Synthesis of 3β, 7α, 11α-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives and their cytotoxic activities

Article abstract of DOI:10.1016/j.bmcl.2009.10.019

A series of novel 3β, 7α, 11α-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives were synthesized and characterized by NMR, HRMS. The pregnenolone (1) was first biotransformed by Mucor circinelloides var lusitanicus to 3β, 7α, 11α-trihydroxy-pregn-5-

Full text of DOI:10.1016/j.bmcl.2009.10.019

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6637–6639
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 3b, 7a, 11a-trihydroxy-pregn-21-benzylidene-5-en-20-one
derivatives and their cytotoxic activities
b
c
a
c
Li-Hong Shan a, Hong-Min Liu a, , Ke-Xue Huang , Gui-Fu Dai , Chen Cao , Rui-Jing Dong
*
a New Drug Research and Development Center of Zhengzhou University, Zhengzhou 450001, China
b College of Life Science, Hunan Normal University, Changsha 410081, China
c Bioengineering Department of Zhengzhou University, Zhengzhou 450001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 3b, 7
a
, 11
a
-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives were synthe-
Received 18 February 2009
Revised 18 September 2009
Accepted 5 October 2009
Available online 14 October 2009
sized and characterized by NMR, HRMS. The pregnenolone (1) was first biotransformed by Mucor circi-
nelloides var lusitanicus to 3b, 7 , 11 -trihydroxy-pregn-5-en-20-one (3), then 3 was treated with
various benzaldehydes to produce 3b, 7 , 11 -trihydroxy-pregn-21-benzylidene-5-en-20-one deriva-
a
a
a
a
tives. These derivatives showed remarkable activity against EC109 cells. The absolute configuration of
3 was also confirmed by signal-crystal X-ray analysis.
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Biotransformation
Pregnenolone
Hydroxylation
Mucor circinelloides lusitanicus
Claisen–Schimidt condensation
Cytotoxicity
It is well known that a variety of steroids are widely used as
anti-inflammatory, diuretic, anabolic, contraceptive, antiandrogen-
ic, progestational, anticancer agents as well as in other applica-
tions.1 The distinct structural steroids have aroused great
interest, and extensive research have been carried out to develop
the methods to synthesize and characterize steroids.2 Since Peter-
our interests focus on the improvement of the yields for important
metabolites as well as preparation of new steroids that are difficult
to synthesize by chemical approaches.
The fungus, Mucor circinelloides var lusitanicus, was isolated from
local soil and identified by Microbial ID (Newark, USA). The family
Mucor are widely distributed in nature. Mucor circinelloides was used
to hydrolyze carboxymethyl cellulose, insoluble cellulose substrates
to produce valuable soluble cellodextrins.17–20 But no report on
using it to biotransform steroids was previously reported.
Our studies showed that Mucor circinelloides var lusitanicus can
son and Murray reported the 11a-hydroxylation of progesterone
by Rhizopus arrhizus in 1952,3 microbial reactions for the transfor-
mation of steroids have proliferated, and specific microbial trans-
formation steps have been incorporated into numerous partial
syntheses of new steroids for evaluation as drugs and hormones.4,5
Pregnenolone (1) is a major hormone mainly present in human
nerve tissues,6 and its therapeutic role in repairing neurons has
transform various 5-en-3b-ol steroids to their 7a-hydroxy deriva-
tives. We were interested in the biotransformation of compound 1
(Scheme 1), mainly because compound 3 could be easily crystallized
from the mixture with high yield (46.38%). This made it possible to
been well documented.7 Recent studies indicated that 7
a-hydrox-
ylated metabolite of 1 has many profound activities, such as aug-
menting immune response in mouse,8 possessing antiglucorticoid
potencies,9 improving spatial memory,10 and acting as a neuronal
activator to stimulate locomotor activity.11 Compound 1 was also
synthesize 3b, 7a, 11a-trihydroxy-pregn-21-benzylidene-5-en-20-
onederivatives5a–k (Table1)from3inlargequantitiesthroughCla-
isen–Schimidt reaction. In the current studies, the cytotoxic effects
of 3 and 5a–k on EC109 cells were evaluated.
biotransformed to its 7
a
-hydroxylated, 11
a
-hydroxylated or 7
a
,
In order to obtain compound 3, the fermentation media was
optimized,21 which resulting in an improvement of the transfor-
mation efficiency of 1 from 70% to 94%. Furthermore, the recovery
yield of 3 reached 46.38% by crystallizing from the extract mixture
directly with methanol and chloroform (1:3). The recovery yield of
3 was higher than that of the reported yield 19.6%22 and 35.0%.23
Product 2 was purified by silica gel chromatography with a yield
of 24.38%. The optimization process showed that the concentration
11a
-dihydroxylated derivatives with low yield,12–15 and these
metabolites are not commercially available. This hinders further
studies on their structure modification and activities evaluation.16
Although introduction of new steroids into market is now limited,
* Corresponding author. Tel./fax: +86 371 67781739.
E-mail address: liuhm@zzu.edu.cn (H.-M. Liu).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.019

6638
L.-H. Shan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6637–6639
O
O
O
HO
Mucor circinelloides l.
+
HO
HO
OH
HO
OH
3
1
2
Scheme 1. Biotransformation of pregnenolone 1 to compound 2 and 3 by Mucor circinelloides var lusitanicus.
Table 1
Introduction of aromatic group into steroids have led to new
physiological activity.25 Hydroximino derivatives of 16E-arylide-
nosteroids showed good antineoplastic activity,26 and 16-(4-
substituted benzylidene) derivatives of androst-5-ene also had
antiproliferative activity against cancer cells.27 So we introduced
benzylidene groups into 3 and evaluated their cytotoxic activity.
Through the Claisen–Schimidt condensation reaction, compound
3 was reacted with benzaldehyde 4a–k and compounds 5a–k were
produced (Scheme 2). Compared to Na2CO3, K2CO3 and 1 mol/L of
NaOH solution, solid NaOH catalyzed the reaction more efficiently.
The effect of solvents in the reaction was also investigated. Only in
strong polar protolytic solvent, such as methanol and ethanol, was
the reaction performed successfully. When ethanol (95%) was used
as the reaction solvent, the reaction was completed under room
temperature in short period. The structures of compounds 5a–k
and the reaction conditions are presented in Table 1. The reaction
time and yield revealed that the electron-withdrawing group on
the benzene ring was favorable to the reaction. Taking 5a as an
example to illustrate how the structures of 5a–k were determined:
From the 1H NMR spectrum,28 the signal of 21-methyl (d 2.07) of 3
disappeared, instead of that, two proton signals at d 6.92 (d), 7.54
(d) were found, and the two relevant protons shared a same J con-
stant (16.07 Hz). Thus they were assigned to the 21 and 22 proton,
respectively. Through the J Constant, we concluded that the substi-
tutes on the C21 and C22 were E-type. The chemical shifts at d
130.5 and 141.1 in 13C NMR spectrum of 5a confirmed that the de-
sired carbonyl group C@C had been formed. The HRMS spectrum of
5a also confirmed the formula of 5a was C28H36O4. Together with
other signals in the 1H, 13C NMR spectrum, 5a was defined as 3b,
Structures of compound 5a–k and the yields
Entry
R
Reaction time (h)
Product
Yield (%)
1
2
3
4
5
6
7
8
9
H
3-NO2
3-Cl
4-Cl
4-F
4-OCH3
2-OCH3
4-OH
4-NH2
4-N(CH3)2
2,4,6-(OCH3)3
8
8
8
8
8
12
12
24
24
24
24
5a
5b
5c
5d
5e
5f
5g
5h
5i
87
88
85
85
89
74
68
63
58
51
50
10
11
5j
5k
of Mg2+ and the initial pH (pH 6.0) in the fermentation media were
critical for the transformation of 1 to 3.
The 1H NMR spectrum24 of 3 showed three hydroxyl-bearing
methine proton signals at d 3.80 (m, 1H), 3.57 (m, 1H), 3.34 (m,
1H). These values corresponded to the chemical shift of proton at-
tached to a carbon atom bearing a hydroxyl group. Also the 1H
NMR spectrum showed a downfield shift of the C-19 methyl pro-
tons (d 1.12) when compared to the spectrum of the substrate 1
(d 1.00). This is due to the presence of a hydroxyl group at the
11
field shift (d 5.44) and split (J = 5.3 Hz) of 6 proton implied a hydro-
xyl group at 7 -position and assignment of multiple d 3.57 to 7b
proton. Finally, d 3.34 was attributed to the 3 proton. Based on
the NMR, HRMS results, together with the literature data,23 com-
pound 3 was assigned the structure of 7 , 11 -dihydroxy-pregn-
a-position. So d 3.80 was attributed to 11b proton. The down-
a
a
a
a
5-en-20-one. The absolute configuration of 3 was established by
7a, 11a-trihydroxy-pregn-21-benzylidene-5-en-20-one. To our
X-ray crystallographic analysis (Fig. 1), which confirmed the hy-
knowledge, this is the first report of the synthesis of compounds
droxyl group at 7 and 11 position was in the axial (a) orientation.
5a–k.
The in vitro cytotoxicity29 of this novel series of compounds
against the human esophageal cancer cell line, EC109, was evalu-
ated. Table 2 shows the inhibitory effects on cell proliferation. Ori-
donin, an efficacious component of antitumour extracted from
Chinese herb Rabdisia rubeseas (hemsl) Hara, was used to treat tu-
mor of digestive tract on clinical.30 We selected oridonin as the po-
sitive contral. From the results, we can find that compound 1 only
show faintish activity to EC109. Compared to 1, both compound 3
and 5a–k show stronger inhibition activity. This indicated that the
7a- and 11a-hydroxyl group and the structure of benzylidene are
essential for the cytotoxic activity we examined. Moreover, the
majority of 5a–k produced a stronger suppressive activity than
their parent compound (3), and the cytotoxicity response was sen-
Figure 1. The X-ray diffraction analysis diagram of compound 3.
O
O
R
HO
HO
HOC
+
R
HO
OH
HO
OH
4a-k
5a-k
3
Scheme 2. Synthesis of compound 5a–k. Universal reagent and conditions: solid NaOH, 95% ethanol, rt.

L.-H. Shan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6637–6639
6639
Table 2
4. Fernandes, P.; Cruz, A.; Angelova, B.; Pinheiro, H. M.; Cabral, J. M. S. Enzyme
Microb. Technol. 2003, 32, 688.
5. Herbert, L. H. Steroids 1999, 64, 178.
6. Morfin, R.; Young, J.; Corpechot, C. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 6790.
7. Baulieu, E. E.; Robel, P.; Fellous, A.; Duchossoy, Y.; Fontaine-Lenoir, V.; David, S.
J. Mol. Neurosci. 2004, 24, 63.
8. Morfin, R.; Courchay, G. J. Steroid Biochem. Mol. Biol. 1994, 50, 91.
9. Morfin, R.; Calvez, D.; Malewiak, M. I. Inl. Congr. Hormonal Steroids (Dallas,
Texas, USA), 1994, poster 103.
10. Yau, J. L. W.; Noble, J.; Graham, M.; Seckl, J. R. J. Neurosci. 2006, 26, 11034.
11. Matsunaga, M.; Ukena, K.; Baulieu, E.; Tsutsui, K. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 17282.
12. Andrew, S. C.; William, A. D.; Sir Ewart, R. H. J.; Denis Meakins, G.; John, T. P. J.
C. S Perkin I. 1973, 2137.
13. Madyastha, K. M. Pro. India Acad. Sci. (Chem. Sci.) 1994, 106, 1203.
14. Schaaf, O.; Dettner, K. J. Steroid Biochem. Mol. Biol. 2000, 75, 187.
15. Muhammad, I. C.; Iffat, B.; Syed, A. A. S.; Sarfraz, A. N.; Atta, U. R. Chem. Pharm.
Bull. 2005, 53, 1455.
16. Anne-Cécile, C.; Robert, M. J. Steroid Biochem. Mol. Biol. 1999, 68, 229.
17. Saha, B. C. Process Biochem. 2004, 39, 1871.
Inhibitory effects of compounds against EC109 cells at concentration of 50 lg/mL
Compd
EC109
1
3
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
13.6
21.72
59.79
72.37
76.43
80.25
76.82
55.29
47.42
45.66
50.21
38.97
35.26
94.65
7.5E-5
5k
Oridonin
NCa
18. Szczesna-Antczak, M.; Antczak, T.; Piotrowicz-Wasiak, M.; Rzyska, M.;
Binkowska, N.; Bielecki, S. Enzyme Microb. Technol. 2006, 39, 1214.
19. Antczak, T.; Graczyk, J.; Szczesna-Antczak, M.; Bielecki, S. J. Mol. Catal. B: Enzym.
2002, 19–20, 287.
a
Negative control.
20. Navarro, E.; Sandmann, G.; Torres-Martinez, S. Exp. Mycology. 1995, 19, 186.
21. The optimized medium consisted of maltose 4.3%, peptone 2.3%, yeast 0.1%,
K2HPO4 0.9%, MgSO4ꢀ7H2O 0.9 % and pH 6.0. Flasks (500 ml) containing 100 ml
medium were inoculated with spore suspension (1.0 ml, 2.2 ꢁ 108 spores) from
a two-day-old culture grown on the above slants, and incubated at 28 °C on a
rotary shaker (220 rpm) for 48 h. After this growth period, 100 mg substrate in
1 ml acetone or DMSO was added to each flask and the incubation was
continued for four days. Biotransformation of the substrates was monitored by
TLC.
sitive to a variety of functional groups and positions on the ben-
zene ring. Furthermore, the results indicate that when there was
an electron-withdrawing group on the benzene ring, cytotoxic
activity was much higher than when compared to the presence
of an electron-donating group on the benzene ring.
In conclusion, we have synthesized a series of new 3b, 7a, 11a-tri-
22. Ge, W. Z.; Wang, S. M.; Shan, L. H.; Li, N.; Liu, H. M. J. Mol. Catal. B: Enzym. 2008,
55, 37.
hydroxy-pregn-21-benzylidene-5-en-20-one derivatives through
biotransformation and chemical synthesis, and evaluated their cyto-
toxicity against EC109 cells. The synthesis procedure was simple
and efficient, and the results indicated that Mucor circinelloides var
lusitanicus can be used to biotransform other steroids to produce no-
vel active steroids. Additionally, other activities of these compounds
and further structure modification to improve the bioactivity are
under investigation.
23. Madyastha, K. M.; Joseph, T. Appl. Microbiol. Biotechnol. 1995, 44, 339.
24. Compound 3: 1H NMR (400.1 MHz, DMSO-d6, 25 °C, TMS): d 0.50 (s, 3H, C(18)-
CH3), 1.12 (s, 3H, C(19)-CH3), 2.07 (s, 3H, C(21)-CH3), 3.34 (m, 1H, C(3)-H), 3.57
(m, 1H, C(7)-H), 3.80 (m, 1H, C(11)-H), 5.44 (d, 1H, J = 5.3 Hz, C(6)-H); 13C NMR
(100.6 MHz, DMSO-d6, 25 °C, TMS): d 14.1, 17.7, 22.6, 23.7, 31.3, 31.6, 37.1,
38.4, 38.5, 42.8, 43.6, 47.8, 49.1, 49.5, 62.8, 63.5, 67.2, 70.2, 124.3, 144.6, 208.7;
HRMS: calcd for C21H32O4 [M+Na]+ requires 371.2198, and 371.2201 was
found.
25. Dubey, S.; Piplani, P.; Jindal, D. P. Lett. Drug Des. Discovery 2005, 2, 537.
26. Chattopadhaya, R.; Jindal, D. P.; Minu, M.; Gupta, R. Arzneim.-Forsch. 2004, 54,
551.
Acknowledgment
27. Dubey, S.; Piplani, P.; Jindal, D. P. Chem. Biodivers. 2004, 1, 1529.
28. Compound 5a: 1H NMR (400.1 MHz, DMSO-d6, 25 °C, TMS): d 0.51 (s, 3H, C(18)-
CH3), 0.99 (s, 3H, C(19)-CH3), 3.29 (m, 1H, C(3)-H), 3.61 (d, 1H, J = 2.2 Hz, C(7)-
H), 3.79 (m, 1H, C(11)-H), 5.45 (d, 1H, J = 5.4 Hz, C(6)-H), 6.92 (d, 1H,
This research work was supported by NSFC of China (No.
20572103).
2
J = 16.07 Hz, C(21)-H), 7.44 (t, 2H, J = 3.6, C(30)-H and C(50)-H), 7.45 (s, 1H,
2
C(40)-H), 7.54 (d, J = 16.07 Hz, 1H, C(22)-H), 7.71 (t, 2H, J = 3.7, C(20)-H and
C(60)-H); 13C NMR (100.6 MHz, DMSO-d6, 25 °C, TMS): d 14.4, 17.6, 22.6, 23.9,
31.6, 37.2, 38.3, 38.5, 42.9, 44.6, 47.8, 49.2, 49.6, 60.4, 63.5, 67.2, 70.1, 124.2,
127.4, 128.5(2C), 129.1(2C), 130.5, 134.8, 141.1, 144.6, 199.9; HRMS: calcd for
C28H36O4 [M+H]+ requires 437.2692, and 437.2675 was found.
Supplementary data
Supplementary data (CCDC-668534 (compound 3) contains the
supplementary crystallographic data for this paper. These data can
be obtained free of charge via www. ccdc. com. ac.uk/data_request/
cif, by emailing data_request@ccdc.cam.ac.uk, or by contacting the
Cambridge Crystallographic Data Center, 12 Union Road, Cam-
bridge CB2 1EZ, UK [fax: +44 1223 336033]) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2009.10.019.
29. In vitro cytotoxicity studies: EC109 cell lines were obtained from National
Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of
Medical Science & Peking Union Medical College. Culture medium was RPMI-
1640 (GIBCO Co., Grand Island, NY) supplemented with 10% (v/v) fetal calf
serum, 100 IU/mL penicillin and 100 lg/mL streptomycin(Sigma Chemical Co.,
St. Louis, MO) at 37 °C in humidified air atmosphere of 5% CO2 (Binder, CB150,
Germany). Cell cytotoxicity was assessed by MTT assay. Briefly, cells were
plated into 96-well-plate (7000 cells/well). The next day compound at 100
mL diluted in culture medium was added (200 L/well) to the wells. 48 h later,
20 L of MTT (Sigma Chemical Co., St. Louis, MO) (0.5 mg/mL MTT in PBS) was
added and cells were incubated for a further 4 h. 200 L DMSO was added to
lg/
l
l
l
References and notes
each culture to dissolve the reduced MTT crystals. The MTT-formazan product
dissolved in DMSO was estimated by measuring absorbance at 570 nm with a
microplate reader (Biotech, Power Wave, CA). Then the inhibitory percentage
of each compound was calculated.
1. Mahato, S. B.; Garai, S. Steroids 1997, 62, 332.
2. Reese, P. B. Steroids 2001, 66, 481.
3. Peterson, D. H.; Murry, H. C.; Eppstein, S. H.; Reineke, L. M.; Weintraub, A.;
Meister, P. D.; Leigh, H. M. J. Am. Chem. Soc. 1952, 74, 5933.
30. Li, D.; Wu, L. J.; Tashiro, S. I. Biol. Pharm. Bull. 2007, 30, 254.