Synthetic studies on cyclic octapeptides: Yunnanin F and Hymenistatin

Article abstract of DOI:10.1016/j.ejmech.2004.11.013

Two biologically active cyclic peptides, Yunnanin F 8 and Hymenistatin 16 were synthesized and the structures were established on the basis of analytical, IR, NMR and mass spectral data. The newly synthesized compounds were screened for their antimicrobia

Full text of DOI:10.1016/j.ejmech.2004.11.013

European Journal of Medicinal Chemistry 40 (2005) 407–412
www.elsevier.com/locate/ejmech
Short communication
Synthetic studies on cyclic octapeptides:Yunnanin F and Hymenistatin
Boja Poojary a,*, Shiddappa L. Belagali b
a Department of Post-Graduate Studies and Research in Chemistry, Mangalore University, Mangalagangotri 574 199, Mangalore, India
b Department of Environmental Sciences, University of Mysore, Mysore 575 006, India
Received 29 June 2004; received in revised form 20 November 2004; accepted 23 November 2004
Available online 04 February 2005
Abstract
Two biologically active cyclic peptides, Yunnanin F 8 and Hymenistatin 16 were synthesized and the structures were established on the
basis of analytical, IR, NMR and mass spectral data. The newly synthesized compounds were screened for their antimicrobial and pharma-
cological activities. These cyclic octapeptides have shown moderate to good growth inhibition against bacterial strains and weak activity
against fungal strains more than that of the standard drug against only Pseudomonas aeruginosa but weak to moderate activity against
remaining three bacterial strains. They have shown very weak activity against fungal strains.Yunnanin F possessed good anthelmintic activity
while Hymenistatin possessed very low activity, but both showed moderate anti-inflammatory activity.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Cyclic peptides; Yunnanin F; Hymenistatin; Antimicrobial activity; Pharmacological activity; p-Nitrophenyl ester method
1. Introduction
was elucidated by extensive spectroscopic evidences and
chemical degradations.Yunnanin F exhibited cytotoxic activ-
ity. Another cyclic octapeptide, Hymenistatin active against
the P388 leukemia cell line was isolated by Pettit et al. [9]
from the Western Pacific ocean sponge Hymeniacidon sp. The
structural determination of this peptide was accomplished uti-
lizing NMR FABMS/MS techniques followed by chromato-
graphic analysis. The structure of Hymenistatin was also con-
firmed by the solid phase synthesis [10].
In continuation of our research work of synthesizing natu-
ral cyclic peptides of biological interest [11,12], an attempt
was made towards the synthesis ofYunnanin F and Hymeni-
statin. Keeping in view of significant biological activities
exhibited by various cyclic peptides, the above synthetic pep-
tides were further subjected to antibacterial and pharmaceu-
tical activity studies.
In the past two decades, a wide variety of naturally occur-
ring bioactive cyclic peptides have been isolated from plants,
marine sponges and tunicates [1]. Recently, a large number
of these cyclic peptides are emerging as an important class of
organic compounds due to their unique structure and biologi-
cal activities. The wide spread increase of bacterial resis-
tance towards conventional antibiotics encourages the explo-
ration of novel antimicrobial molecules with unexploited
mechanisms of action. Initially discovered as a defensive sys-
tem in invertebrates and vertebrates, antimicrobial peptides
are attracting increased interest as potential therapeutics [2–4].
Unlike classical antibiotics, which must penetrate the tar-
get cell, the principle mode of action of peptides involves
perturbation and permeability of the cell membrane. This
mechanism confers activity towards a broad spectrum of
microbial cells, but is also responsible for undesired lytic
activity against mammalian cells such as erythrocytes [5–7].
In the continued investigation of the roots of Stellaria yun-
nanensis, Morita et al. [8] isolated a new biologically active
cyclic octapeptide, Yunanin F. The structure of this peptide
2. Chemistry
For the synthesis of these cyclic octapeptides, disconnec-
tion approach was used. In order to carry out the total synthe-
sis ofYunnanin F, cyclo(Gly-Val-Thr-Trp-Tyr-Pro-Ser-Ser-),
it was disconnected into four dipeptide units, Boc-Gly-Val-
OMe 1, Boc-Thr-Trp-OMe 2, Boc-Tyr-Pro-OMe 3 and Boc-
Ser-Ser-OMe 4. The required dipeptides were prepared by
* Corresponding author.
E-mail address: bojapoojary@yahoo.com (B. Poojary).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.11.013

408
B. Poojary, S.L. Belagali / European Journal of Medicinal Chemistry 40 (2005) 407–412
coupling Boc-amino acids with the respective amino acid ester
hydrochlorides using DCC, HOBt and N-methyl morpholine
according to Bodanszky and Bodanszky [13] procedure with
suitable modifications [14]. The ester group of dipeptide 1
was removed with LiOH and the Boc-group of dipeptide 2
was removed with trifluoroacetic acid. Both the deprotected
units were coupled to get the tetrapeptide, Boc-Gly-Val-Thr-
Trp-OMe 5. The remaining two dipeptides (3 and 4) were
also coupled similarly to obtain the another tetrapeptide, Boc-
Tyr-Pro-Ser-Ser-OMe 6. These tetrapeptides were then
coupled after proper deprotection using DCC, HOBt and
NMM to get the octapeptide, Boc-Gly-Val-Thr-Trp-Tyr-Pro-
Ser-Ser-OMe 7. The methyl ester group of 7 was deprotected
with LiOH and p-nitrophenyl (pnp) ester group was intro-
duced by treating it with p-nitrophenol in presence of DCC.
The Boc-group of resulting Boc-Gly-Val-Thr-Trp-Tyr-Pro-
Ser-Ser-Opnp was removed by treating it with trifluoroacetic
acid in chloroform. The solution of Boc-deprotected octapep-
tide pnp ester was diluted with chloroform and allowed to
cyclize [15] in presence of pyridine to obtainYunnanin F 8 as
depicted in Fig. 1. In the same way, Hymenistatin [cyclo(-Ile-
Pro-Pro-Tyr-Val-Pro-Leu-Ile-)] was also disconnected into
four dipeptide units of Boc-Ile-Pro-OMe 9, Boc-Pro-Tyr-
OMe 10, Boc-Val-Pro-OMe 11 and Boc-Leu-Ile-OMe 12. The
tetrapeptide units, Boc-Ile-Pro-Pro-Tyr-OMe 13 and Boc-Val-
Pro-Leu-Ile-OMe 14 of were prepared as per the above pro-
Fig. 2. Synthesis of Hymenistatin.
cedure by condensing the required dipeptides (9–12) after
proper deprotection. The resulting tetrapetides (13 and 14)
were then coupled after proper deprotection using DCC, HOBt
and NMM to get the octapeptide, Boc-Ile-Pro-Pro-Tyr-Val-
Pro-Leu-Ile-OMe 15, the linear segment of Hymenistatin.
Finally, the cyclization of the linear segment (Fig. 2) was car-
ried out by the pnp ester method to obtain Hymenestatin 16.
3. Biological activity studies
The synthesized cyclic peptides,Yunnanin F and Hymeni-
statin were also screened for its antibacterial, antifungal, anti-
inflammatory and anthelmintic activity. The antibacterial and
antifungal activity are carried out against four bacterial (Sta-
phylococcus aureus, Bacillus subtilis, Pseudomonas aerugi-
nosa and Escherichia coli) and two fungal strains (Candida
albicans and Aspergillus niger). These activity studies were
carried out according to disc diffusion method [16]. Penicil-
lin and griseofulvin were used as standards against bacteria
and fungal strains at 10 and 25 µg per disc, respectively. The
results are summarized in Table 1. The anti-inflammatory
activity was carried out according to the method of Winter et
al. [17] using ibuprofen as the standard and the results are
presented in Table 2. The anthelmintic activity was carried
out against the earthworms (pontoscotex corethruses) accord-
Fig. 1. Synthesis of Yunnanin F.

B. Poojary, S.L. Belagali / European Journal of Medicinal Chemistry 40 (2005) 407–412
409
Table 1
Antimicrobial activity data
Compound
Diameter of zone of inhibition
Antibacterial activity studies
Antifungal activity studies
P. aeruginosa
E. coli
08
B. subtilis
S. aureus
C. albican
A. niger
Yunnanin F 8
Hymenistatin 16
Penicillin
14
14
12
–
10
16
18
–
16
08
18
–
10
08
–
08
09
–
10
12
Greseofulvin
–
20
20
Table 2
inflammatory data reveals that both of them are less active as
compared with the standard drug, ibuprofen (Table 2). Yun-
nanin F possessed good anthelmintic activity while Hymeni-
statin possessed very low activity as compared to the stan-
dard.
Anti-inflammatory activity data
Compound
Increase in paw volume
after 3 h S.E. (ml)
Percentage
inhibition of oedema
after 3 h S.E.
Yunnanin F 8
Hymenistatin 16
Ibuprofen
0.74 0.03
0.77 0.03
0.55 0.03
0.90 0.02
17.78
14.44
40.50
–
5. Conclusions
Control
Table 3
Anthelmintic activity data
We have successfully synthesized two cyclic octapep-
tides,Yunnanin F and Hymenistatin by solution phase method
in good yield. Also, we have studied their antimicrobial and
pharmacological activities. The antimicrobial results indi-
cated the moderate to good antibacterial activity of synthetic
cyclic peptides when compared to the reference drug penicil-
lin. Evaluation of anti-inflammatory activity revealed very
weak activity of these cyclic octapeptides as compared to the
standard drug, ibuprofen. Among the two synthesized cyclic
peptides, onlyYunnanin F showed a good anthelmintic activ-
ity.
Compound
Concentration
of the compound paralyzing time time
(mg)
100
200
Mean
Mean death
(min) S.E.
28.50 2.20
14.64 1.90
98.24 1.20
85.18 1.03
18.01 2.01
12.55 1.02
–
(min) S.E.
55.30 1.10
29.05 1.60
118.3 1.10
106.6 2.12
35.20 2.00
32.01 1.10
–
Yunnanin F 8
Hymenistatin 16 100
200
Mebendazole
Control
100
200
–
ing to Garg and Atal [18] method (Table 3) using mebenda-
zole as standard drug.
6. Experimental protocols
6.1. General chemistry
4. Results and discussion
Melting points were taken in open capillary and are uncor-
rected. IR spectra (in CHCl3) were recorded on a Perkin–
Elmer infrared spectrophotometer. NMR spectra were re-
corded in CDCl3/DMSO-d6 on a 300 MHz spectrophotometer
using TMS as an internal standard. The mass spectra were
recorded on a FAB mass spectrometer. The progresses of the
reactions were checked by TLC on silica gel G plates and the
products were purified by silica gel column chromatography.
Eight dipeptides, Boc-Gly-Val-OMe 1, Boc-Thr-Trp-
OMe 2, Boc-Tyr-Pro-OMe 3, Boc-Ser-Ser-OMe 4, Boc-Ile-
Pro-OMe 9, Boc-Pro-Tyr-OMe 10, Boc-Val-Pro-OMe 11 and
Boc-Leu-Ile-OMe 12 were prepared and these dipeptides were
used for the preparation of four tetrapeptides (Fig. 1), Boc-
Gly-Val-Thr-Trp-OMe 5, Boc-Tyr-Pro-Ser-Ser-OMe 6, Boc-
Ile-Pro-Pro-Tyr-OMe 13 and Boc-Val-Pro-Leu-Ile-OMe 14
after proper deprotection using LiOH and trifluoroacetic acid
according to Bodanszky procedure with suitable modifica-
tions [12]. The tetrapetides (5) and (6), were then condensed
as per the procedure used for the preparation of tetrapeptides
to obtain octapetide, Boc-Gly-Val-Thr-Trp-Tyr-Pro-Ser-Ser-
OMe 7, the linear segment ofYunnanin F. The linear segment
The intermediate and final products were purified by col-
umn chromatography using chloroform–methanol system and
recrystallized from EtOAc–petroleum ether. The newly syn-
thesized compounds were analyzed for C, H, N and the struc-
tures were confirmed by IR, NMR and mass spectral data.
The characteristic IR and NMR spectra of all the intermedi-
ate compounds were analyzed. The characteristic IR absorp-
tion bands of –CO–NH– moiety were present in the cyclized
product. The NMR spectra of all the cyclized products clearly
indicated the presence of all respective amino acid moieties.
Further more, the mass spectra these cyclic octapeptidesYun-
nanin F and Hymenistatin showed the [M+ + H] peak at m/z
878 and 893, which are consistent with their molecular for-
mulae, C42H55N9O12 and C47H72N8O9, respectively.
The antibacterial activity data presented in Table 1 indi-
cates that both synthesized cyclic octapeptides have shown
the growth inhibition more than that of the standard drug
against only P. aeruginosa but weak to moderate activity
against remaining three bacterial strains. They have shown
very weak activity against fungal strains. The anti-

410
B. Poojary, S.L. Belagali / European Journal of Medicinal Chemistry 40 (2005) 407–412
of Hymenistatin, Boc-Ile-Pro-Pro-Tyr-Val-Pro-Leu-Ile-
OMe 15 was obtained by coupling tetrapeptides (13) and (14)
according to above procedure.
(m, 3H, a-CH), 3.75 (s, 3H, OCH3), 3.6–3.4 (m, 2H, N–CH2),
2.4–2.0 (m, 4H, –CH2–CH2), 1.9–1.6 (m, 4H, b-CH2 and
b-CH), 1.45 (s, 9H, C(CH3)3), 1.3–1.1 (m, 3H, c-CH2 and
c-CH), 1.0 (doublet overlapped with triplet, 18H, CH3 and
C(CH3)2); C28H50N4O7 (554): Calc. C 60.65, H 9.03, N 10.11;
Found: C 60.78, H 9.12, N 10.21.
6.1.1. Boc-Gly-Val-Thr-Trp-OMe 5
Yield: 79%. IR(CHCl3): m 3580 (O–H str.), 3295 (N–H
str.), 3010 (=C–H str.), 2955 (C–H str.), 2860 (C–H str.), 1730
(C=O str. ester), 1690 (C=O str. amide), 1655 (C=O str.
amide), 1650 (C=O str. amide), 1530, 1450, 1390, 1265, 1160,
1090, 890 cm–1; 1H NMR (300 MHz, CDCl3): d 9.0 (s, 1H,
NH), 8.5 (br.s, 2H, NH), 8.1 (br.s, 2H, NH), 7.8–7.2 (m, 5H,
Ar-H), 5.2 (m, 1H, OH), 4.9–4.7 (m, 2H, ␣-CH), 4.6–4.5 (m,
1H, a-CH), 4.3–4.2 (m, 1H, a-CH), 4.1–4.0 (m, 2H, a-CH2),
3.7(s, 3H, O–CH3), 3.5–3.3 (m, 2H, b-CH2), 1.6–1.5 (m, 1H,
b-CH), 1.4 (s, 9H, C(CH3)3), 1.2 (d, 3H, J = 6.5 Hz, CH3),
0.95 (d, 6H, J = 6.5 Hz); C28H41N5O8(575): Calc. C 58.43, H
7.13, N 12.17; Found: C 58.58, H 7.25, N 12.30.
6.1.5. Boc-Gly-Val-Thr-Trp-Tyr-Pro-Ser-Ser-OMe 7
Yield: 68%. IR (CHCl3): m 3590 (O–H str.), 3320 (N–H
str.), 3040 (=C–H str.), 2985 (C–H str.), 2850 (C–H str.), 1730
(C=O str. ester), 1680 (C=O str. amide), 1675 (C=O str.
amide), 1665 (C=O str. amide), 1600, 1530, 1470, 1320, 1240,
1160, 1080, 840 cm–1; 1H NMR (300 MHz (CDCl3)): d 9.0
(br.s, 1H), 6.9 (m, 9H, Ar-H), 5.2 (s, 1H, –OH), 5.18 (s, 1H,
–OH), 4.8–4.7 (m, 4H, a-CH), 4.6–4.5 (m, 5H, a-CH), 4.4–
4.2 (m, 5H, a-CH2 and a-CH), 3.7 (s, 3H, OCH3), 3.6–3.4
(m, 2H, N–CH2), 3.2–3.0 (m, 4H, b-CH2), 2.2–1.7 (m, 4H,
–CH2–CH2–), 1.6–1.5 (m, 1H, b-CH), 1.4 (s, 9H, C(CH3)3),
1.3 (d, 3H, J = 6.5 Hz, CH3), 0.95 (d, 6H, J = 6.5 Hz,
–C(CH3)2); C48H67N9O15(1009): Calc. C 57.09, H 6.64, N
12.49; Found: C 57.16, H 6.62, N 12.61.
6.1.2. Boc-Tyr-Pro-Ser-Ser-OMe 6
Yield: 84.5%. IR(CHCl3): m 3490 (O–H str.), 3320 (N–H
str.), 3090 (=C–H str.), 2940 (C–H str.), 2851 (C–H str.), 1730
(C=O str. ester), 1690 (C=O str. amide), 1680 (C=O str.
amide), 1650 (C=O str. amide), 1622 (N–H def.), 1570, 1500,
6.1.6. Boc-Ile-Pro-Pro-Tyr-Val-Pro-Leu-Ile-OMe 15
Yield: 69.3%. IR(CHCl3): m 3550 (O–H str.), 3460 (N–H
str.), 3010 (=C–H str.), 2980 (C–H str.), 2920 (C–H str.), 1730
(C=O str. ester), 1690(C=O str. amide), 1670 (C=O str. amide),
1630 (C=O str. amide), 1610 (N–H def.), 1540 (C–N str.),
1510, 1450 (C–H def.), 1380 (C–H def.), 1260 (C–O str.),
1100 (C–O str.), 970 (C–H def.) cm–1; 1H NMR (300 MHz
(CDCl3)): d 9.2 (br.s, 1H, OH), 8.4 (br.s, 1H, NH), 7.5 (br.s,
1H, NH), 7.0 (d, 2H, 2,6-Ar-H), 6.9 (2H, d, J = 8.0 Hz, 3,5-
Ar-H), 6.8 (br.s, 2H, NH), 6.3 (br.s, 2H, NH), 4.7–4.4 (m,
4H, a-CH), 4.3–4.0 (m, 4H, a-CH), 3.75 (s, 3H, OCH3), 3.6–
3.4 (m, 6H, N–CH2), 3.2–3.0 (m, 2H, b-CH2), 2.4–2.0 (m,
12H, –CH2–CH2), 1.9–1.6 (m, 5H, b-CH2 and b-CH), 1.4 (s,
9H, C(CH3)3), 1.3–1.1 (m, 5H, c-CH2 and c-CH), 0.95 (dou-
blet overlapped with triplet, 24H, J = 6.5 Hz, CH3 and
C(CH3)2); C53H84N8O12 (1024): Calc. C 62.11, H 8.20, N
10.94; Found: C 62.16, H 8.12, N 10.88.
1
1430, 1370, 1240, 1160, 895 cm–1; H NMR (300 MHz,
CDCl3): d 9.3 (br.s, 1H, OH), 8.7 (br.s, 2H, NH), 8.3 (br.s,
1H, NH), 7.8 (br.s, 1H, NH), 7.0 (d, 2H, J = 8.0 Hz, 2,6-Ar-
H), 6.85 (d, 2H, J = 8.0 Hz, 3,5-Ar-H), 5.2–5.1 (m, 2H, OH),
4.9–4.7 (m, 4H, a-CH), 4.5–4.2 (m, 4H, a-CH2), 3.7 (s, 3H,
O–CH3), 3.6–3.4 (m, 2H, –N–CH2), 3.2–3.0 (m, 2H, b-CH2),
2.2–1.7 (m, 4H, –CH2–CH2–), 1.4 (s, 9H, C(CH3)3);
C26H38N4O10(566): Calc. C 55.12, H 6.71, N 9.89; Found: C
55.24, H 6.58, N 9.73.
6.1.3. Boc-Ile-Pro-Pro-Tyr-OMe 13
Yield: 86.6%. IR(CHCl3): m 3510 (O–H str.), 3480 (N–H
str.), 3015 (m, =C–H str.), 2980(C–H str.), 2840 (C–H str.),
1720 (C=O str. ester), 1670(C=O str. amide), 1650 (C=O str.
amide), 1610 (N–H def.), 1500 (C–N str.), 1300 (C–H def.),
1260 (C–O str.), 1015 (C–H def.), 970 (C–H def.) cm–1; 1H
NMR (300 MHz, CDCl3): d 9.25 (br.s, 1H, OH), 7.0 (d, 2H, J
= 8.0 Hz, 2,6-Ar-H), 6.85 (d, 2H, J = 8.0 Hz, 3,5-Ar-H), 6.8
(br.s, 1H, NH), 6.2 (br.s, 1H, NH), 4.7–4.5 (m, 2H, a-CH),
4.4–4.1 (m, 2H, a-CH), 3.75 (s, 3H, OCH3), 3.5–3.3 (m, 4H,
N–CH2), 3.2–3.1 (m, 2H, b-CH2), 2.3–1.7 (m, 9H, –CH2–
CH2 and b-CH), 1.45 (s, 9H, C(CH3)3), 1.3–1.1(m, 2H,
c-CH2), 1.0 (doublet overlapped with triplet, 6H, CH3).
–C31H46N4O8 (602): Calc. C 61.79, H 7.64, N 9.30; Found:
C 61.76, H 7.89, N 9.90.
6.1.7. General procedure for the preparation of cyclic
octapeptides, Yunnanin F 8 and Hymenistatin 16
To the solution of Boc-heptapeptide 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 sulfate. To the resulting Boc-
deprotected peptide-pnp ester in THF (15 ml), pyridine
(1.4 ml, 2 mmol) was added and kept at 4 °C for 7 days. The
reaction mixture was washed with 10% sodium bicarbonate
solution until the byproduct p-nitrophenol was removed com-
pletely and finally washed with 5% HCl (5 ml). The organic
layer was dried over anhydrous sodium sulfate. THF and pyri-
dine were distilled under reduced pressure to get cyclic pep-
tide. The crude product was purified by silica gel column chro-
matography using the dichloromethane–methanol system and
finally recrystallized from EtOAc–n-hexane.
6.1.4. Boc-Val-Pro-Leu-Ile-OMe 14
Yield: 79.9%. IR(CHCl3): m 3520 (N–H str.), 3450 (N–H
str.), 2960 (C–H str.), 2920 (C–H str.), 2880 (C–H str.), 1740
(C=O str. ester), 1690 (C=O str. amide), 1630 (C=O str.
amide), 1500, 1437 (C–N str.), 1311 (C–H def.), 1245, 1159,
1
1080, 892 cm–1; H NMR (300 MHz (CDCl3)): d 6.6 (br.s,
2H, NH), 5.9 (br.s, 1H, NH), 4.6–4.5 (m, 1H, ␣-CH), 4.3–4.0

B. Poojary, S.L. Belagali / European Journal of Medicinal Chemistry 40 (2005) 407–412
411
6.1.8. Yunnanin F 8
19.5 (q, –CH3), 19.4 (q, –CH3), 18.5 (q, –CH3), 17.8 (q,
–CH3); FAB mass: m/z 893 [M+ + H]; C47H72N8O9(71): Calc.
C 63.23, H 8.07, N 12.56; Found: C 63.15, H 8.21, N 12.50.
Yield: 56%; m.p.: 245–248 °C. IR(CHCl3): m 3600 (O–H
str.), 3320 (N–H str.), 3030 (=C–H str.), 2940 (C–H str.), 2850
(C–H str.), 1680 (C=O str. amide), 1650 (C=O str. amide),
1630 (C=O str. amide), 1605(N–H def.), 1530 (C–N str.), 1430
(C–H def.), 1150 (C–H def.), 1075 (C–H def.), 975 (C–H
def.) cm–1; 1H NMR (300 MHz, DMSO-d6): d 9.2 (1H, br.s,
OH), 9.0 (1H, br.s, NH), 8.8 (br.s, 2H, NH), 8.5 (br.s, 2H,
NH), 8.2 (br.s, 3H, NH), 7.9 (br.s, 1H, NH), 7.8–7.2 (m, 7H,
Ar-H andAr(Trp)–H), 6.95 (2H, d, J = 8.0 Hz, 3,5-Ar-H), 5.2
(s, 1H, OH), 5.1 (s, 1H, OH), 4.8–4.7 (m, 4H, b-CH2), 4.6–
4.5 (m, 5H, a-CH and b-CH), 4.4–4.0 (m, 5H, a-CH2 and
a-CH), 3.6–3.4 (m, 2H, N–CH2), 3.2–3.0 (m, 4H, b-CH2),
2.2–1.8 (m, 4H, –CH2–CH2–), 1.5–1.4 (m, 1H, b-CH), 1.3
(d, 3H, J = 6.5 Hz, –CH), 0.9 (d, 6H, J = 6.5 Hz, –C(CH3)2);
13C NMR (DMSO-d6): d 174.8 (s, C=O), 173.5 (s, C=O),
173.0 (s, C=O), 172.4 (s, C=O), 170.9 (s, C=O), 170.8 (s,
C=O), 170.1(s, C=O), 169.4 (s, C=O), 154.8 (s,Ar-4-C), 137.4
(s, Trp-8-C), 130.1 (d, Ar-2,6-C), 129.0 (s, Ar-1-C), 128.8 (s,
Trp-9-C), 125.5 (d, Trp-2-C), 122.2 (d, Trp-6-C), 119.9 (d,
Trp-5-C), 119.3 (d, Trp-4-C), 113.9 (d, Ar-3,5-C), 112.6 (s,
Trp-3-C), 110.1 (d, Trp-7-C), 62.9 (t, b-CH2) 62.2 (t, b-CH2),
61.0 (d, a-CH), 60.1 (t, b-CH2), 59.5 (d, a-CH), 56.7 (d,
a-CH), 46.4 (t, a-CH), 57.2 (d, a-CH), 57.0 (d, a-CH), 55.4(d,
a-CH), 53.2(d, a-CH), 48.3 (t, d(N)–CH2), 36.9 (t, b-CH2),
27.4 (t, b-CH2), 30.1 (d, b-CH), 28.5 (t, b-CH2), 25.3(t,
c-CH2), 21.5 (q, CH3), 19.4 (q, CH3), 19.0 (q, CH3); FAB
mass: m/z 878 [M+ + H]; C42H55N9O12 (877): Calc. C 57.47,
H 6.27, N 14.37; Found: C 57.60, H 6.15, N 14.42.
6.2. Biological experimental section
6.2.1. Antibacterial and antifungal activity
The synthesized cyclic octapeptides were screened for their
antibacterial activity against S. aureus, B. subtilis, P. aerugi-
nosa, E. coli and antifungal activity against C. albicans and
A. niger by disc diffusion method. The discs measuring 6 mm
in diameter were punched from Whatman No. 1 filter paper.
Batches of 100 discs were dispensed to each screw capped
bottles and sterilized by dry heal at 140 °C for 1 h. The solu-
tions of test compounds were prepared in dimethyl sulfox-
ide. The samples of test compounds were tested for antibac-
terial and antifungal activity at 10 and 25 µg level, respectively.
One milliliter containing 100 times the amount of chemical
in each disc was added to each bottle, which contains
100 discs. The discs of each concentration were placed in
triplicate in nutrient agar medium seeded with fresh bacteria
and fungi separately. The incubation was carried out at 37 °C
for 24 h. Penicillin, streptomycin and griseofulvin were used
as standard drugs for antibacterial and antifungal activity
study. The solvent and growth controls were kept and zones
of inhibition were noted. The results of such studies are given
in Table 1.
6.2.2. Anti-inflammatory activity
Winter’s hind paw method was used in the present study
for the evaluation of the anti-inflammatory activity on albino
rats. Carragenin at a concentration of 1 mg ml–1 was injected
subcutaneously into the hind paw of the rat, to produce the
oedema. Forty healthy rats of both sexes (body weight 100–
200 g), pregnant females excluded, were selected and made
in to four groups of 10 animals each. One group of animals
was kept as control that received 2% w/v acacia mucilage,
which was used to suspend the sample. Another group
received the standard drug ibuprofen, 20 mg kg–1 body weight
intraperitonially. Remaining two groups of animals were given
a dose of different test compounds (20 mg kg–1 body weight).
After 30 min, 0.1 ml of w/v carragenin was injected subcu-
taneously in to the right hind paw and the paw volume was
measured by a mercury plethysmometer and then measured
again after 3 h. The mean increase of paw volume was com-
pared with that of the control group and percent inhibition
values were calculated. The experimental results are fisted in
Table 2.
6.1.9. Hymenistatin 16
Yield: 58.9%; m.p.: 180–181 °C (Ref. [9] 180–182 °C)).
IR(CHCl3): m 3580 (O–H str.), 3270 (N–H str.), 3005 (=C–H
str.), 2985 (C–H str.), 2930 (C–H str.), 1685 (C=O str. amide),
1660 (C=O str. amide), 1610 (N–H def.), 1530 (C–N str.),
1500 (C–H def.), 1450 (C–H def.), 1170 (C–H def.), 970 (C–H
def.) cm–1; 1H NMR (300 MHz, DMSO-d6): d 9.25 (br.s, 1H,
OH), 7.6 (br.s, 1H, NH), 7.0 (d, 2H, 2,6-Ar-H), 6.9 (2H, d,
J = 8.0 Hz, 3,5-Ar-H), 6.6 (br.s, 2H, NH), 6.2 (br.s, 2H, NH),
4.8–4.5 (m, 4H, a-CH), 4.4–4.1 (m, 4H, a-CH), 3.6–3.2 (m,
4H, N–CH2), 3.2–3.0 (m, 2H, b-CH2), 2.4–2.1 (m, 12H,
–CH2–CH2–), 1.8–1.5 (m, 5H, b-CH2 and b-CH), 1.3–1.2
(m, 5H, c-CH2 and b-CH), 1.3–1.2 (m, 5H, c-CH2 and c-CH),
1.0 (doublet overlapped with triplet, 24H, CH3 and C(CH3)2);
13C NMR (DMSO-d6): d 173.5 (s, C=O), 173.2 (s, C=O),
172.9 (s, C=O), 172.8 (s, C=O), 172.5 (s, C=O), 172.0 (s,
C=O), 171.6 (s, C=O), 171.5 (s, C=O), 157.6 (s, Ar-1-C),
130.7 (d, Ar-2,6-C), 127.1 (s, Ar-4-C), 115.9 (d, Ar-3,5-C),
62.8 (t, b-CH2), 61.8 (d, a-CH), 60.7 (d, a-CH), 60.5 (d,
a-CH), 59.9 (d, a-CH), 58.4 (d, a-CH), 57.7 (d, a-CH), 57.6
(d, a-CH), 56.7 (d, a-CH), 47.2 (t, d(N)–CH2), 47.0 (t, d(N)–
CH2), 46.9 (t, d(N)–CH2), 40.0 (t, b-CH2), 39.3 (t, b-CH),
39.2 (t, b-CH), 36.8 (t, b-CH2), 31.6(t, b-CH2), 29.3 (d,
b-CH), 28.8 (t, b-CH2), 28.5 (t, b-CH2), 28.2 (t, b-CH2), 25.7
(t, c-CH), 25.2 (t, c-CH2), 25.1 (t, c-CH2), 24.9 (t, c-CH2),
24.6 (t, c-CH), 24.5 (t, c-CH), 20.3 (q, –CH3), 19.8 (q, –CH3),
6.2.3. Anthelmintic activity
Anthelmintic activity studies were carried out against earth-
worms, Pomoscolex corethrusus by Garg’s method. Suspen-
sions of the samples were prepared by triturating the samples
with 0.5% tween 80 and distilled water. The suspensions were
diluted to contain 0.1% and 0.2% w/v of the test samples.
0.1% and 0.2% w/v suspensions of the standard drug,

412
B. Poojary, S.L. Belagali / European Journal of Medicinal Chemistry 40 (2005) 407–412
[2] M. Zosloff, Nature 415 (2002) 389.
mebendazole were also prepared in a similar way. Ten earth-
worms of similar size were placed in a Petri plate of 4 in.
diameter containing 50 ml of suspension of the test standard
drug at room temperature.Another set of 10 earth worms were
kept as control in 50 ml suspension of distilled water and
0.5% tween 80. Fifty milliliters each of the suspensions of
the test compounds were added in to separate Petri plates con-
taining 10 earthworms in each. The time required for the
paralysis and deaths of the earthworms were noted. The death
time was ascertained by placing the earthworms in warm water
at 50 °C, which stimulated the movement if the worm was
alive. The results are summarized in Table 3.
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Acknowledgements
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The authors are grateful to Professor M.V. Ramana, Depart-
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Science, Mangalore and Ms. Beena Mascarenhas, KMC Hos-
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