Novel isoniazid-spirooxindole derivatives: design, synthesis, biological evaluation, in silico ADMET prediction and computational studies

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Novel isoniazid-spirooxindole derivatives: design, synthesis,

biological evaluation, in silico ADMET prediction and computational

studies

Mayuri A. Borad , Divya J. Jethava , Manoj N. Bhoi ,

Chirag N. Patel , Himanshu A. Pandya , Hitesh D. Patel

PII:

S0022-2860(20)31206-0

DOI:

Reference:

https://doi.org/10.1016/j.molstruc.2020.128881

MOLSTR 128881

To appear in:

Journal of Molecular Structure

Received date:

Revised date:

Accepted date:

11 May 2020

1 July 2020

9 July 2020

Please cite this article as: Mayuri A. Borad , Divya J. Jethava , Manoj N. Bhoi , Chirag N. Patel ,

Himanshu A. Pandya , Hitesh D. Patel , Novel isoniazid-spirooxindole derivatives: design, synthesis,

biological evaluation, in silico ADMET prediction and computational studies, Journal of Molecular Struc-

ture (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128881

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Highlights:



Novel isoniazid containing spirooxindole derivatives were synthesized as potential anti-

mycobacterial agent.

Compound 6ab proves to be best anti-mycobacterial agent against H₃₇Rv strain and

MDR-TB.

The synthesized molecules docked with isoniazid-resistant I21V enoyl-ACP(COA)

reductase mutant enzyme from Mycobacterium tuberculosis in Complex with NADH-

INH (PDB entry: 2IE0).



Molecular dynamics study for the structural integrity and stability of the complex.

In silico ADMET prediction of synthesized compounds for good pharmacokinetic

properties.

Novel isoniazid-spirooxindole derivatives: design, synthesis, biological evaluation, in silico

ADMET prediction and computational studies

Mayuri A. Borad^a, Divya J. Jethava^a, Manoj N. Bhoi^a*, Chirag N. Patel^b, Himanshu A. Pandya^b,

and Hitesh D. Patel^a*

^aDepartment of Chemistry, School of Sciences, Gujarat University, Ahmedabad, India.

^bDepartment of Bioinformatics, Applied Botany Centre (ABC), School of Sciences, Gujarat

University, Ahmedabad 380009, Gujarat, India

E-mail: mayuri.borad@gmail.com, divyajethva187@gmail.com, manoj18bhoi@gmail.com,

hitesh13chem@rediffmail.com Tel.: +91-079-26300969; fax: +91-079-26308545.

Abstract

In the present scenario, the Synthesis of new and desired antimycobacterial agent has an eternal

demand to resist Mycobacterium tuberculosis (MTB). The design and identification of new

molecules for the treatment of tuberculosis is an important task in organic as well as medicinal

chemistry research. In the present study, we have reported the combination of the desired

compound using two versatile and significant moieties, isoniazid and spirooxindole derivatives.

A series of novel isoniazid-spirooxindole hybrid molecules (6a-6ao) were designed, synthesized,

and well-characterized by various spectroscopic methods. We have evaluated for their in vitro

antimycobacterial activity against Mycobacterium tuberculosis H₃₇Rv (MTB) strain and MDR-

TB. Among them, Compound 6ab was found to be the most effective compare to other

compounds. ADMET-related descriptors were to be calculated of all the compounds to predict

the pharmacokinetic properties for the selection of the effective and bioavailable compounds. In

addition, molecular docking and molecular dynamics studies reveal that the binding modes of all

the compounds in the active site of isoniazid-resistant enoyl-ACP(COA) reductase, which helped

to establish a structural basis of inhibition of Mycobacterium tuberculosis.

Keywords: ADMET properties, Antimycobacterial activity, Isatin, Isoniazid-spirooxindole,

Molecular docking, Molecular dynamics

1. Introduction

Microbial infections are a growing problem in contemporary medicine, yet only a few

antimicrobial agents are used in clinical practice. Mycobacterium tuberculosis (MTB) is a

pathogenic bacterial species in the genus Mycobacterium and the causative agent of most cases

of tuberculosis (TB).[1] It typically affects the lungs (pulmonary TB) but can affect other sites as

well (extra-pulmonary TB). The disease is spread in the air when people who are sick with

pulmonary TB expel bacteria, for example by coughing. Overall, a relatively small proportion (5-

15%) of the estimated 2-3 billion people infected with M. tuberculosis will develop TB disease

during their lifetime. However, the probability of developing TB is much higher among people

infected with HIV.[2] As stated by the World Health Organization (WHO) Global Tuberculosis

Report 2015, Around 9.6 million people are estimated to have tumbled ill with TB, and 1.5

million people deaths from the disease were reported in 2014.[3] Keeping insight into the above

statistics, WHO declared TB as a global health emergency and aimed at saving 14 million lives

between 2006 and 2015.[4] Streptomycin, Isoniazid, Rifampin, Ethambutol, and Pyrazinamide

are the first-line drugs for TB control while, second-line drugs for treatments are p-

aminosalicylic acid, Ethionamide, Cycloserine, macrolide antibiotics, such as Azithromycin,

Fluoroquinolones, etc.. There was an extreme deterioration in the number of TB cases, after the

discovery of many effective anti-TB drugs during the 1950s and 1970s. But, due to the

emergence of multi-drug resistant tuberculosis (MDR-TB), drug-resistant tuberculosis (XDR-

TB) and totally drug-resistant tuberculosis (TDR-TB), the number of TB cases has been

increasing rapidly throughout the world since the 1980s.[5] The main problem accompanying

antimycobacterial drugs is poor compliance with prolonged treatment and regimens used to treat

TB that is badly tolerated, relatively ineffective, expensive, and must be taken for at least 2

years.[6] So, there is an exigent need for new anti-tuberculosis drugs that are active towards the

broad spectrum of species of the Mycobacterium genus become obvious.

A number of anti-tubercular (anti-TB) drugs (Fig. 1) have been established over the past

decades. However, the drug-resistance issue has not been resolved as yet. Thus, there is a

tremendous need to develop new anti-TB drugs that are active against both acute and chronic

growth phases of mycobacterium to stop all forms of drug resistant-TB.[7] In recent years

several, new drug candidates or repurposed drugs namely linezolid, TMC207, Moxifloxacin,

OPC67683, PA824, Gatifloxacin, Rifapentine, PNU100480, AZD5847, SQ109, etc.[8] have

been still developed and some of them are also in the improvement stages of clinical trials for the

treatment of tuberculosis.[9,10] Among them, only one drug that is bedaquiline has been recently

approved by the FDA for its use in drug-resistant tuberculosis.[11]

Due to the global impact of this infectious disease, a great deal of research work is being

devoted to recognizing newer molecular entities that are dynamic against the microbial strains.

Several different approaches such as molecular alterations of the known drugs in combinatorial

chemistry, targeting bacterial virulence, structure-based drug discovery, high-throughput

screening have been discovered to search innovative and novel biologically important

molecules.[12,13] The molecular modification approach has been found to be very promising,

among all these strategies. Several drugs that are available in the market have been developed by

using this strategy. The main aim of molecular modification is to do chemical change in a

molecule for the enhancement of its pharmaceutical, pharmacokinetics or pharmacodynamics

properties.

Isoniazid (INH) has been widely used as a frontline anti-tubercular drug for the treatment

of TB for the past 40 years.[14] It is a prodrug and must be activated by bacterial catalase. It is

activated by catalase-peroxidase hemoproteins, KatG, which couples the isonicotinic acyl with

nicotinamide adenine dinucleotide (NADH) to form the isonicotinic acyl-NADH complex [15],

that binds with the enoyl-acyl carrier protein (ACP) reductase InhA, which is involved during the

mycolic acid synthesis.[16] INH inhibits the synthesis of mycolic acid which is required for the

mycobacterial cell wall. It is metabolized in the liver to form compounds such as hydrazine,

which is toxic to the central nervous system and other body organs.[17] Recent reports indicate

that the incorporation of hydrophobic moieties into the basic structure of INH increases

penetration of the drug into the highly lipophilic cell wall of the bacterium. Several researchers

have introduced the amidoether functionality into biologically active molecules and the resulting

hybrids exhibited good biological activities.[18,19] Additionally, spirocyclic scaffolds are being

progressively utilized in drug discovery. The construction of a spiro heterocyclic framework has

always been a challenging endeavor for synthetic organic chemists as it frequently requires

synthetic design based on specific strategies. Spiro cyclic building blocks have been subjected to

make more pharmaceutically active molecules. Spirooxindole and its derivatives have attracted

considerable interest to researchers in the area of synthetic organic chemistry as well as

medicinal chemistry because they are present in many natural products and exhibits highly

noticeable biological activities.[20, 21] It has been reported that the sharing of the C-3 atom for

the synthesis of spirooxindoline derivatives can highly enhance biological activity.[22] Amongst

them, some of the spirooxindoles derivatives are recognized for their antimycobacterial

properties and have been shown comparable or even better activities than some of the first-line

TB drugs. [23,24]

Inspiration from the previous studies and in continuation of our efforts towards the

synthesis of new anti-tuberculosis agents, we have here reported spirooxindole derivatives

linkage with isoniazid drug to form a new series of isoniazid-spirooxindole derivatives. All the

derivatives were synthesized and well-characterized with various spectroscopic techniques. The

synthesized compounds were screened for their in vitro antimycobacterial activity against

Mycobacterium tuberculosis H₃₇Rv (MTB) and also against MDR-TB. Additionally, we have

carried out computational studies such as molecular docking, molecular dynamics, and in silico

ADMET prediction of our compounds.

2. Materials and methods

2.1. Chemistry

All Starting materials and other reagents were purchased from commercial suppliers

(Merk, sigma Aldrich, Avra, spectrochem, etc.) and were used without any further purification

unless otherwise indicated. The reactions were examined by thin-layer chromatography (TLC)

and terminated as arbitrated by the consumption of starting material. TLC was performed on

silica gel G 60 F254 (Merck) plate. It was visualized by UV radiation or exposure to iodine

vapors. Gravitational column chromatography was conducted over silica gel 60 (60-120 µm).

CEM Discover microwave system (model no.: 908010; make up CEM Matthews. Inc, USA) was

used for synthesis. The nomenclature of the compounds was performed using

CambridgeSoft.ChemOffice.2010. v12. The melting points were recorded on an optimelt

automated melting point system that was uncorrected. IR spectra were recorded at room

temperature in the region from 500-3950 (cm^-1) on a Perkin-Elmer 377 spectrophotometer in

1

13

KBr with absorption in cm^-1. H-NMR and C-NMR spectra were recorded on Bruker AV 400

and 100 MHz using DMSO-d6 as solvent and TMS as an internal standard. Chemical shifts are

reported in parts per million (δ in ppm) relative to the designated referenced peaks: DMSO-d₆at

2.5, 3.33 ppm. When peak multiplicities are reported, the following abbreviations are used: s =

singlet, d = doublet, t = triplet q = quartet, m = multiplet, br = broadened, dd = doublet of

1

doublets. Coupling constants (J) were reported in (Hertz) Hz. H NMR and ¹³C NMR were

recorded at 300 K temperature and other details as mentioned in NMR spectra. Mass spectra

were recorded on Advion Expression CMS, USA, using ACN: Water: Formic acid (70: 30:

0.1%) as mobile phase. Important parameter of Mass spectra: DL temperature: C, heat

lock: C, drying gas flow: 20 Litter/Minute, run time: 1 minute, flow: 0.5mL/Min., range

mass: 0-1500 m/z, oven temperature: C nebulizing gas flow: 4.4 Litter/minute, cooler

temperature: C and detector: UV. ESI Mass analysis parameter: capillary temperature: C

source temperature: C capillary voltage: volts, MeOH: Water: Formic acid (90: 10:

0.1%) as mobile phase. Elemental analysis was performed as per standard protocol on vario

MICRO cube, elementar CHNS analyzer serial no.: 15084053. Molecular docking and Molecular

dynamics were done using the YASARA commercial package. Pose visualization of molecular

docking and molecular dynamics was carried out using Schrodinger Maestro 11, LLC, New

York, 2015. ADMET properties of novel compounds were foretold using the Accelrys Discovery

Studio 2.1 software. DruliTo software was used to get drug-likeness properties (Lipinski’s Rule

of Five).

2.2. General procedure for the synthesis of compound (5a-5ao):

In 25 ml round-bottomed flask, a mixture of 2-(3-(4-substituted phenyl)-4,5-dihydro-1H-

pyrazol-5-yl)phenol derivatives 4a-4o (1 eq.) and isatin derivatives (1 eq.) with 5 ml DMF were

subjected to microwave (CEM microwave) at 450W (150 C) for 6-12 minutes in the presence of

p-TSA (1 eq.). The progress of the reaction was monitored by TLC (30% ethyl acetate-hexane).

The reaction mixture was allowed to cool at room temperature. The reaction mixture was then

poured into ice water and the resulting solid residues were filtered off, dried to get the crude

product which was further recrystallized from methanol or ethanol to yield 2-(4-substituted

phenyl)-1,10b dihydrospiro [benzo[e]pyrazolo[1,5-c][1,3]oxazine-5,3'-indolin]-2'-one (5a-5ao)

in very good yields with good purity.[25]

2.3. General procedure for the synthesis of compound (6a-6ao):

To a stirred solution of 2-(substituted phenyl)-1,10bdihydrospiro[benzo[e]pyrazolo[1,5-

c][1,3] oxazine-5,3'-indolin]-2'-one (5a-5ao) (1 eq.) in acetonitrile (10V) was added

triethylamine (2 eq.) at room temperature. To this, chloroacetyl chloride (1.2 eq.) was added

dropwise at 0 °C to the reaction mixture. The resulting solution was stirred at room temperature

for 3h. The reaction mixture was then poured into water, and extracted with DCM. The

combined organic layer was washed with 1M aq. solution of NaOH, & brine solution and dried

over Na₂SO₄, filtered and evaporated to afford chloroacetyl adduct of (5a-5ao). Furthermore,

Isoniazid (1 eq) and triethylamine (0.26 mmol, 1.5 equiv.) were refluxed with above obtained

chloroacetyl adduct of (5a-5ao, 1 eq.) for 4h. The progress of the reaction was monitored by

TLC. After completion of the reaction, the reaction mixture was transferred onto ice/cold water

and the separated solid was then filtered, washed with water, and dried well in a rotary

evaporator. The crude product was further purified by gravitational column chromatography

using 15-30% Ethyl acetate: hexane as eluent. Pure fractions were evaporated to dryness. The

column purified compound further triturated using 10-20% diethyl ether in n-pentane to afford

highly pure (>95%) title compound (6a-6ao) with good practical yield.

Characterizations Data:

2.3.1. N'-(2-oxo-2-(2'-oxo-2-phenyl-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]oxazine-

5,3'-indolin]-1'-yl)ethyl)isonicotinohydrazide (6a):

Brown solid; IR (KBr) (υmax cm^-1): 3255 (C-H Aromatic), 1616 (C=N_str), 1724 (C=O_str), 1213

(C-O ether); ¹H NMR ( MHz DMSO) δ 86 (s H) 8 9 (d J = 7.5 Hz, 2H), 7.95 (d, J =

7.2 Hz, 2H), 7.79 (dd, J = 7.4, 1.4 Hz, 1H), 7.56 (dd, J = 7.6, 1.5 Hz, 1H), 7.54 - 7.46 (m, 2H),

7.44 (dd, J = 7.6, 1.5 Hz, 1H), 7.35 (dd, J = 7.4, 1.4 Hz, 1H), 7.27 (dtdd, J = 12.9, 7.2, 5.9, 1.6

Hz, 5H), 7.10 (td, J = 7.6, 1.5 Hz, 1H), 6.98 (dd, J = 7.4, 1.4 Hz, 1H), 5.49 (t, J = 6.4 Hz, 1H),

4.17 (s, 1H), 3.90 (s, 1H), 3.74 (s, 1H), 2.67 (dd, J = 12.4, 6.4 Hz, 1H), 2.42 (dd, J = 12.4, 6.4

Hz, 1H); ¹³C NMR ( MHz DMSO) δ 7 67 8 6 99 86

143.94, 139.99, 134.71, 132.76, 130.29, 129.32, 128.59, 127.52, 127.25, 125.81, 123.85, 123.03,

120.06, 119.58, 116.20, 111.55, 60.70, 53.75, 38.96; ESI-MS: m/z Calculated 544.57, found

[M+H]⁺545.5; Anal. Calcd for C₃₁H₂₄N₆O₄: C, 68.37; H, 4.44; N, 15.43; O, 11.75%; found C,

68.45; H, 4.82; N, 15.20%.

2.3.2. N'-(2-(2-(4-chlorophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]oxazi

ne-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6b):

Saddle Brown solid; IR (KBr) (υmax cm^-1): 3150 (C-H Aromatic), 1688 (C=N_str), 1720 (C=O_str),

1212 (C-O ether); ¹H NMR ( MHz DMSO) δ 8 9 (d J = 7.5 Hz, 2H), 8.52 (s, 1H), 7.91 (d,

J = 7.5 Hz, 2H), 7.78 (dd, J = 7.6, 1.5 Hz, 1H), 7.48 (tdd, J = 15.2, 7.6, 1.5 Hz, 4H), 7.32 (dd, J =

7.4, 2.5 Hz, 3H), 7.24 (ddd, J = 16.3, 7.6, 1.4 Hz, 2H), 7.07 (td, J = 7.6, 1.5 Hz, 1H), 6.97 (dd, J

= 7.6, 1.5 Hz, 1H), 5.29 (t, J = 6.1 Hz, 1H), 4.15 (s, 1H), 4.11 (s, 1H), 3.90 (s, 1H), 2.67 (dd, J =

12.4, 6.0 Hz, 1H), 2.42 (dd, J = 12.5, 6.1 Hz, 1H); ¹³C NMR ( MHz DMSO) δ 7

167.84, 164.23, 154.15, 150.99, 145.86, 143.94, 139.99, 134.78, 132.53, 130.29, 129.32, 129.05,

127.77, 127.52, 127.25, 123.85, 123.03, 120.06, 119.58, 116.20, 111.55, 60.70, 53.75, 38.96;

ESI-MS: m/z Calculated 579.01, found [M+H]⁺580.0; Anal. Calcd for C₃₁H₂₃ClN₆O₄: C, 64.31;

H, 4.00; Cl, 6.12; N, 14.51; O, 11.05%; found C, 64.69; H, 4.30; N, 14.35%.

2.3.3. N'-(2-(2-(4-bromophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]oxazi

ne-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6c):

Burly Wood solid; IR (KBr) (υmax cm^-1): 3200 (C-H Aromatic), 1675 (C=N_str), 1812 (C=O_str),

1200 (C-O ether); ¹H NMR ( MHz DMSO) δ 8 9 (d J = 7.5 Hz, 2H), 8.58 (s, 1H), 7.90 (d,

J = 7.5 Hz, 2H), 7.79 (dd, J = 7.4, 1.4 Hz, 1H), 7.62 - 7.53 (m, 1H), 7.50 (d, J = 26.5 Hz, 1H),

7.47 - 7.32 (m, 4H), 7.26 (dddd, J = 15.1, 11.2, 7.6, 1.5 Hz, 3H), 7.07 (td, J = 7.5, 1.4 Hz, 1H),

6.97 (dd, J = 7.4, 1.4 Hz, 1H), 5.09 (t, J = 5.8 Hz, 1H), 4.13 (s, 1H), 4.02 (s, 1H), 3.96 (s, 1H),

2.67 (dd, J = 12.4, 6.0 Hz, 1H), 2.42 (dd, J = 12.5, 5.8 Hz, 1H); ¹³C NMR ( MHz DMSO) δ

175.32, 167.84, 164.23, 154.15, 150.99, 145.86, 143.94, 139.99, 134.71, 131.98, 131.57, 130.29,

129.32, 127.52, 127.25, 126.33, 123.85, 123.03, 121.78, 120.06, 119.58, 116.20, 111.55, 60.70,

53.75, 38.96; ESI-MS: m/z Calculated 623.47, found [M+H]⁺624.4; Anal. Calcd for

C₃₁H₂₃BrN₆O₄: C, 59.72; H, 3.72; Br, 12.82; N, 13.48; O, 10.26%; found C, 59.89; H, 3.58; N,

13.27%.

2.3.4. N'-(2-(2-(4-aminophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]oxazi

ne-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6d):

Saddle Brown solid; IR (KBr) (υmax cm^-1): 3100 (C-H Aromatic), 1610 (C=N_str), 1700 (C=O_str),

1

1100 (C-O ether); H NMR ( MHz DMSO) δ (s H) 8 9 (d J = 7.5 Hz, 2H), 7.97

(d, J = 7.5 Hz, 2H), 7.87 - 7.72 (m, 1H), 7.63 – 7.48 (m, 1H), 7.45 (td, J = 7.6, 1.5 Hz, 1H), 7.40

- 7.33 (m, 3H), 7.25 (ddd, J = 15.6, 7.7, 1.5 Hz, 2H), 7.10 (td, J = 7.5, 1.4 Hz, 1H), 7.04 – 6.90

(m, 1H), 6.67 (d, J = 7.5 Hz, 2H), 5.72 (t, J = 7.1 Hz, 1H), 5.29 (s, 2H), 4.13 (s, 1H), 3.93 (s,

1H), 3.72 (s, 1H), 3.67 (dd, J = 12.4, 7.1 Hz, 1H), 2.42 (dd, J = 12.4, 7.1 Hz, 1H); ¹³C NMR (100

MHz DMSO) δ 7 67 8 6 99 7 86 9 9 99 7

130.29, 129.32, 127.52, 127.25, 126.93, 123.85, 123.03, 122.22, 120.06, 119.58, 116.20, 113.58,

111.55, 60.70, 53.75, 38.96; ESI-MS: m/z Calculated 599.59, found [M+H]⁺600.5; Anal. Calcd

for C₃₁H₂₅N₇O₄: C, 66.54; H, 4.50; N, 17.52; O, 11.44%; found C, 66.28; H, 4.43; N, 17.37%.

2.3.5. N'-(2-(2-(4-hydroxyphenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]

oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6e):

Gray solid; IR (KBr) (υmax cm^-1): 3132 (C-H Aromatic), 1593 (C=Nstr), 1702 (C=Ostr), 1300

(C-O ether) ; ¹H NMR ( MHz DMSO) δ (s H) 8 9 (d J = 7.5 Hz, 2H), 8.37 (s, 1H),

7.94 (d, J = 7.5 Hz, 2H), 7.77 (dd, J = 7.4, 1.4 Hz, 1H), 7.62 – 7.46 (m, 1H), 7.44 (dt, J = 7.3, 3.6

Hz, 1H), 7.40 - 7.29 (m, 3H), 7.25 (dd, J = 10.8, 4.3 Hz, 2H), 7.10 (td, J = 7.4, 1.4 Hz, 1H), 6.98

(dd, J = 7.4, 1.4 Hz, 1H), 6.79 (d, J = 7.5 Hz, 2H), 5.69 (t, J = 6.2 Hz, 1H), 4.42 (s, 1H), 3.74 (s,

1H), 3.44 (s, 1H), 2.67 (dd, J = 12.6, 6.2 Hz, 1H), 2.42 (dd, J = 12.4, 6.2 Hz, 1H); ¹³C NMR (100

MHz DMSO) δ 7 67 8 6 9 8 99 86 9 9 99 7

130.29, 129.32, 128.09, 127.52, 127.25, 124.84, 123.85, 123.03, 120.06, 119.58, 116.20, 115.78,

111.55, 60.70, 53.75, 38.96; ESI-MS: m/z Calculated 560.57, found [M+H]⁺561.5; Anal. Calcd

for C₃₁H₂₄N₆O₅: C, 66.42; H, 4.32; N, 14.99; O, 14.27%; found C, 66.82; H, 4.63; N, 14.40%.

2.3.6. N'-(2-oxo-2-(2'-oxo-2-(p-tolyl)-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]oxazine-

5,3'-indolin]-1'-yl)ethyl)isonicotinohydrazide (6f):

Brown solid; IR (KBr) (υmax cm^-1): 3245 (C-H Aromatic), 1623 (C=N_str), 1810 (C=O_str), 1298

1

(C-O ether); H NMR ( MHz DMSO) δ 8 9 (d J = 7.5 Hz, 2H), 8.53 (s, 1H), 7.91 (d, J =

7.5 Hz, 2H), 7.78 (dd, J = 7.4, 1.4 Hz, 1H), 7.57 - 7.47 (m, 3H), 7.44 (td, J = 7.4, 1.4 Hz, 1H),

7.32 (dd, J = 7.4, 1.4 Hz, 1H), 7.24 (ddd, J = 16.3, 7.6, 1.5 Hz, 2H), 7.17 (d, J = 7.5 Hz, 2H),

7.08 (td, J = 7.4, 1.4 Hz, 1H), 6.97 (dd, J = 7.4, 1.4 Hz, 1H), 5.30 (t, J = 6.2 Hz, 1H), 4.16 (s,

1H), 4.11 (s, 1H), 3.90 (s, 1H), 2.67 (dd, J = 12.4, 6.0 Hz, 1H), 2.42 (dd, J = 12.5, 6.1 Hz, 1H),

2.29 (s, 3H); ¹³C NMR ( MHz DMSO) δ 7 67 8 6 99 86

143.94, 139.99, 139.17, 134.71, 131.77, 130.29, 129.32, 128.97, 127.52, 127.25, 125.43, 123.85,

123.03, 120.06, 119.58, 116.20, 111.55, 60.70, 53.75, 38.96, 21.12; ESI-MS: m/z Calculated

558.60, found [M+H]⁺559.6; Anal. Calcd for C₃₂H₂₆N₆O₄: C, 68.81; H, 4.69; N, 15.05; O,

11.46%; found C, 68.42; H, 4.34; N, 15.25%.

2.3.7. N'-(2-(2-(4-fluorophenyl)-2'-oxo-1,10b-dihydrospiro [benzo[e] pyrazolo [1,5-c][1,3]

oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6g):

Sienna Brown solid; IR (KBr) (υmax cm^-1): 3214 (C-H Aromatic), 1685 (C=Nstr), 1728

1

(C=Ostr), 1110 (C-O ether); H NMR ( MHz DMSO) δ 8 9 (d J = 7.5 Hz, 2H), 8.79 (s,

1H), 7.90 (d, J = 7.5 Hz, 2H), 7.76 (dd, J = 7.6, 1.3 Hz, 1H), 7.54 (dd, J = 7.6, 1.5 Hz, 1H), 7.51

- 7.47 (m, 2H), 7.44 (td, J = 7.6, 1.4 Hz, 1H), 7.31 (dd, J = 7.4, 1.4 Hz, 1H), 7.25 (tdd, J = 7.5,

4.6, 1.4 Hz, 2H), 7.08 (td, J = 7.4, 1.4 Hz, 1H), 7.04 (t, J = 7.8 Hz, 2H), 6.97 (dd, J = 7.4, 1.4 Hz,

1H), 5.12 (t, J = 5.7 Hz, 1H), 4.34 (s, 1H), 4.09 (s, 1H), 4.02 (s, 1H), 2.67 (dd, J = 12.6, 5.7 Hz,

1H), 2.42 (dd, J = 12.4, 5.7 Hz, 1H); ¹³C NMR ( MHz DMSO) δ 7 67 8 6

163.10, 160.48, 154.15, 150.99, 145.86, 143.94, 139.99, 134.71, 130.29, 129.29, 128.32, 127.52,

127.25, 123.85, 123.03, 120.06, 119.58, 116.37, 111.55, 60.70, 53.75, 38.96; ESI-MS: m/z

Calculated 562.56, found [M+H]⁺563.5; Anal. Calcd for C₃₁H₂₃FN₆O₄: C, 66.19; H, 4.12; F,

3.38; N, 14.94; O, 11.38%; found C, 66.30; H, 4.41; N, 14.85%.

2.3.8. N'-(2-(2-(3-bromophenyl)-2'-oxo-1,10b-dihydrospiro [benzo[e]pyrazolo [1,5-c][1,3]

oxazi ne-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6h):

Brown solid; IR (KBr) (υmax cm^-1): 3135 (C-H Aromatic), 1600 (C=N_str), 1721 (C=O_str), 1285

1

(C-O ether); H NMR ( MHz DMSO) δ 8 9 (d J = 7.5 Hz, 2H), 8.53 (s, 1H), 7.91 (d, J =

7.5 Hz, 2H), 7.84 - 7.76 (m, 2H), 7.61 - 7.44 (m, 3H), 7.43 (dd, J = 2.9, 1.5 Hz, 1H), 7.32 (dd, J

= 7.4, 1.4 Hz, 1H), 7.24 (ddd, J = 14.5, 7.4, 1.4 Hz, 2H), 7.19 (t, J = 7.6 Hz, 1H), 7.08 (td, J =

7.6, 1.5 Hz, 1H), 6.97 (dd, J = 7.4, 1.4 Hz, 1H), 5.29 (t, J = 6.1 Hz, 1H), 4.15 (s, 1H), 4.11 (s,

1H), 3.90 (s, 1H), 2.67 (dd, J = 12.4, 6.0 Hz, 1H), 2.42 (dd, J = 12.4, 6.0 Hz, 1H); ¹³C NMR (100

MHz DMSO) δ 7 67 8 6 99 8 9 9 99 69 9

130.05, 129.80, 129.32, 127.61, 127.25, 124.63, 123.85, 123.03, 120.06, 119.58, 116.20, 111.55,

60.70, 53.75, 38.96; ESI-MS: m/z Calculated 623.47, found [M+H]⁺624.4; Anal. Calcd for

C₃₁H₂₃BrN₆O₄: C, 59.72; H, 3.72; Br, 12.82; N, 13.48; O, 10.26%; found C, 59.39; H, 3.38; N,

13.27%.

2.3.9. N'-(2-(2-(4-nitrophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]

oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6i):

Burly Wood solid; IR (KBr) (υmax cm^-1): 3155 (C-H Aromatic), 1612 (C=N_str), 1642 (C=O_str),

1202 (C-O ether); ¹H NMR ( MHz DMSO) δ 8 9 (d J = 7.2 Hz, 2H), 8.75 (s, 1H), 8.23 (d,

J = 7.5 Hz, 2H), 7.90 (d, J = 7.4 Hz, 2H), 7.76 (d, J = 7.5 Hz, 2H), 7.52 - 7.34 (m, 3H), 7.28 -

7.22 (m, 2H), 7.09 (td, J = 7.5, 1.5 Hz, 1H), 6.97 (dd, J = 7.4, 1.5 Hz, 1H), 5.45 (t, J = 6.0 Hz,

1H), 4.38 (s, 1H), 4.32 (s, 1H), 3.57 (s, 1H), 2.67 (dd, J = 12.4, 6.0 Hz, 1H), 2.42 (dd, J = 12.5,

6.0 Hz, 1H); ¹³C NMR ( MHz DMSO) δ 7 67 8 6 99 6

145.86, 143.94, 139.99, 136.93, 134.71, 130.29, 129.32, 127.52, 127.25, 125.51, 123.85, 123.03,

120.06, 119.58, 116.20, 111.55, 60.70, 53.75, 38.96; ESI-MS: m/z Calculated 589.57, found

[M+H]⁺590.5; Anal. Calcd for C₃₁H₂₃N₇O₆: C, 63.15; H, 3.93; N, 16.63; O, 16.28%; found C,

63.58; H, 3.63; N, 16.95%.

2.3.10. N'-(2-(2-(2-hydroxyphenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]

oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6j):

Greenish Brown solid; IR (KBr) (υmax cm^-1): 3125 (C-H Aromatic), 1618 (C=N_str), 1678

1

(C=O_str), 1268 (C-O ether); H NMR ( MHz DMSO) δ (s H) 8 96 (d J = 7.4 Hz,

2H), 8.64 (s, 1H), 7.90 (d, J = 7.4 Hz, 2H), 7.52 – 7.44 (m, 2H), 7.44 - 7.29 (m, 3H), 7.24 (dtd, J

= 17.5, 7.4, 1.6 Hz, 2H), 7.10 (dtd, J = 10.3, 7.5, 1.5 Hz, 2H), 7.01 (dd, J = 7.5, 1.4 Hz, 1H), 6.87

(td, J = 7.5, 1.4 Hz, 1H), 6.78 (dd, J = 7.5, 1.4 Hz, 1H), 5.52 (t, J = 9.0 Hz, 1H), 4.91 (s, 1H),

3.93 (s, 1H), 3.69 (s, 1H), 2.74 - 2.65 (m, 1H), 2.44 - 2.36 (m, 1H); ¹³C NMR (100 MHz,

DMSO) δ 7 67 8 6 3, 157.79, 154.15, 150.99, 143.94, 139.99, 138.27, 134.71,

130.29, 129.84, 129.32, 127.81, 127.52, 127.25, 123.85, 123.03, 121.25, 120.06, 119.58, 118.65,

116.20, 115.54, 111.55, 60.70, 53.75, 38.89; ESI-MS: m/z Calculated 560.57, found [M+H]⁺

561.5; Anal. Calcd for C₃₁H₂₄N₆O₅: C, 66.42; H, 4.32; N, 14.99; O, 14.27%; found C, 66.65; H,

4.61; N, 14.49%.

2.3.11. N'-(2-(2-(4-methoxyphenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]

oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6k):

Saddle Brown solid; IR (KBr) (υmax cm^-1): 3254 (C-H Aromatic), 1612 (C=N_str), 1671 (C=O_str),

1145 (C-O ether); ¹H NMR ( MHz DMSO) δ 8 9 (d J = 7.4 Hz, 2H), 8.53 (s, 1H), 7.90 (d,

J = 7.4 Hz, 2H), 7.78 (dd, J = 7.4, 1.5 Hz, 1H), 7.48 (tdd, J = 15.0, 7.5, 1.5 Hz, 4H), 7.42 – 7.20

(m, 3H), 7.10 (td, J = 7.4, 1.5 Hz, 1H), 7.02 - 6.82 (m, 3H), 5.34 (t, J = 6.8 Hz, 1H), 4.63 (s, 1H),

13

4.18 (s, 1H), 3.78 (s, 4H), 2.67 (dd, J = 12.4, 6.7 Hz, 1H), 2.42 (dd, J = 12.5, 6.8 Hz, 1H); C

NMR (100 MHz, DMSO) δ 7 67 8 6 9 7 99 86 9

139.99, 134.71, 130.29, 129.32, 127.36, 126.78, 123.85, 123.03, 120.06, 119.58, 116.20, 114.84,

111.55, 60.70, 56.03, 53.75, 38.96; ESI-MS: m/z Calculated 574.60, found [M+H]⁺575.6; Anal.

Calcd for C₃₂H₂₆N₆O₅: C, 66.89; H, 4.56; N, 14.63; O, 13.92%; found C, 66.47; H, 4.93; N,

14.47%.

2.3.12. N'-(2-(2-(2-chlorophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]

oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6l):

Gray solid; IR (KBr) (υmax cm^-1): 3165 (C-H Aromatic), 1617 (C=N_str), 1759 (C=O_str), 1123

(C-O ether); ¹H NMR ( MHz DMSO) δ 8 9 (d J = 7.4 Hz, 2H), 7.90 - 7.81 (m, 3H), 7.81 -

7.63 (m, 1H), 7.62 – 7.48 (m, 2H), 7.45 (td, J = 7.5, 1.4 Hz, 1H), 7.39 - 7.16 (m, 6H), 7.12 (td, J

= 7.4, 1.5 Hz, 1H), 7.00 (dd, J = 7.5, 1.4 Hz, 1H), 5.31 (t, J = 7.7 Hz, 1H), 4.20 (s, 1H), 3.93 (s,

1H), 3.63 (s, 1H), 2.69 - 2.65 (m, 1H), 2.44 - 2.40 (m, 1H); ¹³C NMR ( MHz DMSO) δ

175.32, 167.84, 164.23, 154.15, 150.99, 144.48, 143.94, 139.99, 134.71, 133.42, 131.78, 130.20,

129.32, 127.82, 127.52, 127.25, 126.84, 123.85, 123.03, 120.06, 119.58, 116.20, 111.55, 60.70,

53.75, 38.89; ESI-MS: m/z Calculated 579.01, found [M+H]⁺580.0; Anal. Calcd for

C₃₁H₂₃ClN₆O₄: C, 64.31; H, 4.00; Cl, 6.12; N, 14.51; O, 11.05%; found C, 64.67; H, 4.30; N,

14.38%.

2.3.13. N'-(2-(2-(3-nitrophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]

oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6m):

Brown solid; IR (KBr) (υmax cm^-1): 3102 (C-H Aromatic), 1660 (C=N_str), 1704 (C=O_str), 1071

1

(C-O ether); H NMR ( MHz DMSO) δ 8 9 (d J = 7.4 Hz, 2H), 8.62 (s, 1H), 8.53 (t, J =

1.4 Hz, 1H), 8.19 (dt, J = 7.5, 1.4 Hz, 1H), 7.98 - 7.82 (m, 3H), 7.80 (dd, J = 7.5, 1.5 Hz, 1H),

7.55 (t, J = 7.5 Hz, 1H), 7.49 (dd, J = 7.5, 1.5 Hz, 1H), 7.47 - 7.39 (m, 2H), 7.24 (dtd, J = 9.0,

7.4, 1.4 Hz, 2H), 7.10 (td, J = 7.5, 1.5 Hz, 1H), 6.80 (dd, J = 7.5, 1.4 Hz, 1H), 5.17 (t, J = 6.4 Hz,

1H), 4.49 (s, 1H), 3.66 (s, 1H), 2.98 (s, 1H), 2.67 (dd, J = 12.5, 6.4 Hz, 1H), 2.42 (dd, J = 12.5,

6.4 Hz, 1H); ¹³C NMR ( MHz DMSO) δ 7 67 8 6 99 7 7

145.08, 143.94, 139.99, 135.81, 134.71, 130.36, 129.30, 127.52, 127.25, 124.10, 123.85, 123.03,

121.84, 120.06, 119.58, 116.20, 111.55, 60.70, 53.75, 38.96; ESI-MS: m/z Calculated 589.57,

found [M+H]⁺590.5; Anal. Calcd for C₃₁H₂₃N₇O₆: C, 63.15; H, 3.93; N, 16.63; O, 16.28%;

found C, 63.37; H, 3.44; N, 16.41%.

2.3.14. N'-(2-(2-(2,4-dichlorophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]

oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6n):

Brown solid; IR (KBr) (υmax cm^-1): 3269 (C-H Aromatic), 1640 (C=N_str), 1724 (C=O_str), 1081

1

(C-O ether); H NMR ( MHz DMSO) δ 8 9 (d J = 7.4 Hz, 2H), 7.94 - 7.78 (m, 3H), 7.69

(dd, J = 7.5, 1.4 Hz, 1H), 7.54 (dd, J = 7.5, 1.4 Hz, 1H), 7.45 - 7.31 (m, 4H), 7.31 - 7.08 (m, 4H),

7.01 (dd, J = 7.4, 1.5 Hz, 1H), 5.08 (t, J = 6.9 Hz, 1H), 4.22 (s, 1H), 3.71 (s, 1H), 3.32 (s, 1H),

2.67 (dd, J = 12.4, 6.9 Hz, 1H), 2.42 (dd, J = 12.4, 6.9 Hz, 1H); ¹³C NMR ( MHz DMSO) δ

175.32, 167.84, 164.23, 154.15, 150.99, 144.48, 143.94, 139.99, 134.71, 133.42, 132.99, 132.69,

130.55, 130.29, 129.32, 128.41, 127.86, 127.52, 127.25, 123.85, 123.03, 120.06, 119.58, 116.20,

111.55, 60.70, 53.75, 38.89; ESI-MS: m/z Calculated 613.46, found [M+H]⁺614.4; Anal. Calcd

for C₃₁H₂₂Cl₂N₆O₄: C, 60.70; H, 3.61; Cl, 11.56; N, 13.70; O, 10.43%; found C, 60.45; H, 3.24;

N, 13.55%.

2.3.15. N'-(2-(2-(2-methoxyphenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]

oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6o):

Saddle Brown solid; IR (KBr) (υmax cm^-1): 3240 (C-H Aromatic), 1681 (C=N_str), 1729 (C=O_str),

1207 (C-O ether); ¹H NMR ( MHz DMSO) δ 8 9 (d J = 7.4 Hz, 2H), 8.61 (s, 1H), 7.91 (d,

J = 7.4 Hz, 2H), 7.79 (dd, J = 7.4, 1.5 Hz, 1H), 7.66 - 7.42 (m, 3H), 7.40 - 7.19 (m, 4H), 7.06

(ddd, J = 14.5, 7.4, 1.4 Hz, 2H), 6.98 (ddd, J = 11.4, 7.5, 1.5 Hz, 2H), 5.13 (t, J = 5.3 Hz, 1H),

4.33 (s, 1H), 4.14 (s, 1H), 3.87 (s, 1H), 3.76 (s, 3H), 2.67 (dd, J = 12.5, 5.3 Hz, 1H), 2.42 (dd, J

= 12.5, 5.3 Hz, 1H); ¹³C NMR ( MHz DMSO) δ 7 67 8 6 8

150.99, 143.94, 139.99, 136.73, 134.71, 130.29, 129.32, 128.31, 127.52, 127.26, 123.77, 123.03,

121.85, 120.06, 119.58, 116.20, 114.88, 111.55, 60.70, 56.78, 53.75, 38.89; ESI-MS: m/z

Calculated 574.60, found [M+H]⁺575.6; Anal. Calcd for C₃₂H₂₆N₆O₅: C, 66.89; H, 4.56; N,

14.63; O, 13.92%; found C, 66.37; H, 4.38; N, 14.22%.

2.3.16. N'-(2-(5'-chloro-2'-oxo-2-phenyl-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]

oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6aa):

Gray solid; IR (KBr) (υmax cm^-1): 3155 (C-H Aromatic), 1630 (C=N_str), 1738 (C=O_str), 1227

(C-O ether); ¹H NMR ( MHz DMSO) δ 67 (s H) 8 9 (d J = 7.4 Hz, 2H), 7.85 (d, J =

7.2 Hz, 2H), 7.74 (d, J = 7.5 Hz, 1H), 7.64 (d, J = 1.4 Hz, 1H), 7.57 - 7.45 (m, 2H), 7.45 - 7.35

(m, 1H), 7.35 - 7.23 (m, 5H), 7.10 (td, J = 7.5, 1.5 Hz, 1H), 6.89 (dd, J = 7.4, 1.5 Hz, 1H), 5.07

(t, J = 5.8 Hz, 1H), 4.24 (s, 1H), 3.59 (s, 1H), 3.46 (s, 1H), 2.67 (dd, J = 12.4, 5.8 Hz, 1H), 2.42

(dd, J = 12.4, 5.8 Hz, 1H); ¹³C NMR ( MHz DMSO) δ 7 67 8 6

150.99, 145.86, 143.98, 139.99, 136.05, 134.33, 132.76, 130.29, 128.59, 127.52, 126.17, 125.81,

123.85, 123.12, 120.06, 116.20, 113.30, 60.70, 53.75, 38.9;. ESI-MS: m/z Calculated 579.01,

found [M+H]⁺580.0; Anal. Calcd for C₃₁H₂₃ClN₆O₄: C, 64.31; H, 4.00; Cl, 6.12; N, 14.51; O,

11.05%; found C, 64.68; H, 4.33; N, 14.96%.

2.3.17. N'-(2-(5'-chloro-2-(4-chlorophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e] pyrazolo[1,5-

c][1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6ab):

Saddle Brown solid; IR (KBr) (υmax cm^-1): 3270 (C-H Aromatic), 1614 (C=N_str), 1769 (C=O_str),

1

1200 (C-O ether); H NMR ( MHz DMSO) δ 9 66 (s H) 8 96 (d J = 7.4 Hz, 2H), 7.75

(dd, J = 8.9, 4.4 Hz, 3H), 7.48 (dd, J = 7.5, 1.4 Hz, 1H), 7.45 - 7.29 (m, 5H), 7.29 - 7.22 (m, 2H),

7.07 (td, J = 7.6, 1.5 Hz, 1H), 7.01 (dd, J = 7.3, 1.4 Hz, 1H), 4.63 (t, J = 5.8 Hz, 1H), 3.86 (s,

1H), 3.80 (s, 1H), 3.74 (s, 1H), 2.89 (dd, J = 12.4, 5.8 Hz, 1H), 2.42 (dd, J = 12.4, 5.8 Hz, 1H);

¹³C NMR ( MHz DMSO) δ 7 67 8 6 154.15, 150.99, 145.86, 143.98, 139.99,

136.05, 134.85, 134.33, 132.53, 130.29, 129.05, 127.77, 127.52, 126.17, 123.85, 123.12, 120.06,

116.20, 113.30, 60.70, 53.75, 38.96; ESI-MS: m/z Calculated 613.46, found [M+H]⁺614.4;

Anal. Calcd for C₃₁H₂₂Cl₂N₆O₄: C, 60.70; H, 3.61; Cl, 11.56; N, 13.70; O, 10.43%; found C,

60.62; H, 3.37; N, 13.49%.

2.3.18. N'-(2-(2-(4-bromophenyl)-5'-chloro-2'-oxo-1,10b-dihydrospiro[benzo[e] pyrazolo[1,5-

c][1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6ac):

Dark rown solid; IR (KBr) (υmax cm^-1): 3275 (C-H Aromatic), 1610 (C=N_str), 1770 (C=O_str),

1

1213 (C-O ether); H NMR ( MHz DMSO) δ 9 6 - 8.93 (m, 2H), 8.91 (s, 1H), 7.99 - 7.81

(m, 2H), 7.70 (s, 1H), 7.54 - 7.30 (m, 7H), 7.27 (s, 1H), 7.12 (s, 1H), 6.99 (s, 1H), 5.18 (s, 1H),

13

4.90 (s, 1H), 3.85 (s, 1H), 3.73 (s, 1H), 2.67 (s, 1H), 2.42 (s, 1H); C NMR (100 MHz, DMSO)

δ 7 67 8 6 99 86 98 9 99 6 98

131.57, 130.29, 127.52, 126.25, 123.85, 123.12, 121.78, 120.06, 116.20, 113.30, 60.70, 53.75,

38.96; ESI-MS: m/z Calculated 657.91, found [M+H]⁺658.9; Anal. Calcd for C₃₁H₂₂BrClN₆O₄:

C, 56.59; H, 3.37; Br, 12.15; Cl, 5.39; N, 12.77; O, 9.73%; found C, 56.22; H, 3.15; N, 12.42%.

2.3.19. N'-(2-(2-(4-aminophenyl)-5'-chloro-2'-oxo-1,10b-dihydrospiro[benzo[e] pyrazolo[1,5-

c][1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6ad):

Olive green solid; IR (KBr) (υmax cm^-1): 3125 (C-H Aromatic), 1616 (C=N_str), 1778 (C=O_str),

1204 (C-O ether); ¹H NMR ( MHz DMSO) δ 8 9 (d J = 7.4 Hz, 2H), 8.20 (s, 1H), 7.88 (d,

J = 7.4 Hz, 2H), 7.62 (d, J = 1.3 Hz, 1H), 7.46 - 7.31 (m, 5H), 7.27 (td, J = 7.5, 1.5 Hz, 1H), 7.12

(td, J = 7.5, 1.5 Hz, 1H), 7.00 (dd, J = 7.5, 1.4 Hz, 1H), 6.64 (d, J = 7.5 Hz, 2H), 5.48 (s, 2H),

5.35 (t, J = 9.2 Hz, 1H), 3.82 (s, 1H), 3.73 (s, 1H), 3.53 (s, 1H), 2.67 (dd, J = 12.4, 9.1 Hz, 1H),

2.42 (dd, J = 12.4, 9.1 Hz, 1H); ¹³C NMR ( MHz DMSO) δ 7 67 8 6

150.99, 147.40, 145.86, 143.98, 139.99, 136.05, 134.33, 130.29, 127.52, 126.93, 126.17, 123.85,

123.12, 122.22, 120.06, 116.20, 113.58, 113.30, 60.70, 53.75, 38.96; ESI-MS: m/z Calculated

594.03, found [M+H]⁺595.0; Anal. Calcd for C₃₁H₂₄ClN₇O₄: C, 62.68; H, 4.07; Cl, 5.97; N,

16.51; O, 10.77%; found C, 62.34; H, 4.14; N, 16.97%.

2.3.20. N'-(2-(5'-chloro-2-(4-hydroxyphenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-

c][1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotino hydrazide (6ae):

Saddle Brown solid; IR (KBr) (υmax cm^-1): 3136 (C-H Aromatic), 1615 (C=N_str), 1749 (C=O_str),

1299 (C-O ether); ¹H NMR ( MHz DMSO) δ 8 9 (d J = 7.4 Hz, 2H), 8.67 (s, 1H), 8.35 (s,

1H), 7.88 (d, J = 7.4 Hz, 2H), 7.71 (d, J = 7.5 Hz, 1H), 7.60 - 7.44 (m, 2H), 7.30 (qd, J = 7.8, 1.6

Hz, 4H), 7.14 (td, J = 7.5, 1.5 Hz, 1H), 6.91 - 6.68 (m, 3H), 5.00 (t, J = 9.1 Hz, 1H), 4.39 (s, 1H),

13

3.61 (s, 1H), 3.21 (s, 1H), 2.67 (dd, J = 12.4, 9.1 Hz, 1H), 2.42 (dd, J = 12.4, 9.1 Hz, 1H); C

NMR ( MHz DMSO) δ 7 67 8 6 9 8 99, 145.86, 143.98,

139.99, 136.05, 134.33, 130.29, 128.09, 127.52, 126.17, 124.84, 123.85, 123.12, 120.06, 116.20,

115.78, 113.30, 60.70, 53.75, 38.96; ESI-MS: m/z Calculated 595.01, found [M+H]⁺596.01;

Anal. Calcd for C₃₁H₂₃ClN₆O₅: C, 62.58; H, 3.90; Cl, 5.96; N, 14.12; O, 13.44%; found C,

62.20; H, 3.40; N, 14.28%.

2.3.21. N'-(2-(5'-chloro-2'-oxo-2-(p-tolyl)-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]

oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6af):

Saddle Brown solid; IR (KBr) (υmax cm^-1): 3255 (C-H Aromatic), 1620 (C=N_str), 1786 (C=O_str),

1

1241 (C-O ether); H NMR ( MHz DMSO) δ 9 (s H) 8 9 (d J = 7.4 Hz, 2H), 7.88

(d, J = 7.2 Hz, 2H), 7.63 (dd, J = 4.4, 3.1 Hz, 2H), 7.55 - 7.35 (m, 4H), 7.31 (td, J = 7.5, 1.5 Hz,

1H), 7.21 (dd, J = 7.5, 1.5 Hz, 1H), 7.19 - 7.10 (m, 3H), 5.08 (t, J = 9.2 Hz, 1H), 3.93 (s, 1H),

3.65 (d, J = 17.2 Hz, 2H), 2.67 (dd, J = 12.4, 9.1 Hz, 1H), 2.42 (dd, J = 12.4, 9.1 Hz, 1H), 2.28

(s, 3H); ¹³C NMR ( MHz DMSO) δ 7 67 8 6 154.15, 150.99, 145.86, 143.98,

139.99, 139.17, 136.05, 134.33, 131.77, 130.29, 128.97, 127.52, 126.17, 125.43, 123.85, 123.12,

120.06, 116.20, 113.30, 60.70, 53.75, 38.96, 21.12; ESI-MS: m/z Calculated 593.04, found

[M+H]⁺594.0; Anal. Calcd for C₃₂H₂₅ClN₆O₄: C, 64.81; H, 4.25; Cl, 5.98; N, 14.17; O, 10.79%;

found C, 64.43; H, 4.47; N, 14.56%.

2.3.22. N'-(2-(5'-chloro-2-(4-fluorophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e] pyrazolo[1,5-

c][1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6ag):

Greenish Brown solid; IR (KBr) (υmax cm^-1): 3230 (C-H Aromatic), 1625 (C=N_str), 1727

(C=O_str), 1265 (C-O ether); ¹H NMR ( MHz DMSO) δ 8 9 (d J = 7.4 Hz, 2H), 8.69 (s, 1H),

7.81 (d, J = 7.4 Hz, 2H), 7.68 (d, J = 7.5 Hz, 1H), 7.61 (d, J = 1.4 Hz, 1H), 7.44 (ddd, J = 7.5,

5.0, 1.3 Hz, 2H), 7.41 - 7.17 (m, 3H), 7.11 (td, J = 7.5, 1.4 Hz, 1H), 7.05 - 6.92 (m, 3H), 5.00 (t,

J = 9.1 Hz, 1H), 4.71 (s, 1H), 3.97 (s, 1H), 3.76 (s, 1H), 2.67 (dd, J = 12.4, 9.1 Hz, 1H), 2.42 (dd,

13

J = 12.4, 9.1 Hz, 1H); C NMR ( MHz DMSO) δ 7 67 8 6 6 6 8

154.15, 150.99, 145.86, 143.98, 139.99, 136.05, 134.33, 130.29, 129.28, 128.32, 127.52, 126.17,

123.85, 123.12, 120.06, 116.37, 113.30, 60.70, 53.75, 38.96; ESI-MS: m/z Calculated 597.00,

found [M+H]⁺598.0; Anal. Calcd for C₃₁H₂₂ClFN₆O₄: C, 62.37; H, 3.71; Cl, 5.94; F, 3.18; N,

14.08; O, 10.72%; found C, 62.64; H, 3.44; N, 14.19%.

2.3.23. N'-(2-(2-(3-bromophenyl)-5'-chloro-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-

c][1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6ah):

Saddle Brown solid; IR (KBr) (υmax cm^-1): 3055 (C-H Aromatic), 1616 (C=N_str), 1730 (C=O_str),

1175 (C-O ether); ¹H NMR ( MHz DMSO) δ 8 96 (d J = 7.4 Hz, 2H), 8.76 (s, 1H), 7.90 (d,

J = 7.4 Hz, 2H), 7.77 (t, J = 1.4 Hz, 1H), 7.66 (d, J = 1.5 Hz, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.45

(ddt, J = 7.4, 4.6, 1.2 Hz, 3H), 7.32 (dd, J = 7.4, 1.5 Hz, 1H), 7.26 (td, J = 7.5, 1.6 Hz, 1H), 7.19

(t, J = 7.4 Hz, 1H), 7.10 (td, J = 7.5, 1.4 Hz, 1H), 6.98 (dd, J = 7.4, 1.5 Hz, 1H), 5.17 (t, J = 5.8

Hz, 1H), 4.00 (s, 1H), 3.88 (s, 1H), 3.75 (s, 1H), 2.67 (dd, J = 12.4, 5.8 Hz, 1H), 2.42 (dd, J =

12.4, 5.8 Hz, 1H); ¹³C NMR ( MHz DMSO) δ 7 67 8 6 99

145.08, 143.98, 139.99, 136.05, 134.66, 134.33, 130.29, 130.05, 129.80, 127.61, 126.17, 124.63,

123.85, 123.12, 120.06, 116.20, 113.30, 60.70, 53.75, 38.96; ESI-MS: m/z Calculated 657.91,

found [M+H]⁺658.9; Anal. Calcd for C₃₁H₂₂BrClN₆O₄: C, 56.59; H, 3.37; Br, 12.15; Cl, 5.39; N,

12.77; O, 9.73%; found C, 56.46; H, 3.96; N, 12.41%.

2.3.24. N'-(2-(5'-chloro-2-(4-nitrophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c]

[1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6ai):

Saddle Brown solid; IR (KBr) (υmax cm^-1): 3152 (C-H Aromatic), 1632 (C=N_str), 1761 (C=O_str),

1085 (C-O ether); ¹H NMR ( MHz DMSO) δ 8 96 (d J = 7.4 Hz, 2H), 8.60 (s, 1H), 8.19 (d,

J = 7.5 Hz, 2H), 7.89 (dd, J = 12.6, 7.5 Hz, 3H), 7.73 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 1.6 Hz,

1H), 7.53 (dd, J = 7.5, 1.6 Hz, 1H), 7.32 (dd, J = 7.4, 1.5 Hz, 1H), 7.25 (td, J = 7.5, 1.5 Hz, 1H),

7.10 (td, J = 7.5, 1.4 Hz, 1H), 7.05 - 6.87 (m, 1H), 5.09 (t, J = 9.1 Hz, 1H), 3.96 (s, 1H), 3.69 (s,

1H), 3.36 (s, 1H), 2.67 (dd, J = 12.4, 9.1 Hz, 1H), 2.42 (dd, J = 12.4, 9.1 Hz, 1H); ¹³C NMR (100

MHz DMSO) δ 7 67 8 6 99 6 86 98 9 99 6 9

136.05, 134.33, 130.29, 127.52, 126.17, 125.51, 123.85, 123.12, 120.06, 116.20, 113.30, 60.70,

53.75, 38.96; ESI-MS: m/z Calculated 624.01, found [M+H]⁺625.0; Anal. Calcd for

C₃₁H₂₂ClN₇O₆: C, 59.67; H, 3.55; Cl, 5.68; N, 15.71; O, 15.38%; found C, 59.54; H, 3.24; N,

15.34%.

2.3.25. N'-(2-(5'-chloro-2-(2-hydroxyphenyl)-2'-oxo-1,10b-dihydrospiro[benzo [e]pyrazolo[1,5-

c][1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotino hydrazide (6aj):

Sienna Brown solid; IR (KBr) (υmax cm^-1): 3251 (C-H Aromatic), 1684 (C=N_str), 1727 (C=O_str),

1140 (C-O ether); ¹H NMR ( MHz DMSO) δ (s H) 8 9 (d J = 7.4 Hz, 2H), 8.33 (s,

1H), 7.84 (d, J = 7.4 Hz, 2H), 7.71 (d, J = 1.6 Hz, 1H), 7.65 (d, J = 7.5 Hz, 1H), 7.45 (dd, J =

7.5, 1.4 Hz, 1H), 7.43 - 7.21 (m, 3H), 7.13 (td, J = 7.5, 1.5 Hz, 1H), 7.07 (td, J = 7.5, 1.6 Hz,

1H), 7.04 - 6.91 (m, 2H), 6.85 (dd, J = 7.4, 1.5 Hz, 1H), 5.74 (t, J = 5.4 Hz, 1H), 5.61 (s, 1H),

4.18 (s, 1H), 3.69 - 3.50 (m, 2H), 2.42 (dd, J = 12.4, 5.4 Hz, 1H); ¹³C NMR (100 MHz, DMSO)

δ 7 67 8 6 7 79 99 98 9 99 8 7 6

130.29, 129.84, 127.8, 127.52, 126.17, 123.85, 123.12, 121.25, 120.06, 118.65, 116.20, 115.54,

113.30, 60.70, 53.75, 38.89; ESI-MS: m/z Calculated 595.01, found [M+H]⁺596.0; Anal. Calcd

for C₃₁H₂₃ClN₆O₅: C, 62.58; H, 3.90; Cl, 5.96; N, 14.12; O, 13.44%; found C, 62.44; H, 3.40; N,

14.37%.

2.3.26. N'-(2-(5'-chloro-2-(4-methoxyphenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo

[1,5-c][1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6ak):

Saddle Brown solid; IR (KBr) (υmax cm^-1): 3155 (C-H Aromatic), 1641 (C=N_str), 1737 (C=O_str),

1243 (C-O ether); ¹H NMR ( MHz DMSO) δ 8 9 (d J = 7.4 Hz, 2H), 8.73 (s, 1H), 7.91 (d,

J = 7.4 Hz, 2H), 7.73 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 1.4 Hz, 1H), 7.58 - 7.41 (m, 3H), 7.41 -

7.20 (m, 2H), 7.07 (td, J = 7.5, 1.4 Hz, 1H), 7.04 - 6.82 (m, 3H), 5.26 (t, J = 6.2 Hz, 1H), 4.10 (d,

J = 19.4 Hz, 2H), 3.84 (s, 1H), 3.78 (s, 3H), 2.67 (dd, J = 12.5, 6.2 Hz, 1H), 2.42 (dd, J = 12.3,

6.2 Hz, 1H); ¹³C NMR ( MHz DMSO) δ 7 67 8 6 9 7 99

145.86, 143.98, 139.99, 136.05, 134.33, 130.29, 127.42, 126.78, 126.17, 123.85, 123.12, 120.06,

116.20, 114.84, 113.30, 60.70, 56.03, 53.75, 38.96; ESI-MS: m/z Calculated 609.04, found

[M+H]⁺610.0; Anal. Calcd for C₃₂H₂₅ClN₆O₅: C, 63.11; H, 4.14; Cl, 5.82; N, 13.80; O, 13.13%;

found C, 63.33; H, 4.34; N, 13.46%.

2.3.27. N'-(2-(5'-chloro-2-(2-chlorophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c]

[1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6al):

Saddle Brown solid; IR (KBr) (υmax cm^-1): 3200 (C-H Aromatic), 1670 (C=N_str), 1676 (C=O_str),

1260 (C-O ether); ¹H NMR ( MHz DMSO) δ 8 9 (d J = 7.4 Hz, 2H), 8.21 (s, 1H), 7.83 (d,

J = 7.4 Hz, 2H), 7.60 (d, J = 1.3 Hz, 1H), 7.52 - 7.23 (m, 6H), 7.18 - 7.08 (m, 3H), 6.98 (dd, J =

7.4, 1.5 Hz, 1H), 5.01 (t, J = 9.1 Hz, 1H), 3.92 (d, J = 9.0 Hz, 2H), 3.79 (s, 1H), 2.67 (dd, J =

12.4, 9.1 Hz, 1H), 2.42 (dd, J = 12.4, 9.1 Hz, 1H); ¹³C NMR ( MHz DMSO) δ 7

167.84, 164.23, 154.15, 150.99, 144.48, 143.98, 139.99, 136.05, 134.33, 133.42, 131.78, 130.20,

127.82, 127.52, 126.84, 126.17, 123.85, 123.12, 120.06, 116.20, 113.30, 60.70, 53.75, 38.89;

ESI-MS: m/z Calculated 613.46, found [M+H]⁺614.4; Anal. Calcd for C₃₁H₂₂Cl₂N₆O₄: C, 60.70;

H, 3.61; Cl, 11.56; N, 13.70; O, 10.43%; found C, 60.50; H, 3.64; N, 13.58%.

2.3.28. N'-(2-(5'-chloro-2-(3-nitrophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-c]

[1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6am):

Dark rown solid; IR (KBr) (υmax cm^-1): 3241 (C-H Aromatic), 1691 (C=N_str), 1800 (C=O_str),

1

1271 (C-O ether); H NMR ( MHz DMSO) δ 8 89 (d J = 7.4 Hz, 2H), 8.57 (t, J = 1.4 Hz,

1H), 8.17 (dt, J = 7.5, 1.5 Hz, 1H), 8.01 (s, 1H), 7.95 - 7.74 (m, 3H), 7.64 (dd, J = 16.4, 4.4 Hz,

2H), 7.56 (t, J = 7.5 Hz, 1H), 7.40 (ddd, J = 11.0, 7.5, 1.4 Hz, 2H), 7.25 (td, J = 7.5, 1.5 Hz, 1H),

7.09 (td, J = 7.5, 1.4 Hz, 1H), 6.97 (dd, J = 7.5, 1.4 Hz, 1H), 5.96 (t, J = 6.0 Hz, 1H), 5.28 (s,

1H), 4.30 (s, 1H), 3.69 (s, 1H), 2.67 (dd, J = 12.4, 6.1 Hz, 1H), 2.42 (dd, J = 12.5, 6.0 Hz, 1H);

¹³C NMR ( MHz DMSO) δ 7 67 8 6 99 7 7 8 98

139.99, 136.05, 135.81, 134.33, 130.36, 129.29, 127.52, 126.17, 124.10, 123.85, 123.12, 121.84,

120.06, 116.20, 113.30, 60.70, 53.75, 38.96; ESI-MS: m/z Calculated 624.01, found [M+H]⁺

625.0; Anal. Calcd for C₃₁H₂₂ClN₇O₆: C, 59.67; H, 3.55; Cl, 5.68; N, 15.71; O, 15.3 %; found C,

59.56; H, 3.26; N, 15.34%.

2.3.29. N'-(2-(5'-chloro-2-(2,4-dichlorophenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo

[1,5-c][1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6an):

Olive green solid; IR (KBr) (υmax cm^-1): 3245 (C-H Aromatic), 1645 (C=N_str), 1690 (C=O_str),

1

1200 (C-O ether); H NMR ( MHz DMSO) δ 8 9 (d J = 7.4 Hz, 2H), 8.48 (s, 1H), 7.90 (d,

J = 7.4 Hz, 2H), 7.71 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 1.4 Hz, 1H), 7.44 (dd, J = 7.8, 1.8 Hz, 1H),

7.43 - 7.31 (m, 3H), 7.31 - 7.13 (m, 2H), 7.11 (td, J = 7.5, 1.5 Hz, 1H), 6.98 (dd, J = 7.5, 1.4 Hz,

1H), 5.58 (t, J = 9.0 Hz, 1H), 4.47 (s, 1H), 4.08 (s, 1H), 3.80 (s, 1H), 2.74 - 2.65 (m, 1H), 2.44 -

13

2.36 (m, 1H); C NMR ( MHz DMSO) δ 7 67 8 6 99 8

143.98, 139.99, 136.05, 134.33, 133.42, 132.99, 132.69, 130.55, 130.29, 128.41, 127.86, 127.52,

126.17, 123.85, 123.12, 120.06, 116.20, 113.30, 60.70, 53.75, 38.89; ESI-MS: m/z Calculated

647.90, found [M+H]⁺648.9; Anal. Calcd for C₃₁H₂₁Cl₃N₆O₄: C, 57.47; H, 3.27; Cl, 16.41; N,

12.97; O, 9.88%; found C, 57.67; H, 3.47; N, 12.46%.

2.3.30. N'-(2-(5'-chloro-2-(2-methoxyphenyl)-2'-oxo-1,10b-dihydrospiro[benzo[e]pyrazolo[1,5-

c][1,3]oxazine-5,3'-indolin]-1'-yl)-2-oxoethyl)isonicotinohydrazide (6ao):

Saddle Brown solid; IR (KBr) (υmax cm^-1): 3260 (C-H Aromatic), 1630 (C=N_str), 1763(C=O_str),

1089 (C-O ether); ¹H NMR ( MHz DMSO) δ 7 (s H) 9 - 8.86 (m, 2H), 8.02 - 7.83

(m, 2H), 7.73 (s, 1H), 7.67 (s, 1H), 7.48 (d, J = 30.2 Hz, 2H), 7.30 (s, 1H), 7.24 (d, J = 1.2 Hz,

2H), 7.07 (s, 1H), 6.97 (d, J = 0.5 Hz, 2H), 6.90 (s, 1H), 5.25 (s, 1H), 4.20 (s, 1H), 4.08 (s, 1H),

3.82 - 3.77 (m, 3H), 3.73 (s, 1H), 2.67 (s, 1H), 2.42 (s, 1H); ¹³C NMR ( MHz DMSO) δ

175.32, 167.84, 164.23, 158.35, 154.15, 150.99, 143.98, 139.99, 136.73, 136.05, 134.33, 130.29,

128.31, 127.52, 127.27, 126.17, 123.77, 123.12, 121.85, 120.06, 116.20, 114.88, 113.30, 60.70,

56.78, 53.75, 38.89; ESI-MS: m/z Calculated 609.04, found [M+H]⁺610.0; Anal. Calcd for

C₃₂H₂₅ClN₆O₅: C, 63.11; H, 4.14; Cl, 5.82; N, 13.80; O, 13.13%; found C, 63.40; H, 4.25; N,

13.40%.

2.4. Antimycobacterial activity

2.4.1. Method 1: Lowenstein-Jensen (L-J) method for the zone of inhibition

All the newly synthesized compounds (6a-6ao) were screened for their in vitro

antimycobacterial activity against Mycobacterium tuberculosis H₃₇Rv using Lowenstein-Jensen

(L-J) method. The anti-mycobacterial assay was performed in the L-J medium. An antibiotic

standard used was isoniazid. Determination of Colony-forming units (c.f.u) on L-J in brief, the

ten-fold dilution of standard 1 mg/mL, M. tuberculosis suspension[26] were streaked on L-J

medium for determining c.f.u in the presence and absence of compound. An M. tuberculosis

suspension of 1 mg/ml is equivalent to the MacFarland standard.[27] One loopful (6 µl) of this

suspension was streaked on the L-J slants using a 3 mm external diameter loop. Reagents of L-J

media included potassium dihydrogen phosphate anhydrous (Qualigens), magnesium sulfate

anhydrous (Qualigens), magnesium citrate (Loba Chemie), L-asparagine (Hi-media, Mumbai),

glycerol (Fisher Scientific, Mumbai), and malachite green (Hi-Media, Mumbai). The drug was

incorporated in the medium at a concentration of 2 percent v/v and 4 percent v/v of the drug was

dissolved into 100 ml of culture medium prior to inspissation. The medium set inoculated with

the standard bacterial suspension and incubated at 37 °C for 42 days. Reading was taken weekly.

For comparison, drug-free control slants were used. Susceptibility testing of H₃₇Rv isolates was

also performed against standard drugs like isoniazid. Each test was done in triplicate. Percentage

inhibition was calculated by the mean reduction in the number of colonies on drug-containing as

compared to drug-free controls.

Where, c = control, t = test.

2.4.2. Method 2: Lowenstein–Jensen (L-J) method for MIC value

Drug susceptibility and determination of MIC of the test compounds (6a-6ao) against

Mycobacterium tuberculosis H₃₇Rv were performed by Lowenstein-Jensen (L-J) MIC

method.[28,29] Where primary 1000, 500, 250, and secondary 200, 100, 62.5, 50, 25, 12.5, 6.25,

μg mL⁻¹dilutions of each test compound was added to liquid Lowenstein–Jensen medium.

The standard strain M. tuberculosis H₃₇Rv was tested with known drug isoniazid. Further,

compounds 6a, 6b, 6e, 6g, 6h, 6o, 6ab, 6ad, and 6ag were tested for MDR-TB.

2.5. Statistical analysis

The determination assay for each Antimycobacterial activity result was carried out in

thrice to minimize the percentage of error. Statistical analysis was performed using the Statistical

Package for Social Science (IBM SPSS Statistics 20 for Windows, SPSS Inc., Chicago, IL,

USA).

2.6. In silico ADMET properties

In silico pharmacokinetic properties and toxicities were predicted using Accelrys

Discovery Studio 2.1. All the compounds were prepared in neutralized form for the calculation

of pharmacokinetic properties by the Marvin Suite program saved in SD format. The module

uses six mathematical models, to predict properties quantitatively by a set of keys (Table 1) that

specify threshold ADMET characteristics for the chemical structure of the molecules based on

available drug information. For the ADMET human intestinal absorption[6], the model was

developed using 199 compounds in the training set based on the calculations AlogP

(ADMET_AlogP98) and polar surface area (PSA_2D). ADMET aqueous solubility predicts the

solubility of all compounds in water at 25 °C. The model is based on a genetic partial least

squares method on a training set of 784 compounds with experimentally measured

solubilities.[30] ADMET Blood-Brain Barrier (BBB) model predicts blood-brain penetration of a

molecule after oral administration. This model was derived from a quantitative linear regression

model, over 800 compounds that are known to enter the CNS after oral administration.[31]

ADMET Plasma Protein Binding (PPB) model predicts whether a compound is likely to be

highly bound to carrier proteins in the blood. Binding levels predicted by the marker similarities

are modified according to conditions on calculated logP [32] ADMET CYP2D6 binding predicts

cytochrome P4502D6 enzyme inhibition using the 2D chemical structure as input and a probable

estimate for the prediction. Predictions are based on a training set of 100 compounds with known

CYP2D6 inhibitions.[33] ADMET hepatotoxicity predicts the potential human hepatotoxicity for

a varied range of structurally different compounds. Predictions are based on a model of 382

training compounds known to exhibit liver toxicity or to trigger dose-related elevated

aminotransferase levels in more than 10% of the human population.[34]

2.7. Molecular Docking

Molecular docking and Molecular dynamics were performed using the YASARA (Yet

Another Scientific Artificial Reality Application) commercial package. The experiments were

accomplished using Autodock Vina 1.0 [35] configured in YASARA Structure [36] with eight

central processing units in a Microsoft Windows™ workstation equipped with Intel® Xeon™

E5620 processor. Docking analysis is a significant step for the selection of potential hits in

virtual screening. Consequently, grid points with 0.35 Å spacing were enumerated using

Autodock Vina default optimization parameters. The experimentally verified active site residues

were demarcated as a docking area. The torsional angles of the ligands and active site residues

side chains have been made flexible due to obtain a favorable binding conformation. For

considering standard docking procedure, 100 genetic algorithm runs per ligand with an initial

population of 150 randomly placed individually. The maximum number of energy evaluations

was limited to 2.5 × 10⁵. The rate of gene mutation and crossover was controlled to 0.02 and 0.8.

The docking poses from each experiment (100 solutions) were gathered on the basis of docking

energy (kcal/mol) and the root mean squared deviation (RMSD). The most noticeable thing is

that higher positive energies mean better binding, and negative energies mean no binding

according to YASARA energy parameters. The docking energy was calculated by the following

equation.

ΔG = ΔG_vdw+ ΔG_Hbond+ ΔG_elec+ ΔG_tor+ ΔG_desolv

Where,

ΔG_vdw= van der Waals term for docking energy

ΔG_Hbond= H bonding term for docking energy

ΔG_elec= electrostatic term for docking energy

ΔG_tor= torsional free energy term for ligand when the ligand transits

from unbounded to bounded state

ΔG_desolv= desolvation term for docking energy

2.7.1. Preparation of Ligands

Three-dimensional structures of ligand input for all docking experiments were generated

using the Marvin Suite applications (Marvin Sketch) followed by adding explicit hydrogen with

cleaning, energy minimization, and alignment process. The generated structures were saved as

MOL2/SDF files by energy minimization using the MMFF94 force field.

2.7.2. Preparation of Receptors

To investigate the ligand-protein interactions of synthesized compounds, docking studies

were performed on all compounds against isoniazid-resistant I21V enoyl-ACP(COA) reductase

mutant enzyme from Mycobacterium tuberculosis in Complex with NADH-INH (PDB entry:

2IE0)[37] Crystal structure of 2IE0 was retrieved from RCSB Protein Data Bank

(http://www.pdb.org). The structure having the depth of resolution 2.2 Å, crystal structure

includes Oxidoreductase enzyme 268 amino acids chain length, ligand placed isonicotinic-

acetyl-nicotinamide-adenine dinucleotide (ZID) which are effective in recognition of its active

binding sites. The 3D structures determined by X-ray diffraction of Escherichia coli (PDB ID:

2IE0). The protein structure was treated using standard molecular mechanics protocol which

includes removal of crystallographic waters, the addition of polar hydrogens, assignment of bond

orders, and Kollman charge [38]. The protein structure with bound isonicotinic-acetyl-

nicotinamide-adenine dinucleotide was atom-typed with the Amber03 force field [39] and energy

minimized using steepest descent technique (100 iterations) in YASARA Structure program.[36]

The interaction profiles for all generated hits have been studied. The active side of amino acid

residues was determined which is being used to dock molecules for automated docking and

scoring. The active site residues of the enoyl-ACP(COA) reductase enzyme are Ile194, Gly96,

Trp222, Gly14, Lys165, Tyr158, Asp64, Val21, Met147, Pro193, Leu63, Phe41, Ala191,

Phe149, Ser94, Ile122, Met199, Ile95, Val65, Thr196, Asp148, Gly192, Ile15, Ser20, Ile16.

2.8. Molecular Dynamics

The residence time and binding stability of the designed ligands in complex with

isoniazid-resistant enoyl-ACP (COA) reductase mutant enzyme from Mycobacterium

Tuberculosis were examined using molecular dynamic simulations. The dock poses of these

receptor-ligand complexes were energy minimized due to remove unfavorable atomic contacts

and subsequently specified as starting conformation for dynamic simulations in YASARA

structure software. [36] The hydrogen-bonding network in the complexes was optimized as well

as atom typed using the AMBER03 force field [36] intended for its application in aqueous

solution. The acid dissociation constant (pKa) values of titratable amino acids were assigned and

chosen orthorhombic simulation box with other default parameters of periodic boundary

conditions. molecular dynamics simulation were performed by following steps: the temperature

at 298 K, pressure of 1 bar, pH 7.0, coulomb electrostatics at a cutoff of 7.86, solvated by water

model (density 0.997 gL⁻¹) inside the simulation cell, 0.9 % as ion concentration of NaCl to

neutralize the system and to minimize the energy using steepest gradient approach (100 cycles)

and all atoms were mobile. The 2IE0 protein was cleaned, hydrogens were added and water

molecules were removed. 10 ns production time had been chosen for the analysis of protein

structure evaluations by every 2500 ps. The generated time trajectories of the selected protein-

ligand complex were analyzed for the stabilization of protein viz. RMSD parameter. The protein-

ligand interaction patterns of protein-ligand complex generated at the starting time and ending

time through the Protein-Ligand Interaction Profiler server. The average structures were

determined from the simulations and were used to calculate the RMSDs for the respective model.

3. Results and discussion

3.1. Chemistry

The molecular hybridization is a strategy of rational drug design of new ligands or

prototypes based on the recognition of pharmacophoric sub-unities in the molecular structure of

two or more known bioactive derivatives which, through the adequate fusion of these sub-

unities, lead to the design of new hybrid architectures that maintain pre-selected characteristics

of the original templates. Furthermore, this strategy can result in compounds presenting modified

selectivity profiles, different and/or dual modes of action, and reduced undesired side effects. In

our previous work(25), we have reported synthesis of five compound series of 2-phenyl-

1,10bdihydrospiro [benzo[e]Pyrazolo[1,5-c][1,3]oxazine-5,3'-indolin]-2'-ones derivatives (5a-5e)

by conventional as well as microwave methods. These compounds were showed good

antimycobacterial activities. In an effort to prepare a better therapeutic agent for the treatment of

tuberculosis and in continuation of our efforts, we have attempted to design novel

antimycobacterial agents by hybridizing isoniazid linker to spirooxindole scaffold. Incorporation

of two biologically versatile heterocyclic scaffolds like isoniazid and spirooxindole in a single

molecular platform was undertaken. Therefore, the designed compound possessed two

pharmacophoric units, Isoniazid, a linker stacked between spirooxindole core as shown in (Fig.

2).

A series of targeted compounds dihydrospiro[benzopyrazolo[1,5-c][1,3]oxazine-5,3'-

indolin]isonicotinohydrazide derivatives (6a-6ao) were synthesized by stepwise reactions that

have been demonstrated in Scheme 1. In the first step, we have described an efficient, one-pot

synthesis of 2-phenyl-1,10b-dihydrospiro[benzo[e]Pyrazolo[1,5-c][1,3]oxazine-5,3'-indolin]-2'-

ones (5a-5ao) by the reaction between 2-(3-(4-substituted phenyl)-4,5-dihydro-1Hpyrazol-5-yl)

phenol derivatives (4a-4o) and isatin derivative using p-TSA as a prompt catalyst under

conventional as well as microwave methods, with better yield and short reaction time. In the next

step, compound (5a-5ao) react with chloroacetyl chloride in the presence of triethylamine at 0°C

temperature and followed by react with isoniazid to give N'-(2-oxo-2-(2'-oxo-2-phenyl-1,10b-

dihydrospiro[benzo[e]pyrazolo

[1,5-c][1,3]oxazine-5,3'-indolin]-1'-yl)ethyl)isonicotine

hydrazide (6a-6ao) with good 60-80% practical yield. A comparison of yield and melting point

for the synthesis of 6a-6ao was mentioned in Table 2.

Designed series of isoniazid-spirooxindole derivatives (6a-6ao) were characterized by

elemental analysis and different spectroscopic techniques like IR, ¹H NMR, ¹³C NMR, and mass

spectrometry and were in full agreement with proposed synthesized structures. The IR spectrum

of 6a exhibited four main peaks situated at 3255, 1616, 1724, and 1213 cm^-1, which were

assigned to the (C-H_str) aromatic, (C=N_str) imine, (C=O_str) ketone, (C-O_str) ether respectively.

1

The H NMR spectrum of 6a showed a singlet at 86 (s H) δ value is due to amidic N-H

proton of isoniazid. Another N-H proton of isoniazid gives singlet at 9 (s H) δ ppm Chiral

carbon (C-H) of pyrazole ring displayed a triplet at δ 9 (t J = 6.4 Hz, 1H) and two

characteristic peaks of neighboring chemically non-equivalent, diastereotopic protons showed as

double doublet at 2.42 (dd, J = 12.4, 6.4 Hz, 1H) and 2.67 (dd, J = 6 Hz H) δ ppm All

the protons of aromatic regions appeared in the range of 6.9 - 8 9 δ ppm Sidechain contained

pyridine ring’s proton which resonates at 8 9 (d J = 7.5 Hz, 2H), 7.95 (d, J = 7 Hz H) δ

ppm. The ¹³C NMR spectrum of 6a showed the carbonyl carbons of isatin, isoniazid, and the side

chain of isatin displays signals at 175, 164, and 167 ppm, correspondingly. The -CH and -CH₂

carbons of pyrazole ring resonated at 60 and 38.9 ppm, respectively. Another CH₂carbon of

isatin linkage showed a signal at 53 ppm. Values between 111-139 ppm are due to aromatic

carbons present in all the aromatic rings in compound 6a. Mass spectrum of 6a gave a molecular

ion peak at m/z 545.5 due to [M+H]⁺thereby confirming the structure. In compound 6a, the

1

characteristic peaks for protons and carbons in H-NMR and ¹³C-NMR appeared at specific

chemical shift values were in good agreement with the molecular formula of the synthesized

compounds.

3.2. In vitro antimycobacterial activity

Because of the increased resistance diversity of tuberculosis in contradiction of standard

drugs, there is an urgent requirement for the development of novel drug moiety that could

susceptibly kill Mycobacterium species. This approach was laid for the development of such

compounds with increased efficiency in terms of lower MIC values to kill H₃₇Rv with a

maximum percent inhibition. The synthesized isoniazid-spirooxindole derivatives (6a-6ao) have

been evaluated for anti-tubercular activity against Mycobacterium tuberculosis H₃₇Rv as shown

in Table 3, (Fig. 3). Compound 6ab which has two chloro (-Cl) groups at the para position of a

benzene ring and fifth position of isatin proves to be the best anti-tubercular agent (highest %

inhibition value 81.88) shows MIC value 12.5 μg mL^-1. Compound 6a (% inhibition 72.25), 6c

(% inhibition 76.30), 6f (% inhibition 65.89), 6h (% inhibition 71.09), 6l (% inhibition 58.69),

6n (% inhibition 52.89), 6af (% inhibition 58.67), 6ai (% inhibition 46.28), 6am (% inhibition

46.28) and 6ao (% inhi ition 6 8) are known to show same MIC value ( μg mL^-1) with

variation in percent inhibition values of the targeted pathogenic strain of Mycobacterium.

Compounds 6a (R= 4-H), 6c (R= 4-Br), 6g (R= 4-F) and 6h (R= 3-Br) were found to be more

active in comparison to other compounds with %inhibition in the range of 70-77%. Compounds

6o (R = 2-OCH₃) and 6ad (R = 4-NH₂, R₁= Cl) having electron-donating and electro

withdrawing groups showed etter antimyco acterial activity at MIC value μg mL^-1.

Compounds 6e (R = 4-OH) and 6ag (R = 4-F) also have good MIC value 6 μg mL^-1.

Compounds 6g, 6k, 6ac, 6ah, 6an, and 6ah inhibit the M.tuberculosis at μg mL^-1which

displayed moderate to good activity. Compound 6a ( μg mL^-1), 6b ( μg mL^-1), 6e ( μg

mL^-1), 6g ( μg mL^-1), 6h ( μg mL^-1), 6o (6 μg mL^-1), 6ab ( μg mL^-1), 6ad (6 μg

mL^-1), 6ag ( μg mL^-1) were to be screened for MDR-TB using Kanamycin (6 μg mL^-1) as

a standard drug (Table 4). Out of them, compound 6ab showed good MIC value μg mL^-1.

The presence of the isoniazid and spirooxindole ring as pharmacophores and different derivatives

of acetophenone increase the lipophilic character of the molecule. The presence of electro

donating and electro withdrawing groups on acetophenone and isatin derivative facilitates the

crossing through the biological membrane of the microorganism thereby inhibiting their growth.

3.3. In silico ADMET Prediction and evaluation of Lipinski's ‘Rule of 5’

ADME/Toxicity properties, pharmacokinetics, and toxicity are a major indication for the

failure of drugs in the drug development process. Thus, the initial assessment of ADMET

(Adsorption, Distribution, Metabolism, Excretion, and Toxicity) is necessary for an effective

drug development process. We have predicted the ADMET properties of test compounds with

reference compound isoniazid for the pharmaceutically relevant properties to evaluate the drug-

likeness and pharmacokinetic properties (Table 5). In the theory of ADMET descriptors, various

protocols generate models to evaluate the pharmacokinetic properties. Human Intestinal

Absorption [6] model predicts human intestinal absorption after oral administration. A well-

absorbed compound is one that is absorbed in least 90% into the bloodstream in humans.

Isoniazid has good absorption than all the compounds. Compound 6a and 6g show moderate

absorption, on the other hand, compounds 6i, 6m, 6ai and 6am showed very low absorption.

Other compounds absorb very poorly. According to the model, a compound should have

optimum cell permeability criteria (PSA < 140 Å²and AlogP98 < 5).[40] Most of the

compounds showed a polar surface area (PSA) < 140 Å². Considering the AlogP98 criteria, all

compounds had AlogP98 value <5, except four compounds. There was no blood-brain

penetration of any compound is to be defined after oral administration because of the BBB

(blood-brain barrier) level is 4. The aqueous solubility plays an important role in the

bioavailability of the drug candidate. Categorical solubility level is extremely low of compound

6an and also all of the other compounds than standard. Further, all compounds have been

predicted to have a hepatotoxicity level of 1, except isoniazid. The hepatotoxicity model predicts

potential organ toxicity for a wide range of structurally diverse compounds. Isoniazid is non-

toxic and unlikely to cause dose-dependent liver injuries. Moreover, all the compounds are

seemed to be toxic to the liver. ADMET CYP2D6, the model predicts cytochrome P4502D6

enzyme inhibition. Compounds 6m, 6n, 6ai, 6aj, 6al, 6an, and isoniazid are non-inhibitor and

unlikely to inhibit the CYP2D6 enzyme but other compounds are able to inhibit the enzyme. The

Plasma Protein Binding (PPB) model predicts whether a compound is likely to be highly bound

to a carrier protein in the blood. Compound 6d, 6e, and 6j bind at a level of less than 90% having

AlogP98 less than 4.0, other compounds have binding >90%, clearly suggesting that have good

bioavailability and are not likely to be high binding to carrier proteins in the blood.

The DruliTo software was used to obtain new drug-like leads generated hits were

su jected to Lipinski’s Rule of Five.[41] As per Lipinski’s rule-of-five, an orally administrated

drug should have log P ≤ molecular weight (MW) < Daltons hydrogen ond donor (HBD)

≤ , and hydrogen bond acceptor (HBA) ≤ All the compounds have Lipinski’s violation of

molecular weight as shown in Table 6. Lipophilicity is a property that has a major effect both on

the absorption, distribution, metabolism, excretion and toxicity properties, and pharmacological

activity since many drugs cross biological membranes through passive transport, which strongly

depends on their lipophilicity. Lipophilicity has been studied and applied as an important drug

property for eras. Compounds 6d showed the lowest lipophilicity, while compounds 6an

demonstrated the highest. It was found that all the ligands followed the Lipinski rule with

maximum ligands (except 6d, 6e, 6j, 6k, 6o, 6ad, 6ae, 6aj, 6ak, and 6ao) showed no violation of

the above criteria. Therefore, these ligands have good potential for ensuring development as an

oral agent and can be potentially active drug candidates. The number of hydrogen bond acceptor

value was beyond the Lipinski limit in case some compounds have not affected the bioactivity of

the synthesized hybrids but they are highly effective against MTB.

3.4. Molecular docking study

In order to gain insight into the plausible mechanism of action of compounds docking

simulations were performed. In silico docking studies were carried out with crystal structure of

isoniazid-resistant I21V enoyl-ACP(COA) reductase mutant enzyme From Mycobacterium

tuberculosis in Complex with NADH-INH (PDB entry: 2IE0) with native ligand isonicotinic-

acetyl-nicotinamide-adenine dinucleotide (ZID) (Fig. 4 (a)). All the synthesized compounds were

docked into active sites of isoniazid-resistant I21V enoyl-ACP(COA) reductase mutant enzyme

has been reported as the target receptors for docking studies in finding the suitable drug

candidates against the bacteria. A prerequisite to any successful experiment is the validation step.

The validation of the docking procedure was performed by re-docking co-crystallized native

ligand (ZID) into the active site of 2IE0 using YASARA software. In this analysis, the re-docked

2IE0 reproduced the binding pose with binding energy 11.4310 kcal/mol was found in Fig. 4 (c).

which shows hydrogen bonding interactions, hydrophobic interaction, Van der Waals

interactions, and π-π interactions were observed with peptides like Gly14, Ile16, Ser20, Val21,

Ala22, Phe41, Leu63, Asp64, Val65, Gln66, Ser94, Ile95, Gly96, Phe97, Met98, Ile122, Ser123,

Met147, Asp 148, Phe149, Tyr158, Met161, Lys165, Ala191, Gly192, Pro193, Thr196, Leu197,

Ala198, Met199, and Leu218 were found as responsible key residues. The docked position was

compared to the crystal structure position by calculating RMSD values 0.52 Å (Fig. 4 (d)). In

general, RMSD values smaller than 2.0 Å indicates that the docking protocol is capable of

accurately predicting the binding orientation of the co-crystallized ligand. In this study, RMSD

values were within 2.0 Å, indicating our docking methods are validated for the given structures

and YASARA software, therefore deemed reliable for docking of isoniazid-spirooxindole

derivatives (6a-6ao) into the inhibitor binding cavity of 2IE0. The stereochemistry was measured

of protein 2IE0 using the Ramachandran plot [42] implemented in RAMPAGE [43] (RAMPAGE

server) exhibited 92.9% in ψ-ϕ core areas (Fig. 4 (b)). The occupancy of residues in most

favored, additional allowed, and generally allowed regions with an exception of seven amino

acids excluding general, Gly and Proline occupied in disallowed regions.

As it was possible to observe, all isoniazid-spirooxindole derivatives (6a-6ao) were

docked into the defined binding pocket with the given docking input constraints. The best-

docked poses were chosen on the basis of scoring function and their binding characteristics

comparing with the reference ligand. Simplifying the docking result for compound 6m, we

described here the interaction map in Fig. 5 with the highest energy 12.6080 Kcal/mol. The nitro

group of compound 6m showed H-bonding with amino acids Phe41 (2.89 Å). The nitrogen of

the pyridine ring shows H-bonding with amino acid Ile194 (3.10 Å). In addition, Van der Waals

contacts and weak π-alkyl π-sigma and other interactions were observed with peptides like

Gly14, Ile15, Ile16, Thr17, Ser20, Val21, Ala22, Thr39, Gly40, Phe41, Ile47, Ser94, Ile95,

Gly96, Phe97, Met103, Ile122, Met147, Phe149, Tyr158, Met161, Lys165, Pro193, Ile194,

Thr196, Leu197, Ala198, and Met199. In contrast, Compound 6ao has possessed the lowest

binding energy of 11.0700 Kcal/mol. Similarly, the other representative compound 6ab (Fig. 6)

which showed the highest MIC value binds via two crucial H-bond strongly with Gly96 (1.83 &

1.91 Å) through -NH and -CO of the side chain. The nitrogen of the pyridine ring shows three H-

bonding with aminoacids Ile16 (4.69 Å), Leu197 (5.35 Å), and Ala198 (3.80 Å). The compound

6ab was observed to be stabilized within the active site through strong van der Waals

interactions, electrostatic interactions, weak π-alkyl π-sigma and other interactions observed

with residues Gly14, Ile16, Thr17, Ser19, Ser20, Phe41, Asp42, Arg43, Ser94, Ile95, Gly96,

Phe97, Met98, Met103, Ile122, Thr196, Leu197, Ala198, Ile202, and Leu 7 The π stacking

was observed with Phe97 at an angle of 70.73 (5.03 and 4.60 Å) (Fig. 6). The amino acid Gly96

formed H-bond with -NH of isoniazid in compounds 6ab, 6ad, 6ae, 6af, 6ag, 6ah, 6aj, 6ak, 6al,

6am, 6an, 6ao at a distance of 3.03, 3.16, 2.82, 3.05, 3.22, 3.26, 3.21, 3.14, 3.14, 3.12, 3.00 and

2.92 Å, respectively. The information from the docking studies and In vitro anti-tubercular

activity proved the newly synthesized compounds as potent towards the bacterial pathogens.

3.5. Molecular Dynamics simulations

Molecular dynamics simulations were used to study the binding affinity of selected

ligands, in coherence with conformational changes of the isoniazid-resistant enoyl-ACP (COA)

reductase-6m compound complex and the isoniazid-resistant enoyl-ACP (COA) reductase-6ab

compound complex. This simulation study was executed at the physiological condition to

account for the following strategies. The binding affinity of the selected ligands was obtained by

docking experiments after that the molecular dynamics were performed on the target Isoniazid-

resistant enoyl-ACP (COA) reductase-6m compound complex through the 10 ns production time

to measure the binding stability of the ligand (Fig. 7). Energy and RMSD is an average taken

over the residue given time scale. The average energy of the protein-ligand complex calculated

over the simulation trajectory showed that (2IE0) developed effective interactions with the

complete ligand dataset as their energies were in the range of -592099.596 kJ mol^-1to -

777052.693 kJ mol^-1(Fig. 7). Various energy results were measured during molecular dynamics

simulations, -777052.693 kJ mol^-1retrieved at the initial start while -592099.596 kJ mol^-1at 10

ns time trajectory and the average of energy has been recognized with -594320.779 kJ mol^-1.

Different types of interactions were formed after the end of the dynamics simulation.

The simulation analyzed from the initial (0 ps) and final (10 ns) conformations, RMSD of

common target trajectories highlighted the importance of H bonds and hydrophobic interactions

conferred by hot spot residues. The protein-ligand interaction maps generated using protein-

ligand interaction profiler showed that the dominance of the residue Phe41 and Phe149 exhibited

less structural motions and acted as anchoring sites for ligand binding at the initial level of

dynamics simulation. The diverse pattern of H-bonding with main and side chains of

experimental hot spot residues (Ile16, Val21, Phe41, Val65, Ile95, Phe97, Ile122, Ile194,

Met147, Leu197, Ala198,) was obtained after the completion of the 10 ns target trajectories. The

corresponding RMSD values for the Gly96 residue represent large fluctuations with more than 2

Å and may act as ligand binding determinants. The isoniazid-resistant Enoyl-ACP (COA)

reductase-6m compound-complex was used in receptor-based superposition to study the

ensemble of simulation poses which shows the diverse pattern of the ligand interacted with the

selected receptor (Fig. 8).

When we have performed molecular dynamics study using potent and hit biological

active compound 6ab with Isoniazid-resistant enoyl-ACP (COA) reductase protein through the

10 ns production time for conformational changes and binding stability of the ligand (Fig. 9-10).

-763265.702 kJ mol^-1energy observed at the initial stage (0 ns) and -581658.933 kJ mol^-1at 10

ns time trajectory during the molecular dynamics simulations. The average energy has been

documented -583951.885 kJ mol^-1during the entire MD of protein-6ab. An overall interaction

diagram of protein residues, energy, and RMSD variation during molecular dynamic simulations

is illustrated in Fig. 9-10.

4. Conclusion

Many antimycobacterial agents are available, still with limitations with respect to their

activity due to increasing reports of multi-drug resistance. Hence, research on novel anti-TB drug

agents is now intensively pursued to identify molecular targets and their modes of action. The

present study involved the synthesis of a novel series of N'-(2-oxo-2-(2'-oxo-2-phenyl-1,10b-

dihydrospiro[benzo[e]pyrazolo[1,5-c][1,3]oxazine-5,3'-indolin]-1'-yl)ethyl)isonicotinohydrazide

derivatives. The novelty of these compounds is the most biologically active spirooxindole

scaffold is to be linkage with very well-known tuberculosis drug isoniazid for the purpose of

increasing activity. These new chemical entities displayed significant in vitro antimycobacterial

activity against Mycobacterium tuberculosis H₃₇Rv with the comparison of first-line drug

isoniazid. Among them, compound 6ab was found to be an active antimycobacterial agent.

Further, the same compound 6ab showed excellent MDR-TB activity out of some selected

compounds. Additionally, all compounds were docked into the binding pocket of Isoniazid-

resistant I21V Enoyl-ACP(COA) reductase mutant enzyme from Mycobacterium tuberculosis

(PDB entry: 2IE0). Compound 6m bind with highest energy 12.6080 Kcal/mol and of course,

compound 6ab also showed good binding with protein, in spite of showing only one violation of

Lipinski's rule. Compound 6m was analyzed for the molecular dynamics on the target Isoniazid-

resistant Enoyl-ACP (COA) reductase-6m compound complex through the 10 ns production time

to measure the binding stability of the ligand. Biological activity and molecular docking

observations conclude that compounds 6ab and 6m would help in designing new strategies for

the development of novel therapeutics, or improvement of existing pharmacotherapies against

tuberculosis, a major burden for global health.

Acknowledgment

The authors are thankful to UGC-Info net and INFLIBNET Gujarat University for providing e-

source facilities. The authors are thankful to the department of chemistry, Gujarat University for

providing necessary facilities. The authors (M.N.B) and (M.A.B) are thankful to the UGC-BSR

fellowship for financial support. Author is also very thankful to Edwin. A. Pithawala for

biological activity support for this research work.

Supplementary data

Supplementary data associated with this article can be found, in the online version.

Author Contributions

The manuscript was written through the contributions of all authors. All authors have given

approval to the final version of the manuscript.

Declaration of interests

The authors declare that they have no known competing financial interests or personal

relationships that could have appeared to influence the work reported in this paper. The authors

declare the following financial interests/personal relationships which may be considered as

potential competing interests:

The authors have declared that there is not any conflict of interest.

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Abbreviations

ADMET

BBB

CADD

FAS

Absorption, distribution, metabolism, elimination, and toxicity

blood brain barrier

computer-aided drug design

Fatty Acid Synthase

FDA

INH

Food and Drug Administration

Isoniazid

MD

Molecular dynamics

MDR

MIC

Multi-drug-resistant

Minimum inhibitory concentration

Mycobacterium tuberculosis

Nuclear magnetic resonance

Protein data bank

MTB

NMR

PDB

PSA

Polar surface area

RMSD

RMSF

TB

Root mean squared deviation

Root mean squared fluctuation

Tuberculosis

TLC

Thin layer chromatography

World Health Organization

Extensively drug-resistant

WHO

XDR

YASARA

Yet Another Scientific Artificial Reality Application

Article Doi

Novel isoniazid-spirooxindole derivatives: design, synthesis, biological evaluation, in silico ADMET prediction and computational studies

DOI: 10.1016/j.molstruc.2020.128881

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