Comparison of anti amoebic activity of stereoisomeric diamino and monoamino pregnene alkaloids and their N-methylated analogs

Article abstract of DOI:10.1007/s12039-012-0346-x

The steroidal alkaloid 3β, 20α-diamino-pregn-5-ene (kurchamine) obtained from the stem bark of Holarrhena antidysenterica is reported to have appreciable amoebicidal activity. Its three stereoisomers namely 3α, 20β-diamino-pregn-5-ene, 3β, 20β-diamino-pregn-5-ene and 3α,20α-diamino-pregn-5-ene and their intermediate stereoisomeric monoamino pregnene alkaloids namely 3β-amino-pregn-5-ene-20-one, 3α-amino-pregn-5-ene-20-one, 20α -amino-pregn-5-ene-3β-ol, 20β-amino-pregn-5-ene-3β-ol were synthesized. The natural stereoisomer and synthesized diamino and monoamino stereoisomers were N-methylated and all the compounds were evaluated for amoebicidal activity comparison. The natural stereoisomer 3β,20α-diamino-pregn-5-ene (kurchamine) was found to be superior than other stereoisomers and N-methylation was found to have insignificant effect on amoebicidal activity of stereoisomers.

Full text of DOI:10.1007/s12039-012-0346-x

J. Chem. Sci. Vol. 125, No. 1, January 2013, pp. 183–185. ꢀc Indian Academy of Sciences.
Comparison of anti amoebic activity of stereoisomeric diamino
and monoamino pregnene alkaloids and their N-methylated analogs
RAJ M VAID and K K BHUTANIb
a,∗
a
Organic Chemistry Division, Regional Research Laboratory, Indian Institute of Integrative Medicine (IIIM),
Canal Road, Jammu 180 001, India
b
National Institute of Pharmaceutical Education and Research, (NIPER),
Sector 67, SAS Nagar, (Mohali) 160 062, India
e-mail: RajMVaid@yahoo.com; director@niper.ac.in
MS received 25 February 2012; accepted 25 May 2012
Abstract. The steroidal alkaloid 3β, 20α-diamino-pregn-5-ene (kurchamine) obtained from the stem bark of
Holarrhena antidysenterica is reported to have appreciable amoebicidal activity. Its three stereoisomers namely
3
α, 20β-diamino-pregn-5-ene, 3β,20β-diamino-pregn-5-ene and 3α, 20α-diamino-pregn-5-ene and their inter-
mediate stereoisomeric monoamino pregnene alkaloids namely 3β-amino-pregn-5-ene-20-one, 3α-amino-
pregn-5-ene-20-one, 20α -amino-pregn-5-ene-3β-ol, 20β-amino-pregn-5-ene-3β-ol were synthesized. The natu-
ral stereoisomer and synthesized diamino and monoamino stereoisomers were N-methylated and all the com-
pounds were evaluated for amoebicidal activity comparison. The natural stereoisomer 3β,20α-diamino-pregn-
5
-ene (kurchamine) was found to be superior than other stereoisomers and N-methylation was found to have
insignificant effect on amoebicidal activity of stereoisomers.
Keywords. Holarrhena antidysenterica; amoebiasis; kurchamine; conessine; diamino pregnene;
monoamino pregnene; stereoisomers.
1
. Introduction
2. Experimental
Stem bark of Holarrhena antidysenterica (Kurchi) has 2.1 Materials and methods
been traditionally used for the treatment of amoebi-
1,2
Melting points were determined in open capillaries
and are uncorrected. IR were recorded for KBr discs
on Perkin-Elmer instrument. H NMR spectra were
asis. A number of steroidal alkaloids belonging to
conenine and pregnene series have been reported from
1
3
4,5
this plant. As already reported comparative eva-
lution of antiamoebic activity of both series of alka-
loids, pregnene alkaloid 3β, 20α-diamino-pregn-5-ene
recorded on Varian 60 MHz instrument in CDCl with
3
TMS as an internal standard and chemical shifts are
given in parts per million (ó scale). Column chro-
(kurchamine) was found to be most active even bet-
matography was carried out in basic Al O . TLC was
ter than the conenine principal alkaloid conessine. This
prompted us to evaluate the other known stereoisomers
of kurchamine as well as their intermediate stereoiso-
meric monoamino pregnenes with the aim to find a bet-
ter amoebicidal compound as compared to kurchamine
and to know the quantum of change in the amoebici-
dal behaviour of intermediate mono amino pregnenes as
these are transformed into diamino pregnene stereoiso-
mers. It was also planned to N-methylate, the natural
and synthesized mono and diamino stereoisomers so as
to enhance their amoebicidal activity as was observed
in coenine alkaloids of this plant.
2
3
run on silica gel coated glass plates. In Benzene-
Ethylacetate-Et NH (6:3:1) and spots were developed
2
by dragendorffs reagent. Authentic samples of 16-DPA,
conessine, conarrhimine, 3β 20α- diamino-pregn-5-ene
(kurchamine) were obtained inhouse.
2
.1a 3 α–Amino –pregn-5-ene -20-one (1): It was
obtained as crystalline solid. Yield: −60.0%,
m.p. 130–132 C (lit m.p. 135–136 C). H NMR
60 MHz,CDCl ): ó 0.75 (s,3H), 1.09 (s, 3H), 2.05 (s,
◦
9
◦
1
(
3
3
H) and 5.32 (brs, 1H).
2
.1b 3β–Amino –pregn-5-ene -20-one (2): It was
◦
obtained as HCl salt. Yield: −45.0%, m.p. >300 C,
∗
−1
1
For correspondence
IR : 3250,2920,1715, and 810 cm ; H NMR
83
1

184
Raj M Vaid and K K Bhutani
(
60 MHz,CDCl ): ó 0.68 (s, 3H), 0.90 (s, 3H), 2.08 (s, (2), 20α -amino –pregn-5-ene-3β-ol (3), 20β-amino
3
3
H) and 5.32 (brs,1H).
–pregn-5-ene-3β-ol (4), 3α, 20α -diamino- pregn-5-ene
6), 3β, 20β-diamino-pregn-5-ene (7) and 3α, 20β-
.1c 20α - Amino-pregn-5-ene-3β-ol (3): It was diamino-pregn-5-ene (8). The synthesized compounds
(
2
obtained as crystalline solid. Yield: −2.6%, m.p. 164– were confirmed by comparison of spectral data with the
◦
9
◦
1
9
6
6 C (lit m.p. 177 C). H NMR (60 MHz,CDCl ): ó literature values. To complete the series stereoisomer
3
0
.68 (s, 3H), 0.98 (s, 3H), 1.16 (d, 3H, J 6 Hz) and 5.32 3β, 20α-diamino-pregn-5-ene (kurchamine) (5) was
(
brs, 1H).
isolated from the methanolic extract of powdered stem
bark of kurchi (H. antidysenterica) plant by column
chromatography in Al O (basic) by gradient elution
2
.1d 20 β -Amino-pregene-5-ene-3 β-ol (4): It was
2
3
using pet.ether (60–80)- ethylacetate-methanol.10 All
the synthesized compounds and natural stereoisomer
(5) were subjected to amoebicidal activity tests in
obtained as crystalline solid. Yield: −18.35%, m.p. 230–
◦
9
◦
3
1
1
5 C (lit m.p. 218–20 C), IR :3450,2935,1465 and
−
1 1
050 cm ; H NMR (60 MHz,CDCl ): ó 0.68 (s, 3H),
3
11
.01(s, 3H), 1.03 (d,3H, J 7Hz) and 5.32 (brs, 1H).
vitro. The results obtained in terms of MIC are shown
in table 1. A glance at the results shows that that among
the diamino stereoisomers 3β, 20α –diamino-pregn-5-
ene (5) the natural stereoisomer (kurchamine) with 3β,
2
.1e 3 β, 20α- Diamino-pregn-5-ene (5): It was
obtained as crystalline solid. Yield: −0.12%, m.p.
◦
9
◦
1
1
45–47 C (lit m.p. 148 C). IR:3400,2900,1410 and
2
0α disposition of amino grs is most active there by cor-
−
1
1
1
320 cm ; H NMR (60 MHz,CDCl ) H NMR
4,5
3
roborating the earlier findings followed by 3α, 20α
diamino-pregn-5-ene (6) whose activity was found to
(60 MHz,CDCl ): ó 0.68 (s,3H), 1.00 (s, 3H), 1.10 (d,
3
–
3
(
H, J 6Hz). 2.0 (s, 4H,D O- exchangeable) and 5.32
2
brs,1H).
be half of natural stereoisomer (5) and equal to cones-
sine (16), the stereoisomer 3β, 20β-diamino-pregn-5-
ene (7) had activity half of (6) and the stereoisomer
3 α, 20 β-diamino-pregn-5-ene (8) was found to be four
times less active as compared to the stereoisomer (7).
The results also show that the introduction of one amino
gr at C-20 position in α orientation in pregnene nucleus
induces appreciable activity in 20 α-amino-pregn-5-ene
2
.1f 3α, 20α-Diamino-pregn-5-ene (6): It was
◦
obtained as a solid. Yield: −68%, m.p. 148–150 C
9
◦
(
lit m.p. 149–151 C), IR: 3400,2900,1550,1390 and
−1
1
7
0
1
80 cm ; H NMR (60 MHz,CDCl ): ó 0.68 (s,3H),
3
.97 (s, 3H), 1.16 (d,3H, J 6Hz), 1.86(m,4H), 3.16 (brs,
H) and 5.32 (brs, 1H).
(
3) and is comparable to 3 α 20 α –diamino isomer (6)
and conessine (16) but half as active as (5). The other
mono amino stereoisomer (4) with C- 20 amino gr in
β orientation was found to have four times less activity
2
.1g 3β, 20β-Diamino-pregn-5-ene (7): It was
◦
9
obtained as solid. Yield: −54.7%, m.p. 145–46 C (lit
◦
1
m.p. 145–47 C). H NMR (60 MHz,CDCl ): ó 0,70
3
(
(
s,3H), 0.98 (s, 3H), 1.05 (d, 3H, J 6.5Hz) and 5.32
brs,1H).
Table 1. Amoebicidal activity results of mono and
diamino pregnene stereoisomers, conarrhimine and their N-
methylated analogs.
2
.1h 3α, 20β-Diamino-pregn-5-ene (8): It was
obtained as crystalline solid. Yield: −86.15%, m.p. 154–
Compound R1
R2
R3
MIC(μg/ml)
◦
9
◦
1
5
6 C (lit m.p. 158 C). H NMR (60 MHz,CDCl ): δ
3
0
.68 (s,3H), 1.03 (s, 6H) and 5.32 (brs, 1H).
1
2
3
4
5
6
7
8
9
1
1
α-NH2
β-NH2
β-OH
–
–
=O
NA
NA
62
250
31.75
62.50
125
500
=O
H
β-NH2
H
H
β-NH2
β-NH2
H
β-NMe2
H
α-NH2
H
α-NH2
α-NH2
H
3
. Results and discussion
β-OH
β-NH2
α-NH2
β-NH2
α-NH2
β-OH
β-OH
β-NMe2
α-NMe2
Except for few alkaloids such as 3β, 20α-diamino-pregn-
5
-ene (kurchamine) (5)6,7 and 3β, 20α- bisdimethylamino-
8
H
pregn-5-ene (kurchessine) (11) other stereoisomeric
diamino and monoamino pregnene alkaloids are not
reported from this plant (H. antidysenterica). To carryout
the study, the stereoisomeric di and mono amino preg- 12
nene alkaloids were synthesized starting from 16-DPA 13
α-NMe2 NA
H NA
α-NMe2 125
α-NMe2 NA
H
H
0
1
H
β-NMe2 β-NMe2
α-NMe2 β-NMe2
H
500
NA
NA
14
(Dehydropregnenolone acetate) following the literature
9
15
H
Me
H
Me
methodology with few modifications wherever neces-
sitated. The compounds synthesized were 3α- amino-
16
Me
62.50
10
pregn-5-ene-20-one (1), 3β –amino-pregn-5-ene-20-one NA –No activity was observed up to 1000 (μg/ml)

Comparison of anti amoebic activity of pregnene alkaloids
185
as compared to the 20 α –amino isomer (3). The C-3 C-3β and C-20α sites which enables better interaction
amino α and β stereoisomers (1 and 2) were found to and annihilation of amoebae and makes the natural ste-
be inactive at the tested concentrations.
reoisomer superior to others. To comment on the exact
Keeping in view of the conenine series of alkaloids mechanism involved is beyond the scope of this work.
of this plant where amoebicidal activity increases as
the degree of methylation of nitrogens at C-3 and C-20
4
. Conclusion
increases as this was observed in conarrhimine (15) the
parent base not active but fully N-methylated parent
base conessine (16) has appreciable activity. The syn-
thesized pregnene stereoisomers (3, 4, 6–8) and natural
stereoisomer (kurchamine) (5) were N methylated with
The stereoisomers of kurchamine were found to have
amoebicidal activity but were not comparable to natu-
ral stereoisomer and the order of activity was found to
be 3β, 20α -diamino-pregn-5-ene (5) > 3α, 20α-diamino-
pregn-5-ene (6) > 3β, 20β-diamino-pregn-5-ene (7)
10
HCHO-HCOOH thus yielding 20 α–dimethylamino
–
–
pregn-5-ene -3β-ol (9), 20 β–dimethylamino
pregn-5-ene -3β-ol (10), 3 β, 20α-bis dimethylamino-
>
3α, 20β-diamino-pregn-5-ene (8). Among the mono
amines 20α-amino isomer (3) was found to have appre-
ciable activity comparable to 3α, 20α- diamino stereo-
isomer (6) whereas 20β mono amino isomer (4) was found
to have four times less activity as compared to 20α-
amino isomer (3). N-methylation of all the synthesized
stereoisomers and kurchamine resulted in remarkable
fall in amoebicidal activity of respective compounds.
pregn-5-ene (11)), 3α, 20α-bisdimethylamino-pregn-
-ene (12), 3β, 20β -bis dimethylamino- pregn-5-ene
13), 3α, 20β-bisdimethyiamino-pregn-5-ene (14) and
5
(
their amoebicidal activity was evaluated in vitro.
The results (table 1) indicate that N-methylation of
stereoisomers has resulted in appreciable fall in activity
instead of enhancement of activity as was observed
in conenine series. The amoebicidal activity results
indicate that the stereoisomer (5) has superior activity
as compared to other stereoisomers. The better activity
of (5) appears to be due to the presence of two amino
groups at less hindered
Acknowledgements
The authors are thankful to Dr. G L Sharma and Ravinder
Kour of Organic Chemistry Division, Regional Re-
search Laboratory, Jammu (RRL) for biological testing
of the compounds. RMV is thankful to the Council of
Scientific and Industrial Research (CSIR) for the award
of Junior Research Fellowship (JRF).
Me
R2
R 3
Me
Me
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