Synthesis and biological activity of N-aryl-2-aminothiazoles: Potent pan inhibitors of cyclin-dependent kinases

Article abstract of DOI:10.1016/j.bmcl.2004.02.105

N-Aryl aminothiazoles 6-9 were prepared from 2-bromothiazole 5 and found to be CDK inhibitors. In cells they act as potent cytotoxic agents. Selectivity for CDK1, CDK2, and CDK4 was dependent of the nature of the N-aryl group and distinct from the CDK2 se

Full text of DOI:10.1016/j.bmcl.2004.02.105

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2973–2977
Synthesis and biological activity of N-aryl-2-aminothiazoles: potent
pan inhibitors of cyclin-dependent kinases
*
Raj N. Misra, Hai-yun Xiao, David K. Williams, Kyoung S. Kim, Songfeng Lu,
Kristen A. Keller, Janet G. Mulheron, Roberta Batorsky, John S. Tokarski,
à
John S. Sack, S. David Kimball, Francis Y. Lee and Kevin R. Webster
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA
Received 1 February 2004; accepted 27 February 2004
Abstract—N-Aryl aminothiazoles 6–9 were prepared from 2-bromothiazole 5 and found to be CDK inhibitors. In cells they act as
potent cytotoxic agents. Selectivity for CDK1, CDK2, and CDK4 was dependent of the nature of the N-aryl group and distinct from
the CDK2 selective N-acyl analogues. The N-2-pyridyl analogues 7 and 19 showed pan CDK inhibitory activity. Elaborated
analogues 19 and 23 exhibited anticancer activity in mice against P388 murine leukemia. The solid-state structure of 7 bound to
CDK2 shows a similar binding mode to the N-acyl analogues.
Ó 2004 Elsevier Ltd. All rights reserved.
Cyclin-dependent kinases (CDKs) are a prominent
family of protein kinases, which play a key role in the
growth, development, proliferation, and death of
CH3
N
tBu
1
eukaryotic cells. Along with their regulatory subunit
OH
O
O
H
N
cyclins, CDKs serve the function of orderly coordinat-
ing events which move cells through the cell cycle and
insure the genetic integrity of daughter cells. Due to
their identified role as drivers of aberrant cell growth
and division in a number of cancers, drug discovery
programs have directed a major effort toward the iden-
tification of small molecule inhibitors of CDKs as
HO
S
S
R
N
Cl
N
O
OH
O
2
: R=CH
3
2
3: R=CH -3-pyridyl
1
2;3
a
potential antitumor therapeutic agents.
Table 1. Structure and biological activity of CDK inhibitors
Compds CDK1/cycB CDK2/cycE CDK4/cycD A2780 Cytotox
Flavopiridol, 1, is a pioneering benchmark CDK
inhibitor, which shows inhibitory activity against a
broad range of kinases including CDK1, CDK2, and
CDK4 and is currently in Phase 2 clinical trials as an
antitumor agent. We recently reported on novel N-acyl-
2-aminothiazoles 2 and 3, which were potent CDK
5
inhibitors. In contrast to flavopiridol, 2 and 3 exhibited
IC50, nM
IC50, nM
IC50, nM
IC50, nM
1
2
3
30
40
80
170
5
100
690
71
50
5
1090
240
4
a
See Ref. 5b for description of biological assays.
selective inhibition of CDK2. Shown in Table 1 is the
inhibitory activity of 1–3 in cell-free enzyme assays and
their cytotoxic activity against a human ovarian (A2780)
cancer cell line.
*
à
Corresponding author. Mailing address: 12 Eaton Place, Hopewell,
NJ 08525, USA; e-mail: raj_n_misra@hotmail.com
Present address: Lexicon Pharmaceuticals, 350 Carter Road, Prince-
ton, NJ 08540, USA.
Herein we report on the synthesis, in vitro CDK
inhibitory activity and in vivo anticancer activity against
P388 murine leukemia of a series of N-aryl amino-
thiazoles, which show a CDK selectivity profile distinct
from N-acyl analogues such as 2 and 3.
Present address: Astra-Zeneca R&D Boston, 35 Gatehouse Drive,
Waltham, MA02451, USA.
0
960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.105

2974
R. N. Misra et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2973–2977
tBu
tBu
H
N
H N
N
2
a, b
X
S
S
N
N
O
O
a
N
N
N
S
S
10
1
1: X = H
S
S
12: X = Br
NH2
Br
c
tBu
4
N
5
N
O
H
O
S
N
tBu
tBu
SAc
N
N
N
1
3
O
7
b
N
S
Scheme 2. Synthesis of N-aryl analogue 7. Reagents and conditions:
a) NaH (1 equiv), THF, 55 °C then 2-bromothiazole (0.33 equiv), 0–
55 °C, 53%; (b) Br (1.2 equiv), HOAc, 25 °C, 95%; (c) NaOH,
NaHCO
, aq MeOH, 25–60 °C, 68%.
S
(
Ar
N
H
2
N
3
6
-9
Scheme 1. Synthesis of N-aryl analogues 6–9. Reagents and condi-
tions: (a) CuBr
(1.2 equiv), t-BuONO (1.5 equiv), MeCN, 0–25 °C,
3%; (b) NaH (3–5 equiv), ArNH
(3–5 equiv)/THF, 55 °C then 0–
5 °C, see Table 2 for yields.
H
N
2
H N
N
2
a, b
X
S
N
4
2
2
N
Br
Br
R
1
4
1
1
5: X = H
6: X = Br
Table 2. Structure and synthesis of N-aryl aminothiazoles
c, d
tBu
O
H
N
Compd
Ar
Yield, %
O
N
S
S
tBu
N
SAc
N
6
7
8
9
Phenyl
17
20
14
40
N
2-Pyridyl
3-Pyridyl
4-Pyrimidinyl
1
3
1
7: R = Br
18: R = CHO
tBu
e
O
H
N
S
S
N
N
The N-aryl analogues 6–9 were prepared from the pre-
viously reported amine 4 via intermediate bromide 5 as
H
N
N
OH
5;6
1
9
shown in Scheme 1. As indicated, treatment of 4 with
tert-butyl nitrite in the presence of copper(II) bromide
7
Scheme 3. Synthesis of N-aryl analogue 19. Reagents and conditions:
a) NaH (1 equiv), THF, 55 °C then 2-bromothiazole (0.33 equiv), 25–
5 °C, 33%; (b) Br (1.2 equiv), HOAc, 25 °C, 98%; (c) NaOH, aq
afforded the 2-bromo derivative 5 in moderate yield.
(
Bromide 5 was coupled with simple aryl amines by ini-
tially generating excess amine anion with sodium hy-
dride in THF at 55 °C and then addition of 5 at 0–25 °C.
The yields as indicated in Table 2 were low to moderate.
The major side products appeared to result from com-
petitive deprotonation of the acidic protons on the
methylene group adjacent to the sulfur.
6
2
MeOH, 25 °C then DMF, 60 °C, 57%; (d) t-BuMgCl (1.2 equiv) then
t-BuLi (2.9 equiv), THF, )78 °C followed by DMF, 44%; (e) 3-amino-
2
,2-dimethylpropanol (8 equiv), NaBH(OAc)
3
(13 equiv), HOAc,
THF, 70%.
O N
2
R
a, b
H
N
Alternatively, as shown in Scheme 2, the 2-pyridyl
analogue 7 was prepared in improved yield by initially
coupling excess 2-aminopyridine anion with 2-bromo-
thiazole to afford 11. Thiazole 11 was then brominated
selectively at the 5-position to give 12. Substitution at
the 5-position with the in situ generated thiolate from
OH
SO Cl
SO
2
2
2
0
2
1: R = NO
2
2
22: R = NH
tBu
c
8
acetate 13 afforded 7 in 34% overall yield. The 2-pyridyl
O
H
N
S
inhibitor 19 was available in a similar sequence from
commercial 2-amino-5-bromopyridine, 14, as the start-
ing amine. As shown in Scheme 3, 14 was converted to
bromide 17. Deprotonation followed by metal–halogen
exchange and quenching the resulting anion with DMF
afforded aldehyde 18 in moderate yield. Aldehyde 18
was then treated with excess 3-amino-2,2-dimethylpro-
panol under standard reductive amination conditions to
S
N
H
N
N
OH
SO2
2
3
Scheme 4. Synthesis of substituted N-phenyl aminothiazole 23. Re-
agents and conditions: (a) 2-aminoethanol (1.3 equiv), Et
70%; (b) 10% Pd/C, H2 (1 atm), HOAc/THF (1:1), 84%; (c) bromo-
3
N, 0–25 °C,
thiazole 5 (0.2 equiv), DMA, 145 °C, 5.5 h, 57%.
9
give 19.
Scheme 4, by heating an excess of free amine 22 with
bromide 5 in dimethylacetamide (DMA). The reaction
conditions are a modification of the initial low yield
Finally, the 4-sulfonamide substituted N-phenyl anal-
ogue 23 was prepared in 57% yield, as shown in

R. N. Misra et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2973–2977
2975
conditions in which the amine anion was generated prior
to addition of the bromide 5. Amine 22 was available in
two-steps from phenylsulfonyl chloride 20 by coupling
with 2-aminoethanol followed by reduction of the nitro
group under palladium catalyzed hydrogenation condi-
tions.
The three-dimensional solid-state structure of 7 in
complex with CDK2 was determined by X-ray crystal-
lography. Crystals were obtained by incubating the
inhibitor (72 h) with crystalline protein in the absence of
cyclin. The crystal structure, shown in the upper panel
of Figure 1, confirmed that 7 binds in the ATP-binding
site in the same folded conformation seen previously
with the N-acyl aminothiazole chemotype (see overlay in
5;10
the lower panel of Fig. 1).
In this conformation the
tert-butyl oxazole ring folds back toward the thiazole
core and into the ribose pocket rather than extending
toward Phe-80. Clear hydrogen bonding interactions are
not seen between the oxazole ring and the protein.
Important hydrogen bonds between the amide backbone
atoms of Leu-83 and both the thiazole nitrogen and
exocyclic amine proton are clearly discernable. Finally
and importantly, the N-aryl ring extends toward the
exterior of the protein, which should allow for variable
substitution para to the amino group. The origins for
the differences in CDK enzyme inhibitory selectivity
between the N-acyl and N-aryl series appear to be subtle
and are not conclusively apparent from the structural
data. However, they clearly are not the result of a
change in hydrogen bonding interactions or a major
change in binding mode.
Figure 1. Upper panel: Solid-state structure of N-aryl aminothiazole 7
bound in the ATP-pocket of CDK2 (no cyclin). The inhibitor carbon
atoms are shown in green, the nitrogen atoms are shown in blue,
oxygen atoms are shown in red, and the sulfur atoms are shown in
yellow. The protein carbons are in gray. Hydrogen bonds are shown by
the magenta dotted lines. Lower panel: Overlay of N-aryl amino-
thiazole 7 with N-acyl aminothiazole 2 (t-butyl group replaced by an
ethyl group). Inhibitor 7 is depicted with aqua colored carbon atoms
and the ethyl analogue of 2 is depicted with green carbon atoms.
N-Aryl aminothiazoles were initially evaluated in a cell-
free enzyme assay for inhibition of CDK1/cyclin B,
CDK2/cyclin E, and CDK4/cyclin D induced phos-
phorylation of RB protein and subsequently for cyto-
toxicity in whole cells. An ovarian cancer cell line,
A2780, was utilized in the cellular cytotoxicity assay.
The results of these assays are shown in Table 3. Com-
pounds 6–9 were all potent inhibitors of CDKs in the
enzyme assay with a selectivity profile distinct from the
N-acyl analogues (e.g., 2 and 3). Whereas the N-acyl
analogues exhibited selectivity for CDK2 over both
CDK1 and CDK4, the N-aryl analogues generally
showed diminished selectivity for CDK2 over CDK1
and CDK4. In the case of 2-pyridyl analogue 7, potent
inhibition of CDK1, CDK2, and CDK4 was observed.
leukemia. In the P388 model, drug was dosed intraper-
itoneally (ip) in immunocompetent mice immediately
after tumor cells were implanted (ip), then once a day
for 7 days (qdx7). The anticancer efficacy was measured
as an increase in lifespan and the data are expressed as a
ratio of lifespan of drug treated group (T) versus control
group (C). The results indicated that despite its highly
potent in vitro CDK inhibitory and cytotoxic activity, 9
was not active in vivo (%T/C ¼ 110 for inhibitor 9, active
is defined as %T/C > 125) when increased lifespan was
utilized as a criteria. Based on work with the N-acyl
analogues we speculated that these compounds may be
highly protein bound resulting in low levels of circulat-
ing free drug. In addition, poor solubility may be a
Aminothiazoles 6–9 also exhibited potent cytotoxic
activity in A2780 cells. Compound 9 was evaluated at
its maximum tolerated dose (MTD) of 45 mg/kg for
in vivo anticancer efficacy in mice against P388 murine
a
Table 3. CDK inhibitory activity of N-aryl aminothiazoles 6–9
Compd
Ar
CDK1/cycB IC50, nM CDK2/cycE IC50, nM CDK4/cycD IC50, nM A2780 Cytotox IC50, nM
6
7
8
9
Phenyl
12
4
4
2
3
1
78
9
140
10
50
3
2-Pyridyl
3-Pyridyl
4-Pyrimidinyl
10
7
130
30
a
See Ref. 5b for description of biological assays.

2976
R. N. Misra et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2973–2977
a
Table 4. CDK inhibitory and anticancer activity of N-aryl aminothiazoles
b
Compd
CDK1/cycBIC50
nM
,
CDK2/cycEIC50
nM
,
CDK4/cycDIC50
nM
,
A2780 CytotoxIC50
nM
,
% Protein binding
P388 %T/C
1
2
9
3
18
10
3
2
26
233
3
69
90
97
156 @ 4 mg/kg
180 @ 90 mg/kg
a
See Ref. 5b for description of biological assays.
Determined by equilibrium dialysis in mouse serum.
b
significant factor in the lack of anticancer activity for
this agent. Following a strategy employed for the N-acyl
aminothiazoles we sought to introduce polar functional
groups into the molecule as a method of both increasing
Acknowledgements
We thank Dr. Bang-Chi Chen, Mr. Rulin Zhao, and
Mr. Mark S. Bednarz for preparation of chemical
intermediates and Ms. Ming Chang for serum protein
binding data. We also thank Dr. John T. Hunt for sci-
entific guidance.
5
solubility and exposure of free drug. Modeling and
crystallographic studies (see above) had indicated that
substituents para to the amino group on the aryl ring
would be directed toward the exterior of the protein and
thus should be compatible with maintaining CDK
inhibitory activity. Based, in part, on modeling experi-
ments compounds 19 and 23 with basic amine and
alcohol side chains, respectively, were prepared and
found to mirror the in vitro CDK inhibitory activity of
their unsubstituted counterparts 6 and 7 (see Table 4).
Analysis indicated serum protein binding at 90% and
References and notes
1. (a) Hunter, T.; Pines, J. Cell 1994, 79, 573; (b) Sherr, C.
Science 1996, 274, 1672; Review: (c) Pines, J. Seminars
Cancer Biol. 1994, 5, 305.
. (a) Webster, K. R. Exp. Opin. Invest. Drugs 1998, 7, 1; (b)
Webster, K. R.; Kimball, S. D. Emerging Drugs 2000, 5,
2
9
7% for 19 and 23, respectively. Examination in the
4
5; (c) Kimball, S. D.; Webster, K. R. Annu. Rpt. Med.
Chem. 2001, 36, 139; (d) Sausville, E. A. Trends Mol. Med.
002, 8(4, Suppl.), S32; (e) Huwe, A.; Mazitschek, R.;
Giannis, A. Angew. Chem., Int. Ed. 2003, 42, 2122.
P388 model showed that in contrast to 9, both 19 and 23
were effective in significantly increasing lifespan.
Administered at their MTD 2-pyridyl analogue 19
increased lifespan by 56% while 2-phenyl analogue 23
increased lifespan by 80%.
2
3. (a) Vesely, J.; Havlicek, L.; Strnad, M.; Blow, J.; Donella-
Deana, A.; Pinna, L.; Letham, D. S.; Kato, J.; Detivaud,
L.; Leclerc, S.; Meijer, L. Eur. J. Biochem. 1994, 224, 771;
(
b) Abraham, R.; Acquarone, M.; Andersen, A.; Asensi,
In summary, N-aryl aminothiazoles 6–9 were prepared
and found to be inhibitors in vitro of CDK1, CDK2,
and CDK4. In cells they acted as potent cytotoxic
agents. Selectivity for CDK1, CDK2, and CDK4 was
dependent on the nature of the N-aryl group and was
distinct from the CDK2 selective N-acyl analogues. The
N-2-pyridyl analogues 7 and 19 exhibited a pan-like
CDK inhibitory profile. Analogues 19 and 23 with polar
substituents on the aryl ring afforded inhibitors that
were also efficacious in vivo as anticancer agents. For
example, 19 (BMS-357075) was a potent inhibitor of
CDK1, CDK2, and CDK4 and produced a 56%
increase in survival time versus untreated control against
P388 murine leukemia in mice (Fig. 2).
A.; Belle, R.; Berger, F.; Bergounioux, C.; Brun, G.;
Buquet-Fagot, C.; Fagot, D.; Glab, N.; Goudeau, H.;
Goudeau, M.; Guerrier, P.; Houghton, P.; Hendriks, H.;
Kloareg, B.; Lippai, M.; Marie, D.; Maro, B.; Meijer, L.;
Mester, J.; Mulner-Lorillon, O.; Poulet, S.; Schierenberg,
E.; Schutte, B.; Vaulot, D.; Verlhac, M. Biol. Cell 1995, 83,
1
05; (c) Havlicek, L.; Hanus, J.; Vesely, J.; Leclerc, S.;
Meijer, L.; Shaw, G.; Strand, M. J. Med. Chem. 1997, 40, 408.
4. (a) Kaur, G.; Stetler-Stevenson, M.; Sebers, S.; Worland,
P.; Sedlacek, H.; Myers, C.; Czeck, J.; Naik, R.; Sausville,
E. J. Nat. Cancer Inst. 1992, 84, 1736; (b) Carlson, B.;
Dubay, M. M.; Sausville, E. A.; Brizuela, L.; Worland, P.
J. Cancer Res. 1996, 56, 2973; (c) Sedlacek, H. H.; Czech,
J.; Naik, R.; Kaur, G.; Worland, P.; Losiewicz, M.;
Parker, B.; Carlson, B.; Smith, A.; Senderowicz, A.;
Sausville, E. Int. J. Oncol. 1996, 9, 1143; (d) Senderowicz,
A . M.Invest. New Drugs 1999, 17, 313; (e) Stadler, W. M.;
Vogelzang, J.; Amato, R.; Sosman, J.; Taber, D.; Liebo-
witz, D.; Vokes, E. E. J. Clin. Oncol. 2000, 18, 371;
(f) Zhai, S.; Senderowicz, A. M.; Sausville, E. A.; Figg, W.
D. Ann. Pharmacother. 2002, 36, 905.
. (a) Misra, R. N.; Xiao, H. Y.; Kim, K. S.; Lu, S.; Han,
W. C.; Barbosa, S. A.; Hunt, J. T.; Rawlins, D. B.; Shan,
W.; Ahmed, S. Z.; Qian, L.; Chen, B. C.; Zhao, R.;
Bednarz, M. S.; Kellar, K. A.; Mulheron, J. G.; Barorsky,
R.; Roongta, U.; Kamath, A.; Marathe, P.; Ranadive,
S. A.; Sack, J. S.; Tokarski, J. S.; Pavletich, N. P.; Lee,
F. Y.; Webster, K. R.; Kimball, S. D. J. Med. Chem.
2004, 47, 1719; (b) Kim, K. S.; Kimball, S. D.; Misra,
R. N.; Rawlins, D. B.; Hunt, J. T.; Xiao, H.-Y.; Lu, S.;
Qian, L.; Han, W.-C.; Shan, W.; Mitt, T.; Cai, Z.-W.;
Poss, M. A.; Zhu, H.; Sack, J. S.; Tokarski, J. S.; Chang,
C.-Y.; Pavletich, N.; Kamath, A.; Humphreys, W. G.;
tBu
O
H
N
S
S
N
N
H
N
5
N
OH
1
9 (BMS-357075)
CDK1/cycB IC = 18 nM
5
0
CDK2/cycE IC50 = 3 nM
CDK4/cycD IC50 = 26 nM
A2780 cytotox IC50 = 3 nM
P388 %T/C= 156
Figure 2. Summary of biological data for aminothiazole pan-CDK
inhibitor 19.

R. N. Misra et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2973–2977
2977
Marathe, P.; Bursuker, I.; Kellar, K. A.; Roongta, U.;
Batorsky, R.; Mulheron, J. G.; Bol, D.; Fairchild, C. R.;
Lee, F. Y.; Webster, K. R. J. Med. Chem. 2002, 45, 3905.
10. In solving the X-ray structure of 7 complexed to CDK2 it
was found that the nitrogen in the pyridine ring could be
placed in two alternative positions because the electron
density was indiscriminate. The pyridine nitrogen could
(1) face the hinge region or (2) be oriented on the same side
as the sulfur of the thiazole ring. Energy calculations of
the different orientations of the pyridine ring reveal that
the orientation of the nitrogen on the same side as the
sulfur is ꢀ2 kcal lower in energy than the alternative
orientation with the pyridine and thiazole rings being near
planar. The starting coordinates of the structures were
generated using CONCORDe. The evaluated conforma-
tions were subjected to 30 steps of DFT energy optimi-
zations as implemented in Jaguar (Jaguar 4.0,
Schrodinger, Inc., Portland, OR, 1991–2000) at B3LYP/
G-31G** and a single-point energy was calculated for the
6
. Synthetic compounds were characterized by HPLC, 400 or
1
5
00 MHz H NMR, and LC/MS.
7
. (a) Doyle, M. P.; Siegfried, B.; Dellaria, J. F. J. Org.
Chem. 1977, 42, 2426; (b) Hodgetts, K. J.; Kershaw, M. T.
Org. Lett. 2002, 4, 1363.
8
. Prepared by addition of 5-tert-butyl-2-chloromethyl-ox-
azole to a solution of thioacetic acid (2equiv) and DIPEA
2 equiv) in THF at room temperature. The reaction
5a
(
mixture was stirred for 0.5 h, concentrated in vacuo then
filtered through silica gel and eluted with diethyl ether.
The crude material, which discolored upon concentration
was obtained in quantitative yield and used in this form.
. Abdel-Magid, A. F.; Carson, A. G.; Harris, B. D.;
Maryanoff, C. A.; Shah, R. D. J. Org. Chem. 1996, 61, 3849.
9
þ
resulting geometries using B3LYP/cc-PVTZ(-f) .