Bioorganic & Medicinal Chemistry Letters 23 (2013) 6816–6821

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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Amino acids derived benzoxazepines: Design, synthesis and antitumor activity Shailendra Kumar Dhar Dwivedi a,⇑, Krishnananda Samanta c, Manisha Yadav b, Amit Kumar Jana c, Abhishek Kumar Singh b, Bandana Chakravarti a, Sankalan Mondal c, Rituraj Konwar a, Arun Kumar Trivedi b, Naibedya Chattopadhyay a, Sabyasachi Sanyal b, Gautam Panda c,⇑ a b c

Division of Endocrinology, CSIR-Central Drug Research Institute, 10 Janakipuram Extension, Sitapur Road, Lucknow 226031, UP, India Division of Drug Target Discovery and Development, CSIR-Central Drug Research Institute, 10 Janakipuram Extension, Sitapur Road, Lucknow 226031, UP, India Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, 10 Janakipuram Extension, Sitapur Road, Lucknow 226031, UP, India

a r t i c l e

i n f o

Article history: Received 23 April 2013 Revised 23 September 2013 Accepted 5 October 2013 Available online 11 October 2013 Keywords: Amino acids Benzoxazepine Mitsunobu reaction Anticancer activity Capasase

a b s t r a c t Two series of new benzoxazepines substituted with different alkyl amino ethyl chains were synthesized comprising synthetic steps of inter and intramolecular Mitsunobu reaction, lithium aluminium hydride (LAH) reduction, debenzylation, bimolecular nucleophilic substitution (SN2) reaction. The present study investigates the effect of a tyrosine-based benzoxazepine derivative in human breast cancer cells MCF7 and MDA-MB-231 and in breast cancer animal model. The anti-proliferative effect of 15a on MCF-7 cells was associated with G1 cell-cycle arrest. This G1 growth arrest was followed by apoptosis as 15a dose dependently increased phosphatidylserine exposure, PARP cleavage and DNA fragmentation that are hallmarks of apoptotic cell death. Interestingly, 15a activated components of both intrinsic and extrinsic pathways of apoptosis characterized by activation of caspase-8 and -9, mitochondrial membrane depolarization and increase in Bax/Bcl2 ratio. However, use of selective caspase inhibitors revealed that the caspase-8-dependent pathway is the major contributor to 15a-induced apoptosis. Compound 15a also significantly reduced the growth of MCF-7 xenograft tumors in athymic nude mice. Together, 15a could serve as a base for the development of a new group of effective breast cancer therapeutics. Ó 2013 Elsevier Ltd. All rights reserved.

Breast cancer is the most frequently diagnosed life-threatening disease in women and more than 1 million women are diagnosed with breast cancer every year, accounting for 10% of all new cancers and 23% of all female cancer cases. In India the prevalence of this cancer is estimated around 2.5 million, with over 0.8 million new cases and 0.5 million deaths occurring each year.1 Current available therapies suffer from several drawbacks including resistance and toxic side effects.2,3 We have previously shown that the benzoxazepine derivatives are active against breast cancer and are non-toxic in normal cell lines tested4 the present study is designed to investigate the effect of new compounds synthesized as lead optimization process. Benzoxazepines are well-known pharmacophore in medicinal chemistry with promising activity against various diseases such as antipsychotic, central nervous system activity along with anticancer profile against breast cancer cells.5 One of these groups of benzoxazepines has been identified to target microtubule assembly to induce anti-cancer activity.5 In our previous study, we have synthesized and explored the amino acid based benzoxazepines6a–d

⇑ Corresponding authors. E-mail address: [email protected] (G. Panda). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.013

OBn

TsHN

+ OH

R1

R

3, 4, 5

c CH 2OH R

OBn Ts N

R1

2

OBn Ts N 6, 7, 8

a

R

1 R1 = H, OMe

1

CO 2Me

R1

OH Ts N 9, 10, 11

d

R O

R1 CH2OH R

b CO 2Me

R NTs

12, 13a-b, 14

Scheme 1. Reagents and conditions: (a) 1, 2, DEAD, PPh3, THF, 0 °C (2 h) to rt (10 h), N2, 60–75%; (b) LAH, THF, 0 °C, 1 h, 59–70%; (c) H2, 10% Pd/C, MeOH, rt, 2 h, 50 psi, 61–70%; (d) DEAD, PPh3, THF, 0 °C (1 h) to rt, (14 h), N2, 63–76%.

with antitumor activity. Thus, in search of new lead molecule, we synthesized two different series of benzoxazepine (A, B) from amino acids. Easy availability of amino acid-based benzoxazepines promoted us to evaluate them for anticancer activity. We followed the same methodology developed in our laboratory for the synthesis of benzoxazepines (Scheme 1). N-tosylated S-amino acid esters and substituted benzyl alcohol were used as building blocks for the construction of benzannulated chiral heterocycles. The intermolecular Mitsunobu reaction of S-amino acid

6817

S. K. D. Dwivedi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6816–6821 OR2

OH O

O

a R1

R1

Table 1 IC50 (lM) value of benzoxazepine series of compounds in breast cancer cell lines using MTT assay after 24 h treatment

NTs

NTs

OR2

15a-b 16a-e

12 14

O R1 NR

Scheme 2. Reagents and conditions: (a) anhyd K2CO3, R2Cl, dry acetone, reflux, 5 h, 60–70%.

15a

Ts

H

15b

Ts

H

R2

R2 = alkyl amino ethyl chain

13a

Figure 1. Two different types of benzoxazepine.

Br

OH

OBn H N

b

OBn

OBn a

ClH . H2N

CO2Me

17

13b

3OMe 3OMe 2OMe

Ts

OMe

OBn Boc N

d

OMe

OMe OMe O

f

21

g NBoc OMe

OMe

24

9.62

10.2

R

Inactive

Inactive

R

5

Inactive

Inactive

R

3

Inactive

Inactive

Inactive

Inactive

Inactive

Inactive

Inactive

Inactive

Inactive

Inactive

2OMe

N

16b

Ts

2OMe

N

16c

Ts

2OMe

N

16d

Ts

16e

Ts

22 23 25

Boc H R3

26a

R3

N

26b

R3

N

R3

N

R3

N

R3

N

26c

N

O

2OMe 2OMe H H H

Inactive

Inactive

Me Me Me

Inactive Inactive 10.34

Inactive Inactive 13.21

H

Me

Inactive

Inactive

H

Me

14.5

18.6

H

Me

Inactive

Inactive

H

Me

12.45

15.67

H

Me

N N

OH 25

OR2

26e 4OH-T

OMe O N

N HCl 4

Ts

26d Br

7.2

16a

O

h OTBDMS

23

6.78

22

OMe O

N HCl

Ts

e OH

20

19

OH

MDA-MB231

14

CO 2Me

OMe

NH

Ts

18

OBn Boc CO 2Me N

MCF-7

OR2

B

A R1 = H, 2-OMe, 3-OMe R2 = alkyl amino ethyl chain

i

R1

N

NTs

OH Boc N

R

O

O R1

c

Compd no. OMe

OR2

1a

Benzoxazepine

26a, R2 = CH 2CH2N(CH 3)2 26b, R2 = CH2CH 2N(CH2CH 3)2 26c, R2 = CH 2CH2-piperidine 26d, R2 = CH2CH 2-pyrolidine 26e, R2 = CH 2CH2-azepane

26

Scheme 3. Reagents and conditions: (a) PPh3, CBr4, DCM; (b) K2CO3, DMF, 2 h, 70% in two steps; (c) NaHCO3, Boc2O, EtOH, 74%; (d) LiBH4, THF, 85%; (e) H2, 10% Pd–C, 50 psi, 75%; (f) PPh3, DEAD, 2 h, 75%; (g) 6 N HCl, MeOH, 70 °C, 2 h, 70%; (h) K2CO3, DMF, 74%; (i) R2Cl, K2CO3, acetone, 70–80%.

derivatives 2 with 1 provided the esters 3, 4, 5 in 60–75% yield. The Lithium aluminium hydride (LAH) reduction of 3, 4, 5 afforded the corresponding alcohols 6, 7, 8 which on subsequent debenzylation by H2/Pd (10% on carbon) gave 9, 10, 11 containing free alcoholic and phenolic hydroxyl groups in 60–70% yield in two steps. Exposure of 9, 10, 11 to Mitsunobu reaction conditions7, that is, diethylazodicarboxylate (DEAD), triphenylphosphine (TPP) at 0 °C in tetrahydrofuran as solvent resulted in the formation of desired enantiomerically pure benzoxazepine derivatives 12, 13 and 14 in 63–76% yield. The biological results of the S-amino acid derived benzoxazepines showed the compounds having benzene ring in side chain exhibiting better activity and the compound derived from tyrosine

Inactive

Inactive

7.2

10.4

Results were calculated from three independent experiments with triplicate of each observation. (i) Compound with IC50 more than 20 lM were considered inactive R3 =

OH (iii) R4 =

H N

(iv) R5 = CH2CHMe2.

was most potent.6d This observation influenced us to further derivatize the tyrosine-based benzoxazepine compound. In this context we made various amino alkyl chain derivatives 15a–b, 16a–e (see Scheme 2).

Table 2 IC50 value (lM) of compounds in MCF-7 and MDA-MB-231 cells Treatment

Time (h)

MCF-7

MDA-MB-231

15a

24 48 72 24 48 72

6.78 3.55 1.83 9.62 4.31 3.23

7.2 5.45 4.6 10.2 5.12 4.2

15b

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Figure 2. Effect of novel Benzoxazepines on breast cancer and ‘non-cancer’ cells. (A–C) MDA-MB-231 and MCF-7 cells were treated with vehicle (V), 15a or 4OHT (positive control) as indicated in the figure and cell death was determined by MTT assay. % cell death compared to vehicle is plotted. (D) Vero, Hek 293 and primary rat calvarial osteoblast cells were treated with vehicle or different concentrations of 15a for 24 h and % cell death compared to V is plotted. (E) MCF cells were treated with different concentrations of 15a and LDH release in the media was measured. Data represent mean ± SEM from three independent experiments.

In an attempt to further explore the biological properties of benzoxazepines as active pharmacophore, we further modified the scheme to prepare new kind of tyrosine based benzoxazepine derivatives with alkyl amino ethyl chains (Fig. 1B). The synthesis of the new kind of core is outlined in Scheme 3. First, benzyl alcohol 1a was converted to its bromo derivative 17 and proceeds next step without purification. It was reacted with tyrosine derivative in presence of K2CO3/DMF to obtain 18 in good overall yield. Secondary amine of 18 was protected with Boc-group, followed by lithium borohydride reduction to afford alcohol 20. Next, debenzylation of 20, followed by Mitsunobu reaction afforded benzoxazepane derivative 22. Boc-group of 22 was deprotected under acidic condition (6 N HCl in MeOH) to obtain 23. 23 was treated with 24 furnishing 25. Different kinds of amino alkyl chain were introduced in 25 to obtain 26a–e. The newly synthesized compounds were evaluated for anti-proliferative activity in both MCF-7 (ER+ve) and MDA-MB-231 (ER-ve) cells using MTT assay. Five compounds (15a, 15b, 25, 26b and 26d) were found to inhibit growth of MCF-7 and MDA-MB-231 cells at

Table 3 Safety index and therapeutic potential of compounds at 72 h Activity/ treatments

MCF-7 (EC50)

HEK-293 (IC50)

Safety index (IC50/EC50)

Therapeutic potential versus THBP4

15a THBP

1.83 10

30 30

16.39 3

5.46 1

IC50 of less than 20.0 lM., Tables 1 and 2. Out of these compounds, 15a exhibited highest cytotoxicity in breast cancer cells MCF-7 and MDA-MB-231 (Fig. 2A–C) Comparison of 15a with the previously published THBP4 is given in Table 3. Cytotoxicity towards normal cells: Specific cytotoxicity against cancer cells without affecting normal cell growth is a key safety feature of cancer chemotherapy. Five active compounds of the series were evaluated for their cytotoxicity in normal cells, (Vero, HEK-293 and primary rat calvarial osteoblasts) and 15a mediated cytotoxicity in these cells were visible at a much higher dose than in MCF-7 and MFA-MB-231 cells, signifying that the molecules have selectivity for inducing growth inhibition of cancer cells (Table 3 and Fig. 2D). Next, effect of 15a on membrane integrity in MCF-7 cell line was assessed by LDH release. At IC50 value, 15a did not significantly affect membrane integrity of MCF-7 cells (Fig. 2E), while it did cause LDH release at higher doses, indicating that although at high concentration 15a affected membrane integrity, the 15a-induced cytotoxicity was probably independent of its effect on membrane integrity. Compound 15a induces apoptosis in MCF-7 cells: To determine whether cell death induced by 15a was apoptotic in nature, cellular processes associated with apoptosis rather than necrosis such as: phosphatidylserine exposure (Annexin V-PI; Flow cytometry), DNA fragmentation and PARP Cleavage were assessed. As evident from Figure 3, 15a concentration dependently increased phosphatidylserine exposure (Fig. 3A). Compound 15a also induced PARP cleavage (Fig. 3B) and DNA fragmentation (Fig. 3C). Effect of 15a on breast cancer cell cycle progression: Effect of 15a on breast cancer cell line (MDA-MB-231and MCF-7) cell cycle

S. K. D. Dwivedi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6816–6821

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Figure 3. Compound 15a induces Apoptosis in MCF-7 cells. (A) Compound 15a increases phosphatidylserine exposure- MCF-7 cells were treated either with vehicle (V) or indicated concentrations of 15a for 24 h and phosphatidylserine exposure was measured by flow cytometry using FITC labelled Annexin V & PI staining. Representative dot plots and relative apoptotic cells are shown. (B) Effect of 15a treatment on PARP Cleavage—15a treated MCF-7 cell lysates were immunoblotted against PARP, Densitometry data of cleaved PARP is plotted. (C) Effect of 15a treatment on DNA Fragmentation—cells were treated with 15a and 4 OHT as indicated and DNA fragmentation after 24 h of treatment was quantified. Graph represents relative DNA fragmentation taking +ve control (PC) as 100% (#). Data represent mean ± SEM from three independent experiments; ⁄P