Substituted 4-(thiazol-5-yl)-2-(phenylamino)pyrimidines are highly active CDK9 inhibitors: Synthesis, X-ray crystal structures, structure-activity relationship, and anticancer activities

Article abstract of DOI:10.1021/jm301475f

Cancer cells often have a high demand for antiapoptotic proteins in order to resist programmed cell death. CDK9 inhibition selectively targets survival proteins and reinstates apoptosis in cancer cells. We designed a series of 4-thiazol-2-anilinopyrimidine derivatives with functional groups attached to the C5-position of the pyrimidine or to the C4-thiazol moiety and investigated their effects on CDK9 potency and selectivity. One of the most selective compounds, 12u inhibits CDK9 with IC₅₀ = 7 nM and shows over 80-fold selectivity for CDK9 versus CDK2. X-ray crystal structures of 12u bound to CDK9 and CDK2 provide insights into the binding modes. This work, together with crystal structures of selected inhibitors in complex with both enzymes described in a companion paper,(34) provides a rationale for the observed SAR. 12u demonstrates potent anticancer activity against primary chronic lymphocytic leukemia cells with a therapeutic window 31- and 107-fold over those of normal B- and T-cells.

Full text of DOI:10.1021/jm301475f

Article
pubs.acs.org/jmc
Substituted 4‑(Thiazol-5-yl)-2-(phenylamino)pyrimidines Are Highly
Active CDK9 Inhibitors: Synthesis, X‑ray Crystal Structures,
Structure−Activity Relationship, and Anticancer Activities
Hao Shao,† Shenhua Shi,† Shiliang Huang,† Alison J. Hole,§ Abdullahi Y. Abbas,† Sonja Baumli,§
Xiangrui Liu,† Frankie Lam,†,‡ David W. Foley,† Peter M. Fischer,† Martin Noble,§,∥ Jane A. Endicott,§,∥
Chris Pepper,⊥ and Shudong Wang*,†,‡
†School of Pharmacy and Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
‡Shool of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5001, Australia
§Department of Biochemistry, University of Oxford, Oxford OX1 3QU, U.K.
∥Northern Institute for Cancer Research, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, U.K.
⊥Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, U.K.
S
* Supporting Information
ABSTRACT: Cancer cells often have a high demand for antiapoptotic proteins in order to resist programmed cell death. CDK9
inhibition selectively targets survival proteins and reinstates apoptosis in cancer cells. We designed a series of 4-thiazol-2-
anilinopyrimidine derivatives with functional groups attached to the C5-position of the pyrimidine or to the C4-thiazol moiety
and investigated their effects on CDK9 potency and selectivity. One of the most selective compounds, 12u inhibits CDK9 with
IC50 = 7 nM and shows over 80-fold selectivity for CDK9 versus CDK2. X-ray crystal structures of 12u bound to CDK9 and
CDK2 provide insights into the binding modes. This work, together with crystal structures of selected inhibitors in complex with
both enzymes described in a companion paper,34 provides a rationale for the observed SAR. 12u demonstrates potent anticancer
activity against primary chronic lymphocytic leukemia cells with a therapeutic window 31- and 107-fold over those of normal B-
and T-cells.
regulatory roles of CDKs has been challenged.2,3 The
observations that cancer cell lines and some embryonic
INTRODUCTION
■
Cyclin-dependent kinases (CDKs) can generally be classified
into two main groups based on whether their primary role is in
the control of cell cycle progression or regulation of
fibroblasts lacking CDK2 proliferate normally and that CDK2
knockout mice are viable4,5 suggest that this CDK performs a
nonessential role in cell-cycle control. Furthermore, redun-
transcription. Multiple CDKs control the cell cycle and are
dancy of CDK4 and CDK6 was also suggested in cells that
considered essential for normal proliferation, development, and
enter the cell cycle normally.6 It has been demonstrated that
homeostasis. CDK4/cyclin D, CDK6/cyclin D, and CDK2/
cyclin E facilitate the G1-S phase transition by sequentially
mouse embryos deficient in CDKs 2, 3, 4, and 6 develop to
mid-gestation, as CDK1 can form complexes with their cognate
phosphorylating the retinoblastoma protein (Rb), while
cyclins and subsequently phosphorylate Rb protein. Inactiva-
CDK1/, CDK2/cyclin A, and CDK1/cyclin B are essential
for S-phase progression and G2-M transition, respectively.1
tion of Rb in turn activates E2F-mediated transcription of
proliferation factors.7 In cells depleted of CDK1/cyclin B,
Most CDK inhibitors have been developed as potential
CDK2/cyclin B is readily detectable and can facilitate G2/M
cancer therapeutics based on the premise that they might
counteract the uncontrolled proliferation of cancer cells by
targeting the cell-cycle regulatory functions of CDKs. However
in recent years, this understanding of the cellular functions and
Received: October 11, 2012
Published: January 9, 2013
© 2013 American Chemical Society
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progression.3 These studies suggest that specifically targeting
individual cell-cycle CDKs may not be an optimal therapeutic
approach because of a high level of functional redundancy and
compensatory mechanisms.
gatekeeper residue Phe103 and the ribose-binding pocket, we
prepared a series of 5-substituted-2-anilino-4-(thiazol-5-yl)-
pyrimidines (type II, Figure 1) and 4-(4-substituted-thiazol-5-
yl)-N-phenylpyrimidin-2-amines (type III, Figure 1).
By contrast, the hypothesis that inhibition of transcriptional
CDKs might be an effective anticancer strategy has gained
considerable support following the observation that many cells
rely on the production of short-lived mitotic regulatory kinases
and apoptosis regulators such as Mcl-1 for their survival.2,8 The
transcriptional CDKs, particularly CDK9/cyclin T and CDK7/
cyclin H, are involved in the regulation of RNA transcription.
CDK7/cyclin H is a component of transcription factor IIH
(TFIIH) that phosphorylates the serine-5 residues within the
heptad repeats of RNA polymerase II (RNAPII) C-terminal
domain (CTD) to initiate transcription.9,10 CDK9/cyclin T, the
catalytic subunit of positive transcription elongation factor P-
TEFb,11,12 phosphorylates two elongation repressors, i.e., the
DRB-sensitive-inducing factor (DSIF) and the negative
elongation factor (NELF), and position serine-2 of the CTD
heptad repeat to facilitate productive transcription elonga-
tion.2,13 While CDK7 is also recognized as a CDK-activating
kinase (CAK),10 CDK9 appears to have a minimal effect on
cell-cycle regulation.14
Here, we report the synthesis, SAR, crystal structural analysis
and biological evaluation of a novel class of 5-substituted-2-
anilino-4-(thiazol-5-yl)pyrimidines and 4-(4-substituted-thiazol-
5-yl)-N-phenylpyrimidin-2-amines. Structure−activity relation-
ship (SAR) analysis reveals the importance of the C5-group of
the pyrimidine core, in the context of a bulky substituted aniline
moiety, for CDK9 potency and selectivity. A nanomolar Ki
inhibitor of CDK9, 12u demonstrates excellent selectivity with
over 80-fold selectivity for CDK9 versus CDK2. This
compound inhibits cellular CDK9-mediated RNA polymerase
II transcription, reduces the expression level of Mcl-1
antiapoptotic protein, and subsequently triggers apoptosis in
human cancer cell lines and primary CLL cells. In a companion
paper34 we also give a detailed structural analysis of several 5-
substituted-2-anilino-4-(thiazol-5-yl)pyrimidines bound to
CDK2 and CDK9.
CHEMISTRY
■
Synthesis of 2-anilino-4-thiazolpyrimidine type I compounds
shown in Table 1 was carried out according to the methods
described previously.32,35 The chemistry for synthesis of 5-
substituted-2-anilino-4-(thiazol-5-yl)pyrimidines (type II) is
outlined in Scheme 1. Treatment of 1-methylthiourea with
ethyl 2-chloro-3-oxobutanoate in pyridine resulted in ethyl 4-
methyl-2-(methylamino)thiazole-5-carboxylate (1). The 2-
methylamino group in 1 was found to interfere in subsequent
reactions and was therefore masked as the tert-butoxycarbonate
(2). Alkylation of 2 with cyanomethanide afforded tert-butyl 5-
(2-cyanoacetyl)-4-methylthiazol-2-yl(methyl)carbamate (3, R′
= CN) in a 72% yield. Converting 3 to enaminone (4, R′ =
CN) was achieved conveniently by refluxing in N,N-
dimethylformamide−dimethylacetal (DMF−DMA).35 4 (R′ =
CN) can also be obtained by bromination of 1-(4-methyl-2-
(methylamino)thiazol-5-yl)ethanone (5), followed by treat-
ment with sodium cyanide and then DMF−DMA. However,
because of the requirement for highly toxic sodium cyanide, this
procedure is not recommended for routine synthesis.
Enaminone (6) was treated with SelectFluor36,37 in methanol
at 0 °C, producing 3-(dimethylamino)-2-fluoro-1-(4-methyl-2-
(methylamino)thiazol-5-yl)prop-2-en-1-one (4, R′ = F). The
analogue 2-chloro-3-(dimethylamino)-1-(4-methyl-2-
(methylamino)thiazol-5-yl)prop-2-en-1-one (4, R′ = Cl) was
obtained by treating 6 with N-chlorosuccinimide.38 Preparation
of alkyl-substituted enaminones (4, R′ = Me, Et, or Pr) started
from tert-butyl methyl(4-methylthiazol-2-yl)carbamate (8),
followed by treatment with alkylaldehyde (R′′CH2CHO) in
the presence of LDA to yield 9, which was then oxidized with
manganese dioxide.39,40 Compound 8 was obtained by
condensation reaction between 1-chloropropan-2-one (7) and
1-methylthiourea.
During the past decade an intensive search for pharmaco-
logical CDK inhibitors has led to the development of several
clinical candidates and to the realization that inhibition of the
transcriptional CDKs underlies their antitumor activity.2,15
Flavopiridol (alvocidib), the first CDK inhibitor to enter clinical
trials, is the most potent CDK9 inhibitor identified to date and
has demonstrated marked antitumor activity in chronic
lymphocytic leukemia (CLL).16,17 Flavopiridol has been
shown to inhibit multiple CDKs18 and other kinases,19 but
the primary mechanism responsible for its observed antitumor
activity in CLL appears to be the CDK9-mediated down-
regulation of transcription of antiapoptotic proteins.20,21
R-Roscovitine (seliciclib) is the first orally bioavailable CDK
inhibitor that targets CDK2, CDK7, and CDK9 (IC50 ≈ 0.1,
0.5, and 0.8 μm, respectively).22−24 During evaluation in phase
I oncology monotherapy and combination chemotherapy
clinical trials it was shown to be well tolerated and some
evidence of disease stabilization was reported.15 Phase II clinical
trials are underway in non-small-cell lung cancer (NSCLC)
patients. R-Roscovitine has demonstrated selective induction of
apoptosis in cancer cells by down-regulation of antiapoptotic
proteins through transcriptional CDK inhibition.25,26 Other
CDK inhibitors including AZD5438,27 R547,28,29 and AT51930
have also been evaluated in clinical trials.
While there are several pan-CDK inhibitors in clinical
studies,27,29−31 CDK9 inhibitors with good potency and
selectivity have only recently emerged.32,33 To further exploit
the sensitivity of the 4-hetertoarylpyrimidine pharmacophore
(type I, Figure 1) that specifically targets the CDK9-ATP
Reaction conditions required for preparing 4 greatly varied
depending on the R′ group of the intermediates 3 and 10. With
R′ as a carbonitrile, i.e., an electron withdrawing group, 4 (R′ =
CN) was conveniently obtained under mild reaction con-
ditions. Conversely, an electron donating R′ group slowed the
rate of reaction, requiring heat for an extended period of time.
The enaminones (4, R′ = Me, Et, or n-propyl), for instance,
were obtained under high temperature microwave conditions
with low yields. The reaction became more difficult with a
Figure 1. 4-(Thiazol-5-yl)-2-(phenylamino)pyrimidine derivatives.
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Table 1. Structure and Biological Activity Summary
a
kinase inhibition Ki (nM)
b
compd
R′
R
CDK9T1
CDK1B
CDK2A
CDK7H
cytotoxicity GI50 (μM), HCT-116
Ia
H
m-NO2
2
5
3
417
260
>5000
1960
114
>5000
91
0.09
0.04
3.20
0.05
0.55
2.03
<0.01
0.03
0.30
3.81
4.50
0.22
0.23
0.82
0.03
0.93
0.35
0.31
0.16
0.26
0.64
0.33
0.42
0.66
12a
12b
Ib
CN
OH
H
m-NO2
6
6
1
m-NO2
932
1424
6
>5000
4
m-SO2NH2
m-SO2NH2
m-SO2NH2
m-SO2NH2
m-SO2NH2
m-SO2NH2
m-SO2NH2
m-SO2NH2
2
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
12t
12u
Ic
CN
OH
F
6
12
>5000
4
4
>5000
>5000
3
4
Cl
11
19
62
788
ND
43
42
45
79
35
26
24
18
88
47
91
94
195
10
685
1176
1285
ND
92
Me
Et
5
34
98
845
>5000
43
Pr
>5000
CN
CN
CN
CN
CN
F
m-4-acetylpiperazin-1-yl
m-piperazin1-yl
7
5
56
68
p-4-acetylpiperazin-1-yl
p-piperazin-1-yl
22
6
26
316
71
39
m-piperidin-1-yl
9
42
286
302
193
473
155
111
210
46
m-4-acetylpiperazin-1-yl
m-piperazin-1-yl
7
42
F
4
20
F
m-morpholin-4-yl
3
20
Cl
m-piperazin-1-yl
4
45
F
m-1,4-diazepan-1-yl
m-4-acetyl-1,4-diazepan-1-yl
m-1,4-diazepan-1-yl
m-1,4-diazepane-1-yl
5
85
CN
CN
H
7
131
568
320
7
19
433
a
The ATP concentrations used in these assays were within 15 μM of Km, i.e., 45, 45, 90, and 45 μM for CDK1/cyclin B, CDK2/cyclin A, CDK7/
cyclin H/MAT1, and CDK9/cyclin T1, respectively. The data given are mean values derived from two replicates. Apparent inhibition constants (Ki)
b
were calculated from IC50 values and the appropriate Km (ATP) values for each kinase.35 Antiproliferative activity by MTT 48 h assay. The data
given are mean values derived from at least three replicates.
a
Scheme 1. Synthesis of 3-(Dimethylamino)-2-(4-methyl-2-(methylamino)thiazole-5-carbonyl)acrylonitrile and Derivatives
a
Reagents and conditions: (a) di-tert-butyl dicarbonate, 4-dimethylaminopyridine (DMAP), DCM, rt, 1 h, 93%; (b) LDA, MeCN, THF, −78 °C, 1.5
h, 72%; (c) N,N-dimethylformamide−dimethylacetal (DMF−DMA), reflux, overnight or microwave, Δ, 20−45 min, 30−80%; (d) SelectFluor,
MeOH, 0 °C, 1 h; or NCS, MeOH, rt 0.5 h, 30%; (e) 1-methylthiourea, MeOH, pyridine, rt 4 h, 88%; (f) LDA, R′CH2CHO, MeCN, THF, −78 °C,
1−1.5 h, 53−77%; (g) MnO2, CHCl3, Δ, 3 h, 65−85%.
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bulkier alkyl group; thus, attempting the synthesis of 4, where
R′ = isopropyl, failed even under harsher reaction conditions
because of unfavorable electronic and steric effects of the bulky
isopropyl group. Pyrimidine ring formation reaction was
performed under conditions similar to those we have developed
for the synthesis of 2-anilinoamino-4-(heteroaryl)pyrimidines.35
The limiting factor in the preparation of type II analogues was
the efficiency of the condensation reactions between the
substituted phenylguanidines (11) and 4 (Scheme 2). In
In order to extend the SARs, we prepared another series of
compounds with functional group R′ at the C4-position of the
thiazol ring system (type III, Figure 1); the chemistry is
outlined in Scheme 3. 2-Methylaminothiazol-5-ylethanone
derivative 14 was obtained by bromination of 5 in the presence
of PTSA to afford 13, which was then treated with di-tert-butyl
dicarbonate, followed by reaction with methyl 2,2-difluoro-2-
(fluorosulfonyl)acetate in the presence of a catalytic amount of
CuI.41 Converting 14 to the corresponding enaminone 15 (R1
= Boc, R′′ = CH2CF3) was achieved by reacting 14 with
DMF−DMA as described above. To prepare analogue 17,
1,1,1-trifluoropentane-2,4-dione was treated with hydroxy-
(tosyloxy)iodobenzene,42 followed by reaction with 1-methyl-
thiourea and then di-tert-butyl dicarbonate. tert-Butyl (5-
acetylthiazol-2-yl)(methyl)carbamate (20) was obtained by
cyclization reaction between chloroacetone chloride and N′-
carbamothioyl-N,N-dimethylformimidamide (18),43 giving 19,
followed by treating the latter with di-tert-butyl dicarbonate. To
prepare tert-butyl(5-acetyl-4-cyclopropylthiazol-2-yl)(methyl)-
carbamate (24), bromination of 1-cyclopropylethanone (21)
yielded 2-bromo-1-cyclopropylethanone (22), which was
subsequently reacted with 1-methylthiourea and then di-tert-
butyl dicarbonate to afford 4-cyclopropyl-N-methylthiazol-2-
amine (23). Acetylation was achieved by LDA-mediated
alkylation reaction between 23 and acetaldehyde,39 followed
by oxidation of the resulting thiazol-5-ylethanol intermediate
with MnO2. 4-Phenylthiazol derivative (26) was prepared by
condensation reaction between 2-chloro-1-phenylethanone and
Scheme 2. Synthesis of 4-(4-Methylthiazol-5-yl)-2-
(phenylamino)pyrimidine-5-carbonitrile and Derivatives
a
a
Reagents and conditions: (a) 2-methoxyethanol, microwave, 200−
300 W, Δ, 20−45 min.
general, microwave-aided protocols were more effective in
terms of reducing reaction times and improving yields in the
pyrimidine condensation reactions compared to conventional
methodology. Analogues 12b and 12d, where R′ = OH, were
obtained from condensation of 4 (R′ = Cl) with corresponding
phenylguanidines 11, followed by in situ hydrolysis.
a
Scheme 3. Synthesis of N-Methyl-5-(2-(phenylamino)pyrimidin-4-yl)-4-thiazol-2-amine Derivatives
a
Reagents and conditions: (a) pentane-2,4-dione, pyridine, EtOH, rt 4 h; (b) NBS, p-tolenensulfonic acid (PTSA), CHCl3, 0−5 °C, 1 h, 56%; (c) di-
tert-butyl dicarbonate, DMAP, DCM, rt 2 h; (d) methyl 2,2-difluoro-2-(fluorosulfonyl)acetate, CuI, DMF, Δ, 12 h; (e) DMF−DMA, Δ, overnight,
or microwave, Δ, 45 min; (f) (i) 1,1,1-trifluoropentane-2,4-dione, hydroxy(tosyloxy)iodobenzene, MeCN, Δ, 1 h; (ii) 1-methylthiourea, Δ, 4 h, 53%;
(g) DMF−DMA, CHCl3, Δ, overnight, 98%; (h) chloroacetone chloride, MeCN, Δ, 4 h, 79%; (i) Br2, 0 °C to rt 4 h, 77%; (j) (i) LDA,
acetaldehyde, THF, −78 °C, 2 h, 49%; (ii) MnO2, CHCl3, Δ, 4 h, 55%; (k) acetyl chloride/AlCl3, rt, 2 h, 67%; (l) 11, 2-methoxyethanol, microwave
200−300 W, Δ, 20−45 min.
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1-methylthiourea to afford N-methyl-4-phenylthiazol-2-amine,
followed by the Friedel−Crafts acylation reaction. Finally, the
enaminones (15) were converted to the corresponding
pyrimidines (27a−l) upon treatment with the appropriate
phenylguanidines32,35 under microwave irradiation conditions.
potency and selectivity of a series of 5-substituted 2-anilino-4-
(thiazol-5-yl)-pyrimidines against CDKs and characterized their
cellular antitumor activity. The results are summarized in Table
1.
Compound Ia (R′ = H, R = m-NO2) is a highly potent pan-
CDK inhibitor. Substitution of hydrogen at C5-pyrimidine in Ia
with a carbonitrile group results in compound 12a (R′ = CN, R
= m-NO2) that exhibits a similar potency and selectivity profile.
Both compounds inhibit CDK9, CDK1, and CDK2 potently
with Ki values ranging from 1 to 6 nM but are significantly less
active toward CDK7. Both compounds are highly effective
antiproliferative agents with respective GI50 values of 90 and 40
nM in the HCT-116 human colon cancer cell line. Replacement
of the C5-carbonitrile with a C5-hydroxyl group in 12b (R′ =
OH, R = m-NO2) results in over 155-fold and 230-fold loss in
CDK9 and CDK1 inhibition, respectively. This replacement
also abolishes CDK2 and CDK7 inhibitory activity and
significantly reduces cellular antiproliferative activity. A
compound containing the m-sulfonamideaniline ring, 12c (R′
= CN, R = m-SO2NH2), shows similar potencies against CDK1,
CDK2, and CDK9 but a 17-fold or a 10-fold loss in CDK7
inhibition and cellular toxicity, respectively, compared to Ib (R′
= H, R = m-SO2NH2). Again, introducing a hydroxyl group at
C5-pyrimidine, in the context of m-sulfonamideaniline, is not
tolerated; 12d (R′ = OH, R = m-SO2NH2) shows little
biological activity in the enzymatic and cellular assays. These
results demonstrate the importance of C5-substitution of the
pyrimidine and that a protic or hydrogen bond donating
function at this position has a detrimental effect on biological
activity. Compound 12e (R′ = F, R = m-SO2NH2), a potent
pan-CDK inhibitor (Ki = 3−7 nM), is the most potent
antiproliferative agent of this series, with GI50 < 10 nM against
HCT-116 cells. Analogue 12f, where R′ = Cl, R = m-SO2NH2,
however, displays a >3-fold reduced CDK inhibitory activity
and cellular potency compared to 12e. A more interesting trend
toward CDK9 selectivity is observed with C5-alkylpyrimidines;
12g (R′ = Me, R = m-SO2NH2) exhibits a CDK9 inhibitory
potency similar to that of 12e with Ki = 5 nM but enhances
selectivity for CDK9 with >7-fold lower effectiveness against
other CDKs. However, this selectivity results in 12g showing
over 30-fold reduced cytotoxicity in HCT-116 cells compared
to 12e. With further introduction of a bulkier alkyl group,
CDK9 inhibitory activity dramatically decreases; thus, 12h (R′
= Et, R = m-SO2NH2) is 20-fold less potent against CDK9 than
12g, while 12i (R′ = Pr, R = m-SO2NH2) is not active against
CDKs at concentrations up to 5 μM. As expected, 12h and 12i
are also less cytotoxic in cancer cells with respective GI50 values
of 3.81 and 4.50 μM.
RESULTS AND DISCUSSION
■
Inhibitor Design and SAR Analysis. We previously
identified a series of 2-herteroaryl-4-anilinopyrimidine CDK
inhibitors.32,35,44 Many of these compounds showed potent
CDK9 inhibitory activity. The lead compounds demonstrated
excellent pharmaceutical properties and in vivo antitumor
efficacy.32 However, CDK9 specificity was not achieved, as they
cross-reacted with other cell cycle CDKs, in particular CDK2.
Previously established SARs of 2-anilino-4-(thiazol-5-yl)-
pyrimidines (type I, Figure 1) with respect to CDK2 suggested
the importance of substituents at the C2-position in the
thiazole ring.32,35 It was found that introduction of amino
functions, in the context of either meta- or para-substituted
anilines at the C2-pyrimidine ring, resulted in a significant
increase of inhibitory activity not only against CDK2 but also
against CDK9. Cocrystal structures of some of these inhibitors
bound to CDK2 revealed that the thiazole C2-amino group
interacted strongly with the Asp145 residue of CDK2,
enhancing the hydrophobic interactions of the thiazol C4-
methyl group with the Phe80 gatekeeper residue of CDK2
(Phe103 in CDK9). Additional hydrogen bonding interactions
between the thiazole C2-amino groups and Gln131 and Asp86
of CDK2 were also observed. Substitution of the thiazole C2-
amino group with a C2-methylamino or C2-ethylamino
appeared to have a detrimental effect on CDK2 and CDK4
activity while having only a minimal effect on CDK9
potency.32,35 A bulkier group, such as phenyl, pyridyl, or
other heterocycles at this position, however, led to significantly
reduced activity against all CDKs. We therefore designed a
series of 5-substituted-4-thiazolpyrimidines in the context of the
C2-methylamino to improve potency and selectivity against
CDK9.
The SAR analysis of the pyrimidinyl C2-aniline moiety was
previously described.32,35 It was shown that many meta- or
para-substitutions of the aniline, in the context of the 4-
thiazolpyrimidine, were well tolerated and manipulation of
these substituents led to a number of inhibitors possessing
varying CDK selectivity profiles. In many cases, meta-
substituted anilines gave rise to selectivity for CDK9 over
CDK2 compared with their para-substituted aniline analogues.
However, substituents in the ortho position abolished CDK-
inhibitory activity in all cases.
Retaining the C5-carbonitrile pyrimidine core but replacing
the m-sulfonamide with a bulkier 1-(piperazin-1-yl)ethanone or
piperazine leads to the corresponding 12j (R′ = CN, R = m-1-
(piperazin-1-yl)ethanone) or 12k (R′ = CN, R = m-piperazine).
This not only maintains CDK9 potency but also increases
selectivity (∼10-fold) over CDK2 compared to 12c, indicating
the tolerance of a large ring system in the corresponding CDK9
binding region. Compounds 12m−r, bearing heterocyclic
piperidine, 1-(piperazin-1-yl)ethanone, piperazine, or morpho-
line at the meta- or para-position of the aniline in the context of
a C5-carbonitrile or C5-halogen pyrimidine moiety, display
favorable CDK9 inhibitory activity with low nanomolar
potencies and possesses over 4-fold selectivity for CDK9.
The exception is compound 12l (R′ = CN, R = p-1-(piperazin-
1-yl)ethanone), which shows a >3-fold loss of potency against
It is recognized that the ATP-binding sites are highly
conserved among kinases,45 but the nonconserved hydrophobic
region, which is not occupied by ATP, and the so-called
“gatekeeper” region can be exploited for inhibitor desig-
n.4647−50A cocrystal structure of flavopiridol bound to CDK9
showed that the hydrophobic region that accommodates the
chlorophenyl ring of flavopiridol is more open in CDK9 than
CDK2. CDK9 Gly112 takes the place of CDK2 Lys89 and
creates a less crowded and a different electrostatic environ-
ment.51 Analysis of the previously published 2-anilino-4-
(thiazol-5-yl)pyrimidine CDK2-bound crystal structures and
their corresponding models of CDK9 binding32,35 suggested
that an appropriate functional group at either the C5-
pyrimidine or the C4-thiazol moiety might enhance interactions
with the CDK9 gatekeeper region. We thus investigated the
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Table 2. Structure and Biological Activity Summary
a
kinase inhibition Ki (nM)
b
compd
R′
R
CDK9T1
CDK1B
CDK2A
CDK7H
cytotoxicity GI50 (μM), HCT-116
27a
27b
27c
27d
27e
27f
27g
27h
27i
Ph
m-NO2
m-NO2
m-OH
p-OH
>5000
134
245
278
156
2
>5000
>5000
556
334
154
1.5
>5000
>5000
282
347
226
2
>5000
>5000
3474
1543
1984
47
0.06
0.90
2.60
3.70
0.91
0.05
0.01
0.13
0.02
0.41
0.08
0.77
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CF3
m-SO2NH2
m-SO2NH2
p-SO2NH2
CF3
3
0.5
1.5
1
64
H
p-SO2NH2
5
1
54
H
m-SO2NH2
m-SO2NH2
p-SO2NH2
3
4
5
40
27j
cyclopropyl
cyclopropyl
cyclopropyl
16
8
18
2
273
255
464
27k
27l
2
13
m-1,4-diazepan-1-yl
66
176
326
a
The ATP concentrations used in these assays were within 15 μM of Km. The data given are mean values derived from two replicates. Apparent
inhibition constants (Ki) were calculated from IC50 values and the appropriate Km (ATP) values for each kinase.35 Antiproliferative activity by MTT
b
48 h assay. The data given are mean values derived from at least three replicates.
CDK9. All these analogues demonstrate excellent antiprolifer-
ative activity with GI50 values ranging from 0.03 to 0.93 μM.
Introduction of a bulkier heterocylic (1,4-diazepan-1-yl)-
ethanone or 1,4-diazepane at the meta-position of the aniline
affords 12s−u, displaying appreciable selectivity for CDK9
versus CDK2. Compound 12u, in particular, shows a >80-fold
enhanced CDK9 selectivity over CDK2. Compounds 12s−u
effectively inhibit tumor cell growth with GI50 values of 0.64,
0.33, and 0.42 μM, respectively. Replacement of the C5-
carbonitrile or C5-fluoride with a C5-hydrogen affords Ic (R′ =
H, R = m -1,4-diazepane). However, this replacement results in
a >2-fold loss in CDK9 inhibitory activity but a more significant
drop in CDK2 selectivity when compared with 12s and 12u.
These further support the role of the carbonitrile or fluoride
substitution at the C5-pyrimidine in favoring potency and
selectivity against CDK9 over CDK2.
In general, all C5-substituted pyrimidine analogues are also
potent CDK1 inhibitors, with activity comparable to that of
CDK2 as shown in Table 1. An exception is compound 12u
which targets CDK1 and CDK2 with Ki values of 94 and 568
nM, respectively, being 6-fold more potent for CDK1 than for
CDK2. It is apparent that this combined inhibition of CDK9,
CDK1, and CDK2 results in significant cytotoxicity in cancer
cells. 12e, a nanomolar CDK9, CDK1, and CDK2 inhibitor, for
example, is the most potent cytotoxic agent of this chemical
class, with GI50 < 10 nM against HCT-116 cells. This is
consistent with the finding that cancer cells expressing shRNA
targeting a combination of CDK2, CDK1, and CDK9 were
most effective in induction of apoptosis of cancer cells, and
targeting CDK9, CDK1, and CDK2 has been proposed as an
anticancer strategy.3
irrespective of modest CDK7 inhibition (Table 1). Compounds
Ib and 12g, for example, inhibit CDK7 with Ki > 1 μM, but
both exhibit excellent antiproliferative activity with GI50 = 0.05
and 0.30 μM, respectively.
In order to assess whether modification of the C4-methyl of
the thiazole is tolerated, we prepared a series of substituted C4-
thiazolpyrimidine derivatives; the SAR is summarized in Table
2. Replacement of the C4-methyl with phenyl (27a, R′ = Ph, R
= m-NO2) is not tolerated, and no inhibitory activity against
CDKs is detected up to 5 μM. However, this compound
exhibits potent antiproliferative activity with a GI50 of 60 nM,
indicating potential non-CDK kinase targets. Substitution of
C4-trifluoroethyl, in the context of m-nitroanilinopyrimidine,
yields 27b (R′ = CH2CF3, R = m-NO2). This compound
exhibits excellent selectivity for CDK9 (Ki = 0.134 μM), being
inactive against other CDKs at concentrations up to 5 μM.
Despite its high selectivity, 27b still displays good antiprolifer-
ative activity with GI50 < 1 μM in HCT-116 cancer cells.
However, analogues 27c (R′ = CH2CF3, R = m-OH), 27d (R′
= CH2CF3, R = p-OH), and 27e (R′ = CH2CF3, R = m-
SO2NH2) reduce CDK9 inhibition and selectivity when
compared to 27b. Keeping the benzenesulfonamide moiety
but replacing the 4C-trifluoroethyl with a C4-trifluoromethyl,
C4-hydrogen, or C4-cyclopropyl affords compounds 27f−k. All
of these compounds possess significantly enhanced inhibitory
activity not only against CDK9 but also against other CDKs. As
expected, these compounds are extremely cytotoxic to cancer
cells with GI50 values in the range of 0.01−0.41 μM. However,
substitution of the benzenesulfonamide with 1,4-diazepan-1-
ylaniline yields 27l (R′ = cyclopropyl, R = m-1,4-diazepane).
This analogue shows a significant loss of activity which suggests
that the benzenesulfonamide moiety is a key contributor to
optimal CDK inhibition and cellular potency of this series.
Cocrystal Structures of 12u Bound to CDK9/Cyclin T
and CDK2/Cyclin A. As one of the most selective CDK9
inhibitors in the chemical series, 12u was cocrystallized with
Most of the analogues described here are significantly less
effective as CDK7 inhibitors when compared to their activity
against other CDKs, suggesting that CDK7 inhibition is not a
requirement for the observed cellular cytotoxicity: many
compounds demonstrate excellent antiproliferative activity
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Table 3. Crystallographic Parameters of CDK2/Cyclin A/12u and CDK9/Cyclin T/12u
CDK2/cyclin A/12u
CDK9/cyclin T/12u
Data Collection
beamline
Diamond I-03
P212121
Diamond I-03
H3
space group
unit cell (Å)
a = 73.81; b = 134.55; c = 149.17
α = β = γ = 90
29.83−2.26 (2.38−2.26)
308028 (43211)
69656 (9807)
a = b = 172.80; c = 98.88
α = β = 90; γ = 120
86.40−3.08 (3.25−3.08)
70331 (10036)
20079 (2956)
0.058 (0.511)
3.5 (3.4)
unit cell (deg)
resolution (highest resolution shell) (Å)
total observations
unique observations
Rmerge
0.071 (0.496)
multiplicity
4.4 (4.4)
mean I/σI
13.4 (2.8)
15.4 (2.4)
completeness (%)
Refinement Statistics
highest resolution shell (Å)
total number of atoms
total number of waters
R
99.2 (96.6)
99.0 (99.6)
2.29−2.26
9233
3.09−3.17
4649
363
16
18.10 (23.09)
21.71 (28.66)
0.003
16.11 (28.6)
19.72 (34.2)
0.011
Rfree
rms bonds (Å)
rms angles (deg)
0.702
1.433
Figure 2. Cocrystal structures of 12u bound to CDK9/cyclin T1 (PDB code 4BCG) and CDK2/cylin A (PDB code 4BCP). The structures of
CDK9/T1/12u (A, C) are shown. Compound 12u bound to CDK2/cyclin A (B, D) and showing two binding orientations. Electron density around
12u is shown as a wire mesh (A, B). Selected CDK9 and CDK2 residues are drawn in ball-and-stick representation. Hydrogen bonds in all panels are
depicted by dotted lines.
CDK9/cyclin T and CDK2/cyclin A in order to explain the
observed SAR. The crystal structure and refinement data are
summarized in Table 3. A more thorough rationalization of the
SARs provided by the determination of five additional inhibitor
cocrystal structures bound to CDK9/cyclin T and CDK2/
cyclin A is provided in the companion paper.34 As shown in
Figure 2, 12u adopts a similar binding mode within the CDK9
and CDK2 ATP binding sites located between the N- and C-
terminal lobe, and the thiazole, pyrimidine, and aniline moieties
occupy similar positions. In both CDK9 and CDK2, 12u
hydrogen-bonds with the kinase hinge regions. The N1-
pyrimidine accepts a hydrogen bond from the peptide nitrogen
of Cys106 (Leu83 in CDK2), while the C2-NH of the
pyrimidine ring donates a hydrogen bond to the peptide
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carbonyl of Cys106. At the back of the ATP binding site the
C5-carbonitrile group exploits the hydrophobic region close to
the gatekeeper residue Phe103 (Phe80 in CDK2) to form a
favorable lone pair−π interaction. The CDK2/cyclin A/12u
structure was determined at a higher resolution and shows a
water molecule trapped in a pocket behind the C5-carbonitrile
(Figure 2B and Figure 2D). This water molecule forms a
hydrogen-bond network with the backbone of residue Asp145
and with the side chain of Glu51. In the adenine site the
pyrimidine ring is sandwiched between the hydrophobic side
chains of Ala46 (Ala31 in CDK2) and Leu156 (Leu134 in
CDK2), with which it forms extensive van der Waals
interactions. The hydrogen of the C2-methylaminothiazole
binds to Asp167 in CDK9 and to the corresponding residue
Asp145 in CDK2. At the front of the ATP binding pocket, the
aniline ring is contacted from above by Ile25 (Ile10 in CDK2)
to make favorable van der Waals interactions with both
enzymes.
The very weak electron density of the 1,4-diazepan-1-
ylaniline moiety of 12u suggests that it is not bound tightly to
CDK2. Two conformations of 12u were consistent with the
observed electron density and represent possible alternative
binding modes. Neither of these modes suggests either
favorable or unfavorable interactions, correlating to the relative
absence of electron density for the 1,4-diazepane ring. In the
CDK9 complex, however, the 1,4-diazepane ring clearly adopts
an “inward” conformation orientated toward the thiazole ring.
This may be more favorable for 12u because of a reduced
solvent exposure of the hydrophobic 1,4-diazepane ring in this
orientation.
Although the inhibitor interactions with CDK2 and CDK9
are mostly conserved, there are, however, two significant
differences. First, as seen for other CDK9/cyclin T inhibitor
structures described in the companion paper,34 the binding of
the inhibitor induces a lowering of the glycine-rich loop into
the ATP binding site. The loop adopts several conformations,
and this inherent flexibility is reflected in the higher b-factors.
By contrast, the conformation of the glycine rich loop in
CDK2/cyclin A appears to be relatively unaltered on inhibitor
binding (PDB 2JGZ). Second, in comparison with the apo
structure of CDK9/cyclin T (PDB 3BLH)51 the backbone of
the hinge region adapts to inhibitor binding by shifting away
from 12u. This shift enables a hydrogen bond to form between
the C2-NH of the pyrimidine with the peptide carbonyl of
Cys106 at an optimal length of 2.8 Å. These two observations
support the hypothesis that CDK9 has a more flexible ATP
binding pocket than CDK2.
We propose that the greater flexibility of the ATP-binding
site of CDK9 enables the large flexible anilino-1,4-diazepine of
12u, in the context of the C5-carbonitrilepyrimidine moiety, to
be well accommodated by CDK9. In contrast, the crystal
structure of 12u bound to CDK2 shows that this ring adopts an
orientation either “inward” or “outward”, suggesting that the
CDK2 binding pocket is too crowded for 12u. This variation in
the ability of the kinases to adapt and readily accommodate
inhibitors offers an explanation for the high potency and
selectivity of 12u toward CDK9.
Table 4. Inhibitory Activity of 12u against Protein Kinases
a
protein kinases
inhibition IC50 (nM)
CDK1/cyclin B
CDK2/cyclin A
CDK6/cyclin D3
CDK7/cyclin H
CDK9/cyclin T1
BCR-Abl
188
1126
>5000
92
14
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
CaMK1
IKKβ
Lck
MAPK2
PKA
PKBα
PKCα
cSRC
a
The ATP concentrations used in these assays were within 15 μM of
Km. The data given are mean values derived from two replicates.
panel of nine tumor cell lines and three nontranformed cell
lines were examined using a 48 h MTT assay as summarized in
Table 5A. To investigate cell-type sensitivity, we included
HCT-116 colon carcinoma (wild-type and mutant p53,
respectively), MCF-7 breast carcinoma (wild-type p53, pRb
positive, ER positive and containing CDK4/cyclin D and
CDK6/cyclin D), and MDA-MB-468 breast carcinoma cells
(mutant p53, pRb negative, ER negative and lacking CDK4/
cyclin D and CDK6/cyclin D),52 and other cell lines. Similar
sensitivity is observed for cells with different p53, Rb, and
CDK4/6 status. Compound 12u suppresses tumor cell
proliferation with GI50 values ranging from 0.38 to 0.78 μM,
irrespective of the tumor cell type. However, all non-
transformed cell lines, i.e., microvascular endothelial cell line
HMEC-1 and embryonic lung fibroblasts MRC-5 and WI-38,
are significantly less sensitive to 12u treatment (GI50 = 3.12−
5.96 μM). The time-course assays were performed using A2780
ovarian cancer and MRC-5 and HMEC-1 nontransformed cell
lines. As shown in Table 5B, 24 h treatment with 12u, as well as
with flavopiridol, is sufficient to achieve maximal growth
inhibition of 12u in A2780 cancer cells. Again, 12u is
significantly less toxic in the HMEC-1 and MRC-5 non-
transformed cells. In contrast, flavopiridol fails to demonstrate
any significant differential effects between the cancer cells and
noncancerous cell lines.
Compound 12u Effectively Induces Cancer Cell
Apoptosis. Cell death induced by therapeutic agents can
occur through caspase-dependent or -independent apoptosis or
by necrosis. To assess whether apoptosis is contributing to the
cytotoxic effect of 12u, we used annexin V/PI (propidium
iodide) surface staining in A2780 cancer cells following
treatment with 12u for 48 h (Figure 3A). Compound 12u
induced cell apoptosis at the GI50 (the concentration of 12u
required to inhibit 50% of cell proliferation by MTT assay) in a
dose-dependent manner. At the GI50 concentration 12u causes
38% annexin V-positive cells and the percentage increases to
50% at 5GI50. With flavopiridol the same treatments results in
39% and 54% apoptotic cells at GI50 and 5GI50, respectively.
Concurrent treatment with 5GI50 of either 12u or flavopiridol
together with 50 μM of the pan-caspase inhibitor Z-VAD-fmk
suppresses apoptosis, suggesting a caspase-dependent mecha-
nism of apoptosis induction.18
Compound 12u Is a Potent Antiproliferative Agent.
Compound 12u was screened against a panel of kinases using
biochemical assays and showed no inhibitory activity at
concentrations up to 5 μM against a panel of kinases, including
BCR-Abl, CaMK1, IKK, Lck, MARK2, PKA, PKB, PKC, and
cSRC (Table 4). The antiproliferative effects of 12u against a
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Table 5. Antiproliferative Activity of 12u and Flavopiridol against a Panel of Human Humour and Nontranformed Cell Lines
a
(A) and Activity by MTT Time-Course Experiments (B)
A
human cell line
origin
designation
48 h MTT, GI50 SD (μM)
colon carcinoma
HCT-116 (p53wt, pRb+)
0.420 0.020
0.780 0.060
0.385 0.091
0.690 0.020
0.402 0.026
0.320 0.010
0.630 0.140
0.747 0.076
0.590 0.078
3.120 0.320
5.960 0.720
5.490 0.630
HCT-116 (p53null)
HCC 2998 (p53wt, pRb+)
breast carcinoma
MCF-7 (p53wt, pRb+, ER+)
MDA-MB468 (p53mut, pRb−, ER−)
ovarian carcinoma
A2780
HeLa
cervical carcinoma
renal carcinoma
TK 10
PANC-1
HMEC-1
MRC-5
WI-38
B
pancreatic carcinoma
microvascular endothelial
embryonic lung fibroblast
antiproliferative activity, GI50 SD (μM)
cell line
A2780
compd
12u
24 h
48 h
72 h
0.493 0.048
0.023 0.001
6.250 0.515
0.049 0.004
7.500 1.020
0.061 0.001
0.320 0.010
0.031 0.008
5.960 0.720
0.039 0.012
3.120 0.320
0.062 0.004
0.288 0.020
0.029 0.001
5.670 0.410
0.028 0.001
4.500 0.310
0.066 0.002
flavopiridol
12u
MRC-5
flavopiridol
12u
HMEC-1
flavopiridol
a
The data given are mean values derived from at least three replicates SD.
Activation of caspase-3 activity by 12u was confirmed in
CTD, reduction of Mcl-1 and HDM2, and induction of cleaved
PARP being observed.
A2780 cancer cell following exposure to drug for 24 h and was
used to compare that in HMEC-1 untransformed endothelial
cells (Figure 3B). Compound 12u significantly activates
caspase-3 activity in the tumor cells starting at GI50
concentration (p < 0.001), and the effect is further enhanced
at higher concentrations. In contrast, no such activity is
detected in the HMEC-1 cells up to 10GI50 concentrations of
12u. These results confirm that the cytotoxicity induced by 12u
is mediated through the preferential induction of apoptosis in
cancer cell lines and corroborates the MTT cytotoxic potency.
As 12u showed potent CDK1 inhibition in biochemical
kinase assays, we next investigated its effects on cell cycle
progression. A2780 cells were treated with 12u (or flavopiridol)
for a period of 24 h at GI50 and 5GI50 concentrations,
respectively (Figure 3C). The cells showed no alteration in cell
cycle distribution at concentrations less than 5GI50 12u, at
which concentration accumulation of cells in G2/M phase of
the cell cycle was detected. This confirms that 12u has a lower
cellular CDK1 inhibitory activity compared to that of CDK9. A
similar cell cycle profile is observed with flavopiridol (Figure
3C).
Compound 12u Inhibits CDK9 Activity and Down-
Regulates Mcl-1 in Cancer Cells. We next investigated the
cellular mode of action of 12u by Western blot analysis (Figure
3D). Treatment of A2780 cells with 12u for a period of 24 h
showed that phosphorylation at Ser-2 CTD of RNAPII was
reduced at the GI50 and abrogated at 5GI50, confirming cellular
CDK9 inhibition. The same treatment caused down-regulation
of Mcl-1 and HDM2 but had little effect on the levels of Bcl-2
expression. Induction of apoptosis was indicated by PARP
cleavage. Analogous results were obtained with flavopiridol,
with inhibition of the phosphorylation of Ser-2 of RNAPII
Ex Vivo Antitumor Activity in Primary Chronic
Lymphocytic Leukemia Cells. The potency and selectivity
of 12u were further evaluated in patient-derived CLL cells
(Table 6), as well as age-matched normal B-cells and T-cells,
using an annexin V-FITC apoptosis assay. As shown in Figure
4A, the compound exhibits excellent activity with a mean LD50
of 2.60 μM 1.1 μM against CLL cells (the concentration of
12u required to kill 50% of the CLL cells following exposure
for 48 h). Figure 4B shows that 12u induces a dose-dependent
increase in apoptosis in CLL cells as denoted by an increased
annexin V positivity. In contrast, little toxicity is observed in the
normal B- and T-cells with LD50 of >80 and >280 μM,
respectively (Figure 4A). To determine whether the cytotox-
icity induced by 12u is caspase-dependent, primary CLL cells
were incubated with various concentrations of 12u for 24 h,
followed by flow cytometric assessment of active caspase-3. As
shown in Figure 4C, the caspase-3 activity is significantly
induced at 1.0 μM 12u (P < 0.05) and is further enhanced in a
dose-dependent manner at 5 μM (P < 0.0001) and 10 μM (P <
0.0001) when compared with untreated controls. These data
support the conclusion that 12u-induced cytotoxicity is
mediated via the activation of the effector caspase-3.
CLL cells are characterized by resistance to apoptosis
mediated by up-regulation of Bcl-2 family proteins. Mcl-1 is
the most important antiapoptotic member of the Bcl-2 protein
family and is overexpressed in the majority of patients with
CLL at baseline. Increased levels of Mcl-1 are associated with
both drug resistance and inferior survival.53,54 Down-regulation
of Mcl-1 is sufficient to induce apoptosis in CLL cells.55
A
correlation between lower Mcl-1 protein and mRNA levels with
known biologic prognostic markers and improved outcomes in
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Table 6. Clinical Characteristics of the CLL Patients (n =
10) in This Study
patient characteristics
mean age (years)
number
68
sex (male/female)
7/3
10/0
8/2/0
8/2
7/3
7/3
previously untreated/treated
Binet stage (A/B/C)
IGHV gene mutation (mutated/unmutated)
CD38 expression (<20%/≥20%)
ZAP-70 expression (<20%/≥20%)
0.0001) irrespective of stages of the disease. The change in Mcl-
1 protein expression precedes evidence of apoptosis induction
suggesting that the inhibition is a trigger for apoptosis rather
than a consequence of apoptosis induction.
CONCLUSION
■
In this communication we describe the synthesis and SAR of a
series of 5-substituted-4-(thiazol-5-yl)-2-(phenylamino)-
pyrimidines and 4-(4-substituted-thiazol-5-yl)-N-phenylpyrimi-
din-2-amines. Many compounds inhibit CDK9 activity at low
nanomolar concentrations and exhibit very potent antiprolifer-
ative activity in tumor cells. Optimization led to the discovery
of 12u, one of the most selective CDK9 inhibitors in the series,
being >80-fold more potent for CDK9 versus CDK2. The
cocrystal structures of 12u bound in CDK9/cyclin T and
CDK2/cyclin A provide a rationale for the observed potency
and selectivity. Compound 12u was examined in more detail
regarding its cellular mode of action. The study demonstrates
that by inhibiting cellular RNAPII transcriptional activity, 12u
mediates down-regulation of the antiapoptotic protein Mcl-1,
thereby rendering cells sensitive to apoptosis. Significantly, 12u
exhibits excellent antitumor activity in primary CLL cells but
shows little toxicity in healthy normal B- and T-cells. In keeping
with this finding, Mcl-1 is not detectable in normal B- and T-
cells (data not shown), indicating that Mcl-1 may not be a
critical regulator of survival in normal lymphocytes. In contrast,
CLL cells appear to have a requirement for this protein in order
to maintain viability.53 This study provides a rationale for
further development of CDK9 inhibitors for the treatment of
CLL and other human malignancies.
Figure 3. Cellular mode of action of 12u. (A) A2780 cells were
exposed to 12u, flavopiridol, or 12u (or flavopiridol) + 25 μM Z-VAD-
fmk for 48 h and analyzed by annexin V/PI staining. The percentage of
cells undergoing apoptosis was defined as the sum of early apoptosis
(annexin V-positive cells) and late apoptosis (annexin V-positive and
PI-positive cells). (B) 12u induces caspase-3 activity in A2780 after
treatment for a period of 24 h, but 12u is less effective in
nontransformed endothelial cell line HMEC-1 upon the same
treatment. Vertical bars represent the mean SD of two independent
experiments. Values significantly different from DMSO vehicle control
are marked with asterisks: (∗) P < 0.05; (∗∗) P < 0.0001. (C) Cell-
cycle analysis of A2780 cells treated with 12u or flavopiridol for 24 h.
(D) Western blot analysis of A2780 cells treated with 12u or
flavopiridol for 24 h. DMSO diluent was used as control in each
experiment, and β-actin antibody was used as an internal control.
EXPERIMENTAL SECTION
Chemistry. Chemical reagents and solvents were obtained from
■
commercial sources. When necessary, solvents were dried and/or
1
purified by standard methods. H NMR and 13C NMR spectra were
obtained using a Bruker 400 Ultrashield spectrometer at 400 and 100
MHz, respectively. These were analyzed using the Bruker TOPSPIN
2.1 program. Chemical shifts are reported in parts per million relative
to internal tetramethylsilane standard. Coupling constants (J) are
quoted to the nearest 0.1 Hz. The following abbreviations are used: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. High
resolution mass spectra were obtained using a Waters 2795 single
quadrupole mass spectrometer/micromass LCT platform. Purity for
final compounds was greater than 95% and was measured using Waters
high performance liquid chromatography (Waters 2487 dual λ
absorbance detector) with Phenomenex Gemini-NX 5u C18 110A
250 mm × 4.60 mm column, UV detector at 254 nm, using system A
(10% MeOH containing 0.1% TFA for 4 min, followed by linear
gradient 10−100% MeOH over 6 min at a flow rate of 1 mL/min),
system B (10% MeCN containing 0.1% TFA for 2 min, followed by
linear gradient 10−100% over 10 min at a flow rate of 1 mL/min), and
system C (10% MeCN containing 0.1% TFA for 4 min, followed by
linear gradient 10−100% over 10 min at a flow rate of 1 mL/min).
patients with CLL has been reported.53,56 In the present study,
primary CLL cells derived from 10 patients were cultured with
1 μM 12u for 8 h and examined for the effect on Mcl-1 protein.
Figure 4D shows that the levels of Mcl-1 protein expression are
consistently inhibited by 12u in all CLL patient samples (P <
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Figure 4. Compound 12u shows selective toxicity against CLL cells and induces its effects through the induction of apoptosis. Primary CLL cells and
normal B-cells were cultured in the presence of increasing concentrations of 12u for 48 h. (A) Sigmoidal dose−response curves for 12u in CLL cells
and normal B-cells and T-cells. (B) Compound 12u caused a dose-dependent increase in annexin V-positive cells, and this was preceded by (C) a
dose-dependent increase in caspase-3 activation after 24 h in culture ((∗) P < 0.05, (∗∗) P < 0.0001). (D) Mcl-1 protein expression was significantly
inhibited by 12u at 8 h in all the primary CLL samples tested (P < 0.0001).
Flash chromatography was performed using either a glass column
packed with silica gel (200−400 mesh, Aldrich Chemical) or
prepacked silica gel cartridges (FlashMaster systems, Biotage). Melting
points were determined with an Electrothermal melting point
apparatus.
Ethyl 4-Methyl-2-(methylamino)thiazole-5-carboxylate 1.
To a solution of ethyl 2-chloroacetoacetate (13.8 mL, 100 mmol) in
100 mL of MeOH were added 1-methylthiourea and 3 mL of pyridine,
and the mixture was stirred at room temperature for 4 h. The mixture
was concentrated and the precipitate was washed with saturated
NaHCO3 solution, filtered, and dried to offer the title compound as a
white solid (17.46 g, 85% yield), mp 88−90 °C. 1H NMR (CDCl3): δ
1.35 (t, 3H, J = 7.2 Hz, CH3), 2.53 (s, 3H, CH3), 2.99 (s, 3H, CH3),
4.28 (q, 2H, J = 7.2 Hz, CH2). 13C NMR (CDCl3): δ 14.78, 17.67,
31.27, 60.28, 107.47, 159.79, 162.37, 171.08. HR-MS (ESI+): m/z [M
+ H]+ calcd for C8H13 N2O2S, 201.0698, found 201.0463.
were added 4-dimethylaminopyridine (DMAP) (1.0 g) and di-tert-
butyl dicarbonate (12.0 g, 55.0 mmol), and the reaction was continued
for 8 h at room temperature. After completion of the reaction, the
mixture was washed with 5% aqueous HCl, followed by saturated
NaHCO3 solution, brine, dried over MgSO4, and filtered. The organic
solution was concentrated to dryness, and the title compound was
obtained via recrystallization from hexane as a white solid (14 g, 93%),
mp 148−150 °C. 1H NMR (CDCl3): δ 1.34 (t, 3H, J = 7.2 Hz, CH3),
1.60 (s, 9H, 3 × CH3), 2.64 (s, 3H, CH3), 3.55 (s, 3H, CH3), 4.29 (q,
2H, J = 7.2 Hz, CH2). 13C NMR (CDCl3): δ 14.35, 17.37, 28.14,
33.94, 60.54, 83.85, 116.19, 153.13, 156.56, 162.68, 163.10. HR-MS
(ESI+): m/z [M + H]+ C13H21N2O4S, 301.1222, found 301.1312.
tert-Butyl 5-(2-Cyanoacetyl)-4-methylthiazol-2-yl(methyl)-
carbamate 3. To a solution of 2 (6.0 g, 20.0 mmol) in 6 mL of
anhydrous THF was added 1.50 mL of acetonitrile (1.3 mmol). The
mixture was cooled at −78 °C, and LDA was added dropwise over 10
min. The reaction was continued for 2 h. After completion of the
reaction, 10 mL of H2O was added and the mixture was acidified with
dilute HCl solution and extracted with CHCl3 (3 × 50 mL). The
Ethyl 2-(tert-Butoxycarbonyl(methyl)amino)-4-methylthia-
zole-5-carboxylate 2. To a solution of ethyl 4-methyl-2-
(methylamino)thiazole-5-carboxylate (10.0 g, 50.0 mmol) in DCM
650
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combined organic phase was washed with brine, dried over MgSO4,
and concentrated to dryness. The mixture was purified by using PE/
EtOAc as elutant to afford the title compound as a white solid (4.25 g,
72%), mp 119−121 °C. 1H NMR (CDCl3): δ 1.61 (s, 9H, 3 × CH3),
2.68 (s, 3H, CH3), 3.59 (s, 3H, CH3), 3.86 (s, 2H, CH2). 13C NMR
(CDCl3): δ 18.71, 28.09, 32.23, 34.26, 84.76, 113.76, 122.37, 153.11,
159.36, 163.14, 179.25. HR-MS (ESI+): m/z [M + H]+ calcd for
C13H18 N3O3S, 296.1069, found 296.1130.
tert-Butyl (5-(2-Cyano-3-(dimethylamino)acryloyl)-4-methyl-
thiazol-2-yl)(methyl)carbamate (4, R1 = Boc, R′ = CN). The
compound was prepared by the method described previously.35 Yellow
solid (80% yield), mp 174−176 °C. 1H NMR (CDCl3): δ 1.59 (s, 9H,
3 × CH3), 2.56 (s, 3H, CH3), 3.28 (s, 3H, CH3), 3.46 (s, 3H, CH3),
3.54 (s, 3H, CH3), 7.86 (s, 1H, CH). 13C NMR (CDCl3): δ 18.33,
28.18, 34.09, 38.97, 48.17, 81.56, 83.89, 119.49, 124.63, 152.80,
153.07, 158.71, 161.97, 182.43. HR-MS (ESI+): m/z [M + H]+ calcd
for C16H23N4O3S, 351.1491, found 351.1281.
3-(Dimethylamino)-2-fluoro-1-(4-methyl-2-(methylamino)-
thiazol-5-yl)prop-2-en-1-one (4, R′ = F). To a well-stirred solution
of 3-(dimethylamino)-1-(4-methyl-2-(methylamino)thiazol-5-yl)prop-
2-en-1-one (6)35 (5.0 mmol) in MeOH under ice bath was added
SelecFluor (7.5 mmol), and the mixture was stirred for 1 h. After
completion of the reaction, the mixture was concentrated and purified
by column chromatography using EtoAc/MeOH to yield the title
compound. Yellow solid (30%). 1H NMR (DMSO-d6): δ 2.40 (s, 3H,
CH3), 2.83 (d, 3H, J = 4.8 Hz, CH3), 3.04 (apparent s, 6H, 2 × CH3),
6.88 (d, 1H, J = 30.4 Hz, CH), 8.04 (d, 1H, J = 4.4 Hz, NH). HR-MS
(ESI+): m/z [M + H]+ calcd for C10H15FN3OS, 244.0920, found
244.0849.
(s, 3H, CH3), 3.51 (s, 3H, CH3), 4.81 (t, 1H, J = 7.2 Hz, CH). HR-MS
(m/z): calcd for C13H22N2O3S, 287.1429; found 287.1503 [M + H]+.
tert-Butyl (5-(1-Hydroxybutyl)-4-methylthiazol-2-yl)-
(methyl)carbamate (9, R′ = Et). Light yellow liquid (77%). 1H
NMR (CDCl3): δ 0.94 (t, 3H, J = 7.2 Hz, CH3), 1.24−1.48 (m, 2H,
CH2), 1.59 (s, 9H, 3 × CH3), 1.67−1.94 (m, 3H, OH and CH2), 1.94
(s, 1H, OH), 2.30 (s, 3H, CH3), 3.51 (s, 3H, CH3), 4.89 (t, 1H, J = 7.2
Hz, CH). HR-MS (ESI+): m/z [M + H]+ calcd for C14H25N2O3S,
301.1586, found 301.1650.
tert-Butyl (5-(1-Hydroxypentyl)-4-methylthiazol-2-yl)-
(methyl)carbamate (9, R′ = Pr). Light yellow liquid (53%). 1H
NMR (CDCl3): δ 0.91 (t, 3H, J = 7.2 Hz, CH3), 1.23−1.43 (m, 4H, 2
× CH2), 1.59 (s, 9H, 3 × CH3), 1.71−1.98 (m, 3H, CH2 and OH),
2.31 (s, 3H, CH3), 3.52 (s, 3H, CH3), 4.89 (t, 1H, J = 7.2 Hz, CH).
HR-MS (ESI+): m/z [M + H]+ calcd for C15H27N2O3S, 315.1742,
found 315.1776.
General Procedure for Preparation of 10 (R′ = Me, Et, or Pr).
A solution of corresponding 9 in CHCl3 (1.5 mmol/mL) was treated
with MnO2 (5.0 equiv), and the mixture was refluxed for 3 h. Upon
completion of the reaction, the mixture was filtered through Celite,
and the filtrate was concentrated to dryness.
tert-Butyl Methyl(4-methyl-5-propionylthiazol-2-yl)-
1
carbamate (10, R′ = Me). Yellow solid (85%), mp 97−99 °C. H
NMR (CDCl3): δ 1.20 (t, 3H, J = 7.2 Hz, CH3), 1.61 (s, 9H, 3 ×
CH3), 2.66 (s, 3H, CH3), 2.78 (q, 2H, J = 7.2 Hz, CH2), 3.57 (s, 3H,
CH3). 13C NMR (CDCl3): δ 8.36, 18.40, 28.15, 34.08, 36.01, 84.03,
125.02, 153.14, 155.40, 161.74, 194.13. HRMS (ESI+): m/z [M + H]+
C13H21N2O3S, 285.1273, found 285.1183.
tert-Butyl (5-Butyryl-4-methylthiazol-2-yl)(methyl)-
carbamate (10, R′ = Et). Yellow solid (81%), mp 101−103 °C.
1H NMR (CDCl3): δ 0.99 (t, 3H, J = 7.2 Hz, CH3), 1.61 (s, 9H, 3 ×
CH3), 1.70−1.82 (m, 2H, CH2), 2.67 (s, 3H, CH3), 2.76 (t, 2H, J =
7.2 Hz, CH2), 3.57 (s, 3H, CH3). 13C NMR (CDCl3): δ 13.79, 17.92,
18.35, 28.15, 34.12, 44.93, 84.06, 125.21, 153.14, 155.37, 161.72,
193.63. HR-MS (ESI+): m/z [M + H]+ C14H23N2O3S, 299.1429,
found 299.1459.
(2-Chloro-3-(dimethylamino)-1-(4-methyl-2-(methylamino)-
thiazol-5-yl)prop-2-en-1-one (4, R′ = Cl). To an ice-cooled
solution of 6 (2.0 mmol) in 50 mL of methanol was added N-
chlorosuccinmide (20 mmol) over 10 min dropwise. After being
stirred at room temperature for 30 min, the mixture was concentrated
and purified by column chromatography using EtoAc to yield the title
1
compound as a light yellow solid (43%), mp 141−143 °C. H NMR
(CDCl3): δ 2.38 (s, 3H, CH3), 2.99 (s, 3H, CH3), 3.26 (s, 6H, 2 ×
CH3), 6.44 (s, 1H, NH), 7.42 (s, 1H, CH). 13C NMR (CDCl3): δ
18.28, 31.98, 43.33, 101.88, 117.86, 149.13, 153.84, 171.91, 180.77.
HR-MS (ESI+): m/z [M + H]+ calcd for C10H15ClN3OS, 260.0624,
found 260.0541.
tert-Butyl Methyl(4-methyl-5-pentanoylthiazol-2-yl)-
1
carbamate (10, R′ = Pr). Yellow solid (65%), mp 82−84 °C. H
NMR (CDCl3): δ 0.92 (t, 3H, J = 7.2 Hz, CH3), 1.30−1.43 (m, 2H,
CH2), 1.59 (s, 9H, 3 × CH3), 1.62−1.72 (m, 2H, CH2), 2.64 (s, 3H,
CH3), 2.75 (t, 2H, J = 7.2 Hz, CH2), 3.55 (s, 3H, CH3). 13C NMR
(CDCl3): δ 13.88, 18.38, 22.37, 26.54, 28.14, 34.07, 42.73, 84.01,
125.16, 153.16, 155.50, 161.67, 193.78. HR-MS (ESI+): m/z [M + H]+
C15H25N2O3S, 313.1586, found 313.1620.
tert-Butyl Methyl(4-methylthiazol-2-yl)carbamate (8). N,4-
Dimethylthiazol-2-amine was obtained from 1-methylthiourea and 1-
chloropropan-2-one using the method for preparing 1 as off-white
solid (88%), mp 68−70 °C. 1H NMR (CDCl3): δ 2.96 (d, 3H, J = 1.2
Hz, CH3), 2.96 (s, 3H, CH3), 6.06 (q, 1H, J = 1.2 Hz, CH), 6.15 (brs,
1H, NH). 13C NMR (CDCl3): δ 17.36, 31.95, 100.06, 148.66, 171.61.
HR-MS (ESI+): m/z [M + H]+ calcd for C5H9N2OS, 129.0486, found
129.0372. The title compound was obtained from N,4-dimethylthiazol-
2-amine and di-tert-butyl dicarbonate by the method for preparing 2 as
light yellow liquid (55%). 1H NMR (CDCl3): δ 1.59 (s, 9H, 2 × CH3),
2.35 (d, 3H, J = 1.2 Hz, CH3), 3.55 (s, 3H, CH3), 6.48 (q, 1H, J = 0.8
Hz, CH). 13C NMR (CDCl3): δ 17.41, 28.20, 34.32, 82.91, 108.39,
147.11, 153.13, 161.20. HR-MS (ESI+): m/z [M − tert-butyl + H]+
calcd for C6H9N2O2S, 173.0385, found 173.0224.
General Procedure for Preparation of 9 (R′ = Me, Et, or Pr).
To a solution of 8 (10.0 mmol) in 15 mL of anhydrous THF cooling
at −78 °C was added 25.0 mmol of LDA dropwise. After the mixture
was stirred for 30 min the corresponding aldehyde (12.0 mmol) was
added and the reaction was continued for a further 1 h. After
completion of the reaction, 10 mL of water was added and the mixture
was washed with 2 M aqueous HCl solution. After removal of THF,
the mixture was extracted with CHCl3 (3 × 30 mL) and the combined
organic layers were washed with brine, dried over MgSO4, filtered, and
concentrated to dryness. The title compound was obtained by column
chromatography using EtOAc/PE as eluant.
Preparations of 4 (R1 = Boc, R′ = Me, Et or Pr) were done by
heating 10 in DMF−DMA using the method described previously35 or
by heating in a Discovery microwave at 140 °C for 45 min. The
mixture was concentrated and used for the pyrimidine formation
reaction without further purification.
General Procedure for Preparation of Ic and 12a−u. A
mixture of the appropriate 3-(dimethylamino)-1-(4-methyl-2-
(methylamino)thiazol-5-yl)prop-2-en-1-one or 4 and 1-phenylguani-
dine (11)32,35 (2 equiv mmol) in 2-methoxyethanol (0.2 mL/mmol)
was heated in a microwave at 100−140 °C for 20−45 min. When the
mixture was cooled, the residue was purified by flash chromatography
using appropriate mixtures of EtoAc/PE or EtoAc/MeOH as the
eluant. The products were further purified by crystallization from
EtOAc−MeOH mixtures.
4-(4-Methyl-2-(methylamino)thiazol-5-yl)-2-(3-
nitrophenylamino)pyrimidine-5-carbonitrile (12a). 12a was
obtained from tert-butyl (5-(2-cyano-3-(dimethylamino)acryloyl)-4-
methylthiazol-2-yl)(methyl)carbamate and 1-(3-nitrophenyl)guanidine
hydrochloride. Yellow solid (39%); mp 304−305 °C. Anal. RP-HPLC:
1
tR 12.94 min (method A), 12.34 min (method C), purity 99%. H
NMR (DMSO-d6): δ 2.45 (s, 3H, CH3), 2.91 (d, 3H, J = 4.4 Hz,
CH3), 7.63 (t, 1H, J = 8.0 Hz, Ph-H), 7.91 (dt, 1H, J = 8.0, 1.6 Hz, Ph-
H), 8.06 (d, 1H, J = 7.6 Hz, Ph-H), 8.34 (q, 1H, J = 4.4 Hz, NH), 8.84
(s, 1H, Ph-H), 8.88 (s, 1H, Py-H), 10.68 (s, 1H, NH). 13C NMR
(DMSO-d6): δ 20.03, 31.39, 95.05, 114.44, 117.74, 117.99, 126.51,
130.35, 140.84, 148.49, 156.48, 159.11, 161.43, 164.19, 170.99. HR-
tert-Butyl 5-(1-Hydroxypropyl)-4-methylthiazol-2-yl-
1
(methyl)carbamate (9, R′ = Me). Light yellow liquid (63%). H
NMR (CDCl3): δ 0.94 (t, 3H, J = 7.2 Hz, CH3), 1.28−1.48 (m, 2H,
CH2), 1.58 (s, 9H, 3 × CH3), 1.67−1.94 (m, 3H, CH2 and OH), 2.30
651
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MS (ESI+): m/z [M + H]+ calcd for C16H14N7O2S, 368.0930, found,
368.0840.
no)-2-methyl-1-(4-methyl-2-(methylamino)thiazol-5-yl)prop-2-en-1-
one. Light orange solid (76%); mp 224−226 °C. Anal. RP-HPLC: tR
11.02 min (method A), 8.87 (method B), purity 99%. 1H NMR
(DMSO-d6): δ 2.20 (s, 3H, CH3), 2.24 (s, 3H, CH3), 2.86 (d, 1H, J =
4.8 Hz, CH3), 7.28 (s, 1H, NH2), 7.37 (dt, 1H, J = 8.0, 1.2 Hz, Ph-H),
7.45 (t, 1H, J = 8.0 Hz, Ph-H), 7.78 (q, 1H, J = 4.8 Hz, NH), 7.93−
8.00 (m, 1H, Ph-H), 8.30 (s, 1H, J = 2.0 Hz, Ph-H), 8.37 (s, 1H, Py-
H), 9.76 (s, 1H, NH). 13C NMR (DMSO-d6): δ 16.71, 18.19, 31.29,
114.68, 115.60, 118.36, 119.07, 121.56, 129.48, 141.75, 144.94, 150.52,
158.26, 158.94, 160.21, 169.57. HRMS (ESI+): m/z [M + H]+ calcd
for C16H19N6O2S2, 391.1011, found 391.1020.
4-(4-Methyl-2-(methylamino)thiazol-5-yl)-2-((3-
nitrophenyl)amino)pyrimidin-5-ol (12b). 12b was obtained from
2-chloro-3-(dimethylamino)-1-(4-methyl-2-(methylamino)thiazol-5-
yl)prop-2-en-1-one and 1-(3-nitrophenyl) guanidine. Yellow solid
(6%); mp 210−211 °C. Anal. RP-HPLC: tR 10.95 min (method A),
1
10.78 min (method C), purity 97%. H NMR (DMSO-d6): δ 2.32 (s,
3H, CH3), 2.86 (d, 3H, J = 4.8 Hz, CH3), 6.42 (s, 2H, NH and OH),
7.53 (s, 1H, Py-H), 7.71 (dt, 2H,, J = 8.0, 1.6 Hz, Ph-H), 7.75 (t, 1H, J
= 8.0 Hz, Ph-H), 8.12 (t, 1H, J = 2.0 Hz, Ph-H), 8.19 (q, 1H, J = 4.8
Hz, NH), 8.30−8.26 (m, 1H, Ph-H). 13C NMR (DMSO-d6): δ 18.89,
31.31, 116.43, 122.88, 123.22, 128.78, 130.97, 134.79, 137.80, 139.01,
148.64, 154.79, 157.72, 170.63, 172.04. HR-MS (ESI+): m/z [M + H]+
calcd for C15H15N6O3S, 359.0926, found, 359.0688.
3-((5-Ethyl-4-(4-methyl-2-(methylamino)thiazol-5-yl)-
pyrimidin-2 yl)amino)benzenesulfonamide (12h). 12h was
obtained from tert-butyl (5-(2-((dimethylamino)methylene)-
butanoyl)-4-methylthiazol-2-yl)(methyl)carbamate and 3-guanidino-
benzenesulfonamide. Light yellow powder (30%); mp 183−185 °C.
Anal. RP-HPLC: tR 11.32 min (method A), 9.12 min (method B),
3-(5-Cyano-4-(4-methyl-2-(methylamino)thiazol-5-yl)-
pyrimidin-2-ylamino)benzenesulfonamide (12c). 12c was ob-
tained from tert-butyl (5-(2-cyano-3-(dimethylamino)acryloyl)-4-
methylthiazol-2-yl)(methyl)carbamate and 3-guanidinobenzenesulfo-
namide. Gray solid (76%); mp 314−315 °C. Anal. RP-HPLC: tR 11.25
1
purity 100%. H NMR (DMSO-d6): δ 1.10 (t, 1H, J = 7.6 Hz, CH3),
2.20 (s, 3H, CH3), 2.59 (q, 2H, J = 7.6 Hz, CH2), 2.85 (d, 1H, J = 4.8
Hz, CH3), 7.28 (s, 1H, NH2), 7.38 (dt, 1H, J = 8.0, 1.6 Hz, Ph-H),
7.45 (t, 1H, J = 8.0 Hz, Ph-H), 7.74 (q, 1H,, J = 4.8 Hz, NH), 7.93−
8.00 (m, 1H, Ph-H), 8.31 (t, 1H, J = 1.6 Hz, Ph-H), 8.43 (s, 1H, Py-
H), 9.82(s, 1H, NH). 13C NMR (DMSO-d6): δ 15.46, 17.72, 22.93,
31.28, 113.99, 115.61, 118.40, 121.54, 125.64, 129.49, 141.70, 144.95,
149.80, 158.26, 158.46, 159.40, 169.20. HR-MS (ESI+): m/z [M + H]+
calcd for C17H21N6O2S2, 405.1167, found 405.0863.
3-((4-(4-Methyl-2-(methylamino)thiazol-5-yl)-5-propylpyri-
midin-2-yl)amino)benzenesulfonamide (12i). 12i was obtained
from tert-butyl (5-(2-((dimethylamino)methylene)pentanoyl)-4-meth-
ylthiazol-2-yl)(methyl)carbamate and 3-guanidinobenzenesulfona-
mide. Yellow solid (30%); mp 160−162 °C. Anal. RP-HPLC: tR
11.52 min (method A), 11.57 min (method C), purity 100%. 1H NMR
(DMSO-d6): δ 0.85 (t, 1H, J = 7.6 Hz, CH3), 1.42−1.54 (m, 2H,
CH2), 2.21 (s, 3H, CH3), 2.55 (t, 2H, J = 7.6 Hz, CH2), 2.85 (d, 1H, J
= 4.8 Hz, CH3), 7.28 (s, 1H, NH2), 7.37 (dt, 1H, J = 8.0, 1.6 Hz, Ph-
H), 7.45(t, 1H, J = 8.0 Hz, Ph-H), 7.92−8.00 (m, 1H, Ph-H), 8.30 (t,
1H, J = 1.6 Hz, Ph-H), 8.40 (s, 1H, Py-H), 9.81(s, lH, NH). 13C NMR
(DMSO-d6): δ 14.11, 17.71, 23.80, 31.26, 31.64, 114.08, 115.59,
118.40, 121.55, 124.04, 129.49, 141.68, 144.94, 149.80, 158.29, 158.69,
159.76, 169.15. HR-MS (ESI+): m/z [M + H]+ calcd for
C18H23N6O2S2, 419.1324, found 419.0733.
1
min (method A), 11.25 (method C), purity 99%. H NMR (DMSO-
d6): δ 2.45 (s, 3H, CH3), 2.90 (d, 3H, J = 4.8 Hz, CH3), 7.36 (s, 2H,
NH2), 7.50−7.57 (m, 2H, 2 × Ph-H), 7.88−7.95 (m, 1H, Ph-H), 8.25
(s, 1H, Ph-H), 8.29 (q, 1H, J = 4.8 Hz, NH), 8.82 (s, 1H, Py-H), 10.49
(s, 1H, NH). 13C NMR (DMSO-d6): δ 20.07, 31.37, 94.51, 117.71,
118.16, 120.62, 123.80, 129.73, 139.85, 145.06, 156.63, 159.30, 161.44,
164.17, 170.89. HR-MS (ESI+): m/z [M + H]+ calcd for
C16H16N7O2S2, 402.0870, found, 402.0970.
3-((5-Hydroxy-4-(4-methyl-2-(methylamino)thiazol-5-yl)-
pyrimidin-2-yl)amino)benzen esulfonamide (12d). 12d was
obtained from 2-chloro-3-(dimethylamino)-1-(4-methyl-2-
(methylamino)thiazol-5-yl)prop-2-en-1-one and 3-guanidinobenzene-
sulfonamide. Gray solid (6%); mp 254−256 °C. Anal. RP-HPLC: tR
1
10.12 min (method A), 10.10 (method C), purity 100%. H NMR
(DMSO-d6): δ 2.32 (s, 3H, CH3), 2.86 (d, 3H, J = 4.8 Hz, CH3), 6.26
(s, 2H, NH & OH), 7.41−7.52 (m, 4H, Ph-H and Py-H and NH2),
7.63 (t, 1H, J = 1.6 Hz, Ph-H), 7.67 (t, 1H, J = 8.0 Hz, Ph-H), 7.84 (dt,
1H, J = 8.0, 1.6 Hz, Ph-H), 8.19 (q, 1H, J = 4.8 Hz, NH). 13C NMR
(DMSO-d6): δ 18.88, 31.31, 116.64, 124.25, 125.43, 128.92, 130.51,
131.17, 137.11, 138.85, 145.31, 154.70, 157.60, 170.61, 172.07. HR-
MS (ESI+): m/z [M + H]+ calcd for C15H17N6O3S2, 393.0804, found,
393.0883.
3-(5-Fluoro-4-(4-methyl-2-(methylamino)thiazol-5-yl)-
pyrimidin-2-ylamino)benzenesulfonamide (12e). 12e was ob-
tained from 3-guanidinobenzenesulfonamide and 3-(dimethylamino)-
2-fluoro-1-(4-methyl-2-(methylamino)thiazol-5-yl)prop-2-en-1-one.
Yellow solid (24%); mp 268−270 °C. Anal. RP-HPLC: tR 11.45 min
(method A), 9.12 min (method B), purity 99%. 1H NMR (DMSO-d6):
δ 2.48 (s, 3H, CH3), 2.88 (d, 3H, J = 4.8 Hz, CH3), 7.29 (s, 2H, NH2),
7.40 (d, 1H, J = 8.0 Hz, Ph-H), 7.47 (t, 1H, J = 8.0 Hz, Ph-H), 7.89 (d,
1H, J = 8.0 Hz, Ph-H), 8.13 (brs q, 1H, J = 4.8 Hz, NH), 8.25 (s, 1H,
Ph-H), 8.47 (d, 1H, J = 3.2 Hz, Py-H), 9.83 (s, 1H, NH). 13C NMR
(DMSO-d6): δ 19.43 (d, J = 5 Hz), 31.33, 109.97 (d, J = 8 Hz),
115.81, 118.78, 121.89, 129.51, 141.46, 144.94, 145.97 (d, J = 25 Hz),
147.63 (d, J = 12 Hz), 147.94 (d, J = 248 Hz), 155.66, 156.04, 171.34.
HR-MS (ESI+): m/z [M + H]+ calcd for C15H16FN6O2S2, 395.0760,
found 395.0641.
2-(3-(4-Acetylpiperazin-1-yl)phenylamino)-4-(4-methyl-2-
(methylamino)thiazol-5-yl)pyrimidine-5-carbonitrile (12j). 12j
was obtained from 1-(3-acetylpiperazin-1-yl)phenyl)guanidine and
tert-butyl (5-(2-cyano-3-(dimethylamino)acryloyl)-4-methylthiazol-2-
yl)(methyl)carbamate. Yellow solid (58%); mp 241−243 °C. Anal.
RP-HPLC: tR 12.10 min (method A), 9.02 min (method B), purity
1
100%. H NMR (DMSO-d6): δ 2.05 (s, 3H, CH3), 2.40 (brs, 3H,
CH3), 2.89 (d, 3H, J = 4.4 Hz, CH3), 3.10 (apparent t, 2H, J = 5.2 Hz,
CH2), 3.17 (apparent t, 2H, J = 5.2 Hz, CH2), 3.54−3.62 (m, 4H, 2 ×
CH2), 6.62−6.73 (m, 1H, Ph-H), 7.12−7.24 (m, 2H, 2 × Ph-H), 7.40
(br s, 1H, Ph-H), 8.25 (brq, 1H, J = 4.8 Hz, NH), 8.77 (s, 1H, Py-H),
10.12 (brs, 1H, NH). 13C NMR (DMSO-d6): δ 19.93, 21.65, 31.35,
41.12, 45.90, 48.87, 49.31, 94.05, 108.65, 111.62, 112.38, 118.28,
129.47, 140.09, 151.66, 159.46, 161.56, 163.89, 168.80, 170.68. HR-
MS (ESI+): m/z [M + H]+ calcd for C22H25N8OS, 449.1872, found
449.1727.
4-(4-Methyl-2-(methylamino)thiazol-5-yl)-2-(3-(piperazin-1-
yl)phenylamino)pyrimidine-5-carbonitrile (12k). A mixture of
12j in methanol (5 mL) and 2 M HCl (4 mL) was heated under reflux
for 3 h. After completion of the reaction, the mixture was neutralized
by NaOH solution, extracted with chloroform, and purified by column
chromatography using chloroform/MeOH as the eluant to get the
final product. Yellow solid (57%); mp 160−162 °C. Anal. RP-HPLC:
tR 11.09 min (method A), 8.65 min (method B), purity 98%. 1H NMR
(DMSO-d6): δ 2.39 (s, 3H, CH3), 2.83 (apparent t, 4H, J = 4.4 Hz, 2
× CH2), 2.89 (s, 3H, CH3), 3.04 (apparent t, 2H, J = 4.4 Hz, 2 ×
CH2), 6.55−7.70 (t, 1H, J = 2.0 Hz, Ph-H), 7.02−7.22 (m, 2H, 2 ×
Ph-H), 7.36 (br s, 1H, Ph-H), 8.25 (br s, 1H, NH), 8.77 (s, 1H, Py-H),
10.08 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 19.92, 31.35, 46.08,
3-((5-Chloro-4-(4-methyl-2-(methylamino)thiazol-5-yl)-
pyrimidin-2-yl)amino)benzenesulfonamide (12f). 12f was ob-
tained from 3-guanidinobenzenesulfonamide and 2-chloro-4-methyl-1-
(4-methyl-2-(methylamino)thiazol-5-yl)pent-2-en-1-one. Yellow solid
(10%); mp 165 °C (dec). Anal. RP-HPLC: tR 11.45 min (method A),
1
11.40 min (method C), purity 98%. H NMR (MeOH-d4): δ 2.44 (s,
3H, CH3), 2.99 (s, 3H, CH3), 7.46 (t, 1H, J = 8.0 Hz, Ph-H), 7.52−
7.57 (m, 1H, Ph-H), 7.81−7.87 (m, 1H, Ph-H), 8.41 (t, 1H, J = 1.2
Hz, Ph-H), 8.42 (s, 1H, Py-H). HRMS (ESI+): m/z [M + H]+ calcd
for C15H16ClN6O2S2, 411.0465; found 411.0530.
3-((5-Methyl-4-(4-methyl-2-(methylamino)thiazol-5-yl)-
pyrimidin-2-yl)amino)benzenesulfonamide (12g). 12g was
obtained from 3-guanidinobenzenesulfonamide and 3-(dimethylami-
652
dx.doi.org/10.1021/jm301475f | J. Med. Chem. 2013, 56, 640−659

Journal of Medicinal Chemistry
Article
50.01, 93.99, 108.06, 111.10, 111.71, 118.30, 129.32, 140.01, 152.56,
159.46, 161.58, 163.85, 170.66. HR-MS (ESI+): m/z [M + H]+ calcd
for C20H23N8S, 407.1766, found 407.1874.
Hz, CH3), 2.81−2.90 (m, 7H, 2 × CH2 and CH3), 3.04 (apparent t,
4H, J = 4.8 Hz, 2 × CH2), 6.53 (dd, 1H, J = 8.0, 1.6 Hz, Ph-H), 7.09
(t, 1H, J = 8.0 Hz, Ph-H), 7.17 (d, 1H, J = 8.0 Hz, Ph-H), 7.33 (s, 1H,
Ph-H), 8.10 (br q, 1H, J = 4.8 Hz, NH), 8.43 (d, 1H, J = 3.6 Hz, Py-
H), 9.32(s, 1H, NH). 13C NMR (DMSO-d6): δ 19.20 (d, J = 6 Hz),
31.31, 45.92, 49.91, 106.52, 109.54, 110.31, 110.68 (d, J = 8 Hz),
129.18, 141.65, 146.24 (d, J = 25 Hz), 147.22 (d, J = 12 Hz), 146.67
(d, J = 247 Hz), 152.49, 154.54, 156.50, 170.99. HR-MS (ESI+): m/z
[M + H]+ calcd for C19H23FN7S, 400.1720, found 400.1722.
5-(5-Fluoro-2-((3-morpholinophenyl)amino)pyrimidin-4-yl)-
N,4-dimethylthiazol-2-amine 12q. 12q was obtained from 3-
(dimethylamino)-2-fluoro-1-(4-methyl-2-(methylamino)thiazol-5-yl)-
prop-2-en-1-one and 1-(3-morpholinophenyl)guanidine. Light pink
solid (28%); mp 225−227 °C. Anal. RP-HPLC: tR 11.89 min (method
A), 11.32 min (method B), purity 98%. 1H NMR (DMSO-d6): δ 2.44
(d, 3H, J = 3.2 Hz, CH3), 2.88 (d, 3H, J = 4.8 Hz, CH3), 3.09
(apparent t, 4H, J = 4.8 Hz, 2 × CH2), 3.75 (apparent t, 4H, J = 4.8
Hz, 2 × CH2), 6.55 (dd, 1H, J = 8.0, 2.0 Hz, Ph-H), 7.12 (t, 1H, J =
8.0 Hz, Ph-H), 7.20 (dd, 1H, J = 8.0, 0.8 Hz, Ph-H), 7.36 (t, 1H, J =
2.0 Hz, Ph-H), 8.10 (q, 1H, J = 4.8 Hz, NH), 8.43 (d, 1H, J = 3.6 Hz,
Py-H), 9.36 (s, 1H, NH). 13C NMR (DMSO-d6): δ 19.21 (d, J = 6
Hz), 31.29, 49.23, 66.59, 106.22, 109.23, 110.68, 110.73, 129.26,
141.73, 146.23 (d, J = 25 Hz), 147.24 (d, J = 11 Hz), 147.69 (d, J =
248 Hz), 151.96, 154.57, 156.49 (d, J = 2 Hz), 170.99 (d, J = 4 Hz).
HR-MS (ESI+): m/z [M + H]+ calcd for C19H22FN6OS, 401.1560,
found 401.1647.
2-(4-(4-Acetylpiperazin-1-yl)phenylamino)-4-(4-methyl-2-
(methylamino)thiazol-5-yl)pyrimidine-5-carbonitrile (12l). 12l
was obtained from 1-(4-acetylpiperazin-1-yl)phenyl)guanidine and
tert-butyl (5-(2-cyano-3-(dimethylamino)acryloyl)-4-methylthiazol-2-
yl)(methyl)carbamate. Yellow solid (23%); mp 226−228 °C. Anal.
RP-HPLC: tR 11.69 min (method A), 8.67 min (method B), purity
1
98%. H NMR (DMSO-d6): δ 2.05 (s, 3H, CH3), 2.38 (br s, 3H,
CH3), 2.89 (d, 3H, J = 4.8 Hz), 3.05 (apparent t, 2H, J = 4.8 Hz, CH2),
3.12 (apparent t, 2H, J = 4.8 Hz, CH2), 3.51−3.63 (m, 4H, 2 × CH2),
6.95 (d, 2H, J = 9.2 Hz, 2 × Ph-H), 7.55 (d, 2H, J = 8.8 Hz, 2 × Ph-
H), 8.21 (br q, 1H, J = 4.8 Hz, NH), 8.71 (s, 1H, Py-H), 10.08 (br s,
1H, NH). 13C NMR (DMSO-d6): δ 19.91, 21.65, 31.29, 41.14, 45.95,
49.21, 49.63, 93.28, 116.57, 118.48, 122.40, 124.24, 131.47, 147.63,
155.52, 159.44, 161.47, 163.91, 168.74, 170.49. HR-MS (ESI+): m/z
[M + H]+ calcd for C22H25N8OS, 449.1872, found 449.1940.
4-(4-Methyl-2-(methylamino)thiazol-5-yl)-2-(4-(piperazin-1-
yl)phenylamino)pyrimidine-5-carbonitrile (12m). 12m was
obtained from 2-(4-(4-acetylpiperazin-1-yl)phenylamino)-4-(4-meth-
yl-2-(methylamino)thiazol-5-yl)pyrimidine-5-carbonitrile. Yellow solid
(80%); mp 192−194 °C. Anal. RP-HPLC: tR 10.92 min (method A),
8.35 min (method B), purity 96%. 1H NMR (DMSO-d6) δ: 2.39
(s.3H, CH3), 2.88 (d, 3H, J = 4.8 Hz), 3.08−3.17 (m, 4H, 2 × CH2),
3.20−3.28 (m, 4H, 2 × CH2), 6.96 (d, 2H, J = 8.8 Hz, 2 × Ph-H), 7.56
(d, 2H, J = 8.8 Hz, 2 × Ph-H), 8.24 (q, 1H, J = 4.8 Hz, NH), 8.71 (s,
1H, Py-H), 10.09 (bs, 1H, NH). 13C NMR (DMSO-d6): δ 19.92,
31.28, 43.52, 46.96, 93.28, 114.50, 116.63, 118.46, 122.40, 131.86,
147.02, 155.53, 159.40, 161.47, 163.79, 170.48. HR-MS (ESI+): m/z
[M + H]+ calcd for C20H23N8S, 407.1766, found 407.1814.
5-(5-Chloro-2-((3-(piperazin-1-yl)phenyl)amino)pyrimidin-4-
yl)-N,4-dimethylthiazol-2-amine 12r. 12r was obtained from 1-(4-
(3-((5-chloro-4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-
yl)amino)phenyl)piperazin-1-yl)ethanone. Yellow solid (70%); mp
108−110 °C. Anal. RP-HPLC: tR 11.39 min (method A), 8.75 min
1
4-(4-Methyl-2-(methylamino)thiazol-5-yl)-2-((3-(piperidin-1-
yl)phenyl)amino)pyrimidine-5-carbonitrile (12n). 12n was ob-
tained from 1-(3-piperidine-1-yl)phenyl)guanidine and tert-butyl (5-
(2-cyano-3-(dimethylamino)acryloyl)-4-methylthiazol-2-yl)(methyl)-
carbamate. Pale yellow solid (20%); mp 254−256 °C. Anal. RP-
HPLC: tR 11.09 min (method A), 10.89 min (method C), purity
(method B), purity 99%. H NMR (DMSO-d6): δ 2.33 (s, 3H, CH3),
2.83 (apparent t, 4H, J = 4.8 Hz, 2 × CH2), 2.86 (d, 3H, J = 4.8 Hz,
CH3), 3.02 (apparent t, 4H, J = 5.2 Hz, 2 × CH2), 6.55 (dd, 1H, J =
8.0, 1.2 Hz, Ph-H), 7.10 (t, 1H, J = 8.0 Hz, Ph-H), 7.16 (d, 1H, J = 8.4
Hz, Ph-H), 7.36 (s, 1H, Ph-H), 7.97 (q, 1H, J = 4.8 Hz, NH), 8.47 (s,
1H, Py-H), 9.52 (s, 1H, NH). 13C NMR (DMSO-d6): δ 19.26, 31.30,
46.10, 50.09, 106.96, 109.83, 110.60, 112.65, 116.27, 129.20, 141.17,
152.56, 153.47, 156.64, 158.14, 158.47, 170.21. HR-MS (ESI+): m/z
[M + H]+ calcd for C19H23ClN7S, 416.1624, found 416.1615.
1-(4-(3-((5-Chloro-4-(4-methyl-2-(methylamino)thiazol-5-yl)-
pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)ethanone. The
compound was obtained from 2-chloro-3-(dimethylamino)-1-(4-
methyl-2-(methylamino)thiazol-5-yl)prop-2-en-1-one and 1-(3-acetyl-
piperazin-1-yl)phenyl)guanidine. Yellow solid (42%); mp 193−195
°C. Anal. RP-HPLC: tR 11.42 min (method C), purity 99%. 1H NMR
(DMSO-d6): δ 2.05 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.87 (d, 3H, J =
4.4 Hz, CH3), 3.08 (apparent t, 2H, J = 4.8 Hz, CH2), 3.14 (apparent t,
2H, J = 4.8 Hz, CH2), 3.53−3.62 (m, 4H, 2 × CH2), 6.59 (dd, 1H, J =
8.0, 1.6 Hz, Ph-H), 7.13 (t, 1H, J = 8.0 Hz, Ph-H), 7.16 (dd, 1H, J =
8.0, 1.2 Hz, Ph-H), 7.40 (t, 1H, J = 2.0 Hz, Ph-H), 7.98 (q, 1H, J = 4.8
Hz, NH), 8.48 (s, 1H, Py-H), 9.56 (s, 1H, NH). 13C NMR (DMSO-
d6): δ 19.30, 21.67, 31.29, 41.14, 45.92, 48.98, 49.39, 107.48, 110.32,
111.22, 112.66, 116.34, 129.35, 141.28, 151.66, 153.51, 156.64, 158.11,
158.52, 168.73, 170.20. HR-MS (ESI+): m/z [M + H]+ calcd for
C21H25ClN7OS, 458.1580, found 458.1157.
1
100%. H NMR (DMSO-d6): δ 1.48−1.58 (m, 2H, CH2), 1.58−1.67
(m, 4H, 2 × CH2), 2.40 (br s, 3H, CH3), 2.89 (d, 3H, J = 4.8 Hz,
CH3), 3.14 (apparent t, 4H, J = 4.8 Hz, 2 × CH2), 6.10−6.20 (m, 1H,
Ph-H), 7.19−7.24 (m, 2H, 2 × Ph-H), 7.37 (br s, 1H, Ph-H), 8.24 (br
q, 1H, J = 4.8 Hz, NH), 8.77 (s, 1H, Py-H), 10.07 (s, 1H, NH). 13C
NMR (DMSO-d6): δ 19.90, 24.41, 25.69, 31.36, 50.12, 93.97, 108.55,
111.44, 111.54, 118.30, 129.31, 139.99, 152.47, 155.25, 159.45, 161.57,
163.82, 170.67. HR-MS (ESI+): m/z [M + H]+ calcd for C21H24N7S,
406.1688, found 406.1871.
1-(4-(3-(5-Fluoro-4-(4-methyl-2-(methylamino)thiazol-5-yl)-
pyrimidin-2-ylamino)phenyl)piperazin-1-yl)ethanone (12o).
12o was obtained from 1-(3-acetylpiperazin-1-yl)phenyl)guanidine
and 3-(dimethylamino)-2-fluoro-1-(4-methyl-2-(methylamino)thiazol-
5-yl)prop-2-en-1-one. Yellow solid (15%); mp 184−186 °C. Anal. RP-
HPLC: tR 11.87 min (method A), 8.90 min (method B), purity 100%.
1H NMR (DMSO-d6): δ 2.05 (s, 3H, CH3), 2.44 (d, 3H, J = 2.8 Hz,
CH3), 2.87 (d, 3H, J = 4.8 Hz, CH3), 3.08 (apparent t, 2H, J = 5.2 Hz,
CH2), 3.15 (apparent t, 2H, J = 5.2 Hz, CH2), 3.53−3.62 (m, 4H, 2 ×
CH2), 6.57 (dd, 1H, J = 8.0, 1.6 Hz, Ph-H), 7.12 (t, 1H, J = 8.0 Hz, Ph-
H), 7.21 (d, 1H, J = 8.0 Hz, Ph-H), 7.38 (s, 1H, Ph-H), 8.10 (q, 1H, J
= 4.4 Hz, NH), 8.43 (d, 1H, J = 3.6 Hz, Py-H), 9.36 (s, 1H, NH). 13C
NMR (DMSO-d6): δ 19.21 (d, J = 6 Hz), 21.65, 31.31, 41.17, 45.96,
49.04, 49.48, 107.01, 109.98, 110.71 (d, J = 8 Hz), 110.87, 129.30,
141.74, 146.23 (d, J = 25 Hz), 147.23 (d, J = 12 Hz), 147.69 (d, J =
248 Hz), 151.70, 154.59, 156.47 (d, J = 2 Hz), 168.77, 171.03 (d, J = 3
Hz). HR-MS (ESI+): m/z [M + H]+ calcd for C21H25FN7OS,
442.1825, found 442.1917.
5-(2-((5-(1,4-Diazepan-1-yl)-2-fluorophenyl)amino)-
pyrimidin-4-yl)-N,4-dimethylthiazol-2-amine 12s. 12s was ob-
tained from 1-(4-(3-((5-fluoro-4-(4-methyl-2-(methylamino)thiazol-5-
yl)pyrimidin-2-yl)amino)phenyl)-1,4-diazepan-1-yl)ethanone. Yellow
solid (37%); mp 180−182 °C. Anal. RP-HPLC: tR 11.19 min (method
1
A), 10.98 min (method C), purity 100%. H NMR (MeOH-d4): δ
1.95−2.04 (m, 2H, CH2), 2.48 (d, 3H, J = 2.8 Hz, CH3), 2.88
(apparent t, 2H, J = 5.2 Hz, CH2), 2.97 (s, 3H, CH3), 3.07 (apparent t,
2H, J = 5.2 Hz, CH2), 3.58−3.67 (m, 4H, 2 × CH2), 6.43 (dd, J = 8.0,
2.0 Hz, Ph-H), 6.93 (dd, J = 8.0, 1.6 Hz, Ph-H), 7.10 (t, 1H, J = 8.0
Hz, Ph-H), 7.15 (t, 1H, J = 2.0 Hz, Ph-H), 8.24 (d, 1H, J = 3.6 Hz, Py-
H). 13C NMR (DMSO-d6): δ 19.16 (d, J = 6 Hz), 29.39, 31.29, 47.72,
47.85, 48.43, 52.40, 102.42, 105.66, 106.82, 110.75, 129.46, 141.96,
146.14 (d, J = 26 Hz), 147.27 (d, J = 12 Hz), 147.65 (d, J = 248 Hz),
5-(5-Fluoro-2-(3-(piperazin-1-yl)phenylamino)pyrimidin-4-
yl)-N,4-dimethylthiazol-2-amine 12p. 12p was obtained from 1-
(4-(3-(5-fluoro-4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidin-2-
ylamino)phenyl)piperazin-1-yl)ethanone. Yellow solid (48%); mp
134−136 °C. Anal. RP-HPLC: tR 11.23 min (method A), 8.62 min
(method B), purity 99%. 1H NMR (DMSO-d6): δ 2.44 (d, 3H, J = 3.2
653
dx.doi.org/10.1021/jm301475f | J. Med. Chem. 2013, 56, 640−659

Journal of Medicinal Chemistry
Article
149.08, 154.40, 156.64, 170.98. HR-MS (ESI+): m/z [M + H]+ calcd
for C20H25FN7S [M + H]+, 414.1876, found, 414.1866.
CH3), 3.46 (s, 3H, CH3), 4.19 (q, 2H, J = 11.2 Hz, CH2), 5.31 (d, 1H,
J = 12.0 Hz, CH), 7.71 (d, 1H, J = 12.0 Hz, CH). 13C NMR (CDCl3):
δ 28.16, 34.01, 34.78 (q, J = 30 Hz), 37.45, 45.12, 83.66, 95.10, 125.60
(q, J = 273 Hz), 129.99, 143.94 (q, J = 3 Hz), 153.38, 153.72, 160.06,
180.77. HR-MS (ESI+): m/z [M + H]+ calcd for C16H23F3N3O3S,
394.1412, found 394.1359.
2-(3-(4-Acetyl-1,4-diazepan-1-yl)phenylamino)-4-(4-methyl-
2-(methylamino)thiazol-5-yl)pyrimidine-5-carbonitrile (12t).
12t was obtained from tert-butyl (5-(2-cyano-3-(dimethylamino)-
acryloyl)-4-methylthiazol-2-yl)(methyl)carbamate and 1-(3-(4-acetyl-
1,4-diazepan-1-yl)phenyl)guanidine. Yellow solid (30%); mp 129−131
°C. Anal. RP-HPLC: tR 12.15 min (method A), 11.55 (method C),
purity 100%. 1H NMR (DMSO-d6): δ 1.76−1.92 (m, 3.6 H, CH2 and
CH3), 1.92 (s, 1.4 H, CH3), 2.39 (s, 3H, CH3), 2.89 (d, 3 H, J = 4.0
Hz, CH3), 3.28−3.34 (m, 2 H, CH2), 3.46−3.59 (m, 6 H, 3 × CH2),
6.48 (d, 1 H, J = 8.0 Hz, Ph-H), 7.02−7.18 (m, 3 H, 3 × Ph-H), 8.23
(q, 1H, J = 4.8 Hz, NH), 8.76 (d, 1 H, J = 0.8 Hz Py-H), 10.01 (s, 1 H,
NH). 13C NMR (DMSO-d6, (∗) minor rotamer): δ 19.82, 19.87*,
21.40, 21.73*, 24.42, 26.31*, 31.31, 44.33, 44.83*, 47.27, 47.35*,
47.59, 48.70*, 49.41, 50.04*, 94.00, 94.08*, 104.30, 107.50, 108.92,
118.29, 129.78, 140.42, 147.74, 148.06*, 159.49, 161.58, 161.65*,
163.78, 163.82*, 169.47, 169.71*, 170.56, 170.60*. HR-MS (ESI+): m/
z [M + H]+ calcd for C23H27N8OS, 463.2029, found, 463.1791.
2-(3-(1,4-Diazepan-1-yl)phenylamino)-4-(4-methyl-2-
(methylamino)thiazol-5-yl)pyrimidine-5-carbonitrile (12u). 12u
was obtained from 2-(3-(4-acetyl-1,4-diazepan-1-yl)phenylamino)-4-
(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidine-5-carbonitrile. Yel-
low solid (34%); mp 210−212 °C. Anal. RP-HPLC: tR 11.19 min
(method A), 10.94 min (method C), purity 100%. 1H NMR (DMSO-
d6): δ 1.79−1.92 (m, 2H, CH2), 2.40 (s, 3H, CH3), 2.77 (apparent t,
2H, J = 5.2 Hz, CH2), 2.89 (d, 3H, J = 4.0 Hz, CH3), 2.97 (apparent t,
4H, J = 4.8 Hz, CH2), 3.53 (apparent t, 4H, J = 5.2 Hz, 2 × CH2), 6.45
(dd, 1H, J = 8.0, 2.0 Hz, Ph-H), 6.99−7.15 (m, 3H, 3 × Ph-H), 8.24
(br q, 1H, J = 5.2 Hz, NH), 8.76 (s, 1H, Py-H), 10.01 (s, 1H, NH).
13C NMR (DMSO-d6): δ 19.87, 28.10, 31.31, 47.06, 47.65, 50.51,
t e r t - B u t y l ( 5 - ( 3 - ( D i m e t h y l a m i n o ) a c r y l o y l ) - 4 -
(trifluoromethyl)thiazol-2-yl)(methyl)car bamate (15, R1
=
Boc, R′ = CF3). 15 (R1 = Boc, R′ = CF3) was obtained from tert-
butyl (5-acetyl-4-(trifluoromethyl)thiazol-2-yl)(methyl)carbamate and
DMF−DMA. Orange solid (77%); mp 116−118 °C. 1H NMR
(CDCl3): δ 1.61 (s, 9H, 3 × CH3), 2.91 (s, 3H, CH3), 3.16 (s, 3H,
CH3), 3.57 (s, 3H, CH3), 5.44 (d, 2H, J = 12.4 Hz, CH), 7.70 (d, 1H, J
= 12.4 Hz, CH). HR-MS (ESI+): m/z [M + H]+ calcd for
C15H21F3N3O3S, 380.1256, found 380.1038.
tert-Butyl (5-(3-(Dimethylamino)acryloyl)thiazol-2-yl)-
(methyl)carbamate (15, R1 = Boc, R′ = H). 15 (R1 = Boc, R′ =
H) was obtained from tert-butyl (5-acetylthiazol-2-yl)(methyl)-
carbamate and DMF−DMA. Yellow solid (92%); mp 170−172 °C.
1H NMR (CDCl3): δ 1.61 (s, 9H, 3 × CH3), 2.92 (brs, 3H, CH3), 3.14
(brs, 3H, CH3), 3.57 (s, 3H, CH3), 5.48 (d, 2H, J = 12.4 Hz, CH),
7.77 (d, 1H, J = 12.4 Hz, CH), 7.95 (s, 1H, CH). 13C NMR (CDCl3):
δ 28.18, 34.04, 37.45, 44.99, 83.76, 92.58, 135.91, 139.87, 152.94,
153.16, 164.20, 180.55. HR-MS (ESI+): m/z [M + H]+ calcd for
C14H22N3O3S, 312.1382, found 312.1168.
tert-Butyl (4-Cyclopropyl-5-(3-(dimethylamino)acryloyl)-
thiazol-yl)(methyl)carbamate (15, R1 = Boc, R′ = cycloprop).
15 (R1 = Boc, R′ = cycloprop) was obtained from tert-butyl (5-acetyl-
4-cyclopropylthiazol-2-yl)(methyl)carbamate and DMF−DMA. Or-
1
ange solid (61%); mp 151−153 °C. H NMR (CDCl3): δ 0.89−0.97
(m, 4H, 2 × CH2), 1.52 (s, 9H, 3 × CH3), 2.86 (bs, 3H, CH3), 2.94−
3.02 (m, 1H, CH), 3.13 (br s, 3H, CH3), 3.35 (s, 3H, CH3), 5.35 (d,
1H, J = 12.0 Hz, CH), 7.64 (d, 1H, J = 12.4 Hz. CH). 13C NMR
(CDCl3): δ 9.30, 11.93, 28.19, 33.77, 37.35, 45.00, 83.31, 95.77,
126.02, 152.85, 153.27, 157.99, 160.12, 182.16. HRMS (ESI+): m/z
[M + H]+ calcd for C17H26N3O3S, 352.1695, found 352.1647.
93.97, 104.21, 107.41, 108.70, 115.00, 118.31, 129.65, 140.35, 148.98,
154.98, 159.50, 161.61, 163.80, 170.57. HR-MS calcd for C21H25N8S,
419.1878, found, 419.1862.
1-(4-(Bromomethyl)-2-(methylamino)thiazol-5-yl)ethanone
(13). To an ice-cooled solution of 1-(4-methyl-2-(methylamino)-
thiazol-5-yl)ethanone (3.4 g, 20.0 mmol) in 50 mL of CHCl3 was
added N-bromosuccinimide (3.5 g, 20.0 mmol). After being stirred at
room temperature for 3 h, the mixture was washed with saturated
aqueous NaHCO3, and the organic layer was dried over MgSO4,
filtered, and concentrated. The precipitates were collected and washed
with MeOH to afford the title compound as a white solid (2.76 g,
tert-Butyl (5-(3-(Dimethylamino)acryloyl)-4-phenylthiazol-2-
yl)(methyl)carbamate (15, R1 = Boc, R′ = Ph). 15 (R1 = Boc, R′ =
Ph) was obtained from tert-butyl (5-acetyl-4-phenylthiazol-2-yl)-
(methyl)carbamate and DMF−DMA. Yellow solid (66%); mp 166−
1
167 °C. H NMR (CDCl3): δ 1.61 (s, 9H, 3 × CH3), 2.53 (br s, 3H,
CH3), 3.02 (br s, 3H, CH3), 3.58 (s, 3H, CH3), 5.11 (d, 1H, J = 12.4
Hz, CH), 7.32−7.43 (m, 3H, 3 × Ph-H), 7.58 (d, 1H, J = 12.4 Hz,
CH), 7.64−7.72 (m, 2H, 2 × Ph-H). 13C NMR (CDCl3): δ 28.22,
33.92, 36.88, 44.89, 83.62, 95.71, 127.91, 128.34, 129.86, 131.15,
136.16, 150.34, 152.91, 153.10, 161.23, 182.16. HR-MS (ESI+): m/z
[M + H]+ calcd for C20H26N3O3S [M + H]+, 388.1695, found,
388.1595. tert-Butyl (5-acetyl-4-phenylthiazol-2-yl)(methyl)carbamate
was obtained from 1-(2-(methylamino)-4-phenylthiazol-5-yl)ethanone
and di-tert-butyl dicarbonate. Colorless liquid (81%). 1H NMR
(CDCl3): δ 1.62 (s, 9H, 3 × CH3), 2.20 (s, 3H, CH3), 3.59 (s, 3H,
CH3), 7.43−7.50 (m, 3H, 3 × Ph-H), 7.56−7.65 (m, 3H, 2 × Ph-H).
HRMS (ESI+): m/z [M + H]+ calcd for C17H21N2O3S [M + H]+,
333.1273, found, 333.1247.
1
56%), mp 149 °C (dec). H NMR (DMSO-d6): δ 2.41 (s, 3H, CH3),
2.87 (d, 3H, J = 4.8 Hz, CH3), 4.74 (s, 2H, CH2), 8.57 (d, H, J = 4.4
Hz, NH). HR-MS (ESI+): m/z [M + H]+ calcd for C7H10BrN2OS,
250.9677, found 250.9607. tert-Butyl 5-acetyl-4-(bromomethyl)thiazol-
2-yl(methyl)carbamate was obtained by reacting 13 and di-tert-butyl
dicarbonate as white solid (98%), mp 84−86 °C. 1H NMR (CDCl3): δ
1.61 (s, 9H, 3 × CH3), 2.54 (s, 3H, CH3), 3.60 (s, 3H, CH3), 5.34 (s,
2H, CH2). HR-MS (ESI+): m/z [M + H]+ calcd for C12H18Br N2O3S,
351.0210, found 351.0338.
tert-Butyl 5-Acetyl-4-(2,2,2-trifluoroethyl)thiazol-2-yl-
(methyl)carbamate (14). A solution of tert-butyl 5-acetyl-4-
(bromomethyl)thiazol-2-yl(methyl)carbamate (8.45 g, 24.0 mmol) in
DMF was treated with methyl 2,2-difluoro-2-(fluorosulfonyl)acetate
(5.7 mL, 45.0 mmol) and CuI (2.9 g, 15.0 mmol). The mixture was
heated at 80 °C for 12 h. After completion of the reaction, the mixture
was purified by chromatography using EtoAc/PE to afford the title
1-(2-(Methylamino)-4-(trifluoromethyl)thiazol-5-yl)-
ethanone (16). To a solution of 1,1,1-trifluoropentane-2,4-dione (1.0
g, 6.4 mmol) in 15 mL of acetonitrile was added hydroxy(tosyloxy)-
iodobenzene (3.0 g, 7.7 mmol), and the mixture was refluxed for 1 h.
When the mixture was cooled, 1-methylthiourea (0.69 g, 7.7 mmol)
was added and the mixture was heated under reflux for 4 h. The
mixture was concentrated and purified using PE/EtOAc as eluant to
yield the title compound as a white solid (2.66 g, 53%), mp 133−135
1
compound as a white solid (4.13g, 51%), mp 93−95 °C. H NMR
(CDCl3): δ 1.61 (s, 9H, 3 × CH3), 2.53 (s, 3H, CH3), 3.59 (s, 3H,
CH3), 4.03 (q, 2H, J = 10.4 Hz, CH2). 13C NMR (CDCl3): δ 28.10,
30.84, 34.12, 35.00 (q, J = 30 Hz), 84.36, 124.15 (q, J = 276 Hz),
127.96, 146.63 (q, J = 3 Hz), 153.26, 161.96. 190.21. HRMS (ESI+):
m/z [M + H]+ calcd for C13H18F3N2O3S, 339.0990, found 339.1058.
tert-Butyl 5-(3-(Dimethylamino)acryloyl)-4-(2,2,2-
trifluoroethyl)thiazol-2-yl(methyl)carbamate (15, R1 = Boc, R′
= CH2CF3). 15 (R1 = Boc, R′ = CH2CF3) was obtained from tert-butyl
5-acetyl-4-(2,2,2-trifluoroethyl)thiazol-2-yl(methyl)carbamate and
1
°C. H NMR (DMSO-d6) δ: 2.42 (d, 3H, J = 0.8 Hz, CH3), 2.88 (d,
3H, J = 4.8 Hz, CH3), 8.86 (bs, 1H, NH). 13C NMR (DMSO-d6): δ
29.87 (d, J = 3 Hz), 31.59, 120.44 (q, J = 271 Hz), 125.57, 141.93 (q, J
= 36 Hz), 171.05, 187.48. HR-MS (ESI+): m/z [M + H]+ calcd for
C7H8 F3N2OS, 225.0309, found 225.0309.
tert-Butyl (5-Acetyl-4-(trifluoromethyl)thiazol-2-yl)(methyl)-
carbamate (17). 17 was obtained from 1-(2-(methylamino)-4-
(trifluoromethyl)thiazol-5-yl)ethanone and di-tert-butyl dicarbonate.
1
DMF−DMA. Light yellow solid (69%); mp 150−152 °C. H NMR
(DMSO-d6): δ 1.54 (s, 9H, 3 × CH3), 2.89 (s, 3H, CH3), 3.15 (s, 3H,
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Article
1
White solid (94%). 1H NMR (CDCl3): δ 1.62 (s, 9H, 3 × CH3), 2.60
(d, 3H, J = 0.8 Hz, CH3), 3.59 (s, 3H, CH3). HR-MS (ESI+): m/z [M
− (tert-butyl) + H]+ calcd for C8H8F3N2O3S, 269.0208, found
269.0265.
(3.51 g, 79%), mp 89−90 °C. H NMR (CDCl3): δ 0.98−1.08 (m,
2H, CH2), 1.09−1.18 (m, 2H, CH2), 1.60 (s, 9H, 3 × CH3), 2.51 (s,
3H, CH3), 2.98−3.10 (m, 1H, CH), 3.49 (s, 3H, CH3). 13C NMR
(DMSO-d6): δ 10.12, 12.10, 28.14, 30.92, 33.86, 83.95, 124.81, 153.15,
161.43, 162.18, 190.96. HR-MS (ESI+): m/z [M + H]+ calcd for
C14H21N2O3S, 297.1273, found 297.1296.
N,N-Dimethyl-N′-(methylcarbamothioyl)formimidamide
(18). A mixture of N-methylthiourea (9.0 g, 100 mmol) and DMF−
DMA (12 mL, 120 mmol) in 50 mL of CHCl3 was refluxed overnight.
The mixture was concentrated and the resulting precipitate was
collected by filtration to afford 18 as white solid (14.3 g, 98%), mp
1-(2-(Methylamino)-4-phenylthiazol-5-yl)ethanone (26). To
a solution of N-methyl-4-phenylthiazol-2-amine (1.90 g, 10 mmol) and
anhydrous AlCl3 (6.7 g, 50 mmol) in 100 mL of CH2Cl2 under
nitrogen gas was added acetyl chloride (1.42 mL, 20 mmol) over 15
min. The reaction was continued for 2 h. The mixture was cooled in an
ice bath and quenched using MeOH. After being concentrated, the
mixture was purified by column chromatography using EtOAc/PE as
eluant to affort the title compound as a white solid (1.55g, 67%), mp
229−231 °C. 1H NMR (DMSO-d6): δ 1.90 (s, 1H, CH3), 2.88 (d, 1H,
J = 4.8 Hz, CH3), 7.42−7.50 (m, 3H, 3 × Ph-H), 7.50−7.56 (m, 2H, 2
× Ph-H), 8.52 (br q, 1H, J = 4.8 Hz, NH). 13C NMR (DMSO-d6): δ
28.64, 31.40, 124.06, 128.53, 129.49, 129.80, 136.19, 159.09, 171.46,
189.10. HR-MS: m/z [M + H]+ calcd for C12H13N2OS, 233.0749,
found, 233.0658. N-Methyl-4-phenylthiazol-2-amine was obtained
from benzoyl chloride and 1-methylthiourea as a white solid
(100%), mp 143−144 °C. 1H NMR (DMSO-d6): δ 2.99 (s, 3H,
CH3), 7.18 (s, 1H, thiazol-H), 7.36−7.43 (m, 1H, Ph-H), 7.43−7.50
(m, 2H, 2 × Ph-H), 7.75−7.81 (m, 2H, 2 × Ph-H), 8.73 (br s, 1H,
NH). HRMS: m/z [M + H]+ calcd for C10H11N2S, 191.0643; found
191.0512.
N-Methyl-5-(2-(3-nitrophenylamino)pyrimidin-4-yl)-4-phe-
nylthiazol-2-amine (27a). 27a was obtained from 1-(3-
nitrophenyl)guanidine and tert-butyl (5-(3-(dimethylamino)acryloyl)-
4-phenylthiazol-2-yl)(methyl)carbamate. Yellow solid (18%); mp
215−216 °C. Anal. RP-HPLC: tR 13.84 min (method A), 13.45 min
(method B), purity 97%. 1H NMR (DMSO-d6): δ 2.92 (d, 3H, J = 4.8
Hz, CH3), 6.33 (d, 1H, J = 5.2 Hz, Py-H), 7.40−7.60 (m, 6H, 6 × Ph-
H), 7.76−7.84 (m, 1H, Ph-H), 7.97−8.06 (m, 1H, Ph-H), 8.16 (d, 1H,
J = 5.6 Hz, Py-H), 8.27 (q, 1H, J = 4.8 Hz, NH), 8.98 (t, 1H, J = 3.0
Hz, Ph-H), 10.05 (s, 1H, NH). 13C NMR (DMSO-d6): δ 31.49,
107.90, 112.77, 115.97, 119.41, 125.04, 129.12, 129.37, 130.09, 136.47,
142.37, 148.65, 154.89, 157.58, 159.21, 159.57, 170.52. HR-MS: m/z
[M + H]+ calcd for C20H17N6O2S, 405.1134; found, 405.1118.
N-Methyl-5-(2-((3-nitrophenyl)amino)pyrimidin-4-yl)-4-
(2,2,2-trifluoroethyl)thiazol-2-amine (27b). 27b was obtained
from 1-(3-nitrophenyl)guanidine and tert-butyl 5-(3-(dimethylamino)-
acryloyl)-4-(2,2,2-trifluoroethyl)thiazol-2-yl(methyl)carbamate. Yellow
solid (47%); mp 265−267 °C. Anal. RP-HPLC: tR 13.94 min (method
A), 11.85 min (method B), purity 99%. 1H NMR (DMSO-d6): δ 2.89
(d, 3H, J = 4.4 Hz, CH3), 4.11 (q, 2H, J = 11.2 Hz, CH2), 7.01 (d, 2H,
J = 5.2 Hz, Py-H), 7.58 (t, 1H, J = 8.0 Hz, Ph-H), 7.78−7.85 (m, 1H,
Ph-H), 8.05 (dd, 1H, J = 5.2, 1.6 Hz, Ph-H), 8.33 (q, 1H, J = 4.8 Hz,
NH), 8.48 (d, 1H, J = 5.6 Hz, Py-H), 8.59 (t, 1H, J = 2.0 Hz, Ph-H),
10.07 (s, 1H, NH). 13C NMR (DMSO-d6): δ 31.52, 35.33 (q, J = 29
Hz), 109.05, 113.11, 116.22, 121.42, 126.21 (q, J = 276 Hz), 125.34,
130.18, 142.18, 144.49, 148.60, 158.26, 159.03, 159.45, 170.07. HR-
MS (ESI+): m/z [M + H]+ calcd for C16H14F3N6O2S, 411.0851, found
411.1019.
1
109−110 °C. H NMR (CDCl3): δ 3.02 (d, 0.9H, J = 5.2 Hz, CH3),
3.04 (s, 2.1H, CH3), 3.13 (s, 0.9H, CH3), 3.15 (s, 2.1H, CH3), 3.19 (s,
0.9H, CH3), 3.21 (d, 2.1H, J = 5.2 Hz, CH3), 6.88 (brs, 1H, NH), 8.85
(s, 0.3H, CH), 8.88 (s, 0.7H, CH). HR-MS (ESI+): m/z [M + H]+
calcd for C5H12N3S, 146.0752; found 146.0638.
1-(2-(Methylamino)thiazol-5-yl)ethanone (19). A mixture of
N,N-dimethyl-N′-(methylcarbamothioyl)formimidamide (3.62 g, 25.0
mmol) and chloroacetone chloride (2 mL, 25.0 mmol) in 50 mL of
acetonitrile was refluxed for 4 h. After completion of the reaction, the
mixture was concentrated, neutralized by saturated NaHCO3 solution,
and dried over air to yield the title compound as a white solid (3.10 g,
1
79%), mp 163−164 °C. H NMR (DMSO-d6): δ 2.35 (s, 3H, CH3),
2.88 (s, 3H, CH3), 8.00 (s, H, thiazol-H), 8.54 (br s, 1H, NH). 13C
NMR (DMSO-d6): δ 26.03, 31.55, 127.66, 150.04, 175.29, 188.75.
HR-MS (ESI+): m/z [M + H]+ calcd for C6H9N2OS, 157.0436; found
157.0269.
tert-Butyl (5-Acetylthiazol-2-yl)(methyl)carbamate (20). 20
was obtained from 1-(2-(methylamino)thiazol-5-yl)ethanone and di-
1
tert-butyl dicarbonate. White solid (95%). H NMR (CDCl3): δ 1.61
(s, 9H, 3 × CH3), 2.53 (s, 3H, CH3), 3.59 (s, 3H, CH3), 8.00 (s, 1H,
thiazol-H). HR-MS (ESI+): m/z [M + H]+ calcd for C11H17N2O3S,
257.0960; found 257.0994.
tert-Butyl (4-Cyclopropylthiazol-2-yl)(methyl)carbamate
(23). A solution of 1-cyclopropylethanone (8.41 g, 100 mmol) in 30
mL of methanol was cooled in an ice bath, and bromine (5.15 mL, 100
mmol) was added dropwise. The mixture was stirred for 1 h before
being warmed to room temperature. After the mixture was stirred for a
further 3 h, 50 mL of water was added. The mixture was extracted by
diethyl ether (3 × 100 mL) and the combined organic phase was
washed with brine, dried over MgSO4, and concentrated to yield 22 as
1
a colorless oil (12.49 g, 77%). H NMR (CDCl3): δ 0.98−1.04 (m,
2H, CH2), 1.08−1.14 (m, 2H, CH2), 2.14−2.23 (m, 1H, CH), 4.02 (s,
2H, CH2). A solution of 22 (12.49 g, 76.0 mmol) in 30 mL of
methanol was treated with 1-methylthiourea (6.84 g, 76.0 mmol), and
an amount of 2 mL of pyridine was added. After being stirred at room
temperature overnight, the mixture was concentrated and basified with
saturated NaHCO3 solution. The precipitate was filtered and dried
over air. 4-Cyclopropyl-N-methylthiazol-2-amine was obtained as a
1
white solid after recrystallization from PE/EtOAc (5.70 g, 49%). H
NMR (CDCl3): δ 0.76−0.81 (m, 2H, CH2), 0.81−0.87 (m, 2H, CH2),
1.81−1.90 (m, 1H, CH), 2.95 (s, 3H, CH3), 5.46 (br s, 1H, NH), 7.07
(s, 1H, thiazol-H). HR-MS (ESI+): m/z [M + H]+ calcd for C7H11N2S,
155.0643; found 155.0452. The latter was treated with di-tert-butyl
dicarbonate to afford the title compound 23 as a brown liquid (55%).
1H NMR (CDCl3) δ: 0.81−0.90 (m, 4H, 2 × CH2), 1.58 (s, 9H, 3 ×
CH3), 1.89−1.99 (m, 1H, CH), 3.52 (s, 3H, CH3), 6.45 (s, 1H,
thiazol-H). HR-MS (ESI+): m/z [M + H]+ calcd for C12H19N2O2S,
255.1167; found 255.1184.
tert-Butyl (5-Acetyl-4-cyclopropylthiazol-2-yl)(methyl)-
carbamate (24). A solution of 23 (3.81g, 15.0 mmol) in 20 mL of
anhydrous THF at −78 °C was treated with 25.0 mmol of LDA. After
the mixture was stirred for 30 min, acetaldehyde (20.0 mmol, 1.12
mL) was added and the reaction was continued for 2 h. After
completion of the reaction, 20 mL of water was added and the mixture
was treated with 2 M aqueous HCl solution. After concentration, the
mixture was extracted with CHCl3 (3 × 50 mL). The combined
organic phase was washed with brine, dried over MgSO4, filtered, and
concentrated to dryness. The resulting mixture was dissolved in 30 mL
of CHCl3 and then treated with MnO2 (10 equiv). The mixture was
heated under reflux for 4 h. The reaction mixture was purified using
PE/EtOAc as elutant to afford the title compound as a yellow solid
3-((4-(2-(Methylamino)-4-(trifluoromethyl)thiazol-5-yl)-
pyrimidin-2-yl)amino)benzenesulfonamide (27f). 27f was ob-
tained from tert-butyl (5-(3-(dimethylamino)acryloyl)-4-
(trifluoromethyl)thiazol-2-yl)(methyl)carbamate and 3-guanidinoben-
zenesulfonamide. Yellow solid (10%); mp 279−281 °C. Anal. RP-
HPLC: tR 13.00 min (method A), 11.32 (method B), purity 100%. 1H
NMR (DMSO-d6): δ 2.91 (d, 3H, J = 4.8 Hz, CH3), 7.03 (dt, 1H, J =
5.2, 1.2 Hz, Py-H), 7.31 (s, 2H, NH2), 7.44 (dt, 1H, J = 8.0, 1.2 Hz,
Ph-H), 7.49 (t, 1H, J = 8.0 Hz, Ph-H), 7.88 (d, 1H, J = 8.0 Hz, Ph-H),
8.39 (s, 1H, Ph-H), 8.50 (q, 1H, J = 4.8 Hz, NH), 8.56 (d, 1H, J = 5.2
Hz, Py-H), 10.05 (s, 1H, NH). 13C NMR (DMSO-d6): δ 31.63,
109.34, 116.19, 119.18, 121.28 (q, J = 276 Hz), 122.27, 125.72, 129.54,
137.80 (q, J = 35 Hz), 141.04, 145.03, 156.11, 159.63, 159.74, 170.54.
HR-MS (ESI+): m/z [M + H]+ calcd for C15H14F3N6O2S2, 431.0572,
found 431.0766.
655
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Article
3-((4-(2-(Methylamino)thiazol-5-yl)pyrimidin-2-yl)amino)-
benzenesulfonamide (27i). 27i was obtained from tert-butyl (5-(3-
(dimethylamino)acryloyl)thiazol-2-yl)(methyl)carbamate and 3-guani-
dinobenzenesulfonamide. Yellow solid (13%); mp 295−297 °C. Anal.
RP-HPLC: tR 11.09 min (method A), 8.72 (method B), purity 99%.
1H NMR (DMSO-d6): δ 2.90 (d, 3H, J = 4.8 Hz, CH3), 7.19 (d, 1H, J
= 5.2 Hz, Py-H), 7.29 (s, 2H, NH2), 7.44 (dt, 1H, J = 8.0, 1.6 Hz, Ph-
H), 7.46 (t, 1H, J = 8.0 Hz, Ph-H), 7.88−7.95 (m, 1H, Ph-H), 8.08 (s,
1H, thiazol-H), 8.20 (q, 1H, J = 4.8 Hz, NH), 8.34 (d, 1H, J = 5.2 Hz,
Py-H), 8.43 (t, 1H, J = 1.6 Hz, Ph-H), 9.81 (s, 1H, NH). 13C NMR
(DMSO- d6): δ 31.50, 106.60, 116.00, 118.66, 121.92, 124.27, 129.44,
141.55, 143.64, 144.96, 157.64, 159.07, 159.97, 173.00. HR-MS
(ESI+): m/z [M + H]+ calcd for C14H15N6O2S2, 363.0698, found
363.0666.
3-((4-(4-Cyclopropyl-2-(methylamino)thiazol-5-yl)pyrimidin-
2-yl)amino)benzenesulfonamide (27j). 27j was obtained from
tert-butyl (4-cyclopropyl-5-(3-(dimethylamino)acryloyl)thiazol-2-yl)-
(methyl)carbamate and 3-guanidinobenzenesulfonamide. Off-white
solid (15%); mp 258−260 °C. Anal. RP-HPLC: tR 11.55 min (method
A), 9.22 min (method B), purity 100%. 1H NMR (DMSO-d6): δ
0.93−1.04 (m, 4H, 2 × CH2), 2.52−2.62 (m, 1H, CH), 2.83 (d, 1H, J
= 4.8 Hz, CH3), 7.09 (d, 1H, J = 5.6 Hz, Py-H), 7.28 (s, 2H, NH2),
7.40 (d, 1H, J = 8.0 Hz, Ph-H), 7.46 (t, 1H, J = 8.0 Hz, Ph-H), 7.96 (d,
1H, J = 8.0 Hz, Ph-H), 8.05 (q, 1H, J = 4.8 Hz, NH), 8.33 (s, 1H, Ph-
H), 8.36 (d, 1H, J = 5.2 Hz, Py-H), 9.75 (s, 1H, NH). 13C NMR
(DMSO-d6): δ 9.27, 13.07, 31.46, 108.32, 116.08, 117.37, 118.67,
122.04, 129.41, 141.56, 144.97, 158.08, 158.50, 159.41, 159.73, 170.37.
HR-MS (ESI+): m/z [M + H]+ calcd for C17H19N6O2S2, 403.1011,
found 403.0900.
Crystallography. CDK9330 (residues 1−330)/cyclin T1 (residues
1−259, Q77R, E96G, F241L) compounds were expressed, purified,
and crystallized as described previously.51 Crystals were grown by
vapor diffusion against a reservoir containing 14% PEG1000, 100 mM
sodium potassium phosphate, pH 6.2, 500 mM NaCl, 4 mM TCEP. A
crystal was soaked in mother liquor containing also 1 mM 12u and
15% glycerol for 45 min before cryocooling in liquid nitrogen.
CDK2/cyclin A was expressed and purified as described
previously.57 Purified protein was incubated with 12u, filtered, and
cocrystallized in 1.25 M ammonium sulfate, 0.5 M potassium chloride,
100 mM Hepes, pH 7.0, 5 mM DTT at 4 °C. Crystals were
cryoprotected and frozen in 7 M sodium formate in the presence of 1
mM 12u.
HEPES, 1% 200 mM L-glutamine, and 1% penicillin. Other cell lines
were maintained in RPMI-1640 with 10% FBS.
Proliferation Assays. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, Sigma) assays were performed as
reported previously.35 Compound concentrations required to inhibit
50% of cell growth (GI50) were calculated using nonlinear regression
analysis.
Caspase-3/7 Assay. Activity of caspase 3/7 was measured using
the Apo-ONE homogeneous caspase-3/7 kit (Promega G7790).18
Cell Cycle Analysis and Detection of Apoptosis. Cells (4×
105) were cultured for 48 h in medium alone or with varying
concentrations of inhibitor. Cell cycle status was analyzed using a
Beckman Coulter EPICS-XL MCL flow cytometer, and data were
analyzed using EXPO32 software. Apoptosis was also confirmed using
FITC annexin V/PI (propidium idodide) staining after cells were
cultured in medium only or with varying concentrations of inhibitors
according to the protocols (BD Bioscience). The annexin V/PI-
positive apoptotic cells were enumerated using flow cytometry. The
percentage of cells undergoing apoptosis was defined as the sum of
early apoptosis (annexin V-positive cells) and late apoptosis (annexin
V-positive and PI-positive cells). The pan-caspase inhibitor Z-Val-Ala-
Asp-(OMe)-CH2F (Z-VAD-fmk, Sigma) was dissolved in DMSO and
used at 25 μM.
Detection of Apoptosis in Primary CLL Cells. Freshly isolated
primary CLL cells and normal B- and T-cells were cultured in RPMI
with 10% fetal calf serum and L-glutamine, penicillin, and
streptomycin. Cells were maintained at 37 °C in an atmosphere
containing 95% air and 5% CO2 (v/v). CLL cells (106/mL) were
treated with inhibitor for 48 h. Subsequently, cells were labeled with
CD19-APC (Caltag) and then resuspended in 200 μL of binding
buffer containing 4 μL of annexin V-FITC (Bender Medsystems,
Vienna, Austria). Apoptosis was quantified in the CD19+ CLL cells,
CD19+ normal B-cells, and CD3+ normal T-cells using an Accuri C6
flow cytometer and FlowJo software (TreeStar). LD50 values were
calculated from line-of-best-fit analysis of the sigmoidal dose−response
curves.
Western Blots. Western blotting was performed as described.20
Antibodies used were as follows: total RNAP-II (8WG16),
phosphorylated RNAP-II Ser-2 (Covance), Bcl-2 (Dako, Denmark
A/S), MDM2 and β-actin (Sigma-Aldrich), Mcl-1, PARP (Cell
Signaling Technology). Both anti-mouse and anti-rabbit immunoglo-
bulin G (IgG) horseradish peroxidase-conjugated antibodies were
obtained from Dako.
Diffraction data for the CDK9/cyclin T1/12u and CDK2/cyclin A/
12u were collected from single crystals at Diamond Light Source
beamline I03. Diffraction data for CDK9/cyclin T/12u were processed
with XDS58 and SCALA (CCP4).59 PHENIX.refine60 was used for
rigid body refinement with a model derived from 3BLH as the initial
model. REFMAC61 was used for subsequent TLS and restrained
refinement. Jelly body restraints to an external model (3BLH) were
used during refinement. CDK2/cyclin A data were processed using
XDS58 and SCALA.59 Molecular replacement was performed by the
program PHASER62 using a search model derived from PDB entry
3DDQ. Ligand restraints were defined using PHENIX, and structures
were refined and rebuilt using PHENIX.refine and COOT.63
Kinase Assay. Inhibition of CDKs and other kinases was measured
by radiometric assay Millipore’s KinaseProfiler according to the
protocols detailed at http://www.millipore.com/drugdiscovery/dd3/,
where ATP concentration for each specific kinase assay was set within
15 μM of the apparent Km for ATP where determined. Half-maximal
inhibition (IC50) values were calculated from 10-point dose−response
curves, and apparent inhibition constants (Ki) were calculated from
the IC50 values and Km (ATP) values for the kinases in question as
described.35 The assay details can also be found in the Supporting
Information.
Statistical Analysis. All experiments were performed in triplicate
and repeated at least twice, representative experiments being selected
for figures. Statistical significance of differences for experiments was
determined using one-way analysis of variance (ANOVA), with a
minimal level of significance at p < 0.01.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis and characterization of compounds Ic, 27c−e, 27g,-h,
and 27k,l, and kinase assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
Accession Codes
PDB codes are the following: CDK9/cyclin T/12u, 4BCG;
CDK2/cyclin A/12u, 4BCP.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +61883022372. E-mail: shudong.wang@unisa.edu.au.
Author Contributions
Cell Culture. All cancer cell lines were obtained from the cell bank
at the Centre for Biomolecular Sciences, University of Nottingham,
U.K. The HMEC-1 cell line was purchased (ECACC), and cells were
cultured in essential medium with 10% fetal bovine serum (FBS), 7.5%
sodium bicarbonate, 1% 0.1 mM nonessential amino acids, 1% 1 M
Overall research design and writing of the manuscript: Wang.
Chemistry experiments: Shao, Shi, Huang, and Foley. Biological
experiments: Abbas, Liu, and Lam. Ex vivo CLL experiments:
Pepper. Crystallographic experiments: Hole, Noble, Endicott,
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Article
expressed throughout the cell cycle, and its steady-state expression is
independent of SKP2. Mol. Cell. Biol. 2003, 23, 5165−5173.
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development for the treatment of cancer. Expert Opin. Invest. Drugs
2003, 12, 955−970.
and Baumli. Contributions to or assistance in preparation of the
manuscript: Shao, Pepper, Hole, Noble, Endicott, and Fischer.
Notes
The authors declare no competing financial interest.
(16) Byrd, J. C.; Lin, T. S.; Dalton, J. T.; Wu, D.; Phelps, M. A.;
Fischer, B.; Moran, M.; Blum, K. A.; Rovin, B.; Brooker-McEldowney,
M.; Broering, S.; Schaaf, L. J.; Johnson, A. J.; Lucas, D. M.; Heerema,
N. A.; Lozanski, G.; Young, D. C.; Suarez, J. R.; Colevas, A. D.; Grever,
M. R. Flavopiridol administered using a pharmacologically derived
schedule is associated with marked clinical efficacy in refractory,
genetically high-risk chronic lymphocytic leukemia. Blood 2007, 109,
399−404.
ACKNOWLEDGMENTS
■
This study was supported by Cancer Research UK Grants
C21568/A8988 and C21568/A12474.
ABBREVIATIONS USED
■
ATP, adenosine triphosphate; Bcl-2, B-cell lymphoma 2;
CaMK1, calcium/calmodulin-dependent protein kinase type
1; DCM, dichloromethane; DMF, N,N-dimethylformamide;
DMSO, dimethylsulfoxide; DTT, dithiothreiol; HRMS, high
resolution mass spectrometry; IKKβ, inhibitor of nuclear factor
κB kinase subunit β; Lck, lymphocyte-specific protein tyrosine
kinase; LDA, lithium diisopropylamide; MeCN, acetonitrile;
MAPK2, mitogen-activated protein kinase 2; Mcl-1, myeloid
cell leukemia sequence 1; NBS, N-bromosuccinimide; NCS, N-
chlorosuccinimide; PDB, Protein Data Bank; PARP, poly ADP-
ribose polymerase; PKA, protein kinase A; PKB, protein kinase
B; PKC, protein kinase C; RNAPII, RNA polymerase II; SRC,
sarcoma kinase; SelectFluor, 1-chloromethyl-4-fluoro-1,4-
diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate)
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