Synthesis and biological evaluation of pseudostellarin B

Article abstract of DOI:10.1016/S0014-827X(01)01031-X

A new potent bioactive cyclic peptide pseudostellarin B has been synthesised. The structure was elucidated by elemental analyses, IR, ¹H NMR, ¹³C NMR and FAB mass spectral data. The synthesised compound was also screened for its antibacterial, antifungal, antiinflammatory and anthelmintic activities. Copyright

Full text of DOI:10.1016/S0014-827X(01)01031-X

www.elsevier.nl/locate/farmac
Il Farmaco 56 (2001) 331–334
Short Communication
Synthesis and biological evaluation of pseudostellarin B
Boja Poojary, K. Harish Kumar, Shiddappa L. Belagali *
Department of Post-Graduate Studies and Research in Chemistry, Mangalore Uni6ersity, Mangalagangothri 574199, India
Received 5 April 2000; accepted 10 October 2000
Abstract
A new potent bioactive cyclic peptide pseudostellarin B has been synthesised. The structure was elucidated by elemental
analyses, IR, 1H NMR, 13C NMR and FAB mass spectral data. The synthesised compound was also screened for its antibacterial,
antifungal, antiinflammatory and anthelmintic activities. © 2001 Elsevier Science S.A. All rights reserved.
Keywords: Pseudostellarin B; Spectral studies; Bioactivities
1. Introduction
2. Chemistry
Recently a number of cyclic peptides with unique
structures and biological activities have been isolated
from natural sources [1]. Peptides appear to be impor-
tant potential drugs: cyclotheonamides serve as a model
compound for antithrombin drugs, discodermins are
potential antitumor promoting drugs and calyculins are
useful biochemical reagents [1]. A new potent tyrosinase
inhibitory cyclic peptide, pseudostellarin B, has been
isolated from the roots of Pseudostellaria heterophylla
and the structure was elucidated by extensive 2D NMR
methods, chemical and enzymatic degradations and
tandem MS spectroscopic analyses by Hiroshi Morita et
al. [2].
For the present work, the cyclic octapeptide was
disconnected into four dipeptides 1–4. These dipeptides
were coupled after proper deprotection using dicyclo-
hexyl carbodiimide (DCC) as a coupling agent to get
tetrapeptides 5 and 6. These two tetrapeptides were
coupled to get octapeptide 7. Finally cyclisation of 7
employing the p-nitrophenyl ester method resulted in
expected product 8 (Scheme 1).
For the protection of the amino group of the
acids, we used di-tert-butyl pyrocarbonate to get N-tBoc
amino acids [4]. The carboxyl group of -amino acids
L-amino
L
was protected by esterification in the presence of thionyl
chloride. Dipeptides 1–4, tetrapetides 5 and 6 and
octapeptide 7 were prepared by the Bodanszky method
[5]. All these intermediates were purified by recrystallisa-
tion from CHCl3–n-hexane. CF3COOH–CHCl3 was
used for the removal of the tBoc group and LiOH–H2O
for the removal of the ester group. Cyclisation of linear
segment 7 was carried out by using p-nitrophenyl ester
method [6] to yield pseudostellarin B 8.
As part of our ongoing study on the synthetic aspects
of bioactive cyclic peptides [3], we planned to synthesise
pseudostellarin B and study its biological activities. In
this paper, we describe the synthesis, characterisation
and biological studies of pseudostellarin B.
The newly synthesised compound was characterised
1
by elemental analyses, IR, H NMR, and mass spectral
data (Table 1). The infrared spectrum of 8 shows
absorption bands that are characteristic of the sec-
ondary amide groups, no absorption bands appear for
free amino groups or carboxyl groups. Extensive appli-
cation of NMR techniques was used to determine the
* Corresponding author.
E-mail address: bojapoojary@yahoo.com (S.L. Belagali).
0014-827X/01/$ - see front matter © 2001 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01031-X

332
B. Poojary et al. / Il Farmaco 56 (2001) 331–334
identity of these amino acid units. 1H NMR clearly
showed the presence of all the protons of eight amino
acids. Moreover the mass spectrum of 8 showed the
M++H peak at m/z 683, which corresponds to the
molecular formula C33H48N8O8. The relative intensity
of the molecular ion peak suggested the cyclic nature of
the octapeptide.
3.1. Pseudostellarin B 8
To the solution of Boc-octapeptide-pnp ester (1.2
mmol) in chloroform (15 ml), trifluoroacetic acid (0.274
g, 2.4 mmol) was added, stirred for 1 h at room
temperature and washed with 10% sodium bicarbonate
solution. The organic layer was dried over anhydrous
sodium sulphate. To the Boc-deprotected peptide-pnp
ester in THF (15 ml), pyridine (1.4 ml, 2 mmol) was
added and kept at 4°C for seven days. The reaction
mixture was washed with 10% sodium bicarbonate so-
lution until the byproduct p-nitrophenol was removed
completely and finally washed with 5% HCl (5 ml). The
organic layer was dried over anhydrous sodium sul-
phate. THF and pyridine were distilled to get pseudos-
tellarin B 8. The crude product was purified by silica gel
column chromatography using the dichloromethane–
methanol system and finally recrystallised from
EtOAc–n-hexane.
3. Experimental
Melting points were taken in open capillary tubes
and are uncorrected. IR spectra (in CHCl3) were
recorded on a Perkin–Elmer infrared spectrophotome-
ter. NMR spectra were recorded in CHCl3-d/DMSO-d6
on a 300 MHz spectrophotometer using tetramethyl
silane (TMS) as an internal standard. The mass spectra
were recorded on a FAB mass spectrometer. The purity
of all compounds was checked by thin layer chromatog-
raphy (TLC) on silica gel G plates.
Scheme 1.

B. Poojary et al. / Il Farmaco 56 (2001) 331–334
333
Table 1
Characterisation of 1–8
Compound
Physical state
M.p. (°C)
Yield (%)
Molecular formula
C14H26N2O5
Anal. (%N), found (calc.)
1 a
2
3
Semisolid mass
Semisolid mass
Viscous liquid
Pale white solid
Semisolid mass
Semisolid mass
Semisolid mass
White solid
–
–
–
82.8
71.8
76.9
79.8
75.6
68.2
70.0
61.0
9.34 (9.27)
11.43 (11.38)
9.84 (9.79)
C10H18N2O5
C13H22N2O5
C20H28N2O5
C18H32N4O7
C27H38N4O7
C39H58N8O11
C33H46N8O8
4 b
5 c
6 d
7 e
8 f
55–56
–
–
–
7.40 (7.44)
13.44 (13.46)
10.60 (10.56)
13.80 (13.76)
16.48 (16.42)
167–169
a IR (cm−1): 3320 (gNꢀH), 2930 (gCꢀH), 1710 (gCꢁO ester), 1670 (gCꢁO amide), 1051 (gCꢀO), 860 (gCꢀH); 1H NMR (CHCl3-d): l 8.2 (br s, 1H, NH),
t
6.9 (br s, 1H, NH), 4.4–4.3 (m, 1H, a-CH), 4.1–3.9 (m, 2H, a-CH2), 3.7 (s, 3H, ꢀOCH3), 1.7–1.6 (m, 1H, g-CH), 1.4 (s, 9H, Boc), 1.3–1.1 (m,
2H, b-CH2), 0.95 (doublet overlapped with triplet, 6H, 2CH3).
b IR (cm−1): 3540 (gNꢀH), 3310 (gNꢀH), 3010 (gꢁCꢀH), 2975 (gCꢀH), 1735 (gCꢁO ester), 1675 (gCꢁO amide), 1660 (gCꢁO amide), 1595 (gCꢁC), 1020
(gCꢀO), 740 (gCꢀH); 1H NMR (CHCl3-d): l 7.25 (m, 5H, ArH), 5.1–4.7 (br s, 1H, NH), 4.5–4.1 (m, 2H, 2a-CH), 3.7 (s, 3H, ꢀOCH3), 3.4–3.0 (m,
t
4H, NꢀCH2 and b-CH2), 2.2–1.6 (m, 4H, ꢀCH2ꢀCH2ꢀ), 1.4 (s, 9H, Boc).
1
c IR (cm−1): 3315 (gNꢀH), 2910 (gCꢀH), 1740 (gCꢁO ester), 1690 (gCꢁO amide), 1670 (gCꢁO amide), 1420 (gCꢀH), 1100 (gCꢀO), 970 (gCꢀH); H NMR
(CHCl3-d): l 8.2–7.8 (br s, 3H, 3NH), 6.9 (br s, 1H, NH), 4.4–4.3 (m, 3H, a-CH and a-CH2), 4.2–3.9 (m, 4H, 2a-CH2), 3.7 (s, 3H, ꢀOCH3),
t
1.8–1.6 (m, 1H, ꢀCH), 1.4 (s, 9H, Boc),1.3–1.1 (m, 2H, g-CH2), 0.98 (doublet overlapped with triplet, 6H, 2CH3).
d IR (cm−1): 3300 (gNꢀH), 3020 (gꢁCꢀH), 2950 (gCꢀH), 1720 (gCꢁO ester), 1675 (gCꢁO amide), 1620 (gCꢁC), 1320 (gCꢀN), 1140 (gCꢀO), 970 (gCꢀH);
1H NMR (CHCl3-d): l 8.3 (br s, 1H, NH), 7.8 (br s, 1H, NH), 7.4–7.1 (m, 5H, ArH), 4.7–4.5 (m, 3H, 3a-CH), 4.2–4.0 (m, 2H, a-CH2), 3.7 (s,
t
3H, ꢀOCH3), 3.6–3.4 (m, 4H, NꢀCH2), 3.3–3.0 (m, 2H, b-CH2), 2.2–1.6 (m, 8H, 2-CH2ꢀCH2ꢀ), 1.4 (s, 9H, BOc).
e IR (cm−1): 3310 (gNꢀH), 3150 (gNꢀH), 3010 (gꢁCꢀH), 2950 (gCꢀH), 1720 (gCꢁO ester), 1690 (gCꢁO amide), 1685 (gCꢁO amide), 1675 (gCꢁO amide),
1620 (gCꢁC), 1470 (gCꢀH), 1280 (gCꢀO), 970 (gCꢀH); 1H NMR (CHCl3-d): l 8.4–8.2 (br s, 4H, 4NH), 8.0–7.7 (br s, 2H, 2NH), 7.4–7.1 (m, 5H, ArH),
4.8–4.5 (m, 4H, a-CH), 4.3–4.0 (m, 6H, a-CH2), 3.7 (s, 3H, ꢀOCH3), 3.5–3.1 (m, 6H, 2N ꢀCH2 and b-CH2), 2.2–1.7 (m, 8H, ꢀCH2ꢀCH2ꢀ), 1.6–1.5
t
(m, 1H, b-CH), 1.4 (s, 9H, BOC), 1.3–1.1 (m, 2H, g-CH2), 0.95 (doublet overlapped with triplet, 6H, 2CH3).
f IR (cm−1): 3350 (gNꢀH), 3015 (gꢁCꢀH), 2985 (gCꢀH), 1690 (gCꢁO amide), 1670 (gCꢁO amide), 1615 (gCꢁC), 1510 (gCꢀN), 1445 (gCꢀH), 975 (gCꢀH);
1H NMR (DMSO): l 8.3–8.1 (br s, 4H, 4NH), 8.0–7.7 (br s, 2H, 2NH), 7.3–7.0 (m, 5H, ArH), 4.8–4.6 (m, 4H, 4a-CH), 4.4–4.1 (m, 6H, 3a-CH2),
3.6–3.4 (m, 4H, 2-NꢀCH2), 3.3–3.1 (m, 2H, b-CH2) 2.2–1.8 (m, 8H, 2-CH2ꢀCH2ꢀ), 1.7–1.6 (m, 1H, b-CH), 1.4–1.2 (m, 2H, g-CH2); FABMS (m/z):
(M++H) 683.
Table 2
Antibacterial and antifungal activity data
Comp. no.
Zone of inhibition (in mm) at 10 mg ml−1 concentration
P. aeruginosa
E. coli
B. subtilis
S. aureus
C. albicans
A. niger
8
–
12
16
–
14
12
16
–
16
18
12
–
–
18
12
–
11
–
–
–
–
–
Penicillin
Streptomycin
Griseofulvin
20
18
4. Biological activity studies
activity against Escherichia coli, Staphylococcus aureus,
Bacillus subtilis, Pseudomonas aeruginosa, C. albicans
and A. niger were carried out by employing the disc
diffusion method [7]. The results are summarised in
Table 2. The anti-inflammatory activity study was car-
ried out according to the method of Winter et al. [8]
(Table 3) and the anthelmintic activity study according
to the method of Garg and Atal [9] (Table 4).
The newly synthesised compound 8 was screened for
its in vitro antibacterial, antifungal, anti-inflammatory
and anthelmintic activity. Antibacterial and antifungal
Table 3
Anti-inflammatory activity data
Comp. no.
Increase in Paw volume
(ml)9S.E.
Percentage inhibition of
oedema
5. Results and discussion
8
0.8590.04
0.9090.02
0.5590.04
5.56
–
40.50
Pseudostellarin B showed moderate antibacterial ac-
tivity against E. coli, B. subtilis, and antifungal activity
against C. albicans, whereas it showed very less anti-
Control
Ibuprofen

334
B. Poojary et al. / Il Farmaco 56 (2001) 331–334
Mean paralysing time (min)9S.E
Table 4
Anthelmintic activity data
Comp. no.
% Conc of comp. (mg)
Mean death time (min)9S.E
8
100
200
65.30+2.09
56.58+2.05
95.17+2.03
85.24+2.16
Control
–
–
–
–
–
–
Mebendazole
100
200
18.01+2.01
12.55+1.02
35.20+2.00
32.01+1.09
[4] S.L. Belagali, T. Mathew, M. Himaja, P. Kocienski, Ind. J. Chem.
34B (1995) 45.
[5] M. Bodanszky, A. Bodanszky, The Practice of Peptide Synthesis,
Springer, New York, 1984 (p. 145).
inflammatory and anthelmintic activity as compared
with the standard drug.
[6] M. Hothe, K.D. Stifen, I. Rother, Angew Chem., Int. Ed. Engl. 4
(1965) 356.
References
[7] R. Crucickshank, J.P Dugnid, B.P. Marnion, R.H.A. Swain,
Medicinal Microbiology, vol. vol. II, Churchill Livingstone, New
York, 1975 (p. 190).
[8] C.A. Winter, E.A. Risely, G.W. Nuss, Proc. Soc. Exp. Biol. Med.
111 (1962) 544.
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