Total synthesis and biological potential of psammosilenin A

Article abstract of DOI:10.1002/ardp.200800006

The present work reports the synthesis of a plant-originated cyclooctapeptide - psammosilenin A 8 by cyclization of linear peptide fragment phe-pro-phe-phe-ala-pro-leu-pro-Opfp which was prepared by coupling of tetrapeptide units Boc-phe-pro-phe-phe-OH an

Full text of DOI:10.1002/ardp.200800006

502
Arch. Pharm. Chem. Life Sci. 2008, 341, 502–509
Full Paper
Total Synthesis and Biological Potential of Psammosilenin A
Rajiv Dahiya
Department of Pharmaceutical Chemistry, Rajiv Academy for Pharmacy, Mathura, India
The present work reports the synthesis of a plant-originated cyclooctapeptide – psammosilenin
A 8 by cyclization of linear peptide fragment phe-pro-phe-phe-ala-pro-leu-pro-Opfp which was
prepared by coupling of tetrapeptide units Boc-phe-pro-phe-phe-OH and ala-pro-leu-pro-OMe fol-
lowed by proper deprotection of terminal groups and activation of the acid functionality. Dur-
ing synthesis, dicyclohexylcarbodiimide (DCC) and N,N-diisopropylcarbodiimide (DIPC) were
used as the coupling agents and N-methylmorpholine (NMM)/triethylamine (TEA) were used as
bases. Structure of the synthesized peptide was elucidated by spectral as well as elemental data.
The newly synthesized peptide was subjected to biological screening and found to possess potent
cytotoxic activity against DLA and EAC cell lines with IC50 value of 7.93 and 17.06 lM, respec-
tively. Furthermore, good anthelmintic activity against earthworms M. konkanensis and Eudrilus
species at 1 and 2 mg/mL was also observed for the synthesized cyclic peptide. Studies indicated
that N-methylmorpholine was a more useful base for cyclization of linear peptide unit in com-
parison to pyridine.
Keywords: Cyclic octapeptide / Macrocyclization / Psammosilenin A / Pharmacological activities / Psammosilene tuni-
coides /
Received: January 7, 2008; accepted: April 9, 2008
DOI 10.1002/ardp.200800006
antimalarial activity [9] and tyrosinase inhibitory activity
Introduction
[10]. A natural cyclic octapeptide – psammosilenin A has
been isolated from the roots of higher plant Psammosilene
tunicoides which is used as anodyne and haemastatic
agent. The structure of isolated cyclopeptide was eluci-
dated using FABMS (Fast Atom Bombardment Mass Spec-
The literature is enriched with several reports proving
the significant role of natural products in the pharma-
ceutical research as biomedically useful agents or as lead
compounds for drug development. Among these, cyclo-
polypeptides and related congeners having unique struc-
tures and a wide pharmacological profile emerged as
vital organic congeners which may prove better candi-
dates to overcome the problem of the widespread
increase of resistance towards conventional agents [1–3].
Plant-derived natural cyclic peptides exhibit a wide range
of pharmacological activities such as cytotoxic activity
[4, 5], vasorelaxant activity [6, 7], estrogen-like activity [8],
1
troscopy) and 2D NMR techniques including H-1H COSY
(Correlated SpectroscopY), HMQC (Heteronuclear Multi-
ple Quantum Coherence) and HMBC (Heteronuclear Mul-
tiple Bond Coherence) [11]. In continuation of our syn-
thetic work on natural cyclopolypeptides [12–15], the
present investigation was aimed at synthesis of natural
cyclopeptide – psammosilenin A. Keeping in view of sig-
nificant bioactivities possessed by cyclopeptides [16, 17],
the above synthetic peptide was further subjected to cyto-
toxic and anthelmintic screening.
Correspondence: Rajiv Dahiya, Assistant Professor, Department of
Pharmaceutical Chemistry, Rajiv Academy for Pharmacy, PO Chattikara,
Mathura – 281 001 (UP), India.
Results and discussion
E-mail: rajivdahiya04@yahoo.co.in, rajivdahiya77@rediffmail.com
Abbreviations: dicyclohexylcarbodiimide (DCC); N,N-diisopropylcarbo-
diimide (DIPC); Dalton's lymphoma ascites (DLA); Ehrlich's ascites carci-
noma (EAC); N-methylmorpholine (NMM); pentafluorophenyl (pfp); trie-
thylamine (TEA)
Chemistry
In the present work, disconnection strategy was
employed to carry out the first total synthesis of psammo-
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Arch. Pharm. Chem. Life Sci. 2008, 341, 502–509
Synthetic and Biological Studies on Psammosilenin A
503
Reagents and conditions: a) LiOH, THF : H2O (1 : 1), r.t., 1 h; b) CF3COOH, CHCl3, r.t. 1 h; c) DCC/DIPC, TEA/NMM, CHCl3/THF/DMF, r.t., 24 – 36 h; d) DCC, pfp, r.t. 12 h; e)
NMM/pyridine, 7 days, 08C.
Scheme 1. Synthetic route to psammosilenin 8.
silenin A. The cyclic octapeptide molecule was split into employing DCC/DIPC as coupling agent according to the
four dipeptide units Boc-phe-pro-OMe 1, Boc-phe-phe- Bodanszky-and-Bodanszky method [18] with certain mod-
OMe 2, Boc-ala-pro-OMe 3 and Boc-leu-pro-OMe 4. The ifications. The ester group of dipeptide 1 was removed by
required dipeptide units 1–4 were prepared by coupling alkaline hydrolysis with LiOH, and the Boc-group of
of Boc-amino acids viz. Boc-phe-OH, Boc-ala-OH, and Boc- another dipeptide 2 was removed using CF3COOH. Both
leu-OH with corresponding amino acid methyl ester deprotected units were coupled with each other using
hydrochlorides such as pro-OMe.HCl and phe-OMe.HCl DCC and triethylamine (TEA) as base, to get the first tetra-
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504
R. Dahiya
Arch. Pharm. Chem. Life Sci. 2008, 341, 502–509
peptide unit Boc-phe-pro-phe-phe-OMe 5. Similarly, elemental analysis of compound 8 afforded values
dipeptide 3, after deprotection at carboxyl terminal, was (l 0.03) strictly in accordance to the molecular composi-
coupled with dipeptide 4, after deprotection at amino tion.
end, to get another tetrapeptide Boc-ala-pro-leu-pro-OMe
The absolute configuration of the amino acid residues
6. After removal of the ester and Boc-groups of the tetra- was determined by acid hydrolysis of 8 followed by deri-
peptides 5 and 6, those deprotected units were coupled vatization with Marfey's reagent [N-(3-fluoro-4,6-dinitro-
to get the linear octapeptide Boc-phe-pro-phe-phe-ala-pro- phenyl)-L-alaninamide, FDAA] and subsequent HPLC anal-
leu-pro-OMe 7. The ester group of the linear fragment ysis. By comparing the chromatograms with those of
was removed using LiOH, and pentafluorophenyl (pfp) derivatives of commercially available amino acids, it was
ester group was introduced. The Boc-group was removed found that cyclopeptide 8 contained L-ala, L-leu, L-phe,
using CF3COOH and the deprotected linear fragment was and L-pro in the ratio 1.1/1/3.1/3.2, showing the presence
now cyclized by keeping the whole contents at 08C to get of one L-ala, one L-leu, three L-phe, and three L-pro in the
cyclo (phe-pro-phe-phe-ala-pro-leu-pro) 8 (Scheme 1).
Synthesis of psammosilenin A 8 was carried out suc-
structure.
DCC was found to be a yield-effective coupling agent
cessfully with good yield (A85%). Cyclization of the linear giving a highly insoluble by-product dicyclohexylurea
octapeptide unit was indicated by the disappearance of (DCU), in comparison to DIPC. Pentafluorophenyl ester
absorption bands at 1747, 1270 cm– 1 and 1389, was proved to be better for the activation of the acid func-
1366 cm– 1 (corresponding to C-O stretching of the ester tionality of the linear octapeptide unit and NMM was
and C-H deformation of the tert-Butyl group), and the found to be a good base for intramolecular cyclization of
presence of additional Amide-I and Amide-II bands of the the linear peptide fragment, in comparison to pyridine.
-CO-NH- moiety at 1638–1635 cm– 1 and 1531–1528 cm– 1
in the IR spectra of compound 8. Formation of a cyclopep- Pharmacology
tide was further confirmed by the disappearance of sin- Results of pharmacological activity studies are summar-
glets at 79.8 and 29.0 ppm corresponding to alpha and ized in Tables 1–3. Comparison of cytotoxic activity data
beta carbons of the tert-Butyl group of Boc and one singlet suggested that synthesized cyclooctapeptide 8 exhibited
at 53.4 ppm corresponding to the carbon of the methyl a high level of cytotoxic activity against Dalton's lym-
ester n the 13C-NMR spectrum. Also, a singlet at 1.54 ppm phoma ascites (DLA) and Ehrlich's ascites carcinoma
disappeared, corresponding to nine protons of the Boc- (EAC) cell lines with IC50 values of 7.93 and 17.06 lM (7.26
tert-Butyl group and a singlet at 3.62 ppm, corresponding and 15.63 lg/mL respectively) in comparison to the stand-
to three protons of the methyl ester in 1H-NMR spectrum ard drug – 5-fluorouracil (IC50 values – 37.36 and
1
of compound 8. Furthermore, the H-NMR and 13C-NMR 90.55 lM), whereas 8 showed no toxic effect against nor-
spectra of the synthesized cyclooctapeptide showed char- mal human hepatoma cells (HeP2). A possible mechanism
acteristic peaks confirming the presence all the 64 pro- of cytotoxic action may be through apoptosis via induc-
tons and 51 carbon atoms. Presence of the [M+1]+ ion tion of early cell death, nuclear fragmentation, and inter-
peak at m/z 917.5, corresponding to the molecular for- nucleosomal DNA scission. Anthelmintic activity data
mula C51H64N8O8 in the mass spectrum of compound 8, revealed that compound 8 showed higher activity against
along with other fragment-ion peaks resulting from M. konkanensis and Eudrilus species at 1 and 2 mg/mL con-
,
,
,
cleavage at leu-pro’, phe-pro’, and ala-pro’ amide bond centration, in comparison to standard drugs albendazole
levels, showed the exact sequence of the attachment of and mebendazole. However, anthelmintic activity pos-
all the eight amino acid moieties in a chain. The presence sessed by compound 8 against P. corethruses was greater
of the immonium-ion peaks at m/z 120.2 (phe), 86.2 (leu), than that possessed by mebendazole but lower than that
70.1 (pro), and 44.1 (ala) further confirmed the presence showed by albendazole. Antimicrobial activity data indi-
of these amino acid moieties in the cyclopeptide struc- cated that compound 8 exhibited higher antibacterial
ture.
activity than reference drug – gatifloxacin, against
Mild acid hydrolysis (1.2 N HCl, 1058C, 1 h) of the cyclic pathogenic Gram-negative bacteria P. aeruginosa and K.
peptide followed by C18-HPLC led to the recovery of the pneumoniae at 6 lg/mL concentration. The mechanism of
full-length cyclic peptide, which resulted from cleavage antibacterial action may involve cell lysis, breakdown of
,
of the leu–pro-3’ peptide bond and four smaller frag- the cytoplasmic membrane barrier or interaction with a
,
,
,
ments ( pro-phe’ and pro-phe-phe-ala-pro-leu’) and ( pro- cytoplasmic target such as DNA. Furthermore, synthe-
,
phe-pro-phe-phe-ala’ and pro-leu’) formed by a subse- sized cyclopeptide showed moderate antifungal activity
quent cut at phe-1–pro-1’ and ala–pro-2’ linkages, against dermatophytes, in comparison to the standard
,
,
respectively, as evidenced by LC-MS analysis. In addition, drug – griseofulvin. However, compound 8 displayed no
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2008, 341, 502–509
Synthetic and Biological Studies on Psammosilenin A
505
Table 1. In-vitro cytotoxicity of compound 8 against DLA, EAC, and HeP2 cell lines.
Compound Conc.
(lg mL– 1
DLA cells
EAC cells
HeP2 cells
% growth
)
b)
No. of
% growth
IC50
No. of
% growth
dead cells Inhibition (lM)
IC50
No. of
IC50
dead cells Inhibitiona) (lM)
dead cells inhibition (lM)
8
62.5
38
36
29
20
13
100.0
94.74
76.32
52.63
34.21
28
25
14
8
100.0
89.29
50.00
28.57
31.58
0
0
0
0
0
–
–
–
–
–
31.25
15.63
7.81
7.93
17.06
–
3.91
4
Control
62.5
0
0
0
0
0
–
0
0
0
0
0
–
–
–
–
–
0
0
0
0
0
–
–
–
–
–
31.25
15.63
7.81
–
–
–
–
–
–
–
3.91
Standard
(5-FU)
62.5
38
38
28
25
16
100.0
100.0
73.68
65.79
42.11
28
28
17
9
100.0
100.0
60.71
32.14
17.86
0
0
0
0
0
–
–
–
–
–
31.25
15.63
7.81
37.36
90.55
–
3.91
5
a)
% growth inhibition = 100 – [{(Celltotal – Celldead)6100}/Celltotal].
IC50 = cytotoxic concentration inhibiting 50% of percentage growth.
b)
Table 2. Anthelmintic activity of compound 8 against three earthworm species.
Compound
Concentration
(mg)
Earthworm species
M. konkanensis
P. corethruses
Eudrilus species
Mean paraly-
Mean death
Mean paraly-
zing time (min) time (min)
Mean death
Mean paraly-
zing time (min) time (min)
Mean death
zing time (min)a) time (min)a)
8
100
200
100
200
08.26 l 0.40
05.08 l 0.22
11.25 l 0.18
06.40 l 0.37
13.85 l 0.64
08.21 l 0.48
–
14.28 l 0.11
10.14 l 0.34
19.44 l 0.40
13.12 l 0.83
22.90 l 0.53
15.11 l 0.28
–
15.05 l 0.41
10.34 l 0.36
13.20 l 0.54
08.60 l 0.50
17.85 l 0.48
12.44 l 0.21
–
26.09 l 0.17
18.53 l 0.25
24.22 l 0.11
18.04 l 0.19
29.64 l 0.73
21.05 l 0.39
–
10.29 l 0.48
06.44 l 0.30
12.32 l 0.33
07.37 l 0.28
13.54 l 0.45
10.18 l 0.77
–
21.05 l 0.39
14.59 l 0.17
23.35 l 0.42
16.11 l 0.21
24.05 l 0.62
18.52 l 0.30
–
Albendazole
Mebendazole 100
200
Control
–
a)
Data are given as mean l S.D. (n = 3).
Table 3. Antimicrobial activity of compound 8 against eight pathogenic bacteria and fungi.
Compound
Diameter of zone of inhibition (mm)
Bacterial strains
Fungal strains
B. subtilis
S. epiderm.
P. aeruginosa K. pneumoniae C. albicans
M. audouinii A. niger
T. mentagro.
8
10 (25)a)
–
20 (12.5)
–
9 (12.5)
–
28 (6)
–
28 (6)
–
24 (6)
–
27 (6)
–
25 (6)
–
8 (12.5)
–
–
13 (6)
–
–
10(25)
–
–
17 (6)
–
–
Control
Gatifloxacin
Griseofulvin
20 (6)
17 (6)
18 (12.5)
22 (6)
a)
Values in brackets are MIC values (lg mL– 1).
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Arch. Pharm. Chem. Life Sci. 2008, 341, 502–509
evaporated in vacuo. The crude product was recrystallized from a
mixture of chloroform and petroleum ether (b.p. 40–608C) fol-
lowed by cooling at 08C to get compounds 1, 3, and 4 as pale yel-
low semisolid masses (1: Yield 79%, [a]D = –39.48 (c = 0.25 M,
MeOH), Rf = 0.71 (CHCl3 : MeOH/7 : 3), Anal. calcd. for C20H28N2O5:
C, 63.81; H, 7.50; N, 7.44. Found: C, 63.79; H, 7.54; N, 7.45. 3: Yield
83%, [a]D = –113.68 (c 0.25, MeOH), Rf = 0.68 (CHCl3 : MeOH/7 : 3).
Anal. calcd. for C14H24N2O5: C, 55.99; H, 8.05; N, 9.33. Found: C,
56.02; H, 8.05; N, 9.35. 4: Yield 87%, [a]D = -2.88 (c = 0.25 M, MeOH),
Rf = 0.77 (CHCl3 : MeOH/7 : 3), Anal. calcd. for C17H30N2O5: C,
59.63; H, 8.83; N, 8.18. Found: C, 59.61; H, 8.85; N, 8.22). Simi-
larly, 2.16 g (0.01 mol) of L-phenylalanine methyl ester hydro-
chloride was coupled with 2.65 g (0.01 mol) of Boc-L-phe-OH
using DCC/DIPC as coupling agents, to afford the desired com-
pound 2 (Yield 76%; [a]D = +63.58 (c = 0.25 M, MeOH); Rf = 0.59
(CHCl3 : MeOH/7 : 3), Anal. calcd. for C24H30N2O5: C, 67.59; H, 7.09;
N, 6.57. Found: C, 67.60; H, 7.12; N, 6.58).
significant activity against Gram positive bacteria and
other pathogenic fungi tested. On passing toxicity tests,
cyclic octapeptide 8 may prove a good candidate for clini-
cal studies and could be a novel cytotoxic, anthelmintic,
and antibacterial drug of the future.
The author is grateful to U.S.I.C., DU, Delhi and R.S.I.C., I.I.T.,
Delhi (India) and C.D.R.I., Lucknow (India) for spectral anal-
ysis. Also, thanks to J.S.S. College of Pharmacy, Ooty (India) for
carrying out the cytotoxicity studies and C.P.C.R.I., Kasaragod,
Kerala (India) for providing earthworms for testing anthelmin-
tic activity.
The author has declared no conflict of interest.
Deprotection of dipeptide units at carboxyl terminal
To a solution of the compound 1 (3.76 g, 0.01 mol) in THF : H2O
(1 : 1, 36 mL), 0.36 g (0.015 mol) of LiOH was added at 08C. The
mixture was stirred at r.t. for 1 h and then acidified to pH = 3.5
with 1 N H2SO4. The aqueous layer was extracted with three
equal proportions of 25 mL of Et2O. The combined organic
extracts were dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The crude product was crystallized
from methanol and ether to get Boc-phe-pro-OH 1a as viscous
liquid (Yield 86%, Rf = 0.57 (CHCl3 : MeOH/7 : 3), Anal. calcd. for
C19H26N2O5: C, 62.97; H, 7.23; N, 7.73. Found: C, 62.98; H, 7.22; N,
7.75). Similarly, compound 3 (3.0 g, 0.01 mol) was hydrolyzed
under alkaline conditions to obtain Boc-ala-pro-OH 3a (Yield
89%, Rf = 0.79 (CHCl3 : MeOH/7 : 3), Anal. calcd. for C13H22N2O5: C,
54.53; H, 7.74; N, 9.78. Found: C, 54.56; H, 7.75; N, 9.76).
Experimental
Melting point was determined by open capillary method and is
uncorrected. Dicyclohexylcarbodiimide (DCC), trifluoroacetic
acid (TFA), triethylamine (TEA), N-methylmorpholine (NMM),
pyridine (C5H5N) were purchased from SpectroChem Pvt. Ltd.,
Mumbai (India). Boc-amino acids, amino acid methyl ester
hydrochlorides, N,N-diisopropylcarbodiimide (DIPC), and penta-
fluorophenol (pfp) were procured from RANKEM RFCL Ltd., New
Delhi (India). IR spectra were recorded on Shimadzu 8700 FTIR
spectrophotometer (Shimadzu, Japan) using a thin film sup-
ported on KBr or CHCl3 as solvent. 1H-NMR and 13C-NMR spectra
were recorded on Bruker AC NMR spectrometer (300 MHz)
(Brucker, USA) using CDCl3/DMSO-d6 as solvent and tetramethyl-
silane (TMS) as internal standard. FABMS was recorded on JMS-
DX 303 Mass spectrometer (Jeol, Tokyo, Japan) operating at 70 eV
using fast atom bombardment technique and electron spray ion-
ization mass spectra were obtained on a Hewlett-Packard HP
1100 integrated LC-MS system (Hewlett Packard, USA). Elemental
analyses of all compounds were performed on Vario EL III ele-
mental analyzer (Elementar, Germany). Optical rotation was
measured on automatic polarimeter (Optics Tech, Ghaziabad,
India) in a 2 dm tube at 258C using sodium-D light. Purity of all
compounds was checked by TLC on precoated silica gel G plates
(Kieselgel 0.25 mm, 60G F254, Merck, Germany) utilizing CHCl3/
MeOH (9 : 1/7 : 3) and CHCl3/AcOH/H2O (3 : 2 : 5) as developing
solvents.
Deprotection of dipeptide units at amino terminal
Compound 2/compound 4 (4.27 g/3.42 g, 0.01 mol) was dissolved
in 15 mL of CHCl3 and treated with 2.28 g (0.02 mol) of trifluoro-
acetic acid. The resulting solution was stirred at r.t. for 1 h,
washed with 25 mL of saturated NaHCO3 solution. The organic
layer was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The crude product was purified by crystalliza-
tion from CHCl3 and petroleum ether (b.p. 40–608C) to get pure
phe-phe-OMe 2a/leu-pro-OMe 4a as semisolid masses (2a: Yield
74%, Rf = 0.73 (CHCl3 : MeOH/7 : 3), Anal. calcd. for C19H22N2O3: C,
69.92; H, 6.79; N, 8.58. Found: C, 69.93; H, 6.77; N, 8.60; 4a: Yield
77%, Rf = 0.55 (CHCl3 : MeOH / 7 : 3), Anal. calcd. for C12H22N2O3: C,
59.48; H, 9.15; N, 11.56. Found: C, 59.45; H, 9.17; N, 11.56).
General procedure for the preparation of dipeptide
General procedure for the preparation of tetrapeptide
fragments 1–4
fragments 5, 6
1.66 g (0.01 mol) of L-proline methyl ester hydrochloride was dis-
solved in 20 mL of CHCl3. To this solution, 2.8 mL (0.021 mol) of
TEA was added at 08C and the resulting mixture was stirred for
15 min. Then 2.65 g/1.89 g/2.31 g (0.01 mol) of Boc-L-phe-OH/Boc-
L-ala-OH/Boc-L-leu-OH dissolved in 20 mL of CHCl3 and 2.1 g/
1.26 g (0.01 mol) of DCC/DIPC were added to above mixture with
stirring. Stirring was continued for 24 h, after which the reac-
tion mixture was filtered and the residue was washed with
25 mL of CHCl3 and added to the filtrate. The filtrate was washed
with 25 mL each of 5% NaHCO3 and saturated NaCl solutions.
The organic layer was dried over anhydrous Na2SO4, filtered, and
3.62 g (0.01 mol) of compound 1a was dissolved in 25 mL of DMF
and solution was neutralized with 2.21 mL (0.021 mol) of NMM
at 08C and the resulting mixture was stirred for 15 min. 3.26 g
(0.01 mol) of compound 2a were dissolved in 25 mL of DMF and
resulting solution with 2.1 g/1.26 g (0.01 mol) of DCC/DIPC were
added to the above mixture. Stirring was first done for 1 h at 0–
58C and then further for 24 h at r.t. After completion of the reac-
tion, the reaction mixture was diluted with an equal amount of
water. The precipitated solid was filtered, washed with water,
and recrystallized from a mixture of chloroform and petroleum
ether (b.p. 40–608C) followed by cooling at 08C to get compound
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Arch. Pharm. Chem. Life Sci. 2008, 341, 502–509
Synthetic and Biological Studies on Psammosilenin A
507
5 as yellowish semisolid mass (Yield 71%, [a]D = +22.78 (c = 0.5 M,
MeOH), Rf = 0.81 (CHCl3 : MeOH/9 : 1), Anal. calcd. for C38H46N4O7:
C, 68.04; H, 6.91; N, 8.35. Found: C, 68.05; H, 6.89; N, 8.38). Simi-
larly, compounds 3a and 4a (2.86 g/2.42 g, 0.01 mol) were
coupled using DCC/DIPC to yield compound 6 (Yield 82%, [a]D = –
77.38 (c = 0.5 M, MeOH), Rf = 0.67 (CHCl3 : MeOH/9 : 1), Anal. calcd.
for C25H42N4O7: C, 58.81 H, 8.29; N, 10.97. Found: C, 58.85; H,
8.26; N, 10.99).
H-a, phe-1), 4.46 (t, J = 6.85 Hz, 1H, H-a, pro-1), 4.25-4.18 (m, 2H, H-
a, phe-3 and H-a, ala), 4.11 (t, J = 6.9 Hz, 1H, H-a, pro-2), 3.92 (t, J =
6.85 Hz, 1H, H-a, pro-3), 3.75 (t, J = 7.2 Hz, 2H, H-d, pro-1), 3.62
(3H, s, OCH3), 3.39 (t, J = 7.15 Hz, 2H, H-d, pro-2), 3.32 (t, J =
7.15 Hz, 2H, H-d, pro-3), 2.89–2.78 (m, 6H, H-b, phe-1, phe-2 and
phe-3), 2.69–2.65 (m, 4H, H-b, pro-1 and pro-2), 2.04–1.99 (m, 4H,
H-b and H-c, pro-3), 1.95–1.89 (m, 4H, H-c, pro-1 and pro-2), 1.78
(t, J = 4.6 Hz, 2H, H-b, leu), 1.54 (9H, s, butyl-t), 1.47 (d, J = 5.85 Hz,
3H, H-b, ala), 1.46–1.42 (m, 1H, H-b, leu), 0.98 (d, J = 6.2 Hz, 6H, H-
d, leu). 13C-NMR (300 MHz, DMSO-d6) d (ppm): 177.2 (C=O, phe-3),
174.5 (C=O, pro-2), 173.4 (C=O, pro-1), 170.9 (C=O, phe-1), 169.6
(C=O, phe-2), 169.2 (C=O, ala), 168.6 (C=O, pro-3), 168.0 (C=O, leu),
153.2 (C=O, boc), 136.6 (C-c, phe-2), 134.2 (C-c, phe-3), 132.7 (C-c,
phe-1), 130.7 (2C, C-m, phe-1), 130.1 (2C, C-o, phe-3), 129.5 (2C, C-o,
phe-2), 128.6 (2C, C-m, phe-2), 128.0 (2C, C-m, phe-3), 127.7 (2C, C-
o, phe-1), 127.2 (C-p, phe-1), 126.9 (C-p, phe-2), 125.6 (C-p, phe-3),
79.8 (C-a, boc), 58.3 (C-a, pro-3), 56.1 (C-a, pro-2), 54.4 (C-a, pro-1),
53.4 (OCH3), 51.8 (C-a, phe-1), 49.8 (C-a, ala), 49.2 (C-a, phe-3), 48.8
(C-d, pro-1), 48.2 (C-a, phe-2), 47.5 (C-d, pro-2), 46.9 (C-a, leu), 45.4
(C-d, pro-3), 39.7 (C-b, leu), 38.3 (C-b, phe-1), 38.0 (C-b, phe-2), 37.6
(C-b, phe-3), 29.9 (C-b, pro-3), 29.0 (3C, C-b, boc), 26.8 (C-b, pro-2),
25.7 (C-b, pro-3), 25.1 (C-c, pro-3), 24.8 (C-c, pro-2), 24.2 (C-c, pro-
1), 22.9 (C-c, leu), 22.2 (2C, C-d, leu), 17.9 (C-b, ala). Anal. calcd. for
C57H76N8O11: C, 65.25; H, 7.30; N, 10.68. Found: C, 65.26; H, 7.28;
N, 10.65.
Deprotection of tetrapeptide units at the carboxyl and
amino terminals
Hydrolysis of compound 5 (6.71 g, 0.01 mol) was carried out
using 0.36 g (0.015 mol) of lithium hydroxide and the boc-group
of compound 6 (5.11 g, 0.01 mol) was removed using 2.28 g
(0.02 mol) of trifluoroacetic acid according to the procedures
which were adopted for preparation of compound 1a and 3a
from compound 1 and 3 respectively, to afford compound 5a
and 6a as pale yellow semisolid masses (5a: Yield 79%, [a]D
=
+52.68 (c = 0.5 M, MeOH), Rf = 0.60 (CHCl3 : MeOH/9 : 1), Anal.
calcd. for C37H44N4O7: C, 67.67; H, 6.75; N, 8.53. Found: C, 67.65;
H, 6.79; N, 8.52; 6a: Yield 76%, [a]D = -37.28 (c = 0.5 M, MeOH), Rf =
0.78 (CHCl3 : MeOH / 9 : 1), Anal. calcd. for C20H34N4O5: C, 58.52 H,
8.35; N, 13.65. Found: C, 58.53; H, 8.35; N, 13.69).
Procedure for the preparation of linear octapeptide
unit 7
Synthesis of the cyclic octapeptide – psammosilenin
A 8
4.11 g (0.01 mol) of compound 6a was dissolved in 35 mL of THF.
To this solution, 2.8 mL (0.021 mol) of TEA was added at 08C and
the resulting mixture was stirred for 15 min. 6.57 g (0.01 mol) of
compound 5a was dissolved in 35 mL of THF and 2.1 g/1.26 g
(0.01 mol) of DCC/DIPC were added to above mixture with stir-
ring. Stirring was continued for 36 h, after which the reaction
mixture was filtered and the filtrate was washed with 30 mL
each of 5% NaHCO3 and saturated NaCl solutions. The organic
layer was dried over anhydrous Na2SO4, filtered and evaporated
in vacuo. The crude product was recrystallized from a mixture of
chloroform and petroleum ether (b.p. 40–608C) followed by
cooling at 08C to get compound 7 as yellowish semisolid mass.
To synthesize compound 8, the linear octapeptide unit 7 (5.25 g,
0.005 mol) was deprotected at the carboxyl end using LiOH
(0.18 g, 0.0075 mol) to get Boc-L-phe-L-pro-L-phe-L-phe-L-ala-L-pro-L-
leu-L-pro-OH. The deprotected octapeptide unit (5.18 g,
0.005 mol) was now dissolved in 50 mL of CHCl3 at 08C. To this
solution, 0.0067 mol of pentafluorophenol (1.23 g) and DCC
(1.06 g, 0.005 mol) were added and stirring was done at r.t. for
12 h. The reaction mixture was filtered and the filtrate was
washed with 10% NaHCO3 solution (3625 mL) and finally
washed with 5% HCl (2620 mL) to get the corresponding penta-
fluorophenyl ester Boc-L-phe-L-pro-L-phe-L-phe-L-ala-L-pro-L-leu-L-
pro-Opfp. To this compound (4.8 g, 0.004 mol) dissolved in
35 mL of CHCl3, 0.91 g (0.008 mol) of CF3COOH was added,
stirred at r.t. for 1 h and washed with two proportions each
25 mL of 10% NaHCO3 solution. The organic layer was dried over
anhydrous Na2SO4 to get L-phe-L-pro-L-phe-L-phe-L-ala-L-pro-L-leu-L-
pro-Opfp which was dissolved in 25 mL of CHCl3 and NMM/
C5H5N (2.21 mL/1.61 mL, 0.021 mol) was added. Then, whole con-
tents were kept at 08C for seven days. The reaction mixture was
washed with 10% NaHCO3 (3625 mL) and 5% HCl (2620 mL) sol-
utions. The organic layer was dried over anhydrous Na2SO4 and
the crude cyclized product was crystallized from CHCl3/n-hex-
ane to get compound 8 as white crystals (m.p. 193–1958C).
tert-Butyloxycarbonyl-L-phenylalanyl-L-prolyl-L-
phenylalanyl-L-phenylalanyl-L-alanyl-L-prolyl-L-leucyl-L-
proline methyl ester 7
Yield 83%, [a]D = –59.18 (c = 0.3 M, MeOH), Rf = 0.83 (CHCl3 :
MeOH/9 : 1). FT-IR (CHCl3) mmax (cm– 1): 3125, 3122, 3117 (m, N-H
str, amide), 2999, 2995, 2992 (m, C-H str, CH2, pro), 2966, 2963,
2929-2924 (m, C-H str, asym, CH3 and CH2), 2875, 2844–2839 (m,
C-H str, sym, CH3 and CH2), 1747 (s, C=O str, ester), 1666–1663,
1647, 1642, 1641, 1639 (s, C=O str, amide), 1589, 1582, 1478–
1470 (m, skeletal bands, rings), 1538-1533, 1527–1523 (m, N-H
def, amide), 1389, 1366 (s, C-H def, butyl-t), 1382, 1359 (s, C-H def,
propyl-i), 1270 (s, C-O str, ester), 720–715, 698–694 (s, C-H bend,
out-of-plane, rings). 1H-NMR (300 MHz, CDCl3) d (ppm): 8.85 (brs,
1H, NH, phe-1), 8.20 (brs, 1H, NH, phe-2), 7.53 (tt, J = 7.15, 4.35 Hz,
2H, H-m, phe-1), 7.29 (brs, 1H, NH, leu), 7.22-7.14 (m, 4H, H-m,
phe-2 and phe-3), 7.08–7.01 (m, 2H, H-p, phe-2 and phe-3), 6.92 (t,
J = 6.15 Hz, 1H, H-p, phe-1), 6.87 (dd, J = 8.75, 2H, 4.2 Hz, H-o, phe-
1), 6.83 (dd, J = 8.8, 4.15 Hz, 2H, H-o, phe-2), 6.80 (dd, J = 8.75,
4.15 Hz, 2H, H-o, phe-3), 6.54 (brs, 1H, NH, phe-3), 6.44 (brs, 1H,
NH, ala), 4.82–4.79 (td, J = 7.45, 3.8 Hz, 1H, H-a, phe-2), 4.68–4.63
(td, J = 6.2, 3.6 Hz, 1H, H-a, leu), 4.60-4.55 (td, J = 7.5, 3.75 Hz, 1H,
Cyclo (L-phe-L-pro-L-phe-L-phe-L-ala-L-pro-L-leu-L-pro) 8
Yield 3.94 g (86%, NMM), 3.25 g (71%, C5H5N), [a]D = –108.28 (c =
0.4 M, MeOH), Rf = 0.63 (CHCl3 : AcOH : H2O/3 : 2 : 5). FT-IR (KBr)
mmax (cm– 1): 3128, 3123, 3115 (m, N-H str, amide), 2998, 2996,
2992 (m, C-H str, CH2 (cyclic)), 2967, 2964 (m, C-H str, asym, CH3),
2928, 2925 (m, C-H str, asym, CH2), 2877 (m, C-H str, sym, CH3),
2845, 2836 (m, C-H str, sym, CH2), 1668, 1662, 1645, 1638–1635
(s, C=O str, amide), 1588, 1585, 1475, 1469 (m, skeletal bands,
rings), 1538, 1534, 1531-1528, 1525 (m, N-H def, amide), 1383,
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www.archpharm.com

508
R. Dahiya
Arch. Pharm. Chem. Life Sci. 2008, 341, 502–509
1358 (s, C-H def, propyl-i), 722–717, 698–693 (s, C-H bend, out-of-
plane, rings). H-NMR (300 MHz, CDCl3) d (ppm): 9.88 (brs, 1H,
(min) and relative peak area (%) of the observed peaks of the
FDAA derivatized hydrolysis product of cyclopeptide 8 were as
follows: L-ala (6.45, 2.62%), L-leu (14.96, 2.38%), L-phe (17.44,
7.39%), L-pro (18.93, 7.52%).
1
NH, phe-2), 9.75 (brs, 1H, NH, phe-1), 9.22 (brs, 1H, NH, leu), 7.75
(brs, 1H, NH, phe-3), 7.56 (brs, 1H, NH, ala), 7.23 (tt, J = 6.75,
4.45 Hz, 2H, H-m, phe-2), 7.19 (tt, J = 6.8, 4.5 Hz, 2H, H-m, phe-1),
7.16 (tt, J = 6.75, 4.5 Hz, 2H, H-m, phe-3), 7.04 (t, J = 6.15 Hz, 1H, H-
p, phe-3), 7.01 (t, J = 6.2 Hz, 1H, H-p, phe-3), 6.98 (t, J = 6.1 Hz, 1H,
H-p, phe-3), 6.86 (dd, J = 8.8, 4.15 Hz, 2H, H-o, phe-1), 6.84 (dd, J =
8.75, 4.15 Hz, 2H, H-o, phe-3), 6.81 (dd, J = 8.8, 4.2 Hz, 2H, H-o,
phe-2), 6.04–5.98 (m, 1H, H-a, ala), 5.69–5.65 (td, J = 7.4, 3.75 Hz,
1H, H-a, phe-3), 5.17–5.12 (td, J = 6.15, 3.6 Hz, 1H, H-a, leu), 4.44–
4.39 (td, J = 7.45, 3.8 Hz, 1H, H-a, phe-1), 4.23-4.19 (td, J = 7.5,
3.75 Hz, 1H, H-a, phe-2), 3.94 (t, J = 6.85 Hz, 1H, H-a, pro-2), 3.94 (t,
J = 6.9 Hz, 1H, H-a, pro-1), 3.94 (t, J = 6.8 Hz, 1H, H-a, pro-3), 3.24 (t,
J = 7.2 Hz, 2H, H-d, pro-1), 3.21 (t, J = 7.15 Hz, 2H, H-d, pro-3), 3.18
(t, J = 7.1 Hz, 2H, H-d, pro-2), 2.72 (q, J = 5.9 Hz, 2H, H-b, pro-2),
2.69 (q, J = 5.85 Hz, 2H, H-b, pro-1), 2.65 (q, J = 5.9 Hz, 2H, H-b, pro-
3), 2.63 (d, J = 5.6 Hz, 2H, H-b, phe-2), 2.59 (d, J = 5.55 Hz, 2H, H-b,
phe-1), 2.56 (d, J = 5.6 Hz, 2H, H-b, phe-3), 1.89–1.85 (q, 2H, H-c,
pro-2), 1.84–1.78 (m, 4H, H-c, pro-1 and pro-3), 1.72 (t, J = 4.55 Hz,
2H, H-b, leu), 1.43 (d, J = 5.9 Hz, 3H, H-b, ala), 0.99 (d, J = 6.15 Hz,
6H, H-d, leu), 0.89–0.75 (m, 1H, H-c, leu). 13C-NMR (300 MHz,
DMSO-d6) d (ppm): 173.0 (C=O, phe-3), 172.8 (C=O, leu), 171.6
(C=O, pro-3), 171.1 (C=O, pro-3), 170.7 (C=O, pro-1), 170.2 (C=O,
phe-2), 169.6 (C=O, phe-1), 168.3 (C=O, ala), 140.1 (C-c, phe-2),
139.8 (C-c, phe-1), 137.5 (C-c, phe-3), 131.5 (2C, C-o, phe-1), 130.3
(2C, C-o, phe-3), 129.1 (2C, C-o, phe-2), 128.9 (2C, C-m, phe-1), 128.4
(2C, C-m, phe-2), 127.8 (2C, C-m, phe-3), 127.3 (C-p, phe-1), 127.0
(C-p, phe-2), 126.7 (C-p, phe-3), 59.4 (C-a, pro-2), 58.9 (C-a, pro-3),
58.4 (C-a, pro-1), 57.2 (C-a, phe-2), 50.7 (C-a, phe-1), 49.6 (C-a, ala),
49.1 (C-a, phe-3), 48.3 (C-a, leu), 48.0 (C-d, pro-1), 47.8 (C-d, pro-2),
47.5 (C-d, pro-3), 44.3 (C-b, leu), 39.1 (C-b, phe-2), 38.2 (C-b, phe-1),
37.5 (C-b, phe-3), 32.3 (C-b, pro-1), 31.9 (C-b, pro-2), 31.5 (C-b, pro-
3), 28.2 (C-c, leu), 22.7 (2C, C-d, leu), 21.5 (C-c, pro-1), 21.0 (C-c,
pro-2), 20.6 (C-c, pro-3), 15.7 (C-b, ala). FAB MS m/z [rel. int.]: 917.5
[M + 1]+, (100), 889.5 (21), 846.4 (42), 818.4 (17), 804.4 (33), 770.4
(67), 707.4 (56), 699.3 (23), 679.4 (19), 673.3 (41), 636.3 (32), 560.3
(27), 552.3 (69), 489.3 (27), 461.3 (17), 455.3 (48), 427.3 (22), 392.2
(31), 364.3 (11), 342.3 (23), 308.2 (19), 280.2 (12), 245.2 (28), 217.2
(10), 211.2 (15), 183.2 (21), 120.2 (29), 98.2 (17), 91.2 (28), 86.2 (14),
70.1 (21), 65.1 (21), 57.1 (19), 44.1 (9), 43.1 (6), 42.1 (16), 15.1 (7).
Anal. calcd for C51H64N8O8: C, 66.79; H, 7.03; N, 12.22. Found: C,
66.80; H, 7.02; N, 12.25.
Partial hydrolysis of cyclopeptide 8 and LC-MS analysis
Compound 8 (0.1 mg) was dissolved in 200 lL of 1.2 N HCl and
heated at 1058C for 1 h. The solution was then diluted with
600 lL of H2O and stored frozed at –208C prior to analysis. 50 lL
of hydrolyzate was chromatographed in duplicate by linear
ingredient C18-HPLC (Dynamax, 4.66250 mm, 8 lm) employing
0–50% MeCN in H2O (0.1% TFA) over 30 min (1.5 mL min– 1). Indi-
vidual peaks were collected and chromatographed by LC-MS to
identify the linear peptide fragments: [M + H]+ m/z 691.8, appro-
,
priate for pro-phe-phe-ala-pro-leu’, tR = 12.8 min; [M + H]+ m/z
,
725.8, for pro-phe-pro-phe-phe-ala’, tR = 14.1 min; [M + H]+ m/z
,
,
263.3, for pro-phe’, tR = 15.6 min; [M + H]+ m/z 229.3, for pro-
,
leu’, tR = 17.2 min; [M + H]+ m/z 936.2, for pro-phe-pro-phe-phe-
ala-pro-leu’, tR = 18.4 min.
Biology
Cytotoxic activity assay
The synthesized cyclopeptide 8 was subjected to a short-term in-
vitro cytotoxicity study [19] at 62.5–3.91 lg/mL using 5-fluorour-
acil (5-FU) as reference compound. Activity was assessed by deter-
mining the percentage inhibition of DLA, EAC, and HeP2 cells.
Human hepatoma cells (HeP2) were isolated from rat liver by per-
fusion method. Besides, DLA and EAC cells were cultured in the
peritoneal cavity of healthy albino mice by injecting the suspen-
sion of cells (16106 cells/mL) intraperitoneally. After 15–20
days, cells were withdrawn from the peritoneal cavity of the
mice with help of sterile syringe and counted using haemocy-
tometer and adjusted to 6106 cells/mL. The stock solution of the
tested compound 8 was prepared in DMSO and was used for
serial dilutions ranging from 62.5–3.91 lg/mL in Dulbeccos
minimum essential medium and 0.1 mL of diluted test com-
pound was added to 0.1 mL of DLA cells (16106 cells/mL) and
EAC cells (16106 cells/mL). Resulted suspensions were incubated
at 378C for 3 h. After 3 h, tryphan blue dye exclusion test was
performed and percentage growth inhibition was calculated.
IC50 values were determined by graphical extrapolation method.
Controls were also tested at 62.5–3.91 lg/mL against all three
cell lines. The results of cytotoxic activity studies are summar-
ized in Table 1.
Preparation and analysis of Marfey derivatives
0.5 mg of synthesized cyclooctapeptide 8 was hydrolyzed by
heating in 1 mL of 6 M HCl at 1108C for 24 h. After cooling, the
solution was evaporated to dryness and redissolved in 50 lL of
water. 100 lL of a 1% w/v solution of FDAA (Marfey's reagent) in
acetone was added to the acidic hydrolyzate solution (or to 50 lL
of a 50 mM solution of the respective amino acid). After addition
of 20 lL of 1 M NaHCO3 solution, the mixture was incubated for
1 h at 408C. The reaction was stopped by the addition of 10 lL of
2 M HCl. Finally, the solvents were evaporated to dryness and
the residue was dissolved in 1 mL of MeOH. An aliquot of this sol-
ution (20 lL for cyclopeptide 8 and 10 lL for standards) was ana-
lyzed by HPLC (Phenomenex Luna C18, 4.66250 mm, 5 lm, sol-
vents: (A) water + 0.05% TFA, (B) MeCN, linear gradient: 0 min
35% B, 30 min 45% B, 1 mL min– 1, 258C). Retention times (min)
of the FDAA amino acid derivatives used as standards were as fol-
lows: L-ala (6.42), D-ala (7.67), L-leu (14.94), D-leu (20.58), L-phe
(17.41), D-phe (22.32), L-pro (18.91), D-pro (20.22). Retention times
Anthelmintic activity assay
Three earthworm species namely Megascoplex konkanensis, Ponto-
scotex corethruses, and Eudrilus sp. were selected for anthelmintic
testing of compound 8 at 1 and 2 mg/mL concentration [20]. Sus-
pensions of samples were prepared by triturating synthesized
compound (100/200 mg) with tween 80 (0.5%) and distilled
water and the resulting mixtures were stirred using a mechani-
cal stirrer for 30 min. The suspensions were diluted to contain
0.1% and 0.2% w/v of the test sample. Suspension of reference
drugs, albendazole, and mebendazole were prepared with the
same concentration in a similar way. Three sets of five earth-
worms of almost similar sizes (2 inch in length) were placed in
petri plates (4 inch in diameter) containing 50 mL of suspension
of the test sample and reference drug at room temperature.
Another set of five earthworms was kept as control in 50 mL sus-
pension of distilled water and tween 80 (0.5%). The paralyzing
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www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2008, 341, 502–509
Synthetic and Biological Studies on Psammosilenin A
509
and death times were noted and their mean was calculated for
triplicate sets. The death time was ascertained by placing the
earthworms in warm water (508C) which stimulated the move-
ment, if the worm was alive. The results of anthelmintic studies
are given in Table 2.
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i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com