WO2012104793A1

WO2012104793A1 - An antibacterial and antiviral compound

Info

Publication number: WO2012104793A1
Application number: PCT/IB2012/050463
Authority: WO
Inventors: Vanaja KUMAR; Mukesh DOBLE; Balagurunathan RAMASAMY; Suresh Ganesan; Radhakrishnan MANIKKAM; Luke Elizabeth HANNA; Sowmya Swaminathan; Selvakumar NAGAMIAH
Original assignee: Indian Council Of Medical Research
Priority date: 2011-02-02
Filing date: 2012-02-01
Publication date: 2012-08-09

Abstract

The invention provides a compound represented by Formula (I) 5-(4-bromobutyl)-N-(but-3-enyl)dodec-11-enamide or a derivative thereof. Said compound is effective against bacterial and viral pathogens.

Description

"AN ANTIBACTERIAL AND ANTIVIRAL COMPOUND"

Field of the invention:

The present invention is in the field of medicine. The invention primarily relates to a compound effective against bacterial and viral pathogens.

Background of the invention:

The incidence of infections caused by drug resistant bacteria continues to increase and remains a serious threat to human health (Asolkar et al, 2010). Disease causing bacteria such as Mycobacterium tuberculosis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa gradually develop resistance to drugs. Out of all these the drug resistance developed by Mycobacterium tuberculosis against the commonly used antibiotics is of major concern. Tuberculosis remains one among the leading causes of infectious disease worldwide. One third of the world population is infected with Mycobacterium tuberculosis and hence at risk of developing active TB (Boogoard et al, 2009).

The current first line TB regimen is more than 40 years old and consists primarily of rifampicin and isoniazid. These antibiotics are effective in active drug susceptible TB, provided that patients complete the course of treatment. However, there is a poor patients' compliance due to the cost of drugs, adverse effects, the long time required for completion of treatment (6-12 months) and the required number of drug doses. Non-compliance has contributed to the emergence of multi drug resistant (MDR) and extensively drug resistant (XDR) TB strains. MDR TB (strains resistant to isoniazid and rifampicin) often takes longer time to treat with second line drugs. XDR-TB (MDR TB resistant to second line drugs including fluoroquinolones and any one of the injectable drugs such as capreomycin, kanamycin and amikacin) is virtually incurable. Furthermore, HIV/AIDS antiretroviral therapies are not always compatible with the current TB regimen because of shared drug toxicities and drug interactions (Rivers and Mancera, 2008). In this context, there is an urgent need for developing novel antiTB drugs with less toxic side effects, improved pharmacokinetic properties with extensive and potent activity against resistant strains and to reduce the total duration of treatment (De Sousa, 2006).

Actinomycetes are the most economically valuable prokaryotes which are well known to produce chemically diverse metabolites with wide range of biological activities. It has been estimated that about half of the microbial bioactive metabolites notably antibiotics, antitumor agents, immuno suppressives and enzyme inhibitors have been isolated from actinomycetes (Balagurunathan and Radhakrishnan, 2010). Recently the rate of discovering new compounds from terrestrial actinomycetes has decreased but the rate of re-isolation of known actinomycetes and antibiotics is on the increase. This has led researchers to explore unique and extreme habitats such as marine environment for potentially new biosynthetic diversity. Marine actinomycetes are the promising source for secondary metabolites (Lam, 2006). In the past 10 years, 659 marine bacterial compounds have been described in which 256 compounds have originated from actinomycetes (Williams, 2008).

From the discovery of streptomycin from Streptomyces griseus, actinomycetes derived antibiotics are still in use for the treatment of tuberculosis. Due to the emergence of MDR and XDR TB cases, search for novel antibiotics is still continuing.

The present team of inventors has isolated for the first time a novel strain of Actinomycetes which produce a chemical compound effective against various bacterial as well as viral pathogens. The inventors have also isolated and characterized a novel compound effective against various bacterial and viral pathogens.

Objective of the invention:

The primary objective of the invention is to provide a compound which is effective against bacterial and viral pathogens.

Another objective of the invention is to provide a process of preparing the compound.

Yet another objective of the invention is to provide a novel strain of Actinomycetes which produces the chemical compound having activity against bacterial and viral pathogens.

Summary of the invention:

(I)

5-(4-bromobutyl)-A^-(but-3-enyl)dodec-l l-enamide

or a derivative thereof. Said compound is effective against bacterial and viral pathogens. Description of the accompanying drawings:

Figure 1 : ^lH NMR Expansion spectrum of the compound of formula (I)

Figure 2: ^lH NMR Expansion spectrum of the compound of formula (I)

Figure 3 : ^lH NMR spectrum of the compound of formula (I)

Figure 4: HSQC NMR spectrum of the compound of formula (I)

Figure 5: DEPT ¹³C NMR spectrum of the compound of formula (I)

Figure 6: ¹³C NMR spectrum of the compound of formula (I)

Figure 7: 'H- 'H COSY NMR spectrum of the compound of formula (I)

Figure 8 : Showing the number of compounds present in the crude extract obtained from Actinomycete strain MTCC 5597

Figure 9: Effect of crude extract and INH and Rif against drug sensitive and MDR isolate of latent tubercle bacilli

Figure 10: Inhibitory effect of compound of purified compound of formula (I), INH and Rif against MDR and XDR isolate of latent tubercle bacilli

Figure 1 1 : Effect of purified compound of formula (I) on various clades of HIV- 1

Detailed description of the invention:

sented by formula (I)

5-(4-bromobutyl)-A^-(but-3-enyl)dodec-l l -enamide

or a derivative thereof.

The chemical structure of the compound is elucidated based on its spectral data. The molecular formula of the compound is derived as C₂oH₃₄ONBr. The structure of the compound is as elucidated as 5-(4-bromo butyl)-N-(but-3-enyl) dode-1 1 -enamide, a brominated compound.

The derivatives of the compound are selected from triazol, Chloro, lodo, O-alkylation and N- alkylation products. The compound of the invention is effective against bacterial pathogens such as Mycobacterium tuberculosis, Bacillus subtilis, Bacillus pumilus, Bacillus cereus, Staphylococcus aureus, and Acinetobacter baumanii. The compound is also effective against viral pathogens such as Human Immuno Deficiency Virus (HIV). The compound is effective against multiple drug resistant and extensively drug resistant strains of Mycobacterium tuberculosis. The compound also shows good activity against SHRE (Streptomycin, Isoniazid, Rifampicin, and Ethambutol) sensitive and SHRE resistant strains of Mycobacterium tuberculosis.

The invention further provides a composition comprising the compound of formula (I) along with pharmaceutically acceptable additives, excipients and adjuvants. The composition can be formulated in various forms such as liquid, solid, powder and lozenges. Suitable excipients are mixed in composition to improve the stability of the composition. Such excipients are selected from the group comprising liquid or solid carrier, disintegrator, coating agents etc. The excipients are further useful for improving efficacy of composition for controlling the bacterial and viral pathogens. The pharmaceutically acceptable additives and excipients are selected from the group comprising glycerol, lactic acid, poly ethylene glycol (PEG), salts such as KC1, cationic surfactants, anionic surfactants and natural surfactants, lactose, sucrose, dextrose, sorbitol and mannitol; dextrins; polycarboxylic acids, chitosan, vitamin C; polyethylene glycols, polyvinyl pyrrolidone, benzyl alcohol and polyvinyl acetate.

The invention further provides use of compound of formula (I) against bacterial and viral pathogens. The invention further provides a method of using compound of formula (I) against bacterial and viral pathogens.

The invention further provides a process of preparing the compound of formula (I), said process comprising the steps of:

(i) inoculating Actinomycetes strain MTCC 5597 onto a suitable agar based media;

(ϋ) incubating the agar plate(s) at a temperature of 20 °C to 40 °C for a period of 3 to 10 days and obtaining mycelial growth;

(iii) removing the mycelial growth from the agar plate(s) and obtaining the agar medium containing the compound of formula (I);

(iv) optionally cutting the media into pieces;

(v) adding the media pieces into a suitable solvent; (vi) incubating the media dissolved in the solvent at a temperature of 23 °C to 30 °C for a period of 3 to 18 hours and extracting the compound of formula (I);

(vii) collecting the solvent part and concentrating the same;

(viii) obtaining the concentrate containing the compound of formula (I); and

(ix) purifying the compound.

The media for the growth and inoculation is selected from the group comprising yeast extract malt extract agar, glycerol asparagine agar, oatmeal agar, czapek's dox agar and tyrosine agar

In one of the preferred embodiments, the agar plates are incubated for a period of 7 days at a temperature of 28 °C which is an ideal temperature for the growth of Actinomycetes. After obtaining sufficient growth, the mycelia are removed from the medium. The compound of the invention is secreted extracellularly by the Actinomycete strain. Therefore, the compound is easily extracted from the medium used for the growth of the Actinomycete.

After removing the mycelia from the media, the media is dissolved in a suitable solvent for the extraction of the compound of formula (I). The media can be cut into small pieces for easy and better dissolution.

The solvent used for dissolving the media for the purpose of extraction of the compound is an organic solvent. Preferably, the solvent used for the purpose of the invention is selected from the group comprising methanol, chloroform, dichloromethane, diethyl ether, ethyl acetate and n-hexane.

It is preferred that the media dissolved in the solvent is incubated for a period of 24 hours and at a temperature of 28°C.

The concentrate obtained in step (viii) of the process is either directly subjected to the process of purification or is stored at a suitable temperature for further usage.

The preferred temperature for the purpose of storing the concentrate is 4 °C to 25 °C. More preferably, the temperature for the purpose of storing the concentrate is 4 °C.

The purification of the compound is done by various methods such as chromatography or crystallization methods. Chromatography involves methods such as thin layer chromatography and column chromatography. Column chromatography is useful for large scale production of the compound. Crystallization involves methods such as single solvent recrystallization, multi solvent recrystallization. The invention further provides an Actinomycetes strain with accession no MTCC 5597. The strain is useful for producing the compound of formula (I). The Actinomycetes strain of the invention is isolated from coral reef marine ecosystem of, Rameswaram, Tamil Nadu, India.

The invention also provides a biologically active agent comprising a compound of formula (I), wherein the agent is effective against bacterial and viral pathogens. The invention also provides a kit comprising a compound of formula (I) along with an instruction manual. The kit of the invention may also comprise the composition along with an instructions manual.

The invention is illustrated further by the following examples which are only meant to illustrate the invention and not act as limitations. The embodiments which may be apparent to a person skilled in the art are deemed to fall within the scope of the present invention.

Example 1. Sample collection and isolation of actinomycete strain of the invention

i. Collecting sediment samples from Coral reef ecosystem of Rameswaram, South India;

ii. Drying the sediment sample at room temperature for five days;

iii. Keeping the sample at 55°C in hot air oven for 10 minutes;

iv. Serially diluting the sediment sample using sterile distilled water;

v. Plating the diluted sample on nalidixic acid (20 μg/ml) and cycloheximide (100 g/ml) supplemented Starch Casein agar prepared in 50% filtered sea water;

vi. Incubating the plates at 28°C for one month;

vii. Isolating the colonies with actinomycete morphology and subculturing on YEME (ISP 2) agar medium prepared in 50% seawater;

viii. Maintaining the stock cultures of actinomycete strain YEME agar slants, 30%> glycerol stock as well as in lyophilized form.

Morphologically distinct actinomycete colonies are observed on starch casein agar medium after 5 days of incubation. Actinomycete strain of the invention produced small colonies with powdery consistency, with yellow colour soluble pigment production. Good growth of actinomycete strain is observed on YEME agar medium.

Example 2. Antibacterial activity of actinomycete strain of the invention - agar plug method

I. Culturing the actinomycete strain on YEME agar medium at 28°C for 10 days; II. Preparing the cell suspensions of test bacterial cultures using nutrient broth and Sabouraud Dextrose broth, respectively, and adjusting the turbidity to 0.5 McFarland standards;

III. Inoculating the cell suspensions on Muller Hinton Agar (MHA) plates using sterile cotton swabs;

IV. Removing the mycelial growth of actinomycete strain R2 from YEME agar plates using sterile spatula;

V. Preparing the agar plugs with 5 mm diameter using sterile well cutter;

VI. Placing the agar plugs over the surface of each MHA plates seeded with test bacterial cultures;

VII. Incubating the MHA plates at 37°C for 24 hours for bacteria and 48 - 72 hours for fungi;

VIII. Measuring the zone of inhibition of bacterial cultures around the agar plug and expressing in millimetre in diameter;

Table 1 provides the results of the antibacterial activity of the actinomycete strain by agar plug method.

Table 1

Test cultures Zone of inhibition

[expressed in millimetre in

diameter]

Bacillus subtilis NCIM 2063 20

Bacillus pumilus NCIM 2327 18

Bacillus cereus NCIM 2106 23

Staphylococcus aureus NCIM 2079 19

Staphylococcus aureus (clinical) 11

Staphylococcus aureus (clinical; methicillin and 12

vancomycin resistant)

Bacillus subtilis MTCC 10

Acinetobacter baumanii (clinical; ESBL 14

producing)

Acinetobacter baumanii (clinical; ESBL 15

producing)

Example 3: Preparation of the crude extract of compound of formula (I):

Preparation of crude extracts of compound of formula (I) by agar plate culture:

The process of preparing the compound of formula (I) comprises following steps:

(i) inoculating a loopful of Actinomycetes strain R2 grown on yeast extract malt extract agar slants onto yeast extract malt extract agar plates (20ml/plate) in 50 plates by continuous streaking; (ii) incubating the agar plates at a temperature of 28°C for a period of 7 days and obtaining mycelial growth;

(iii) removing the mycelial growth from the agar plates using a sterile spatula;

(iv) obtaining the agar medium containing the compound of formula (I);

(v) cutting the media into small pieces;

(vi) adding the agar media pieces into

(vii) adding the media pieces into beakers, wherein each beaker contains 100ml of methanol as solvent;

(viii) incubating the beakers for a period of 24 hours at a temperature of 28°C and extracting the compound of formula (I);

(ix) collecting the solvent portion and concentrating the same using rotary evaporator; and

(x) storing at 4 °C;

(xi) Quantifying the crude extract using electronic balance.

Each 100ml quantity of methanol used as solvent provides approximately 40mg of crude extract.

Example 4: Antibacterial activity of crude extract of the Actinomycete strain

i. Preparing 10 mg/ml concentration of crude ethyl acetate extract of actinomycete strain R2 using ethyl acetate;

ii. Preparing crude extract discs by adding 10 μΐ of crude extract into each 5mm diameter filter paper disc in order to get 100μg/disc concentration;

iii. Drying the discs in laminar air flow cabinet;

iv. Testing the antibacterial activity of crude extract by disc diffusion method;

v. Preparing the cell suspensions of test bacterial cultures using nutrient broth and Sabouraud Dextrose broth, respectively, and adjusting the turbidity to 0.5 McFarland standards;

vi. Inoculating the cell suspensions on Muller Hinton Agar (MHA) plates using sterile cotton swabs;

vii. Placing the crude extract impregnated paper discs over the surface of each MHA plates seeded with test bacterial cultures

viii. Incubating the MHA plates at 37°C for 24 hours for bacteria;

ix. Measuring the zone of inhibition of bacterial cultures around the agar plug and expressing in millimetre in diameter;

Table 2 provides the results of the antibacterial activity of crude ethyl acetate extract of the actinomycete strain by disc diffusion method. Table 2

S. Test cultures Zone of inhibition

No [expressed in millimetre in diameter]

1 Bacillus subtilis NCIM 2063 20

2 Bacillus pumilus NCIM 2327 21

3 Bacillus cereus NCIM 2106 25

4 Staphylococcus aureus NCIM 2079 20

5 Staphylococcus aureus (clinical) 14

6 Staphylococcus aureus (clinical; methicillin and 18

vancomycin resistant)

7 Bacillus subtilis MTCC 15

8 Acinetobacter baumanii (clinical; ESBL 18

producing)

9 Acinetobacter baumanii (clinical; ESBL 19

producing)

Example 5: Purification of compound of formula (I):

A. Thin Layer Chromatography:

(i) the crude extract (compound extracted in ethyl acetate) is dissolved completely in chloroform;

(ii) taking ready made silica gel coated alumina sheet (20 X 20 cm).

(iii) spotting the crude extract at the bottom of the TLC sheet using glass capillary tube.

(iv) running TLC using chloroform: methanol (9:1) solvent systems; and

(v) visualizing the spot with spraying reagent KMn0₄;

(vi) obtaining a single white color spot with Rf value 0.5.

B. Column chromatography:

(i) preparing crude extract slurry by mixing with chloroform and small amount of neutral alumina powder;

(ϋ) taking a glass column (40cm X 7cm) packed with neutral alumina as stationary phase and chloroform: methanol (9:1) as mobile phase, wherein the he alumina powder is packed in the column using chloroform;

(iii) adding the slurry on the column and running the chromatography;

( (iivv)) increasing the polarity of the solvent by adding methanol;

(v) obtaining a yellow colour fraction; (vi) eluting the fraction and concentrating by drying.

Example 6: HPLC analysis of crude extract and purified compound of formula (I)

i. analyzing the crude extract and purified compound of formula (I) using Shimadzu (Japan) RID- 1 OA gradient high-performance liquid chromatographic instrument, equipped with two LC-20AD pumps controlled by a CBM-10 inter- face module. ii. using Refractive Index Detector RID 10A (Shimadzu) for peak detection

iii. prefiltering the solvents by using a Millipore system

iv. performing the analysis on a Luna 5u Cig (2) 100 A reversed- phase column with 150X4.6mm size. The column temperature is 30°C.

v. filtering the mobile phase solvents through 0.2 Ώ μ membrane filter and degassing by sonication before use.

vi. Achieving the separation with a two-pump gradient program for pump A (0.1% Acetic acid in Methanol) and pump B (0.1% Acetic acid in H₂0) as follows 2%> solvent A with flow rate 1 ml/min;

vii. increasing gradually to 10%> until 60 minutes.

viii. Injecting about 20 μΐ of sample and the detecting the peaks with PDA detector.

Figure 8 is showing the number of compounds present in the crude extract of actinomycete strain. Totally 12 compounds are present in the crude extract.

Example 7: Characterization of purified compound of formula (I):

• Solubility of purified compound is tested by adding the purified compound in 100 μΐ of solvents such as methanol, chloroform, dichloromethane, diethyl ether, ethyl acetate and n-hexane.

· Melting point of the purified compound is tested using Tempo instrument and is determined as 247-248°C.

• UV spectra of the purified compound is determined using Techem 8500 UV spectrophotometer at a wavelength of 190 - 900 nm. The UVmax at 448 nm shows the presence of conjugation moiety.

· FT IR analysis is performed using Perkin Elmer, N017-1159 FT IR spectrophotometer with KBr pellet.

• The scan is performed over 4000 - 450 cm^"1 at a resolution of 1.0 cm^"1.

• LC MS analysis is performed using HP API AGILENT 1200 series 300 single quadrupole MS equipped with a ESI. About 100 μΐ of sample is dissolved in 1 ml of methanol. The column is packed with 3 micrometer BDS-H persil CI 8 Silica. The mobile phase is composed of MeCN with 2 mM of ammonium formate and 50 mM of formic acid at a flow rate of 0.25 ml/min; flow ratio of 35:65 and 100 μΐ injection volumes. The column effluent is split, so that 10 % is taken by the mass spectrometer. The operation conditions of MS is positive polarity, ion source voltage of 5000; ion source temperature of 450°C and ESI mode H and ¹³C nuclear magnetic resonance (NMR) spectra are recorded on a Bruker model AVANCE III 500MHz (AV500) spectrometer using tetramethylsilane (TMS) as internal reference. Deuterated chloroform (CDCI₃) from Aldrich Chemical Company is used as solvent. The chemical shift is referenced to residual solvent, CDCI₃ (^lH-7.26, ¹³C-77). The spectra are collected at 25°C in 5mm NMR tube. The 2D proton homo nuclear correlation data is observed with the varian COSY pulse sequences. Single and multiple bond hetero nuclear connectivity data are observed using the HSQC and HMBC pulse sequences respectively. Elemental analysis is carried out using Perkin-Elmer Series II 2400 CHN model.

• The compound is obtained as yellowish oily substance. In silica gel coated TLC plates, the yellow fraction shows single spot with Rf value 0.5 cm and it develops white color spot when sprayed with KMn0₄ reagent. This indicates the presence of amide functional group.

• The crude extracts resulting from column chromatography on neutral alumina with CHCk/MeOH (9: 1) yields one major yellow pigmented fraction.

Table 3 provides the results as physicochemical properties of the compound of formula (I).

Table 3

Physical state Yellow color, oily substance

TLC (Rf) 0.5

Reaction with KMn0₄ White-yellow spot

Soluble in Water, methanol, acetone, chloroform, ethyl acetate

Insoluble in n-hexane

Melting point 247-248°C

LC-MS (m/z) (molecular weight) 385.90

IR (KBr) Vmax (cm ¹) 3368, 1738

CHN analysis - calculated

found) C 62.7 (61.97 %)

H 9.39 (9.72%)

N 3.62 (3.68%)

Molecular formula C₂oH₃₄ONBr

Example 8: Studying the NMR spectral data of the compound of formula (I):

The proton nuclear magnetic resonance (^lH NMR) spectrum of the isolated compound indicates identical sets of allylic protons, which are observed as multiplets at 5.3ppm and 4.2ppm. The -NH proton broad singlet appear at 1.8ppm. The rest of the aliphatic chain protons appear at 3.9, 3.6, 2.7, 2.3, 2.06, and 1.6ppm. ¹³C NMR spectrum indicate the presence of sp2 carbon atom (alkene) substituted by one allyl group (110-150ppm). The two peaks at 115.90 and 130.23 ppm, are assigned to allylic carbons. Furthermore, the peaks at 41.07, 36.69, 35.52, 34.99, 34.33, 34.13, 33.23, 32.53, 31.51, 29.08, 27.19, 25.62, and 24.88ppm aliphatic chain carbon. The carbonyl carbon of the amide (CONH) peak appears at 174.35 ppm. The DEPT NMR peaks indicate 16 methylene carbons, 3 methine carbons and one quaternary carbon. The COSY and HSQC NMR eliminates short range coupling (i.e., one bond), proton - carbon couplings, and hence can indicate the specific carbon that correlates with specific proton. The peak at 1.64ppm indicate two methylene protons and peak at 2.06ppm correlates with methine and methylene carbon.

Table 4 provides the details of NMR spectral data of the compound of formula (I)

Table 4

S.NO ^JH ppm ¹³C ppm DEPT ^H ppm HSQC ppm

ppm

1. 5.39 (m) 130.00, 2CH Hi-Hi,Hi-H₂,Hi-H₇ 130.00, 130.23

130.23

2. 4.21(dddd) 127.89, 2CH₂ H₂-H₂,H2-H1 127.89, 128.08

(Jl=5Hz,J2= 128.08

6.5Hz)

3. 3.95 (t) (J = 41.07 CH₂ 33.23

7Hz)

4. 3.65 (t) (J = 35.52, CH₂ ¾-¾,¾-¾ 41.07

6Hz) 36.69

5. 2.7 (t) (J = 34.99 CH₂ ¾-¾,¾-¾ 36.69 6.5Hz)

6. 2.3 (m) 34.33 C¾ Ηβ-Ηβ,Ηβ-Η _ΐ,Ηβ-Ηι 34.13

7. 2.0 (m) 34.13 2CH₂, H₇-H7,H7-Hi,H₇- 34.13, 33.23

1.6 (m) 33.23,32.53 2C1¾,CH Hg-Hg,Hg-H7,Hg- 24.88,29.08,35.52

9. 1.3 (m) 31.51, 3CH₂ !¾-]¾,]¾-]¾,]¾- 32.53,27.19,25.62

Ηιο,Η₉-Η₇

10. 0.9 (m) 29.08 3CH₂ Hio-Hio,Hio-H9,Hio- 34.99,34.33

H₈

11. 27.19

12. 25.62

13. 24.88

14. 174.30

15.

Example 9: Effect of solvents on the extraction of the compound of formula (I):

The process as elaborated in example 1 is carried our using solvents methanol, chloroform, dichloromethane, diethyl ether and ethyl acetate.

Table 5 gives the results of the effect of solvents on the extraction of the compound.

Table 5

Solvent extracts Quantity of crude extract

(mg/100 ml)

Methanol 40

Chloroform 41

Dichloromethane 40

Diethyl ether 10

Ethyl acetate 9

The results shows that the compound is extracted well in methanol, chloroform and dichloromethane compared to diethyl ether and ethyl acetate. Extracts in methanol, chloroform and dichloromethane gives better colour intensity as compared to the extract in diethyl ether and ethyl acetate. However, the extracts with methanol, chloroform and dichloromethane extracts shows presence of salt crystals and other debris. Ethyl acetate and diethylether extracts does not show any such salt crystals and debris.

Example 10: Antimycobacterial activity of compound of formula (I):

Stock preparation:

Adding 10 mg of crude extract into 1 ml of 10% Dimethyl Sulfoxide (DMSO) and sterilizing the extract by filtration using 0.45μ filter.

Preparing cell suspension:

i. Adding standard strain Mycobacterium tuberculosis H37Rv growing on Lowenstein Jenson (LJ) slopes in to 5 ml of sterile glycerol 7H9 (G7H9) broth and mixing using vortex mixer for 2 minutes.

ii. Allowing the cell suspension to stand for few minutes for settling the clumps of bacteria.

Luciferase Reporter Phage (LRP) Assay:

i. Taking each 350 μΐ of G7H9 broth in seven cryo vials.

ii. Adding 50 μΐ of different solvent extracts into first five vials to give final concentration of 100μg/ml.

iii. Adding 50 μΐ of 1% DMSO in to the sixth and seventh vials

iv. Adding 100 μΐ of M. tuberculosis H37Rv cell suspension in to all the vials.

v. Incubating all the vials at 37°C for 72 hours.

vi. adding 50 μΐ of high titre phage phAE129 and 40 μΐ of 0.1M CaCl₂ into all the vials. vii. Incubating all the vials at 37°C for 4 hours.

viii. taking 100 μΐ of reaction mixture in cuvettes and adding D-luciferin.

ix. measuring relative light units (RLU) immediately in the luminometer using 10 second integration time

x. Calculating the percentage of reduction in RLU by using the following formula

Control RLU - Test RLU X 100

Control RLU

Extracts resulting in more than 50%> reduction in RLU are considered as active against M. tuberculosis.

Table 6 provides the results of the antimycobacterial activity of different solvent extracts Table 6

Solvent extracts % reduction in RLU

Methanol 58.31

Chloroform 18.07

Dichloromethane 22.71

Ethyl acetate 74.23

Diethyl ether 83.4

The results clearly indicate that among the different solvent extracts diethyl ether and ethyl acetate extract exhibits maximum activity.

The activity of the crude extracts is also tested on different strains of Mycobacterium tuberculosis.

Table 7 provides the results of the activity of crude extract on different strains of

Mycobacterium tuberculosis.

Table 7

Test organisms % RLU reduction

M. tuberculosis H37Rv 98.96 M. tuberculosis SHRE sensitive 98.46 M. tuberculosis SHRE resistant 97.49

The results clearly show that more than 95% RLU reduction is achieved. This indicates good activity against all the three M. tuberculosis strains tested.

Example 11: Minimum inhibitory concentration of the compound of formula (I) against Mycobacterium tuberculosis:

The active fraction is dissolved in 1 ml of 10% DMSO (lOmg/ml) and is used as stock solution. Minimal inhibitory concentration of the purified fraction is tested at different concentration ranging from 50, 25, 12.5, 6.25, 3.125, 1.5 and 0.75μg/ml against standard strain M. tuberculosis H37Rv and clinical isolates of SHRE sensitive, multi drug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis by LRP assay.

Table 8 provides the results for minimum inhibitory concentration (MIC) of the purified 5 compound against different strains of Mycobacterium tuberculosis.

Table 8

Organisms (strains) MIC ^g/ml)

M. tuberculosis H37Rv < 1

M. tuberculosis (SHRE sensitive) 1.5

M. tuberculosis (SHRE resistant) 6.25

M. tuberculosis (XDR) 6.25

The results clearly indicate that the compound is effective against all the strains of Mycobacterium tuberculosis. However, the best activity is observed against Mycobacterium lOtuberculosis H37Rv.

Example 12: Activity of crude and purified compound of formula (I) against latent TB bacilli

i. Preparing the stock solutions of crude extract and purified compound of formula (I) in 15 10% DMSO;

ii. Determining the inhibition of the growth of dormant tubercle bacilli grown under hypoxic condition according to Wayne's dormant model by the crude and purified compound of formula (I) at lOC^g/ml and 10 μ^ιηΐ, respectively in sealed containers with moderate agitation;

0 iii. Finding the difference in the colony forming units before and after addition of crude and purified compound of formula (I).

Reduction in the CFU in M. tuberculosis cultures with purified compound of formula (I) is noticed in comparison with that of the CFU without the compound 5 Figure 9 provides the effect of crude extract and also the standard drugs INH and Rif against drug sensitive and MDR isolate of latent tubercle bacilli. INH was used as negative control and was resistant to dormant bacilli Figure 10 provides the effect of purified compound of formula (I) and also the standard drugs ΓΝΗ and Rif against MDR and XDR isolate of latent tubercle bacilli.

Example 13. Inhibitory activity of purified compound of formula (I) against MTB biofilm

i. preparing the cell suspension of SHRE sensitive, MDR and XDR isolates of M. tuberculosis using 7H9 broth;

ii. developing the biofilm of M. tuberculosis isolates on 24 well tissue culture plates;

iii. adding 2 ml of Sautons medium (without Tween 80) and inoculating 20 μΐ of saturated planktonic culture of M. tuberculosis isolates;

iv. Adding 100 μg/ml of the compound of formula (I) in to the first wells, Rif and ΓΝΗ into the second and third wells, respectively;

v. Wrapping the plates with parafilm and incubating without shaking at 37°C for 5 weeks in humidified conditions;

vi. Observing the plates for biofilm formation by M. tuberculosis isolates;

vii. Adding the purified compound of formula (I), Rif and ΓΝΗ into the 4^th, 5^th and 6^th wells containing the biofilms;

viii. Determining the viable counts of tubercle bacilli from the wells before and after adding the compound of formula (I), Rif and ΓΝΗ;

Biofilm formation is observed in the wells containing M .tuberculosis alone. In the wells containing M .tuberculosis cells and the compound of formula (I) there is no biofilm formation.

CFU is determined at the end of 2 months and after the treatment of wells containing M. tuberculosis cells with compound of formula (I). There are no viable colonies found in the wells containing the compound, whereas the CFU determined before addition of the compound is 2 x 10⁶ /ml.

Example 14: Minimum inhibitory concentration (MIC) of the compound of formula (I) against other bacterial pathogens:

The minimum inhibitory concentration (MIC) of the compound of formula (I) is determined for other bacterial pathogens Staphylococcus aureus (NCIM5021), Pseudomonas aeruginosa (NCIM5029) and Escherichia coli (NCIM2931). The minimum inhibitory concentration (MIC) is determined by micro dilution broth assay method with modifications using resazurin as an indicator as follows:

(i) dissolving the compound of formula (I) in absolute ethanol to a concentration of lOmg/ml; (ii) Serially diluting the compound and adding to successive wells in a 96 well microtiter plate and incubating with the bacterial pathogens for 18 hours at 37°C;

(iii) maintaining the growth and sterility controls during the experiment;

(iv) adding 10 μΐ of 0.01% resazurin solution and incubating for 2 hours;

(v) visually assessing the color change.

Blue colour indicates inhibition of growth, indicating MIC.

The results of the activity are provided in table 9.

Table 9

Organisms (strains) MIC ^ /ml)

Staphylococcus aureus (NCIM5021 ) 138.88 Escherichia coli (NCIM2931) 17.36 Pseudomonas aeruginosa (NCIM5029) 17.36

The results clearly indicate that the active compound shows good activity against all the three bacterial pathogens tested.

This clearly establishes that the compound of the invention is not merely effective against Mycobacterium tuberculosis, but is also effective in controlling the growth of other bacterial pathogens. Therefore, the compound is also useful against other bacterial pathogens.

Example 15: Anti-HIV activity of crude extract and compound of formula (I):

Activity of crude extract:

i. Testing the in vitro antiviral activity of the crude extract on an infectious laboratory adapted subtype B strain of HIV- 1 ;

ii. Infecting the activated healthy donor PBMC with 100TCID₅₀ of the virus per 1 x 10⁶ cells and cultured in the presence of varying concentrations of the crude extract (100μg/ml,

50μg/ml, 25μg/ml and 10μg/ml);

iii. Determining the HIV-1 p24 antigen production on day 7 as an indirect measure of viral replication in the culture supernatants using the Alliance HIV-1 p24 ELISA kit (Perkin

Elmer, USA).

Viral inhibition is observed at all concentrations tested. Complete inhibition of growth of HIVvirus is observed at all concentrations tested. Activity of purified compound of formula (I):

Virus production by transfection of 293T cells: 293T cells are plated at a concentration of lx 10⁶ cells/ml in a 100mm culture dish and grown at 37°C in a CO₂ incubator for 24 hours. Cells are 5transfected with 20μg of HIV IIIB plasmid DNA using the mammalian cell transfection kit (Millipore). The culture supernatant is collected at 48 hours post-transfection, clarified by centrifugation and stored in liquid nitrogen.

Titration of virus stock: Seven serial four-fold dilutions of virus stock, ranging from 1 :16 to 1 :65,635 are titrated in triplicate in a 96-well flat bottomed tissue culture plate containing 200,000 lOcells/well (PBMC stimulated with PHA for 72 hours). After 7 days of culture at 37°C in a C0₂ incubator, the titration assay is terminated and the culture supernatants are tested for HIV-1 p24 antigen. The TCID50 (tissue culture infection dose50) is calculated employing the Spearman- Kaber method.

Testing for anti-HIV activity of compound: HIV IIIB is used as a representative clade B virus and 15lndie-Cl as a representative clade C virus. Healthy donor PBMC (Peripheral blood mononuclear cells) activated through PHA (Phyto heme agglutinin) stimulation for 72 hours are incubated with I OOTCID₅₀ of the virus per 1 x 10⁶ cells for 2 hours at 37°C. The cells are washed twice to get rid of the unadsorbed virus and plated at a concentration of 200,000 cells/well in a 96-well tissue culture plate. Varying concentrations of the compound are added to triplicate wells 0(concentrations tested were

and

Control cultures are set up without addition of the compound. Cultures are maintained for 7 days at 37°C in a CO₂ incubator. On day 7, culture supernatants are tested for HIV-1 p24 antigen.

Measurement of HIV-1 p24 antigen: HIV-1 p24 antigen production is measured as an indirect 5measure of viral replication in the culture supernatants using the Alliance HIV-1 p24 ELISA kit (Perkin Elmer, USA).

Virus growth is determined by measuring p24 concentrations in culture supernatants. Table 10 below provides the results for the anti-HIV activity of the compound.

Table 10

Compound ^g/ml) P24 antigen (pg/ml)

Clade B Clade C

0 2394 406

0.001 1603 390

0.01 337 310

0.1 163 344

1 144 302 5 147 295

10 163 296

Reduction in p24 levels indicates the level of inhibition. The results clearly indicate that the compound of the invention is effective against HIV.

Activity of purified compound of formula (I) against different clades of HIV-1

I. Examining the activity of the compound of formula (I) on different HIV-1 subtypes;

II. The virus isolates tested were:

Subtype A: 92RW020

Subtype B: JR-FL

Subtype C: 92BR025

Subtype D: 92UG001

Subtype E: 92TH021

Subtype A/C: 92RW009

III. Infecting the activated donor PBMC with 100TCID₅₀ of primary clinical isolates representing different HIV-1 clades (clades A, B, C, D, E, A/E), as well as nevirapine resistant and AZT resistant strains, in the presence of purified compound of formula

(i);

IV. Measuring the activity of the purified compound of formula (I) by measuring p24 antigen produced upon culture for 7 days;

Figure 2 provide the effect of the purified compound of formula (I) on various clades of HIV- 1. The purified compound of formula (I) has activity on all the different strains of HIV-1 tested.

Example 16: Cytotoxicity of the compound of formula (I):

Cytotoxicity of the compound is measured by adopting MTT assay (Mosmann, 1983) as follows: (i) preparing the sample by inoculating 3T3 cells in 5 x 10⁴ concentrations in each well of 96 well microtiter plates with in Dulbecco's modified Eagles medium (DMEM) containing 10% FBS, 100 U/ml Penicillin 100 g/ml Streptomycin;

(ii) incubating for a period of 2 days at a temperature of 37 °C in 5% CO₂ atmosphere (Astec Japan);

(iii) adding the compound of formula (I) in four different dilutions of 25, 50, 75, 100 μg/ml in DMSO to the medium and incubating the cells for another 12 hours;

(iv) Discarding the growth medium in the plates and washing the wells with phosphate buffer saline (PBS); (v) Adding MTT in growth medium at a final concentration of 0.5 mg/ml and incubating for 4 hours;

(vi) solublizing the insoluble formazan crystals with 0.04N HC1 in isopropylalcohol and measuring the absorption on a Spectramax Plus384 spectrophotometer (Molecular Devices, CA, USA) at 570 nm.

For each of the samples evaluated, the test is performed in triplicate. The control cells are treated with PBS. Overnight experiment is done with DMSO alone as a control. The results for cytotoxicity of the compound of formula (I) is given in table 11.

Table 11

Concentration of the compound Average % Viability SD

Control 100.00 0.00

DMSO 97.48 5.56

10 M 93.00 4.66

25 M 86.01 2.93

50 M 73.88 4.00

100 M 66.13 2.05

The results clearly show the viability of the cells for various concentrations of the compound. It is evident from the results that the compound of formula (I) shows very poor cytotoxic activity even at 100 M concentration.

Example 17: Synthesis of purified compound of formula (I) predictable derivatives - in silico approach

The in silico derivatives (n=27+251) of compound of formula (I) are subjected to QIKPROP module of SHROEDINGER software output.

Table 12. The in silico derivatives of compound of formula (I) are subjected to QIKPROP module of SHROEDINGER software output.

Table 12

Hum Percent

m Q an Rul ol vo do ac QP Q PI met Oral Human e Rul lu no cp log PP og abo abso Oral Of e Of

M m rH tH Po Ca B lis rptio absorpt Fiv Thr molecule W e B B ct CO B m n ion e ee Methylketone 37 14 15. 59 -

7.6 86 56 7.2 1.9

isopropanol

09 .7 0 4 1 21 63 7 1 100 1 compound of 47

formula (I)

Allyl Isopropyl

compound of 15 16

37 20 14. 01. - formula (I)

5.6 .7 32 71 1.5

37 08 0 2 2 3 5 100

Methylketone

ethyl compound

of formula (I)

1.9

82 83 0 4 1 96 81 6 1 100

Allyl ethyl

compound of 14

- , _/τ 36 72 13.

1 91 0 2 4 75 61 6 1 100 1 1

Methylketone

Silicon

compound of 14

3 02 2 4 1 37 98 6 1 94.058 1 0

14 13

1_{1 1} · ι · 36 63 21. -

Allyl Silicon ^ ₃ ^ _{Q6 1 ?}

57 43 2 2 5 7 16 6 1 100 1 1 13

38 71 12. 14

compound of 6.4 .6 74 36. 1.3

formula (I) 14 01 0 2 6 25 62 4 1 100 1 1

QikProp's use of whole-molecule descriptors that have a straightforward physical interpretation (as opposed to fragment-based descriptors) could provide a useful pathway for medicinal chemists to modify ADME properties. QikProp has been thoroughly evaluated at many major pharmaceutical companies and found to be extremely useful in the context of both high-throughput library screening and lead optimization.

Schrodinger's QikProp is an extremely fast ADME properties prediction program. It provides the following benefits:

• Wide range of predicted properties: QikProp predicts the widest variety of pharmaceutically relevant properties - octanol/water and water/gas log Ps, log S, log BB, overall CNS activity, Caco-2 and MDCK cell permeabilities, human oral absorption,

log Khsa for human serum albumin binding, and log IC50 for HERG K+-channel blockage - so that decisions about a molecule's suitability can be made based on a thorough analysis. • Lipinski Rule-of-Five and Jorgensen Rule-of-Three: QikProp has the ability to check for Lipinski Rule-of-Five and Jorgensen Rule-of-Three violations to provide an at-a-glance measure of whether a compound is drug-like.

• Lead generation: QikProp rapidly screens compound libraries for hits. QikProp identifies molecules with computed properties that fall outside the normal range of known drugs, making it simple to filter out candidates with unsuitable ADME properties.

These shortlisted derivatives can be synthesized to optimize lead compound from compound of formula (I)

• Improving accuracy: QikProp computes over twenty physical descriptors, which can be used to improve predictions by fitting to additional or proprietary experimental data, and to generate alternate QSAR models.

The 27 and 251 derivatives were narrowed down to 6 due to the ADME filters , the results depicted in the above Table enabled the selection of six possible derivatives of compound of formula (I).

The druglikeliness of the six derivatives of compound of formula (I) based on the ADME results of QIKPROP validates its claim for synthesis.

Advantages of the invention:

1. The compound of the invention is effective against multiple drug resistant and extensive drug resistant strains of Mycobacterium tuberculosis.

2. The compound of the invention is also effective against other bacterial pathogens.

3. The compound of the invention is effective against Human Immuno Deficiency Virus (HIV).

4. The process of producing the compound of the invention is a simple process and does not require complex laboratory set-up. Therefore, the process of production of compound is economically viable.

5. The compound of the invention is a natural product. Also, the compound is produced through naturally occurring microorganisms. Therefore, the compound itself or the process of producing the same are eco-friendly and does not pose any threat to environment.

6. The compound shows very poor cytotoxic activity. Therefore, the compound can be effectively used to manufacture pharmaceutical formulations against bacterial and viral pathogens.

Claims

The claims:

A compound represented by formula (I)

5-(4-bromobutyl)-A^-(but-3-enyl)dodec-l l -enamide or a derivative thereof, wherein the compound is effective against bacterial and viral pathogens.

2. The compound as claimed in claim 1 , wherein the compound is brominated.

3. The compound as claimed in claim 1 , wherein the compound is 5-(4-bromo butyl)-N-

(but-3-enyl) dode-1 1 -enamide.

4. The compound as claimed in claim 1, wherein the compound is effective against bacterial pathogens such as Mycobacterium tuberculosis, Bacillus subtilis, Bacillus pumilus, Bacillus cereus, Staphylococcus aureus, and Acinetobacter baumanii.

The compound as claimed in claim 4, wherein the compound is effective against multiple drug resistant and extensively drug resistant strains of Mycobacterium tuberculosis.

The compound as claimed in claim 4, wherein the compound is effective against SHRE sensitive and SHRE resistant strains of Mycobacterium tuberculosis.

The compound as claimed in claim 1, wherein the compound is effective against Human Immuno Deficiency Virus (HIV).

A composition comprising the compound of claim 1 in an amount of 0.1 to 5C^g/ml along with pharmaceutically acceptable additives, excipients and adjuvants.

The composition as claimed in claim 8, wherein the composition is formulated in various forms such as liquid, solid, powder and lozenges.

A process of preparing the compound as claimed in claim 1 , said process comprising the steps of:

(i) inoculating Actinomycetes strain MTCC 5597 onto a suitable agar based media; (ii) incubating the agar plate(s) at a temperature of 20 °C to 40 °C for a period of 3 to 10 days and obtaining mycelial growth;

(iv) optionally cutting the media into pieces;

(v) adding the media pieces into a suitable solvent;

(vi) incubating the media dissolved in solvent at a temperature of 23 °C to 30 °C for a period of 3 to 18 hours and extracting the compound of formula

(i);

(vii) collecting the solvent part and concentrating the same;

(viii) obtaining the concentrate containing the compound of formula (I); and

(ix) purifying the compound.

11. The process as claimed in claim 10, wherein the media is selected from the group comprising yeast extract malt extract agar, glycerol asparagine agar, oatmeal agar, czapek's dox agar and tyrosine agar.

12. The process as claimed in claim 10(ii), wherein the agar plate(s) are preferably incubated at a temperature of 28 °C.

13. The process as claimed in claim 10(ii), wherein the agar plate(s) are preferably incubated for a period of 7 days.

14. The process as claimed in claim 10(vi), wherein the media dissolved in solvent is preferably incubated at a temperature of 28 °C.

15. The process as claimed in claim 10(vi), wherein the media dissolved in solvent is preferably incubated for a period of 24 hours.

16. The process as claimed in claim 10(vii), wherein the solvent part is concentrated using a rotary evaporator.

17. The process as claimed in claim 10, wherein the concentrate containing the compound of formula (I) is optionally stored at a temperature of 4°C to 25°C before purification step.

18. The process as claimed in claim 17, wherein the concentrate is stored at a temperature of 4 °C.

19. The process as claimed in claim 10(ix), wherein the purification is done by chromatography methods.

20. The process as claimed in claim 19, wherein the purification is done by thin layer chromatography and column chromatography.

21. The process as claimed in claim 10(ix), wherein the purification is done by crystallization methods such as single solvent recrystallization or multi solvent recrystallization.

22. The process as claimed in claim 10, wherein the solvent is an organic solvent.

23. The process as claimed in claim 22, wherein the solvent is selected from the group comprising methanol, chloroform, dichloromethane, diethyl ether, ethyl acetate and n-hexane.

24. An Actinomycete strain with accession no MTCC 5597 useful for producing the compound of formula (I) as claimed in claim 1.

25. A biologically active agent comprising a compound of formula (I) as claimed in claim 1 , wherein the agent is effective against bacterial and viral pathogens.

26. A kit comprising a compound of formula (I) as claimed in claim 1 along with an instruction manual.

27. A kit comprising the composition as claimed in claim 9 along with an instruction manual.

28. Use of compound of formula (I) against bacterial and viral pathogens.

29. Use as claimed in claim 27, wherein bacterial pathogens are selected from Mycobacterium tuberculosis, Bacillus subtilis, Bacillus pumilus, Bacillus cereus,

Staphylococcus aureus, and Acinetobacter baumanii.

30. Use as claimed in claim 27, wherein viral pathogen is Human Immuno Deficiency Virus (HIV).

31. A method of using compound of formula (I) against bacterial and viral pathogens.