2-Aminomethylthieno[3,2-d]pyrimidin-4(3H)-ones bearing 3-methylpyrazole hinge binding moiety: Highly potent, selective, and time-dependent inhibitors of Cdc7 kinase

Article abstract of DOI:10.1016/j.bmc.2017.04.044

In order to increase the success rate for developing new Cdc7 inhibitors for cancer therapy, we explored a new chemotype which can comply with the previously-constructed pharmacophore model. Substitution of a pyridine ring of a serendipitously-identified Cdc7 inhibitor 2b with a 3-methylpyrazole resulted in a 4-fold increase in potency and acceptable kinase selectivity, leading to the identification of thieno[3,2-d]pyrimidin-4(3H)-one as an alternative scaffold. Structure-activity relationship (SAR) study revealed that incorporation of a substituted aminomethyl group into the 2-position improved kinase selectivity. Indeed, a pyrrolidinylmethyl derivative 10c was a potent Cdc7 inhibitor (IC₅₀?=?0.70?nM) with high selectivity (Cdk2/Cdc7?≥?14,000, ROCK1/Cdc7?=?200). It should be noted that 10c exhibited significant time-dependent Cdc7 inhibition with slow dissociation kinetics, cellular pharmacodynamic (PD) effects, and COLO205 growth inhibition. Additionally, molecular basis of high kinase selectivity of 10c is discussed by using the protein structures of Cdc7 and Cdk2.

Full text of DOI:10.1016/j.bmc.2017.04.044

Accepted Manuscript
2-Aminomethylthieno[3,2-d]pyrimidin-4(3H)-ones Bearing 3-Methylpyrazole
Hinge Binding Moiety: Highly Potent, Selective, and Time-Dependent Inhibi-
tors of Cdc7 Kinase
Osamu Kurasawa, Misaki Homma, Yuya Oguro, Tohru Miyazaki, Kouji Mori,
Noriko Uchiyama, Kenichi Iwai, Akihiro Ohashi, Hideto Hara, Sei Yoshida,
Nobuo Cho
PII:
S0968-0896(17)30310-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.044
BMC 13714
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 February 2017
27 April 2017
28 April 2017
Please cite this article as: Kurasawa, O., Homma, M., Oguro, Y., Miyazaki, T., Mori, K., Uchiyama, N., Iwai, K.,
Ohashi, A., Hara, H., Yoshida, S., Cho, N., 2-Aminomethylthieno[3,2-d]pyrimidin-4(3H)-ones Bearing 3-
Methylpyrazole Hinge Binding Moiety: Highly Potent, Selective, and Time-Dependent Inhibitors of Cdc7 Kinase,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.04.044
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2-Aminomethylthieno[3,2-d]pyrimidin-4(3H)-ones Bearing 3-Methylpyrazole Hinge Binding
Moiety: Highly Potent, Selective, and Time-Dependent Inhibitors of Cdc7 Kinase
Osamu Kurasawa,*,a Misaki Homma,a Yuya Oguro,a Tohru Miyazaki, a Kouji Mori,a Noriko Uchiyama,a
Kenichi Iwai,a Akihiro Ohashi,a Hideto Hara,a Sei Yoshida,a and Nobuo Choa
a Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., 26-1, Muraoka-Higashi
2-chome, Fujisawa, Kanagawa 251-8555, Japan
*To whom correspondence should be addressed. Tel: +81-466-32-1177. Fax: +81-466-29-4456. E-mail:
osamu.kurasawa@takeda.com.
Abstract
In order to increase the success rate for developing new Cdc7 inhibitors for cancer therapy, we
explored a new chemotype which can comply with the previously-constructed pharmacophore model.
Substitution of a pyridine ring of a serendipitously-identified Cdc7 inhibitor 2b with a 3-methylpyrazole
resulted in a 4-fold increase in potency and acceptable kinase selectivity, leading to the identification of
thieno[3,2-d]pyrimidin-4(3H)-one as an alternative scaffold. Structure-activity relationship (SAR) study
revealed that incorporation of a substituted aminomethyl group into the 2-position improved kinase
selectivity. Indeed, a pyrrolidinylmethyl derivative 10c was a potent Cdc7 inhibitor (IC50 = 0.70 nM) with
high selectivity (Cdk2/Cdc7 = >14000, ROCK1/Cdc7 = 200). It should be noted that 10c exhibited
significant time-dependent Cdc7 inhibition with slow dissociation kinetics, cellular pharmacodynamic
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(PD) effects, and COLO205 growth inhibition. Additionally, molecular basis of high kinase selectivity of
10c is discussed by using the protein structures of Cdc7 and Cdk2.
Graphical Abstract
Keywords
Cell division cycle 7 (Cdc7)
Time-dependent Cdc7 inhibition
Thieno[3,2-d]pyrimidin-4(3H)-one
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Introduction
Cell division cycle 7 (Cdc7) is a serine/threonine kinase that plays an important role in initiation
and maintenance of DNA replication in eukaryotic cells. The Cdc7 kinase is activated by binding of a
regulatory subunit Dumbbell former 4 (Dbf4), which controls transition from G1 phase to S phase by
phosphorylation of Ser40 and Ser53 in minichromosome maintenance 2 (MCM2).1-2 Cdc7 and Dbf4 are
overexpressed in various cancer cells and significantly involve in the proliferation of tumor.3 In cancer
cells, suppression of Cdc7 expression by siRNA causes a prolonged S phase and subsequent
p53-independent apoptotic cell death. By contrast, in normal cells, the suppression induces G1 arrest in a
p53-dependent fashion with maintaining cell viability.4 Therefore, a selective Cdc7 inhibitor is expected
to be a safer agent for the treatment of cancers than existing cell cycle inhibitors.
We previously reported the 2,3-dihydrothieno[3,2-d]pyrimidin-4(1H)-ones as a new series of
Cdc7 inhibitors that displayed potent enzymatic inhibitory activity and high selectivity for Cdc7 over
ROCK1 and Cdk2/cycE.5 To increase the success rate for developing Cdc7 inhibitors as a novel
anticancer agent, we next searched for a new chemotype which can comply with the
previously-constructed pharmacophore model.5 The key feature of the scaffold which could fit in the
model was a bicyclic aromatic carboxamide bearing a heteroaryl group as a hinge binding moiety. We
thus focused on a thieno[3,2-d]pyrimidin-4(3H)-one 2b which was serendipitously obtained as a
by-product for the preparation of the 2,3-dihydrothieno[3,2-d]pyrimidin-4(1H)-one 2a probably due to
steric hindrance of the ketone (Figure 1). Even though 2b showed moderate Cdc7 inhibitory activity (IC50
= 290 nM), the scaffold has a cyclic amide which can interact with the salt bridge formed by Lys90 and
Asp196 residues in the Cdc7 protein. This finding led us to modify the hinge binding moiety based on the
structure-activity relationships (SARs) of the 2,3-dihydrothieno[3,2-d]pyrimidin-4(1H)-one series.
Replacement of the pyridine ring of 2b with the 3-methylpyrazole significantly enhanced the potency,
giving a potent Cdc7 inhibitor 7a (IC50 = 75 nM). Moreover, acceptable kinase selectivity for Cdc7 over
Cdk2 and ROCK1 and chemical tractability of 7a encouraged us to explore new Cdc7 inhibitors
possessing the scaffold.
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Figure 1. Design of novel 6-(3-substituted-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4(3H)-ones
In this report, we describe synthesis and biological evaluation of
6-(3-substituted-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4(3H)-one derivatives as a novel selective Cdc7
kinase inhibitor showing time-dependent inhibition and slow dissociation property. Furthermore, on the
basis of the protein structures of Cdc7 and Cdk2, we discuss how high selectivity of the representative
compound 10c is achieved.
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Chemistry
The general synthetic route of 6-heteroarylthieno[3,2-d]pyrimidin-4(3H)-one derivatives
3,7a-e,f',g,h possessing various substituents at the 2-position is described in Scheme 1. Acylation of the
carboxamide 1 with chloroacetyl chloride, followed by substitution with pyrrolidine, and subsequent ring
closure by heating with 2 M NaOH gave the 6-pyridyl analog 3. Saponification of the ester 46 and
subsequent neutralization exclusively provided a decarboxylation byproduct instead of the desired
carboxylic acid. As reported previously,5 the problem was solved by direct condensation of the sodium
salt of the carboxylic acid with ammonium chloride, affording the desired amide 5 in 76% yield. The
thieno[3,2-d]pyrimidin-4(3H)-one scaffold was constructed by a two-step procedure, i.e. condensation of
5 with acid chlorides or carboxylic acids, followed by cyclization under basic conditions, to furnish 6a-f.
The pyrrolidinylethyl derivative 6g was prepared from 5 via a three-step protocol similar to that described
for the preparation of 3. Alternatively, construction of the thienopyrimidinone scaffold was achieved by a
facile one-step reaction. Specifically, compound 4 was reacted with morpholine-4-carbonitrile under
acidic conditions to give an amidine intermediate, which was easily cyclized to provide the target
compound 6h. Suzuki coupling reaction of 6a-c,f with a Boc-protected methylpyrazolyl boronic acid
proceeded smoothly with simultaneous removal of the Boc group on the pyrazole ring to yield the desired
products 7a-c,f. In the case of 7d,e,g,h, the crude Suzuki reaction mixtures were treated with 8 M NaOH
to remove the residual Boc groups because complete removal did not occur in the coupling step. With
regard to 7f, deprotection of the Boc group on the basic nitrogen using 4 M HCl in EtOAc produced the
corresponding 7f' as a dihydrochloride salt.
Scheme 1
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O
N
O
a
S
S
NH
NH2
NH2
N
N
N
1
3
d
O
N
O
O
O
N
b
c
e
S
S
S
S
HN
N
NH
OMe
NH2
NH2
NH2
NH
Br
Br
Br
R
R
Me
4
5
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
7g
7h
7f'
[R = Me]
[R = Me]
[R = cyclopentylmethyl]
[R = cyclohexyl]
[R = Ph]
[R = CH2OEt]
[R = CH2N(Boc)Me]
[R = 2-(pyrrolidin-1-yl)eth-1-yl]
[R = morpholin-1-yl]
[R = CH2NHMe, 2HCl]
[R = cyclopentylmethyl]
[R = cyclohexyl]
[R = Ph]
[R = CH2OEt]
[R = CH2N(Boc)Me]
6g
6h
[R = 2-(pyrrolidin-1-yl)eth-1-yl]
[R = morpholin-1-yl ]
f
Reagents and conditions: (a) ClCH2COCl, TEA, THF, rt, then pyrrolidine, 80 °C, then 2 M NaOH,
100 °C, 28%; (b) NaOH, MeOH–water, 70 °C, then NH4Cl, TEA, HOBt, EDCI, DMF, rt, 76%; (c)
RCOCl, TEA or pyridine, with or without DMAP, THF, rt or 70 °C, then 2 M NaOH, with or without
EtOH, 70 °C or 120 °C, 59–89% for 6a,c-e; RCOOH, HATU, DIPEA, DMF, 70 °C, then 4 M NaOH,
EtOH, 70 °C, 59% for 6b; RCOOH, i-BuOCOCl, TEA, 60 °C, then 2 M NaOH, EtOH, 70 °C, 97% for
6f; ClCH2CH2COCl, TEA, THF, rt, then pyrrolidine, rt, then 2 M NaOH, 100 °C, 74% for 6g; (d)
morpholine-4-carbonitrile, 4 M HCl in CPME, 110 °C for 6h; (e) tert-butyl
3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate, PdCl2(dppf),
Na2CO3 or Cs2CO3, DME–water, 80–100 °C, 36–63% for 7a-c,f; tert-butyl
3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate, PdCl2(dppf),
Na2CO3, DME–water, 100 °C, then 8 M NaOH, 100 °C, 42–58% for 7d,e,g,h; (f) 4 M HCl in EtOAc,
MeOH, rt, 13% (2 steps).
Chemical modification of the basic group at the 2-position and the substituent on the pyrazole ring
was performed according to the synthetic route outlined in Scheme 2. The thienopyrimidinone scaffold
was constructed from the ester 4 by the above-mentioned one-step protocol using chloroacetonitrile to
provide the 2-chloromethyl derivative 8 as a key intermediate. Incorporation of various amines into the
2-position furnished the aminomethyl derivatives 9a,c,d,f,g,i-k. In the case of compound 9b,h,
microwave synthesizer was used for the substitution reaction and the crude reaction mixtures were
subjected to the next reaction without further purification. Treatment of 8 with 2,5-dihydro-1H-pyrrole
didn’t give the corresponding amination product. Instead, LC-MS data of the reaction mixture suggested
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further oxidation proceeded to yield the pyrrol-1-ylmethyl analog 9e; therefore, the mixture was also used
directly without any purification in the next step. Suzuki coupling of the tertiary amines 9b-k with
properly-protected pyrazolylboronic acid esters gave the corresponding coupling products 10b-m. In the
case of 10c,f,g,j,k, treatment of 8 M NaOH was needed to complete Boc deprotection. By contrast,
Suzuki coupling of 9a was unsuccessful probably due to deactivation of the palladium catalyst by the
secondary amine group. On the basis of the result, protection of the amine was attempted utilizing
intramolecular acetalization. Compound 9a was readily reacted with formaldehyde to afford the tricyclic
compound 11, which was converted to the desired product 10a via Suzuki coupling and subsequent
de-acetalization with trifluoroacetic acid (TFA). Finally, the free amines 10b,h,j,m were treated with HCl
solution to afford the dihydrochlorides 10b',h',j',m', respectively, with simultaneous deprotection of
sulfamoyl group in the case of 10m.
Scheme 2
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Reagents and conditions: (a) chloroacetonitrile, 4 M HCl in CPME, 70 °C, 100%; (b) RH (amine)a,
K2CO3, NaI, DMF or DMA, 70 °C, 37–97% for 9a,c,d,f,g,i-k; (c) pyrazolylboronic acid derivativeb,
PdCl2(dppf), Na2CO3, DME–water, 100 °C, 32–58% for 10d,i,l,m,11; (d) pyrazolylboronic acid
derivativeb, PdCl2(dppf), Na2CO3, DME–water, 100 °C then 8 M NaOH, 100 °C, 20–48% for 10c,f,g,j,k;
(e) RH, with or without K2CO3, with or without NaI, DMF or DMA, 70 °C or 100 °C (microwave was
used for 9b,h.) then pyrazolylboronic acid derivativeb, PdCl2(dppf), Na2CO3 or Cs2CO3, DME–water,
reflux or 100 °C, 23% (10e) for 10b,e,h; (f) 37% formaldehyde aq., THF, rt, 54%; (g) TFA, MeOH, 70 °C,
44% for 10a; (h) 10% HCl in MeOH, MeOH, 13–24% (3 steps); (i) 4 M HCl in EtOAc, MeOH, 65% (2
steps); (j) 1 M HCl in Et2O, MeOH, 60 °C, 75% (2 steps).
a2,5-Dihydro-1H-pyrrole was used as RH for the preparation of 10e.
bBoronic acid ester A was used for the preparation of 10b-k,12. Boronic acid ester B and C were used
for the preparation of 10l,m.
Incorporation of an additional substituent into the benzylic position of the 2-substituent of 10c was
conducted by the route shown in Scheme 3. The amide 5 was converted to compound 13a,b using
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α-haloacyl chlorides by a three-step protocol, i.e. acylation of the primary amine, reaction with
pyrrolidine, and subsequent cyclization, described in Scheme 1. Finally, the 3-methylpyrazole group was
introduced into the 6-position of 13a,b by Suzuki coupling reaction to afford the target compounds 14a,b.
Scheme 3
Reagents and conditions: (a) MeBrCHCOCl or PhClCHCOCl, TEA, THF, rt, then pyrrolidine, 70 °C,
then 2 M NaOH, 120–150 °C, 80–84%; (b) tert-butyl
3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate, PdCl2(dppf),
Na2CO3, DME–water, 100 °C, 53–73%.
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Results and Discussion
Biology. All synthesized compounds were evaluated for the Cdc7 kinase inhibitory activity to investigate
SAR. To assess kinase selectivity profiles, inhibitory activities against Cdk2/cyclinE kinase and ROCK1
were also examined. For in vitro pharmacodynamic (PD) analysis, phosphorylation status of Ser40 in
MCM2 was measured using HeLa cells (cervix adenocarcinoma cell line). In cell growth inhibition assay,
COLO205 cells (colorectal adenocarcinoma cell line) were used.
To investigate SARs of the 2-substituent of the lead compound 7a, modification of the substituent
was performed with maintaining the 3-methylpyrazol-4-yl group as a hinge binder (Table 1, 2).
Introduction of a bulky cyclohexyl group (7c) to the 2-position produced 28-fold increase in Cdc7
inhibitory activity and remarkable improvement in kinase selectivity compared with the methyl group
(7a). By contrast, introduction of a phenyl group (7d) led to 6-fold less potent activity relative to the
cyclohexyl group (7c). The finding indicates that an aliphatic group is preferable to an aromatic group for
the 2-substituent. Other 2-aliphatic analogs having one or two hetero atoms (7e,f',h) tended to show one
or two digit nanomolar activities. In particular, N-methylaminomethyl derivative 7f' displayed potent
Cdc7 inhibitory activity (IC50 = 1.4 nM) with 3800-fold kinase selectivity over Cdk2/cycE. The result
suggests that aminomethyl motif is more favorable than alkylamino and alkoxymethyl congeners as the
2-substituent in terms of both potency and kinase selectivity (Table 1).
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Table 1. Effects of 2-substituent on Cdc7 kinase inhibition and kinase selectivity (I)
Enzyme inhibition: IC50 (nM)a
Selectivity
Compound
R
Cdc7
Cdk2/cycE
ROCK1
Cdk2/Cdc7
ROCK1/Cdc7
7a
75 (55–100)
990 (790–1300)
1500 (1200–1800)
x13
x810
x27
x20
Me
7c
7d
7h
7e
7f'
2.7 (1.8–4.0)
15 (14–16)
2200 (1700–2800)
410 (270–630)
940 (790–1100)
x350
x110
x410
x84
1600 (1200–2100)
4.1 (3.1–5.4)
25 (19–34)
1100 (1000–1200) 1700 (1500–1900)
3600 (3100–4200) 2100 (1800–2500)
x270
x140
x3800
1.4 (1.0–1.8)
5300 (4800–5800)
360 (310–420)
x260
aNumbers in parentheses represent 95% confidence interval.
Next, the 2-substituent with the aminomethyl motif was further examined and the results are
presented in Table 2. Replacement of the N-methyl group of 7f' with an N-benzyl group resulted in ca.
4-fold decrease in Cdc7 inhibitory activity (10a). Since introduction of an additional methyl group to the
nitrogen atom of 7f' gave comparable activity (10b'), secondary cyclic amine was incorporated into the
2-methyl group (10c-d). As a result, pyrrolidine and piperidine derivatives 10c,d turned out to be potent
and selective Cdc7 inhibitors. On the other hand, pyrrole and cyclopentane derivatives 10e,7b also
exhibited potent Cdc7 inhibitory activity but diminished kinase selectivity. Moreover, one carbon chain
extension of the 2-substituent of 10c resulted in 200-fold drop in activity concomitant with decreased
kinase selectivity (7g). These results strongly suggested that the basic nitrogen placed at the beta position
of the 2-substituent is crucial for potent Cdc7 inhibition as well as kinase selectivity at least against
Cdk2/cycE. Accordingly, the pyrrolidinylmethyl moiety was the most favorable 2-substituent. As
discussed later, the docking study data with the structure of Cdc7 protein also supports the SAR results.
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Table 2. Effects of 2-substituent on Cdc7 kinase inhibition and kinase selectivity (II)
Enzyme inhibition: IC50 (nM)a
Selectivity
Compound
R
Cdc7
Cdk2/cycE
ROCK1
Cdk2/Cdc7
ROCK1/Cdc7
7f'
1.4 (1.0–1.8)
5300 (4800–5800)
360 (310–420)
x3800
x670
x260
10a
10b'
10c
10d
10e
7b
5.1 (3.9–6.7)
2.2 (0.94–5.3)
0.7 (0.51–0.96)
2.4 (2.0–3.0)
4.7 (3.3–6.6)
1.5 (0.48–4.7)
140 (110–180)
3400 (3000–3900)
6800 (6300–7300)
>10000
610 (500–750)
260 (220–300)
140 (130–160)
200 (170–220)
370 (320–420)
400 (340–460)
1600 (1300–1800)
x120
x120
x200
x83
x3100
x14000
x4200
x170
>10000
810 (720–920)
560 (500–620)
>10000
x79
x370
x270
x11
7g
x70
aNumbers in parentheses represent 95% confidence interval.
Introduction of an additional substituent into the 2-pyrrolidinylmethyl group was subsequently
executed (Table 3). Incorporation of a methyl or phenyl group at the benzylic position of 10c was
detrimental to both Cdc7 inhibitory activity and kinase selectivity (14a,b). A similar tendency was
observed in the case of introduction of a phenyl group into the pyrrolidine ring (10i); however, the
decrease in Cdc7 inhibition and kinase selectivity is not noticeable when compared to 14b. In contrast,
various small substituents such as methyl, hydroxymethyl, and fluoro groups at the 2- or 3-position of the
pyrrolidine ring (10f,g,h',j',k) were tolerated for the potency but the overall in vitro profiles were not
basically improved relative to 10c (data not shown). As a consequence, the non-substituted
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pyrrolidinylmethyl group (10c) proved to be the best as the 2-substituent in terms of both Cdc7 kinase
inhibition and kinase selectivity in this series.
Table 3. Effects of additional substituent of the 2-pyrrolidinylmethyl group on Cdc7 kinase inhibition and
kinase selectivity
Enzyme inhibition: IC50 (nM)a
Selectivity
Compound
R1
R2
R3
Cdc7
Cdk2/cycE
>10000
ROCK1
Cdk2/Cdc7
x14000
x3700
x27
ROCK1/Cdc7
x200
10c
14a
14b
10h'
10j'
10f
H
Me
Ph
H
H
H
H
H
H
H
H
H
0.70 (0.51–0.96)
2.7 (2.2–3.2)
130 (110–160)
1.2 (0.98–1.5)
3.9 (3.2–4.8)
2.7 (1.3–5.6)
1.6 (1.3–2.0)
7.0 (3.9–12)
1.1 (0.90–1.3)
140 (130–160)
40 (34–47)
>10000
x15
3500 (3100–3800)
>10000
4.1 (3.6–4.7)
160 (140–180)
x0.03
x130
Me
x8300
x2300
x3700
x6300
x1400
x4100
(S)
H
hydroxymethyl
8900 (7500–11000) 1200 (1000–1400)
x310
H
H
H
H
H
Me
>10000
>10000
540 (490–600)
420 (360–480)
170 (150–190)
770 (690–860)
x200
(R)
(S)
10g
10i
H
Me
x260
H
Ph
>10000
x24
10k
H
F
4500 (4100–5000)
x700
aNumbers in parentheses represent 95% confidence interval.
The hinge binding moiety was investigated with maintaining N-pyrrolidinylmethyl group as the
2-substituent (Table 4). Replacement of the methylpyrazolyl group of 10c with a 4-pyridyl group led to a
17-fold drop in Cdc7 inhibitory activity (3); nevertheless, compound 3 was 24 times more potent than the
corresponding 2-methyl derivative 2b (IC50 = 290 nM; see Figure 1). Deletion of the methyl group of 10c
significantly reduced activity and kinase selectivity (10l). Elongation of the methyl group of 10c also
attenuated both Cdc7 inhibition and selectivity (10m'). These SAR studies clearly demonstrated that
compound 10c is the best Cdc7 inhibitor among compounds prepared. Thus, we examined further in vitro
characterization of 10c along with the related analogs.
Table 4. Effects of hinge binding moiety on Cdc7 kinase inhibition and kinase selectivity
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Enzyme inhibition: IC50 (nM)a
Cdk2/cycE
Selectivity
Compound
R
Cdc7
ROCK1
Cdk2/Cdc7
ROCK1/Cdc7
3
12 (9.8–15)
>10000
>10000
180 (160–210)
x830
x2200
x14000
x4000
x15
10l
10c
4.5 (3.0–6.9)
820 (740–920)
140 (130–160)
830 (750–910)
x180
x200
x330
0.70 (0.51–0.96)
2.5 (2.1–2.9)
>10000
>10000
10m'
aNumbers in parentheses represent 95% confidence interval.
To characterize in vitro profiles of 10c, cell-free Cdc7 inhibition under various conditions,
inhibition of phosphorylation of Ser40 in MCM2 (PD effect), and growth inhibition of COLO205 cells
were investigated with the 10c-related analogs 3,7b,10b',10l (Table 5). There is a significant difference in
ATP concentrations between the standard cell-free inhibition assay (1 µM, equal to the Km value) and
cellular assay (a few mM); thus, we evaluated the selected compounds in high ATP concentrations (50 µM,
50-fold of the Km value). All compounds showed more than 29-fold decreased activity in high ATP
concentrations without pre-incubation when compared to the IC50 values evaluated in the standard assay
conditions. Pre-incubation of the compounds with Cdc7 kinase in high ATP concentrations generally
caused time-dependent inhibition of kinase activity, suggesting slow binding behavior. It is noteworthy
that compound 7b,10b',c showed nanomolar Cdc7 inhibitory activities even in high ATP concentrations
after a 60 min pre-incubation. From the results, it was revealed that the methylpyrazole significantly
contributed to time-dependency (7b,10b',c vs. 3,10l) and incorporation of the basic nitrogen into the
2-substituent enhanced time-dependency (10c vs. 7b) as well. Dissociation kinetics of compound 3,10c,1
to Cdc7/Dbf4(ASK) were measured in the Proteros reporter displacement assay.7-8 Values and order of
equilibrium dissociation constant (KD), dissociation kinetics (Koff) and residence time are well correlated
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with the above-mentioned enzyme assay data. When compared with 10b',c,l, the 3-methyl group of the
pyrazole ring is, in particular, important for not only high potency but also time-dependency and slow
dissociation property. Cell-based PD effects, inhibition of phosphorylation of Ser40 in MCM2, were
found to be in the order of 3, 10l << 10b' < 10c < 7b. Furthermore, COLO205 cell growth inhibition were
consistent with the PD effects, both of which reflect the cell-free activities in high ATP concentrations
after a 60 min pre-incubation rather than those in the standard assay conditions. Although the discrepancy
between the cell-free and cellular activities of 7b,10c remains obscure, it is suggested that the greater
growth inhibition of 7b,10c, at least in part, results from the time-dependency and slow dissociation
property. Judging from the overall in vitro profiles, compound 10c is superior to 7b, especially in terms of
kinase selectivity (see Table 2). The results indicate that compound 10c provides a promising starting
point for further optimization of this series.
Table 5. Effect of hinge binding moiety and 2-substituent on Cdc7 inhibition, time-dependency,
phosphorylation of Ser40 in MCM2, and cancer cell growth
Cdc7 inhibition: IC50 (nM)
Dissociation Kinetics
pMCM2
COLO205
Compound
Het
R
a
M
b
residence time
(min)
µ
µ
M
ATP 1
ATP 50
0 minc
d
KD
e
Koff (sec-1
)
IC50 (nM) EC50 (nM)
10 minc
60 minc
5.41 x 10-10
4.94 x 10-4
10c
7b
0.70
190
70
1.7
34
NT
NT
6
250
67
1100
900
1.5
2.2
4.5
12
5.6
1.5
77
NT
NT
10b'
10l
3
140
190
350
NT
NT
350
3200
7500
9500
1.26 x 10-9
2.80 x 10-9
2.67 x 10-3
2.15 x 10-3
>1000
>1000
150
8
aATP concentration (Km) in the standard cell-free assay conditions.
bATP concentration (x50 Km).
cPre-incubation time with a tested compound. dEquilibrium dissociation constant.
eDissociation rate constant.
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Docking study
To gain insight into molecular basis of the high kinase selectivity of compound 10c, docking
studies were conducted by using the published Cdc7 co-crystal structure (4F9C)9 and Cdk2 protein
structure (3FZ1).10 Compound 10c was docked into both the Cdc7 and Cdk2 protein structures by using
Glide (Schrödinger, Inc.). The putative binding modes are shown in Figure 6. The docking study
suggested that the pyrrolidinyl nitrogen of 10c could interact with Asn182 or Asp196 in the Cdc7 kinase,
forming a hydrogen bond network with the neighboring lactam NH (Figure 6a). In the case with Cdk2,
similar hydrogen bond interactions were not detected despite having Asn132 and Asp145 which are
equivalent to Asn182 and Asp196 of the Cdc7 protein (Figure 6b). This is presumably due to steric
repulsion between the pyrrolidnyl group and Gln131, whose corresponding residue in the Cdc7 kinase is
less bulky Ser181. In the both models, there are similar interactions, i.e. between the two pyrazole
nitrogens and Pro135, Leu137 (Cdc7) / Glu81, Leu83 (Cdk2) and between the carbonyl oxygen and
Lys90 (Cdc7) / Lys33 (Cdk2). Although a hydrogen bond between the pyrrolidnyl nitrogen and Gln131
was observed in the Cdk2 docking model, it is speculated that the high selectivity for Cdc7 over Cdk2 is
mainly attributed to the robust hydrogen bond network formed by the pyrrolidnyl nitrogen, lactam NH,
and carbonyl oxygen with the above-mentioned amino acid residues of the Cdc7 kinase (Figure 6c).
Compound 10c exhibited time-dependent Cdc7 inhibition with slow dissociation kinetics as described
above; however, notable conformational change of the protein structure such as DFG-out and αc-helix
movement11-12 was not observed in the docking model. These findings suggested uniqueness of compound
10c as a nontypical, ATP-competitive, Type I kinase inhibitor with slow dissociation property.
(a)
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Pro135
Lys90
Met134
Asp196
Asn182
Leu137
(b)
Asp145
Phe80
Lys33
Asn132
Glu81
Leu83
Gln131
(c)
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Lys33 (Cdk2)
Lys90 (Cdc7)
Asp145 (Cdk2)
Asp196 (Cdc7)
Asn132 (Cdk2)
Asn182 (Cdc7)
Gln131 (Cdk2)
Ser181 (Cdc7)
Figure 6. (a) Docking study on compound 10c with the Cdc7 crystal structure (PDB: 4F9C). (b)
Docking study on compound 10c with the Cdk2 crystal structure (PDB: 3FZ1). (c) Superposition of
docking model with Cdk2 (white) and that with Cdc7 (yellow).
Conclusion
We have succeeded in the identification of the thieno[3,2-d]pyrimidin-4(3H)-one scaffold as a
novel series of Cdc7 inhibitors, which could fit in the previously-constructed pharmacophore model.
Starting from the serendipitously-identified 2b, incorporation of pyrrolidinylmethyl and
3-methylpyrazolyl groups into 2- and 6-positions of the scaffold remarkably enhanced Cdc7 inhibitory
activity and kinase selectivity, providing a new, highly potent, and selective Cdc7 inhibitor (10c).
Combination of the two substituents significantly contributed to exerting the potent PD effects and
COLO205 cell growth, mainly thanks to potent and time-dependent Cdc7 kinase inhibition with slow
dissociation property. The docking study using the 3-dimensional Cdc7 and Cdk2 protein structures
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suggested that the robust hydrogen bond network formed by the pyrrolidnyl nitrogen, lactam NH, and
carbonyl oxygen with the amino acid counterparts of the Cdc7 kinase could endow 10c with high kinase
selectivity. The results clearly demonstrated that compound 10c is a promising starting point to develop a
potential anticancer agent and further optimization study will be reported elsewhere.
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Experimental
Chemistry
General
Starting materials, reagents, and solvents for reactions were reagent grade and used as purchased. Thin
layer chromatography (TLC) analyses were carried out using Merck Kieselgel 60 F254 plates or Fuji
Silysia Chemical Ltd. TLC plate NH. Chromatographic purification was carried out using silica gel
(Merck, 70–230 mesh) or amino silica gel (Fuji Silysia, aminopropyl-coated, 100–200 mesh) or
Purif-Pack (SI 60 µM or NH 60 µM, Fuji Silysia Chemical, Ltd.) or Combi-Flash. The proton nuclear
magnetic resonance (1H NMR) spectra were recorded on Bruker AVANCE II (300 MHz), Bruker AV 300
(300 MHz), or Bruker AV (500 MHz) instruments. Chemical shifts are given in parts per million (ppm)
with tetramethylsilane as an internal standard. Abbreviations are used as follows: s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, dd = doublets of doublet, br = broad, br s = broad singlet. Coupling
constants (J values) are given in hertz (Hz). HPLC with Corona charged aerosol detector (CAD) was used
to confirm > 95% purity of each compound. The column used was Capcell Pak C18AQ (3.0 mm i.d. × 50
mm, Shiseido, Japan) or L-column 2 ODS (2.0 mm i.d. × 30 mm, CERI, Japan) with a temperature of 50
oC and a flow rate of 0.5 mL/min. Mobile phase A and B under neutral conditions were a mixture of 50
mmol/L ammonium acetate, water, and MeCN (1:8:1, v/v/v) and a mixture of 50 mmol/L ammonium
acetate and MeCN (1:9, v/v), respectively. The ratio of mobile phase B was increased linearly from 5% to
95% over 3 min, 95% over the next 1 min. Mobile phase A and B under acidic conditions were a mixture
of 0.2% formic acid in 10 mmol/L ammonium formate and 0.2% formic acid in MeCN, respectively. The
ratio of mobile phase B was increased linearly from 14% to 86% over 3 min, 86% over the next 1 min.
MS spectra were recorded using a Shimadzu LCMS-2020 or Agilent 6130 Quadrupole LCMS with
electrospray ionization (ESI or APCI). Elemental analysis and high resolution mass spectrometry
(HRMS) were measured by Takeda Analytical Research Laboratories, Ltd.
Abbreviations:
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CPME: cyclopentyl methyl ether
DCM: dichloromethane
DMA: N,N-dimethylacetamide
DMAP: 4-dimethylaminopyridine
DME: 1,2-dimethoxyethane
DMF: N,N-dimethylformamide
DMSO: dimethyl sulfoxide
EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
HOBt: 1-hydroxybenzotriazole
HATU: 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V)
NH: amino silica gel
PdCl2(dppf): 1,1’-bis(diphenylphosphino)ferrocenepalladium (II) dichloride dichloromethane adduct
PTSA: p-toluenesulfonic acid monohydrate
TFA: trifluoroacetic acid
THF: tetrahydrofuran
2-Methyl-6-(pyridin-4-yl)thieno[3,2-d]pyrimidin-4(3H)-one (2b). A mixture of 15 (0.108 g, 0.500
mmol), 3-methyl-2-butanone (3.0 mL, 27.9 mmol), PTSA (9.5 mg, 0.050 mmol) and AcOH (3.0 mL) in a
sealed tube was stirred overnight at 70 °C. The mixture was poured into saturated NaHCO3 aq., and
extracted with EtOAc–THF. The organic layer was dried over MgSO4, filtered and concentrated in vacuo.
The residue was purified by column chromatography on silica gel (EtOAc/MeOH, 100:0 to 90:10, v/v) to
afford 2b (21.9 mg, yield 18%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 2.39 (3H, s), 7.83 (2H,
dd, J = 4.5, 1.5 Hz), 8.05 (1H, s), 8.69 (2H, dd, J = 4.5, 1.8 Hz), 12.54 (1H, br s). ESI MS m/z 244
[C12H9N3OS+H]+.
6-(Pyridin-4-yl)-2-(pyrrolidin-1-ylmethyl)thieno[3,2-d]pyrimidin-4(3H)-one (3). To a stirred
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suspension of 1 (219 mg, 1.0 mmol), Et3N (0.152 mL, 1.1 mmol) and THF (20 mL) was added
chloroacetyl chloride (0.087 mL, 1.1 mmol). After 10 min, pyrrolidine (1.65 mL, 20 mmol) was added
and the mixture was stirred at 80 °C for 1 h. The mixture was concentrated in vacuo, and the residual oil
was mixed with 2 M NaOH (10 mL). The mixture was stirred at 100 °C for 1 h. The mixture was
concentrated in vacuo and the residue was poured in to saturated NaHCO3 aq., and extracted with
EtOAc/THF. The organic layer was dried over MgSO4, filtered and concentrated in vacuo. The residue
was purified by column chromatography on amino silica gel (EtOAc/MeOH, 100:0 to 80:20, v/v) to
afford light yellow solid. The solid was triturated with EtOAc and the precipitate was collected by
filtration to afford 3 (87 mg, yield 28%) as white solid. 1H NMR (300 MHz, DMSO-d6) δ 1.70–1.74 (4H,
m), 2.55–2.59 (4H, m), 3.60 (2H, s), 7.82–7.85 (2H, m), 8.11 (1H, s), 8.68–8.70 (2H, m), 12.32 (1H, br s).
HRMS: Calcd for C16H17N4OS [M+H]+: 313.1118. Found: 313.1090.
6-Bromo-2-methylthieno[3,2-d]pyrimidin-4(3H)-one (6a). To a solution of 55 (0.200 g, 0.90 mmol) and
Et3N (0.190 mL, 1.36 mmol) in THF (4 mL) was added AcCl (0.096 mL, 1.36 mmol) at 0 °C. The
mixture was stirred at room temperature for 0.5 h. The mixture was quenched with water and extracted
with EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated in
vacuo. To the residue were added 2 M NaOH (0.450 mL, 0.90 mmol) and EtOH (2.0 mL), and the
mixture was stirred at 70 °C for 2 h. The mixture was neutralized with 1 M HCl (1.3 mL) at 0 °C. After
addition of water (2.0 mL), the precipitate was collected by filtration to give 6a (131 mg, yield 59%) as a
colorless solid. 1H NMR (300 MHz, DMSO-d6) δ 2.36 (3H, s), 7.54 (1H, s), 12.55 (1H, br s).
6-Bromo-2-(cyclopentylmethyl)thieno[3,2-d]pyrimidin-4(3H)-one (6b). To a solution of 5 (0.200 g,
0.90 mmol) and 2-cyclopentylacetic acid (0.113 mL, 0.90 mmol) in DMF (2.0 mL) were added
N-ethyl-N-isopropylpropan-2-amine (0.316 mL, 1.81 mmol) and HATU (0.413 g, 1.09 mmol) at 0 °C.
The mixture was stirred at 70 °C for 20 h. The mixture was quenched with water and extracted with
EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo.
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The residue was purified by column chromatography on amino silica gel (n-hexane/EtOAc, 85:15 to
50:50, v/v) to give yellow oil. To the oil were added 4 M NaOH (1.751 mL, 3.50 mmol) and EtOH (3.0
mL). The mixture was stirred at 70 °C for 2 h. The mixture was neutralized with 1 M HCl (2.3 mL) at
0 °C. After addition of water (2.0 mL), the precipitate was collected by filtration to give 6b (165 mg,
yield 60%) as a colorless solid. 1H NMR (300 MHz, DMSO-d6) δ 1.12–1.28 (2H, m), 1.42–1.75 (6H, m),
2.24–2.35 (1H, m), 2.60 (2H, d, J = 7.6 Hz), 7.57 (1H, s), 12.52 (1H, br s).
6-Bromo-2-cyclohexylthieno[3,2-d]pyrimidin-4(3H)-one (6c). Compound 6c was prepared in 89%
yield from 5 by a procedure similar to that described for 6a as a colorless solid. 1H NMR (300 MHz,
DMSO-d6) δ 1.14–1.38 (3H, m), 1.44–1.62 (2H, m), 1.63–1.95 (5H, m), 2.55–2.67 (1H, m), 7.57 (1H, s),
12.45 (1H, br s).
6-Bromo-2-phenylthieno[3,2-d]pyrimidin-4(3H)-one (6d). Compound 6d was prepared in 67% yield
from 5 by a procedure similar to that described for 6a as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ
7.50–7.61 (3H, m), 7.70 (1H, s), 8.07–8.14 (2H, m), 12.87 (1H, br s).
6-Bromo-2-(ethoxymethyl)thieno[3,2-d]pyrimidin-4(3H)-one (6e). Compound 6e was prepared in 89%
yield from 5 by a procedure similar to that described for 6a as a pale yellow solid. 1H NMR (300 MHz,
DMSO-d6) δ 1.14 (3H, t, J = 7.0 Hz), 3.53 (2H, q, J = 7.0 Hz), 4.30 (2H, s), 7.48 (1H, s), 12.60 (1H, br s).
tert-Butyl (6-bromo-4-oxo-3,4-dihydrothieno[3,2-d]pyrimidin-2-yl)methyl(methyl)carbamate (6f).
Isobutyl chloroformate (614 mg, 4.49 mmol) was added to a solution of Et3N (0.741 mL, 5.35 mmol) and
2-(tert-butoxycarbonyl(methyl)amino)acetic acid (850 mg, 4.49 mmol) in THF (5.0 mL) at 0 °C. The
mixture was stirred at room temperature for 1 h. To the mixture was added 5 (473 mg, 2.14 mmol). The
resulting mixture was stirred at 60 °C for 15 h. The mixture was extracted with EtOAc, washed with
water, brine, dried over MgSO4 and concentrated in vacuo. The residue was dissolved in 2 M NaOH (5.35
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mL, 10.70 mmol) and EtOH (10 mL), and the mixture was stirred at 70 °C for 15 h. The mixture was
neutralized with 1 M HCl at 0 °C and extracted with EtOAc. The organic layer was washed with water
and brine, dried over MgSO4, filtered and concentrated in vacuo. The residual solid was collected and
washed with EtOAc–hexane to give 6f (780 mg, yield 97%) as a white solid. 1H NMR (300 MHz,
DMSO-d6) δ 1.31–1.49 (9H, m), 2.77–2.87 (3H, m), 3.79–3.90 (2H, m), 7.60 (1H, s), 12.63 (1H, br s).
6-Bromo-2-(2-pyrrolidin-1-ylethyl)thieno[3,2-d]pyrimidin-4(3H)-one (6g). To a suspension of 5 (120
mg, 0.543 mmol) and Et3N (0.083 mL, 0.60 mmol) in THF (3.0 mL) was added 3-chloropropanoyl
chloride (0.057 mL, 0.60 mmol) at room temperature. After stirring at that temperature for 10 min,
pyrrolidine (0.227 mL, 2.72 mmol) was added. The mixture was stirred at room temperature for 10 min.
After removal of the solvent, 2 M NaOH (1.0 mL) was added. The mixture was stirred at 100 °C for 1 h.
The organic materials were extracted with EtOAc. The organic layer was washed with brine, dried over
Na2SO4, filtered and concentrated in vacuo to give 6g (131 mg, yield 74%) as a pale yellow solid. 1H
NMR (300 MHz, DMSO-d6) δ 1.63–1.70 (4H, m), 2.43–2.49 (4H, m), 2.78–2.82 (4H, m), 7.57 (1H, s),
12.61 (1H, br s).
6-Bromo-2-morpholin-4-ylthieno[3,2-d]pyrimidin-4(3H)-one (6h). A mixture of 46 (200 mg, 0.847
mmol), morpholine-4-carbonitrile (0.17 mL, 1.69 mmol) and 4 M HCl in cyclopentylmethylether (3.0
mL) was stirred at 110 °C for 4 h. The reaction mixture was concentrated under reduced pressure, and
saturated NaHCO3 aq. was added. The precipitate was collected by filtration and washed with water and
EtOAc/hexane (1:1) to give 6h (144 mg, yield 54%) as a pale yellow solid. 1H NMR (300 MHz,
DMSO-d6) δ 3.51–3.60 (4H, m), 3.61–3.71 (4H, m), 7.29 (1H, s), 11.50 (1H, br s).
2-Methyl-6-(3-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4(3H)-one (7a). A mixture of 6a (130
mg, 0.53 mmol), tert-butyl
3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate (327 mg, 1.06
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mmol), Na2CO3 (169 mg, 1.59 mmol), PdCl2(dppf) (43.3 mg, 0.053 mmol), DME (3.0 mL) and water
(1.5 mL) was stirred at 100 °C for 3 h under Ar. The organic materials were extracted with EtOAc, dried
over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography on amino
silica gel (EtOAc/MeOH, 100:0 to 90:10, v/v) to give pale yellow solid. The solid was triturated with
MeOH, and the precipitate was collected by filtration to afford 7a (74.0 mg, yield 57%) as a colorless
solid. 1H NMR (300 MHz, DMSO-d6) δ 2.36 (3H, s), 2.44 (3H, s), 7.31 (1H, s), 7.98 (1H, br s), 12.30
(1H, br s), 12.96 (1H, br s). HRMS: Calcd for C11H11N4OS [M+H]+: 247.0648. Found: 247.0625.
2-(Cyclopentylmethyl)-6-(3-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4(3H)-one (7b).
Compound 7b was prepared in 36% yield from 6b by a procedure similar to that described for 7a as a
colorless solid. 1H NMR (300 MHz, DMSO-d6) δ 1.09–1.32 (2H, m), 1.40–1.78 (6H, m), 2.25–2.40 (1H,
m), 2.45 (3H, br s), 2.61 (2H, d, J = 7.4 Hz), 7.34 (1H, s), 7.73–8.37 (1H, m), 12.26 (1H, br s), 12.95 (1H,
br s). Anal. Calcd for C16H18N4OS: C, 61.12; H, 5.77; N, 17.82. Found: C, 60.84; H, 5.82; N, 17.73.
2-Cyclohexyl-6-(3-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4(3H)-one (7c). Compound 7c was
prepared in 63% yield from 6c by a procedure similar to that described for 7a as a pale yellow solid. 1H
NMR (300 MHz, DMSO-d6) δ 1.11–1.97 (10H, m), 2.45 (3H, s), 2.54–2.68 (1H, m), 7.35 (1H, s), 7.95
(1H, br s), 12.20 (1H, br s), 12.96 (1H, br s). Anal. Calcd for C16H18N4OS·1.1H2O: C, 57.50; H, 6.09; N,
16.76. Found: C, 57.56; H, 6.33; N, 16.30.
6-(3-Methyl-1H-pyrazol-4-yl)-2-phenylthieno[3,2-d]pyrimidin-4(3H)-one (7d). A mixture of 6d (111
mg, 0.358 mmol), tert-butyl
3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate (492 mg, 1.60
mmol), Na2CO3 (331 mg, 1.07 mmol), PdCl2(dppf) (29 mg, 0.036 mmol), DME (3.0 mL) and water (1.5
mL) was stirred at 100 °C for 2 h under Ar. Then 8 M NaOH (1.0 mL) was added. After stirring at 100 °C
for 0.5 h, the organic materials were extracted with EtOAc/THF. The combined extracts were dried over
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Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on amino silica
gel (n-hexane/EtOAc, 80:20 to 0:100, v/v). The residue was triturated with MeOH–EtOAc and the
precipitate was collected by filtration to afford 7d (51 mg, yield 46%) as a pale yellow solid. 1H NMR
(300 MHz, DMSO-d6) δ 2.49 (3H, br s), 7.49 (1H, s), 7.51–7.65 (3H, m), 7.94 (0.6H, br s), 8.10–8.20 (2H,
m), 8.29 (0.4H, br s), 12.65 (1H, br s), 13.02 (1H, br s). Anal. Calcd for C16H12N4OS·0.1H2O: C, 61.96; H,
3.96; N, 18.06. Found: C, 61.82; H, 3.97; N, 17.97.
2-(Ethoxymethyl)-6-(3-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4(3H)-one (7e). Compound
7e was prepared in 42% yield from 6e by a procedure similar to that described for 7d as a pale brown
solid. 1H NMR (300 MHz, DMSO-d6) δ 1.17 (3H, t, J = 7.0 Hz), 2.38–2.50 (3H, m), 3.56 (2H, q, J = 7.0
Hz), 4.38 (2H, s), 7.39 (1H, s), 7.89 (0.6H, br s), 8.26 (0.4H, br s), 12.34 (1H, br s), 12.77–13.18 (1H, m).
Anal. Calcd for C13H14N4O2S: C, 53.78; H, 4.86; N, 19.30. Found: C, 53.78; H, 4.91; N, 19.09.
6-(3-Methyl-1H-pyrazol-4-yl)-2-((methylamino)methyl)thieno[3,2-d]pyrimidin-4(3H)-one
dihydrochloride (7f'). A mixture of 6f (400 mg, 1.07 mmol), tert-butyl
3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate (659 mg, 2.14
mmol), Cs2CO3 (696 mg, 2.14 mmol), PdCl2(dppf) (44.0 mg, 0.054 mmol), DME (5.0 mL) and water (0.5
mL) was stirred at 80 °C overnight. The mixture was diluted with water and extracted with EtOAc. The
combined extracts were washed with brine, dried over MgSO4 and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (n-hexane/EtOAc, 9:1 to 1:1, v/v). The residue was
dissolved in MeOH (4.0 mL) and 4 M HCl in EtOAc (1.0 mL, 4.00 mmol). The mixture was stirred at
room temperature for 3 h. The resulting solid was collected and washed with EtOAc to give 7f' (50.0 mg,
yield 13%) as a colorless solid. 1H NMR (300 MHz, DMSO-d6) δ 2.47 (3H, s), 2.69 (3H, br s), 4.23 (2H,
br s), 7.31–8.30 (5H, m), 9.54 (2H, br s). Anal. Calcd for C12H15N5OSCl2·0.15H2O: C, 41.07; H, 4.39; N,
19.96. Found: C, 41.29; H, 4.58; N, 19.67.
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6-(3-Methyl-1H-pyrazol-4-yl)-2-(2-pyrrolidin-1-ylethyl)thieno[3,2-d]pyrimidin-4(3H)-one (7g).
Compound 7g was prepared in 53% yield from 6g by a procedure similar to that described for 7d as a
pale yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 1.51–1.78 (4H, m), 2.40–2.49 (7H, m), 2.75–2.89
(4H, m), 7.34 (1H, s), 8.02 (1H, br s), 11.93–13.36 (2H, m). Anal. Calcd for C16H19N5OS: C, 58.34; H,
5.81; N, 21.26. Found: C, 58.32; H, 5.80; N, 21.07.
6-(3-Methyl-1H-pyrazol-4-yl)-2-morpholin-4-ylthieno[3,2-d]pyrimidin-4(3H)-one (7h). Compound
7h was prepared in 58% yield from 6g by a procedure similar to that described for 7d as a pale brown
solid. 1H NMR (300 MHz, DMSO-d6) δ 2.33–2.47 (3H, m), 3.50–3.61 (4H, m), 3.61–3.72 (4H, m), 7.11
(1H, s), 7.83 (0.6H, br s), 8.19 (0.4H, br s), 11.35 (1H, br s), 12.79–13.07 (1H, m). Anal. Calcd for
C14H15N5O2S·0.5H2O: C, 51.52; H, 4.94; N, 21.46. Found: C, 51.33; H, 4.98; N, 21.18.
6-Bromo-2-(chloromethyl)thieno[3,2-d]pyrimidin-4(3H)-one (8). A mixture of 4 (15 g, 62.7 mmol),
chloroacetonitrile (12 mL, 188 mmol) and 4 M HCl in cyclopentylmethylether (100 mL) was stirred at
room temperature for 2 h, and stirred at 70 °C for 1 h. The mixture was concentrated under reduced
pressure, and saturated NaHCO3 aq. was added to the residue. The precipitate was collected by filtration
and washed with water. The obtained solid was dried at 80 °C for 8 h under reduced pressure to give 8 (18
g, yield 100%) as a pale brown solid. 1H NMR (300 MHz, DMSO-d6) δ 4.51 (2H, s), 7.56 (1H, s), 13.00
(1H, br s).
2-[(Benzylamino)methyl]-6-bromothieno[3,2-d]pyrimidin-4(3H)-one (9a). To a suspension of
1-phenylmethanamine (0.586 mL, 5.37 mmol), K2CO3 (495 mg, 3.58 mmol), sodium iodide (27 mg,
0.179 mmol) and DMF (5.0 mL) was added 8 (500 mg, 1.79 mmol). The mixture was stirred at 70 °C for
1 h. The mixture was purified by column chromatography on amino silica gel (n-hexane/EtOAc, 85:15 to
0:100, v/v, then EtOAc/MeOH, 100:0 to 93:7, v/v) to give 9a (234 mg, yield 37%) as a pale yellow solid.
1H NMR (300 MHz, DMSO-d6) δ 3.67 (2H, s), 3.72 (2H, s), 7.17–7.40 (5H, m), 7.47–7.87 (2H, m). 1H
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of amide or amine portion was not observed independently.
6-Bromo-2-(pyrrolidin-1-ylmethyl)thieno[3,2-d]pyrimidin-4(3H)-one (9c). Compound 9c was
prepared in 84% yield by a procedure similar to that described for 9a as a pale yellow solid. 1H NMR
(300 MHz, DMSO-d6) δ 1.65–1.77 (4H, m), 2.52–2.60 (4H, m), 3.57 (2H, s), 7.60 (1H, s), 12.24 (1H, br
s).
6-Bromo-2-(piperidin-1-ylmethyl)thieno[3,2-d]pyrimidin-4(3H)-one (9d). Compound 9d was prepared
in 83% yield by a procedure similar to that described for 9a as a pale yellow solid. 1H NMR (300 MHz,
DMSO-d6) δ 1.31–1.42 (2H, m), 1.45–1.56 (4H, m), 2.35–2.46 (4H, m), 3.40 (2H, s), 7.61 (1H, s), 12.20
(1H, br s).
6-Bromo-2-{[(3R)-3-methylpyrrolidin-1-yl]methyl}thieno[3,2-d]pyrimidin-4(3H)-one (9f).
Compound 9f was prepared in 81% yield by a procedure similar to that described for 9a as a pale yellow
solid. 1H NMR (300 MHz, DMSO-d6) δ 0.98 (3H, d, J = 6.6 Hz), 1.17–1.37 (1H, m), 1.85–2.02 (1H, m),
2.03–2.26 (2H, m), 2.53–2.61 (1H, m), 2.62–2.71 (1H, m), 2.77–2.86 (1H, m), 3.55 (2H, s), 7.59 (1H, s),
12.30 (1H, br s).
6-Bromo-2-{[(3S)-3-methylpyrrolidin-1-yl]methyl}thieno[3,2-d]pyrimidin-4(3H)-one (9g).
Compound 9g was prepared in 88% yield by a procedure similar to that described for 9a as a pale yellow
solid. 1H NMR (300 MHz, DMSO-d6) δ 0.98 (3H, d, J = 6.6 Hz), 1.21–1.36 (1H, m), 1.86–2.03 (1H, m),
2.05–2.25 (2H, m), 2.53–2.61 (1H, m), 2.61–2.74 (1H, m), 2.76–2.88 (1H, m), 3.55 (2H, s), 7.59 (1H, s),
12.19 (1H, br s).
6-Bromo-2-[(3-phenylpyrrolidin-1-yl)methyl]thieno[3,2-d]pyrimidin-4(3H)-one (9i). Compound 9i
was prepared in 72% yield by a procedure similar to that described for 9a as a yellow solid. 1H NMR
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(300 MHz, DMSO-d6) δ 1.71–1.85 (1H, m), 2.18–2.32 (1H, m), 2.55–2.63 (1H, m), 2.75–2.85 (2H, m),
3.04 (1H, t, J = 8.4 Hz), 3.26–3.40 (1H, m), 3.65 (2H, s), 7.14–7.22 (1H, m), 7.24–7.34 (4H, m), 7.61 (1H,
s), 12.39 (1H, br s).
6-Bromo-2-{[(2R)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}thieno[3,2-d]pyrimidin-4(3H)-one (9j).
Compound 9j was prepared in 55% yield by a procedure similar to that described for 9a as a colorless
solid. 1H NMR (300 MHz, DMSO-d6) δ 1.53–1.91 (4H, m), 2.23–2.38 (1H, m), 2.61–2.71 (1H, m),
2.83–2.98 (1H, m), 3.20–3.55 (3H, m), 4.01 (1H, d, J = 14.7 Hz), 4.33–5.67 (1H, m), 7.60 (1H, s),
11.13–12.68 (1H, m).
6-Bromo-2-{[(3S)-3-fluoropyrrolidin-1-yl]methyl}thieno[3,2-d]pyrimidin-4(3H)-one (9k). Compound
9k was prepared in 83% yield by a procedure similar to that described for 9a as a pale yellow solid. 1H
NMR (300 MHz, DMSO-d6) δ 1.75–2.28 (2H, m), 2.44–2.54 (1H, m), 2.67–2.98 (3H, m), 3.61 (2H, s),
4.96–5.48 (1H, m), 7.62 (1H, s), 12.42 (1H, br s).
6-Benzyl-2-bromo-6,7-dihydroimidazo[1,5-a]thieno[3,2-d]pyrimidin-9(5H)-one (11). A mixture of 9a
(185 mg, 0.528 mmol), 37% aqueous formaldehyde (1 mL) and THF (2 mL) was stirred at room
temperature for 1 h. The precipitate was collected by filtration, and washed with water and EtOAc to give
11 (104 mg, yield 54%) as a pale yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 3.87 (2H, s), 4.04 (2H,
s), 4.84 (2H, s), 7.24–7.45 (5H, m), 7.63 (1H, s).
6-Benzyl-2-(3-methyl-1H-pyrazol-4-yl)-6,7-dihydroimidazo[1,5-a]thieno[3,2-d]pyrimidin-9(5H)-one
trifluoroacetic acid (10a). A mixture of 11 (100 mg, 0.276 mmol), tert-butyl
3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate (255 mg, 0.828
mmol), PdCl2(dppf) (23 mg, 0.028 mmol), Na2CO3 (66 mg, 1.10 mmol), DME (3.0 mL) and water (1.5
mL) was stirred at 100 °C for 1.5 h. The mixture was diluted with water and extracted with EtOAc. The
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