Bioorganic & Medicinal Chemistry Letters 24 (2014) 1366–1372

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Bioorganic & Medicinal Chemistry Letters

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Synthesis and in vitro anti-proliferative effects of 3-(hetero)arylsubstituted 3-[(prop-2-ynyloxy)(thiophen-2-yl)methyl]pyridinederivatives on various cancer cell lines

N

OS

Hetaryl

cf ref7 anticancerproperties

ref 2-6

newanticanceragents?

OH

A B

Hetaryl

C

Ar2

Ar1

Figure 1. Design of novel small molecules based on prop-2-ynyloxy fram

http://dx.doi.org/10.1016/j.bmcl.2014.01.0440960-894X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel.: +91 40 6657 1500; fax: +91 40 6657 1581 (M.P.).E-mail address: manojitpal@rediffmail.com (M. Pal).

Upendar Reddy Chamakura a, E. Sailaja b, Balakrishna Dulla a, Arunasree M. Kalle c, S. Bhavani d,D. Rambabu e, Ravikumar Kapavarapu f, M. V. Basaveswara Rao b,⇑, Manojit Pal a,⇑a Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Hyderabad 500046, Andhra Pradesh, Indiab Department of Chemistry, Krishna University, Machilipatnam 521001, Andhra Pradesh, Indiac Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad 500046, Andhra Pradesh, Indiad Department of Chemistry, K.L. University, Vaddeswaram, Guntur 522502, Andhra Pradesh, Indiae Department of Chemistry and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be’er-Sheva 84105, Israelf Doctoral Programme in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal

a r t i c l e i n f o

Article history:Received 19 November 2013Revised 29 December 2013Accepted 14 January 2014Available online 26 January 2014

Keywords:AlkyneCouplingPd/CCancerAnti-proliferation

a b s t r a c t

A series of 3-(hetero)aryl substituted 3-[(prop-2-ynyloxy)(thiophen-2-yl)methyl]pyridine derivativeswere designed as potential anticancer agents. These compounds were conveniently prepared by usingPd/C–Cu mediated Sonogashira type coupling as a key step. Many of these compounds were found tobe promising when tested for their in vitro anti-proliferative properties against six cancer cell lines. Allthese compounds were found to be selective towards the growth inhibition of cancer cells with IC50 val-ues in the range of 0.9–1.7 lM (against MDA-MB 231 and MCF7 cells), comparable to the known antican-cer drug doxorubicin.

� 2014 Elsevier Ltd. All rights reserved.

ework.

Cancer remains the second leading cause of death1 worldwideafter cardiovascular diseases, according to WHO. Indeed, leukemia,neuroblastoma [a malignant (cancerous) tumor that develops fromnerve tissue] and hepatocarcinoma (a primary malignancy of theliver) along with colon, and breast cancers cause the most cancerdeaths worldwide each year. Thus, it is highly desirable to discoverand develop suitable agents that are promising for the potentialtreatment of various types of cancer especially the breast cancer.Since the antiproliferative and cytotoxic agents play a major rolein cancer therapy whether used alone or in combination with othertreatment options (e.g. surgery, radiation and biological therapy)discovery and development of such agents have attracted enor-mous interest among medicinal chemists over the years.

Alkynes possessing a heteroaryl substituent for example uracil,2

pyrone,3 purin,4 adenosine,5 quinolines6 etc. have been explored aspotential anticancer agents. Some of them for example 5-ethynyluracil was identified as a potential anti-cancer drug and underwentclinical trials. The diphenylmethanol derivatives7 on the other

hand have also been explored as potential antiproliferative agents.These reports and our continuing interest in identification of po-tential anti-cancer agents prompted us to build a library of smallmolecules based on prop-2-ynyloxy framework (Fig. 1). We envi-sioned that combining the structural features of diarylmethanol

U. Reddy Chamakura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1366–1372 1367

(A) and alkyne (B) in a single molecule might provide a templatefor the discovery of novel anticancer agents. Herein, we reportour initial findings on the synthesis and in vitro pharmacologicalevaluation of a series of 3-(hetero)aryl substituted 3-[(prop-2-ynyloxy)(thiophen-2-yl)methyl]pyridine derivatives C. To the bestof our knowledge the synthesis and in vitro pharmacological prop-erties of this class of compounds have not been explored earlier.

While the library model of target compounds, as shown in Fig-ure 1, has several centers for the introduction of diversity into thecompound C our major focus however was particularly on the aryl/heteroaryl substituents attached to the alkynyl moiety as thisplayed a key role in anticancer activities of various 5-alkynyl uracilderivatives as inhibitors of thymidylate synthease.2 We thereforewere in need of a convenient, straightforward, and inexpensivemethodology to prepare our target molecules. The alkynylationof (hetero)arene via the Sonogashira coupling8 has been termedas a booming methodology9 in organic synthesis because of itsremarkable applications in C–C bond forming reactions underPd–Cu catalysis. While this methodology has been employed forthe preparation of a large variety of internal alkynes its applicationfor the synthesis of C or similar class of compounds are not com-mon in the literature. The use of Pd/C–CuI–PPh3 as a less expensivecatalyst system for efficient Sonogashira coupling has been ex-plored earlier.10 The advantages associated with the use of catalystPd/C are that it is stable and easy to handle as well as separablefrom the product. Moreover, it is recyclable.10a Due to the simplic-ity, advantages and versatility of this methodology we decided touse the Pd/C-based methodology as a key step for the synthesisof our target compounds C or 3 (Scheme 1). The synthesis of fewother analogues of compound 3 (e.g. 6 and 7) was also undertaken(Scheme 1).

The starting alkyne that is 2-chloro-3-[(prop-2-ynyloxy)(thio-phen-2-yl)methyl]pyridine (1) required for the synthesis of com-pound 3 was prepared following a two-step method as shown inScheme 1. The Grignard reagent prepared from 2-bromothiophenewas reacted with 2-chloro pyridine-3-carboxaldehyde (4) to afford(2-chloropyridin-3-yl)(thiophen-2-yl)methanol11 (5) which onpropargylation with propargyl bromide furnished the desired al-kyne 1. The alkyne 1 was then reacted with iodoarenes or iodohe-teroarenes (2) in the presence of 10% Pd/C, PPh3, CuI andtriethylamine in EtOH to give 3-(hetero)aryl substituted 3-[(prop-2-ynyloxy)(thiophen-2-yl)methyl]pyridine derivatives (3)(Table 1).

The reactions proceeded well irrespective of the presence ofgroups like I, NO2, OH, NH2, CO2H or MeO (Table 1, entries 1–7),on the aryl ring of 2 affording the desired compound 3. In case of

N Cl

H

O

N Cl

OH

S

4 5

Mg, LiCl, THF0oC, 30 minrt, 30 min75%

S BrBr

K2CO3, acetort, 12h90%

N

O

S

Cl

K2CO3

Acetonert, 12h90%

BrCH2CO2Et

Scheme 1. Pd/C-mediated synthesis of 3-(hetero)aryl substituted 3-[(prop-2-ynyloxy)(thcompound 3.

2-iodobenzoic acid (2e) the reaction afforded a low yield of the de-sired product (3e) along with a mixture of side products perhapsgenerated due to the intramolecular cyclization of 3e.12,13 Simi-larly, the formation of side product that is 1,4-alkynyl substitutedbenzene derivatives (3l) due to the Sonogashira coupling14 at C-1and C-4 of 2a was also observed under the reaction conditionstested and was isolated in low yield (�20%). The use of iodo hetero-arenes (Table 1, entries 8–10) or 5-iodoindoline-2,3-dione (Table 1,entry 11) was also successful. Moderate to good yields of productswere obtained in all these cases except in case of 2e (Table 1, entry5).15 It is worthy to mention that 5–10% of dimeric product of al-kyne 1 that is 1,6-bis[(2-chloropyridin-3-yl)(thiophen-2-yl)-methoxy]hexa-2,4-diyne (3m) was isolated as a side product inall the cases that is known to form via Glaser coupling as a sidereaction.

Having prepared a number of 3-(hetero)aryl substituted 3-[(prop-2-ynyloxy)(thiophen-2-yl)methyl]pyridine derivatives (3)we then decided to synthesize few of its other analogues requiredfor the in vitro pharmacological studies (Scheme 1). Thus the alco-hol 5 was reacted with ethyl bromoacetate in the presence ofK2CO3 to give the ester 6 which on hydrolysis afforded the corre-sponding acid 7.

All the target compounds 3 prepared based on the template C(Fig. 1) were then evaluated for their potential anti-cancer proper-ties in vitro. The cells used for our in vitro studies include humanmetastatic breast cancer cells that is MDA-MB 231, human chronicmyeloid leukemia cells that is K562, human colon carcinoma cellsthat is Colo-205, human neuroblastoma cells that is IMR-32, andnon-cancerous human embryonic kidney cells that is HEK293.The effect of test compounds on cell viability was measured usinga colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide] assay16 after 24 h of treatment in culture med-ium containing PBS. The percentages of cell viability for mostpromising test compounds at 10 lM are presented in Table 2.The compounds that showed >50% activities were considered asactive. It is evident from Table 3 that compounds 3f, 3h–k showedsignificant activities against K562 Leukemia cells whereas 3a, 3c,3d, 3g, and 3k showed encouraging anti-proliferation againstMDA-MB 231 cells. Compounds 3c, 3d, 3g, and 3k also showedanti-proliferation against MCF7 cells. The compounds 3a, 3b and3k were found to be active against IMR-32 cells whereas 3a wasfound to be the only compound that showed >50% anti-prolifera-tion against HepG2 cells. Notably, all these compounds were foundto be selective towards the growth inhibition of cancer cells asnone of these compounds showed any significant effects whentested against HEK293 cells. We also tested the compounds 5, 6

N

Cl

OS

1

ne 10% Pd/CCuI, Et3NPPh3, EtOHreflux

ArI (2)

N

Cl

OS

3

Ar

OEt

ON

O

S

Cl

OH

O6 7

LiOH.H2OTHF-H2O

25 oC, 2.5 h81%

iophen-2-yl)methyl]pyridine derivatives (3) and the synthesis of other analogues of

Table 1Synthesis of 3-(hetero)aryl substituted 3-[(prop-2-ynyloxy)(thiophen-2-yl)methyl]pyridine derivatives (3)

N Cl

O

S

1

10% Pd/CCuI, Et3N

PPh3, EtOHreflux

N Cl

O

S

3

+ I (Het)Ar

2

(Het)Ar

Entry Anilides (2) Products (3) Yielda (%)

1

I

I

2a

N

O

S

ClI

3a

75b

2

INO2

2b N

O

S

Cl

NO2

3b

88

3

IOH

2c N

O

S

Cl

OH

3c

83

4

INH2

2d N

O

S

Cl

NH2

3d

79

5

ICO2H

2e N

O

S

Cl

COOH

3e

26

6

I

CO2H

2f

N

O

S

ClCOOH

3f

76

1368 U. Reddy Chamakura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1366–1372

Table 1 (continued)

Entry Anilides (2) Products (3) Yielda (%)

7

I

OMe

2g

N

O

S

ClO

3g

92

8

S

O

O

I

2hN

O

S

ClS

O

O3h

89

9I

N NH

O O

2i

N

O

S

ClN NH

O O

3i

84

10

SO

O

I

2j

3j

N

O

S

ClSO

O

85

11

I

NH

O

O

2kN

O

S

ClNH

O

O

3k

88

a Isolated yields.b 1,4-Bis(3-[(2-chloropyridin-3-yl)(thiophen-2-yl)methoxy]prop-1-ynyl)benzene (3l) was isolated as a side product in this case.

U. Reddy Chamakura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1366–1372 1369

and 7 in vitro using the cell lines as mentioned above. However,none of these compounds showed significant anti-proliferationagainst these cells indicating the possible key role played by the(hetero)aryl alkynyl moiety in compound 3 in their in vitroactivities.

We then determined the IC50 values (Table 3) of some selectedcompounds for example 3c, 3d, 3g and 3k based on their promisingin vitro data against MDA-MB 231 and MCF7 cells (�70% inhibitionin growth of these cells) and compared with standard drug doxoru-bicin17 which is primarily used to treat breast cancer. The Table 3indicated that the compound 3d and 3k were marginally betterthan other compounds in terms of IC50 values and comparable tothat of doxorubicin against MDA-MB 231 cells. While all thesecompounds showed similar IC50 values against MCF7 cells theywere found to be slightly less effective than doxorubicin in thisin vitro assay. Nevertheless, the present class of compoundsseemed to have potential medicinal value18 as the use of doxorubi-cin is associated with the adverse side effects17 including cardiactoxicity.

A series of purine derivatives possessing alkynyl moiety at C-2have been reported as potent inhibitors of cyclin-dependent

kinases (CDKs).19 The structural studies of two most active com-pounds suggested that the alkynyl moiety played an important rolein their inhibitory activities by occupying the ribose binding pocketof ATP. This was further supported by the fact that a partial or com-plete reduction of the alkynyl moiety affected the activities ofthese compounds.18a These reports prompted us to assess theCDK inhibitory properties of the alkynes 3d and 3k in silico. TheCDK2 protein in complex with the known lead inhibitor roscovitine(PDB code-2A4L) was used as the receptor for docking studies andthe molecular docking simulation was performed with the Chemi-cal Computing Group’s Molecular Operating Environment (MOE)software 2008.10 version, ‘DOCK’ application module. Both 3dand 3k were docked into the CDK2 protein and their respectivedocking scores and interactions were observed (Fig. 2, see alsoESI). The dock score of 3d (�24.17 K cal/mol) was comparable withthat of roscovitine (�25.61 K cal/mol) and marginally better than3k (�21.90 K cal/mol). The study also indicated that both 3d and3k utilized the conserved active binding site of ATP pocket inCDK2 protein complex and participated in H-bonding interactionsmainly with the active site residue for example Leu83 and Lys89,respectively. Indeed, the –NH group of 3d formed H-bond with

Table 2In vitro antiproliferative properties of compounds 3 against various cells

Compounds % Inhibition in growth of cancer cell lines by compounds 3 and 5 at 10 lMa

K562Leukemia

Colo-205Colon

MDA-MB 231 ER –vebreast cancer

MCF7 ER +ve breastcancer

IMR-32Neuroblastoma

HepG2 Hepato-carcinoma

HEK293b Non-cancerous

N Cl

O

S

Ar

3 ; Ar =3a; 48.0 39.0 52.2 45.6 60.0 54.2 0C6H4I-p3b; 18.9 16.5 48.0 34.8 53.2 46.2 0.1C6H4NO2-o3c; 40.6 28.5 70.8 67.7 34.4 13.6 6C6H4OH-o3d; 43.8 37.8 73.3 69.3 47.6 36.2 3.2C6H4NH2-o3e; 40.2 34.7 47.9 35.9 38.1 33.1 2.7C6H4CO2H-o3f; 57.7 44.0 38.9 35.9 39.6 38.2 4.8C6H4CO2H-p3g; 33.8 31.6 70.1 64.7 20.7 13.9 �1.1C6H4OMe-p

3h;

S

EtO2C

53.0 32.1 46.4 31.8 22.5 29.5 �0.9

3i;

N NH

CO2Et54.8 45.8 35.3 33.8 30.9 34.3 0.4

3j;

SEtO2C

65.1 46.0 37.9 25.0 25.2 21.9 �1.6

3k;

NH

O

O

61.1 49.3 72.3 71.9 58.4 44.8 0.8

a Data represent the mean values of three independent determinations.b HEK293 cell line was used as non cancerous cell line.

Table 3IC50 values of compound 3

Compound IC50a (lM)

MDA-MB 231 MCF7

3c 1.10 ± 0.29 1.51 ± 0.153d 0.95 ± 0.14 1.29 ± 0.213g 1.16 ± 0.21 1.72 ± 0.183k 0.99 ± 0.11 1.05 ± 0.19Doxorubicin 0.65 ± 0.37 0.44 ± 0.13

a IC50 represent the concentration of compound that causes a 50% growth inhi-bition to untreated cells using an MTT assay.

1370 U. Reddy Chamakura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1366–1372

Leu83 whereas the C@O groups of 3k formed H-bond with Lys89.Overall, the docking results suggested that CDK could be the possi-ble molecular target for this class of molecules.

In summary, a series of 3-(hetero)aryl substituted 3-[(prop-2-ynyloxy)(thiophen-2-yl)methyl]pyridine derivatives were designedand explored as new and potential anti cancer agents. All these com-pounds were synthesized by using Pd/C–Cu mediated Sonogashiratype coupling as a key step. Thus the starting alkyne that is 2-chloro-3-[(prop-2-ynyloxy)(thiophen-2-yl)methyl]pyridine pre-pared via a two-step method was reacted with a range of iodo are-nes/heteroarenes in the presence of 10% Pd/C, PPh3, CuI andtriethylamine in EtOH to give the desired products. Six cancer celllines including human chronic myeloid leukemia cells (K562), hu-man colon carcinoma cells (Colo-205), breast cancer cells (MDA-MB 231 and MCF7), human neuroblastoma cells (IMR-32) and hep-ato-carcinoma (HepG2) as well as noncancerous cell line for exam-ple HEK293 were used to evaluate anti-proliferative properties ofthese compounds in vitro. These compounds were found to be selec-tive towards the growth inhibition of cancer cells with IC50 values in

the range of 0.9–1.7 lM (against MDA-MB 231 and MCF7 cells),comparable to the known anticancer drug doxorubicin. The possiblerole of (hetero)aryl alkynyl moiety in anti-proliferative properties ofthese compounds is also indicated. Overall, the alkyne frameworkdescribed here could be an interesting template for the identifica-tion of small molecule based potential anticancer agents and thePd/C–Cu based synthetic strategy could be handy for this purpose.

Figure 2. Binding mode of 3d in the active site of CDK2 protein (PDB code-2A4L).

U. Reddy Chamakura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1366–1372 1371

Acknowledgment

The authors thank management of DRILS for encouragementand support.

Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.01.044.

References and notes

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21, 1681.15. Synthesis of compound 3: A mixture of iodo compound 1 (0.33 mmol), 10% Pd/C

(0.0022 mmol), PPh3 (0.0086 mmol), CuI (0.022 mmol) and Et3N (0.54 mmol)in EtOH (5 mL) was stirred for 30 min under a nitrogen atmosphere. To thismixture was added the terminal alkyne 2 (0.33 mmol) slowly and the mixturewas refluxed for 12 h. The progress of the reaction was monitored by TLC. Aftercompletion of the reaction the mixture was cooled to room temperature andfiltered through celite. The ethanol layer was collected and concentrated underreduced pressure. The residue was diluted with water (50 mL) and extracted

with ethyl acetate (3 � 25 mL). The organic layers were collected, combined,washed with water (2 � 25 mL), dried over anhydrous sodium sulphate,filtered and concentrated. The residue thus obtained was purified by columnchromatography on silica gel to afford the desired compound.

16. MTT assay: Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (5 � 103 cells/well) wereseeded to 96-well culture plate and cultured with or without compounds at10 lM concentration (five different concentrations i.e., 10, 5, 1, 0.5, 0.1 and0.01 lM for dose response study) in duplicates for 24 h in a final volume of200 ll. After treatment, the medium was removed and 20 ll of MTT (5 mg/mlin PBS) was added to the fresh medium. After 3 h incubation at 37 �C, 100 ll ofDMSO was added to each well and plates were agitated for 1 min. Absorbancewas read at 570 nm on a multi-well plate reader (Victor3, Perkin Emler).Percent inhibition of proliferation was calculated as a fraction of control(without compound).

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18. In a preliminary study we evaluated the mode of cell death and cell cycle arrestin breast cancer cell lines (MCF-7 and MDA-MB-231) by the compound 3d.Thus the compound 3d was assessed for its effects on the cell cycle of MCF-7and MDA-MB-231 cells at different concentrations. More than 20% increase inthe population of MCF-7 cells at the G2/M phase as compared to the controlgroup after treatment with 5 lM of 3d for 24 h was observed. On the otherhand a 20–50% increase in the accumulation of MDA-MB-231 cells at the sub-G1/cell death phase without cell cycle arrest after treatment with 3d (2.5–10 lM) as compared to the control group indicated induction of cell deaththrough apoptosis.

19. (a) Legraverend, M.; Ludwig, O.; Bisagni, E.; Leclerc, S.; Meijer, L. Bioorg. Med.Chem. Lett. 1998, 8, 793; (b) Legraverend, M.; Tunnah, P.; Noble, M.; Ducrot, P.;Ludwig, O.; Grierson, D. S.; Leost, M.; Meijer, L.; Endicott, J. J. Med. Chem. 2000,43, 1282.