Synthesis of coumarin-theophylline hybrids as a new class of anti-tubercular and anti-microbial agents

Article abstract of DOI:10.1016/j.ejmech.2018.01.025

A series of novel coumarin-theophylline hybrids were synthesized and examined for their anti-tubercular activity in vitro against Mycobacterium tuberculosis H₃₇Rv, anti-microbial activity in vitro against gram-positive bacteria (Staphylococcus aureus) and gram-negative bacterias (Escherichia coli, Salmonella typhi) as well as fungi (Candida albicans). The compound (3a) has shown excellent anti-tubercular activity with MIC of 0.12 μg/mL. Electron donating compounds (3a, 3f) have displayed significant anti-microbial activity. The compounds have also been precisely elucidated using single crystal X-ray diffraction techniques. Molecular docking study has been performed against 4DQU enzyme of Mycobacterium tuberculosis showed good binding interactions and is in agreement with the in vitro results.

Full text of DOI:10.1016/j.ejmech.2018.01.025

Accepted Manuscript
Synthesis of coumarin-theophylline hybrids as a new class of anti-tubercular and anti-
microbial agents
Sumitra N. Mangasuli, Kallappa M. Hosamani, Hirihalli C. Devarajegowda, Mahantesh
M. Kurjogi, Shrinivas D. Joshi
PII:
S0223-5234(18)30025-4
10.1016/j.ejmech.2018.01.025
EJMECH 10102
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 October 2017
Revised Date: 21 December 2017
Accepted Date: 8 January 2018
Please cite this article as: S.N. Mangasuli, K.M. Hosamani, H.C. Devarajegowda, M.M. Kurjogi, S.D.
Joshi, Synthesis of coumarin-theophylline hybrids as a new class of anti-tubercular and anti-microbial
agents, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.025.
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ACCEPTED MANUSCRIPT
Synthesis of coumarin-theophylline hybrids as a new class of anti-
tubercular and anti-microbial agents
Sumitra N. Mangasulia, Kallappa M. Hosamani*, a, Hirihalli C. Devarajegowdab, Mahantesh
M. Kurjogic, Shrinivas D. Joshid
aDepartment of Studies in Chemistry, Karnatak University, Dharwad-580003, India.
bDepartment of Physics, Yuvaraja’s College, University of Mysore, Mysore-570005, India.
cDepartment of Studies in Microbiology & Biotechnology, Karnatak University, Dharwad-
580003, India.
dNovel Drug Design and Discovery Laboratory, Department of Pharmaceutical
Chemistry, S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-580002,
Karnataka, India.
* Corresponding Author E-mail address: dr_hosamani@yahoo.com (K.M. Hosamani).
Tel.: +91-836-2215286; fax: +91- 836-2771275 & +91-836-2747884;
Graphical abstract
Bioactive Pharmocore
O
O
N
N
N
O
N
N
O
HN
N
N
Br
Anhy. K2CO3, Acetone
@ rt 6-8 hrs
R
O
O
R
R = 6-CH3, 7-CH3, 5,6-benzo, 7,8-benzo, 5,7-diCH3,
6-OCH3, 6-Cl, 6-Br, 7-OH, 6-ter but.
O
O
Anti-TB and
anti-microbial
agent

ACCEPTED MANUSCRIPT
Synthesis of coumarin-theophylline hybrids as a new class of anti-
tubercular and anti-microbial agents
Sumitra N. Mangasulia, Kallappa M. Hosamani*, a, Hirihalli C. Devarajegowdab, Mahantesh
M. Kurjogic, Shrinivas D. Joshid
aDepartment of Studies in Chemistry, Karnatak University, Dharwad-580003, India.
bDepartment of Physics, Yuvaraja’s College, University of Mysore, Mysore-570005, India.
cDepartment of Studies in Microbiology & Biotechnology, Karnatak University, Dharwad-
580003, India.
dNovel Drug Design and Discovery Laboratory, Department of Pharmaceutical
Chemistry, S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-580002,
Karnataka, India.
* Corresponding Author E-mail address: dr_hosamani@yahoo.com (K.M. Hosamani).
Tel.: +91-836-2215286; fax: +91- 836-2771275 & +91-836-2747884;
Abstract
A series of novel coumarin-theophylline hybrids were synthesized and examined for
their anti-tubercular activity in vitro against Mycobacterium tuberculosis H37Rv, anti-
microbial activity in vitro against gram-positive bacteria (Staphylococcus aureus) and gram-
negative bacterias (Escherichia coli, Salmonella typhi) as well as fungi (Candida albicans).
The compound (3a) has shown excellent anti-tubercular activity with MIC of 0.12 µg/mL.
Electron donating compounds (3a, 3f) have displayed significant anti-microbial activity. The
compounds have also been precisely elucidated using single crystal X-ray diffraction
techniques. Molecular docking study has been performed against 4DQU enzyme of
Mycobacterium tuberculosis showed good binding interactions and is in agreement with the
in vitro results.
Key words: Coumarin-theophylline hybrids; Molecular docking; Anti-tuberculosis; Anti-
microbial; X-ray structure.

ACCEPTED MANUSCRIPT
1. Introduction
Tuberculosis (TB) is an infectious disease caused by the pathogenic bacterium
Mycobacterium tuberculosis and remains a major global health problem till date. In the year
2015, the World Health Organization (WHO), has estimated TB prevails one of the top 10
causes of death worldwide and about 6.1 million new TB cases with 1.4 million TB deaths [1,
2]. At times, the drug resistant being one of the reasons as the patient doesn’t respond to
treatment with anti-TB drug [3]. Despite the availability of effective anti-TB drugs, such as
Isoniazid and rifampicin, mortality rates due to TB is still increasing [4]. Thus, there is an
emergent need of novel drug entities to counter Multi-drug resistant (MDR) and extensively
drug resistant (XDR) mycobacterium tuberculosis strains which compromises longer
resistance and recovery of the immune compromised patients [5]. Anti-TB drugs consist of
two groups: first-line drugs which are usually used for the treatment of TB patients and
second-line drugs are used for the treatment of MDR-TB. Second-line drugs have many more
adverse effects than the first-line anti-TB drugs [6]. Development of new anti-TB agents that
reduce toxicity, side effects with ability to penetrate host cells as well as effectively threat
MDR and XDR are in great interest and would have major impact on public health [4, 7].
Coumarins are the elite class of oxygen-containing fused heterocycles, which are
widely distributed in plants [8]. Many of them have been extensively recognized as the key
subunits to design synthetic drug candidates in terms of their dynamic pharmacological
activities such as anti-inflammatory [9], anti-cancer [10], anti-HIV [11] and anti-malarial
[12]. In recent studies naturally occurring coumarins have been reported to possess potent
antimycobacterial activity. Noteworthy, (+) Calanolide A was found to be extremely active
against M. tuberculosis H37Rv and number of MDR-TB stains (A, Figure 1) [13]. Some
coumarin derivatives conjugated with nitrogen containing heterocyclic moieties such as
pyridine, thiazolyl, Pyrazolo and pyrazole pyrimidine exhibited antimicrobial and anti-
tubercular activity profiles. Thus, the hybrids of coumarin nucleus with other moieties have
afforded new molecules with improved activity [14]. Recently, 2-mercaptobenzimidazole
linked coumarinyl triazoles and coumarin containing thiazole derivatives have been reported
as potential anti-tubercular agents (B, C Figure 1) [15, 16]. Purines are nitrogen containing
heterocycles which are found abundantly in nature. Purines and its derivatives play an
important role in biological activity [17]. In recent years purine derivatives are found to be an
important class of drug candidate against Mycobacterium tuberculosis (MTB). Yulian
Voynikov and co-workers synthesized amino acid derived from theophylline 7-acetamides

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which substituent’s (R= H, Me, iPr, iBu, 2-CH2-indole), varying at α-position are found to be
highly active against MTB bacilli (H37Rv) (D, Figure 1) [18]. Some purine derivatives like 6-
(2-furyl) purines are found to be potent MTB. It is demonstrated that nature of the N-9
substituent is vital for activity against MTB. The compounds with the N-9 substituent such as
N-benzylated purine are highly potent. Alkylation at N-9 position are essentially inactive,
when the length of alkyl side chain was increased the activity was decreased (E, F and G,
Figure 1) [19, 20].
The design concept of the presently synthesised compounds inherently comes by
taking into account of the bioactivity related to nitrogen heterocycles linked to various other
pharmacophoric moieties as reported in literature, hence clubbing of theophylline with
coumarin will serve an interesting bioactive template for anti-microbial and tuberculosis
screening.
Insert Figure 1
2. Results and Discussion
2.1. Chemistry
The substituted 4-bromomethyl coumarins 1(a-j) were synthesized using Pechman
cyclisation of phenols with 4-bromoethylacetoacetate using sulphuric acid as the condensing
agent [21]. Condensation of 4-bromomethyl coumarin 1(a-j) with theophylline (2) in
anhydrous K2CO3 using acetone as solvent afforded 1,3-dimethyl-9-[(substituted-2-oxo-2H-
chromen-4-yl)methyl)-1H-purine-2,6(3H,9H]-dione derivatives 3(a-j). Synthesis of target
compounds was carried out as outlined in Scheme 1. The newly synthesized compounds were
1
13
characterized H NMR, C NMR, mass and elemental analysis. The spectral data of newly
synthesized compounds 3(a-j) are provided in the experimental section, and in accordance
with the assigned structures of the compounds.
Insert Scheme 1
Formation of compounds 3(a-j) was supported by spectroscopic data. In case of
compound (3j), (R = ter but). The IR Spectrum exhibited band at 1725 cm-1 assignable to
lactone carbonyl stretching, and 1706cm-1 and 1664cm-1 are assignable to theophylline
(C=O) carbonyls and 1550 cm-1 assignable to diazine (–C=N) of theophylline. Formation of
1
product was further established by H NMR spectrum, wherein (N1–CH3) of theophylline
resonated as singlet at 3.42 ppm and (N3–CH3) of theophylline resonated as singlet at 3.32

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ppm. C3–H of coumarin and C4–CH2 resonated at 5.68 and 5.86 ppm as a singlet respectively.
Methyl protons of coumarin resonated at 1.30 ppm as a singlet, H–C=N of the azomethine
ring appeared at 8.17 ppm as a singlet and C5–H of coumarin ring appeared at 7.70 ppm as a
doublet (Jmeta=2.8Hz). C7–H of coumarin resonated at 7.73 ppm as a doublet of doublet
(Jortho=8.8Hz, Jmeta=2.8Hz), C8–H of coumarin resonated at 7.36 as a doublet (Jortho= 8.8Hz).
Further, molecular ion peak in EI-MS at m/z 394 (100%), lent an additional support to the
architecture of compound (3j). Rest all the compounds are in good agreement with the
expected molecular weight and are an indication of the stability of ion.
3. X-Ray Diffraction Studies
Single crystals for compounds (3c), (3d) and (3e) were developed by slow
evaporation of chloroform at room temperature. Compounds (3c) and (3d) crystallized under
a monoclinic system with the space group C2/c and P21/n respectively and the compound
(3e) crystallized under a triclinic system with the space group P-1. The unit cell dimensions
of compound (3c) are as follows: a = 19.8675(9) Å, b = 8.3781(3) Å, c = 22.2513(8) Å, α =
90°, β = 108.902(3)°, γ =90°, Z = 8. For compound (3d) the unit cell dimensions are: a =
10.8667(16) Å, b = 13.567(2) Å, c = 12.4117(18) Å, α = 90°, β = 104.347(4)°, γ = 90(6)°, Z
= 4. And the unit cell dimensions of compound (3e) are a = 7.9181(6) Å, b = 8.4627(8) Å, c =
14.2273(13) Å, α = 97.649(5)°, β = 90.998(5)°, γ = 117.355(4)°, Z = 4. Data collection and
reduction were performed using CrysAlisPro (version 1.171.36.32) [22], OLEX2 (version
1.2) [23] and SHELXS-97 [24] was used to solve and refine the crystal structures.
The crystal structure for compound (3c). The compound 1,3-dimethyl-7-[(3-oxo-3H-
benzo[f]chromen-1-yl)methyl]-3,7-dihydro-1H-purine-2,6-dione was crystallized using
chloroform by slow evaporation at room temperature and crystalline state is characterized by
a long range, well defined three dimensional orders. The asymmetric unit contains only one
independent molecule is depicted in Figure 2. In the crystal structure, intermolecular C---
H…O and weak intramolecular C—H...N & C---H…O hydrogen bonds are observed. In the
crystal, π-π interaction with C---H…π are stabilized by crystal packing. Bond lengths and
angles are within the normal ranges [R]. The X-ray structure parameters and refinement for
compound (3c) are presented in Table 1. The ORTEP and packing diagrams of compound
(3c) are portrayed in Figure 2.
Insert Table 1
Insert Figure 2

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The crystal structure for compound (3d). From the crystal data it was observed that
the asymmetric unit of compound 1,3-dimethyl-9-[(2-oxo-2H-benzo[h]chromen-4-
yl)methyl]-3,9-dihydro-1H-purine-2,6-dione was crystallized using chloroform by slow
evaporation at room temperature and crystalline state is characterized by a long range, well
defined three dimensional orders. The asymmetric unit contains only one independent
molecule is depicted in Figure 3. In the crystal structure, intermolecular C--H...N & C---
H…O and intramolecular C--H...N & C---H…O hydrogen bonds are observed. In the crystal,
2
inversion related C---H...O interactions generate an R2 (8) ring pattern and link pair of
independent molecules into dimers. In addition, π–π interactions between inversions related
molecules and C---H…π is stabilized by crystal packing. Bond lengths and angles are within
the normal ranges [R]. The crystal structure contains weak intramolecular C---H...N and C---
H...O hydrogen bonds. The X-ray structure parameters and refinement for compound (3d)
are presented in Table 2. The ORTEP and packing diagram of compound (3d) are portrayed
in Figure 3.
Insert Table 2
Insert Figure 3
The crystal structure for compound (3e). The compound 9-[(5,7-dimethyl-2-oxo-2H-
chromen-4-yl)methyl]-1,3-dimethyl-3,9-dihydro-1H-purine-2,6-dione was crystallized using
chloroform by slow evaporation at room temperature and crystalline state is characterized by
a long range, well defined three dimensional orders. The asymmetric unit contains only one
independent molecule is depicted in Figure 4. In the crystal structure, intermolecular C---
H…O and weak intramolecular C—H...N & C---H…O hydrogen bonds are observed. In the
crystal, π-π interaction with C---H…π are stabilized by crystal packing. Bond lengths and
angles are within the normal ranges [R]. The X-ray structure parameters and refinement for
compound (3e) are presented in Table 3. The ORTEP and packing diagram of compound
(3e) are portrayed in Figure 4.
Insert Table 3
Insert Figure 4

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4. Biological activity
All the compounds prepared herein were screened for their potential in-vitro anti-
tubercular activity against MtbH37Rv strain by MicroplateAlamar Blue Assay (MABA) [25].
Anti-bacterial activity against Gram positive and Gram negative bacteria and also fungal
activity against Candida albicans by MIC method [26].
4.1. Anti-tubercular activity
All the compounds 3(a-j) were primarily screened at a single concentration of 7.8 µg/
mL against MtbH37Rv (ATCC-27294) in BACTEC 12 B medium, using a Microplate Alamar
Blue Assay (MABA). The results are summarized in Table 4. Compounds exhibiting >90%
inhibition in the primary test were further screened using 2-fold dilution to determine the
exact MIC values of the test compounds. In the primary screening, seven compounds (3a, 3b,
3c, 3e, 3g, 3h and 3j) demonstrated 92–96% inhibition of Mtb. In the secondary level of
screening, these seven compounds (3a, 3b, 3c, 3e, 3g, 3h and 3j) inhibited Mtb with a MIC
0.12, 1.95, 1.95, 0.97, 3.9, 3.9 and 0.48 µg/mL respectively. Compared to that of control
Isoniazid with 100 % inhibitions of Mtb at 0.06 µg/mL. We were found a good number of
active anti-TB compounds (3a, 3b, 3c, 3e, 3g, 3h and 3j).
4.1.1. Structure Activity Relationship (SAR) Studies for Anti-tuberculosis
It was observed from structural activity relationship studies, the methyl substitution at
C-6 position of the coumarin moiety showed excellent activity with MIC of 0.12 µg/mL.
Second line of activity was observed by tertiary butyl at C-6 position of the coumarin with
MIC of 0.48 µg/mL. Whereas, the dimethyl substitution at C-5,7 position of coumarin
showed good activity with MIC of 0.97µg/mL. Methyl substitution at C-7 and benzo
substitution at C-5,6 position of coumarin moiety have shown similar activity with MIC of
1.95µg/mL. It was also observed that the halogen substituted compounds at C-6 of coumarin
moiety (Cl and Br) were also found to exhibit similar MIC value of 3.9 µg/mL. Whereas, the
other substitutions i.e., 6-OCH3, 7,8-benzo and 7-OH showed no activity against MtbH37Rv.
From the overall structural activity relationship study it was observed that the mono
substituted methyl group at C-6 position of coumarin enhances the biological activity of the
compound [27]. It was also observed that tertiary butyl substitution at C-6 position exhibit
significant anti-TB activity. Dimethyl substituted compound (5,7-diCH3) exhibited good
activity. Further the methyl at C-7 position was found to exhibit less activity compared to
methyl substitution at C-6 of coumarin. The second line of anti-Tb activity were flaunted by

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7-CH3 and 5.6-benzo substituted coumarin derivative exhibiting good activity. Whereas, the
halogen substituted compounds (Cl and Br) at C-6 of coumarin was found to exhibit
moderate activity with MIC of 3.9µg/mL. Whereas, the other substituted compounds -OCH3,
7,8-benzo and 7-OH was found to be less active.
Insert Table 4
4.2. In-vitro antimicrobial studies
The investigation of antibacterial screening of all the tested compounds 3(a-j) against
both gram positive and gram negative human pathogenic bacterial strains showed moderate to
excellent activity and was compared with the standard antibiotics like tetracyclin. Screening
results are summarized in Table 5 The best antibacterial effect has compound 6-CH3 (3a)
with MIC–3.9µg/mL against S. aureus, E. coli and 7.8 µg/mL against S. typhi. Moreover,
compound 5,6-Benzo (3c), 6-OCH3 (3f), and 6-ter but (3j) also showed good antibacterial
activity with MIC–7.8µg/mL against S. aureus and for E. Coli, these compounds (3c, 3f and
3j) showed the activity with MIC 7.8, 7.8 and 3.9µg/mL respectively, whereas the
antibacterial activity of these compounds against S. typhi was shown to be 15.6, 7.8 and 7.8
µg/mL respectively. Compounds (3b, 3d, 3e, 3g, 3h and 3i) showed moderate antibacterial
activity against both gram positive and gram negative bacteria.
The antifungal activity was evaluated against fungal strain such as Candida albicans.
Ampoterecin B was used as a standard for the comparison of antifungal activity. It is evident
from the result that, compounds 6-CH3 (3a), 7-CH3 (3b), and 6-OCH3 (3f) were observed as
most active against Candida albicans with MIC value 31.3 µg/mL. Especially, compounds
(3c, 3d, 3i, and 3j) exhibited sensible activity against Candida albicans (MIC 62.5 µg/mL).
While compounds (3g) and (3h) showed moderate activity against the tested organism. The
test compound 5,7-diCH3 (3e) was fairly active against Candida albicans.
4.2.1. Structure Activity Relationship (SAR) Studies for Anti-bacterial
The coumarin-theophylline hybridsweretested forbroad diversity regarding growth
inhibitory activity with minimum inhibitory concentrations ranging from 3.9 to
250µg/mL.The presence of electron donating groups in the coumarin skeleton is found to
contribute positive effect to the antibacterial activity. Methylsubstitution, at C-6 of coumarin
has shown maximum activity with MIC of 3.9 µg/mL against Gram positive bacterial stain S.
aureus, Gram negative bacteria E. coli, and MIC of 7.8µg/mL for Gram negative bacteria S.

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typhi. Further substitution -OCH3 at C-6 f coumarin has shown MIC of 7.8µg/mL for all
bacterial stainsS. aureus, E.coliand S. typhi. The -ter but group at C-6 of coumarin has shown
MIC of 7.8µg/mL for S. aureus and S. typhi and MIC of 3.9µg/mL for E. coli. For halogens (-
Cl and -Br), -Br has shown MIC of 15.6µg/mL for S. aureusand MIC of 31.3µg/mL for E.
coli and S. typhi. And -Cl has shown MIC of 31.3µg/mL for S. areus and E. coliand MIC of
62.5 µg/mL for S. typhi. Among the halogens -Br has shown maximum activity for S. aureus
and S. typhi. The efficacy of substituent at C-6 position decreased in the orderof -CH3>ter but
> OCH3. 5,6-benzo have shown MIC of 7.8 µg/mL for S. aureus and Gram negative E. coli
and MIC of 15.6µg/mL for S. typhi. Also the remaining substitutions like -CH3at C-7, 5,7-
diCH3, 7,8-benzo, -OH at C-7 position of coumarin moiety have shown moderate activity.
4.2.2. Structure Activity Relationship (SAR) Studies for Anti-fungal
SAR studies on coumarin-theophylline hybrids have carried out using fungal strain
(Candida albicans) with reference to standard Amphotericin B. It is observed the methyl,
methoxy at C-6 position and methyl at C-7 position of coumarin have shown maximum
activity with MIC of 31.3µg/mL. Followed by ter but at C-6 position, 5,6-benzo, 7,8-benzo
and hydroxyl group at C-7 position with MIC of 62.5µg/mL. Halogens (-Cl and -Br) at C-6
of coumarin with MIC of 125µg/mL. Finally 5,7-dimethyl of coumarin has shown least
activity with MIC of 250µg/mL.
Insert Table 5
5. Molecular Docking studies
Molecular docking study was performed to support the interaction and preferred binding
mode of coumarin-theophylline derivatives with enzyme. All the 10 inhibitors were docked
into the active site of enzyme as shown in Figure 5 (A & B). The predicted binding energies
of all the compounds are listed in Table 6 which shows that the compound (3a) exhibits the
highest C-score value of 6.27 which is in agreement with our observed in vitro anti-TB
results. As depicted in Figure 6 (A-D), compound (3a) makes four hydrogen bonding
interactions with amino acid residues, among them one bonding interaction raised from the
oxygen atom of coumarin moiety with the hydrogen of ILE21 (O ---- H-ILE21, 1.98 4Å),
three interactions were of oxygen atom of keto group of coumarin ring with hydrogen of
ILE21, SER94 and ALA22 (-C=O------ H-ILE21, 2.42 Å; SER94, 2.72Å and H-ALA22,
1.81 Å).

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As depicted in Figure 7 (A-D), compound (3e), makes five hydrogen bonding
interactions at the active site of the enzyme (PDB ID: 4DQU), -C=O group of coumarin ring
makes three binding interactions (-C=O------H-LEU197, 2.73 Å; THR196, 2.39 Å and H-
ALA198, 2.01 Å), one bonding interaction raised from the oxygen atom of coumarin moiety
with the hydrogen of THR196 (O ---THR196, 1.85 Å) and remaining another hydrogen
bonding interaction raised from the carbonyl group of theophylline (purine) ring with
hydrogen of LYS165 (-C=O----- H-LYS165, 1.75 Å). Anti-tubercular assays against M.
tuberculosis H37Rv strain shows excellent results with compounds (3a) and (3j) with -CH3
and -(CH3)3 groups at C-6 position. In addition, coumarin C4-bridged theophylline moiety
has increased the binding ability of the new molecular entities at the active site as revealed by
in vitro studies.
Insert Figure 5
Insert Figure 6
Insert Figure 7
Insert Table 6
6. Experimental section
6.1. Instrumentation
Melting points were determined using an open capillary method on a Buchi apparatus and
are uncorrected. IR spectra were recorded on a Nicolet 5700 FT-IR instrument (Nicolet,
Madison. WL, USA) using KBr discs. 1H NMR spectra were recorded on a Bruker 400 MHz
and Geol 400 MHz spectrometers using CDCl3 and DMSO as solvents and TMS as an
internal standard. All chemical shifts are reported as δ values (ppm). EI-MS were recorded
using a shimadzu GCMSQP2010S. The elemental analyses were carried out using a Hereaus
CHN rapid analyzer. The microwave irradiation syntheses were carried out using a CEM-
Discover Focused Microwave system. All the synthesized derivatives were subjected to TLC
analysis to ensure the completion of the reaction. The X-ray single crystal structures of
compounds (3c, 3d and 3e) were recorded using CrysAlisPro (version 1.171.36.32), OLEX2
(version 1.2) and SHELXS-97.
6.2. General procedure for the preparation of compounds 3(a–j)

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A mixture of Theophylline (0.01 mol) and powdered anhydrous K2CO3 (0.02 mol) with
the substituted 4-bromomethyl coumarin (0.01 mol) in 10 mL acetone was stirred at room
temperature for 6-8 h. The progress of the reaction was monitored by TLC. After completion
of the reaction the reaction mixture was quenched in crushed-ice. Separated solid was filtered
and recrystallized from suitable solvent.
6.2.1.
1,3-dimethyl-9-((6-methyl-2-oxo-2H-chromen-4-yl)methyl)-1H-purine-2,6(3H,9H)-
o
dione (3a). Colourless crystals; Yield 85%; m.p: 272-275 C; IR (KBr) (υmax/cm-1): 1726
(C=O of lactone), 1700 (C=O of theophylline), 1666 (C=O of theophylline), 1545 (C=N
1
diazine of theophylline); H NMR (400 MHz, CDCl3, δ ppm): δ 2.44 (s, 3H, CH3), 3.38 (s,
3H, CH3), 3.65 (s, 3H, CH3), 5.67 (s, 2H, CH2), 5.72 (s, 1H, Ar-H), 7.40 (s, 1H, Ar-H), 7.41
(d, 1H, Ar-H, J=8.6Hz), 7.29 (d, 1H, Ar-H, J=8.6 Hz), 7.66 (s, 1H, CH); 13C NMR (100
MHz, CDCl3, δ ppm): δ 21.18, 28.12, 29.83, 46.21, 106.94, 112.72, 116.69, 117.54, 122.99,
133.86, 134.76, 141.68, 149.17, 149.74, 151.67, 151.84, 155.29, 160.40; MS m/z (%): 352
[M]+ (10.2 %); Anal. Calcd for C18H16N4O4: C, 61.36; H, 4.58; N, 15.90; O, 18.16 Found: C,
61.84; H, 4.14; N, 16.01; O, 18.02%.
6.2.2.
1,3-dimethyl-9-((7-methyl-2-oxo-2H-chromen-4-yl)methyl)-1H-purine-2,6(3H,9H)-
o
dione (3b). Colourless crystals; Yield 84%; Mp: 220-222 C; IR (KBr) (υmax/cm-1): 1722
(C=O of lactone), 1700 (C=O of theophylline) 1621 (C=O of theophylline), 1569 (C=N
1
diazine of theophylline); HNMR (400 MHz, CDCl3, δ ppm ) δ 2.45 (s, 3H, CH3), 3.35 (s,
3H, CH3), 3.62 (s, 3H, CH3), 5.63(s, 1H, Ar-H), 5.69 (s, 1H, CH2), 7.14 (s, 1H, Ar-H), 7.17
13
(d, 1H, Ar-H, J=4.8Hz), 7.47 (d, 1H, Ar-H, J=8Hz), 7.65 (s, 1H, CH); C NMR (100 MHz,
CDCl3) 21.64, 27.92, 29.84, 46.00, 106.77, 111.85, 114.44, 117.74, 122.71, 125.93, 141.50,
144.11, 148.98, 149.56, 151.49, 153.69, 155.13, 160.23; MS m/z (%): 352 [M]+ (10%); Anal.
Calcd for C18H16N4O5 : C, 61.36; H, 4.58; N, 15.90; O, 18.16; Found: C, 61.12; H, 4.62; N,
15.19; O, 18.06%.
6.2.3.
1,3-dimethyl-9-((3-oxo-3H-benzo[f]chromen-1-yl)methyl)-1H-purine-2,6(3H,9H)-
o
dione (3c). Colourless crystals; Yield 80%; m.p: 288-292 C; IR (KBr) (υmax/ cm-1): 1740
(C=O of lactone), 1703 (C=O of theophylline), 1657 (C=O of theophylline) 1549 (C=N
1
diazine of theophylline); H NMR (400 MHz, DMSO-d6, δ ppm), δ 3.14 (s, 3H, CH3), 3.43
(s, 3H, CH3), 5.73 (s, 1H, Ar-H), 5.90 (s, 2H, CH2), 8.34 (s, 1H, Ar-H, J=2.4Hz), 8.21 (s, 1H,

ACCEPTED MANUSCRIPT
13
CH), 8.06 (s, 1H, Ar-H, J=2.4Hz), 7.89-7.91 (m, 2H, Ar-H), 7.70-7.52 (m, 2H, Ar-H); C
NMR (100 MHz, DMSO-d6, δ ppm): δ 28.06, 31.24, 46.84, 106.68, 111.51, 113.10, 120.58,
122.26, 122.70, 125.04, 128.29, 128.62, 129.65, 135.00, 143.73, 149.21, 150.41, 151.68,
153.06, 154.97, 159.96; MS m/z (%): 388 [M]+ (20%); Anal. Calcd for C21H16N4O4: C,
64.94; H, 4.15; N, 14.43; O, 16.48; Found: C, 64.68; H, 4.18; N, 14.234; O, 16.02%.
6.2.4.
1,3-dimethyl-9-((2-oxo-2H-benzo[h]chromen-4-yl)methyl)-1H-purine-2,6(3H,9H)-
o
dione (3d). Colourless crystals; Yield 83%; m.p: 290-293 C; IR (KBr) (υmax/ cm-1): 1722
(C=O of lactone), 1699 (C=O of theophylline), 1680 (C=O of theophylline), 1548 (C=N
diazine of theophylline); 1H NMR (400 MHz, CDCl3, δ ppm) δ 3.36 (s, 3H, CH3), 3.65 (s,
3H, CH3), 5.69 (s, 2H, CH2), 6.21 (s, 1H, Ar-H), 7.51 (d, 1H, Ar-H, J= 8Hz), 7.64 (dd, 1H,
Ar-H, J=8Hz, 4Hz), 7.70 (dd, 1H, Ar-H, J=8Hz, 4Hz), 7.71 (s, 1H, CH ), 8.06 (d, 1H, Ar-H,
J=8Hz); 13C NMR (100 MHz, DMSO-d6, δ ppm): δ 28.50, 34.06, 46.01, 105.12, 111.05,
115.09, 120.11, 121.02, 122.07, 125.49, 128.18, 129.11, 130.82, 135.50, 143.31, 149.26,
150.35, 151.39, 153.19, 154.05, 159.11; MS m/z (%): 388 [M]+ (22%); Anal. Calcd for
C21H16N4O4: C, 64.94; H, 4.15: N, 14.43; O, 16.48; Found: C, 64.28; H, 4.25; N, 14.85; O,
16.12%.
6.2.5. 1,3-dimethyl-9-((5,7-dimethyl-2-oxo-2H-chromen-4-yl)methyl)-1H-purine-2,6(3H,9H)-
o
dione (3e). Colourless crystal; Yield 84%; m.p: 277-280 C; IR (KBr) (υmax/cm-1): 1715
(C=O of lactone), 1703 (C=O of theophylline), 1672 (C=O of theophylline), 1548 (C=N
diazine of theophylline); 1H NMR (400 MHz, CDCl3, δ ppm) δ 2.40 (s, 3H, CH3), 2.73 (s,
3H, CH3), 3.36 (s, 3H, CH3), 3.64 (s, 3H, CH3), 5.37 (s, 1H, Ar-H), 5.86 (s, 2H, CH2), 6.96
13
(s, 1H, Ar-H), 7.04 (s, 1H, Ar-H ), 7.67(s, 1H, CH); C NMR (100 MHz, CDCl3, δ ppm): δ
21.23, 24.61, 27.92, 29.91, 49.44, 106.59, 111.49, 114.42, 116.56, 130.28, 135.04, 141.49,
143.07, 149.06, 151.54, 152.17, 155.10, 155.17, 160.08; MS m/z (%): 366 [M]+ (100%);
Anal. Calcd for C19H18N4O4: C, 62.29; H, 4.95: N, 15.29; O, 17.47; Found: C, 62.56; H, 4.35;
N, 15.30; O, 17.45%.
6.2.6. 1,3-dimethyl-9-((6-methoxy-2-oxo-2H-chromen-4-yl)methyl)-1H-purine-2,6(3H,9H)-
o
dione (3f). Colourless crystals; Yield 80%; m.p: 275-277 C; IR (KBr) (υmax/cm-1): 1729
(C=O of lactone), 1697(C=O of theophylline), 1659 (C=O of theophylline), 1548 (C=N
1
diazine of theophylline); H NMR (400 MHz, CDCl3, δ ppm) δ 3.38 (s, 3H, CH3), 3.64 (s,
3H, CH3), 3.86 (s, 3H, CH3), 5.71 (s, 2H, CH2), 5.78 (s, 1H, Ar-H), 7.05 (d, 1H, Ar-H,

ACCEPTED MANUSCRIPT
J=2.8Hz ), 7.18 (dd, 1H, Ar-H, J=8.8Hz, 2.8Hz), 7.33(d, 1H, Ar-H, J=9.2Hz), 7.67 (s, 1H,
CH); 13C NMR (100 MHz, CDCl3, δ ppm): δ 28.12, 30.07, 46.23, 56.09, 106.40, 106.91,
113.74, 117.39, 118.79, 119.94, 141.64, 148.18, 149.18, 149.35, 151.65, 155.37, 156.52,
160.30; MS m/z (%): 368 [M]+ (42%); Anal. Calcd for C18H16N4O5; C, 58.69; H, 4.38; N,
15.21; O, 21.72; Found: C, 58.20; H, 4.48; N, 15.11; O, 21.74%.
6.2.7.
1,3-dimethyl-9-((6-chloro-2-oxo-2H-chromen-4-yl)methyl)-1H-purine-2,6(3H,9H)-
o
dione (3g). Colourless crystals; Yield 75%; m.p: 278-280 C; IR (KBr) (υmax/cm-1): 1726
(C=O of lactone), 1708 (C=O of theophylline), 1666 (C=O of theophylline), 1551 (C=N
diazine of theophylline); 1H NMR (400 MHz, DMSO-d6 , δ ppm) δ 3.17 (s, 3H, CH3), 3.44 (s,
3H, CH3), 5.76 (s, 1H, Ar-H), 5.78 (s, 2H, CH2), 8.01 (d, 1H, Ar-H, J=2.4Hz), 7.69 (dd, 1H,
Ar-H, J= 8.8Hz, 2.4Hz), 7.45 (d, 1H, Ar-H, J=9.2Hz), 8.14 (s, 1H, CH); 13C NMR (100
MHz, DMSO-d6, δ ppm) δ 27.01, 29.21, 46.19, 106.74, 113.67, 119.02, 124.49, 129.42,
132.74, 139.75, 144.12, 149.32, 150.19, 151.04, 152.21, 155.03, 159.39; MS m/z (%): 372
[M]+ (100%), 374 [M+2] (33%); Anal. Calcd for C17H13ClN4O4: C, 54.78; H, 3.52; Cl, 9.51:
N, 15.03; O, 17.17; Found: C, 54.68; H, 3.50; Cl, 9.23; N, 15.05; O, 17.52%.
6.2.8.
1,3-dimethyl-9-((6-bromo-2-oxo-2H-chromen-4-yl)methyl)-1H-purine-2,6(3H,9H)-
o
dione (3h). Colourless crystals; Yield 73%; m.p: 276-280 C; IR (KBr) (υmax/cm-1): 1727
(C=O of lactone), 1709 (C=O of theophylline), 1665 (C=O of theophylline), 1551(C=N
diazine of theophylline); 1H NMR (400 MHz, DMSO-d6, δ ppm) δ 3.18 (s, 3H, CH3), 3.44 (s,
3H, CH3), 5.76 (s, 1H, Ar-H), 5.77 (s, 2H, CH2), 7.39 (d, 1H, Ar-H, J=9.2Hz), 7.80 (dd, 1H,
Ar-H, J=8.8Hz, 2.8Hz), 8.11 (d, 1H, Ar-H, J=2.8Hz), 8.12 (s, 1H, CH); 13C NMR (100 MHz,
DMSO-d6, δ ppm) δ 27.03, 29.12, 46.09, 105.22, 113.24, 117.11, 119.21, 120.12, 129.22,
132.23, 144.11, 145.13, 150.21, 151.16, 153.12, 154.11, 160.02; MS m/z (%): 416 [M]+
(100%), 418 [M+2] (100%); Anal. Calcd for C17H13BrN4O4: C, 48.94; H, 3.14; Br, 19.15: N,
13.43; O, 15.34; Found: C, 45.06; H, 3.24; Br, 19.75; N, 13.13; O, 15.32%.
6.2.9. 1,3-dimethyl-9-((7-hydroxy-2-oxo-2H-chromen-4-yl)methyl)-1H-purine-2,6(3H,9H)-
o
dione (3i). Colourless crystals; Yield 74%; m.p: 250-255 C; IR (KBr) (υmax/cm-1): 1721
(C=O of lactone), 1701 (C=O of theophylline), 1655 (C=O of theophylline), 1504 (C=N
1
diazine of theophylline), 3411(OH of Coumarin); H NMR (400 MHz, DMSO-d6, δ ppm) δ
3.13 (s, 3H, CH3), 3.41 (s, 3H, CH3), 5.75 (s, 2H, CH2), 5.95 (s, 1H, Ar-H), 7.17 (d, 1H, Ar-
H, J=8.8Hz, 2Hz ), 7.28 (s, 1H, Ar-H), 7.41 (d, 1H, Ar-H, J=8.8Hz), 8.17 (s, 1H, CH), 3.65

ACCEPTED MANUSCRIPT
(s, 1H, Ar-OH); 13C NMR (100 MHz, DMSO-d6, δ ppm): 28.06, 30.10, 46.40, 102.81,
106.67, 111.38, 113.56, 126.21, 143.75, 149.13, 151.67, 154.91, 155.25, 157.52, 160.38,
161.72, 162.01; MS m/z (%): 354 [M]+ (72%); Anal. Calcd for C17H14N4O5: C, 57.63; H,
3.98; N, 15.81; O, 22.58; Found: C, 57.67; H, 3.78; N, 15.15; O, 21.52%.
6.2.10. 1,3-dimethyl-9-((6-tert-butyl-2-oxo-2H-chromen-4-yl)methyl)-1H-purine-2,6(3H,9H)-
o
dione (3j). Colourless crystals; Yield 84%; m.p: 200-202 C; IR (KBr) (υmax/cm-1): 1725
(C=O of lactone), 1706 (C=O of theophylline), 1664 (C=O of theophylline), 1550 (C=N
diazine of theophylline); 1H NMR (400 MHz, DMSO-d6 , δ ppm) δ 1.30 (s, 9H, CH3), 3.32 (s,
3H, CH3), 3.42 (s, 3H, CH3), 5.68 (s, 2H, CH2), 5.86 (s, 1H, Ar-H), 7.36 (d, 1H, Ar-H,
J=8.8Hz ), 7.71 (dd, 1H, Ar-H, J=8.8Hz, 2.8Hz), 7.74 (d, 1H, Ar-H, J=2.8Hz), 8.17 (s, 1H,
13
CH); C NMR (100 MHz, DMSO-d6, δ ppm) δ 28.03, 29.99, 31.37, 34.85, 46.24, 106.84,
112.96, 116.28, 117.23, 119.23, 130.54, 141.77, 148.07, 149.03, 150.01, 151.58, 151.69,
155.27, 160.37; MS m/z (%): 394 [M]+ (100%); Anal. Calcd for C21H22N4O4: C, 63.95; H,
5.62: N, 14.20; O, 16.23; Found: C, 63.90; H, 5.22; N, 14.22; O, 16.35%.
6.3. Docking study
Surflex-Dock was used to investigate detailed intermolecular interactions between the
ligand and the target protein. Three dimensional structure information on the target protein
was taken from the PDB entry 4FDO. The proteins were prepared for docking by adding
polar hydrogen atom with Gasteiger-Huckel charges and water molecules were removed. The
3D structure of the ligands was generated by the SKETCH module implemented in the
SYBYL program (Tripos Inc., St. Louis, USA) and its energy-minimized conformation was
obtained with the help of the Tripos force field using Gasteiger-Huckel [28] charges and
molecular docking was performed with Surflex-Dock program that is interfaced with Sybyl-
X 2.0.[29] and other miscellaneous parameters were assigned with the default values given by
the software.
6.4. Anti-tubercular assay
Initially all the compounds 3(a-j) were screened against M. tuberculosis H37Rv (ATCC-
27294) at a concentration of 7.8 µg/mL using the Microplate Alamar Blue Assay (MABA).
Compounds exhibiting >90% inhibition were further taken for re-test against MtbH37Rv at
lower concentrations to evaluate the minimum inhibitory concentration in the MABA. The

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experiments were carried out in triplicate using a 96-well plate; to each well 100 µL of
Middlebrook 7H9 broth was added and a serial dilution of the compound was made directly
on the plate using a two-fold serial dilution method. Isoniazid was included as a positive drug
control. The plates were sealed with parafilm and incubated at 37 °C for 5 days. After this, 25
µL of a freshly prepared 1: 1 mixture of Alamar Blue reagent and 10% tween 80 was added
to the plate and incubated for 24 h. Inhibition of bacterial growth was indicated by blue color
in the wells while a pink color indicated as growth.
6.5. Antimicrobial assay
Two bacteria viz., S. aureus (Gram-positive) and E. coli, S. typhi (Gram-negative) and
one fungi Candida albicans were used to screen the antimicrobial activity of synthesized
compounds by evaluating the minimum inhibitory concentration of the all the compounds.
All the synthesized compounds 3(a-j) were screened for their potential in-vitro antimicrobial
activity in triplicate. Minimum inhibitory concentration (MIC) was determined by broth
micro dilution method using 96 well microplate (Andrews, 2001). The microbial inoculum
was prepared from 24 h broth cultures using 0.5 McFarland standards. The test compounds
3(a-j) were dissolved in DMSO at 1 mg/mL, and were then diluted in tryptone broth to
achieve a twofold serial dilution of the stock samples (concentration of 1000, 500, 250, 125,
62.5, 31.3, 15.6 7.8 and 3.9 µg/mL). The inoculums (100 µL) were added to each well and a
final volume 200 µL was obtained in each well. A number of wells were reserved in each
plate for positive and negative controls. Tetracyclin and nystatin was used as positive control
for bacteria and fungi respectively. The wells containing 100 µL of sterile broth and 100 µL
o
of inoculum was used as a negative control. The microplates were incubated at 37 C for 24
h. After that, resazurin (30 µL) in aqueous solution (0.01%) was added to the microplates, to
indicate the microorganism viability (Palomino et al., 2002). The MIC was determined as the
lowest concentration of the compound capable of inhibiting microorganism growth.
7. Conclusions
In conclusion, various substituted C-4 bridged coumarin-thyophilline hybrids 3(a-j)
were synthesised and evaluated for their anti-microbial and anti-tubercular properties. Single
crystals x-ray confirmed the proposed structure of the synthesised compounds. Compound 3a
showed highest anti-TB activity, whereas 3a, 3f and 3j showed excellent anti-bacterial
activity. Further 3a, 3b and 3f proved good candidates as anti-fungal agents. In silico
molecular docking shows the hydrogen bond interactions and further supports the obtained

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results. These compounds can act as lead compounds for design and synthesis of potent anti-
microbial and anti-tubercular drugs in future.
Acknowledgement
The authors acknowledge NMR Research Centre, Indian Institute of Science (IISc),
Bengaluru, India and University Scientific Instrumentation Centre (USIC), Karnatak
University, Dharwad for carrying out the spectral analyses. One of the authors acknowledges
the Karnatak University, Dharwad for awarding UGC-UPE fellowship.
Appendix A. Supplementary data
IR, NMR and Mass spectral data of new compounds 3(a-j).
Crystallographic data had been deposited with the Cambridge Crystallographic Data Centre
as supplementary publication for the compounds (3c, 3d and 3e) with CCDC No. 1588777,
1506761 and 1589963 respectively. This data can be obtained free of charge via the URL
http://26 www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road,
Cambridge CB21EZ, UK; fax: (+44) 1223 336033; e-mail: deposit@ccdc.cam.ac.uk)
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1
N
O
2
6
O
3
N
4
5
N
N
9
Br
7
O
N
8
N
N
Activated K2CO3, Acetone
RT/ 6-8h
R
R
N
H
O
O
O
O
O
1(a-j)
2
3(a-j)
R = a) 6-CH3, b)7-CH3, c) 5,6-benzo, d)7,8-benzo, e) 5,7-diCH3,
f) 6-OCH3, g) 6-Cl, h) 6-Br, i) 7-OH, j) 6-ter but.
Scheme 1. Schematic representation for the synthesis of the desired compounds.
Tables
Table 1. Crystal data, Data collection and Structure refinement of compound 3c.
Empirical formula
Formula weight
Temperature/K
Crystal system
C21H16N4O4
388.38
297.49
monoclinic

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Space group
C2/c
a/Å
19.8675(9)
8.3781(3)
22.2513(8)
90
b/Å
c/Å
α/°
β/°
108.902(3)
90
γ/°
Volume/Å3
3504.0(2)
8
Z
ρcalcg/cm3
µ/mm-1
F(000)
Crystal size/mm3
Radiation
1.472
0.105
1616.0
0.21 × 0.19 × 0.17
MoKα (λ = 0.71073)
2Θ range for data collection/°
Index ranges
4.784 to 56.632
-26 ≤ h ≤ 26, -11 ≤ k ≤ 11, -29 ≤ l ≤ 29
72910
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indexes [I>=2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole / e Å-3
CCDC No.
4356 [Rint = 0.0460, Rsigma = 0.0161]
4356/0/264
1.176
R1 = 0.0527, wR2 = 0.1569
R1 = 0.0720, wR2 = 0.1773
0.66/-0.70
1588777
Table 2. Crystal data, Data collection and Structure refinement of compound 3d.
Empirical formula
Formula weight
Temperature
C21 H16 N4 O4
388.38
293(2) K
Monoclinic
P 21/n
Crystal system
Space group

ACCEPTED MANUSCRIPT
a/Å
10.8667(16)Å
13.567(2)Å
12.4117(18)Å
90°
b/Å
c/Å
α/°
β/°
104.347(4)°
90(6)°
γ/°
Volume/Å3
1772.8(5)
4
Z
ρcalcg/cm3
Crystal size/mm3
1.455
0.22 × 0.15 × 0.12
Absorption coefficient
F(000)
0.859mm-1
808
Crystal form
Prism, colourless
fine-focus sealed tube
Cu Kα
Radiation source
Radiation type
Radiation monochromator
Graphite
Criterion for observed reflection I > 2σ(I)
Data collection
Diffractometer
Bruker SMART CCD area-detector
Data collection method
Absorption correction
2Θ range for data collection/°
Index ranges
ω- χ scans
multi-scan
5.318 to 64.651
-11<=h<=12, -15<=k<=15, -14<=l<=13
12825 / 2908 [R(int) = 0.0291]
97.2 %
Reflections collected / unique
Completeness to theta
Max. and min. transmission
Tmax = 1.000, Tmin = 0.790
Refinement
Refinement method
Data / restraints / parameters
Goodness-of-fit on F2
Final R indices [I>2σ(I)]
Full-matrix least-squares on F2
2908 / 0 / 262
1.071
R1 = 0.0427, wR2 = 0.1178

ACCEPTED MANUSCRIPT
R indices (all data)
R1 = 0.0442, wR2 = 0.1196
(∆/σ)max
< 0.001
Extinction coefficient
Largest diff. peak and hole
CCDC No.
0.039(4)
0.236 and -0.238 e.Ǻ-3
1506761
Table 3. Crystal data, Data collection and Structure refinement of compound 3e.
Empirical formula
C19H18N4O4
Formula weight
366.37
Temperature/K
298.99
Crystal system
triclinic
Space group
P-1
a/Å
7.9181(6)
b/Å
8.4627(8)
c/Å
14.2273(13)
α/°
97.649(5)
β/°
90.998(5)
γ/°
117.355(4)
Volume/Å3
835.86(13)
Z
2
ρcalcg/cm3
1.456
µ/mm-1
0.105
F(000)
384.0
Crystal size/mm3
0.25 × 0.23 × 0.21
MoKα (λ = 0.71073)
5.49 to 49.984
Radiation
2Θ range for data collection/°
Index ranges
Reflections collected
Independent reflections
Data/restraints/parameters
-9 ≤ h ≤ 9, -10 ≤ k ≤ 10, -16 ≤ l ≤ 16
24267
2935 [Rint = 0.0743, Rsigma = 0.0410]
2935/0/248

ACCEPTED MANUSCRIPT
Goodness-of-fit on F2
1.492
Final R indexes [I>=2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole / e Å-3
CCDC No.
R1 = 0.0987, wR2 = 0.3420
R1 = 0.1191, wR2 = 0.3535
0.55/-0.43
1589963
Table 4. In vitro anti-tubercular screening data of the compounds 3(a-j) against MtbH37Rv.
% inhibition at
a concentration of 7.8 µg/mL
96
MICa values
µg/mL
Compounds
R
3a
6CH3
0.12
3b
7CH3
92
92
16
94
56
92
92
15
96
100
1.95
1.95
ND
0.97
ND
3.9
3c
5,6-Benzo
7,8-Benzo
5,7-diCH3
6-OCH3
6-Cl
3d
3e
3f
3g
3h
3i
6-Br
3.9
7-OH
ND
0.48
0.06
3j
6-ter but
--
Isoniazid
aMinimum inhibitory concentration against the H37Rv strain of Mtb (µg/mL).
N.D – not determined.
Table 5. Minimum inhibition concentration of Compounds 3(a-j) using standard antibiotics
for antimicrobial activity.
Micro-organisms used for Antimicrobial
Activity (MIC values µg/mL)
Compounds
R
Gram positive
Gram negative
Candida
albicans
Staphylococcus Escherichia Salamonella
aureus
coli
typhi
3a
3b
6-CH3
7-CH3
3.9
3.9
7.8
31.3
31.3
62.5
31.3
125

ACCEPTED MANUSCRIPT
3c
3d
3e
3f
3g
3h
3i
5,6-Benzo
7,8-Benzo
5,7-diCH3
6-OCH3
6-Cl
7.8
15.6
15.6
7.8
7.8
15.6
125
7.8
15.6
62.5
250
7.8
62.5
62.5
250
31.3
125
31.3
15.6
15.6
7.8
31.3
31.3
125
3.9
62.5
31.3
31.5
7.8
6-Br
125
7-OH
62.5
62.5
3j
6-ter but
Tetracyclin
--
--
3.9
--
3.9
--
3.9
--
--
Amphoterecin B
0.97
Table 6. Surflex Docking score (kcal/mol) of the coumarin derivatives 3(a-j)
Crash
Polar
PMF
Entry
C Scorea
D Scored
G Scoref ChemScoreg
Scoreb
Scorec
Scoree
3a*
3b
3c
3d
3e
3f
6.27
5.63
5.29
5.10
5.78
4.78
5.15
4.40
5.83
5.34
-1.33
-1.20
-1.42
-2.18
-1.48
-1.70
-2.37
-0.69
-0.57
-2.30
2.05
1.99
1.16
1.05
3.06
2.49
0.93
3.09
2.11
2.18
-118.030
-114.194
-115.041
-116.011
-91.620
-55.890
-58.178
-8.974
-219.991
-213.297
-211.385
-229.890
-181.545
-188.143
-228.685
-140.315
-150.021
-203.667
-22.030
-22.486
-21.843
-31.307
22.486
-40.860
-10.523
-30.188
-35.416
-23.043
-10.490
2.279
-101.345
-122.356
-80.910
-23.770
-17.088
-22.243
-19.977
-22.427
3g
3h
3i
-91.388
3j
-100.623
*Asterisk indicates compound with higher C score.
aCScore (Consensus Score) integrates a number of popular scoring functions for ranking the
affinity of ligands bound to the active site of a receptor and reports the output of total score.
b Crash-score revealing the inappropriate penetration into the binding site. Crash scores close
to 0 are favorable. Negative numbers indicate penetration.

ACCEPTED MANUSCRIPT
c Polar indicating the contribution of the polar interactions to the total score. The polar score
may be useful for excluding docking results that make no hydrogen bonds.
d D-score for charge and van der Waals interactions between the protein and the ligand.
e PMF-score indicating the Helmholtz free energies of interactions for protein-ligand atom
pairs (Potential of Mean Force, PMF).
f G-score showing hydrogen bonding, complex (ligand-protein), and internal (ligand-ligand)
energies.
gChem-score points for H-bonding, lipophilic contact, and rotational entropy, along with an
intercept term.
Figures
S
NH
O
S
N
N
N
S
N
O
O
N
O
Cl
OH
O
O
O
O
MIC= 0.09µg/mL
MIC= 3.13µg/mL
MIC= 3.8µmol/mL
C
B
A
O
O
N
COOH R= H, 6.78µg/mL
R= H, 6.25µg/mL
CH3, 6.25µg/mL
O
N
Me, 0.65µg/mL
iPr, 5.93µg/mL
iBu, 5.69µg/mL
N
N
N
N
N
R
H
N
CH2Ph, 3.13µg/mL
CH2CH2Ph, 6.25µg/mL
N
O
2-CH2-indole, 0.47µg/mL
R
E
D
SC10H23
N
SC10H23
N
N
N
N
N
O
N
N
H
O
MIC90 = 3.13µg/mL
MIC90 = 1.56µg/mL
G
F

ACCEPTED MANUSCRIPT
Figure 1. Structurally related anti-tubercular coumarins and purines.
Figure 2. ORTEP and packing diagram for compound 3c.
Figure 3. ORTEP and packing diagram for compound 3d.

ACCEPTED MANUSCRIPT
Figure 4. ORTEP and packing diagram for compound 3e.
B
A
Figure-5: Docked view of all the compounds 3(a-j) at the active site of the enzyme
PDB ID: 4DQU.
A
B
O
N
O
O
A
N
N
N
SER94.HG
H
1
.
8
1
A
O
H
2.42Ao
1.98Ao
ALA22.H
H
ILE21.H
D
C

ACCEPTED MANUSCRIPT
Figure-6: Docked view of compound (3a) at the active site of the enzyme PDB: 4DQ
LYS165.HZ2
o
A
H
A
B
5
7
.
O
1
N
A
O
N
N
N
ALA198.H
LEU197.H
o
H
A
1
0
.
2
2
.
7
o
3
A
O
O
2.39Ao
H
1.85Ao
H
THR196.HG1
D
C
C
Figure-7: Docked view of compound (3e) at the active site of the enzyme PDB: 4DQU.

ACCEPTED MANUSCRIPT
Synthesis of coumarin-theophylline hybrids as a new class of anti-
tubercular and anti-microbial agents
Sumitra N. Mangasulia, Kallappa M. Hosamani*, a, Hirihalli C. Devarajegowdab, Mahantesh
M. Kurjogic, Shrinivas D. Joshid
aDepartment of Studies in Chemistry, Karnatak University, Dharwad-580003, India.
bDepartment of Physics, Yuvaraja’s College, University of Mysore, Mysore-570005, India.
cDepartment of Studies in Microbiology & Biotechnology, Karnatak University, Dharwad-
580003, India.
dNovel Drug Design and Discovery Laboratory, Department of Pharmaceutical
Chemistry, S.E.T’s College of Pharmacy, Sangolli Rayanna Nagar, Dharwad-580002,
Karnataka, India.
* Corresponding Author E-mail address: dr_hosamani@yahoo.com (K.M. Hosamani).
Tel.: +91-836-2215286; fax: +91- 836-2771275 & +91-836-2747884;
Highlights
•
•
•
•
•
A series of novel Coumarin-Theophylline hybrids were prepared.
The structures of 3c, 3d, 3e were confirmed by single-crystal X-ray diffraction.
Compound 3a exhibited the highest anti-TB activity against MtbH37Rv.
Most compounds displayed good anti-tubercular and antimicrobial activity.
Molecular docking studies have shown good binding interactions and are in good
agreement with in vitro anti-TB.