Development of novel inhibitors targeting HIF-1α towards anticancer drug discovery

Article abstract of DOI:10.1016/j.bmcl.2010.09.083

Hypoxia-inducible factor-1α (HIF-1α) is a critical regulatory protein of cellular response to hypoxia, and regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, vascularization, and invasion/metastasis. HIF-1α has been implicated in the regulation of genes involved in angiogenesis, for example, VEGF and is associated with tumor progression. In the last decade, over expression of HIF-1α has been demonstrated in many common human cancers and emerging as a validated target for anticancer drug discovery. Here we report the discovery of newly designed and synthesized pyridylpyrimidine based potent and selective inhibitors of HIF-1α. P2630 has been found as potent antiproliferative, antiangiogenic and orally efficacious compound in PC-3 xenograft mice model.

Full text of DOI:10.1016/j.bmcl.2010.09.083

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6426–6429
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of novel inhibitors targeting HIF-1
drug discovery
a towards anticancer
Nilambari Yewalkar a, Vijaykumar Deore a, Amol Padgaonkar b, Sonal Manohar b, Bichismita Sahu a,
Pramod Kumar a, Archana Jalota-Badhwar b, Kalpana S. Joshi b, Somesh Sharma b, Sanjay Kumar a,
⇑
a Department of Medicinal Chemistry, Piramal Life Sciences, 1-Nirlon Complex, Off Western Express Highway, Goregaon (E), Mumbai 400 063, India
b Department of Pharmacology, Piramal Life Sciences, 1-Nirlon Complex, Off Western Express Highway, Goregaon (E), Mumbai 400 063, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2010
Revised 6 September 2010
Accepted 14 September 2010
Available online 18 September 2010
Hypoxia-inducible factor-1
regulates the transcription of many genes involved in key aspects of cancer biology, including immortali-
zation, maintenance of stem cell pools, cellular dedifferentiation, vascularization, and invasion/metastasis.
a
(HIF-1
a
) is a critical regulatory protein of cellular response to hypoxia, and
HIF-1
a
has been implicated in the regulation of genes involved in angiogenesis, for example, VEGF and is
has been demonstrated
associated with tumor progression. In the last decade, over expression of HIF-1
a
in many common human cancers and emerging as a validated target for anticancer drug discovery. Here
we report the discovery of newly designed and synthesized pyridylpyrimidine based potent and selective
Keywords:
Hypoxia
inhibitors of HIF-1
a. P2630 has been found as potent antiproliferative, antiangiogenic and orally efficacious
HIF-1
a
compound in PC-3 xenograft mice model.
Small molecule
Anticancer
Ó 2010 Elsevier Ltd. All rights reserved.
Antiproliferative
Drug discovery
Pyridylpyrimidine
Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of
a
and b
availability and activity of HIF-1
of HIF-1 protein.8 HIF-1
is now considered as a potential target for
many common solid tumors.2 Various advance molecules targeting
aprotein determines the bioactivity
transcriptional factor, and a master regulator of transcriptional re-
sponse to oxygen deficiency. HIF-1 has been implicated in the regu-
lation of genes involved in tumorigenesis, angiogenesis, glycolysis,
survival, growth, invasion, metastasis, to treatment resistance.1–4
a
HIF-1a
are under clinical trials, for example, Topotecan,9 PX478,10
YC-1.11 Recently boron-containing phenoxyacetanilide based
molecule (GN26361),12 AC1-00113 and its benzimidazole derivative
The over expression of HIF-1
stage and poor prognosis in many common human cancers.5 The
HIF-1 subunit is degraded rapidly in normoxic conditions and sta-
bilized under hypoxic conditions, while HIF-1b is constitutively ex-
pressed as a nuclear protein. Under normoxia HIF-1 protein
undergoes hydroxylation at specific prolyl residue by prolylhydrox-
ylase (PHD), this hydroxylated HIF-1 is recognized by von Hippel-
a is associated with advanced disease
AC-00414 have been reported as lead molecules targeting HIF-1
a
. In
the present study, we herein report the design, synthesis, and dis-
covery of pyridylpyrimidine based inhibitors of HIF-1 . In our quest
a
a
a
for finding potential therapeutics, we choose pyridylpyrimidine as a
basic scaffold. The substituted amino-pyridyl and pyridylpyrimi-
dine scaffold is a very common structural motif that can be found
in many natural products and in several pharmacologically interest-
ing compounds.15–17 Therefore, the synthesis of pyridylpyrimidine
derivatives, with the objective of developing new drugs, is an active
area of research.18 Based on the above rationale we designed,
synthesized and characterized various pyridylpyrimidine based
molecules18 (3a–g, 4a–h, 5a–e, 6a–e, and 7) as out lined in Scheme
1. In brief 5-aminopicolinonitrile (1) was treated with ethanolic-HCl
followed by ethanolic-ammonia till basic pH. This reaction mixture
was stirred at room temperature for about 12–15 h to get 4-amino-
picolinimidamide hydrochloride (2). Compound 2 on treatment
with substituted b-ketoester in ethanol/water (1:2) mixture and
Na2CO3 at room temperature for 22 h yielded corresponding
2-(5-aminopyridin-2-yl)pyrimidin-4-ol based compounds (3a–g)
in fairly good yield (Scheme 1). Compounds 3a–g were subjected
a
Lindau (VHL) tumor suppressor protein and undergoes for proteaso-
mal degradation.6 Under hypoxia, PHD is not activated and hence
the hydroxylation by PHD is compromised, which leads to HIF-1
a
stabilization. Stabilized HIF-1 translocates to the nucleus and
a
forms a heterodimeric complex with HIF-1b. This complex further
binds to hypoxia response element (HRE) DNA sequence with co-
activators to activate various genes involved in angiogenesis
(through VEGF), erythropoietin (EPO), glycolysis etc.1–4,7 In addition
to hypoxia, the activation of oncogenes and/or inactivation of tumor
suppressor genes can also lead to HIF-1 activation.1–4 In general, the
⇑
Corresponding author. Tel.: +91 22 30818317; fax: +91 22 30818334.
E-mail
addresses:
sanjay.kumar@piramal.com,
sanjaykrai71@gmail.com
(S. Kumar).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.083

N. Yewalkar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6426–6429
6427
OH
R1
NH
NH2.HCl
N
CN
N
R2
(a)
(b)
3a: R1 = H, R2 = CF3; 3b: R1 = Cl, R2 = Me
3c: R1 = Me, R2 = Me; 3d: R1 = Et, R2 = Me
3e: R1 = H, R2 = -CH2Cl; 3f: R1 = -CH2Ph, R2 = Me
3g: R1 = -F, R2 = Me
N
N
N
H2N
H2N
H2N
(3a-g)
1
2
(d)
(c)
R4
O
R4
Cl
N
OH
N
O
N
R1
R2
R1
R2
R1
R2
R1
R2
N
N
N
N
(c)
N
(e)
R3
N
R3
N
R3
N
N
N
N
H
N
H
H2N
H
7
(4a-h)
(5a-e)
(6a-e)
4a: R1 = H, R2 = CF3, R3 = -COCH2Cl
4b: R1 = Cl, R2 = Me, R3 = -COCH2Cl
4c: R1 = Me, R2 = Me, R3 = -COCH2Cl
4d: R1 = Et, R2 = Me, R3 = -COCH2Cl
5a: R1 = Et, R2 = Me, R4= Allyl
6a: R1 = Et, R2 = Me, R3 = -COCH2Cl, R4= Allyl
6b: R1 = H, R2 = CF3, R3 = -COCH2Cl, R4= Allyl
6c: R1 = H, R2 = CF3, R3 = -COCH2Cl, R4= Me
6d: R1 = Cl, R2 = Me, R3 = -COCH2Cl, R4= Allyl
6e: R1 = H, R2 = CF3, R3 = -COCH2Cl, R4= Et
R1 = H, R2 = CF3,
5b: R1 = H, R2 = CF3, R4= Allyl
5c: R1 = H, R2 = CF3, R4= Me
5d: R1 = Cl, R2 = Me, R4= Allyl
5e: R1 = H, R2 = CF3, R4= Et
R3 = -COCH2Cl
4e: R1 = H, R2 = -CH2Cl, R3 = -COCH2Cl
4f: R1 = -CH2Ph, R2 = Me, R3 = -COCH2Cl
4g: R1 = -F, R2 = Me, R3 = -COCH2Cl
4h: R1 = H, R2 = CF3, R3 = -COCF2Cl
Scheme 1. Reagents and conditions: (a) ethanol, HCl gas till saturation, stir at rt for 30 min, then addition of ethanolic-ammonia at À20 °C till basic pH, then stir at rt for
about 12–15 h; (b) substituted b-ketoester, ethanol/water (1:2), Na2CO3, rt for 22 h; (c) triethyl amine, substituted acetyl chloride in THF, rt, 1 h; (d) alkyl halide, K2CO3, DMF,
rt, 1 h; (e) benzyl triethyl ammonium chloride, dimethylaniline, POCl3, CH3CN, reflux, 3 h.
Table 1
HIF-1
to acylation of amine using various acetyl chlorides in THF at room
a inhibitory activity of new compounds 3a–g, 4a–h, 5a–e, 6a–e, and 7 under
hypoxia and normoxia
temperature for 1 h, yielded compounds 4a–h. Compound 4a was
further refluxed in acetonitrile for 3 h using benzyltriethylammo-
nium chloride and POCl3 to get compound 7. In another set of
reaction 3a–g were treated with various alkyl halides and K2CO3 in
DMF at room temperature for 1 h to get compounds 5a–e. Com-
pounds 5a–e on further acylation yielded compounds 6a–e. All these
newly synthesized compounds were characterized by 1H NMR, MS,
HRMS and purity (>99%) by HPLC.
Compd No.
HIF-1aa (hypoxia)
Normoxiaa
SI
3a
>30
>30
>30
>30
6
>30
>30
0.8
5.5
>30
1.5
1.5
4
ND
ND
ND
ND
22
ND
ND
8
ND
ND
5.5
5.5
10
ND
ND
ND
ND
3.6
ND
ND
10
ND
ND
3.6
3.6
2.5
3
3b
3c
3d
3e
3f
3g
These newly synthesized compounds were screened against
4a (P2630)
4b
4c
4d
4e
HIF-1
normoxic (21% O2) conditions.9 Compounds 4a, 4d, 4e, 6d and 7
have shown significant inhibition of HIF-1 under hypoxia
(61.5 M). Further, these HIF-1 active compounds were subjected
a by reporter gene based assay under hypoxic (1% O2) and
a
l
a
4f
4g
to reporter gene based assay using U251-pGL3 cell line under nor-
moxia to evaluate their specificity index (SI) as shown in Table 1.
The higher SI indicates the specificity of compounds in hypoxia with
respect to normoxia. Compound 4a was found as the most potent
2
6
4h
5a
5b
5c
5d
5e
>30
>30
>30
>30
>30
>30
1.8
2
3.5
1.5
8
1.4
0.06
ND
ND
ND
ND
ND
ND
22
30
15
8
ND
10
ND
ND
ND
ND
ND
ND
12.2
15
4.3
5.3
ND
7.1
>50
and specific HIF-1
to as P2630. The inhibition of HIF-1
a
inhibitor with 10-fold SI and further referred
expression was further con-
a
firmed by Western blot analysis using U251-HRE and PC-3 cell lines
6a
in a dose dependent manner19 (Fig. 1).
6b
6c
6d
6e
After confirming the HIF-1a activity, compound 4a (P2630) was
subjected for antiproliferative activity using 3H-thymidine incor-
poration assay across different cancer cell lines such as prostate
cancer (PC-3 and DU-145), glioblastoma (U251), colon cancer
(HCT-116), ovarian cancer (Ovcar-3), pancreatic cancer (Panc-1),
and normal cell lines like MRC-5 and WI-38 (Table 2).
7
Topotecan
>3
a
IC50 values in lM; ND: not done; SI: specificity index. Topotecan evaluated as
positive control.
P2630 has shown the best antiproliferative activity in PC-3 cell
line (IC50; 1
WI-38 (normal cells) were >10 and 6.5
data suggest that P2630 has selective potential to inhibit HIF-1
expression under hypoxia over normoxia and specifically inhibits
the proliferation of cancer cells. Since it is known that HIF-1 regu-
l
M) whereas the antiproliferative IC50 in MRC-5 and
lM, respectively. These
lates the genes involved in angiogenesis, we thought to evaluate its
effect on VEGF mRNA expression under hypoxia in PC-3 cells. The
inhibition of VEGF mRNA expression by P2630 in dose dependent
a

6428
N. Yewalkar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6426–6429
Figure 1. Western blot analysis showing the effect of P2630 on HIF-1
a protein expression in U251-HRE and PC-3 cell lines. P2630 specifically inhibited HIF-1a protein
accumulation under hypoxia in a dose dependent manner in both the cell lines.
Table 2
Anti-proliferative activity (IC50 in lM) of P2630 against human cancer cell lines & normal cell lines (MRC-5 & WI-38)
Compound
PC-3
HCT-116
U251
Ovcar-3
DU-145
Panc-1
MRC-5
WI-38
P2630
Topotecan
1.0
1.0
0.8
ND
1.5
0.5
3.5
ND
1.2
1.2
2.0
ND
>10
ND
6.5
ND
ND: not done.
thelial tube formation assay as per published procedure.20 Topo-
tecan was used as positive control in this study (Fig. 3). Result
shows that P2630 significantly inhibits the HUVEC tube formation
at 3 and 5 lM in dose dependent manner.
Considering the antiproliferative potential of P2630 in PC-3 can-
cer cells and its antiangiogenic potential, the in-vivo efficacy was
evaluated in PC-3 xenograft model using SCID mice. P2630 admin-
istered 50 mg/kg, bid, po for 19 days, which shows significant
tumor growth inhibition (TGI) as compared to vehicle treated ani-
mals (Fig. 4A). The mice treated with P2630 have no significant
weight loss during course of treatment (Fig. 4B).
In conclusion, we report the discovery of P2630,21 a structurally
novel inhibitor of HIF-1a. Compound P2630 displays very good
potency in both mechanistic and antiproliferative cellular assays.
Compound P2630 also exhibits significant oral in-vivo efficacy in
PC-3 xenograft model without any significant weight loss. The
further mechanistic studies of this molecule are warranted and will
be published in due course of time.
Figure 2. RT-PCR analysis showing the down regulation of VEGF mRNA by P2630
under hypoxia in PC-3 cell line in dose dependent manner at 3 and 5
was used as a positive control.
lM. Topotecan
manner was confirmed by RT-PCR as shown in Figure 2. The
antiangiogenic potential of P2630 was evaluated by 3D gel endo-
Figure 3. The antiangiogenic potential of P2630 by endothelial tube formation assay. P2630 was demonstrated to have significant inhibitory effect on HUVEC tube formation
in a dose dependent manner (3 and 5 M) Topotecan has been taken as positive control.
l

N. Yewalkar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6426–6429
6429
Figure 4. In vivo efficacy of P2630 in PC-3 human tumor xenograft. (A) SCID mice were dosed twice a day for 19 days. Tumor size (median tumor volume) was measured on
5th, 7th, 12th, and 19th day and compared w.r.t tumor volume of vehicle treated mice. (B) Percentage weight profile of P2630 treated and vehicle treated mice during course
of treatment.
12. Shimizu, K.; Maruyama, M.; Yasui, Y.; Minegishi, H.; Ban, H. S.; Nakamura, H.
Acknowledgments
Bioorg. Med. Chem. Lett. 2010, 20, 1453.
13. Lee, K.; Lee, J. H.; Boovanahalli, S. K.; Jin, Y.; Lee, M.; Jin, X.; Kim, J. H.; Hong, Y.
We are thankful to department of analytical chemistry for pro-
viding NMR, mass, and HPLC for characterization of all novel
molecules.
S.; Lee, J. J. J. Med. Chem. 2007, 50, 1675.
14. Won, M. S.; Im, N.; Park, S.; Boovanahalli, S. K.; Jin, Y.; Jin, X.; Chung, K. S.; Kang,
M.; Lee, K.; Park, S. K.; Kim, H. M.; Kwon, B. M.; Lee, J. J.; Lee, K. Biochem.
Biophys. Res. Commun. 2009, 385, 16.
15. Deore, V.; Yewalkar, N.; Bhatia, D.; Desai, N.; Gupte, R. D.; Dadarkar, S. S.;
Jadhav, M. G.; Tannu, A. A.; Bhatt, P.; Nemmani, K. V.; Vishwakarma, R. A.;
Sharma, S.; Roychowdhury, A.; Dagia, N. M.; Bhonde, M. R.; Kumar, S. Bioorg.
Med. Chem. Lett. 2009, 19, 2949.
16. Cee, V. J.; Cheng, A. C.; Romero, K.; Bellon, S.; Mohr, C.; Whittington, D. A.; Bak,
A.; Bready, J.; Caenepeel, S.; Coxon, A.; Deak, H. L.; Fretland, J.; Gu, Y.; Hodous,
B. L.; Huang, X.; Kim, J. L.; Lin, J.; Long, A. M.; Nguyen, H.; Olivieri, P. R.; Patel, V.
F.; Wang, L.; Zhou, Y.; Hughes, P.; Geuns-Meyer, S. Bioorg. Med. Chem. Lett.
2009, 19, 424.
17. Musonda, C. C.; Whitlock, G. A.; Witty, M. J.; Brun, R.; Kaiser, M. Bioorg. Med.
Chem. Lett. 2009, 19, 401.
18. Kumar, S.; Deore, V.; Yewalkar, N.; Joshi, K.; Rathos, M.; Padgaonkar, A.;
Bhonde, M. Pyridyl derivatives, their preparation and use, WO 2009 019656 A1,
2009.
References and notes
1. Harris, A. L. Nat. Rev. Cancer 2002, 2, 38.
2. Semenza, G. L. Nat. Rev. Cancer 2003, 3, 721.
3. Giaccia, A.; Siim, B. G.; Johnson, R. S. Nat. Rev. Drug Disc. 2003, 2,
803.
4. Semenza, G. L. Expert Opin. Ther. Targets 2006, 10, 267.
5. Zhong, H.; De Marzo, A. M.; Laughner, E.; Lim, M.; Hilton, D. A.; Zagzag, D.;
Buechler, P.; Isaacs, W. B.; Semenza, G. L.; Simons, J. W. Cancer Res. 1999, 59,
5830.
6. Huang, L. E.; Gu, J.; Schau, M.; Bunn, H. F. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
7987.
7. Jiang, B. H.; Rue, E.; Wang, G. L.; Roe, R.; Semenza, G. L. J. Biol. Chem. 1996, 271,
17771.
8. Nagle, D. G.; Zhou, Y. D. Curr. Drug Targets 2006, 7, 355.
9. Rapisarda, A.; Uranchimeg, B.; Scudiero, D. A.; Selby, M.; Sausville, E. A.;
Shoemaker, R. H.; Melillo, G. Cancer Res. 2002, 62, 4316.
10. Koh, M. Y.; Spivak-Kroizman, T.; Venturini, S.; Welsh, S.; Williams, R. R.;
Kirkpatrick, D. L.; Powis, G. Mol. Cancer Ther. 2008, 7, 90.
11. Yeo, E. J.; Chun, Y. S.; Cho, Y. S.; Kim, J.; Lee, J. C.; Kim, M. S.; Park, J. W. J. Natl.
Cancer Inst. 2003, 95, 516.
19. Mottet, D.; Dumont, V.; Deccache, Y.; Demazy, C.; Ninane, N.; Raes, M.;
Michiels, C. J. Biol. Chem. 2003, 278, 31277.
20. Jang, Y. J.; Kim, D. S.; Jeon, O. H.; Kim, D. S. J. Biochem. Mol. Biol. 2007, 40, 439.
21. Spectral data for P2630 (4a): 2-chloro-N-(6-(4-hydroxy-6-(trifluoromethyl)-
pyrimidin-2-yl)pyridin-3-yl)acetamide: 1H NMR (DMSO-d6, 300 MHz): d:
12.80 (br s, 1H), 10.93 (s, 1H), 8.97 (s, 1H), 8.25–8.34 (m, 2H), 6.90 (s, 1H),
4.37 (s, 2H); MS: m/e (ESÀ) 331.02 (MÀ1); HRMS calcd for C12H8ClF3N4O2
332.0288; found: 333.0361 (M+H); HPLC purity 99.88%.