Discovery of a new class of 16-membered (2: Z,11 Z)-3,11-di(aryl/naphthyl)-1,13-dioxa-5,9-dithia-2,12-diazacyclohexadeca-2,11-dienes as anti-tumor agents

Article abstract of DOI:10.1039/c6ra15140j

A series of new 16-membered small macrocyclic compounds, (2Z,11Z)-3,11-di(aryl/naphthyl)-1,13-dioxa-5,9-dithia-2,12-diazacyclohexadeca-2,11-dienes (1a-k) were designed and developed by a simple and practical synthetic route from readily available substrates using simple organic transformations. Evaluation of in vitro anti-tumor activities on human triple negative breast cancer cells MDAMB-231 cell lines reveal that the macrocycles, 1a, 1f, 1g, 1i and 1k are promising anti-tumor compounds as evidenced from inhibition of cell migration and proliferation, upregulation of anti-tumor genes p53, MDA7 and TRAIL. The anti-proliferative effect of macrocycles is specific to cancer cells but no cytotoxic effect on normal breast epithelial cells has been observed (MCF10A). The developed synthetic route is free from metals, protecting groups and air-free techniques. The structure of macrocycle (1e) is confirmed by single crystal XRD studies.

Full text of DOI:10.1039/c6ra15140j

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2016, DOI: 10.1039/C6RA15140J.
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Discovery of a new class of 16-membered (2Z, 11Z)-3,11-di(aryl/naphthyl)-
1,13-dioxa-5,9-dithia-2,12-diazacyclohexadeca-2,11-dienes as novel anti-
tumor agents
Mohan Reddy Bodireddy,a Ranjeet Singh Mahla,c P. Md. Khaja Mohinuddin,a
G.Thrivikram Reddy,a D. Vijaya Raghava Prasad,b Himanshu Kumar,c, d,* N. C. Gangi
Reddya,*
aDepartment of Chemistry, School of Physical Sciences, Yogi Vemana University, Kadapa-
516 003, Andhra Pradesh, India.
bDepartment of Microbiology, School of Life Sciences, Yogi Vemana University, Kadapa-516
003, Andhra Pradesh, India.
cDepartment of Biological Sciences, Indian Institute of Science Education and Research
(IISER), Bhopal-462023, Madhya Pradesh, India.
dLaboratory of Host Defense, WPI Immunology Frontier Research Centre, Osaka University,
Osaka, Japan.
Corresponding authors: ncgreddy@yogivemanauniversity.ac.in, hkumar@iiserb.ac.in
Abstract: A series of new 16ꢀmembered small macrocyclic compounds, (2Z,11Z)ꢀ3,11ꢀ
di(aryl/naphthyl)ꢀ1,13ꢀdioxaꢀ5,9ꢀdithiaꢀ2,12ꢀdiazacyclohexadecaꢀ2,11ꢀdienes (1a-k) were
designed and developed by a simple and practical synthetic route from readily available
substrates using simple organic transformations. Evaluation of in vitro antiꢀtumor activities
on human triple negative breast cancer cells MDAMBꢀ231 cell lines reveal that macrocycles
(1a, 1f, 1g, 1i and 1k) are promising antiꢀtumor compounds as evidenced from inhibition of
cell migration and proliferation, upregulation of antiꢀtumor genes p53, MDA7 and TRAIL.
The antiꢀproliferative effect of macrocycles is specific to cancer cells but no cytotoxic effect
on normal breast epithelial cells has been observed (MCF10A). The developed synthetic
route is free from metals, protecting groups and airꢀfree techniques. The structure of
macrocycle (1e) is confirmed by single crystal XRD studies.
Keywords: Macrocycles, MDAMBꢀ231 cells, p53, TRAIL, MDA7 & STATꢀ3 genes,
Antiꢀtumor agents, Apoptosis.
Introduction
Design, synthesis and development of new antiꢀtumor agents has always been a challenging
task for the scientific community as the cancer is a major public health problem in many parts
of the world1ꢀ4 particularly in developing countries increasingly, an adoption of cancerꢀ
associated lifestyle options including smoking, westernized diets and lack of adequate
exercise.5 Globally every 8th death is caused due to cancer.6 Many of the available antiꢀtumor
drugs have adverse effects on human health, therefore, there is a great need to develop new

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class of drugs for the treatment of cancer that are cost effective and chemically stable with
low toxicity to normal cells.
Macrocyclic molecules played a key role in drug discovery as evidenced from natural
products.7 Among the macrocycles, the heteroꢀatoms incorporated macrocyclic molecules 7
might be the best option as they possess unusual characteristics such as i) structural
complexity, regiogenicity, stereogenicity and rigidity, ii) potential hydrogen bonding to
interact with biomolecules such as RNA, DNA, proteins (enzymes),8,9 iii) high degree of
structural preꢀorganization which leads to the interaction of the key functional groups across
extended protein’s active site and iv) ‘drugꢀlike’ physicoꢀchemical as well as
pharmacokinetic properties.10, 11 For instance, small macrocyclic molecules derived from
natural products (I)12 and many synthetic macrocycles (II-IV)13, 14, 15 displayed apoptosis and
15‐membered macrocycles (V)16 exhibited the highest levels of cytotoxicity (Figure 1).
Further, macrocyclic molecules exhibited various pharmacological properties which include
antiꢀbacterial, antiꢀfungal, antiꢀtumor and antiꢀHIV activities.17
H
O
O
N
N
O
N
CN
OH
HN
N
H
O
N
O
O
N
S
n
HO
O
N
N
n = 1, 2, 3
H
Ixempra® (ixabepilone)
SB1317/TG02
Activity: CDK2/JAK2/FLT3 kinase
Activity: Microtubulin stabilizer
(I)
Activity: CHK1 inhibitor
(III)
inhibitor
(II)
O
O
O
O
O
CH2 NH
CH2 NH
3
CH2
NH2
3
O
Dihydromotuporamine C derivative
ditert-butyldibenzo-18-crown-6
Activity: Strong G1 arrest and apoptosis
Activity: Cytotoxicity and invasion inhibition
(V)
(IV)
Figure 1. Representative examples of biologically active macrocyclic molecules.
However, disinclination to investigate and develop macrocycles as new drug candidates in
the academic and pharmaceutical industry was observed owing to poor yields during

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macrocyclization, difficulties in analog synthesis and unproductive side reactions during the
joining of ends of a linear precursor molecule.18 Later, metal template methods were
developed,19 but these methods were also associated with few disadvantages such as
difficulties in choosing the correct template metal ion and its removal.20 Other methods were
nonꢀtemplate, but required high dilution conditions21 which lead to huge organic waste
generation along with low yields of desired product and also these methods require lengthy
separations to remove oligomeric byꢀproducts. Later, ring closing metathesis,22 Glaser–
Eglinton–Hayꢀtype sp–sp coupling23 and head to tail macrocyclizations (Build/Couple/Pair
strategies)24 methods have emerged. But most of these methods suffer from one or more
disadvantages as discussed above. Keeping in view of various pharmacological and
biomedical applications and difficulties in the synthesis of macrocycles, we aimed to design
and synthesize a new class of nonꢀpeptidic 16ꢀmembered macrocycles i.e. (2Z,11Z)ꢀ3,11ꢀ
di(aryl/naphthyl)ꢀ1,13ꢀdioxaꢀ5,9ꢀdithiaꢀ2,12ꢀdiazacyclohexadecaꢀ2,11ꢀdienes(1) from readily
available substrates using simple organic transformations. The following factors formed the
basis for the present design and synthesis: i) high probability of cyclic oximeꢀethers in the
synthesis of biologically active synthetic molecules25 and ii) accessibility of diverse
topologies26 by the presence of sulfur atoms which were amiable to disrupt proteinꢀprotein
interactions (PPIs).27
Retro-synthetic analysis: The retrosyntheic analysis of target 16ꢀmembered (2Z,11Z)ꢀ3,11ꢀ
di(aryl/naphthyl)ꢀ1,13ꢀdioxaꢀ5,9ꢀdithiaꢀ2,12ꢀdiazacyclohexadecaꢀ2,11ꢀdienes (1) is outlined
in scheme 1.
O
O
N
S
Br
Br
S
R
(9)
+
OH
N
OH
N
N
R
S
(8)
R
R
S
FGI
1
R= Aryl/Naphthyl
2
H2N OH
(7)
SH SH
(5)
O
O
+
O
R
2
Br
FGI
R
R
O
S
S
R
(2)
(6)
(4)
Scheme 1. Retrosynthetic analysis for the synthesis of (2Z,11Z)ꢀ3,11ꢀdi(aryl/naphthyl)ꢀ1,13ꢀ
dioxaꢀ5,9ꢀdithiaꢀ2,12ꢀdiazacyclohexadecaꢀ2,11ꢀdienes (1).

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Herein, a simple, efficient and practical synthetic route is developed for the synthesis of
structurally diverse nonꢀpeptidic 16ꢀmembered 2Z,11Z)ꢀ3,11ꢀdi(aryl/naphthyl)ꢀ1,13ꢀdioxaꢀ5,9ꢀ
dithiaꢀ2,12ꢀdiazacyclohexadecaꢀ2,11ꢀdienes (1) (1a-k) from readily available substrates such as
aralkyl ketones (2a-k), Nꢀbromosuccinimide (3), 1,3ꢀpropanedithiol (5), NH2OH.HCl (7) and
1,3ꢀdibromopropane (9) using simple organic transformations involving αꢀbromination, CꢀS bond
construction, oxime formation followed by macrocyclization (CꢀO bond formation) as shown in
1
scheme 2. Further, the synthesized macrocyclic molecules (1a-k) are well characterized by H,
13C NMR, HRMS and single crystal XRD studies and evaluation of their antiꢀtumor activity is
duly undertaken.
O
O
O
O
ii
i
Br
S
S
R
R
R
R
R
R
4a-k
2a-k
6a-k
iii
S
S
R
S
S
iv
N
R
N
N
N
O
OH
HO
O
8a-k
1a-h
R=
Cl
Br
F
O
Br
Br
Reaction conditions: (i) 10% (w/w) Silicagel, NBS (3), MeOH, reflux, 15ꢀ20 min; (ii) 1,3ꢀ
propanedithiol (5), K2CO3, acetonitrile, 15ꢀ20°C, 85ꢀ95 min, (iii) NH2OH.HCl (7), NaOAc, reflux, 2
h; (iv) 1,3ꢀdibromopropane (9), KOH in a mixture of H2OꢀDMSO (1:5 ratio), 5ꢀ10°C, 25ꢀ35 min.
Scheme 2. Synthesis of (2Z,11Z)ꢀ3,11ꢀdi(aryl/naphthyl)ꢀ1,13ꢀdioxaꢀ5,9ꢀdithiaꢀ2,12ꢀdiaza
cyclohexadecaꢀ2,11ꢀdienes (1).
Results and Discussion
Chemistry
The synthetic route of title compounds i.e. (2Z,11Z)ꢀ3,11ꢀdi(aryl/naphthyl)ꢀ1,13ꢀdioxaꢀ5,9ꢀ
dithiaꢀ2,12ꢀdiazacyclohexadecaꢀ2,11ꢀdienes (1) was accomplished via a fourꢀstep process as
shown in scheme 2. Accordingly, first 2ꢀbromoꢀ1ꢀ(4ꢀchlorophenyl)ethanone (4a) was
prepared from 4ꢀchloroacetophenone (2a) by αꢀbromination using NBS (3) in MeOH in the
presence of silica gel under reflux condition in high yield (91%) within a short period of
time.28 The same procedure was applied for the preparation of other αꢀbromoaralkyl ketones

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(4b-k) from various acetophenones and acetonaphthones (2a-k) and the obtained results were
presented in scheme 3.
Scheme 3. Synthesis of αꢀbromoaralkyl ketones (4)a,b
O
O
10% Silicagel
Br
R
R
NBS (3), MeOH
2a-k
4a-k
Reflux, 15ꢀ20 min
O
O
O
O
O
O
O
Br
Br
Br
Br
Br
Br
Br
Br
Cl
4a
Br
4b
Br
O
F
4c
4d
4e
4f
4g
O
O
O
Br
Br
O
Br
Br
4h
4i
4k
4j
aReagents and conditions: Aralkyl ketones, 2a-k (10.0 mmol), NBS (12.0 mmol) (3), 10%
b
(w/w) Silica gel, MeOH, reflux, 15ꢀ20 min; Isolated yields : 4a (91%), 4b (87%), 4c (81%),
4d (74%), 4e (94%), 4f (87%), 4g (92%), 4h (88%), 4i (95%), 4j (86%), 4k (91%).
Later, the reaction conditions for the synthesis of 2,2'ꢀ(propaneꢀ1,3ꢀdiylbis(sulfanediyl))bis(1ꢀ(4ꢀ
chlorophenyl)ethanone) (6a) were optimized. Towards this direction, firstly, 1, 3ꢀpropanedithiol
was added drop wise to the αꢀbromo compound, 2ꢀbromoꢀ1ꢀ(4ꢀchlorophenyl)ethanone (4a) in
presence of Na2CO3 (3.0 equiv.) in ethanol at 20ꢀ25°C and obtained 45% yield of the product
(6a). To improve the yield further, the effect of various solvents such as dichloromethane,
methanol, acetonitrile, THF, DMF and DMSO was investigated and 42%, 54%, 65%, 49%, 58%
and 53% yields of product (6a) were obtained respectively. Preliminary screening of solvent
study revealed that acetonitrile was the best option for maximum conversion of product (6a). To
improve the yield of the product (6a) further, various bases such as K2CO3 (3.0 equiv.), Et3N (3.0
equiv.) and pyridine (3.0 equiv.) have been employed in acetonitrile which resulted in 85%, 65%
and 55% yields of the product (6a) respectively. Finally, the effect of temperature on the course
of CꢀS bond formation was also studied at various temperatures ranging from 5 to 25°C. The
study indicated that 15ꢀ20°C was the optimum temperature for maximum yield of product 6a
(98%). With the help of optimized reaction conditions, the substrate scope was tested for the
synthesis
of
various
2,2’ꢀ(propaneꢀ1,3ꢀdiylbis(sulfanediyl)ꢀbis(1ꢀ
aryl/naphthylethanone)derivatives (6b-k) using αꢀbromoketones (4b-k) as substrates and the
obtained results were presented in scheme 4.

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Scheme 4. Synthesis of various 2,2’ꢀ(propaneꢀ1,3ꢀdiylbis(sulfanediyl))bis(1ꢀaryl/naphthyl
ethanone) derivatives (6)a,b
O
O
O
K2CO3
Br
S
S
HS
SH
R
+
R
R
Acetonitrile
4a-k
15ꢀ20°C, 85ꢀ95 min
5
6a-k
Br
O
Br
O
O
O
Cl
O
O
O
S
S
S
S
S
S
Br
6c
6a
6b
6e
Br
O
Cl
O
F
O
O
O
O
Br
S
S
S
Br
S
S
S
6f
6d
O
F
O
O
O
S
S
O
O
S
O
S
S
S
6h
6i
6g
O
S
S
O
O
S
S
6k
6j
O
aReagents and conditions: αꢀbromoaralkyl ketones, 4a-k (10.0 mmol), 1,3ꢀpropanedithiol (5.0
mmol) (5), K2CO3, acetonitrile, 15ꢀ20°C, 85ꢀ95 min; bIsolated yields : 6a (98%), 6b (96%), 6c
(93%), 6d (86%), 6e (97%), 6f (95%), 6g (96%), 6h (95%), 6i (95%), 6j (94%), 6k
(98%).
The third step was the oxime formation step which involves, the reaction between compound (6a)
and NH2OH.HCl (7) in the presence of NaOAc in ethanol under reflux conditions29 to produce
the precursor of the pharmacophore, (1Z,1'Z)ꢀ1ꢀ(4ꢀchlorophenyl)ꢀ2ꢀ(3ꢀ((Z)ꢀ2ꢀ(4ꢀchlorophenyl)ꢀ2ꢀ
(hydroxyimino)ꢀethylthio) propylthio) ethanone oxime (8a) in excellent yield (98%). The same
procedure was followed for the preparation of (1Z,1'Z)ꢀ1ꢀ(ayl/naphthyl)ꢀ2ꢀ(3ꢀ((Z)ꢀ2ꢀ
(ayl/naphthyl)ꢀ2ꢀ(hydroxyimino)ꢀethylthio)propylthio) ethanone oximes (8b-k) using various
substrates, 6b-k and the obtained results were presented in scheme 5.

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Scheme 5. Preparation of various (1Z, 1’Z)ꢀ1ꢀ(aryl/naphthyl)ꢀ2ꢀ(3ꢀ((Z)ꢀ2ꢀ(aryl/naphthyl)ꢀ2ꢀ
(hydroxyimino) ethylthio) propylthio) ethanone oxime (8)a,b
OH
HO
N
N
O
O
NH2OH.HCl (7), NaOAc
S
S
S
S
R
R
R
R
EtOH, Reflux, 2.0 h
6a-k
8a-k
HO N
HO N
HO N
Br
S
HO N
S
S
HO N
S
HO N
Br
Cl
S
S
Br
HO N
Cl
Br
8b
8a
8c
HO N
HO N
Br
S
HO N
S
HO N
S
S
Br
HO N
O
F
S
S
O
F
8f
8d
8e
HO N
HO N
HO N
S
HO N
S
HO N
S
HO N
S
S
S
8g
8i
8h
HO N
S
HO N
S
S
N OH
S
8j
8k
N OH
aReagents and conditions: Substrates, 6a-k (10.0 mmol), NH2OH.HCl (40.0 mmol) (7), NaOAc
(30.0 mmol w. r. t. NH2OH.HCl), ethanol, reflux, 2h; bIsolated yields : 8a (98%), 8b (97%), 8c
(95%), 8d (88%), 8e (98%), 8f (97%), 8g (98%), 8h (96%), 8i (97%), 8j (96%), 8k
(98%).
Then, the attention was focused on macrocyclization step to synthesize the title compound,
(2Z,11Z)ꢀ3,11ꢀbis(4ꢀchlorophenyl)ꢀ1,13ꢀdioxaꢀ5,9ꢀdithiaꢀ2,12ꢀdiazacyclohexadecaꢀ2,11ꢀdiene
(1a) by the reaction of pharmacophore, (1Z,1'Z)ꢀ1ꢀ(4ꢀchlorophenyl)ꢀ2ꢀ(3ꢀ((Z)ꢀ2ꢀ(4ꢀ
chlorophenyl)ꢀ2ꢀ(hydroxyimino)ꢀethylthio)propylthio)ethanoneoxime (8a) with linker i.e. 1,3ꢀ
dibromopropane (9). Towards this direction, initially, a reaction was carried out by using the
substrate, 8a and 1,3ꢀdibromopropane (9) in MeOH in the presence of NaOH at 20ꢀ25°C and
lower yield (15%) of product, 1a was obtained. To improve the yield, the reaction conditions for
the preparation of target 16ꢀmembered macrocycle 1a have been optimized using different
solvents and bases. Initially, the effect of solvent or mixtures of solvents was studied for their
impact on the course of macrocyclization. It was found that the solvents like MeOH, EtOH,
acetonitrile, DMSO and DMF were provided 15%, 20%, 10%, 50% and 35% yields, respectively.

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From this study, it was found that DMSO provided considerable yield (50%) of the product 1a.
For the improvement of the yield of the product 1a further, the effect of mixture of solvents was
investigated. Towards this direction, a mixture of H2O and DMSO was selected and the study
revealed that 1:5 ratio of H2O and DMSO provided good yield (75%) of product 1a. Further, the
effect of temperature was also studied and found that 5ꢀ10°C was the optimum temperature for
maximum yield of product 6a (86%). The effect of various bases such as Na2CO3, K2CO3,
NaOH, KOH, Et3N and pyridine on the course of macrocyclization was studied. From this study,
it was concluded that KOH was the best in obtaining maximum isolated yield (94%) of the
desired product 1a in presence of 1:5 ratio of H2O and DMSO at 5ꢀ10oC. Encouraged by the
productive results the same procedure was applied for the synthesis of a series of novel nonꢀ
peptidic
16ꢀmembered
(2Z,11Z)ꢀ3,11ꢀdi(aryl/dinaphthyl)ꢀ1,13ꢀdioxaꢀ5,9ꢀdithiaꢀ2,12ꢀ
diazacyclohexadecaꢀ2,11ꢀdiene derivatives (1b-k) from various oximes (8b-k) and 1,3ꢀ
dibromopropane (9) and the yields obtained were shown in scheme 6.
The synthesized compounds were characterized by 1HꢀNMR, 13CꢀNMR and HRMS data and the
structure of one of the macrocyclic compound, (2Z,11Z)ꢀ3,11ꢀbis(4ꢀfluorophenyl)ꢀ1,13ꢀdioxaꢀ
5,9ꢀdithiaꢀ2,12ꢀdiazacyclohexadecaꢀ2,11ꢀdiene (1e) was confirmed by single crystal Xꢀray
diffraction studies as shown in figure.2.
Scheme 6. Synthesis of (2Z, 11Z)ꢀ3,11ꢀdi(aryl/dinaphthyl)ꢀ1,13ꢀdioxaꢀ5,9ꢀdithiaꢀ2,12ꢀ
diazacyclohexadecaꢀ2,11ꢀdiene derivatives (1)a,b,c
.
S
S
OH
HO
N
N
R
KOH
Br
Br
S
S
N
O
R
R
R
H2OꢀDMSO
(1:5 ratio)
N
O
5ꢀ10°C, 25ꢀ35 min
8a-k
9
1a-k
S
S
S
S
S
S
O
S
Br
Cl
Br
Br
Cl
N
N
N
N
N
N
Br
O
O
O
O
O
1a
1b
1c
S
S
S
S
Br
S
Br
F
F
O
O
N
N
N
N
N
N
O
O
O
O
O
O
1d
1e
1f

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S
S
S
S
S
S
N
N
N
O
N
N
N
O
O
O
O
O
1i
1g
1h
S
S
S
S
N
N
N
N
O
O
O
O
1j
1k
aReagents and conditions: Substrates, 8a-k (10.0 mmol), 1,3ꢀdibromopropane (10.0 mmol)
(9), KOH (20.0 mmol) in a mixture of H2OꢀDMSO (1:5 ratio), 5ꢀ10°C, 25ꢀ35 min; bIsolated
yields : 1a (94%), 1b (94%), 1c (91%), 1d (86%), 1e (95%), 1f (92%), 1g (94%), 1h (94%),
1i (95%), 1j (89%), 1k (95%); cOverall Yield: (%): 82,76, 65, 48, 85, 74, 81, 75, 83, 69 and
83.
Figure 2. ORTEP view of (2Z, 11Z)ꢀ3,11ꢀbis(4ꢀfluorophenyl)ꢀ1,13ꢀdioxaꢀ5,9ꢀdithiaꢀ2,12ꢀ
diazacyclohexadecaꢀ2,11ꢀdiene (1e).
Anti-tumor activity studies
The antiꢀtumor activity of macrocyclic compounds (1a-k) was evaluated in-vitro using triple
negative breast cancer cells (TNBC) MDAMBꢀ231. The antiꢀtumor effects of macrocyclic
compounds were evaluated using standard antiꢀtumor assays, including wound healing assays
(Figure 3) and colonogenic assay (Figure 4), depicting effect on cancer cells metastatic and
cell division potential, respectively. For understanding the underlying molecular mechanism
involved in antiꢀtumor effects of macrocyclic compounds (1a-k), transcription levels of key
antiꢀtumor genes was measured by quantitative (q) RTꢀPCR (Figure 5). Fragmentation of
genomic DNA representing induction of apoptosis was analyzed by DNA laddering (Figure

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6) in agarose gel electrophoresis. The cytotoxic effects of these compounds (1a-k) were
measured on normal breast epithelial cells (MCF10A) (Figure 7).
Macrocycles inhibit migration of cancer cells
Tumor cells possess the property of cell migration and invasion that can be measured by
standard wound healing assay measuring effect of antiꢀtumor drugs on migration cells in
cancer cell line exꢀvivo.30 The inhibitory effect of macrocyclic compounds was evaluated in
comparison to known antiꢀcancer drug 17ꢀNꢀAllylaminoꢀ17ꢀdemthoxygeldanamycin
(17AAG). The macrocyclic compounds 1a, 1f, 1g and 1i strongly reduced migration of
TNBC MDAMBꢀ231 and healing of the wound delayed (Figure 3), compounds 1b, 1e and 1j
showed negligible effects on cell migration, while compound 1k had moderate effect on
migration of cancer cells (Figure 3). These results suggested that macrocycles 1a, 1f, 1g, 1i
and 1k are antiꢀtumor in their nature.
Figure 3: Effect of macrocyclic compounds in cell migration: Macrocyclic compounds
(1a, 1b, 1e, 1f, 1g, 1i, 1j and 1k) differentially inhibit the migration of TNBC MDAMBꢀ231
in the wound. Compounds 1a, 1f, 1g, 1i and 1k possess substantial antiꢀproliferative
properties.
Macrocycles hampers proliferation of cancer cells
The cell division potential of cancer cells was measured by colonogenic assay besides
measuring the effect of antiꢀtumor drug on survival and proliferation of cancer cells in exꢀ

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vivo system. The antiꢀproliferative effect of macrocyclic compounds (1a, 1b, 1e, 1f, 1g, 1i, 1j
and 1k) was evaluated on MDAMBꢀ231 cells. Except 1b and 1j all the tested macrocycles
possess varying degree of antiꢀproliferative potential (Figure 4), compared with positive
controls 17AAG and hydroxyl urea (HU). Macrocycles 1a, 1e, 1f, 1g, 1i and 1k effectively
inhibited colonogenic survival of MDAMBꢀ231 cells. Similar results were observed with
IMR32 cells (data not shown). In comparison to macrocycles 1e, 1f, 1g and 1k, macrocycles
1b and 1j showed least response on colonogenic survival of cancer cells, while macrocycles
1a and 1i had intermediate effect. These results suggest that macrocycles 1a, 1e, 1f, 1g, 1i
and 1k are antiꢀtumor in their nature and the results are identical to wound healing assay.
Figure 4: Effect of macrocyclic compounds on colonogenic survival of tumor-cells:
Macrocyclic compounds (1a, 1b, 1e, 1f, 1g, 1i, 1j and 1k) possess differential effect on
colonogenic survival and proliferation of MDAMBꢀ231 cells. Compounds 1e, 1f, 1g, and 1k
substantially inhibited colonogenic survival of tumor cells.
Macrocyclic compounds induces transcription of anti-tumor genes
The antiꢀtumor agents are known to induce array of tumor suppressor genes such as p53,
TRAIL, MDA7, STATꢀ3 etc. These genes exert antiꢀtumor activity by induction of apoptosis
through variety of mechanisms, upregulation of tumor suppressor protein have direct impact
on tumor cells survival.31 Transcriptional upregulation of p53, TRAIL and MDA7 was
observed for MDAMBꢀ231 cells, treated with macrocyclic compounds 1a, 1i, 1f, 1g and 1k.
Compounds 1b and 1j did not show significant induction of antiꢀtumor genes. Previous
studies clearly establish the role of TRAIL mediated induction of caspase dependent
apoptotic cell death of tumor cells.32, 33 Macrocycles compounds 1a, 1f, 1g, 1i and 1k,

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upregulate transcription of TRAIL might be leading to activation of caspase dependent
apoptotic cell death of MDAMBꢀ231 cells. Macrocycles 1a, 1i and 1k synergistically induce
transcripts for TRAIL, MDA7 and p53 (Figure 5) genes, higher than observed with
compounds 1f and 1g. The tumor suppressor gene p53 regulates transcription of TRAIL34,
also regulates MDA7 activities.35. The MDA7 is an antiꢀcancer gene, which can be induced
by retroviral mediated restriction of tumor growth, both in vivo and in vitro 35, 36. Among 8
tested macrocycles, compounds 1a, 1f, 1g, 1i and 1k induced transcription of MDA7. Both
MDA7 and TRAIL belong to ILꢀ10 family of cytokines which are known to induce apoptosis
in tumor cells in caspaseꢀ3 dependent manner that has been considered as model genes for
studying antiꢀtumor activities and gene therapy.37 Earlier report suggests that MDA7 induces
apoptotic cells death of tumor cells in type –I interferon dependent manner,31b however, we
have not found any induction of IFNꢀβ gene (data not shown). The study suggests that antiꢀ
cancer activities might be interferon independent. Further it establishes that the down
regulation of proꢀcancer gene STAT3 concurrently elevates TRAIL mediatedꢀinduction of
38
cell death and two compounds 1a and 1i follow similar trend38 (Figure 5). Induction of
STAT3 in 1f, 1g and 1k treatment groups might be antagonizing TRAIL effect and that might
be the reason for the absence of DNA laddering in groups treated with these compounds
(Figure 6).

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Figure 5: Effect of macrocyclic compounds on expression of anti-tumor genes:
Macrocyclic compounds 1a, 1i and 1k robustly induced the transcription of p53, MDA7 and
TRAIL, while 1f and 1g have moderate effect, however 1b and 1j fail to induce antiꢀtumor
genes. Expression of genes is represented in fold change where as compared solvent only
treated control groups and were measured after 24hrs treatment with respective compounds.
Macrocycles induces apoptosis in tumor cells
Among the investigated macrocyclic compounds (1a, 1b, 1e, 1f, 1g, 1i, 1j and 1k), only two
compounds 1a and 1i effectively induced DNA laddering in MDAMBꢀ231 cells (Figure 6).
The ladder like impression of genomic DNA in agarose gel represents fragmentation of DNA
into nucleosome monomers (consisting of 146bp DNA) and oligomers, a caspaseꢀ3
dependent cellular event, observed during induction of apoptosis. These results suggest that
compounds 1a and 1i induces caspaseꢀ3 mediated activation of apoptosis.
Figure 6: Effect of macrocyclic compounds on integrity of genomic DNA: Macrocyclic
compounds 1a and 1i induce genomic DNA fragmentation in MDAMBꢀ231 cells as
evidenced from observed DNA ladder like impression of genomic DNA in agarose gel.
Macrocyclic compounds have no observed cytotoxic effect on normal breast cells.
None of the investigated macrocyclic compounds 1a, 1b, 1e, 1f, 1g, 1i, 1j and 1k exerted
cytotoxic effect on breast epithelia cells (MCF10A) (Figure 7) were observed till 72 hours.
These results suggest that macrocyclic compounds 1a, 1f, 1g, 1i, and 1k, which restrict
proliferation of tumor cells (Figure 4) have no cytotoxic effect on primary cells and can be
considered for further validation in animal system.

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Figure 7: Cytotoxic activity studies on primary human breast MCF10A cells: Investigated
macrocyclic compounds 1a, 1b, 1e, 1f, 1g, 1i, 1j and 1k have no cytotoxic effect on normal
human breast epithelial cells (MCF10A).
Conclusion
We designed and synthesized a new class of small macrocyclic molecules, (2Z,11Z)ꢀ3,11ꢀ
di(aryl/naphthyl)ꢀ1,13ꢀdioxaꢀ5,9ꢀdithiaꢀ2,12ꢀdiazacyclohexadecaꢀ2,11ꢀdienes (1a-k) from
readily available substrates using simple organic transformations. These macrocycles were
evaluated for their antiꢀtumor effect on triple negative breast cancer cell line, MDAMBꢀ231.
Compounds 1a, 1f, 1g, 1i and 1k restrict cell invasion, compounds 1e, 1f, 1g, and 1k restrict
cell proliferation and colonogenic survival, compounds 1a, 1i and 1k induces TRAIL, MDA7
and p53 antiꢀtumor genes. None of the tested compound was found to be cytotoxic for normal
breast epithelial cells. Further, it would be of great interest to evaluate these macrocyclic
compounds in animal system, especially the compounds 1a and 1i to establish its effect under
physiological condition. Other notable advantages are (i) accessibility of diverse topologies
due to presence of two ‘S’ atoms that are amiable to disrupt proteinꢀprotein interactions
(PPIs) and (ii) the presence of pharmacophore cyclic oximeꢀether functionalities (C=NꢀOꢀ)
which are suitable for further ‘FG’ transformations holds the potential to access lead analogs
other than the present reported macrocyclic molecules.

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Materials and methods
General information
All commercially available starting materials, reagents and solvents were purchased from
Sigma Aldrich, Acros organics and Merck were used without further purification. Melting
points of various obtained products were determined and uncorrected. NMR spectra were
recorded on a Varian, Jeol/AL and Bruker 400 MHz. Chemical shifts were expressed in parts
per million (ppm), coupling constants were expressed in Hertz (Hz). Splitting patterns
describe apparent multiplicities and were designated as s (singlet), br s (broad singlet), d
(doublet), t (triplet), q (quartet), quintet, m (multiplet). Highꢀresolution mass spectra (MS)
and compound purity data were acquired on a Waters LCT premier XE TOF HRMS and
single quadrupole system equipped with electro spray ionization (ESI) source and Exactive
orbitrap mass spectrometer (Thermo Scientific). Single crystal XRD analysis was carried out
on a Bruker Apex ii diffractometer with CCD detector MoKα radiation (λ = 0.71069 Å)
operating ωꢀ2θ scanning mode at room temperature. Thinꢀlayer chromatography was
performed on 0.25 mm Merck silica gel plates and visualized with UV light. Column
chromatography was performed on silica gel. The structures of isolated compounds were
elucidated on the basis of spectroscopic data (1H &13CꢀNMR, HRMS) and single crystal
XRD analysis.
Cell culture
Human triple negative breast cancer (TNBC) MDAMB231ꢀcells (ATCC; cat no. HTBꢀ26™)
and neuroblastoma cell, IMRꢀ32 were grown in 10% fetal bovine supplemented (FBS)
(GIBCO, Life technologies, USA) complete Dulbecco’s modified Eagles medium (cDMEM)
(Sigma Aldrich, USA). The primary human breast epithelial cells (MCF10) were cultured in
MEBM (Lonza/clonetics MEGM Kit cat# CCꢀ3150), supplemented with 100 ng/ml cholera
toxin, 2 ml/liter bovine pituitary extract (cat# CC4009G), 0.5 ml/liter gentamicin sulfate (cat#
4081G), 0.5 ml/liter recombinant growth factor human (cat# CCꢀ4017G), recombinant
insulin human (cat# CCꢀ4021G), 0.5ml/liter hypo cortisone (cat# CCꢀ4031G). All the culture
were maintained at 37°C in humidified chamber (Thermo Fisher Scientific USA), supplied
with supplied with 5% CO2. For subculture and seeding, TNBC MDAMBꢀ231, IMRꢀ32 cells
were harvested with 0.01% trypsinꢀEDTA and MCF10A with 0.05% trypsinꢀEDTA, setting
the cells density as per experimental requirements.

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Wound healing assay
For wound healing assay TNBC MDAMBꢀ231 cells were seeded in six well plate, at density
of 1 x 106 cells/well in cDMEM. The 95% monolayer of cells carefully wounded using 20ꢀ
200 µl micro tips. Cells were washed with DMEM for removing cell debris. Wounded
monolayer of cells was treated with 1a, 1b, 1e, 1f, 1g, 1i, 1j and 1k (5µM each) for 24 hrs.
17AAG (5 µM) and solvent only treated cells were used as positive and negative control.
Migration of cells into wounded area was captured by phase contrast microscope (Zeiss
Axiovert model) at 0 hr and 24 hrs time points.
Colonogenic assay
The antiꢀproliferative effect of macrocycles (1a, 1b, 1e, 1f, 1g, 1i, 1j and 1k) were tested by
this assay. TNBC MDAMBꢀ231 cells were seeded in six well plates at density of 1 x 103
cells/well and left in culture for 10 days for formation of colonies. Than cells were treated
with test macrocyclic compounds 1a, 1b, 1e, 1f, 1g, 1i, 1j and 1k (5µM each), positive
control 17AAG (5µM) or hydroxyl urea (1mM) for 24 hrs. Followed by cells were washed
with 1XꢀPBS and fixed using 70:30 solution of ethanol acetic acid for 10 minutes. Fixed cells
were stained with 1% methylene blue for 15 min. Extra stain was drained by washing with
water. Dried plates were scanned for representation of effect of macrocycles on colonogenic
survival of cells.
Quantitative-PCR analysis
The MDAMBꢀ231 cells were seeded in six well plate at density of 0.2 x 106. Next day, cells
were treated with macrocycles 1a, 1b, 1e, 1f, 1g, 1i, 1j and 1k (5µM each) for 24 hrs. The
cells were washed with 1XꢀPBS and total RNA was extracted TRIzol reagent (Invitrogen
USA) and first strand cDNA was synthesized for 1µg RNA, using iScriptꢀcDNA synthesis kit
(BIORAD USA), according to manufacturers instruction. Relative quantification of antiꢀ
tumor genes (p53, MDA7, and TRAIL) and transcription factor STAT3 was performed with
SYBR green master mix, using 18S as housekeeping control. Following, initial denaturation
step at 95°C for 10 min, 40 cycles of PCR amplification were performed with denaturation
for 15 sec and annealing at 60°C for 1 min. In order to confirm amplification as single
specific product, melt curve analysis was done as 95°C for 15 sec, 60°C for 1 min, 95°C for
15 sec. The sequences of primer used in RNA quantification is given is Table 1.

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Table 1: Primer used for quantitative PCR
Primer Direction Sequence
Size bp Amplicon
S18
Forward
Reverse
Forward
Reverse
ATCACCATTATGCAGAATCCACG
GACCTGGCTGTATTTTCCATCC
GCCATCTACAAGCAGTCACAG
TCATCCAAATACTCCACACGC
AGCAATGCCACTTTTGGAGT
TTCACAGTGCTCCTGCAGTC
TTTGTCAGCGATGGAGTACG
TGTTGACGGGTCTGAAGTTG
GAGGAACACGAGACTGAGAG
TCCAGAGAAGCAGGGTAAAAC
23
22
21
21
20
20
20
20
20
21
93 bp
P53
143 bp
120 bp
168 bp
116bp
TRAIL Forward
Reverse
STAT3 Forward
Reverse
MDA7 Forward
Reverse
DNA fragmentation assay
Human TNBC MDAMBꢀ231 and IMR32 cells were seeded in 6 well culture dish at density
of 0.2 × 106 cells/well. Next day cells were treated with macrocyclic compounds 1a, 1b, 1e,
1f, 1g, 1i, 1j and 1k. Followed to 48 hrs incubation, cells were harvested by trypsinꢀEDTA
and pellet down at 200g for 2 min. Cells washed with 1XꢀPBS and reꢀsuspended lysis buffer
(10 mM TrisꢀCl: pH 8.0, 10 mM: EDTA, 0.5% Triton Xꢀ100) and nuclei pellet was collected
at 13000g for 20 min at 4°C. Obtained nuclei pellet, reꢀsuspended in lysis buffer and treated
DNAseꢀfree RNAse (0.1 mg/ml) for 1 h at 37°C. Then proteinase K (0.2 mg/ml) and SDS
(final concentration of 1%) were added and a resultant mixture was incubated further for 2 h
at 50°C. DNA from these samples was extracted using phenol chloroform isoamyl alcohol
(25:24:1). DNA precipitation was done using 3.5 volume of 100% ethanol and 0.1 volume of
CH3COOK (pH 5.2) at ꢀ80°C. The DNA pellet was resuspended in 40µl of MQ and equal
quantity of DNA was analyzed in 2% agarose gel.
Cell cytotoxicity assay
The cytotoxic effect of newly synthesized macrocyclic compounds 1a, 1b, 1e, 1f, 1g, 1i, 1j
and 1k was analyzed on human breast epithelial cells MCF10A. Cells were seeded in 24 well
plates at density of 0.5 x 106 cells/well, cultured till >90% cell density and treated with
macrocyclic compounds 1a, 1b, 1e, 1f, 1g, 1i, 1j and 1k (200 µM each) for 24hrs. Cells
micrographs were taken before and after treatment at 0h and 24h time points using phase
contrast microscopy.

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General experimental procedure
Synthesis of 2-bromo-1-(4-chlorophenyl)ethanone (4a): In a 100ꢀmL two necked roundꢀ
bottom flask, 1ꢀ(4ꢀchlorophenyl)ethanone (2a) (10.0 mmol), Nꢀbromosuccinimide (3) (12.0
mmol), 10% (w/w) Silica gel in methanol (20 mL) were stirred under reflux conditions for 19
min. The progress of the reaction was monitored by TLC. After completion of the reaction,
the stirring was stopped and cooled to RT. The solvent was removed under reduced pressure
using rotary evaporator. To the crude product dichloromethane and distilled water were
added. The organic layer was collected and again washed with water (2x50 mL) twice. The
layers were separated and the organic layer was collected and it was dried using anhydrous
Na2SO4. Again, the solvent was evaporated using rotary evaporator. The crude was purified
over column chromatography using Silica gel (99:1 ratio of nꢀhexane and EtOAc) and pure
product 3a was obtained in 91% yield. The same experimental procedure was adopted for the
preparation of other αꢀbromoketones (4b-k).
Synthesis of 2,2'-(propane-1,3-diylbis(sulfanediyl))bis(1-(4-chlorophenyl)ethanone (6a):
In a 100ꢀmL two necked round bottom flask, 2ꢀbromoꢀ1ꢀ(4ꢀchlorophenyl)ethanone (4a) [10.0
mmol] in acetonitrile (15 mL) was taken. The temperature of the reaction mixture was cooled
to 15ꢀ20oC and then K2CO3 (30 mmol) was added. Then, the propaneꢀ1,3ꢀdithiol (5)[5.0
mmol] was taken in acetonitrile (15 mL) and it was added drop wise using dropping funnel at
15ꢀ20oC. The reaction mixture was stirred for 90 min at the same temperature up to the
disappearance of the substrate (4a). The completion of the reaction was confirmed by TLC.
Then, the reaction mass was filtered and then the filtrate was concentrated under reduced
pressure. The obtained crude product (6a) was purified by column chromatography using
silica gel (4:1 ratio of nꢀhexane and ethyl acetate). The same experimental procedure was
adopted for the preparation of other 2,2’ꢀ(propaneꢀ1,3ꢀdiylbis(sulfanediyl))bis(1ꢀ
aryl/naphthyl)ethanone derivatives (6b-k).
Synthesis
of
(1Z,1'Z)-1-(4-chlorophenyl)-2-(3-((Z)-2-(4-chlorophenyl)-2-
(hydroxyimino)ethylthio)propylthio)ethanoneoxime (8a): In a 100ꢀmL two necked round
bottom flask, substrate (6a) [10.0 mmol] and hydroxyl ammonium chloride (NH2OH.HCl) (7)
[40.0 mmol] were dissolved in ethanol (30 mL) and then NaOAc (30.0 mmol w.r.t
NH2OH.HCl) was added. Then, the reaction mixture was stirred at reflux temperature for 2 h.
The progress of the reaction was monitored by TLC. After completion of the reaction,
reaction mixture was cooled to RT and then filtered, washed thoroughly with ethanol. The
filtrate was collected and the solvent was removed under reduced pressure. To the crude

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reaction mass, water (50 mL) was added and the product (8a) was extracted with EtOAc
(2x25 mL). Then organic layer was collected and washed with water (2x25 mL) twice.
Again, the organic layer was collected and dried over anhydrous Na2SO4 and the solvent was
removed under reduced pressure. The crude product (8a) was recrystallized from the ethanol
(10 mL) and nꢀhexane (30 mL) mixture. The same experimental procedure was followed for
the preparation of other ketoxime derivatives (8b-k).
Synthesis
of
(2Z,11Z)-3,11-bis(4-chlorophenyl)-1,13-dioxa-5,9-dithia-2,12-
diazacyclohexadeca-2,11-diene (1a): In a 100ꢀmL two necked round bottom flask,
compound 8a (10.0 mmol) was dissolved in DMSO (15 mL). The reaction mass was cooled
to 5 to 10°C. At this temperature, aqueous KOH solution (20.0 mmol of KOH was dissolved
in 5 mL of water) was added slowly and stirred at 5 to 10°C for 15 min. TLC was checked to
confirm the disappearance of substrate (it convert to dipotassium salt form). Then, 1,3ꢀ
dibromopropane (9) [10.0 mmol] was taken in 10 mL of DMSO and was added slowly at 5 to
10°C and the reaction mass was stirred at the same temperature for 10 min. The reaction
progress was monitored by TLC. After completion of the reaction, the reaction mixture was
quenched in crushed Ice (100 mL) and then the product (1a) was extracted with diethyl ether
(2x50 mL) twice and the solvent was evaporated under reduced pressure. The obtained crude
product (1a) was purified by column chromatography using silica gel (99:1 ratio of nꢀhexane
and ethyl acetate). The same experimental procedure has been followed for the preparation of
other macrocyclic compounds (1b-k). The synthesized compounds were characterized by 1H,
13C NMR and HRMS spectral data and the structure of compound 1e was confirmed by
single crystal XRD data.
Supporting information:
Physical and spectral characterization data and single crystal XRD analysis data are available
at
Single crystal XRD data:
Crystallographic data of macrocycle 1e was deposited in CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK, CCDC 1046587. The detailed information and copies can be obtained free of
charge at http://www.ccdc.cam.ac.uk.
Acknowledgments:
The authors acknowledge the financial support for this work from the Council of Scientific
and Industrial Research (CSIR), New Delhi, Government of India under a major research
project (No. 01 (2391)/10/EMRꢀII) and Department of Atomic EnergyꢀBoard of Research in

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Nuclear Sciences (DAEꢀBRNS), Mumbai, Government of India through a major research
project (No. 2011/37C/52/BRNS/2264). The study related to the analysis of biological
activities is also supported by research grants number SR/S2/RJNꢀ55/2009,
BT/PR6009/GBD/27/382/2012 from Department of Science and Technology (DST) and
Department of Biotechnology (DBT), Government of India respectively. Authors also thank
Dr. R.V.J. Kashyap, Dept. of English, Yogi Vemana University, for critical reading of the
manuscript to check the linguistic corrections.
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Discovery of a new class of 16-membered (2Z,11Z)-3,11-di(aryl/naphthyl)-1,13-dioxa-5,9-
dithia-2,12-diazacyclohexadeca-2,11-dienes as novel anti-tumor agents
Mohan Reddy Bodireddy, Ranjeet Singh Mahla, P. Md. Khaja Mohinuddin, G.Thrivikram Reddy, D. Vijaya
Raghava Prasad, Himanshu Kumar,* N. C. Gangi Reddy*
Abstract: A series of new 16-membered macrocyclic compounds were synthesized from readily
available substrates using simple organic transformations. Evaluation of in vitro anti-tumor
activities on MDAMB-231 cell lines revealed that macrocycles (1a, 1f, 1g, 1i and 1k) are
promising anti-tumor compounds, evidenced from inhibition of cell migration and proliferation,
upregulation of anti-tumor genes p53, MDA7 and TRAIL.

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132x60mm (300 x 300 DPI)