Title of Invention	NOVEL COMPOUND NAMELY METHYL SUBSTITUTED DIOXA-TETRACYCLO HEPTADECA-2,4,6,11,13,15- HEXAENE
Abstract	Methyl substituted dioxa-tetracyclo heptadeca-2, 4, 6, 11, 13, 15-hexaene having antibacterial, antifungal, antihelicobatel pylori and antitubercular activity represented by formula (I) R] CH-0 CH3

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FORM 2 THE PATENTS ACT, 1970 (39 of 1970) COMPLETE SPECIFICATION (See section 10) "NOVEL COMPOUND NAMELY METHYL SUBSTITUTED DIOXA-TETRACYCLO HEPTADECA-2,4,6,11,13,15-HEXAENE" SAURASHTRA UNIVERSITY, an Indian University, of University Road, Rajkot 360 005, Gujarat, India, The following specification (particularly) describes the nature of the invention and the manner in which it is to be performed. lovel compound namely methyl substituted dioxa-tetracyclo heptadeca-2,4,6,11,13,15-hexaene FIELD OF THE INVENTION The present invention relates to novel compounds namely methyl substituted dioxa-tetracyclo heptadeca-2,4,6,ll,13,15-hexaenes, having antibacterial, antifungal, antihelicobacter pylori and antitubercular activity. BACKGROUND AND PRIOR ART REFERENCES. Pechmann condensation between phenols and p-ketonic esters like methyl acetoacetate under catalytic effect of various condensing agents lead to the formation of coumarins. Thus, it provides one of the excellent methods by which coumarin formation takes place. The otheT well known methods for coumarin synthesis is Perkin reaction [W. H. Perkin (1868), J. Chem. Soc, 21, 53&181; W. H. Perkin (1877), J. Chem. Soc, 3_L 388; J. R. Johnson (1942), Org. React., I, 210; H. O. House (1972),Modern Synthetic Reactions(W. A. Benjamin, Menlo Park, California, 2nd Edition pp 660-663;N. Poonia etal (1980), Bull. Chem. Soc. Japan, 53, 3338; T. Rosen (1991), Comp. Org. Syn., 2, 395-408]. For Pechmann condensation numbers of condensing agents are employed. In most of the cases, a considerable amount of the condensing agents has been employed. The iodine, bromine, concentrated sulphuric acid, phosphorous oxychloride, phosphorous trichloride, phosphorous pentoxide , aluminium chloride, zinc chloride and other Lewis acids may be employed at room temperature (28-32 C) for 24, 48 or more hours with or without a solvent [H. von Pechmann and C. Duisberg (1883), Ber., 16, 2119; D. W. Hill (1936), Chem. Rev., 19, 27; S. M. Sethna and N. M. Shah (1945), Chem. Rev., 36(1); S. S. Israelstam and E. Barris(1958), Chem. Ind., 1430 ; V. M. Thakor and A. B. Acharya (1971) Jour. Indian Chem. Soc.,48(5|. The reaction of dihydric phenols and trihydric phenols with 1,3-diketones like benzoylacetone was investigated earlier in presence of Pechmann condensing agents and the outcome of products are much studied and a comprehensive review has indicated some ambiguity in structures of reaction product also [H. von Pechmann and Duisberg (1883), Ber., 16, 2119; D. W. Hill (1936), Chem. Rev., 19, 27; B. N. Ghosh( 1915), J. Chem. Soc, 107, 739]. The reactivity of aromatic phenolic compounds with P-ketonic ester are much studied and many reviews has been given fairly good account of products formed, reagents employed, phenol used[R. C. Gupta et al (1982), Ind. J. Chem., 2KB), 344-7; C. Bulow and H. Wanger(1901), Ber., 34, 1189] None of the literature or prior art suggests the type and structure product formation from conditions similar to Pechmann condensation to a mono substituted phenol like m-cresol, p-cresol or disubstituted phenols like 2,3-xylenol or 2,4-xylenol. SUMMARY OF THE INVENTION The present invention provides novel compounds namely methyl substituted dioxa-tetracyclo heptadeca-2, 4,6,11,13,15-hexaenes having anti-microbial properties. The invention also provides for the first time novel reaction ie condensation of phenols with 1,3-diketones occurring in presence of Zinc Chloride as condensing agent. DETAILED DESCRIPTION OF THE INVENTION The present invention basically relates to novel compounds namely methyl substituted dioxa-tetracyclo heptadeca-2,4,ll,13,15-hexaene of formula as shown below: D„ p, R4 Methyl substituted dioxa-tetracyclo heptadeca-2,4-6,11,13,15-hexaenes, of the present invention show anti-microbial properties such as antibacterial , antifungal, antihelicobacter pylori and antitubercular activity. The preferred compounds arc selected from 1,4,9,14-tctramethyl-8,10-dioxa- Y tetracyc Jheptadeca-2,4,6,11,13,15-hexaene, 1,5,9,13- " ' tetrametnyi-5,iu-aioxa-tetracyc[6|7.7.1.0.0 3 exaene, 1,5,6,9,12,13-hexamethyl-8,10-dioxa-tetracyclof [7.7.1.0-2,7.0.11.1] heptadeca-2,4,6,11,13,15-hexaene and 1,4,6,9,12,13-hexamethyl-8,10-dioxa-tetracyclo.[7.7.1.0-2,7.0.11.1]1ieptadeca-2,4,6,11,13,15-heaenc. Another embodiment of the invention relates to a process for preparing above compounds by reacting phenols and acetylacetone in presence of zinc chloride as a condensing agent. In the process of the present invention, the applicants claims that when an alkyl phenol where methyl groups is present in the meta or para position (m-cresol, or p-cresoi) in this case or dialkyl phenol, where two methyl groups are present in vicinity to phenol (2,3-dimethyl phenol or in case of 2,4-dimethyl phenol) then only the said reaction occurs. In the present case, that mono/di alkyl phenol has been condensed with a 1,3 diketone i.e. acetyl acetone in presence of Zinc chloride ands in unusual mode of reaction sequence depicted below, the reaction proceeds by taking up phenolic moiety in two steps leading to the formation of a special structure of compound as below: Structure Rl R2\ ,J\ .OH R3^ v + CH3COCH2COCH3 ^R5 Lewis acid »■ Room Temperature 24-48 hours Where; Rl,R4-H R^ = CH3 Rl,R2 = (CH3)2 R3 = CH3 R1,R3=(CH3)2 following are typical examples of reaction products and their IUPAC nomenclatures Example-1 Synthesis of l,4,9,t4-tetramethyl-8,10-dioxa-tetracyclo[7.7.1.0-2,7.0.11.16] heptadcca-2,4,6,11,13,15-hcxaene. 4-Methyl phenol (0.05 mole) was mixed with acetyl acetone (0.05 mole) in presence of anhydrous zinc chloride (0.15 mole). The reaction mixture was mixed well and protected from moisture. It was heated on steam-bath with gradual stirring for 12 hours. It was then poured over crushed ice with constant stirring, the solid product l,4,9,14-tetramethyl-8,10-dioxa-tetracyclo 1 [[7.7.1.0-2,7.0.11.16] heptadeca-2,4,6,11,13,15-hexaene obtained, was filtered, washed with water, followed by dilute sodium hydroxide solution (18%) and finally with DM water. It was dried and crystallized from ethanol, as colorless needles. Yield: 18%, Melting Point 220°C; Molecular Formula: Ci9H20O2 Calculated: C: 81.42, H: 7.14, O: 11.42; Found: C: 81.56, H: 7.22, O: 11.23; IR (KBr) cm"1 2937, 2933 [Alkane (-CH3) C-H str. (sym. & asym.)], 2857 [CH2, C-H str.], 1052, 1131 [Ether, C-O-C str.]; H]-NMR (300 MHz, DMSO-d6) 5 ppm: 1.80 (s, 3H, -CH3), 1.81 (s, 3H, -CH3), 2.11 (s, 2H, -CH2), 2.20 (s, 6H, -CH3), 6.66-6.68 (m, 4H, Ar-H), 7.16 (d, 2H, J=8.3Hz, Ar-H); Mass: 280 (M\23.1%), 265 (100%). ExampIe-2 Synthesis of 1,5,9,13-tetramrthy1-8,10-dioxa-tetracy[7.7.1.0-2,7.0.11.1] heptadcca-2,4,6,11,13,15-hexaene 3-Methyl phenol (0.05 mole) was mixed with acetyl acetone (0.05 mole) in presence of anhydrous zinc chloride (0.15 mole). The reaction mixture was mixed well and protected from moisture. It was heated on steam-bath with gradual stirring for 12 hours. It was then poured over crushed ice with constant stirring, the solid product l,5,9,13-tetramethyl-8,10-dioxa-tetracyclo [7.7.1.0-2,7.0.11.1] heptadeca-2,4,6,11,13,15-hexaene obtained, was filtered, washed with water followed by dilute sodium hydroxide solution (18%) and finally with DM water. It was dried and crystallized from ethanol, as colorless needles. Yield: 24%, Melting Point 169°C; Molecular Formula: C19.,H2()02 Calculated: C: 81.42, H: 7.14, O: 11.42; Found: C: 1.54, H: 7.27, 0: 11.19; IR (KBr) cm"' 2972, 2940 [Alkane (-CH3) C-H str. (sym. & asym.)l, 2862 [CH2, C-H str.], 1043, 1127 [Ether, C-O-C str.]; H^NMR (300 MHz, DMSO-d6) 5 ppm: 1.81 (s, 3H, -CH3), 1.82 (s, 3H, -CH3), 2.10 (s, 2H, -CH2), 2.24 (s, 6H, -CH3), 6.71 (d, 2H, J=8.2Hz, Ar-H), 6.82 (dd, 2H, J=8.3,1.7Hz, Ar-H); Mass: 280 (M+,25%), 265 (100%). Example-3 Synthesis of l,5,6,9,12,13-hexamethyl-8,10-dioxa-tetracyclo ,3-Dimethyl phenol (0.05 mole) was mixed with acetyl acetone (0.05 mole) in presence of anhydrous zinc chloride (0.15 mole). The reaction mixture was mixed well and protected from moisture. It was heated on steam-bath with gradual stirring for 12 hours. It was then poured over crushed ice with constant stirring, the solid product l,5,6,9,12,13-hexamethyl-8,10-dioxa-tetracyclo (7.7.1.0.01, heptadeca-2,4,6,ll,13,15-hexaene obtained, was filtered, washed with water, followed by dilute sodium hydroxide solution (18%) and finally with DM water. It was dried and crystallized from ethanol, as colorless needles. Yield: 33%, Melting Point 194 C; Molecular Formula: C21H24O2 Calculated: C: 81.81, H: 7.79, O: 10.38; Found: C: 81.50, H: 8.02, O: 10.30; IR (KBr) cm4 2975, 2935 [Alkane (-CH3) C-H str. (sym. & asym.)], 2862 [CH2, C-H srr.]? 1052, 1127 [Ether, C-O-C str.J; H'-NMR (300 MHz, DMSO-d6) 5 ppm: 1.79 (s, 3H, -CH3), 1.86 (s, 3H, -CH3), 2.07 (s, 2H, -CH2), 2.11 (s, 6H, -CH3), 2.16 (s, 6H, -CH3), 6.65 (d, 2H, J=7.8Hz, Ar-H), 7.05 (d, 2H, J=7.8Hz, Ar-H); Mass: 308 (M+,23%), 293 (100%), 187 (41.4%). Example-4 Synthesis of l,4,6,9,12,13-hexamethyl-8,10-dioxa-tetracyclo [7.7.1.0-2,7.0.11.16]1 heptadeca-2,4,6,11,13,15-hexacne. 2,4-Dimethyl phenol (0.05 mole) was mixed with acetyl acetone (0.05 mole) in presence of anhydrous zinc chloride (0.15 mole). The reaction mixture was mixed well and protected from moisture. It was heated on steam-bath with gradual stirring for 12 hours. It was then poured over crushed ice with constant stirring the soild product 1,4,6,9,12,13-hexamcthy!-8,10-dioxa-tctracyclo [7.7.1.0.0] heptadeca-2,4,6,11,13,15-hexaene obtained, was filtered, washed with water, followed by dilute sodium hydroxide solution (18%) and finally vyith DM water. It was dried and crystallized from ethanol, as colorless needles. Yield: 13%, Melting Point 262 C; Molecular Formula: C2IH2402 Calculated: C: 81.81, H: 7.79, 0: 10.38; Found: C: 81.79, H: 7.75, 0: 10.33; IR (KBr) cm'1 2975, 2942 [Alkane (-CH3) C-H str. (sym. & asym.)], 2858 [CH2, C-H str.], 1057, 1151 [Ether, C-O-C str.]; H]-NMR (300 MHz, DMSO-d6) 5 ppm: 1.78 (s, 3H, -CH3), 1.82 (s, 3H, -CH3), 2.05 (s, 2H, -CH2), 2.15 (s, 6H, -CH3), 2.21 (s, 6H, -CH3), 6.71 (d, 2H, J=1.3Hz, Ar-H), 6.96 (d, 2H, J=1.2Hz, Ar-H); Mass: 308 (M+,28%), 293 (100%), 187 (43.1%). FOLLOWING ARE BIOLOGICAL ACTrVTTY PROFILE OF NEW COMPOUNDS FOR PHARMACEUTICAL USE AS ANTIBACETRIAL, ANTIHELICOBACETER PYLORI, ANTIFUNGAL AND ANTITUBERCULAR ACTIVITIES. Pharmacologically Experimental example will now be described to illustrate the usefulness of the compounds of the present invention. Experimental method It was according to method of Dastidar, S. G., Chaudhury, A., Annadurai, S. et al., In vitro and in vivo antimicrobial action of fluphenazine. J. Chemother. 7, 201 (1995). 1. Bacteria Gram-positive bacteria {Escherichia coli, Shigella sonnei, Shigella aysenteriae, Salmonella typhi, Salmonella typhimurium, Vibrio cholerae) and Gram-positive bacetira (Staphylococuss aureus, Bacillus licheniformis) were received as freeze-dried ampoules. H.pyiori (ATCC 43504) was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). 2. Measurement of MIC on antibacterial activity. Gram-positive bacteria were grown 'in nutrient broth and Gram-negative bacteria in peptone water (PW, 1.0% bacteriological peptone (Difco) plus 0.5% NaCl) for 18 h; this gave an optimum growth of the test bacteria. Each compound was dissolved in dimethylsulfoxide (DMSO, 5mg/ml) sterilizedd by filtration by usin g 7 sintered glass filter (G-5) and stored at 4 °C. Each agent was then added to molten nurtrient agar in the following concentration (pg/ml): 0 (control), 10, 25, 50, 100, 200, 400, 800 and 1000 and poured into sterile petri dishes. The pH of the media was maintained at 7.2 to 7.4. the inoculum consisted of an overnight grown broth culture of a bacterium diluted in such a manner that a 2mm (internal diameterO loopful of the culture 105 colony hyphen forming units (CFU): These were then spot inoculated on NA plates containing increasing amount of a compound, incubated at 37 °C up to 72 h for determination of the minimum inhibitory concentration (MIC). 3. Measurement of anti-H. pylori activity Evaluation of Antimicrobial activity of Substituted l,9-dimethyI-8, 10-dioxa-tetracycIo[[7.7.1.0-2,7.0.11.1]hepatadeca-2,4,6,11,13,15- The micro-dilution broth method was used to determine the MIC (minimum inhibitory concentration). Mueller-Hilton broth containing 5% fetal bovine serum (FBS) was as the medium, and was cultured in a jar conditioned with Campylo Pack (Dia latron) for 48 hrs. Briefly, H. pylori strains were inoculated on a Brucella agar plate containing 10% jorse serum, and cultured at 37°C for 48 hrs. the bacterial colonies collected were diluted to 107 colony forming unit (UFC)/mL with 0.9% saline. The fractions were dissolved in DMSO, and then diluted with Mueller-Hilton broth. To the solution of the fractions, a suspension solution of the bacteria was added to make 106 CFU/100 mL/well. The mixture was incubated at 37°C for 48 hrs. MIC values of fractions tested were determined by observation. Table: Compd. No. hexaene ^*w_ _,. .» »*,»~ I II III IV V VI VII VIII IX X XI XII XIII XIV XV 1 25 25 25 1000 200 800 800 200 200 25 800 800 50 50 >100 2 25 25 100 1000 800 100 200 800 800 800 50 50 25 25 >100 3 50 20 50 1000 200 100 200 25 100 50 50 20 50 20 >100 4 50 50 30 1000 50 50 100 25 400 25 3'2 50 30 43 49 Me ND ND ND ND ND ND ND ND ND ND ND ND ND ND 74 C1 ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1.9 Er ND ND ND ND ND ND ND \ ND ND ND ND ND ND ND 1.8 I: S. aureus NCTC 8530; II: S. aureus NCTC 8531; III: 5. aureus ML 36; IV: B. lithruformis 10341; V: Shigella Sonnie 56; VI: Sh. dysenteriael; VII: Sk dysenteriae 2; VHI: Salmonella typhi 57; DC: S. typhimurium 74; X: E. coli Row; XI: V. Chloerae 517; XII: V. Chloerae 865; XIII: V. Chloerae 945; XIV: V. Chloerae 976; XV: Helicobacter pylori ATCC 43504. Me: Metronidazole; C1: Clarithromycin; Er: Erythromycin 4. Measurement of antifungal activity It was according to method of Bentley, A. C, In; Rawlins, E. A., Eds., Bentleny's Text book of Pharamaceutics., 8th Ed., ELBS, London, 517 (1980). For antifungal activity the compounds were tested at 5mg/ml concentration. The method used to study antifungal activity was the disc-plate method against Candida albicans and A. niger. Table: Evaluation of Antifunga Lactiyity of ubstituted l,9-dimethyl-8, 10- dioxa-tetracycl([7.7.1.0-2,7.0.11.1]heptadeca-2,4,6,ll,13,15-hexaene Compd. No. Candida albicans A. Niger 1 6.2 9.4 2 10.0 8.1 3 6.2 8.5 4 9.5 6.8 Clotrimazole 2.0 2.5 5. Measurement of antitubercular activity It was according to method of Collins, L.; Anitirnicrob. Agent Chemother., 41, 1004-1009 (1997). Antitubercular activity was determined using the BACTEC 460 system as modified below. Stock solutions as test compounds were prepared in dimethylsulfoxide (DMSO) at l mg/ml and sterilized by passage through 0.22 pm PFTE filters (Millex-FG, Millipore, Bedford MA). Fifty microliters was added to 4 ml radiometric 7H,2 broth (BACTEC 12B; Becton Dickinson Diagnostic Instrument System, Sparks, MD) to achieve a final concentration of 6.25 ng/ml. Controls 9 received 50 ul DMSO. Rifampin (Sigma Chemical Co., St. Louis, MO) was included as a positive drug control. Rifampin was solubilized and diluted in DMSO and added to BACTEC-12 broth to achieve a range of concentration for determination of minimum inhibitory (MIC, lowest concentration inhibiting 99% of the inoculum). M. Tuberculosis H37RV (ATCC 27294; American type culture collection, Rockville, MD) was cultured at 37°C on a rotary shaker in middle brook 7H9 broth (Difco Laboratories, Detroiet, M1) supplemented with 0.2 v/v glycerol and 0.05% v/v Tween 80 until the culture turbidity achieved an optical density of 0.45-0.55 at 550 run. Bacteria were then pelleted by centrifugation, washed twice and resuspended In one fifth the original volume in dulbecoo's phosphate buffered saline (PBS, Irvine Scientific Santa, Nalgene, Rochester, NY) and aliquots were frozen at 80°C. Cultures were showed and an appropriate dilution performed such that a BACTEC-12B vial inoculated with a 0.1 ml would reach a growth index (GI) of 999 in 5 days. One tenth of the diluted inoculum was used to inoculate 4 ml fresh BACTEC 12B broth containing the test compounds. An additional control vial was included which received a further 1:100 diluted inoculum (as well as 50 ul DMSO) for use in calculating the MIC of Rifampin, respectively by establishing procedures. Cultures were incubated in 37°C and the GI determined daily until control cultures achieved a GI of 999. Assays were usually completed in 5-8 days. Percent inhibition was defined as-1- (GI of test sample/GI of control) xlOO. Minimum inhibitory concentration of compound effecting a reduction in daily change in GI, which was less than that, observed with a 1:100 diluted control culture on day the latter reached a GI of at least 30. Table: Evaluation of antitubercular activity of Substituted l,9-dimethyI-8 10-dioxa-tetracyclo[7.7.1.0-2,7.0.11.1]hepatadeca-2,4,6,11,13,15- hexaene Compd. No. Assay MIC (pg/ml) % inhibition 1 BACTEC 12.5 23 2 BACTEC 12.5 41 3 BACTEC (>12.5 55 4 BACTEC \>12.5 / 91 The compounds of the invention can be used) as active ingredients in novel pharmaceutical compositions having anti-bacterial antifungal, anti-helicobacter pylori and antitubercular activity. These compositions are prepared according to processes known in the art. 10 WE CLAIM: 1. Methyl substituted dioxa-tetracyclo heptadeca-2, 4, 6, 11, 13, 15-hexaene having antibacterial, antifungal, antihelicobatel pylori and antitubercular activity represented by formula (I) R] CH-0 CH3 Formula (I) 2. Methyl substituted dioxa-tetracyclo heptadeca-2, 4, 6,11,13,15-hexaene as claimed in claim 1, wherein the compound is 1, 4, 9, 14-tetramethyl-8,10-dioxa-tetracyclo heptadeca-2, 4,6,11,13,15-hexaene. 3. Methyl substituted dioxa-tetracyclo heptadeca-2, 4,6,11,13, 15-hexaene as claimed in claim 1, wherein the compound is 1, 5, 9, 13-tetramethyl-8, 10-dioxa-tetracyclo heptadeca-2, 4,6,11,13,15-hexaene. 4. Methyl substituted dioxa-tetracyclo heptadeca-2, 4,6,11, 13, 15-hexaene as claimed in claim I, wherein the compound is 1,5,6,9,12, 13-hexamethyl-8, 10-dioxa-tetracyclo heptadeca-2, 4,6,11,13,15-hexaene. 5. Methyl substituted dioxa-tetracyclo heptadeca-2, 4,6, 11, 13, 15-hexaene as claimed in claim 1, wherein the compound is 1,4,6,9,12, 13-hexamethyl-8, 10-dioxa-tetracyclo heptadeca-2, 4,6,11,13,15-hexaene. Dated this 30th March, 2001 ANUPAM TRIVEDI OF K& S PARTNERS AGENT OF THE APPLICANTS

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