Synthesis and VEGF inhibitory activity of 16,17-pyrazo-annulated steroids

Article abstract of DOI:10.1016/j.cclet.2010.12.043

Eight 16,17-pyrazo-annulated steroidal derivatives were synthesized and evaluated in vitro vascular endothelial growth factor (VEGF) inhibitory activity with 2-methoxyestradiol (2-ME) as the reference compound. Most of the compounds showed potent VEGF inh

Full text of DOI:10.1016/j.cclet.2010.12.043

Available online at www.sciencedirect.com
Chinese Chemical Letters 22 (2011) 757–760
www.elsevier.com/locate/cclet
Synthesis and VEGF inhibitory activity
of 16,17-pyrazo-annulated steroids
Hong Shan Liu a, Hu Ling Zheng a, Mei Ge b, Peng Xia a, Ying Chen a,
*
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China
b Laiyi Biological Drug Research and Development Center Co., Ltd., Shanghai 201203, China
Received 12 October 2010
Available online 16 April 2011
Abstract
Eight 16,17-pyrazo-annulated steroidal derivatives were synthesized and evaluated in vitro vascular endothelial growth factor
(VEGF) inhibitory activity with 2-methoxyestradiol (2-ME) as the reference compound. Most of the compounds showed potent
VEGF inhibitory activity with EC50 values of micromolar or submicromolar range. Among them, the compounds 3 and 8 exhibited
similar EC50 values and obviously better TI values compared with 2-ME.
# 2010 Ying Chen. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Pyrazo-annulated steroid; 2-Methoxyestradiol (2-ME); VEGF inhibitory activity
Angiogenesis is a crucial process in tumor pathogenesis as it provides malignant cells with nutrients and oxygen.
Tumor cells can secrete several growth factors including VEGF, which triggers endothelial cells to form new
capillaries. Preventing the expansion of new blood vessel networks can result in reduced tumor size and metastases.
VEGF can obviously induce angiogenesis under hypoxic conditions [1]. It was reported that steroidal heterocycles
containing annulated rings in the steroidal moiety have prompted a great interest in the biological study of many
heterocyclic compounds [2]. Y. Kashiwada et al. reported their work on betulinic acid derivatives as potent anti-HIV
agents and found that esterification of C-3 hydroxyl group of betulinic acid with 2,2-dimethylbutanedioic anhydride
gave 3-O-(3,3-dimethylsuccinyl)betulinic acid with extremely potent anti-HIVactivity [3]. As our research focused on
the synthesis of new steroids with anti-tumor or anti-virus activities, we designed and synthesized a series of 3-O-(3,3-
dimethylsuccinyl)-16,17-pyrazo-annulated steroidal derivatives (Fig. 1) and screened their biological activities. 8
newly synthesized compounds were evaluated for the VEGF inhibitory activity with 2-methoxyestradiol (2-ME) as the
reference compound [4–6], and most of them exhibited promising results. Among them, the compounds 3 and 8
showed potent VEGF inhibitory activities with EC50 values of 0.05 and 0.03 mmol/L and four- and two-fold better TI
values of 19.6 and 12.67 than 2-ME (TI = 5), respectively.
Using epiandrosterone and dehydroepiandrosterone as the starting materials, via C-16 formylation, condensation
with monosubstituted hydrazines and esterification of C-3 hydroxyl group with 2,2-dimethylbutanedioic anhydride, 8
different 16,17-pyrazo-annulated steroidal derivatives were synthesized (Scheme 1). 16-formylepiandrosterone
* Corresponding author.
E-mail address: yingchen71@fudan.edu.cn (Y. Chen).
1001-8417/$ – see front matter # 2010 Ying Chen. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2010.12.043

[()TD$FIG]
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H.S. Liu et al. / Chinese Chemical Letters 22 (2011) 757–760
R2
N
R2
N
N
N
or
R1O
R1O
Fig. 1. 16,17-Pyrazo-annulated steroids.
[()TD$FIG]
R
N
O
O
N
O
a
b
H
HO
HO
R
HO
epiandrosterone
R = -CH
3
1 R = -C(CH )
3 3
N
N
2 R = -C H
6
5
O
c
O
HOOC
3 R = -CH
3
O
O
4 R = -C H
6
5
O
H
b
a
HO
R
HO
N
N
dehydroepiandrosterone
R
N
O
N
c
O
HOOC
7 R = -CH
3
R = -CH
8 R = -C H
3
6
5
5 R = -C(CH )
3 3
HO
6 R = -C H
6
5
Scheme 1. (a) Ethyl formate, C2H5ONa, CH2Cl2, r.t., 90%–93% (b) NH2NHR, C2H5OH, r.t. or reflux, 84%–96% (c) 2,2-dimethylbutanedioic
anhydride, pyridine, DMAP, reflux, 78%–92%.
reacted with different monosubstituted hydrazines in ethanol at room temperature or by refluxing for 1–2 h. The crude
products were filtered and recrystallized from methanol to give compounds 1 or 2 [11]. Compound 2, 2,2-
dimethylbutanedioic anhydride and catalytic amount DMAP in dry pyridine were refluxed for 10 h. The crude product
was filtered and recrystallized from acetic ether to give compound 4 [11]. The compounds 5, 6, 7 and 8 [11] were
prepared in the same way as mentioned above.
Delphine S. Fischer, Gillian M. Allan et al. reported that monosubstituted hydrazines reacted with steroids
containing 16-formyl group and 17-ketone group to give sole product with its R group lying in 20-N position (as
showed in Scheme 1) [7]. We selected compound 6 to measure its crystal structure by X-ray diffraction analysis
(Fig. 2), which coincided with the reported conclusion.
The 8 synthesized pyrazo-annulated steroidal derivatives were tested for VEGF inhibitory activity against the
human breast cancer cell MDA-MB-231 [8–10] in comparison with 2-ME and the results are presented in Table 1. It
clearly showed that most of the compounds displayed significantly potent VEGF inhibitory activities and compounds 3
and 8 had similar activities and improved selectivity in comparison with 2-ME. The activity data of compounds 3, 4, 7

[()TD$FIG]
H.S. Liu et al. / Chinese Chemical Letters 22 (2011) 757–760
759
Fig. 2. X-ray crystal structure of compound 6.
Table 1
VEGF inhibitory results of pyrazo-annulated steroidal derivativesa.
Compounds
R1
R2
IC50 (mmol/L)
EC50 (mmol/L)
TI value
1
–H
–H
–C(CH3)3
–C6H5
–CH3
1.48
1.28
0.99
0.08
0.02
1.42
0.03
0.38
0.05
1.48
1.02
0.05
0.03
0.17
0.17
0.007
0.03
0.01
1
2
1.25
19.6
2.67
ꢀ
3
–COCH2C(CH3)2COOH
–COCH2C(CH3)2COOH
–H
4
–C6H5
–C(CH3)3
–C6H5
–CH3
5
6
–H
8.35
4.28
12.67
5
7
–COCH2C(CH3)2COOH
–COCH2C(CH3)2COOH
8
2-ME
–C6H5
a
EC50 is the concentration of sample for 50% VEGF inhibitory rate; IC50 is the concentration of sample for 50% cell growth inhibitory rate; TI
(therapeutic index) = IC50/EC50
.
and 8 suggested that esterification of C-3 hydroxyl group with 2,2-dimethylbutanedioic anhydride can improve the
VEGF inhibitory activity. Due to the limited compound activity data, continued work is needed to elucidate the
structure-activity relationship (SAR).
In conclusion, 8 synthesized pyrazo-annulated steroidal derivatives exhibited fine in vitro VEGF inhibitory activity.
It implies this kind of heterocyclic steroidal derivatives merit further investigation as new VEGF inhibitory agents.
Acknowledgments
This research was supported by grants from the National Natural Science Foundation of China awarded to P. Xia
(No. 20272010) and Y. Chen (No. 30200348 and 30873164), respectively. Thanks are also due to the National Drug
Innovative Program (No.2009ZX09301-011) for partial support.
References
[1] J. Fayette, J.C. Soria, J.P. Armand, et al. Eur. J. Cancer 41 (2005) 1109.
[2] D.G. Rivera, K. Peseke, et al. Molecules 8 (2003) 444.
[3] Y. Kashiwada, F. Hashimoto, et al. J. Med. Chem. 39 (1996) 1016.
[4] D. Chauhan, L. Catley, T. Hideshima, et al. Blood 100 (2002) 2187.
[5] N.J. Mabjeesh, D. Escuin, T.M. LaVallee, et al. Cancer Cell 3 (2003) 363.
[6] S.J. Stafford, J. Schwimer, C.T. Anthony, et al. J. Surg. Res. 125 (2005) 104.
[7] D.S. Fischer, G.M. Allan, et al. J. Med. Chem. 48 (2005) 5749.
[8] VEGF bioactivity detection: The single human breast cancer cell MDA-MB-231 suspension in 1640 medium with 10% foetal bovine serum at a
concentration of 104–105 cells per milliliter was prepared. Each sample to be detected was resolved in distilled water or 0.5% DMSO and
diluted to multi-proportion on concentration. 90 mL of cell suspension and 10 mL of sample solution were mixed and cultivated in each well of
a 96 well-micro plate at 37 8C with 5% CO2 for 3 d. The IC50 was obtained by detecting the cell growth inhibitory rate directly using 10 mL of
medium as the blank control. Then the VEFG inhibitory rate was detected using VEGF ELISA kit (SHENZHEN YINUOJIN Biotechnology
Company). The EC50 was obtained by calculation from the diagram of inhibitory rate vs. sample concentration.

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H.S. Liu et al. / Chinese Chemical Letters 22 (2011) 757–760
[9] D. Chelouche-Lev, C.P. Miller, C. Tellez, et al. Eur. J. Cancer 40 (2004) 2509.
[10] Human VEGF Quantitative Reagent Kit, http://www.innogent-cn.com.
[11] The data of compounds 1–8. 1: white needle crystals; yield: 92.0%; mp: 245–247 8C; [a]D20 +11 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3):
d 0.86 (s, 3H, H-18), 1.03 (s, 3H, H-19), 1.59 (s, 9H, –C(CH3)3), 3.60 (m, 1H, H-3), 7.14 (s, 1H, N CH–), 0.76–2.47 (m, 21H); 13C NMR
(400 MHz, CDCl3): d 155.82, 131.14, 126.10, 71.19, 63.00, 58.84, 54.20, 44.82, 43.78, 38.08, 37.00, 36.73, 35.57, 34.48, 31.42, 30.65, 28.52,
23.63, 21.17, 17.37, 12.27; HRMS(EI): Calcd. for C24H38N2O: 370.2984; Found: 370.2986; 2: white needle crystals; yield: 84.9%; mp: 217–
219 8C; [a]D ꢁ167 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3): d 0.85 (s, 3H, H-18), 1.06 (s, 3H, H-19), 3.59 (m, 1H, H-3), 7.41 (s, 1H, –
20
N
CH–), 7.33–7.52 (m, 5H, Ar-H), 0.76–2.56 (m, 21H); 13C NMR (400 MHz, CDCl3): d 157.15, 140.52, 135.20, 128.85, 127.08, 126.17,
123.49, 71.16, 63.02, 54.48, 44.88, 42.69, 38.09, 36.70, 35.64, 35.00, 34.29, 31.45, 31.40, 28.47, 24.11, 20.72, 17.76, 12.28; HRMS(EI): Calcd.
for C26H34N2O: 390.2671; Found: 390.2668; 3: white solid; yield: 86.7%; mp: 232–234 8C; [a]D ꢁ91 (c 0.1, CHCl3); 1H NMR (500 MHz,
20
CDCl3): d 0.85 (s, 3H, H-18), 0.96 (s, 3H, H-19), 1.30 (s, 6H, H-30 C(CH3)2), 2.58 (s, 2H, H-20), 3.78 (s, 3H, N-CH3), 4.71 (m, 1H, H-3), 7.12 (s,
1H, –N CH–), 0.78–2.63 (m, 20H); 13C NMR (400 MHz, CDCl3): d 181.75, 170.73, 157.09, 132.86, 123.64, 73.73, 62.54, 54.46, 44.73,
44.55, 41.32, 40.49, 36.62, 36.47, 35.68, 34.08, 33.93, 33.81, 31.34, 28.29, 27.29, 25.37, 25.24, 24.24, 20.61, 17.33, 12.18; HRMS(ESI): Calcd.
for C27H40N2O4: 456.2988; Found: 457.3061(M + H+); 4: white solid; yield: 88.2%; mp: 216–218 8C; 1H NMR (400 MHz, CDCl3): d 0.85 (s,
3H, H-18), 1.03 (s, 3H, H-19), 2.61 (d, 2H, J = 12.1 Hz, H-20), 4.70 (m, 1H, H-3), 7.38 (s, 1H, –N CH–), 7.30–7.52 (m, 5H, Ar-H), 0.78–2.20
20
(m, 20H); HRMS(EI): Calcd. for C32H42N2O4: 518.3145; Found: 518.3147; 5: white needle crystals; yield: 95.5%; mp: 253–255 8C; [a]D
ꢁ80 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3): d 1.08 (s, 6H, H-18, H-19), 1.58 (s, 9H, –C(CH3)3), 3.54 (m, 1H, H-3), 5.40 (d, 1H,
J = 4.64 Hz, H-6), 7.15 (s, 1H, –N CH–), 1.13–2.50 (m, 18H); 13C NMR (400 MHz, CDCl3): d 155.68, 140.88, 131.14, 126.10, 121.27, 71.62,
63.06, 58.86, 49.93, 43.50, 42.20, 37.06, 36.85, 36.61, 31.54, 31.20, 30.97, 30.65, 23.67, 20.89, 19.29, 17.19; HRMS(EI): Calcd. for
C24H36N2O: 368.2828; Found: 368.2832; 6: white needle crystals; yield: 83.8%; mp: 232–235 8C; [a]D ꢁ232 (c 0.1, CHCl3); 1H NMR
20
(500 MHz, CDCl3): d 1.05 (s, 3H, H-18), 1.09 (s, 3H, H-19), 3.53 (m, 1H, H-3), 5.40 (t, 1H, J = 2.20 Hz, J = 2.93 Hz, H-6), 7.38 (s, 1H, –
N
CH–), 7.31–7.52 (m, 5H, Ar-H), 1.07–2.51 (m, 18H); 13C NMR (400 MHz, CDCl3): d 157.02, 140.99, 140.51, 135.19, 128.86, 127.11,
126.16, 123.49, 121.13, 71.61, 63.12, 50.17, 42.41, 42.22, 37.03, 36.65, 34.81, 31.53, 31.19, 30.73, 24.16, 20.40, 19.30, 17.56; HRMS(EI):
20
1
Calcd. for C26H32N2O: 388.2515; Found: 388.2513; 7: white solid; yield: 77.9%; mp: 217–220 8C; [a]D ꢁ250 (c 0.1, CHCl3); H NMR
(500 MHz, CDCl3): d 1.01 (s, 3H, H-18), 1.05 (s, 3H, H-19), 1.31 (s, 6H, H-30 C(CH3)2), 2.60 (s, 2H, H-20), 3.79 (s, 3H, N-CH3), 4.61 (m, 1H,
H-3), 5.42 (m, 1H, H-6), 7.13 (s, 1H, –N CH–), 0.78–2.94 (m, 17H); 13C NMR (400 MHz, CDCl3): d 170.60, 157.00, 139.81, 132.87, 123.67,
122.06, 73.94, 62.58, 50.16, 44.53, 41.06, 40.37, 37.91, 36.76, 36.65, 33.83, 31.14, 30.48, 29.66, 27.58, 25.37, 25.29, 25.24, 24.30, 20.26,
19.21, 17.08; HRMS(ESI): Calcd. for C27H38N2O4: 454.2832; Found: 455.2904(M + H+); 8: white solid; yield: 91.6%; mp: 216–218 8C;
[a]D –104 (c 0.1, CHCl3); 1H NMR (500 MHz, CDCl3): d 1.05 (s, 6H, H-18, H-19), 1.31 (s, 6H, H-30 C(CH3)2), 2.57 (m, 2H, H-20), 4.62 (m,
20
1H, H-3), 5.42 (s, 1H, H-6), 7.38 (s, 1H, –N CH–), 7.31–7.51 (m, 5H, Ar-H), 1.10–2.34 (m, 17H); 13C NMR (400 MHz, CDCl3): d 176.19,
170.51, 140.41, 139.79, 135.18, 128.86, 127.16, 123.53, 122.08, 74.04, 73.96, 62.96, 50.01, 44.49, 42.39, 37.90, 37.77, 36.71, 34.75, 31.16,
30.67, 27.57, 27.40, 25.36, 25.25, 24.15, 20.32, 19.24, 19.18, 17.54; HRMS(ESI): Calcd. for C32H40N2O4: 516.2988; Found:
517.3061.(M+H+).