1,3-Oxazine-2-one derived dual-targeted molecules against replicating and non-replicating forms of Mycobacterium tuberculosis

European Journal of Medicinal Chemistry 208 (2020) 1 12835

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

1,3-Oxazine-2-one derived dual-targeted molecules against

replicating and non-replicating forms of Mycobacterium tuberculosis

,

Anand Babu Velappan ^a, Dhanunjaya Kesamsetty ^a, Dhrubajyoti Datta ^{b 1}, Rui Ma ^c,

,

*

Natarajan Hari ^d, Scott G. Franzblau ^c, Joy Debnath ^a

^aDepartment of Chemistry, SCBT, SASTRA Deemed to Be University, Tamilnadu, 613401, India

^bDepartment of Chemistry, Indian Institute of Science Education and Research, Pune, Maharashtra, 411008, India

^cInstitute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood St, Chicago, IL, 60612, USA

^dNMR Laboratory, SCBT, SASTRA Deemed to Be University, Tamilnadu, 613401, India

a r t i c l e i n f o

a b s t r a c t

Article history:

The high mortality rate and increasing prevalence of resistant Mtb are the major concerns for the

Tuberculosis (TB) treatment in this century. To curtail the prevalence of resistant Mtb, we have prepared

1,3-oxazine-2-one based dual targeted molecules. Compound 67 and 68 were found to be equally active

Received 29 April 2020

Received in revised form

2 August 2020

Accepted 7 September 2020

Available online 13 September 2020

against replicating and non-replicatiing form of Mtb (MIC_MABA3.48 and 2.97

2.15 g/ml respectively). They had found to suppress the biosynthesis of alfa, methoxy and keto-mycolate

completely, as well as inhibit enzymatic activity of MenG (IC₅₀¼ 9.11 and 6.25 g/ml respectively for

g/ml respectively for M smegmatis).

mg/ml; MIC_LORA2.94 and

m

H37Ra; IC₅₀¼ 11.76 and 10.88

m

Keywords:

1,3-Oxazine-2-one

Mycobacterium tuberculosis

Mycolic acid

MenG enzyme

1. Introduction

Among the several other targets of anti-tuberculosis drugs,

arguably the most important target is the cell wall biosynthesis for

replicating Mtb. Isoniazid and ethambutol (1st line antibiotics)

exhibit early bactericidal activity against actively metabolizing

bacilli by inhibition of mycolic acid biosynthesis and arabinoga-

lactan biosynthesis respectively [9]. However, it is not sufﬁcient to

arrest Mtb as it is capable to undergo in the dormant form and re-

emerges under favorable condition [10]. Thus, electron transport

system of Mtb is a vivid target for the development of new drugs to

combat with non-replicating bacilli. Several drug like molecules

like SQ109 [1 1], Piericidin A [12,13], Stigmatellin [14], Aurachin RE

[15], Aurachin E [16], Aurachin C [15] and Polyalthidin [1 7] have

been successfully tested for their inhibitory activity on the electron

transport system. Among them SQ109 draw a signiﬁcant attention,

it inhibits the activity of MenA and MenG enzymes which in turn

inhibit the biosynthesis of Menaquinone (MQ) [18]. Therefore, it is

important to come up with molecules which are capable to eradi-

cate both the replicating and non-replicating form of Mtb.

Tuberculosis (TB) is one of the major threat for mankind, solely

responsible for worldwide death of 1.6 million people in the year of

2017 including HIV positive and negative patients [1]. The new

incidence caused by Mycobacterium tuberculosis (Mtb) is estimated

about 10 million in 2017 [1]. The complete destruction of this

pathogen is challenging because of the emergence of new resis-

tance strains and their ability to survive in persisted form [2,3]. In

an effort to shrink the morbidity and mortality rates different

strategies have been adopted to confront the resistance issue;

namely, virulence inhibition [4], combination therapy [5] and use of

multi-targeted drugs or polypharmachology [6]. Among the others

multi-targeted drug discovery programme has drawn signiﬁcant

attention for reducing the propensity of high-level endogenous

resistance development [7]. In general, multi-targeted drug mole-

cules work through three mechanisms; series inhibition, parallel

inhibition and network inhibition [8].

From our previous study, we observed that diaryl urea mole-

cules are capable for selective inhibition of epoxy-mycolate

biosynthesis [19]. However, these molecules did not show signiﬁ-

cant activity against the non-replicating Mtb. Therefore, in an effort

to develop active molecular scaffold with dual activity, we

* Corresponding author.

E-mail address: joydebnath@scbt.sastra.edu (J. Debnath).

1

Alnylam Pharmaceuticals, Inc. 675 W Kendall St., Cambridge, MA 02142, USA.

https://doi.org/10.1016/j.ejmech.2020.1 12835

A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

Fig. 1. (a) Energy minimized conformation of Benzo[d][1,3]oxazine-4-one. (b) Rationale of the designing.

Scheme 1. Reagents and condition: i) a. TEA, rt, 3 h; b. EDCI.HCl, rt, 3 h; ii) HBr/AcOH, 70 ^ꢀC, 12 h; iii) 3-Chloro-1-propanol, TPP, DIAD, THF, rt, 6 h; iv) Corresponding alcohols, TPP,

DIAD, THF, rt, 6e8 h; v) R₂Cl, K₂CO₃, DMF, 80 ^ꢀC, 8e12 h; vi) KMnO₄, NaH₂PO₄, Acetonitrile/water, rt, 2 h; vii) 3-Chloro-1-propanol, DCC, DMAP, rt, 6 h.

2

A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

Scheme 2. Reagents and condition: i) R₁X, K₂CO₃, DMF, 70 ^ꢀC, 8 h; ii) (R₂CO)₂O, Pyridine, Chloroform, rt, 5 h; iii) R₃COOH, EDCI.HCl, DMAP, THF, rt, 3 h; iv) 4e7 or 18e27, K₂CO₃,

DMF, 70 ^ꢀC, 6 h.

restricted the conformational ﬂexibility of urea linkage through 1,3-

benzoxazin-2-one ring formation and chemically attached various

aliphatic chain to mimic the structure of demethylmenaquinone

(DMMQ) (Fig .1a). The cyclization of eNC(O)N-linkage (1,3-oxazine-

2-one ring system) induced a decent redox activity in the molecule

(SI-II), which made it structurally and functionally resemble with

DMMQ (Fig. 1b).

Based on these observations we had prepared several molecules

with various ﬂanking chains and checked their activity against Mtb

(H37Rv, H37Ra) and M. smegmatis (mc²155). We also determined

their cytotoxic effect on PBMC cells. Among the three different sets

scaffold, we started with anthranilic acid and 3-methoxyphenyl

isocyanate. Initially, we tied to form the urea linkage between an-

thranilic acid and isocyanate by stirring them together at room

temperature as well as under heating. The yield of the reaction was

found to be compromised because of the hydrolysis of isocyanate to

the corresponding carbamic acid. Thereafter, we tried the reaction

in presence of bases like pyridine, triethylamine and DBU at tem-

perature 30e100 ^ꢀC. The reaction was found slow and on pro-

longing the reaction time we observed signiﬁcant amount of

carbamic acid formation. So we tried one pot strategy to make the 1,

3-benzoxazin-4-one from anthranilic acid and 3-methoxyphenyl

isocyanate in presence of triethylamine and EDCI.HCl. The urea

formation and cyclization together were carried out in one pot

reaction. However, immediate addition of EDCI.HCl was found to

accelerate self coupling reaction of anthranilic acid, therefore, we

added EDCI.HCl after 3 h to get a quantitative yield of the required

1,3-benzoxazin-4-one scaffold 1 (Scheme 1). Intermediate 2 was

obtained by HBr/AcOH (33%) mediated demethylation of com-

pound 1 at 80 ^ꢀC [24].

of molecules, 10 molecules showed anti-tubercular activity <20

ml. Out of them six best molecules against the replicating form of

Mtb (comp. 1, 39, 40, 41, 48, and 49 with MIC value < 10 g/ml in

mg/

m

MABA assay) were chosen to evaluate their effect on the biosyn-

thesis of mycolic acid and menaquinone (MQ). For this purpose

H37Ra (Mtb) was used as a model system.

2. Results and discussion

Intermediate

3 was prepared by the esteriﬁcation of 4-

2.1. Synthesis of the target molecules

hydroxybenzoic acid with 3-chloro-1-propanol. To begin with we

used DCC as the coupling reagent, but we observed formation of

more than one product. So we tried Mitsunobu conditions for the

esteriﬁcation [25] and obtained the desired compound in quanti-

tative yield. Thereafter, compounds 4 to 8 were prepared by making

the ether linkage with their corresponding alcohols in presence of

TPP/DIAD (Scheme 1). The third set of intermediates, p-

Benzo[d][1,3]oxazine-4-one is a biologically important scaffold

and many methodologies have been reported for the synthesis of

the same [20e23]. However, in the reported methods one need to

go through multistep synthesis or need to use expensive catalyst. In

an effort to obtain a convenient method to synthesize the active

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A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

Table 1

The antitubercular activity and cytotoxicity (against PBMC cell) of the synthesized molecules.

* Microplate Alamer Blue Assay (for replicating form of Mtb); ** Low Oxygen Recovery Assay (for non-replicating form of Mtb); ^aINH: Isoniazid; ^bRIF: Refampin.

alkoxybenzaldehydes 9e17 were synthesized by the nucleophilic

substitution reaction of p-hydroxybenzaldehyde with the corre-

sponding alkyl bromides in presence of K₂CO₃in DMF at 70 ^ꢀC

(Scheme 1). Thereafter, 27e36 were prepared in two steps, oxida-

tion of the aldehyde to corresponding carboxylic acid (18e26) in

presence of KMnO₄followed by esteriﬁcation of the acid with 3-

chloro-1-prpanol using Steglich esteriﬁcation condition with DCC

and catalytic amount of DMAP (Scheme 1).

The ﬁrst set of hybrid molecules; the ether series (37e43)

were prepared using the phenolic group of the intermediate 2. In

case of nucleophilic substitution reaction, there was a problem

with the selectivity as both the eNH and the eOH groups are

susceptible towards the alkylation reaction. Only at 60 ^ꢀC the

reaction was found to be favored towards O-alkylation than N-

alkylation. We also tried Mitsunobu condition, but under this

condition the yield was very low ~10e19% despite the use of

different azodicarboxylates DIAD, DBAD, DNAD [26,27]. The part

of second set of target molecules 44e46 were synthesized by

treating the corresponding anhydrides with the phenolic inter-

mediate 2 [28]. The reactions were carried out in chloroform and

pyridine at room temperature (Scheme 2). Other molecules of

this series (47e54) were prepared by esteriﬁcation reaction in

presence of EDCI.HCl and DMAP in THF (Scheme 2) [29]. The third

set of target molecules 55e69 are the esters, where 2 was

connected with the aliphatic chains via a linker. These molecules

were prepared by nucleophilic substitution reaction between 2

and the intermediates 4e8 and 27e36 in presence of K₂CO₃in

DMF under heating (Scheme 2).

2.2. Evaluation of anti mycobacterial activity and cytotoxicity

The in-vitro anti-tubercular activity of the synthesized molecule

was determined by its Minimum Inhibitory Concentration (MIC)

[30]. For all the three series of 1, 3-benzoxazin-4-one derivatives,

MIC was evaluated against Mtb (H37Rv and H37Ra strains) and

M. smegmatis (mc²155 strain). We determined the activity against

the replicating Mtb by MABA (Microplate Alamar Blue Assay) [31]

method, whereas, the activity on the non-replicating Mtb was

determined by LORA (Low Oxygen Recovery Assay) method

(Table 1) [32].

Cytotoxicity is an unavoidable criterion to access the utility of a

molecule as a potential drug. The PBMC (Peripheral Blood Mono-

nuclear Cells) cells majorly composed of lymphocytes and mono-

cytes was used to determine the cytotoxicity. The cytotoxicity of

these 1, 3-benzoxazin-4-one derivatives was evaluated by MTT

based cell viability assay. The IC₅₀values were calculated from a

dose-response curve plotted against percentage of viability versus

log₁₀concentration (Table 1) [33].

4

A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

12.50 to >100

m

g/ml (for replicating mc²155). Interestingly, we

observed improved activity against non-replicating H37Rv for

compounds 40, 41, 42 and 43 (10.33, 7.83, 5.14 and 3.76 g/ml

m

respectively). Compound 42 and 43 were found to be active only

against non-replicating form of Mtb, on the contrary 40 and 41

showed moderate activity against replicating Mtb but compara-

tively better activity was found against non-replicating Mtb. This

observation indicated that the chain length plays a signiﬁcant role

for shifting the activity from replicating to non-replicating Mtb.

In series 2, we increased the chain length up to 9-carbon atoms

(44e52) besides another two molecules with 15-carbon (53) and

17-carbon (54) chain length. Except compounds 52 and 54 all the

ester derivatives were found almost inefﬁcient against the both

form of Mtb. We presume that the replacement of the ether linkage

with ester was responsible for diminishing their activity against

replicating as well as non-replicating Mtb.

Series 3 molecules (55e69), where we connected the benzox-

azinone core to 4-alkoxybenzoic acids through a 1, 3-dioxopropyl

linkage showed much better activity compared to series 1 and se-

ries 2 molecules. This set of molecules showed improved activity

against H37Rv in both MABA and LORA method. Improved efﬁcacy

against mc²155 strain was also observed for these molecules.

Compounds 67 and 68 showed the best activity proﬁle against both

replicating and non-replicating Mtb (H37Rv and H37Ra) and

M. smegmatis (mc²155). Altogether, we obtained 7 molecules from

this series where the MIC values were <20

mg/ml for all the

different mycobacterium species.

2.3. Structure-activity relationship

The anti-tubercular activity of the synthesized molecules was

found to be dependent on the carbon numbers in the aliphatic

chain and also on the type of linkage with benzo[d][1,3]oxazine-4-

one scaffold.

For the molecules belonging to series 1, the activity against

replicating Mtb was found to increase with concomitant increase

of carbon number till 4-carbon atoms. Thereafter, further incre-

ment to 5-carbon and 6-carbon improved the activity to moderate

level, but the activity completely diminished for 10-carbon and

Fig. 2. Relation between the activity and the carbon number in the aliphatic chain. (A)

Activity against H37Rv replicating form and (B) Activity against H37Rv non-replicating

form.

15-carbon (MIC > 100

the molecules except 52 and 54 showed MIC <50

m

g/ml) (Fig. 2A). On the other hand, none of

g/ml with 9-

m

carbon and 17-carbon atoms respectively. This poor activity is

presumably because of the alteration of the polarity caused by

ester linkage. Compared to series 1 molecules, series 3 molecules

showed better activity against the replicating Mtb. In this set of

molecules the activity was found <50

mg/ml for all the chain

length (1-carbon to 18-carbon atoms) except 2-carbon (56), 7-

carbon (61) and 18-carbon (65). The best activity was found for

compound 68 with farnesyl group (15-carbon) (Fig. 2A). For

H37Ra and mc²155 strains we also observed similar type of cor-

relation between the activity and carbon number in the aliphatic

chain.

The activity proﬁle of the compounds of series 1 showed a def-

inite trend against non-replicating Mtb. Compounds 40, 41, 42 and

43 with increasing number of carbon atom showed gradual

improvement in their activity and reached to the limiting activity

for 10-carbon (42) and 15-carbon (43) (Fig. 2B). However, for series

2 molecules we did not observed any activity (<50 mg/ml) against

the non-replicating Mtb, irrespective of the various chain lengths.

On the contrary, most of the molecules of series 3 showed activity at

Fig. 3. Production of four different mycolic acids (for H37Ra) in presence of the syn-

thesized molecules 68, 67, 60, 59, 58 and 1 (in DMSO). The concentration of the

molecules was used 5 times of their corresponding MIC values against H37Ra.

For series 1 molecules the alkyl chain was extended up to 6-

carbon atoms (37e41) and for other two molecules (42 and 43)

geranyl and farnesyl were attached through ether linkage. The MIC

a concentration <20 mg/ml. The activity improved signiﬁcantly by

increasing the chain length from 1-carbon to 6-carbon atoms

(except 56), then it again diminished with increase of carbon

number 8-carbon to 10-carbon. Finally, it reached to a static level

for 16-carbon (69) and 18-carbon (65) chains. However, it is

values were found in the range of 28.41 to >100

mg/ml (for repli-

cating H37Rv), 27.51to >100 g/ml (for replicating H37Ra) and

m

5

A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

Scheme 3. Reagents and conditions: (i) BF₃$Et₂O, EtOAc/Dioxane (1:1), 70 ^ꢀC, 4 h; (ii) 10% aqueous Na₂S₂O₄, rt, 3 h. Conversion of A (substrate) to B (product) was achieved in

presence of the membrane fraction of H37Ra and SAM as a methyl transferring agent.

Fig. 4. Retention time for compounds A and B measured at 250 nm with a ﬂow rate of

1 ml/min in the solvent system of ACN/H₂O (90:10).

pertinent to note that for 10-carbon (67, geranyl) and 15-carbon

(68, farnesyl) they showed the optimum activity (Fig. 2B).

From the structure activity relationship, we can conclude that

Fig. 5. The IC₅₀curve for compound 49 and 48 with H37Ra and mc²155. Substrate A

the length of aliphatic side chain along with its position from the

(500 mM) was mixed with 500 mM SAM and 100 ml membrane fraction (H37Ra and M

smegmatis separately) and incubated for 3 h at 37 ^ꢀC.

main scaffold play a crucial role to offer the activity against repli-

cating and non-replication Mtb. Comparing the activity of 1 with

42, 43, 67 and 68 it is clear that geranyl and farnesyl chains plays a

deﬁnite role to kill the Mtb in its latent state.

(Mtb) was used as the model system. In this assay we had used a

chromophoric benzyl moiety instead of ¹⁴C-radio labled acetate

(Fig. 1) [34]. The individual mycolic acids were characterised by

2.4. Effect of the hybrid molecules on mycolic acid biosynthesis

MALDI-TOF in presence of

a reference molecule C₈₃H₁₅₄O₃

We had selected six best molecules 1, 55, 58, 59, 60, 67 and 68

(Mass ¼ 1200.2; Benzyl ester of mycolic acid C₇₆H₁₄₈O₃) (shown in

SIeI) [35]. Molecules belong to series 3 (67 and 68) completely inhibit

the production of alfa, methoxy and keto-mycolates. Whereas,

compound 60 abolished the production of alfa and keto-mycolates

from all the three series which showed MIC <10

mg/ml (MABA)

against replicating Mtb. They were subjected for the evaluation of

their effect on the mycolic acid biosynthesis. For this purpose H37Ra

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A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

Table 2

Effect of the synthesized molecules on the MenG inhibition.

Fig. 6. Effect of the comp. 68 and 69 against non-replicating H37Ra: Growth of the non-replicating H37Ra in absence and in presence of Vit-K2 (1 mM) at 0^thand 10th day (Left

panel); Growth of the non-replicating H37Ra in absence and in presence of Vit-K2 (1 mM) and comp. 67 and 68 at 3 and 30 mg/ml on 10th day followed by 27 days after CFU

counting on 7H10 agar plate (Right panel).

Table 3

The MIC values of the compounds 67 and 68 against H37Rv drug resistance strains.

Entry

Strains

MIC (

67

m

g/ml)

68

INH^a

RIF^b

1

2

3

H37Rv (ATCC 27294)

H37RveINHeR (ATCC 35822)

H37Rv-RIF-R (ATCC 35838)

3.5

4.4

2.6

2.9

2.3

1.8

0.4

>8

0.25

0.01

0.03

>2

a

INH: Isoniazid.

b

RIF: Refampin, The MIC value were calculated by agar plate dilution method.

(51%), methoxy (29%) and keto-mycolates (68%) (Fig. 3). Comparing

the effect of these molecules on mycolic acid biosynthesis it is

pertinent to note that the presence of geranyl and fernasyl group

enhances their inhibitory effect on mycolic acid biosynthesis. How-

ever, these molecules showed insigniﬁcant or no effect on the fatty

acid biosyjthesis similar to our previous study with unsymmetrical

diaryl derivatives [19]. It may be attributed because of their selective

interaction with FAS-II enzymes rather than FAS-I [36].

Fig. 7. Effect of Comp. 68 on the ATP synthesis against non-replicating H37Ra on 3rd,

6th and 9th day at 20, 50 and 80 g/ml.

m

2.5. Effect of the hydride molecules on MenG

along with 61% reduction of methoxy-mycolates (calculated using

ImageJ software) compared with the control set. For compound 58

and 59, we observed complete inhibition of alfa-mycolate biosyn-

thesis and partial inhibition of methoxy (48% for 59; 36% for 58) and

keto-mycolates (37% for 59; 31% for 58). However, for the base

molecule 1, unlike the others we observed partial inhibition of alfa

To understand the plausible reason of their activity against non-

replication Mtb, we tested active molecules against the respiratory

system of dormant Mtb. Hence, our synthesized molecules have

structural resemblance with MenG substrate, we performed the

MenG inhibition study with compounds 41, 58, 59, 60, 67 and 68

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A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

(LORA MIC < 10

m

g/ml; MIC_MABA< 10

m

g/ml). The MenG inhibition

system of Mtb simultaneously. Out of them compound 67 and 68

showed the best activity where the 1,3-oxazine-2-one and the

aliphatic chains are connected through a linker. The relatively

weaker activity of compound 1 against the non-replicating Mtb

proved the necessity of prenyl chains for the recognition by the

MenG. In Vit-K2 rescue study compound 67 and 68 with geranyl

and farnesyl group respectively, clearly showed their inhibitory

effect on the electron transport system which get revived in pres-

ence of Vit-K2. Similarly, the ATP/ADP ratio get diminished in

presence of these compounds, which plausibly conﬁrms that these

molecules kill the Mtb in its non-replication form by affecting its

electron transport system. On the other hand 1,3-oxazine-2-one

scaffold was found to be responsible for the inhibition of mycolic

acid biosynthesis. The dual-targeted best molecules 67 and 68 are

also active against the INH and RIF resistant strains. This work

shows that by customizing the designing of hybrid molecules we

can perturb more than one target of Mtb, in an effort to counteract

the emergence of resistant strain.

values (IC₅₀) were determined by HPLC based assay, which is quite

similar to the ManA inhibition assay where we used DMMQ and

SAM (methyl transferring agent) [10]. MenG is the ﬁnal enzyme in

the menaquinone biosynthesis pathway, which converts the

DMMQ to MQ in presence of SAM. The MQ accepts two electrons

from NADH and get converted to its reduced form, ﬁnally it again

reversibly converted to MQ by transferring the electrons to the

other electron acceptors present in the electron transport system.

Therefore, inhibition of this enzyme is expected to perturb the

respiratory system of Mtb through which it harnesses its required

energy in the latent state [37].

The substrate and the product were prepared separately

(Scheme 3) [38e40] for calibration and identiﬁcation purpose. The

retention times for comp. A and comp. B were 5.713 min and

6.400 min respectively, which was found good enough to deter-

mine the IC₅₀value using HPLC system (Fig. 4).

The membrane fraction (according to the reported protocol) [18]

from H37Ra and mc²155 was used as a source of membrane bound

MenG. However, no signiﬁcant conversion of A to B was observed

under in vitro condition and it was apparently because of the use of

A in oxidized form. Therefore, to overcome this issue we had used a

reducing agent DTT to convert A in the reduced form and it makes A

chemically feasible to undergo the methylation (i. e., comp. B).

Representative IC₅₀proﬁle for comp. 67 and 68 is shown in Fig. 5.

The IC₅₀calculation was based on the conversion of A with respect

to the control set (without inhibitor molecule) and given in Table 2.

In this assay the conversion of the model substrate (comp. A) by

MenG to the corresponding product (comp. B) was monitored at

250 nm (by HPLC) in presence of 4 different concentrations of the

synthesized molecules (41, 58, 59, 60, 67 and 68) and the corre-

sponding IC₅₀values were calculated by non-linear regression

analysis within the error limit of 0.5% (Table 2).

4. Experimental

4.1. Reagents and instrumentation

All the reagents were purchased from sigma-Aldrich, Alfa-Aesar

and Merck chemicals. Solvents were dried according to the stan-

dard protocols. TLC was performed on Merck silica gel 60, f₂₅₄pre-

coated aluminium plates and spots were visualized under UV lamp

or stained using 10% PMA in Ethanol, 5% Sulfuric acid in methanol

or Iodine. Column chromatographic separations were performed

using silica gel (100e200 mesh). NMR spectra were run on Bruker

AVANCE II instrument using TMS as internal standard for ¹H (300

and 400 MHz) and ¹³C (75 and 100 MHz) experiments. Chemical

shifts were given in ppm (d scale), UVevis measurements were

To probe the signiﬁcance of the MQ biosynthesis inhibition in the

dormant form, we had prepared the dormant H37Ra following the

reported protocol [41] and performed the Vit-K2 rescue study. In this

experiment we observed the growth of the H37Ra (dormant form)

did not signiﬁcantly affected in presence of Vit-K2 (1 mM) till 10 days

of exposure (Fig. 6; left panel). However, in presence of compound 67

made using a PerkinElmer UVevis spectrophotometer (Model

Lambda 25). Mass spectra had been recorded using Waters Mass

Spectrometer (model XevoG2QTof). HPLC experiments were per-

formed using an Agilent 1200 inﬁnity series with silica C₁₈column

(4.6 ꢁ 250mm and 5

mm). ImageJ app was used for calculating the

TLC intensity proﬁle from the image.

and 68 (30 mg/ml) the growth was diminished by 2.9 and 4.1 fold

respectively (logCFU/ml) compared to the set with 1 mM Vit-K2

(Fig. 6; right panel). This provided strong evidence that these mole-

cules inhibit the biosynthesis of MenG and in turn reduced their

growth in dormant state by blocking the respiratory chain of H37Ra.

Next, we determined the ATP production in presence of comp.

68 in the dormant state for H27Ra. The ATP/ADP ratio was varied in

the range of 1.02e1.40 for the control set from day 3 to day 9

(Fig. 7). Whereas, for the co-culture of the dormant H37Ra with

4.1.1. Synthesis of 2-((3-ethoxyphenyl)amino)-4H-benzo[d][1,3]

oxazin-4-one (37)

The phenolic intermediate 2 (0.1 g, 0.39 mmol) was dissolved in

DMF and to that potassium carbonate (0.163 g, 1.18 mmol) was

added under stirring condition. Then bromoethane (0.035 mL,

0.47 mmol) was added and the reaction mass was heated at 70 ^ꢀ

C

for 8 h. After completion of reaction (checked by TLC), the reaction

mass was added into water and extracted with ethyl acetate (3 ꢁ 30

mL). Then the combined organic layer was washed with water

followed by brine solution. It was dried over sodium sulfate and

evaporated. The compound was puriﬁed by column chromatog-

raphy where the product was eluted at 30% ethyl acetate in hex-

anes. Yield: 0.063 g, 57.3%, sticky white solid, ¹H NMR (400 MHz,

comp. 68 (20, 50 and 80 mg/ml), we observed continuous reduction

in their ATP production level. It conﬁrms that the compound 68

eradicates Mtb in the dormant state by inhibiting the MenG

biosynthesis and as a whole by reducing its ATP production.

2.6. Effect on resistant strains of Mtb

DMSO‑d₆):

d

1.25 (t, J ¼ 7.2 Hz, 3H), 4.16 (q, J ¼ 7.2 Hz, 2H), 6.71 (d,

J ¼ 8.8 Hz, 2ArH), 6.82e6.85 (m, 1ArH), 7.21e7.33 (m, 2ArH), 7.55 (d,

Finally, we tested the best molecules 67 and 68 (MIC < 4 mg/ml)

J ¼ 8.4 Hz, 1ArH), 7.81 (t, J ¼ 7.2 Hz, 1ArH), 8.07 (dd, J ¼ 1.6, 8.0 Hz,

against the isoniazide and refampin resistant H37Rv strains (ATCC-

35822 and ATCC-35838 respectively). Both of these compounds

showed slightly better MIC values against the resistance strains as

compared to the non-resistance strain (Table 3).

1ArH), 9.65 (s, 1 NeH). ¹³C NMR (100 MHz, DMSO‑d₆):

d 12.9, 38.8,

114.9, 115.6, 116.1, 116.6, 119.8, 123.1, 128.7, 129.9, 136.0, 137.5, 140.3,

150.6, 158.2, 161.7. HRMS (ESI^þ): m/z calculated for C₁₆H₁₅N₂O₃

[MþH]^þ: 283.1083; found: 283.1088.

3. Conclusion

4.1.2. Synthesis of 2-((3-propoxyphenyl)amino)-4H-benzo[d][1,3]

oxazin-4-one (38)

In this study, we have designed and synthesized 33 hybrid

molecules to target the cell wall biosynthesis and the respiratory

The phenolic intermediate 2 (0.1 g, 0.39 mmol) was dissolved in

DMF and to that potassium carbonate (0.163 g, 1.18 mmol) was

8

A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

added under stirring condition. Then 1-bromopropane (0.043 mL,

added under stirring condition. Then 1-Bromohexane (0.066 mL,

0.47 mmol) was added and the reaction mass was heated at 70 ^ꢀ

C

0.47 mmol) was added and the reaction mass was heated at 70 ^ꢀ

C

for 8 h. After completion of reaction (checked by TLC), the reaction

mass was added into water and extracted with ethyl acetate (3 ꢁ 30

mL). Then the combined organic layer was washed with water

followed by brine solution. It was dried over sodium sulfate and

evaporated. The compound was puriﬁed by column chromatog-

raphy where the product was eluted at 30% ethyl acetate in hex-

anes. Yield: 0.061 g, 52.6%, white solid, m.p: 188e190 ^ꢀC. ¹H NMR

for 8 h. After completion of reaction (checked by TLC), the reaction

mass was added into water and extracted with ethyl acetate (3 ꢁ 30

mL). Then the combined organic layer was washed with water

followed by brine solution. It was dried over sodium sulfate and

evaporated. The compound was puriﬁed by column chromatog-

raphy where the product was eluted at 30% ethyl acetate in hex-

anes. Yield: 0.076 g, 57.6%, white solid, m.p: 140e142 ^ꢀC. ¹H NMR

(400 MHz, DMSO‑d₆):

d

0.96 (t, J ¼ 7.6 Hz, 3H), 1.64e1.77 (m, 2H),

(400 MHz, DMSO‑d₆):

d

0.88 (t, J ¼ 6.4 Hz, 3H), 1.18e1.39 (m, 6H),

4.06 (t, J ¼ 7.6 Hz, 1H), 6.70 (d, J ¼ 8.8 Hz, 2ArH), 6.82e6.85 (m,

1ArH), 7.21e7.33 (m, 2ArH), 7.55 (d, J ¼ 8.4 Hz, 1ArH), 7.82 (t,

1.62e1.67 (m, 2H), 4.09 (t, J ¼ 7.2 Hz, 2H), 6.71 (d, J ¼ 8.4 Hz, 2ArH),

6.82e6.84 (m, 1ArH) 7.23e7.30 (m, 1ArH), 7.53 (d, J ¼ 8.4 Hz, 1ArH),

7.82 (t, J ¼ 7.6 Hz, 1ArH), 8.07 (d, J ¼ 7.6 Hz, 1ArH), 9.63 (s, 1 NeH).

J ¼ 7.6 Hz, 1ArH), 8.07 (dd, J ¼ 1.6, 7.6 Hz, 1ArH), 9.64 (s, 1NeH). ¹³

C

NMR (100 MHz, DMSO‑d₆):

d

11.4, 20.7, 45.0, 115.1,115.6, 116.0, 116.5,

¹³C NMR (100 MHz, DMSO‑d₆):

d14.4, 22.5, 27.2, 29.1, 31.4, 43.5,

119.8, 123.1, 129.9, 136.0, 140.5, 150.6, 158.2, 161.7. HRMS (ESI^þ): m/z

115.1, 115.6, 116.0, 116.5, 119.8, 123.1, 128.7, 129.9, 136.0, 137.6, 140.4,

150.6, 158.3, 161.6. HRMS (ESI^þ): m/z calculated for C₂₀H₂₃N₂O₃

[MþH]^þ: 339.1708; found: 339.1711.

calculated for C₁₇H₁₇N₂O₃[MþH]^þ: 297.1239; found: 297.1240.

4.1.3. Synthesis of 2-((3-butoxyphenyl)amino)-4H-benzo[d][1,3]

oxazin-4-one (39)

The phenolic intermediate 2 (0.1 g, 0.39 mmol) was dissolved in

DMF and to that potassium carbonate (0.163 g, 1.18 mmol) was

added under stirring condition. Then 1-Bromobutane (0.051 mL,

4.1.6. Synthesis of (E)-2-((3-((3,7-dimethylocta-2,6-dien-1-yl)oxy)

phenyl)amino)-4H-benzo[d][1,3]oxazin-4-one (42)

The phenolic intermediate 2 (0.1 g, 0.39 mmol) was dissolved in

DMF and to that potassium carbonate (0.163 g, 1.18 mmol) was

added under stirring condition. Then Geranylchloride (0.087 mL,

0.47 mmol) was added and the reaction mass was heated at 70 ^ꢀ

C

for 8 h. After completion of reaction (checked by TLC), the reaction

mass was added into water and extracted with ethyl acetate (3 ꢁ 30

mL). The combined organic layer was washed with water followed

by brine solution. It was dried over sodium sulfate and evaporated.

The compound was puriﬁed by column chromatography where the

product was eluted at 30% ethyl acetate in hexanes. Yield: 0.065 g,

53.7%, white solid, m.p: 166e168 ^ꢀC. ¹H NMR (400 MHz, DMSO‑d₆):

0.47 mmol) was added and the reaction mass was heated at 70 ^ꢀ

C

for 8 h. After completion of reaction (checked by TLC), the reaction

mass was added into water and extracted with ethyl acetate (3 ꢁ 30

mL). Then the combined organic layer was washed with water

followed by brine solution. It was dried over sodium sulfate and

evaporated. The compound was puriﬁed by column chromatog-

raphy where the product was eluted at 30% ethyl acetate in hex-

anes. Yield: 0.098 g, 64.4%, white solid, m.p: 178e180 ^ꢀC. ¹H NMR

d

0.93 (t, J ¼ 7.2 Hz, 3H),1.39e1.47 (m, 2H),1.61e1.66 (m, 2H), 4.10 (t,

J ¼ 7.6 Hz, 2H), 6.72 (d, J ¼ 8.4 Hz, 2ArH), 6.82e6.84 (m, 1ArH),

7.21e7.33 (m, 2ArH), 7.53 (d, J ¼ 8.4 Hz, 1ArH), 7.82 (t, J ¼ 7.6 Hz,

1ArH), 8.07 (dd, J ¼ 1.6, 7.6 Hz, 1ArH), 9.63 (s, 1 NeH). ¹³C NMR

(300 MHz, CDCl₃):

d 1.63e1.64 (m, 6H), 1.84 (s, 3H), 2.01e2.09 (m,

4H), 4.81 (d, J ¼ 6.0 Hz, 2H), 5.02 (t, J ¼ 6.6 Hz,1H), 5.22 (t, J ¼ 6.0 Hz,

1H), 6.49 (bs, 1 NeH), 6.63 (t, J ¼ 2.1 Hz, 1ArH), 6.79 (td, J ¼ 1.8,

7.8 Hz, 2 ArH), 7.21e7.29 (m, 1ArH), 7.30e7.35 (m, 2ArH), 7.71 (td,

J ¼ 1.5, 7.5 Hz, 1ArH), 8.27 (dd, J ¼ 1.5, 7.8 Hz, 1ArH) ¹³C NMR

(100 MHz, DMSO‑d₆):d14.2, 29.5, 31.2, 43.3, 115.1, 115.6, 116.1, 116.5,

119.8, 123.1, 128.7, 129.9, 136.0, 137.6, 140.4, 150.6, 158.2, 161.6.

HRMS (ESI^þ): m/z calculated for C₁₈H₁₉N₂O₃[MþH]^þ: 311.1395;

found: 311.1396.

(100 MHz, CDCl₃):

d 16.9, 17.9, 25.9, 26.4, 39.7, 42.7, 114.6, 116.0,

116.2, 116.7, 118.3, 119.9, 123.4, 123.8, 129.7, 130.5, 132.1, 135.7, 136.2,

4.1.4. Synthesis of 2-((3-(pentyloxy)phenyl)amino)-4H-benzo[d]

[1,3]oxazin-4-one (40)

140.5, 140.8, 151.1, 157.7, 162.4. HRMS (ESI^þ): m/z calculated for

C

₂₄H₂₇N₂O₃[MþH]^þ: 391.2022; found: 391.2026.

The phenolic intermediate 2 (0.1 g, 0.39 mmol) was dissolved in

DMF and to that potassium carbonate (0.163 g, 1.18 mmol) was

added under stirring condition. Then 1-bromopentane (0.056 mL,

4.1.7. Synthesis of 2-((3-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-

trien-1-yl)oxy)phenyl)amino)-4H-benzo[d][1,3]oxazin-4-one (43)

The phenolic intermediate 2 (0.1 g, 0.39 mmol) was dissolved in

DMF and to that potassium carbonate (0.163 g, 1.18 mmol) was

added under stirring condition. Then farnesyl chloride (0.123 g,

0.47 mmol) was added and the reaction mass was heated at 70 ^ꢀ

C

for 8 h. After completion of reaction (checked by TLC), the reaction

mass was added into water and extracted with ethyl acetate (3 ꢁ 30

mL). Then the combined organic layer was washed with water

followed by brine solution. It was dried over sodium sulfate and

evaporated. The compound was puriﬁed by column chromatog-

raphy where the product was eluted at 30% ethyl acetate in hex-

anes. Yield: 0.069 g, 54.3%, waxy solid, ¹H NMR (400 MHz,

0.47 mmol) was added and the reaction mass was heated at 70 ^ꢀ

C

for 8 h. After completion of reaction (checked by TLC), the reaction

mass was added into water and extracted with ethyl acetate (3 ꢁ 30

mL). Then the combined organic layer was washed with water

followed by brine solution. It was dried over sodium sulfate and

evaporated. The compound was puriﬁed by column chromatog-

raphy where the product was eluted at 30% ethyl acetate in hex-

anes. Yield: 0.121 g, 68.0%, white solid, m.p: 76e78 ^ꢀC. ¹H NMR

DMSO‑d₆):

d

0.88 (t, J ¼ 6.8 Hz, 3H), 1.32e1.38 (m, 4H), 1.66 (quintet,

J ¼ 7.2 Hz, 2H), 4.09 (t, J ¼ 8.0 Hz, 2H), 6.71 (d, J ¼ 8.4 Hz, 2ArH),

6.82e6.84 (m, 1ArH) 7.21e7.35 (m, 2ArH), 7.53 (d, J ¼ 8.8 Hz, 1ArH),

7.79e7.84 (m, 1ArH) 8.07 (dd, J ¼ 1.6, 7.6 Hz, 1ArH), 9.62 (s, 1 NeH).

¹³C NMR (100 MHz, DMSO‑d₆):

d14.3, 22.4, 28.2, 28.8, 43.5, 115.1,

(300 MHz, CDCl₃): d 1.57e1.58 (m, 6H), 1.63e1.67 (m, 3H), 1.85 (s,

115.6, 116.0, 116.5, 119.8, 123.1, 128.7, 129.9, 136.0, 137.6, 140.4, 150.6,

158.2, 161.6. HRMS (ESI^þ): m/z calculated for C₁₉H₂₁N₂O₃[MþH]^þ:

325.1552; found: 325.1555.

3H), 2.00e2.10 (m, 8H), 4.81 (d, J ¼ 6.0 Hz, 2H), 5.05 (t, J ¼ 1.2 Hz,

2H), 5.18e5.23 (m, 1H) 6.61e6.62 (m, 2ArH), 6.75e6.80 (m, 2H),

7.21e7.34 (m, 3ArH), 7.64e7.72 (m, 1ArH), 8.27 (d, J ¼ 7.8 Hz, 1ArH)

¹³C NMR (100 MHz, CDCl₃):

d 16.3, 16.9, 17.9, 23.6, 26.4, 26.9,

4.1.5. Synthesis of 2-((3-(hexyloxy)phenyl)amino)-4H-benzo[d][1,3]

oxazin-4-one (41)

The phenolic intermediate 2 (0.1 g, 0.39 mmol) was dissolved in

DMF and to that potassium carbonate (0.163 g, 1.18 mmol) was

32.2,39.9, 42.7, 114.5, 116.0, 116.2, 116.7, 118.2, 118.3, 119.9, 123.3,

123.7, 124.5, 129.7, 130.5, 135.7, 135.9, 136.3, 140.5, 141.0, 151.1, 157.7,

162.4. HRMS (ESI^þ): m/z calculated for C₂₉H₃₅N₂O₃[MþH]^þ:

459.2648; found: 459.2650.

9

A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

4.1.8. Synthesis of 3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)amino)

phenyl acetate (44)

325.1190.

The phenolic intermediate 2 (0.1 g, 0.39 mmol) was dissolved in

chloroform and stirred at room temperature. To that pyridine

(0.063 mL, 0.78 mmol) was added and cooled to 0 ^ꢀCe5 ^ꢀC and

stirred at that temperature for 15 min. Acetic anhydride (0.056 mL,

0.59 mmol) was added dropwise and stirred at room temperature

for 5 h. After completion of reaction water was added and

neutralized with 5% hydrochloric acid and the organic layer

collected (3 ꢁ 30 ml). Combined organic layers washed with water

and brine solution. It was dried over sodium sulfate and evapo-

rated. The compound was puriﬁed by column chromatography

where the product was eluted at 30% ethyl acetate in hexanes.

Yield: 0.071 g, 61.7%, white solid, m.p: >240 ^ꢀC$¹H NMR (300 MHz,

4.1.11. Synthesis of 3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)amino)

phenyl pentanoate (47)

Valeric acid (0.052 mL, 0.47 mmol) was dissolved in THF. To that

DMAP (0.005 g, 0.04 mmol) and EDCI.HCl (0.09 g, 0.47 mmol) were

added and stirred at room temperature. After 30 min the phenolic

intermediate 2 (0.1 g, 0.39 mmol) was added and stirred for 3 h at

room temperature. After completion of reaction (monitored by

TLC), volatiles evaporated and the crude mass was dissolved in

ethyl acetate and washed with 10% sodium bicarbonate, water and

ﬁnally with brine solution. It was dried over sodium sulfate and

evaporated. The compound was puriﬁed by column chromatog-

raphy where the product was eluted at 25% ethyl acetate in hex-

anes. Yield: 0.102 g, 77.9%, white solid, m.p: 190e192 ^ꢀC. ¹H NMR

DMSO‑d₆):

d

2.29 (s, 3H), 7.18 (t, J ¼ 1.8 Hz, 1ArH), 7.20e7.26 (m,

4ArH), 7.51 (t, J ¼ 7.8 Hz, 1ArH), 7.71 (td, J ¼ 1.5, 9.1 Hz, 1ArH), 7.95

(400 MHz, CDCl₃):

d

0.95 (t, J ¼ 7.2 Hz, 3H), 1.43 (sextet, J ¼ 7.2 Hz,

(dd, J ¼ 1.2, 9.1 Hz, 1ArH), 11.60 (bs, 1 NeH). ¹³C NMR (100 MHz,

2H), 1.73 (quintet, J ¼ 7.6 Hz, 2H), 2.56 (t, J ¼ 7.2 Hz, 2H), 6.89 (d,

J ¼ 8.0 Hz, 1ArH), 7.15 (t, J ¼ 1.6 Hz, 1ArH), 7.18e7.27 (m, 3ArH), 7.52

CDCl₃þ DMSO‑d₆):

d 20.0, 113.3, 114.6, 120.7, 121.4, 121.7, 125.2,

126.9, 128.4, 134.1, 135.2, 138.9, 149.4, 149.9, 161.4, 167.6. HRMS

(ESI^þ): m/z calculated for C₁₆H₁₃N₂O₄[MþH]^þ: 297.0875; found:

297.0879.

(t, J ¼ 8.0 Hz, 2ArH), 8.11 (d, J ¼ 7.6 Hz, 1ArH), 10.45 (s, 1 NeH). ¹³

C

NMR (100 MHz, CDCl₃):

d 13.7, 22.2, 26.9, 34.1, 114.6, 115.7, 122.1,

122.3, 123.5, 126.0, 128.5, 129.3, 135.6, 138.8, 151.3, 151.7, 162.4,

171.8. HRMS (ESI^þ): m/z calculated for C₁₉H₁₉N₂O₄[MþH]^þ:

339.1345; found: 339.1347.

4.1.9. Synthesis of 3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)amino)

phenyl propionate (45)

The phenolic intermediate 2 (0.1 g, 0.39 mmol) was dissolved in

chloroform and stirred at room temperature. To that pyridine

(0.063 mL, 0.78 mmol) was added and cooled to 0 ^ꢀCe5 ^ꢀC and

stirred at that temperature for 15 min. Propionic anhydride

(0.075 mL, 0.59 mmol) was added dropwise and stirred at room

temperature for 5 h. After completion of reaction water was added

and neutralized with 5% hydrochloric acid and the organic layer

collected (3 ꢁ 30 ml). Combined organic layers washed with water

and brine solution. It was dried over sodium sulfate and evapo-

rated. The compound was puriﬁed by column chromatography

where the product was eluted at 30% ethyl acetate in hexanes.

Yield: 0.092 g, 76.0%, m.p: > 240 ^ꢀC. ¹H NMR (300 MHz, DMSO‑d₆):

4.1.12. Synthesis of 3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)amino)

phenyl hexanoate (48)

Hexanoic acid (0.059 mL, 0.47 mmol) was dissolved in THF. To

that DMAP (0.005 g, 0.04 mmol) and EDCI.HCl (0.09 g, 0.47 mmol)

were added and stirred at room temperature. After 30 min the

phenolic intermediate 2 (0.1 g, 0.39 mmol) was added and stirred

for 3 h at room temperature. After completion of reaction (moni-

tored by TLC), volatiles evaporated and the crude mass was dis-

solved in ethyl acetate and washed with 10% sodium bicarbonate,

water and ﬁnally with brine solution. It was dried over sodium

sulfate and evaporated. The compound was puriﬁed by column

chromatography where the product was eluted at 25% ethyl acetate

in hexanes. Yield: 0.099 g, 72.2%, white solid, m.p: 196e198 ^ꢀC. ¹H

d

1.14 (t, J ¼ 7.5 Hz, 3H), 2.62 (q, J ¼ 7.5 Hz, 2H), 7.17 (t, J ¼ 2.1 Hz,

1ArH), 7.20e7.26 (m, 4ArH), 7.51 (t, J ¼ 7.8 Hz, 1ArH), 7.71 (td,

J ¼ 1.5, 8.7 Hz, 1ArH), 7.94 (dd, J ¼ 1.5, 8.1 Hz, 1ArH), 11.59 (bs,

NMR (300 MHz, CDCl₃):

d

0.92 (t, J ¼ 6.9 Hz, 3H), 1.36e1.41 (m, 4H),

1.67e1.77 (m, 2H), 2.55 (t, J ¼ 7.5 Hz, 2H), 7.02 (d, J ¼ 8.1 Hz,

1ArH),7.14 (t, J ¼ 1.8 Hz, 1ArH), 7.18e7.21 (m, 1ArH), 7.24e7.27 (m,

2ArH), 7.53 (t, J ¼ 8.1 Hz, 1ArH), 7.60 (td, J ¼ 1.2, 8.4 Hz, 1ArH), 8.14

(d, J ¼ 7.8 Hz, 1ArH), 9.82 (bs, 1NH). ¹³C NMR (75 MHz, CDCl₃):

1 NeH). ¹³C NMR (100 MHz, CDCl₃þ DMSO‑d₆):

d 7.5, 25.8, 112.9,

114.0, 120.2, 121.1, 124.9, 126.3, 127.9, 133.6, 134.9, 138.4, 148.8,

149.4, 160.8, 170.6. HRMS (ESI^þ): m/z calculated for C₁₇H₁₅N₂O₄

[MþH]^þ: 311.1032; found: 311.1033.

d

13.9, 22.3, 24.6, 31.2, 34.4, 114.7, 115.6, 122.1, 122.3, 123.6, 126.0,

128.6, 129.8, 135.5, 135.6, 138.8, 151.3, 151.6, 162.4, 171.8. HRMS

(ESI^þ): m/z calculated for C₂₀H₂₁N₂O₄[MþH]^þ: 353.1501; found:

353.1504.

4.1.10. Synthesis of 3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)amino)

phenyl butyrate (46)

The phenolic intermediate 2 (0.1 g, 0.39 mmol) was dissolved in

chloroform and stirred at room temperature. To that pyridine

(0.063 mL, 0.78 mmol) was added and cooled to 0 ^ꢀCe5 ^ꢀC and

stirred at that temperature for 15 min. Butanoic anhydride

(0.096 mL, 0.59 mmol) was added dropwise and stirred at room

temperature for 5 h. After completion of reaction water was added

and neutralized with 5% hydrochloric acid and the organic layer

collected (3 ꢁ 30 ml). Combined organic layers washed with water

and brine solution. It was dried over sodium sulfate and evapo-

rated. The compound was puriﬁed by column chromatography

where the product was eluted at 30% ethyl acetate in hexanes.

Yield: 0.089 g, 70.6%, m.p: 232e234 ^ꢀC. ¹H NMR (300 MHz,

4.1.13. Synthesis of 3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)amino)

phenyl heptanoate (49)

Heptanoic acid (0.067 mL, 0.47 mmol) was dissolved in THF. To

that DMAP (0.005 g, 0.04 mmol) and EDCI.HCl (0.09 g, 0.47 mmol)

were added and stirred at room temperature. After 30 min the

phenolic intermediate 2 (0.1 g, 0.39 mmol) was added and stirred

for 3 h at room temperature. After completion of reaction (moni-

tored by TLC), volatiles evaporated and the crude mass was dis-

solved in ethyl acetate and washed with 10% sodium bicarbonate,

water and ﬁnally with brine solution. It was dried over sodium

sulfate and evaporated. The compound was puriﬁed by column

chromatography where the product was eluted at 25% ethyl acetate

in hexanes. Yield: 0.111 g, 78.2%, white solid, m.p: 196e198 ^ꢀC. ¹H

DMSO‑d₆):

d

0.98 (t, J ¼ 7.5 Hz, 3H), 1.67 (sextet, J ¼ 7.5 Hz, 2H), 2.58

(t, J ¼ 7.2 Hz, 2H), 7.18 (t, J ¼ 1.8 Hz, 1ArH), 7.19e7.26 (m, 4ArH), 7.52

(t, J ¼ 8.1 Hz, 1ArH), 7.71 (td, J ¼ 1.5, 8.1 Hz, 1ArH), 7.94 (dd, J ¼ 1.2,

8.1 Hz, 1ArH), 11.59 (bs, 1 NeH). ¹³C NMR (100 MHz, CDCl₃þ

NMR (300 MHz, CDCl₃):

d

0.90 (t, J ¼ 6.9 Hz, 3H), 1.28e1.42 (m, 6H),

1.72 (q, J ¼ 7.5 Hz, 2H), 2.55 (t, J ¼ 7.5 Hz, 2H), 7.03 (d, J ¼ 8.1 Hz,

1ArH), 7.14 (t, J ¼ 1.8 Hz, 1ArH), 7.17e7.22 (m, 1ArH), 7.25e7.26 (m,

2ArH), 7.53 (t, J ¼ 8.1 Hz, 1ArH), 7.61 (td, J ¼ 1.5 Hz, 8.1 Hz, 1ArH),

8.14 (d, J ¼ 8.1 Hz, 1ArH), 9.61 (s, 1NH). ¹³C NMR (75 MHz, CDCl₃):

DMSO‑d₆):

d 12.4, 17.0, 34.7, 113.2, 114.4, 120.6, 121.3, 121.5, 125.1,

126.7, 128.2, 134.0, 135.1, 138.7, 149.2, 149.8, 161.2, 170.1. HRMS

(ESI^þ): m/z calculated for C₁₈H₁₇N₂O₄[MþH]^þ: 325.1188; found:

10

A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

d

14.0, 22.4, 24.7, 29.6, 31.3, 34.3, 114.5, 115.5, 121.7, 122.1, 122.8,

1.69e1.76 (m, 2H), 2.55 (t, J ¼ 7.5 Hz, 2H), 7.04 (d, J ¼ 8.1 Hz, 1ArH),

7.14 (t, J ¼ 1.8 Hz, 1ArH), 7.18e7.22 (m, 1ArH), 7.24e7.26 (m, 2ArH),

7.53 (t, J ¼ 8.1 Hz, 1ArH), 7.62 (td, J ¼ 1.5, 8.7 Hz, 1ArH), 8.15 (d,

125.9, 128.2, 129.5, 135.1, 135.9, 139.7, 150.8, 151.2, 162.6, 171.5.

HRMS (ESI^þ): m/z calculated for C₂₀H₂₃N₂O₄[MþH]^þ: 367.1658;

found: 367.1659.

J ¼ 7.2 Hz, 1ArH), 9.56 (bs, 1NH). ¹³C NMR (75 MHz, CDCl₃):

d 14.1,

22.7, 24.9, 29.1, 29.3, 29.4, 29.7, 31.9, 34.4, 114.7, 115.6, 122.1, 122.3,

123.6, 126.0, 128.6, 129.8, 135.5, 135.6, 138.8, 151.3, 151.7, 162.4, 171.

HRMS (ESI^þ): m/z calculated for C₂₄H₂₉N₂O₄[MþH]^þ: 409.2127;

found: 409.2128.

4.1.14. Synthesis of 3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)amino)

phenyl octanoate (50)

Octanoic acid (0.074 mL, 0.47 mmol) was dissolved in THF. To

that DMAP (0.005 g, 0.04 mmol) and EDCI.HCl (0.09 g, 0.47 mmol)

were added and stirred at room temperature. After 30 min the

phenolic intermediate 2 (0.1 g, 0.39 mmol) was added and stirred

for 3 h at room temperature. After completion of reaction (moni-

tored by TLC), volatiles evaporated and the crude mass was dis-

solved in ethyl acetate and washed with 10% sodium bicarbonate,

water and ﬁnally with brine solution. It was dried over sodium

sulfate and evaporated. The compound was puriﬁed by column

chromatography where the product was eluted at 25% ethyl acetate

in hexanes. Yield: 0.106 g, 71.6%, white solid, m.p: 202e204 ^ꢀC. ¹H

4.1.17. Synthesis of 3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)amino)

phenyl palmitate (53)

Palmitic acid (0.121 g, 0.47 mmol) was dissolved in THF. To that

DMAP (0.005 g, 0.04 mmol) and EDCI.HCl (0.09 g, 0.47 mmol) were

added and stirred at room temperature. After 30 min the phenolic

intermediate 2 (0.1 g, 0.39 mmol) was added and stirred for 3 h at

room temperature. After completion of reaction (monitored by

TLC), volatiles evaporated and the crude mass was dissolved in

ethyl acetate and washed with 10% sodium bicarbonate, water and

ﬁnally with brine solution. It was dried over sodium sulfate and

evaporated. The compound was puriﬁed by column chromatog-

raphy where the product was eluted at 25% ethyl acetate in hex-

anes. Yield: 0.132 g, 68.8%, white solid, m.p: 192e194 ^ꢀC ¹H NMR

NMR (300 MHz, CDCl₃):

d

0.89 (t, J ¼ 6.9 Hz, 3H), 1.25e1.59 (m, 8H),

1.74 (q, J ¼ 6.9 Hz, 2H), 2.55 (t, J ¼ 7.5 Hz, 2H), 7.03 (d, J ¼ 7.8 Hz,

1ArH), 7.4 (t, J ¼ 1.8 Hz, 1ArH), 7.20e7.22 (m, 1ArH), 7.25e7.27 (m,

2ArH), 7.53 (t, J ¼ 8.1 Hz, 1ArH), 7.62 (td, J ¼ 1.2, 8.4 Hz, 1ArH), 8.15

(dd, J ¼ 1.2, 8.1 Hz, 1ArH), 9.49 (bs, 1NH). ¹³C NMR (75 MHz, CDCl₃):

(300 MHz, CDCl₃):

d

0.88 (t, J ¼ 6.9 Hz, 3H), 1.25e1.39 (m, 22H),

d

14.0, 22.4, 24.7, 28.7, 28.8, 31.5, 34.2,114.4,115.5,121.7,122.1, 122.7,

1.69e1.79 (m, 4H), 2.55 (t, J ¼ 7.5 Hz, 2H), 7.04 (d, J ¼ 8.1 Hz, 1ArH),

7.14 (t, J ¼ 1.8 Hz, 1ArH), 7.18e7.20 (m, 1ArH), 7.22e7.25 (m, 2ArH),

7.53 (t, J ¼ 8.1 Hz, 1ArH), 7.62 (td, J ¼ 1.2, 8.1 Hz, 1ArH), 8.15 (d,

125.9,128.1, 129.5,135.1, 135.9, 139.7,150.6,151.1,162.5,171.4. HRMS

(ESI^þ): m/z calculated for C₂₂H₂₅N₂O₄[MþH]^þ: 381.1814; found:

381.1817.

J ¼ 4.2 Hz, 1ArH), 10.05 (bs, 1NH). ¹³C NMR (75 MHz, CDCl₃):

d 14.1,

22.7, 24.9, 29.1, 29.3, 29.4, 29.5, 29.6, 29.7, 29.7, 31.9, 34.4, 114.6,

115.7,122.1, 122.3,123.5,126.0,128.5,129.8,135.6, 138.8,151.3, 151.7,

162.4, 171.8. HRMS (ESI^þ): m/z calculated for C₃₀H₄₁N₂O₄[MþH]^þ:

493.3066; found: 493.3067.

4.1.15. Synthesis of 3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)amino)

phenyl nonanoate (51)

Nonanoic acid (0.083 mL, 0.47 mmol) was dissolved in THF. To

that DMAP (0.005 g, 0.04 mmol) and EDCI.HCl (0.09 g, 0.47 mmol)

were added and stirred at room temperature. After 30 min the

phenolic intermediate 2 (0.1 g, 0.39 mmol) was added and stirred

for 3 h at room temperature. After completion of reaction (moni-

tored by TLC), volatiles evaporated and the crude mass was dis-

solved in ethyl acetate and washed with 10% sodium bicarbonate,

water and ﬁnally with brine solution. It was dried over sodium

sulfate and evaporated. The compound was puriﬁed by column

chromatography where the product was eluted at 25% ethyl acetate

in hexanes. Yield: 1.11 g, 76.0%, white solid, m.p: 210e212 ^ꢀC. ¹H

4.1.18. Synthesis of 3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)amino)

phenyl oleate (54)

Oleic acid (0.148 mL, 0.47 mmol) was dissolved in THF. To that

DMAP (0.005 g, 0.04 mmol) and EDCI.HCl (0.09 g, 0.47 mmol) were

added and stirred at room temperature. After 30 min the phenolic

intermediate 2 (0.1 g, 0.39 mmol) was added and stirred for 3 h at

room temperature. After completion of reaction (monitored by

TLC), volatiles evaporated and the crude mass was dissolved in

ethyl acetate and washed with 10% sodium bicarbonate, water and

ﬁnally with brine solution. It was dried over sodium sulfate and

evaporated. The compound was puriﬁed by column chromatog-

raphy where the product was eluted at 25% ethyl acetate in hex-

anes. Yield: 0.156 g, 77.2%, white waxy solid, ¹H NMR (300 MHz,

NMR (300 MHz, CDCl₃):

d

0.88 (t, J ¼ 6.9 Hz, 3H),1.23e1.45 (m,10H),

1.74 (q, J ¼ 7.2 Hz, 2H), 2.55 (t, J ¼ 7.5 Hz, 2H), 7.02 (d, J ¼ 8.1 Hz,

1ArH), 7.15 (t, J ¼ 1.8 Hz, 1ArH), 7.18e7.22 (m, 1ArH), 7.24e7.26 (m,

2ArH), 7.53 (td, J ¼ 0.9, 9.0 Hz, 1ArH), 7.61 (td, J ¼ 1.2, 8.1 Hz, 1ArH),

7.14 (dd, J ¼ 0.9, 7.8 Hz, 1ArH), 9.67 (bs, 1NH). ¹³C NMR (75 MHz,

CDCl₃):

d

0.88 (t, J ¼ 6.9 Hz, 3H), 1.25e1.41 (m, 20H), 1.60e1.79 (m,

CDCl₃):

d

14.1, 21.1, 22.6, 24.8, 29.1, 29.6, 31.8, 34.4, 114.5, 115.6,

4H), 2.02e2.06 (m, 2H), 2.55 (t, J ¼ 7.5 Hz, 2H), 5.33e5.38 (m, 2H),

7.00 (d, J ¼ 8.1 Hz, 1ArH), 7.14 (t, J ¼ 1.8 Hz, 1ArH), 7.15e7.27 (m,

3ArH), 7.53 (t, J ¼ 8.1 Hz, 1ArH), 7.60 (td, J ¼ 1.5, 7.2 Hz, 1ArH), 8.13

(d, J ¼ 7.8 Hz, 1ArH), 10.09 (bs, 1NH). ¹³C NMR (75 MHz, CDCl₃):

121.8, 122.1, 122.9, 125.9, 128.3, 129.6, 135.2, 135.9, 139.6, 150.9,

151.1, 162.6, 171.5. HRMS (ESI^þ): m/z calculated for C₂₃H₂₇N₂O₄

[MþH]^þ: 395.1971; found: 395.1974.

d

14.1, 22.7, 24.9, 27.2, 29.1, 29.2, 29.3, 29.5, 29.7, 31.9, 34.4, 114.7,

4.1.16. Synthesis of 3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)amino)

phenyl decanoate (52)

115.5, 122.1, 122.3, 123.6, 125.9, 128.1, 128.6, 129.8, 130.0, 135.5,

138.7, 151.3, 151.6, 162.3, 171.7. HRMS (ESI^þ): m/z calculated for

Decanoic acid (0.091 mL, 0.47 mmol) was dissolved in THF. To

that DMAP (0.005 g, 0.04 mmol) and EDCI.HCl (0.09 g, 0.47 mmol)

were added and stirred at room temperature. After 30 min the

phenolic intermediate 2 (0.1 g, 0.39 mmol) was added and stirred

for 3 h at room temperature. After completion of reaction (moni-

tored by TLC), volatiles evaporated and the crude mass was dis-

solved in ethyl acetate and washed with 10% sodium bicarbonate,

water and ﬁnally with brine solution. It was dried over sodium

sulfate and evaporated. The compound was puriﬁed by column

chromatography where the product was eluted at 25% ethyl acetate

in hexanes. Yield: 0.102 g, 64.2%, white solid, m.p: 222e224 ^ꢀC. ¹H

C

₃₂H₄₃N₂O₄[MþH]^þ: 519.3223; found: 519.3225.

4.1.19. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-methoxybenzoate (55)

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

stirring. Finally intermediate 27 (0.089 g, 0.39 mmol) was added

ꢀ

and maintained at 70 C for 8 h. After completion of reaction, the

reaction mass was poured into water and extracted with ethyl ac-

etate (3 ꢁ 150 ml). Combined organic layer was thoroughly washed

with water followed by brine solution. The organic portion was

then evaporated and puriﬁed by column chromatography on silica

NMR (300 MHz, CDCl₃):

d

0.88 (t, J ¼ 6.9 Hz, 3H),1.26e1.45 (m,12H),

11

A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

gel (100e200 mesh) using 25% ethyl acetate in hexanes to afford

the compound. Yield: 0.123 g, 70.1%, white solid, m.p: 210e212 ^ꢀC.

acetate (3 ꢁ 150 ml). Combined organic layer was thoroughly

washed with water followed by brine solution. The organic portion

was then evaporated and puriﬁed by column chromatography on

silica gel (100e200 mesh) using 25% ethyl acetate in hexanes to

afford the compound. Yield: 0.126 g, 66.3%, white wax, ¹H NMR

¹H NMR (300 MHz, CDCl₃):

d 2.27 (m, 2H), 3.85 (s, 3H), 4.37 (t,

J ¼ 7.2 Hz, 2H), 4.45 (t, J ¼ 6.0 Hz, 2H), 6.30 (s, 1H NeH), 6.64 (t,

J ¼ 2.1 Hz, 1ArH), 6.75 (m, 1ArH), 6.83 (dd, J ¼ 2.4, 8.4 Hz, 1ArH),

6.87e6.93 (m, 2ArH), 7.26e7.35 (m, 3ArH), 7.68 (td, J ¼ 1.5, 8.7 Hz,

1ArH), 7.91e7.98 (m, 2ArH), 8.26 (dd, J ¼ 1.5, 7.8 Hz, 1ArH). ¹³C NMR

(300 MHz, CDCl₃):

d

0.98 (t, J ¼ 7.2 Hz, 3H), 1.44e1.51 (m, 2H),

1.74e1.83 (m, 2H), 2.27 (quintet, J ¼ 6.9 Hz, 2H), 4.00 (t, J ¼ 6.6 Hz,

2H), 4.36 (t, J ¼ 6.9 Hz, 2H), 4.44 (t, J ¼ 6.0 Hz, 2H), 6.47 (bs, 1H

NeH), 6.63 (t, J ¼ 2.1 Hz, 1ArH), 6.74 (d, J ¼ 7.8 Hz, 1ArH), 6.82 (dd,

J ¼ 1.5, 8.1 Hz, 1ArH), 6.86e6.91 (m, 2ArH), 7.25e7.34 (m, 3ArH),

7.67 (td, J ¼ 1.8, 7.5 Hz, 1ArH), 7.89e7.94 (m, 2ArH), 8.26 (dd, J ¼ 1.5,

(100 MHz, CDCl₃):

d 26.4, 40.9, 52.2, 62.0, 113.5, 115.5, 116.5, 119.5,

121.0,122.7,128.9,129.6,131.2,135.3,136.4,139.7,150.6,157.3,161.5,

163.3, 165.8. HRMS (ESI^þ): m/z calculated for C₂₅H₂₃N₂O₆[MþH]^þ:

447.1556; found: 447.1558.

8.1 Hz, 1ArH). ¹³C NMR (100 MHz, CDCl₃):

d 14.0, 19.4, 27.0, 31.3,

4.1.20. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-ethoxybenzoate (56)

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

41.6, 62.4, 68.2, 113.8, 114.4, 116.0, 116.3, 116.7, 120.0, 122.1, 123.5,

129.9, 130.5, 131.7, 135.8, 136.2, 140.1, 151.2, 157.5, 162.1, 163.4, 166.5.

HRMS (ESI^þ): m/z calculated for C₂₈H₂₉N₂O₆[MþH]^þ: 489.2026;

found: 489.2028.

stirring. Finally intermediate 28 (0.095 g, 0.39 mmol) was added

ꢀ

and maintained at 70 C for 8 h. After completion of reaction, the

reaction mass was poured into water and extracted with ethyl ac-

etate (3 ꢁ 150 ml). Combined organic layer was thoroughly washed

with water followed by brine solution. The organic portion was

then evaporated and puriﬁed by column chromatography on silica

gel (100e200 mesh) using 25% ethyl acetate in hexanes to afford

the compound. Yield: 0.116 g, 64.4.0%, white wax, ¹H NMR

4.1.23. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-(pentyloxy)benzoate (59)

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

stirring. Finally intermediate 31 (0.111 g, 0.39 mmol) was added and

ꢀ

maintained at 70 C for 8 h. After completion of reaction, the re-

(300 MHz, CDCl₃):

d

1.43 (t, J ¼ 6.9 Hz, 3H), 2.27 (quintet, J ¼ 6.9 Hz,

action mass was poured into water and extracted with ethyl acetate

(3 ꢁ 150 ml). Combined organic layer was thoroughly washed with

water followed by brine solution. The organic portion was then

evaporated and puriﬁed by column chromatography on silica gel

(100e200 mesh) using 25% ethyl acetate in hexanes to afford the

compound. Yield: 0.126 g, 64.3%, white solid, m.p: 138e140 ^ꢀC. ¹H

2H), 4.07 (q, J ¼ 6.9 Hz, 2H), 4.36 (t, J ¼ 6.9 HZ, 2H), 4.44 (t,

J ¼ 6.0 Hz, 2H), 6.90 (bs, 1H NeH), 6.63 (t, J ¼ 2.1 Hz, 1ArH),

6.72e6.75 (m, 1ArH), 6.80e6.83 (m, 1ArH), 6.86e6.89 (m, 2ArH),

7.26e7.33 (m, 3ArH), 7.67 (td, J ¼ 1.5, 7.5 Hz, 1ArH), 7.92 (dt, J ¼ 2.1,

6.9 Hz, 2ArH), 8.26 (dd, J ¼ 1.5, 7.8 Hz, 1ArH). ¹³C NMR (100 MHz,

CDCl₃):

d

14.9, 27.0, 41.6, 62.4, 64.0, 113.7, 114.4, 116.0, 116.3, 116.7,

NMR (400 MHz, CDCl₃):

d

0.86 (t, J ¼ 5.1 Hz, 3H), 1.31e1.37 (m, 4H),

120.0, 122.2, 123.5, 129.9, 130.5, 131.8, 135.8, 136.2, 140.1, 151.2,

1.71e1.7 (m, 2H), 2.17e2.21 (m, 2H), 3.92 (t, J ¼ 6.4 Hz, 2H), 4.29 (t,

J ¼ 6.4 Hz, 2H), 4.37 (t, J ¼ 6.0 Hz, 2H), 6.55 (t, J ¼ 2.0 Hz, 1ArH), 6.66

(d, J ¼ 8.0 Hz, 1ArH), 6.73 (d, J ¼ 2.0 Hz, 1ArH), 6.79e6.82 (m, 3ArH),

7.20e7.25 (m, 3ArH), 7.60 (t, J ¼ 6.0 Hz, 1ArH), 7.84 (d, J ¼ 6.8 Hz,

2ArH), 8.18 (dd, J ¼ 1.2, 7.2 Hz, 1ArH). ¹³C NMR (100 MHz, CDCl₃):

157.5, 162.1, 163.2, 166.4. HRMS (ESI^þ): m/z calculated for

C

₂₆H₂₅N₂O₆[MþH]^þ: 461.1713; found: 461.1716.

4.1.21. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-propoxybenzoate (57)

d

14.2, 22.6, 26.9, 28.3, 28.9, 41.6, 62.3, 68.4, 113.7, 114.3, 115.9, 116.2,

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

116.6, 119.9, 122.0, 123.4, 129.8, 130.5, 131.7, 135.8,136.1, 140.0,151.2,

157.5, 162.1, 163.3, 166.4. HRMS (ESI^þ): m/z calculated for

stirring. Finally intermediate 29 (0.1 g, 0.39 mmol) was added and

C

₂₉H₃₁N₂O₆[MþH]^þ: 503.2182; found: 503.2186.

ꢀ

maintained at 70 C for 8 h. After completion of reaction, the re-

action mass was poured into water and extracted with ethyl acetate

(3 ꢁ 150 ml). Combined organic layer was thoroughly washed with

water followed by brine solution. The organic portion was then

evaporated and puriﬁed by column chromatography on silica gel

(100e200 mesh) using 25% ethyl acetate in hexanes to afford the

compound. Yield: 0.118 g, 63.8%, white solid, m.p: 134e136 ^ꢀC. ¹H

4.1.24. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-(hexyloxy)benzoate (60)

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

stirring. Finally intermediate 32 (0.117 g, 0.39 mmol) was added and

ꢀ

NMR (400 MHz, CDCl₃):

d

0.97 (t, J ¼ 2.8 Hz, 3H), 1.74 (m, 2H), 2.18

maintained at 70 C for 8 h. After completion of reaction, the re-

(m, 2H), 3.88 (t, J ¼ 6.8 Hz, 2H), 4.28 (t, J ¼ 7.2 Hz, 2H), 4.36 (t,

J ¼ 6.0 Hz, 2H), 6.56 (t, J ¼ 2.0 Hz, 1ArH), 6.64 (d, J ¼ 8.0 Hz, 1ArH),

6.73 (d, J ¼ 2.0 Hz,1ArH), 6.80e6.82 (m, 3ArH), 7.19e7.23 (m, 3ArH),

7.59e7.60 (m, 1ArH), 7.84 (dd, J ¼ 2.0, 7.2 Hz, 2ArH), 8.18 (d,

action mass was poured into water and extracted with ethyl acetate

(3 ꢁ 150 ml). Combined organic layer was thoroughly washed with

water followed by brine solution. The organic portion was then

evaporated and puriﬁed by column chromatography on silica gel

(100e200 mesh) using 22% ethyl acetate in hexanes to afford the

compound. Yield: 0.132 g, 65.7%, white solid, m.p: 130e132 ^ꢀC. ¹H

J ¼ 6.0 Hz, 1ArH). ¹³C NMR (100 MHz, CDCl₃):

d 10.6, 22.6, 26.9, 41.5,

62.3, 69.9, 113.7, 114.2, 115.9, 116.2, 116.6, 119.8, 122.0, 123.4, 129.8,

130.4,131.7, 135.8,136.1, 140.0,151.2,157.6, 162.1,163.3,166.4. HRMS

(ESI^þ): m/z calculated for C₂₇H₂₇N₂O₆[MþH]^þ: 475.1865; found:

475.1867.

NMR (400 MHz, CDCl₃):

d

0.85 (t, J ¼ 5.6 Hz, 3H), 1.18e1.25 (m, 2H),

1.26e1.28 (m, 2H), 1.68e1.73 (m, 4H), 2.18 (sextet, J ¼ 6.4 Hz, 2H),

3.91 (t, J ¼ 6.4 Hz, 2H), 4.28 (t, J ¼ 6.8 Hz, 2H), 4.36 (t, J ¼ 6.0 Hz, 2H),

6.55 (t, J ¼ 2.0 Hz, 1ArH), 6.64 (d, J ¼ 8.0 Hz, 1ArH), 6.72 (d,

J ¼ 2.0 Hz, 1ArH), 6.79e6.81 (m, 3ArH), 7.19e7.23 (m, 3ArH), 7.59 (t,

J ¼ 6.0 Hz, 1ArH), 7.83 (dd, J ¼ 2.0, 6.8 Hz, 2ArH), 8.18 (dd, J ¼ 1.2,

4.1.22. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-butoxybenzoate (58)

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

7.6 Hz, 1ArH). ¹³C NMR (100 MHz, CDCl₃):

d 14.2, 22.7, 25.8, 26.9,

29.2, 31.7, 41.5, 62.3, 68.4,113.7,114.2,115.9,116.2,116.6,119.7,122.0,

123.4, 129.8, 130.4, 131.6, 131.7, 135.8, 136.0, 140.0, 151.2, 157.7, 162.1,

163.3, 166.4. HRMS (ESI^þ): m/z calculated for C₃₀H₃₃N₂O₆[MþH]^þ:

517.2338; found: 516.2343.

stirring. Finally intermediate 30 (0.106 g, 0.39 mmol) was added

ꢀ

and maintained at 70 C for 8 h. After completion of reaction, the

reaction mass was poured into water and extracted with ethyl

12

A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

4.1.25. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-(heptyloxy)benzoate (61)

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

1ArH), 6.83e6.91 (m, 3ArH), 7.25e7.36 (m, 3ArH), 7.68 (td, J ¼ 1.5,

7.2 Hz, 1ArH), 7.91e8.02 (m, 2ArH), 8.26 (dd, J ¼ 1.5, 6.3 Hz, 1ArH).

¹³C NMR (100 MHz, CDCl₃):

d 14.3, 22.9, 26.2, 27.0, 29.3, 29.5, 29.6,

29.7, 32.1, 41.6, 62.3, 68.5, 113.8, 114.4, 115.9, 116.3, 116.7, 119.9,

122.0, 123.5, 129.9, 130.5, 131.7, 135.9, 136.1, 140.1, 151.2, 157.6, 162.2,

163.4, 166.5. HRMS (ESI^þ): m/z calculated for C₃₃H₃₉N₂O₆[MþH]^þ:

559.2808; found: 559.2811.

stirring. Finally intermediate 33 (0.122 g, 0.39 mmol) was added

ꢀ

and maintained at 70 C for 8 h. After completion of reaction, the

reaction mass was poured into water and extracted with ethyl ac-

etate (3 ꢁ 150 ml). Combined organic layer was thoroughly washed

with water followed by brine solution. The organic portion was

then evaporated and puriﬁed by column chromatography on silica

gel (100e200 mesh) using 22% ethyl acetate in hexanes to afford

the compound. Yield: 0.138 g, 66.7%, white solid, m.p: 114e116 ^ꢀC.

4.1.28. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-(decyloxy)benzoate (64)

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

¹H NMR (300 MHz, CDCl₃):

d

0.90 (t, J ¼ 6.6 Hz, 3H), 1.25e1.38 (m,

stirring. Finally intermediate 36 (0.138 g, 0.39 mmol) was added

ꢀ

4H), 1.40e1.46 (m, 4H), 1.80 (quintet, J ¼ 6.6 Hz, 2H), 2.27 (quintet,

J ¼ 6.3 Hz, 2H), 4.00 (t, J ¼ 6.6 Hz, 2H), 4.36 (t, J ¼ 7.2 Hz, 2H), 4.44 (t,

J ¼ 5.7 Hz, 2H), 6.13 (bs, 1 NeH), 6.64 (t, J ¼ 2.1 Hz, 1ArH), 6.76 (dd,

J ¼ 1.2, 7.8 Hz, 1ArH), 6.84 (dd, J ¼ 1.8, 8.4 Hz, 1ArH), 6.88 (d,

J ¼ 9.0 Hz, 2ArH), 7.25e7.36 (m, 3ArH), 7.68 (td, J ¼ 1.8, 8.7 Hz,

and maintained at 70 C for 8 h. After completion of reaction, the

reaction mass was poured into water and extracted with ethyl ac-

etate (3 ꢁ 150 ml). Combined organic layer was thoroughly washed

with water followed by brine solution. The organic portion was

then evaporated and puriﬁed by column chromatography on silica

gel (100e200 mesh) using 20% ethyl acetate in hexanes to afford

the compound. Yield: 0.141 g, 63.2%, white solid, m.p: 122e124 ^ꢀC.

1ArH), 7.92 (d, J ¼ 8.7 Hz, 2ArH), 8.26 (dd, J ¼ 1.5, 7.8 Hz, 1ArH). ¹³

C

NMR (100 MHz, CDCl₃): d 14.1, 22.6, 26.0, 26.8, 29.0, 30.3, 31.8, 41.4,

62.1, 68.3, 113.6, 114.2, 115.8, 116.1, 116.6, 119.6, 121.8, 123.3, 129.7,

130.4, 131.5, 135.7, 135.9, 139.9, 151.1, 157.5, 162.0, 163.2, 166.3.

HRMS (ESI^þ): m/z calculated for C₃₁H₃₅N₂O₆[MþH]^þ: 531.2495;

found: 531.2498.

¹H NMR (400 MHz, CDCl₃):

d

0.81 (t, J ¼ 6.0 Hz, 3H), 1.20e1.25 (m,

10H), 1.36e1.38 (m, 2H), 1.67e1.73 (m, 4H), 2.16e2.19 (m, 2H), 3.91

(t, J ¼ 6.4 Hz, 2H), 4.28 (t, J ¼ 6.0 Hz, 2H), 4.36 (t, J ¼ 6.0 Hz, 2H), 6.54

(t, J ¼ 2.0 Hz, 1ArH), 6.64 (d, J ¼ 8.0 Hz, 1ArH), 6.71 (d, J ¼ 2.0 Hz,

1ArH), 6.79e6.81 (m, 3ArH), 7.19e7.24 (m, 3ArH), 7.59 (t, J ¼ 6.4 Hz,

1ArH), 7.83 (d, J ¼ 6.8 Hz, 2ArH), 8.17 (d, J ¼ 7.2 Hz, 1ArH). ¹³C NMR

4.1.26. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-(octyloxy)benzoate (62)

(100 MHz, CDCl₃): d 14.3, 22.8, 26.1, 27.0, 29.2, 29.4, 29.5, 29.7, 32.0,

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

41.6, 62.3, 68.4, 113.7, 114.3, 115.9, 116.2, 116.7, 119.7, 122.0, 123.4,

129.8,130.5,131.6,135.8,136.0,140.0,151.2, 157.7, 162.1,163.3,166.4.

HRMS (ESI^þ): m/z calculated for C₃₄H₄₁N₂O₆[MþH]^þ: 573.2965;

found: 573.2966.

stirring. Finally intermediate 34 (0.127 g, 0.39 mmol) was added

ꢀ

and maintained at 70 C for 8 h. After completion of reaction, the

reaction mass was poured into water and extracted with ethyl ac-

etate (3 ꢁ 150 ml). Combined organic layer was thoroughly washed

with water followed by brine solution. The organic portion was

then evaporated and puriﬁed by column chromatography on silica

gel (100e200 mesh) using 22% ethyl acetate in hexanes to afford

the compound. Yield: 0.156 g, 73.6%, colorless liquid, ¹H NMR

4.1.29. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-(octadecyloxy)benzoate (65)

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

stirring. Finally int_ꢀermediate 4 (0.171 g, 0.39 mmol) was added and

maintained at 70 C for 8 h. After completion of reaction, the re-

action mass was poured into water and extracted with ethyl acetate

(3 ꢁ 150 ml). Combined organic layer was thoroughly washed with

water followed by brine solution. The organic portion was then

evaporated and puriﬁed by column chromatography on silica gel

(100e200 mesh) using 20% ethyl acetate in hexanes to afford the

compound. Yield: 0.148 g, 57.8%, white solid, m.p: 112e114 ^ꢀC. ¹H

(300 MHz, CDCl₃):

d

0.89 (t, J ¼ 6.9 Hz, 3H), 1.25e1.32 (m, 8H),

1.40e1.48 (m, 2H),1.75e1.84 (m, 2H), 2.27 (q, J ¼ 6.6 Hz, 2H), 3.99 (t,

J ¼ 6.6 Hz, 2H), 4.36 (t, J ¼ 7.5 Hz, 2H), 4.44 (t, J ¼ 6.0 Hz, 2H), 6.32

(bs, 1NH), 6.64 (t, J ¼ 2.1 Hz, 1ArH), 6.75 (d, J ¼ 7.8 Hz, 1ArH), 6.83

(dd, J ¼ 1.8, 8.1 Hz, 1ArH), 6.87e6.91 (m, 2ArH), 7.25e7.35 (m,

3ArH), 7.68 (td, J ¼ 1.5, 8.7 Hz, 1ArH), 7.90e7.93 (m, 2ArH), 8.26 (dd,

J ¼ 1.5, 6.3 Hz, 1ArH). ¹³C NMR (100 MHz, CDCl₃):

d 14.3, 22.9, 26.2,

26.9, 29.3, 29.4, 29.5, 32.0, 41.6, 62.3, 68.5, 113.8, 114.4, 115.9, 116.3,

116.7, 119.9, 122.0, 123.5, 129.9, 130.5, 131.7, 135.8, 136.1, 140.1, 151.2,

157.6, 162.2, 163.4, 166.5. HRMS (ESI^þ): m/z calculated for

NMR (300 MHz, CDCl₃):

d

0.88 (t, J ¼ 6.9 Hz, 3H), 1.18e1.38 (m,

28 H), 1.42e1.46 (m, 2H),1.75e1.83 (m, 2H), 2.26e2.32 (m, 2H), 4.01

(t, J ¼ 6.6 Hz, 2H), 4.37 (t, J ¼ 7.8 Hz, 2H), 4.45 (t, J ¼ 6.0 Hz, 2H),

6.73e6.78 (m, 2ArH), 6.89e6.95 (m, 3ArH), 7.10 (bs, 1NH),

7.25e7.28 (m, 1ArH), 7.31e7.37 (m, 2ArH), 7.68 (td, J ¼ 1.5, 8.7 Hz,

C

₃₂H₃₇N₂O₆[MþH]^þ: 545.2652; found: 545.2655.

4.1.27. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-(nonyloxy)benzoate (63)

1ArH), 7.95 (d, J ¼ 8.7 Hz, 2ArH), 8.26 (dd, J ¼ 1.5, 7.8 Hz, 1ArH). ¹³

C

NMR (100 MHz, CDCl₃): d 14.3, 22.9, 26.2, 29.3, 29.6, 29.7, 29.8, 29.9,

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

32.1, 41.6, 62.3, 68.5, 113.8, 114.4, 115.9, 116.3, 116.7, 120.0, 122.0,

123.5, 129.9, 130.6, 131.7, 135.8, 136.2, 140.1, 151.2, 157.5, 162.1, 163.4,

166.5. HRMS (ESI^þ): m/z calculated for C₄₂H₅₇N₂O₆[MþH]^þ:

685.4217; found: 685.4221.

stirring. Finally intermediate 35 (0.133 g, 0.39 mmol) was added

ꢀ

and maintained at 70 C for 8 h. After completion of reaction, the

reaction mass was poured into water and extracted with ethyl ac-

etate (3 ꢁ 150 ml). Combined organic layer was thoroughly washed

with water followed by brine solution. The organic portion was

then evaporated and puriﬁed by column chromatography on silica

gel (100e200 mesh) using 20% ethyl acetate in hexanes to afford

the compound. Yield: 0.132 g, 60.8%, white solid, m.p: 120e122 ^ꢀC.

4.1.30. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-((3-methylbut-2-en-1-yl)oxy)benzoate

(66)

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

¹H NMR (300 MHz, CDCl₃):

d

0.88 (t, J ¼ 6.9 Hz, 3H), 1.26e1.28 (m,

stirring. Finally intermediate 5 (0.11 g, 0.39 mmol) was added and

ꢀ

10H), 1.40e1.46 (m, 2H), 1.75e1.84 (m, 2H), 2.27 (q, J ¼ 6.9 Hz, 2H),

4.00 (t, J ¼ 6.6 Hz, 2H), 4.36 (t, J ¼ 7.2 Hz, 2H), 4.44 (t, J ¼ 6.0 Hz, 2H),

6.29 (bs, 1NH), 6.65 (t, J ¼ 2.1 Hz, 1ArH), 6.76 (dt, J ¼ 0.9, 6.9 Hz,

maintained at 70 C for 8 h. After completion of reaction, the re-

action mass was poured into water and extracted with ethyl acetate

(3 ꢁ 150 ml). Combined organic layer was thoroughly washed with

13

A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

water followed by brine solution. The organic portion was then

evaporated and puriﬁed by column chromatography on silica gel

(100e200 mesh) using 20% ethyl acetate in hexanes to afford the

compound. Yield: 0.125 g, 64.1%, white solid, ¹H NMR (300 MHz,

4.1.33. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl (E)-4-(octadec-9-en-1-yloxy)benzoate (69)

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

stirring. Finally int_ꢀermediate 8 (0.181 g, 0.39 mmol) was added and

maintained at 70 C for 8 h. After completion of reaction, the re-

action mass was poured into water and extracted with ethyl acetate

(3 ꢁ 150 ml). Combined organic layer was thoroughly washed with

water followed by brine solution. The organic portion was then

evaporated and puriﬁed by column chromatography on silica gel

(100e200 mesh) using 15% ethyl acetate in hexanes to afford the

compound. Yield: 0.145 g, 54.5%, white solid, m.p: 90e92 ^ꢀC. ¹H

CDCl₃):

d

1.76 (s, 3H), 1.81 (s, 3H), 2.27 (q, J ¼ 6.0 Hz, 2H), 4.36 (t,

J ¼ 6.6 Hz, 2H), 4.44 (t, J ¼ 5.7 Hz, 2H), 4.56 (d, J ¼ 6.9 Hz, 2H), 5.48

(td, J ¼ 1.5, 5.4 Hz, 1H), 6.59 (bs, 1NH), 6.63 (t, J ¼ 2.1 Hz, 1ArH), 6.74

(td, J ¼ 0.9, 7.8 Hz, 1ArH), 6.80e6.83 (m, 1ArH), 6.87e6.91 (m,

2ArH), 7.25e7.34 (m, 3ArH), 7.67 (dt, J ¼ 1.5, 8.7 Hz, 1ArH),

7.89e7.96 (m, 2ArH), 8.26 (dd, J ¼ 1.5, 7.8 Hz, 1ArH). ¹³C NMR

(100 MHz, CDCl₃):

d 18.5, 26.0, 27.0, 41.6, 62.4, 65.2, 113.8, 114.6,

116.0, 116.3, 116.7, 119.2, 120.1, 122.2, 123.5, 129.9, 130.5, 131.7, 135.8,

136.2, 139.1, 140.1, 151.2, 157.5, 162.1, 163.1, 166.4. HRMS (ESI^þ): m/z

calculated for C₂₉H₂₉N₂O₆[MþH]^þ: 501.2026; found: 501.2029.

NMR (300 MHz, CDCl₃):

d

0.88 (t, J ¼ 6.9 Hz, 3H), 1.26e1.32 (m,

22H), 1.43e1.46 (m, 2H), 1.80 (q, J ¼ 6.6 Hz, 2H), 2.01e2.02 (m, 2H),

2.23e2.32 (m, 2H), 4.00 (t, J ¼ 6.6 Hz, 2H), 4.37 (t, J ¼ 7.5 Hz, 2H),

4.45 (t, J ¼ 6.0 Hz, 2H), 5.35 (td, J ¼ 1.2, 7.8 Hz, 2H), 5.62 (bs, 1NH),

6.67 (t, J ¼ 2.1 Hz, 1ArH), 6.79 (qd, J ¼ 0.9, 7.8 Hz, 1ArH), 6.87e6.91

(m, 3ArH), 7.28e7.32 (m, 2ArH), 7.35 (t, J ¼ 7.8 Hz, 1ArH), 7.68 (td,

J ¼ 1.8, 7.5 Hz, 1ArH), 7.91e7.95 (m, 2ArH), 8.27 (dd, J ¼ 1.5, 7.8 Hz,

4.1.31. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl (E)-4-((3,7-dimethylocta-2,6-dien-1-yl)oxy)

benzoate (67)

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

stirring. Finally int_ꢀermediate 6 (0.137 g, 0.39 mmol) was added and

maintained at 70 C for 8 h. After completion of reaction, the re-

action mass was poured into water and extracted with ethyl acetate

(3 ꢁ 150 ml). Combined organic layer was thoroughly washed with

water followed by brine solution. The organic portion was then

evaporated and puriﬁed by column chromatography on silica gel

(100e200 mesh) using 20% ethyl acetate in hexanes to afford the

compound. Yield: 0.156 g, 70.6%, white wax, ¹H NMR (300 MHz,

1ArH). ¹³C NMR (100 MHz, CDCl₃):

d 14.3, 22.9, 26.2, 27.4, 29.5, 29.7,

29.7, 30.0, 32.1, 41.6, 62.3, 68.5, 113.8, 114.4, 115.9, 116.3, 116.7, 119.8,

122.0,123.5,129.9,130.0,130.2,130.5,131.7, 135.8,136.1, 140.1,151.2,

157.7, 162.2, 163.3, 165.5. HRMS (ESI^þ): m/z calculated for

C

₄₂H₅₅N₂O₆[MþH]^þ: 683.4060; found: 683.4061.

4.2. Drug susceptibility on replicating M. tuberculosis H37Rv by

MABA assay

The evaluation of MIC for the synthesized molecules against

replicating H37Rv was performed by MABA assay. Brieﬂy the bac-

teria were grown using the 7H12 media instead of the 7H9 media

supplemented with glycerol, casitone and OADC. Luciferase re-

porter strains of H37Rv were used for compounds with intrinsic

background ﬂuorescence and the intracellular ATP levels measured.

CDCl₃):

d 1.61 (s, 3H), 1.70 (s, 3H), 1.75 (s, 3H), 2.06e2.12 (m, 4H),

2.27 (q, J ¼ 6.0 Hz, 2H), 4.37 (t, J ¼ 7.8 Hz, 2H), 4.45 (t, J ¼ 6.0 Hz, 2H),

4.59 (d, J ¼ 6.3 Hz, 2H), 5.09e5.1 (m,1H), 5.48 (t, J ¼ 8.4 Hz,1H), 6.00

(bs, 1NH), 6.63e6.70 (m, 1ArH), 6.77 (d, J ¼ 7.5 Hz, 1ArH), 6.85e6.90

(m, 1ArH), 6.91e6.93 (m, 2ArH), 7.25e7.37 (m, 3ArH), 7.68 (td,

J ¼ 1.5, 7.2 Hz, 1ArH), 7.92e7.95 (m, 2ArH), 8.27 (dd, J ¼ 1.5, 7.8 Hz,

Cultures were incubated in 200 ml medium in 96-well plates for 7

1ArH). ¹³C NMR (100 MHz, CDCl₃):

d 17.0, 17.9, 25.3, 25.9, 26.5, 39.8,

days at 37 ^ꢀC. Alamar Blue and Tween 80 were added and incu-

bation was continued for 24 h at 37 ^ꢀC. Fluorescence was deter-

mined at excitation/emission wavelengths of 530/590 nm

respectively. The MIC was calculated as the lowest concentration

effecting a reduction in ﬂuorescence (or luminescence) of 90%

relative to controls. Isoniazid and rifampin were run as the refer-

ence drug.

62.3, 65.34, 113.8, 114.7, 116.0, 116.4, 116.9, 119.0, 120.4, 122.2, 123.4,

123.9,129.9,130.6,131.7,135.8, 136.2,139.2,140.1,151.4, 157.6,162.0,

163.4, 166.5. HRMS (ESI^þ): m/z calculated for C₃₄H₃₇N₂O₆[MþH]^þ:

569.2652; found: 569.2655.

4.1.32. Synthesis of 3-(3-((4-oxo-4H-benzo[d][1,3]oxazin-2-yl)

amino)phenoxy)propyl 4-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-

trien-1-yl)oxy)benzoate (68)

4.3. Drug susceptibility on non replicating M. tuberculosis H37Rv

by LORA assay

Intermediate 2 (0.1 g, 0.39 mmol) was dissolved in DMF and to

that potassium carbonate (0.162 g, 1.17 mmol) was added under

stirring. Finally int_ꢀermediate 7 (0.163 g, 0.39 mmol) was added and

maintained at 70 C for 8 h. After completion of reaction, the re-

action mass was poured into water and extracted with ethyl acetate

(3 ꢁ 150 ml). Combined organic layer was thoroughly washed with

water followed by brine solution. The organic portion was then

evaporated and puriﬁed by column chromatography on silica gel

(100e200 mesh) using 15% ethyl acetate in hexanes to afford the

compound. Yield: 0.158 g, 63.7%, white wax, ¹H NMR (300 MHz,

Low Oxygen Recovery Assay was used to determine the anti

mycobacterial efﬁciency of the synthesized molecule against the

non replicating bacilli. The strain used for LORA is a recombinant

H37Rv strain pFCA-LuxAB which makes direct luminescence mea-

surements to determine the MIC. Brieﬂy the strain was cultured in

7H12 media under humidiﬁed atmosphere (37 ^ꢀC, 5% CO₂). Cultures

were incubated in 200 ml medium in 96-well plates evacuated in an

anaerobic jar for producing hypoxic environment using anaerobic

gas mixture of 10% H₂, 5% CO₂and 85% N₂for 10 days at 37 ^ꢀC. Then

the culture was subjected to a recovery phase in 5% CO₂and 95%

humidity in a CO₂incubator. Then 1% n-decanal was added and

luminescence measured in a luminometer.

CDCl₃):

d 1.60 (s, 6H), 1.65 (s, 3H), 1.68 (s, 3H), 1.95e2.18 (m, 8H),

2.27 (q, J ¼ 6.0 Hz, 2H), 4.36 (t, J ¼ 7.5 Hz, 2H), 4.44 (t, J ¼ 6.0 Hz, 2H),

4.58 (d, J ¼ 6.6 Hz, 2H), 5.06e5.11 (m, 2H), 5.48 (t, J ¼ 6.3 Hz, 1H)

6.31 (bs, 1NH), 6.64 (t, J ¼ 2.1 Hz, 1ArH), 6.75 (dt, J ¼ 0.9, 7.8 Hz,

1ArH), 6.82e6.85 (m, 1ArH), 6.87e6.92 (m, 2ArH), 7.25e7.35 (m,

3ArH), 7.68 (td, J ¼ 1.5, 7.5 Hz, 1ArH), 7.93 (d, J ¼ 9.0 Hz, 2ArH), 8.26

4.4. Drug susceptibility on replicating M. tuberculosis H37Ra by

MABA assay

(dd, J ¼ 1.5, 6.3 Hz, 1ArH). ¹³C NMR (100 MHz, CDCl₃):

d 16.3, 17.0,

17.9, 25.9, 27.0, 29.9, 39.9, 41.6, 62.4, 65.3, 113.8, 114.6, 116.0, 119.0,

120.2, 122.2, 123.4, 123.8, 124.5, 129.9, 130.5, 131.7, 135.8, 136.3,

140.1, 151.2, 157.3, 162.1, 163.1, 166.4. HRMS (ESI^þ): m/z calculated

for C₃₉H₄₅N₂O₆[MþH]^þ: 637.3278; found: 637.3279.

The MIC evaluation of the synthesized molecules against repli-

cating H37Ra was performed by MABA assay. Brieﬂy the bacteria

were grown using the 7H9 media supplemented with OADC. at

14

A.B. Velappan, D. Kesamsetty, D. Datta et al.

European Journal of Medicinal Chemistry 208 (2020) 112835

37 ^ꢀC. For minimum inhibitory concentration (MIC), we prepared

different drug concentration in optically clear round bottom 96-

well plates. An equivalent volume of mid loge phase H37Ra cul-

ture (diluted to an optical density at 570 nm of 0.01 was added to

tedious. In order to avoid the difﬁculties we modiﬁed the methyl

esteriﬁcation protocol with benzyl and thereby the production of

UV active mycolic acid esters became visible on the TLC plate which

was then quantiﬁed using ImageJ software.

achieve a ﬁnal drug concentration range of 100e0.78

m

g/ml in 7H9

The H37Ra cultures were grown on 7H9 media supplemented

with OADC till the mid log phase. 5 ml cultures of OD₅₇₀¼ 0.5 were

incubated with the test molecules for 7 days at 37 ^ꢀC. The con-

centration of the molecules was used 5 times of their MIC value.

One control experiment and one set with DMSO treated experi-

ment were also carried out. After 7 days of incubation, the bacilli

were collected by centrifugation (3000 rpm for 10 min) followed by

washing with PBS buffer (3 times). Then the bacilli were subjected

to cell wall hydrolysis using 40% tetrabutyl ammonium hydroxide

solution (5 mL) at 100 ^ꢀC for overnight. Then dichloromethane was

broth, with a control. Plates were incubated in ambient air at 37 ^ꢀC

for 7 day. Thereafter, Alamar Blue and Tween 80 were added and

incubation was continued for 24 h at 37 ^ꢀC. Fluorescence was

determined at excitation/emission wavelengths of 530/590 nm

respectively. The MIC was calculated as the lowest concentration

effecting a reduction in ﬂuorescence (or luminescence) of 95%

relative to controls. Isoniazid and rifampin were run as the refer-

ence drug.

4.5. Drug susceptibility on M. Smegnatis mc²155 strain

added and stirred, to that 20 mL benzylbromide was added and

stirred further at room temperature for 3 h. The organic layer

collected from each experiment, dried and evaporated. Stock so-

M. smegmatis (mc²155) was cultured in Middlebrook 7H9 broth

supplemented with 10% (v/v) albuminedextrose complex and

0.03% (v/v) tween 80 at 37 ^ꢀC. For minimum inhibitory concen-

tration (MIC) testing, two fold serial dilutions of compounds were

lutions of the benzyl esters were made by dissolving them in 15

mL

of DCM. Exactly 5 L of the samples were used for TLC, and the TLC

m

plates were eluted 3 times with 3% ethyl acetate in hexanes. Finally

the images were recorded using a Nikon camera and the images

were quantiﬁed by using ImageJ app.

prepared in 100

100

l of mid logephase mc²155 culture (diluted to an optical

density at 570 nm of 0.01 was added to achieve a ﬁnal drug con-

ml in optically clear, round bottom 96-well plates.

m

centration range of 100e0.78

m

g/ml in 7H9 broth, with a control.

4.9. MenG inhibition assay

Plates were incubated in ambient air at 37 ^ꢀC for 48 h, at which

point MICs were recorded at the lowest concentration of compound

that prevented any visual growth.

The inhibitory activity of the synthesized molecules against

MenG was evaluated by a HPLC based assay by monitoring S-Ade-

nosyl Methionine (SAM) mediated methylation of Comp A (repre-

sentative molecule for DMMQ) in presence of the molecule under

4.6. Cytotoxicity study

investigation. In brief for 200

(20 l) was incubated with 5

and 0.1% CHAPS (20

l) in 100 mM Tris buffer (pH ¼ 8). To that

500 M SAM (10 l) was added along with the inhibitor molecule

with varing concentration (10 l) and the reaction was initiated by

adding 100 l of H37Ra membrane bound protein (obtained by

disrupting H37Ra cell using probe sonicator) and 100 l of M

smegmatis membrane bound protein separately. The mixture was

incubated for 3 h and then quenched by adding 0.1 M acetic acid in

methanol. The reaction mixture was then extracted with ether and

injected to the HPLC (Agilent) and quantiﬁed the conversion of

Comp A to Comp B. The same experiment was repeated with the

synthesized molecules in increasing concentrations (5, 10, 20 and

m

l assay mixture, 500

m

M Comp A

Cytotoxicity studies were performed against the PBMC cells

isolated from blood. The PBMC cells were propagated in RPMI

media in a humidiﬁed incubator (37 ^ꢀC, 5% CO₂). After scrapping the

cells with a cell scraper, they were collected by centrifugation

m

M (20 l) MgCl₂, 5 mM DTT (20

m

ml)

m

(1000 rpm for

5

min), re-suspended in fresh medium at

m

~1 ꢁ 10⁶cells/mL, dispensed into 96-well microplates (100

ml/well)

m

and incubated for 24 h at 37 ^ꢀC before being used for cytotoxicity

assays. Test compounds were subsequently added at concentra-

tions ranging from 400 to 0.2 mg/mL and incubation continued for

another 72 h before the cytopathic effects of compounds was

determined using the MTT Cell Proliferation Assay. The cytotoxic

IC₅₀deﬁned as the concentration causing 50% reduction in PBMC

cell viability, was obtained from a doseeresponse curve plotted

from percentage activity versus log₁₀concentration.

40

mg/ml) and the corresponding IC₅₀was determined from non-

linear regression analysis. We had used HyperClone 5

mm BDS

C18 130 Å column from Phenomenex (250 ꢁ 4.6 mm) with the

solvent system ACN/Water (90:10) with 0.05% TFA. The ﬂow rate

was maintained at 1 ml/min.

4.7. Preparation of the dormant H37Ra

To obtain the dormant H37Ra, we had taken 2 ꢁ 10⁶bacteria per

ml in a ﬂat bottom glass vial with 30 ml capacity (sample volume

28 ml). Thereafter, we sealed those glass vials (24 individual vials)

and incubated at 37 ^ꢀC for total 24 days with 25 rpm. We took 1 ml

bacterial culture from each vial for all the 24 days and measures the

OD₆₀₀and plated the same on agar plate (7H10 admixed with 5%

ADC) and calculated the CFU.

4.10. Vit-K2 rescue assay

The activity of Comp. 67 and 68 against non-replicating Mtb

(H37Ra) was determined by using resazurin reduction assay. The

effects of Vit-K2 supplementation were investigated using medium

supplemented with 1 mM Vit-K2 (as a control set). The growth was

measured by plating and counting the CFU from 0 to 10 days of

culture. Similarly, the growth in presence of comp. 67 and 68 (3 and

4.8. Inhibition of mycolic acid biosynthesis

30 mg/ml) was measured at 10th day with 1 mM Vit-K2 supple-

The inhibitory capability of these 1,3-Benzoxazi-4-one de-

rivatives was evaluated by comparing the production of the

different mycolic acid components of Mtb (H37Ra) bacilli in control

and drug treated culture of H37Ra. In all the available literature

methods, it was done by radio-labelled [1e¹⁴C] acetate assay or

converting the mycolic acid to its methyl esters or evaluating them

in TLC by PMA staining. However the use of scintillation counters

for radio-labelled assay or the PMA staining for TLC method are

mentation. In this case the cultures (incubated with 67 and 68

individually þ 1 mM Vit-K2) were grown for 10 days followed by 27

days after CFU counting on 7H10 agar plates.

4.11. Determination of the ATP production

H37Ra was cultured for 12 days (with 25 rpm at 37 ^ꢀC) in 7H9

broth till OD₆₀₀

¼

0.8. Thereafter, we incubated H37Ra

15

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Declaration of competing interest

There is no conﬂict of interest with others.

Acknowledgements

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16

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1,3-Oxazine-2-one derived dual-targeted molecules against replicating and non-replicating forms of Mycobacterium tuberculosis

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