Synthesis and biological study of 2-amino-4-aryl-5-chloropyrimidine analogues as inhibitors of VEGFR-2 and cyclin dependent kinase 1 (CDK1)

Article abstract of DOI:10.1016/j.bmcl.2007.01.086

The series of 2-amino-4-aryl-5-chloropyrimidines was discovered to be potent for both VEGFR-2 and CDK1. Described here are the chemistry for analogue synthesis, SAR study, and its kinase selectivity prolifing. The full rat PK data and in vivo efficacy stu

Full text of DOI:10.1016/j.bmcl.2007.01.086

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2179–2183
Synthesis and biological study of 2-amino-4-aryl-5-
chloropyrimidine analogues as inhibitors of VEGFR-2
and cyclin dependent kinase 1 (CDK1)
Shenlin Huang,a,* Ronghua Li,a Peter J. Connolly,a Stuart Emanuel,a
Angel Fuentes-Pesquera,a Mary Adams,a Robert H. Gruninger,a Jabed Seraj,a
Steven A. Middleton,a Jeremy M. Davisb and David F. C. Moffatb
aJohnson & Johnson Pharmaceutical Research & Development, L.L.C, 1000 Route 202, Raritan, NJ 08869, USA
bUCB Pharma, 216 Bath Road, Slough, SL14EN, UK
Received 31 December 2006; revised 23 January 2007; accepted 24 January 2007
Available online 2 February 2007
Abstract—The series of 2-amino-4-aryl-5-chloropyrimidines was discovered to be potent for both VEGFR-2 and CDK1. Described
here are the chemistry for analogue synthesis, SAR study, and its kinase selectivity prolifing. The full rat PK data and in vivo efficacy
study are also included.
Ó 2007 Elsevier Ltd. All rights reserved.
Angiogenesis, the formation of new capillaries from the
endothelium of an existing vascular network, plays a
crucial role in tumor growth. Solid tumors cannot grow
larger than several cubic millimeters until they establish
a blood supply because cells must be within 100–200 lm
of a blood vessel to survive.1 The molecular mechanisms
underlying angiogenesis have been studied and are well
established due to the pharmaceutical potential of
antiangiogenesis as cancer therapy. Although several
growth factors play important role in angiogenesis, vas-
cular endothelial growth factor (VEGF) and its tyrosine
kinase receptor (VEGFR-2) are of particular interest be-
cause of the magnitude of their effects and the potential
for therapeutic application.2 Inhibition of VEGF activity
or VEGFR-2 kinase has been shown to suppress tumor
angiogenesis and tumor growth in tumor xenograft
studies. FDA approval of the anti-VEGF antibody
bevacizumab for the treatment of colorectal cancer
provides valuable proof-of-concept in a clinical setting.3
Recently, two small molecule inhibitors of VEGFR-2
kinase, sorafenib (BAY-43-9006)4 and sunitinib
(SU-11248),5 were approved by the FDA for renal and/
or gastrointestinal cancer. Numerous other small
molecules have progressed to the clinical evaluation
stage.6,7 Cyclin dependent kinase 1 (CDK1) is a member
of a family of serine/theronine kinases that plays an
important function in regulation of the cell cycle.8 Be-
cause abnormal CDK control of the cell cycle has been
linked to the molecular pathology of cancer, attention
has focused on CDKs as potential targets for cancer ther-
apy.9 CDK1 is an especially attractive target due to its
crucial role in regulating the cell cycle at the G2 and mito-
sis stages. Although inhibition of CDK has yet to be val-
idated in cancer patients, several CDK1 inhibitors have
progressed into clinical trials, among them flavopiridol,
UCN-01, CYC202, and SNS-032 (BMS-387032).10
We recently reported the in vitro anti-angiogenic and
in vivo anti-tumor activity of a 5-cyanopyrimidine deriv-
ative that is a potent, selective inhibitor of VEGFR-2 ki-
nase.11 In this communication, we disclose a related
series (Fig. 1) of 2-amino-4-aryl-5-chloropyrimidines
NC
N
O
Cl
R1
N
N
N
N
H
R2
N
N
H
NH2
Figure 1.
Keywords: CDK1; VEGFR-2; 2-Amino-4-aryl-5-chloropyrimidine.
*
Corresponding author. E-mail: shenlinhuang@hotmail.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.086

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S. Huang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2179–2183
that are potent inhibitors of VEGFR-2 and CDK1.12
This series also displays antiproliferative activity against
cancer cells and therefore could more effectively inhibit
tumor growth. Herein we describe the synthesis, initial
structure–activity relationships (SAR), and PK proper-
ties of this novel series.
N-substituted 4-(2-aminoethyl)phenylamines provided
target molecules 9a–k.
All compounds were evaluated for their ability to
inhibit VEGFR-2 kinase, CDK1, and proliferation of
three tumor cell lines: malignant melanoma (A375),
cervical adenocarcinoma (HeLa), and colon carcinoma
(HCT116).11 Biological activities of the 4-(cumylamino)
analogues are presented in Table 1. With the exception
of the 3-bromo (6a) and morpholin-4-ylethyl (7d)
derivatives, all of the cumylamino compounds were po-
tent VEGFR-2 inhibitors, having IC50 values <0.1 lM.
Neutral polar group (2-hydroxyethyl, 6b) was compat-
ible with good activity while basic substitution, incor-
porated to improve aqueous solubility, conferred the
highest VEGFR-2 potency. Interestingly, the basicity
of the terminal amino group seemed to correlate with
potency, with the more basic methylamine (7a), dim-
ethylamine (7b), and pyrrolidine (7c) analogues having
the lowest IC50 values (0.019–0.032 lM), N-methylpip-
erazine (7e) having an intermediate value, and the least
basic morpholine (7e) having the highest IC50. CDK1
inhibition and cellular antiproliferative activity did
not follow the same trend, however. CDK1 potency
was 19- to 60-fold lower than VEGFR-2 for the amine-
containing compounds, but only 3- to 10-fold lower for
the neutral derivatives. Cellular antiproliferative activity
was poor (IC50 > 1 lM) to moderate (IC50 0.3–1 lM)
with no obvious correlation to either VEGFR-2 or
CDK1.
Our chemistry efforts began with an exploration of the
2-arylamino substituent on a subset of 4-(cumylami-
no)-5-chloropyrimidines. These derivatives were pre-
pared using the procedure shown in Scheme 1, starting
with addition of methylcerium chloride to 4-bro-
mobenzonitrile to generate 4-bromocumylamine (1).13
After protection of the amino group with Boc, bromide
2 was converted to boronate 3 using bis(pinacola-
to)diboron in the presence of Pd(II).14 Compound 3
was then coupled with 2,4,5-trichloropyrimidine to af-
ford the 4-aryl-2,5-dichloropyrimidine 4. This step was
highly regio-selective and proceeded in good yield
(80%). Next, the 2-chloro substituent of compound 4
was displaced with various arylamines to produce
2-amino intermediates 5a–b, which were in turn treated
with HCl to afford final products 6a–b. One of the inter-
mediates (5b) was also used to generate analogues
containing amino side chains; the chemistry is shown
in Scheme 2. Compound 5b was reacted with metha-
nesulfonyl chloride, followed by displacement of the
mesyl group with aliphatic amines. Finally, removal of
the Boc protecting group with HCl generated com-
pounds 7a–e. To further expand SAR in the series, the
cumylamino substituent at the 4-position was replaced
by selected heterocylic groups. As indicated in Scheme
3, various arylboronates were coupled with 2,4,5-trichlo-
ropyrimidine to generate 4-aryl-2,5-dichloropyrimidines
8.15 Displacement of the 2-chloro of compounds 8 with
VEGFR-2 activity was found to be quite sensitive to the
nature of the 4-substituent on the 5-chloropyrimidine
core, as seen in Table 2. For example, thien-2-yl ana-
logue 9a was 8-fold less potent (IC50 = 0.16 lM) than
(a)
(b)
Br
Br
CN
Br
NH2
NHBOC
1
2
Cl
O
B
N
O
(c)
N
Cl
(d)
NHBOC
NHBOC
3
4
Cl
Cl
N
N
Ar
(e)
(f)
Ar
N
N
H
N
N
H
NHBOC
NH2
5a-b
6a-b
Scheme 1. Synthesis of compounds 6a–b. Reagents: (a) CeCl3, MeLi, THF, 95%; (b) (Boc)2O, toluene, 96%; (c) KOAc, bis(pinacolato)diboron,
Pd(dppf)2Cl2, THF, 82%; (d) 2,4,5-trichloropyrimidine, 1,2-dimethoxyethane, 80%; (e) ArNH2, 2-methoxyethanol, 80–90%; (f) HCl, MeOH, 90–
100%.

S. Huang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2179–2183
2181
R2
N
Cl
OH
Cl
R1
N
N
(a), (b), (c)
N
N
N
N
H
H
NHBOC
NH2
5b
7a-e
Scheme 2. Synthesis of compounds 7a–e. Reagents: (a) MsCl, Et3N, CH2Cl2; (b) HNR1R2, DMF; (c) HCl, MeOH, 60–80%.
R2
Cl
Cl
Cl
Cl
Ar
N
(a)
R1
(b)
N
N
N
O
O +
B
N
Cl
Ar
N
Cl
N
N
H
Ar
8
9a-k
Scheme 3. Synthesis of compounds 9a–k. Reagents: (a) Pd(PPh3)4, 1,2-dimethoxyethane, 60–85%; (b) 4-[2-(N-R1,R2-amino)ethyl]aniline,
2-methoxyethanol, 60–80%.
Table 1. Kinase and cellular antiproliferative activities of 4-(cumylamino) derivatives
Cl
N
R1
N
N
H
NH2
Compound
R1
Kinase inhibition
(IC50, lM)
Cell proliferation
(IC50, lM)
VEGFR-2
CDK1
A375
HCT116
HeLa
6a
6b
7a
7b
7c
7d
7e
3-Bromophenyl
4-(HOCH2CH2)phenyl
0.348
0.069
0.033
0.019
0.026
0.148
0.076
1.040
0.687
1.060
1.140
0.808
2.770
1.610
2.610
1.370
0.175
0.163
0.247
3.170
0.868
3.690
1.600
0.885
1.680
0.359
3.220
1.300
3.350
2.920
0.542
0.500
0.339
3.010
0.811
4-(MeNHCH2CH2)phenyl
4-(Me2NCH2CH2)phenyl
4-(Pyrrolidin-1-yl-CH2CH2)phenyl
4-(Morpholin-4-yl-CH2CH2)phenyl
4-(1-Me-piperazin-4-yl-CH2CH2)phenyl
the corresponding 4-cumylamino derivative 7b and the
2,4-dimethylthiazol-5-yl analogue was 42-fold less po-
tent (IC50 = 0.80 lM). At the other extreme, the indol-
3-yl (9c, 9i) and indol-6-yl (9f, 9j, 9k) analogues were
extremely potent VEGFR-2 inhibitors (IC50 values ran-
ged from 0.007 to 0.036 lM). Attachment at other posi-
tions of the indole was detrimental, however, with
VEGFR-2 potency decreasing in order from indol-5-yl
(9e) > indol-4-yl (9d) > indol-7-yl (9g). VEGFR-2 activ-
ity was significantly reduced by substituting the indol-3-
yl bicyclic system with the isosteric indazol-3-yl moiety
(9h, IC50 = 0.314 lM), suggesting an important binding
role for the indole N–H. With the exception of com-
pounds 9f, 9j, and9k, the 4-heteroaryl-5-chloropyrimi-
dines were relatively non-selective with respect to
CDK1, having VEGFR-2/CDK1 IC50 ratios less than
8. Notably, the weakly potent thiazole derivative 9b
was a better inhibitor of CDK1 than VEGFR-2. In con-
trast, 9f, 9j, and 9k were 34- to 120-fold selective for
VEGFR-2, with CDK1 IC50 values in excess of
1.2 lM. Cellular antiproliferative activities were general-
ly modest, but the indol-3-yl derivative 9c distinguished
itself as a highly potent antiproliferative agent in all
three tumor cell lines (IC50 values ranged from 0.050
to 0.084 lM). The other indo-3-yl derivative (9i),
although equipotent against VEGFR-2, had much
weaker antiproliferative activity, perhaps as a result of
poorer membrane permeability due to the doubly ioniz-
able piperazine group. To determine general kinase
selectivity for the series, compound 9i was evaluated in
a panel of tyrosine and serine/threonine kinases.16 Out
of 100 kinases tested, 9i exhibited >80% inhibition of
62 kinases at a concentration of 1 lM, indicating that
the 2-arylamino-4-(indol-3-yl)-5-chloropyrimidine scaf-
fold is a relatively promiscuous kinase pharmacophore.
Indeed, examples of the non-chloro version of this scaf-
fold have been reported to be inhibitors of protein ki-
nase C and Bcr-abl kinase.17

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S. Huang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2179–2183
Table 2. Kinase and cellular antiproliferative activities of 4-heteroaryl derivatives
R2
Cl
N
R1
N
N
H
Compound
R1
R2
Kinase inhibition
(IC50, lM)
Cell proliferation
(IC50, lM)
VEGFR-2
CDK1
A375
HCT116
HeLa
9a
9b
9c
9d
9e
9f
2-thienyl
Me2N
Me2N
Me2N
Me2N
Me2N
Me2N
Me2N
Me2N
0.159
0.797
0.007
0.216
0.103
0.015
0.455
0.314
0.007
0.016
0.037
0.461
0.439
0.048
0.540
0.847
1.770
0.334
0.781
0.18
0.016
0.399
0.050
0.210
0.030
0.336
0.774
3.970
3.930
1.970
2.570
0.228
0.391
0.084
0.084
0.321
0.446
0.470
3.760
3.050
1.680
2.450
1.240
0.431
0.075
0.292
0.596
0.472
1.290
3.060
3.330
0.973
2.080
2,4-dimethyl-thiazol-5-yl
Indol-3-yl
Indol-4-yl
Indol-5-yl
Indol-6-yl
Indol-7-yl
9g
9h
9i
Indazol-3-yl
Indol-3-yl
Indol-6-yl
1-Me-piperazin-4-yl
Pyrrolidin-1-yl
1-Et-piperazin-4-yl
9j
9k
1.54
1.24
Indol-6-yl
Table 3. Pharmacokinetic properties of compounds 6b, 9j, and 9k in
Sprague–Dawley rats
erative activity. SAR at the 2- and 4-positions of the
5-chloropyrimidine core was studied, leading to the
synthesis of many potent analogues having (2-aminoeth-
yl)phenylamino at the 2-position and cumylamino, in-
dol-3-yl, or indol-6-yl at the 4-position. Potent CDK1
inhibition and antiproliferative activity were found in
several analogues, notably those having indo-3-yl at
the 4-position. The mechanism for the antiproliferative
activity did not appear to correlate with either VEG-
FR-2 or CDK1 inhibition. Several derivatives also dis-
played acceptable pharmacokinetic behavior in rat but
were not orally active in an A375 xenograft experiment
in nude mice.
Compound
6b
9j
9k
Oral bioavailability
Dose (mg/kg), poa
t1/2 (h), po
55%
10
70%
10
40%
10
4.5
2.6
4.6
Cmax (lM), po
AUC (lM h), po
Dose (mg/kg), iv
1.11
7.65
2
0.72
5.74
2
0.59
6.12
2
b
t1/2 (h), iv
4.9
3.9
5.8
Cmax (lM), iv
AUC (lM h), iv
Clearance (mL/min/kg)
Vss (L/kg)
1.43
2.77
34
0.67
1.63
51
0.96
3.06
25
10.7
16.3
10.1
a Vehicle = 0.5% MethocelÒ.
b Vehicle = 10% SolutolÒ.
References and notes
Pharmacokinetic parameters for several analogues were
determined in male Sprague–Dawley rats. As shown in
Table 3, compounds from the 4-(cumylamino) series
(6b) and the 4-(indol-6-yl) series (9j and 9k) were orally
bioavailable. These derivatives achieved useful plasma
concentrations (0.6–1.1 lM) and had reasonable plasma
half-lives (2.6–4.6 h) after oral administration at 10 mg/
kg, but also had high clearance values (25–51 mL/min/
kg). Based on these results and their in vitro kinase and
cell proliferation profiles, compounds 6b, 9j, and 9k were
evaluated for their ability to inhibit the growth of A375
xenograft implanted in the hind flank of nude mice.
Unfortunately, all three compounds failed to demon-
strate significant anti-tumor activity when dosed orally
at 100 mg/kg for 28 days, although a trend toward tumor
growth inhibition was observed. This outcome is surpris-
ing in light of the potent VEGFR-2 inhibition shown by
all compounds and the robust antiproliferative activity
exhibited by 6b. It is possible that high clearance values
indicate that the compounds are not resident in the tumor
long enough to show the desired effect.
1. (a) Carmeliet, P.; Jain, R. K. Nature 2000, 407, 249; (b)
Folkman, J. Nat. Med. 1995, 1, 27.
2. Ferrara, N.; Gerber, H. P.; LeCouter, J. Nat. Med. 2003,
9, 669.
3. Dvorak, H. F. J. Clin. Oncol. 2002, 20, 4368.
4. Strumberg, D. Drugs Today 2005, 41, 773.
5. Motzer, R. J.; Michaelson, M. D.; Redman, B. G.; Hudes,
G. R.; Wilding, G.; Figlin, R. A.; Ginsberg, M. S.; Kim, S.
T.; Baum, C. M.; DePrimo, S. E.; Li, J. Z.; Bello, C. L.;
Theuer, C. P.; George, D. J.; Rini, B. I. J. Clin. Oncol.
2006, 24, 16.
6. (a) Connell, R. D. Expert Opin. Ther. Patent 2002, 12,
1763; (b) Boyer, S. J. Curr. Top. Med. Chem. 2002, 2, 973.
7. Traxler, P. Expert. Opin. Ther. Targets 2003, 2, 215.
8. Harper, J. W.; Adams, P. D. Chem. Rev. 2001, 101, 2511.
9. (a) Sielecki, T. M.; Boylan, J. F.; Benfiled, P. A.; Trainor,
G. T. J. Med. Chem. 2000, 43, 1; (b) Toogood, P. I. Med.
Res. Rev. 2001, 21, 487.
10. (a) Fischer, P. M.; Gianella-Borradori, A. Expert Opin.
Investig. Drugs 2003, 12, 955; (b) Sausville, E. A. Curr.
Med. Chem.—Anti-Cancer Agents 2003, 3, 47; (c) Misra,
R. N.; Xiao, H.; Kim, K. S.; Lu, S.; Han, W.; Barbosa, S.
A.; Hunt, J. T.; Rawlins, D. B.; Shan, W.; Ahmed, S. J.;
Qian, L.; Chen, B.; Zhao, R.; Bednarz, M. S.; Kellar, K.
A.; Mulheron, J. G.; Batorsky, R.; Roongta, U.; Kamath,
A.; Marathe, P.; Ranadive, S. A.; Sack, J. S.; Tokarski, J.
In summary, we identified a novel series of potent
VEGFR-2 kinase inhibitors with CDK1 and antiprolif-

S. Huang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2179–2183
2183
S.; Pavletich, N. P.; Lee, F. Y. F.; Webster, K. R.;
Kimball, S. D. J. Med. Chem. 2004, 47, 1719.
14. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem.
1995, 60, 7508.
11. Emanuel, S.; Gruninger, R. H.; Fuentes-Pesquera, A.;
Connolly, P. J.; Seamon, J. A.; Hazel, S.; Tominovich, R.;
Hollister, B.; Napier, C.; D’Andrea, M. R.; Reuman, M.;
Bignan, G.; Tuman, R.; Johnson, D.; Moffatt, D.;
Batchelor, M.; Foley, A.; O’Connell, J.; Allen, R.; Perry,
M.; Jolliffe, L.; Middleton, S. A. Mol. Pharmacol. 2004,
66, 635.
12. Davis, J. M.; Moffatt, D. F. C. U.S. Patent 6,600,037, 2003.
13. Imamoto, T.; Kusumoto, T.; Tawarayama, Y.; Sugiura,
Y.; Mita, T.; Hatanaka, Y.; Yokoyama, M. J. Org. Chem.
1984, 49, 3904.
15. The boronates were made from the corresponding bro-
mides using the procedure described in Ref. 14.
16. Upstate Cell Signaling Solutions, Serologicals Corporation,
706 Forest Street, Suite 1, Charlottesville, VA 22903, USA.
17. (a) Zimmermann, J.; Caravatti, G.; Mett, H.; Meyer, T.;
Mueller, M.; Lydon, N. B.; Fabbro, D. Archiv. der
Pharmazie (Weinheim, Germany) 1996, 329, 371; (b)
Zimmermann, J.; Buchdunger, E.; Mett, H.; Meyer, T.;
Lydon, N. B. Bioorg. Med. Chem. Lett. 1997, 7, 187; (c)
Huang, S.; Li, R.; Connolly, P.; Emanuel, S.; Middleton,
S. Bioorg. Med. Chem. Lett. 2006, 16, 4818.