A facile synthesis of novel indole derivatives as potential antitubercular agents

Accepted Manuscript Title: A facile synthesis of some novel indole derivatives as potential antitubercular agents Author: Gulzar A. Khan Javeed A. War Arun Kumar Imtiyaz A. Sheikh Arti Saxena Ratnesh Das PII: DOI: Reference:

S1658-3655(16)30069-3 http://dx.doi.org/doi:10.1016/j.jtusci.2016.09.002 JTUSCI 329

To appear in: Received date: Revised date: Accepted date:

14-12-2015 31-8-2016 4-9-2016

Please cite this article as: G.A. Khan, J.A. War, A. Kumar, I.A. Sheikh, A. Saxena, R. Das, A facile synthesis of some novel indole derivatives as potential antitubercular agents, Journal of Taibah University for Science (2016), http://dx.doi.org/10.1016/j.jtusci.2016.09.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ac ce pt e

d

M

an

us

cr

ip t

Graphical Abstract

Highlights •

Synthesis of novel of 5,5-Dimethyl-11-phenyl-4b,5,5a,10,10a,11,11a,12-octahydro10,11,12-triaza-indeno[2,1-b]fluorenes 3a-3l.

•

With simple precursors novel indoles were prepared via a green one pot synthetic route.

•

Using of organocatalyst in acetic acid at mild conditions for improvement of yields and reaction times than the reported methods. 1

Page 1 of 32

•

Both theoretical as well as in vitro studies have uncovered the antitubercular nature of

ip t

synthesized indoles (3a-l).

cr

A facile synthesis of some novel indole derivatives as potential antitubercular agents

us

Gulzar A Khana*, Javeed A Warb, Arun Kumarc, Imtiyaz A Sheikhd, Arti Saxenaa and Ratnesh

Heterocyclic synthesis and Electroanalytical Laboratory, Department of Chemistry, HariSingh

d

Gour Central University, Sagar, India.

M

a

an

Dasa

b

Ac ce pt e

Synthetic Organic Chemistry & Molecular Modelling Laboratory, Department of Chemistry,

HariSingh Gour Central University, Sagar, India. c

Neuroscience and Endocrinology Laboratory, Department of Zoology, HariSingh Gour Central

University, Sagar, India. d

Microbial Technology Laboratory, Department of Botany, HariSingh Gour Central University,

Sagar, India.

Corresponding author: Gulzar Ahmad Khan, 2

Page 2 of 32

Telephone: +918966829653 Fax: NA

cr

ip t

E-mail Id: [email protected]/[email protected]

us

Abstract

A novel series of 5,5-Dimethyl-11-phenyl-4b,5,5a,10,10a,11,11a,12-octahydro-10,11,12-triaza-

using dibutylamine as a organocatalyst via

an

indeno[2,1-b]fluorenes3a-3lhas been prepared by reacting oxindole, aryl amines and acetone Knoevenagel and Michael type reactions

M

simultaneously. The present protocol is environmentally benign and highly compatible as it was conveniently carried out in ethanol at mild conditions. The structure of new compounds were

d

determined by spectroscopic techniques like IR, 1H NMR,

13

C NMR and LCHRMS. Docking

Ac ce pt e

studies against enoyl acyl carrier protein reductase predicts that the compounds possess high binding affinity towards the target. The compound 3k(MIC, 40 µg/mL) shows comparable activity in reference with Isoniazid at the same concentrationsagainst MT H37 Rv. Keywords:

Green

synthesis,Knoevenagel–Michael

addition

reaction,Dibutylamine,

Antitubercular activity, Molecular Docking.

1. Introduction Tuberculosis (TB) is regarded as intractable disease and as such a major cause of death worldwide. On an average about 100 million people become affected with TB annually. As a 3

Page 3 of 32

consequence, it accounts about 3 million deaths globally every year[1]. The tremendous increase in the number of TB cases is its resurgence greatly dependent on two factors; development of TB 100-fold among HIV infected patients and drug resistance by some bacterial strains. Some

ip t

researchers have reported the drug resistance to as many as nine drugs [2]. Primary anti-TB drugs like Isonicotinic acid hydrazide (INH) in combination with streptomycin, ethambutol etc

cr

becomes inactive while dealing with some bacterial strains and these strains evolve into extreme

us

drug resistant (XDR-TB) and multidrug resistant (MDR-TB) virulent forms due to partial/inadequate drug therapy. Ultimately, the terrible situation has prompted WHO to declare

an

TB a major global killer [3, 4]. The mechanism of action of antitubercular drugs is related to their ability to inhibit cell wall growth of target bacterial strains[5]. Such drugs blocks the

M

biosynthesis of mycoloic fatty acids of type II (a vital component in bacterial cell wall) by

Ac ce pt e

formation [6].

d

controlling the activity of specific enzymes (NADH enoylase-reductase) necessary for cell wall

Natural products are the main source of inspiration to design and synthesize new molecules for drug development. Most of these natural products consist of a heterocyclic core.Among nitrogen containing heterocycles, indole is a ubiquitous structural motifof a number of natural products like rutaecarpine, horsfilline, spirotryprostatin B, cryptosnguinolentine etc. (Fig. 1). Indole moiety has been employed in the designing of new heterocyclic compounds with diverse biological and pharmacological properties like antimicrobial, antitubercular, antimalarial, antitubulin, α-glucosidase inhibitors, antioxidant and fluorescent metal probes to sense molecular recognitions [7-13].However, indole derivatives bearing ferrocene moiety and carboxylate chains have been found to exhibit potent anticancer, cytotoxic and antiviral [7, 14-17]properties.

4

Page 4 of 32

Multi-component reactions (MCR's) are of immense importance in the field of medicinal chemistry. MCR's facilitates to assemble several reacting precursors in a single step transformation with no need of intermediate isolation and their purification ultimately affords a

ip t

desirablebioactive heterocyclic product [15, 17, 18, 19].High atom-economy, mild conditions, structural complexity and environmentally benign synthesis of some valuable heterocyclic

cr

scaffolds are one of the most advantageous features encountered in MCRs[5, 20]. The aforesaid

us

applications encouraged us to synthesize a novel series of 5,5-Dimethyl-11-phenyl4b,5,5a,10,10a,11,11a,12-octahydro-10,11,12-triaza-indeno[2,1-b]fluorenes3a-3l (Scheme 1) as

an

molecular templates which can withstand present as well as forthcoming challenges in the medicinal chemistry.

M

There is a global need to develop new drugs which could be effective against resistant bacterial strains. Keeping this challenge in mind, here we report the synthesis and antitubercular nature of

d

the compounds (3a-l). Amongst the various targets for anti TB drugs enoyl-acyl carrier protein

Ac ce pt e

(ACP) reductase being a well established target [21, 22] was selected as the target for docking simulations. ACP reductase is a key enzyme for the synthesis of the type II fatty acids. 2. Experimental

2.1. Chemicals and apparatus

Sigma-Aldrich, Himedia, CDH and Merck India purchased chemicals were used without purification to carry out this work. Melting points were determined using open capillary tube melting point apparatus and are presented without any correction. The infrared (IR) spectra were recorded on a FTIR Shimadzu-8400S spectrometer using KBr pellets. The 1HNMR and

13

C

NMR spectra were recorded on Bruker Avance 500 spectrometer using tetramethylsilane (TMS) as the internal standard and CDCl3 as solvent. LCHRMS were determined on Bruker Microtoff5

Page 5 of 32

QII 10330 mass spectrometer. The purity of the compounds were checked by TLC using Merck pre-coated silica gel GF aluminium plates and methanol: chloroform:ethyl acetate (1 : 3 : 5) as solvent system.

ip t

2.2General one pot synthesis of 5,5-Dimethyl-11-phenyl-4b,5,5a,10,10a,11,11a,12-octahydro-

cr

10,11,12-triaza-indeno[2,1-b]fluorenes(3a-l)

Ethanolic solution of oxindole (2 mmol) and aniline (1mmol) in presence of dibutylamine

us

(10 mol %) was refluxed forrequired time (or stirred at room temperature). Then acetone (4 mL) was added. The mixture was again refluxed (or stirred at room temperature) till the completion of

an

reaction (monitored by TLC).The precipitate obtained was filtered and washed successively with water and hexane. It was then recrystallized from ethanol followed by drying under vacuum to

HNMR, 13CNMR and LCHRMS.

d

1

M

afford the pure product (3a-l). The structural elucidation of the productwas confirmed by IR,

Ac ce pt e

2.2.1. 5,5-Dimethyl-11-phenyl-4b,5,5a,10,10a,11,11a,12-octahydro-10,11,12-triaza-indeno[2,1b]fluorenes (3a)

IR (KBr) (νmax cm-1): 1090 (C-C), 1247 (C-N), 1560 (C=C), 3090 (C-H), 3405 (N-H).1HNMR (500 MHZ, CDCl3, TMS, δppm): 1.8 (s, 6H, CH3), 3.4 (s, 2H, CH), 3.9 (s, 2H, CH), 6.6 (d, J = 5 Hz, 2H, HAr), 6.9 (m, 5H, HAr), 7.4 (d, J = 4.5 Hz, 2H, HAr), 7.5 (t, J = 12 Hz, 2H, HAr),7.7 (t, J = 8 Hz, 2H, HAr), 8.9 (s, 2H, NH);

13

CNMR (125 MHZ, CDCl3, δppm) δc: 26.7, 35.9, 56.7,

76.7,109.4,112.5, 115.5, 121.5,123.1, 124.3, 127.5, 136.4, 139.4, 155.5. LCHRMS (ESI): m/z [M+H]+:368.2059 (Supplementary; Fig S1, Fig.S2&Fig S3). .

6

Page 6 of 32

2.2.2.5,5-Dimethyl-11-o-tolyl-4b,5,5a,10,10a,11,11a,12-octahydro-10,11,12-triaza-indeno[2,1b]fluorene (3b) IR (KBr) (νmax cm-1): 1093 (C-C), 1242 (C-N), 1552 (C=C), 3090 (C-H), 3405 (N-H). 1HNMR

ip t

(500 MHZ, CDCl3, TMS, δppm): 1.8 (s, 6H, CH3), 2.1 (s, 3H, CH3),3.4 (s, 2H, CH), 3.9 (s, 2H,

cr

CH), 6.6 (d, J = 5 Hz, 2H, HAr), 6.8 (d, J = 9.2 Hz, 1H, HAr), 6.9 (t, J= 3.7 Hz, 1H, HAr), 7.1 (t, J = 6.3 Hz, 1H, HAr), 7.1 (t, J= 5.1 Hz, 1H, HAr), 7.4 (d, J = 4.5 Hz, 2H, HAr), 7.5 (t, J = 12 Hz, 2H, 13

CNMR (125 MHZ, CDCl3, δppm) δc: 24.3

us

HAr), 7.7 (t, J = 8 Hz, 2H, HAr), 8.9 (s, 2H, NH);

26.7, 35.9, 56.7, 76.7,109.4,114.1, 115.5, 119.1,123.1, 124.3, 129.4, 136.4, 140.7, 155.5.

an

HRLCMS (ESI): m/z [M+H]+:382.2459.

indeno[2,1-b]fluorene (3c)

M

2.2.3.11-(2-Ethyl-phenyl)-5,5-dimethyl-4b,5,5a,10,10a,11,11a,12-octahydro-10,11,12-triaza-

d

IR (KBr) (νmax cm-1): 1090 (C-C), 1242 (C-N), 1565 (C=C), 3095 (C-H), 3405 (N-H).1HNMR

Ac ce pt e

(500 MHZ, CDCl3, TMS, δppm): 1.3 (t, J = 9.1 Hz, 3H, CH3), 1.8 (s, 6H, CH3), 2.1 (q, J = 5.9 Hz, 2H, HAr), 3.4 (s, 2H, CH), 3.9 (s,2H, CH), 6.6 (d, J = 5 Hz, 2H, HAr), 6.9 (d, J = 3.7 Hz, 1H, HAr), 7.1 (t, J = 8.1 Hz, 1H, HAr), 7.2 (d, J = 6.1 Hz,1H,HAr), 7.4 (d, J = 4.5 Hz, 2H, HAr), 7.5 (t, J = 12 Hz, 2H, HAr), 7.7 (t, J = 8 Hz, 2H, HAr), 7.9 (t, J = 5.3 Hz, 1H, HAr), 8.9 (s, 2H, NH); 13

CNMR (125 MHZ, CDCl3, δppm) δc: 26.7, 35.9, 56.7, 76.7, 109.4, 111.9, 113, 115.5, 121.4,

122.8, 123.1, 124.3, 125.3, 136.4, 141.3, 155.5. LCHRMS (ESI): m/z [M+H]+:396.2459. 2.2.4.11-(4-Chloro-phenyl)-5,5-dimethyl-4b,5,5a,10,10a,11,11a,12-octahydro-10,11,12-triazaindeno[2,1-b]fluorene (3d)

7

Page 7 of 32

IR (KBr) (νmax cm-1): 744 (C-Cl), 1097 (C-C), 1247 (C-N), 1568 (C=C), 3090 (C-H), 3405 (NH).1HNMR (500 MHZ, CDCl3, TMS, δppm): 1.8 (s, 6H, CH3), 3.4 (s, 2H, CH), 3.9 (s, 2H, CH), 6.6 (d, J = 5 Hz, 2H, HAr), 6.7 (d, J = 11.3 Hz, 2H, HAr), 7.4 (d, J = 4.5 Hz, 2H, HAr), 7.5 (t, J =

13

ip t

12 Hz, 2H, HAr), 7.6 (d, J = 8.4 Hz,2H, HAr), 7.7 (t, J = 8 Hz, 2H, HAr), 8.9 (s, 2H, NH); CNMR (125 MHZ, CDCl3, δppm) δc: 26.7, 35.9, 56.7, 76.7,109.4,116.4, 115.5, 123.1, 124.3,

cr

126.3,131.8, 136.4, 148, 155.5. LCHRMS (ESI): m/z [M+H]+:402. 2459.

us

2.2.5.5,5-Dimethyl-11-(4-nitro-phenyl)-4b,5,5a,10,10a,11,11a,12-octahydro-10,11,12-triaza-

an

indeno[2,1-b]fluorene(3e)

IR (KBr) (νmax cm-1): 1103 (C-C), 1247 (C-N), 1571 (C=C), 3090 (C-H), 3405 (N-H). 1HNMR

M

(500 MHZ, CDCl3, TMS, δppm): 1.8 (s, 6H, CH3), 3.4 (s, 2H, CH), 3.9 (s, 2H, CH), 6.6 (d, J = 5 Hz, 2H, HAr), 7.0 (d,J = 9.5 Hz, 2H, HAr), 7.4 (d, J = 4.5 Hz, 2H, HAr), 7.5 (t, J = 12 Hz, 2H,

d

HAr), 7.7 (t, J = 8 Hz, 2H, HAr), 7.9 (d, J =6.9 Hz, 2H, HAr), 8.9 (s, 2H, NH); 13CNMR (125 MHZ,

Ac ce pt e

CDCl3, δppm) δc: 26.7, 35.9, 56.7, 76.7,109.4,114.1, 115.5, 121.5, 123.1, 124.3, 136.4, 142.1,152.8, 155.5. LCHRMS (ESI): m/z [M+H]+:413. 2459. 2.2.6.5,5-Dimethyl-11-(2-nitro-phenyl)-4b,5,5a,10,10a,11,11a,12-octahydro-10,11,12-triazaindeno[2,1-b]fluorene (3f)

IR (KBr) (νmax cm-1): 1090 (C-C), 1247 (C-N), 1569 (C=C), 3090 (C-H), 3405 (N-H). 1HNMR (500 MHZ, CDCl3, TMS, δppm): 1.8 (s, 6H, CH3), 3.4 (s, 2H, CH), 3.9 (s, 2H, CH), 6.6 (d, J = 5 Hz, 2H, HAr), 6.7 (d, J = 4.3 Hz, 1H, HAr), 6.8 (t, J = 8.3 Hz, 1H, HAr), 7.0 (t, J = 6.4 Hz, 1H, HAr), 7.4 (d, J= 4.5 Hz, 2H, HAr), 7.5 (t, J = 12 Hz, 2H, HAr), 7.7 (t, J = 8Hz, 2H, HAr), 7.9 (d, J= 7.6 Hz, 1H, HAr), 8.9 (s, 2H, NH);

13

CNMR (125 MHZ, CDCl3, δppm) δc: 26.7, 35.9, 56.7,

8

Page 8 of 32

76.7,109.4,112.8, 115.5, 119.7,123.1, 124.3, 128.5, 137.3,136.4, 139.1, 139.6, 155.5. LCHRMS (ESI): m/z [M+H]+:413. 2459. 2.2.7.2,8-Dichloro-5,5-dimethyl-11-phenyl-4b,5,5a,10,10a,11,11a,12-octahydro-10,11,12-triaza-

ip t

indeno[2,1-b]fluorene (3g)

cr

IR (KBr) (νmax cm-1): 744.9 (C-Cl), 1090 (C-C), 1247 (C-N), 1560 (C=C), 3090 (C-H), 3405 (N-

us

H). 1HNMR (500MHZ, CDCl3, TMS, δppm): 1HNMR (500 MHZ, CDCl3, TMS, δppm): 1.8 (s, 6H, CH3), 3.4 (s, 2H, CH), 3.9 (s, J = 75 Hz, 2H, CH), 6.7(s, 2H, HAr), 6.9 (m, 5H, HAr), 7.3 (d, J

an

= 8.1 Hz, 2H, HAr), 7.6 (d, J = 4.3 Hz, 2H, HAr), 8.9 (s, 2H, NH); 13CNMR (125 MHZ, CDCl3, δppm) δc:26.7, 35.9, 56.7, 76.7,110.8,112.5, 117.4, 121.5,122.9, 127.5, 134.7, 138.2, 139.4,

M

156.3. LCHRMS (ESI): m/z [M+H]+: 436.1459.

Ac ce pt e

indeno[2,1-b]fluorene (3h)

d

2.2.8.2,8-Dichloro-5,5-dimethyl-11-o-tolyl-4b,5,5a,10,10a,11,11a,12-octahydro-10,11,12-triaza-

IR (KBr) (νmax cm-1): 744.9 (C-Cl), 1093 (C-C), 1242 (C-N), 1552 (C=C), 3090 (C-H), 3405 (NH),. 1HNMR (500 MHZ, CDCl3, TMS, δppm): 1HNMR (500 MHZ, CDCl3, TMS, δppm): 1.8 (s, 6H, CH3), 2.1 (s, 3H, CH3), 3.4 (s, 2H, CH), 3.9 (s, 2H, CH), 6.7(s, 2H, HAr),6.8 (d, J = 9.2 Hz, 1H, HAr), 6.9 (t, J = 3.7 Hz, 1H, HAr), 7.1 (t, J= 6.3 Hz, 1H, HAr), 7.1 (t, J = 5.1 Hz, 1H, HAr), 7.3 (d, J = 8.1 Hz, 2H, HAr), 7.6 (d, J = 4.3 Hz, 2H, HAr), 8.9 (s, 2H, NH),;13CNMR (125MHZ, CDCl3, δppm) δc: 24.3 26.7, 35.9, 56.7, 76.7,110.8,114.1, 117.4, 119.1,122.9, 129.4, 134.7, 138.2, 140.7, 156.3. LCHRMS (ESI): m/z [M+H]+:450.1759. 2.2.9.2,8-Dichloro-11-(2-ethyl-phenyl)-5,5-dimethyl-4b,5,5a,10,10a,11,11a,12-octahydro10,11,12-triaza-indeno[2,1-b]fluorene (3i)

9

Page 9 of 32

IR (KBr) (νmax cm-1): 744.9 (C-Cl), 1090 (C-C), 1242 (C-N), 1565 (C=C), 3095 (C-H), 3405 (NH). 1HNMR (500 MHZ, CDCl3, TMS, δppm): 1HNMR (500 MHZ, CDCl3, TMS, δppm): 1.3 (t, J = 9.1, 3H, CH3), 1.8 (s, 6H, CH3), 2.1 (q, J = 5.9 Hz, 2H, HAr), 3.4 (s, 2H, CH), 3.9 (s,2H, CH),

ip t

6.7(s, 2H, HAr),6.9 (d, J= 3.7 Hz, 1H, HAr), 7.1 (t, J= 8.1 Hz, 1H, HAr), 7.2 (d, J = 6.1 Hz, 1H,HAr), 7.3 (d, J = 8.1 Hz, 2H, HAr), 7.6 (d, J = 4.3 Hz, 2H, HAr), 7.9 (t, J = 5.3 Hz, 1H, HAr),

cr

8.9 (s, 2H, NH);13CNMR(125 MHZ, CDCl3, δppm) δc: 26.7, 35.9, 56.7, 76.7, 110.8, 113, 117.4,

us

111.9, 121.4, 122.8, 122.9, 125.3, 134.7, 138.2, 141.3, 156.3. LCHRMS (ESI): m/z [M+H]+:464.1859.

an

2.2.10.2,8-Dichloro-11-(4-chloro-phenyl)-5,5-dimethyl-4b,5,5a,10,10a,11,11a,12-octahydro-

M

10,11,12-triaza-indeno[2,1-b]fluorene (3j)

IR (KBr) (νmax cm-1): 744 (C-Cl), 1097 (C-C), 1247 (C-N), 1572 (C=C), 3090 (C-H), 3405 (N-

d

H). 1HNMR (500 MHZ, CDCl3, TMS, δppm):1.8 (s, 6H, CH3), 3.4 (s, 2H, CH), 3.9 (s, 2H, CH),

Ac ce pt e

6.7(s, 2H, HAr), 6.7 (d, J = 11.3 Hz, 2H, HAr), 7.3 (d, J = 8.1 Hz, 2H, HAr),7.6 (d, J= 4.3 Hz, 2H, HAr), 7.6 (d, J =8.4 Hz, 2H, HAr), 8.9 (s, 2H, NH); 13CNMR (125 MHZ, CDCl3, δppm) δc: 26.7, 35.9, 56.7, 76.7,110.8,116.4, 117.4, 126.3,122.9, 131.8, 134.7, 138.2, 148, 156.3. LCHRMS (ESI): m/z [M+H]+: 470.1159.

2.2.11.2,8-Dichloro-5,5-dimethyl-11-(4-nitro-phenyl)-4b,5,5a,10,10a,11,11a,12-octahydro10,11,12-triaza-indeno[2,1-b]fluorene(3k)

IR (KBr) (νmax cm-1): 744.9 (C-Cl), 1103 (C-C), 1247 (C-N), 1571 (C=C), 3090 (C-H), 3405 (NH).1HNMR (500 MHZ, CDCl3, TMS, δppm): 1.8 (s, 6H, CH3), 3.4 (s, 2H, CH), 3.9 (s, 2H, CH), 6.7(s, 2H, HAr),7.0 (d, J = 9.5 Hz, 2H, HAr7.3 (d, J = 8.1 Hz, 2H, HAr),7.6 (d, J = 4.3 Hz, 2H, HAr), 7.9 (d, J =6.9 Hz, 2H, HAr), 8.9 (s, 2H, NH); 13CNMR (125 MHZ, CDCl3, δppm) δc: 26.7, 10

Page 10 of 32

35.9, 56.7, 76.7,110.8,114.1, 117.4, 121.5, 122.9, 134.7, 138.2, 142.1, 152.8, 156.3. LCHRMS (ESI): m/z [M+H]+:481.1459. 2.2.12.2,8-Dichloro-5,5-dimethyl-11-(2-nitro-phenyl)-4b,5,5a,10,10a,11,11a,12-octahydro-

ip t

10,11,12-triaza-indeno[2,1-b]fluorene (3l)

cr

IR (KBr) (νmax cm-1): 744.9 (C-Cl), 1090 (C-C), 1247 (C-N), 1569 (C=C), 3090 (C-H), 3405 (N-

us

H). 1HNMR (500 MHZ, CDCl3, TMS, δppm): 1.8 (s, 6H, CH3), 3.4 (s, 2H, CH), 3.9 (s, 2H, CH), 6.7(s, 2H, HAr), 6.7 (d, J = 4.3 Hz, 1H, HAr), 6.8 (t, J = 8.3 Hz, 1H, HAr), 7.0 (t, J = 6.4 Hz, 1H,

2H, NH);

13

an

HAr), 7.3 (d, J = 8.1 Hz, 2H, HAr),7.6 (d, J = 4.3 Hz, 2H, HAr), 7.9 (d, J = 7.6 Hz, 1H, HAr), 8.9 (s, CNMR (125 MHZ, CDCl3, δppm) δc: 26.7, 35.9, 56.7, 76.7,110.8,112.8, 117.4,

M

119.7,122.9, 128.5, 134.7, 137.3,138.2, 139.1, 139.6, 156.3. LCHRMS (ESI): m/z [M+H]+: 481.1459.

Ac ce pt e

2.3.1.Antitubercular activity

d

2.3. In vitro biological evaluation assay:

Mycobacterium tuberculosis bacterial strain( MTCC CODE 300)was purchased from Institute of Microbial Technology (MTCC), Chandigarh (India) and were cultured in blood nutrient agar medium in late logarithmic (A600 nm = 1) fashion. Bacterial strains possessing a plasmid adhering Isoniazid resistance marker were cultured in the same medium containing 100 µg/mL Isoniazid. It was shortly followed by streaking of 0.5 µL bacterial spread on LB agar plates ( 25 mL agar medium ± 90 µg/ mL Isoniazid discs over 9 cm Petri plates) as control. Filter discs (5mm diameter) of Whatman range were treated with 5µL of compound solutions including reference as mentioned in (table 3). After this, discs were air-dried for 7-10 minutes and kept

11

Page 11 of 32

over plates. These plates were incubated at 37 0C for about 48 h in a humid chamber[23]. Following this bacterial zone, inhibition diameters were observed and measured carefully. 2.4.Docking study

ip t

High resolution crystal structure of Enoyl-ACP reductase was downloaded from the RCSB PDB

cr

website (PDB ID: 1QG6)[24]. All molecular docking calculations were performed on AutoDockVina software [25].The protein was prepared for docking by removing the co-crystallized

us

ligands, waters and co-factors. The AutoDockTools (ADT) graphical user interface was used to calculate Kollman charges and polar hydrogens. The ligand was prepared for docking by

an

minimizing its energy with MMFF94s force field. Partial charges were calculated by Geistenger

M

method. The active site of the enzyme was defined to include residues of the active site within the grid size of 40Å x 40Å x 40Å. The most popular algorithm, Lamarckian Genetic Algorithm

d

(LGA) available in AutoDock was employed for docking. The docking protocol was tested by

Ac ce pt e

extracting co-crystallized inhibitor from the protein and then docking the same. The docking protocol predicted the same conformation as was present in the crystal structure with RMSD value well within the reliable range of 2Å(Fig 2). Amongst the docked conformations, one which binds well at the active site was analysed for detailed interactions in Discover Studio Visualizer 4.

3.Results and discussion 3.1. Synthesis

To the best of our knowledge, this new synthetic strategy provides the first example of a facile three

component

protocol

for

the

preparation

of

novel

5,5-Dimethyl-11-phenyl-

4b,5,5a,10,10a,11,11a,12-octahydro-10,11,12-triaza-indeno[2,1-b]fluorenes (3a-l). To get the

12

Page 12 of 32

best experimental conditions, the reaction conditions were optimized for the preparation of 3a chosen as model reaction for both at room temperature and at reflux conditions. As depicted in Scheme 1 oxindole and acetone as reacting materials were treated with different aryl amines. A

ip t

number of solvents in presence of a variety of catalysts (with/without suitable additive) were employed to design a desirable reaction medium. Initially dichloroethane (DCE), a nonpolar

cr

solvent in presence of anh.FeCl3 furnished 3a with poor yield including prolonged reaction time

us

(Table 1; entry 1, 16%)[26]. Afterwards, reaction was performed in polar aprotic solvents like tetrahydrofuran, dimethyl sulphoxide (DMSO), dimethyl formamide (DMF) and acetonitrile

an

(ACN) using different metal catalysts like Li(OH)2, CuO/ I2, CuI/ K2CO3 and Cs2CO3. It was observed that product yield was obtained in 19%, 12%, 23%, 19% and < 7% (Table 1; Entry 2 to

M

6) but prolonged reaction time was still a challenge [15, 27-30]. Inspired by these results the reaction was then carried in polar protic solvents like in methanol (MeOH), ethanol (EtOH) and glycol

(PEG)

using

catalysts

d

polyethylene

pyrollidine,

anh.ZnCl2,

AcOH,

4-

Ac ce pt e

dimethylaminopyridine (DMAP) and KH2PO4. In this context, reaction occurred smoothly and improved product yield as well as reaction time. As a result we were able to isolate the product yield in 21%, 35%, 39%, 44%, 26%, 53% and 47% (Table 1; Entry7, 8, 9, 10, 11, 12)[31, 32, 7, 33, 34, 18]. The aim behind this strategy involving screening of above solvents was to design a desirable solvent in which the aforesaid problems regarding product yield and reaction time can be addressed to a desirable extent. Finally, ethanol was taken as a best medium choice which in presence of a suitable organocatalyst (dibutylamine) afforded 3a(Table 1; Entry 13, 83%) in highest yield than all other solvents[35]. The plausible mechanism explaining the above mentioned result is depicted in Scheme 2. It is proposed that carbonyl group of oxindole1 gets converted into its enol form, results in the 13

Page 13 of 32

formation of 2. Intermediate 2 undergoes a facile nucleophilic addition from aryl amine producing 4which tautomerizes to 5which on dehydration gives imino intermediate6.On the other hand, oxindole condensed with acetone to give intermediate 7via Knoevenagel reaction in

ip t

the presence of a catalytic amount of dibutyl amine.However 7being a Michael acceptor is susceptible to nucleophilic addition from 6 as Michael donorto yield the corresponding adduct 8.

cr

Ultimately, intermediate 8on subsequent dehydration affords the desirable compound 3.

us

The synthesized compounds were fully characterized by different spectral techniques like IR, NMR and HRLCMS. IR spectrum of compound 3a exhibited sharp bands at 1090 cm-1 and 1247

an

cm-1 assigned to C-C and C-N is stretching frequencies respectively.The band at 1560 cm-1was attributed to C=C stretching whereas aromatic C-H stretching was observed at 3005 cm-1. In IH

M

NMR spectrum of 3a three singlets were recorded at 1.8, 3.4 and δ 3.9 δ ppm due to protons of

d

C6/C7-CH3, C8-CH and C15-CH supporting the existence of piperidine ring. The proton at

Ac ce pt e

C13'- position shows a doublet at 6.6 δ ppm whereas aromatic protons of phenyl ring showed a resonance multiplet from 6.9-7.2 δ ppm. However, singlet due to indolic proton was recorded at 8.9 δ ppm.13C NMR of compound 3a agrees with the number of carbons. It exhibited signals around 26-76 δ ppm attributed to methyl and methine carbon atoms. Peaks between 111- 136 δppm were assigned to aromatic carbon atoms. The two peaks observed at 139 and 155 δppm might be due to carbon atoms at C20' and C14' positions. The above results were further confirmed by mass spectra exhibited the molecular ion peak m/z 368 [M+H]+, in agreement with the molecular weight of the compound.

With the optimum conditions in mind, a number of reactive ketones (butanone, 1, 3diphenylacetone) in place of acetone were used but the out-put of these findings were quite unsatisfactory. During the course of such study, it was noticed that present strategy can tolerate 14

Page 14 of 32

both electron withdrawing and electron donating substituents as the reactions proceeded smoothly to afford corresponding products with desirable yields. It was also observed that electron donating alkyl-substituted anilines gave better yields than those of corresponding

ip t

electron withdrawing chloro and nitro substituted anilines (Table 2). This may be due to good nucleophilicity of aryl amines bearing electron donating substituents as compared to aryl amines

cr

having electron withdrawing substituents.

us

3.2.Docking study

Docking study reveals that the synthesized molecules bind at the same site where the co-

an

crystallized drug Triclosan is attached (Fig.2). Ala95 and Tyr156 form H-bonds of length 2.06

M

and 2.97Å lengths respectively with 3e, the highest scored molecule (Fig.3). Met159, Ala196, Ile200, Pro191 and Ile20 are involved in alkyl-π interactions with the π electron cloud of the

d

molecule (Fig. 3)[36]. These interactions together with other weak forces are responsible for

Ac ce pt e

formation of strong ligand-macromolecule complex. These findings predict that the molecules have high affinity towards bacterial Enoyl-ACP reductase. Docking studies predicted binding affinity values greater than -9 kcal/mol for all the ligand-ACP-reductase complexes which further corroborates the findings (Table 3). Compound 3i and 3k scored high after compound 3e. The computational predictions were complemented by the in vitro antitubercular activity evaluation.

3.3.In vitro anti tubercular activity

As is evident from Table 3, compound 3d,3e and 3k are able to inhibit the bacterial strain to appreciable levels with respective MIC values of 40, >40 and 40 µg/mL in reference to Isoniazid taken as standard against mycobacterium tuberculosis. However rest of the tested compounds

15

Page 15 of 32

showed MIC values ranging from 80 to 160 µg/mL(Table & Fig.4). No clear relation was observed between lipophilicity (calculated logP) and MIC. Compounds 3d, 3e, 3f, 3j, 3k and 3l being most active carry chloro and nitro substituents at para and ortho positions of the phenyl

ip t

ring.In particular nitro derivatives show better activity as compared to chloro, methyl and ethyl derivatives. Thus electron withdrawing groups reinforced the antitubercular activity

cr

ofsynthesized indole derivatives (3a-l).Amongst the nitro derivatives (3e, 3f, 3k & 3l) the para

us

nitro substituted indole derivatives (3e, 3k) are more active as compared to ortho substituted ones. The present study clearly supports the volume as well as substituent’s position as

an

significant for a compound in its biological evaluation. The reason behind the observed activity trend seems to be the way in which the molecules bind at the target active site as is predicted by

M

docking (Fig 3). The compound 3k shows better inhibition than all other compounds at the same

Ac ce pt e

4. Conclusion

d

concentrations.

We report here a mild and sustainable approach for the synthesis of 5,5-Dimethyl-11-phenyl4b,5,5a,10,10a,11,11a,12-octahydro-10,11,12-triaza-indeno[2,1-b]fluorene

derivatives

employing a MCR protocol. It has several advantageous features in terms of operational simplicity, better yields, reduced reaction times and ease of product isolation. Docking studies and in vitro antitubercular evaluation show that compounds (3a-l) exhibit moderate to good activity against Mycobacterium tuberculosis strain MT H37 Rv. Further structural modification if carried could generate a library of antitubercular analogs which may exhibit enhancedselectivity. Moreover, the present study supports the presence as well as relative position of nitro group (3e, 3k) as significant for triaza-indeno[2,1-b] fluorenes to behave as novel inhibitors of Enoyl-ACP reductase. 16

Page 16 of 32

Acknowledgement Gulzar A Khan, Arun Kumar, Arti Saxena and Imtiyaz A Sheikhacknowledge UGC, New Delhi for financial support. One of the authors (Javeed A War) would like to acknowledge the financial

ip t

support of DST, New Delhi in the form of INSPIRE fellowship (IF-120399). Authors are also

cr

thankful to SIL of Dr. Hari Singh Gour Central University, Sagar (M.P) and IISER Bhopal, India

us

for providing instrumental facilities.

an

References

M

[1] M. Basangouda, V.B. Jambagi, N.N. Barigidad, S.S. Laxmeshwar, V. Devaru, Narayanchar, Synthesis, Structure-activity relationship of iodinated-4-aryloxymethyl-coumarins as potential

d

anticancer and antimycobacterial agents, Eur. J. Med. Chem. 74(2014) 225-233.

Ac ce pt e

[2] K. Sayed, P. Bartyzel, X.Y. Shen, T.L. Perry, J.K. Kjawiony, M.T. Hamann, Marine Natural Products as Antituberculosis Agents, Tetrahedron,56 (2000)949. [3] M. Jeyachandran, P. Ramesh, D. Sriram, P. Senthikumar, P. Yogeswari, Synthesis and invitro antitubercular activity of 4-aryl/alkyl sulphonylmethylcoumarins as inhibitors of mycobacterium tuberculosis, Bioorg. Med. Chem. Lett. 22(2012) 4807- 4809. [4] Z.Q. Xu, W.W. Barrow, W.J. Suling, L. Westbrook, E. Barrow, Y.M. Lin, M.T. Flavin, Anti-HIV natural product (þ)-calanolide A is active against both drug-susceptible and drugresistant strains of Mycobacterium tuberculosis, Bioorg. Med. Chem. 12 (2004) 1199-1207.

17

Page 17 of 32

[5] G.A. Khan, J.A. War, G.A. Naikoo, U.J. Pandit, R. Das, Porous CuO catalysed green synthesis of some novel 3-alkylated indoles as potent antitubercular agents, J. Saudi Chem. Soc. (2016), http://dx.doi.org/10.1016/j.jscs.2016.03.009.

ip t

[6]K. Johnnson, D.S. King, P.G. Schultz, Studies on the Mechanism of Action of Isoniazid and

cr

Ethionamide in the Chemotherapy of Tuberculosis, J. Am. Chem. Soc.117(1995) 5009-5010.

[7] R. Gali, J. Banothu, R. Gondru, R. Bavantula, Y. Velivela, P.A. Crooks,One-pot

us

multicomponent synthesis of indole incorporated thiazolylcoumarins and their antibacterial,

an

anticancer and DNA cleavage studies, J. Bioorg. Med. Chem. Lett. 25 (2015) 106-112. [8]R.P. Karuvalam, R. Pakkath, K.R. Haridas, R. Rishikesan, N.S. Kumari,Synthesis, and

QSAR

studies

of

new

(1H-indol-3-yl)-alkyl-3-(1H-indol-3-

M

characterization

yl)propanamidederivatives as possible antimicrobial and antitubercular agents, Med. Chem. Res.

d

22 (2013) 4437–4454.

Ac ce pt e

[9] C. Desiree, Schuck, A.K. Jordao, M. Nakabashi, A.C. Cunha, V.F. Ferreira, C.R.S. Garcia, Synthetic indole and melatonin derivatives exhibit antimalerial activity on the cell cycle of the human malaria parasite Plasmodium falciparum, Eur. J. Med. Chem. 78 (2014) 375-382. [10] Q. Guan, C. Han, D. Zuo, M. Zhai, Z. Li,Q. Zhang, Y. Zhai, X. Jiang, K. Bao, Y. Wu, W. Zhang, Synthesis and evaluation of benzimidazolecarbamates bearing indole moieties for antiproliferative and antitubulin activities, Eur. J. Med. Chem. 87 (2014) 306-315. [11] S. Naureen, S. Noreen,A. Nazeer, M. Ashraf, U. Alam, M.A. Munawar, M.A. Khan, Triarylimidazoles-synthesis of 3-(4,5-diaryl-1H-imidazol-2-yl)-2-phenyl-1H-indole derivatives as potent a-glucosidase inhibitors, Med. Chem. Res. 24 (2015) 1586–1595. [12]J.S.Biradar, B.S.Sasidhar, R.Parveen, Synthesis, antioxidant and DNA cleavage activities of novel indole derivatives, Eur. J. Med. Chem.45 (2010) 4074-4078. 18

Page 18 of 32

[13] A. Rocha, M. Manuel, B. Marques, C.Lodeiro, Synthesis and characterization of novel indole-containing half-crowns as new emissive metal probes, Tetrahedron Lett. 50 (2009) 4930– 4933.

ip t

[14] N.S. Radulovic, D.B. Zlatkovic, K.V.Mitic, P.J.Randjelovic, N.M. Stojanovic, Synthesis, spectral characterization, cytotoxicity and enzyme-inhibiting activity of new ferrocene-indole

cr

hybrids, Polyhedron, 80 (2014) 134-141.

us

[15] B.V.S. Reddy, A.V. Ganesh, M. Vani, T.R. Murthy, S.V. Kalivendi, J.S. Yadav, Theecomponent, one-pot synthesis of hexahydroazepino [3,4-b]indole and tetrahydro-1H-pyrido[3,4-

an

b]indole derivatives and evaluation of their cytotoxicity, Bioorg. Med. Chem. Lett. 24 (2014) 4501-4503.

M

[16] S. Xue, L. Ma, R. Gao, Y. Lin, Z.Li, Synthesis and antiviral activity of some novel indole-2-

d

carboxylate derivatives, Acta Pharm.Sinica B, 4 (2014) 313–321.

Ac ce pt e

[17]S. Paul, A.R. Das, Dual role of the polymer supported catalyst PEG-OSO3H in aqueous reaction medium: synthesis of highly substituted structurally diversified coumarins and uracil fused spirooxindoles, Tetrahedron Lett. 54 (2013) 1149-1145. [18] L. Wang, M. Huang, X. Zhu, Y. Wan, Polyethylene glycol (PEG-200)-promoted sustainable one-pot three-component synthesis of 3-indole derivatives in water, App Catalysis A: General, 454 (2013) 160– 163.

[19] P.P Ghosh, A.R Das, Nano crystalline ZnO: a competent and reusable catalyst for one pot synthesis of novel benzylamino coumarin derivatives in aqueous media, Tetrahedron Lett. 53 (2012) 3140-3143.

19

Page 19 of 32

[20]Y. Gu, Multicomponent reactions in unconventional solvents: state of the art, Green Chem. 14 (2012) 2091-2128. [21] Heath, J. Richard, Y. Yuen-Tsu, M.A. Shapiro, E. Olson, C.O. Rock,Broad spectrum

ip t

antimicrobial biocides target the FabI component of fatty acid synthesis, J. Bio. Chem.273

cr

(1998) 30316-30320.

us

[22]Sacchettini, C. James, E.J. Rubin, J.S. Freundlich, Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis, Nature Rev. Micro bio.6 (2008) 41-52.

an

[23]J.G.B. Sanchez, V.V. Kouznetsov, Antimycobacterial susceptibility testing methods for

M

natural products research, Braz. J.Microbiol. 41 (2010) 270-277.

[24] W.H.J. Ward, G.A. Holdgate, S. Rowsell, E.G. McLean, R.A. Pauptit, E. Clayton, W.W.

d

Nichols, J.G. Colls, C.A. Minshull, D.A. Jude, A. Mistry, D. Timms, R. Camble, N.J. Hales, C.J.

Ac ce pt e

Britton, I.A.F. Taylor, Kinetic and structural characteristics of the inhibition of enoyl (acyl carrier protein) reductase by triclosan, Biochem. 38 (1999) 12514-12525. [25] T. Oleg, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading,J. Computational Chem. 31(2010) 455-461.

[26] J.S. Yadav, B.V.S. Reddy, K. Praneeth, FeCl3 – catalyzed alkylation of indoles with 1, 3dicarbonyl compounds: an expedient synthesis of 3-substituted indoles, Tetrahedron Lett.49 (2008) 199-202. [27] R. Meesala, R. Nagarajan, A short route to the synthesis of pyrroloacridines via Ullmann– Goldberg condensation, Tetrahedron Lett. 51 (2010) 422-424. 20

Page 20 of 32

[28] F.F. Gao, W.J. Xue, J.G. Wang, A.Wu, Logical design and synthesis of Indole-2,3-diones and 2-hydroxy- 3(2H)-benzofuranones via one-pot intramolecular cyclization, Tetrahedron, 70 (2014) 4331-4335.

ip t

[29] R. Elayaraja, R.J. Karunakaran, Cesium carbonate mediated synthesis of 3-(α-hydroxyaryl)

cr

indoles, Tetrahedron Lett. 53 (2012) 6901-6904.

[30] M.L. Deb, P.J. Bhuyan, Uncatalyzed Michael addition of indoles: Synthesis of some novel

us

3-alkylated indoles via a three component reaction in solvent-free conditions, Tetrahedron Lett.

an

48 (2007) 2159-2163.

Carbohydrate Res. 344 (2009) 1028-1031.

M

[31] S. Nagarajan, T.M. Das, Facile one-pot synthesis of sugar–quinoline derivatives,

d

[32] S.S. Ganesan, A.Ganesan, ZnCl2 promoted efficient, one-pot synthesis of 3-arylmethyl and

Ac ce pt e

diarylmethyl indoles, Tetrahedron Lett. 55 (2014) 694–698. [33]P. Dai,G. Zha,X. Lai,W.L.Q. Gan, Y. Shen, Inorganic base catalyzed synthesis of (2-amino3-cyano-4H-chromene-4-yl) phosphonates derivatives via multi-component reaction under mild and efficient conditions, RSC Advances, 4 (2014) 63420-63424. [34] Han, W. Meng, H. Liu, Y. Liu, J.Tao, DMAP-catalyzed four-component one-pot synthesis of

highly

functionalized

spirooxindole-1,4-dihydropyridines

derivatives

in

aqueous

ethanol,Tetrahedron 70 (2014) 8768-8774.

[35] R.M.N.Kalla, J.S. Choi, J.W. Yoo, S.J. Byeon, M.S. Heo, I.I. Kim,Synthesis of 2-amino-3cyano-4H-chromen-4-ylphosphonates and their anticancer properties, Eur. J. Med. Chem. 76 (2014) 61-66. 21

Page 21 of 32

[36] R.A.Laskowski, M.B. Swindells,LigPlot+: multiple ligand-protein interaction diagrams for

Fig.1: Structures of some natural products containing Indole nucleus.

ip t

drug discovery, J. Chem. Inf. Model51 (2011) 2778-2786.

cr

Fig.2: (a) The docked ligand 3e (yellow) binds at the same site where co-crystallized triclosan (pink) binds. (b) Superimposition of docked conformation (pink) over the co-crystallized

us

conformation(yellow) of triclosan shows RMSD value close to zero, confirming the reliability of

an

docking protocol. (c) Detailed interactions of 3k with the inhibitor residues. Dotted lines represent the interactions. H bonds, π-π stacking and alkyl- π interactions are represented by

M

green, pink and violet dotted lines respectively.

Fig.3: Schematic representation for the docked conformation of the ligand 3k at the active site of

d

Enoyl-ACP reductase constructed by Ligplus.

Ac ce pt e

Fig.4: Effect on absorbance of Mycobacterium tubercular culture with different concentrations (5, 10, 20, 40, 80 & 160; µg/ml) used to calculate MIC values of compounds (3a-3l) with reference to Isoniazid taken as Standard.

Scheme 1: Dibutyl amine catalyzed one pot three component reaction of oxindole, aryl amine and acetone.

Scheme 2: Plausible reaction mechanism for one pot synthesis of novel 5,5-Dimethyl-11Phenyl-4b,5,10,11,11a,12-hexahydro-10,11,12-triaza-indeno[2,1-b] fluorenes (3a-3l).

Figure 1:

22

Page 22 of 32

ip t cr us an M d Ac ce pt e Figure 2:

23

Page 23 of 32

ip t cr us an M d Ac ce pt e Figure 3:

24

Page 24 of 32

ip t cr us an M d Ac ce pt e Figure 4:

25

Page 25 of 32

ip t cr us an M d Ac ce pt e Scheme 1:

26

Page 26 of 32

27

Page 27 of 32

d

Ac ce pt e us

an

M

cr

ip t

Ac ce pt e

d

M

an

us

cr

ip t

Scheme 2:

TABLES

28

Page 28 of 32

Table 1: Optimized catalyst (or additives) and solvent effects on the reaction rate and %yield of 5,5-Dimethyl-11-Phenyl-4b,5,10,11,11a,12-hexahydro-10,11,12-triaza-indeno[2,1-b]

fluorene

(1a).

ip t

Table 2: Synthesis of Phenyl-hexahydro- triaza-indeno [2,1-b] fluorenes (3a-l) in ethanol at mild reaction conditions.

cr

Table 3: Lipscomb’s parameters, binding affinity and diameter of zone of inhibition (mm) for

us

Phenyl-hexahydro- triaza-indeno [2,1-b] fluorenes (3a-l).

2

3

4

5

6

DCE

-

THF/HOH

-

Catalyst

M

Additive

Anh.FeCl3

d

1

Solvent

Ac ce pt e

Entry

an

Table 1:

DMSO

DMSO

DMF

CH3CN

K2CO3

I2

-

-

Li(OH)2

CuI

CuO

Cs2 CO3

-

Time (h)

% Yield

P

10

16

Q

17

11

P

11

19

Q

17

13

P

14

12

Q

-

-

P

17

23

Q

-

-

P

14

19

Q

-

-

P

15

<7

Q

-

-

Ref.

[26]

[15]

[27]

[28]

[29]

[30]

29

Page 29 of 32

9

MeOH

10

EtOH

11

EtOH/HOH

PEG/HOH

Pyrollidine

Anh.ZnCl2

-

AcOH

-

DMAP

-

KH2PO4

-

M

12

-

P

-

Dibutylamine

d

EtOH

Ac ce pt e

13

10

21

Q

14

18

P

7

35

Q

10

32

P

7

Q

12

P

[31]

[32]

ip t

EtOH

P

39

9

[7]

31

cr

8

-

-

us

MeOH

26

Q

13

16

P

9

53

Q

14

38

P

8

47

Q

-

-

an

7

[33]

[34]

[18]

P

1.44

83

Present

Q

8.30

79

Work / [35]

Reflux condition, QRoom temperature

Table 2:

30

Page 30 of 32

Entry R

R’

Protocol

Time (hr)

Product

(%)

yield

m.p. (0C)

Isolated

5

6

7

8

9

H

H

p-ClC6H5

p-NO2C6H5

P

8.43

Q

1.50

P

9.14

Q

1.22

P

8.72

Q

1.54

P

H

Cl

Cl

Cl

o-NO2C6H5

C6H5

o-CH3C6H5

o-C2H5C6H5

310-313

3b

86

298-301

ip t

1.44

83

cr

Q

3a

89

319-322

3d

85

329-332

3e

76

287-290

3f

82

307-310

3g

80

326-329

3h

92

314-317

89

31 339-342

3c

us

o-C2H5C6H5

8.30

an

4

H

o-CH3C6H5

P

M

3

H

C6H5

d

2

H

Ac ce pt e

1

8.78

Q

1.36

P

8.92

Q

1.62

P

8.17

Q

1.68

P

8.25

Q

1.32

P

8.35

Q

1.48

3i

Page 31 of 32

P

Room temperature, Q Reflux condition.

code

clogP

affinity

H-

H-bond

bond

acceptors weight

donors

0

3b

-10.8

5.9443

4

0

3c

-10.3

6.4733

4

3d

-11.3

6.3291

4

3e

-11.2

5.5543

7

3k 3l

Isoniazid

(µg/mL)

368.24

13.10±0.18

>160

382.24

11.2±0.52

>160

0

396.24

7.68±0.33

>160

0

402. 24

20.19±0.92

40

0

20.60±0.96

>40

d

3j

Concentration

413. 24

-9.9

5.5543

7

0

413. 24

18.44±0.89

80

-10.8

3.4713

6

2

436.14

16.18±0.23

> 160

-10.3

3.9704

6

2

450.17

17.32±0.90

> 160

-10.8

4.4993

6

2

464.18

9.11±0.22

> 160

-10.8

4.3551

6

2

470.11

20.66±1.86

40

-11.6

3.5803

9

2

481.14

21.82±1.35

40

-10.1

3.5803

9

2

481.14

18.39±0.88

80

-6

-0.69

2

2

137.14

25.70±0.64

10

Ac ce pt e

3i

Inhibitory

Inhibition

us

4

3h

Minimum

an

5.4453

M

-10.7

3g

Zone of

(mm)*

3a

3f

Molecular

cr

Compound Binding

ip t

Table 3:

*Values are given as mean±standard deviation (n=3).

32

Page 32 of 32