Design and synthesis of a new class of cryptophycins based tubulin inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.11.068

Tubulin binding compounds represent one of the most attractive targets for anticancer drug development. They broadly fall into two categories viz., tubulin polymerization inhibitors, which block microtubule growth and destabilize microtubules like vinca alkaloids and cryptophycins, and the others, which polymerize microtubules into hyperstable forms represented by family of taxanes. In this context, we aimed at design and synthesis of cryptophycins based macrocyclic depsipeptides, which are synthetically more accessible, however have the basic information to target tubulins and establish structure activity relationship (SAR). Thus, a new class of cryptophycins based marocyclic depsipeptides with a truncated epoxide chain were synthesized as potential tubulin inhibitors. The resultant lead analogues 15a and 16a exhibited good anti-cancer activity, induced apoptosis, caused block/delay in cell cycle as well as significantly reduced the expression of α- and β-tubulins. Molecular modelling studies show that 15a and 16a bind in the same domain as that of cryptophycins.

Full text of DOI:10.1016/j.ejmech.2014.11.068

European Journal of Medicinal Chemistry 93 (2015) 55e63
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of a new class of cryptophycins based tubulin
inhibitors
,
,
Arvind Kumar a b, Manjeet Kumar a b, Simmi Sharma b, Santosh Kumar Guru c,
Shashi Bhushan c, Bhahwal Ali Shah a
, b, *
a Academy of Scientific and Innovative Research (AcSIR), India
b Natural Product Microbes, CSIR-Indian Institute of Integrative Medicine Jammu, J & K, 180001, India
c Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine Jammu, J & K, 180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Tubulin binding compounds represent one of the most attractive targets for anticancer drug develop-
ment. They broadly fall into two categories viz., tubulin polymerization inhibitors, which block micro-
tubule growth and destabilize microtubules like vinca alkaloids and cryptophycins, and the others, which
polymerize microtubules into hyperstable forms represented by family of taxanes. In this context, we
aimed at design and synthesis of cryptophycins based macrocyclic depsipeptides, which are synthetically
more accessible, however have the basic information to target tubulins and establish structure activity
relationship (SAR). Thus, a new class of cryptophycins based marocyclic depsipeptides with a truncated
epoxide chain were synthesized as potential tubulin inhibitors. The resultant lead analogues 15a and 16a
exhibited good anti-cancer activity, induced apoptosis, caused block/delay in cell cycle as well as
Received 9 October 2014
Received in revised form
10 November 2014
Accepted 30 November 2014
Available online 28 January 2015
Keywords:
Asymmetric aldol
Cryptophycins
Macrocyclic depsipeptides
Anti-cancer
significantly reduced the expression of
a- and b-tubulins. Molecular modelling studies show that 15a and
16a bind in the same domain as that of cryptophycins.
Tubulin inhibitors
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
way higher than the parent analogues having IC50 values as low as
10 pM. This implies that reducing the toxicities of these molecules
is desirable; even if it means they have high IC50 values, as such
molecules might be better option for targeted drug delivery and
may well decipher new insights into their mechanism of action.
Thus, in our continued efforts to develop novel anti-cancer leads
[11,12], we initiated a program directed toward the synthesis of
macrocylic depsipeptides based on cryptophycins with an aim to
find minimal structure for tubulin inhibition and hence, establish
structure activity relationship (SAR). Though the SAR [5] of cryp-
tophycins is largely known, but still to the best of our knowledge
none of the studies have so far reported analogues with a truncated
epoxide chain. However, the importance of epoxide moiety has
been established by comparing its activity with analogues having
aziridine, thiirane, olefin, chlorohydrin and dihydorxyl moieties
instead [5]. Thus, we proposed two structural variants 1 and 2 sans
epoxide chain. The structure 1 has a shorter carbon side chain
(pruned epoxide moiety), whereas, 2 in addition has variable ste-
reochemistry at C-10, to understand its effect on tubulin inhibition
Cryptophycins, a group of macrocyclic depsipeptides produced
by cyanobacteria of the genus Nostoc [1], like colchicine, com-
bretastatins and vinca alkaloids inhibit the polymerization of
tubulin by blocking microtubule growth and destabilizing them [2].
They are the most potent compounds known in the dynamics of
tubulin and display remarkable cytotoxicity against multi drug
resistance cancer cell lines. They had been a subject of extensive
research in past, prompting myriad total synthesis [3,4] and
structural modifications [5]. The result of these efforts was the
development of cryptophycin-52 (C-52), a synthetic analogue of
cryptophycins, which reached phase 2 clinical trials [6,7]. Studies
continued to develop new cryptophycin analogues through syn-
thetic or biotechnological methods [8,9] and recently Sanofi-
eAventis published two patents on cryptophycin antibody-drug
conjugates (ADCs) for treatment of lung cancer [10]. Notably, the
cryptophycins-ADCs had IC50 value as high as 0.710 nM, which is
as naturally occurring cryptophycin-46 has L-tyrosine at C-10 po-
sition (Fig. 1). Retrosynthetic disconnection of 1 or 2 shows that
they can be assembled from ester 7 (northern half) and peptide 11
* Corresponding author. Natural Product Microbes, CSIR-Indian Institute of
Integrative Medicine Jammu, J & K, 180001, India.
E-mail address: bashah@iiim.ac.in (B.A. Shah).
http://dx.doi.org/10.1016/j.ejmech.2014.11.068
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

56
A. Kumar et al. / European Journal of Medicinal Chemistry 93 (2015) 55e63
Fig. 1. Approach to simplified analogues of Cryptophycins.
or 12 (southern half). Further disconnection of 11 or 12 reveals that
the fragment comprises of three segments i.e., leucic acid, -alanine
and D-O-methyltyrosine. The ester 7 can be synthesized by Wittig
reaction of -hydroxy aldehyde. However, the bottleneck of the
synthesis was the synthesis of -hydroxy aldehyde, which as it
Boc-bealanine using EDCI and DMAP in DCM at room temperature
for 12 h, resulting in the synthesis of diester 10. The resultant
diester 10 was deprotected using TFA in DCM to give the free amine,
b
b
which was then condensed with N-Boc-O-methyl-D-tyrosine and
b
N-Boc-O-methyl- -tyrosine using EDCI, DMAP and HOBt followed
L
turns out required the use of acetaldehyde in cross aldol conden-
sation, deemed impossible until recently owing to large number of
side product formations [13]. This may have been a reason that
analogues without epoxide chain haven't been reported so far. We
reasoned that our method employing vinyl acetate as a precursor to
by debenzylation via hydrogenation in the presence of 5% Pd/C
under H2 atmosphere in ethyl acetate to obtain southern halves 11
and 12 respectively. The coupling of 11 and 12 with 7aef were
carried out through esterification using DIC and DMAP in DCM to
give 13aef and 14aef respectively, thus setting the stage for final
ring closure via macrolactamization. The Boc protecting group and
tert-butyl ester of each coupled products 13aef and 14aef were
cleaved in a single step with TFA and the resultant amines and
carboxylic acids were macrocyclized using HBTU and Hunig's base
in acetonitrile to afford macrocyclic depsipeptides 15aef and 16aef
respectively (Scheme 1).
acetaldehyde to access
[14,15].
b-hydroxy aldehyde could prove handy
2. Results and discussion
2.1. Chemistry
Thus, our efforts commenced with the synthesis of northern half
from cross aldol product of acetaldehyde (Scheme 1). To synthesize
the enantioselective cross-aldol product we followed our meth-
odology wherein the reaction involves lipase-catalysed in situ
generation of acetaldehyde from vinyl acetate which in presence of
2.2. Biological evaluation
The synthesized depsipeptides were then screened for cell
viability assay against human cancer cell lines; PC-3, HeLa and A549
(Table 1). Out of 12 molecules, two molecules, 15a & 16a displayed
organocatalyst (R-
a
,a
-Bis[3,5-bis (trifluormethyl) phenyl]-2-
low IC50 value in A549 cells viz., 10 and 11 mM, respectively,
pyrolidinemethanol) reacts with aromatic aldehydes to give high-
ly enantiopure cross-aldol products 4aef [14,15]. The hydroxy
groups of cross aldol products were protected as their TBS ethers
using TBDMSCl and imidazole in DCM at 0 ꢀC to give 5aef. The
primary alcohols in 5aef were selectively deprotected using HF-
Pyridine in THF, followed by oxidation with Dess-Martin period-
inane in DCM to give 6aef, which were then subjected to Wittig
reaction with (tert-butoxycarbonylmethylene) triphenylphosphor-
ane in DCM, followed by deprotection of secondary alcohol using
TBAF in THF resulting in synthesis of the northern halves 7aef.
We then focused on construction of southern half. Thus, we
whereas 15a was found to be more cytotoxic when compared to 16a
in PC-3 and HeLa cells. Also, it was observed that the compounds
show selective cytotoxicity against human cancer cell lines as their
IC50 values in FR-2 (human normal breast epithelial) cells were
considerably high. As both the compounds 15a & 16a showed
relative low IC50 in A549 cell, so we chose this cell line for further
studies at different concentrations Fig. 2.
Apoptotic bodies' formation is a characteristic feature of cells
undergoing apoptosis [16]. These are membrane enclosed vesicles
consisting of damaged organelles and DNA. Induction of apoptotic
bodies by chemotherapeutic agents has always been a preferred
choice in developing anti-cancer therapeutics. Apoptosis induction
by 15a and 16a were analysed by visualizing cellular and nuclear
morphology through phase contrast microscopy and Hoechst
staining. Phase contrast microscopy of 15a and 16a treated cells
showed cellular blebs, cell wall deformation, shrinkage of cell size,
and formation of multiple apoptotic bodies, which were shown by
started with the synthesis of
L
-leucic acid from -leucine using
L
NaNO2 and H2SO4, which was O-benzylated by treatment with
Cs2CO3 in MeOH/H2O (1:1) at room temperature for 30 min, fol-
lowed by removal of the solvents and subsequent reaction with
BnBr in DMF at 0 ꢀC to room temperature for 12 h to give benzyl
(ꢁ)-2-hydroxyisocaproate 9. The compound 9 was coupled to N-

A. Kumar et al. / European Journal of Medicinal Chemistry 93 (2015) 55e63
57
Scheme 1. Synthesis of cryptophycin based macrocyclic depsipeptides.
arrows, while the nuclei of untreated cells are intact and round in
shape (Fig. 3A, B). The number of apoptotic bodies increased with
increased concentration of 15a and 16a. Therefore, the mode of
A549 cell death induced by depsipeptides was apoptosis.
DNA fragmentation constitutes one biochemical hallmark of
apoptosis [17]. Thus, measurement of DNA content makes it
possible to identify and quantify the apoptotic cells population,
and cell cycle phase specificity. The relative DNA content was
analyses through flow cytometry by using fluorescent DNA
binding dye, propidium iodide (PI), which becomes highly fluo-
rescent after binding to DNA. After cellular permeabilization, PI
binds to DNA in cells at all stages of the cell cycle, and the

58
A. Kumar et al. / European Journal of Medicinal Chemistry 93 (2015) 55e63
Table 1
fluorescence). A-549 cells treated with 15a and 16a for 24 h
exhibited concentration dependent increase in hypo diploid sub-
G0 DNA fraction (apoptotic, <2nDNA, Fig. 4). The sub-G0 fraction
(apoptotic) was 5% in control cells, which increased to 26% and
IC50 values of different compounds against human cancer cell lines.
Compound
Cell type
HeLa
A549
PC-3
FR-2
49% after 30
the cell cycle G2/M phase was significantly blocked by 16a at
30 m/ml from 5% to 19% indicating that it produced mitotic
mM of 15a and 16a treatments, respectively. Further,
IC50 values (mM)
15a
15b
15c
15d
15e
15f
16a
16b
16c
16d
16e
16f
28
>100
29
90
78
55
40
35
47
10
>100
15
100
30
60
11
90
13
15
>100
67
100
70
97
m
>100
>100
>100
>100
87
78
93
90
>100
79
block or caused delay in cell cycle (Fig. 4). Whereas, 15a did not
block any phase of A-549 cells cycle. Paclitaxel, a known G2/M
blocker, at 1 mM significantly block G2/M phase from 5% to 45%
(data not shown) [18].
As cryptophycins are known tubulin inhibitors, we were
intrigued to test whether the synthesized macrocycles would do
60
70
100
100
32
100
68
the same. Both 15a and 16a significant inhibit the expression of
tubulin and -tubulin in A549 cells. The 16a was found to be a
strong inhibitor of and -tubulin than 15a. The 16a inhibits the
expression of -tubulin very strongly, which was supported by
blockade of G2/M phase of A549 cells (Figs. 4 and 5). Also this is a
significant observation as 16a with -tyrosine not only had com-
a-
33
100
60
45
>100
56
b
>100
16
a
b
Paclitaxel
0.010
0.025
0.012
b
Note: HeLa ¼ Prostate Cancer; A549 ¼ Lung Cancer; PC-3 ¼ Cervix Cancer; Normal
FR-2 ¼ Breast Epithelial.
L
parable IC50 values but also proved to be a better inhibitor of
tubulin polymerization in contrast to most of the naturally occur-
ring cryptophycins. Further mechanistic studies on 16a will be
needed to platform its role as anticancer therapeutic. The obser-
vations can direct the future course of work.
fluorescent intensity is directly proportional to its DNA content.
The results of the measurement are illustrated in a histogram,
where the number of cells (counts) is plotted against the relative
fluorescence intensity of PI (FL-2;
lem: 585 nm; red
Fig. 2. Cytotoxic profile of 15a and 16a in different panel of human cancer cell line (Prostate, Lung and Cervix). The cells were grown in 96-well culture plate and treated with
indicated concentrations of 15a & 16a for 48 h. Thereafter, cells were incubated with MTT solution for 4 h and optical density of formazon crystals was measured as described in
Materials and Methods. Data are Mean SD (n ¼ 8 wells), and representative of one of three similar experiments. IC50 values were calculated through non linear regression with
variable slope (Sigmoid curve) by using GraphPAD Prism software version 5.0.
Fig. 3. Effect of 15a & 16a on the cellular and nuclear morphology of A549 cells. Cells were treated with 30 mM concentrations of 15a & 16a for 24 h, visualized for cellular
morphology and simultaneously stained with DNA binding Hoechst 33258 (as described in Materials and Methods). Nuclear morphology and apoptotic bodies' formation were
visualised on fluorescent microscope. Condensed nuclei and the apoptotic bodies are indicated by white arrows. Data are representative of one of three similar experiments and
magnification of the pictures was 30ꢂ on Olympus 1X 70 inverted microscope.

A. Kumar et al. / European Journal of Medicinal Chemistry 93 (2015) 55e63
59
Fig. 4. DNA cell cycle analysis in A549 cells exposed to indicated concentration of 15a & 16a for 24 h. After treatment cells were stained with Propidium iodide, PI (10
mg/ml) to
determine DNA fluorescence and cell cycle phase distribution as described in Materials and Methods. Data were analysed by Modfit software (Verity Software House Inc., Topsham,
ME) for the proportions of different cell cycle phases. The fraction of cells from apoptosis, G1, S and G2 phases analysed from FL2- A vs. cell counts are shown in %.
To determine the binding orientation and mode of inhibition of
15a and 16a, molecular modelling studies were carried out. Cryp-
tophycins are known to inhibit tubulin-dependent GTP hydrolysis
unit in a/b tubulin dimer of microtubule. Beside hydrophobic in-
teractions, 15a and 16a also displayed H-bonding interactions with
polar side chain residues (15a with Thr343 and 16a with Arg215),
close to nucleotide binding site as shown in Fig. 6. Based on these
[19] as well as nucleotide exchange on
consequence, inhibit tubulin assembly. The binding site of crypto-
phycins was proposed to be in a hydrophobic pocket on -tubulin
b-tubulin [20] and, as a
interactions it could be proposed that 15a and 16a binds to
b-
b
tubulin subunit of microtubule at tubulin dimer interphase, and
a/b
surface consisting of following residues Ser172, Lys174, Val175,
Asp177, Asn204, Glu205, Tyr208, Asp209, Phe212, Pro220 and
Tyr222 [21]. Therefore, based on above observations, 15a and 16a
binding site was defined considering these residues as centroid of
grid for molecular docking in vicinity of vinblastine binding site and
GTP hydrolysis site. Molecular modelling were performed with
tubulin (PDB ID: 3RYF) containing buried GTP and Mg2þ ion [22].
Molecular docking results demonstrate that 15a and 16a like
therefore inhibit tubulin polymerization by hindering GTP nucle-
otide hydrolysis and ring formation; thereby restricting the
microtubule conformation to straight form and further polymeri-
zation process.
3. Conclusion
In conclusion, a new class of macrocyclic depsipeptides based on
cryptophycins were synthesized and screened for cytotoxicity
cryptophycins bind in vicinity of the GTP nucleotide of b-tubulin
Fig. 5. Effect of 15a & 16a on the expression of proteins involve in cytoskeleton. A549 cells at 70e80% confluent stage were treated with 15a & 16a at indicated concentrations for
24 h. Cell lysates were prepared and equal amount of protein was loaded on SDS-PAGE gel for western blot analysis (as described in Supporting information). -actin was used as
internal control. Specific antibodies were used for detection of - and -Tubulin. Data are representative of one of three similar experiments.
b
a
b

60
A. Kumar et al. / European Journal of Medicinal Chemistry 93 (2015) 55e63
Fig. 6. Molecular modelling of synthesized macrocyclic depsipeptides with
b-tubulin at a/b tubulin interphase; (A) interactions for 15a; (B) interactions for 16a. GTP is represented
by colour spheres. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
against a panel of human cancer cell lines. The lead analogues
induced apoptosis, blocked/delayed G2/M cell cycle as well as
133.9, 132.9, 130.9, 129.8, 127.9, 117.9, 78.1, 75.1, 59.1, 53.7, 45.1, 43.6,
39.1, 37.9, 36.2, 28.2, 26.4, 25.5; HRMS calcd. for C30H35N3O9 [MþH]
significantly reduced the expression of
silico studies reveal that these molecules bind to
of microtubule at tubulin dimer interphase, same as that of
a-tubulin and
b
-tubulin. In
þ 582.2373, found 582.2441.
b
-tubulin subunit
a/b
4.1.2. (3S,10R,16S,E)-3-isobutyl-10-(4-methoxybenzyl)-16-(3-
nitrophenyl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-
cryptophycins. Further studies aimed at optimizing their activity,
elucidate their mode of inhibition are underway.
tetraone (15b)
23
Yield 72%, [
a
]
ꢁ3.0 (c ¼ 1.00, CHCl3); 1H NMR (500 MHz,
¼
D
4. Material and methods
CDCl3) d 8.23 (m, 2H), 7.73 (m, 1H), 7.60 (m, 1H), 7.13 (m, 2H), 6.81
(m, 3H), 6.16 (m, 1H), 5.94 (d, J ¼ 15.1 Hz, 1H), 5.08 (dd, J ¼ 8.9,
5.1 Hz,1H), 4.68 (m,1H), 3.77 (s, 3H), 3.50 (m, 2H), 3.19 (m,1H), 2.91
(m, 2H), 2.70 (m, 1H), 2.63 (m, 2H), 1.68 (m, 1H), 1.57 (m, 1H), 1.46
1H and 13C NMR spectra were recorded on 400 and 500 MHz
spectrometers with TMS as the internal standard. Chemical shifts
are expressed in parts per million (
d
ppm). Silica gel coated
(m, 1H), 0.88 (m, 6H); 13C NMR (100 MHz, CDCl3)
d 172.5, 171.3,
aluminium plates were used for TLC. Final products were purified
by column chromatography on silica gel (60-120/100-200 mesh)
using petroleum ethereethyl acetate as the eluent to obtain the
pure products. Exact Mass of all products were analysed by using
HRMS with QTOF analyser. Reagents used were purchased from
Sigma Aldrich.
170.3, 166.1, 158.3, 148.4, 141.0, 139.9, 132.2, 129.9, 129.9, 128.9,
125.9, 123.4, 120.9, 113.9, 74.0, 71.1, 55.1, 54.7, 41.1, 39.7, 35.1, 33.9,
þ
32.2, 24.4, 22.4, 21.6; HRMS calcd. for C30H35N3O9 [MþH]
582.2373, found 582.2444.
4.1.3. (3S,10R,16S,E)-3-isobutyl-10-(4-methoxybenzyl)-16-(2-
nitrophenyl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-
4.1. General procedure for macrocyclization 15aef & 16aef
tetraone (15c)
Yield 71%,[
CDCl3)
23
a
]
ꢁ6.3 (c ¼ 1.00, CHCl3); 1H NMR (400 MHz,
¼
D
Ester 13a or 14a (50 mg, 0.07 mmol) was dissolved in CH2Cl2
(3 ml), and TFA (0.5 ml) was added. After 1 h, toluene (2 ml) was
added, and the reaction mixture was concentrated in vacuum. The
resulting acid was dissolved in CH3CN (3 ml), and DIEA (0.03 ml,
0.02 mmol) and HBTU (30 mg, 0.08 mmol) were added. The reac-
tion was stirred at room temperature for 45 min. Saturated aqueous
NaHCO3 solution was added, and the aqueous phase was extracted
with CH2Cl2. The organic layer was dried with Na2SO4 and the
residue was purified by column chromatography to obtain 15a or
16a respectively.
d
8.02 (d, J ¼ 8.1 Hz, 1H), 7.71 (dd, J ¼ 19.5, 7.5 Hz, 2H),
7.52 (t, J ¼ 7.5 Hz, 1H), 7.15 (d, J ¼ 8.1 Hz, 2H), 6.82 (d, J ¼ 8.0 Hz,
2H), 6.67 (d, J ¼ 10.5 Hz, 1H), 6.03 (d, J ¼ 8.0 Hz, 1H), 5.89 (d,
J ¼ 15.2 Hz, 1H), 5.05 (dd, J ¼ 8.9, 4.7 Hz, 1H), 4.77 (m, 1H), 3.78
(s, 3H), 3.52 (m, 2H), 3.19 (m, 1H), 3.04 (m, 1H), 2.96 (m, 1H), 2.61
(m, 3H), 1.67 (m, 1H), 1.56 (m, 1H), 1.41 (m, 1H), 0.86 (m, 6H); 13
C
NMR (100 MHz, CDCl3)
147.0, 140.4, 134.5, 133.8, 130.2, 129.2, 128.7, 127.9, 125.9, 124.9,
114.0, 71.2, 70.9, 55.3, 54.1, 40.8, 39.6, 35.1, 34.2, 32.5, 24.3, 22.6,
d 172.7, 170.9, 169.7, 165.6, 158.5,
þ
21.7; HRMS calcd. for C30H35N3O9 [MþH]
582.2373, found
582.2563.
4.1.1. (3S,10R,16S,E)-3-isobutyl-10-(4-methoxybenzyl)-16-(4-
nitrophenyl)-1,4-dioxa-8,11-diaza cyclohexadec ꢁ13-ene-2, 5, 9, 12-
4.1.4. (3S,10R,16S,E)-16-(2,4-dichlorophenyl)-3-isobutyl-10-(4-
methoxybenzyl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-
tetraone (15a)
23
Yield ¼ 70%, [
a
]
ꢁ1.3 (c ¼ 1.00, CHCl3); 1H NMR (400 MHz,
¼
tetraone (15d)
D
ꢁ3.5 (c ¼ 1.00, CHCl3); 1H NMR (400 MHz,
¼
23
CDCl3)
d
8.25 (d, J ¼ 8.8 Hz, 2H), 7.53 (d, J ¼ 8.7 Hz, 2H), 7.11 (d,
Yield 70%, [
a
]
D
J ¼ 32.9, 7.1 Hz, 2H), 6.79 (m, 3H), 6.23 (m, 1H), 5.92 (d, J ¼ 15.2 Hz,
1H), 5.06 (d, J ¼ 3.9 Hz, 1H), 4.69 (m, 1H), 3.78 (s, 3H), 3.54 (m,2H),
3.45e3.11 (m, 3H), 3.05e2.84 (m, 1H), 2.82e2.46 (m, 2H), 1.68 (m,
1H), 1.57 (m, 1H), 1.45 (m, 1H), 1.31 (m,1H), 0.89 (m, 6H); 13C NMR
(100 MHz, CDCl3) 176.5, 175.5, 174.2, 170.2, 162.3, 151.7, 149.9, 143.9,
CDCl3) d 7.42 (m, 2H), 7.30 (m, 1H), 7.12 (m, 2H), 6.84 (m, 2H), 6.44
(m, 1H), 6.09 (d, J ¼ 8.2 Hz, 1H), 5.87 (d, J ¼ 15.5 Hz, 1H), 5.01 (dt,
J ¼ 14.9, 7.4 Hz,1H), 4.77 (m,1H), 3.79 (m, 3H), 3.52 (m,1H), 3.19 (m,
1H), 3.01 (m, 1H), 2.78 (m, 2H), 2.54 (m, 2H), 1.71 (m, 1H), 1.59 (m,
1H), 1.46 (m, 1H), 0.87 (m 6H); 13C NMR (100 MHz, CDCl3)
d 172.8,

A. Kumar et al. / European Journal of Medicinal Chemistry 93 (2015) 55e63
61
170.8, 169.8, 165.5, 158.5, 140.3, 139.2, 135.4, 134.8, 132.8, 130.2,
4.1.9. (3S,10S,16S,E)-3-isobutyl-10-(4-methoxybenzyl)-16-(2-
nitrophenyl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-
129.7,128.7,127.9,127.6,125.8,114.1, 71.5, 71.3, 55.2, 54.4, 39.6, 39.3,
35.2, 34.2, 32.5, 24.4, 22.6, 21.7; HRMS calcd. for C30H34Cl2N2O7
[MþH] þ 605.1743, found 605.1922.
tetraone (16c)
23
Yield 71%,[
a
]
ꢁ16.2 (c ¼ 1.00, CHCl3); 1H NMR (400 MHz,
¼
D
CDCl3)
d
8.06 (d, J ¼ 8.1 Hz, 1H), 7.73 (m, 2H), 7.55 (m, 1H), 7.18 (d,
J ¼ 7.7 Hz, 2H), 6.84 (d, J ¼ 7.9 Hz, 2H), 6.73 (d, J ¼ 10.7 Hz, 1H), 6.65
(m, 1H), 6.08 (m, 1H) 5.89 (d, J ¼ 15.2 Hz, 1H), 5.04 (m, 1H), 4.86 (dd,
J ¼ 14.4, 8.3 Hz, 1H), 3.78 (s, 3H), 3.72 (m, 1H), 3.24 (m, 2H), 2.95 (m,
2H), 2.77 (m, 1H), 2.60 (m, 1H), 2.47 (m, 1H), 1.63 (m, 2H), 1.40 (m,
4.1.5. (3S,10R,16S,E)-3-isobutyl-10-(4-methoxybenzyl)-16-
(perfluorophenyl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-
2,5,9,12-tetraone (15e)
23
Yield 71%,[
CDCl3)
a
]
ꢁ2.3 (c ¼ 1.00, CHCl3); 1H NMR (400 MHz,
¼
D
1H), 0.88 (m, 6H); 13C NMR (100 MHz, CDCl3)
d 172.3, 171.2, 170.7,
d
7.10 (d, J ¼ 8.4 Hz, 2H), 6.80 (m, 3H), 6.44 (m, 1H), 6.13
166.6, 158.4, 147.1, 137.6, 134.8, 134.1, 130.2, 129.3, 129.1, 127.8, 125.0,
(d, J ¼ 8.0 Hz, 2H), 5.95 (d, J ¼ 15.4 Hz, 9H), 4.99 (dd, J ¼ 9.4,
5.0 Hz, 1H), 4.76 (dd, J ¼ 13.8, 7.9 Hz, 2H), 3.81e3.72 (m, 4H),
3.62e3.47 (m, 2H), 3.25e3.09 (m, 1H), 3.10e2.91 (m, 2H),
2.66e2.56 (m, 2H), 1.74e1.59 (m, 1H), 1.59e1.51 (m, 1H),
1.46e1.37 (m, 1H), 0.91e0.80 (m, 6H); 13C NMR (100 MHz, CDCl3)
114.1, 71.4, 71.2, 55.2, 53.1, 39.4, 38.9, 34.5, 34.4, 32.5, 24.4, 22.7,
þ
21.6; HRMS calcd. for C30H35N3O9 [MþH]
582.2373, found
582.2424.
4.1.10. (3S,10S,16S,E)-16-(2,4-dichlorophenyl)-3-isobutyl-10-(4-
methoxybenzyl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-
d
172.4, 171.0, 166.3, 158.4, 146.2, 145.8, 143.6, 142.6, 140.2, 136.7,
130.1, 129.0, 128.2, 114.0, 77.3, 77.0, 76.7, 71.1, 66.1, 55.2, 53.1, 39.3,
36.1, 34.4, 32.3, 29þ.7, 24.5, 22.6, 21.5; HRMS calcd. for
tetraone (16d)
Yield 73%, [
CDCl3)
23
a
]
ꢁ9.9 (c ¼ 1.00, CHCl3); 1H NMR (400 MHz,
¼
D
C
30H31F5N2O7 [MþH] 627.2051, found 627.2128.
d
7.43 (m, 2H), 7.33 (m, 1H), 7.18 (m, 2H), 6.80 (dt, J ¼ 14.3,
6.8 Hz, 2H), 6.58 (m, 1H), 6.43 (m, 1H), 5.98 (m, 1H), 5.03 (dd, J ¼ 9.7,
4.7 Hz,1H), 4.85 (dd, J ¼ 14.9, 8.6 Hz, 1H), 3.75 (m, 3H), 3.56 (m, 1H),
3.22 (m, 2H), 2.98 (m, 1H), 2.77 (m, 2H), 2.53 (m, 2H), 1.66 (m, 2H),
4.1.6. (3S,10R,16S,E)-16-(4-bromophenyl)-3-isobutyl-10-(4-
methoxybenzyl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-
tetraone (15f)
Yield 73%, [
CDCl3)
1.44 (m, 1H), 0.90 (m, 6H); 13C NMR (100 MHz, CDCl3)
d 172.4, 171.1,
23
a]
ꢁ5.5 (c ¼ 1.00, CHCl3); 1H NMR (400 MHz,
¼
170.9, 166.6, 158.3, 137.3, 135.2, 134.9, 132.6, 130.1, 129.6, 129.6,
129.1, 127.9, 127.7, 114.0, 71.9, 71.1, 55.4, 52.8, 39.5, 37.7, 34.2, 32.5,
D
d
7.44 (d, J ¼ 8.4 Hz, 2H), 7.15 (d, J ¼ 8.4 Hz, 2H), 7.06 (d,
þ
J ¼ 8.5 Hz, 2H), 6.73 (m, 3H), 6.00 (dd, J ¼ 9.5, 4.1 Hz, 1H), 5.77 (d,
J ¼ 15.3 Hz, 1H), 4.93 (dd, J ¼ 9.3, 5.1 Hz, 1H), 4.69 (dd, J ¼ 13.6,
7.6 Hz,1H), 3.70 (s, 3H), 3.49 (m, 1H), 3.39 (m, 1H), 3.11 (m, 1H), 2.98
(m, 1H), 2.88 (m, 1H), 2.59 (m, 1H), 2.50 (m, 2H), 1.60 (m, 2H), 1.50
(dd, J ¼ 13.5, 6.7 Hz, 1H), 0.78 (m, 6H); 13C NMR (125 MHz, CDCl3)
29.5, 24.4, 22.8, 21.5; HRMS calcd. for C30H34Cl2N2O7 [MþH]
605.1743, found 605.1811.
4.1.11. (3S,10S,16S,E)-3-isobutyl-10-(4-methoxybenzyl)-16-
(perfluorophenyl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-
2,5,9,12-tetraone (16e)
d
173.6, 171.9, 171.1, 166.7, 159.3, 141.4, 138.9, 132.8, 131.3, 131.0,
23
129.7, 128.6, 126.6, 123.2,114.9, 75.5, 72.1, 56.1, 55.4, 40.5, 40.0, 39.4,
36.1, 33.4, 25.2, 23.4, 22.6; HRMS calcd. for C30H35BrN2O7 [MþH] þ
615.1628, found 615.1688.
Yield 72%,[
CDCl3)
a]
ꢁ13.0 (c ¼ 1.00, CHCl3); 1H NMR (400 MHz,
¼
D
d
7.16 (d, J ¼ 8.4 Hz, 2H), 6.83 (d, J ¼ 8.5 Hz, 2H), 6.62e6.52
(m, 1H), 6.47 (dd, J ¼ 12.0, 2.2 Hz, 1H), 5.99 (d, J ¼ 16.0 Hz, 1H), 5.89
(d, J ¼ 9.1 Hz, 1H), 5.00 (dd, J ¼ 9.4, 4.6 Hz, 1H), 4.84 (dd, J ¼ 14.7,
8.7 Hz, 1H), 3.81e3.68 (m, 4H), 3.32e3.19 (m, 1H), 3.15 (m, 1H),
3.11e3.01 (m, 1H), 2.96 (m, 1H), 2.84e2.70 (m, 1H), 2.61e2.42 (m,
2H), 1.70e1.57 (m, 2H), 1.43e1.37 (m, 1H), 0.87 (dd, J ¼ 12.7, 6.5 Hz,
4.1.7. (3S,10S,16S,E)-3-isobutyl-10-(4-methoxybenzyl)-16-(4-
nitrophenyl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-
tetraone (16a)
6H); 13C NMR (100 MHz, CDCl3)
d 172.3, 171.0, 166.3, 158.4, 146.2,
23
Yield 73%, [
a
]
ꢁ15.5 (c ¼ 1.00, CH3OH); 1H NMR (400 MHz,
¼
D
145.8, 143.6, 142.6, 140.2, 136.7, 130.2, 129.0, 128.2, 114.0, 77.3, 77.0,
76.7, 71.1, 66.1, 55.2, 53.1, 39.3, 36.1, 34.4, 32.3, 29.7, 24.5, 22.6, 21.5;
HRMS calcd. for C30H31F5N2O7 [MþH] þ 627.2051, found 627.2125.
CDCl3)
d
8.27 (d, J ¼ 8.7 Hz, 2H), 7.54 (d, J ¼ 8.7 Hz, 2H), 7.16 (d,
J ¼ 8.5 Hz, 2H), 6.83 (d, J ¼ 8.6 Hz, 2H), 6.58 (dt, J ¼ 15.8, 6.9 Hz, 1H),
6.20 (m, 1H), 5.99 (d, J ¼ 15.6 Hz, 1H), 5.04 (m, 1H), 4.74 (m, 1H),
3.81 (s, 3H), 3.26 (m, 1H), 3.17 (m, 1H), 2.94 (m, 1H), 2.87e2.76 (m,
2H), 2.75e2.59 (m, 2H), 2.49 (m, 1H),1.66 (m, 2H),1.44 (m, 1H), 0.92
4.1.12. (3S,10S,16S,E)-16-(4-bromophenyl)-3-isobutyl-10-(4-
methoxybenzyl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-
tetraone (16f)
(m, 6H); 13C NMR (100 MHz, CDCl3)
d 172.4, 171.1, 166.4,158.4,147.9,
145.7, 137.0, 130.1, 129.1, 127.8, 126.9, 124.2, 114.0, 74.4, 71.2, 55.2,
53.1, 39.6, 39.3, 34.4, 32.4, 29.7, 24.5, 22.7, 21.6; HRMS calcd. for
Yield 77%, 1H NMR (400 MHz, CDCl3)
d
7.44 (d, J ¼ 8.4 Hz, 2H),
7.17 (d, J ¼ 7.8 Hz, 2H), 7.07 (d, J ¼ 8.4 Hz, 2H), 6.76 (d, J ¼ 8.6 Hz,
2H), 6.47 (m, 1H), 5.98 (dd, J ¼ 11.3, 2.6 Hz, 1H), 5.88 (d, J ¼ 16.1 Hz,
1H), 4.94 (dd, J ¼ 9.4, 4.6 Hz,1H), 4.78 (m,1H), 3.69 (m, 4H), 3.20 (m,
1H), 3.10 (m, 1H), 2.89 (m, 1H), 2.63 (m, 3H), 2.40 (m, 1H), 1.57 (m,
C
30H35N3O9 [MþH] þ 582.2373, found 582.2311.
4.1.8. (3S,10S,16S,E)-3-isobutyl-10-(4-methoxybenzyl)-16-(3-
nitrophenyl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-
2H), 1.34 (m, 1H), 0.79 (s, 6H); 13C NMR (125 MHz, CDCl3)
d 173.3,
tetraone (16b)
172.1, 172.0, 167.5, 159.2, 138.7, 138.6, 132.9, 131.1, 130.1, 128.7, 128.3,
123.5, 114.9, 75.8, 72.2, 56.1, 54.0, 40.4, 40.2, 35.4, 35.3, 33.3, 25.3,
23.6, 22.5; HRMS calcd. for C30H35BrN2O7 [MþH] þ 615.1628, found
615.1699.
23
Yield 74%,[
a
]
ꢁ13.7 (c ¼ 1.00, CHCl3); 1H NMR (400 MHz,
¼
D
CDCl3)
d
8.22 (dd, J ¼ 16.9, 8.0 Hz, 2H), 7.69 (d, J ¼ 7.7 Hz, 1H), 7.61
(m, 1H), 7.16 (d, J ¼ 8.6 Hz, 2H), 6.83 (d, J ¼ 8.6 Hz, 2H), 6.61 (m, 1H),
6.20 (m, 2H), 6.01 (d, J ¼ 16.1 Hz, 1H), 5.05 (dt, J ¼ 16.8, 8.2 Hz, 1H),
4.84 (m, 1H), 3.75 (s, 3H), 3.26 (m, 1H), 3.15 (m, 1H), 2.98 (m, 1H),
2.82 (m, 2H), 2.76 (m, 2H), 2.49 (m, 1H), 1.67 (m, 2H), 1.45 (m, 1H),
4.2. Biological assay
0.88 (m, 6H); 13C NMR (100 MHz, CDCl3)
d
172.4, 171.2, 171.1, 166.5,
Human prostate cancer cell line (PC-3), Lung Cancer cell line
(A549), cervical cancer cell line (HeLa) and Normal Breast epithelial
cells (FR-2) were obtained from ECACC (Sigma Aldrich); The cells
were grown in RPMI-1640 medium containing 10% FCS, 100 units
158.3, 148.5, 140.9, 137.1, 131.9, 130.2, 130.0, 129.1, 127.8, 123.5, 121.1,
114.0, 74.3, 71.3, 55.2, 53.2, 39.6, 39.2, 38.6, 34.4, 32.3, 24.5, 22.6,
þ
21.6; HRMS calcd. for C30H35N3O9 [MþH]
582.2373, found
582.2407.
pencillin/100 mg/ml streptomycin, in a CO2 incubator (Thermocon

62
A. Kumar et al. / European Journal of Medicinal Chemistry 93 (2015) 55e63
Electron Corporation, USA) at 37 ꢀC with 95% humidity and 5% CO2
gas environment. Cells were treated with 15aef & 16aef (1, 10, 30
4.2.4. Cell cycle analysis
About 0.5 ꢂ 106 A549 cells were seeded in 6-well plate. After
& 100
mM), which were dissolved in DMSO while the untreated
attachment of cells were treated with 15a & 16a (10 & 30 mM) for
cultures received only the vehicle (DMSO, < 0.1%, v/v). RPMI-1640,
3-(4,5, -dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide
24 h. After completion of 24hr, cells were collected, centrifuge at
400g for 5min. and washed with PBS. The pellet was fixed in 70%
ethanol overnight at ꢁ20 ꢀC, followed by washing with PBS and
incubated with RNAse for 90 min at 37 ꢀC. Cells were then stained
with PI and acquired in FACS as described earlier. Resulting DNA
distributions were analysed by Modfit (Verity Software House Inc.,
Topsham, ME) for the proportions of cells in G0-G1, S- phase, and
G2-M phases of the cell cycle.
(MTT), penicillin, streptomycin,
ruvic acid, Tween 20, protease inhibitor cocktail were purchased
from Sigma chemical Co. -Tubulin and -tubulin primary antibody
L-glutamine, Hoechst-33258, py-
a
b
was acquired from Cell Signalling Technology, USA. Electrophoresis
reagents, Protein estimation kit and protein marker were from Bio-
Rad Laboratories (Hercules, CA).
4.2.5. Cell lysates preparation for western blots analysis
4.2.1. Cell proliferation assay
Human prostate cancer cell line (PC-3), Lung Cancer cell line
(A549) and cervical cancer HeLa cells were plated in 96 well plates
at the density of 10000 cells per well/200
tures were treated with different concentrations (1, 10, 30 &
Human lung cancer A549 cells (2 ꢂ 106) were treated with
10 mM and 30 mM concentrations of 15a & 16a for 24 h. Cells were
collected by centrifugation at 400 ꢂ g at 4 ꢀC, washed with PBS
twice and processed for preparation of whole cell lysates. Cells
were lysed with cold Cell lysis buffer (RIPA, Sigma with 50 mM NaF,
0.5 mM NaVO4, 2 mM PMSF and 1% protease inhibitor cocktail) for
40 min. Cells were centrifuged at 12000 ꢂ g for 15 min at 4 ꢀC and
the supernatant was collected as whole cell lysates for western blot
analysis [25].
ml of the medium. Cul-
100
mM) of 15a and 16a for 48 h. After that, 20
ml of MTT (3-
(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium
bromide)
(2.5 mg/ml in PBS) solution was added to each well and incubated
at 37 ꢀC for 2 h. The plates were centrifuged at 2000 rpm for 15 min
and supernatant were discarded and the formazen crystals were
dissolved in 150 ml of DMSO. Plates were shaken on shaker for 3 min
and then incubated at 37 ꢀC for 5 min. The OD was measured at
570 nm and percentage of cell viability and growth inhibitions were
calculated according to the following equations [23].
4.2.6. Western blot analysis
Protein was measured using Bio-Rad protein assay kit and pro-
tein lysates (60 mg) were subjected to SDS-PAGE analysis. They are
Absorbance of treated cells ꢁ Absorbance of blank
Aborbance of control cells ꢁ Absorbance of blank
The % of cell viability ¼
ꢂ 100
transferred to PVDF membrane for 2 h at 4 ꢀC at 100V using Bio-Rad
electrode assembly. The membrane was blocked by incubation with
3% BSA or 5% non-fat milk in Tris-buffered saline containing 0.1%
Tween-20 (TBST) for 1 h at room temperature to prevent any non-
% Growth inhibition ¼ 100 ꢁ % cell viability
All in vitro experiments were repeated at least three times with
quadruplicate in each experiment.
specific binding. After an hour the blots were incubated with a-
tubulin primary antibodies for 3e4 h at room temperature. They are
washed three times with TBST and were incubated with horse-
radish peroxidase conjugated secondary antibodies for another 1 h.
At the end, membranes were washed three times with TBST buffer
with 15 min interval and signals detected using ECL plus chem-
iluminescence's kit on X-ray film.
4.2.2. Phase contrast microscopy
Phase contrast microscopy was done to assess the morpholog-
ical changes induce by 15a and 16a. Human lung cancer A549 cells
were incubated in six well plates and treated with 10 mM and 30 mM
concentrations of 15a and 16a for 24 h, after completion of time
cells were subjected to photography. Apoptosis was assessed by
using microscope (1X70, Olympus), photographs were taken by
using Olympus DP-12 camera.
4.2.7. Protocol for molecular modelling
GTP-Tubulin: RB3 Stathmin-like domain complex (PDB ID:
3RYF) was retrieved from protein data bank, and prepared in pro-
tein preparation wizard in default mode. This protein carries 4
chains (two sets of identical Alpha and beta chains). For 20 0A grid
preparation binding site was defined with chain B residues namely
Ser172, Lys174, Val175, Asp177, Asn204, Glu205, Tyr208, Asp209,
Phe212, Pro220, and Tyr222. Similarly, ligand structures were
4.2.3. Hoechst 33258 staining of cells for nuclear morphology
Human lung cancer A549 cells were treated with 10 and 30 mM
concentrations of 15a and 16a for 24 h. Cells were centrifuged at
400 g for 5 min and washed twice with PBS. Cells were then stained
with 1 mL of staining solution (Hoechst 33258, 10 mg/ml of 0.01M
citric acid and 0.45 M disodium phosphate containing 0.05% Tween
20) under subdued light at room temperature. After staining the
sketched using maestro, Ionized at pH 7.0
2.0 using Epik and
minimized using Macromodel. Finally cyclic peptides were docked
to beta tubulin grid using Glide XP docking.
cells were resuspended in 50
ml of mounting fluid (PBS: glycerol, 1/
1) and 10 l of mounting solution containing cells was spread on
m
Acknowledgements
clean glass slides and covered with the cover slips. The slides were
then observed for any nuclear morphological alterations and
apoptotic bodies under inverted fluorescence microscope
(Olympus 1ꢂ70, magnification 30X) using UV excitation [24].
We thank DST, India (SR/FT/CS-028/2010) for financial assis-
tance. M.K. and S.G. thank CSIR for the award of Research Fellow-
ships. IIIM communication No. IIIM/1686/2014.

A. Kumar et al. / European Journal of Medicinal Chemistry 93 (2015) 55e63
Biochemistry 44 (2005) 13457e13466;
63
Appendix A. Supplementary data
(c) J.D. White, H. Smits, E. Hamel, Synthesis of cryptothilone 1, the first
cryptophycin-epothilone hybrid, Org. Lett. 8 (2006) 3947e3950.
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(b) H. Bouchard, M. P. Barun A. Commercon, J. Zhang, US Pat. 2012/0225089,
2012.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.11.068.
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