Design, synthesis and structure-activity relationship studies of novel 4 (1-adamantyl) phenyl analogues as hif-1α inhibitors

Article abstract of DOI:10.2174/1573406412666151109113007

Hypoxia inducible factor-1 (HIF-1) is a key mediator during cancer cells to adapt tumor hypoxic condition. In this study, a series of adamantane-based compounds were synthesized and evaluated as potential inhibitors of HIF-1α. Examination of their structu

Full text of DOI:10.2174/1573406412666151109113007

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Medicinal Chemistry, 2016, 12, 338-346
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Design, Synthesis and Structure-Activity Relationship Studies of
Novel 4 (1-adamantyl) Phenyl Analogues as HIF-1ꢀ Inhibitors
Yan Xiaa, Qiong Duana, Bao-Hua Zhaoa, Dong-Feng Lia,* andꢀ Rui-Bin Houa,b,*
aLaboratory of Organic Chemsitry, College of Chemistry and Life Science,
Changchun University of Technology; Changchun, Jilin Province 130012, P.
R. China; bLaboratory of Photoelectric Material, Advanced Institute of Mate-
rials Science, Changchun University of Technology; Changchun, Jilin Prov-
ince 130012, P. R. China
Abstract: Hypoxia inducible factor-1 (HIF-1) is a key mediator during cancer cells to
adapt tumor hypoxic condition. In this study, a series of adamantane-based com-
pounds were synthesized and evaluated as potential inhibitors of HIF-1ꢀ. Examination
of their structure-activity relationship (SAR) identified the adamantane-containing
indole derivative 20a as a potent inhibitor of HIF-1ꢀ in Hep3B cell lines under tumor hypoxia (IC50 = 0.02 ꢁM). The
study herein may provide valuable information for the development of novel therapeutics against cancer and tumor angio-
genesis.
Keywords: Adamantane, hypoxia inducible factor-1, structure-activity relationship, tumor angiogenesis.
Received: May 13, 2015
Revised: October 08, 2015
Accepted: October 26, 2015
1. INTRODUCTION
Our studies have focused on the development of small
molecule inhibitors of HIF-1ꢀ, including compounds 1–3, as
potential anticancer agents (Fig. 1) [14-18]. To identify more
potent and efficient HIF-1ꢀ inhibitors, we performed struc-
tural modifications of 4-(1-adamantyl) phenol, including
adamantyl moiety and phenyl ring moiety substituted with
adamantyl group. Here, we present data from the synthesis
and evaluation of 4-(1-adamantyl) phenol analogues as po-
tential HIF-1ꢀ inhibitors. Interestingly, these compounds
were identified in a structure-activity relationship study us-
ing a cell-based, hypoxia reporter assay. Particularly, the 4-
(1-adamantyl) phenyl analogue 20a displayed potent inhibi-
tion of HIF-1ꢀ in human cancer cell lines with no significant
cytotoxicity.
Tumor hypoxia is considered as a general characteristic
of solid tumors and a negative prognostic factor for treat-
ment responses and cancer patient survival [1]. Hypoxia in-
ducible factor-1 (HIF-1), which is a transcription factor can
be activated in response to intratumoral hypoxia and activate
oncogenes and inactivate tumor suppressor genes as a result
of genetic alterations. It plays a pivotal role in adaptation of
tumor cells to hypoxia by activating the transcription of tar-
get genesꢁ These genes can regulate several biological proc-
esses including angiogenesis, cell proliferation and survival,
glucose metabolism, pH regulation, and migration. The ac-
tivity of HIF-1 in tumors mainly depends on the HIF-1ꢀ
subunit availability, whose levels can increase by hypoxic
conditions and during activation of oncogenes and/or inacti-
vation of tumor suppressor genes. Overexpression of oxy-
gen-regulated HIF-1ꢀ induces tumor cell proliferation as
well as resistances against chemotherapy and radiotherapy
[2]. Consequently, inhibiting HIF-1ꢀ activity has a crucial
role to play in cancer therapy [3].
2. EXPERIMENTAL METHODS
2.1. Chemistry
All reagents were purchased from commercial sources
and used without further purification. Solvents were dried
with standard procedures. 1H NMR spectra were recorded on
a Varian (300 MHz) or a Bruker Avance-500 (500 MHz)
spectrometer and 13C NMR spectra were determined on a
Bruker Avance-400 (400 MHz) spectrometer. IR spectra
were recorded on a Perkin Elmer 2400 instrument (KBr
pressed disc method). Chemical shifts (ꢀ) are reported in
parts per million (ppm) relative to tetramethylsilane used as
an internal standard. Mass spectra (MS) were obtained from
a JMS-700LC/MS Spectrometry Services. The thermogra-
vimetry analysis (TGA) was checked on a Pyris Diamond
2920 instrument. The purity of all the compounds was con-
ducted by using reversed phase high-pressure liquid chroma-
tography (RP-HPLC) on a YMC Hydrosphere C18 (HS-302)
column (5ꢁm particle size, 12 nm pore size), 4.6 mm dia. x
Recently, considerable effort has been directed to the dis-
covery of HIF-1 inhibitors among in house chemical libraries
and natural products [4-7]. All of these inhibitors can lower
HIF-1ꢀ protein levels, inhibit the expression of HIF-1 target
genes, like VEGF and EPO, and hinder tumor growth in
vivo. Furthermore, numerous inhibitors have been reported
to target regulation and signal transduction pathways of HIF
[8-13].
*Address correspondence to these authors at the Department of Chemistry,
College of Chemistry and Life Science, Changchun University of Technol-
ogy, 130012, Changchun, P. R. China; and Laboratory of Photoelectric
Material, Advanced Institute of Materials Science, Changchun University of
Technology, 130012, Changchun, P. R. China; Tel/Fax: +86-431-85716671;
E-mails: hrb1018@163.com; lidongfeng@ccut.edu.cn
1875-6638/16 $58.00+.00
© 2016 Bentham Science Publishers

Synthesis and Antitumor Study of HIF-1ꢀ Inhibitor
Medicinal Chemistry, 2016, Vol. 12, No. 4 339
O
OCH3
O
O
H
N
O
O
N
N
H
O
N
H
N
O
1
2
OH
O
O
HO
OH
3
Fig. (1). Structures of HIF-1ꢂ inhibitors.
150 mm with a flow rate of 1.0 mL/min. Analytic HPLC was
equipped with a UV detector at 254 nm. The mobile phases
employed were A: twice-diluted H2O containing 0.05 %
TFA, and B: CH3CN, respectively. The purity of the tested
compounds was reported as ꢀ 95% evaluated by utilizing one
of the following systems, System A: gradient 20 % B to 100
% B, running time 30 min; System B: gradient 25 % B to
100 % B, running time 30 min.
adamantan-1-ylmethanone (7b) was obtained according to
general procedure from 6a as white foam (24.1 mg, yield
27 %). mp 315 °C; IR (KBr): 3200, 1715, 1680, 1530, 1260
1
cm-1; H NMR (CDCl3, 300 MHz) ꢀ: 8.38 (s, 1H), 8.07 (s,
1H), 8.00-8.03 (m, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.56-7.60
(m, 2H), 7.43-7.48 (m, 3H), 7.33 (d, J = 8.7 Hz, 2H), 6.96
(d, J = 8.7 Hz, 2H), 4.61 (s, 2H), 3.93 (s, 3H), 3.71 (m, 4H),
3.55 (s, 2H), 2.43 (m, 4H), 2.25 (m, 2H), 2.00-2.09 (m, 6H),
1.87 (m, 4H), 1.73 (m, 2H); 13C NMR (CDCl3, 100 MHz) ꢀ:
175.37, 166.76, 155.20, 144.68, 137.27, 131.25, 129.48,
129.33, 126.49, 126.04, 125.46, 125.42, 124.67, 121.04,
114.74, 67.93, 62.47, 53.52, 52.43, 45.51, 44.97, 42.97,
42.52, 38.42, 36.66, 35.87, 29.30; MS (EI) m/z 689 (M+);
HRMS (EI) m/z calcd for C39H42F3N3O5 [M+] 689.3077,
found: 689.3076; Purity = 100 % (as determined by
RPHPLC, System A).
2.2. General Coupling Reaction Procedure Under PPAA
Condition for Synthesis of 7a-e and 8a-b
The compound 6a-b (1.0 equiv) was dissolved in the 10
mL ofꢀ acetonitrile and to the solution Et3N (4.0 equiv) and
50 % PPAA (1.2 equiv) were added. The reaction solution
was kept and stirred at rt. for 30 min and to round flask was
added amine (1.2 equiv) and continued to stir overnight. The
volatile solvent was removed on rotavap and purification of
the resulting residue by utilizing column chromatography on
silica gel formed 7a-e and 8a-b.
2.2.3. 3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-metho-
xy]-phenyl}-[4-(4-fluorobenzyl) piperazin-1-yl]-adama-
ntan-1-ylmethanone (7c)
2.2.1. 3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-
methoxy]-phenyl}-[4-(3,4-methylendioxybenzyl)piperazin-
1-yl]-adamantan-1-ylmethanone (7a)
3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-methoxy]-
phenyl}-[4-(4-fluorobenzyl) piperazin-1-yl]-adamantan-1-
ylmethanone (7c) was obtained according to general proce-
1
dure from 6a as white foam (24 mg, yield 35 %). H NMR
3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-methoxy]-
phenyl}-[4-(3,4-methylendioxybenzyl)piperazin-1-yl]-adam-
antan-1-ylmethanone (7a) was obtained according to general
(DMSO-d6, 300 MHz) ꢀ: 10.31 (s, 1H), 8.33 (s, 1H), 7.90 (d,
J = 8.1 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 8.1 Hz,
1H), 7.29-7.35 (m, 4H), 7.13 (t, J = 8.7 Hz, 2H), 6.94 (d, J =
8.7 Hz, 2H), 4.69 (s, 2H), 3.85 (s, 3H), 3.58 (b, 4H), 3.44 (s,
2H), 2.30 (b, 4H), 2.14 (b, 2H), 1.84-1.92 (m, 8H), 1.60-1.76
(m, 4H); MS (EI) m/z 639 (M+); HRMS (EI) m/z calcd for
C38H42FN3O5 [M+]ꢀ 639.3108, found: 639.3110; Purity > 98
% (as determined by RPHPLC, System B).
1
procedure from 6a as white foam (38 mg, yield 54 %). H
NMR (DMSO-d6, 300 MHz) ꢀ: 10.31 (s, 1H), 8.33 (s, 1H),
7.90 (d, J = 9 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.48 (t, J =
7.8 Hz, 1H), 7.30 (d, J = 9 Hz, 2H), 6.94 (d, J = 9.3 Hz, 2H),
6.85 (s, 1H), 6.82 (s, 1H), 6.73 (d, J = 9 Hz, 1H), 5.98 (s,
2H), 4.69 (s, 2H), 3.85 (s, 3H), 3.57 (b, 4H), 3.36 (s, 2H),
2.28 (b, 4H), 2.14 (b, 2H), 1.68-1.92 (m, 12H); MS (EI) m/z
665 (M+); HRMS (EI) m/z calcd for C39H43N3O7 [M+]
665.3101, found: 665.3101; Purity = 100 % (as determined
by RPHPLC, System B).
2.2.4. 3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-metho-
xy]-phenyl}-[4-(4-methoxybenzyl)piperazin-1-yl]-
adamantan-1-ylmethanone (7d)
3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-methoxy]-
phenyl}-[4-(4-methoxybenzyl)piperazin-1-yl]-adamantan-1-
ylmethanone (7d) was obtained according to general proce-
dure from 6a as white foam (12.9 mg, yield 18 %). 1H NMR
(CDCl3, 300 MHz) ꢀ: 8.39 (s, 1H), 8.07 (s, 1H), 8.01 (d, J =
9.0 Hz, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.45 (t, J = 7.8 Hz,
2.2.2. 3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-met-
hoxy]-phenyl}-[4-(4-trifluoromethylbenzyl)piperazin-1-yl]-
adamantan-1-ylmethanone (7b)
3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-methoxy]-
phenyl}-[4-(4-trifluoromethylbenzyl)piperazin-1-yl]-

340 Medicinal Chemistry, 2016, Vol. 12, No. 4
Xia et al.
1H), 7.33 (d, J = 8.7 Hz, 2H), 7.21 (d, J = 7.8 Hz, 2H), 6.96
(d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.1 Hz, 2H), 4.61 (s, 2H),
3.93 (s, 3H), 3.80 (s, 3H), 3.69 (m, 4H), 3.43 (s, 2H), 2.40
(m, 4H), 2.24 (m, 2H), 2.00-2.09 (m, 6H), 1.87 (m, 4H), 1.72
(m, 2H); MS (EI) m/z 651 (M+); HRMS (EI) m/z calcd for
C39H45N3O6 [M+] 651.3308, found: 651.3308; Purity = 100
% (as determined by RPHPLC, System A).
2.3. General Procedure for Alkylation of 14a-b with
Morpholine to Synthesize 15a-b
The compound 14a-b (1.0 equiv) was dissolved in the 10
mL of acetonitrile and to the solution was added K2CO3 (3.0
equiv), Cs2CO3 (0.5 equiv), KI (0.5 equiv) and morpholine
(1.2 equiv). The resulting mixture was refluxed overnight.
Aqueous workup followed by extracted with EA. The or-
ganic phase was dried over anhy. MgSO4. Before purifica-
tion by column chromatography on silica gel to give 15a-b,
filtered and evaporated under vacuum.
2.2.5. 3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-metho-
xy]-phenyl}-[4-(3-methoxybenzyl)piperazin-1-yl]-adaman-
tan-1-ylmethanone (7e)
2.3.1. 3-[4-(3-Morpholinopropoxy)phenyl]-[4-(4-methoxy-
benzyl)piperazin-1-yl]-adamantan-1-ylmethanone (15a)
3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-methoxy]-
phenyl}-[4-(3-methoxybenzyl)piperazin-1-yl]-adamantan-1-
ylmethanone (7e) was obtained according to general proce-
dure from 6a as white foam (17.4 mg, yield 20 %). 1H NMR
(CDCl3, 300 MHz) ꢀ: 8.39 (s, 1H), 8.08 (m, 1H), 8.00-8.02
(m, 1H), 7.83 (d, J = 7.2 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H),
7.33 (d, J = 9.6 Hz, 2H), 7.21 (d, J = 7.8 Hz, 1H), 6.96 (d, J
= 8.7 Hz, 2H), 6.88-6.90 (m, 2H), 6.80 (dd, J = 3.0 Hz, 7.7
Hz, 1H), 4.61 (s, 2H), 3.93 (s, 3H), 3.80 (s, 3H), 3.70 (m,
4H), 3.47 (s, 2H), 2.42 (t, J = 5.1 Hz, 4H), 2.24 (m, 2H),
2.00-2.09 (m, 6H), 1.87 (m, 4H), 1.72 (m, 2H); MS (EI) m/z
651 (M+); HRMS (EI) m/z calcd for C39H45N3O6 [M+]
651.3308, found: 651.3310; Purity > 97 % (as determined by
RPHPLC, System A).
3-[4-(3-Morpholinopropoxy)phenyl]-[4-(4-methoxy-
benzyl)piperazin-1-yl]-adamantan-1-ylmethanone (15a) was
obtained according to general procedure (40 mg, yield 80
%). 1H NMR (CDCl3, 300 MHz) ꢀ: 7.25 (d, J = 8.7 Hz, 2H),
7.21 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 5.1 Hz, 2H), 6.84 (d, J
= 4.5 Hz, 2H), 4.00 (t, J = 6.6 Hz, 2H), 3.80 (s, 3H), 3.70-
3.73 (m, 8H), 3.43 (s, 2H), 2.51 (t, J = 7.5 Hz, 2H), 2.46 (t, J
= 5.1 Hz, 4H), 2.39 (t, J = 5.1 Hz, 4H), 2.22 (b, 2H), 2.07 (s,
2H), 2.00 (b, 4H), 1.95 (m, 2H), 1.85 (b, 4H), 1.71 (b, 2H);
MS (EI) m/z 587 (M+); HRMS (EI) m/z calcd for
C36H49N3O4 [M+] 587.3723, found: 587.3721; Purity = 100
% (as determined by RPHPLC, System A).
2.3.2. 3-[4-(3-Morpholinopropoxy)phenyl]-[4-(4-tri-fluoro-
methylbenzyl)piperazin-1-yl]-adamantan-1-ylmethanone
(15b)
2.2.6. 3-{4-[(3-Trifluoromethylphenylcarbamoyl)-methoxy]-
phenyl}-{4-[4-(trifluoromethyl)benzyl]piperazin-1-yl}-
adamantan-1-ylmethanone (8a)
3-[4-(3-Morpholinopropoxy)phenyl]-[4-(4-trifluoro-
methylbenzyl)piperazin-1-yl]-adamantan-1-ylmethanone
(15b) was obtained according to general procedure (125 mg,
yield 87 %). 1H NMR (CDCl3, 300 MHz) ꢀ: 7.57 (d, J = 9.0
Hz, 2H), 7.44 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 7.2 Hz, 2H),
6.85 (d, J = 8.7 Hz, 2H), 4.00 (t, J = 6.6 Hz, 2H), 3.70-3.73
(m, 8H), 3.54 (s, 2H), 2.51 (t, J = 7.5 Hz, 2H), 2.46 (t, J =
4.2 Hz, 4H), 2.42 (t, J = 4.5 Hz, 4H), 2.23 (b, 2H), 2.07 (s,
2H), 2.00 (b, 4H), 1.95 (m, 2H), 1.86 (b, 4H), 1.71 (b, 2H);
MS (EI) m/z 625 (M+); HRMS (EI) m/z calcd for
C36H46F3N3O3 [M+] 625.3491, found: 625.3493; Purity > 98
% (as determined by RPHPLC, System B).
3-{4-[(3-Trifluoromethylphenylcarbamoyl)-methoxy]-
phenyl}-{4-[4-(trifluoromethyl)benzyl]piperazin-1-yl}-
adamantan-1-ylmethanone (8a) was obtained according to
general procedure from 6b as white foam (65 mg, yield 44
%). 1H NMR (DMSO-d6, 300 MHz) ꢀ: 10.40 (s, 1H), 8.13 (s,
1H), 7.88 (d, J = 8.7 Hz, 1H), 7.68 (d, J = 8.1 Hz, 2H), 7.58
(d, J = 8.1 Hz, 1H), 7.54 (d, J = 7.2 Hz, 2H), 7.43 (d, J = 8.1
Hz, 1H), 7.30 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.7 Hz, 2H),
4.70 (s, 2H), 3.59 (b, 4H), 3.56 (s, 2H), 2.33 (b, 4H), 2.14 (b,
2H), 1.84-1.92 (m, 8H), 1.60-1.76 (m, 4H); MS (EI) m/z 699
(M+); HRMS (EI) m/z calcd for C38H39F6N3O3 [M+]
699.2896, found: 699.2894; Purity = 100 % (as determined
by RPHPLC, System B).
2.4. General Alkylation Procedure for Synthesis of 20a-c
and 22
2.2.7. 3-{4-[(3-Trifluoromethylphenylcarbamoyl)-methoxy]-
phenyl}-[4-(4-methoxybenzyl)piperazin-1-yl]-adamantan-1-
ylmethanone (8b)
The mixture of compound 19a-c or 17 (1.0 equiv) and
compound 21 (1.5 equiv) was dissolved in the 10 mL of ace-
tone or DMF. The K2CO3 (2.0 equiv), Cs2CO3 (0.5 equiv)
and KI (0.5 equiv) were subsequently added under a nitrogen
atmosphere. After stirring at room temperature for 6-12 h,
the reaction was checked by TLC. Aqueous workup fol-
lowed by extracted with EA. After drying over anhydrous
MgSO4 and removing solvent under reduced pressure, puri-
fication of dryness by silica gel column chromatography
obtains corresponding compounds.
3-{4-[(3-Trifluoromethylphenylcarbamoyl)-methoxy]-
phenyl}-[4-(4-methoxybenzyl)piperazin-1-yl]-adamantan-1-
ylmethanone (8b) was obtained according to general proce-
1
dure from 6b as white foam (73 mg, yield 52 %). H NMR
(DMSO-d6, 300 MHz) ꢀ: 10.40 (s, 1H), 8.13 (s, 1H), 7.89 (d,
J = 8.7 Hz, 1H), 7.57 (t, J = 8.1 Hz, 1H), 7.44 (d, J = 7.5 Hz,
1H), 7.30 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 9.0 Hz, 2H), 6.94
(d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.1 Hz, 2H), 4.70 (s, 2H),
3.72 (s, 3H), 3.56 (b, 4H), 2.28 (b, 4H), 2.14 (b, 2H), 1.84-
1.92 (m, 8H), 1.60-1.76 (m, 4H); MS (EI) m/z 661 (M+);
HRMS (EI) m/z calcd for C38H42F3N3O4 [M+] 661.3127,
found: 661.3127; Purity = 100 % (as determined by
RPHPLC, System B).
2.4.1. (E)-Methyl-3-(2-{2-[3-(1H-indol-3-yl)acrylamido]-4-
(adamantan-1-yl)phenoxy}acetamido)benzoate (20a)
(E)-Methyl-3-(2-{2-[3-(1H-indol-3-yl)acrylamido]-4-(ad-
amantan-1-yl)phenoxy}acetamido)benzoate (20a) was ob-
tained according to general procedure as pale yellow solid (6
mg, yield 21 %). IR (KBr): 3400, 3190, 1679, 1668, 1524,

Synthesis and Antitumor Study of HIF-1ꢀ Inhibitor
Medicinal Chemistry, 2016, Vol. 12, No. 4 341
1254 cm-1; 1H NMR (DMSO-d6, 300 MHz) ꢀ: 11.65 (b, 1H),
10.54 (s, 1H), 9.69 (s, 1H), 8.47 (b, 1H), 8.04 (d, J = 8.1 Hz,
1H), 7.98 (m, 2H), 7.82 (d, J = 15.3 Hz, 1H), 7.81 (d, J = 2.1
Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.45-7.50 (m, 2H), 7.15-
7.25 (m, 2H), 7.08 (dd, J = 2.1 Hz, 8.7 Hz, 1H), 7.02 (d, J =
15.9 Hz, 1H), 7.01 (s, 1H), 4.83 (s, 2H), 3.82 (s, 3H), 2.06
(b, 3H), 1.86 (m, 6H), 1.73 (b, 6H); 13C NMR (CDCl3, 100
MHz) ꢀ: 168.79, 166.79, 165.29, 148.85, 146.38, 138.83,
138.50, 137.56, 137.24, 132.44. 131.40, 129.32, 128.75,
126.11, 125.52, 124.38, 123.58, 123.56, 121.73, 120.86,
120.63, 114.78, 113.89, 113.39, 111.89, 68.30, 52.38, 43.19,
36.61, 36.22, 28.88; MS (EI) m/z 603 (M+); HRMS (EI) m/z
calcd for C37H37N3O5 [M+] 603.2733, found: 603.2733; Pu-
rity = 100 % (as determined by RPHPLC, System A).
1.26 mmol) and 50 % PPAA (0.22 mL, 0.38 mmol) were
added followed by stirring for 30 min at room temperature
and treated with compound 23 (150 mg, 0.34 mmol). After
stirring overnight and removing volatile solvent under re-
duced pressure to give dry residue which was through purifi-
cation by column chromatography on silica gel to provide
pale yellow solid (145 mg, yield 73 %). 1H NMR (DMSO-d6,
300 MHz) ꢀ: 10.29 (s, 1H), 9.64 (s, 1H), 8.30 (b, 1H), 8.04
(d, J = 1.5 Hz, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 7.2
Hz, 1H), 7.52 (d, J = 2.1 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H),
7.37 (dd, J = 2.1 Hz, 8.7 Hz, 1H), 7.20 (d, J = 9.0 Hz, 1H),
7.10 (dd, J = 1.5 Hz, 8.7 Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H),
4.92 (s, 2H), 4.81 (s, 2H), 3.84 (s, 3H), 2.04 (b, 3H), 1.82-
1.83 (m, 6H), 1.72 (m, 6H); MS (EI) m/z 636 (M+); HRMS
(EI) m/z calcd for C34H34Cl2N2O6 [M+] 636.1794, found:
636.1794; Purity = 100 % (as determined by RPHPLC, Sys-
tem A).
2.4.2. Methyl-3-(2-{4-(adamantan-1-yl)-2-[3-(benzo[d][1,3
]dioxol-5-yl)propanamido]phenoxy}acetamido)benzoate
(20b)
2.6. Biological Methods
Methyl-3-(2-{4-(adamantan-1-yl)-2-[3-(benzo[d][1,3]di-
oxol-5-yl)propanamido]phenoxy}acetamido)benzoate (20b)
was obtained according to general procedure as pale yellow
solid (126 mg, yield 83 %). H NMR (DMSO-d6, 300 MHz)
ꢀ: 10.35 (s, 1H), 9.60 (s, 1H), 8.38 (s, 1H), 7.95 (s, 1H), 7.67
(d, J = 8.1 Hz, 1H), 7.62 (d, J = 2.4 Hz, 1H), 7.47 (t, J = 8.1
Hz, 1H), 7.09 (dd, J = 2.4 Hz, 9.0 Hz, 1H), 6.98 (d, J = 9.0
Hz, 1H), 6.84 (s, 1H), 6.77 (d, J = 7.2 Hz, 1H), 6.71 (d, J =
7.8 Hz, 1H), 5.92 (s, 2H), 4.76 (s, 2H), 3.83 (s, 3H), 2.89 (t,
J = 7.2 Hz, 2H), 2.69 (t, J = 7.2 Hz, 2H), 2.04 (b, 3H), 1.80-
1.81 (m, 6H), 1.71 (m, 6H); MS (EI) m/z 610 (M+); HRMS
(EI) m/z calcd for C36H38N2O7 [M+] 610.2679, found:
610.2677; Purity > 96 % (as determined by RPHPLC, Sys-
tem B).
2.6.1. Cell Culture
1
Human hepatocellular carcinoma cell lines Hep3B cells
(American Type Culture Collection, Manassas, VA) were
maintained in DMEM (Invitrogen, Grand Island, NY). This
media were supplemented with 10% (V:V) fetal bovine se-
rum (Hyclone, Logan, UT), penicillin, and streptomycin un-
der a 37 °C humidified 5% CO2 atmosphere. Hypoxic cul-
tures were in a gas-controlled chamber (Thermo Electron
Corp., Marietta, OH) which was kept at 37 °C, 1 % O2, 94 %
N2, and 5 % CO2.
2.6.2. Reporter Assay
The inhibiting hypoxia-inducible factor-1 of compounds
was tested by a HRE-dependent reporter assay as previously
described [19]. Generally, at 60-80 % confluence, Hep3B
cells, which were harbored with the vectors for pGL3-HRE-
luciferase plasmid with six copies of HREs from the human
VEGF gene and with pRL-CMV (Promega, Madison, WI)
employing Lipofectamine Plus reagent (Invitrogen). Follow-
ing 24 h incubation, the cells were treated with the synthe-
sized compounds with various concentrations and incubated
for 16 h upon hypoxic conditions (1 % O2, 94 % N2, and 5 %
CO2). The luciferase assay was conducted by a dual-
luciferase reporter assay system followed by the manufac-
turer’s protocol (Promega). Luciferase activity was measured
in a Microlumat Plus luminometer (EG&G Berthold, Bad
Wildbad, Germany) through injecting 25-50 ꢃL of assay
buffer which had luciferin and checking light emission for
10 seconds. The results were normalized to the activity of
renilla expressed by cotransfected Rluc gene under the con-
trol of a constitutive promoter.
2.4.3. Methylꢀ 3-{2-[4-(Adamantan-1-yl)-2-(3,5-dimethoxy-
benzamido)phenoxy]acetamido}benzoate (20c)
Methyl 3-{2-[4-(Adamantan-1-yl)-2-(3,5-dimethoxy-
benzamido)phenoxy]acetamido}benzoate (20c) was obtained
according to general procedure as pale yellow solid (141mg,
1
yield 72 %). H NMR (DMSO-d6, 300 MHz) ꢀ: 10.42 (s,
1H), 10.10 (s, 1H), 8.38 (s, 1H), 7.93 (d, J = 8.1 Hz, 1H),
7.71 (d, J = 2.1 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.48 (t, J =
8.1 Hz, 1H), 7.22 (s, 1H), 7.21 (s, 1H) 7.18 (d, J = 2.1 Hz,
1H), 7.09 (d, J = 9.0 Hz, 1H), 6.72 (s, 1H), 4.84 (s, 2H), 3.85
(s, 3H), 3.79 (s, 6H), 2.05 (b, 3H), 1.85 (m, 6H), 1.73 (m,
6H); MS (EI) m/z 598 (M+); HRMS (EI) m/z calcd for
C35H38N2O7 [M+] 598.2679, found: 598.2680; Purity = 100
% (as determined by RPHPLC, System A).
2.4.4. Methyl 3-{2-[4-(adamantan-1-yl)-2-nitrophenoxy]
acetamido}benzoate (22)
300 mg, yield 59 %. 1H NMR (CDCl3, 300 MHz) ꢀ: 9.31
(b, 1H), 8.38 (s, 1H), 8.06 (d, J = 2.4 Hz, 1H), 7.94 (d, J =
8.1 Hz, 1H), 7.85 (d, J = 7.2 Hz, 1H), 7.65 (dd, J = 2.4 Hz,
9.0 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 9.0 Hz,
1H), 4.76 (s, 2H), 3.94 (s, 3H), 2.14 (b, 3H), 1.90-1.91 (m,
6H), 1.73-1.84 (m, 6H); MS (EI) m/z 464 (M+).
2.6.3. Cytotoxicity Assay
Cell viability was measured by a MTT assay. Briefly,
cells were seeded in a 96-well plate at 4 ꢀ 104 cells per well
and incubated for 3 h at 37 °C. Then cells were treated by
synthesized compounds with various concentrations under
normoxic or hypoxic conditions which were incubated for
24h followed by the addition of MTT to the cells. Before
formazan crystals dissolving in 100 ꢃL of DMSO, removed
supernatant, and the optical densities were performed on a
microplate reader.
2.5. Methyl 3-{2-[4-(Adamantan-1-yl)-2-[2-(2,4-dichloro-
phenoxy)acetamido]phenoxy]acetamido}benzoate (24)
To a solution of 2,4-dichlorophenoxyacetic acid (69 mg,
0.31 mmol) in absolute acetonitrile (10 mL) Et3N (0.17 mL,

342 Medicinal Chemistry, 2016, Vol. 12, No. 4
Xia et al.
O
R
O
O
O
O
6 steps
a
O
HN
O
OH
BnO
BnO
HO
ref.
5a: R = CO2CH3
5b: R = CF3
4
N
O
CF3
O
R
O
HN
O
R2
b
O
HN
O
HN
N
c
HO
R2
N
6a-b
8a: R2 = CF3
8b: R2 = OCH3
c
O
CO2CH3
O
O
HN
N
N
7a-e
R1
O
O
7c: R1 =
7a: R1
7d: R1
=
7b: R1
=
F
CF3
OCH3
7e: R1
=
=
OCH3
Scheme 1.
2.6.4. Statistical Analysis
to convert the hydroxyl group to the corresponding ether
followed by palladium-mediated reduction leading to acidic
compound 11. The intermediates 12a and b were derived
from a coupling reaction of 11 with two kinds of substituted
benzyl piperazines, which gave rise to phenols 13a and b by
exposure to TBAF. 1,3-Dibromopropane employed as a
linker between the phenol and morpholine was obtained
from commercial sources. Compounds 14a and b were
formed by base-mediated, mono-substituted alkylation of
13a and b, which were reacted with morpholine to obtain
derivatives 15a and b.
Each experiment was conducted at least three times, and
representative data were displayed in the tables which were
given as mean values
separate experiments. Means were tested for statistical dif-
ferences through the Student’s t-test applying error prob-
abilities of p < 0.05.
standard deviation obtained from
3. RESULTS AND DISCUSSION
3.1. Chemistry
The various building blocks could be combined to form
the desired adamantyl phenol intermediates or related com-
pounds as shown in Scheme 3. Regioselective nitration of
phenol 16 using nitric acid resulted in a good yield of 17.
Subsequent reduction of the nitro group to the corresponding
aniline by catalytic hydrogenation provided an excellent
yield of amino phenol 18. Coupling reactions of 18 with the
appropriate acid formed intermediates 19a–c. Finally, alkyla-
tion of 19a–c with intermediate 21 under basic condition,
which can be synthesized by acylation of commercially
available 3-aminobenzoic acid methyl ester, was performed
to obtain compounds 20a–c. The compound 20a was charac-
Schemes 1–3 illustrate the routes used for the synthesis
of thirteen adamantane-substituted analogues with modifica-
tions of their benzene rings and side chains. The synthetic
routes are straightforward and differ mainly by the introduc-
tion of two moieties to the 4-(1-adamantyl) phenol precursor.
In Scheme 1, synthesis of 7a–e, and 8a and b was under-
taken to explore the effects of adamantane substitution on
inhibition of HIF-1. Compound 4 was obtained using ada-
mantane 1-carboxylic acid as the starting material through
six steps as described previously [20]. Compound 4 was re-
acted with different amines to produce compounds 5a and b
under a coupling reaction condition using PyBOP and
DMAP. Subsequent debenzylation proceeded with palladium
carbon to yield 6a and b. Piperazine-substituted analogues
7a–e were prepared from 6a via coupling with substituted
piperazines using PPAA and Et3N. Compounds 8a and b
were synthesized by coupling reactions of 6b with substi-
tuted benzyl piperazines.
1
terized by H-NMR, 13C-NMR, IR, MS and HRMS. The
derivative 24 was prepared by another route staring from 14
that was first reacted with intermediate 21 to form compound
22. Reduction of the nitro group yielded amino compound 23
with a quantitative yield. Synthesis of compound 24 was
achieved under coupling conditions by employing PPAA and
Et3N.
In Scheme 2, the construction of adamantyl phenol with
morpholine moiety derivatives was achieved by starting
from compound 9 according to the described previously pro-
cedures [20]. Silylation of 9 with TBDMSCl was performed
3.2. Biological Activity Evaluation
The synthesized compounds were assessed their inhibi-
tion of HIF-1 activation induced by hypoxia (1% O2, 94%

Synthesis and Antitumor Study of HIF-1ꢀ Inhibitor
Medicinal Chemistry, 2016, Vol. 12, No. 4 343
O
O
O
3 steps
ref.
a
OTBDMS
OH
Br
BnO
BnO
HO
9
10
R3
O
NH
O
OTBDMS
N
N
b
OTBDMS
HO
R3
N
c
12a: R3 = OCH3
12b: R3 = CF3
11
O
O
Br
Br
d
OH
O
Br
N
e
N
R3
R3
N
N
14a-b
13a-b
O
HN
O
O
N
O
N
e
R3
N
15a-b
Scheme 2.
R5
O
CO2CH3
22: R5 = NO2
23: R5 = NH2
b
e
O
HN
Cl
24: R5 = NHCOR6
R6
=
O
d
Cl
R4
O
NO2
NH2
NH
R4CO2H
c
b
a
OH
OH
OH
OH
16
17
18
O
O
19b: R4
=
19a: R4
=
O
N
H
H
N
R4
H3CO
Cl
O
O
H3CO
19c: R4
=
NH
O
CO2CH3
21
O
HN
d
OCH3
20a-c
Scheme 3.
N2, and 5% CO2) employing a cell-based reporter assay in
the human hepatoblastoma Hep3B cell line (as shown in
Tables 1 and 2). All biological assays were made under hy-
poxic conditions according to a formerly depicted protocol
[21]. Cell viability, which was checked by a 3-(4,5-
Our initial study focused on exploring variants of the
adamantyl carboxylic acid and phenol moieties of 3-(4-
hydroxyphenyl)adamantane-1-carboxylic acid (Table 1). As
shown in Table 1, compound 7a containing a 3,4-
methylendioxy group was more potent than analogues bear-
ing one methoxy group, compounds 7d and 7e. Modification
by replacing the methyl ester group in 7b with a trifluoro-
methyl group to form 8a increased the potency of HIF-1 in-
hibition. While the improved potency observed between 7d
dimethylthiazole-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) assay, displayed that most of synthesized compounds
had no remarkably cytotoxicity at concentrations that can
effectively inhibit HIF-1 activity.

344 Medicinal Chemistry, 2016, Vol. 12, No. 4
Xia et al.
Table 1. In vitro inhibiting HIF-1 activity of piperazine-substituted adamantane derivativesa.
A
O
Cell
O
Pre-ADME (Solubility
Hep3B
N
Viability
Cd
Pre-ADME (Log P)
N
(mg/L))
IC50 (ꢀM)
R1
(ꢀM)
R1
A
O
O
O
O
O
H
N
O
OMe
OMe
OMe
OMe
OMe
7a
7b
7c
7d
7e
3.437
6.332
5.595
5.373
5.373
0.217
0.23 0.04
>30
>30
>30
>30
>30
O
O
O
O
O
O
H
N
2.203ꢁ10-3
6.223ꢁ10-3
8.690ꢁ10-3
9.145ꢁ10-3
>30
CF3
H
N
>30
F
H
N
>30
OCH3
OCH3
H
N
>30
H
N
CF3
8a
8b
7.419
6.460
7.923ꢁ10-4
3.130ꢁ10-3
0.04 0.02
5.16 1.45
>30
>30
O
CF3
H
N
CF3
O
OCH3
O
15a
15b
4.770
5.729
1.245
0.318
>30
>30
>30
>30
N
N
OCH3
O
CF3
aValues are the mean of three experiments.
and 8b reflected the importance of the increase in the elec-
tron-withdrawing ability. 3,4-Methylendioxybenzyl 7a
among compounds 7a–e was the only analogue that dis-
played potent inhibition of HIF-1. Compound 7a had an IC50
of 0.23 ꢄꢅ in the cellular assay. This result indicates that an
electronic effect is possibly beneficial for achieving good
cellular potency. The compound with ethylenedioxy in the
left and CF3 in the right may be more active on HIF-1, which
will be obtained in following study. Cells were more sensi-
tive to modification of electron-withdrawing group in the
benzene ring as observed by the range of cellular activities
displayed by 7a–e, and 8a and b. The position of methoxy
group in compounds 7e and 8b can influence the activity
based on the IC50 values of HIF-1. Changing more rigid ben-
zoyl moiety into flexible there-carbon linker to form com-
pounds 15a-b reduced the inhibition activity of HIF-1. On
the other hand, increasing the compounds’ solubility by in-
troducing a morpholine moiety also led to a loss of cellular
potency (compounds 15a and 15b).
As summarized in Table 2, a brief examination of modi-
fications to the benzene ring, which serves to link the ada-
mantyl moiety, revealed a dramatic effect on HIF-1 inhibi-
tion. Replacement of the linkage in 20c with a carbon and
oxygen atom chain resulted in a decrease in cellular potency
(24, IC50 = 26.8 ꢄꢅ). Compared with 24, the derivative
compound 20b with a two carbon atom linkage displayed an
increase in potency (IC50 = 4.89 ꢄꢅ), possibly because of
the aromatic substituent solubility. Furthermore, a double
bond linkage was introduced between the amide and indole
(20a, IC50 = 0.02 ꢄꢅ), leading to a dramatic increase in the
inhibitive effect on HIF-1.
It is noteworthy that compounds fit for the rule of five
had better pharmacokinetic properties. One of the shortcom-
ings of previous lead compounds (e.g., LW6) was their poor
water solubility, a common issue with synthetic chemicals,
which often leads to difficulties in further studies of such
compounds. Generally, the partition coefficient Log P should

Synthesis and Antitumor Study of HIF-1ꢀ Inhibitor
Medicinal Chemistry, 2016, Vol. 12, No. 4 345
Table 2. In vitro inhibiting HIF-1 activity of adamantyl phenyl derivativesa.
O
B
O
OCH3
Pre-ADME
(solubility(mg/L))
Hep3B
IC50 (ꢁM)
Cell Viability
(ꢁM)
Cd
Pre-ADME (Log P)
O
HN
O
2.670ꢀ10-5
1.480ꢀ10-4
0.02 0.14
4.89 1.07
>30
>30
N
H
20a
20b
6.261
5.759
N
H
O
O
O
N
H
O
OCH3
N
H
20c
24
5.467
6.664
5.050ꢀ10-4
10.2 0.21
26.8 1.68
>30
>30
OCH3
Cl
O
O
1.800ꢀ10-5
N
H
Cl
aValues are the mean of three experiments.
be less than 5. These data can be calculated by using
PreADME software designed by Bioinformation & Molecu-
lar Design Research Center. As shown in Tables 1 and 2,
compound 8a showed more potency than that of 7a. While
its Log P was better and its solubility in water was quite
good according to data as pre-ADME. Likewise, a more po-
tent compound, 20a, had a slightly higher log P value and
poor solubility in water as shown in Table 2. Therefore,
these data as pre-Log P and solubility may provide a basis
for designing compounds that are suitable for the rule of
five.
TBDMSCl
TBAF
ADME
PMA
=
=
=
=
=
=
=
=
tert-Butylchlorodimethylsilane
Tetrabutylammonium Fluoride
Olumn Chromatography
Phosphomolybdic Acid
Trifluoroacetic Acid
TFA
EA
Ethyl acetate
DCM
Dichloromethane
HBTU
N,N,N',N'-Tetramethyl-O-(benzotriazol-
1-yl)uronium Hexafluorophosphate
CONCLUSION
DIPEA
VEGF
EPO
=
=
=
=
=
=
=
N,N-Diisopropylethylamine
Vascular Endothelial Growth Factor
Erythropoietin
Based on this preliminary structure-activity relationship
study involving structural modifications, we have defined the
structural requirements for HIF-1 inhibition by 4-(1-
adamantyl) phenol analogues and identified several highly
potent HIF-1 inhibitors. More detailed studies are in progress
to investigate mechanisms of the effects of 20a on HIF-1ꢂ-
mediated tumor progression and angiogenesis. Herein the
compounds synthesized may provide worthy information to
design and develop more potent anticancer agents.
IC50
50% Inhibitory concentration
Dimethylsulfoxide
DMSO
DMEM
THF
Dulbecco's Modified Eagle Medium
Tetrahydrofuran
CONFLICT OF INTEREST
LIST OF ABBREVIATIONS
The authors confirm that this article content has no con-
flict of interest.
PyBOP
=
1H-Benzotriazol-1-
yloxytripyrrolidinophosphonium Hex-
afluorophosphate
ACKNOWLEDGEMENTS
DMAP
PPAA
DMF
=
=
=
4-Dimethylaminopyridine
Propylphosphonic Anhydride
N,N-Dimethylformamide
This study was supported by the National Science Foun-
dation of China (No. 21442004 and No. 21502008), the Na-
tional Natural Science Foundation of Jilin Province (grant
No. 20101548).

346 Medicinal Chemistry, 2016, Vol. 12, No. 4
Xia et al.
logue inhibitors of HIF prolyl hydroxylase. Bioorg. Med. Chem.
Lett., 2003, 13, 2677-2680.
SUPPLEMENTARY MATERIAL
[9]
Epstein, A.C.; Gleadle, J.M.; McNeill, L.A.; Hewitson, K.S.;
O’Rourke, J.; Mole, D.R.; Mukherji, M.; Metzen, E.; Wilson, M.I.;
Dhanda, A.; Tian, Y.M.; Masson, N.; Hamilton, D.L.; Jaakkola, P.;
Barstead, R.; Hodgkin, J.; Maxwell, P.H.; Pugh, C.W.; Schofield,
C.J.; Ratcliffe, P.J. C. elegans EGL-9 and mammalian homologs
define a family of dioxygenases that regulate HIF by prolyl hy-
droxylation. Cell, 2001, 107, 43-54.
Supplementary material is available on the publishers
Web site along with the published article.
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