Description:

FORM-2

THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003 COMPLETE SPECIFICATION
(See section 10 and rule 13)

Novel antimicrobial acetamide and synthesis thereof

APPLICANT-

1. MR. AMIT KUMAR,

2. DR. SUSHIL KUMAR,

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED

FIELD OF INVENTION:

The present invention belongs to the field of synthetic pharmaceutical science. More, particularly the invention pertains to Synthesis and Biological Activity of novel acetamides. The novel process is for synthesis of acetamides for antimicrobial potential.
BACKGROUND OF THE INVENTION:

Acetamide (systematic name: ethanamide) is an organic compound with the formula CH3CONH2. It is the simplest amide derived from acetic acid. It finds some use as a plasticizer and as an industrial solvent. The related compound N,N-dimethylacetamide (DMA) is more widely used, but it is not prepared from acetamide. Acetamide can be considered an intermediate between acetone, which has two methyl (CH3) groups either side of the carbonyl (CO), and urea which has two amide (NH2) groups in those locations. Acetamide is also a naturally occurring mineral with the IMA symbol: Ace.
Acetamide can be produced in the laboratory from ammonium acetate by dehydration:

[NH4][CH3CO2] ? CH3C(O)NH2 + H2O

Alternatively acetamide can be obtained in excellent yield via ammonolysis of acetylacetone under conditions commonly used in reductive amination.
It can also be made from anhydrous acetic acid, acetonitrile and very well dried hydrogen chloride gas, using an ice bath, alongside more valuable reagent acetyl chloride. Yield is typically low (up to 35%), and the acetamide made this way is generated as a salt with HCl.
Acetamide is used as a plasticizer and an industrial solvent. Molten acetamide is good solvent with a broad range of applicability. Notably, its dielectric constant is higher than most organic solvents, allowing it to dissolve inorganic compounds with solubilities closely analogous to that of water. Acetamide has uses in electrochemistry and the organic synthesis of pharmaceuticals, pesticides, and antioxidants for plastics. It is a precursor to thioacetamide.

Acetamide has been detected near the center of the Milky Way galaxy. This finding is potentially significant because acetamide has an amide bond, similar to the essential bond between amino acids in proteins. This finding lends support to the theory that organic molecules that can lead to life (as we know it on Earth) can form in space.
Acetamide, also known as ethanamid or acetic acid amide, belongs to the class of organic compounds known as carboximidic acids. These are organic acids with the general formula RC(=N)-OH (R=H, organic group). Acetamide is a mousy tasting compound. Acetamide has been detected, but not quantified in, red beetroots (Beta vulgaris var. rubra). This could make acetamide a potential biomarker for the consumption of these foods. Acetamide is formally rated as a possible carcinogen (by IARC 2B) and is also a potentially toxic compound.
Acetamide is classified as a potentially carcinogenic substance. It also is a mild irritant. In the past, acetamide was used as a plasticiser and as a stabiliser. Molten acetamide was frequently used as a solvent in chemical synthesis. It also acts as a solubiliser; its mere addition renders many sparingly soluble compounds more soluble in water.
In view of the foregoing, anti-microbial compound is synthesized, wherein said compounds are acetamides meeting the following objectives are desired.
OBJECTIVE OF THE INVENTION:

1. It is an object of the invention to provide an antimicrobial acetamide compound represented by the formula:

HN COCH2 O COCH3

Formula-I

2. Another object of the present invention is to provide method for synthesis of antimicrobial acetamide compound 2-(2-acetylphenoxy)-N-cyclohexylacetamide.
3. Another object of the present invention is to provide method for synthesis of intermediate compound 2-chloro-N-cyclohexylacetamide.

NH2 ClCOCH2Cl HN COCH2Cl
i

(1) (2)

DETAILED DESCRIPTION:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a

non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or systems or elements or structures or components proceeded by "comprises... a" does not, without more constraints, preclude the existence of other devices or other systems or other elements or other structures or other components or additional devices or additional systems or additional elements or additional structures or additional components.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.

The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items.

The terms “having”, “comprising”, “including”, and variations thereof signify the presence of a component.

Now the present invention will be described below in detail with reference to the following embodiment.

Generally speaking, the present invention provides a novel Process for Synthesis of Newer Derivatives of Thiobarbituric Acid for Anticonvulsant Potential.

Example-1
1. Synthesis and Characterization of Compounds

Step I. Synthesis of 2-chloro-N-cyclohexylacetamide (2) Step II. Synthesis of target compounds (4a-d)
a. Synthesis of 2-(4-acetylphenoxy)-N-cyclohexylacetamide (4a)

b.Synthesis of 2-(2-acetylphenoxy)-N-cyclohexylacetamide (4b)
c. Synthesis of 2-((4-acetylphenyl) amino)-N-cyclohexylacetamide (4c)
d. Synthesis of 2-((3-acetylphenyl) amino)-N-cyclohexylacetamide (4d)
Step I.Synthesis of 2-chloro-N-cyclohexylacetamide (2)

NH2 ClCOCH2Cl HN COCH2Cl
i

(1) (2)

Scheme 1. Reagents and conditions: (i) NaOH, Dichloromethane, 0 oC temperature.

Procedure

Cyclohexylamine 1 (4.61 ml, 0.04mol) in 2N aqueous sodium hydroxide (150 ml) at 0oC temperature was treated with chloroacetylchloride (3.18ml, 0.04mol) as asolution in dichloromethane (100 ml). After 1h, the layers were separated and the aqueous phase was extracted with additional portion of dichloromethane. The organic phases were combined, washed with an aqueous solution of 1N hydrochloric acid, saturated sodium bicarbonate, dried over sodium sulphate. Removal of the solvent afforded the compound 2-chloro-N- cyclohexylacetamide2.
The physical parameters were of the synthesized compound Percentage yield : 70.20 %
Melting range : 98-102oC
Rf value : 0.75

Mobile phase : hexane: ethyl acetate (1:1)
Molecular formula : C8H14Cl

Table 1.IR Data of 2-chloro-N-cyclohexylacetamide 2

Wave number (cm-1) Group Assignment 3414 N-H Str
2937 C-H Str Aliphatic

1658 C=O Str

1219 C-N Str
668 C-Cl Str

Step II. Synthesis of final compounds (4a-d)

4a. Synthesis of 2-(2-acetylphenoxy)-N-cyclohexylacetamide (4b)
Procedure: 2-Chloro-N-cyclohexylacetamide 2 (0.005mol) is dissolved in 100 ml of acetonitrile in a 250 ml round bottom flask. Anhydrous K2CO3 (0.005mol), catalytic amount of potassium iodide and appropriate orthohydroxyacetophenone 3 (0.005 mol) are added into above solution. The mixture is allowed to reflux with continuous stirring on magnetic stirrer for 12 h. After completion of reaction, reaction mixture is filtered and solvent is removed by vacuum distillation afforded the crude product which is recrystallized from ethanol to afford final compounds (4b).
Synthesis of 2-(2-acetylphenoxy)-N-cyclohexylacetamide (4b)

Scheme 1. Synthesis of the target compounds. Reagents and conditions: (i) Acetonitrile, K2CO3, KI, Reflux.

Physical parameters of 2-(2-acetylphenoxy)-N-cyclohexylacetamide (4b)
a) Percentage yield : 55.45 %
b) Melting Point : Semisolid
c) Rf value : 0.52
d) Mobile Phase : Ethyl acetate: n-Hexane (1:1)
e) Molecular formula : C15H21NO3

Table 2: IR data of 2-(2-acetylphenoxy)-N-cyclohexylacetamide (4b)

S. no ? (cm-1) Functional Group Assignment
1. 3292 NH str.
2. 2914 C-H str. (Aromatic)
3. 2849 C-H str. (Aliphatic)
4. 1649 C=O str.
5. 1545 C=C str.
6. 1233 CN str.

Table 3: NMR data of 2-(2-acetylphenoxy)-N-cyclohexylacetamide (4b)

S. No. Chemical shift(d)(ppm) No. of Protons Inferences
1. 7.28 1H NH
2. 6.37 - 8.10 4H Ar-H
3. 4.54 2H COCH2
4. 2.59 3H COCH3
5. 1.13-196 11H CH

EXAMPLE-2

Biological studies

There are two methods for determining antimicrobial activity

1) Paper-disc-plate technique (Disc diffusion method).

2) Tube-dilution technique (Broth micro dilution technique).

Paper-disc-plate technique (Disc diffusion method)

Sensitivity testing is done to determine the range of microorganisms that are susceptible to the compound under specified conditions. It can be done by disk diffusion method. This method is suitable for the organisms that grow well overnight such as most of the common aerobes and facultative anaerobes and rapidly growing fungi. This method is solely depends upon the diffusion of the drug substance from a disc to an extent such that the observed growth of the microorganism is prevented totally in a zone just around the disc impregnated in a solution of drug substances. A zone of inhibition (a clear area) around the disc indicates that the organism was inhibited by the drug, which diffused into the agar from the disc.
Tube-dilution technique (Broth micro dilution technique)

Dilution susceptibility testing methods are used to determine the minimal concentration, usually expressed in units or microorganisms/ml., of an agent required to inhibit or kill a microorganism. Procedures for determining antimicrobial activity are carried out by either agar or broth-based methods. By this method, one can determine the smallest.
amount of drug substances required to inhibit the growth of the organism in vitro, this method is referred to as the MIC (Minimum inhibitory concentration). MIC can be determined by tube dilution technique. Antimicrobial agents are usually tested at 10 g. sub (2) (two-fold) serial dilutions & the lowest concentration that inhibits visible growth of an organism recorded as the MIC.

Determination of antibacterial activity
To determine the antimicrobial activity of target compounds zone of inhibition (a clear area) is measured around the disc which indicates that the organism was inhibited by the drug, which diffused into the agar from the disc. To determine zone of inhibition Paper disc plate technique was used.
Methodology: -

Paper disc plate technique (Determination of zone of inhibition) Preparation of solution of synthesized compound
Accurately weighed 25 mg of each synthesized compound was transferred to 100 ml volumetric flask. These compounds were then dissolved in 2 ml. DMSO and volumes in each flask were made up to 100 ml with sterile distilled water. These suspension (each having conc.100 ?g/ml) were used as stock suspension. 1 ml and 2 ml of these suspensions were transferred to two 10 ml volumetric flasks and further dilutions were made to 10 ml mark with sterile distilled water. The final suspension contained 50 and 100 ?g of each compound per ml of the suspension.
Preparation of stock solution of standard drug (Ciprofloxacin)

Ciprofloxacin (25 mg) was accurately weighed and transferred to 100 ml volumetric flask. The drug was dissolved in 2 ml DMSO and diluted up to 100 ml with sterile distilled water. The final solution contained 100 ?g/ml of the standard drug Ciprofloxacin. This solution in volume of 1 ml and 2 ml were transferred to two 10 ml volumetric flasks and further dilutions were made to
10 ml mark with sterile distilled water. The final solutions contained 50 and 100 ?g Ciprofloxacin per ml of the solution.
Petri dishes, pipettes and culture tubes were washed with distilled water, dried in oven, packed in brown paper & then autoclaved at 15 lb /inch2pressure (1210 C) for 15 min.
Test strains:

B. subtilis E.coli
Composition of media

Nutrient agar was used for the purpose which contains the constituents as presented in the

Peptone 0.5g
Beef Extract 0.5 g
NaCl 2.5 g
Agar 10 g
Distilled water Up to 500 ml
Composition of nutrient agar media Procedure for preparation of media: -
Peptone (5gms), beef extract (5gm) and sodium chloride (2.5gm) (All of biological grades) were weighed and dissolved in 400 ml of distilled water in a 500 ml volumetric flask and warmed. 10 gm agar was dissolved in 50 ml of warm distilled water. The two solutions were mixed and the volume in volumetric flask was made up to 500 ml with warm distilled water. This nutrient agar media was sterilized in an autoclave at 15 lb /inch2 pressure (1210 C) for 15 min.
Following steps were followed for determination of antibacterial activity of synthesized compounds:
• Laminar airflow bench was swapped with 70 % alcohol and UV lamp was switched on. After 30 min, the UV lamp was switched off.
• All the reagents, media, inoculums and glassware were placed in laminar airflow bench and aseptic condition were maintained.
• The plates were inoculated within minutes of the preparation of suspension, so that the density did not change. A sterile cotton swab over was dipped into the suspension and the medium was inoculated by even streaking of the swab over the entire surface of the plate in three directions. After the inoculums had dried, the drug solution was poured on the disk & then disk was placed on the agar plate with the help of a forceps. After inoculation at 37 0C for 48 hours. The zone of inhibition was measured using mm scale.
• Negative controlled plate- In this plate, only nutrient agar medium was poured i.e., it did not contain drug dilution and inoculums.

• Positive controlled plate- In this plate, nutrient agar medium was poured and after its solidification, inoculums spread over the surface of culture plate. But this petriplate did not contain drug solution.
ANTIBACTERIAL ACTIVITY

All the target compounds (4a-d) were evaluated for their in-vitro antibacterial activity against both Gram-positive (Bacillus subtilis) and Gram-negative bacteria (Escherichia coli) using Ciprofloxacin as standard drug at concentration of 100?g/ml. Paper disc plate method was used for the determination of the preliminary antibacterial activity. The compound 4b showed potent activity 18 mm against B.subtilis and 18 mm against E.coli,respectively as compared to ciprofloxacin. The solvent (control) i.e., DMSO did not show any activity.The results were observed for each tested compounds are shown in Table.
Table 10: Antibacterial activity of target compounds (4a-d) compounds against gram positive B.subtilis and gram negative and E.coli.

Zone of inhibition is 18 or greater than 18 mm the
compound considers as active.

While the invention has been described with respect to specific compounds which include presently preferred modes of carrying out the invention, those skilled in the art will appreciate that there are numerous variations and permutations of the above described embodiments that fall within the spirit and scope of the invention. It should be understood that the invention is not limited in its application to the details of construction and arrangements of the components set forth herein.
Variations and modifications of the foregoing are within the scope of the present invention. Accordingly, many variations of these embodiments are envisaged within the scope of the present invention.
The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching.
The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, and to thereby enable others skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions and substitutions of equivalents are contemplated as circumstances may suggest or render expedient, but such omissions and substitutions are intended to cover the application or implementation without departing from the spirit or scope of the present invention.

We claim

1. An antimicrobial acetamide compound represented by the formula:

Formula-I

2. The antimicrobial acetamide compound as claimed in claim 1, wherein IUPAC name of said compound is 2-(2-acetylphenoxy)-N-cyclohexylacetamide.

3. The antimicrobial acetamide compound as claimed in claim 1, wherein molecular formula of said compound is C15H21NO3.

4. A method for synthesis of antimicrobial acetamide compound as claimed in claim 1 comprising the step of;
Step I. Synthesis of 2-chloro-N-cyclohexylacetamide

NH2 ClCOCH2Cl HN COCH2Cl
i

(1) (2)

(a) 4.61 ml of 0.04 mol Cyclohexylamine and 150 ml of 2N aqueous sodium hydroxide is treated with chloroacetylchloride (3.18ml, 0.04mol) as a solution in dichloromethane (100 ml) at 0oC temperature,
(b) After 1h, the layers were separated and the aqueous phase was extracted with additional portion of dichloromethane.
(c) The organic phases were combined, washed with an aqueous solution of 1N hydrochloric acid, saturated sodium bicarbonate and dried over sodium sulphate.
(d) Removal of the solvent to obatin the intermediate compound 2-chloro-N- cyclohexylacetamide.

Step II: Synthesis of 2-(2-acetylphenoxy)-N-cyclohexylacetamide

(e) 0.005 mol of intermediate compound obtained in step (d) of step I is dissolved in 100 ml of acetonitrile in a 250 ml round bottom flask.
(f) 0.005 mol of anhydrous K2CO3 and catalytic amount of potassium iodide and appropriate para hydroxy acetophenone 3 (0.005 mol) are added into above solution (e).
(g) The mixture is allowed to reflux with continuous stirring on magnetic stirrer for 12 h.
(h) After completion of reaction, reaction mixture is filtered and solvent is removed by vacuum distillation afforded the crude product which is re-crystallized from ethanol to obtain final compound 2-(2-acetylphenoxy)-N-cyclohexylacetamide.

MR. AMIT KUMAR, DR. SUSHIL KUMAR, Mr. Anesh Sagar

Mr. Vivek Kumar Mr. Satendra Kumar Mr. Rahul Chauhan

Mr. Raj Kumar Bharti Dr. Abhishek Suman

ABSTRACT

Novel antimicrobial acetamide and its method of Synthesis
The present invention belongs to the field of synthetic pharmaceutical science. More, particularly the invention pertains to Synthesis and Biological Activity of novel acetamide 2-(2-acetylphenoxy)- N-cyclohexylacetamide. The novel process is for synthesis of acetamides for antimicrobial potential.
Formula-I

Claims:1. An antimicrobial acetamide compound represented by the formula:

Formula-I

2. The antimicrobial acetamide compound as claimed in claim 1, wherein IUPAC name of said compound is 2-(2-acetylphenoxy)-N-cyclohexylacetamide.

3. The antimicrobial acetamide compound as claimed in claim 1, wherein molecular formula of said compound is C15H21NO3.

4. A method for synthesis of antimicrobial acetamide compound as claimed in claim 1 comprising the step of;
Step I. Synthesis of 2-chloro-N-cyclohexylacetamide

NH2 ClCOCH2Cl HN COCH2Cl
i

(1) (2)

(a) 4.61 ml of 0.04 mol Cyclohexylamine and 150 ml of 2N aqueous sodium hydroxide is treated with chloroacetylchloride (3.18ml, 0.04mol) as a solution in dichloromethane (100 ml) at 0oC temperature,
(b) After 1h, the layers were separated and the aqueous phase was extracted with additional portion of dichloromethane.
(c) The organic phases were combined, washed with an aqueous solution of 1N hydrochloric acid, saturated sodium bicarbonate and dried over sodium sulphate.
(d) Removal of the solvent to obatin the intermediate compound 2-chloro-N- cyclohexylacetamide.

Step II: Synthesis of 2-(2-acetylphenoxy)-N-cyclohexylacetamide

(e) 0.005 mol of intermediate compound obtained in step (d) of step I is dissolved in 100 ml of acetonitrile in a 250 ml round bottom flask.
(f) 0.005 mol of anhydrous K2CO3 and catalytic amount of potassium iodide and appropriate para hydroxy acetophenone 3 (0.005 mol) are added into above solution (e).
(g) The mixture is allowed to reflux with continuous stirring on magnetic stirrer for 12 h.
(h) After completion of reaction, reaction mixture is filtered and solvent is removed by vacuum distillation afforded the crude product which is re-crystallized from ethanol to obtain final compound 2-(2-acetylphenoxy)-N-cyclohexylacetamide.