2,4-Disubstituted pyrimidines: A novel class of KDR kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(03)00244-0

2,4-Disubstituted pyrimidines were synthesized as a novel class of KDR kinase inhibitors. Evaluation of the SAR of the screening lead compound 1 (KDR IC₅₀=105 nM, Cell IC₅₀=8% inhibition at 500 nM) led to the potent 3,5-dimethylaniline derivative 2d (KDR IC₅₀=6 nM, cell IC₅₀=19 nM).

Full text of DOI:10.1016/S0960-894X(03)00244-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1673–1677
2,4-Disubstituted Pyrimidines: A Novel Class of KDR
Kinase Inhibitors
Peter J. Manley,* Adrienne E. Balitza, Mark T. Bilodeau, Kathleen E. Coll,
George D. Hartman, Rosemary C. McFall, Keith W. Rickert, Leonard D. Rodman
and Kenneth A. Thomas
Departments of Medicinal Chemistry and Cancer Research, Merck Research Laboratories, West Point, PA 19486, USA
Received 7 January 2003; revised 10 March 2003; accepted 11 March 2003
Abstract—2,4-Disubstituted pyrimidines were synthesized as a novel class of KDRkinase inhibitors. Evaluation of the SARof the
screening lead compound 1 (KDRIC 50=105 nM, Cell IC50=8% inhibition at 500 nM) led to the potent 3,5-dimethylaniline deri-
vative 2d (KDRIC 50=6 nM, cell IC50=19 nM).
# 2003 Elsevier Science Ltd. All rights reserved.
Neovascularization plays an important role in the
pathology of several diseases including diabetic retino-
pathy,1 rheumatoid arthritis,2 psoriasis,3 and cancer.4
The process, know as angiogenesis, is controlled by a
number of endogenous mitogenic proteins including
vascular endothelial growth factor (VEGF). Up-regula-
tion of VEGF and its subsequent signaling through the
receptor tyrosine kinase KDR(VEGFR-2) is an impor-
tant requirement for tumor growth and proliferation.5,6
Interruption of this signaling cascade with anti-VEGF7
and anti-KDR8 antibodies as well as small molecule
Initially, we sought to investigate the SARof the aniline
portion of compound 1 with a focus on increasing
intrinsic potency (Fig. 2). Table 1 describes the initial
SARtrends of 2a–l. In general, ortho- or para-substitu-
tion of the aniline ring of compound 1 decreased
intrinsic activity regardless of the nature of the sub-
stituent. meta-Substitution, on the other hand,
enhanced intrinsic potency relative to compound 1.
Symmetrical di-substitution with a slightly electron
donating group (2d) or electron-rich (2h) substituents
resulted in a further enhancement of intrinsic activity
9
inhibitors of the KDRkinase domain has resulted in
the inhibition of angiogenesis as observed in tumor
xenograft models. The interest in using small molecules
as anti-angiogenic agents in human cancers has
increased in recent years. Clinical trials have been initi-
ated for a number of KDRkinase inhibitors from dif-
ferent structural classes, including indolin-2-ones,
phthalazines, and quinazolines.10
Figure 1.
In our efforts to discover novel small molecule inhibitors
of the KDRkinase domain, 11 a screening of the corporate
sample collection resulted in the lead compound 1 (Fig. 1).
While 1 exhibited moderate intrinsic potency (KDR
IC50=105 nM)12 it lacked any significant cellular activity
(cell IC50=8% inhibition at 500 nM).13
*Corresponding author. Tel.: +1-215-652-9466; fax: +1-215-652-
7310; e-mail: peter_manley@merck.com
Figure 2.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00244-0

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P. J. Manley et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1673–1677
Table 1. KDRkinase activity of compounds 212
Compd
R1
KDRIC 50 (nM)
3080
Compd
R1
KDRIC 50 (nM)
289
Compd
R1
KDRIC 50 (nM)
2a
2e
2i
25Æ18
2b
2c
2d
18Æ3
279
2f
2g
2h
20Æ9
392
2j
2k
2l
26Æ10
10Æ1
6Æ1
5Æ1
77Æ17
relative to their mono-substituted analogue (2b and 2f,
respectively). However, symmetrical di-substitution with
electron-withdrawing substituents (2j and 2l) had little
effect on intrinsic potency relative to their mono-sub-
stituted analogues (2i and 2k).
hydroxymethyl to an aminomethyl, as in 3d, resulted in
a moderate loss of potency.
In a further effort to increase the overall polarity of the
series, heterocyclic amines were used in place of aniline
derivatives. Of those examined, all but the pyridine
analogues (Table 3) resulted in a dramatic loss of
intrinsic activity. Compound 4a, as well as all the other
heterocycles examined that contained a heteroatom
adjacent to the pyrimidine core, had intrinsic potencies
greater than 1 mM (data not shown). Potency enhance-
ment of the most active compound in this series (4c) was
achieved by the sequential addition of methyl groups, as
in 4e and 4f, to mimic 2b and 2d; however, none of these
analogues was as potent as the lead 1.
Table 2 describes the SARof unsymmetrically di-sub-
stituted aniline derivatives of 1. The combination of a
slightly electron donating substituent with an electron-
withdrawing one, as in 3a, had no effect on intrinsic
potency relative to the corresponding symmetrically
substituted analogue 2d. However,
a substantial
enhancement was observed when compared to the sym-
metrically substituted analogue 2l. Combination of an
electron-rich substituent with an electron-withdrawing
one, as in 3b, astonishingly resulted in a severe loss of
intrinsic potency. In an effort to increase the overall
polarity of the series, the addition of a hydroxyl group
to one of the methyl substituents of 2d resulted in 3c,
which suffered little loss in potency. Conversion of the
The synthesis of 2a–l, 3a–d, and 4a–f is found in Scheme
1. Nucleophilic aromatic substitution of commercially
available 4-chloro-2-methylthiopyrimidine with the
sodium salt of 2-phenylimidazole in DMF at 100 ꢀC
gave 2-(methylthio)-4-(2-phenyl-1H-imidazol-1-yl)pyri-
midine. Oxidation of the thioether with sodium tung-
state dihydrate and 30% H2O2 in EtOAc gave 2-
(methylsulfonyl)- 4- (2- phenyl-1H -imidazol -1-yl)pyr-
imidine. Nucleophilic aromatic substitution of the
methyl sulfone with the various aniline derivatives gave
2a–l, 3a–d, and 4a–f.
Table 2. KDRkinase activity of compounds 312
Compd
R1
KDRIC 50 (nM)
3a
5Æ3
Next, we focused our attention on the synthesis and
SARof the 2-position of the imidazole ring in 2d.
Nucleophilic substitution of the thiomethyl group in
2-(methylthio)pyrimidin-4(3H)-one with 3,5-dimethyla-
niline in diglyme at 170 ꢀC gave 2-[(3,5-dimethylphenyl)
amino]pyrimidin - 4(3H) - one. Conversion of the pyr-
imidinone functionality to 4-chloro-N-(3,5-dimethyl-
phenyl)pyrimidin-2-amine was accomplished in refluxing
POCl3. Nucleophilic aromatic substitution of the chlo-
ride with imidazole derivatives and Cs2CO3 in DMA at
140 ꢀC gave compounds 5a–j (Scheme 2).
3b
3c
19,000
9Æ1
3d
32Æ5
Table 4 describes the SARof these compounds. While
all suffered a loss in intrinsic potency relative to 2d,
compounds 5d, 5e, 5g, and 5j showed comparable

P. J. Manley et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1673–1677
Table 3. KDRkinase activity of compounds 412 Table 4. KDRkinase activity of compounds 512
1675
Compd
R1
KDRIC 50 (nM)
6830
Compd
R2
KDRIC 50 (nM)
1610
4a
5a
4b
4c
4d
4e
4f
340
273
5b
5c
5d
5e
2390
1410
149
3570
276.5
135
45Æ7
5f
5g
5h
5i
1870
60
765
4220
232
5j
intrinsic activity to the lead 1. These data suggest
that the unsubstituted phenyl ring of 2d might fit
tightly in the H-1 hydrophobic pocket of the ATP
15
active site of the KDRkinase domain
since even
the relatively small electronic perturbation found in
compound 5e resulted in nearly a 10-fold loss in
potency. Conversely, the addition of functionality
onto one of the methyl groups of the aniline ring in
2d, as in 3c and 3d, had little to no effect on intrinsic
potency. This suggests that the aniline portion of 2d
is exposed to solvent and is consistent with our
binding hypothesis.
Scheme 1. Synthesis of compounds 2a–l, 3a–d, and 4a–f.14
Other modifications of 2d included movement of the
aniline portion of the molecule from the 2-position to
the 6-position of the pyrimidine core (6), deletion of N-3
in the pyrimidine ring (7), and the addition of a methyl
group in the 4-position of the imidazole ring (8, Fig. 3).
In each case a drastic loss in intrinsic potency was
observed relative to 2d (KDRIC 50=1808, 892, and 450,
respectively).
The cellular activity of a number of potent compounds can
be found in Table 5. As with intrinsic activity, the addition
of meta-functionality on the aniline ring of 1 increased
potency. The addition of polar functionality gave mixed
results. Alcohol 3c maintained its cellular potency while
the amine 3d suffered a 20-fold loss in potency relative to
2d. The latter is contrary to reports in other series from
our laboratories11 where the addition of a basic amine
had little effect on intrinsic potency but did have a
beneficial effect on cellular potency.
Scheme 2. Synthesis of compounds 5a–j.

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P. J. Manley et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1673–1677
Acknowledgements
We thank Dr. Art Coddington, Dr. Chuck Ross, and
Dr. Harri Ramjit for mass spectral analyses, and Matt
Zrada for logP determinations. We also thank Dr. Cory
Theberge and Irma Escudero for their synthesis of
intermediates.
References and Notes
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Figure 3. 16,17
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5. (a) Hanahan, D.; Folkman, J. Cell 1996, 86, 353. (b)
Holmgen, L.; O’Reilly, M. S.; Folkman, J. Nat. Med. 1995, 1,
149.
Table 5. Comparison on intrinsic and cellular potency of compounds
1, 2b, 2d, 2f, 2h, 2k, 3a, 3c, and 3d12,13
Compd
KDRIC 50 (nM)
Cell IC50 (nM)
6. (a) Veikkola, T.; Karkkainen, M.; Claesson-Welsh, L.;
Alitalo, K. Cancer Res. 2000, 60, 203. (b) Thomas, K. A. J.
Biol. Chem. 1996, 271, 603.
7. Kim, K. J.; Li, B.; Winer, J.; Armanini, M.; Gillett, N.;
Phillips, H. S.; Ferrara, N. Nature 1993, 362, 841.
8. Witte, L.; Hicklin, D.; Zhu, Z.; Pytowski, B.; Kotanides,
H.; Rockwell, P.; Bohlen, P. Cancer Metastasis Rev. 1998, 17,
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9. (a) Fong, T. A. T.; Shawyer, L. K.; Sun, L.; Tang, C.; App,
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W.; Ullrich, A.; Hirth, K. P.; McMahaon, G. Cancer Res.
1999, 59, 99. (b) Drevs, J.; Hofmann, I.; Hugenschmidt, H.;
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G.; Unger, C.; Marme, D. Cancer Res. 2000, 60, 4819.
10. Bilodeau, M. T.; Fraley, M. E.; Hartman, G. D. Expert
Opin. Invest. Drugs 2002, 11, 737.
11. (a) Fraley, M. E.; Hoffman, W. F.; Rubino, R. S.; Hungate,
R. W.; Tebben, A. J.; Rutledge, R. Z.; McFall, R. C.; Huckle,
W. R.; Kendall, R. L.; Coll, K. E.; Thomas, K. A. Bioorg. Med.
Chem. Lett. 2002, 12, 2767. (b) Fraley, M. E.; Rubino, R. S.;
Hoffman, W. F.; Hambaugh, S. R.; Arrington, K. A.; Hun-
gate, R. W.; Bilodeau, M. T.; Tebben, A. J.; Rutledge, R. Z.;
Kendall, R. L.; McFall, R. C.; Huckle, W. R.; Coll, K. E.;
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1
105Æ7
18Æ3
6Æ1
8% inhibition @ 500
2b
2d
2f
2h
2k
3a
3c
3d
151
19
88
22
303
26
23
396
20Æ9
5Æ1
10Æ1
5Æ3
9Æ1
32Æ5
Table 6. Selectivity profile of compounds 1, 2b, and 2d
Compd PDGFRb FLT-1 FLT-4 FGFR-1 FGFR-2 SRC
1
2b
2d
nd
22
6
13
103
30
5
13
5
295
1023
606
nd
686
261
11
11
10
nd, not determined.
The selectivity profile of 1, 2b, and 2d is found in Table
6. The data, expressed as a ratio of IC50 to KDR
kinase, suggests modest selectivity versus the highly
homologous kinases PDGFRb, Flt-1, Flt-4, and SRC
kinases and high selectivity versus FGFR-1 and
FGFR-2. These data are representative for the entire
series.
12. The KDRIC value represents biochemical inhibition of
50
phosphorylation of a poly-Glu/Tyr (4:1) peptide substrate by
isolated KDRkinase (cloned and expressed as a GST-fusion
protein); see: Kendall, R. L.; Rutledge, R. Z.; Mao, X.; Teb-
ben, A. L.; Hungate, R. W.; Thomas, K. A. J. Biol. Chem.
1999, 274, 6453. Values are reported as single determinations
or as the average of at least two determinationsÆstandard
deviation.
13. The Cell IC50 value represents the inhibition of VEGF-
stimulated mitogenesis as determined in human umbilical vein
endothelial cells.
14. All target compounds were fully characterized by 1H
NMRand mass spectroscopy.
In this series, the optimized compound 2d exhibits
excellent intrinsic activity and cell potency with modest
kinase selectivity. 2d has the following physical and
pharmacokinetic properties: a logP of 3.7418 with a high
clearance (Cl=30.10Æ1.27 mL/min/kg), volume of dis-
tribution (Vdss=1.11Æ0.01 L/kg), and short half-life
(t1/2=0.71Æ0.01 h) in dogs.
15. For a description of the ATP binding region of protein
tyrosine kinases, see: Traxler, P.; Furet, P. Pharmacol. Ther.
1999, 82, 195.
In conclusion we have investigated the SARfor KDR
inhibition of lead compound 1. meta-Substitution of the
aniline portion of compound 1 led to potency enhance-
ments both with the enzyme and in cells.
16. 6 was synthesized according to Scheme 1 starting with
commercially available 4,6-dichloropyrimidine. 8 was synthe-
sized according to Scheme 2 using 4(5)-methyl-2-phenylimid-
azole as the imidazole in the last step.

P. J. Manley et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1673–1677
1677
17.
18. Partition coefficients were determined by HPLC analysis
in octanol/phosphate buffer (pH 7.4).