Pyridylmethylthio derivatives as VEGF inhibitors. Part 1

Article abstract of DOI:10.1016/j.bmcl.2010.10.096

Optimization from compound 4a, having intramolecular S-O nonbonded interaction, led to discover compounds 4m and 4n. They were highly active in vitro (VEGF induced HUVEC proliferation assay) and showed efficacies in three disease models in vivo (cancer, R

Full text of DOI:10.1016/j.bmcl.2010.10.096

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7234–7238
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyridylmethylthio derivatives as VEGF inhibitors. Part 1
Hisashi Tajima a,b, , Takahiro Honda a,b, Kenji Kawashima a, Yoshimasa Sasabuchi a, Minoru Yamamoto a,b
,
⇑
Masakazu Ban a,b, Kazuyoshi Okamoto a, Kenji Inoue a, Takaaki Inaba a, Yuriko Takeno a, Hiroyuki Aono a,b
a Research and Development Center, Santen Pharmaceutical Co. Ltd, 8916-16 Takayama-cho Ikoma-shi, Nara 630-0101, Japan
b Graduate School of Materials Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho Ikoma-shi, Nara 630-0192, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimization from compound 4a, having intramolecular S–O nonbonded interaction, led to discover com-
pounds 4m and 4n. They were highly active in vitro (VEGF induced HUVEC proliferation assay) and
showed efficacies in three disease models in vivo (cancer, RA, AMD).
Received 18 August 2010
Revised 5 October 2010
Accepted 20 October 2010
Available online 26 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
VEGF
Pyridylmethylthio
RA
Cancer
AMD
Angiogenesis is closely related to tumor proliferation or
metastasis,1 and also pathogenesis and the progression of rheuma-
toid arthritis (RA)2 and age-related macular degeneration (AMD).3
The pathway of signal transduction through vascular endothelial
growth factor (VEGF) plays a very important role in pathological
angiogenesis. It is considered that VEGF receptor 2 (VEGFR2) inhib-
itors are likely to be useful agents for these diseases. And recently,
the anti-VEGF antibody, bevacizumab (Avastin), was filed by the
FDA as a therapeutic agent for cancer and AMD.4
Several small molecule VEGFR2 inhibitors have also been re-
ported as the orally available drugs or candidates for the treatment
of these diseases. They inhibit VEGFR2 kinase (KDR) selectively or
non-selectively and have various scaffolds, such as indolinones,5
quinazolines,6 quinolines,7 and phthalazines. PTK-787 (1), which
has a phthalazine ring, has been developed in clinical studies8
(Fig. 1). AAL-993 (2)9 and AMG-706 (3),10 categorized anthranil
amide derivatives, demonstrated efficacy in vitro and in vivo in
preclinical studies. Furthermore, it was reported that compound
2 has similar conformation to 1 by its intramolecular hydrogen
bond.11
of the selected compounds. We utilized the cell based assay (VEGF
induced human umbilical vein endothelial cell (HUVEC) prolifera-
tion assay)13 as evaluation of in vitro growth inhibitory activity
Cl
HN
CF3
HN
N
N
N
O
intramolecular
hydrogen bond
H
N
N
1
2
Cl
N
H
HN
S
HN
Recently we reported that 4-pyridylmethylthio derivative 4a
was discovered, its conformation was controlled by S–O intramo-
lecular nonbonded interaction, and has similar conformation to
compounds 1 and 2.12
O
O
intramolecular
non-bonded
S-O interaction
N
NH
In this Letter, we report the syntheses, in vitro assay of the var-
ious derivatives of compound 4a, and in vivo biological evaluations
N
N
3
4a
⇑
Corresponding author. Tel.: +81 743 79 4527.
E-mail address: hisashi.tajima@santen.co.jp (H. Tajima).
Figure 1. Chemical structures of compounds 1–3 and 4a.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.096

H. Tajima et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7234–7238
7235
Table 1
part C
Activity of novel 4-pyridylmethylthio derivatives
Cl
Cl
HN
O
HN
part A
N
S
O
A
part B
N
N
a
Figure 2. Strategy of optimization from 4a.
Compd
A
IC50 (nM)
33
1
for structural development. In our strategy these derivatives were
divided into three parts of compound 4a as shown in Figure 2.
At first we investigated nicotinic acid moiety defined as part A
(Table 1). Compound 5a, which had a benzene ring as a replace-
ment of the pyridine ring, showed moderate potency. However,
compounds 6–8, on which substituents were introduced, showed
diminished activity. These results indicated that the introduction
of substituents at each position on the benzene ring might be not
preferable. Compounds 9 and 10 did not show activity at all,
though these compounds had pyridine rings like 4a. In the same
way, though compounds 11a, 12, and 13 had thiophene rings, only
13 did not show the activity. It is difficult to explain these differ-
ences of activity only with the angle by two substituents on the
rings. Compound 14 did not show activity, and its sulfoxide group
was considered to prevent from conforming S–O intramolecular
interaction like 4a.
4a
5a
230
480
N
S
S
F
6
7
8
1500
S
>2000
>2000
Cl
S
S
S
Cl
N
Next we optimized part B and assay results are shown in Table
2. Changing from 4-pyridyl group to 2-pyridyl and 3-pyridyl group,
like compounds 15 and 16, respectively, resulted in diminishing
potency. The other modifications, compounds 17–19, also led to re-
duce activity. These results indicated the 4-pyridylmethylthio
group was the most suitable to show the activity.
9
>2000
>2000
240
N
10
11a
S
On the other hand, the investigation of the substitution on the
benzene ring defined as part C led to interesting results (Table 3).
The activity of N-(3-chlorophenyl)nicotinamide 4b got better,
however N-(2-chlorophenyl)nicotinamide 4c did not show activity
at all. In the same way, compound 4f showed much less potency
than compounds 4d and 4e. The substitution on 2-position seemed
to affect the conformation of the derivatives. The activity of unsub-
stituted compound 4g was not so potent, so functional groups were
considered to be required on 3 or 4 position. Then we designed and
synthesized several 3 and/or 4-substituted derivatives. As a result,
we found compounds, such as 4i, 4k, 4l and 4m, have the most po-
tent activity. Interestingly, not only electron withdrawing groups
but also electron donating groups seemed to be acceptable as sub-
stituents. And these results indicated 3-position was more prefer-
able than 4-position as the point of substitution. As a further
investigation, the combination of favorable groups was explored
in each part, and highly potent compounds 5k and 5m were found
(Table 4).
S
S
12
13
14
510
S
S
S
>2000
>2000
S
O
N
S
a
IC50 values are the half inhibition concentrations (nM) of VEGF induced HUVEC
proliferation.
These derivatives can be synthesized generally in three routes
(Scheme 1). In the first route, S-alkylation of 2-mercaptonicotinoic
acid (20) with 4-(bromomethyl)pyridine hydrochloride (21) under
basic condition gave desirable carboxylic acids 22 without
purification. Compound 4a was obtained by the condensation of
intermediate 22 with 4-chloroaniline (23) in the presence of
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetrauronium hexafluorophos-
phate (HATU).14 However, in some cases the yields of the conden-
sation step were low because of the less nucleophilicity of aniline
with the electron withdrawing groups such as trifluoromethyl
group and chlorine atom. At that time the second route was used.
In this route, for the amide bond formation of compound 25,
4-chloroaniline (23) was carried out at first by treating with
2-chloronicotinic acid chloride (24) under basic condition. By the
following reaction with 4-(mercaptomethyl)pyridine hydrochlo-
ride (26) in the presence of potassium carbonate, compound 4a
could be furnished via the sulfide bond formation. On the other
hand, in the syntheses of benzanilide like 5k the second route
could not be applied. So another route was explored, and
palladium coupling was adopted to introduce 4-(mercaptomeh-
tyl)pyridine hydrochloride (26) as shown in the third route.

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H. Tajima et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7234–7238
Table 2
Table 3
Activity of various 4-arylmethylthio derivatives
Activity of R-group variants of novel 4-pyridylmethylthio derivatives
3
Cl
4
2
R
HN
O
5
HN
S
6
O
N
S
R
N
a
N
Compd
R
IC50 (nM)
a
Compd
R
IC50 (nM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4-Cl
3-Cl
2-Cl
4-OMe
3-OMe
2-OMe
H
4-Me
3-Me
4-CF3
3-CF3
3,4-di-Me
3,5-di-Me
4-OCF3
230
29
4a
230
N
>2000
230
200
1800
370
140
<2.7
48
11
22
6.2
54
15
16
>2000
N
>2000
>2000
>2000
>2000
N
3-OCF3
27
17
18
19
a
IC50 values are the half inhibition concentrations (nM) of VEGF induced HUVEC
proliferation.
F
O
N
AMD. These results suggested that compounds 4m and 4n had
favorable properties for drugs against above mentioned
diseases.
Table 4
Activity of centered ring variants of novel 4-pyridylmethylthio derivatives
a
IC50 values are the half inhibition concentrations (nM) of VEGF induced HUVEC
proliferation.
R
HN
O
Thiophene-2-carboxamide derivatives 11a, 11k, and 11m were
ring
also synthesized with this palladium catalyzed coupling.
S
Several derivatives were evaluated in mouse xenograft model15
,
and among them compound 4m showed a potent effect at 100 mg/
kg/day (Table 5). On the other hand, compounds 4i, 4k and 5m
showed only moderate efficacy in spite of its potent activity in cell
based assay. Especially, 5k did not show efficacy. Conversely com-
pound 4n showed potent efficacy in comparison with its activity in
vitro.
N
a
Compd
Ring
R
IC50 (nM)
4k
3-CF3
11
N
Then compounds 4m and 4n showed efficacy in rat adjuvant
arthritis model16 at 10 mg/kg/day, po as the model of RA (Table
6). Moreover these compounds also showed efficacy in rat laser in-
duced choroidal neovascularization (CNV) model17 at 30 mg/kg/
day, po, which is the disease model of AMD (Table 7). These results
indicated that compounds 4m and 4n have potential as a therapeu-
tic drug for cancer, RA and AMD clinically with oral administration.
In conclusion we designed, synthesized and evaluated the novel
4-pyridylmethylthio derivatives from 4a to discover effective VEG-
FR2 inhibitors. As a result, several compounds showed activity in
the cell based assay (VEGF induced HUVEC proliferation assay).
Among them compounds 4i, 4k, 4m, 5k and 5m showed highly po-
tent activity. And compounds 4m and 4n showed efficacy in all
three animal models; one is xenograft model reflecting cancer, an-
other is rat adjuvant arthritis model revealing the RA, and the other
is rat laser induced CNV model known as the indicating model of
5k
3-CF3
3-CF3
3.3
S
11k
400
4m
3,5-di-Me
6.2
N
S
5m
3,5-di-Me
3,5-di-Me
8.7
11m
240
a
IC50 values are the half inhibition concentrations (nM) of VEGF induced HUVEC
proliferation.

H. Tajima et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7234–7238
7237
Br
O
HBr
O
OH
Cl
21
N
OH
N
S
H2N
b
N
SH
a
23
N
20
Cl
22
HN
S
O
N
Cl
N
4a
Cl
SH
HCl
N
O
H2N
23
d
HN
Cl
26
O
c
N
Cl
N
Cl
24
25
SH
HCl
CF3
28
H2N
CF3
HN
S
N
26
O
I
CF3
HN
I
O
Cl
O
e
f
29
27
N
5k
Scheme 1. Reagents and conditions: (a) triethylamine, DMF, rt, 6 h, 61%; (b) HATU, DIEA, DMF, rt, 3 h, 91%; (c) DIEA, THF, rt, 3 h, 82%; (d) K2CO3, EtOH, 65 °C, 8 h, 37%; (e)
DIEA, THF, rt, 24 h, quant.; (f) Pd2(dba)3, dppf, DIEA, DMA, 60 °C, 21 h, 76%.
Table 5
Supplementary data
Efficacy on mouse xenograft model (100 mg/kg/day, po)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.096.
Compd
Inhibition% of tumor tissue weight
4i
40
35
71
69
9
4k
4m
4n
5k
5m
References and notes
1. Shibuya, M. Folia Pharmacol. Japonica 2003, 122(6), 498.
2. Sone, H.; Kawakami, Y.; Sakauchi, M.; Nakamura, Y.; Takahashi, A.; Shimano,
H.; Okuda, Y.; Segawa, T.; Suzuki, H.; Yamada, N. Biochem. Biophys. Res.
Commun. 2001, 281, 562.
49
3. Kvanta, A.; Algvere, P. V.; Berglin, L.; Seregard, S. Invest. Ophthalmol. Vis. Sci.
1996, 37, 1929.
4. Ferrara, N.; Hillan, K. J.; Gerber, H. P.; Novotny, W. Nat. Rev. Drug Disc. 2004, 3,
391.
Table 6
Efficacy on rat adjuvant arthritis model (10 mg/kg/day, po)
5. Sun, L.; Liang, C.; Shirazian, S.; Zhou, Y.; Miller, T.; Cui, J.; Fukuda, J. Y.; Chu, J. Y.;
Sistla, A.; Luu, T. C.; Tang, F.; Wei, J.; Tang, C. J. Med. Chem. 2003, 46, 1116.
6. Hennequin, L. F.; Stokes, E. S. E.; Thomas, A. P.; Johnstone, C.; Ple, P. A.; Ogilvie,
D. J.; Dukes, M.; Wedge, S. R.; Kendrew, J.; Curwen, J. O. J. Med. Chem. 2002, 45,
1300.
Compd
Inhibition% of the paw edema
4m
4n
46
60
7. Kubo, K.; Shimizu, T.; Ohyama, S.; Murooka, H.; Iwai, A.; Nakamura, K.;
Hasegawa, K.; Kobayashi, Y.; Takahashi, N.; Takahashi, K.; Kato, S.; Izawa, T.;
Isoe, T. J. Med. Chem. 2005, 48, 1359.
Table 7
8. (a) Bold, G.; Altmann, K. H.; Jorg, F.; Lang, M.; Manley, P. W.; Traxler, P.;
Wietfeld, B.; Bruggen, J.; Buchdunger, E.; Cozens, R.; Ferrari, S.; Furet, P.;
Hofmann, F.; Martiny-Baron, G.; Mestan, J.; Rosel, J.; Sills, M.; Stover, D.; Masso,
E.; Roth, R.; Schlachter, C.; Vetterli, W.; Wyss, D.; Wood, J. J. Med. Chem. 2000,
43, 2310; (b) Bold, G.; Altmann, K. H.; Jorg, F.; Lang, M.; Manley, P. W.; Traxler,
P.; Wietfeld, B.; Bruggen, J.; Buchdunger, E.; Cozens, R.; Ferrari, S.; Pascal, F.;
Hofmann, F.; Martiny-Baron, G.; Mestan, J.; Rosel, J.; Sills, M.; Stover, D.; Masso,
E.; Roth, R.; Schlachter, C.; Vetterli, W.; Wyss, D.; Wood, J. J. Med. Chem. 2000,
43, 3200.
Efficacy on rat laser induced choroidal neovascularization model
(30 mg/kg/day, po)
Compd
Inhibition% of neovascularization incidence rate
4m
4n
22
51

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H. Tajima et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7234–7238
9. Manley, P. W.; Furet, P.; Bold, G.; Brueggen, J.; Mestan, J.; Meyer, T.; Schnell, C.
R.; Wood, J. J. Med. Chem. 2002, 45, 5687.
10. Polverino, A.; Coxon, A.; Starnes, C.; Diaz, Z.; DeMelfi, T.; Wang, L.; Bready, J.;
Estrada, J.; Cattley, R.; Kaufman, S.; Chen, D.; Gan, Y.; Kumar, G.; Meyer, J.;
Neervannan, S.; Alva, G.; Talvenheimo, J.; Montestruque, S.; Tasker, A.; Patel,
V.; Radinsky, R.; Kendall, R. Cancer Res. 2006, 66, 8715.
11. Manley, P. M.; Bold, G.; Brüggen, J.; Fendrich, G.; Furet, P.; Mestan, J.; Schnell,
C.; Stolz, B.; Meyer, T.; Meyhack, B.; Stark, W.; Strauss, A.; Wood, J. Biochim.
Biophys. Acta, Proteins Proteomics 2004, 1697, 17.
12. Honda, T.; Tajima, H.; Kaneko, Y.; Ban, M.; Inaba, T.; Takeno, Y.; Okamoto, K.;
Aono, H. Bioorg. Med. Chem. Lett. 2008, 18, 2939.
13. (a) Annie, T.; Fong, T.; Shawver, L. K.; Sun, L.; Tang, C.; App, H.; Powell, T. J.;
Kim, Y. H.; Schreck, R.; Wang, X.; Risau, W.; Ullrich, A.; Hirth, K. P.; McMahon,
G. Cancer Res. 1999, 59, 99; (b) HUVEC was cultured into collagen coated well
plate. After one day incubation VEGF and a test compound were added. After
three days incubation, cell number was counted. Compared with control, cell
growth rate was calculated; see also Supplementary data.
Cancer Res. 1999, 59, 5209; (b) B16 cell suspension was s.c. injected into the
C57BL/6 mouse (female, six weeks old) under anesthesia. The suspension of a
test compound was administrated orally once a day every day. Ten days after
injection, the weight of extirpated tumor tissue was measured. Compared with
control, anti-cancer effect was calculated; see also Supplementary data.
16. The adjuvant was injected subcutaneously into a left hind paw sole of Lewis
rats (male, nine weeks old) to induce arthritis. The suspension of a test
compound was administrated orally once a day every day. Twenty one days
after induction, the paw volume was measured. Compare with control, paw
edema inhibition rate was calculated; see also Supplementary data.
17. (a) Xianjin, X.; Ogata, N.; Komada, M.; Yamamoto, C.; Takahashi, K.; Omori, K.;
Uyama, M. Graefe’s Arch. Clin. Exp. Ophthalmol. 1997, 235, 313; (b) Under
anesthesia and mydriasis, photocoagulation was performed in
a Bruch’s
membrane of Brown Norway rats (male, eight weeks old) with a krypton
laser. The suspension of a test compound was administrated orally once a day
every day. Seven days after photocoagulation, fluorescein solution was injected
into
a tail vein. After fluorescein fundus photography was performed,
14. Carpino, L. Z. J. Am. Chem. Soc. 1993, 115, 4397.
15. (a) Prewett, M.; Huber, J.; Li, Y.; Santiago, A.; O’Connor, W.; King, K.;
Overholser, J.; Hooper, A.; Pytowski, B.; Witte, L.; Bohlen, P.; Hicklin, D. J.
fluorescence leak was counted. Compared with control, Neovascularization
incidence rate was calculated; see also Supplementary data.