in saline (iv) or 0.5% methyl cellulose (po); 1 mg/kg iv, 3 mg/kg po in DMSO/

PEG400/H O-20/60/20; n = 3.

2

and 69% in rats and dogs). Consequently, human dose

predictions for optimized ureas were inferior (180 mg qd with

a P/T of 27 for optimized urea 3k).

With the success of methyl ether-substituted cyclobutyl

amides 3g and 3h, we explored the larger cyclic ethers 3m, 3n,

methylpyrimidine series summarized in Table 2 but was

signiﬁcantly less polar with the cLogP and HPLC Log D values

about 1 unit greater in head-to-head comparisons. At the

outset, we hoped that this physical property space would

render inhibitors with a higher unbound volume of distribution

and consequently more optimizable to longer rat and dog half-

lives. In fact, the triﬂuoromethyl pyridines bearing the

cyclopropyl amide, 4a and 4b, did have larger unbound

volumes (e.g., V_d_u= 8.8, 3.9 L/kg for 4a); however, they also

had higher plasma clearances, and so half-lives of only 0.7−1.2

h were achieved. Furthermore, NH-linked analogs prepared in

this series were potent inhibitors of adenosine uptake in HeLa

cells. For example, compound 4a inhibited adenosine uptake in

HeLa cells with an IC₅₀of 1.2 μM. This was consistent with

our previous observation that increased lipophilicity often led

to adenosine uptake activity, especially for NH-linked

pyrrolidines. For this reason, we continued optimization in

this subseries with O-linked pyrrolidines only.

To rebalance the polarity and lower lipophilicity, we again

turned to ether-containing amides. cis-3-Methoxycyclobutyl

amide 4c did have signiﬁcantly improved PK, with promising

half-lives (3.3, 2.6 h) that were consistent with the higher

unbound volumes (13, 2.5 L/kg) as well as good bioavailability

(39, 100%) in rats and dogs. 3-Methoxyazetidine urea 4d had

lower unbound volume of distribution in both rats and dogs

and shorter half-lives in both species (t₁_/₂= 2.0 and 0.7 h in

rats and dogs), as observed for the 2-methylpyrimidine series

in Table 2. Hence, while the human dose prediction for 4c was

promising (90 mg qd with P/T = 10), the prediction for the

urea 4d was not (130 mg qd with P/T = 43) owing to a shorter

predicted half-life in human for 4d. It was the tetrahydrofuran

and oxazole amides 4e and 4f that oﬀered the best PK in the

triﬂuoromethyl pyridine series. They were polar amides within

3

o, and 3p. For tetrahydrofuran amides 3m and 3n, the

potency was maintained versus 3g and 3h, and plasma intrinsic

clearances were low; however, the half-lives were suboptimal

(t₁_/₂= 1.4 and 3.8 in rats and dogs for 3n) due to lower

unbound volumes in both species than the methoxycyclobutyl

amides. This lower V_d_uis consistent with lower HPLC Log D

values (0.3 units lower than 3g and 3h). To close the loop with

starting point 1, we prepared the tetrahydropyran amides as

well; however, we noted inferior potency and short half-lives.

This was not a surprise given the results of the bile duct

cannulation with 1, indicating metabolism of the tetrahy-

dropyran ring. In addition to alkyl and cycloalkyl amides, we

also explored a variety of aromatic amides. Generally few

aromatic amides could match the potency achieved with small

cycloalkyl amides, with one somewhat successful example

being 4-oxazole amides 3q and 3r. Biochemical potency was

good for both, and half-lives for 3r in particular were

reasonable (t₁_/₂= 1.7 and 1.8 h for rats and dogs); however,

dose predictions were not competitive since the human whole

blood potency was inferior.

As part of the optimization process to identify additional

inhibitors to mitigate any potential issues that we may

encounter later in development, we cross-checked our best

amide and urea pyrrolidine capping groups with several purine

C(8) aromatic groups identiﬁed earlier in the campaign.

Shown in Table 3 is the SAR generated for one such series, the

6

-triﬂuoromethylpyridine substituent at purine C(8). As a

whole, the triﬂuoromethyl pyridine series was as potent as the

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Figure 4. Reduction of regulatory T (Treg) cells in Foxp3-GDL reporter mice following treatment with 3c at 30 and 100 mg/kg. Diphtheria toxin

2

(

DT) was used as a control. Radiance is expressed as photons/s/cm /steradian.

the 2-methylpyrimidine series, having lower HPLC Log D

values and unbound volumes; however, they were optimal

when combined with the 2-triﬂuoromethylpyridine moiety.

Plasma intrinsic clearances were low for 4e and 4f, and this

resulted in excellent half-lives for both 4e (t₁_/₂= 2.3 and 3.6 h

in rats and dogs) and 4f (t₁_/₂= 6.0 and 2.5 h in rats and dogs)

with excellent bioavailability. The unbound volumes for 4e and

Compound 3c was not an inhibitor of potassium hERG,

sodium Nav1.5, or calcium Cav1.2 ion channels (IC₅₀> 50

μM). It was not an inhibitor of major CYP enzymes, CYP-1A2,

2B6, 2C8, 2C9, 2C19, 2D6, and 3A4 (IC > 50 μM); hence, it

5

0

is unlikely to be a perpetrator of CYP-mediated DDI.

Furthermore, no signiﬁcant induction response was observed

for CYP-3A4, 2B6, and 1A2, suggesting low potential for DDI.

Lastly, 3c was not a time-dependent inhibitor of CYP-3A4.

Compound 3c was stable when incubated in hepatocytes

across all species (Cl_i_n_t< 2 mL/min/kg, rats, dogs, monkeys,

humans). Renal clearance was expected to be the major route

of elimination in humans given the stability to hepatocytes as

well as the observed routes of elimination in rats and dogs. In

bile duct cannulated (BDC) studies in rats and dogs dosed

4

f were comparable to optimized analogs in the 2-

methylpyrimidine series, indicating that we had optimized to

a similar overall physical property space and were again unable

to achieve longer half-lives using an unbound volume strategy.

These PK proﬁles supported low predicted human doses of 8

and 20 mg qd for 4e and 4f, respectively, with a low P/T = 4

for both.

3

With the promising preliminary potency and PK proﬁles of

with [2- H-purine]-3c, the majority of the radioactivity was

3

c, we completed characterization of this inhibitor, shown in

excreted in urine as the unchanged parent compound. More

speciﬁcally, 75% of the recovered radioactivity in excreta

following iv dose in rats was the unchanged parent with renal

excretion as the major clearance mechanism (Cl_r_e_n_a_l2× GFR;

indicating active secretion). Following a po dose in dogs, 65%

of the recovered radioactivity was the unchanged parent as

renal and biliary excretion (Cl_r_e_n_a_l= GFR; indicating passive

ﬁltration). The balance of radioactivity in rat and dog BDC

studies was eliminated in bile and urine as oxidative

metabolites and numerous pyrrolidine products. Consistent

with these observations, the compound had good permeability

Figure 3. A crystal structure of 3c bound to PI3Kδ conﬁrmed a

binding mode very similar to what was observed for 1; the

purine makes hydrogen bond contacts to the kinase hinge

region while the pyrrolidine amide gears the cyclopropane

toward Trp760 residue. Lead compound 3c has excellent

potency in the Ramos Burkitt’s lymphoma-derived B cell line

(

AKT pSer473 IC 20 nM) and in human whole blood (B-cell

50

activation biomarker CD69 IC₅₀= 65 nM, basophil activation

biomarker CD63 IC₅₀= 50 nM). The enzyme potency was

maintained from humans to mice, rats, and dogs, as expected.

Lead 3c was then evaluated in numerous in vitro safety panels.

In a 280-kinase panel at Invitogen (Thermo Fisher Scientiﬁc),

no kinase activity was found within 100× of the PI3Kδ IC₅₀

value of 1.6 nM. The closest identiﬁed oﬀ-targets were PI3Kα

−

6

in the LLC-PK1 assay (Papp of 22 × 10 cm/s), with

transport observed in the MDCKII transport assay expressing

MDR1 PGP (BA/AB 16); however, 3c was not an inhibitor of

PGP (IC > 300 μM). In addition, 3c was not an inhibitor of

5

0

(

150× selectivity), PI3Kβ (1370×), and PI3Kγ (3690×). No

OATP1B1.

activity was identiﬁed in the Euroﬁns Panlabs safety

pharmacology panel of 116 receptors and enzymes at 10 μM.

Compound 3c was isolated as a stable non-hygroscopic

crystalline monohydrate with good solubility in simulated

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intestinal ﬂuid (14 mg/mL) and neutral water (15 mg/mL). It

upregulation of basophil degranulation CD63 surface bio-

marker following anti-IgE activation. The CD63 biomarker is

more relevant for the treatment of respiratory diseases

predominately mediated by type 2 T helper (TH2) cells. For

compound 3c, the human whole blood CD63 inhibition

potency was IC₅₀= 50 nM (unbound uIC₅₀= 36 nM),

consistent with the inhibition of CD69 surface biomarker

upregulation by anti-CD76b. We used the Brown Norway rat

model for allergic rhinitis and asthma, and results with 3c was

has a pK of 3.06 with protonation on the purine ring,

a

moderate MW (392.47 g/mol), HPLC Log D (0.8), and PSA

(

105 Å). The solubility in the preclinical formulation of 0.5%

methocel was 150 mg/mL.

IN VIVO PHARMACOLOGY

■

Lead compound 3c (MSD-496486311) was evaluated in

rodent models to corroborate potency in B-cell and basophil

activation whole blood assays. Two models described here are

the Foxp3-GFP mouse model to monitor the population and

localization of regulatory T cells and the Brown Norway rat

ovalbumin model for allergic rhinitis and asthma PK/PD

readouts.

2

0

published in greater detail elsewhere (where 3c was

identiﬁed as MSD-496486311). Rats were presensitized for

1

4 days by daily treatment with ovalbumin (0.020 mg ip).

These rats were then administered with 3c (0.3−30 mg/kg po)

for 2 days, and then after 1 h following the second dose, they

were treated with ovalbumin. After 48 h, the BAL (pulmonary

bronchoalveolar lavage) inﬂammation eosinophil cell count

was assessed. The data is shown in Figure 5 . The 10 mg/kg

Tumor-induced immunosuppression constitutes a signiﬁcant

obstacle for eﬀective cancer immunotherapy. Regulatory T

(Treg) cells play key roles in the maintenance of immune

homeostasis, and expression of the transcription factor

15

forkhead box P3 (Foxp3) is a widely used Treg marker.

Cancer patients have elevated numbers of Treg cells within the

blood and lymphoid tissues as well as the tumor micro-

environment. For some patients, increased Treg populations

correlate with a poor prognosis, and Tregs have been proposed

to aid tumors in evading immune surveillance. In vitro studies

indicate that PI3K/AKT signaling is required for Tregs to

15

mature and suppress anticancer immune responses, and

inhibitors of PI3Kδ have been shown to impair the

immunosuppressive function of Tregs in mice while leaving

16−18

cytotoxic T cell responses intact.

To conﬁrm the impact

of PI3Kδ inhibition on regulatory T cell proliferation and

localization in mice with our inhibitor, we used the C57BL/6-

Foxp3 reporter mouse model available from Jackson

Laboratory. These Foxp3-GFP mice co-express GFP (green

ﬂuorescent protein) and the regulatory T cell-speciﬁc tran-

scription factor Foxp3, such that GFP expression accurately

identiﬁes the Foxp3+ T cell population.

Figure 5. Attenuation of allergic-mediated pulmonary inﬂammation in

ovalbumin-sensitized and challenged Brown Norway rat by treatment

with 3c.

1

9

^d^o^s^e^wⁱ^t^h^Ctrough = 14 nM (unbound = 8 nM given Brown

Norway rat PPB 41%) approximates a 50% reduction in the

^B^A^L^c^o^uⁿ^t^,^w^hⁱ^l^e^a³⁰^m^g^/^k^g^d^o^s^e^wⁱ^t^h^Ctrough = 103 nM

(unbound 62 nM) elicits a full response by BAL count. This

potency is consistent with what we observed in the human

CD63 whole blood assay.

Foxp3-GFP mice were treated for up to 7 days with PI3Kδ

inhibitor 3c at 30 and 100 mg/kg/day. Bioluminescence

imaging was performed 10 min following ip injection of 3 mg

of luciferin using an IVIS Spectrum imaging system, and

2

radiance was quantiﬁed in units of photons/s/cm /steradian.

Diphtheria Toxin (DT) was used as a positive control, as DT is

cytotoxic and completely ablates Treg. Consistent with the

literature, a dose-dependent decrease of Treg population was

observed in mice upon treatment with 3c (Figure 4).

Interestingly, a plateau of 70% Treg reduction was observed

with 3c, unlike DT, which also induces spontaneous

autoimmunity. Hence, PI3Kδ inhibition might be a safer

modulator of Treg cells. The exposure for 3c in mice when

dosed at 100 mpk was AUC = 133 μM h, C_m_a_x= 33 μM, and

C_t_r_o_u_g_h= 110 nM. This equated to an unbound uC_m_a_x= 16 μM

and an unbound uC_t_r_o_u_g_h= 52 nM. The unbound uC_t_r_o_u_g_hlevel

in mice required to achieve 70% Treg reduction is comparable

to the human whole blood CD69 unbound uIC₅₀value of 47

nM. Note that these studies were performed in accordance to

the guidelines of the Institute for Laboratory Animal Research

SYNTHESIS

■

The 9-alkyl-8-aryl-6-chloropurine precursors used to access

new inhibitors were prepared via the routes outlined in Scheme

1: either through condensation of arylaldehydes with a

diaminopyrimidine or through a Suzuki coupling with 9-

alkyl-8-iodopurines. For example, N-ethyl-1H-5-methyl-pyra-

zole-4-carboxaldehyde I was condensed under oxidative

conditions with FeCl and air to 6-chloro-N4-methylpyrimi-

3

dine-4,5-diamine II, giving 6-chloro-8-(1-ethyl-5-methyl-1H-

pyrazol-4-yl)-9-methyl-9H-purine (III). Alternatively, 6-chlor-

opurine was selectively alkylated at N(9) with K CO and

ethyl iodide and then iodinated at C(8) with LDA and I₂

giving VII. Suzuki coupling with 2-methylpyrimidine-5-boronic

ester gave 6-chloro-9-ethyl-8-(2-methylpyrimidin-5-yl)-9H-pu-

rine (X).

2

3

(

LAR). All studies were part of an institutional animal care and

use committee (IACUC)-approved protocol and animals were

housed in an AAALAC international accredited research

facility.

In addition to monitoring the impact of PI3Kδ inhibition on

the B-cell surface biomarker CD69 in whole blood, we

monitored PI3Kδ inhibitor-mediated inhibition of the

Hydroxypyrrolidine inhibitors containing ether linkers were

prepared by treatment of a chloropurine precursor from

Scheme 1 with an alcohol and NaH. Aminopyrrolidines were

prepared from the corresponding primary amine with iPr NEt

2

or K CO at 80 °C (Scheme 2). In some cases, a selectivity

2

3

motif was introduced bearing a protective group (e.g., an N-

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a

Scheme 1. Synthesis of Chloropurine Precursors

the metabolic stability and half-life in preclinical species. The

gearing eﬀect of the (S)-pyrrolidine amide toward Trp is

critical for kinome and isoform selectivity, with small amides

adequate and preferred for excellent selectivity. Optimization

gave N-linked compound 3c representing a structurally distinct

PI3Kδ inhibitor with excellent potency in whole blood and

selectivity over the PI3K family. Taken together with good

half-lives in preclinical species and solubility, inhibitor 3c is

predicted to have a low once-daily predicted human dose of 35

mg qd to maintain CD69 IC₅₀coverage, or 76 mg bid, to

maintain an IC coverage, with a dose number of <1. With the

9

0

low volume of distribution generally observed in the purine

pyrrolidine series, we resorted to driving intrinsic clearance as

low and potency as high as possible to achieve acceptable

predicted human doses. Concurrent with the characterization

of 3c, we identiﬁed several additional inhibitors with low

predicted human dose, such as N- and O-linked 3-

methoxycyclobuyl amides 3g and 3h. Reoptimization with

the triﬂuoromethylpyridine purine substituent led to O-linked

4

e and 4f bearing tetrahydrofuran and oxazole amides to

introduce greater structural diversity in our portfolio of

pyrrolidine PI3Kδ inhibitors. While lowering intrinsic clear-

ance was the more successful strategy, small boosts in unbound

volume also contributed to increasing the half-life and lowering

the projected human doses. Compound 3c (MSD-496486311)

oﬀers a distinct structure from currently approved PI3Kδ

inhibitors for the treatment of hematologic malignancies driven

by the AKT pathway.

a

Reagents and conditions: (i) FeCl ·6H O, air, DMF, 85 °C, 75−

3

2

8

8%; (ii) EtI, K CO , DMSO, 53%; (iii) LDA, I , THF, −78 °C, 62%;

2

3

2

(

iv) Pd(dppf)Cl ·DCM, K CO , dioxane, water, 90 °C, 56%.

2 2 3

Scheme 2. General assembly of amino- and

hydroxypyrrolidine purines from chloropurine

a

intermediates

EXPERIMENTAL SECTION

■

General Synthetic Chemistry Methods. Reactions were

performed in dried round-bottom ﬂasks or capped vials with stirring

under an inert atmosphere of nitrogen unless stated otherwise.

Solvents in septum-sealed bottles and reagents were obtained from

commercial suppliers and used as received. All temperatures are in

degrees Celsius (°C), and ambient temperature is 20 °C. Microwave

reactions were performed with a Biotage Initiator Series microwave.

Most compounds were puriﬁed by reverse-phase preparative HPLC or

MPLC on silica gel. The course of the reactions was followed by LC/

MS (30 mm × 2 mm, 2 μm column; 3 to 98% MeCN/water with

0

.05% TFA gradient over 2.3 min; 0.9 mL/min ﬂow rate; ESI; UV

detection at 254 nm). Products were analyzed by NMR, LC/MS, and

HRMS. NMR spectra were recorded on either a 400, 500, or 600

MHz Varian spectrometer, chemical shifts are reported in parts per

million (ppm) relative to tetramethylsilane and referenced to residual

solvent. Coupling constants are reported in Hz. HRMS was obtained

with a Waters Acquity UPLC coupled to a Waters Xevo G2 QTof

using ESI+ detection. The purity of all compounds screened in the

biological assays was examined by LC/MS analysis (100 mm × 3 mm

C18 column; 3 to 98% MeCN/water with 0.05% TFA gradient over 5

min run; UV detection at 215 nm) and was found to be ≥95%.

a

Reagents and conditions: (i) NaH, THF, 0 °C to RT; (ii) iPr NEt or

2

K CO , DMF or t-BuOH, 80 °C; (iii) TFA, DCM or HCl, dioxane;

2

3

(iv) RCOCl, iPr NEt, DMF or RCO H, HATU, DMF.

2

Boc group), then after the nucleophilic displacement step, the

protective group was removed using standard conditions (e.g.,

TFA or HCl) and the pyrrolidine was capped (e.g., amide

formation with acid chloride or acid and HATU). Speciﬁc

synthetic details are available in the Experimental Section for

all compounds.

6

-Chloro-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-pu-

rine (III). A 1000 mL three-neck ﬂask ﬁtted with a thermometer was

charged with N-ethyl-1H-5-methyl-pyrazole-4-carboxaldehyde (I, 68.5

g, 496 mmol), anhydrous N,N-dimethylformamide (400 mL), and 6-

chloro-N-methylpyrimidine-4,5-diamine (II, 71.5 g, 451 mmol). The

reaction mixture was stirred at RT for 10 min to ensure dissolution of

all solid materials. Iron(III) chloride hexahydrate (122 g, 451 mmol)

was then added over the course of 5 min, resulting in the formation of

a dark brown solution. A stream of air was continuously bubbled

through the stirred reaction mixture, which was heated to 85 °C and

stirred for 14 h. The mixture was poured into 2500 mL of crushed ice

and water, and the resulting pale orange precipitate was collected by

vacuum ﬁltration and dried on the ﬁlter. The solid was triturated with

ethanol (500 mL) at 50 °C for 2 h and at RT overnight. The residue

was collected by vacuum ﬁltration and dried on the ﬁlter to give 93.5 g

CONCLUSIONS

■

In summary, beginning with pyrrolidine 1, we learned that

growing from an N-Me-purine core to an N-Et-purine core

gave an 8× boost in potency by ﬁlling the hydrophobic cavity

below gatekeeper residue Ile825 and above Tyr813 that is

unique to lipid kinases. We next learned that replacing the N-

ethyl-pyrazole ring with a 2-methylpyrimidine ring improved

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(

75% yield) of intermediate 6-chloro-8-(1-ethyl-5-methyl-1H-pyrazol-

H), 7.95−7.97 (m, 1 H), 4.09 (s, 3 H); MS (EI) calcd for

1

+

4-yl)-9-methyl-9H-purine (III) as a pale yellow solid. H NMR (400

MHz, DMSO-d ) δ 8.67 (s, 1 H), 8.09 (s 1 H), 4.18 (q, 2 H), 3.89 (s,

C H ClF N [M + H] , 314; found, 314.

1

2

8

3

5

6-Chloro-9-ethyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-9H-purine

(XII). A solution of 6-chloro-9-ethyl-8-iodo-9H-purine (3.0 g, 9.7

mmol) in dioxane (40 mL) and water (4 mL) was treated with

6

3

H), 2.63 (s, 3 H), 1.35 (t, 3 H); MS (EI) calcd for C H ClN [M

1

2

14

6

+

H] , 277; found 277.

-Chloro-9-ethyl-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9H-purine

IV). Intermediate IV was prepared following a synthetic sequence

6

K CO (4.0 g, 29 mmol), Pd(dppf)Cl (1.1 g, 1.5 mmol), and (6-

2

3

2

(

(triﬂuoromethyl)pyridin-3-yl)boronic acid (2.0 g, 11 mmol). The

mixture was stirred for 12 h at 90 °C, then quenched by the addition

of water (50 mL), and extracted with DCM (3 × 100 mL). The

analogous to that used for the synthesis of III, obtaining 88% yield of

IV. H NMR (600 MHz, CDCl ) δ 8.65 (s, 1 H), 7.75 (s, 1 H), 4.42

1

3

(

q, 2 H), 4.20 (q, 2 H), 2.62 (s, 3 H), 1.50 (t, 3 H), 1.45 (t, 3 H); MS

EI) calcd for C H ClN [M + H] , 291; found 291.

organic layers were combined, dried (MgSO ), and ﬁltered. The

4

+

ﬁltrate was concentrated and puriﬁed by chromatography on SiO₂

(10% EtOAc/petroleum ether) to aﬀord 6-chloro-9-ethyl-8-(6-

13

16

6

-Chloro-9-ethyl-8-iodo-9H-purine (VII). Into a 10 L four-neck

round-bottom ﬂask was placed a solution of 6-chloro-9H-purine (400

g, 2.59 mol), iodoethane (405 g, 2.60 mol), and potassium carbonate

(triﬂuoromethyl)pyridin-3-yl)-9H-purine (1.2 g, 3.7 mmol, 38%

1

yield) as a white solid. H NMR (300 MHz, DMSO-d ) δ 9.26 (s,

6

(

430 g, 3.11 mol) in DMSO (4 L). The resulting solution was stirred

1 H), 8.89 (s, 1 H), 8.62 (m, 1 H), 8.20 (m, 1 H), 4.50 (m, 2 H), 1.40

+

overnight at RT, diluted with ice water, and extracted with MTBE.

(m, 3 H); MS (EI) calcd for C H ClF N [M + H] , 328; found,

1

3

10

3

5

The organic extracts were combined, dried (MgSO ), and

328.

4

concentrated under vacuum. The residue was puriﬁed by chromatog-

(S)-(3-((8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-

6-yl)oxy)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone (1).

A solution of (S)-pyrrolidin-3-ol (15.0 g, 172 mmol) and triethyl-

amine (19.2 g, 189 mmol) in 100 mL of DCM was treated dropwise

at 0 °C with a solution of tetrahydro-2H-pyran-4-carbonyl chloride

(26.4 g, 177 mmol) in 75 mL of DCM. The mixture was stirred for 2

h and concentrated. The residue was taken up in 1 L of EtOAc, stirred

overnight, and then ﬁltered to remove amine salts. The mother liquor

was concentrated to provide 36 g (100%) of (S)-(3-hydroxypyrro-

raphy on SiO (1:10 to 1:1 EtOAc/petroleum ether) providing 250 g

2

(

53%) of 6-chloro-9-ethyl-9H-purine. Into a 10 L four-neck round-

bottom ﬂask purged and maintained with an inert atmosphere of

nitrogen was placed a solution of diisopropylamine (200 g, 1.98 mol)

in THF (1.2 L). This was followed by addition of a 2.5 M solution of

n-BuLi (736 mL, 1.40 equiv) at −78 °C. After stirring for 30 min, a

solution of 6-chloro-9-ethyl-9H-purine (240 g, 1.31 mol) in THF (1.2

L) was added dropwise with stirring at −78 °C. The solution was

stirred for 5 min at −78 °C, followed by addition of a solution of I

(

for an additional 10 min at −78 °C and then quenched by the

addition of 200 mL of aqueous NH Cl. The organic layer was washed

1

lidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone. H NMR (600

2

467 g, 1.84 mol) in THF (1.2 L) at −78 °C. The solution was stirred

MHz, CDCl ) δ 4.52 (d, J = 6.0 Hz, 1 H), 3.99−4.03 (m, 2 H),

3

3.38−3.70 (m, 6 H), 2.50−2.64 (m, 1 H), 1.98−2.06 (m, 2 H), 1.86−

1.93 (m, 2 H), 1.60−1.71 (m, 2 H). A solution of (S)-(3-

hydroxypyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone (4.32

g, 21.7 mmol) in 60 mL of THF was treated at 0 °C with a 60%

mineral oil suspension of sodium hydride (1.01 g, 25.2 mmol)

followed by 6-chloro-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-

9H-purine (Intermediate III, 5.00 g, 18.1 mmol). The reaction

mixture was stirred for 2 h and allowed to warm to RT. The crude

reaction mixture was concentrated, and the residue was puriﬁed by

4

with aqueous Na S O , dried (MgSO ), and concentrated under

2

3

4

vacuum. The solid was washed with 2 × 200 mL of ethyl ether to give

1

2

50 g (62%) 6-chloro-9-ethyl-8-iodo-9H-purine (VII). H NMR (400

MHz, DMSO-d ) δ 8.73 (s, 1 H), 4.28 (q, J = 7.2 Hz, 2 H), 1.37 (t, J

6

+

=

7.2 Hz, 3 H); MS (EI) calcd for C H N ClI [M + H] , 309; found,

7 7 4

3

09.

6

-Chloro-9-methyl-8-(2-methylpyrimidin-5-yl)-9H-purine (IX).

Intermediate IX was prepared following a synthetic sequence

chromatography on SiO (gradient of 0−20% MeOH/DCM) to give

2

1

analogous to that used for the synthesis of X, obtaining 68% yield

5.0 g (63%) of 1. [α] = +29° (c 0.58, CHCl ); H NMR (600 MHz,

D

3

1

of IX. H NMR (300 MHz, CDCl ) δ 9.18 (s, 2 H), 8.88 (s, 1 H),

CDCl ) δ 8.48 (d, J = 7.0 Hz, 1 H), 7.75 (d, J = 6.3 Hz, 1 H), 5.93 (s,

3

+

3

.97 (s, 3 H), 2.89 (s, 3 H); MS (EI) calcd for C H ClN [M + H] ,

1 H), 4.16−4.20 (m, 2 H), 3.93−4.04 (m, 3 H), 3.87, 3.89 (2 s, 3 H),

3.70−3.86 (m, 3 H), 3.35−3.45 (m, 2 H), 2.52−2.65 (m, 1 H), 2.56,

2.60 (2 s, 3 H), 2.45−2.50 (m, 1 H), 2.23−2.40 (m, 2 H), 1.86−1.94

1

10

6

2

61; found, 261.

-Chloro-9-ethyl-8-(2-methylpyrimidin-5-yl)-9H-purine (X). Into

6

1

3

a 5 L four-neck round-bottom ﬂask purged and maintained with an

inert atmosphere of nitrogen was placed a solution of 6-chloro-9-

ethyl-8-iodo-9H-purine (200 g, 648 mmol), 2-methyl-5-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (180 g, 818 mmol),

(m, 2 H), 1.60−1.66 (m, 2 H), 1.45 (t, J = 6 Hz, 3 H); C NMR

(125 MHz, DMSO-d ) δ 172.9, 158.6, 154.2, 150.9, 148.6, 139.9,

6

138.0, 120.8, 108.7, 76.7, 75.2, 66.7, 52.0, 44.1, 39.0, 38.9, 31.9, 30.7,

+

30.1, 28.8, 15.4, 10.6; HRMS (EI) calcd for C H N O [M + H] ,

2

30

7

3

potassium carbonate (134 g, 970 mmol), and Pd(dppf)Cl -DCM (30

440.2410; found, 440.2414.

2

g, 39 mmol) in dioxane (2.0 L) and water (400 mL). The resulting

solution was stirred for 6 h at 90 °C. The reaction mixture was cooled

to RT, diluted with water, and then extracted with EtOAc. The

(S)-1-(3-(8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9H-purin-6-

ylamino)pyrrolidin-1-yl)propan-1-one (2a). Prepared in a fashion

similar to that used for the synthesis of 2b, in which precursor 6-

organic extracts were combined, dried (MgSO ), and concentrated

chloro-N4-methylpyrimidine-4,5-diamine is replaced by 6-chloropyr-

4

1

under vacuum. The residue was puriﬁed by chromatography on SiO₂

imidine-4,5-diamine. H NMR (500 MHz, DMSO-d

6

) δ 8.18 (br, 1

(

1:1:1 petroleum ether/DCM/EtOAc) providing 100 g (56%) of 6-

H), 8.00 (s, 1 H), 7.56 (br m, 1 H), 4.78 (m, 1 H), 4.14 (q, J = 7.2

Hz, 2 H), 3.82 (m, 1 H), 3.63−3.74 (m, 1 H), 3.55 (m, 1 H), 3.46

(m, 1 H), 3.38 (m, 1 H), 2.72 (s, 3 H), 2.21−2.31 (m, 2 H), 1.99−

1

chloro-9-ethyl-8-(2-methylpyrimidin-5-yl)-9H-purine (X). H NMR

300 MHz, CDCl ) δ 9.12 (s, 2 H), 8.80 (s, 1 H), 4.49 (q, J = 7.2 Hz,

(

2

3

H), 2.89 (s, 3 H), 1.51−1.57 (t, J = 7.2 Hz, 3 H). MS (EI) calcd for

2.18 (m, 2 H), 1.34 (t, J = 7.2 Hz, 3 H), 0.99 (m, 3 H); HRMS (EI)

+

C H N Cl [M + H] , 275; found, 275.

calcd for C18

H

N

25

8

O [M + H] , 369.2151; found, 369.2152.

12

6

Chloro-9-methyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-9H-purine

(S)-1-(3-((8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-

purin-6-yl)amino)pyrrolidin-1-yl)propan-1-one (2b). A solution of

6-chloro-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purine

(

XI). A solution of 6-chloro-N4-methylpyrimidine-4,5-diamine (300

mg, 1.89 mmol) in DMF (1 mL) was treated with iron(III) chloride

hexahydrate (204 mg, 0.756 mmol), followed by 6-(triﬂuoromethyl)-

nicotinaldehyde (364 mg, 2.08 mmol). The reaction mixture was

heated at 85 °C with air bubbling through the reaction mixture for 48

h. Next, the mixture was cooled to RT and water was added. The

mixture was extracted with DCM, dried (Na SO ), ﬁltered, and

(III, 500 mg, 1.81 mmol) in DMF (18 mL) was treated with iPr NEt

2

(0.80 mL, 4.6 mmol) and (S)-tert-butyl-3-aminopyrrolidine-1-

carboxylate (0.55 mL, 3.1 mmol). The mixture was stirred at 80 °C

for 72 h. The mixture was diluted with EtOAc, washed with water and

brine, dried (Na SO ), and concentrated. The residue was puriﬁed by

2

4

2

4

concentrated. The residue was puriﬁed by chromatography on SiO₂

chromatography on SiO₂(gradient of 0−10% MeOH/DCM) to

(

MeOH/DCM; 1/60 to 1/40) to give 6-chloro-9-methyl-8-(6-

triﬂuoromethyl)pyridin-3-yl)-9H-purine as a white solid. H NMR

aﬀord (S)-tert-butyl-3-((8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-

1

methyl-9H-purin-6-yl)amino)pyrrolidine-1-carboxylate (345 mg,

1

400 MHz, CDCl ) δ 9.25 (s, 1 H), 8.85 (s, 1 H), 8.49−8.51 (m, 1

45%). H NMR (600 MHz, DMSO-d ) δ 8.19 (s, 1 H), 7.89 (s, 1

3

6

5

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+

H), 7.73 (s, 1 H), 4.13 (q, J = 7.2 Hz, 2 H), 3.72 (s, 3 H), 3.54−3.65

Hz, 3 H). HRMS (EI) calcd for C H N O [M + H] , 411.2621;

21 31 8

found, 411.2627.

(m, 1 H), 3.40−3.50 (m, 1 H), 3.18−3.35 (m, 3 H), 2.52 (s, 3 H),

2

.05−2.20 (m, 1 H), 1.90−2.04 (m, 1 H), 1.33−1.38 (2s, 9 H), 1.32

1-((S)-3-(9-(2,2-Diﬂuoroethyl)-8-(1-ethyl-5-methyl-1H-pyrazol-4-

yl)-9H-purin-6-ylamino)pyrrolidin-1-yl)propan-1-one (2e). Prepared

in a fashion similar to that used for the synthesis of 2d. H NMR (500

MHz, DMSO-d ) δ 8.27 (s, 1 H), 7.98 (br, 1 H), 7.83 (s, 1 H), 6.39

+

(t, J = 7.3 Hz, 3 H); MS (EI) calcd for C H N O [M + H] , 427;

21 31 8 2

1

found 427.

A solution of (S)-tert-butyl-3-((8-(1-ethyl-5-methyl-1H-pyrazol-4-

yl)-9-methyl-9H-purin-6-yl)amino)pyrrolidine-1-carboxylate (345

mg, 0.809 mmol) in dioxane (2 mL) was treated with 4 M HCl in

dioxane (1 mL, 4 mmol). The mixture was stirred at RT for 2 days

and concentrated giving (S)-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-

methyl-N-(pyrrolidin-3-yl)-9H-purin-6-amine, HCl (323 mg, 100%

6

(m, 1 H), 4.81 (m, 1 H), 4.64 (m, 2 H), 4.18 (q, J = 7.2 Hz, 2 H),

3.82 (m, 1 H), 3.61−3.73 (m, 1 H), 3.50 (m, 1 H), 3.37 (m, 1 H),

2.47 (m, 3 H), 2.24 (m, 2 H), 2.00−2.20 (m, 2 H), 1.36 (t, J = 7.2 Hz,

3 H), 0.98 (q, J = 7.5 Hz, 3 H); HRMS (EI) calcd for C20

H

F

N

O

8

27

2

+

[M + H] , 433.2276; found, 433.2271.

+

yield). MS (EI) calcd for C H N [M + H] , 327; found 327.

1-((S)-3-(8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-(2,2,2-triﬂuor-

oethyl)-9H-purin-6-ylamino)pyrrolidin-1-yl)propan-1-one (2f). A

mixture of (S)-1-(3-(8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9H-purin-

6-ylamino)pyrrolidin-1-yl)propan-1-one (2a; 100 mg, 0.27 mmol)

and K CO (75 mg, 0.54 mmol) in DCM (5 mL) was cooled to 0 °C

and then treated with 2,2,2-triﬂuoroethyl triﬂuoromethanesulfonate

(74 mg, 0.32 mmol). The reaction mixture was stirred for 16 h at 25

°C under an N atmosphere. The mixture was ﬁltered and

concentrated. The residue was puriﬁed by reverse-phase chromatog-

raphy (gradient of MeCN/water with 0.05% TFA) to provide 1-((S)-

3-(8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-(2,2,2-triﬂuoroethyl)-9H-

1

6

23

8

A solution of (S)-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-

N-(pyrrolidin-3-yl)-9H-purin-6-amine, HCl (20 mg, 0.046 mmol) in

DMF (0.5 mL) was treated with propionyl chloride (5 μL, 0.06

mmol) and TEA (0.05 mL, 0.36 mmol). The mixture was stirred at

RT for 16 h, ﬁltered, and puriﬁed by reverse-phase chromatography

2 3

(gradient of water/MeCN with 0.1% TFA) to aﬀord (S)-1-(3-((8-(1-

ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)amino)-

2

1

pyrrolidin-1-yl)propan-1-one (21 mg, 90% yield). H NMR (600

MHz, DMSO-d ) δ 8.32 (s, 1 H), 7.93 (s, 1 H), 4.14 (q, J = 7.2 Hz, 2

6

H), 3.76 (s, 3 H), 3.64−3.70 (m, 1 H), 3.56−3.64 (m, 1 H), 3.44−

3

.54 (m, 2 H), 3.30−3.42 (m, 1 H), 2.52 (s, 3 H), 1.95−2.30 (m, 4

purin-6-ylamino)pyrrolidin-1-yl)propan-1-one (2f, 12 mg; 9%) as a

1

H), 1.32 (t, J = 7.2 Hz, 3 H), 0.94 (t, J = 7.4 Hz, 3 H); HRMS (EI)

calcd for C H N O [M + H] , 383.2308; found 383.2309.

white solid. H NMR (400 MHz, CD

3

OD) δ 8.42 (s, 1 H), 7.91 (s, 1

+

H), 5.14 (s, 2 H), 4.31 (t, J = 7.2 Hz, 2 H), 3.89−4.02 (m, 1 H),

3.61−3.82 (m, 3 H), 2.53 (s, 3 H), 2.37−2.47 (m, 3 H), 2.15−2.27

(m, 2 H), 1.49 (q, J = 7.2 Hz, 3 H), 1.17 (q, J = 7.2 Hz, 3 H); HRMS

(EI) calcd C H F N O [M + H] , 451.2182; found, 451.2184.

(S)-1-(3-((9-Cyclopropyl-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-

9H-purin-6-yl)amino)pyrrolidin-1-yl)propan-1-one (2g). Prepared

19

27

8

9

-Ethyl-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-N-[(3S)-1-propa-

noylpyrrolidin-3-yl]-9H-purin-6-amine (2c). Prepared in a fashion

similar to that used for the synthesis of 2b, in which precursor

intermediate III is replaced by intermediate IV; 6-chloro-9-ethyl-8-(1-

+

2

0

25

3

8

ethyl-5-methyl-1H-pyrazol-4-yl)-9H-purine. [α]_D= +22° (c 0.5,

1

MeOH); H NMR (600 MHz, DMSO-d ) δ 8.24 (br, 1 H), 7.87

in a fashion similar to that used for the synthesis of 2d. H NMR

(600 MHz, DMSO-d ) δ 8.34 (s, 1 H), 8.05 (s, 1 H), 4.15 (q, J = 6

6

(br, 1 H), 7.83 (br, 1 H), 4.78 (br, 1 H), 4.22 (br, 2 H), 4.18 (q, J = 6

6

Hz, 2 H), 3.81 (m, 1 H), 3.63−3.71 (m, 2 H), 3.44−3.55 (m, 2 H),

Hz, 2 H), 3.78 (m, 1 H), 3.67 (m, 1 H), 3.52−3.62 (m, 2 H), 3.47

(m, 2 H), 3.37 (m, 1 H), 2.56 (s, 3 H), 2.16−2.24 (m, 2 H), 2.09 (m,

3

.36 (m, 1 H), 2.20−2.28 (m, 3 H), 1.99−2.18 (m, 2 H), 1.37 (t, J =

6

Hz, 3 H), 1.29 (t, J = 6 Hz, 3 H), 0.98 (m, 3 H); ¹³C NMR (150

1 H), 2.00 (m, 1 H), 1.33 (t, J = 6 Hz, 3 H), 1.11 (m, 2 H), 0.94 (m, 3

+

MHz, DMSO-d ) δ 171.7, 153.9, 153.8, 152.1, 144.7, 139.4, 137.5,

H), 0.88 (m, 2 H), HRMS (EI) calcd for C21

H

29

F

2

N

8

O [M + H] ,

6

1

9

3

24.7, 109.2, 44.8, 44.2, 44.2, 40.5, 38.3, 27.4, 26.9, 15.5, 15.4, 10.5,

409.2464; found, 409.2471.

+

.4; HRMS (EI) calcd for C H N O [M + H] , 397.2464; found,

1-((S)-3-(9-(Cyclopropylmethyl)-8-(1-ethyl-5-methyl-1H-pyrazol-

2

0

29

8

97.2464.

-((S)-3-(8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-propyl-9H-

purin-6-ylamino)pyrrolidin-1-yl)propan-1-one (2d). A mixture of

,6-dichloropyrimidin-5-amine (3.0 g, 18 mmol) and propan-1-amine

2.7 g, 46 mmol) in IPA (20 mL) and water (10 mL) was heated at

0 °C for 16 h. The reaction mixture was concentrated and puriﬁed

4-yl)-9H-purin-6-ylamino)pyrrolidin-1-yl)propan-1-one (2h). Pre-

1

pared in a fashion similar to that used for the synthesis of 2d. H

NMR (500 MHz, DMSO-d ) δ 8.23 (s, 1 H), 7.75−7.99 (m, 2 H),

6

4

(

8

4.79 (s, 1 H), 4.18 (q, J = 7.2 Hz, 2 H), 4.03−4.15 (m, 2 H), 3.67 (m,

1 H), 3.41−3.60 (m, 2 H), 3.37 (m, 1 H), 2.49 (s, 3 H), 2.24 (m, 2

H), 1.94−2.19 (m, 2 H), 1.36 (t, J = 7.2 Hz, 3 H), 1.12 (m, 1 H), 0.99

(m, 3 H), 0.40 (m, 2 H), 0.28 (m, 2 H); HRMS (EI) calcd for

by chromatography on silica gel (20:1 DCM/MeOH) to give 6-

chloro-N4-propylpyrimidine-4,5-diamine (3.0 g, 88%) as a yellow

+

C H N O [M + H] , 423.2621; found, 423.2614.

2

31

8

+

solid. MS (EI) calcd for C H ClN [M + H] , 187; found, 187. To a

(S)-1-(3-(8-(1-Ethyl-2-methyl-1H-pyrrol-3-yl)-9-methyl-9H-purin-

7

12

4

solution of 6-chloro-N4-propylpyrimidine-4,5-diamine (600 mg, 3.2

mmol) and 6-methylnicotinaldehyde (1.3 g, 9.6 mmol) in DMF (10

mL) was added FeCl ·6H O (216 mg, 0.8 mmol) slowly at RT. The

6-ylamino)pyrrolidin-1-yl)propan-1-one (2i). A solution of 6-chloro-

N4-methylpyrimidine-4,5-diamine (2.0 g, 13 mmol) and 1-ethyl-2-

methyl-1H-pyrrole-3-carbaldehyde (1.7 g, 13 mmol) in DMF (15

3

2

resulting mixture was heated at 80 °C under air for 16 h. The reaction

was quenched with water (10 mL) and extracted with DCM. The

organic layer was washed with brine, dried (Na SO ), ﬁltered, and

mL) was treated with FeCl ·6H O (1.0 g, 3.8 mmol) at RT. The

3 2

resulting mixture was heated at 90 °C for 16 h. The reaction was

quenched with water and extracted with EtOAc. The organic layers

2

4

concentrated. The residue was puriﬁed by chromatography on silica

gel (10:1 DCM/MeOH) to give 6-chloro-8-(1-ethyl-5-methyl-1H-

pyrazol-4-yl)-9-propyl-9H-purine (500 mg, 50%) as a yellow solid.

were washed with brine, dried over Na SO , ﬁltered, and

2 4

concentrated. The residue was puriﬁed by column chromatography

on silica gel (3:1 petroleum ether/EtOAc) to give 6-chloro-8-(1-ethyl-

+

MS (EI) calcd for C H N [M + H] , 305; found, 305.

2-methyl-1H-pyrrol-3-yl)-9-methyl-9H-purine (1.4 g, 37%) as a

1

4

18

6

+

6

-Chloro-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-propyl-9H-purine

yellow solid. MS (EI) calcd for C13

276.

H15ClN

5

[M + H] , 276; found,

(

200 mg, 0.65 mmol) and (S)-1-(3-aminopyrrolidin-1-yl)propan-1-

one (370 mg, 2.62 mmol) were dissolved in a mixture of t-BuOH/

To a solution of 6-chloro-8-(1-ethyl-2-methyl-1H-pyrrol-3-yl)-9-

methyl-9H-purine (50 mg, 0.18 mmol) in t-BuOH (5 mL) were

added (S)-1-(3-aminopyrrolidin-1-yl)propan-1-one (51 mg, 0.36

mmol) and DIPEA (0.16 mL, 0.9 mmol) at RT. The reaction

DIPEA (1:1, 4 mL). The reaction mixture was heated to 80 °C for 36

h under an N atmosphere. The reaction mixture was cooled to RT,

2

and the solvent was removed under reduced pressure. The residue

was puriﬁed by reverse-phase chromatography (gradient of MeCN/

water with 10 mM NH HCO ) to provide 1-((S)-3-(8-(1-ethyl-5-

mixture was heated to 80 °C for 36 h under a N atmosphere. The

2

reaction mixture was concentrated, and the residue was puriﬁed by

reverse-phase chromatography (gradient of MeCN/water with 10

mM NH CO ) to provide 2i (40 mg, 0.10 mmol, 59%) as a white

4

3

methyl-1H-pyrazol-4-yl)-9-propyl-9H-purin-6-ylamino)pyrrolidin-1-

1

yl)propan-1-one (2d). H NMR (400 MHz, DMSO-d ) δ 8.25 (s, 1

6

4

3

1

H), 7.81−7.90 (m, 2 H), 4.70 (s, 1 H), 4.10−4.25 (m, 4 H), 3.30−

solid. H NMR (400 MHz, DMSO-d ) δ 8.20 (s, 1 H), 7.65−7.75 (m,

6

3

.85 (m, 4 H), 2.50(s, 3 H), 2.00−2.30 (m, 4 H), 1.15−1.25 (m, 2

1 H), 6.85−6.94 (m, 1 H), 6.43 (s, 1 H), 4.85 (br s, 1 H), 3.95 (q, J =

7.2 Hz, 2 H), 3.36−3.83 (m, 7 H), 2.40−2.50 (m, 3 H), 2.00−2.28

H), 1.36 (t, J = 7.2 Hz, 3 H), 0.95 (t, J = 7.2 Hz, 3 H), 0.75 (t, J = 8.0

5

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(

m, 4 H), 1.31 (t, J = 7.2 Hz, 3 H), 0.98 (q, J = 7.4 Hz, 3 H); HRMS

mmol) and triethylamine (4.34 g, 43.0 mmol) in dry DCM (17 mL)

portionwise at 0 °C. The reaction mixture was allowed to warm to RT

and stirred for 3 h. Then, water (30 mL) was added, the mixture was

extracted with DCM, and the organic layer was dried (Na SO ),

ﬁltered, and concentrated to obtain (S)-tert-butyl-(1-propionylpyrro-

lidin-3-yl)carbamate, which was used in the next step without further

puriﬁcation. A solution of (S)-tert-butyl-(1-propionylpyrrolidin-3-

yl)carbamate (4.5 g, 18 mmol) in dioxane (5 mL) was treated with

+

EI) calcd for C H N O [M + H] , 382.2355; found, 382.2358.

20

28

7

(

S)-1-(3-((8-(2-(tert-Butyl)thiazol-5-yl)-9-methyl-9H-purin-6-yl)-

amino)pyrrolidin-1-yl)propan-1-one (2j). Prepared in a fashion

similar to that used for the synthesis of 2b. H NMR (600 MHz,

2

4

1

DMSO-d ) δ 8.36 (br, 1 H), 8.31 (s, 1 H), 8.30 (s, 1 H), 4.60−4.74

6

(

3

1

m, 1 H), 3.90 (s, 3 H), 3.75 (m, 1 H), 3.60−3.65 (m, 1 H), 3.43−

.49 (m, 2 H), 3.34 (m, 1 H), 2.17−2.24 (m, 2 H), 2.09 (m, 1 H),

.40 (s, 9 H), 0.93 and 0.95 (2 t, J = 6 Hz, 3 H); HRMS (EI) calcd for

1

N HCl in dioxane (10 mL), and the mixture was stirred for 2 h at

+

C H N OS [M + H] , 414.2079; found, 424.2079.

20

28

7

RT. The pH of the mixture was adjusted to 9−10 with saturated

9

-Methyl-8-(2-methylpyrimidin-5-yl)-N-[(3S)-1-propanoylpyrroli-

aqueous Na CO . The aqueous layer was dried in vacuo and the

2

3

din-3-yl]-9H-purin-6-amine (2k). Prepared in a fashion similar to that

used for the synthesis of 2b, in which intermediate IX was used as the

chloropurine precursor. [α] = +42° (c 0.5, MeOH); H NMR (600

MHz, DMSO-d ) δ 9.19 (s, 2 H), 8.41−8.09 (m, 2 H), 4.77 (m, 1 H),

3

residue was extracted with MeOH and concentrated to obtain (S)-1-

3-aminopyrrolidin-1-yl)propan-1-one, which was used in the next

step without further puriﬁcation.

-Chloro-9-methyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-9H-purine

XI, 50 mg, 0.16 mmol) and (S)-1-(3-aminopyrrolidin-1-yl)propan-1-

(

1

D

6

.87 (s, 3 H), 3.79, 3.50 (m, 1 H), 3.73−3.62 (m, 1 H), 3.51 (m, 1

(

H), 3.36 (m, 1 H), 2.74 (s, 3 H), 2.24 (m, 2 H), 2.10 (m, 1 H), 2.05

one (28 mg, 0.19 mmol) were added to a mixture of 1:1 t-BuOH/

DIEA (2 mL). The reaction mixture was heated at 75 °C for 3 days.

The resulting mixture was cooled to RT, and the solvent evaporated

1

3

(m, 1 H), 0.99 (m, 3 H); C NMR (150 MHz, DMSO-d ) δ 171.7,

6

1

4

+

68.4, 157.0 (2C), 154.4, 153.1, 151.4, 145.6, 122.1, 119.8, 50.8, 44.7,

4.1, 30.5, 27.4, 27.0, 26.2, 9.4; HRMS (EI) calcd for C H N O [M

1

8

23

8

to aﬀord the crude residue which was puriﬁed by chromatography on

+

H] , 367.1995; found, 367.1999.

S)-1-(3-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)-

1

SiO (MeOH/DCM = 1/20) to give 2o as a white solid. H NMR

2

(

400 MHz, CDCl ) δ 9.19 (s, 1 H), 8.49 (m, 1 H), 8.35 (m, 1 H),

3

amino)pyrrolidin-1-yl)propan-1-one (2l). Prepared in a fashion

similar to that used for the synthesis of 2b, in which intermediate X

7

3

.91 (m, 1 H), 5.99 (m, 1 H), 4.99 (s, 1 H), 3.97−4.00 (m, 3 H),

.76−3.95 (m, 1 H), 3.63−3.70 (m, 2 H), 3.48−3.51 (m, 1 H), 2.28−

was used as the chloropurine precursor. [α] = +28° (c 0.5, MeOH);

D

1

2.48 (m, 3 H), 2.16−2.20 (m, 1 H), 1.16−1.21 (m, 3 H); HRMS (EI)

calcd for C H F N O [M + H] , 420.1759; found, 420.1764.

H NMR (600 MHz, DMSO-d ) δ 9.08 (s, 2 H), 8.58 (s, 1 H), 8.35

6

+

1

9

21

3

7

(

1

s, 1 H), 4.69 (m, 1 H), 4.28 (m, 2 H), 3.75 (m, 1 H), 3.59−3.66 (m,

8

-(6-Methoxypyridin-3-yl)-9-methyl-N-[(3S)-1-propanoylpyrroli-

H), 3.44−3.49 (m, 1 H), 3.32−3.37 (m, 1 H), 2.71 (s, 3 H), 2.16−

din-3-yl]-9H-purin-6-amine (2p). Prepared in a fashion similar to

2

0

1

3

+

.23 (m, 2 H), 2.08 (m, 1 H), 2.01 (m, 1 H), 1.27 (t, J = 10 Hz, 3 H),

1

that used for the synthesis of 2k. H NMR (500 MHz, DMSO-d ) δ

_.₉₄₍_m_,₃_H₎_;13C NMR (125 MHz, DMSO-d ) δ 171.7, 168.6,

6

8

.68 (s, 1 H), 8.30 (s, 1 H), 8.17−8.22 (m, 1 H), 8.10 (br d, 1 H),

57.0 (2C), 154.2, 153.2, 150.8, 145.0, 124.7, 122.3, 51.2, 44.7, 44.1,

7.03 (d, J = 8.6 Hz, 1 H), 4.80 (br, 1 H), 3.95 (s, 3 H), 3.86 (s, 3 H),

8.9, 27.4, 26.9, 26.2, 15.5, 9.4; HRMS (EI) calcd for C H N O [M

1

9

25

8

+

3.61−3.73 (m, 1 H), 3.43−3.56 (m, 2 H), 3.35−3.40 (m, 1 H), 2.24

m, 2 H), 1.96−2.19 (m, 2 H), 0.98 (q, J = 7.5 Hz, 3 H); HRMS (EI)

H] , 381.2151; found, 381.2154.

-(2-Methylpyrimidin-5-yl)-N-[(3S)-1-propanoylpyrrolidin-3-yl]-

-propyl-9H-purin-6-amine (2m). Prepared in a fashion similar to

(

8

+

calcd for C H N O [M + H] , 382.1991; found, 382.2003.

1

9

24

7

2

9

1

8-(5-Fluoro-6-methoxypyridin-3-yl)-9-methyl-N-[(3S)-1-propa-

that used for the synthesis of 2l. H NMR (500 MHz, DMSO-d ) δ

9

2

(

6

noylpyrrolidin-3-yl]-9H-purin-6-amine (2q). Prepared in a fashion

.13 (s, 2 H), 8.16−8.39 (m, 2 H), 4.77 (m, 1 H), 4.26 (t, J = 7.3 Hz,

1

similar to that used for the synthesis of 2k. H NMR (500 MHz,

H), 3.62−3.85 (m, 1 H), 3.51 (m, 2 H), 3.35−3.41 (m, 1 H), 2.75

DMSO-d ) δ 8.50 (s, 1 H), 8.31 (s, 1 H), 8.18 (m, 2 H), 4.79 (s, 1

6

s, 3 H), 2.24 (m, 2 H), 1.99−2.19 (m, 2 H), 1.69 (m, 2 H), 0.98 (t, J

H), 4.05 (s, 3 H), 3.83 (s, 3 H), 3.62−3.73 (m, 1 H), 3.50 (m, 2 H),

=

7.5 Hz, 3 H), 0.75 (t, J = 7.3 Hz, 3 H); HRMS (EI) calcd for

+

3.35 (m, 1 H), 2.24 (m, 2 H), 2.08 (m, 2 H), 0.98 (t, J = 7.6 Hz, 3

C H N O [M + H] , 395.2308; found, 395.2311.

20

27

8

+

H); HRMS (EI) calcd for C H FN O [M + H] , 400.1897; found,

1

9

23

7

2

9

-Methyl-8-(6-methylpyridin-3-yl)-N-[(3S)-1-propanoylpyrroli-

4

00.1906.

din-3-yl]-9H-purin-6-amine (2n). A solution of 6-chloro-N4-methyl-

pyrimidine-4,5-diamine (150 mg, 0.95 mmol) and 6-methylnicoti-

naldehyde (126 mg, 1.0 mmol) in DMF (3 mL) was treated with

FeCl ·6H O (64 mg, 0.24 mmol). The mixture was heated to 90 °C

for 16 h. The reaction was quenched with water and extracted with

DCM. The organic layer was washed with brine, dried (Na SO ),

ﬁltered, and concentrated. The residue was puriﬁed by chromatog-

raphy on SiO (10:1 DCM/MeOH) to give 6-chloro-9-methyl-8-(6-

methylpyridin-3-yl)-9H-purine (80 mg, 33%) as a yellow solid. MS

(

S)-1-(3-((8-(6-Methoxy-5-methylpyridin-3-yl)-9-methyl-9H-

purin-6-yl)amino)pyrrolidin-1-yl)propan-1-one (2r). Prepared in a

fashion similar to that used for the synthesis of 2b. H NMR (600

1

3

2

MHz, DMSO-d ) δ 8.63 (br, 1 H), 8.48 (s, 1 H), 8.34 (s, 1 H), 8.01

6

(

3

2

s, 1 H), 4.61−4.71 (br m, 1 H), 3.93 (s, 3 H), 3.81 (s, 3 H), 3.60−

2

4

.75 (m, 1 H), 3.44−3.50 (m, 2 H), 3.35 (m, 1 H), 2.19 (s, 3 H),

.16−2.25 (m, 2 H), 2.00−2.20 (m, 2 H), 0.95, 0.93 (2 t, J = 6 Hz, 3

2

+

H); HRMS (EI) calcd for C H N O [M + H] , 396.2148; found,

20 26

7

2

+

396.2148.

(EI) calcd for C H ClN [M + H] , 260; found, 260.

12

11

5

(

S)-1-(3-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)oxy)-

6

-Chloro-9-methyl-8-(6-methylpyridin-3-yl)-9H-purine (80 mg,

pyrrolidin-1-yl)propan-1-one (3b). To a solution of (S)-tert-butyl-3-

0

.31 mmol) and (S)-1-(3-aminopyrrolidin-1-yl)propan-1-one (48

hydroxypyrrolidine-1-carboxylate (4.0 g, 21 mmol) in THF (80 mL),

mg, 0.34 mmol) were added to a mixture of t-BuOH/DIPEA (1:1,

mL). The reaction mixture was heated to 80 °C for 36 h. The

60% NaH in mineral oil (1.5 g, 38 mmol) was added. The mixture

3

was stirred at 0 °C for 30 min, and then intermediate X (5.0 g, 18

mmol) was added. The solution was stirred at RT for 15 h, then

cooled, diluted with water, and extracted with EtOAc. The combined

organic layers were concentrated under reduced pressure to give (S)-

tert-butyl-3-(9-ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yloxy)-

pyrrolidine-1-carboxylate (7.0 g, 90%). MS (EI) calcd for

reaction mixture was cooled to RT and concentrated. The residue was

puriﬁed by reverse-phase chromatography (gradient of MeCN/water

with 10 mM NH HCO ) to provide 9-methyl-8-(6-methylpyridin-3-

4

3

yl)-N-[(3S)-1-propanoylpyrrolidin-3-yl]-9H-purin-6-amine (20 mg,

1

8%) as a yellow solid. H NMR (400 MHz, CD OD) δ 9.2 (s, 1

3

H), 8.74 (d, J = 8.0 Hz, 1 H), 8.46 (d, J = 4.0 Hz, 1 H), 7.94 (d, J =

+

C H N O [M + H] , 426; found 426.

8

3

8

.0 Hz, 1 H), 5.50−5.60 and 4.70−4.80 (m, 1 H), 4.05 (s, 3 H),

21 28

7

3

To a solution of (S)-tert-butyl-3-(9-ethyl-8-(2-methylpyrimidin-5-

yl)-9H-purin-6-yloxy)pyrrolidine-1-carboxylate (7.0 g, 16 mmol) in

DCM (80 mL) was added TFA (20 mL), and the reaction stirred at

.69−3.74 (m, 4 H), 2.85 (s, 3 H), 2.38−2.45 (m, 4 H), 1.50 (t, J =

+

.0 Hz, 3 H). HRMS (EI) calcd for C H N O [M + H] , 366.2042;

1

9

24

7

found, 366.2044.

(

S)-1-(3-((9-Methyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-9H-purin-

RT for 2 h. The mixture was cooled, saturated aqueous NaHCO was

3

6

-yl)amino)pyrrolidin-1-yl)propan-1-one (2o). A solution of pro-

added, and the mixture was extracted with DCM. The combined

organic extracts were concentrated under reduced pressure to give

(S)-9-ethyl-8-(2-methylpyrimidin-5-yl)-6-(pyrrolidin-3-yloxy)-9H-pu-

pionyl chloride (2.19 g, 23.6 mmol) in dry DCM (3 mL) was treated

with a solution of (S)-tert-butyl-pyrrolidin-3-ylcarbamate (4.00 g, 21.5

5

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+

rine (5 g, 93%). MS (EI) calcd for C H N O [M + H] , 326; found,

methylmorpholine (0.034 mL, 0.31 mmol). The mixed solution was

stirred at RT for 15 min, then (S)-9-ethyl-8-(2-methylpyrimidin-5-yl)-

6-(pyrrolidin-3-yloxy)-9H-purine (50 mg, 0.15 mmol) was added, and

the solution was stirred at RT for 15 h. The mixture was cooled, water

was added, and the mixture was extracted with EtOAc. The combined

organic extracts were concentrated under reduced pressure. The

residue was puriﬁed by preparative reverse-phase HPLC, eluting with

1

6

20

7

326.

A solution of (S)-9-ethyl-8-(2-methylpyrimidin-5-yl)-6-(pyrrolidin-

-yloxy)-9H-purine (35 mg, 0.088 mmol) in DMF (1 mL) was

treated with Hunig’s base (0.10 mL, 0.57 mmol) followed by

propionyl chloride (15 mg, 0.16 mmol), stirred for 1 h, ﬁltered, and

puriﬁed by reverse-phase chromatography (gradient of MeCN/water

with 0.1% TFA) to provide 3b (19 mg, 44%). H NMR (600 MHz,

CD OD) δ 9.10 (s, 2 H), 8.55 (s, 1 H), 6.00 (m, 1 H), 4.42 (m, 2 H),

.70−3.90 (m, 4 H), 2.80 (s, 3 H), 2.30−2.45 (m, 4 H), 1.43 (t, J = 9

Hz, 3 H), 1.12 (m, 3 H); MS (EI) calcd for C H N O [M + H] ,

83; found, 383.

S)-Cyclopropyl(3-((9-ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-

-yl)amino)pyrrolidin-1-yl)methanone (3c). A solution of (S)-tert-

3

1

MeCN/water with 0.05% TFA, to give 3d (49 mg, 81%). [α] = +34°

D

1

3

(c 0.5, MeOH); H NMR (400 MHz, DMSO-d ) δ 9.23 (s, 2 H),

6

3

8.56 (s, 1 H), 5.87−5.98 (m, 1 H), 4.37 (m, 2 H), 3.70−4.08 (m, 2

+

1

9

25

7

2

H), 3.39−3.67 (m, 2 H), 2.76 (s, 3 H), 2.24−2.51 (m, 2 H), 1.75−

13

3

1.85 (m, 1 H), 1.35 (m, 3 H), 0.57−0.67 (m, 4 H); C NMR (150

(

MHz, DMSO-d ) δ 171.6, 169.0, 159.6, 157.2, 154.1, 152.1, 148.2,

6

1

46.5, 124.8, 121.8, 76.3, 54.6, 44.7, 33.7, 30.8, 26.2, 15.5, 12.4, 7.6,

+

butyl-pyrrolidin-3-ylcarbamate (100 g, 537 mmol) in EtOAc (1 L)

7.3; HRMS (EI) calcd for C H N O [M + H] , 394.1991; found,

2

0

24

7

2

was treated dropwise at 0 °C over a 1 h period with NEt (79 mL, 569

3

394.1993.

mmol) followed by cyclopropanecarbonyl chloride (50 mL, 548

mmol) while maintaining an internal temperature of less than 10 °C.

The mixture was stirred for another 15 min and then washed with

water. The aqueous layer was extracted with EtOAc, and the

combined organic layers dried (Na SO ) and ﬁltered. The organic

(

S)-Cyclobutyl(3-((9-ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-

6-yl)amino)pyrrolidin-1-yl)methanone (3e). A 250 mL ﬂask

containing 6-chloro-9-ethyl-8-(2-methylpyrimidin-5-yl)-9H-purine

(5.0 g, 18 mmol) and tert-butyl-(S)-3-aminopyrrolidine-1-carboxylate

(4.8 g, 22 mmol) in DMF (100 mL) was treated with Hunig’s base

(20 mL, 120 mmol). The mixture was stirred at 65 °C for 48 h. The

solvent was evaporated, and the residue was puriﬁed by chromatog-

2

4

layer was then treated with TsOH·H O (112 g, 591 mmol), and the

2

mixture was stirred at 47 °C for 15 h. The precipitate that then

formed was ﬁltered, collected, and dried with a ﬂow of nitrogen giving

raphy on SiO (gradient of 0−10% MeOH/DCM) to aﬀord (S)-tert-

butyl-3-((9-ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)amino)-

pyrrolidine-1-carboxylate (6.7 g, 87% yield). H NMR (600 MHz,

DMSO-d ) δ 9.08 (s, 2 H), 8.28 (s, 1 H), 8.18 (s, 1 H), 4.67 (m, 1

H), 4.26 (m, 2 H), 3.56 (m, 1 H), 3.42 (m, 1 H), 3.25 (m, 2 H), 2.70

(s, 3 H), 2.11 (m, 1 H), 1.99 (m, 1 H), 1.34 and 1.36 (2s, 9 H), 1.26

(m, 3 H); MS (EI) calcd for C H N O [M + H] , 425; found, 425.

A mixture of (S)-tert-butyl-3-((9-ethyl-8-(2-methylpyrimidin-5-yl)-

9H-purin-6-yl)amino)pyrrolidine-1-carboxylate (6.7 g, 16 mmol) in

dioxane (80 mL) was treated with a 4 M solution of HCl in dioxane

(14 mL, 56 mmol). The resulting slurry was stirred overnight and

concentrated to dryness to provide (S)-9-ethyl-8-(2-methylpyrimidin-

2

1

4

53 g (87%) of (S)-(3-aminopyrrolidin-1-yl)(cyclopropyl)methanone

1

-methylbenzenesulfonate salt. H NMR (600 MHz, CD OD) δ 7.68

3

(

d, J = 10 Hz, 2 H), 7.21 (d, J = 10 Hz, 2 H), 4.02 (m, 1 H), 3.81−

6

3

.91 (m, 2 H), 3.72 (m, 1 H), 3.52 (m, 1 H), 2.43 (m, 1 H), 2.35 (s, 3

H), 2.14 (m, 1 H), 1.78 (m, 1 H), 0.81−0.91 (m, 4 H).

+

A mixture containing 6-chloro-9-ethyl-8-(2-methylpyrimidin-5-yl)-

2

1

29

8

2

9

(

H-purine (X, 120 g, 437 mmol), (S)-(3-aminopyrrolidin-1-yl)-

cyclopropyl)methanone-4-methylbenzenesulfonate salt (157 g, 481

mmol), and Na CO (162 g, 1530 mmol) in tert-amyl-OH (1 L) was

2

3

stirred at 90 °C for 42 h. The mixture was poured into 1.6 L of water

and extracted with DCM (3 × 450 mL). The organic layer was dried

(

Na SO ) and concentrated. The residue was then taken-up in 1.6 L

2 4

5-yl)-N-(pyrrolidin-3-yl)-9H-purin-6-amine, 2HCl (6.4 g, 16 mmol,

00% yield). MS (EI) calcd for C H N [M + H] , 325; found, 325.

+

of hexane, stirred overnight, and ﬁltered. The solid was dried

overnight with a ﬂow of nitrogen, giving 150 g (88%) of (S)-

cyclopropyl(3-((9-ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)-

1

6

21

8

A mixture of (S)-9-ethyl-8-(2-methylpyrimidin-5-yl)-N-(pyrrolidin-

-yl)-9H-purin-6-amine, 2 HCl (120 mg, 0.30 mmol), cyclo-

butanecarboxylic acid (64 mg, 0.63 mmol), HATU (140 mg, 0.36

mmol), DMF (2.5 mL), and Hunig’s base (0.40 mL, 2.3 mmol) was

stirred at RT for 18 h. The mixture was ﬁltered and puriﬁed by

reverse-phase chromatography (gradient of MeCN/water with 0.1%

TFA) to aﬀord (S)-cyclobutyl(3-((9-ethyl-8-(2-methylpyrimidin-5-

3

amino)pyrrolidin-1-yl)methanone (3c). [α] = +42° (c 2.0, MeOH);

D

1

H NMR (600 MHz, CD OD) δ 9.08 (s, 2 H), 8.31 (m, 1 H), 4.80−

3

4

.95 (m, 1 H), 4.37 (m, 2 H), 3.90−4.15 (m, 1 H), 3.79−3.87 (m, 1

H), 3.64−3.74 (m, 1 H), 3.52−3.59 (m, 1 H), 2.78 (s, 3 H), 2.30−

2

6

1

5

.40 (m, 1 H), 2.07−2.24 (m, 1 H), 1.73−1.84 (m, 1 H), 1.40 (t, J =

1

3

Hz, 3 H), 0.78−0.88 (m, 4 H); C NMR (125 MHz, DMSO-d ) δ

yl)-9H-purin-6-yl)amino)pyrrolidin-1-yl)methanone (3e, 73 mg, 46%

yield). H NMR (600 MHz, DMSO-d ) δ 9.08 (s, 2 H), 8.51 (br, 1

6

1

71.4, 168.6, 157.0, 154.4, 153.2, 150.9, 145.0, 144.9, 122.3, 120.0,

6

1.4, 50.1, 44.8, 38.9, 30.5, 26.2, 15.5, 12.3, 7.5, 7.4; HRMS (EI) calcd

for C H N O [M + H] , 393.2151; found, 393.2153.

H), 8.33 (s, 1 H), 4.67 (m, 1 H), 4.27 (q, J = 6 Hz, 2 H), 3.64 (m, 1

H), 3.45−3.55 (m, 1 H), 3.38 (m, 1 H), 3.33 (m, 1 H), 3.15−3.25

(m, 1 H), 2.70 (s, 3 H), 1.90−2.15 (m, 6 H), 1.85 (m, 1 H), 1.69 (m,

+

20

25

8

(

S)-Cyclopropyl(3-((9-ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-

-yl)oxy)pyrrolidin-1-yl)methanone (3d). To a solution of (S)-tert-

butyl-3-hydroxypyrrolidine-1-carboxylate (4.0 g, 21 mmol) in THF

80 mL), 60% NaH in mineral oil (1.5 g, 38 mmol) was added. The

6

1

H), 1.27 (t, J = 7 Hz, 3 H); HRMS (EI) calcd for C H N O [M +

21 27 8

+

H] , 407.2308; found, 407.2309.

(

S)-Cyclobutyl(3-((9-ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-

mixture was stirred at 0 °C for 30 min, and then intermediate X (5.0

g, 18 mmol) was added. The solution was stirred at RT for 15 h, then

cooled, diluted with water, and extracted with EtOAc. The combined

organic layers were concentrated under reduced pressure to give (S)-

tert-butyl-3-(9-ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yloxy)-

pyrrolidine-1-carboxylate (7.0 g, 90%). MS (EI) calcd for

6-yl)oxy)pyrrolidin-1-yl)methanone (3f). Compound 3f was pre-

pared via a sequence analogous to the route used for the preparation

of 3d, replacing cyclopropyl carboxylic acid with cyclobutyl carboxylic

acid. H NMR (400 MHz, DMSO-d ) δ 9.14 (s, 2 H), 8.62 (s, 1 H),

1

6

5.87 (m, 1 H), 4.39 (m, 2 H), 3.22−3.80 (m, 5 H), 2.76 (s, 3 H),

1.85−2.20 (m, 8 H), 1.33 (t, J = 7.2 Hz, 3 H); HRMS (EI) calcd for

+

C H N O [M + H] , 426; found 426.

C H N O [M + H] , 408.2148; found, 408.2144.

21

28

7

3

21 26

7

2

To a solution of (S)-tert-butyl-3-(9-ethyl-8-(2-methylpyrimidin-5-

cis-((S)-3-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)-

amino)pyrrolidin-1-yl)((1S,3R)-3-methoxycyclobutyl)-methanone

(3g). A mixture of (S)-9-ethyl-8-(2-methylpyrimidin-5-yl)-N-(pyrro-

lidin-3-yl)-9H-purin-6-amine, 2HCl (79 mg, 0.20 mmol), 3-

methoxycyclobutanecarboxylic acid (26 mg, 0.20 mmol), HATU

(84 mg, 0.22 mmol), DMF (1 mL), and Hunig’s base (0.24 mL, 1.4

mmol) was stirred at RT for 16 h. The mixture was ﬁltered and

puriﬁed by SFC (ES Industries Pyridyl Amide column, 21 × 250 mm;

70 mL/min; 10% MeOH in CO with 0.25% Me NEt) to aﬀord the

yl)-9H-purin-6-yloxy)pyrrolidine-1-carboxylate (7.0 g, 16 mmol) in

DCM (80 mL) was added TFA (20 mL), and the reaction was stirred

at RT for 2 h. The mixture was cooled, saturated aqueous NaHCO₃

was added, and the mixture was extracted with DCM. The combined

organic extracts were concentrated under reduced pressure to give

(

S)-9-ethyl-8-(2-methylpyrimidin-5-yl)-6-(pyrrolidin-3-yloxy)-9H-pu-

+

rine (5 g, 93%). MS (EI) calcd for C H N O [M + H] , 326; found,

1

6

20

7

3

26.

To a solution of cyclopropanecarboxylic acid (27 mg, 0.31 mmol)

in DMF (2 mL) was added HATU (88 mg, 0.23 mmol) and 4-

2

cis- and trans-methoxycyclobutane products (22 and 24 mg,

respectively). Data for the cis isomer 3g, the second isomer eluted

5

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1

on the abovementioned column: [α]_D= +32° (c 0.5, MeOH); H

was dried (MgSO ) and concentrated. The residue was dissolved in

4

NMR (600 MHz, DMSO-d ) δ 9.10 (s, 2 H), 8.30 (m, 1 H), 8.24 (m,

DMSO and puriﬁed by reverse-phase chromatography (gradient of

MeCN/water with 0.1% TFA) to provide the desired product 3k (38

mg, 26%). [α]_D= +38° (c 0.5, MeOH); H NMR (600 MHz,

6

1

H), 4.71 (m, 1 H), 4.28 (m, 2 H), 3.40−3.75 (m, 4 H), 3.34 (m, 1

1

H), 3.07 and 3.09 (2 s, 3 H), 2.72 (s, 3 H), 2.73 (m, 1 H), 2.35 (m, 2

H), 2.15−2.30 (m, 1 H), 2.07 (m, 1 H), 1.85−2.00 (m, 2 H), 1.28

DMSO-d ) δ 9.11 (s, 2 H), 8.32 (m, 1 H), 8.23 (m, 1 H), 4.69 (m, 1

6

(

m, 3 H); ¹³C NMR (150 MHz, DMSO-d ) δ 171.8, 168.6, 157.0

2C), 154.5, 154.3, 153.2, 150.9, 122.3, 120.0, 70.1, 54.7, 51.0, 50.9,

H), 4.29 (m, 2 H), 4.10 (m, 1 H), 4.02 (m, 2 H), 3.67 (m, 2 H), 3.59

6

(m, 1 H), 3.47 (m, 1 H), 3.31 (m, 2 H), 3.17 (s, 3 H), 2.74 (s, 3 H),

1

3

4

4.4, 38.9, 32.9, 32.8, 28.7, 28.4, 26.2, 15.5; HRMS (EI) calcd for

1.98−2.15 (m, 2 H), 1.30 (m, 3 H); C NMR (150 MHz, DMSO-

+

C H N O [M + H] , 437.2413; found, 437.2419.

d ) δ 168.6, 160.8, 157.0 (2C), 154.4, 153.2, 150.9, 144.9, 122.3,

22

29

8

2

6

(

(S)-3-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)oxy)-

120.0, 69.2, 57.4, 55.7, 51.8, 50.1, 45.2, 38.9, 30.7, 26.2, 26.0, 15.5;

pyrrolidin-1-yl)((1S,3R)-3-methoxycyclobutyl)-methanone (3h). A

mixture containing cis-3-methoxycyclo-butanecarboxylic acid (22 mg,

+

HRMS (EI) calcd for C H N O [M + H] , 438.2366; found,

2

1

28

8

2

4

38.2372.

0

.17 mmol), Hunig’s base (0.029 mL, 0.17 mmol), HATU (76 mg,

.20 mmol), and (S)-9-ethyl-8-(2-methylpyrimidin-5-yl)-6-(pyrroli-

(

S)-(3-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)oxy)-

pyrrolidin-1-yl)(3-methoxyazetidin-1-yl)methanone (3l). To a sol-

ution of 3-methoxyazetidine, HCl (0.020 g, 0.17 mmol) in DCM (3.3

mL) and TEA (0.092 mL, 0.66 mmol) was added triphosgene (0.030

g, 0.10 mmol). The solution was stirred at RT for 1 h. In a separate

reaction vessel, (S)-9-ethyl-8-(2-methylpyrimidin-5-yl)-6-(pyrrolidin-

3-yloxy)-9H-purine (intermediate prepared for the synthesis of 3d;

din-3-yloxy)-9H-purine (60 mg, 0.17 mmol) in 2 mL of DMF was

stirred for 4 h at RT and puriﬁed by reverse-phase chromatography

(

gradient of MeCN/water with 0.1% TFA) to provide the desired

1

product, 3h (37 mg, 48%). [α] = +36° (c 0.5, MeOH); H NMR

D

(600 MHz, DMSO-d ) δ 9.10 (s, 2 H), 8.58 (s, 1 H), 5.85 (m, 1 H),

6

4

3

4

.33 (m, 2 H), 3.81 (m, 1 H), 3.37 (m, 1 H), 3.51−3.74 (m, 3 H),

0.060 g, 0.17 mmol) was suspended in DCM (0.50 mL) along with

.05 and 3.07 (2 s, 3 H), 2.78 (m, 1 H), 2.71 (s, 3 H), 2.13−2.39 (m,

TEA (0.050 mL, 0.36 mmol). The solution was stirred at RT for 1 h.

This suspension was then added via a syringe to the ﬁrst vessel, and

the reaction was stirred at RT for 16 h. The solvent was then

evaporated, and the crude reaction mixture was then resuspended in

DMF (1.0 mL). An additional portion of TEA (0.10 mL) was added,

and the reaction was heated to 50 °C for 72 h. The reaction vial was

then cooled, diluted with DMSO (0.90 mL), and puriﬁed by reverse-

phase preparative HPLC to aﬀord the TFA salt of 3l. The TFA salt

was then dissolved in methanol and eluted through a 1g SiliPrepTM

1

3

H), 1.84−1.94 (m, 2 H), 1.29 (m, 3 H); C NMR (125 MHz,

DMSO-d ) δ 172.0, 169.1, 159.5, 157.2, 154.1, 152.1, 148.1, 121.9,

6

1

2

21.4, 77.0, 75.5, 70.1, 54.7, 51.9, 44.2, 33.1, 32.7, 30.1, 28.6, 28.5,

+

6.2, 15.3; HRMS (EI) calcd for C H N O [M + H] , 438.2253;

2

28

7

3

found, 438.2256.

(

S)-(3,3-Diﬂuorocyclobutyl)(3-((9-ethyl-8-(2-methylpyrimidin-5-

yl)-9H-purin-6-yl)amino)pyrrolidin-1-yl)methanone (3i). Prepared

1

analogously to compound 3e. Analytical data for 3i: H NMR (600

MHz, DMSO-d ) δ 9.08 (s, 2 H), 8.51 (s, 1 H), 8.32 (s, 1 H), 4.73

1

6

silicon-carbonate cartridge to aﬀord neutral 3l (9.9 mg, 14%). H

(

m, 1 H), 4.26 (m, 2 H), 3.72 (m, 1 H), 3.67 (m, 1 H), 3.57 (m, 1

H), 3.46 (m, 2 H), 3.38 (m, 1 H), 3.09 (m, 1 H), 2.74 (m, 2 H), 2.70

s, 3 H), 1.97−2.22 (m, 3 H), 1.27 (m, 3 H); HRMS (EI) calcd for

NMR (600 MHz, DMSO-d ) δ 9.13 (s, 2 H), 8.59 (s, 1 H), 5.82 (s, 1

6

H), 4.35 (q, J = 7.2 Hz, 2 H), 4.04−4.17 (m, 2 H), 3.92−4.04 (m, 1

H), 3.61−3.78 (m, 3 H), 3.53 (d, J = 12.0 Hz, 1 H), 3.37−3.49 (m, 2

H), 3.16 (s, 3 H), 2.74 (s, 3 H), 2.11−2.31 (m, 2 H), 1.32 (t, J = 7.2

(

+

C H F N O [M + H] , 443.2119; found, 443.2132.

21

25

2

8

(

S)-(3,3-Diﬂuorocyclobutyl)(3-((9-ethyl-8-(2-methylpyrimidin-5-

+

Hz, 3 H). MS (EI) calcd for C H N O [M + H] 439; found 439.

2

1

27

8

3

yl)-9H-purin-6-yl)oxy)pyrrolidin-1-yl)methanone (3j). Prepared

1

(3(S)-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)amino)-

pyrrolidin-1-yl)(tetrahydrofuran-3(R or S)-yl) methanone (3m). A

solution of (S)-9-ethyl-8-(2-methylpyrimidin-5-yl)-N-(pyrrolidin-3-

yl)-9H-purin-6-amine (50 mg, 0.15 mmol) in DMF (2 mL) was

treated with tetrahydrofuran-3-carboxylic acid (18 mg, 0.15 mmol),

HATU (59 mg, 0.15 mmol), and Hunig’s base (20 mg, 0.15 mmol).

The mixture was stirred for 10 h and then puriﬁed by reverse-phase

analogously to compound 3h. Analytical data for 3j: H NMR (600

MHz, DMSO-d ) δ 9.10 (s, 2 H), 8.57 (s, 1 H), 5.87 (m, 1 H), 4.33

6

(

m, 2 H), 3.38−3.86 (m, 5 H), 3.09−3.19 (m, 2 H), 2.64−2.79 (m, 2

H), 2.72 (s, 3 H), 2.15−2.35 (m, 2 H), 1.29 (m, 3 H); HRMS (EI)

calcd for C H F N O [M + H] , 444.1959; found, 444.1959.

+

21

24

2

7

2

(

S)-(3-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)-

amino)pyrrolidin-1-yl)(3-methoxyazetidin-1-yl)methanone (3k). A

mixture of (S)-9-ethyl-8-(2-methylpyrimidin-5-yl)-N-(pyrrolidin-3-

yl)-9H-purin-6-amine, 2HCl (200 mg, 0.503 mmol) and CDI (163

mg, 1.01 mmol) was taken-up in THF (2.5 mL), and Hunig’s base

chromatography (gradient of MeCN/water with 0.05% NH OH to

4

provide a diastereomeric mixture, which was then resolved using

chiral column chromatography (Chiralpak IA column, eluting with

MeOH/CO ) to provide diastereomer 1 (15 mg, 46%) and

(

2

0.26 mL, 1.5 mmol) was added. The reaction was stirred at 75 °C for

2

diastereomer 2 (17 mg, 52%)). Analytical data for the ﬁrst, more

h. Next, 3:1 chloroform/IPA and sat’d NH Cl were added. The

4

1

potent diastereomer 3m: H NMR (400 MHz, CD OD) δ 9.13 (s, 2

combined organics were washed with brine, dried (MgSO ), and

3

4

H), 8.36 (s, 1 H), 4.40 (m, 2 H), 4.02 (m, 1 H), 3.50−3.90 (m, 7 H),

concentrated. Puriﬁcation by chromatography on SiO (50−100% 3:1

2

3

.10 (m, 1 H), 2.82 (s, 4 H), 2.10−2.22 (m, 4 H), 1.44 (m, 3 H);

EtOAc:EtOH with 1% NH in hexanes gave (S)-(3-((9-ethyl-8-(2-

4

+

methylpyrimidin-5-yl)-9H-purin-6-yl)amino)pyrrolidin-1-yl)(1H-imi-

HRMS (EI) calcd for C21

423.2263.

H N O [M + H] , 423.2257; found,

27 8 2

dazol-1-yl)methanone (132 mg, 0.315 mmol, 63% yield). MS (EI)

+

calcd for C H N O [M + H] , 419; found, 419.

((S)-3-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)oxy)-

pyrrolidin-1-yl)(tetrahydrofuran-3(R or S)-yl)methanone (3n). A

solution of tetrahydrofuran-3-carboxylic acid (39 mg, 0.33 mmol) in

DMF (2 mL) was treated with HATU (150 mg, 0.40 mmol), (S)-9-

ethyl-8-(2-methylpyrimidin-5-yl)-6-(pyrrolidin-3-yloxy)-9H-purine,

HCl (120 mg, 0.332 mmol), and Hunig’s base (0.058 mL, 0.33

mmol). The mixture was stirred for 4 h and then puriﬁed by reverse-

phase chromatography (gradient of MeCN/water with 0.1% TFA to

provide a diastereomeric mixture, which was then resolved using

chiral column chromatography (Chiralpak AD-H column, eluting with

20

23 10

A vial was charged with (S)-(3-((9-ethyl-8-(2-methylpyrimidin-5-

yl)-9H-purin-6-yl)amino)pyrrolidin-1-yl)(1H-imidazol-1-yl)-

methanone (132 mg, 0.315 mmol), MeCN (4 mL) followed by

iodomethane (0.079 mL, 1.3 mmol). The reaction mixture was stirred

for 1 h at RT. The solvent was removed in vacuo, and the solid was

triturated in ether and ﬁltered to give an orange solid (S)-1-(3-((9-

ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)amino)pyrrolidine-1-

carbonyl)-3-methyl-1H-imidazol-3-ium, HCl (142 mg, 0.302 mmol,

+

9

6% yield). MS (EI) calcd for C H N O [M + H] , 433; found,

2

1

25 10

4

33.

S)-1-(3-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)-

amino)pyrrolidine-1-carbonyl)-3-methyl-1H-imidazol-3-ium (142 mg,

.328 mmol) and 3-methoxyazetidine-HCl (34 mg, 0.39 mmol) in

40% MeOH/CO

2

) to provide diastereomer 1 (42 mg, 28%) and

(

diastereomer 2 (13 mg, 9%)). Analytical data for the second, more

1

potent diastereomer 3n: H NMR δ (600 MHz, DMSO-d ) δ 9.10,

6

0

9.11 (2 s, 2 H), 8.58 (2 s, 1 H), 5.87 (m, 1 H), 4.33 (m, 2 H), 3.56−

3.92 (m, 6 H), 3.41 (m, 1 H), 3.20 (m, 1 H), 2.72 (s, 3 H), 1.89−2.36

(m, 5 H), 1.29 (m, 3 H); HRMS (EI) calcd for C H N O [M +

DMA (3 mL) were treated with Hunig’s base (0.11 mL, 0.66 mmol)

and stirred at 55 °C overnight. The mixture was taken-up in 3:1

2

1

26

7

3

+

chloroform/IPA and washed with sat’d NaHCO . The organic layer

H] , 424.2097; found, 424.2100.

3

5

151

https://doi.org/10.1021/acs.jmedchem.1c00237

J. Med. Chem. 2021, 64, 5137−5156

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(

S)-(3-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)-

H), 3.55−3.75 (m, 2 H), 2.25−2.40 (m, 1 H), 2.18−2.25 (m, 1 H),

1.70−1.82 (m, 1 H), 1.30−1.40 (m, 3 H), 0.65−0.75 (m, 4 H);

amino)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone (3o).

A solution of (S)-9-ethyl-8-(2-methylpyrimidin-5-yl)-N-(pyrrolidin-3-

yl)-9H-purin-6-amine (200 mg, 0.617 mmol), tetrahydro-2H-pyran-4-

carboxylic acid (120 mg, 0.925 mmol), Hunig’s base (0.32 mL, 1.9

mmol), and HATU (258 mg, 0.678 mmol) in DMF (6 mL) was

+

HRMS (EI) calcd for C H F N O [M + H] 447.1756; found

2

1

22

3

6

2

447.1754.

((S)-3-((9-Ethyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-9H-purin-6-yl)-

oxy)pyrrolidin-1-yl)((1S,3R)-3-methoxycyclobutyl)methanone (4c).

A solution of (S)-tert-butyl-3-hydroxypyrrolidine-1-carboxylate (430

mg, 2.30 mmol) in dioxane (10 mL) was treated with a 60%

suspension of NaH (120 mg, 3.00 mmol), stirred for 10 min, then

treated with 6-chloro-9-ethyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-9H-

purine (500 mg, 1.53 mmol), stirred at 20 °C for 16 h, diluted with

stirred overnight. 3:1 chloroform/IPA and saturated NH Cl were

added. The products were extracted into 3:1 chloroform/IPA. The

combined organic layers were washed with brine, dried (MgSO ), and

concentrated. Puriﬁcation by chromatography on SiO (25−100% 3:1

EtOAc/EtOH with 1% NH in hexanes) gave (S)-(3-((9-ethyl-8-(2-

methylpyrimidin-5-yl)-9H-purin-6-yl)amino)pyrrolidin-1-yl)-

tetrahydro-2H-pyran-4-yl)methanone (3o; 155 mg, 58% yield) as a

4

2

4

DCM, and washed with water. The organic layer was dried (Na

and concentrated to a colorless oil. Chromatography on SiO

2

SO )

2 4

(

1

white solid. H NMR (600 MHz, DMSO-d ) δ 9.13 and 9.14 (2 s, 2

(gradient of 0−30% MeOH/DCM) gave the desired intermediate,

(S)-tert-butyl-3-((9-ethyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-9H-

6

H), 8.62 (2 s, 1 H), 5.90 (m, 1 H), 4.37 (m, 2 H), 3.76−3.88 (m, 3

H), 3.58−3.70 (m, 2 H), 3.29−3.42 (m, 3 H), 2.75 (s, 3 H), 2.60−

purin-6-yl)oxy)pyrrolidine-1-carboxylate (670 mg, 92% yield) as a

1

2

.70 (m, 2 H), 2.18−2.40 (m, 2 H), 1.50−1.65 (m, 4 H), 1.33 (m, 3

colorless oil. H NMR (600 MHz, DMSO-d

) δ 9.18 (s, 1 H), 8.60 (s,

6

+

H); HRMS (EI) calcd for C H N O [M + H] 437.2413; found

1 H), 8.53 (d, J = 8.0 Hz, 1 H), 8.11 (d, J = 8.1 Hz, 1 H), 5.82 (m, 1

H), 4.36 (q, J = 7.1 Hz, 2 H), 3.59−3.69 (m, 1 H), 3.43−3.51 (m, 2

H), 3.18−3.26 (m, 1 H), 3.30−3.41 (m, 1 H), 2.19−2.25 (m, 1 H),

1.37 (m, 9 H), 1.32 (t, J = 7.2 Hz, 3 H); MS (EI) calcd for

2

29

8

2

4

37.2413.

(

S)-(3-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)oxy)-

pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone (3p). A sol-

ution of (S)-9-ethyl-8-(2-methylpyrimidin-5-yl)-6-(pyrrolidin-3-

yloxy)-9H-purine, 2HCl (35 mg, 0.088 mmol) in DMF (1 mL) was

treated with Hunig’s base (0.10 mL, 0.57 mmol) followed by

tetrahydro-2H-pyran-4-carbonyl chloride (15 mg, 0.10 mmol), stirred

for 1 h, ﬁltered, and puriﬁed by reverse-phase chromatography

+

C H F N O [M + H] 479; found 479.

2

26

3

6

3

A solution containing (S)-tert-butyl-3-((9-ethyl-8-(6-

(triﬂuoromethyl)pyridin-3-yl)-9H-purin-6-yl)oxy)pyrrolidine-1-car-

boxylate (670 mg, 1.40 mmol) in DCM (10 mL) was treated with a 4

M solution of HCl in dioxane (1.0 mL, 4.0 mmol). The reaction

mixture was stirred for 4 h and then concentrated to dryness to

(

gradient of MeCN/water with 0.1% TFA) to provide the TFA salt of

p (19 mg, 32%). H NMR (600 MHz, CD OD) δ 9.09 (s, 2 H),

1

3

8

3

2

provide the desired product as a white amorphous solid, the HCl salt.

3

+

.57 (m, 1 H), 6.00 (m, 1 H), 4.41 (m, 2 H), 3.70−4.06 (m, 6 H),

MS (EI) calcd for C17

H

F

18

N

3

6

O [M + H] 379; found 379.

.39−3.50 (m, 2 H), 2.83 (m, 1 H), 2.80 (s, 3 H), 2.75 (m, 1 H),

A solution of (S)-9-ethyl-6-(pyrrolidin-3-yloxy)-8-(6-

(triﬂuoromethyl)pyridin-3-yl)-9H-purine, HCl (250 mg, 0.603

mmol) in DMF (3 mL) was treated with Hunig’s base (0.30 mL,

1.7 mmol), HATU (300 mg, 0.79 mmol), and ﬁnally (1S,3S)-3-

methoxycyclobutanecarboxylic acid (120 mg, 0.922 mmol). The

reaction mixture was stirred overnight at 20 °C. The mixture was

ﬁltered and puriﬁed by reverse-phase chromatography (gradient of

.29−2.45 (m, 2 H), 1.70−1.78 (m, 2 H), 1.63−1.68 (m, 2 H), 1.43

+

(

m, 3 H); HRMS (EI) calcd for C H N O [M + H] 438.2254;

2

28

7

3

found 438.2261.

(

S)-(3-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)-

amino)pyrrolidin-1-yl)(oxazol-4-yl)methanone (3q). Compound 3q

was prepared via a sequence analogous to that used for the synthesis

of 3e. MS (EI) calcd for C H N O [M + H] 420; found 420.

+

MeCN/water with 0.1% NH OH) to provide 4c (150 mg; 51%).

2

0

22

9

2

4

1

(

S)-(3-((9-Ethyl-8-(2-methylpyrimidin-5-yl)-9H-purin-6-yl)oxy)-

[α] = +44° (c 0.5, MeOH); H NMR (600 MHz, DMSO-d ) δ 9.17

D

6

pyrrolidin-1-yl)(oxazol-4-yl)methanone (3r). A mixture of (S)-9-

ethyl-8-(2-methylpyrimidin-5-yl)-6-(pyrrolidin-3-yloxy)-9H-purine

(s, 1 H), 8.60 (s, 1 H), 8.52 (d, J = 8 Hz, 1 H), 8.11 (d, J = 8 Hz, 1

H), 5.85 (m, 1 H), 4.33−4.39 (m, 2 H), 3.60−3.83 (m, 3 H), 3.54

(m, 1 H), 3.37 (m, 1 H), 3.04, 3.06 (2s, 3 H), 2.75 (m, 1 H), 2.13−

(

137 mg, 0.421 mmol), oxazole-4-carboxylic acid (48 mg, 0.42

mmol), and HATU (176 mg, 0.463 mmol) in DMF (3.5 mL) was

treated with Hunig’s base (0.22 mL, 1.3 mmol). The mixture was

stirred for 16 h, diluted with EtOAc and sat’d NH Cl, extracted with

EtOAc, dried (Na SO ), ﬁltered, and concentrated. Chromatography

on SiO (0−7% MeOH/DCM) gave the desired product 3r (83 mg,

7%): H NMR (600 MHz, DMSO-d ) δ 9.12 and 9.13 (2s, 2 H),

13

2.40 (m, 4 H), 1.85−1.95 (m, 2 H), 1.32 (m, 3 H); C NMR (125

MHz, DMSO-d ) δ 172.0, 159.7, 154.2, 152.3, 150.4, 149.0, 147.7,

6

4

139.5, 129.5, 123.0, 121.4, 121.3, 76.2, 70.1, 54.7, 51.9, 44.1, 33.0,

32.7, 31.8, 30.1, 28.6, 15.3; HRMS (EI) calcd for C H F N O [M

2

4

23 26

3

6

3

+

2

+ H] 491.2018; found 491.2020.

1

4

8

(S)-(3-((9-Ethyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-9H-purin-6-yl)-

oxy)pyrrolidin-1-yl)(3-methoxyazetidin-1-yl)methanone (4d). A

mixture containing (S)-pyrrolidin-3-ol (2.0 g, 23 mmol) and 4-

nitrophenyl 3-methoxyazetidine-1-carboxylate (6.9 g, 28 mmol) in n-

BuOH (57 mL) was heated to 100 °C, stirred for 4 h, diluted with

6

.64 and 8.66 (2s, 1 H), 8.60 and 8.62 (2s, 1 H), 8.47 and 8.51 (2s, 1

H), 5.93 (m, 1 H), 4.35 (m, 1 H), 4.23 (m, 1 H), 3.58−3.98 (m, 4

H), 2.73 (s, 3 H), 2.24−2.39 (m, 2 H), 1.31 (m, 3 H); HRMS (EI)

+

calcd for C H N O [M + H] 421.1737; found 421.1739.

20

21

8

3

(

S)-Cyclopropyl(3-((9-ethyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-

H-purin-6-yl)amino)pyrrolidin-1-yl)methanone (4a). A mixture of

S)-(3-aminopyrrolidin-1-yl)(cyclopropyl)methanone (94 mg, 0.61

EtOAc, washed with water, dried (Na SO ), and concentrated to

2 4

9

(

provide (S)-(3-hydroxypyrrolidin-1-yl)(3-methoxyazetidin-1-yl)-

+

methanone (4.75 g). MS (EI) calcd for C H N O [M + H] 201;

9

17

2

3

mmol), 6-chloro-9-ethyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-9H-pu-

rine (100 mg, 0.31 mmol), and DIEA (0.27 mL, 1.5 mmol) in

DMF (2 mL) was stirred overnight at 90 °C. The crude reaction

mixture was puriﬁed by reverse-phase chromatography (gradient of

found 201.

A solution of (S)-(3-hydroxypyrrolidin-1-yl)(3-methoxyazetidin-1-

yl)methanone (3.74 g, 18.7 mmol) in 50 mL of THF was treated at 0

°C with a 60% suspension of NaH (934 mg, 23.4 mmol). The mixture

was stirred for 5 min and then treated with 6-chloro-9-ethyl-8-(6-

(triﬂuoromethyl)pyridin-3-yl)-9H-purine (5.10 g, 15.6 mmol). The

reaction mixture was stirred for 2 h, diluted with EtOAc, and washed

with water. The organic layer was dried (Na

Chromatography on SiO (gradient of 3−15% MeOH/EtOAc) gave

the desired product (S)-(3-((9-ethyl-8-(6-(triﬂuoromethyl)pyridin-3-

yl)-9H-purin-6-yl)oxy)pyrrolidin-1-yl)(3-methoxyazetidin-1-yl)-

methanone 4d (6.1 g, 80%). [α]

(600 MHz, CDCl ) δ 9.10 (s, 1 H), 8.56 (s, 1 H), 8.34 (d, J = 6.5 Hz,

1 H), 7.87 (d, J = 8.1 Hz, 1 H), 5.87 (m, 1 H), 4.41 (q, J = 7.2 Hz, 2

H), 4.06−4.18 (m, 4 H), 3.81−3.92 (m, 3 H), 3.55−3.73 (m, 2 H),

3.26 (s, 3 H), 2.02−2.25 (m, 2 H), 1.49 (d, J = 7.2 Hz, 3 H);

MeCN/water with 0.1% TFA) to provide 50 mg (37%) of the desired

1

product 4a. H NMR (600 MHz, CD OD) δ 9.18 (s, 1 H), 8.48 (s, 1

3

H), 8.42 (m, 1 H), 8.03 (m, 1 H), 4.40−4.44 (m, 2 H), 4.20 (m, 1

H), 3.80−3.95 (m, 2 H), 3.60−3.65 (m, 1 H), 2.30−2.40 (m, 1 H),

SO ) and concentrated.

2

4

2

0

4

.10−2.25 (m, 1 H), 1.70−1.85 (m, 2 H), 1.40−1.50 (m, 3 H), 0.78−

2

+

.98 (m, 4 H); HRMS (EI) calcd for C H F N O [M + H]

2

1

23

3

7

46.1916; found 446.1918.

S)-Cyclopropyl(3-((9-ethyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-

H-purin-6-yl)oxy)pyrrolidin-1-yl)methanone (4b). Synthesized ac-

1

(

D

= +42° (c 0.5, MeOH); H NMR

9

3

1

cording to the procedure followed for the synthesis of 4c. H NMR

600 MHz, DMSO-d ) δ 9.18 (s, 1 H), 8.62 (s, 1 H), 8.55 (m, 1 H),

(

6

1

3

8.10 (m, 1 H), 5.87 (m, 1 H), 4.35−4.40 (m, 2 H), 3.82−4.05 (m, 2

C

5

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NMR (150 MHz, DMSO-d ) δ 160.8, 159.8, 154.2, 152.3, 150.4,

Ramos AKT-pSer473 Assay. The phosphorylation status of

serine 473 of AKT in the Ramos lymphoma-derived B cell line is

driven by PI3Kδ, hence a good measure of cellular activity of PI3Kδ

inhibition. The Ramos cell line expresses cell surface IgM and

responds to IgM cross-linking by activating PI3Kδ-dependent

signaling. The AlphaScreen SureFire Akt p-Ser473 assay was used

to measure the phosphorylation of endogenous AKT in cellular

lysates. B lymphocyte Ramos cells (ATCC catalog #CRL-1596) were

split 1:3 every 3 to 4 days and maintained between 100,000 and

1,000,000 cells/mL. Cells were diluted in assay media (DMEM, high

glucose, HEPES, No phenol red supplemented with sodium pyruvate)

to a concentration of 10,000,000 cells/mL and plated (6 μL, 60,000

cells per well) using a BioRaptor. Compounds were serially diluted (3-

fold in 100% DMSO) for a 10 concentration dose response, and 10

nL was added to the wells. The mixture was preincubated for 20 min

at RT. Anti-IgM (2 μL of 1 μg/mL) was then added, and the plates

were incubated at 37 °C for 30 min. The cells were then treated with

lysis buﬀer (2 μL of 5×) and incubated for 30 min at RT. In the dark,

acceptor beads (8 μL of 0.039 mg/mL) were added with Ser473

reaction buﬀer and the plates were incubated for 2 h. Donor beads (3

μL of 0.036 mg/mL) were added in the dark and again incubated for

2 h. The plates were then read on an Envision plate reader.

SKBr3 AKT-pThr308 Assay. The phosphorylation status of

threonine 308 of AKT in the breast cancer cell line SKBR3 is driven

by PI3Kα, hence a good measure of cellular activity of PI3Kα

inhibition. The protocol is identical to that used in the Ramos AKT-

pSer473 assay, with the following modiﬁcations. SKBR3 cells are used

and are stimulated instead by heregulin (2 μL of 0.05 μg/mL). In

addition, Thr308 reaction buﬀer was used with the acceptor beads.

Human Whole Blood B-Cell Activation CD69 Biomarker

Assay. B cell activation is linked to a broad number of diseases

including oncology, arthritis, and lupus. Activation of B-cells can be

induced ex vivo by stimulation with antibodies that recognize

components of the B-cell receptor. These antibodies cross-link

receptors on the surface of the B cell, inducing a receptor signaling

cascade that drives cell activation. The B-cell receptor is composed of

three subunits: a transmembrane IgM for antigen recognition, and

CD79a and CD79b, with small cell surface epitopes and prominent

intracellular domains containing ITAM signaling subunits. Human

whole blood was obtained from healthy volunteer donors at Merck &

Co., Inc., Kenilworth, NJ, USA. Using an Echo liquid handler, 120 nL

of the compound in DMSO at varying concentrations in a 96-well

plate was then treated with 100 μL of blood and incubated for 60 min

at 37 °C. To each well was added 11 μL of anti-CD76b antibody (BD

Biosciences), and the mixture was incubated for 3 h at 37 °C. The

reaction was stopped by placing on wet ice for 5 min. Each well was

treated with 50 μL of staining cocktail (CD45-V450, 5 μL; CD3-APC,

5 μL; CD20-PerCP-Cy5-5, 7.5 μL; CD69-FITC, 20 μL in FACS

Buﬀer, 12.5 μL; all from BD Biosciences). The mixture was incubated

for 30 min at 4 °C. The red blood cells were lysed by the addition of

1.8 mL of FACS lysis buﬀer to each well, followed by incubation for

20 min at 20 °C. The plate containing the cells was spun at a rate of

1000 rpm for 5 min, the resulting supernatant was removed, and an

aborbent pad was used to collect any excess liquid. The pellet was

resuspended in each well in 250 μL of FACS reading buﬀer (1× BD

stain buﬀer, 0.5% Pluronic F68, 0.2 mg/mL human IgG), and cells

were transferred to a clean 384 well U-bottom Greiner plate. Fixed

and stained cells are kept at 4 °C before analyzing ﬂuorescence with a

Fortessa A FACS machine. Gating: Gate1 = Lymphocyte, based on

CD45 and side scatter; Gate2 = Singlet (from gate1), based on

forward scatter A&H; Gate3 = B cell (from gate2), which are CD3-

APC negative and CD20-PerCP Cy5.5 positive; Gate4 = Activated B

cell (from gate3), which are CD69-FITC positive.

6

1

5

48.9, 147.7, 139.5, 129.5, 122.9, 121.5, 121.4, 121.0, 76.5, 69.2, 57.5,

7.4, 55.7, 52.8, 45.0, 31.2, 15.3; HRMS (EI) calcd for C H F N O

3

2

25

3

7

+

[

M + H] 492.1971; found 492.1964.

(S)-3-((9-Ethyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-9H-purin-6-yl)-

oxy)pyrrolidin-1-yl)((S or R)-tetrahydrofuran-3-yl)methanone (4e).

solution of (S)-9-ethyl-6-(pyrrolidin-3-yloxy)-8-(6-

triﬂuoromethyl)pyridin-3-yl)-9H-purine (500 mg, 1.32 mmol) in

(

A

(

dioxane (10 mL) was treated with Hunig’s base (0.50 mL, 2.9 mmol),

HATU (700 mg, 1.84 mmol), and tetrahydrofuran-3-carboxylic acid

(

200 mg, 1.72 mmol). The reaction mixture was stirred overnight at

0 °C, then diluted with DCM, and washed with water. The organic

layer was dried (Na SO ) and concentrated to dryness. Chromatog-

2

4

raphy on SiO (0−30% MeOH/DCM using 120 g of silica) gave the

2

desired product as a set of diastereomers. The colorless oil (550 mg)

was then dissolved in 8 mL of MeOH and resolved using chiral

column chromatography (column 21 × 250 mm Chiralpak AD-H; 70

mL/min 30% MeOH/CO₂with 0.25% Me NEt; 0.4 mL per

2

injection) to provide the desired diastereomer (225 mg, t_R= 3.49

min) and undesired diastereomer (275 mg, t = 5.15 min). [α]

=

R

D

1

+

46° (c 0.5, MeOH); H NMR (600 MHz, DMSO-d ) δ 9.17 (s, 1

6

H), 8.60 (s, 1 H), 8.52 (m, 1 H), 8.12 (d, J = 8.1 Hz, 1 H), 5.84−5.93

(

m, 1 H), 4.36 (m, 2 H), 3.82−3.94 (m, 2 H), 3.74−3.80 (m, 1 H),

3

.56−3.73 (m, 2 H), 3.41 (m, 1 H), 3.15−2.25 (m, 2 H), 2.98 (m, 1

H), 2.15−2.37 (m, 2 H), 1.90−2.10 (m, 2 H), 1.32 (2 t, J = 7.2 Hz, 3

H); ¹³C NMR (150 MHz, DMSO-d ) δ 171.7, 159.8, 154.2, 152.4,

6

1

5

50.4, 148.9, 147.6, 139.5, 129.5, 122.8, 121.4, 121.0, 76.2, 70.1, 68.2,

2.3, 44.7, 42.5, 31.7, 30.2, 29.9, 15.3; HRMS (EI) calcd for

+

C H F N O [M + H] 477.1862; found 477.1873.

22

24

3

6

3

(

S)-(3-((9-Ethyl-8-(6-(triﬂuoromethyl)pyridin-3-yl)-9H-purin-6-yl)-

oxy)pyrrolidin-1-yl)(oxazol-4-yl)methanone (4f). A solution of (S)-

9

-ethyl-6-(pyrrolidin-3-yloxy)-8-(6-(triﬂuoromethyl)pyridin-3-yl)-

H-purine (250 mg, 0.661 mmol) in DMF (3 mL) was treated with

Hunig’s base (0.30 mL, 1.7 mmol), HATU (300 mg, 0.789 mmol),

and oxazole-4-carboxylic acid (120 mg, 1.06 mmol). The reaction

mixture was stirred at 20 °C overnight, ﬁltered, and puriﬁed by

reverse-phase chromatography (gradient of MeCN/water with 0.1%

NH4OH) to provide the desired product. [α]_D= +52° (c 0.5,

1

MeOH); H NMR (600 MHz, DMSO-d ) δ 9.17 (m, 1 H), 8.59−

6

8

1

.65 (m, 2 H), 8.51 (m, 1 H), 8.46 (m, 1 H), 8.10 (m, 1 H), 5.94 (m,

H), 4.36 (q, J = 7.1 Hz, 2 H), 4.08−4.26 (m, 2 H), 3.79−3.95 (m, 2

H), 3.75 (m, 1 H), 3.60 (m, 1 H), 2.23−2.42 (m, 2 H), 1.31 (q, J =

7

1

.0 Hz, 3 H); ¹³C NMR (125 MHz, DMSO-d ) δ 160.0, 159.8, 154.2,

6

52.4, 152.3, 150.4, 149.0, 147.8, 144.1, 143.9, 139.5, 136.4, 129.5,

21.4, 121.3, 76.1, 53.5, 45.8, 32.2, 29.4, 15.3; HRMS (EI) calcd for

C H F N O [M + H] 473.1501; found 473.1512.

+

21

19

3

7

3

HTRF PI3K Biochemical Assay. PI3K family biochemical

potencies in the phosphorylation of PIP2 (phosphatidylinositol

4,5)-bisphosphate) to PIP3 (phosphatidylinositol (3,4,5)-trisphos-

(

phate) were measured using an HTRF assay. PI3K biochemical assays

were optimized from an Upstate (Millipore) HTRF kit. Brieﬂy,

compounds were serially diluted (3-fold in 100% DMSO) for a 10

concentration dose response. A PI3K reaction buﬀer was prepared by

dilution of stock with DI water and then treated with DTT, PIP2, and

biotin-PIP3 at ﬁnal concentrations of 5 μM, 5 μM and 25 nM,

respectively. Enzyme and the compounds were added at RT for a 15

min preincubation. Reactions were initiated by addition of substrate

solution (PIP2 and ATP) and incubated at RT for 1 h before

quenching with EDTA. The detection solution (streptavidin-APC

with Eu-labeled anti-GST plus GST-tagged PH-domain) was added

and incubated in the dark for 1 h followed by measurement of the

HTRF signal with an Envision plate reader (330 nm excitation and

dual emission detection at 620 nm (Eu) and 665 nm (APC)). The

individual kinases were purchased from Upstate (PI3Kα 14-602,

PI3Kβ 14-603, PI3Kγ 14-558, and PI3Kδ 14-604). The assay format

was the same for all four isoforms, and the diﬀerences lie in the

concentration of enzyme and ATP used. The PI3Kα, PI3Kβ, PI3Kδ,

and PI3Kγ assays were run with 0.5, 1, 0.3, and 5 nM of enzyme,

respectively. The ATP concentration was 100 μM in the PI3Kα,

PI3Kβ, and PI3Kδ assays and 50 μM in the PI3Kγ assay.

Human Whole Blood Basophil Activation CD63 Biomarker

Assay. Basophils are the least common leukocyte but are an

important eﬀector cell population in allergy, with CD63 being one of

the best described biomarkers of basophil activation. Human whole

blood was obtained from healthy volunteer donors at Merck & Co.,

Inc., Kenilworth, NJ, USA. Using an Echo liquid handler, 120 nL of

the compound in DMSO at varying concentrations was then treated

5

153

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Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

with 100 μL of blood and incubated for 30 min at 37 °C. To each well

was added 20 μL of reagent B (Basotest kit; Gylcotope

Biotechnology, cat#10-0500), and the mixture was incubated for 30

min at 37 °C. Separately, basophil cell activating goat anti-human IgE

antibodies (0.7 μL; Bethyl Laboratories) were prepared to a ﬁnal

concentration of 50 ng/mL in reagent A (200 μL; also from Basotest

kit). Each well was treated with 10 μL of the activating anti-IgE, and

the plates were incubated for 20 min at 37 °C. The reaction was

stopped by placing on wet ice for 5 min. Each well was treated with 30

μL of reagent F stain (anti-human CD63-FITC and anti-human IgE-

PE reagent solution; 10 mL, anti-human CD203c-APC; 1500 μL,

anti-human CD45-V450; 313 μL, in FACS dilution buﬀer; 5 mL).

The mixture was incubated for 20 min at 4 °C. The red blood cells

were lysed by the addition of 1.9 mL of FACS lysis buﬀer to each well,

followed by incubation for 15 min at 20 °C. The plates were spun at

Meredeth A. McGowan − Discovery Chemistry, Merck & Co.,

Inc., Boston, Massachusetts 02115, United States

Neville John Anthony − Discovery Chemistry, Merck & Co.,

Inc., Boston, Massachusetts 02115, United States

Matthew Christopher − Discovery Chemistry, Merck & Co.,

Inc., Boston, Massachusetts 02115, United States

Yudith Garcia − Discovery Chemistry, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States

Abdelghani Achab − Discovery Chemistry, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States

Kathryn Lipford − Discovery Chemistry, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States

Benjamin Wesley Trotter − Discovery Chemistry, Merck &

Co., Inc., Boston, Massachusetts 02115, United States;

orcid.org/0000-0002-6780-0358

1500 rpm for 5 min, and the supernatant aspirated. The pellet was

suspended in 70 μL of FACS reading buﬀer (1× phosphate buﬀered

saline, 0.2% bovine serum albumin, 0.5% pluronic F-68, 0.2 mg/mL

human IgG) and the plate maintained at 4 °C. Plates were read using

LSR Fortessa ﬂow cytometer. An anti-human IgE gate was used to

count basophil cells, and a CD63+ gate was used to analyze for

^a^c^tⁱ^v^a^t^e^d^b^a^s^o^p^hⁱ^l^c^e^l^l^s^.^T^h^e^I^C⁵0 values were calculated from percent

inhibition values of CD63 activated basophils using ADA software to

ﬁt to a sigmoidal dose response.

Michael D. Altman − Computational and Structural

Chemistry, Merck & Co., Inc., Boston, Massachusetts 02115,

United States

Xavier Fradera − Discovery Chemistry, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States; orcid.org/

0000-0002-6118-075X

Charles A. Lesburg − Discovery Chemistry, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States; orcid.org/

HeLa Cell Adenosine Uptake (AdU) Inhibition Assay.

Dilution plates with the compound in DMSO were prepared as a

0000-0001-7245-7331

1

0-point titration and diluted with HBSS with 5% FBS to reach

concentrations of 25,000 to 0.8 nM of the compound. HeLa cells

ATCC) were thawed and seeded at 25,000 cells/well in Cytostar T

Chaomin Li − Process Chemistry, Merck & Co., Inc., Boston,

Massachusetts 02115, United States

(

plates overnight in MEM and 10% FBS. The growth media were

removed by ﬂicking, 40 μL of HBSS with 5% FBS was added, and

then 40 μL of the compound in buﬀer was added to the cells. The

solution was incubated for 30 min. 20 μL of radiolabeled 100 nM 3H-

adenosine (ARC; 40 Ci/mmol speciﬁc activity and 1 mCi/mL

concentration from 25,000 nM stock concentration) in HBSS with 5%

FBS was added, and the solution was incubated for 60 min. A total

volume of 100 μL was reached, with compound concentrations of

Stephen Alves − Discovery Biology, Merck & Co., Inc., Boston,

Massachusetts 02115, United States

Craig P. Chappell − Discovery Biology, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States

Renu Jain − Discovery Biology, Merck & Co., Inc., Boston,

Massachusetts 02115, United States

Ruban Mangado − Discovery Biology, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States

Elaine Pinheiro − Discovery Biology, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States

Sybill M. G. Williams − Discovery Biology, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States

1

0,000 to 0.32 nM. To measure the uptake of radioactive adenosine,

plates were read with a PerkinElmer TopCount NXT HTS plate

reader. Data was analyzed using ADA Logic to ﬁt to a 4-parameter ﬁt

to provide IC₅₀values.

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00237.

Peter Goldenblatt − In Vitro Pharmacology, Merck & Co.,

Inc., Boston, Massachusetts 02115, United States

Armetta Hill − In Vitro Pharmacology, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States

Lynsey Shaﬀer − In Vitro Pharmacology, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States

■

*

Substitution about the pyrrolidine cyclopropane amide

selectivity motif of 3c, crystallographic data collection

and reﬁnement statistics for 3c, molecular formula

strings, and purity HPLC traces for lead compounds

Dapeng Chen − Preclinical Pharmacokinetics and Drug

Metabolism, Merck & Co., Inc., Boston, Massachusetts

02115, United States

(

PDF)

Crystallographic data of 3c (CSV)

Accession Codes

Vincent Tong − Preclinical Pharmacokinetics and Drug

Metabolism, Merck & Co., Inc., Boston, Massachusetts

02115, United States

Robbie L. McLeod − In Vivo Pharmacology, Merck & Co.,

Inc., Boston, Massachusetts 02115, United States

Hyun-Hee Lee − In Vivo Pharmacology, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States

Accession codes have been deposited in the RCSB Protein

Data Bank; Compound 3c (MSD-496486311) ID: 7LM2.

AUTHOR INFORMATION

Hongshi Yu − Discovery Pharmaceutical Sciences, Merck &

Co., Inc., Boston, Massachusetts 02115, United States

Sanjiv Shah − In Vitro Pharmacology, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States

Jason D. Katz − Discovery Chemistry, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States

■

Corresponding Author

Joey L. Methot − Discovery Chemistry, Merck & Co., Inc.,

Boston, Massachusetts 02115, United States; orcid.org/

0

000-0003-3374-181X; Email: joey_methot@merck.com

Authors

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00237

Hua Zhou − Discovery Chemistry, Merck & Co., Inc., Boston,

Massachusetts 02115, United States

5

154

https://doi.org/10.1021/acs.jmedchem.1c00237

J. Med. Chem. 2021, 64, 5137−5156

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Notes

Recent Inhibitor Design Strategies: A Medicinal Chemistry

Perspective. J. Med. Chem. 2019, 62, 4815−4850. (c) Perry, M. W.

D.; Abdulai, R.; Mogemark, M.; Petersen, J.; Thomas, M. J.; Valastro,

B.; Eriksson, A. W. Evolution of PI3K γ and δ Inhibitors for

Inflammatory and Autoimmune Diseases. J. Med. Chem. 2019, 62,

4783−4814.

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors would like to thank Dr. Scott K. Pruitt and Peter

Fuller for assistance in preparing this manuscript.

(9) (a) Williams, O.; Houseman, B. T.; Kunkel, E. J.; Aizenstein, B.;

Hoffman, R.; Knight, Z. A.; Shokat, K. M. Discovery of dual inhibitors

of the immune cell PI3Ks p110 δ and p110 γ : a prototype for new anti-

inflammatory drugs. Chem. Biol. 2010, 17, 123−134. (b) Winkler, D.

J.; Faia, K. L.; DiNitto, J. P.; Ali, J. A.; White, K. F.; Brophy, E. E.;

Pink, M. M.; Proctor, J. L.; Lussier, J.; Martin, C. M.; Hoyt, J. G.;

Tillotson, B.; Murphy, E. L.; Lim, A. R.; Thomas, B. D.; MacDougall,

J. R.; Ren, P.; Liu, Y.; Li, L.-S.; Jessen, K. A.; Fritz, C. C.; Dunbar, J.

L.; Porter, J. R.; Rommel, C.; Palombella, V. J.; Changelian, P. S.;

Kutok, J. L. PI3K-δ and PI3K-γ Inhibition by IPI-145 Abrogates

Immune Responses and Suppresses Activity in Autoimmune and

Inflammatory Disease Models. Chem. Biol. 2013, 20, 1364−1374.

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