Design, Synthesis, and Biological Evaluation of Dimorpholine Substituted Thienopyrimidines as Potential Class I PI3K/mTOR Dual Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b00357

Dysfunctional signaling of the PI3K/AKT/mTOR pathway in cancer and its crucial role in cell growth and survival have made it a much desired target for cancer therapeutics. A series of dimorpholine substituted thienopyrimidine derivatives had been prepared and evaluated in vitro and in vivo. Among them, compound 14o was identified as a dual Class I PI3K and mTOR kinase inhibitor, which had an approximately 8-fold improvement in mTOR inhibition relative to the class I PI3K inhibitor 1 (pictilisib, GDC-0941). Western blot analysis confirmed the 14o mechanistic modulation of the cellular PI3K/AKT/mTOR pathway through inhibiting phosphorylation of both AKT and S6 in human cancer cell lines. In addition, 14o demonstrated significant efficacy in SKOV-3 and U87MG tumor xenograft models without causing significant weight loss and toxicity.

Full text of DOI:10.1021/acs.jmedchem.7b00357

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Article
Design, Synthesis, and Biological Evaluation of Dimorpholine Substituted
Thienopyrimidines as Potential Class I PI3K/ mTOR Dual Inhibitors
Miao Zhan, Yufang Deng, Lifeng Zhao, Guoyi Yan, Fangying
Wang, Ye Tian, Lanxi Zhang, Hongxia Jiang, and Yuanwei Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00357 • Publication Date (Web): 14 Apr 2017
Downloaded from http://pubs.acs.org on April 15, 2017
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Design, Synthesis and Biological Evaluation of
Dimorpholine Substituted Thienopyrimidines as
Potential Class I PI3K/ mTOR Dual Inhibitors
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1
,†
1,†
2
1
1
1
Miao Zhan , Yufang Deng , Lifeng Zhao *, Guoyi Yan , Fangying Wang , Ye Tian , Lanxi
1
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1,3
Zhang , Hongxia Jiang , Yuanwei Chen *
1
State Key Laboratory of Biotherapy, Collaborative Innovation Center of Biotherapy and Cancer
Center, West China Hospital of Sichuan University, Chengdu, 610041, China
2
Chengdu University, Sichuan Industrial Institute of Antibiotics, Chengdu 610052, China
3
Hinova Pharmaceuticals Inc., Suite 402, Building B, #5 South KeYuan Road, Chengdu,
610041, China
KEYWORDS : Class I PI3K/ mTOR Inhibitor; PI3K/AKT/mTOR pathway; Morpholine;
Antitumor.
ABSTRACT: Dysfunctional signaling of the PI3K/AKT/mTOR pathway in cancer, and its
crucial role in cell growth and survival have made it a much desired target for cancer
therapeutics. A series of dimorpholine substituted thienopyrimidine derivatives had been
prepared and evaluated in vitro and in vivo. Among them, compound 14o was identified as a dual
Class I PI3K and mTOR kinase inhibitor, which had an approximately 8ꢀfold improvement in
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mTOR inhibition relative to the class I PI3K inhibitor 1 (pictilisib, GDCꢀ0941). Western blot
analysis confirmed that 14o could mechanistic modulation of cellular PI3K/AKT/mTOR
pathway through inhibiting phosphorylation of both AKT and S6 in human cancer cell lines. In
addition, 14o demonstrated significant efficacy in SKOVꢀ3 and U87MG tumor xenograft models
without causing significant weight loss and toxicity.
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Introduction
The PI3K/AKT/mTOR pathway is one of the most frequently dysregulated signaling
cascades in human malignancies, and plays a central role in cell proliferation, survival,
1
migration, and metabolism.
There are eight mammalian PI3K enzymes, which are grouped into IA, 1B, II, and III
according to their sequence, homology, and substrate preferences. Class IA PI3Ks are
heterodimers that contain one of three PI3K catalytic subunits (p110α, p110β and p110δ) and a
p85 regulatory subunits, whereas Class IB PI3Ks consist of a p110γ catalytic subunit and
2
regulatory subunit p101 or p87. Class I PI3Ks are responsible for the conversion of
3
phosphatidylinositol 4,5ꢀdiphosphate (PIP2) to phosphatidylinositol 3,4,5ꢀtriphosphate (PIP3).
The generation of PIP3 leads to the downstream activation of serineꢀthreonine kinase AKT,
4
which integrates multiple signaling pathways to regulate cell growth and metabolism. Moreover,
the gene PIK3CA that encode the p110α subunit, is overexpressed or mutated in a wide variety of
5ꢀ8
cancers including ovarian, colorectal, glioblastoma, and gastric cancers. The phosphatase and
tensin homologue gene (PTEN), which serves as a critical negative regulator of PI3K signaling
by converting PIP3 back to PIP2, is among the most frequently lost or mutated tumour
9
suppressor genes. In addition, in cancer cells expressing normal PI3K and PTEN genes, other
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lesions are present that can activate the PI3K signaling pathway (mutation and/or amplification
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of genes encoding RTKs, RAS and AKT). Active PI3K signaling is probably an important
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mechanism of resistance to various targeted cancer therapies.
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The kinase mTOR, a member of the PIKK (phosphatidylinositol like kinase) family, has a
high degree of active site similarity with PI3Ks. mTOR can be activated by AKT and lead to
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increased protein synthesis and cell growth.
The relevancy between mTOR and cancers has
been validated by several approved drugs such as everolimus and temsirolimus for the treatment
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of advanced renal cell carcinoma.
Dual PI3K/mTOR inhibitors appear to offer an augment
on better efficacy and less likelihood to induce drug resistance, as they target the pathway at two
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nodal points.
In recent years, many PI3K/mTOR dual inhibitors were discovered including
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PFꢀ04691502 , PIꢀ103 , voxtalisib (XL765) , apitolisib (GDCꢀ0980) , dactolisib (BEZ235)
and so on (Figure 1).
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Figure 1. Examples of PI3K inhibitors
It has been reported that the morpholine ring is the most common feature of PI3K inhibitors
Figure 1), and the morpholine oxygen may form critical hydrogen bond with amino acid
(
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2
residues.
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The potent class I PI3K inhibitor 1 , currently in phase II clinical trials for the treatment of
cancer, served as a useful template to explore new PI3K inhibitors. Starting with 1, considerable
structure−activity relationship (SAR) investigations have been focused on the substituents of
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thienopyrimidine core, and several advanced compounds were developed such as CNXꢀ1351
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and GNEꢀ477 . However, for the Cꢀ2 position, these explorations are restricted to the
replacement of the indazole with aryl or heteroaryl group, and the alkyl, cycloalkyl group or
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saturated heterocycles substituents have barely been reported.
It was conceivable that the
potency as well as other drugꢀlike properties could be further optimized through modification of
1
(Figure 2).
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O
N
O
N
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S
N
S
6
N
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R1
N
N
N
N
R2
O
S
N
NH
R = Alkyl, Aryl
R2 = Secondary amines
O
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Figure 2. Design of series derivatives based on 1.
It is worth mentioning that, several PI3K inhibitors were designed by incorporation of the
second morpholine group into the triazine or pyrimidine core to improve the solubility or
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potency, such as ZSTK474 , buparlisib (NVPꢀBKM120) and gedatolisib (PFꢀ05212384, PKIꢀ
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87) (Figure 1). Herein we disclose the preparation and biological evaluation of a series of
dimorpholine substituted thienopyrimidine derivatives that demonstrate potent inhibition of
PI3K/AKT/mTOR passway, culminating in the discovery of 14o.
CHEMISTRY
As depicted in Scheme 1, the desired 2,4ꢀdimorpholine substituted pyrimidine derivatives
3
2ꢀ34
(3aꢀ3c) were prepared from 2ꢀchloroꢀ4ꢀmorpholinopyrimidine derivatives
2 via amination on
the Cꢀ2 position with morpholine.
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Scheme 1. Synthesis of Compounds 3aꢀ3c
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Scheme 2 describes the general procedure for preparing thieno[3,2ꢀd]pyrimidine derivatives.
Lithiation of the common intermediate 2a followed by acylation with DMF or DMAc provided
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aꢀ4b. Reduction of 4aꢀ4b by NaBH and then nucleophilic substitution with appropriate amine
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yielded compounds 6aꢀ6h. Alternatively, the lithium anion of 7 could be trapped with aldehyde
to give the secondary alcohol derivatives 12a and 12b. Ketone derivative 11 could be easily
obtained through trapping of lithium anion of 7 with DMAc. Oxidization of aldehyde 8 with mꢀ
CPBA resulted in the carboxylic acid derivative 9. And then, the corresponding primary amide
10 was obtained through amidation. Reductive amination of 8 using appropriate amine and
sodium triacetoxyborohydride provided the compound 13. Also in Scheme 2, the intermediate 7
could be converted to the arylated derivatives 14aꢀ14s through iodization and Suzuki coupling
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(For 14c, Stille coupling was used). It was worth noting that 14q was prepared similarly to 14o
from the methyl substituted starting material.
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Scheme 2. Synthesis of thienopyrimidine analogues
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RESULTS AND DISCUSSION
KinaseꢀGlo Plus luminescent assay and Lance Ultra Assay were used for evaluating the
inhibitory activity of the compounds against PI3Kα and mTOR, respectively.
As presented in Table 1, the hit compound 3a showed promising activity against PI3Kα
isoform. Then replacement of the core structure of 3a with purine (3b) or pyrrolo[2,3ꢀ
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d]pyrimidine (3c) caused a decrease in activity, suggesting that thieno[3,2ꢀd]pyrimidine
analogues were worthy of further optimization, although poor mTOR inhibitory activities were
observed.
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Table 1. IC Values for Enzymatic Inhibition of PI3Kα and mTOR
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a
IC50 (nM)
Compd
X
Y
PI3Kα
mTOR
>2000
>2000
>2000
3
a
S
N
CH
NH
NH
1875
>10000
6184
3b
3
c
CH
a
IC50 values for enzymatic inhibition of PI3Kα and mTOR. The IC50 values are the mean of at least three
experiments, with typical variations of less than 20%.
Subsequent efforts were focused on the substitutions of the Cꢀ6 position of thieno[3,2ꢀ
d]pyrimidine ring due to the synthetic accessibility of this position (Table 2). Compared with
compound 3a, 6b substituted with a methylol group in the Cꢀ6 position showed a dramatic
increase in PI3Kα inhibitory activity. Similarly, the carboxylic acid derivative 9 showed
comparable potency to 6b. In contrast, ketone 11 and tertiary amine 13 derivatives displayed
decreased inhibitory activities toward PI3Kα.
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Table 2. In Vitro PI3Kα and mTOR Inhibitory Activities
b
b
IC50 (nM)
IC50 (nM)
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a
a
Compd
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Compd
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R2
PI3Kα
mTOR
PI3Kα
mTOR
HO
N
O
6
6
a
122
632
>10000
>10000
>2000
HO
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N
216
237
675
>2000
>2000
>2000
O
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9
N
N
HO
O
11
14g
14h
14i
165
9468
24
70
>2000
424
O
S
N
N
O
N
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3
4817
>2000
O
HO
N
N
6c
1034
279
>2000
>2000
O
HO
HO
6
d
S
N
N
O
6e
S
N
249
681
603
336
1
4j
>10000
2203
>2000
109
N
HO
HO
HO
6
f
O
1
4k
N
O
6
g
216
299
191
463
335
865
1
4l
3562
222
6h
O
N
1
4m
>10000
>2000
O
N
1
0
H2N
O
N
1
4n
4o
4p
4185
15
172
16
HO
1
2a
239
>2000
O
N
1
HO
1
1
1
2b
4a
4b
2050
>10000
>10000
694
>2000
>2000
225
Ph
O
N
1
1242
>2000
O
N
c
OH
14q
197
995
73
O
N
1
4r
427
N
1
4c
270
O
N
1
4s
1
4955
4.8
>2000
134
N
1
4d
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612
O
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Unless otherwise noted, R
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= H. IC50 values for enzymatic inhibition of PI3Kα and mTOR. The IC50 values are
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the mean of triplicate measurements, with typical variations of less than 20%. R = Methyl.
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Next, we conducted an SAR study by replacing the C2ꢀmorpholine of 6b with other isosteres.
The piperazine derivatives 6c and 6d showed decreased potency when compared to 6b. By contrast,
the thiomorpholine analogue 6e showed slightly decrease in PI3Kα inhibitory activity relative to 6b,
but an improved potency toward mTOR. Modifications to the C2ꢀmorpholine revealed that
compound 6g with cisꢀ2,6ꢀdimethyl substituent on morpholine ring showed improved inhibitory
activity against mTOR and maintained PI3Kα inhibitory activity when compared to 6b and 6e. Next,
we continued to modify the Cꢀ6 position of thieno[3,2ꢀd]pyrimidine core, mainly because of the
greatest degree of flexibility of this region in terms of SAR.
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Upon introducing a methyl group to the α position of primary alcohol 6g, 6a displayed slightly
higher activity against PI3Kα with an IC50 of 122 nM. However, other secondary alcohols 12a and
1
2b did not improve the inhibitory activity in comparison with 6g. 10 with a primary amide group at
Cꢀ6 position displayed slightly lower activities against PI3Kα and mTOR when compared to 6a.
Meanwhile, we concentrated our efforts to modify the Cꢀ6 position of the thienopyrimidine core
with aromatic and heteroaromatic rings. Phenyl substituted analogues 14a and 14b dramatically
decreased PI3Kα inhibitory activity with IC50 values of over 10 ꢁM. 14cꢀ14e were characterized by
replacing the Cꢀ6 benzene of 14a with pyridine. To our delight, metaꢀsubstituted pyridine 14d
showed promising inhibition activities toward PI3Kα and mTOR when compared with orthoꢀ
and paraꢀsubstituted pyridine derivatives (14c and 14e). 2ꢀAminopyridyl derivative 14g exhibited 9ꢀ
fold improvement in mTOR inhibition relative to 14d. Further optimization of 14g followed, 14i
1
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with aminopyrimidine group displayed stronger inhibitory activity on PI3Kα with IC50 value of 24
nM.
Fiveꢀmembered heteroaromatic rings were introduced at Cꢀ6 position for further SAR
investigations. The PI3Kα inhibition activities decreased dramatically when pyrrole, furan, indazole
and thiophene (14jꢀ14n) were installed. In contrast, upon introducing 5ꢀpyrazole led to a more potent
compound 14o, which inhibits PI3Kα with a remarkable IC50 of 15 nM. It is worth mentioning that,
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4o displayed stronger inhibitory activity on mTOR with IC50 values of 16 nM in comparison to 1
with 134 nM. For PI3Kα enzyme potency, the Nꢀmethyl substituted analogue 14p was about 80ꢀfold
less potent than the corresponding NH analogue 14o. Substitution with a methyl group in the Cꢀ7
position, compound 14q showed a potent reduction in activities on PI3Kα and mTOR relative to 14o,
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possibly because of the methyl group induces a conformational change. Replacement of the 5ꢀ
pyrazolyl group with 4ꢀpyrazolyl (14r and 14s) resulted in a large reduction in potency. These results
suggested that the pyrazole NH of 14o forms an important hydrogen bonding interaction with the
target protein.
The activities of 14i and 14o against the Class I PI3Ks and mTOR were presented in Table 3.
Interestingly, 14i also showed an IC50 value of 35 nM against γꢀisozyme, which was 2ꢀfold higher
activity than 14o. It was noteworthy that, compound 14o was almost equipotent against PI3Kα,
PI3Kδ and mTOR, while displaying modest levels of selectivity against PI3Kβ and PI3Kγ, making it
a promising Class I PI3K/mTOR dual inhibitor.
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Table 3. Activities of 14i and 14o against Class I PI3Ks and mTOR (IC50 Values in nM)
Compd
PI3Kα
24
PI3Kβ
317
175
33
PI3Kγ
35
PI3Kδ
171
10
mTOR
424
14i
14o
15
75
16
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4.8
46
3.3
134
a
IC50 values are the mean of triplicate measurements.
36, 37
Genetic aberrations of PI3K pathway frequently occur in various human cancers.
Inhibition
of cell proliferation was measured using the CCKꢀ8 assay with the U87MG (PTEN null) human
glioblastoma cell line, LNCaP (PTEN null) human prostate cancer cell line, PCꢀ3 (PTEN null)
1
7, 38ꢀ
human prostate cancer cell line and SKOVꢀ3 (PIK3CA mutation) human ovarian cancer cell line
40
,
and 1 was employed as positive control (Table 4). Compounds with IC values less than 500 nM
50
on PI3Kα or mTOR were subjected to these cell lines. In general, these results correlated with their
effects on kinases activities.
14o showed somewhat more potent than 1 in these cancer cell lines with submicromolar IC50s,
possibly due to the increased mTOR inhibition activity. Unexpectedly, 14i performed well on kinase
but exhibited weak cellular inhibition. Hence, 14o was selected for further antitumor evaluation in
vitro and in vivo.
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Table 4. Antiproliferative Activities Various Cell Lines.
a
IC50 (ꢁM)
Compd
PCꢀ3
1.38
6.00
14.99
2.26
2.67
1.05
1.32
>25
LNCaP
U87MG
3.29
17.47
>25
SKOVꢀ3
1.22
6
a
2.59
2.06
6.16
4.2
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60
6
b
d
8.24
6
23.33
2.18
6e
>25
6
f
1.82
1.78
2.63
23.55
3.27
1.60
2.38
4.43
3.0
21.2
6.59
>25
2.89
6g
4.99
6h
9
2.17
2.44
>25
>25
10
4.32
2.97
2.49
2.75
12.85
7.00
3.67
>25
7.11
1
2a
14b
4c
>25
9.12
9.88
4.45
>25
1.80
1
0.99
1
4d
4g
15.68
2.90
1
1.13
4.53
2.64
6.77
2.39
0.21
1.10
4.05
0.34
>25
14i
4k
>25
10.02
5.91
1
12.80
>25
14l
5.92
3.05
0.446
1.74
>25
21.77
18.88
0.504
0.83
14n
>25
14o
4q
0.43
8.76
>25
1
1
4r
10.12
0.864
1
0.457
0.55
a
IC , the mean value of triplicate measurements.
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The intracellular PI3K pathway inhibitory activities of 14o were evaluated by Western blot
analysis. We observed that 14o could inhibit the PI3K/AKT/mTOR pathway inside the cancer cells,
as indicated by the doseꢀdependent decreases in phosphorylation of AKT and its downstream target
S6 in U87MG and SKOVꢀ3 cell lines (Figure 3).
0
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2
3
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8
9
0
Figure 3. Effects of 14o on pAKT, AKT, pS6 and S6.
To examine the possible binding modes of 14o, docking analysis was performed using Autodock
4
1
42
43
4.0 . The protein crystal structure of PI3Kα (PDB 4L23) and mTOR (PDB 4JT5) was used,
followed by molecular dynamics simulations to study the binding modes using the Amber 12
44
package .
Three hydrogen bonds interaction between 14o and PI3Kα were observed. The oxygen of C4
morpholine formed a hydrogen bond with Lys802, while C2 dimethylmorpholine oxygen formed an
additional hydrogen bond interaction with the hydroxy group of Ser773. The pyrazole moiety
interacted with the hinge Val851 through a hydrogen bond and formed hydrophobic interactions with
the side chains of Val850, Val851, Met922, Phe930 and Ile932 (Figure 4A). Docking modes of 14o
with mTOR showed that, cisꢀ2,6ꢀdimethylmorpholine has the hydrophobic contacts with Leu2185,
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Trp2239 (hinge region) and Ile2237 (Figure 4B). More importantly, the pyrazole group formed two
hydrogen bonds to the residues of Lys2187 and Glu2190, which may contributed to the improved
mTOR inhibition activity.
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The binding modes of 14o suggested that the newly introduced cisꢀ2,6ꢀdimethylmorpholine and
pyrazole moieties had hydrophobic and/or hydrogenꢀbond interactions with PI3Kα and mTOR
binding sites, which were in accordance with the SAR study in Table 2.
A
B
Figure 4. (A) Proposed binding mode of compound 14o in PI3Kα model. (B) Docking modes for
1
4o into protein crystal structure of mTOR.
The pharmacokinetic properties of 14o were evaluated in SpragueꢀDawley rats (Table 5).
Moderate plasma clearance and volume of distribution were found after intravenous administration at
2
mg/kg. Acceptable oral bioavailability (41%) was achieved in this species.
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Table 5. Pharmacokinetic Data for Compound 14o in Rat
0
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0
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8
9
0
a
b
IV (2 mg/kg)
Cl
PO (10 mg/kg)
V
ss
T
1/2
C
max
AUC
(ꢁM·h)
5.2
F
(
mL/min/kg)
29
(L/kg)
4.4
h
(ꢁM)
1.4
%
41
1.5
a
Male Sprague−Dawley rats were dosed intravenously with 2 mg/kg of 14o prepared in 40% PEG400/ 5% DMSO/
5% Saline. Compound 14o was administered PO at the 10 mg/kg in 0.5% sodium carboxymethylcellulose with
0.2% Tween 80.
5
On the basis of the appealing biochemical and cellular activity and pharmacokinetic properties,
the antitumor efficacy of 14o was evaluated in vivo.
BALB/c nude mice bearing SKOVꢀ3 and U87MG xenograft tumors were treated with 14o by
intragastric administration for 24 and 18 days, respectively. As shown in Figure 5A, the growth of
xenograft tumors was significantly inhibited by 14o in a doseꢀdependent manner. The tumor growth
inhibition (TGI) of 89.6%, 75.9%, 48.5% were observed in the SKOVꢀ3 model at the dose of 60, 30
and 15 mg/kg, respectively. For the U87MG model, administration of 14o at doses of 40 and 20
mg/kg caused respective TGI of 81.2% and 69.2% (p < 0.05). In contrast, 1 as positive control
showed less potent than 14o in both SKOVꢀ3 and U87MG models at the same dose. Moreover, 14o
did not cause significant weight loss and toxicity during the treatment period (See SI).
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Figure 5. Pharmacodynamic profile of 14o in vivo. (A) Growth inhibitory effect of 14o on established U87MG and
SKOVꢀ3 xenografts in female BALB/c nude mice (N = 10 per group, mean ± SD). (B) Immunohistochemical
staining of pAKT(S473), pS6, Ki67 and TUNEL in vehicle or 14oꢀtreated tumors of SKOVꢀ3 xenograft model.
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To further confirm that 14o blocked the PI3K and mTOR activity in vivo, immunohistochemical
(
IHC) analyses of SKOVꢀ3 tumors collected at the end of the dosing period were carried out. As
S473
shown in Figure 5B, 14o caused a doseꢀdependent decrease of pAKT
and pS6 in vivo. In addition,
0
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doseꢀdependent decreases in staining for Ki67 were observed, which confirmed a pronounced
decrease in tumor cell proliferation. Furthermore, increased apoptosis judged by TUNEL staining
was observed in SKOVꢀ3 tumors.
CONCLUSIONS
Structural modifications of 1 led to the discovery of 14o, which had been shown to be a potent
Class I PI3K and mTOR kinase dual inhibitor. Compound 14o showed a strong inhibitory effect on
the growth of U87MG and SKOVꢀ3 xenografts in nude mice. IHC analyses of 14oꢀtreated SKOVꢀ3
S473
tumors demonstrated a doseꢀdependent decrease of pAKT
and pS6, confirming that 14o blocked
the PI3K/AKT/mTOR passway in vivo. By far, the existing data indicated that 14o was a promising
Class I PI3K/mTOR dual inhibitor for oncology indications. The antitumor properties and other
biological activities of 14o in vivo are presently being investigated.
EXPERIMENTAL SECTION
All solvents and reagents obtained from commercial sources were used without further purification.
1
Flash column chromatography was performed using silica gel from Qingdao Haiyang. H NMR and
1
3
C NMR spectra were recorded on a Bruker AMX 400 spectrometer and were calibrated using TMS
1
or residual deuterated solvent as an internal reference (CDCl
3
: 7.26 ppm for H NMR and 77.16 ppm
1
3
1
13
for C NMR; d
6
ꢀDMSO: 2.50 ppm for H NMR and 39.52 ppm for C NMR). The purity of final
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compounds (>95%) were determined by highꢀperformance liquid chromatography (HPLC) analysis
performed on Waters 2695 Series chromatographs. HRMS spectra were recorded on a Shimadzu
LCMSꢀITꢀTOF.
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23
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59
60
General Procedure A for the Synthesis of 3aꢀ3c
To a solution of compound 2 (1 mmol) in 1ꢀbutanol (2 mL) was added morpholine (174 mg, 2
mmol), followed by trifluoroacetic acid (342 mg, 3 mmol). The solution was stirred at 125 °C
overnight. The reaction solution was concentrated and then EtOAc (10 mL) was added. The resulting
mixture was washed with saturated NaHCO
3
(10 mL) and brine (10 mL). The organic phase was
then dried (Na SO ), filtered, and concentrated under reduced pressure. Pure product was obtained
2
4
after flash column chromatography.
4
,4'-(Thieno[3,2-d]pyrimidine-2,4-diyl)dimorpholine (3a) was obtained from 4ꢀ(2ꢀchlorothieno[3,2ꢀ
1
d]pyrimidinꢀ4ꢀyl)morpholine (2a) and morpholine following the General Procedure A. H NMR
3
(400 MHz, CDCl ) δ: 7.60 (d, J = 5.5 Hz, 1H), 7.17 (d, J = 5.5 Hz, 1H), 3.93 – 3.87 (m, 4H), 3.86 –
1
3
3
.75 (m, 12H); C NMR (101 MHz, CDCl ) δ: 164.03, 160.48, 158.76, 131.55, 124.39, 105.78,
3
+
6
7.13, 66.91, 46.43, 45.03. HRMS (DARTꢀTOF) calculated for C14
H
19
4
N O
2
S [M+H] m/z 307.1223,
found 307.1199.
4
,4'-(9H-Purine-2,6-diyl)dimorpholine (3b) was obtained from 4ꢀ(2ꢀchloroꢀ9Hꢀpurinꢀ6ꢀ
1
yl)morpholine (2b) and morpholine following the General Procedure A. H NMR (400 MHz, d
6
ꢀ
1
3
DMSO) δ: 12.43 (s, 1H), 7.78 (s, 1H), 4.33 – 3.94 (m, 4H), 3.72 – 3.56 (m, 12H); C NMR (101
MHz, d
6
ꢀDMSO) δ: 158.16, 153.64, 153.16, 135.73, 113.38, 66.22, 66.10, 44.76. HRMS (DARTꢀ
+
TOF) calculated for C13
H
19
N
6
O
2
[M+H] m/z 291.1564, found 291.1551.
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
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3
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4
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4
,4'-(7H-Pyrrolo[2,3-d]pyrimidine-2,4-diyl)dimorpholine (3c) was obtained from 4ꢀ(2ꢀchloroꢀ7Hꢀ
pyrrolo[2,3ꢀd]pyrimidinꢀ4ꢀyl)morpholine (2c) and morpholine following the General Procedure A.
1
H NMR (400 MHz, CDCl
3
) δ: 9.69 (s, 1H), 6.75 (dd, J = 3.5, 2.3 Hz, 1H), 6.35 (dd, J = 3.5, 2.0 Hz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
1
H), 3.93 – 3.87 (m, 4H), 3.86 – 3.81 (m, 4H), 3.81 – 3.76 (m, 4H), 3.76 – 3.69 (m, 4H); C NMR
(101 MHz, CDCl
3
) δ: 158.83, 157.67, 155.01, 117.78, 101.69, 96.58, 67.13, 66.96, 46.00, 45.31.
+
HRMS (DARTꢀTOF) calculated for C14
H
20
N
5
O
2
[M+H] m/z 290.1612, found 290.1664.
General Procedure B for the Synthesis of 6aꢀ6h
To a suspension of 4ꢀ(2ꢀchlorothieno[3,2ꢀd]pyrimidinꢀ4ꢀyl)morpholine (2a, 1.28 g, 5 mmol) in dry
tetrahydrofuran (20 mL) at ꢀ78 °C was added a 2.5 M solution of nꢀbutyllithium in hexanes (2.4 mL,
1.2 equiv, 6 mmol). After stirring for 1 h, dry N,Nꢀdimethylformamide (548 mg, 1.5 equiv, 7.5
mmol, for 4b) or N,NꢀDimethylacetamide (653 mg, 1.5 equiv, 7.5 mmol, for 4a) was added. The
reaction mixture was stirred for 1 h at ꢀ78 °C and then warmed slowly to room temperature and
stirred for another 2 h. The reaction mixture was poured onto ice/water and extracted with EtOAc.
The organic phase was then dried (Na
2 4
SO ), filtered, and concentrated under reduced pressure. 4a or
4
b was obtained after flash column chromatography.
A solution of 4a (596 mg, 2 mmol) or 4b (567 mg, 2 mmol) in MeOH (10 mL) at 0 °C was treated
with sodium borohydride (757 mg, 20 mmol). The solution was allowed to warm to room
temperature and stirred for 30 min. The reaction mixture was quenched with cold water and extracted
with ethyl acetate. The organic layer dried (Na SO ), filtered, and concentrated under reduced
2
4
pressure to give the crude product 5a or 5b. The reduced product 5a or 5b (0.5 mmol) was dissolved
in 1ꢀbutanol (2 mL), and appropriate amine (1 mmol) was added, followed by trifluoroacetic acid
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(
170 mg, 1.5 mmol). The mixture was stirred at 125 °C overnight and monitored by TLC. The
reaction solution was concentrated and then EtOAc (10 mL) was added. The resulting mixture was
washed with saturated NaHCO (10 mL) and brine (10 mL). The organic phase was then dried
Na SO ), filtered, and concentrated under reduced pressure. Pure product 6aꢀ6h was obtained after
flash column chromatography.
3
10
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14
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(
2
4
1
-(2-(cis-2,6-Dimethylmorpholino)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)ethanol
(6a)
was
obtained from 5a and cisꢀ2,6ꢀdimethylmorpholine following the General Procedure B. H NMR (400
MHz, CDCl ) δ: 6.97 (d, J = 0.7 Hz, 1H), 5.11 (q, J = 6.2 Hz, 1H), 4.48 (dd, J = 12.7, 1.1 Hz, 2H),
.90 – 3.77 (m, 8H), 3.71 – 3.61 (m, 2H), 2.56 (dd, J = 13.0, 10.7 Hz, 2H), 1.59 (d, J = 6.4 Hz, 3H),
1
3
3
1
1
1
3
3
.26 (d, J = 6.2 Hz, 6H); C NMR (101 MHz, CDCl ) δ: 163.78, 160.07, 158.54, 156.42, 119.76,
04.88, 71.94, 66.91, 66.70, 50.11, 46.40, 25.17, 19.11. HRMS (DARTꢀTOF) calculated for
+
C H N O S [M+H] m/z 379.1798, found 379.1777.
18
27
4
3
(
2,4-Dimorpholinothieno[3,2-d]pyrimidin-6-yl)methanol (6b) was obtained from 5b and morpholine
1
following the General Procedure B. H NMR (400 MHz, CDCl ) δ: 7.01 (s, 1H), 4.87 (s, 2H), 3.90 –
3
13
3
.85 (m, 4H), 3.84 – 3.80 (m, 4H), 3.79 – 3.72 (m, 8H); C NMR (101 MHz, CDCl
3
) δ: 163.70,
160.37, 158.47, 150.80, 121.31, 105.60, 67.10, 66.90, 60.74, 46.42, 45.00. HRMS (DARTꢀTOF)
+
calculated for C H N O S [M+H] m/z 337.1329, found 337.1287.
1
5
21
4
3
(2-(4-Methylpiperazin-1-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methanol (6c) was obtained
1
from 5b and 1ꢀmethylpiperazine following the General Procedure B. H NMR (400 MHz, CDCl
3
) δ:
6.99 (s, 1H), 4.85 (s, 2H), 3.83 (dd, J = 14.0, 4.3 Hz, 12H), 2.97 (br s, 1H), 2.51 (t, J = 4.4 Hz, 4H),
13
2
.35 (s, 3H); C NMR (101 MHz, CDCl
3
) δ: 163.77, 160.22, 158.45, 151.10, 121.16, 105.33, 66.90,
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2
2
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5
5
5
6
+
6
0.60, 55.12, 46.40, 46.26, 44.21. HRMS (DARTꢀTOF) calculated for C16
50.1645, found 350.1528.
24 5 2
H N O S [M+H] m/z
3
(
2-(4-(Methylsulfonyl)piperazin-1-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methanol (6d) was
0
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8
9
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8
9
0
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2
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4
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6
7
8
9
0
1
obtained from 5b and 1ꢀ(methylsulfonyl)piperazine following the General Procedure B. H NMR
(400 MHz, CDCl
3
) δ: 6.98 (s, 1H), 5.76 (t, J = 5.7 Hz, 1H), 4.72 (d, J = 5.7 Hz, 2H), 3.94 – 3.76 (m,
1
3
8H), 3.77 – 3.64 (m, 4H), 3.15 (dd, J = 6.1, 2.7 Hz, 4H), 2.87 (s, 3H); C NMR (101 MHz, d
6
ꢀ
DMSO) δ: 163.42, 159.12, 157.81, 153.99, 119.39, 104.03, 65.96, 58.75, 45.84, 45.25, 43.41, 33.70.
+
HRMS (DARTꢀTOF) calculated for C16
H
24
N
5
O
S
4 2
[M+H] m/z 414.1264, found 414.1278.
(
4-Morpholino-2-thiomorpholinothieno[3,2-d]pyrimidin-6-yl)methanol (6e) was obtained from 5b
1
3
and thiomorpholine following the General Procedure B. H NMR (400 MHz, CDCl ) δ: 6.99 (s, 1H),
13
4
.86 (s, 2H), 4.22 – 4.04 (m, 4H), 3.91 – 3.72 (m, 8H), 2.73 – 2.56 (m, 4H); C NMR (101 MHz,
CDCl
3
) δ: 163.89, 159.69, 158.58, 150.66, 121.38, 105.13, 66.89, 60.76, 46.95, 46.44, 27.02. HRMS
+
(DARTꢀTOF) calculated for C15
H
21
N
4
O
2
S
2
[M+H] m/z 353.1100, found 352.1021.
(
2-(2H-Benzo[b][1,4]oxazin-4(3H)-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methanol (6f) was
1
obtained from 5b and 3,4ꢀdihydroꢀ2Hꢀbenzo[b][1,4]oxazine following the General Procedure B. H
NMR (400 MHz, CDCl
3
) δ: 7.96 (dd, J = 8.2, 1.0 Hz, 1H), 7.06 (s, 1H), 6.99 – 6.81 (m, 3H), 4.85 (s,
13
2
H), 4.30 (t, J = 4.2 Hz, 2H), 4.25 (t, J = 4.2 Hz, 2H), 3.94 – 3.72 (m, 8H), 2.74 (br s, 1H); C NMR
3
(101 MHz, CDCl ) δ: 163.29, 158.18, 157.72, 151.29, 146.30, 128.24, 124.11, 123.45, 121.34,
1
19.53, 117.03, 107.01, 66.88, 66.08, 60.63, 46.44, 42.75. HRMS (DARTꢀTOF) calculated for
+
C
19 21 4 3
H N O S [M+H] m/z 385.1329, found 385.1283.
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(
2-(cis-2,6-Dimethylmorpholino)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methanol (6g) was
1
obtained from 5b and cisꢀ2,6ꢀdimethylmorpholine following the General Procedure B. H NMR
(400 MHz, d
6
ꢀDMSO) δ: 6.96 (s, 1H), 5.77 (t, J = 5.2 Hz, 1H), 4.71 (d, J = 4.2 Hz, 2H), 4.44 (dd, J =
10
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14
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60
1
1
1
2.9, 1.5 Hz, 2H), 3.82 – 3.77 (m, 4H), 3.75 – 3.70 (m, 4H), 3.59 – 3.48 (m, 2H), 2.43 (dd, J = 13.0,
1
3
6
0.7 Hz, 2H), 1.15 (d, J = 6.2 Hz, 6H); C NMR (101 MHz, d ꢀDMSO) δ: 163.43, 159.27, 157.79,
53.79, 119.41, 103.81, 71.03, 65.98, 58.75, 49.52, 45.82, 18.82. HRMS (DARTꢀTOF) calculated
+
for C H N O S [M+H] m/z 365.1642, found 365.1642.
17 25
4
3
(
R)-(2-(3-Methylmorpholino)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methanol (6h) was obtained
1
from 5b and (R)ꢀ3ꢀmethylmorpholine following the General Procedure B. H NMR (400 MHz,
CDCl
3
) δ: 6.97 (s, 1H), 4.82 (s, 2H), 4.68 – 4.60 (m, 1H), 4.23 (dd, J = 13.4, 1.9 Hz, 1H), 3.96 (dd, J
=
11.2, 3.2 Hz, 1H), 3.91 – 3.78 (m, 8H), 3.76 – 3.68 (m, 2H), 3.55 (td, J = 12.0, 2.9 Hz, 1H), 3.25
1
3
(td, J = 13.0, 3.6 Hz, 1H), 2.82 (s, 1H), 1.26 (d, J = 6.7 Hz, 3H); C NMR (101 MHz, CDCl
3
) δ:
1
1
63.66, 159.67, 158.44, 151.05, 121.07, 105.24, 71.47, 67.32, 66.84, 60.48, 47.14, 46.36, 39.49,
+
3.56. HRMS (DARTꢀTOF) calculated for C H N O S [M+H] m/z 351.1485, found 351.1354.
1
6
23
4
3
2
,4-Dimorpholinothieno[3,2-d]pyrimidine-6-carboxylic acid (9)
To a suspension of 4,4'ꢀ(thieno[3,2ꢀd]pyrimidineꢀ2,4ꢀdiyl)dimorpholine (3a, 1.54 g, 5 mmol) in dry
tetrahydrofuran (20 mL) at ꢀ78 °C was added a 2.5 M solution of nꢀbutyllithium in hexanes (2.4 mL,
1.2 equiv, 6 mmol). After stirring for 1 h, dry N,Nꢀdimethylformamide (548 mg, 1.5 equiv, 7.5
mmol) was added. The reaction mixture was stirred for 1 h at ꢀ78 °C and then warmed slowly to
room temperature and stirred for another 2 h. The reaction mixture was poured onto ice/water and
2 4
extracted with EtOAc. The organic phase was then dried (Na SO ), filtered, and concentrated under
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6
reduced pressure. 2,4ꢀdimorpholinothieno[3,2ꢀd]pyrimidineꢀ6ꢀcarbaldehyde (8a) was obtained after
flash column chromatography. Compound 8a (334 mg, 1mmol) was suspended in dichloromethane
(5 mL) followed the addition of mꢀCPBA (75%, 230 mg, 1.33 mmol). The resulting mixture was
0
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8
9
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7
8
9
0
heated to reflux for 24 h. Upon completion, the mixture was cooled to room temperature and
1
concentrated. Product 9 was obtained after flash column chromatography (207 mg, 59% yield). H
6
NMR (400 MHz, d ꢀDMSO) δ: 7.21 (s, 1H), 3.84 – 3.77 (m, 4H), 3.77 – 3.70 (m, 4H), 3.67 – 3.59
1
3
(
m, 8H); C NMR (101 MHz, d
6
ꢀDMSO) δ: 164.21, 163.24, 159.48, 158.17, 152.73, 123.22, 106.94,
+
6
6.14, 66.00, 45.91, 44.50. HRMS (DARTꢀTOF) calculated for C15
H
19
4
N O
4
S [M+H] m/z 351.1122,
found 351.1091.
2
-(cis-2,6-Dimethylmorpholino)-4-morpholinothieno[3,2-d]pyrimidine-6-carboxamide (10)
2
ꢀ(cisꢀ2,6ꢀdimethylmorpholino)ꢀ4ꢀmorpholinothieno[3,2ꢀd]pyrimidineꢀ6ꢀcarboxylic acid (9b) was
prepared similarly to 9. To a mixture of 9b (189 mg, 0.5 mmol), 1ꢀhydroxyꢀ7ꢀazabenzotriazole
HOAt, 13.6 mg, 0.1 mmol), Oꢀ(7ꢀazabenzotriazolꢀ1ꢀyl)ꢀ(N,N,N',N'ꢀtetramethyluronium
(
hexafluorophosphate (HATU, 285 mg, 0.75 mmol), and N,Nꢀdiisopropylethylamine (258 mg, 2
mmol) in DMF (1.5 mL) was added ammonium chloride (80 mg, 1.5 mmol). The reaction mixture
was stirred overnight at room temperature. The mixture was diluted with EtOAc, washed with
3 2 4
saturated aqueous NaHCO and brine. The organic phase was then dried (Na SO ), filtered, and
concentrated under reduced pressure. The residue was purified by silica gel chromatography to
1
afford 10 (134 mg, 71% yield). H NMR (400 MHz, CDCl
3
) δ: 7.49 (s, 1H), 6.18 (br s, 2H), 4.50 (dd,
J = 12.9, 1.4 Hz, 2H), 3.94 – 3.80 (m, 8H), 3.66 (dqd, J = 12.5, 6.2, 2.4 Hz, 2H), 2.58 (dd, J = 13.1,
13
1
3
0.7 Hz, 2H), 1.27 (d, J = 6.2 Hz, 6H); C NMR (101 MHz, CDCl ) δ: 163.76, 162.91, 160.15,
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58.71, 142.25, 124.89, 108.60, 71.90, 66.81, 49.98, 46.48, 19.10. HRMS (DARTꢀTOF) calculated
+
for C H N O S [M+H] m/z 378.1594, found 378.1591.
17
24
5
3
1
-(2,4-Dimorpholinothieno[3,2-d]pyrimidin-6-yl)ethanone (11)
10
11
12
13
14
15
16
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18
19
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To a suspension of 4,4'ꢀ(thieno[3,2ꢀd]pyrimidineꢀ2,4ꢀdiyl)dimorpholine (3a, 153 mg, 0.5 mmol) in
dry tetrahydrofuran (2 mL) at ꢀ78 °C was added a 2.5 M solution of nꢀbutyllithium in hexanes (0.24
mL, 1.2 equiv, 0.6 mmol). After stirring for 1 h, dry N,NꢀDimethylacetamide (65 mg, 1.5 equiv, 0.75
mmol) was added. The reaction mixture was stirred for 1 h at ꢀ78 °C and then warmed slowly to
room temperature and stirred for another 2 h. The reaction mixture was poured onto ice/water and
extracted with EtOAc. The organic phase was then dried (Na SO ), filtered, and concentrated under
2
4
1
reduced pressure. 11 was obtained after flash column chromatography (106 mg, 61% yield). H
13
NMR (400 MHz, CDCl
3
) δ: 7.70 (s, 1H), 3.94 – 3.88 (m, 4H), 3.85 – 3.76 (m, 12H), 2.64 (s, 3H); C
NMR (101 MHz, CDCl
3
) δ: 192.50, 162.96, 160.47, 158.88, 146.97, 129.04, 110.24, 67.02, 66.78,
+
4
6.50, 44.84, 27.01. HRMS (DARTꢀTOF) calculated for C16
H
21
N
4
O
3
S [M+H] m/z 349.1329, found
349.1310.
General Procedure C for the Synthesis of 12aꢀ12b
cis-2,6-Dimethyl-4-(4-morpholinothieno[3,2-d]pyrimidin-2-yl)morpholine (7a) was prepared
similarly to 3a. To a suspension of cisꢀ2,6ꢀdimethylꢀ4ꢀ(4ꢀmorpholinothieno[3,2ꢀd]pyrimidinꢀ2ꢀ
yl)morpholine (7a, 0.17 g, 0.5 mmol) in dry tetrahydrofuran (2 mL) at ꢀ78 °C was added a 2.5 M
solution of nꢀbutyllithium in hexanes (0.24 mL, 1.2 equiv, 0.6 mmol). After stirring for 1 h, dry
aldehyde (1.2 equiv, 0.6 mmol) was added. The reaction mixture was stirred for 1 h at ꢀ78 °C and
then warmed slowly to room temperature and stirred for another 2 h. The reaction mixture was
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5
5
5
6
2 4
poured onto ice/water and extracted with EtOAc. The organic phase was then dried (Na SO ),
filtered, and concentrated under reduced pressure. Compound 12 was obtained after flash column
chromatography.
0
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1-(2-(cis-2,6-Dimethylmorpholino)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)propan-1-ol (12a) was
1
obtained from 7a and propionaldehyde following the General Procedure C. H NMR (400 MHz,
CDCl
3
) δ: 6.92 (s, 1H), 4.79 (t, J = 6.4 Hz, 1H), 4.47 (d, J = 12.5 Hz, 2H), 3.90 – 3.77 (m, 8H), 3.71
–
3.60 (m, 2H), 3.14 (br s, 1H), 2.55 (dd, J = 12.9, 10.8 Hz, 2H), 1.91 – 1.75 (m, 2H), 1.26 (d, J = 6.2
1
3
Hz, 6H), 0.96 (t, J = 7.4 Hz, 3H); C NMR (101 MHz, CDCl
3
) δ: 163.46, 159.89, 158.42, 155.49
1
20.12, 104.84, 71.90, 71.77, 66.84, 50.07, 46.33, 31.93, 19.06, 9.89. HRMS (DARTꢀTOF)
+
29 4 3
calculated for C19H N O S [M+H] m/z 393.1955, found 393.1900.
1
-(2-(cis-2,6-Dimethylmorpholino)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)-2-phenylethanol
1
(12b) was obtained from 7a and phenylacetaldehyde following the General Procedure C. H NMR
1
(400 MHz, CDCl
3
) δ: H NMR (400 MHz, CDCl
3
) δ 7.34 – 7.21 (m, 5H), 6.99 (s, 1H), 5.16 (dd, J =
8.3, 4.6 Hz, 1H), 4.49 (d, J = 11.6 Hz, 2H), 3.91 – 3.79 (m, 8H), 3.71 – 3.60 (m, 2H), 3.13 (ddd, J =
2.2, 13.7, 6.6 Hz, 2H), 2.56 (dd, J = 13.0, 10.7 Hz, 2H), 2.18 (br s, 1H), 1.26 (d, J = 6.2 Hz, 6H);
2
1
3
3
C NMR (101 MHz, CDCl ) δ: 158.53, 154.24, 136.89, 129.63, 128.86, 127.20, 120.37, 104.96,
7
31 4 3
1.95, 71.58, 66.92, 50.10, 46.44, 45.74, 19.12. HRMS (DARTꢀTOF) calculated for C24H N O S
+
[M+H] m/z 455.2111, found 455.2086.
4
,4'-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidine-2,4-diyl)dimorpholine
(13). A mixture of 8a (167 mg, 0.5 mmol), 1ꢀmethanesulfonylꢀpiperazine hydrochloride (120 mg, 1.2
equiv), and trimethylorthoformate (159 mg, 3 equiv) was stirred in 1,2ꢀdichloroethane (2 mL) for 6 h
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at room temperature. To this mixture was added sodium triacetoxyborohydride (265 mg, 2.5 equiv),
and the reaction mixture was stirred for 24 h at room temperature and then quenched with brine,
extracted with dichloromethane, dried over MgSO
4
, and the solvent removed in vacuo. The crude
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
product was purified by chromatography (104 mg, 43%). H NMR (400 MHz, CDCl ) δ: 7.02 (s,
3
1
3
1
H), 3.93 – 3.68 (m, 18H), 3.34 – 3.22 (m, 4H), 2.79 (s, 3H), 2.68 – 2.59 (m, 4H); C NMR (101
MHz, CDCl
3
) δ: 160.39, 158.43, 148.07, 123.27, 105.86, 67.09, 66.90, 57.49, 52.40, 46.40, 45.94,
+
4
4.97, 34.54. HRMS (DARTꢀTOF) calculated for C20
83.1769.
H
31
N
6
O
4
S
2
[M+H] m/z 483.1843, found
4
General Procedure D for the Synthesis of 14aꢀ14s (14c and 14q were not included)
To a stirred solution of 7a (1.67 g, 5 mmol) in dry THF (20 mL) was added a 2.5 M solution of nꢀ
butyllithium in hexanes (3 mL, 1.5 equiv, 7.5 mmol) at ꢀ78 °C over a period of 10 minutes, stirred
for 2 h at ꢀ40 °C followed by addition of iodine (1.9 g, 7.5 mmol) in THF (5 mL) at ꢀ78 °C. The
mixture was warmed slowly to room temperature and stirred for 8 h. After the completion of reaction
(monitored by TLC), the reaction was quenched with saturated ammonium chloride and extracted
with EtOAc (2 x 50 mL). The organic layer was washed with sodium thiosulphate solution, dried
over anhydrous Na
2 4
SO and concentrated in vacuo. The crude product was purified by
chromatography to afford iodine substituted compound 15 (1.7 g, 75%)
Under N
2
atmosphere, a mixture of iodine substituted compound 15 (230 mg, 0.5 mmol), boronic
acid/ester (1.2 equiv), Na CO (106 mg, 2 equiv), PdCl (dppf) (18 mg, 5 mol%), 1,4ꢀdioxane (1.5
2
3
2
2
ml), and water (0.5 mL) was heated to 100 °C for 16 h, the reaction mixture was cooled, diluted with
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2
2
2
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3
3
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4
4
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5
5
5
5
5
5
5
6
ethyl acetate, washed with brine, dried over Na
2 4
SO , and the solvent was removed in vacuo.
Purification on silica yielded the desired compound 14aꢀ14s (14c and 14q are not included).
cis-2,6-Dimethyl-4-(4-morpholino-6-phenylthieno[3,2-d]pyrimidin-2-yl)morpholine
(14a)
was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
obtained from 15 and phenylboronic acid following the General Procedure D. H NMR (400 MHz,
CDCl
3
) δ: 7.74 – 7.63 (m, 2H), 7.48 – 7.32 (m, 4H), 4.53 (dd, J = 12.9, 1.4 Hz, 2H), 3.94 – 3.82 (m,
8H), 3.69 (dqd, J = 12.3, 6.1, 2.4 Hz, 2H), 2.59 (dd, J = 13.0, 10.7 Hz, 2H), 1.28 (d, J = 6.2 Hz, 6H);
1
3
3
C NMR (101 MHz, CDCl ) δ: 164.48, 160.18, 158.46, 149.18, 133.50, 129.27, 129.18, 126.37,
1
27 4 2
19.90, 105.37, 71.94, 66.92, 50.08, 46.47, 19.12. HRMS (DARTꢀTOF) calculated for C22H N O S
+
[
M+H] m/z 411.1849, found 411.1860.
2-(2-(cis-2,6-Dimethylmorpholino)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)phenol (14b) was
1
obtained from 15 and 2ꢀhydroxyphenylboronic acid following the General Procedure D. H NMR
(400 MHz, CDCl
3
) δ: 7.76 (s, 1H), 7.52 (dd, J = 8.0, 1.3 Hz, 1H), 7.23 (td, J = 8.2, 1.4 Hz, 1H), 6.97
–
6.91 (m, 2H), 4.50 (d, J = 12.2 Hz, 2H), 3.96 – 3.82 (m, 8H), 3.74 – 3.64 (m, 2H), 2.61 (dd, J =
1
3
12.8, 10.8 Hz, 2H), 1.24 (d, J = 6.2 Hz, 6H); C NMR (101 MHz, CDCl ) δ: 159.91, 158.25, 154.17,
3
1
45.75, 130.46, 129.36, 122.78, 120.63, 120.50, 117.02, 105.54, 71.99, 66.94, 50.43, 46.52, 19.09.
+
HRMS (DARTꢀTOF) calculated for C22
H
27
N
4
O
3
S [M+H] m/z 427.1798, found 427.1806.
cis-2,6-Dimethyl-4-(4-morpholino-6-(pyridin-2-yl)thieno[3,2-d]pyrimidin-2-yl)morpholine
Under N atmosphere, a mixture of 15 (230 mg, 0.5 mmol), 2ꢀ(tributylstannyl)pyridine (368 mg, 2
equiv), PdCl (PPh ) (18 mg, 5 mol%), in dry DMF was heated to 100 °C for 2 days, the reaction
(14c)
2
2
3 2
mixture was cooled, diluted with ethyl acetate, washed saturated aqueous NaHCO
3
solution and
brine respectively, dried over Na SO , and the solvent was removed in vacuo. Purification on silica
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Journal of Medicinal Chemistry
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yielded the desired compound 14c (109 mg, 53%). H NMR (400 MHz, CDCl
3
) δ: 8.62 (d, J = 4.7
Hz, 1H), 7.82 – 7.72 (m, 2H), 7.61 (s, 1H), 7.29 – 7.23 (m, 1H), 4.53 (d, J = 12.8 Hz, 2H), 3.97 –
3
6
1
.93 (m, 4H), 3.87 – 3.82 (m, 4H), 3.74 – 3.64 (m, 2H), 2.59 (dd, J = 12.8, 10.9 Hz, 2H), 1.28 (d, J =
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.2 Hz, 6H); C NMR (101 MHz, CDCl ) δ: 164.16, 160.11, 158.65, 151.73, 149.77, 149.30,
3
37.04, 123.60, 120.85, 119.87, 107.08, 71.91, 66.90, 50.04, 46.48, 19.10. HRMS (DARTꢀTOF)
+
calculated for C21
H
26
N
5
O
2
S [M+H] m/z 412.1802, found 412.1763.
cis-2,6-Dimethyl-4-(4-morpholino-6-(pyridin-3-yl)thieno[3,2-d]pyrimidin-2-yl)morpholine
(14d)
was obtained from 15 and 3ꢀpyridineboronic acid pinacol ester following the General Procedure D.
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H NMR (400 MHz, CDCl ) δ: 8.95 (d, J = 1.5 Hz, 1H), 8.61 (d, J = 3.8 Hz, 1H), 7.94 (d, J = 8.0 Hz,
3
1
–
H), 7.44 (s, 1H), 7.37 (dd, J = 7.8, 4.9 Hz, 1H), 4.53 (d, J = 12.2 Hz, 2H), 3.95 – 3.84 (m, 8H), 3.74
1
3
3.64 (m, 2H), 2.60 (dd, J = 12.8, 10.9 Hz, 2H), 1.28 (d, J = 6.2 Hz, 6H); C NMR (101 MHz,
CDCl
3
) δ: 164.25, 160.19, 158.45, 150.11, 147.35, 145.09, 133.43, 129.61, 123.88, 121.12, 105.67,
+
7
1.91, 66.87, 50.01, 46.45, 19.10. HRMS (DARTꢀTOF) calculated for C21
12.1802, found 412.1764.
H
26
N
5
O
2
S [M+H] m/z
4
cis-2,6-Dimethyl-4-(4-morpholino-6-(pyridin-4-yl)thieno[3,2-d]pyrimidin-2-yl)morpholine (14e) was
1
obtained from 15 and pyridineꢀ4ꢀboronic acid following the General Procedure D. H NMR (400
MHz, CDCl
3
) δ: 8.66 (dd, J = 4.6, 1.6 Hz, 2H), 7.55 (s, 1H), 7.53 (dd, J = 4.5, 1.6 Hz, 2H), 4.53 (dd,
J = 13.1, 1.6 Hz, 2H), 3.94 – 3.89 (m, 4H), 3.89 – 3.84 (m, 4H), 3.73 – 3.64 (m, 2H), 2.60 (dd, J =
1
3
1
1
3.2, 10.7 Hz, 2H), 1.28 (d, J = 6.3 Hz, 6H); C NMR (101 MHz, CDCl ) δ: 163.99, 160.17, 158.48,
3
50.69, 145.44, 140.65, 122.24, 120.26, 106.12, 71.88, 66.82, 49.97, 46.43, 19.08. HRMS (DARTꢀ
+
TOF) calculated for C21
H
26
N
5
O
2
S [M+H] m/z 412.1802, found 412.1764.
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