Towards the first total synthesis and anticancer screening of polycarponin C: A cyclic octapeptide

Article abstract of DOI:10.13005/ojc/320159

The present study describes designing, synthesis and anticancer screening of a proline rich cyclic octapeptide polycarponin C, by solution phase synthesis. The synthesis was carried out by coupling a tetrapeptide Boc-Pro-Thr-Leu-Pro-OH with another tetrap

Full text of DOI:10.13005/ojc/320159

ISSN: 0970-020 X
CODEN: OJCHEG
2016, Vol. 32, No. (1):
Pg. 515-521
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Free Access, Peer Reviewed Research Journal
www.orientjchem.org
Towards the First Total Synthesis and Anticancer
Screening of Polycarponin C: A Cyclic Octapeptide
NIRMALA V. SHINdE1*, AVINASH S. dHAkE2 and kISHAN P. HAVAL3
1Department of Pharmaceutical Sciences, Jawaharlal Nehru Technological University,
Kakinada, Andhra Pradesh, India.
2Department of Pharmaceutical Chemistry, S.M.B.T. College of Pharmacy, Nandi Hills,
Dhamangaon. Tah: Igatpuri, Dist: Nashik (MH) India.
3Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada University,
SubcentreOsmanabad(MH) India.
*Corrosponding author E-mail:nirmalampharm@rediffmail.com
http://dx.doi.org/10.13005/ojc/320159
(Received: November 23, 2015; Accepted: March 11, 2016)
AbSTRACT
The present study describes designing, synthesis and anticancer screening of a proline rich
cyclic octapeptide polycarponin C, by solution phase synthesis. The synthesis was carried out by
coupling a tetrapeptide Boc-Pro-Thr-Leu-Pro-OH with another tetrapeptide Boc-Pro-Val-Leu-Phe-
OH, followed by cyclization of the linear octapeptide.The structure of the synthesized compound
was then confirmed by spectral analysis. From the results of biological activity, it was concluded that
the compound has shown moderate activity against SR human tumor cell lines of Leukemia when
compared with Vincristine as a standard.
key words:Cyclic Peptide, Solution phase synthesis,
p-nitro phenyl ester Method, Coupling Agent, Anticancer.
INTROdUCTION
and to obtain a natural bioactive peptide in good
yield, in the present work an attempt has been
made towards the first total synthesis of a proline
rich cyclic octapeptide polycarponin C, cyclo-( Pro-
Thr-Leu-Pro-Pro-Val-Leu-Phe), isolated from whole
plants of Polycarpon prostratum, belonging to family
Caryophyllaceae8. The synthesis was attempted by
solution phase technique9 followed by cyclization of
linear octapeptide by p-nitro phenyl ester method12.
The structure of the synthesized compound was
Anticancer drug development from
natural sources has always been fascinating and
interesting for researchers working in the field
of medicinal chemistry and drug development.
Owing to the various biological activities like
antimicrobial, cytotoxic, anti-HIV, anti-inflammatory,
serine protease and protein tyrosine phosphatase
inhibitory activity possessed by cyclic peptides1-7

516
SHINDE et al., Orient. J. Chem., Vol. 32(1), 515-521 (2016)
confirmed by detailed spectral analysis. The of dipeptide 1 and 3 was then removed by using
synthesized compound was then subjected for LiOH and the Boc group of dipeptide 2 and 4 was
preliminary cytotoxic activity by using Brine shrimp removed by using TFA. The deprotected units were
assay, and further followed by screening against 60 then coupled to get two tetra peptides Boc-Pro-Thr-
human tumor cell lines at NCI, USA. The results of Leu-Pro-OMe 5 and Boc-Pro-Val-Leu-Phe-OMe
anticancer screening showed that the compound is 6. The resulting tetrapeptides were then coupled
moderately active against SR human tumor cell lines together by using DIPC and chloroform to obtain a
of Leukemia when compared with Vincristine as a linear octapeptide, which was then cyclized by using
standard.
p-nitro phenyl ester method to get titled compound.
MATERIALS ANd METHOdS
General method for preparation of di/tetra/linear
All L-amino acids, di-tertbutyldicarbonate octapeptide
(BOC2O),diisopropylcarbodiimide (DIPC), L-Amino acid methyl ester hydrochloride/
trifluoroacetic acid (TFA), triethylamine (TEA), dipeptide methyl ester/tetra peptide methyl ester
pyridine and N-methylmorpholine (NMM) were (10 mmol) was dissolved in chloroform (CCl3, 20
purchased from Spectrochem Limited (Mumbai, ml). To this, TEA (2.8 ml, 20 mmol) was added at 0
India).Melting points were determined by using digital °C and the reaction mixture was stirred for 15 min.
melting point apparatus.
Boc-L-amino acid/Bocdipeptide/ Boctetrapeptide (10
The IR spectra were run on FTIR mmol) in chloroform (20 ml) and DIPC (10 mmol)
spectrophotometer,JASCO 4100 using a thin film were added with stirring. After 24 h, the reaction
supported on KBr pellets or utilizing chloroform mixture was filtered and the residue was washed
and NaCl cells.1H NMR and 13C NMR spectra were with chloroform (30ml) and added to the filtrate.
recorded on Bruker AC NMR spectrometer using The filtrate was was hed with 5% NaHCO3 and
DMSO as a solvent. The mass spectrum of the saturated NaCl solutions.The organic layer was dried
cyclopeptide was recorded on JMS-DX 303 Mass over anhydrous Na2SO4, filtered and evaporated in
spectrometer operating at 70 eV by ESIMS/MS.
vacuum. The crude product was recrystallized from
a mixture of chloroform and petroleum ether (b.p.40-
In order to carry out the total synthesis of 60 °C) followed by cooling at 0 °C. By using above
cyclicpeptide, polycarponin C, cyclo-(Pro-Thr-Leu- procedure, compounds 1-7 were synthesized.
Pro-Pro-Val-Leu-Phe), it was disconnected into four
dipeptide units, BOC-Pro-Thr-OMe 1, Boc-Leu-Pro- Procedure for cyclization of linear
OMe 2, Boc-Pro-Val-OMe 3, Boc-Leu-Phe-OMe 4. octapeptide12:
The dipeptides were obtained by coupling Boc amino
The cyclization of linear octapeptide was
acids with the respective amino acid methyl esters, attempted by using p-nitrophenyl ester method.
by using DIPC as a coupling agent.The ester group The ester group of linear fragment was removed
Table 1: Results for Cytotoxic activity by using brine Shrimp Assay:
Compound
Conc.
Number of Surviving
Total Number
%
(ppm or μg/ml)
Napulii After 24 h
of Survivors
Mortality
T1
T2
T3
1000
500
250
125
62.5
31.25
4
5
5
7
8
6
5
6
6
9
4
5
7
7
8
14
15
18
20
25
30
53.33
50
40
33.33
16.66
0
Poly C
10
10
10
*T=Trial
LC50=460.9 μg/ml

SHINDE et al., Orient. J. Chem., Vol. 32(1), 515-521 (2016)
517
with LiOH and the p-nitrophenyl ester group was
introduced. For introduction of p-nitro phenyl ester
group, the Boc-peptide carboxylic acid(1.5 mmol)
was dissolved in chloroform (15 ml) at 0 °C, to which
p-nitrophenyl(0.27 gm, 2 mmol) was added, and
stirred for 12 hrs at RT. The reaction mixture was
filtered and the filtrate was washed with NaHCO3
solution (10%) until excess of p-nitrophenyl was
removed and finally washed with 5% HCl (5 ml) to
get Boc-peptide-pnp ester.
2.4 mmol) was added, stirred for 1 hour at room
temperature and washed with 10% NaHCO3 solution.
The organic layer was dried over anhydrous Na2SO4.
To the Boc-deprotected peptide-pnp-ester in CHCl3
(15ml), N-methyl morpholine (1.4ml, 2mmol.) was
added and kept at 00C for 7 days. The reaction
mixture was washed with 10% NaHCO3 until the
byproduct p-nitrophenyl was removed completely
and finally washed with 5% HCl (5ml). The organic
layer was dried over anhydrous Na2SO4.Chloroform
and pyridine were distilled off to get the crude
cyclized compound, which was recrystallized from
CHCl3/n-hexane.
To the above Boc-peptide-pnp-ester
(1.2 mmol) in CHCl3 (15ml), CF3COOH (0.274g,
Graph 1: dTP One dose Mean graph for Polycarponin C

518
SHINDE et al., Orient. J. Chem., Vol. 32(1), 515-521 (2016)
Anticancer activity of synthesized compound
Preliminary cytotoxic activity by brine Shrimp with an aquarium air pump.The seawater was put in
Lethality Assay (bSLA)10, 11
a small plastic container (hatching chamber) with a
solution was aerated continuously during incubation
Brine shrimp eggs were obtained from partition for dark (covered) and light areas. Shrimp
the aquarium shop, Nasik. Artificial sea water was eggs were added into the dark side of the chamber
prepared from (1% NaCl) nitrate, phosphate, and while the lamp above the other side (light) will attract
silicate-free sea-salt and distilled water (35 g/l) at the hatched shrimp. Two days were allowed for the
25° C under constant illumination. The saltwater shrimp to hatch and mature as nauplii (larva). After
Graph. 2: Comparison of synthesized Polycarponin C for anticancer screening with data of
vincristine against leukemia human tumor cell lines.
Table 2: %GI shown by Polycarponin C against different human
tumor cell lines
Human Tumor
Cell Line
% GI for
comp. VIII
Human Tumor
Cell Line
% GI for
comp. VIII
Leukemia
CCRF-CEM
HL-60(TB)
K-562
RPMI-8226
SR
Ovarian Cancer
IGROV1
15.33
22.99
39.17
15.09
61.87
13.6
Colon cancer
HCT-116
HCT-15
Renal Cancer
UO-31
12.39
11.79
7.01
15.89
HT29
KM12
13.9
SW-620
8.69
CNS Cancer
SNB-75
Melanoma
LOX IMVI
MALME-3
MDA-MB-435
Breast Cancer
MCF7
18.20
31.78
90.55
61.87
98.09
Mean
Delta
ange
5.59
12.89
26.76

519
SHINDE et al., Orient. J. Chem., Vol. 32(1), 515-521 (2016)
two days, when the shrimp larvae are ready, 4 ml (%M) was also calculated by dividing the number
of the artificial seawater was added to each test of dead nauplii by the total number, and then
tube containing different conc. of drug and 10 brine multiplied by 100%. This is to ensure that the death
shrimps were introduced into each tube. Thus, (mortality) of the nauplii is attributed to the activity of
there were a total of 30 shrimps per dilution. Then the compound. The results of activity are shown in
the volume was adjusted with artificial seawater Table 1.
up to 5 ml per test tube. The test tubes were left
uncovered under the lamp.The number of surviving In vitro cytotoxic activity against Human tumor
shrimps were counted and recorded after 24 hours. cell lines12
The lethality concentration (LC50) was assessed at
The synthesized compoun dwas screened
95% confidence intervals.The percentage mortality for in vitro anticancer assay by National Cancer
OH
O
O
N
OMe
O
O
OH
O
O
boc
N
H
H2
N
OH
OMe
H2N
H2N
boc
OMe
+
boc
N
+
+
H2N
OH
O
O
a,c
boc
N
H
a,c
a,c
OMe
OH
+
O
OMe
O
N
H
O
O
OMe
OH
O
O
boc
N
H
N
H
N
a,c
boc
boc
N
OMe
+
O
N
O
(2)
OMe
+
boc
N
H
(1)
N
(3)
H
a,b,c
(4)
a,b,c
O
O
OMe
O
H
N
O
boc
H
O
N
N
N
H
O
N
O
H
O
N
H
N
+
OH
(5)
O
boc
N
boc
N
H
(6)
OMe
a,b,c
O
OMe
O
OH
N
H
N
H
N
NH
O
O
O
O
NH
N
N
H
O
O
a,b,c,d,e
O
O
N
(7)
O
NH
N
O
NH
NH
O
N
H
O
(8)
O
H
N
N
N
boc
O
H
O
Where: a= DIPC, NMM, CHCl3, RT, 24h, b= TFA, NMM,RT,1h,
c= LiOH, THF:H2O(1:1), reflux, 15 mins d= pnp-, CHCl3, RT, 12h, e= NMM, CHCl3, 0°C, 7days
Scheme 1: Synthetic route for Polycarponin C

520
SHINDE et al., Orient. J. Chem., Vol. 32(1), 515-521 (2016)
Institute (NCI), Bethesda, USA in a panel of 60 human for Cg of Pro2, ä 170.8 for C=O of Pro3, δ 59.2 for Cδ
tumor cell lines. The screening of the compound of Pro3, δ 28.6 for Ca of Pro3, δ 25.8 for Cg of Pro3
operated with In Vitro Cell Line Screening Project
(IVCLSP), which is dedicated service, providing 1H NMR: showed five amide N-H signals
direct support to the DTP anticancer drug discovery
δ 7.2 for HN of Thr, δ 4.55 for HN of Thr, δ
program. The screening was carried out against 60 4.65 for Hb of Thr, δ 1.36 for Hb of Thr, δ 11.04 b
different human tumor cell lines of the leukemia, for HN of Phe, δ 5.02 for Ha of Phe, δ 3.01 for Hb
Non-smallcell lung, colon, CNS, melanoma, ovarian, of Phe, δ 8.7 for HN of Val, δ 4.7 for Ha of Val, δ
renal, Prostrate and breast cancers which was aimed 2.5 for Hb of Val, δ 1.5 for Hb of Val, δ 8 for HN of
in showing selective growth inhibition or cell killing Leu1, δ 5.36 for Ha of Leu1, δ 1.74 for Hb of Leu1, δ
of particular tumor cell lines by specific compound. 1.90 for Hb of Leu1, δ 8.5 for HN of Leu2, δ 5.05 for
The screening begins with the evaluation of selected Ha of Leu2, δ 1.7 for Hb of Leu2, δ 1.94 for Hb of
compounds at asingle dose of 10-5 M.The output from Leu2, δ 4.45 for Ha of Pro1, δ 2.16 for Hb of Pro1,
the single dose screen is reported as a mean graph δ1.8 for Hb of Pro1, δ 4.4 for Ha of Pro2, δ 1.90 for
and is available for analysis by the compare program. Hb of Pro2, δ 1.65 for Hb of Pro2, δ 4.39 for Ha of
The result of anticancer screening is shown in fig. 1 Pro3, δ 1.90 for Hb of Pro3, δ 2.16 for Hb of Pro3,,
and Table 2.
4.08 for Hδ of Pro3. Elemental analysis: C=63.96,
H=8.4, N=13.10, O=14.91
RESULTS ANd dISCUSSION
CONCLUSION
Spectral characterization
Physical state: Semisolid mass
The compound was synthesized with good
IR data: intense C=O absorptions at 3300 yield by using solution phase technique. It showed
and 1650 cm-1.absorptions at 3427 , 1650 cm-1, 1650 moderate activity against SR human tumor cell
cm-1 are due to amino and amide carbonyl groups lines of Leukemia when compared with Vincristine
respectively FABMS showed M+ ion peak at m/z as a standard. In consultation with literature survey,
860.
synthesis of analogs of this molecule may lead to
the development of potent anticancer agents.
13C NMR: showed six amide carbonyl carbon
δ 171 for C=O of Thr, δ 62.5 for Ca of Thr, δ
66.5 for Cb of Thr, δ 22 for Cl of Thr, δ 172 for C=O
of Phe,δ 52.9 for Ca of Phe, δ 38 for Cb of Phe, δ
171.5 for C=O of Val, δ 62 for Ca of Val, δ 31.5 for
ACkNOwLEdGEMENT
Authors are thankful to CSIR, Lucknow-
Cb of Val, δ 19 for Cl of Val, δ 170.5 for C=O of Leu, CDRI, New Delhi for financial assistance. We
a 50 for Ca of Leu, δ 41.5 for Cδ of Leu, δ 25.1 for also extend our thanks to S.M.B.T. College of
Cã of Leu, δ 172.5 for C=O of Leu2, δ 51 for Cb of Pharmacy, Dhamangaon, Nashik for providing
Leu2, δ 43.5 for Cb of Leu2, δ 25for Cy of Leu2, δ necessary facilities to do the research work and
173 for C=O of Pro1, δ 62.2 for Ca of Pro1, δ 32.2 for SAIF, Chandigarh for spectral analysis. Authors are
CY of Pro1, δ 22.2 for Cg of Pro1, δ 171 for C=O of also thankful to NCI, USA for carrying out anticancer
Pro2, δ 61.2 for Cá of Pro2, δ 31 for Cb of Pro2, δ 22 activity.
REFERENCES
1.
Shinde, N. V. ;Himaja, M. ;Bhosale,
S.K.;Ramana, M.V.;Sakarkar, D.M. Indian J 4.
Pharm Sci.2008, 70(6), 695-852
A. Int J. PharmTech Res.2012,4(1), 194-200
Kawagishi H.; Somoto A.;Kuranari J.; Kimura
A.; Chiba S. Tetrahedron1993,34(21), 3439-
3440
Chaudhary S.;Kumar H.;Verma H.;Rajpoot
A. Int Journal of PharmTech Res.2012, 4(1),
2.
3.
Dahiya R.;Gautam H. Bulletin Pharm.
Res.2011,1(1), 1-10
5.
Chaudhary S.; Kumar H.;Verma H.;Rajpoot

521
SHINDE et al., Orient. J. Chem., Vol. 32(1), 515-521 (2016)
9. Bodanszky M. and Bodanszky A., Practice of
194-200
6.
7.
Hernández D. Eur. J. Org. Chem.2008,
3389–3396
Shinde N.V.;Himaja M.;Bhosale S.K.;Ramana 10.
M.V.;Sakarkar D.M.Asian J Chem.2010,22(2),
peptide synthesis;Springer-verlog Publishers.
New York, (1984)
Bussmann R.;Malca G.; Glenn A. J.
Ethnopharmacology.2011,137, 121-140
Houghton P.;Fang R.;Techatanawat I.Science
Direct.2007,42, 377–387.
996-1000
11.
8.
Ding, Z.T.; Zhou, J.;Tan, N.; Cheng,Y.; Deng,
S. ActabotanicaSinica.2001, 43(5), 541-44
www.nci.gov.in