Identification of a new class of potent Cdc7 inhibitors designed by putative pharmacophore model: Synthesis and biological evaluation of 2,3-dihydrothieno[3,2-d]pyrimidin-4(1H)-ones

Article abstract of DOI:10.1016/j.bmc.2017.02.021

Cell division cycle 7 (Cdc7) is a serine/threonine kinase that plays important roles in the regulation of DNA replication process. A genetic study indicates that Cdc7 inhibition can induce selective tumor-cell death in a p53-dependent manner, suggesting that Cdc7 is an attractive target for the treatment of cancers. In order to identify a new class of potent Cdc7 inhibitors, we generated a putative pharmacophore model based on in silico docking analysis of a known inhibitor with Cdc7 homology model. The pharmacophore model provided a minimum structural motif of Cdc7 inhibitor, by which preliminary medicinal chemistry efforts identified a dihydrothieno[3,2-d]-pyrimidin-4(1H)-one scaffold having a heteroaromatic hinge-binding moiety. The structure-activity relationship (SAR) studies resulted in the discovery of new, potent, and selective Cdc7 inhibitors 14a, c, e. Furthermore, the high selectivity of 14c, e for Cdc7 over Rho-associated protein kinase 1 (ROCK1) is discussed by utilizing a docking study with Cdc7 and ROCK2 crystal structures.

Full text of DOI:10.1016/j.bmc.2017.02.021

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of a new class of potent Cdc7 inhibitors designed by
putative pharmacophore model: Synthesis and biological evaluation of
2,3-dihydrothieno[3,2-d]pyrimidin-4(1H)-ones
a
a
a
a
a
Osamu Kurasawa a, , Yuya Oguro , Tohru Miyazaki , Misaki Homma , Kouji Mori , Kenichi Iwai ,
Hideto Hara a, Robert Skene b, Isaac Hoffman b, Akihiro Ohashi a, Sei Yoshida a, Tomoyasu Ishikawa a,
Nobuo Cho a
⇑
a Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
b Takeda California, Inc., 10410 Science Center Drive, San Diego 92121, CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cell division cycle 7 (Cdc7) is a serine/threonine kinase that plays important roles in the regulation of
DNA replication process. A genetic study indicates that Cdc7 inhibition can induce selective tumor-cell
death in a p53-dependent manner, suggesting that Cdc7 is an attractive target for the treatment of can-
cers. In order to identify a new class of potent Cdc7 inhibitors, we generated a putative pharmacophore
model based on in silico docking analysis of a known inhibitor with Cdc7 homology model. The pharma-
cophore model provided a minimum structural motif of Cdc7 inhibitor, by which preliminary medicinal
chemistry efforts identified a dihydrothieno[3,2-d]-pyrimidin-4(1H)-one scaffold having a heteroaro-
matic hinge-binding moiety. The structure-activity relationship (SAR) studies resulted in the discovery
of new, potent, and selective Cdc7 inhibitors 14a, c, e. Furthermore, the high selectivity of 14c, e for
Cdc7 over Rho-associated protein kinase 1 (ROCK1) is discussed by utilizing a docking study with Cdc7
and ROCK2 crystal structures.
Received 13 December 2016
Revised 8 February 2017
Accepted 10 February 2017
Available online xxxx
Keywords:
Cell division cycle 7 (Cdc7)
Pharmacophore model
Dihydrothieno[3,2-d]-pyrimidin-4(1H)-one
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
Cell division cycle 7 (Cdc7), an evolutionally conserved serine/
threonine kinase, is a key regulator for G1/S phase transition
Targeting DNA replication, a fundamental process for cell
proliferation, is one of major strategies for the treatment of cancer.
Various agents targeting DNA replication, platinum DNA cross link-
ers and topoisomerase inhibitors, have potent cancer cell growth
inhibitory activities and are utilized in the clinic. However, in order
to overcome the issue of cancer heterogeneity and emergence of
resistant cancer cells, new agents having novel mechanism of
action are required.
through phosphorylation of one or more residues of minichromo-
some maintenance 2 (MCM2) of the pre-replicative complex.1,2
The Cdc7-dependent phosphorylation causes loading of other
accessory factors and subsequent generation of active replication
forks, ultimately leading to the initiation of DNA replication. Over-
expression of both Cdc7 and the regulatory partner, Dumbbell for-
mer 4 (Dbf4), is observed in various type of cancer cells, suggesting
significant involvement of Cdc7 in tumor progression.3 The Cdc7
kinase is considered to be an attractive target based on the follow-
ing findings: RNA interference silencing of Cdc7 in cancer cells
induces a prolonged S phase, leading to subsequent p53-indepen-
dent apoptotic cell death.4 On the other hand, the silencing in nor-
mal cells evokes G1 arrest in a p53-dependent manner without loss
of viability.4
Abbreviations: CSA, (+/ꢀ)-camphor-10-sulfonic acid; DCM, dichloromethane;
DMA, N,N-dimethylacetamide; DME, 1,2-dimethoxyethane; DMF, N,N-dimethyl-
formamide; DMSO, dimethyl sulfoxide; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide
hydrochloride;
HOBt,
1-hydroxybenzotriazole;
NBS,
N-bromosuccinimide; NH, amino silica gel; PdCl2(dppf), 1,10-bis(diphenylphos-
phino)ferrocenepalladium (II) dichloride dichloromethane adduct; PE, petroleum
ether; PTSA, p-toluenesulfonic acid monohydrate; TFA, trifluoroacetic acid; TFAA,
trifluoroacetic anhydride; THF, tetrahydrofuran.
Several research groups have reported Cdc7 inhibitors and
some of them have entered clinical trials; however, the proof of
concept (POC) in humans has not been demonstrated so far.5–11
We envisioned that it was mainly attributed to the difficulty of
⇑
Corresponding author.
E-mail address: osamu.kurasawa@takeda.com (O. Kurasawa).
http://dx.doi.org/10.1016/j.bmc.2017.02.021
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
identifying the compound with sufficient kinase selectivity and/or
cellular potency, particularly in the case of typical ATP-competitive
Cdc7 kinase inhibitors. We sought, therefore, to discover and
develop a new, potent and selective Cdc7 inhibitor.
In order to design new Cdc7 inhibitors, we firstly constructed a
Cdc7 homology model on the basis of the reported crystal structure
(PDB: 1DAW) of casein kinase 2 (CK2)12 sharing high homology
with Cdc7 in terms of the ATP binding pocket (74% identity). By
utilizing the homology model, a docking study on an ATP-compet-
itive Cdc7 inhibitor (I) reported by Nerviano6 was next carried out
(Fig. 1).
The docking study provided the following clues: (i) The Pro135
and/or Leu137 residues can be exploited as a counterpart of hinge
binding motif of Cdc7 inhibitors. (ii) The Lys90 and Asp196 resi-
dues making a salt bridge in the back pocket are considered to
be important for the interaction with the amide group. (iii) It is
speculated that the sulfur atom of the gate keeper Met134 residue
can be used for an additional interaction with inhibitors. Based on
the clues, we generated a putative pharmacophore model which
suggests a heteroaromatic hinge-binding moiety and an amide
group as two key binding motifs, and appropriate incorporation
of a sulfur atom into the five-membered aromatic ring for an addi-
tional binding interaction. In the model, the aromatic core ring
plays an important role to place the two key binding motifs in ade-
quate positions. The pharmacophore model led us to design bicyc-
lic thiophene-based Cdc7 inhibitors (Fig. 2). A bicyclic scaffold,
cyclic amide fused with the aromatic core ring, was adopted to
fix the amide group to the appropriate direction toward the
Lys90 and Asp196 residues. As a bicyclic scaffold, we chose
thieno[3,2-d]pyrimidinone whose sulfur atom may effectively
interact with the sulfur atom of the Met134 residue. Furthermore,
the docking study suggested that the 2-fluoroethyl group of the
Nerviano inhibitor extends toward solvent-exposed region which
could be utilized to improve potency, kinase selectivity, and
physicochemical properties by introducing desirable substituents
into Rc and/or Rd.
5-fold less potent than dihydrothieno[3,2-d]pyrimidin-4(1H)-one
4a, which may reflect the absence of the non-covalent S-S interac-
tion. Among them, compound 4a with an IC50 value of 26 nM was
the best. In addition, we examined kinase selectivity of compound
4a by using the in-house kinase panel (46 kinases),13 indicating
that overall selectivity of 4a was acceptable (IC50 < 100 nM for
ROCK1 (1/46 kinases), IC50 100–1000 nM for 4/47 kinases, IC50
1000–10,000 nM for 5/46 kinases, IC50 > 10,000 nM for 36/46
kinases). ROCK acting downstream of the small GTPase Rho14 is
known as one of the key players in cell cycle control; however,
the kinase family is involved in a wide range of cellular functions.15
Thus, we considered improvement in selectivity for Cdc7 over
ROCK1 would mitigate off-target effects.
Based on these results, dihydrothieno[3,2-d]pyrimidin-4(1H)-
one was selected as a scaffold and, starting from 4a as a lead com-
pound, we performed further optimization study to identify potent
and selective Cdc7 inhibitors. The preliminary data described in
Fig. 3 suggested that the gem-dimethyl aminal moiety extending
toward the solvent-exposed region, which is significantly spacious
in the Cdc7 homology model, can be potentially modified to
improve kinase selectivity as well as Cdc7 inhibitory activity. Opti-
mization of the hinge-binding moiety would also contribute to
enhancing the in vitro potency.
Herein, we report the synthesis, structure-activity relationships
(SARs), and biological effects of this chemical series. Furthermore,
difference in binding modes of a selected compound with Cdc7
and ROCK in docking models is discussed to elucidate high
selectivity.
2. Chemistry
Dihydrothienopyrimidine derivatives possessing 4-pyridyl
group as a hinge binder (4a–k, 5, 8) were synthesized according
to the procedure in Scheme 1. After N-methylation of methyl 3-
amino-5-(4-pyridyl)thiophene-2-carboxylate 1a, the esters 1a, b
were then hydrolyzed under basic conditions to give the carboxylic
acids 2a, b. Compounds 2a, b were condensed with appropriate
amines to provide the corresponding amides 3a–d. Similarly, ethyl
2-amino-5-(4-pyridyl)thiophene-3-carboxylate 6 was converted to
amide 7 in 38% yield by saponification and subsequent amidation.
The aminothiophenecarboxamides 3a–d, 7 were reacted with var-
ious ketones in the presence of catalytic amount of p-toluenesul-
fonic acid to afford cyclized products 4a–k, 8 in 11–74% yields.
Moreover, the amide 3a was subjected to ring-closure reaction
using urea to yield thienopyrimidin-2,4-dione 5.
Preliminarily, thienopyrimidinones 4a, 5, 8 and a ring-opening
analogue 3a were designed and prepared based on the pharma-
cophore model, and the Cdc7 kinase inhibitory activities were eval-
uated (Fig. 3). As expected, inhibitory activities of compound 4a, 5,
8 were better than that of compound 3a. With regard to the cyclic
lactam ring, the pyrimidin-4-one 4a was more potent inhibitor
than the pyrimidin-2,4-dione 5. Moreover, position of the sulfur
atom in the bicyclic ring system significantly affected the potency.
In fact, dihydrothieno[2,3-d]pyrimidin-4(1H)-one 8 was about
Fig. 1. Docking study on Nerviano inhibitor (I) by utilizing Cdc7 homology model constructed on the basis of the crystal structure of CK2 (PDB: 1DAW).
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O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Fig. 2. (a) Pharmacophore model of Cdc7. (b) Design of bicyclic thiophene-based Cdc7 inhibitors.
Fig. 3. Identification of dihydrothieno[3,2-d]pyrimidin-4(1H)-one as a favorable scaffold of new Cdc7 inhibitors.
Scheme 1. Reagents and conditions: (a) NaH, MeI, DMF, 0 °C to rt, 62%; (b) NaOH, MeOH–water, 70 °C, 92%; (c) LiOH, MeOH–water, 80 °C, quant.; (d) NH4Cl or R2NH2, EDCI,
HOBt, Et3N, DMF, rt, 50–84%; (e) R3COR4, p-TsOHꢁH2O, toluene or AcOH, 70–110 °C, 11–74%; (f) urea, 180 °C, 34%; (g) (1) LiOH, EtOH–water, 80 °C; (2) NH4Cl, EDCI, HOBt, Et3N,
DMF, rt, 38%.
2,3-Dihydro-6-(pyrazol-4-yl)thieno[3,2-d]pyrimidin-4(1H)-ones
possessing various substituents on pyrimidinone and pyrazole
rings (11a–c, 12a–f, 14a–e) were synthesized as outlined in
Scheme 2. Methylation of 9a afforded N-methyl derivative 9b
and both of them were then subjected to ester saponification.
However, attempts to isolate the corresponding carboxylic acids
were unsuccessful because they are prone to decarboxylation.
The result indicates that the electronic nature of the 5-substituent
significantly influences the stability of the 2-carboxylic acid. After
alkaline hydrolysis of 9a, b, the reaction mixture was treated with
6 M hydrochloric acid to maintain pH > 7 and the solvent was
removed by evaporation, yielding corresponding crude sodium
salts. Without further purification, the sodium salts were next sub-
jected to amidation reactions to furnish primary amides 10a, b.
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O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Scheme 2. Reagents and conditions: (a) K2CO3, MeI, DMF, 60 °C, 90%; (b) NaOH, MeOH–water, 70 °C; (c) NH4Cl, EDCI, HOBt, Et3N, DMF, rt, 76–85%; (d) acetone, p-TsOHꢁH2O,
AcOH, 70 °C, 60–91% for 11a, b or methyl 2,2,2-trifluoroethyl ketone, CSA, MgSO4, DMA, 150 °C, 31% for 11c; (e) boronic acid ester A, PdCl2(dppf), DME–water, reflux, 38% for
12a; (f) (1) boronic acid ester B, PdCl2(dppf), DME-water, reflux; (2) NaOH, 32–60% for 12b, c, d; (g) boronic acid ester C, PdCl2(dppf), DME–water, reflux, 36% for 12e; (h) (1)
boronic acid ester D, PdCl2(dppf), DME–water, reflux; (2) TFA, rt; (3) acetone, p-TsOHꢁH2O, MgSO4, DMF, 60 °C, 31% for 12f; (i) 1 M HCl, MeOH, 50 °C, 41%; (j) p-TsOHꢁH2O,
MgSO4, DMF, 80 °C, 41% (2 steps) for 14a; (k) CSA, MgSO4, DMA, 80 °C, 19–34% for 14b-e; The absolute structures were confirmed by X-ray crystallographic analysis (CCDC
1520567 for 14b, CCDC 1520568 for 14c and CCDC 1520566 for 14e).
Cyclization of 10a, b with acetone by using p-toluenesulfonic acid
as a catalyst afforded 11a, b. However, reaction of 10b with
2,2,2-trifluoroethyl methyl ketone didn’t proceed under the same
conditions presumably due to the poor reactivity. To overcome
the problem, reaction in the presence of camphorsulfonic acid
(CSA) and magnesium sulfate in dimethyl acetamide (DMA) was
carried out at higher temperature (150 °C) to successfully produce
cyclized product 11c. Suzuki-Miyaura coupling of 11a–c with pyra-
zole boronic acid esters (A–D), followed by deprotection under
appropriate conditions gave the desired products 12a–f. In the case
of 12f, removal of the dimethylsulfamoyl group by using trifluo-
roacetic acid (TFA) resulted in a mixture of 12f and the monocyclic
byproduct, which was again subjected to cyclization reaction to
generate 12f in a 31% yield. By utilizing the finding conversely,
12b was treated with 1 M hydrochloric acid in methanol to give
ring-opening derivative 13, which was re-cyclized with cyclohex-
Fig. 4. ORTEP of 14b, thermal ellipsoids are drawn at 50% probability.
anones to afford 14a–e. Compounds 14b-e have pseudo-chirality
but were obtained as a single stereoisomer after purification by
column chromatography. The absolute structures of 14b, c, e were
determined by X-ray crystallographic analysis as shown in Figs. 4–
Compounds having a substituent at the 7-position were pre-
pared as described in Scheme 3. Trifluoroacetylation of aminothio-
6. As for 14d, the absolute structure was unambiguously assigned
phene 15, followed by bromination at the 5-position yielded amide
as the antipode of 14e.
16. After N-methylation of 16, removal of trifluoroacetyl group
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O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
produced ester 17. Saponification and subsequent amidation gave
amide 18 as in the case of 10a, b (Scheme 2). Compound 18 was
then subjected to acid-catalyzed cyclization and subsequent
Suzuki coupling to afford 7-methyl derivative 20a. Ionic bromina-
tion of 12a worked well; however, a ring-opening side reaction
partially proceeded presumably due to the strong acidic condi-
tions. Thus, re-cyclization of the mixture was carried out to provide
7-bromo derivative 20b.
3. Results and discussion
3.1. Biological evaluation of 2,3-dihydrothieno[3,2-d]pyrimidin-4
(1H)-ones
All synthesized compounds were evaluated for the ability to
inhibit Cdc7 kinase activity to investigate SARs. To assess kinase
selectivity, the compounds were also evaluated for inhibitory
activities against cyclin-dependent kinase 2 (Cdk2)/cyclin E (cycE)
and ROCK1.
Firstly, starting from 4a (Fig. 3) as a lead compound, we evalu-
ated effects of R1 and R2 groups of the pyrimidine ring on activity
and selectivity, and the results are shown in Table 1. About 3-fold
increase in Cdc7 inhibitory activity was observed by incorporation
of a methyl group to the R1-position (4b vs. 4a) along with 2- to 4-
fold improvement in kinase selectivity. By contrast, incorporation
of a methyl or benzyl group to the R2 -position (4j, k vs. 4a)
resulted in more than 17-fold decrease in Cdc7 inhibitory activity
with decreased kinase selectivity. As expected from the pharma-
cophore model, incorporation of a substituent to the R2-position
was detrimental to Cdc7 inhibition presumably due to disruption
of the interaction with the Lys90 and Asp196 residues forming
the salt bridge (see Fig. 2). On the other hand, the substitution at
the R1-position was well tolerated or rather favorable for the
potency as well as selectivity as speculated in the pharmacophore
model.
Fig. 5. ORTEP of 14c, thermal ellipsoids are drawn at 30% probability.
Next, effects of R3 and R4 groups of the aminal moiety, were
investigated on Cdc7 enzyme inhibition and kinase selectivity,
and the results are presented in Table 2. Replacement of one of
the geminal dimethyl substituents with a bulkier alkyl group
(4c–f) enhanced Cdc7 enzyme inhibitory activity by 3- to 10-fold
compared with that of the lead compound 4a. Compounds 4g, h,
i having a spirocycloalkyl group showed a nanomolar inhibitory
activity and moderate kinase selectivity which were similar to
Fig. 6. ORTEP of 14e, thermal ellipsoids are drawn at 20% probability.
Scheme 3. Reagents: (a) (1) TFAA, pyridine, MeCN, 0 °C to rt; (2) NBS, acetic acid, 80 °C, 60%; (b) (1) MeI, K2CO3, DMF, 60 °C; (2) K2CO3, MeOH–water, rt, 62%; (c) (1) NaOH,
MeOH–water, 70 °C; (2) NH4Cl, Et3N, DMF, WSC, HOBt, 56%; (d) acetone, p-TsOHꢁH2O, MgSO4, DMF, 60 °C, 24%; (e) tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
pyrazole-1-carboxylate pyrazole-4-ylboronic acid, PdCl2(dppf), Cs2CO3, DME–water, 90 °C, 47%; (f) (1) bromine, acetic acid, rt; (2) acetone, p-TsOHꢁH2O, AcOH, 60 °C, 25%.
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O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 1
Effects of N1- and/or N3-substituents on Cdc7 enzyme inhibition and kinase selectivity.
Compound
R1
R2
Enzyme inhibition: IC50 (nM)a
Selectivity
Cdk2/Cdc7
Cdc7
Cdk2/cycE
ROCK1
ROCK1/Cdc7
4a
4b
4j
H
Me
H
H
H
Me
Bn
26 (17–41)
9.9 (5.2–19)
450 (280–700)
>10,000
3200 (6900–9100)
2100 (1800–2600)
>10,000
90 (81–100)
ꢂ120
ꢂ210
ꢂ22
ꢂ3.5
140 (130–150)
240 (210–280)
1.5 (1.3–1.7)
ꢂ14
ꢂ0.53
ꢂ0.00015
4k
H
650 (570–740)
ꢂ0.065
a
Numbers in parentheses represent 95% confidence interval.
Table 2
Effects of 2-substituents on Cdc7 enzyme inhibition and kinase selectivity.
Compound
R3
R4
Enzyme inhibition: IC50 (nM)a
Selectivity
Cdk2/Cdc7
Cdc7
Cdk2/cycE
ROCK1
ROCK1/Cdc7
4a
4c
4d
4e
4f
4g
4h
4i
Me
Et
n-Pr
i-Pr
CH2CF3
A(CH2)4A
A(CH2)5A
A(CH2)6A
Me
Me
Me
Me
Me
26 (17–41)
7.5 (4.4–13)
6.3 (3.3–12)
8.2 (5.9–11)
2.7 (1.8–4.1)
8.2 (5.7–12)
3.0 (2.2–4.0)
3.6 (2.5–5.1)
3200 (2800–3700)
2000 (1800–2600)
2000 (1800–2200)
1400 (1200–1600)
1200 (1000–1500)
4900 (4400–5500)
2000 (1700–2300)
2300 (2100–2500)
90 (81–100)
36 (15–91)
33 (28–39)
35 (31–39)
31 (28–34)
100 (93–110)
53 (43–66)
37 (32–41)
ꢂ120
ꢂ270
ꢂ320
ꢂ170
ꢂ440
ꢂ600
ꢂ670
ꢂ640
ꢂ3.5
ꢂ4.8
ꢂ5.2
ꢂ4.3
ꢂ11
ꢂ12
ꢂ18
ꢂ10
a
Numbers in parentheses represent 95% confidence interval.
those of 4f. The results indicated that the binding pocket accom-
modating the R3 and R4 groups is relatively spacious, which is con-
sistent with our assumption. As shown in Table 2, the 2-methyl-2-
(20,20,20-trifluoro)ethyl group (4f) is the best among the alkyl series.
On the other hand, there is not clear difference in potency and
selectivity of the spiroalkyl series. By considering synthetic feasi-
bility of incorporation of additional substituents into the spiroalkyl
moiety, we selected the spirocyclohexyl (4h) as well as 2-methyl-
2-trifluoroethyl (4f) groups as the 2-substituent.
To further improve Cdc7 inhibitory activity and, particularly
kinase selectivity, modification of the hinge-binding moiety (R5)
and adjacent R6-substituent was carried out and the results are
shown in Table 3. Replacement of the 4-pyridyl group with a 4-
pyrazolyl group (12a) maintained Cdc7 activity but reduced kinase
selectivity over Cdk2/cycE. We next incorporated an alkyl group
onto the pyrazole ring to examine substituent effects on potency
and selectivity. Incorporation of a methyl group to the 3-position
of the pyrazole ring (12c) enhanced the Cdc7 activity by about 3-
fold and partially recovered kinase selectivity over Cdk2/cycE rela-
tive to 12a. Further introduction of a methyl group to the 5-posi-
tion of 12c (12e) or replacement of the 3-methyl group of 12c
with the 3-ethyl group (12f) significantly decreased the Cdc7
inhibitory activity with impaired kinase selectivity. Introduction
of a bromo or methyl group to the R6-position of the scaffold didn’t
significantly affect the Cdc7 activity but led to marked reduction of
kinase selectivity (20a, b vs. 12a). The results strongly suggested
that a spatial limitation exists in the hinge-binding pocket. We
concluded that the 3-methylpyrazol-4-yl moiety was the best
hinge-binding moiety based on the overall in vitro profiles.
Thus, incorporation of the preferable R3 and R4 groups (Tables 1
and 2) to 2,3-dihydro-6-(3-methylpyrazol-4-yl)thienopyrimidin-4-
ones was investigated and the results are summarized in Table 4.
The 2-methyl-2-(20,20,20-trifluoro)ethyl analogue 12d showed the
most potent Cdc7 inhibitory activity (IC50 = 1.0 nM) with moderate
kinase selectivity over ROCK1. Spirocyclohexyl derivative 14a was
found to be almost equipotent to 12d with slightly improved selec-
tivity, which encouraged us to conduct further substitution on the
cyclohexane ring. As described before, the SAR of the 2-substituent
indicated that the binding pocket is relatively spacious. To further
improve kinase selectivity, we attempted to incorporate both polar
and lipophilic substituents to the cyclohexyl moiety, which can
retain desirable lipophilicity (clogP: around 3).16 As a result,
compound 14c possessing 4-fluorophenyl and hydroxyl groups at
the R9- and R10-positions of the cyclohexane ring demonstrated
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O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
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Table 3
Effects of 6- and/or 7-substituents on Cdc7 enzyme inhibition and kinase selectivity.
Compound
R5
R6
H
Enzyme inhibition: IC50 (nM)a
Selectivity
Cdk2/Cdc7
ꢂ210
Cdc7
Cdk2/cycE
ROCK1
ROCK1/Cdc7
4b
9.9 (5.2–19)
2100 (1800–2600)
140 (130–150)
ꢂ14
12a
12c
H
H
8.4 (5.1–14)
2.9 (1.5–5.5)
160 (140–190)
260 (220–310)
130 (110–160)
54 (49–59)
ꢂ19
ꢂ90
ꢂ15
ꢂ19
12e
12f
H
H
290 (180–470)
13 (6.3–25)
1100 (930–1300)
360 (300–420)
2100 (1700–2500)
350 (290–420)
ꢂ3.8
ꢂ28
ꢂ7.2
ꢂ27
20a
20b
Me
Br
14 (9.1–21)
9.5 (7.1–13)
26 (23–31)
26 (23–30)
44 (40–47)
22 (20–25)
ꢂ1.9
ꢂ2.7
ꢂ3.1
ꢂ2.3
a
Numbers in parentheses represent 95% confidence interval.
Table 4
Effects of 2-substituent including cyclohexane analogue on Cdc7 enzyme inhibition, kinase selectivity, MCM2 phosphorylation, and cancer cell growth.
Compound
R9
R10
Enzyme inhibition: IC50 (nM)a
Selectivity
Cdk2/Cdc7
clogPb
pMCM2c
IC50 (nM)
COLO205d
EC50 (nM)
Cdc7
Cdk2/cycE
ROCK1
ROCK1/Cdc7
12d
14a
14b
14c
14d
14e
–
H
–
H
1.0 (0.67–1.6)
1.5 (0.84–2.8)
170 (110–260)
1.2 (0.39–3.8)
380 (150–950)
2.0 (0.80–5.1)
420 (370–490)
1300 (1200–1400)
>10,000
960 (860–1100)
>10,000
22 (20–25)
48 (42–54)
130 (110–160)
820 (710–950)
350 (300–420)
2900 (2500–3400)
ꢂ420
ꢂ870
ꢂ59
ꢂ22
2.82
2.89
2.81
2.81
3.35
3.35
210
290
–
>1000
–
330
470
–
4500
–
ꢂ32
OH
4-F-Ph
OH
4-F-Ph
OH
cyc-Hex
OH
ꢂ0.76
ꢂ680
ꢂ0.92
ꢂ1500
ꢂ800
ꢂ26
cyc-Hex
2200 (2000–2400)
ꢂ1100
>1000
6200
a
b
c
Numbers in parentheses represent 95% confidence interval.
clogP values were calculated by Daylight Software.
Inhibition of phosphorylation of Ser40 in HeLa cell line.
Growth inhibition of COLO205 cell line.
d
significant improvement of selectivity over ROCK1 with maintain-
ing a nanomolar Cdc7 inhibitory activity. On the other hand, the
corresponding pseudo-enantiomer 14b exhibited significantly
reduced potency and selectivity. A similar tendency was observed
in the case of spirocyclohexyl analogues 14d, e and the eutomer
14e proved to show high kinase selectivity with retaining a
nanomolar activity. Accordingly, compound 14c, e were highly
selective Cdc7 inhibitors with low nanomolar potency in this series.
Selected compounds 12d, 14a, c, e were evaluated for their cel-
lular potency (Table 4). To assess Cdc7 kinase inhibition in cells,
phosphorylation status of Ser40 in MCM2 was measured by using
HeLa cells derived from human cervical cancer. COLO205 cells
derived from human colon cancer were also used to evaluate inhi-
bition of cancer cell growth in vitro.
Despite potent Cdc7 inhibitory activity in the cell-free assay, all
the compounds tested showed moderate cellular potency at
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8
O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
mainly contributed to the growth inhibition. However, the gap
between cell-free and cellular Cdc7 inhibition still remains unclear
because there are no significant defect and difference in solubility
and membrane permeability of 12d, 14a, c, e (data not shown).
Consequently, we succeeded in the identification of new,
potent, and selective Cdc7 kinase inhibitors represented by 12d,
14a, c, e designed based on the putative pharmacophore model.
3.2. Molecular modeling studies
In order to elucidate the kinase selectivity profile of compound
14c showing favorable in vitro profiles, docking study with the
reported Cdc7 co-crystal structure (PDB: 4F9C)17 and co-crystal
structural analysis of 14c with ROCK2, of which all the 34 amino
acid residues in the ATP binding pocket are identical to those of
ROCK1, were performed. As shown in Fig. 7a, the docking model
of 14c with the Cdc7 crystal structure17 revealed the following
findings: (i) Two hydrogen bonds between the two pyrazole nitro-
gen atoms and the Pro135/Leu137 residues in the hinge region are
formed. (ii) The oxygen and nitrogen atoms of the lactam moiety
effectively interact with the Lys90 and Asp196 residues, respec-
tively. (iii) The two sulfur atoms of the thiophene ring and the
Met134 residue face to each other, but a direct interaction between
them isn’t observed. (iv) The bulky 4,4-substituted-cyclohexyl
moiety extends toward solvent-exposed region without steric
clash with any amino acid residue in the pocket.
Co-crystal structure of 14c with ROCK2 demonstrates that 14c
forms hydrogen bonding in a similar manner to that observed in
the above-mentioned docking model with Cdc7 (Fig. 7b). The
results don’t clearly explain the high selectivity for Cdc7 over
ROCK1/2. Therefore, superposition of the X-ray co-crystal structure
of 14c with ROCK2 and crystal structure of ROCK2 apo-form was
examined (Fig. 7c). The study suggested that Asp176 and Asp218
residues located in the proximity of the 4-fluorophenyl group of
14c slightly flipped out in comparison with the apo form. This
may be attributed to unfavorable anion-p repulsion between the
two Asp residues and the benzene ring. By considering the high
amino-acid sequence homology between ROCK1 and ROCK2, the
anion-p repulsion may similarly exist, resulting in attenuated
ROCK1 inhibitory activity. As in the case of 14c, compound 14e
bearing a cyclohexane ring on the spirocyclohexyl group displayed
high selectivity for Cdc7 over ROCK1 (see Table 4), which can be
ascribed to steric clash between the bulky cyclohexyl group and
Asp residues. In contrast, no unfavorable factor was observed in
the putative binding mode of the bulky 4,4-disubstituted-cyclo-
hexyl moiety of 14c with any Cdc7 amino acid residue (Fig. 7a),
elucidating the high kinase selectivity.
On the basis of these finding, we concluded that the high selec-
tivity of compounds 14c, e for Cdc7 over ROCK1 mainly results
from the electrostatic repulsion or steric clash between the bulky
4-substituent of the cyclohexyl moiety and the Asp residues of
ROCK1.
4. Conclusion
To search for a new series of potent Cdc7 inhibitors, the putative
pharmacophore model was generated on the basis of the Cdc7
homology model constructed by the crystal structure of CK2. Drug
design based on the model suggested several candidate scaffolds
for new Cdc7 inhibitors, leading to the identification of dihydroth-
ieno[3,2-d]-pyrimidin-4(1H)-one based Cdc7 inhibitor 4a as a lead
compound. Medicinal chemistry program starting from 4a
revealed the following SARs: (i) introduction of substituents to 1-
and/or 2-position was favorable as expected from the pharma-
cophore model; (ii) the 3-methylpyrazol-4-yl moiety was found
Fig. 7. (a) Docking model of compound 14c with the Cdc7 crystal structure (PDB:
4F9C). (b) X-ray co-crystal structure of 14c with ROCK2 (PDB: 5U7R). (c)
Superposition of X-ray co-crystal structure of 14c with ROCK2 (pink) and crystal
structure of ROCK2 apo-form (green, PDB: 5U7Q).
concentrations below 1 lM (phosphorylation) and 10 lM (cancer
cell growth). Among them, compounds 12d, 14a exhibited submi-
cromolar cellular activities. The inhibition of MCM2 phosphoryla-
tion and cell growth of 12d, 14a were observed in the same
concentration range, suggesting that the cellular Cdc7 inhibition
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O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
as the best hinge-binding group in terms of both Cdc7 inhibition
and kinase selectivity. Further SAR studies succeeded in the discov-
ery of new, potent, and cell active Cdc7 selective inhibitors (12d,
14a). Additionally, molecular modeling studies suggested that the
high selectivity of 14c, e for Cdc7 over ROCK1 is mainly due to
the electrostatic repulsion or steric clash of the bulky 4-substituent
of the cyclohexyl moiety with the Asp residues. These results could
pave the way for developing highly potent and selective Cdc7 inhi-
bitors for the treatment of cancer.
bath, then conc. HCl (2.48 mL, 300 mmol) was added to adjust
the pH approx. 4. The resultant yellow precipitate was collected
by filtration, washed with water–MeOH, and dried under reduced
pressure to afford 2a (2.02 g, 92%) as a yellow solid. 1H NMR
(300 MHz, DMSO-d6) d 7.20 (1H, s), 7.62 (2H, dd, J = 4.8, 1.5 Hz),
8.62 (2H, d, J = 6.3 Hz). The amino protons and carboxylic acid pro-
ton were not observed.
5.1.1.3. 3-(Methylamino)-5-pyridin-4-ylthiophene-2-carboxylic acid
(2b). Crude compound 2b was prepared in quantitative yield from
1b by a procedure similar to that described for 2a as a yellow solid.
1H NMR (300 MHz, DMSO-d6) d 2.68 (3H, d, J = 3.0 Hz), 5.59 (1H, br
s), 6.07 (1H, d, J = 1.8 Hz), 7.24 (1H, d, J = 1.8 Hz), 7.51–7.53 (2H, m),
8.52–8.54 (2H, m).
5. Experimental
5.1. Chemistry
5.1.1. General
5.1.1.4. 3-Amino-5-pyridin-4-ylthiophene-2-carboxamide (3a). Com-
pound 3a was prepared in 84% yield from 2a by a procedure similar
to that described for 3b as a yellow solid. 1H NMR (300 MHz,
DMSO-d6) d 6.54 (2H, br s), 7.06 (2H, br s), 7.18 (1H, s), 7.54–
7.58 (2H, m), 8.61 (2H, d, J = 6.3 Hz).
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
5.1.1.5.
3-(Methylamino)-5-pyridin-4-ylthiophene-2-carboxamide
mesh) or Purif-Pack (SI 60 lM or NH 60 lM, Fuji Silysia Chemical,
(3b). To a solution of compound 2b (200 mg, 0.85 mmol), NH4Cl
(93 mg, 1.7 mmol), EDCI (195 mg, 1.02 mmol), HOBt (156 mg,
1.02 mmol) in DMF (20 mL) was added Et3N (258 mg, 2.55 mmol)
at room temperature. The reaction mixture was stirred overnight
at room temperature. After removal of the solvent under reduced
pressure, the residue was purified by column chromatography on
silica gel (DCM/MeOH, 19:1, v/v) to give 3b (110 mg, yield 55%)
as a yellow solid. 1H NMR (300 MHz, DMSO-d6) d 2.94 (3H, d,
J = 5.1 Hz), 7.00–7.09 (2H, m), 7.21–7.28 (1H, m), 7.49 (1H, s),
7.63–7.66 (2H, m), 8.60–8.63 (2H, m).
Ltd.) or Combi-Flash. The proton nuclear magnetic resonance (1H
NMR) spectra were recorded on Bruker AVANCE II (300 MHz), Bru-
ker AV 300 (300 MHz), or Bruker AV (500 MHz) instruments.
Chemical shifts are given in parts per million (ppm) with tetram-
ethylsilane as an internal standard. Abbreviations are used as fol-
lows: 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. ꢂ50 mm, Shiseido, Japan) or L-column
2 ODS
5.1.1.6. 3-Amino-N-methyl-5-pyridin-4-ylthiophene-2-carboxamide
(3c). Compound 3c was prepared in 75% yield from 2a by a proce-
dure similar to that described for 3b as a yellow solid. 1H NMR
(300 MHz, DMSO-d6) d 2.69 (3H, d, J = 4.5 Hz), 6.50 (2H, br s),
7.19 (1H, s), 7.54 (2H, d, J = 6.0 Hz), 7.61–7.62 (1H, m), 8.61 (2H,
d, J = 6.0 Hz).
(2.0 mm i.d. ꢂ30 mm, CERI, Japan) with a temperature of 50 °C
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 ammo-
nium 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.
5.1.1.7. 3-Amino-N-benzyl-5-pyridin-4-ylthiophene-2-carboxamide
(3d). Compound 3d was prepared in 80% yield from 2a by a proce-
dure similar to that described for 3b as a yellow solid. 1H NMR
(300 MHz, DMSO-d6) d 4.38 (2H, d, J = 6.0 Hz), 6.56 (2H, br s),
7.21–7.35 (6H, m), 7.76 (2H, dd, J = 4.5, 1.5 Hz), 8.28 (1H, t,
J = 5.7 Hz), 8.61 (2H, dd, J = 4.5, 1.5 Hz).
5.1.1.8. 2,2-Dimethyl-6-pyridin-4-yl-2,3-dihydrothieno[3,2-d]pyrim-
idin-4(1H)-one (4a). A mixture of 3a (150 mg, 0.68 mmol), PTSA
(50 mg) and acetone (10 mL) in toluene (20 mL) was heated to
reflux overnight. After removal of the solvent under reduced pres-
sure, the residue was purified by column chromatography on silica
gel (DCM/MeOH, 9:1, v/v) to afford 4a (130 mg, yield 74%) as an
orange solid. 1H NMR (300 MHz, DMSO-d6) d 1.42 (6H, s), 7.62–
7.65 (3H, m), 8.59–8.61 (2H, m). HRMS: Calcd for C13H14N3OS [M
+H]+: 260.0852. Found: 260.0834.
5.1.1.1. Methyl 3-(methylamino)-5-pyridin-4-ylthiophene-2-carboxy-
late (1b). To a solution of compound 1a18 (300 mg, 1.29 mmol) in
DMF (25 mL) was added NaH (60% in mineral oil, 63 mg,
1.56 mmol) at 0 °C. After being stirred for 10 min, to the solution
was added dropwise MeI (200 mg, 1.42 mmol), and then the reac-
tion mixture was stirred at room temperature for 3 h. The solvent
was removed under reduced pressure, and the residue was purified
by column chromatography on silica gel (PE/EtOAc, 1:1, v/v) to give
crude 1b (200 mg, 62%) as a yellow solid. This material was used in
the next reaction without further purification.
5.1.1.9.
1,2,2-Trimethyl-6-pyridin-4-yl-2,3-dihydrothieno[3,2-d]
pyrimidin-4(1H)-one (4b). A mixture of 3b (69 mg, 0.30 mmol),
PTSA (5.6 mg) and acetone (2 mL) in acetic acid (1 mL) was stirred
at 80 °C overnight. After removal of the solvent under reduced
pressure, the residue was purified by column chromatography on
silica gel (n-hexane/EtOAc, 95:5 to 0:100, v/v) and triturated with
EtOAc. The precipitate was collected by filtration to afford 4b
(60 mg, yield 74%) as yellow solid. 1H NMR (300 MHz, DMSO-d6)
d 1.42 (6H, s), 2.93 (3H, s), 7.56 (1H, s), 7.68 (2H, dd, J = 4.2,
5.1.1.2. 3-Amino-5-(pyridin-4-yl)thiophene-2-carboxylic acid (2a). A
mixture of 1a18 (2.34 g, 10.0 mmol), sodium methoxide (1.62 g,
30.0 mmol), MeOH (40 mL) and water (10 mL) was refluxed for
4 h. The mixture was allowed to cool to room temperature and stir-
ring was continued overnight. The mixture was cooled under ice-
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O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
1.8 Hz), 8.61 (2H, dd, J = 4.8, 1.8 Hz). MS: Calcd for C14H16N3OS [M
+H]+: 274. Found: 274. Anal. Calcd for C14H15N3OS: C, 61.51; H,
5.53; N, 15.37. Found: C, 61.46; H, 5.60; N, 15.32.
5.1.1.17.
2,2,3-Trimethyl-6-pyridin-4-yl-2,3-dihydrothieno[3,2-d]
pyrimidin-4(1H)-one (4j). Compound 4j was prepared in 32% yield
from 3c by a procedure similar to that described for 4b as yellow
crystals. Mp 231–234 °C (from MeOH–EtOAc). 1H NMR (300 MHz,
DMSO-d6) d 1.47 (6H, s), 2.86 (3H, s), 7.16 (1H, s), 7.18 (1H, br s),
5.1.1.10. 2-Ethyl-2-methyl-6-pyridin-4-yl-2,3-dihydrothieno[3,2-d]
pyrimidin-4(1H)-one (4c). Compound 4c was prepared in 69% yield
from 3a by a procedure similar to that described for 4b as yellow
crystals. Mp 260–263 °C (from EtOAc–DCM). 1H NMR (300 MHz,
DMSO-d6) d 0.89 (3H, t, J = 7.5 Hz), 1.36 (3H, s), 1.63–1.77 (2H,
m), 7.09 (1H, br s), 7.14 (1H, s), 7.59 (1H, br s), 7.62 (2H, dd,
7.64 (2H, d, J = 6.0 Hz), 8.61 (2H, d, J = 6.0 Hz). MS: Calcd for C14
-
H
16N3OS [M+H]+: 274. Found: 274.
5.1.1.18.
3-Benzyl-2,2-dimethyl-6-pyridin-4-yl-2,3-dihydrothieno
[3,2-d]pyrimidin-4(1H)-one (4k). Compound 4k was prepared in
15% yield from 3d by a procedure similar to that described for 4b
as yellow crystals. Mp 201–204 °C (from EtOAc–hexane). 1H NMR
(300 MHz, DMSO-d6) d 1.42 (6H, s), 4.68 (2H, s), 7.19–7.34 (7H,
m), 7.66–7.68 (2H, m), 8.61–8.63 (2H, m). MS: Calcd for C20H20N3-
OS [M+H]+: 350. Found: 350. Anal. Calcd for C20H19N3OS: C, 68.74;
H, 5.48; N, 12.02. Found: C, 68.59; H, 5.52; N, 11.96.
J = 4.5, 1.5 Hz), 8.60 (2H, dd, J = 4.5, 1.5 Hz). MS: Calcd for C14H16
-
N3OS [M+H]+: 274. Found: 274.
5.1.1.11. 2-Methyl-2-n-propyl-6-pyridin-4-yl-2,3-dihydrothieno[3,2-
d]pyrimidin-4(1H)-one (4d). Compound 4d was prepared in 44%
yield from 3a by a procedure similar to that described for 4b as yel-
low crystals. Mp 216–219 °C (from DCM). 1H NMR (300 MHz,
DMSO-d6) d 0.87 (3H, t, J = 7.2 Hz), 1.37–1.42 (5H, m), 1.63–1.67
(2H, m), 7.10 (1H, br s), 7.13 (1H, s), 7.60–7.63 (3H, m), 8.60 (2H,
dd, J = 4.5, 1.5 Hz). MS: Calcd for C15H18N3OS [M+H]+: 288. Found:
288.
5.1.1.19.
6-Pyridin-4-ylthieno[3,2-d]pyrimidine-2,4(1H,3H)-dione
(5). A mixture of 3a (110 mg, 0.50 mmol) and urea (3.00 g,
50.0 mmol) was stirred at 180 °C for 2 h. After cooling to rt, 2 M
NaOH (30 mL) was added. After stirred for 30 min, the precipitate
was collected by filtration to give yellow solid. The obtained solid
was suspended in water (10 mL), and AcOH was added to pH 7. The
precipitate was collected by filtration. The obtained solid was trit-
urated with MeOH (10 mL). The precipitate was collected by filtra-
tion to give product 5 (37 mg, 34%) as an orange solid. 1H NMR
(300 MHz, DMSO-d6) d 7.45 (1H, s), 7.74–7.77 (2H, m), 8.64–8.69
(2H, m, J = 6.2 Hz), 11.16–11.41 (1H, m), 11.58–11.83 (1H, m).
MS: Calcd for C11H8N3O2S [M+H]+: 246. Found: 246. Anal. Calcd
for C11H7N3O2Sꢁ0.4H2O: C, 52.33; H, 3.11; N, 16.64. Found: C,
52.43; H, 3.09; N, 16.64.
5.1.1.12. 2-Isopropyl-2-methyl-6-pyridin-4-yl-2,3-dihydrothieno[3,2-
d]pyrimidin-4(1H)-one (4e). Compound 4e was prepared in 11%
yield from 3a by a procedure similar to that described for 4b as yel-
low crystals. Mp 243–245 °C (from MeOH–EtOAc). 1H NMR
(300 MHz, DMSO-d6) d 0.90–0.94 (6H, m), 1.32 (3H, s), 2.01–2.08
(1H, m), 7.11 (1H, br s), 7.13 (1H, s), 7.60–7.62 (3H, m), 8.60 (2H,
d, J = 6.3 Hz). MS: Calcd for C15H18N3OS [M+H]+: 288. Found: 288.
Anal. Calcd for C15H17N3OS: C, 62.69; H, 5.96; N, 14.62. Found: C,
62.58; H, 5.94; N, 14.48.
5.1.1.13. 2-Methyl-6-pyridin-4-yl-2-(2,2,2-trifluoroethyl)-2,3-dihy-
drothieno[3,2-d]pyrimidin-4(1H)-one (4f). Compound 4f was pre-
pared in 15% yield from 3a by a procedure similar to that
described for 4b as yellow crystals. Mp 238–240 °C (from EtOAc–
hexane). 1H NMR (300 MHz, DMSO-d6) d 1.56 (3H, s), 2.69–2.76
(2H, m), 7.20 (1H, s), 7.42 (1H, br s), 7.65 (2H, d, J = 5.1 Hz), 7.89
(1H, br s), 8.62 (2H, d, J = 4.8 Hz). MS: Calcd for C14H13F3N3OS [M
+H]+: 328. Found: 328.
5.1.1.20. 2-Amino-5-pyridin-4-ylthiophene-3-carboxamide (7). To a
solution of 619 (400 mg, 1.53 mmol) in EtOH (50 mL) was added
a solution of LiOH monohydrate (256 mg, 6.1 mmol) in water
(5 mL). The reaction mixture was heated to 80 oC for 2 h. After
removal of the solvent under reduced pressure, the residue was
dissolved in water and extracted with EtOAc (50 mL). The organic
layer was discarded, and the aqueous layer was treated with 2 M
HCl to pH 5–6. The precipitate was collected by filtration and dried
over MgSO4 to give 2-amino-5-pyridin-4-ylthiophene-3-carboxylic
acid (277 mg, 77%) as an orange solid. [1H NMR (300 MHz, DMSO-
d6) d 7.40 (2H, dd, J = 4.5, 1.5 Hz), 7.59 (1H, s), 7.65–7.72 (2H, m),
8.39–8.43 (2H, m), 12.19–12.27 (1H, m).] To a solution of the car-
boxylic acid (2.20 g, 10 mmol), NH4Cl (1.09 g, 20 mmol), EDCI
(2.30 g, 30 mmol) and HOBt (1.80 g, 12 mmol) in DMF (100 mL)
was added Et3N (3.0 g, 30 mmol) at room temperature. The reac-
tion mixture was stirred overnight at room temperature. After
removal of the solvent, the residue was purified by column chro-
5.1.1.14.
60-Pyridin-4-yl-10H-spiro[cyclopentane-1,20-thieno[3,2-d]
pyrimidin]-40(30H)-one (4g). Compound 4g was prepared in 72%
yield from 3a by a procedure similar to that described for 4a as
an orange solid. 1H NMR (300 MHz, DMSO-d6) d 1.66–1.69 (4H,
m), 1.83–1.89 (4H, m), 7.16 (1H, s), 7.24 (1H, s), 7.62 (2H, d,
J = 6.0 Hz), 7.80 (1H, s), 8.60–8.61 (2H, m). MS: Calcd for C15H16N3-
OS [M+H]+: 286. Found: 286.
5.1.1.15. 60-(Pyridin-4-yl)-10H-spiro[cyclohexane-1,20-thieno[3,2-d]
pyrimidin]-40(30H)-one (4h). Compound 4h was prepared in 36%
yield from 3a by a procedure similar to that described for 4a as yel-
low crystals. Mp 248–250 °C (from MeOH–DCM). 1H NMR
(300 MHz, DMSO-d6) d 1.15–1.30 (1H, m), 1.40–1.66 (7H, m),
1.81–1.93 (2H, m), 7.15 (1H, br s), 7.18 (1H, s), 7.61 (2H, dd,
J = 4.5, 1.5 Hz), 7.63 (1H, br s), 8.61 (2H, dd, J = 4.8, 1.8 Hz). MS:
Calcd for C16H18N3OS [M+H]+: 300. Found: 300.
matography on silica gel (DCM/MeOH, 19:1, v/v) to give 7
(1.10 g, 50%) as a brown solid. 1H NMR (400 MHz, CD3OD) d 7.44
(2H, d, J = 6.0 Hz), 7.79 (1H, s), 8.40 (2H, d, J = 6.4 Hz). MS: Calcd
for C10H10N3OS [M+H]+: 220. Found: 220.
5.1.1.21. 2,2-Dimethyl-6-pyridin-4-yl-2,3-dihydrothieno[2,3-d]pyrim-
idin-4(1H)-one (8). Compound 8 was prepared in 10% yield from 7
by a procedure similar to that described for 4a as an orange solid.
1H NMR (400 MHz, CD3OD) d 1.58 (6H, s), 7.49 (2H, dd, J = 4.8,
5.1.1.16.
60-Pyridin-4-yl-10H-spiro[cycloheptane-1,20-thieno[3,2-d]
1.6 Hz), 7.64 (1H, s), 8.43 (2H, d, J = 6.0 Hz). MS: Calcd for C13H14-
pyrimidin]-40(30H)-one (4i). Compound 4i was prepared in 14%
yield from 3a by a procedure similar to that described for 4b as yel-
low crystals. Mp 233–235 °C (from MeOH–EtOAc). 1H NMR
(300 MHz, DMSO-d6) d 1.52 (8H, m), 1.86–2.02 (4H, m), 7.13 (1H,
s), 7.26 (1H, br s), 7.62 (2H, dd, J = 4.8, 1.8 Hz), 7.74 (1H, br s),
8.61 (2H, dd, J = 4.8, 1.5 Hz). MS: Calcd for C17H20N3OS [M+H]+:
314. Found: 314.
N3OS [M+H]+: 260. Found: 260.
5.1.1.22. Methyl 5-bromo-3-(methylamino)thiophene-2-carboxylate
(9b). A mixture of 9a20 (996 mg, 3.00 mmol), K2CO3 (829 mg,
6.00 mmol), DMF (6 mL) and MeI (0.225 mL, 3.60 mmol) was stir-
red at 60 °C for 4 h. Then, the mixture was poured into saturated
NaHCO3 aq. (100 mL), and extracted with EtOAc (100 mL). The
Please cite this article in press as: Kurasawa O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.021

O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
organic layer was washed with brine, dried over MgSO4, filtered
and concentrated in vacuo. The residue was mixed with K2CO3
(415 mg, 3.00 mmol), MeOH (20 mL) and water (10 mL). The mix-
ture was stirred at room temperature overnight. Then, the reaction
mixture was worked up as described above. The residue was puri-
fied by column chromatography on silica gel (hexane to 70:30 hex-
ane/EtOAc) to afford 9b (674 mg, 90%) as a white solid. 1H NMR
(300 MHz, DMSO-d6) d 2.89 (3H, d, J = 5.1 Hz), 3.70 (3H, s), 6.82–
6.88 (1H, m), 7.05 (1H, s).
was purged with argon gas again. This mixture was refluxed for
18 h. Then, the mixture was poured into saturated NaHCO3 aq.
(100 mL), and EtOAc (100 mL), and the mixture was stirred vigor-
ously. An insoluble materials were filtered off. The organic layer
was separated from the filtrate, washed with brine, dried over
MgSO4, filtered and concentrated under reduced pressure. This
residue was purified by column chromatography on silica gel (n-
hexane/EtOAc, 95:5 to 0:100, v/v, then EtOAc/MeOH, 100:0 to
90:10, v/v) to afford a white solid (60 mg). This solid was triturated
with EtOAc/hexane and collected by filtration to afford 12a
(50.3 mg, 38%) as a white solid. 1H NMR (300 MHz, DMSO-d6) d
1.40 (6H, s), 2.87 (3H, s), 6.97 (1H, s), 7.44 (1H, br s), 7.84 (1H, br
s), 8.16 (1H, br s), 13.12 (1H, br s). HRMS: Calcd for C12H15N4OS
[M+H]+: 263.0961. Found: 263.0931.
5.1.1.23. 3-Amino-5-bromothiophene-2-carboxyamide (10a). A mix-
ture of 9a20 (5.76 g, 24.4 mmol), NaOH (2.94 g, 73.5 mmol), water
(25 mL) and MeOH (100 mL) was stirred at 70 °C overnight. After
cooling under ice-water bath, 6 M HCl (8.17 mL, 49.0 mmol) was
added and the mixture was concentrated in vacuo. NH4Cl (26.3 g,
491 mmol), Et3N (49.7 g, 491 mmol) and DMF (230 mL) were
added to the residue. After being stirred for 5 min at room temper-
ature, EDCI (28.2 g, 147 mmol) and HOBt (19.9 g, 147 mmol) were
added and the stirring was continued for 5 days. The mixture was
poured into saturated NaHCO3 aq., and extracted with EtOAc. The
organic layer was washed with brine, dried over MgSO4, filtered
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (n-hexane/EtOAc) to afford 10a
(4.1 g, 76%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) d
6.56 (2H, br s), 6.70 (1H, s), 6.91 (2H, br s).
5.1.1.29. 2,2-Dimethyl-6-(3-methyl-1H-pyrazol-4-yl)-2,3-dihydroth-
ieno[3,2-d]pyrimidin-4(1H)-one (12b). Compound 12b was pre-
pared in 60% yield from 11a and boronic acid ester B by a
procedure similar to that described for 12a as a pale yellow solid.
1H NMR (300 MHz, DMSO-d6) d 1.40 (6H, s), 2.37 (3H, br s), 6.53
(1H, s), 6.96 (1H, br s), 7.34 (1H, br s), 7.70 (0.6H, br s), 8.06
(0.4H, br s), 12.85 (1H, br s). HRMS: Calcd for C12H15N4OS [M
+H]+: 263.0961. Found: 263.0937.
5.1.1.30.
1,2,2-Trimethyl-6-(3-methyl-1H-pyrazol-4-yl)-2,3-dihy-
drothieno[3,2-d]pyrimidin-4(1H)-one (12c). Compound 12c was
prepared in 32% yield from 11b by a procedure similar to that
described for 12b as a pale yellow solid. 1H NMR (300 MHz,
DMSO-d6) d 1.40 (6H, s), 2.40 (3H, br s), 2.88 (3H, s), 6.86 (1H, s),
7.44 (1H, br s), 7.77 (0.6H, br s), 8.10 (0.4H, br s), 12.86 (1H, m).
Anal. Calcd for C13H16N4OS: C, 56.50; H, 5.84; N, 20.27. Found: C,
56.20; H, 5.87; N, 20.07.
5.1.1.24.
5-Bromo-3-(methylamino)thiophene-2-carboxamide
(10b). Compound 10b was prepared in 85% yield from 9b by a pro-
cedure similar to that described for 10a as a pale brown solid. 1H
NMR (300 MHz, DMSO-d6) d 2.83 (3H, d, J = 5.1 Hz), 6.92 (2H, br
s), 6.98 (1H, s), 7.30–7.33 (1H, m).
5.1.1.25. 6-Bromo-2,2-dimethyl-2,3-dihydrothieno[3,2-d]pyrimidin-4
(1H)-one (11a). Compound 11a was prepared in 90% yield from
10a by a procedure similar to that described for 4b as a brown
solid. 1H NMR (300 MHz, DMSO-d6) d 1.38 (6H, s), 6.69 (1H, s),
7.18 (1H, br s), 7.56 (1H, br s).
5.1.1.31. 1,2-Dimethyl-6-(3-methyl-1H-pyrazol-4-yl)-2-(2,2,2-trifluo-
roethyl)-2,3-dihydrothieno[3,2-d]pyrimidin-4(1H)-one (12d). Com-
pound 12d was prepared in 50% yield from 11c by a procedure
similar to that described for 12b as a pale yellow solid. 1H NMR
(300 MHz, DMSO-d6) d 1.59 (3H, s), 2.40 (3H, br s), 2.55–2.79
(1H, m), 2.84–3.06 (1H, m), 2.93 (3H, s), 6.88 (1H, s), 7.71 (1H, s),
7.79 (0.6H, br s), 8.12 (0.4H, br s), 12.87 (1H, br s). Anal. Calcd
for C14H15F3N4OSꢁH2O: C, 48.83; H, 4.39; N, 16.27. Found: C,
48.84; H, 4.47; N, 16.18.
5.1.1.26.
6-Bromo-1,2,2-trimethyl-2,3-dihydrothieno[3,2-d]pyrim-
idin-4(1H)-one (11b). Compound 11b was prepared in 61% yield
from 10b by a procedure similar to that described for 4b as a yel-
low solid. 1H NMR (300 MHz, DMSO-d6) d 1.39 (6H, s), 2.84 (3H, s),
7.07 (1H, s), 7.64 (1H, br s).
5.1.1.32. 6-(3,5-Dimethyl-1H-pyrazol-4-yl)-1,2,2-trimethyl-2,3-dihy-
drothieno[3,2-d]pyrimidin-4(1H)-one (12e). Compound 12e was
prepared in 36% yield from 11b and boronic acid ester C by a pro-
cedure similar to that described for 12b as a pale yellow solid. 1H
NMR (300 MHz, DMSO-d6) d 1.41 (6H, s), 2.29–2.33 (6H, m), 2.88
(3H, s), 6.65 (1H, s), 7.44 (1H, br s), 12.53 (1H, br s). HRMS: Calcd
for C14H19N4OS [M+H]+: 291.1274. Found: 291.1249.
5.1.1.27.
6-Bromo-1,2-dimethyl-2-(2,2,2-trifluoroethyl)-2,3-dihy-
drothieno[3,2-d]pyrimidin-4(1H)-one (11c). A mixture of 10b
(940 mg, 4.0 mmol), 4,4,4-trifluorobutan-2-one (5.04 g, 40 mmol),
CSA (93 mg, 0.40 mmol), MgSO4 (576 mg, 6.0 mmol) and DMA
(4 mL) was reacted under microwave irradiation at 150 °C for
1 h. The mixture was poured into saturated NaHCO3 aq., and
extracted with EtOAc. The combined extracts were washed with
water and brine, dried over Na2SO4 and filtered. After removal of
the solvent under reduced pressure, the residue was purified by
column chromatography on amino silica gel (n-hexane/EtOAc,
90:10 to 50:50, v/v) to give a yellow solid. The solid was purified
by crystallization from EtOAc/heptane to give 11c (431 mg, 31%)
as a yellow solid. 1H NMR (300 MHz, DMSO-d6) d 1.58 (3H, s),
2.53–2.73 (1H, m), 2.84–3.05 (1H, m), 2.88 (3H, s), 7.10 (1H, s),
7.91 (1H, br s).
5.1.1.33. 6-(3-Ethyl-1H-pyrazol-4-yl)-1,2,2-trimethyl-2,3-dihydroth-
ieno[3,2-d]pyrimidin-4(1H)-one (12f). Compound 12f was prepared
in 31% yield from 11b and boronic acid ester D21 by a procedure
similar to that described for 12a as a pale yellow solid. 1H NMR
(300 MHz, DMSO-d6) d 1.23 (3H, t, J = 7.8 Hz), 1.40 (6H, s), 2.72–
2.88 (5H, m), 6.84 (1H, s), 7.44 (1H, s), 7.76 (0.67H, br s), 8.08
(0.33H, br s), 12.79 (0.33H, br s), 12.88 (0.67H, br s). HRMS: Calcd
for C14H19N4OS [M+H]+: 291.1274. Found: 291.1253.
5.1.1.28. 1,2,2-Trimethyl-6-(1H-pyrazol-4-yl)-2,3-dihydrothieno[3,2-
d]pyrimidin-4(1H)-one (12a). A mixture of 11b (138 mg,
0.50 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazole (291 mg, 1.50 mmol), Na2CO3 (265 mg, 2.50 mmol),
DME (5 mL) and water (2.5 mL) was purged with argon gas. Then,
PdCl2(dppf) (40.8 mg, 0.050 mmol) was added, and the mixture
5.1.1.34. 3-Amino-5-(3-methyl-1H-pyrazol-4-yl)thiophene-2-carbox-
amide (13). A mixture of 12b (1.97 g, 7.51 mmol), 1 M HCl (38 mL)
and MeOH (38 mL) was stirred at 50 °C for 4 h. After removal of the
solvent under reduced pressure, the organic materials were
extracted with EtOAc/THF. The combined extracts were dried over
Na2SO4 and filtered. After removal of the solvent under reduced
Please cite this article in press as: Kurasawa O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.021

12
O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
pressure, the residue was purified by column chromatography on
silica gel (EtOAc/MeOH, 100:0 to 90:10, v/v) to give 13 (1.20 g,
72%) as a green solid. 1H NMR (300 MHz, DMSO-d6) d 2.37 (3H,
br s), 6.44 (2H, br s), 6.59 (1H, s), 6.75 (2H, br s), 7.63 (0.6H, br
s), 7.95 (0.4H, br s), 12.83 (1H, br s).
5.1.1.38. Methyl 5-bromo-4-methyl-3-[(trifluoroacetyl)amino]thio-
phene-2-carboxylate (16). To stirred solution of 15 (50 g,
a
292 mmol) in MeCN (600 mL) were added pyridine (28.6 mL,
350 mmol) and TFAA (58.6 mL, 421 mmol) at 0 °C. After 5 min,
the mixture was allowed to room temperature and the stirring
was continued for 2 h. The mixture was poured into ice-water
(2.5 L) and the mixture was stirred for 20 min. Then, extraction
with EtOAc (1 L), washing with brine, drying over MgSO4, filtration
and concentration under reduced pressure gave a brown oil. This
residue was purified by column chromatography on silica gel (n-
hexane/EtOAc, 100:0 to 50:50, v/v) to afford methyl 4-methyl-3-
[(trifluoroacetyl)amino]thiophene-2-carboxylate (74.8 g, 96%) as
an orange oil. 1H NMR (300 MHz, CDCl3) d 2.24 (3H, s), 3.90 (3H,
s), 7.72 (1H, s), 9.68 (1H, br s). To a stirred this intermediate
(11.7 g, 43.8 mmol) and AcOH (150 mL) was added NBS (15.6 g,
88.0 mmol) at room temperature. Then, this mixture was stirred
at 80 °C for 6 h. After cooling to room temperature, the mixture
was poured into water (600 mL) and brine (300 mL). The precipi-
tate was collected by filtration, washed with water, dried under
vacuum to afford 16 (9.40 g, 62%) as a white solid. 1H NMR
(300 MHz, CDCl3) d 2.15 (3H, s), 3.89 (3H, s), 9.62 (1H, br s).
5.1.1.35. 60-(3-Methyl-1H-pyrazol-4-yl)-10H-spiro[cyclohexane-1,20-
thieno[3,2-d]pyrimidin]-40(30H)-one (14a). Compound 14a was pre-
pared in 41% yield from 12b by a procedure similar to that
described for 11c as a yellow solid. 1H NMR (300 MHz, DMSO-d6)
d 1.17–1.26 (1H, m), 1.43–1.62 (7H, m), 1.85–1.88 (2H, m), 2.38
(3H, m), 6.60 (1H, s), 6.98 (1H, br s), 7.30 (1H, br s), 7.68 (0.6H,
br s), 8.03 (0.4H, br s), 12.77 (0.4H, br s), 12.87 (0.6H, br s). HRMS:
Calcd for C15H19N4OS [M+H]+: 303.1274. Found: 303.1271.
5.1.1.36.
(1r,4r)-4-(4-Fluorophenyl)-4-hydroxy-60-(3-methyl-1H-
3pyrazol-4-yl)-10H-spiro[cyclohexane-1,20-thieno[3,2-d]pyrimidin]-40
(30H)-one (14b) and (1s,4s)-4-(4-Fluorophenyl)-4-hydroxy-60-
(3-methyl-1H-pyrazol-4-yl)-10H-spiro[cyclohexane-1,20-thieno[3,2-d]
pyrimidin]-40(30H)-one (14c). A mixture of 13 (100 mg,
0.450 mmol),
4-(4-fluorophenyl)-4-hydroxycyclohexanone22
(281 mg, 1.35 mmol), CSA (10.5 mg, 0.0450 mmol), MgSO4
(108 mg, 0.900 mmol) in DMA (2 mL) was stirred at 80 °C for
45 min. The mixture was partitioned between EtOAc (20 mL) and
saturated NaHCO3 aq. (10 mL). The aqueous layer was extracted
with EtOAc (5 mL ꢂ 2). The combined organic layers were washed
with brine (5 mL), dried over Na2SO4 and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy on silica gel (EtOAc/MeOH, 100:0 to 95:5, v/v), followed by
crystallization with MeOH–EtOAc to give 14b (less polar isomer,
51.8 mg, 28%) as a yellow solid and 14c (more polar isomer,
35.4 mg, 19%) as a pale yellow solid, respectively. 14b (less polar
isomer): 1H NMR (300 MHz, DMSO-d6) d 1.44–1.55 (2H, m),
1.85–2.06 (4H, m), 2.09–2.23 (2H, br s), 2.37 (3H, s), 4.94 (1H, s),
6.55 (1H, s), 7.04 (1H, s), 7.13 (2H, t, J = 8.9 Hz), 7.70 (1H, br s),
7.65 (2H, dd, J = 8.9, 5.7 Hz), 7.78 (1H, s), 12.82 (1H, br s). Anal.
Calcd for C21H21FN4O2SꢁH2O: C, 58.59; H, 5.39; N, 13.01. Found:
C, 58.75; H, 5.44; N, 12.95. 14c (more polar isomer): 1H NMR
(300 MHz, DMSO-d6) d 1.48–1.58 (2H, m), 1.93–2.06 (6H, m),
2.38 (3H, s), 4.93 (1H, s), 6.65 (1H, s), 7.14 (2H, t, J = 8.9 Hz), 7.27
(1H, s), 7.40 (1H, s), 7.61 (2H, dd, J = 8.9, 5.7 Hz), 7.71 (1H, br s),
12.84 (1H, br s). Anal. Calcd for C21H21FN4O2Sꢁ0.2H2O: C, 60.62;
H, 5.18; N, 13.47. Found: C, 60.73; H, 5.04; N, 13.41. Absolute struc-
tures of 14b and 14c were determined by X-ray crystallographic
analysis.
5.1.1.39. Methyl 5-bromo-4-methyl-3-(methylamino)thiophene-2-
carboxylate (17). A mixture of 16 (9.40 g, 27.2 mmol), K2CO3
(7.51 g, 54.3 mmol), MeI (2.04 mL, 32.6 mmol) and DMF (60 mL)
was stirred at 60 °C for 6 h. The mixture was poured into water
(200 mL) and extracted with EtOAc (200 mL), and the extract was
washed with brine, dried over MgSO4, filtered and concentrated
under reduced pressure. The residue was mixed with K2CO3
(4.13 g, 29.9 mmol), MeOH (200 mL) and water (100 mL). The mix-
ture was stirred at room temperature overnight. Then, the mixture
was concentrated under reduced pressure to a half volume, and
worked up as described above. This residue was purified by col-
umn chromatography on silica gel (n-hexane/EtOAc, 100:0 to
50:50, v/v) to afford 17 (4.46 g, 62%) as a pale yellow solid. 1H
NMR (300 MHz, CDCl3) d 2.08 (3H, s), 3.24 (3H, s), 3.86 (3H, s).
5.1.1.40. 5-Bromo-4-methyl-3-(methylamino)thiophene-2-carboxam-
ide (18). Compound 18 was prepared in 56% yield from 17 by a
procedure similar to that described for 10a as a brown oil. 1H
NMR (300 MHz, DMSO-d6) d 2.26 (3H, s), 2.98 (3H, d, J = 5.4 Hz),
5.35 (2H, br s), 6.82 (1H, br s).
5.1.1.41.
6-Bromo-1,2,2,7-tetramethyl-2,3-dihydrothieno[3,2-d]
pyrimidin-4(1H)-one (19). Compound 19 was prepared in 43% yield
from 18 by a procedure similar to that described for 4a as a pale
orange solid. 1H NMR (300 MHz, DMSO-d6) d 1.39 (6H, s), 2.08
(3H, s), 2.56 (3H, s), 7.87 (1H, br s).
5.1.1.37. (1r,4r)-4-Cyclohexyl-4-hydroxy-60-(3-methyl-1H-pyrazol-4-
yl)-10H-spiro[cyclohexane-1,20-thieno[3,2-d]pyrimidin]-40(30H)-one
(14d) and (1s,4s)-4-cyclohexyl-4-hydroxy-60-(3-methyl-1H-pyrazol-
4-yl)-10H-spiro[cyclohexane-1,20-thieno[3,2-d]pyrimidin]-40(30H)-one
(14e). Compound 14d and 14e were prepared in 24% and 34% yield
from 13 and 4-cyclohexyl-4-hydroxycyclohexanone21 by a proce-
dure similar to that described for 14b and 14c as a yellow crystals,
respectively. 14d (less polar isomer): mp 200–202 °C (from MeOH–
EtOAc). 1H NMR (300 MHz, DMSO-d6) d 0.90–1.27 (6H, m), 1.29–
1.41 (2H, m), 1.53–1.94 (11H, m), 2.36 (3H, s), 3.78 (1H, s), 6.53
(1H, s), 6.98 (1H, s), 7.39 (1H, s), 7.82 (1H, brs), 12.83 (1H, brs).
Anal. Calcd for C21H28N4O2Sꢁ0.1H2O: C, 62.69; H, 7.06; N, 13.93.
Found: C, 62.47; H, 7.04; N, 13.80. 14e (more polar isomer): mp
178–180 °C (from EtOH–water). 1H NMR (300 MHz, DMSO-d6) d
0.90–1.25 (6H, m), 1.31–1.44 (2H, m), 1.45–1.92 (11H, m), 2.37
(3H, s), 3.74 (1H, s), 6.62 (1H, s), 6.93 (1H, s), 7.30 (1H, s), 7.80
(1H, brs), 12.83 (1H, brs). Anal. Calcd for C21H28N4O2SꢁH2O: C,
60.26; H, 7.22; N, 13.39. Found: C, 60.32; H, 7.25; N, 13.34. Abso-
lute structure of 14e was determined by X-ray crystallographic
analysis.
5.1.1.42. 1,2,2,7-Tetramethyl-6-(1H-pyrazol-4-yl)-2,3-dihydrothieno
[3,2-d]pyrimidin-4(1H)-one (20a). Compound 20a was prepared
in 47% yield from 19 by a procedure similar to that described for
12a as a white solid. 1H NMR (300 MHz, DMSO-d6) d 1.39 (6H, s),
2.15 (3H, s), 2.57 (3H, s), 7.70 (1H, s), 7.76 (1H, br s), 8.09 (1H, br
s), 13.21 (1H, br s). HRMS: Calcd for
277.1118. Found: 277.1097.
C
13H17N4OS [M+H]+:
5.1.1.43.
7-Bromo-1,2,2-trimethyl-6-(1H-pyrazol-4-yl)-2,3-dihy-
drothieno[3,2-d]pyrimidin-4(1H)-one (20b). To a stirred solution of
12a (0.111 g, 0.423 mmol) in AcOH (5 mL) was added bromine
(0.030 mL, 0.582 mmol) at room temperature. After 1 h, the precip-
itate was collected by filtration and washed with EtOAc to give a
yellow solid (111 mg). The obtained solid was mixed with acetone
(4 mL), AcOH (1 mL) and PTSA (4.0 mg, 0.021 mmol), and this mix-
ture was stirred at 60 °C for 2 h. The mixture was poured into sat-
urated NaHCO3 aq., and extracted with EtOAc, and the extract was
Please cite this article in press as: Kurasawa O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.021

O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
13
dried over MgSO4 and concentrated under reduced pressure to give
a yellow solid. This residue was mixed with acetone (4 mL), AcOH
(1 mL) and PTSA (4.0 mg, 0.021 mmol). The mixture was stirred at
60 °C overnight. Then, the mixture was worked up as described
above. The residue was purified by column chromatography on sil-
ica gel (95:5 hexane/EtOAc to EtOAc) to give a white solid. This
solid was triturated with EtOAc/hexane and collected by filtration
to afford 20b (36.2 mg, 25%) as a white solid. 1H NMR (300 MHz,
DMSO-d6) d 1.41 (6H, s), 2.68 (3H, s), 7.97–8.33 (3H, m), 13.36
(1H, br s). HRMS: Calcd for C12H14BrN4OS [M+H]+: 341.0066.
Found: 341.0031.
5.1.2. X-ray structure analysis
All measurements were made on a Rigaku R-AXIS RAPID diffrac-
tometer using graphite monochromated Cu K
a radiation. The
structure was solved by direct methods with SHELXS-9723 or SIR-
9224 and was refined using full-matrix least-squares on F2 with
SHELXL-97.23 All non-H atoms were refined with anisotropic dis-
placement parameters.
Fig. 9. ORTEP of 14c, thermal ellipsoids are drawn at 30% probability.
Single crystal of 14b was prepared from DMSO, and analyzed as
follows.
Crystal
data
for
14b:
C
21H21FN4O2S∙0.5C2H6OSꢁH2O,
MW = 469.56; crystal size, 0.20 ꢂ 0.07 ꢂ 0.01 mm; colorless, plate-
let; monoclinic, space group P21/c, a = 21.4974(4) Å, b = 8.9821(2)
Å, c = 12.1415(2) Å,
a = c = 90°, b = 110.3665(12)°, V = 2197.86(7)
Å3, Z = 4, Dx = 1.419 g/cm3, T = 100 K,
Å, R1 = 0.101, wR2 = 0.215 (see Fig. 8).
l
= 2.130 mmꢀ1, k = 1.54187
Single crystal of 14c was prepared from MeOH/EtOAc, and ana-
lyzed as follows.
Crystal data for 14c: C21H21FN4O2S, MW = 412.48; crystal size,
0.26 ꢂ 0.10 ꢂ 0.08 mm; yellow, chip; monoclinic, space group
P21/c, a = 6.97071(17) Å, b = 20.6197(5) Å, c = 13.0525(4) Å,
a
=
c
= 90°,
Dx = 1.527 g/cm3, T = 100 K,
R1 = 0.101, wR2 = 0.274 (see Fig. 9).
b = 107.0450(14)°,
V = 1793.67(8)
Å3,
Z = 4,
l
= 1.934 mmꢀ1
,
k = 1.54187 Å,
Single crystal of 14e was prepared from EtOAc/THF, and ana-
lyzed as follows.
Crystal data for 14e: C21H28N4O2S∙ 0.5 C4H8O, MW = 436.59;
crystal size, 0.20 ꢂ 0.10 ꢂ 0.09 mm; yellow, prism; monoclinic,
space group C2/c, a = 29.7681(5) Å, b = 12.6156(2) Å, c = 23.3913
(4) Å,
Dx = 1.320 g/cm3, T = 100 K,
R1 = 0.099, wR2 = 0.334 (see Fig. 10).
a = c
= 90°, b = 90.6729(8)°, V = 8783.8(3) Å3, Z = 16,
Fig. 10. ORTEP of 14e, thermal ellipsoids are drawn at 20% probability.
l
= 1.551 mmꢀ1
,
k = 1.54187 Å,
CCDC 1520567 for compound 14b, CCDC 1520568 for com-
pound 14c and CCDC 1520566 for compound 14e contain the sup-
plementary crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic Data
Centre via http://www.ccdc.cam.ac.uk/Community/Requestastruc-
ture/Pages/DataRequest.aspx?.
5.2. Biology
5.2.1. Preparation of human-derived MCM2 protein
The genetic engineering methods described below followed the
method described in a book (Maniatis et al., Molecular Cloning,
Cold Spring Harbor Laboratory, 1989) or a method described in
the protocol attached to the reagent. N terminal Histagged recom-
binant human MCM2 protein corresponding to the 10–294th
amino acids from the N terminal was cloned to Escherichia coli
expression vector pET-21. The vector pET21-HH was prepared by
inserting the following 6 ꢂ Histag synthetic DNA
50-TATGCATCATCATCATCATCACGGATCCCATCATCATCATCAT-
CACTGAGC-30 (SEQ ID NO: 1); and
50-GGCCGCTCAGTGATGATGATGATGATGGGATCCGTGATGAT-
GATGATGATGCA-30 (SEQ ID NO: 2)
Fig. 8. ORTEP of 14b, thermal ellipsoids are drawn at 50% probability.
into the Nde I-Not I site of pET-21a(+) (Novagen).
Please cite this article in press as: Kurasawa O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.021

14
O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
The Mcm2(10–294 a.a.) gene encoding the 10–294th amino
acids from the N terminal side of human MCM2 protein was cloned
by PCR using synthetic DNA
was measured. The obtained time-resolved fluorescence resonance
energy transfer signal was measured with EnVision (Perkin Elmer
Inc., MA, USA) by excitation at 320 nm and emission donor at
615 nm or emission acceptor 665 nm, respectively. The inhibitory
rate (%) of the test compound to Cdc7 was calculated by the follow-
ing formula.
50-CGCGGATCCATGGCATCCAGCCCGGCCCA-30 (SEQ ID NO: 3);
and
50-ATTCTTATGCGGCCGCTCACAGCTCCTCCACCAGAGGCA-30
(SEQ ID NO: 4)
Inhibitory rate ð%Þ ¼ ð1 ꢀ ðcount of test compound ꢀ blankÞ
ꢃ ðcontrol ꢀ blankÞÞ ꢂ 100
prepared by reference to the base sequence described in Gen-
Bank accession No.: NM_004526, as a primer set and human testis
cDNA library (TAKARA bio Inc.) as a template. PCR reaction was
performed according to the protocol attached to Pyrobest (TAKARA
bio Inc.).
The obtained 883 bp fragment was digested with restriction
enzymes BamHI and NotI, inserted into the BamHI-NotI site of
pET21-HH, and the inserted base sequence was confirmed to give
pET21-HHhMcm2(10–294) plasmid. The pET21-HHhMcm2(10–
294) plasmid was introduced into Escherichia coli BL21(DE3) cell
line (American Type Culture Collection).
The count of the Cdc7/Dbf4 reaction mixture under compound-
free conditions was taken as the control, and that under com-
pound-free and Cdc7/Dbf4-free conditions was taken as the blank.
5.2.3. Cdk2/CyclinE kinase assay
The Kinase-GloTM (Promega, USA) assay was performed in 384-
well plate format. The enzyme reaction was run in a reaction buffer
consisting of 25 mM HEPES (pH 7.5), 10 mM Mg(OAc)2, 0.01%
bovine serum albumin (BSA), 0.01% Tween 20, and 1 mM DTT.
The final concentrations of substrate Histone H1 and ATP were
100 lg/ml and 500 nM, respectively. The final concentration of
Escherichia coli cells introduced with the above-mentioned
plasmid were cultured in LB medium (1% tripton, 0.5% yeast
extract, 0.5% NaCl) containing 50 mg/L ampicillin, and MCM2
expression was induced by addition of 1 mM isopropyl b-D-1-thio-
galactopyranoside (IPTG) for 6 h. Escherichia coli cells expressing
MCM2 were recovered by centrifugation (6000 rpm, 10 min),
washed with phosphate-buffered saline, and cryopreserved at
ꢀ80 °C. The above-mentioned cryopreserved Escherichia coli cells
were thawed on ice, and suspended in complete ethylenedi-
aminetetraacetic acid (EDTA) (Roche Diagnostics GmbH, Man-
nheim, Germany)-added buffer A (25 mM tris-hydrochloride (pH
7.4), 2.7 mM KCl, 137 mM NaCl). The above-mentioned suspended
Escherichia coli cells were lysed with 1 mg/mL lysozyme, and son-
icated 4 times in Insonator 201 M (Kubota) at 170 W for 30 s while
cooling with ice water. This extract was ultracentrifuged at
15,000 rpm, at 4 °C for 20 min, and the obtained supernatant was
Cdk2/CyclinE (Carnabiosciences, Japan) was 750 ng/ml. After incu-
bation at room temperature for 90 min, the reaction was termi-
nated by the addition of the reagent supplied with the Kinase-
Glo reagent. The luminescence correlated with the amount of
ATP remaining in solution was measured on EnVision (PerkinEl-
mer, MA, USA) after incubation at room temperature for 10 min.
The inhibitory rate (%) of the test compound to Cdc7 was calcu-
lated by the following formula.
Inhibitory rate ð%Þ ¼ ð1 ꢀ ðcount of test compound ꢀ blankÞ
ꢃ ðcontrol ꢀ blankÞÞ ꢂ 100
5.2.4. ROCK1 kinase assay
TR-FRET assay was used to assess ROCK1 (Carnabiosciences,
Japan) enzyme activity (CisBio, France, KinEASE HTRF kit (Cat#
62ST3PEB)). The enzyme reaction was run in a reaction buffer con-
sisting of 50 mM HEPES (pH 7.5), 0.1 mM orthovanadate, 0.01%
BSA, 10 mM MgCl2 and l mM DTT. The assay was done in a 384-
well plate assay format. Before initiation of the enzymatic reaction,
ROCK1, test compounds, and the substrate peptide (Biotin-STK
substrate-2 (Cat# 61ST2BLC)) were incubated in the reaction buf-
fer at room temperature for 5 min. The final concentration of
ROCK1 was 300 ng/mL. The enzymatic reaction was started with
passed through a 0.22 lm filter to give an Escherichia coli cell-
fee cell extract. The Escherichia coli cell-free cell extract was
passed through nickel-NTA Superflow resin, and the resulting resin
was washed with buffer A, and eluted with buffer B (25 mM tris-
hydrochloride (pH 7.4), 2.7 mM KCl, 137 mM NaCl, 10% glycerol,
200 mM imidazole). The eluate was concentrated using Amicon
Ultra 4 (5 K MWCO, Millipore, MA, USA), and purified by gel filtra-
tion using HiLoad 16/60 Superdex 200 pg (GE healthcare, Chalfont
St. Giles, UK) equilibrated with buffer C (25 mM tris-hydrochloride
(pH 7.4), 2.7 mM KCl, 137 mM NaCl, 10% glycerol, 200 mM imida-
zole). The fraction containing MCM2 protein was concentrated as a
purified sample, and cryopreserved at ꢀ80 °C.
the addition of ATP at a final concentration of 2 lM. After incuba-
tion at room temperature for 2 h, the reaction was terminated by
adding 10 mM EDTA in a detection buffer containing 15 nM strep-
tavidin-linked XL665. Time-resolved fluorescence was monitored
with an EnVision Multilabel Plate Reader (PerkinElmer Life
Sciences, Fremont, CA, USA) with an excitation wavelength of
320 nm and emission donor and acceptor wavelengths of 615
and 665 nm, respectively. The total reaction without enzyme as
0% activity and the total reaction as 100% activity were set.
The inhibitory rate (%) of the test compound to Cdc7 was calcu-
lated by the following formula.
5.2.2. Cdc7 kinase assay
Full-length Cdc7 co-expressed with full-length Dbf4 was pur-
chased from Carna Biosciences (Kobe). The enzyme activity of
Cdc7/Dbf4 complex was detected by homogeneous time-resolved
fluorescence method Transcreener ADP assay (Cisbio Inc., MA,
USA). The enzyme reaction was performed in a kinase buffer
(20 mM
(HEPES) pH 7.5, 10 mM Mg(OAc)2, 1 mM dithiothreitol (DTT)) sup-
plemented with 1.0 M ATP, 10 g/mL MCM2, and 0.1 g/mL
4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
Inhibitory rate ð%Þ ¼ ð1 ꢀ ðcount of test compound ꢀ blankÞ
ꢃ ðcontrol ꢀ blankÞÞ ꢂ 100
l
l
l
Cdc7/Dbf4. Prior to the addition of ATP, test compounds and
enzyme were pre-incubated for 10 min. For time dependent inhibi-
tion assay, the enzyme reactions were performed in the kinase buf-
5.2.5. Cell lines
fer containing 1.0
l
M (Km) or 50
l
M (Km ꢂ 50) ATP. Prior to the
HeLa cells from ATCC were cultured in Dulbecco’s modified
eagle medium (DMEM) with 10% fetal bovine serum (FBS).
COLO205 cells from ATCC were cultured in Roswell Park Memorial
Institute (RPMI) medium with 10% FBS. Cell lines were incubated at
37 °C with 5% CO2 gas.
addition of ATP, test compounds and enzyme were pre-incubated
for 0 or 60 min. Free ADP produced by ATP hydrolysis was detected
by Eu3+-Cryptate-labeled anti-ADP monoclonal antibody competi-
tively with d2-labeled ADP, and the production amount thereof
Please cite this article in press as: Kurasawa O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.021

O. Kurasawa et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
15
5.2.6. Cell-based MCM2 phosphorylation
A. Supplementary material
HeLa cells were seeded at 3.0 ꢂ 104 cells/well in a 24-well plate.
After 1-day incubation, the plate was treated with the test com-
pound for 7 h. At end of the incubation, HeLa cells were lysated
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.02.021.
by 200 lL of sodium dodecylsulphate (SDS) buffer. Phosphoryla-
tion level of MCM2 in each sample was determined by Western
blotting. Western blotting was carried out by using the following
antibodies; pSer40 MCM2 (EPITOMICS, Inc., #3378-1), horseradish
peroxidase (HRP)-labeled rabbit IgG polyclonal antibody (Amer-
sham Biosciences, NA9340). Band intensity of each sample was
detected by LAS1000 and the corresponding IC50 value was calcu-
lated by using Prism software.
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We thank to the structural biology group (Dr. Michael Klein, Dr.
Bi-Ching Sang, Dr. Charles Dillard, Dr. Gyorgy Snell and Dr. Wes
Lane) and the management of Takeda California for determining
the X-ray co-crystal structure of compound 14c with ROCK2. The
authors would also like to acknowledge Mr. Motoo Iida, Mr. Youi-
chi Nagano and Ms. Keiko Higashikawa for the X-ray single-crystal
structure analysis of compounds 14b, c, e. We gratefully acknowl-
edge fruitful discussions with the medicinal chemistry group (Dr.
Brian Gregg, Mr. Adam Csakai, Dr. Hong-Jun Wang and Dr. John
Mangetti) of Albany Molecular Research Inc. during collaboration
on the research project. We thank the staff of the Berkeley Center
for Structural Biology (BCSB), Lawrence Berkeley National Labora-
tory, which operates Advanced Light Source beamline 5.0.3, for
their support. BCSB is supported in part by the National Institutes
of Health, National Institute of General Medical Sciences, and the
Howard Hughes Medical Institute. The Advanced Light Source is
supported by the Director, Office of Science, Office of Basic Energy
Sciences, of the U.S. Department of Energy under Contract No. DE-
AC02-05CH11231.
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Please cite this article in press as: Kurasawa O., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.02.021