Design, synthesis and insight into the structure-activity relationship of 1,3-disubstituted indazoles as novel HIF-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.08.120

Design, synthesis and insight into the structure-activity relationship (SAR) of 1,3-disubstituted indazoles as novel HIF-1 inhibitors are described. In particular, the substituted furan moiety on indazole skeleton as well as its substitution pattern turns

Full text of DOI:10.1016/j.bmcl.2011.08.120

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6297–6300
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and insight into the structure–activity relationship
of 1,3-disubstituted indazoles as novel HIF-1 inhibitors
Hongchan An a, Nam-Jung Kim a, Jong-Wha Jung a, Hannah Jang a, Jong-Wan Park b, Young-Ger Suh a,
⇑
a College of Pharmacy, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
b College of Medicine, Seoul National University, 28 Yeongeon-dong, Jongro-gu, Seoul 110-799, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Design, synthesis and insight into the structure–activity relationship (SAR) of 1,3-disubstituted indazoles
as novel HIF-1 inhibitors are described. In particular, the substituted furan moiety on indazole skeleton as
well as its substitution pattern turns out crucial for the high HIF-1 inhibition.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 4 August 2011
Revised 24 August 2011
Accepted 29 August 2011
Available online 1 September 2011
Keywords:
YC-1
HIF-1
a
SAR (structure–activity relationship)
Hypoxia
Anti-cancer agent
Hypoxia-inducible factor 1 (HIF-1) is a transcription factor,
which is heterodimer consisting of a constitutively expressed
regulate HIF-1
any influence to HIF-1
a
expression at the mRNA-translational step without
protein half-life or its mRNA level.15 We
a
HIF-1b and an O2ꢀregulated HIF-1
a
as subunits. Under aerobic
is polyubiquitinated and then degraded by pro-
teasomes, leading to HIF-1 suppression. However, HIF-1 under
hypoxia is not degraded and intact HIF-1 dimerizes with
have also reported that YC-1 inhibits HIF-1 function by stimulating
factor-inhibiting-HIF (FIH) which disturbs the association of HIF-1
complex with p300/CBP (critical co-binders for transcription of
HIF-1 target genes).16 These reports have revealed the mode of
action of YC-1, which can directly inhibit HIF-1 function in biochem-
ical level. In spite of those studies on YC-1 as an anti-cancer agent,
few related report on the inhibitory effects of YC-1 analogues target-
ing HIF-1 or HIF-1 targeted gene expression has been reported.
Considering lack of information as well as significant importance
of the structure–activity relationship between the YC-1 analogues
regarding inhibitory effects on the HIF-1, elucidation of the struc-
ture–activity relationship through syntheses and biological evalua-
tion of YC-1 derived analogues obviously envisages development of
novel HIF-1 inhibitors, which ultimately would be utilized as novel
conditions, HIF-1
a
a
a
HIF-1b, leading to activation of HIF-1, which plays key roles in
physiological processes of not only cell survival1 but also cancer
development including angiogenesis, invasion and metastasis.
Due to hypoxic conditions in the solid tumor cell, which is caused
by its high proliferation, followed by the lack of adequate blood
vessel system, HIF-1 is over expressed in a malignant cancer.2
Therefore HIF-1 has been a central focus as a potential drug target
for cancer treatment.3 Recently, there have been only a few reports
about small molecules targeting HIF-1
a
expression or HIF-1 tran-
inhibitors,
scriptional activity.4,5 Among those reported HIF-1
a
YC-1, 3-(50-hydroxymethyl-20-furyl)-1-benzyl indazole,6,7 has been
considered as one of the most attractive drug candidates due to its
potent inhibitory activity against HIF-1
a ( Fig. 1) and a novel
X2
OH
R2
privileged indazole structure, which was found in a variety of
chemotherapeutics with diverse pharmacological effects such as
antiplatelet,8,9 neuroprotection,10 COX-inhibition,11 and antitumor
activity.6,12–14
In particular, YC-1 was found to inhibit HIF-1 function by
suppressing the PI3K/Akt/mTOR/4E-BP pathway which serves to
X1
O
N
N
N
N
R1
1
(YC-1)
⇑
Corresponding author. Tel.: +82 2 880 7875; fax: +82 2 888 0649.
E-mail address: ygsuh@snu.ac.kr (Y.-G. Suh).
Figure 1. Structure of YC-1 (1) and schematic design of its analogues.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.120

6298
H. An et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6297–6300
chemotherapeutics. Herein, we report design, synthesis and insight
into the structure–activity relationship of the 1,3-disubstituted
indazoles based on YC-1 as novel HIF-1 inhibitors.
scaffolds. For synthesis of analogues 9 and 11a–b, which were not
accessible through the aforementioned procedure, an alternative
synthetic route was pursued. Commercially available indazole-
3-carboxylic acid 7a–b were transformed into the desired interme-
diates such as 8 and 10a–b, respectively, via EDCI coupling of the
requisite amino acid followed by cyclization and optional aromati-
zation. Subsequent DIBAL reduction of the methyl esters afforded
the hydroxymethyl analogues 9 and 11a–b.
The furan analogues such as 1 (YC-1) and 13a–c were conve-
niently synthesized as outlined in Scheme 2. We utilized furan
intermediates 12a–d as key intermediates, which were obtained
by the palladium-catalyzed cross coupling reaction of the inda-
zoles17,18 with 5-formyl-2-furanoboronic acid under microwave
activation instead of thermal reflux. Suzuki cross coupling reaction
was highly facilitated by microwave activation. The aldehydes
were readily reduced by NaBH4 to give the corresponding alcohols
1 and 13a–c, which were further transformed into esters 14a–d by
At first, the diverse analogues consisting of five-membered het-
erocycles as surrogates replacing the furan moiety of YC-1 were
designed and synthesized (Scheme 1). Most of the syntheses of
YC-1 analogues commenced with the palladium-catalyzed aryl cou-
pling reactions of the known indazoles17,18 with diverse heterocyclic
boronic acids or heterocyclic stannanes in DMF, followed by appro-
priate manipulations. For introduction of the hydroxymethyl substi-
tuent to the oxazole intermediates 5a–b, and the thiazole
intermediates 5c–d, they were initially formylated by sequential
lithiation employing t-BuLi and DMF addition. Next, all biaryl inter-
mediates including isoxazoles 3a–b, and the formylated oxazole/
thiazoles prepared from 5a–d were readily reduced with DIBAL or
sodium borohydride, leading to the corresponding hydroxymethyl
substituted analogues with a variety of five-membered heterocycle
OH
MeO2C
N
O
N
O
a
b
N
N
N
N
R
R
4a, R = CH3
3a, R = CH3
3b, Bn
I
4b,
Bn
N
N
OH
R
N
N
2a: R=CH3
X
X
2b:
R=Bn
N
N
a
c
N
N
R
R
X = O; 6a, R = CH3
6b, Bn
X = O; 5a, R = CH3
5b, Bn
X = S; 5c, R = CH3
X = S; 6c, R = CH3
6d,
Bn
5d,
Bn
CO2Me
b
OH
N
N
O
O
d
N
N
N
N
8
9
CO2H
OH
O
N
MeO2C
N
N
R
N
O
7a: R=CH3
7b:
R=Bn
e
b
N
N
N
R
N
R
10a, R = CH3
10b
11a, R = CH3
11b, Bn
,
Bn
Scheme 1. Reagents and conditions: (a) Bu3SnAr, Pd(PPh3)4, DMF, 100 °C, 40–75%; (b) DIBAL-H, CH2Cl2, ꢀ78 °C to rt, 85%ꢁquant.; (c) (i) t-BuLi, DMF, THF, ꢀ78 °C; (ii) NaBH4,
EtOH; 45–53%, for two steps; (d) (i) HClꢂH2N-iso-Ser-OMe, EDCI, HOBt, Et3N, DMF; (ii) DAST, CH2Cl2, ꢀ78 °C to rt, 23%, for two steps; (e) (i) HClꢂH2N-Ser-OMe, EDCI, HOBt,
Et3N, DMF; (ii) DAST, DBU, BrCCl3, CH2Cl2, ꢀ78 °C to rt, 28–30%, for two steps.

H. An et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6297–6300
6299
OR2
d or e or f
R1 = Me, R2 = Ac
O
O
14a,
14b,
14c,
14d,
Bn,
Bn,
Bn,
Ac
OH
COCH(CH3)2
COC(CH3)3
N
N
O
R1
c
N
N
R3
N3
h
R1
O
13a,
13b,
13c,
R1 = H
Me
g
N
N
N
N
SO2Ph
Bn
R1
1 = Me, R3 = NH2
Me, NHAc
R1
1
,
(YC-1)
15a,
15b,
R
1 = Me
Bn
16a,
16b,
R
d
CHO
CO2Me
CONH2
I
O
j
a
O
O
i
N
N
R
N
N
N
N
N
17
N
R
2a:
2b:
2c:
R = CH3
R= Bn
R= H
12a,
12b,
12c,
12d,
R = H
Me
18
b
SO2Ph
Bn
R4
N
R5
19a, R1 = Me, R4/R5 = H, Me
O
19b,
19c,
19d,
Me,
Me,
Me,
Me, Me
(CH2CH2)2O
(CH2CH2)2CH2
k
N
N
R1
Scheme 2. Reagents and conditions: (a) 5-Formyl-2-furanoboronic acid, Pd(PPh3)4, NaHCO3, DME/H2O,
80%; (c) NaBH4, EtOH, quant.; (d) DMAP, Et3N, Ac2O, CH2Cl2, quant.; (e) iPrCOCl, pyridine, 57%; (f) PivCl, pyridine, CH2Cl2, 25%; (g) DPPA, DBU, toluene, 0 °C, 74%; (h) Pd/C, H2,
lW (150 W), 125 °C, 46%; (b) NaOH, BnEt3NꢂCl, PhSO2Cl, CH2Cl2, 0 °C,
MeOH, 57%; (i) MnO2, NaCN, AcOH, MeOH, 38%; (j) NH3/MeOH, 18%; (k) R2NH, Pd/C, H2, THF, 20–80%.
appropriate acylations, respectively. With the intermediates 1 and
13b in hands, we synthesized analogues 15a–b and 16a–b by azide
substitution of 1 and 13b, followed by azide reduction and acety-
lation of the resulting amine. The amide analogue 18 was easily
prepared by transamidation of methyl ester 17, which was
obtained by oxidation and concurrent esterification of the key
aldehyde 12d. The secondary or tertiary amine analogues including
19a–d were synthesized by Pd/C catalyzed reductive amination of
the corresponding aldehydes under H2 atmosphere.
The in vitro activities of the synthesized analogues are summa-
rized in Table 1. The inhibitory activities of analogues were evalu-
ated on hypoxia-induced HIF-1 transcriptional activity by
luciferase reporter assay. IC50s were determined by four parameter
logistic model (Hill-Slope model, Sigma Plot) based on the lucifer-
ase reporter assay.17
It is noteworthy that analogues containing furan moiety as het-
erocyclic core generally exhibited potent HIF-1 inhibition. How-
ever, the analogues consisting of other heterocyclic cores such as
isoxazole, oxazole, thiazole, and oxazoline, in which we are inter-
ested in terms of improved pharmacophoric properties,19 were
not potent or inactive. This is likely due to an inappropriate posi-
tioning of two heteroatoms of the heterocyclic core, which induces
undesired interaction or no interaction with the binding site of tar-
get protein. Thus, furan moiety was considered as an optimal
heterocyclic pharmacophore for potent HIF-1 inhibition.20 On the
basis of preliminary evaluation of the heterocyclic core, we focused
on the furan analogues with diverse substitution pattern including
hydroxy methyl substituent. Generally, analogues with benzyl
substituents on indazole-nitrogen exhibited good or excellent
inhibitory activities as revealed in those of 1 and 13a–b. Phenyl
sulfonyl group on indazole-nitrogen was also beneficial for the
potent inhibitory activity (13c). It also seems to imply that hydro-
gen on indazole-nitrogen is not essential for high HIF-1 inhibitory
activity.
Regarding hydroxymethyl moiety of 1, acetylation of hydroxyl
group slightly decreased activity as shown in the analogues of
14a–b. However the inhibitory activity increased upon introduc-
tion of bulky substituents to the hydroxyl group. In particular, ana-
logue 14c, in which isopropyl ester was introduced to the hydroxyl
moiety, exhibited the most potent activity for HIF-1 inhibition with
IC50 of 0.74 lM. Analogue 14d possessing pivaloyl ester also exhib-
ited potent HIF-1 inhibitory activity. Methyl ester 17 showed
slightly higher potency than YC-1. However, the amide analogue
18 was less potent than methyl ester 17. Analogues including ami-
nomethyl or azidomethyl substituent instead of hydroxymethyl
group completely lost the activities (15a–b, and 16a). The acety-
lated amine analogue was not also active (16b). It is noteworthy
that HIF-1 inhibitory activity increased as the bulkier cyclic amines
were introduced in place of hydroxyl group (19a–c). Especially, the
analogues possessing morpholine (19c) and piperidine (19d)
exhibited quite potent activity for HIF-1 inhibition with IC50 of
1.9–2.0 lM. Interestingly, the similar trend was observed for the
analogue 14b–d, in which HIF-1 inhibitory activity increased as
bulky acyl group was attached to the hydroxyl group. These results
indicate that an appropriate bulky substituent on hydroxyl or ami-
no group of the YC-1 analogues enhances HIF-1 inhibitory activity,

6300
H. An et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6297–6300
Table 1
HIF-1 inhibitory activity of the analogues
R2
A
N
N
R1
Heterocycle A
Analogues
R1
R2
IC50
1.2
(lM)
Figure 2. Western blot analysis showing the effect of 14c on HIF-1
expression in Hep3B cell line.
a protein
1 (YC-1)
Bn
CH2OH
O
for development of the highly potent and novel HIF-1 inhibitors
with appropriate pharmacological properties. Further work on
the therapeutically useful HIF-1 inhibitors based on our current re-
sults is in good progress.
4a
4b
Me
Bn
CH2OH
CH2OH
>30.0
23.9
N
O
O
S
N
N
N
6a
6b
Me
Bn
CH2OH
CH2OH
>30.0
>30.0
6c
6d
Me
Bn
CH2OH
CH2OH
>30.0
>30.0
Acknowledgments
This work was supported by a National Research Foundation of
Korea (NRF) Grant funded by the Korean Government (MEST) (No.
20100028431) and the Research Institute of Pharmaceutical Sci-
ence, Seoul National University.
9
Bn
CH2OH
>30.0
O
11a
11b
Me
Bn
CH2OH
CH2OH
>30.0
>30.0
N
O
O
13a
13b
H
CH3
SO2Ph
CH3
Bn
CH2OH
CH2OH
CH2OH
CH2OAc
CH2OAc
CH2O2CCH(CH3)2
CH2O2C(CH3)3
CH2N3
CH2N3
CH2NH2
5.0
8.9
1.3
16.9
2.3
0.74
0.89
>30.0
>30.0
>30.0
13.4
0.9
References and notes
13c
14a
14b
14c
14d
15a
15b
16a
16b
17
1. Semenza, G. L. Oncogene 2010, 29, 625.
2. Zhong, H.; Marzo, A. M. D.; 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.
3. Semenza, G. L. Nat. Rev. Cancer 2003, 3, 721.
4. Miyata, T.; Takizawa, S.; de Strihou, C. Am. J. Physiol. Cell. Physiol. 2011, 300,
C226.
5. Manolescu, B.; Oprea, E.; Busu, C.; Cercasov, C. Biochimie 2009, 91, 1347.
6. 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.
Bn
Bn
CH3
Bn
CH3
CH3
Bn
CH2NHAc
CO2Me
7. Kim, H.-L.; Yeo, E.-J.; Chun, Y.-S.; Park, J.-W. Int. J. Oncol. 2006, 29, 255.
8. Ko, F.-N.; Wu, C.-C.; Kuo, S.-C.; Lee, F.-Y.; Teng, C.-M. Blood 1994, 84, 4226.
9. Becker, E. M.; Alonso-Alija, C.; Apeler, H.; Gerzer, R.; Minuth, T.; Pleib, U.;
Schmidt, P.; Schramm, M.; Schröder, H.; Schroeder, W.; Steinke, W.; Straub, A.;
Stasch, J.-P. B. M. C. Pharmacology 2001, 1, 13.
18
Bn
CONH2
11.9
7.7
8.5
1.9
2.0
19a
19b
19c
19d
CH3
CH3
CH3
CH3
CH2NHCH3
CH2N(CH3)2
CH2N(CH2CH2)2O
CH2N(CH2CH2)2CH2
10. Lin, Y.-C.; Chou, L.-C.; Chen, S.-C.; Kuo, S.-C.; Huang, L.-J.; Gean, P.-W. Bioorg.
Med. Chem. Lett. 2009, 19, 3225.
11. Chen, C. Y.-C. J. Taiwan Inst. Chem. Eng. 2009, 40, 55.
12. Shin, D. H.; Kim, J.-H.; Jung, Y.-J.; Kim, K.-E.; Jeong, J. M.; Chun, Y.-S.; Park, J.-W.
Cancer Lett. 2007, 255, 107.
13. Pan, S.-L.; Guh, J.-H.; Peng, C.-Y.; Wang, S.-W.; Chang, Y.-L.; Cheng, F.-C.; Chang,
J.-H.; Kuo, S.-C.; Lee, F.-Y.; Teng, C.-M. J. Pharmacol. Exp. Ther. 2005, 314, 35.
14. Chen, C.-J.; Hsu, M.-H.; Huang, L.-J.; Yamori, -T.; Chung, J.-G.; Lee, F.-Y.; Teng,
C.-M.; Kuo, S.-C. Biochem. Pharmacol. 2008, 75, 360.
while hydroxyl methyl group serves as H-bonding acceptor rather
than H-bonding donor. The potent HIF-1 inhibitory activity of ana-
logue 14c was further confirmed by Western blot analysis using
Hep3B cell lines. Compound 14c showed reduction of HIF-1
a
induced by hypoxia condition in a dose dependent manner. In
15. Sun, H.-L.; Liu, Y.-N.; Huang, Y.-T.; Pan, S.-L.; Huang, D.-Y.; Guh, J.-H.; Lee, F.-Y.;
Kuo, S.-C.; Teng, C.-M. Oncogene 2007, 26, 3941.
16. Li, S. H.; Shin, D. H.; Chun, Y.-S.; Lee, M. K.; Kim, M.-S.; Park, J.-W. Mol. Cancer.
Ther. 2008, 7, 3729.
particular, this result implied that 14c had a pronounced effect
on HIF-1a production, which is strongly correlated to the result
of reporter gene assay outlined in Table 1. (Fig. 2)
17. Park, J.-W.; Chun, Y.-S.; Bair, K.; Cho, H. S. WO/2005/030121, 2005.
18. Park, J.-W. WO/2007/065010, 2007.
19. Meanwell, N. A. J. Med. Chem. 2011, 54, 2529.
20. Some of the analogues containing pyrroles as furan isosteres were prepared.
However, those compounds exhibited poor inhibition of HIF-1 expression with
In summary, we have designed and synthesized a new series of
novel HIF-1 inhibitors on the basis of 1 and provided an insight
into structure–activity relationship of 1,3-disubstituted indazoles
as HIF-1 inhibitors. In particular in the course of our studies on
the structure–activity relationship, analogue 14c possessing
isopropyl ester was identified as a highly potent HIF-1 inhibitor
IC50 of more than 20
concentration.
lM and have unexpected cell toxicities at higher
with IC50 of 0.74 lM. Our report would provide an important basis