Synthesis and anticancer activities of proline-containing cyclic peptides and their linear analogs and congeners

Article abstract of DOI:10.1080/00397911.2018.1550201

A solution phase method was adopted for the synthesis of proline-containing cyclic pentapeptide 2 and total synthesis of naturally occurring cyclic heptapeptide Reniochalistatin B 3. For the synthesis of 3, both divergent and convergent strategies were used to improve the overall yield from 12 to 25%. Different N and C terminal modified linear analogs and congeners of 2 and 3 were synthesized. Both cyclic peptides 2 and 3 and their linear analogs/congeners were evaluated for anti-cancer activity against HeLa cell line, among which pentapeptide 2 h and hexapeptide 3n with N-terminal protected hexafluoroisopropyl carbamates (HFIPC) interestingly showed higher cytotoxicity with an IC₅₀ of 2.73 and 4.3 μM, respectively compared to their Boc-protected analogs 2a (IC₅₀ 20 μM) and 3c (IC₅₀ 38.51 μM) and cyclic peptides 2 (>100 μM) and 3 (47 μM). These results were further validated by biological experiments such as colony formation and wound healing assays.

Full text of DOI:10.1080/00397911.2018.1550201

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Synthesis and anticancer activities of proline-
containing cyclic peptides and their linear analogs
and congeners
Keshab Ch Ghosh, Indranil Duttagupta, Chandra Bose, Priyanjalee Banerjee,
Anuran Kumar Gayen & Surajit Sinha
To cite this article: Keshab Ch Ghosh, Indranil Duttagupta, Chandra Bose, Priyanjalee Banerjee,
Anuran Kumar Gayen & Surajit Sinha (2019): Synthesis and anticancer activities of proline-
containing cyclic peptides and their linear analogs and congeners, Synthetic Communications, DOI:
10.1080/00397911.2018.1550201
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SYNTHETIC COMMUNICATIONSV
https://doi.org/10.1080/00397911.2018.1550201
Synthesis and anticancer activities of proline-containing
cyclic peptides and their linear analogs and congeners
Keshab Ch Ghosha, Indranil Duttaguptaa, Chandra Bosea, Priyanjalee Banerjeea,
Anuran Kumar Gayenb, and Surajit Sinhaa
aDepartment of Organic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata,
b
India; Department of Chemistry, North Carolina State University, Raleigh, USA
ABSTRACT
ARTICLE HISTORY
Received 12 September 2018
A solution phase method was adopted for the synthesis of proline-
containing cyclic pentapeptide 2 and total synthesis of naturally
occurring cyclic heptapeptide Reniochalistatin B 3. For the synthesis
of 3, both divergent and convergent strategies were used to
improve the overall yield from 12 to 25%. Different N and C terminal
modified linear analogs and congeners of 2 and 3 were synthesized.
Both cyclic peptides 2 and 3 and their linear analogs/congeners
were evaluated for anti-cancer activity against HeLa cell line, among
which pentapeptide 2 h and hexapeptide 3n with N-terminal pro-
tected hexafluoroisopropyl carbamates (HFIPC) interestingly showed
higher cytotoxicity with an IC50 of 2.73 and 4.3 mM, respectively com-
pared to their Boc-protected analogs 2a (IC50 20 mM) and 3c (IC50
38.51 mM) and cyclic peptides 2 (>100 mM) and 3 (47 mM). These
results were further validated by biological experiments such as col-
ony formation and wound healing assays.
KEYWORDS
Anticancer activities; cell
migration efficiency; cyclic
peptides; linear peptides;
Reniochalistatin;
GRAPHICAL ABSTRACT
Introduction
Peptide-based drugs are known for being highly selective, efficient and at the same time
relatively safe, offering an alternative strategy in the field of rational drug design.[1,2]
CONTACT Surajit Sinha
ocss5@iacs.res.in
Department of Organic Chemistry, Indian Association for the
Cultivation of Science, Jadavpur, Kolkata, 700 032, India.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lsyc.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2019 Taylor & Francis Group, LLC

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K. CH. GHOSH ET AL.
Figure 1. Chemical structure of the cyclic peptides.
Peptide drug moieties, mainly cyclized forms and their derivatives, often lead to the
development of potential drug candidates than their linear counterparts and therefore
continue to seek the attention of the drug industry. Though naturally occurring linear
peptides as therapeutics are limited by their intrinsic weaknesses e.g. poor chemical,
physical and proteolytic stability, however, in some cases linear peptides are considered
better cancer targeting agents than their cyclic counterparts.[3] In general, cyclic peptides
have been broadly exploited over the past 20 years in medicine as active ingredients of
natural extracts (bacteria, fungi, plants, animal venoms). The lack of N and C termini
keeps it from enzymatic degradation and physical denaturation which enhances the bio-
availability.[4–6] Improved structural rigidity, receptor selectivity, biochemical stability
are their characteristics features. Cyclic peptides usually have little to no toxicity due to
their benign amino acid make-up.[7] Target specificity and extra stability makes them
one of the most promising drug candidates or biochemical tools, a few of them being
commercially available as drugs[4,8] e.g. bacitracin, daptomycin, cyclosporine, etc. Owing
to its remarkable properties and growing demand, scientists are in pursuit of novel
cyclic peptides and synthetic strategies for their production.[9–12]
In 2011, Wen Wu et al.[13] isolated a homodetic cyclic peptide 1 from the crude
extract of an endolichenic Xylaria sp, which has shown antifungal activity and we
describe here the synthesis of 2, an analog of 1 where D-amino acids have been
replaced by L-amino acids. In 2014, Kai-Xuan Zhan and et al.[14] isolated
Reniochalistatin B (3), a cyclic heptapeptide from a marine sponge, Reniochalina stalag-
mitis having antitumor activities (Figure 1). While working on this molecule (3), Xu
et al. have reported very recently the first total synthesis of 3 in solid phase method.[15]
Reniochalistatin E, a cyclic octapeptide which is another member of Reniochalistatins
family and its total synthesis was reported in both solid and solution phase methods.
Solution phase synthesis was accomplished by Rafferty et al. in 15 steps with an overall
5.0% yield.[16] The best possible cyclization yield 15% was achieved in macrolactamiza-
tion step when EDC/HOBt was used. On the other hand, Liang et al. have reported
solid phase synthesis of Reniochalistatin E and its conformational isomers with 32%
overall yield.[17] Reniochalistatin E showed cytotoxicity against HeLa cell line with IC50
of 16.9 mM ( 1.9).[16] Fully deprotected linear form of Reniochalistatin E has also
shown cytotoxicity with an almost equal potency having IC50 of 9.5 2.1 mM in U937
cell line.[16] Herein, we report the second total synthesis of Reniochalistatin B (3) using
solution phase method which can be used for large-scale synthesis. In this direction,

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3
Scheme 1. Retrosynthetic analysis of the target cyclic peptide 2.
cyclic peptides 2, 3 and their several linear analogs and congeners have also been used
to evaluate their anticancer activities.
Results and discussion
Chemistry
To achieve the formal synthesis of the desired cyclic peptide 2, we started with cheap
and easily available L- amino acids. Accordingly, the D-isoleucine and D-leucine in 1
were replaced by their L- isomers. The retrosynthetic analysis of 2 was outlined in
Scheme 1. Cyclic peptide was obtained by macrolactamization of linear pentapeptide 2a.
L-Valine was disconnected at the C- terminal to get linear tetrapeptide 2b. Further dis-
connection at the N-terminal and C-terminal gave tripeptide 2c and dipeptide 2d,
respectively. These peptides can be synthesized by stepwise peptide synthesis method
starting from L-proline and L-leucine, respectively (Scheme 1).
The forward synthesis commenced with the condensation of L-proline methyl ester
[18]
hydrochloride with Fmoc-L-Leu-OH to yield the linear dipeptide
2d in 95% yield
which was purified by a flash column chromatography and used in the next step. The
methyl ester of dipeptide 2d was then hydrolyzed under buffered conditions in order to
[19,20]
keep the Fmoc intact. LiOH.H2O mediated hydrolysis in presence of excess CaCl2
afforded the desired product. The corresponding free acid was subsequently treated
with L-leu-OMe.HCl to give the tripeptide 2c in 79% yield. The Fmoc group in the lin-
ear tripeptide 2c was deprotected using 20% piperidine in DMF to yield the free amine
which was then subjected to another coupling with N-Boc-L-Ile yielded the linear tetra-
peptide 2b in 81% yield. Methyl ester of tetrapeptide 2b was hydrolyzed using LiOH,
following another coupling with L-Val-OMe yielded the desired linear pentapeptide ana-
log 2a of 2 in 73% yield. The linear precursor 2a was then treated with LiOH to yield
the corresponding free carboxylic acid. The crude carboxylic acid was then treated with
20% TFA in DCM to yield the NBoc deprotected compound. The deprotected com-
pound underwent an intramolecular condensation in presence of NaHCO3 and BOP in

4
K. CH. GHOSH ET AL.
Scheme 2. Synthesis of cyclic peptide 2. Reagents and conditions: (a) DIPEA, Fmoc-L-Leu-OH, HOBt,
i
EDC, DCM, 12 h, 95%; (b) (i) LiOH, CaCl2, PrOH/THF/H2O (10:3:4), 1 h; (ii) L-Leu-OMe.HCl, DIPEA, EDC,
HOBt, DCM, 12 h, 79%; (c) (i) 20% Piperidine in DMF; (ii) Boc-L-Ile-OH, EDC, HOBT, DCM, 12 h, 81%;
(d) (i) LiOH, THF/H2O/MeOH (10:4:1), 1 h; (ii) L-Val-OMe.HCl, DIPEA, EDC, HOBT, DCM, 12 h, 73%; e) (i).
LiOH, THF/H2O/MeOH (10:4:1), 1 h; (ii) TFA (20%) in DCM; (iii) NaHCO3, BOP, DMF, RT, 14 h, 61%.
Scheme 3. Retrosynthetic analysis of the naturally occurring cyclic peptide 3.
DMF[21] to yield the cyclic peptide 2 in 61% yield (Scheme 2). The overall yield of 2 in
the five steps was 27%.
The retrosynthetic analysis of Reniochalistatin B (3) has been shown in scheme 3.
Cyclic peptide can be obtained by macrolactamization of linear heptapeptide 3a
or 3b3b. Linear peptide 3a and 3b can be synthesized from three major fragments 3c,

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Scheme 4. Synthesis of linear heptapeptide presursor 3a. Reagents and conditions: (a) L-Pro-OMe.HCl,
DIPEA, HOBt, EDC, DCM, 12 h, 85%; (b) (i) LiOH, THF/H2O/MeOH (10:4:1), 1 h; (ii) L-Ile-OMe.HCl, DIPEA,
HOBt, EDC, DCM, 12 h, 62%; (c) (i) LiOH, THF/H2O/MeOH (10:4:1), 1 h; (ii) L-Phe-OMe.HCl, DIPEA, HOBt,
EDC, DCM, 12 h, 92%; (d) (i) LiOH, THF/H2O/MeOH (10:4:1), 1 h; (ii) HOBt, EDC, DCM, 12 h, 64%.
3d, and 3e. Other amino acids are easily recognized as L-Tyrosine, L-Phenylalanine, L-
Isoleucoine, and L-proline. Tripeptide 3e is same as 2c except the change in terminal
protections, hence 3e was synthesized following the procedure of 2c as described in
Scheme 2 (SI Scheme S1).
Synthesis of the cyclic peptide 3 commenced with the coupling of tripeptide 3e and
L-Pro-OMe.HCl to yield the protected tetrapeptide 3d. The methyl ester deprotection of
3d with LiOH followed by EDC coupling with L-Ile-OMe.HCl resulted in the pentapep-
tide 3f. Repeating the sequence of methyl ester hydrolysis and subsequent couplings
with L-Phe-OMe.HCl and L-Tyr-OtBu (the Fmoc group in L-Fmoc-Tyr-OtBu was
deprotected using 20% piperidine in DMF) prior to coupling with 3c yielded the desired
linear heptapeptide 3a, the precursor of cyclic peptide 3 (Scheme 4). The notable drop
in yields during the synthesis of 3f and 3a, compelled us to adopt a convergent route
for the total synthesis of Reniochalistatin B (3). The linear precursor heptapeptide was
synthesized using the convergent route described in Scheme 5.
In this case, heptapeptide precursor 3b was prepared in methyl ester form instead of
tert-butyl ester 3a because of cheap and easily available of L-Tyr-OMe than L-Tyr-OtBu.
Moreover, preparation of tert-butyl ester from Fmoc-L-Tyr-OH was not clean to yield
Fmoc-L-Tyr- OtBu in 40% yield (SI Scheme S2).

6
K. CH. GHOSH ET AL.
Scheme 5. Synthesis of linear tripeptide 3 h and heptapeptide 3b. Reagents and conditions: (a) L-
Phe-OMe.HCl, DIPEA, HOBt, DCC, DCM, 12 h, 88%; (b) (i) LiOH, THF/H2O/MeOH, (10:4:1), 1 h; (ii) L-Tyr-
OMe.HCl, DIPEA, HOBt, EDC, DCM, 12 h, 96%; (c) LiOH, THF/H2O/MeOH, (10:4:1), 1 h; d) TFA (20%) in
DCM; (e) DIPEA, HOBt, EDC, DCM, 88%.
Accordingly, 3b was prepared from 3d and 3h (Scheme 5). Tripeptide 3h was prepared as
follows. DCC-mediated coupling of Boc protected L-isoleucine with L-phenylalanine methyl
ester hydrochloride to give the dipeptide 3g. Methyl ester hydrolysis of 3g using LiOH followed
by coupling with L-Tyr-OMe.HCl in presence of HOBt/EDC the linear tripeptide 3 h was
obtained in 84% overall yield. The amine generated by TFA mediated Boc deprotection of tri-
peptide 3h was subjected to coupling with free acid obtained by methyl ester hydrolysis of tet-
rapeptide 3d to provide the linear heptapeptide precursor 3b (Scheme 5).
Cyclization of 3a or 3b to achieve 3
The Boc group and tert-butyl ester group of linear heptapeptide 3a were simultaneously
deprotected by treating with 20% TFA in DCM. Marcolactamization of the deprotected
compound is done in presence of NaHCO3 and BOP in DMF[21] to yield the cyclic pep-
tide 3 in 39% yield (Scheme 6). Overall yield was 12% from 3e.
On the other hand, the linear precursor 3b was treated with LiOH to yield the corre-
sponding free carboxylic acid. The crude carboxylic acid is then treated with 20% TFA
in DCM to yield the Boc deprotected compound. The fully deprotected compound after
macrolactamization yields the cyclic peptide 3 in 40% yield (Scheme 6). Overall yield
was 25% from 3e.
Synthesis of linear analogs of 2
After first phase screening of 2 and its four linear analogs such as 2a, 2e, 2f and 2g,
compound 2a was found to be the most potent with an IC50 value of 20 mM and

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7
realized that the N-terminal protection has a role in biological activities, hence N-ter-
minal tert-Boc of 2a was replaced by hexafluoroisopropyl carbamate (HFIPC) to obtain
the peptide 2h (Scheme 7). Hexafluoroisopropyl carbamate is chosen because fluorine is
known to improve the lipophilic character, cell-permeability and pharmacokinetics of a
Scheme 6. Synthesis of Reniochalistatin B (3). Reagents and conditions: (a) (i) TFA (20%) in DCM, (ii)
NaHCO3, BOP, DMF, RT, 14 h, 39%; (b) (i) LiOH, THF/H2O/MeOH, (10:4:1): then (iii) TFA (20%) in DCM,
then cyclization using (ii), 40%.
Scheme 7. Synthesis of analogs of 2. Reagents and conditions: (a) TFA (20%) in DCM, 90%; b) tri-
sphosgene, Pyridine, HFIP, Et3N, 15 h, 40%; (c) LiOH, THF/H2O/MeOH, (10:4:1), 1 h, 92%; (d) TFA (20%)
in DCM, 95%.

8
K. CH. GHOSH ET AL.
Scheme 8. Reagents and conditions: (a) LiOH, THF/H2O/MeOH, (10:4:1), 1 h, 94%; (b). TFA (20%) in
DCM, 91%; (c) TFA (20%) in DCM, 97%; (d) Trisphosgene, Pyridine, HFIP, Et3N, 15 h, 62%; e) AllocCl,
Pyridine, 0 ꢀC, 16 h, 87%; (f) LiOH, THF/H2O/MeOH, (10:4:1), 1 h, 95%; (g) Fmoc- L-Tyr-OtBu, Piperidine,
HOBt, EDC, DCM,12h, 78%.
medicinal compound[22–25] HFIPC protected 2h was obtained by reaction of 2e with tri-
sphosgene and hexafluoroisopropanol (HFIP) in presence of pyridine.[26,27]
Synthesis of linear analogs and congeners of 3
Similarly, several linear heptapetide analogs and other short peptides (congeners) were
synthesized based on cyclic peptide 3.
Alloc-protected (Alloc-NH-Leu-Pro-Leu-Pro-OMe) 3i and (Alloc-NH-Leu-Pro-Leu-
Pro-Ile-OMe) 3j were synthesized from (Boc-NH-Leu-Pro-Leu-Pro-OMe) 3d and (Boc-
NH-Leu-Pro-Leu-Pro-Ile-OMe) 3f, respectively using literature procedure[28] (SI
Schemes S3 and S4).
In order to know the role of N-terminal and C-terminals in anticancer activities, sev-
eral linear hexapeptides and heptapeptide were synthesized. Methyl ester hydrolysis of
the linear hexapeptide 3c using LiOH yielded the corresponding free carboxylic acid 3k.
The free acid was then treated with 20% TFA in DCM to afford fully deprotected linear
hexapeptide 3 l. Again Boc group of the linear peptide 3c was deprotected by 20% TFA
in DCM to afford the free N-terminal compound 3m. The compound 3m on reaction
with trisphosgene and hexafluoroisopropanol in presence of pyridine yielded hexafluor-
oisopropyl carbamate[26,27] derivative 3n.
N-Alloc derivatives of linear hexapeptide 3c and heptapeptide 3a were also synthe-
sized. Alloc carbamate 3o was synthesized by reacting free N-terminal compound 3m
with Alloc chloride and pyridine. Methyl ester hydrolysis of N-Alloc protected hexapep-
tide 3o on treatment with LiOH yielded free acid 3p. Coupling of carboxylic acid 3p
with L-Tyr-OtBu (obtained by Fmoc deprotection of L-Fmoc-Tyr-OtBu using 20%
piperidine in DMF) in presence of HOBt/EDC afforded the heptapeptide 3q
(Scheme 8).

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9
Table 1. IC50 values of the cyclic and linear peptides against HeLa cells.
Sl. No.
Compound name
Sequence
IC50 (mM)
1
2
3
4
2
2a
2e
2f
c-(Ile-Leu-Pro-Leu-Val)
Boc-NH-Ile-Leu-Pro-Leu-Val-OMe
NH2-Ile-Leu-Pro-Leu-Val-OMe
>100
20 0.11
>60
Boc-NH-Ile-Leu-Pro-Leu-Val-OH
>60
5
6
7
8
2g
2h
3
3a
3b
3c
3d
3f
NH2-Ile-Leu-Pro-Leu-Val-OH
HFIP-NH-Ile-Leu-Pro-Leu-Val-OMe
c-(Leu-Pro-Leu-Pro-Ile-Phe-Tyr)
Boc-NH-Leu-Pro-Leu-Pro-Ile-Phe-Tyr-OtBu
Boc-NH-Leu-Pro-Leu-Pro-Ile-Phe-Tyr-OMe
Boc-NH-Leu-Pro-Leu-Pro-Ile-Phe-OMe
Boc-NH-Leu-Pro-Leu-Pro-OMe
Boc-NH-Leu-Pro-Leu-Pro-Ile-OMe
Alloc-NH-Leu-Pro-Leu-Pro-OMe
Alloc-NH-Leu-Pro-Leu-Pro-Ile-OMe
Boc-NH-Leu-Pro-Leu-Pro-Ile-Phe-OH
TFA.NH2-Leu-Pro-Leu-Pro-Ile-Phe-OH
TFA.NH2-Leu-Pro-Leu-Pro-Ile-Phe-OMe
HFIP-NH-Leu-Pro-Leu-Pro-Ile-Phe-OMe
Alloc-NH-Leu-Pro-Leu-Pro-Ile-Phe-OMe
Alloc-NH-Leu-Pro-Leu-Pro-Ile-Phe-OH
Alloc-NH-Leu-Pro-Leu-Pro-Ile-Phe-Tyr-OtBu
>60
2.73 0.26
47.17 0.39
>100
50.63 0.48
38.51 0.26
>100
50.79 0.19
>100
51.52 0.38
>60
>60
>60
4.3 0.19
22.59 0.17
>60
9
10
11
12
13
14
15
16
17
18
19
20
21
3i
3j
3k
3l
3m
3n
3o
3p
3q
>100
The bold values highlight the best results obtained for IC50.
Biological activity
Though, cyclic peptide 1 is known to have anti-fungal activity however, the anticancer activ-
ity of its analog 2 has been evaluated as some antifungal compounds have been found to be
potential anti-cancer agents in literature.[29–32] Accordingly, the anti-cancer activity of both
the compounds 2 and 3 and their linear analogs was investigated against HeLa cells (cervical
cancer cell line) by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
method. The experiment was repeated in triplicates for each compound. From the prelimin-
ary evaluation, it was found that cyclic peptide 2 was almost inactive up to 100 mM dose
whereas its linear analog pentapeptide 2a (Boc-NH-Ile-Leu-Pro-Leu-Val-OMe) was active
with an inhibitory concentration (IC50) of 20 mM. Neither partially or fully deprotected pen-
tapeptides 2e, 2f and 2g showed any cytotoxicity with the cell survivability of up to 80–90%
(Table 1, Figure S1). In the case of 3 and its linear peptides, it was found that hexapeptides
3c (Boc-NH-Leu-Pro-Leu-Pro-Ile-Phe-OMe) and 3o (Alloc-NH-Leu-Pro-Leu-Pro-Ile-Phe-
OMe) became more cytotoxic than 3 against the cancer cells with IC50ꢁ of 38.51 0.26 mM
(3c), 22.59 0.17 mM (3o) and 47.17 0.39 mM (3), respectively (Table 1, and Figure S2).
When Boc protection was changed to alloc protection, then 3o became more potent than 3c.
In compare to earlier report,[16] Reniochalistatin B was much more less potent than
Reniochalistatin E. Similarly, in the case of free amine or carboxyl terminal or fully depro-
tected linear peptides namely 3k, 3 l, 3 m and 3p became less potent (IC50 60 mM or even
more) than the linear form of Reniochalistatin E. Neither heptapeptides (3a, 3b, 3q) nor
tetra (3d, 3i) and pentapeptides (3f, 3j) were active even at 50 mM dose.
Hence, based on the preliminary screening of cytotoxic potential, modification at the
N-terminal of 2a and 3o were made with the incorporation of hexafluoroisopropyl car-
bamate (HFIPC) moiety (compounds 2h and 3n), which is expected to have higher cell
penetrating property because of fluorine as described previously.[22–25] Interestingly,
these new compounds 2h and 3n were found to be promising and showed strikingly
higher cytotoxicity against HeLa cell line with remarkably low IC50 values of 2.73 0.26

10
K. CH. GHOSH ET AL.
Figure 2. Cell viability of compounds 2h, 3n and 3o against HeLa cells. Cells were treated with the
compounds at the indicated concentrations for 48 h.
Figure 3. Colony formation of HeLa cells after treatment with compounds 2h (5 mM) and 3n (8 mM).
A representative figure from triplicate experiments is shown.
mM and 4.3 0.19 mM, (Table 1 and Figure 2) respectively. Compared to the parent
compounds 2a and 3o, these two compounds were thus found to be having 7.3 and
5.25 times more activity, respectively.
Colony formation assay
On the basis of the IC50 values found in the cytotoxic assay of the compounds, the col-
ony formation ability[33] of HeLa cells in presence of compounds 2h and 3n was
checked at double concentration of 5 mM and 8 mM, respectively. With respect to the
control, lesser colonies were formed in 2h and 3n as envisioned (Figure 3).
Wound healing assay
Similarly, based on IC50 values evident in the cytotoxic assay of the compounds, the
migration ability[34] of the HeLa cells were tested in presence of compounds 2h and 3n

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11
Figure 4. Wound healing assay in HeLa cells treated with Compounds 2h (5 mM) and 3n (8 mM) with
respect to control.
at concentrations of 5 mM and 8 mM, respectively. Here also the migration of HeLa cells
in both the cases was found to be lesser with respect to control (Figure 4).
Conclusion
In conclusion, we have developed concise routes for the synthesis of cyclic peptides 2
and total synthesis of Reniochalistatin B (3). The low yielding linear route for the syn-
thesis of Reniochalistatin B (3) was replaced by a high yielding convergent route.
Though conventionally, cyclization of peptides confers better therapeutic efficacies by
enhancing selective receptor binding due to constrained geometry, improved stability,
etc, however, cyclization cannot always ensure to produce an improved version of its
linear counterpart. In certain instances, the biological activities of the cyclic and linear
conjugates may be equivalent and even linear peptides with artificial amino acids substi-
tution, hybridization, N- or C- terminal modification can achieve desired rigid conform-
ation and could be more potent compared to its cyclized forms. In this direction, the
linear peptides synthesized in this study have shown more cytotoxicity against HeLa cell
lines owing to the attachment of different groups perhaps due to the better cell-pene-
trating capacity. Interestingly, N-terminal protected with hexafluoroisopropyl carbamate
became more potent and showed IC50 values as low as 2.73 0.26 (2h) and 4.3 0.19
mM (3n). The linear peptide 3n is a new finding which has not been disclosed ear-
lier.[15] Even the colony formation ability and migration property of HeLa cells were
significantly inhibited by these linear analogs which strongly supports their candidature
as a potential therapeutic anticancer agent. In summary, here we report total syntheses
of 2 and 3, several analogs and congeners of cyclic peptides and evaluated their respect-
ive action as anti-cancer agents in in-vitro cell culture model. Further mechanistic inves-
tigation of these peptides is under study and their therapeutic potency in cancer

12
K. CH. GHOSH ET AL.
treatment needs to be further validated in suitable animal models followed by clin-
ical trials.
Experimental section
All reagents were purchased from commercial sources and used without further purifica-
tion, unless otherwise stated. Petroleum ether (PE) refers to the fraction of petroleum boil-
ꢀ
ing between 60–80 C. The following abbreviations are used for DCM = dichloromethane,
DIPEA = N,N-diisopropylethylamine, MeOH = methanol, TFA = trifluoroacetic acid, EDC
=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, HOBt =1-hydroxybenzo-
triazole, DMF = dimethylformamide, EtOAc = ethyl acetate, THF = tetrahydrofuran, BOP
= (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, HFIP =
hexafluoroisopropanol, AllocCl = allyl chloroformate. All reactions were carried out in
oven-dried glassware under an argon atmosphere using anhydrous solvents, standard syr-
inge and septum techniques unless otherwise indicated. Organic extracts were dried over
anhydrous Na2SO4 and then filtered prior to removal of all volatiles under reduced pressure
on rotary evaporation. Chromatographic purification of products was accomplished using
column chromatography on silica gels (mesh 100 ꢁ 200). Thin-layer chromatography
(TLC) was carried out on aluminum sheets, Silica Gel 60 F254 (Merck; layer thickness 0.25
mm). Visualization of the developed chromatogram was performed by UV light and/or
Ceric Ammonium Molybdate (CAM), ninhydrin stains. Optical rotations were measured
25
using a sodium (589, D line) lamp and are reported as follows: [a]D
(c (mg/100 mL),
solvent) H and 13C NMR spectra were recorded at 300 or 400 MHz and 75 or 100 MHz,
respectively using CDCl3 or MeOH-d4 or DMSO-d6 as solvent. Chemical shifts (d) are
given in ppm relative to the solvent residual peak or TMS as internal standard. The follow-
ing abbreviations are used for multiplicity of NMR signals: s = singlet, d = doublet, t = trip-
let, m = multiplet, br = broad. High Resolution Mass Spectra (HRMS) were measured in a
QTOF I (quadrupole-hexapole-TOF) mass spectrometer with an orthogonal Z-spray-elec-
trospray interface on Micro (YA-263) mass spectrometer (Manchester, UK).
1
(3S,6S,9S,12S,17aS)-9-((S)-sec-Butyl)-3,12-diisobutyl-6-isopropyldodecahydro-1H-
pyrrolo[1,2-a][1,4,7,10,13]pentaazacyclopentadecine-1,4,7,10,13-pentaone (2)
(24 mg, 0.036 mmol) was taken in THF: MeOH (1 ml: 0.1 ml) mixture. A solution of
LiOH.H2O (15 mg, 0.36 mmol) in 0.3 ml H2O was added dropwise to attain a molarity
of 1.5 M. The reaction mixture was stirred for 30 min. The solvent was removed, acidi-
ꢀ
fied to pH 4 at 0 C, extracted with ethyl acetate. The organic layer was washed with
water and brine to yield the carboxylic acid as white foamy solid. The crude product so
ꢀ
obtained was dissolved in 20% TFA (0.4 ml) in DCM (1.6 ml) at 0 C and stirred for 2
h. Removal of DCM in vacuo yielded a brownish viscous liquid which was used in the
next step without further purification.
The brownish solid obtained above (21 mg, 0.031 mmol) was dissolved in dry DMF
(32 ml) to attain a concentration of 1 mM. To it NaHCO3 (13 mg, 0.15 mmol) was
added followed by the addition of BOP (20 mg, 0.05 mmol) at room temperature. The
reaction mixture was then stirred overnight at rt. Removal of DMF followed by a flash

R
SYNTHETIC COMMUNICATIONSV
13
column chromatography (5% MeOH-DCM) yielded the desired cyclic peptide 2 as
1
ꢀ
white solid (10 mg, 0.019 mmol) in 61% yield. M.p. 119–121 C; H NMR (300 MHz,
DMSO-d6) d = 8.87 (d, J = 8.7 Hz, 1H), 7.75 (s, 1H), 7.39 (d, J = 9.9 Hz, 1H), 7.07 (d, J
= 7.8 Hz, 1H), 4.46 (m, 1H), 4.28 (d, J = 7.2 Hz, 1H), 4.04–3.98 (m, 2H), 3.84 (t, J =
9.6 Hz, 1H), 3.57– 3.40 (m, 2H), 2.27–2.18 (m, 1H), 2.15–1.99 (m, 2H), 1.86 (br s, 2H),
1.77– 1.70 (m, 2H), 1.55 - 1.30 (m, 7H), 1.10–1.01 (m, 2H), 0.88–0.82 (m, 22H) ppm;
13C NMR (100 MHz, CDCl3:CD3OD (9:1)) d = 172.4, 172.2, 171.7, 171.6, 171.1, 61.7,
59.7, 59.6, 51.7, 51.1, 47.0, 41.9, 41.4, 35.1, 32.2, 26.7, 25.2, 25.0, 24.7, 23.2,22.7, 22.3,
22.2, 22.1, 19.6, 18.5, 15.4, 10.5 ppm; HRMS (ESI): m/z [M þ Na]þ calcd for
C28H49N5O5Na: 558.3632, found 558.3634.
(S)-Methyl 2-((S)-2-((2S,3S)-2-((S)-1-((S)-2-((S)-1-((S)-2-((tert-butoxycarbonyl)amino)-
4-methylpentanoyl)pyrrolidine-2-carboxamido)-4-methylpentanoyl)pyrrolidine-2-
carboxamido)-3-methylpentanamido)-3-phenylpropanamido)-3-(4-hydroxyphenyl)
propanoate (3b)
The compound 3d (202 mg, 0.365 mmol) was taken with THF (5.5 ml) and MeOH (0.6
ml). A solution of LiOH.H2O (154 mg, 3.655 mmol) in H2O (2.4 ml) was added drop-
wise ultimately attaining a molarity of 1.5 M. The reaction ꢀmixture was stirred for about
30 min. The solvent was removed, acidified to pH 4 at 0 C, extracted with ethyl acet-
ate. The organic layer was washed with water and brine to yield the acid (197 mg, 0.365
mmol) in quantitative yield.
The compound 3h (203 mg, 0.365 mmol) was dissolved in dry DCM (4.8 ml) and 1.2
ꢀ
ml TFA was added at 0 C and stirred for 2 h. Removal of DCM in vacuo yielded a
brownish viscous liquid which was used in the next step without further purification.
DIPEA (223 mL, 1.28 mmol) was added dropwise to a stirred suspension of free
amine-TFA salt (0.365 mmol) in dichloromethane (16 mL) at room temperature under
ꢀ
nitrogen. On dissolution, the solution was cooled to 0 C and then the acid obtained
above (197 mg, 0.365 mmol) and 1-hydroxybenzotriazole (55 mg, 0.402 mmol) were
ꢀ
added successively, each in one portion. The suspension was stirred at 0 C for a further
15 min, and then EDC (78 mg, 0.402 mmol) was added in one portion. The mixture
was allowed to warm to room temperature over the course of 12 h, and the solvent was
then evaporated in vacuo. The residue was taken up in ethyl acetate and washed with
cold 0.1 N HCl followed by saturated aqueous sodium bicarbonate solution. The com-
bined organic extracts were dried and evaporated in vacuo to leave the crude product
which was purified by chromatography on silica using 3% MeOH-DCM as eluentꢀ to
give the heptapeptide 3b (312 mg, 0.319 mmol, 88%) as a white solid. M.p. 132–135 C;
1H NMR (300 MHz, CDCl3) d = 8.09 (s, 1H), 7.67 (s, 1H), 7.18–7.14 (m, 6H), 6.88 (d,
J = 8.1 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H), 5.47 (s, 1H), 4.79–4.69 (m, 4H), 4.54–4.46 (m,
2H), 4.28 (s, 1H), 3.77–3.74 (m, 2H), 3.64–3.57 (m, 5H), 3.24 (m, 1H), 2.94–2.86 (m,
3H), 2.53 (s, 1H), 2.19–1.90 (m, 8H), 1.74–1.62 (m, 3H), 1.56–1.32 (m, 13H), 1.09 (s,
1H), 0.95–0.86 (m, 13H), 0.75–0.66 (m, 6H) ppm; 13C NMR (75 MHz, CDCl3) d =
173.4, 172.6, 172.0, 171.7, 171.4, 171.1, 170.9, 156.0, 155.9, 136.9, 130.5, 129.4, 129.4,
128.5, 126.9, 126.8, 115.6, 79.8, 60.5, 59.8, 58.4, 54.2, 53.6, 52.3, 50.4, 49.4, 47.7, 47.4,
42.2, 41.6, 37.7, 37.2, 36.6, 28.5, 28.0, 27.6, 25.3, 24.6, 24.5, 24.3, 23.5, 23.4, 21.9, 21.7,

14
K. CH. GHOSH ET AL.
25
15.6, 11.4 ppm; [a]D = ꢂ54.2 (c 1.44, CHCl3); HRMS (ESI): m/z [M þ Na]þ calcd for
C52H77N7O11Na 998.5579; found 998.5577.
(S)-Methyl 2-((S)-2-((S)-1-((S)-2-((2S,3S)-2-((1,1,1,3,3,3-hexafluoropropan-2-
yloxy)carbonylamino)-3-methylpentanamido)-4-methylpentanoyl)pyrrolidine-2-
carboxamido)-4-methylpentanamido)-3-methylbutanoate (2h)
Triphosgene (96 mg, 0.323 mmol) was added portion wise to the solution of hexafluoroiso-
propanol (HFIP) (69 mL, 0.647 mmol) in dry DCM (3 mL) at 0 ꢀC. Then pyridine (52 mL,
0.647 mmol) was added dropwise and the reaction mixture was stirred at rt for 3.5 h. Et3N
ꢀ
(136 mL, 0.97 mmol) was added slowly to reaction mixture at 0 C and solution became
red. A solution of compound 2e (73.5 mg, 0.108 mmol) was added via cannula and reac-
tion mixture was stirred for 12 h. Excess phosgene was removed by purging Argon and the
solvent was then evaporated in vacuo. The residue was taken up in ethyl acetate and
washed with water followed by brine solution. The combined organic extracts were dried
and evaporated in vacuo to leave the crude product which was purified by chromatography
on silica using 3% MeOH-DCM as eluent to give the pentapeptide 2 h (33 mg, 0.043
mmol, 40%) as a white solid. M.p. 88–90 ꢀC; 1H NMR (300 MHz, CDCl3): d = 7.01 (d, J =
7.2 Hz, 1H), 6.60 (d, J = 8.7 Hz, 1H), 5.82 (td, J = 11.7, 5.7 Hz, 1H), 4.88 (dd, J = 12.1, 3.6
Hz, 1H), 4.56 (dd, J = 8.1, 2.7 Hz, 1H), 4.51–4.46 (m, 2H), 4.31 (m, 1H), 3.71 (s, 3H), 3.59
(t, J = 7.2 Hz, 2H), 2.74 (t, J = 11.4 Hz, 1H), 2.34 (m, 1H), 2.23–2.11 (m, 3H), 2.00(m, 1H),
1.89 (m, 1H), 1.80–1.71 (m, 2H), 1.61–1.41 (m, 6H), 1.02–0.86 (m, 24H) ppm; 13C NMR
(75 MHz, CDCl3): d = 172.4, 171.8, 171.0, 169.0, 151.1, 146.7, 68.4 (q, J = 36 Hz), 62.4,
60.0, 57.2, 53.4, 52.4, 52.2, 47.5, 40.2, 36.6, 35.4, 31.4, 29.8, 26.9, 25.6, 25.5, 25.2, 24.6, 23.2,
21.6, 20.9, 19.0, 17.8, 13.1, 11.8 ppm; HRMS (ESI): m/z [M þ Na]þ calcd for
C33H53N5O8F6Na: 784.3696; found: 784.3698.
Anti-proliferative assay
The anticancer activities of the cyclic peptides, their analogs and congeners were
screened against HeLa cells. They were grown to log phase in 10% fetal bovine serum,
were seeded in 96 well plates at a density of 1.5 ꢃ 104 and grown to a confluency of
ꢀ
60% at 37 C in a 5% CO2 incubator. After 24 hrs, the compounds were added in their
ꢀ
respective doses to 96- well plates in 0.5% FBS media and cultured at 37 C for 48 h.
The MTT solution was prepared in 0.5% FBS media (at a concentration of 1 mꢀg/mL)
and 100 mL of MTT solution was added to each well and incubated for 4 h at 37 C for
violet crystal formation. The suspension was then discarded and 100 mL of dimethyl
sulfoxide (DMSO) was added to each well and the plates were shaken for 30 mins to
dissolve the dark violet formazan crystals. The absorbance was measured at 570 nm
wavelength using a microplate reader. The analysis was repeated for three times with
reproducible results. The IC50 values were calculated from the graphs.
Acknowledgment
K.C.G. is thankful to CSIR, New Delhi for his fellowship. C.B. is grateful to DST-WOS A for her
fellowship. P.B. thanks DBT-RA for financial support.

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SYNTHETIC COMMUNICATIONSV
15
Funding
S.S. thanks Indo-French Centre for promotion of advanced research (CEFIPRA) for financial sup-
port by a grant [IFC/A/5105-2/2014/1060].
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