Design, synthesis and biological evaluation of novel azaspiro analogs of linezolid as antibacterial and antitubercular agents

Article abstract of DOI:10.1016/j.ejmech.2016.07.001

The design, synthesis and antimicrobial evaluation of a novel series of azaspiro analogues of linezolid (1) have been described. Linezolid comprises of a morpholine ring which is known for its metabolism-related liabilities. Therefore, the key modificatio

Full text of DOI:10.1016/j.ejmech.2016.07.001

Accepted Manuscript
Design, synthesis and biological evaluation of novel azaspiro analogs of linezolid as
antibacterial and antitubercular agents
Pradip K. Gadekar, Abhijit Roychowdhury, Prashant S. Kharkar, Vijay Khedkar,
Manisha Arkile, Hardik Manek, Dhiman Sarkar, Rajiv Sharma, V. Vijayakumar, S.
Sarveswari
PII:
S0223-5234(16)30548-7
10.1016/j.ejmech.2016.07.001
EJMECH 8719
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 March 2016
Revised Date: 16 May 2016
Accepted Date: 2 July 2016
Please cite this article as: P.K. Gadekar, A. Roychowdhury, P.S. Kharkar, V. Khedkar, M. Arkile,
H. Manek, D. Sarkar, R. Sharma, V. Vijayakumar, S. Sarveswari, Design, synthesis and biological
evaluation of novel azaspiro analogs of linezolid as antibacterial and antitubercular agents, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.001.
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For Table of Contents Use Only
Design, Synthesis and Biological Evaluation of Novel Azaspiro Analogs of Linezolid as Antibacterial
and Antitubercular Agents
Pradip K. Gadekara, Abhijit Roychowdhurya, Prashant S. Kharkarb, Vijay Khedkarc ,d, Manisha Arkilec,Hardik Manekc, Dhiman Sarkarc, Rajiv Sharmaa,
V.Vijayakumare S. Sarveswarie*
aDepartment of Medicinal Chemistry, Piramal Enterprises Ltd., 1, Nirlon Complex, Goregaon (East), Mumbai –400063, bDepartment of Pharmaceutical
Chemistry, SPP School of Pharmacy and Technology Management, SVKM’s NMIMS, V. L. Mehta Road, Vile Parle (West), Mumbai – 400 056; cCombi
Chem-Bio Resource Centre, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune – 411 008. INDIA.
dSchool of Health Sciences, University of KwaZulu Natal, Westville Campus, Durban 4000, South Africa
eCenter for Organic and Medicinal Chemistry, School of Advanced Sciences, VIT University, Vellore – 632014, Tamil Nadu, INDIA.
_______________________________________________________________________________________________________________
IC50 = 0.8 µg/mL (MtbH37a Active)
IC50 = 0.71 µg/mL (MtbH37a Dormant)
IC50 = 1.56 – 16.34 µg/mL (MtbH37a Active)
IC50 = 2.07 – 22.83 µg/mL (MtbH37a Dormant)

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Design, synthesis and biological evaluation of novel azaspiro analogs of
linezolid as antibacterial and antitubercular agents
Pradip K. Gadekar,a Abhijit Roychowdhurya, Prashant S. Kharkarb, Vijay M. Khedkarc,d, Manisha Arkilec,
Hardik Manekc, Dhiman Sarkarc, Rajiv Sharmaa, V.Vijayakumare and S. Sarveswarie*
aDepartment of Medicinal Chemistry, Piramal Enterprises Ltd., 1, Nirlon Complex, Goregaon
(East), Mumbai –400063. INDIA.
bDepartment of Pharmaceutical Chemistry, SPP School of Pharmacy and Technology
Management, SVKM’s NMIMS, V. L. Mehta Road, Vile Parle (West), Mumbai – 400 056. INDIA.
cCombi Chem-Bio Resource Centre, Division of Organic Chemistry, CSIR-National Chemical
Laboratory, Dr. Homi Bhabha Road, Pune – 411 008. INDIA.
dSchool of Health Sciences, University of KwaZulu Natal, Westville Campus, Durban 4000,
South Africa
eCenter for Organic and Medicinal Chemistry, School of Advanced Sciences, VIT University,
Vellore – 632014, Tamil Nadu, INDIA.
† Electronic Supplementary Information (ESI) available: Spectral data
*Authors for correspondence
Dr. S. Sarveswari, M. Sc., M. Phil., Ph. D.,
Center for Organic and Medicinal Chemistry, School of Advanced Sciences, VIT University,
Vellore – 632014, Tamil Nadu, INDIA
*E-mail: sarveswari@gmail.com
Tel: +91-9042407099

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ABSTRACT
The design, synthesis and antimicrobial evaluation of a novel series of azaspiro analogues of
linezolid (1) have been described. Linezolid comprises of a morpholine ring which is known for
its metabolism-related liabilities. Therefore, the key modification made in the linezolid structure
was the replacement of morpholine moiety with its bioisostere, 2-oxa-6-azaspiro[3.3]heptane.
Furthermore, the replacement of N-acetyl terminal of 1 with various aromatic or aliphatic
functionalities was carried out. The title compounds were evaluated against a panel of Gram-
positive and Gram-negative bacteria and Mycobacterium tuberculosis. Subsequent structure-
activity relationship (SAR) studies identified several compounds with mixed antibacterial and
antitubercular profiles. Compound 22 (IC50 0.72, 0.51, 0.88, 0.49 µg/mL for E. coli, P.
aeruginosa, S. aureus, B. subtilis, respectively) exhibited similar antibacterial profile as 1. The
N-acetyl derivative 18 was similar to 1 in antitubercular profile. Thus, the present study
successfully demonstrated the use of azaspiro substructure in the medicinal chemistry of
antibacterial and antitubercular agents.
KEYWORDS
Azaspiro, MRSA (methicillin-resistant Staphylococcus aureus), bioisostere, morpholine,
antibacterial, antitubercular, VRE (vancomycin-resistant enterococci)

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1. Introduction
The bacterial resistance to established antimicrobial drugs has become a critical global problem.
Infections caused by multidrug-resistant Gram-positive cocci, such as methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and penicillin-
resistant Streptococcus pneumoniae (PNSSP), etc., have emerged as major public health concern
[1]. The number of effective antibiotics has reduced drastically, because microorganisms are able
to create new mechanisms of resistance and rapidly spread the genes encoding them via mobile
genetic elements, mostly plasmids and integrons [2]. Similarly, tuberculosis (TB) also remains a
major global health challenge and has been declared as public health emergency by the World
Health Organization (WHO) [3]. TB affects one-third of the world population. About 9 million
new cases and 1.1 million TB-related deaths were reported in 2013 [3]. Multidrug-resistant
strains of Mycobacterium tuberculosis (Mtb), the causative agent of TB (MDRTB) and
extremely drug-resistant TB (XDRTB) have emerged [4,5]. There is an extremely urgent need of
new antitubercular drugs which can tackle the TB menace.
As a prerequisite, newer antitubercular drugs should be active against the drug-resistant forms of
Mtb. This implies that they must act preferably on molecular targets different than those of the
current drugs. Importantly, such drugs need to be more active against persistent Mtb, which may
lead to further reduction in the treatment duration. The treatment of TB is complicated by the
tendency of its etiological agent, predominantly Mtb, to adopt a nonreplicating persistent state
[6-8].
Linezolid (1, Zyvox®, Figure 1), a newer generation synthetic antibiotic from oxazolidinone
class, was developed by a team at Pharmacia and Upjohn Company (now part of Pfizer) [9]. It
was approved in 2000 for the treatment of serious infections caused by Gram-positive bacteria
resistant to several other antibiotics such as MRSA, VRE (responsible for soft tissue infections
and PNSSN) [10]. Currently, 1 and related compounds are under clinical investigation for the
treatment of TB (Figure 1) [11]. Recently, cytoxazone-linezolid hybrids were shown to induce
apoptosis and senescence in DU145 prostate cancer cell line [12a].
The mechanism of action of 1 involves interaction with 50S A-site pocket at the peptidyl
transferase center (PTC) of the bacterial ribosome, which overlaps with the aminoacyl moiety of
an A-site bound tRNA, thereby inhibiting overall protein synthesis [12b,13]. However, in some

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cases, linezolid-resistant S. aureus and Enterococci were found among the hospital isolates
[14,15]. Toxic effects of linezolid on prolonged use include reversible myelosuppression leading
to anaemia, leucopaenia and thrombocytopaenia. In addition to this, 1 has been shown to inhibit
monoamine oxidase (MAO) enzymes, potentially leading to drug-drug interactions with
adrenergic and serotonergic agents [2]. Overall, newer and potent antibiotics are expected to
possess: i) extended spectrum of antibacterial activity covering fastidious Gram-negative
organisms; ii) activity against linezolid-resistant strains; iii) improved safety profile leading to
circumvention or at least minimization of myelosuppression and iv) activity against drug-
resistant forms of Mtb.
In the present investigation, bioisosteric modifications of 1 were tried in order to develop
potential new treatment against drug-resistant forms of bacteria, including Mtb, with reduced
toxicity profiles. Here, replacement of the morpholine ring in 1 with 2-oxa-6-
azaspiro[3.3]heptane was attempted. Morpholine is one of the common privileged structures
found in several drugs such as linezolid, timolol, gefitinib, moracizine, nimorazole, emorfazone,
etc. It is often used to raise aqueous solubility of drugs.
However, morpholine ring is regularly a target of the oxidative metabolism by non-cytochrome
P450 (CYP) enzyme systems. This is evident from the oxidative metabolites reported for at least
eight marketed drugs containing morpholine [16-18]. The 2-oxa-6-azaspiro[3.3]heptanes, a
bioisostere of morpholine (Figure 2) was selected for combating the oxidative metabolism issues
associated with morpholine. The azaspiro analogue of morpholine, 2-oxa-6-
azaspiro[3.3]heptanes, is better due to its improved chemical stability, lower lipophilicity, higher
solubility and metabolic robustness over morpholine [19]. The present study documents a
systematic structure-activity relationship (SAR) investigation of the azaspiro analogue of 1
(compound 18, Table 1, 2) leading to potent antibacterial and antitubercular agents.
<< Figure 1 >>
<< Figure 2 >>

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2 Results and discussion
2.1 Chemistry
The azaspiro intermediates 9 and 10 were synthesized by reported method [20] in good yield.
Condensation of 10 with 3,4-difluoronitrobenzene (11) under refluxing conditions in presence of
DIPEA in ACN led to compound 12 which was subjected to catalytic hydrogenation to yield
substituted aniline 13. It was further reacted with commercially available 14 to afford secondary
alcohol 15. Use of DIPEA in DMF at 120 °C resulted in moderate yield compared to other bases
and polar solvents (data not shown). Further, 15 was cyclized to oxazolidinone 16 using CDI,
followed by deprotection of the pthalimide-protected 5-methylamino group using hydrazine
hydrate to get alkylamine compound 17.
The systematic exploration of the SAR at the 5-methylamino group of 17 (amide, sulfonamide,
urea and thiourea derivatives) yielded a library of title compounds (see Supporting Information
section) on reaction with substituted acid chlorides, sulphonylchlorides, isocyanates and
thioisocyanates, respectively, at 0-5 °C for 2 hrs. The crude products were purified using flash
chromatography (2-5% MeOH in DCM) to afford title compounds 18-42 (Scheme 1).
Compound 45 was synthesized by condensing commercially available intermediates 43 and 44 at
100 °C in presence of 2M Na2CO3, PdCl2 (dppf)-CH2Cl2 adduct in 1,4-dioxane (Scheme 2).
Further, we have synthesized novel azaspiro compound 52 which was central spiro ring analogue
of 1 (Scheme 3). Compound 47 was synthesized from commercially available compound 46 by
reported method [21]. Compound 49 was synthesized in the presence of lithium perclorate in
ACN. Compound 49 was then cyclised in the presence of CDI to yield compound 50. The
compound 51 was obtained by debenzylation by palladium hydroxide. The acetylation was
carried out with Ac2O in the presence of THF and TEA to yield compound 52.
<< Scheme 1 >>
<< Scheme 2 >>
<< Scheme 3 >>
2.2 Biological evalutions
The present investigation explored the effect of replacement of oxidatively-labile morpholine
ring in 1 with bioisosteric azaspiro substructure (Figure 2). Further, the effect of such structural

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modification on the antibacterial and antitubercular activities of 1 was investigated. The azaspiro
analogue of 1 (compound 18) retained the antibacterial activity profile of 1 except complete
abrogation of anti-E. coli activity (Table 1 and Table S1, Supporting Information section).
Similarly, 18 retained the antitubercular activity of 1 (Table 2, Table S1, and Supporting
Information). Intrigued by these observations, the authors systematically studied the SAR of
compound 18 at the 5-methylamino substituent on the oxazolidinone ring. A total of three series
(amides, thioamides and sulphonamides) were designed, synthesized and tested. The protocols
and results of primary screening (antibacterial and antitubercular) are given in Supporting
Information section (Table S1). Tables 1 and 2 list MIC and IC50 (µg/mL) values of the title
compound which exhibited >75 % inhibition at 3 µg/mL concentration during primary screening
[22].
Careful inspection of the preliminary screening data showed the following activity trends. (Some
of the less active compounds and their details are given in the Supporting Information section -
Table 1S.) Compound 17 is an azaspiro analogue of compound 1 and was as a precursor to
compound 18. Compound 17 retained reasonable activity against E. coli and S. aureus but lost its
activity against other strains. Acetylating compound 17 resulted in compound 18, an exact
azaspiro analogue of compound 1. Compound 18 exhibited activity similar to 1 in all tested
strains except E. coli. Since Linezolid is an acetyl derivative, other acetyl compounds were
synthesized and tested (18, 19, 20, 21, 22, 35, 36 and 37). Generally it was observed that
acetylation of the free amine in compound 17 with bulky substituents (35, 36) led to complete
loss of activity. 2-Acetoxyacetyl substitution (21) or 2-methoxyacetyl substitution (37) led to
significant loss of both antibacterial and antitubercular activity. Upon acetylation with aromatic
substituents led to mixed results. Isoxazole substitution (22) led to encouraging activity trends
across various tested strains but pyridine (19) and CF3-substituted phenyl (20) moieties led to
significant loss of activity across various strains. Compound 19 retained good activity against E.
coli.
Replacing the amide bond in compound 1 with a sulphonamide linkage led to compounds 23, 24,
38, 39 and 40. This was an unfavourable transformation where all compounds exhibited loss of
activity except compound 23 which retained antibacterial activity.

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Different urea and thiourea derivatives of compound 17 were also evaluated. The urea
derivatives such as 25-27 and 41 exhibited loss of antibacterial activity and complete abrogation
of antitubercular activity. Compounds 28-34 and 42 (thiourea derivatives) were retained
antibacterial activity against two or more strains with complete abolition of antitubercular
activity. Compound 34 with polar 4-nitrophenyl substituent completely found to lose both the
activities. To compare urea and thiourea, two compounds (41, 42) were synthesized;
unfortunately both the compounds were found to be inactive in antibacterial and antitubercular
assays. Tables 1 and 2 listed the antibacterial and antitubercular activities (MIC and IC50 values
(µg/mL)) of the compounds showing >75% inhibition at 3 µg/mL concentration [23]. Compound
22 was found to be the best in the series with slightly reduced antitubercular activity compared to
1.
Majority of the compounds showed mixed antibacterial (Gram-positive versus Gram-negative)
profiles. Compound 18 exhibited slightly reduced activity in comparison to 1. A minor change
(morpholine in 1 to azaspiro morpholine in 18) led to marginal reduction in the activity. This
may be due to the linear conformation adopted by azaspiro morpholine as opposed to the chair
conformation of the morpholine ring. It may affect the interaction with its molecular target with
direct effect on the potency. Overall, the present study attempted replacement of oxidatively
labile morpholine ring in 1 with its azaspiro analogue and subsequent SAR investigations.
Among the compounds synthesized using Scheme 3, only compound 51 showed good activities
against E. coli while no activity was shown against other bacterial strains. Unfortunately, it was
also not exhibited antitubercular activity. Compound 52, central spiro analogue of linezolid (1),
was inactive in both antitubercular and antibacterial assays.
<<<<Table 1>>>>
<<<Table 2>>>
In silico ADMET predictions
Nowadays absorption, distribution, metabolism, excretion and toxicity (ADMET) data has got
significant importance in the drug discovery process since several studies have indicated that
approximately 40% of drug candidates fail in clinical trials owing to poor pharmacokinetics and
toxicity properties. These late-stage failures contribute significantly to the cost of new drug

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discovery projects. Therefore, ability to detect problematic candidates in the early stage of drug
discovery significantly reduces the risk of late-stage attrition in optimizing the screening and the
cost of new drug discovery campaign and focuses the lead optimization efforts to enhance the
desired ADMET profiles. Also considering the cost involved in the experiments to obtain the
ADMET data, the in silico approach provides a cost-effective alternative to filter and optimize
the leads in the early phase of drug discovery. With this objective, in silico ADMET predictions
were performed on the novel azaspiro analogs investigated in the present study to gauge their
pharmacokinetic, safety and drug-likeness profile using the QikProp module incorporated in
Small-Molecule Drug Discovery Suite (QikProp, version 4.6, Schrödinger, LLC, New York, NY,
2015). The prediction data is summarized in Table 3. In this study, we have evaluated molecular
weight, number of hydrogen bond acceptor and donor functional groups, logarithm of partition
coefficient (Log P), aqueous solubility, brain/blood partition coefficient, binding to human serum
albumin and % human oral absorption.
Oral bioavailability is often a desirable property for a drug candidate. The molecules investigated
herein have shown moderate-to-high oral bioavailability as reflected from the % human oral
absorption data with very low susceptibility to acid hydrolysis in the stomach. The partition
coefficient values (logPo/w) and prediction of binding to human serum albumin (logKhsa)
suggests that these molecules are hydrophilic in nature which is further confirmed by the logS
values suggesting that these molecules have adequate aqueous solubility. Drugs that act at the
central nervous system are expected to cross the blood-brain barrier (BBB) in order to reach their
site of action while those with peripheral site of actions are expected to have no brain penetration
to avoid related side effects. Low values predicted for brain/blood partition coefficient signify
that these molecules will have very low propensity to cross the BBB, thereby eliminating the
chances of CNS-related toxicity. All of these azaspiro analogs have favorable drug-like
properties and are potentially interesting for future lead optimization.
<<<Table 3>>>
3. Conclusions
The present study successfully attempted the replacement of oxidatively-labile morpholine ring
in a leading antibacterial drug, linezolid (1), with its azapiro analogue. This is an example of the

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successful use of the azaspiro and related substructures in the medicinal chemistry of
therapeutically useful compounds. Novel central azaspiro ring containing analogue of linezolid
was synthesized (compound 52). Further SAR investigations led to identification of several
mixed (Gram-positive and Gram-negative) antibacterial and antitubercular agents. The ADMET
predictions for these molecules were also found to be within tolerance limit qualifying them for
development of oral drug candidates. This study provides novel molecules for further exploration
in our quest for novel antibacterial and antitubercular agents. In our opinion, this is a valuable
investigation with significant impact on antibacterial and antitubercular drug development field.
4. Experimental Section
4.1 General.
Reagents and solvents were obtained from commercial suppliers, Sigma-Aldrich, USA and were
used as received unless otherwise indicated. Solvents were dried, wherever necessary, according
to standard procedures. All reactions were performed under an inert atmosphere (N2) unless
otherwise indicated. Analytical silica gel 60 F254-coated TLC plates were purchased from EMD
1
Chemicals, USA, and were visualized with UV light. The H-NMR spectra were routinely
obtained with a Varian Mercury Plus 300 MHz NMR and 400 MHz (Agilent Technologies,
Santa Clara, USA) and TMS was used as an internal standard. LC-MS spectra were recorded on
6110 AA Series Quadrupole LC/MS system (Agilent Technologies, Santa Clara, USA).
Analytical HPLC studies were carried out with a Kromasil® 100-5C18 column (150 mm X 4.6
mm) on PerkinElmer Series 200 HPLC system with auto sampler and PDA detector
(PerkinElmer, Inc., Waltham, USA). Melting points were recorded using a Veego (VMP)-D
capillary melting point apparatus (Veego Instruments Corp. Mumbai, India) and were
uncorrected.
4.2 Synthesis
4.2.1 Synthesis of 6-tosyl-2-oxa-6-azaspiro[3.3]heptane (9): To
a
solution of
benzenesulphonamide (8) (5.81 g, 36.9 mmol) in 500 mL of EtOH, 3-bromo-2,2-
bis(bromomethyl)propan-1-ol (9) (10 g, 30.8 mmol), KOH (5.53 g, 99 mmol) was added and
heated to reflux at 90 °C for 48 hrs. The solvent was evaporated and 100 mL of 1M KOH was
added and the resultant white suspension was stirred for another 2 hrs at RT. The mixture was

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filtered and the residue was rinsed with water until the filtrate was neutral. The filter cake was
dried under high vacuum to give crude product (4.5 g). Yield: 57.7 %; off-white solid; 1H-NMR
(300 MHz, DMSO-d6) δ (ppm): 7.69 (d, J = 8Hz, 2H, Ar-H), 7.49 (d, J = 8.1Hz, 2H, Ar-H),
4.42 (s, 4H, 2CH2, spiro morpholine), 3.84 (s, 4H, 2CH2, spiro morpholine), 2.42 (s, 3H, CH3);
LC-MS (ESI +ve) m/z 254 [M+H]+; HPLC purity: 99.79 %
4.2.2 Synthesis of of 2-oxa-6-azaspiro[3.3]heptane oxalate (10): To a solution of 9 (7.5 g, 29.6
mmol) in 500 mL MeOH, magnesium turnings (5.04 g, 207 mmol) were added and reaction mass
was sonicated for 1 hr. Then solvent was evaporated and 1 L of diethyl ether was added along
with sodium sulphate decahydrate (4.21 g, 29.6 mmol) and stirred for 1 hr at RT. The reaction
mixture was filtered through celite and dried over anhyd. Na2SO4 and then oxalic acid (1.33 g,
14.80 mmol) dissolved in 2 mL EtOH added and stirred for 5 min. to get oxalate salt of amine
1
(10) (1.5 g). Yield: 51.1% ; off-white solid; H-NMR (300 MHz, DMSO-d6) δ (ppm): 4.65 (s,
4H, 2CH2, spiro morpholine), 4.16 (s, 4H, 2CH2, spiro morpholine); LC-MS (ESI +ve) m/z 100.0
[M+H]+.
4.2.3 Synthesis of 6-(2-fluoro-4-nitrophenyl)-2-oxa-6-azaspiro[3.3] heptane (12): To a
solution of 11 (6.99 mL, 62.9 mmol) in 20 mL of ACN, were added 2-oxa-6-
azaspiro[3.3]heptane (1) (4 g, 27.6 mmol) and DIPEA (32.9 mL, 189 mmol) and the reaction
mixture heated to 90 °C overnight. The cooled to 0 - 5 °C and filtered to get the crude product (8
g). Yield: 53.4 %; yellow solid; 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 7.95 (d, J = 9Hz, 2H,
Ar-H), 6.59 (t, J = 9Hz, 1H, Ar-H), 4.7 (s, 4H, 2CH2, spiro morpholine ), 4.35 (s, 4H, 2CH2, spiro
morpholine); 13C-NMR (50 MHz, DMSO-d6) δ: 150.98, 144.20, 135.41, 122.01(2C), 111.94,
62.64(2C), 56.51(2C), 42.10; LC-MS (ESI +ve) m/z 239.1 [M+H]+; HPLC Purity: 98.88%.
4.2.4 Synthesis of 3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl) aniline (13). To a solution of
12 (5 g, 20.99 mmol) in 25 mL EtOH, Pd-C (2.23 g, 20.99 mmol) was added and reaction mass
was stirred for 4 hrs at 65-80 PSI at RT under H2 atmosphere Parr® shaker. The reaction was
monitored by TLC. After completion, the contents of the flask were filtered through celite and
concerntrated to get pure (13) (4 g). Yield: 92 %; brown solid; 1H-NMR (300 MHz, DMSO-d6)
δ (ppm): 6.31-6.23 (m, 3H, Ar-H), 4.75 (s, 4H, 2CH2, spiro morpholine), 3.81 (s, 4H, 2CH2,
spiro morpholine); 13C-NMR (50 MHz, DMSO-d6) δ: 155.18, 142.50, 129.60, 115.69, 109.85,

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102.28, 79.79(2C), 62.31(2C), 40.76; LC-MS (ESI +ve) m/z 209.1 [M+H]+; HPLC Purity: 99.73
%.
4.2.5 Synthesis of (R)-2-(3-((3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)amino)-2-
hydroxypropyl) isoindoline-1,3-dione (15): To a solution of 13 (2.95 g, 14.17 mmol), (S)2-
(oxiran-2-ylmethyl)isoindoline-1,3-dione (14) (5.4 g, 26.6 mmol) in 10 mL DMF, DIPEA 4.7
mL was added and reaction mass was heated at 120 °C overnight. The solvent was evaporated,
water was added and the mixture was extracted with DCM. It was purified using flash
chromatography (ethyl acetae: pet ether 40:60) to yield pure product (4 g). Yield: 54.9 %; brown
20
1
solid; m.p. 128-130 °C; [
α
]
= -16o (c = 0.1 , CHCl3); H-NMR (300 MHz, DMSO-d6) δ
D
(ppm): 7.85 (d, J = 3.6, 4H, Pthalimide ), 6.42-6.34 (m, 3H, Ar- H), 5.24-5.22 (m, 1H, CH-OH),
5.14 (d, J = 5.1Hz, 1H, NH ), 4.68 (s, 4H, 2CH2, spiro morpholine), 4.0-3.97 (m, 1H, OH ), 3.8
(s, 4H, 2CH2, spiro morpholine), 3.63-3.58 (m, 2H, CH2-pthalimide ), 3.05-3.02 (m, 1H, NHCH-
HOH), 2.94-2.92 (m, 1H, NHCH-HOH); 13C-NMR (50 MHz, DMSO-d6) δ: 168.05 (2C),
154.13, 143.13, 134.20 (2C), 131.79 (2C), 129.52, 122.87(2C), 115, 108, 100, 79.79(2C),
68.57(2C), 62.33(2C), 48.03, 42.39: LC-MS (ESI +ve) m/z 412.1 [M+H]+; HRMS (ESI +ve) m/z
[M+H]+ calcd for C22H22FN3O4: 412.1667, found 412.1672; HPLC Purity: 98.73 %.
4.2.6 Synthesis of (S)-2-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione (16). A reaction mixture containing 15 (4 g,
9.72 mmol), CDI (2.365 g, 14.58 mmol), DMAP (0.594 g, 4.86 mmol) in 10 mL THF was heated
at 60 °C overnight. After completion, the reaction mass was concentrated and water was added
followed by extraction in DCM. The organic layers were dried over sodium sulphate and
purification was done with the help of flash chromatography in ethyl acatete and pet ether
20
(40:60) to get pure product (2.6 g). Yield: 61.1 %; yellow soild; m.p. 203-205 °C; [
α
]
D = -84o
(c = 0.1 , CHCl3); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 7.92-7.84 (m, 4H, Ar-H), 7.32 (d, J
= 15 Hz, 1H, Ar- H), 7.0 (d, J = 8.4Hz, 1H, Ar- H), 6.60 (t, J = 9.3Hz, 1H, Ar- H), 4.8-4.69 (m,
1H, CH oxazolidone ring), 4.69 (s, 4H, 2CH2 spiro morpholine), 4.13 (t, J = 9Hz, 1H, CH2 of
oxazolidone ring), 4.0 (s, 4H, 2CH2 of spiro morpholine), 3.95-3.92 (m, 1H, CH2 of oxazolidone
ring ), 3.91-3.89 (m, 2H, CH2- pthalimide); 13C-NMR (100 MHz, DMSO-d6) δ: 167.77(2C),
153.81, 134.60(2C), 131.48(2C), 123.23 (4C), 114.84 (2), 107.12 (2), 79.71 (3C), 69.93,

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62.27(2C), 48.07, 40.47; LC-MS (ESI +ve) m/z 438.1 [M+H]+; HRMS (ESI +ve): m/z [M+H]+
calcd for C22H22FN3O4: 438.1460, found 438.1465; HPLC Purity: 92.07%.
4.2.7 Synthesis of (S)-5-(aminomethyl)-3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-
yl)phenyl)oxazolidin-2-one (17): To a solution of 16 (3 g, 6.86 mmol) in 10 mL EtOH,
hydrazine (1.722 mL, 54.9 mmol) was added and the reaction mixture refluxed for 2 hrs. The
solvent was evaporated and the residue washed with water and purified on flash chromatography
using DCM and MeOH (95:5) to get pure product (5 g). Yield: 71.2%; off-white solid; m.p.
20
118-120 °C; [
α
]
D = -96o (c = 0.1 , CHCl3); 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 7.42 (d, J
= 15Hz, 1H, Ar- H), 7.14 (d, J = 9Hz, 1H, Ar-H), 6.57 (d, J = 9.9Hz, 1H, Ar- H), 4.7 (s, 4H,
2CH2 spiro morpholine), 4.56- 4.54 (m, 1H, CH of oxazolidone), 3.98-3.95 (s, 5H, 2CH2 spiro
morpholine), 3.81 (t, J = 7.5Hz, 1H, CH2 of oxazolidone ring H), 2.81-2.77 (m, 2H,CH2 attached
to amine), 1.91-1.89 (m, 2H); LC-MS (ESI +ve) m/z 308.1 [M+H]+ ; HRMS (ESI +ve) m/z
[M+H]+ calcd for C15H18FN3O3: 308.1405, found 308.1395; HPLC Purity: 98.49 %.
4.2.8 Synthesis of (S)-N-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)acetamide (18): To a solution of 17 (100 mg, 0.325 mmol) in 5 mL
DCM, TEA (181 µL, 1.302 mmol) was added and the reaction mixture cooled to 0 °C, acetic
anhydride (36.8 µL, 0.390 mmol) was added which was previously cooled to 0 °C and then
stirred at RT for 1 hr. After completion, water was added and the reaction mixture extracted
with DCM. The combined organic layers were which was dried over sodium sulfate and the
solvent evaporated followed by the purification of the residue by flash chromatography using
EtOAc and pet ether (40:60) to get pure product (90 mg). Yield: 79%; off-white solid; m.p. 178-
20
1
180 °C; [
α
]
= - 8o (c = 0.1, CHCl3); H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.25 (t, J =
D
3.6Hz, 1H, amide), 7.38 ( d, J = 7.8Hz, 1H, Ar -H), 7.10 (d, J = 5.1Hz, 1H, Ar-H), 6.60 (d, J =
6Hz, 1H, Ar-H), 4.70-4.65 (m, 5H, CH2 spiro morpholine and CH2 oxazolidone ring), 4.05-4.02
(m, 5H, CH2 spiro morpholine and CH2 oxazolidone ring), 3.67-3.65 (m, 1H, CH2 oxazolidone
ring), 3.39 (t, J = 3Hz, 2H, CH2 attach to amide), 1.83 (s, 3H, methyl ); 13C-NMR (100 MHz,
DMSO-d6) δ: 169, 154, 152, 135, 130, (2)114, 106, (2C)79, 71, (2C) 62, 47, 40, 38, 22; LC-MS
(ESI +ve) m/z 350.1 [M+H]+; HRMS (ESI +ve) m/z [M+H]+ calcd for: C17H20FN3O4; 350.1511,
found 350.1482; HPLC Purity: 99.79 %.
4.2.9 Compounds 19, 20, 21, 22, 35, 36, 37 were synthesized using similar procedure for 18.

ACCEPTED MANUSCRIPT
4.2.10 Synthesis of (S)-N-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)nicotinamide (19): To a solution of 17 (100 mg, 0.325 mmol) in 5
mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled to 0 °C, nicotinoyl
chloride (87 mg, 0.488 mmol) was added and stirred for 2 hrs at RT. Work up was done similar
to compound (18) and purified by flash chromatography in DCM and MeOH (95:5) to get pure
product (76 mg). Yield: 56.6%; off-white solid; m.p. 176-178 °C; 1H-NMR (300 MHz, DMSO-
d6) δ (ppm): 9.0 (m, 2H), 8.72 (d, J = 4.5Hz, 1H), 8.18 (d, J = 8.1Hz, 1H), 7.53 (m, 1H), 7.39
(m, 1H), 7.12 (d, J = 8.4Hz, 1H), 6.60 (t, J = 9.3Hz, 1H), 4.82-4.80 (m, 1H), 4.7 (s, 4H), 4.1 (t, J
13
= 9Hz, 1H), 4.0 (s, 4H), 3.79-3.80 (m, 1H), 3.64-3.62 (m, 2H); C-NMR (50 MHz, CDCl3) δ :
166.68, 154.70, 152.22, 148.33(2C), 135.45(2C), 129.61(2C), 123.47(2C), 114.60, 107.45,
80.96(2C), 71.87, 62.73(2C), 48.12, 42.68, 39.78; LC-MS (ESI +ve) m/z 413.2 [M+H]+ ; HRMS
(ESI +ve) m/z [M+H]+ calcd for: C21H21FN4O4; 413.1620, found 413.1614; HPLC Purity: 99.29
%.
4.2.11 Synthesis of (S)-N-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)-4-(trifluoromethyl)benzamide (20): To a solution of 17 (100 mg,
0.325 mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled to 0
°C, 4-(trifluoromethyl)benzoyl chloride (88 mg, 0.423 mmol) was added and stirred for 2 hrs at
RT. Work up was done similar to compound (18) and purified by flash chromatography in DCM
and MeOH (95:5) to get pure product (70 mg). Yield: 44.88 %; off-white; m.p. 209-210 °C; 1H-
NMR (300 MHz, DMSO-d6) δ (ppm): 9.04-9.02 (m, 1H), 8.04 (d, J = 8.1Hz, 2H), 8.18 (d, J =
8.1Hz, 2H), 7.39-7.34 (m, 1H), 7.12-7.10 (m, 1H), 6.60 (t, J = 9.3Hz, 1H), 4.84-4.80 (m, 1H),
4.7 (s, 4H), 4.1 (t, J = 9Hz, 1H), 4.0 (s, 4H), 3.80 - 3.77 (m, 1H), 3.64-3.63 (m, 2H); 13C-NMR
(50 MHz, CDCl3): δ 166.54, 153.96, 136.79, 129.40, 127.56(5C), 4.67(2C),113.84(2C), 107.20,
106.73, 80.29(2C), 71.02, 62.17(2C), 47.64, 42.26, 39.50; LC-MS (ESI +ve) m/z 480.2 [M+H]+ ;
HRMS (ESI +ve) m/z [M+H]+ calcd for: C23H21FN3O4; 480.1541, found 480.1579; HPLC Purity:
97.77 %.
4.2.12 Synthesis of (S)-2-(((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)amino)-2-oxoethyl acetate (21): To a solution of 17 (100 mg, 0.325
mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) was added and the reaction mixture was
cooled to 0 °C, 2-chloro-2-oxoethyl acetate (66.6 mg, 0.488 mmol) was added and stirred for 2

ACCEPTED MANUSCRIPT
hrs at RT. Work up was done similar to compound (18) followed by purification using flash
chromatography in DCM and MeOH (95:5) to get pure product (69 mg). Yield: 52.1 %; off-
white solid; m.p. 103-105 °C; 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.40 (s, 1H), 7.39 (d, J
= 14.7Hz, 1H), 7.11 (d, J = 8.7Hz, 1H), 6.61 (t, J = 9.Hz, 1H), 4.70 (s, 4H), 4.46 (s, 2H), 4.22-
4.20 (m, 2H), 4.02 (s, 4H), 3.69-3.67 (m, 1H), 3.44-3.40 (m, 2H), 2.06 (s, 3H); HRMS (ESI
+ve) m/z [M+H]+ calcd for C19H22FN3O6; 408.1565, found 408.1549; HPLC Purity: 98.31%.
4.2.13 Synthesis of (S)-N-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)isoxazole-5-carboxamide (22): To a solution of 17 (100 mg, 0.325
mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled to 0 °C,
isoxazole-5-carbonyl chloride (47.2 µL, 0.488 mmol) was added and stirred for 2 hrs at RT.
Work up was done similar to compound (18) and purified by flash chromatography in DCM and
MeOH (95:5) to get pure product (80 mg). Yield: 61.1 %; pale yellow solid; m.p. 164-165 °C;
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.30 (t, J = 8Hz, 1H), 8.75 (d, J = 1.92Hz, 1H), 7.37
(dd, J = 2.4Hz, 1.64 Hz, 1H), 7.10 (d, J = 1.6Hz, 1H), 7.08 (d, J = 2.4Hz, 1H), 6.59 (t, J =
8.80Hz, 1H) 4.81-4.49 (m, 1H), 4.70 (s, 4H), 4.12 (t, J = 9Hz, 1H), 4.02 (s, 4H), 3.80-3.76 (m,
1H), 3.61-3.58 (m, 2H); HRMS (ESI +ve) m/z [M+H]+ calcd for C19H19FN4O5; 403.1412, found
403.1450; HPLC Purity: 92.36 %.
4.2.14
Synthesis
of
(R)-5-chloro-N-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-
yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thiophene-2-sulfonamide (23): To a solution of 17
(100 mg, 0.325 mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was
cooled to 0 °C, 5-chlorothiophene-2-sulfonyl chloride (106 mg, 0.488 mmol) was added and
stirred for 2 hrs at RT. Then water was added and extracted with DCM which was dried over
sodium sulfate. The DCM layer was concentrated and purified by flash chromatography in DCM
and MeOH (95:5) to get pure product (85 mg). Yield: 53.5 %; off-white solid; m.p. 98-100 °C;
1H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.50 (s, 1H), 7.52 (d, J = 4.2Hz, 1H), 7.39 (d, J =
2.1Hz , 1H), 7.34 (d, J = 2.1Hz, 1H), 7.09-7.06 (m, 1H), 6.61 (t, J = 10.2Hz, 1H), 4.70 (s, 5H),
4.02 (s, 5H), 3.71-3.66 (m, 1H), 3.22-3.19 (m, 2H); 13C-NMR (100 MHz, DMSO-d6) δ: 153.91,
152.41, 139.48, 135.54, 134.54, 131.58, 130.04, 127.99, 114.74(2C), 106.91, 79.70(2C), 70.88,
62.25(2C), 47.11, 45.36, 40.12; LC-MS (ESI +ve) m/z 488.1 [M+H]+; HRMS (ESI +ve) m/z
[M+H]+calcd for: C19H19ClFN3O5S2; 488.0511, found 488.0501; HPLC Purity: 99.29 %.

ACCEPTED MANUSCRIPT
4.2.15 Compounds 24, 38, 39, 40 were synthesized similar to 23
4.2.16 Synthesis of (R)-N-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)-3-(trifluoromethyl)benzenesulfonamide (24): To a solution of 17
(100 mg, 0.325 mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was
cooled to 0 °C, 3-(trifluoromethyl)benzene-1-sulfonyl chloride (79 µL, 0.488 mmol was added
and stirred for 2 hrs at RT. Work up and purification was done similar to 23 to get pure product
(76 mg). Yield: 45.32%; white solid; m.p. 98-101 °C; 1H-NMR (300 MHz, DMSO-d6) δ (ppm):
8.44 (d, J = 8.1Hz, 1H), 8.35 (s, 1H), 8.25 (d, J = 7.8Hz, 1H), 7.99 (t, J = 8.1Hz, 2H), 7.41-7.36
(m, 1H), 7.12-7.09 (m, 1H), 6.63 (t, J = 9.3Hz, 1H), 4.96-4.92 (m, 1H), 4.7 (s, 4H), 4.45-4.36
(m, 1H), 4.15-4.09 (m, 2H), 4.03 (s, 4H), 3.74-3.72 (m, 1H); LC-MS (ESI +ve) m/z 516.1
[M+H]+; HRMS (ESI+) m/z [M+H]+ calcd for: C22H21F4N3O5S; 515.1133, found 515.1109;
HPLC Purity: 98.31 %.
4.2.17 Synthesis of (S)-1-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)-3-(4-fluorophenyl)urea (25): To a solution of 17 (100 mg, 0.325
mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled to 0 °C, 4-
flurohenyl isocynate (54.9 µL, 0.488 mmol) was added and stirred for 2 hrs at RT. Then water
was added and extracted with DCM which was dried over sodium sulfate. The DCM layer was
concentrated and purified by flash chromatography in DCM and MeOH (95:5) to get pure
1
product (67 mg). Yield: 46.3%; off-white solid; m.p. 215-217°C; H-NMR (400 MHz, DMSO-
d6) δ (ppm): 8.60 (s, 1H), 7.38-7.34 (m , 3H), 7.11-7.02 (m, 3H), 6.58 (t, J = 9.48Hz, 1H), 6.47-
6.44 (t, J = 5.88, 1H) 4.74-4.735 (m, 1H), 4.69 (s, 4H), 4.08-4.065 (t, J = 9Hz, 1H), 4.01 (s, 4H),
3.72 (m, 1H), 3.45-3.43 (t, J = 8Hz, 2H); LC-MS (ESI +ve) m/z 445.2 [M+H]+; HPLC Purity:
99.55 %.
4.2.18 Compounds 26, 27 and 41 were synthesized similar to 25.
4.2.19 Synthesis of (S)-1-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)-3-(3-(trifluoromethyl)phenyl)urea (26): 17 (100 mg, 0.325 mmol)
in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled to 0 °C, 1-
isocyanato-3-(trifluoromethyl)benzene (91 mg, 0.488 mmol) was added and stirred for 2 hrs at
RT. Work up and purification was done similar to (25) to get pure product (75 mg). Yield: 46.6
1
%; white solid; m.p. 87-90 °C; H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.9 (s, 1H), 7.95 (s,

ACCEPTED MANUSCRIPT
1H), 7.47 (m, 1H), 7.37-7.35 (m, 2H), 7.25 (d, J = 6.3Hz, 1H), 7.13 (d, J = 8.4, 1H), 6.62-6.59
(m, 2H), 4.91-4.90 (m, 1H), 4.7 (s, 4H), 4.11-4.08 (m, 1H), 4.0 (s, 4H), 3.75 (t, J = 6.3Hz, 1H),
3.47-3.46 (m, 2H); 13C-NMR (100 MHz, DMSO-d6) δ: 155.25, 152.42, 150.04, 141.01, 135.51,
130.14, 129.76, 129.58, 129.26, 121.25, 117.50, 114.70, 114.60, 113.65, 106.94, 79.70(2C),
71.77, 62.24(2C), 47.90, 41.94, 39.91; HRMS (ESI+) m/z [M+H]+ calcd for: C23H22F4N4O4.;
495.4650, found 495.1629; HPLC Purity: 96.29 %.
4.2.20 Synthesis of (S)-1-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)-3-(2-fluorophenyl)urea (27): To a solution of 17 (100 mg, 0.325
mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled to 0 °C,
1-fluoro-2-isocyanatobenzene (66 mg, 0.4817. mmol) was added and stirred for 2 hrs at RT.
Work up and purification was done similar to (25) to get pure product (80 mg). Yield: 55.3 %;
white solid; m.p. 1483-205 °C; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.43 (s, 1H), 8.0 (t, J
= 1.6Hz , 1H), 7.40-7.38 (m, 1H), 7.17-6.91 ( m, 5H), 6.91 (t, J = 8.80Hz , 1H), 4.74 - 4.73 (m,
1H), 4.70 (s, 4H), 4.09 (t, J = 9Hz, 1H), 4.01 (s, 4H), 3.80-3.76 (m, 1H), 3.48-3.32 (m, 2H); 13C-
NMR (100 MHz, DMSO-d6) δ: 155.07, 154.113, 152.82, 150.42, 135.53, 130.12, 128.03,
124.35, 121.81, 120.20, 114.88, 114.69, 114.64, 106.97, 79.69(2C), 71.86, 62.24(2C), 47.33,
41.84, 40.13; HRMS (ESI+) m/z [M-H]- calcd for C22H22F2N4O4; 443.1525, found 443.1561;
HPLC Purity: 98.66 %
4.2.21 Synthesis of (S)-1-(4-cyanophenyl)-3-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-
yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thiourea (28): To a solution of 17 (100 mg, 0.325
mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled to 0 °C, 4-
isothiocyanatobenzonitrile (78 mg, 0.488 mmol) was added and stirred for 2 hrs at RT. Then
water was added and extracted with DCM which was dried over sodium sulfate. The DCM layer
was concentrated and purified by flash chromatography in DCM and MeOH (95:5) to get pure
product (70 mg). Yield: 47.9 %; off-white solid; m.p. 196-198 °C; 1H-NMR (300 MHz, DMSO-
d6) δ (ppm): 10.12 (s, 1H), 8.4 (s, 1H), 7.74 (s, 4H), 7.42 (d, J = 14.4Hz, 1H), 7.14 (d, 1H), 6.61
(t, J = 9H, 1H), 4.92-4.90 (m, 1H), 4.7 (s, 4H), 4.01 (s, 4H), 3.92-3.82 (m, 1H), 3.8 (s, 1H), 3.17
(d, J = 5.1Hz, 1H), 2.7 (d, J = 12.36Hz, 1H); 13C-NMR (100 MHz, DMSO- d6) δ : 180.97,
154.03, 152.41, 143.87, 135.56, 132.77(2C), 130.06, 121.56, 119.03, 114.66(2C), 107.2(2C),
105.10, 79.71(2C), 70.92, 62.27(2C), 47.40, 46.38, 40.12; HRMS (ESI+) m/z [M+H]+ calcd for:
C23H22FN5O3S; 468.1500, found 468.1474; HPLC Purity: 98.42 %.

ACCEPTED MANUSCRIPT
4.2.22 Compounds from 29 to 33 and 42 are synthesized similar to 28
4.2.23 Synthesis of (S)-1-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)-3-(3-(trifluoromethyl)phenyl)thiourea (29): To a solution of 17
(100 mg, 0.325 mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was
cooled to 0 °C, 1-isothiocyanato-3-(trifluoromethyl)benzene (99 mg, 0.488 mmol) was added
and stirred for 2 hrs at RT. Work up and purification was done similar to (28) to get pure product
(80 mg). Yield: 48.2 %; white solid; m.p. 180-182 °C; 1H-NMR (300 MHz, DMSO-d6) δ (ppm):
9.95 (s, 1H), 8.27 (s, 1H), 7.79 (s, 1H), 7.65-7.63 (m, 1H), 7.54-7.37 (m, 3H), 7.14 (d, J = 8.7,
1H), 6.59-6.56 (m, 1H), 4.92-4.90 (m, 1H), 4.7 (s, 4H), 4.10-4.09 (m, 1H), 4.0 (s, 4H), 3.90 (s,
13
1H), 3.82-3.80 (m, 1H), 3.17 (d, J = 5.1Hz, 1H); C-NMR (100 MHz, DMSO-d6) δ:181.47,
154.03, 152.42, 140.18, 135.57, 130.09, 129.61, 129.23, 126.44, 125.38, 122.67, 120.42, 119.03,
114.70, 107.03, 79.71(2C), 71.02, 62.26(2C), 47.40, 46.38, 40.28; HRMS (ESI+) m/z [M+H]+
calcd for C23H22F4N4O3S ; 511.1422, found 511.1429; HPLC Purity: 99.71 %.
4.2.24 Synthesis of (S)-1-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)-3-isopropylthiourea (30): To a solution of 17 (100 mg, 0.325
mmol) in 5 mL DCM, TEA (181 µL, 1.302 mmol) was added . Reaction mass was cooled to 0
°C, isopropyl Isocyanate (48.3 µL, 0.488 mmol) was added and stirred for 2 hrs at RT. Work up
and purification was done similar to (28) to get pure product (70 mg). Yield: 52.7 %; white solid;
m.p. 148-150 °C; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 7.51 (br, s, 2H), 7.39 (dd, J = 2.3Hz,
10.98Hz, 1H), 7.11 (dd, J = 1.6Hz, 8.6Hz, 1H), 6.59 (t, J = 8.9Hz, 1H), 4.80 (m, 1H), 4.70 (s,
4H), 4.20-4.18 (m, 1H), 4.06 (s, 5H), 3.79-3.77 (m, 3H); 1.09-1.05 (m, 6H); 13C-NMR (100
MHz, DMSO-d6) δ: 154.07, 152.39, 135.56, 130.09, 114.74(2C), 107.07(2C), 79.69(2C), 71.47,
62.25(2C), 47.27, 46.04, 45.07, 39.92, 22.16(2C); HRMS (ESI+) m/z [M+H]+ calcd for
C19H25FN4O3S; 409.1704, found 409.1752; HPLC Purity: 99.16 %.
4.2.25 Synthesis of (S)-1-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)-3-(4-methoxyphenyl)thiourea (31): To a solution of 17 (100 mg,
0.325 mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled to 0
°C, 1-isothiocyanato-4-methoxybenzene (69.9 mg, 0.423 mmol) was added and stirred for 2 hrs
at RT. Work up and purification was done similar to (28) to get pure product (79 mg). Yield:
1
51.4%; white solid; m.p. 176-178 °C; H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.50 (s, 1H),

ACCEPTED MANUSCRIPT
7.74 (s, 1H), 7.38 (dd, J = 3.72Hz, 14.64Hz, 1H), 7.17 (d, J = 8.84Hz, 2H), 7.11- 7.08 (m, 1H),
6.86 (d, J = 8.92Hz, 2H), 6.58-6.53 (m, 1H), 4.87-4.85 (m, 1H), 4.67 (s, 4H), 4.06 (t, J = 9Hz,
1H), 3.99 (s, 4H), 3.82-3.80 (m, 3H), 3.70 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ : 181.53,
156.68, 154.07, 152.41, 150.03, 135.54, 131.39, 130.12, 125.94, 114.67(2C), 113.30, 92(2C),
79.70(2C), 71.07, 62.26(2C), 55.21, 47.32, 46.53, 40.13; HRMS (ESI+) m/z [M-H]- calcd for
C23H25FN4O4S Na ; 471.1497, found 471.1495; HPLC Purity: 98.95 %.
4.2.26 Synthesis of (S)-1-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)-3-(2-fluorophenyl)thiourea (32): To a solution of 17 (100 mg,
0.325 mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled to 0
°C, 1-fluoro-2-isothiocyanatobenzene (74.8 mg, 0.488 mmol) was added and stirred for 2 hrs at
RT. Work up and purification was done similar to (28) to get pure product (75 mg). Yield: 50.1
1
%; off-white solid; m.p. 189-190 °C; H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.44 (s, 1H),
8.21 (s, 1H), 7.64 (m, 1H), 7.41 (dd, J = 4Hz, 14Hz, 1H), 7.26-7.22 (m, 2H), 7.17-7.16 (m, 2H),
6.60-6.5 (m, 1H), 4.90-4.80 (m , 1H), 4.70 (s, 4H), 4.10 (t, J = 9Hz, 1H), 4.01 (s, 4H), 3.87-3.82
(m, 2 H), 3.82-3.78 (m, 1H); HRMS (ESI+) m/z [M-H]- calcd for C22H22F2N4O3S ; 459.1297,
found 459.1297; HPLC Purity: 99.87 %.
4.2.27 Synthesis of (S)-1-(tert-butyl)-3-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-
yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thiourea (33): To a solution of 17 (100 mg, 0.325
mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction mass was cooled to 0
°C, 2-isothiocyanato-2-methylpropane (56.2 mg, 0.488 mmol) was added and stirred for 2 hrs at
RT. Work up and purification was done similar to (28) to get pure product (80 mg). Yield: 58.2
1
%; off-white solid; m.p. 155-158 °C; H-NMR (400 MHz, DMSO-d6) δ (ppm): 7.53 (t, J =
5.56Hz, 1H), 7.39 (dd, J = 2.36Hz, 14.64 Hz, 2H), 7.11 (dd, J = 2.04Hz, 8.6Hz, 1H), 6.59 (dd, J
= 8.84Hz, 10.12Hz, 1H), 4.80-4.77 (m , 1H), 4.70 (s, 4H), 4.06-4.01 (m, 5H), 4.02 (s, 9H), 3.80-
3.73 (m, 3H); 13C-NMR (50 MHz, DMSO d6) δ: 181.93, 154.09, 152.40, 135.57, 130.10, 114.76
(2C), 107.11, 79.70(2C), 71.65, 62.26(2C), 52.26, 47.25, 45.43, 40.13, 28.54(3C); HRMS (ESI
+ve) m/z [M-H]- calcd for C20H27FN4O3S ; 421.1704, found 421.1628; HPLC Purity: 99.72 %.
4.2.28 Synthesis of (S)-1-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)-3-(4-nitrophenyl)thiourea (34): To a solution of 17 (100 mg,
0.325 mmol) in 5 mL DCM, TEA (181 µL, 1.302 mmol) was added and reaction mass was

ACCEPTED MANUSCRIPT
cooled to 0 °C, 1-isothiocyanato-4-nitrobenzene (76 mg, 0.423 mmol) was added and stirred for
2 hrs at RT. Work up and purification was done similar to (28) to get pure product (87 mg).
Yield: 54.8 %; yellow solid; m.p. 200-202 °C; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 10.53
(s, 1H), 8.53 (s, 1H), 8.18 (d, J = 1.88Hz, 2H), (d , J = 2.92Hz, 2H), 7.42 (dd, J = 2.4Hz, 10.98
Hz, 1H), 7.14 (dd, J = 2Hz, 8.68Hz , 1H), 6.60 (dd, J = 8.9Hz, 10Hz, 1H), 4.94-4.90 (m, 1H),
4.70 (s, 4H), 4.13 (t, J = 9.0Hz, 1H), 4.01 (s, 4H), 3.93-3.89 (m, 2H), 3.82-3.80 (m, 1H), HRMS
(ESI+) m/z [M+Na]+ calcd for C22H22FN5O5S Na ; 510.1218, found 510.1271; HPLC Purity:
99.60%.
4.2.29 Synthesis of (1S,4R)-N-(((S)-3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-
2-oxooxazolidin-5-yl)methyl)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-
carboxamide (35): To a solution of 17 (100 mg, 0.325 mmol) in 5 mL of DCM, TEA (181 µL,
1.302 mmol) added and reaction was cooled to 0 °C, (1S,4R)-4,7,7-trimethyl-3-oxo-2-
oxabicyclo[2.2.1]heptane-1-carbonyl chloride (106 mg, 0.488 mmol) was added and stirred for 2
hrs at RT. Work up was done similar to compound (18) and purified by flash chromatography in
DCM and MeOH (95:5) to get pure product (50 mg). Yield: 31.5 %; white solid; m.p. 103-105
1
°C; H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.39 (t, J = 3.6Hz, 1H), 7.39 ( d, J = 14Hz, 1H),
7.08 (d, J =5.1Hz, 1H), 6.57 (d, J = 6Hz, 1H), 4.7 (s, 4H), 4.02 (s, 4H), 3.78 - 3.76 (m, 1H),
3.53-3.50 (m, 2H), 2.42-2.34 (m, 2H), 1.93-1.91 (m, 3H), 1.53-1.50 (m, 1H), 0.99 (s, 3H), 0.94
(s, 3H), 0.74 (s, 3H); LC-MS (ESI +ve) m/z 488.2 [M+H]+ ; HRMS (ESI +ve) m/z [M+H]+ calcd
for: C25H30FN3O6; 488.2191, found 488.2204; HPLC Purity: 98.44 %.
4.2.30 Synthesis of (S)-N-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)adamantane-1-carboxamide (36): To a solution of 17 (100 mg,
0.325 mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled to 0
°C, adamantane-1-carbonyl chloride (97 mg, 0.488 mmol was added and stirred for 2 hrs at RT.
Work up was done similar to compound (18) and purified by flash chromatography in DCM and
1
MeOH (95:5) to get pure product (90 mg). Yield: 58.9 %; white solid; m.p. 178-180 °C; H-
NMR (300 MHz, DMSO-d6) δ (ppm): 7.72 (t, J = 3.6Hz, 1H), 7.38 ( d, J = 14Hz, 1H), 7.09 (d,
J = 8.7Hz, 1H), 6.57 (d, J = 9.3Hz, 1H), 4.70 (s, 4H), 4.02 (s, 4H), 3.71-3.68 (m, 1H), 3.40-3.34
13
(m, 2H), 1.92 (s, 3H), 1.71-1.61 (m, 14H); C-NMR (100 MHz, DMSO-d6) δ: 177.77, 154.17,
153.20, 135.44, 135.33, 130.19, 114.71, 106.87, 79.70(2C), 71.21, 62.28(2C), 47.48, 41.57,

ACCEPTED MANUSCRIPT
40.14, 36.06(3C), 30.66(3C), 27.58(4C); LC-MS (ESI +ve) m/z 470.3 [M+H]+; HRMS (ESI +ve)
m/z [M+H]+ calcd for: C26H32FN3O4; 470.2450, found 470.2490; HPLC Purity: 99.14 %.
4.2.31 Synthesis of (S)-N-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)-2-methoxyacetamide (37): To a solution of 17 (100 mg, 0.325
mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) was added and reaction mass was cooled to
0 °C, 2-methoxyacetyl chloride (44.56 µL, 0.488 mmol) was added and stirred for 2 hrs at RT.
Work up was done similar to compound (18) and purified by flash chromatography in DCM and
1
MeOH (95:5) to get pure product (70 mg). Yield: 52.7 %; white solid; m.p. 140-142 °C; H-
NMR (400 MHz, DMSO-d6) δ (ppm): 8.14 (t, J = 6Hz, 1H), 7.38 (dd, J = 2.36Hz, 10.98Hz, 1H),
7.11 (dd, J = 1.6Hz, 8.6Hz, 1H), 6.59 (t, J = 8.9Hz, 1H), 4.72 (m, 5H), 4.06-6.55 (m, 5H), 3.82
(s, 2H), 3.75-3.41 (m, 1H), 3.45 (m, 2H), 3.27 (s, 3H); HRMS (ESI +ve) m/z [M+H]+ calcd for
C18H22FN3O5; 380.1616, found 380.1685; HPLC Purity: 97.87 %.
4.2.32 Synthesis of (R)-N-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)-2-
oxooxazolidin-5-yl)methyl)-1,2-dimethyl-1H-imidazole-4-sulfonamide (38): To a solution of
17 (100 mg, 0.325 mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was
cooled to 0 °C, 1,2-dimethyl-1H-imidazole-4-sulfonyl chloride (101 mg, 0.521 mmol) was added
and stirred for 2 hrs RT. Work up and purification was done similar to (23) to get pure product
1
(60 mg). Yield: 39.6 %; white solid; m.p. 182-184 °C; H-NMR (300 MHz, DMSO-d6) δ
(ppm): 7.81 (t, J = 3.6Hz, 1H), 7.64 (s, 1H), 7.33 (d, J = 8.4Hz, 1H ), 7.09 (d, J = 8.7Hz, 1H),
6.57 (d, J = 9.3Hz, 1H), 4.70 (s, 5H), 4.02 (s, 5H), 3.77-3.75 (m, 1H), 3.57 (s, 3H), 3.14-3.12 (m,
13
2H), 2.2 (s, 3H); C-NMR (100 MHz, DMSO- d6) δ: 153.99, 152.41, 146.57, 136.89, 135.51,
135.40, 124.56(2C), 114.74, 106.91, 79.71(2C), 71.07, 62.27(2C), 47.11, 45.16, 40.12, 32.82,
12.43; LC-MS (ESI +ve) m/z 466.1 [M+H]+; HRMS (ESI +ve) m/z [M+H]+ calcd for:
C20H24FN5O5S; 466.1555, found 466.1549; HPLC Purity: 99.14 %.
4.2.33 Synthesis of (R)-3,5-dichloro-N-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-
yl)phenyl)-2-oxooxazolidin-5-yl)methyl)benzenesulfonamide (39): To a solution of 17 (100
mg, 0.325 mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled
to 0 °C, 3,5-dichlorobenzene-1-sulfonyl chloride (120 mg, 0.488 mmol) was added and stirred
for 2 hrs at RT. Work up and purification was done similar to (23) to get pure product (80 mg).
1
Yield: 47 %; White solid; m.p. 105-107 °C; H-NMR (300 MHz, CDCl3) δ (ppm): 8.40 (s, 1H),

ACCEPTED MANUSCRIPT
7.97 (t, J = 2.1Hz, 1H), 7.80 (d, J = 1.8Hz, 2H), 7.38-7.33 (m, 1H), 7.07-7.05 (m, 1H), 6.61 (t, J
13
= 10.2Hz, 1H), 4.70 (s, 5H), 4.02 (s, 5H), 3.70-3.68 (m, 1H), 3.19-3.18 (m, 2H); C-NMR (50
MHz,CDCl3) δ 154.52, 149.54, 142.76, 136.11(2C), 132.77(2C), 129.16, 125.30 (3C), 114.89,
107.66, 81.04(2C), 71.18, 62.78(2C), 47.48, 45.41, 39.81; MS: m/z 516.1 [M+H]+; HRMS (ESI
+ve) m/z [M+H]+ calcd for: C21H20Cl2FN3O5S; 516.0558, found 516.0518; HPLC Purity: 99.29
%.
4.2.34
Synthesis
of
(R)-2-chloro-N-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-
yl)phenyl)-2-oxooxazolidin-5-yl)methyl)-5-nitrobenzenesulfonamide (40): To a solution of 17
(100 mg, 0.325 mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was
cooled to 0 °C, 2-chloro-5-nitrobenzene-1-sulfonyl chloride (108 mg, 0.423 mmol) was added
and stirred for 2 hrs at RT. Work up and purification was done similar to (23) to get pure product
(76 mg). Yield: 44.3 %; brown solid; m.p. 98-100 °C; 1H-NMR (400 MHz, DMSO-d6) δ (ppm):
8.81 (t, J = 8Hz, 1H), 8.62 (d, J = 2.72, 1H), 8.41 (dd, J = 2.72Hz, 8Hz, 1H), 7.89 (d, J = 8Hz,
1H), 7.29 (dd, J = 2.24Hz, 14.64 Hz, 1H), 7.0 (d, J = 10Hz, 1H), 6.57 (d, 8Hz, 1H), 4.70 (s, 4H),
4.63 - 4.58 (m, 1H), 4.02 (s, 4H), 4.0 (d, J = 8Hz, 1H), 3.65 - 3.63 (m, 1H), 3.87-3.32 (m, 2H);
13C-NMR (100 MHz, DMSO-d6) δ: 153.58, 152.34, 145.98, 139.40, 137.21, 135.45, 133.39,
129.97, 128.16, 124.96, 114.66, 114.40, 106.72, 49.70(2C), 71.01, 62.24(2C), 46.88, 45.11,
40.12; HRMS (ESI +ve) m/z [M-H]- calcd for C21H20ClFN4O7S; 525.0642, found 525.0646;
HPLC Purity: 98.49 %.
4.2.35 Synthesis of (S)-1-(4-chlorophenyl)-3-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-
yl)phenyl)-2-oxooxazolidin-5-yl)methyl)urea (41): To a solution of 17 (100 mg, 0.325 mmol)
in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled to 0 °C, 1-chloro-
4-isocyanatobenzene (75.0 mg, 0.488 mmol) was added and stirred for 2 hrs at RT. Work up and
purification was done similar to (25) to get pure product (80 mg). Yield: 53.3 %; off-white solid;
1
m.p. 218-220 °C; H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.72 (s, 1H), 8.74-7.35 (m, 5H),
7.11 (dd, J = 1.9Hz, 8.7Hz, 1H), 6.58-6.50 (m, 2H), 4.75-4.73 (m, 1H), 4.69 (s, 4H), 4.08 (t, J =
18 Hz, 1H), 4.01-4.00 (d, J = 1.6Hz , 4H), 3.72-3.68 (m, 1H), 3.46 (t, J = 5.5Hz, 2H); HRMS
(ESI +ve) m/z [M+H]+ calcd for C22H22ClFN4O4; 461.1431, found 461.1404; HPLC Purity:
98.31 %.

ACCEPTED MANUSCRIPT
4.2.36 Synthesis of (S)-1-(4-chlorophenyl)-3-((3-(3-fluoro-4-(2-oxa-6-azaspiro[3.3]heptan-6-
yl)phenyl)-2-oxooxazolidin-5-yl)methyl)thiourea (42): To a solution of 17 (100 mg, 0.325
mmol) in 5 mL of DCM, TEA (181 µL, 1.302 mmol) added and reaction was cooled to 0 °C, 1-
chloro-4-isothiocyanatobenzene (83 mg, 0.488 mmol) was added and stirred for 2 hrs at RT.
Work up and purification was done similar to (28) to get pure product (80 mg). Yield: 51.5 %;
1
white solid; m.p. 200-202 °C; H-NMR (300 MHz, DMSO-d6) δ (ppm): 9.78 (s, 1H), 8.11 (s,
1H), 7.46 (d, J = 8.7Hz, 1H), 7.36 (d, J = 8.7Hz, 2H), 7.14 (d, J = 8.7Hz, 2H), 6.62 (t, J =
9.3Hz, 1H), 6.62-6.58 (m, 1H), 4.91-4.90 (m, 1H), 4.70 (s, 4H), 4.09-4.06 (m, 1H), 4.0 (s, 4H),
3.88-3.82 (m, 2H), 3.18 (d, J = 5.1Hz, 1H); 13C-NMR (50 MHz, DMSO-d6) δ: 181.36, 154.06,
153.61, 138.08, 135.63, 135.41, 130.16(4C), 124.85, 114.67(2C), 107.14, 79.72(2C), 71.05,
62.29(2C), 47.39, 46.45, 42.10; HRMS (ESI +ve) m/z [M+H]+ calcd for C22H22ClFN4O3S;
477.1158, found 477.1172; HPLC Purity: 98.66 %.
4.2.37 Synthesis of (1R,5S)-tert-butyl 3-(4-((S)-5-(acetamidomethyl)-2-oxooxazolidin-3-yl)-
2-fluorophenyl)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate (45): To a solution of 43 (100 mg,
0.302 mmol) in 5mL of dioxane and 1 mL of water, added ((1R,5S)-8-(tert-butoxycarbonyl)-8-
azabicyclo[3.2.1]oct-2-en-3-yl)boronic acid(42) (99 mg, 0.393 mmol), Added sodium carbonate
(112 mg, 1.057 mmol), Argon gas was purged for 20 minutes. Then added PdCl2 (dppf)-CH2Cl2
adduct (12.33 mg, 0.015 mmol). Again argon gas was purged into reaction mass. Heated the
reaction mass for 100 °C for overnight. Then concentrated the reaction mass. Product was
dissolved into DCM. Saturated sodium bicarbonate solution washing was given to organic layer
followed by water washing was given. The compound was purified by flash chromatography in
DCM and MeOH (95:5) to get pure product (60 mg). Yield: 43.2 %; off-white solid; m.p. 155-
1
158 °C; H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.25 (t, J = 5.56Hz, 1H), 7.48 (d, J = 14Hz,
1H), 7.33-7.27 (m, 2H), 6.32 (s, 1H), 4.71-4.70 (m , 1H), 4.35-4.30 (m, 2H), 4.10 (t, J = 9Hz,
1H), 3.74-3.72 (m, 1H), 3.40 (t, J = 5.4Hz, 2H) 2.92-2.90 (m, 1H), 2.19-2.13 (m , 2H), 1.92-1.91
13
(m, 2H), 1.82 (s, 3H), 1.72-1.70 (m, 1H), 1.39 (s, 9H); C-NMR (50 MHz, CDCl3) δ: 171.23,
162.57, 157.64, 154.17, 138.04, 132.54, 129.67, 124.29, 113.15(2C), 106.43, 79.43, 72.00(2C),
47.38(2C), 41.84(2C), 37.28, 34.29, 28.36(3C), 22.99; LC-MS (ESI +ve) m/z 460.2[M+H]+ ;
HRMS (ESI +ve) m/z [M+H]+ calcd for C24H30FN3O5; 460.2242, found 460.2201; HPLC Purity:
99.32 %.

ACCEPTED MANUSCRIPT
4.2.38 Synthesis of 5-benzyl-1-oxa-5-azaspiro[2.4]heptanes (47): To a solution of
trimethylsulfoxonium iodide (7.4 g) was added to a suspension of NaH (0.81 g) in anhydrous
DMSO (25 mL) at 10 to 15 °C over twenty minutes, followed by stirring at room temperature
under nitrogen atmosphere for 1 hr. A solution of 46 (4.9 g) (commercial available) in anhydrous
DMSO (25 mL) was added thereto at 10 to 15 °C over twenty minutes, followed by stirring at 10
to 24 °C for one hr. Water (50 mL) and EtOAc (50 mL) were added thereto under cooling with
ice-water, and the mixture was separated. The product was extracted with EtOAc (50 mL x 2)
and was combined with the former organic layer. The combined organic layer was sequentially
washed with water (30 mL x 3) and brine (50 mL x 1) and was dried over anhydrous sodium
sulfate. The solvent was removed under reduced pressure, the residue was purified by silica gel
column chromatography (eluent; hexane: EtOAc = 1:1) to yield the title compound (2.25g ) as an
1
oil. Yield: 44.4%; brown semi solid; H-NMR (300 MHz, CDCl3) δ (ppm): 7.34-7.32 (m, 5H),
3.72 (q, J = 12.3Hz, 2H), 2.82-2.80 (m, 2H), 2.63-2.56 (m, 2H), 2.26-2.17 (m, 2H), 1.95-1.88
(m, 2H); LC- MS(ESI +ve) m/z 190 [M+H]+ ; HPLC Purity: 99.39%.
4.2.39 Synthesis of 1-benzyl-3-(((3-fluoro-4-morpholinophenyl)amino)methyl) pyrrolidin-3-
ol (49): To a solution of 47 (601.5 mg, 3.18 mmol) in 10 mL of ACN, added the lithium
perchlorate (676 mg, 6.36 mmol), added 48 (686 mg, 3.50 mmol) and heated the reaction mass
for 90 °C overnight. Then concentrated the reaction mass and purification was done in DCM and
1
MeOH to give the title compound (700 mg). Yield: 57.1%; brown semi solid; H-NMR (300
MHz, CDCl3) δ (ppm): 7.32 (s, 5H), 6.82 (t, J = 9Hz, 1H), 6.48 (d, J = 2.1Hz,1H), 6.34 (d, J =
8.4Hz, 1H), 5.36 (br s, 1H), 4.9 (br s, 1H), 4.0 (m, 1H), 3.69-3.66 (t, J = 4.2 Hz , 5H), 3.16 (d, J
= 5.1Hz, 1H), 3.01 (s, 2H), 2.81-2.72 (t, J = 4.5Hz, 6H), 1.98-1.47 (m , 3H); 13C-NMR (100
MHz, DMSO-d6) δ: 157.60, 146.40, 138.94, 128.86, 128.47(3C), 126.76(2C), 120.41, 107.60,
100.19, 79.16, 66.38(3C), 64.73, 59.63, 52.87, 52.78(2C), 30.66; LC-MS (ESI +ve) m/z 386.2
[M+H]+; HRMS (ESI +ve) m/z [M+H]+ calcd for C22H28FN3O2 : 386.2238, found 386.2242;
HPLC Purity: 98.68%
4.2.40 Synthesis of 7-benzyl-3-(3-fluoro-4-morpholinophenyl)-1-oxa-3,7-diazaspiro[4.4]
nonan-2-one (50): To a solution of 49 (500 mg, 1.297 mmol), added di(1H-imidazol-1-
yl)methanone (421 mg, 2.59 mmol), added 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine
(494 mg, 3.24 mmol). After stirring at 80° C. overnight, the solvent was evaporated under
reduced pressure. The residue was dissolved in EtOAc, and the solution was washed twice with

ACCEPTED MANUSCRIPT
aqueous sodium hydrogen carbonate solution. The organic layer was dried over sodium
sulphate, and the solvent was evaporated under reduced pressure. The residue was purified using
flash chromatography using EtOAc and pet ether to give the title compound (400 mg). Yield:
47.9%; off-white solid, m.p. 148-150 °C; 1H-NMR (300 MHz, CDCl3) δ (ppm): 7.4 (s. 1H), 7.43
(s, 1H), 7.32 (s, 3H), 7.25 (m, 1H), 7.19-7.16 (d, J = 9Hz, 1H), 7.07-7.01 (t, J = 9.6Hz, 1H), 4.0
(s, 2H), 3.7 (s, 3H), 3.6 ( s, 2H), 2.9 (s, 4H), 2.85 (s, 1H), 2.74-2.6 (m, 3H), 2.54 (s, 1H), 2.16
(m, 1H), 1.26 (d, J = 10.8Hz, 1H), 13C-NMR (100 MHz, DMDO-d6) δ: 155.74, 153.55, 138.49,
133.47, 133.37, 128.49(3C), 126.93(2C), 119.19, 114.25, 106.95, 83.98, 66.13(2C), 64.25, 58.98,
54.58, 50.70, 50.67, 37.79, 30.66; LC-MS (ESI +ve) m/z 412.7 [M+H]+; HRMS (ESI +ve) m/z
[M+H]+ calcd for C23H26FN3O3: 412.2038, found 412.2031; HPLC Purity: 98.59%.
4.2.41 Synthesis of 3-(3-fluoro-4-morpholinophenyl)-1-oxa-3,7-diazaspiro[4.4]nonan-2-one
(51): To a solution of 50 (1.5 g, 3.65 mmol), In MeOH, added palladium hydroxide on carbon
(0.358 g, 2.55 mmol). Stirred the reaction mass overnight at rt at hydrogen atmosphere by using
hydrogen balloon. Then filtered the reaction mass and concentrated the reaction mixture. For
further purification was done in DCM and MeOH by using flash chromatography to yield the
title compound (600 mg) Yield: 51.2%; brown solid, m.p. 120-122 °C; 1H-NMR (300 MHz,
CDCl3) δ (ppm): 7.51 (d, d, J = 1.2Hz, 1.2Hz, 1H), 7.21 (d, d, J = 1.2Hz, 1.2Hz, 1H), 7.09 (t, J =
5.7Hz, 1H), 4.10 (q, J = 5.4Hz, 12.3Hz, 2H), 3.74 (m , 4H), 3.14 (d, J = 2Hz, 1H), 3.0-2.93 (m,
7H), 2.17 (m, 1H), 1.99 (m, 1H), 1.90 (s, 1H); 13C-NMR (300 MHz, DMSO-d6) δ: 155.78,
153.66, 135.15, 121.62, 120.27, 119.15, 106.62, 85.95, 66.12(2C), 57.69, 52.53, 50.66(2C),
45.41, 30.60; LC-MS (ESI +ve) m/z 322.3 [M+H]+; HRMS (ESI +ve) m/z [M+H]+ calcd for
C16H20FN3O3: 322.1555, found 322.1561; HPLC Purity: 98.70%.
4.2.42
Synthesis
of
7-acetyl-3-(3-fluoro-4-morpholinophenyl)-1-oxa-3,7-diazaspiro
[4.4]nonan-2-one (52): To a solution of 51 (40 mg, 0.124 mmol) in 5 mL of THF, added the
TEA (52.0 µL, 0.373 mmol), acetic anhydride (23.53 µL, 0.249 mmol) at 0° C, then stirred the
reaction mass for rt for 1h. Then concentrated the reaction mass and water washing was given,
extracted the product with EtOAc and purification was done with DCM and MeOH using flash
chromatography to yield the title compound (30 mg) Yield: 66.3%, off-white solid; m.p. 83-85
1
°C; H-NMR (400 MHz, CDCl3) δ (ppm): 7.51(d, J =14.8Hz, 1H, Ar-H), 7.20(m, 1H),7.09-
7.052(t, J = 9.6, 1H, Ar-H),4.15-4.05(m, 2H), 3.90(d, J = 12H, 1H), 3.79-3.73(m, 5H), 3.57(m,
1H), 3.45(d, J = 13.2Hz, 1H), 2.96(s, 4H), 2.37-2.12(m, 2H), 1.97(d, J = 13.2Hz, 3H), LC-MS

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(ESI +ve) m/z 364.3 [M+H]+ ; HRMS (ESI +ve) m/z [M+H]+ calcd for C18H22FN3O4: 364.1720,
found 364.1667; HPLC Purity: 99.78%;
4.3 Biological evaluation
4.3.1 General
For biological activity, stock solutions of the compounds were prepared in DMSO (10 mg/mL)
and were further used for testing antibacterial activity. Bacterial strains E. coli (NCIM 2688), P.
aeruginosa (NCIM 2036) as Gram-negative and B. subtilus (NCIM 2079), S. aureus (NCIM
2010) as Gram-positive were obtained from NCIM (NCL, Pune) and were grown in Luria
Burtony medi-um from Himedia, India. Antibacterial activity. Bacterial strains were grown to
logarithmic phase (O.D. 1.0 at 620 nm) in a LB medium. Test compound solutions were added
into 96-well plate. DMSO (1%) was used as vehicle control. Initially the compound
concentrations of 30, 10 and 3 µg/mL were used to check the antibacterial activity. The
compounds showing inhibition above 75% for 3 µg/mL concentration were selected for further
analysis by dose-response curve. Dose-response curves were generated by using half dilution
from 100 µg/mL upto 0.78 µg/mL. The OD was measured by Spectramax spectrophotometer at
620 nm.
4.3.2 Antibacterial activity
All bacterial cultures were first grown in LB media at 37 °C at 180 RPM. The assay was
performed in 96-well plates after 8 and 12 hrs. for Gram-negative and Gram-positive bacteria,
respectively. A 0.1 % suspension of OD 1 (620 nm) culture was used for screening. The
inoculated culture was added into each well of 96-well plate containing the compounds to be
tested. Optical density for each plate was measured at 620 nm after 8 hrs for Gram-negative
bacteria and after 12 hrs for Gram-positive bacteria. For antibacterial assay, 2.5 µL of test
compound solution was added in each of the well of 96-well plates, except control and blank
followed by 247.5 µL of inoculated culture to each well except blank to make the final volume
of 250 µL. DMSO (2.5 µL) was used as vehicle control in the blank and control. Sterile media
(248 µL) was added into blank well.
4.3.3 Antimycobacterial activity

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Microbial strains such as Mycobacterium tuberculosis H37Ra (ATCC 25177) and M. bovis BCG
(ATCC 35734) were obtained from AstraZeneca, India. The stock culture was maintained at
-80 oC and subcultured once in a liquid medium before inoculation into an experimental culture.
Cultures were grown in Dubos media (enrichment media). For antimycobacterial assay, M. pheli
medium (minimal essential medium) was used. It contains 0.5 g KH2PO4, 0.25 g trisodium
citrate, 60 mg MgSO4, 0.5 g aspargine and 2 mL glycerol in distilled water (100 mL) followed
by pH adjustment to 6.6.
Cultivation of mycobacteria and antimycobacterial assay. All bacterial stock cultures were first
grown in Dubos media at 37 oC at 150 RPM. It takes at least 8-10 days for OD 1 at 620 nm. The
antimycobacterial assay was performed in 96-well plates for active as well as dormant stages.For
antimycobacterial assay, 1 % of OD 1 culture at 620 nm was used. A total of 2.5 µL of test
compound solution was added into 96-well plates, except control and blank, followed by 250 µL
of inoculated culture to each well except blank. DMSO was used as vehicle control in blank and
control. A total of 250 µL sterile media was added into the blank well. Sterile sealer was kept on
it to achieve dormant condition. After 8 days, all oxygen was used by bacteria and hypoxic
conditions were obtained, resulting in mycobacteria shifting to the dormant stage. XTT (for Mtb)
as well as NR (for M. bovis BCG) assays were performed on 8th day for active stage inhibitor and
on 12th day for dormant stage inhibitor. Primary screening was done at three concentrations 30,
10, 3 µg/mL. Those compounds showing > 80% inhibition at 3 µg/mL concentration were
selected further for generating dose-response curves using half dilution from 100 µg/mL up to
0.78 µg/mL.
4.3.4 XTT assay
For Mtb, XTT assay was performed [22]. For active stage inhibitor, the plate was removed after
8 days. XTT sodium salt powder was used for preparing 1.25 mM stock solution in sterile 1X
PBS and Menadione (Sigma) was prepared as a 6 mM solution in DMSO and was used
immediately. XTT solution was added to make the final concentration to 200 µM. Plates were
o
kept in 37 C incubator for 20 min. Then menadione solution was added to make final
concentration of 60 µM. The plates were incubated for 40 min in the incubator. The OD was
measured at 470 nm. For dormant stage inhibitor, plates were removed after 12 days. All other
procedures were same.

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4.3.5 Nitrate Reductase assay
For M. bovis BCG, NR assay was performed using 1% sulfalic acid in 20% HCl and 1% NEED
(1-naphthyl ethylenediamine dihydrochloride) in water. After 8th and 12th days, the plates were
removed for active and dormant stage inhibitors, respectively. A solution containing one part
each of culture from assay plate, sulfanilic acid solution and NEED solution was prepared.
Purple colour prontosil produced was measured at 540 nm.
Percent inhibition was measured as -
% Inhibition = Comp. OD – Blank
Cont OD – Blank
Acknowledgement
PG thanks Niteshkumar Sahu, Chetan Shah and Ms. Sona Warrier, PhD Students from PSK’s
Research Group at SPPSTM, SVKM’s NMIMS, Mumbai, for their help during preparation of
this manuscript. Authors are also thankful to Schrödinger Inc. for providing the Demo license of
Schrödinger Molecular Modeling Suite and especially Craig Coel for his valuable support that
has tremendously helped in this work. Authors also thankful to SIF VIT University for providing
NMR facility.
Supplementary material
1
Experimental details of synthesis and characterization of representative compounds using H-
NMR, 13C-NMR, Mass spectrometry, HPLC Purity and HRMS are provided.
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20 initiative: pursuing a global commitment to develop 10 new antibacterial drugs by 2020,
Clin. Infect. Dis. 50 (2010), 2081-2083.

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