CLIAMS:WE CLAIM:
1. A novel oxazolidinone antibacterial compound [(S)- N- [[3-[4-fluoro-3-morpholino phenyl]-2-oxooxazolidin-5-yl] methyl] acetamide] of formula –I for prophylaxis and treatment of a disease caused in a subject by gram positive and gram negative pathogens

Compound-I

2. The novel oxazolidinone compound as claimed in claim-1 characterized by IR spectral data: 3335,3085,2984,2900,2743,2697,1736,1671,1609,1597,1542,1512,1474, 1420,1369,1348,1318,1285,1231,1142,1114,1083.

3. The novel oxazolidinone compound as claimed in claim-1 characterized by H-NMR:CDCl3: d2.01(s,3H), 3.06(t,4H), 3.60-3.78(m,4H),3.853.86(t,2H) ,4.03(t,2H), 4.78(m,1H),6.12(s,1H),6.82-6.85(m,1H),6.98 7.03(q,1H),7.32-7.34(dd,1H).

4. The novel oxazolidinone compound as claimed in claim-1 characterized by 13C-NMR:CDCl3:22.7,41.7,47.7,50.5,66.6,71.8,109.6,111.9,116.2,134.3,139.9,140.0,150.8,153.2,154.6,171.2.

5. The novel oxazolidinone compound as claimed in claim-1 characterized by ESI-MS (m/z): 338.37(M+1).

6. The novel oxazolidinone compound as claimed in claim-1 characterized by XRD peaks shown in Figure 5.
7. A process for the preparation of novel oxazolidinone compound (S)-N-((3-(4-fluoro-3-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide of formula –I

Compound-I

comprising the steps of :
a) reduction of 3, 4 –difluoro nitrobenzene compound of formula-II

Compound-II
to give 3,4-difluoro-aniline compound of formula-III;

Compound-III

b) reacting compound-III with (R) epichlorohydrin to obtain (R)-1-chloro-3-((3,4-difluorophenyl)amino)propan-2-ol compound of formula –IV;

Compound-IV
c) coupling the (R)-1-chloro-3-((3,4-difluorophenyl)amino)propan-2-ol compound of formula- IV with Potassium phthalimide to obtain (R)-2-(3-((3,4-difluorophenyl)amino)-2- hydroxyl propyl)isoindoline-1,3-dione compound of formula- V;

Compound-V

d) cyclization of (R)-2-(3-((3,4-difluorophenyl)amino)-2- hydroxyl propyl) isoindoline-1,3-dione compound of formula- V with CDI to obtain (S)-2-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione compound of formula-VI;


Compound-VI
e) opening of phthalimide ring of compound-VI by reacting with Hydrazine hydrate to obtain (S)-5-(amino methyl)-3-(3,4-difluorophenyl)oxazolidin-2-one compound of formula-VII;

Compound-VII
f) acylation of compound-VII with Acetic anhydride to obtain the corresponding (S)-N-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide compound of formula-VIII;

Compound-VIII
g) nitration of compound VIII with nitrating reagents (nitric acid & sulphuric acid ) to give (S)-N-((3-(4,5-difluoro-2-nitrophenyl)-2-oxooxazolidin-5-yl) methyl)acetamide compound of formula IX;

Compound-IX

h) reacting (S)-N-((3-(4,5-difluoro-2-nitrophenyl)-2-oxooxazolidin-5-yl) methyl) acetamide compound-IX with morpholine to give (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide compound formula –X;

Compound-X
i) reduction of (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide compound of formula-X to obtain (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide formula XI;

Compound-XI
j) deamination of (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (compound-XI) to obtain (S)-N-((3-(4-fluoro-3-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide compound of formula –I.

Compound-I

8. The novel oxazolidinone compound as claimed in claim-1, wherein said subject comprises human, animals and birds.

9. The novel oxazolidinone compound as claimed in claim-1, wherein said disease is a disease or pathogenic condition caused by gram positive and gram negative pathogens comprising M. luteus, B. sterothemophilus, S. maltophilia, P. putida, K. pneumonia, P. aeruginosa, E. coli, P. vulgaris, S. typhi, B. subtilis, S. mutans, B. sphaericus, B. circulans, Lysinobacillus, B. cereus, B. megatherium, P. mirabilis, S. paratyphi.

10. A pharmaceutically acceptable salt of the novel oxazolidinone compound-I as claimed in claim-1.

11. A pharmaceutical composition comprising the novel oxazolidinone compound-I or its pharmaceutically acceptable salt.

12. A pharmaceutical formulation comprising oxazolidinone compound-I or a pharmaceutical composition of compound-I or a pharmaceutically acceptable salt of compound-I.

Dated this 5th day of May 2014

,TagSPECI:FIELD OF THE INVENTION
The invention relates to novel oxazolidinone compound [(S)- N- [[3-[4-fluoro-3-morpholino phenyl]-2-oxooxazolidin-5-yl] methyl] acetamide] of Formula-I which was disclosed in Indian Patent Application No. 5063/CHE/2013 and corresponding PCT/IN2014/000018. More particularly, the invention relates to novel oxazolidinone antibacterial compound-I and its broad spectrum antibacterial activities against a large number of gram positive and gram negative pathogens.

Compound-I
BACKGROUND OF THE INVENTION
A large number of oxazolidinone compounds with different structures and different biological activities have been developed so far. The oxazolidinones class of antibacterials possess a unique mechanism of inhibiting bacterial protein synthesis. Linezolid is one of the most important oxazolidinone class of drug having potent antibacterial activity and was the first oxazolidinone to be approved for clinical use with potential antibacterial activity against many important resistant pathogens.
Novel oxazolidinone compound [(S)- N- [[3-[4-fluoro-3-morpholino phenyl]-2-oxooxazolidin-5-yl] methyl] acetamide] of Formula-I was disclosed in Indian Patent Application No. 5063/CHE/2013 and corresponding PCT/IN2014/000018 of the applicant which are incorporated herein by reference.
In further antimicrobial studies on compound-I, it was observed that this compound had shown broad spectrum antibacterial activities and it was effective against a large number of gram positive and gram negative bacteria with broader coverage and better bacteriostatic and bactericidal activities than other known oxazolidinones.
OBJECT OF THE INVENTION
The primary object of the invention is to provide a novel oxazolidinone compound-I for antibacterial use.
Another object of the invention is to identify the antibacterial activities and microbial coverage of novel oxazolidinone compound-I.
SUMMARY OF THE INVENTION
Novel oxazolidinone compound [(S)- N- [[3-[4-fluoro-3-morpholino phenyl]-2-oxooxazolidin-5-yl] methyl] acetamide] of Formula-I was disclosed in Indian Patent Application No. 5063/CHE/2013 and corresponding PCT/IN2014/000018 of the applicant which are incorporated herein by reference.

Compound-I
The compound of formula-I is characterized by following 1H-NMR, C13-NMR, Mass and IR spectral data.
IR spectral data:
3335,3085,2984,2900,2743,2697,1736,1671,1609,1597,1542,1512,1474,1420,1369,1348,1318,1285,1231,1142,1114,1083. ( As shown in Figure-1)
(H-NMR:CDCl3 : d 2.01(s,3H), 3.06(t,4H), 3.60-3.78(m,4H),3.85-3.86(t,2H), 4.03(t,2H) ,4.78 (m,1H), 6.12(s,1H),6.82-6.85(m,1H),6.98-7.03(q,1H),7.32-7.34(dd,1H) (As shown in Figure-2)
13C-NMR:CDCl3:22.7,41.7,47.7,50.5,66.6,71.8,109.6,111.9,116.2,134.3,139.9,140.0, 150.8, 153.2,154.6,171.2. (As shown in Figure-3)
ESI-MS (m/z) :338.37(M+1) (As shown in Figure-4)
The compound of formula-I is further characterized by XRD data shown in figure 5.
The compound-I is prepared by a process comprising the steps of:
a) reduction of 3, 4–difluoro nitrobenzene compound of formula-II

Compound-II
to give 3,4-difluoro-aniline compound of formula-III;

Compound-III
b) reacting compound-III with (R) epichlorohydrin to give (R)-1-chloro-3-((3,4-difluorophenyl)amino)propan-2-ol compound of formula –IV;

Compound-IV
c) coupling (R)-1-chloro-3-((3,4-difluorophenyl)amino)propan-2-ol compound of formula- IV with Potassium phthalimide to obtain (R)-2-(3-((3,4-difluorophenyl)amino)-2- hydroxyl propyl)isoindoline-1,3-dione compound of formula- V;

Compound-V
d) cyclization of (R)-2-(3-((3,4-difluorophenyl)amino)-2- hydroxyl propyl) isoindoline-1,3-dione compound of formula- V with CDI to obtain (S)-2-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione compound of formula-VI;

Compound-VI
e) opening of phthalimide ring of compound-VI by reacting with Hydrazine hydrate to obtain (S)-5-(amino methyl)-3-(3,4-difluorophenyl)oxazolidin-2-one compound of formula-VII;

Compound-VII
f) acylation of compound-VII with Acetic anhydride to obtain the corresponding (S)-N-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide compound of formula-VIII;

Compound-VIII

g) nitration of compound VIII with nitrating reagents (nitric acid & sulphuric acid ) to give (S)-N-((3-(4,5-difluoro-2-nitrophenyl)-2-oxooxazolidin-5-yl) methyl)acetamide compound of formula IX;

Compound-IX
h) reacting (S)-N-((3-(4,5-difluoro-2-nitrophenyl)-2-oxooxazolidin-5-yl) methyl) acetamide compound-IX with morpholine to give (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide compound formula –X;

Compound-X
i) reduction of (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide compound of formula-X to obtain (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide formula XI;

Compound-XI
j) deamination of (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (compound-XI) to obtain (S)-N-((3-(4-fluoro-3-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide compound of formula –I.

Compound-I

To ascertain the antibacterial activities of the compound-I, a thorough antimicrobial study was performed and its coverage spectrum as well as bacteriostatic and bactericidal activities were studied.

Anti bacterial evaluation of compound-I was done against Stenotrophomonas maltophilia on following parameters-
a. Agar-plate assay for different concentrations (10, 50, 100, 150µg)
b. MIC studies
1. With different concentrations (1000 to 5µg/ml - ~ 10 different concentrations)
2. Different time periods (4 to 32 hrs -- 8 different time intervals)
c. Mode of action with relation to survivability of organism
1. Cidal/static with concentration and incubation time based)
2. Evaluation by colony counting/ dye based
d. Susceptibility test (evaluation of >40 antibiotics of different nature)
e. Antibacterial potential studies with other infectious bacteria (~ 20 different strains)

The Antimicrobial activity of compound-I revealed that-
- compound-I is active against S. maltophilia while Linezolid and Vancomycin are not inhibitory
- Concentration based inhibitory activity was noticed against S. maltophilia
- MIC studies suggested 500 and above µg/ml is effective for complete inhibition (bacteriocidal) of S. maltophilia
- Concentration < 500 µg acts as bacteriostatic till 24 hours
- The antibiotic susceptibility profile of S. maltophilia was explored
- compound-I is effective against Xanthomonadaceae (S. maltophilia and Pseudomonas sp.) & some species of Bacillus (Bacillus cereus) family members

During lab analysis it was found that at 10 µg level the compound-I is effective in growth inhibition of S. maltophilia. Also increase of compound-I concentration resulted in increased zone of growth inhibition. However, proportionate growth inhibition zone was not noticed with increase of compound-I concentration on agar plate assay.

It was observed that low dose of compound-I is sufficient to reduce the growth of S. maltophilia. Although, the observation of un-proportionate inhibition zone Vs concentration of compound-I is interesting fact. This may be attributed to mass transfer of compound-I in agar during experimentation which influences the drug and microbial interaction. One of the alternatives to reduce the influence of mass transfer and improve the contact between compound-I and microbial strain was removal of solid barrier or increasing the diffusion of compound in the medium by broth dilution method.

To understand the antimicrobial potential of compound-I, initial lab experiments were planned to evaluate comparative microbial growth inhibition zone against Linezolid and Vancomycin using twenty different bacterial strains consisting of gram-positive and gram-negative nature. The selection of vancomycin for comparison was based on the fact that it is an alternative antibiotic which is generally used for treating the gram-positive bacterial infections. Among all tested different microbial strains, S. maltophilia, P. aeruginosa and one of the strains of B. cereus showed anti bacterial activity against compound-I, but not vancomycin or Linezolid, suggesting compound-I, could be a potential drug candidate to control the infections associated with above organisms more effectively. It is also established from the lab analysis that compound-I is effective against Xanthomonadaceae (S. maltophilia and Pseudomonas sp.) & some species of Bacillus (Bacillus cereus) family members.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure-1 is IP Spectral data of Oxazolidinone compound of formula-I.
Figure-2 is H-NMR Spectral data of Oxazolidinone compound of formula-I.
Figure-3 is 13C-NMR Spectral data of Oxazolidinone compound of formula-I.
Figure-4 is ESI-MS (m/z) spectral data of Oxazolidinone compound of formula-I.
Figures-5A and 5B are XRD data of Oxazolidinone compound of formula-I.
Figure-6 is Agar plate assay for Oxazolidinone compound of formula-I.
Figure 7 is Agar plate showing the zone of inhibition of S.maltophilia against compound-I
Figure 8 is MIC of compound-I by tube dilution method 12 hours.
Figure 9 is Dye based evaluation of cidal / static nature of compound-I to S. Maltophilia
Figure 10 is Agar plates indicating the antimicrobial profile of S. maltophilia for commercially available antibiotics
DETAILLED DESCRIPTION OF THE INVENTION
Detailed embodiments of the present invention are disclosed herein below. However, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. The scope of the invention is not limited to the disclosed embodiments and terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the invention. The invention is defined by claims appended hereto.

Novel oxazolidinone compound [(S)- N- [[3-[4-fluoro-3-morpholino phenyl]-2-oxooxazolidin-5-yl] methyl] acetamide] of Formula-I was disclosed in Indian Patent Application No. 5063/CHE/2013 and corresponding PCT/IN2014/000018 which are incorporated herein by reference.

Compound-I
The compound of formula-I is characterized by following 1H-NMR, C13-NMR, Mass and IR spectral data.
IR spectral data:
3335,3085,2984,2900,2743,2697,1736,1671,1609,1597,1542,1512,1474,1420,1369,1348,1318,1285,1231,1142,1114,1083. ( As shown in Figure-1)
(H-NMR:CDCl3 : d 2.01(s,3H), 3.06(t,4H), 3.60-3.78(m,4H),3.85-3.86(t,2H), 4.03(t,2H) ,4.78 (m,1H), 6.12(s,1H),6.82-6.85(m,1H),6.98-7.03(q,1H),7.32-7.34(dd,1H) (As shown in Figure-2)
13C-NMR:CDCl3:22.7,41.7,47.7,50.5,66.6,71.8,109.6,111.9,116.2,134.3,139.9,140.0, 150.8, 153.2,154.6,171.2. (As shown in Figure-3)
ESI-MS (m/z) :338.37(M+1) (As shown in Figure-4)
The compound of formula-I is further characterized by XRD data shown in figure 5.

The process of synthesis of the novel oxazolidinone compound of formula-I is illustrated in below scheme-I:

Scheme-I
The process for synthesis of the novel oxazolidinone compound [(S)- N- [[3-[4-fluoro-3-morpholino phenyl]-2-oxooxazolidin-5-yl] methyl] acetamide] (formula-I) comprises the following steps:
a) reduction of 3, 4 –difluoro nitrobenzene compound of formula-II

Compound-II
to give 3,4-difluoro-aniline compound of formula-III;

Compound-III

b) reacting compound-III with (R) epichlorohydrin to obtain (R)-1-chloro-3-((3,4-difluorophenyl)amino)propan-2-ol compound of formula –IV;

Compound-IV
c) coupling the (R)-1-chloro-3-((3,4-difluorophenyl)amino)propan-2-ol compound of formula- IV with Potassium phthalimide to obtain (R)-2-(3-((3,4-difluorophenyl)amino)-2- hydroxyl propyl)isoindoline-1,3-dione compound of formula- V;

Compound-V

d) cyclization of (R)-2-(3-((3,4-difluorophenyl)amino)-2- hydroxyl propyl) isoindoline-1,3-dione compound of formula- V with CDI to obtain (S)-2-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione compound of formula-VI;


Compound-VI

e) opening of phthalimide ring of compound-VI by reacting with Hydrazine hydrate to obtain (S)-5-(amino methyl)-3-(3,4-difluorophenyl)oxazolidin-2-one compound of formula-VII;

Compound-VII
f) acylation of compound-VII with Acetic anhydride to obtain the corresponding (S)-N-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide compound of formula-VIII;

Compound-VIII
g) nitration of compound VIII with nitrating reagents (nitric acid & sulphuric acid ) to give (S)-N-((3-(4,5-difluoro-2-nitrophenyl)-2-oxooxazolidin-5-yl) methyl)acetamide compound of formula IX;

Compound-IX

h) reacting (S)-N-((3-(4,5-difluoro-2-nitrophenyl)-2-oxooxazolidin-5-yl) methyl) acetamide compound-IX with morpholine to give (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide compound formula –X;

Compound-X

i) reduction of (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide compound of formula-X to obtain (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide formula XI;

Compound-XI

j) deamination of (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (compound-XI) to obtain (S)-N-((3-(4-fluoro-3-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide compound of formula –I.

Compound-I

Each step of the above process is further elaborated herein below.
Step-a: Reduction of 3, 4 –difluoro nitrobenzene to give 3,4-difluoro-anilene.
3, 4 –difluoro nitrobenzene of formula-I is reduced with pd/C in autoclave, applying Hydrogen gas and maintaining the reaction for 3-4 hrs. After completion of reaction it is filtered and distilled to obtain compound of formula-III.
Step-b: Reaction of 3,4-difluoro-anilene with (R) epichlorohydrin to yield (R)-1-chloro-3-((3,4-difluorophenyl)amino)propan-2-ol of formula –IV.
This step involves reacting 3,4-Difluoro aniline with (R) epichlorohydrin, heating at 50°C-55°C 5.0-6.0 hrs and after completion of reaction distilled under vaccum to obtain (R)-1-chloro-3-((3,4 difluorophenyl)amino)propan-2-ol of formula –IV.
Step-c: Coupling (R)-1-chloro-3-((3,4-difluorophenyl)amino)propan-2-ol of formula- IV with Potassium phthalimide to yield (R)-2-(3-((3,4-difluorophenyl)amino)-2- hydroxyl propyl)isoindoline-1,3-dione of formula- V.
In this step (R)-1-chloro-3-((3,4-difluorophenyl)amino)propan-2-ol of formula- IV is treated with Potassium phthalimide in DMF at 140-150oC for 5.0-6.0hrs. Then cooled, filtered and washed with mixture of water and methanol to yield (R)-2-(3-((3,4-difluorophenyl)amino)-2- hydroxyl propyl)isoindoline-1,3-dione of formula- V.
Step-d: Cyclization of (R)-2-(3-((3,4-difluorophenyl)amino)-2- hydroxyl propyl) isoindoline-1,3-dione of formula- V with CDI to yield (S)-2-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione of formula-VI.
In this step (R)-2-(3-((3,4-difluorophenyl)amino)-2-hydroxypropyl) isoindoline-1,3-dione(formula-V) in ethyl acetate is cyclized with CDI, stirred for 3.0-4.0hrs at room temperature. After completion of reaction washed with ethyl acetate followed by washed with water to get (S)-2-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione of formula-VI.
Step-e: Opening of phthalimide ring of (S)-2-((3-(3,4 difluorophenyl)-2- oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione by reacting with Hydrazine hydrate to yield (S)-5-(amino methyl)-3-(3,4 difluorophenyl)oxazolidin-2-one.of formula-VII.
This step involves reaction of (S)-2-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione with Hydrazine hydrate in methanol and water at room temperature and heated to 65-70oC, stirred for 2.0-3.0 hrs same temperature. After completion of reaction extracted with dichloromethane and distilled to obtain (S)-5-(amino methyl)-3-(3,4 difluorophenyl)oxazolidin-2-one.of formula-VII.
Step-f: Acyclation of (S)-5-(amino methyl)-3-(3,4-difluorophenyl)oxazolidin-2-one of formula-VII with Acetic anhydride yield the corresponding (S)-N-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide of formula-VIII.
In this step (S)-5-(amino methyl)-3-(3,4-difluorophenyl)oxazolidin-2-one of formula-VII reacted with acetic anhydride in dichloromethane at 25-30oC, stirred for 3.0-4.0hrs. After the completion of the reaction, organic layer is distilled and washed with water to obtain (S)-N-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide of formula-VIII.
Step-g: Nitration of (S)-N-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide withnitrating reagent (nitric acid & sulphuric acid ) to give (S)-N-((3-(4,5-difluoro-2-nitrophenyl)-2-oxooxazolidin-5-yl) methyl)acetamide of formula IX.
This step involves addition of (S)-N-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide to a solution of sulphuric acid and nitric acid in dichloromethane, stirred for 30.0 min at 0-5oC, stirred for 3.0-4.0hrs. Then raised the temperature, separated organic layer and extracted with dichloromethane to obtain (S)-N-((3-(4,5-difluoro-2-nitrophenyl)-2-oxooxazolidin-5-yl) methyl)acetamide.
Step-h: (S)-N-((3-(4,5-difluoro-2-nitrophenyl)-2-oxooxazolidin-5-yl) methyl)acetamide reacts with morpholine to give (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide formula X.
In this step to a solution of (S)-N-((3-(4,5-difluoro-2-nitrophenyl)-2-oxooxazolidin-5-yl) methyl)acetamide in isopropyl alcohol, added morpholine, stirred for 1.0-2.0hrs at 70-75oC. Then added water, separated and washed with water to obtain (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide.
Step-i: Reduction of (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide of formula X to give (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5 yl)methyl)acetamide formula XI.
In this step, reduction of (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide with Pd/C in methanol and maintaining at 5.0-6.0Kg/Cm2 of hydrogen gas for 3.0-4.0hrs at 35-40oC. After completion of reaction filtered catalyst and distilled off solvent completely to obtain (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5 yl)methyl)acetamide.
Step-j: Deamination of (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide to yield desired (S)-N-((3-(4-fluoro-3-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide.
This step involves addition of Con. HCl to (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide, maintaining the reaction mass at temperature 0-5°C and then added sodium nitrite in water solution and stirred. After completion of the reaction, added hypo phosphorus (H3PO2) at below 10oC and raised the reaction mass temperature to 30-35oC. After completion of reaction added dichloromethane and extracted with dichloromethane to obtain desired novel (S)-N-((3-(4-fluoro-3-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide.
The process described above for the preparation of novel oxazolidinone derivative compound [(S)- N- [[3-[4-fluoro-3-morpholino phenyl]-2-oxooxazolidin-5-yl] methyl] acetamide] of formula-I is further illustrated by the bellow given examples.
EXAMPLES:
Example-1:Preparation of (R)-1-chloro-3-((3,4-difluorophenyl)amino)propan-2-ol of formula –IV.
Taken 200.0g of 3,4-difluoro nitro benzene, 600.0ml of Toluene and 12.0g of pd/C into autoclave at 25°C-35°C. Apply the Hydrogen gas 2.0-6.0Kg/Cm2 and maintained the reaction for 3-4 hrs at 25°C-35°C Check the reaction mass TLC to conform reaction completion. After reaction complies unload the reaction mass and filter through hyflow bed and wash with toluene (200.0ml). Take the filtrate and distilled under vacuum at below 50°C. the obtained 3,4-Difluoro aniline crude weight 152.0g,Yield:93.8%,purity by HPLC:95.85%.
140.0g of the previous step 3,4-Difluoro aniline, 120.4g of (R)-Epichlorohydrine into a clean round bottom flask. slowly heat to 50°C-55°C and maintain the reaction mass for 5.0-6.0 hrs at 50°C-55°C and check the reaction mass TLC to conform reaction completion. After reaction compiles distill off low boilers under vacuum at below 50°C.to yield the crude compound of formula-III(170.0g,yield:71.0%,purity:73.29%),
Example-2:Preparation of (R)-2-(3-((3,4-difluorophenyl)amino)-2-hydroxypropyl) isoindoline-1,3-dione (Formula-V).
Crude obtained from example-1 treated with 156.0g of potassium phthalimide and 170.0ml of DMF at 140-150oC for 5.0-6.0hrs. the reaction mass checked TLC to conform reaction completion. The reaction mass cooled to Room temperature, added 850.0ml of water and 255.0ml of methanol, stirred for 1.0-2.0hrs at Room temperature. The filtered the solids, washed with mixture of water & methanol(170.0ml). the dried the solids. The obtained solid weight 206.0g, purity by HPLC:63.1%.
Example-3:: Preparation of (S)-2-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione (Formula-VI).
410.0g of (R)-2-(3-((3,4-difluorophenyl)amino)-2-hydroxypropyl) isoindoline-1,3-dione(formula-V),820.0 ml of ethyl acetate and 210.0g of CDI into Round bottom flask. Stirred for 3.0-4.0hrs at room temperature. Checked reaction mass, TLC to conform reaction completion. After reaction complies, filtered the solids, washed with 205.0 ml of ethyl acetate. Then the wet cake and 1800.0ml of water taken into a round bottom flask. Stirred for 1.0-2.0hrs at 40-45oC, filtered the solids, washed with 410.0ml of water. Dried the wet cake, the obtained of (S)-2-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione, weight :272.0g, yield:57.9%, purity by HPLC:94.22%.
Example-4: Preparation of (S)-N-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (Formula-VIII).
370.0g of (S)-2-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)isoindoline-1,3-dione, 370.0ml of methanol and 1480.0ml of water, 216.5g of hydrazine hydrate into a round bottom flask at room temperature. The reaction mass slowly heated to 65-70oC, stirred for 2.0-3.0hrs same temperature. Checked reaction mass TLC to conform reaction completion. After reaction complete, cooled the reaction mass to room temperature, extracted product with dichloromethane. The organic layer distilled off completely to give the crude of formula-VII (208.0g),
Crude obtained above was added 370.0ml of dichloromethane,147.5g of acetic anhydride added slowly above to the reaction mass at 25-30oC, stirred for 3.0-4.0hrs, checked reaction mass TLC to conform reaction completion. After reaction complies, wash the reaction mass with water. The organic layer distilled off completely to get Obtained crude mass to this mass 370.0ml of water was added under stirring maintain for 1hr and filtered the solid, washed with water. Dried to get the (S)-N-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide Formula-VIII ,weight:230.0g, yield:81.6%, purity by HPLC:95.54%.
Example-5: Preparation of (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (Formula-X).
320.3 g of sulphuric acid into round bottom flask. Cooled to 0-5oC,slowly added 88.7g of nitric acid at 0-5oC, stirred for 30 min, added 850.0ml of dichloromethane. Then added 170.0g of (S)-N-((3-(3,4-difluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide lot wise at 0-5oC. stirred for 30.0 min at 0-5oC. raised the reaction mass temperature to room temperature. Stirred for 3.0-4.0hrs. checked TLC to conform reaction completion. After reaction complies, the reaction quenched into 850.0ml of chilled water, separated organic layer. Extracted aqueous layer with dichloromethane. The combined organic layer distilled off solvent. Crude compound (S)-N-((3-(4,5-difluoro-2-nitrophenyl)-2-oxooxazolidin-5-yl) methyl) acetamide of formula IX. (182.0g)
To the crude thus obtained was added182.0ml of isopropyl alcohol and to this 122.4g of Morpholine was added slowly and stirred for 1.0-2.0hrs at 70-75oC.TLC was checked to conform reaction completion. Once complies, added 340.0ml of water to the reaction mass, then cooled to room temperature. The material thus separated was filtered and washed with 170.0ml of water. Dried to get the product obtained (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide of formula-X, weight:189.0g, yield:78.68%,purity by HPLC:91.12%.
Example-6: Preparation (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl) acetamide (Formula-XI).
100.0g of (S)-N-((3-(4-fluoro-5-morpholino-2-nitrophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide, 1500.0ml of methanol and 10.0g of Pd/C are subjected to hydrogenation in an autoclave. At 5.0-6.0Kg/Cm2 of hydrogen gas maintain for 3.0-4.0hrs at 35-40oC.Check for reaction completion by TLC. After reaction complies, filtered the catalyst and distilled off solvent completely. The obtained (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl) acetamide formula-XI ,weight:80.0g, yield:86.9%, purity by HPLC:96.32%.
Example-7: Preparation (S)-N-((3-(4-fluoro-3-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (Formula-I).
To 82.0ml of Con.HCl slowly added 80.0g of (S)-N-((3-(2-amino-4-fluoro-5-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl) acetamide (Formula-XI) maintaining reaction mass temperature 0-5°C . To this were added 23.5g of sodium nitrite in 125.0ml of water solution at 0-5oC,stirred for 30min,checked TLC to conform reaction completion. Once complies added 22.5g of hypo phosphorus (H3PO2) at below 10oC, then raised the reaction mass temperature to 30-35oC, stirred for 30min and checked TLC to conform reaction completion. After reaction complies, cooled the reaction mass to 0-5oC, added 400.0ml of dichloromethane and adjusted pH to 7.0-8.0 with 48%of caustic lye. Separated organic layer and aqueous layer extracted with dichloromethane.Combined organic layer, was distilled off to get the crude.Which is then purified by column chromatography and further re-crystallized with methanol to give (S)-N-((3-(4-fluoro-3-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide formula-I, weight:10.0g, yield:13.0%, purity by HPLC :99.5%.The structure conformed by 1H-NMR,C13-NMR, Mass, IR spectral data.
The structural elucidation of the compound of formula I is confirmed by 1H-NMR, C13-NMR, Mass, IR spectral data etc. stated as below:
IR spectral data:
3335,3085,2984,2900,2743,2697,1736,1671,1609,1597,1542,1512,1474,1420,1369,1348,1318,1285,1231,1142,1114,1083. (Fig -I)
(H-NMR:CDCl3 : d 2.01(s,3H), 3.06(t,4H), 3.60-3.78(m,4H),3.85-3.86(t,2H), 4.03(t,2H) ,4.78 (m,1H), 6.12(s,1H),6.82-6.85(m,1H),6.98-7.03(q,1H),7.32-7.34(dd,1H) (Fig .-II)
13C-NMR:CDCl3:22.7,41.7,47.7,50.5,66.6,71.8,109.6,111.9,116.2,134.3,139.9, 140.0,150.8, 153.2,154.6,171.2. (Fig -III)
ESI-MS (m/z) :338.37(M+1) ; (Fig -IV)
The compound of formula-I is further characterized by XRD data shown in figure 5.
Method of XRD Analysis:
Powder XRD make: Bruker D2 Phaser
LD samples data collected using PMMA holder and scan parameter details as follows:
Two theta range: 3 – 40 º
Step size: 0.012
Time for step: 72 s
Generator KV: 30
Generator mA: 10
Detector: Lynx Eye
Spinner: 15 rpm
X- Ray source: Cu Ka

Antimicrobial Study
To ascertain the antibacterial activities of the compound-I, a thorough antimicrobial study was performed and its coverage spectrum as well as bacteriostatic and bactericidal activities were studied.

Anti bacterial evaluation of compound-I was done against Stenotrophomonas maltophilia on following parameters-
f. Agar-plate assay for different concentrations (10, 50, 100, 150µg)
g. MIC studies
1. With different concentrations (1000 to 5µg/ml - ~ 10 different concentrations)
2. Different time periods (4 to 32 hrs -- 8 different time intervals)
h. Mode of action with relation to survivability of organism
1. Cidal/static with concentration and incubation time based)
2. Evaluation by colony counting/ dye based
i. Susceptibility test (evaluation of >40 antibiotics of different nature)
j. Antibacterial potential studies with other infectious bacteria (~ 20 different strains)

Results

Agar-plate assay
Agar plate assay was performed to assess quickly the antibacterial nature of compound-I using Stenotrophomonas maltophilia, a known causative microbial strain for pulmonary diseases, nosocomial infections, co-morbid illness, bacteremia, meningitis, endocarditis, etc.

In view of the background information provided, the initial agar plate based antibacterial property of compound-I, vancomycin and linezolid was evaluated against S. maltophilia using agar plates and 50 µg of each compound dissolved in DMSO solution. To nullify the effect of DMSO if any on S. maltophilia, DMSO also used as test compound and the data presented in Table 1 and Fig 6.

Table 1: Antimicrobial activity of compound-I against Stenotrophomonas maltophilia

S. No Compound Zone of growth inhibition in mm
1 Vancomycin 00
2 Linezolid 00
3 LDD-01/2013 17
4 DMSO 00

It is clear from above table that the growth of S. maltophilia was inhibited by only compound-I but not by either vancomycin or linezolid suggesting that compound-I has potential in health sector.
Compound-I concentration based growth inhibition studies
It is important to understand the antibacterial efficiency of compound-I before initiating further investigations. To evaluate the same, 0 to 250 µg concentration range used for agar plate assay. Two percent agar plates were prepared and 24 hours grown S. maltophilia was spread on agar plate and incubated at 4oC. After 30 min, the agar plates consisting of S. maltophilia were brought to sterile environment at room temperature and using sterile borer wells were developed. In each well, a known concentration of compound-I dissolved in DMSO was added. After 24 hours of incubation at 37oC, the zone of inhibition was measured. The data presented in Table 2 and Fig 7.

Table 2: Antimicrobial activity of different concentrations of compound-I against Stenotrophomonas maltophilia
S. No Compound-I Conc. (µg/ml) Zone of growth inhibition in mm
1 00 00
2 10 16
3 50 17
4 100 18
5 150 20
6 200 20
7 250 22

The data presented in Table 2 and Fig 7 clearly denote that
i. At 10 µg level the compound-I is effective in growth inhibition of S. maltophilia.
ii. Increase of compound-I concentration resulted in increased zone of growth inhibition.
iii. Proportionate growth inhibition zone was not noticed with increase of compound-I concentration on agar plate assay.
The above data is interesting especially with respect to low dose is sufficient to reduce the growth of S. maltophilia. However, the observation of un-proportionate inhibition zone Vs concentration of compound-I is appealing fact. This may attributed to mass transfer of compound-I in agar during experimentation which influences the drug and microbial interaction. One of the alternatives to reduce the influence of mass transfer and improve the contact between compound-I and microbial strain was removal of solid barrier or increasing the diffusion of compound in the medium by broth dilution method.

MIC studies
In microbiology, minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents. A MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism. In this method the above problem of diffusion is also eliminated.

MIC of compound-I by tube dilution method
To find out the MIC of compound-I concentration required for inhibition of S. maltophilia growth, ten different concentrations of compound-I ranging from 0 to 1000 µg/ml (1000, 500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90 and 0 µg/ml) was supplemented in one ml of sterile medium containing 107 cells. These tubes were incubated at 37oC. The growth of the S. maltophilia was observed after 12 hours. The data presented in the Fig 8.

It is clear from figure-8 that the growth of S. maltophilia noticed in all studied concentrations ranging from 0 to 250 µg/ml however, the growth visibility varied with the concentration of compound-I. In tubes supplemented with 500 and 1000 µg/ml of compound-I, no growth was observed suggesting for this compound MIC is 500 µg with respect to S. maltophilia.

MIC at different time periods
Though the MIC for LD derivate against S. maltophilia is 500 µg/ml, with the time of incubation the growth of S. maltophilia may differ due to metabolic nature of the microbe or physiological properties of the compound. Keeping this in view, the growth was monitored visually at different time intervals. The data is presented as supplement at the end of the report. It is evident from the figures that four hour incubation did not show any growth in any tube supplemented with compound-I at different concentration. However, with the increase in time from four to eight hours, the growth of S. maltophilia could be seen in the tubes supplemented with 62.5, 31.25, 15/62, 7.81, 3.90 and 0 concentration of compound-I. Further increase in incubation time beyond 12 hours i.e. 16, 20 and 24 hours, the growth of S. maltophilia could be visualized in all tubes except 500 and 1000 µg supplemented tubes. This denotes that 500 µg compound-I is effective in inhibiting the growth S. maltophilia in 12 hours.

Mode of action with relation to survivability of organism
Cidal/Static
To evaluate the growth inhibition property of compound-I is associated with killing or arresting the growth of S. maltophilia, the growth pattern of organism in presence of different concentrations of compound-I with respect to incubation time was analyzed. The results were tabulated. It was evident from the data that in any given concentration of compound-I, the growth was not detected till 4 hours of incubation. With the increase in incubation time beyond 4 hours, visible growth was not observed till 16 hours incubation in 62.5, 125 and 250 µg/ml supplemented tubes, however, further extension of growth periods i.e., beyond 20 hours, the growth of S. maltophilia was noticed. This suggests that growth of this organism was temporarily arrested and organism could regain the growth whereas supplementation of compound-I in concentrations of 500µg/ml and beyond, the growth of S. maltophilia was not observed even after 120 hours. This data further confirm that compound-I is effective in eradicating the S. maltophilia if used 500µg/ml. In addition the data also suggest at 500µg/ml and above concentration, the compound-I act as bactericidal and below 500µg/ml it may act as bacteriostatic.

Table 3: MIC of the compound-I against Stenotrophomonas maltophilia with respect to time

S. No Time (hrs) Bacteriostatic
µg/ml Bacteriocidal
µg/ml
1 0 NA NA
2 4 ND ND
3 8 62.5 --
4 12 125 --
5 16 250 --
6 20 -- 500
7 24 -- 500*

*Note: No growth was observed in 500 µg/ml supplemented conditions up to 10 days
Dye based analysis
The antibacterial nature of compound-I has been further confirmed by dye interactive method. The fig 9 indicates the survivability nature of S. maltophilia in the presence of compound-I. It was clear from the figure 9 that supplementation of methylene blue to the S. maltophilia cultures grown in presence of different concentrations of compound-I, the blue colour was noticed with cultures supplemented with 500 and 1000 µg/ml and remaining cultures did not show blue colour. The basic principle associated with the colour change or disappear of blue colour is associated with reducing environment. Methylene blue solution is blue when in an oxidizing environment, but will turn colorless if exposed to a reducing agent. Growth of any living organism is associated with wide range chemical reactions known as metabolism, basically characterized with redox reactions and several reducing cofactors are always exists in actively growing cells. Hence, exposure of methylene blue to such reducing environment (living organism), the dye immediately reduced and losses its colour. Since methylene blue colour is disappeared in the S. maltophilia culture tubes supplemented up to the concentration of 250 µg/ml compound-I, it denote that compound-I concentration at 500 µg/ml or above is detrimental for growth of S. maltophilia.

Susceptibility test profile of S. maltophilia for different antibiotics (Figure 10)
Table 4: Antimicrobial profile of S. maltophilia for commercially available antibiotics

S. No Antibiotic Quantity in µg Zone (mm)
1. Azithromycin 30 12
2. Rifampicin 5 3
3. Penicillin 10 6
4. Piperacillin 100 8
5. Augmentin 30 7
6. Ampicillin/sulbactam 10/10 12
7. Roxithromycin 30 4.5
8. Erythromycin 15 4
9. Ampicillin 10 6.5
10. Cloxacillin 1 3
11. Amoxycillin 10 6.5
12. Vancomycin 30 6.5
13. Chloramphenicol 30 11
14. Ciprofloxacin 5 12
15. Norfloxacin 10 9
16. Lincomycin 2 Resistant
17. Lomefloxacin 30 12
18. Clindamycin 2 Resistant
19. Tetracycline 30 6
20. Levofloxacin 5 15
21. Pefloxacin 5 9
22. Sparfloxacin 5 17
23. Ofloxacin 5 11
24. Doxycycline 30 8
25. Gentamicin 10 7
26. Netillin(Netilmicin sulphate) 30 7
27. Nalidixic 30 7
28. Kanamycin 30 3
29. Amikacin 30 8
30. Co-Trimoxazole 25 10
31. Tobramycin 10 10
32. Clarithromycin 15 Resistant
33. Nitrofurantoin 300 7
34. Streptomycin 10 12
35. Oxytetracycline 30 2
36. Furazolidone 50 Resistant
37. Augmentin 30 8
38. Gatifloxacin 5 15
39. Carbenicillin 100 14
40. Lome floxacin 10 14
41. Imipenem 10 6
42. Teicoplanin 30 4
43. Cefepime 30 9
44. Cefaloridine 30 7
45. Tobramycin 10 9

Antibacterial potential studies with other infectious bacteria (~ 20 different strains)
To understand the antimicrobial potential of compound-I, initial experiments were planned to evaluate comparative microbial growth inhibition zone against Linezolid and Vancomycin using twenty different bacterial strains consisting of gram-positive and gram-negative nature. The selection of vancomycin for comparison was based on the fact it is an alternative antibiotic that is generally used for treating the gram-positive bacterial infections. The results are reported in Table 5.
Table 5: Antimicrobial activity of compound-I with other microorganisms
(Zone of growth inhibition in mm)

S. No Strains Compound-I Linezolid Vancomycin
1 M. luteus 12 45 32
2 B. sterothemophilus 00 00 00
3 S. maltophilia 17 00 00
4 P. putida 00 00 00
5 K. pneumonia 00 35 25
6 P. aeruginosa 12 00 00
7 E. coli 12 35 25
8 P. vulgaris 00 00 00
9 S. typhi 00 40 14
10 B. subtilis 12 40 25
11 S. mutans 00 40 30
12 B. sphaericus 00 30 12
13 B. circulans 00 38 27
14 Lysinobacillus 00 40 30
15 B. cereus (1) 17 00 00
16 B. megatherium 12 37 27
17 B. cereus (2) 00 35 27
18 P. mirabilis 00 35 27
19 B. cereus (3) 00 45 32
20 S. paratyphi 00 00 25

Among all tested different microbial strains, S. maltophilia, P. aeruginosa and one of the strains of B. cereus showed anti bacterial activity against compound-I but not vancomycin or Linezolid suggesting compound-I could be a potential drug candidate to control the infections associated with above organisms more effectively.

Conclusions:
The Antimicrobial activity of compound-I revealed that-
- compound-I is active against S. maltophilia while Linezolid and Vancomycin are not inhibitory
- Concentration based inhibitory activity was noticed against S. maltophilia
- MIC studies suggested 500 and above µg/ml is effective for complete inhibition (bacteriocidal) of S. maltophilia
- Concentration < 500 µg acts as bacteriostatic till 24 hours
- The antibiotic susceptibility profile of S. maltophilia was explored
- compound-I is effective against Xanthomonadaceae (S. maltophilia and Pseudomonas sp.) & some species of Bacillus (Bacillus cereus) family members.

The invention therefore provides novel oxazolidinone compound-I for therapeutic use as a potential antibacterial drug against gram positive and gram negative pathogens.

The compound-I is further converted in a pharmaceutically acceptable salt for use in a pharmaceutical preparation.

The invention also provides pharmaceutical preparations comprising compound-I. The pharmaceutical preparations comprise, and not limited to, oral formulations, injectables, topical preparations and formulations for all other possible routes of administration in human and animals.