Indian Patents. 279922:NITRIC OXIDE RELEASING NITRATE ESTER DERIVATIVES OF PARACETAMOL HAVING ANALGESIC, ANTI-INFLAMMATORY, HEPATOPROTECTIVE ACTIVITY AND PROCESS FOR THEIR PREPARATION

Title of Invention	NITRIC OXIDE RELEASING NITRATE ESTER DERIVATIVES OF PARACETAMOL HAVING ANALGESIC, ANTI-INFLAMMATORY, HEPATOPROTECTIVE ACTIVITY AND PROCESS FOR THEIR PREPARATION
Abstract	The present invention particularly relates to novel nitrate esters of paracetamol. The nitrate esters of paracetamol are prepared by reacting the paracetamol with dihaloalkyl compound and followed by reaction with silver nitrate to obtain the corresponding nitrate ester derivatives. The nitrate esters of paracetamol are useful as analgesic, antiinflammatory agents.

Full Text	FIELD OF THE INVENTION The present invention relates to nitric oxide releasing derivatives of paracetamol The present invention particularly relates to novel nitrate esters of paracetamol The present invention pertains to process of preparation of nitrate esters of paracetamol More particularly, the invention relates to nitric oxide releasing derivatives of paracetamol which show better analgesic and anti-inflammatory activities and decreased liver toxicities as apparent from their biochemical and histopathologic profile BACKGROUND OF THE INVENTION Paracetamol (paracetamol, 1) is among most commonly used analgesics It effectively reduces fever and mild-to moderate pain, and is regarded, in general, as a very safe drug Nevertheless, overdose (deliberate) is a common cause of hepatic injury, accounting for ~ 40% of cases of acute liver failure in the USA [Wu, C C Nitric oxide and inflammation Curr Med Chem - Anti-inflammatory & anti-allergy agents, 2004, 3, 217-222, Joshi, G P NCX-701 NicOx Curr Opin Investig Drugs, 2004, 5, 755-759, and Moore, P K, Marshall, M Nitric oxide releasing paracetamol (nitroparacetamol) Dig Liver Dis, 2003, 35 ] It is an effective analgesic and antipyretic agent and unlike other NSAIDs (Non-steroidal Anti-inflammatory Drugs) has been reported to have little antiinflammatory effects [ Fiorucci, S , Antonelh, E , Mencarelh, A , Palazzetti, B , Alvarez-Miller, L , Muscara, M , del Soldato, P , Sanpaolo, L , Wallace, J L , Morelh, A A NO-releasing derivative of paracetamol spares the liver by acting at several checkpoints in the Fas pathway Br J Pharmacol, 2002, 135, 589-599 ] It is metabolized in liver by three pathways-glucuromdation, sulfonation (both accounting for 95% of metabolism) or via cytochrome P450 enzyme system (5%) in which it is converted to a toxic metabolite (N-acetyl-p-benzoqumone lmine, 2) which further is rendered harmless through an interaction with the endogenous antioxidant glutathione (Formula Removed) But overproduction of this toxic metabolite leads to depletion of glutathione stores in liver and further accumulation of the toxic metabolite causes tissue injury and cell death [ Futter, L E, al-Swayeh, O A, Moore, P K A comparison of the effect of nitroparacetamol and paracetamol on liver injury Br J Pharmacol, 2000, 132, 10-12 ] Numerous derivatives of paracetamol have been synthesized to get a safer non-hepatotoxic drug One of the major achievements has been by Nocox in Italy It has designed NCX-701 (3) by adding NO moiety to paracetamol, via an aliphatic spacer to improve the anti-inflammatory activity of paracetamol, based on the well-demonstrated activity of NO on cytokine synthesis and regulation of proinflammatory mediators [Wallace, J L Paracetamol hepatotoxicity NO to the rescue Br J Pharmacol, 2004,143, 1-2 ] (Formula Removed) NO may modulate spinal and sensory neuron excitability through multiple mechanisms that may improve the anti-nociceptive activity of paracetamol The pharmacological profile of NCX 701 has been reviewed [ Marshall, M , Moore, P K Effect of nitric oxide releasing paracetamol and flurbiprofen on cytokine production in human blood Eur J Pharmacol, 2004, 483, 317-322, and al-Swayeh, O A, Futter, L E, Clifford, R H, Moore, P K Nitroparacetamol exhibits anti-inflammatory and anti-nociceptive activity Br J Pharmacol, 2000, 130, 1453-1456] Compared with NSAIDs, paracetamol possesses little anti-inflammatory activity in humans and it does not induce damage of the gastric mucosa, but it could cause liver damage (or even poisoning) when used in high dosage The aim of coupling paracetamol with NO has, therefore, been to improve its anti-inflammatory activity, and also to exploit the cytoprotective properties of NO to reduce potential liver damage Concerning safety, several studies have highlighted the liver sparing profile of NCX 701 compared to paracetamol The beneficial protective effect on the liver has been supposed to be related to the fact that NO can reduce the synthesis of several pro-inflammatory cytokines [ Wallace, J L Paracetamol hepatotoxicity NO to the rescue Br J Pharmacol, 2004, 143, 1-2] The antiinflammatory activity related to NO release has been demonstrated in vitro and in vivo, [Marshall, M , Moore, P K Effect of nitric oxide releasing paracetamol and flurbiprofen on cytokine production in human blood Eur J Pharmacol, 2004, 483, 317-322 , al-Swayeh, O A, Futter, L E, Clifford, R H, Moore, P K Nitroparacetamol exhibits anti-inflammatory and anti-nociceptive activity Br J Pharmacol, 2000, 130, 1453-1456, AND, Paul Clark, M J, Howat, D , Flower, R J, Moore, P K , Perretti, M Nitroparacetamol (NCX-701) exhibits anti-inflammatory activity in the zymosan air pouch Inflamm Res, 2001, 50, S158]demonstrating that NCX 701 is not only a safer drug than paracetamol, but that it also possesses a wider action due to additive mechanisms Several papers have compared the anti-nociceptive activity of NCX 701 versus paracetamol, [Romero-Sandoval, E A , Mazano, J, Howat, D , Herrero, J F NCX-701 (nitroparacetamol) is an effective antinociceptive agent in rat withdrawal reflexes and wind-up Br J Pharmacol, 2002, 135, 1556-1562 , Romero-Sandoval, A E, Del Soldato, P , Herrero, J F The effects of sham and full spinahzation on the antinociceptive effects of NCX-701 (nitroparacetamol) in monoarthritic rats Neuropharmacology, 2003, 45, 412-419 , and Gaitan, G , Del Soldato, P , Herrero, J F Low doses of nitroparacetamol or dexketoprofen trometamol enhance fentanyl antinociceptive activity Eur J Pharmacol, 2003, 481, 181-188 ] regularly showing the superiority of NCX 701 Moreover, sub-effective doses of NCX 701 have enhanced the anti-nociceptive activity of the u-opioid receptor agonist fentanyl A Phase II study clinical trial has demonstrated that lg NCX 701 provides similar analgesic efficacy to paracetamol lg in post-operative dental pain, therefore with a reduced exposure to paracetamol, confirming a contribution of NO to the anti-nociceptive mechanisms of NCX 701 O-Dealkylation of a homologous series of alkoxy acetanihdes [p-methoxy, p-ethoxy phenacetin, p-(n)-propoxy- and p-(n)-butoxy acetanihdes, 4-7] have shown to release paracetamol The compounds (6 and 7) were structurally related to phenacetin (5), and were synthesized to check whether they release paracetamol (1) upon O-dealkylation, which they did The chain length in the synthesized compounds was varied to study the effect of chain lengthening on the rate of release of paracetamol [Chul-Ho, Y , Miller, G P , Guengench, F P Oxidations of p-Alkoxyacylanihdes Catalyzed by Human Cytochrome P450 1A2 Structure-Activity Relationships and Simulation of Rate Constants of Individual Steps m Catalysisf Biochemistry 2001, 40, 4521-4530] The compounds (4, 6 and 7) are structurally related to phenacetin (5) therefore, on metabolism they will liberate paracetamol (1) (Formula Removed) Release of paracetamol (1) by alkoxy acetamhdes (4-7) There was a need to develop the novel molecules which are having better analgesic and antnnflamatory activity The advantages of the present invention are that these NO releasing compounds have shown better analgesic and antiinflammatory activity than the parent drug, and less liver toxicity in biochemical and histopathological studies OBJECTS OF THE INVENTION The main object of the present invention is to provide novel nitrate esters of paracetamol Another object of the present invention is to provide process for preparation of novel nitrate esters of paracetamol Yet another object of the present invention is to develop nitric oxide releasing derivative of paracetamol that shows better analgesic, anti-inflammatory, biochemical (SGOT, ALP levels) and histopathologic profile Further object of the present invention is to develop a pharmaceutical composition comprising nitric oxide releasing derivatives of paracetamol Still another object of the present invention is to inhibit COX-2 in vitro and release NO in \i\o Compound (13) has shown to have better COX-2 (in vitro) inhibition activity l e 32 5% than the parent drug paracetamol (1) which had 24 0% The compounds 12 and 13 also released NO in vivo SUMMARY OF THE INVENTION The present invention relates to the synthesis of NO-releasing derivatives of paracetamol and their evaluation The compounds showed better analgesic, anti-inflammatory, biochemical and histopathologic profile The present invention particularly relates to novel nitrate esters of paracetamol The nitrate esters of paracetamol are prepared by reacting the paracetamol with dihaloalkyl compound and followed by reaction with silver nitrate to obtain the corresponding nitrate ester derivatives BRIEF DESCRIPTION OF THE FIGURES Figure 1 Analgesic activity of paracetamol (1) and NO-NSAIDs (12 and 13) 10 and 11 are intermediates only which were later converted to -ONO2 moiety on treatment with AgN03 Figure 2 % increase in SGOT levels after 6 hours Figure 3 % increase in ALP levels after 6 hours (Explaination for Figure 2 and Figure 3) Overproduction of toxic metabolite 2 leads to depletion of glutathione stores in liver and further accumulation of the toxic metabolite causes tissue injury and cell death Enzymes like alkaline phosphatase (ALP, EC 3 13 1), serum glutamate pyruvate transaminase (SGPT) or alanine aminotransferase (ALT) (EC 2 6 12) and the total proteins are considered as plasma markers of liver injury Nitric oxide releasing paracetamol derivatives (12 and 13) were evaluated for their effect on the markers of liver injury like enzymes ALP and SGPT/ALT The plasma levels of these enzymes was found to increase in the case of liver toxicity The graph indicates that the rise (with comparison to the basal value) in the case of 1 is much more than in the case of NO releasing derivatives (12 and 13), showing that they cause lesser liver toxicity than the parent drug paracetamol (1) Figure 4 Nitrite ion concentration (umol/L) in serum after 6 hours The graph indicates that the serum NO concentration 6 hours after administration of 12 and 13 was greater than the control (0 9% w/v sodium chloride containing 20% v/v tween-80) confirming that these compounds release NO in vivo Figure 5 Histopathology (hacmatoxylm and eosin) of liver samples Light microscopic analysis of liver samples obtained from mouse treated with control (0 9% w/v sodium chloride containing 20% v/v tween-80), paracetamol (1), (12) and (13), at a dose of 1000 mg/kg, lp is shown in sections (A), (B), (C), and (D), respectively The interpreted results obtained from histopathological studies are summarized below (1) Control did not cause any damage to the liver Only mild portal triad inflammation was observed (n) Paracetamol (1) a proven liver toxic caused ballooning degeneration, sinusoidal decongestion, portal triad inflammation and spoty necrosis in Zone I (in) Just mild ballooning degeneration was observed in the case of 12 (IV) Compound 13 also didn't cause any significant damage to the liver These results were supportive m summarizing that NO-release counteracts the liver toxic side effects of paracetamol (1) DETAILED DESCRIPTION OF THE INVENTION Accordingly the present invention provides nitrate ester derivatives of paracetamol of general formula A, wherein the value of n is selected from 2 to 5 (Formula Removed) In an embodiment of the invention wherein the structural formula A comprising (Formula Removed) In an embodiment of the invention wherein the representative compounds of the general formula A comprising (1) N-[4-(2-Nitrooxy-ethoxy)-phenyl]-acetamide (12) (n) N-[4-(5-Nitrooxy-pentoxy)-phenyl]-acetamide (13) (in) N-[4-(3-Nitrooxy-propoxy)-phenyl]-acetamide (18) (IV) N-[4-(4-Nitrooxy-butoxy)-phenyl]-acetamide (19) In another embodiment of the invention wherein the compound of formula A is having better peripheral analgesic activity and significant anti-inflammatory activity as compared to paracetamol In yet another embodiment of the invention wherein the compounds possess better analgesic and antiinflamatory activity and devoid of toxicity as compared to the parent drug paracetamol (1) In still another embodiment of the invention wherein the compounds 12 and 13 showed 32 68±1 45% and 31 15±1 31% at 2 hr, and, 26 44±0 10% and 28 45±1 28% at 4 hr increase respectively, in the paw volume as compared to 38 37±1 26% and 58 92±1 56% (at 2 hr and 4hr respectively) in case of paracetamol at a dose of l00mg/kg body weight In a further embodiment of the invention wherein the compounds 12 and 13 showed increase in NO serum level 354 2±2 17 and 265 4±2 47 respectively wrt control (182 1±0 68) at a dose of 100mg/kg body weight In an embodiment of the invention wherein the compound 13 showed In vitro cox-2 (ovine) inhibitory activity 32 5% as compared to control 24 0% Accordingly the present invention rovides a process for preparation of nitrate ester derivative of paracetamol comprising the steps a) reacting paracetamol with dibromo alkane in organic solvent in presence of alkali carbonate for a period up to 12 hours, b)filtenng the reaction mixture followed by evaporation of solvent to obtain solid residue, c)dissolving the residue in water immisible solvent and washing with sodium hydroxide solution followed by washing with water,evaprating the solvent to O-alkylated derivative of paracetamol, d)reacting the O-alkylated bromodenvative derivative of paracetamol with sliver nitrate under reflux in an aprotic solvent upto 12 hours,further filtering and evaporating the filterate to obtain the nitrate esters derivative of Paracetamol of general formula A wherein the value of n is selected from 2-5 In an embodiment of the invention wherein dihalo alkane may be selected from a group consisting of 1,2-dibromoethane, 1,3-dibromopropane, 1,4-dibromobutane and 1,5-dibromopentane In an embodiment of the invention wherein the organic solvent used may be selected from a group consisting of ethyl methyl ketone, acetone, acetomtnle and mixture thereof In an embodiment of the invention wherein the aprotic solvent used may be selected from a group consisting of acetomtnle, benzene, hexane and mixture thereof The pharmaceutical composition may be prepared using the compounds of the general formula A along with the pharmaceutical^ acceptable excepients Compounds 12 and 13 are novel and have not been reported earlier Compound (13) has shown to have better COX-2 inhibition activity (in vitro) than the parent drug paracetamol The data of analgesic and anti-inflammatory activites is exactly complimenting and supporting it Therefore, it can be concluded that the NO releasing compound is showing better activity than the parent drug candidate, and moreover, the biochemical and histopathological studies carried out, indicate that the compound is causing less liver toxicity than paracetamol NO-releasing derivatives of paracetamol, (12) and (13) showed good anti-inflammatory activity wrt parent drug, both at 2 hr and 4 hr interval No significant activity was observed in case of paracetamol (1) Oalkylated NO-releasing derivatives (12 and 13) of paracetamol (1) exhibited better response than the parent drug indicated by greater % inhibition in writhings The intermediates 10 and 11, having terminal Br (which was later converted to -ONO2 moiety on treatment with AgNOs) showed very negligible % inhibition in writhings indicating that NO release is responsible for better analgesic activity of the compounds 12 and 13 (having NO releasing -ONO2) moiety In NO releasing activity they also showed significant increase in level of NO The NO release of these compounds could be the acting force behind their better analgesic and antiinflammatory activities than the parent drug Our compound (13) has shown to have better COX-2 (in vitro) than the parent drug paracetamol In the case of biochemical studies paracetamol (1) caused a significant increase in the serum ALP levels as compared to the NO-releasing derivatives (12 and 13), indicating its liver toxicity Both the derivatives were found to increase the levels of ALP but not more than the drug Increase in the levels of SGPT (ALT) δ hr after administration of paracetamol (1) justified its liver toxicity, with % increase more than 80% Increase m the case of both the derivatives (12 and 13) was not that significant In histopathological studies, also the damage caused by 12 and 13, to liver was less m comparison to paracetamol (1) Therefore, compounds 12 and 13 showed better activity and lesser toxicity than the parent drug 1 Paracetamol (1), a proven liver toxic and having no anti-inflammatory activity of its own has been converted to more active and lesser toxic NO releasing compounds 12 and 13 Nitric oxide releasing derivatives of paracetamol are developed (12 and 13) (Figure 2), which have shown better analgesic, anti-inflammatory, biochemical (SGOT, ALP levels) and histopathologic profile Besides these derivatives have shown to inhibit COX-2 in vitro and release NO in vivo The liver toxic drug paracetamol (1) having no antiinflammatory activity has been converted to less liver toxic and more active derivatives Briefly, Paracetamol was stirred in a solution of 1, 2-dibromoethane (8) and 1, 5-dibromopentane (9), in ethyl methyl ketone and K2CO3 for 8-10 hours The resulting solution was filtered, dried and evaporated to get a solid residue The residue was dissolved in CHC13 and washed with 5% NaOH (3 X 100ml) and water (3 X 100ml), dried over anhydrous Na2SO4, filtered and solution evaporated to get O-alkyl derivatives 10 and 11, respectively These were refluxed in a solution of AgN03 and acetomtnle overnight, filtered, and evaporated to get nitrate esters 12 and 13, respectively (Formula Removed) Synthesis of NO releasing derivatives (12 and 13) of paracetamol (1) Following examples are given by way of illustration and should not construed to limit the scope of the invention Example 1 N-[4-(2-Bromo-ethoxy)-phenyl]-acetamide (10) A solution of N-(4-hydroxyphenyl)-acetamide (paracetamol) (1) (1 50 g, 0 01 mol) and 1,2-dibromoethane (8) (10 0 ml) in ethyl methyl ketone (25 0 ml) containing potassium carbonate (5 0 g) was stirred for 8 hr at room temperature The reaction mixture was filtered, residue washed with ethyl methyl ketone and solvent evaporated under reduced pressure to get the residue The residue was dissolved m chloroform (200 0 ml), washed with 5% NaOH solution (3 x 100 ml), water (3 x 50 ml), dried, filtered and crystallised from methanol to get the desired product N-[4-(2-bromo-ethoxy)-phenyl]-acetamide (10) (2 01 g, 78 51 %), mp 126-127° C Anal IR (KBr) 3302, 3024, 1662, 1551,1242, 1029 and 828 cm l 1H NMR (CDC13) δ 2 10 (s, 3H, -NHCOCHc), 3 65 (t, 2H, J = 5 9 Hz, CH2Br), 4 26 (t, 2H, J = 5 9 Hz, -OCH2), δ 83 (d, 2H, Ar-H), 7 49 (d, 2H, Ar-H), 9 42 (s, 1H, -NH Exchangeable with D2O) 13C NMR (CDCI3) δ 23 49 (-NHCOCH3), 29 03 (-CH2Br-), δ7 66 (-OCH2-), 114 40 (2 x 2° Ar-Q, 121 06 (2 x 2° Ar-Q, 132 20 (1 x 3° Ar-Q, 153 72 (1 x 3° Ar-Q and 168 26 (-NHCOCH3) Calcd for Ci0Hi2BrNO2 C, 46 53, H, 4 69, N, 5 43 Found C, 46 77, H, 4 17, N, 5 24 Nr-[4-(2-Nitrooxy-ethoxy)-phenyl]-acetamide(12) A mixture of Ar-[4-(2-bromo-ethoxy)-phenyl]-acetamide (10) (1 30 g, 0 005 mol), silver nitrate (5 0 g) and freshly distilled acetomtnle (50 0 ml) was refluxed for 10 hr, cooled, poured into crushed ice and extracted with chloroform (3 x 100 ml) The combined organic layers were dried, filtered, solvent removed under reduced pressure to obtain a residue which was crystallised from methanol to obtain N-[4-(2-mtrooxy-ethoxy)-phenylj-acetamide (12) (0 91 g, 75 83 %), mp 106-107° C Anal IR (KBr) 3324, 3060, 2920, 1667, 1634, 1522, 1238, 1046 and 861 cm ' 1H NMR (CDC13) δ 2 15 (s, 3H, -NHCOCH3), 4 21 (t, 2H, J = 4 6 Hz, -OCH2-% 4 80 (t, 2H, J = 4 6 Hz, -CH2ONO2), δ 83 (d, 2H, Ar-H), 7 49 (d, 2H, Ar-H) and 7 43 (s, 1H, -NH, Exchangeable with D2O) 13C NMR (CDCI3) δ 24 32 (-NHCOCH3), δ4 46 (-CH2ON02-), 71 09 (-OCH2-), 115 00 (2 x 2° Ar-Q, 122 02 (2 x 2° Ar-Q, 132 03 (1 x 30 Ar-Q, 154 74 (1 x 3° Ar-Q and 168 62 (-NHCOCH3) Calcd for C10H12N2O5 C, 50 00, H, 5 04, N, 11 66 Found C, 49 76, H, 4 60, N, 11 19 Example 2 Nr-[4-(5-Bromo-pentoxy)-phenyl]-acetamide (11) A solution of NL(4-hydroxyphenyl)-acetamide (paracetamol) (1) (1 50 g, 001 mol) and 1,5-dibromopentane (9) (10 0 ml) in ethyl methyl ketone (25 0 ml) containing potassium carbonate (5 0 g) was stirred for 8 hr at room temperature The reaction mixture was filtered, residue washed with ethyl methyl ketone and solvent evaporated under reduced pressure to get the residue The residue was dissolved m chloroform (200 0 ml), washed with 5% sodium hydroxide solution (3 x 100 ml), water (3 * 50 ml), dried, filtered and crystallised from methanol to get the desired product JV-[4-(5-bromo-pentoxy)-phenyl]-acetamide (11) (2 15 g, 72 15 %), mp 110-111° C Anal IR (KBr) 3303, 3031, 2944, 1660, 1549, 1410, 1239, 1045 and 735 cm ' 1H NMR (CDCI3) δ 1 62 (m, 2H, -CH2CH2CH2-), 1 81 (p, 2H, J = 7 4 Hz, - OCH2CH2-), 1 92 (m, 2H, -CH2CH2Br-), 2 11 (s, 3H, -NHCOCH3), 3 43 (t, 2H, J= 6 3 Hz, -CH2Bx), 3 92 (t, 2H, J = 6 3Hz, -OCH2-), δ 81 (d, 2H, Ax-H), 7 37 (d, 2H, Ar-H) and 9 42 (s, 1H, -NH Exchangeable with D20) 13C NMR (CDCI3) δ 24 20 (-NHCOCH3), 24 83 (-CH2CH2CH2-), 28 45 (-OCH2CH2-), 32 49 (-CH2CH2Br-), 33 68 (-CH2Br-), δ7 87 (-OCH2-), 114 72 (2 x 2° Ar-Q, 122 06 (2 x 2° Ar-Q, 131 05 (1 x 3° Ar-Q, 155 84 (1 x 30 Ar-Q and 168 70 (-NHCOCH3) Calcd for Ci3Hi8BrN02 C, 52 01, H, 6 04, N, 4 67 Found C, 52 67, H, 5 54, N, 4 74 N-[4-(5-Nitrooxy-pentoxy)-phenyI]-acetamide(13) A mixture of N-[4-(5-bromo-pentoxy)-phenyl]-acetamide (11) (150 g, 0 005 mol)), silver nitrate (5 0 g) and freshly distilled acetonitnle (50 0 ml) was refluxed for 10 hr, cooled, poured into crushed ice and extracted with chloroform (3 x 100 ml) The combined organic layers were dried, filtered, solvent removed under reduced pressure to obtain a residue which was crystallised from methanol to obtain N-[4-(5-mtrooxy-pentoxy)-phenyl]-acetamide (13) (1 02 g, 72 34 %), mp 96-98° C Anal IR (KBr) 3322, 3025, 2951, 1663, 1547,1236, 1048, 878 and 739 cm ' 1H NMR (CDCI3) δ 1 59 (m, 2H, -CH2CH2CH2-), 1 84 (m, 4H, -CH2CH2CH2-), 2 10 (s, 3H, -NHCOCH3), 3 93 (t, 2H, J = 6 6 Hz, -OCH2-), 4 49 (t, 2H, J = 6 6 Hz, -CH2ON02), δ 80 (d, 2H, Ar-H), 7 45 (d, 2H, Ar-H), 9 10 (s, 1H, -NH, Exchangeable with D20) 13C NMR (CDCI3) δ 22 52 (-NHCOCH3), 24 36 (-CH2CH2CH2-), 26 64 (-OCH2CH2-), 28 83 (-CH2CH2ON02-), δ7 74 (-CH2ON02-), 73 23 (-OCH2-), 114 80 (2 x 2° Ar-C), 122 01 (2 x 2° Ar-Q, 131 11 (1 x 3° Ar-Q, 155 83 (1 x 3° Ar-Q and 168 40 (-NHCOCH3) Calcd for Ci3Hi8N205 C, 55 31, H, 6 43, N, 9 92 Found C, 54 89, H, 6 19, N, 9 47 Compounds 10 and 11 are the intermediates and are synthesized as per literature references (10) A Multi-Mode-Driven Molecular Shuttle Photochemically and Thermally Reactive Azobenzene Rotaxanes Murakami, Hiroto, Kawabuchi, Atsushi, Matsumoto, Rika, Ido, Takeshi, Nakashima, Naotoshi Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Nagasaki, Japan Journal of the American Chemical Society (2005), 127(45), 15891-15899 (11) Antagonists of slow reacting substance of anaphylaxis Synthesis of a series of chromone-2-carboxyhc acids Appleton, R A , Bantick, J R , Chamberlain, T R , Hardern, D N, Lee, T B, Pratt, A D Pharm Div, Fisons Ltd, Loughborough/Leicestershire, UK Journal of Medicinal Chemistry (1977), 20(3), 371-9 Examples of compounds with C3 and C4 chains are given below, (Formula Removed) Example 3 N-[4-(3-Bromo-propoxy)-phenyll-acetamide(16) A solution of N-(4-hydroxyphenyl)acetamide (paracetamol) (1) (1 50 g) and 1,3-dibromobutane (14) (10 0 ml) in ethyl methyl ketone (25 0 ml) containing potassium carbonate (5 0 g) was stirred overnight at room temperature The reaction mixture was filtered, residue washed with ethyl methyl ketone and sovent evaporated under reduced pressure to get the residue The residue was dissolved in chloroform (200 0 ml), washed with 5% NaOH solution (3 x 100 ml), water (3 x 50 ml), dried, filtered and crystallized from methanol to get the desired product N-[4-(3-Bromo-propoxy)-phenylj-acetamide (16) (2 0lg ,75 8%), mp 115-118 C Anal IR (KBr) 3284,1658, 1559, 1410,1239,1032, 832 and 521 cm ' 1H NMR (400 MHz, CDC13) δ 2 14 (s, 3H, -NHCOCH3), δ 2 30 (p, 2H,7=6 1, -CH2CH2Br-), δ 3 59 (t, 2H, J=6 4 Hz, -CH2Br), δ 4 07 (t, 2H, J=5 8 Hz, -OCH2-), δ 6 86 (d, 2H, J=3 0 Hz, Ar-H), δ 7 37 (d, 2H, J=3 0 Hz, Ar-H) and 8 7 27(s, 1H, -NH, Exchangeable with D20) I3C NMR (400 MHz, CDC13) δ 24 40 (-NHCOCH3), δ 30 14 (-CH2CH2CH2Br-), δ 32 36 (-CH2Br-), δ 65 58 (-OCH2-), δ 114 84 (2 x 2° Ar-C), δ 122 (2 x 2° Ar-C), δ 131 23 (1 x 3° Ar-C), δ 155 55 (1 x 30 Ar-C) and 8 168 43 (-NHCOCH3) CHN Calculated C, 48 55, H, 5 19, N, 5 15 Found C, 48 76, H, 5 10, N, 4 36 Nr-[4-(3-Nitrooxy-propoxy)-phenyl]-acetamide(18) A mixture of N-[4-(3-Bromo-propoxy)-phenyl]-acetamide (16) (1 50 g), silver nitrare (5 0 g) and freshly distilled acetomtnle (50 0 ml) was refluxed for 10 hr, cooled, poured into crushed ice and extracted with chloroform (3 x 100 ml) The combined organic layers were dried, filtered, solvent removed under reduced pressure to obtain a residue which was crystallized from methanol to obtain N-[4-(3-Nitrooxy-propoxy)-phenyl]-acetamide (18) (1 09g ,77 30%), mp 89-92 C Anal IR(KBr 3333, 1664, 1548, 1411, 1238, 1057, 839 and 704 cm ' 1H NMR (400 MHz, CDCI3) δ 2 14 (s, 3H, -NHCOCH3), δ 2 19 (p, 2H,J =6 1 -CH2CH2CH2-), δ 4 66 (t, 2H, J =6 3 Hz, -OCH2-), δ 4 03 (t, 2H, J =5 9Hz, -CH2ON02), δ 6 84 (d, 2H, J=3 1 Hz, Ar-H), δ 7 41 (d, 2H, J=3 8 Hz, Ar-H) and 6 7 26(s, 1H, -NH, Exchangeable with D20) 13C NMR (400 MHz, CDC13) δ 24 40 (-NHCOCH3), δ 27 0 (-CH2CH2CH2-), δ 63 80 (-CH2ONOr), δ 70 00 (-OCH2), δ 77 11 (-OCHr), δ 114 76 (2 x 2° Ar-C), δ 121 98 (2 x 2° Ar-C), δ 131 46 (1 x 3° Ar-C), δ 155 27 (1 x 3° Ar-C) and 5 168 46 (-NHCOCH3) CHN Calculated C, 51 97, H, 5 55, N, 11 02 Found C, 48 63, H, 5 37, N, 10 79 Example 4 N-[4-(4-Bromo-butoxy)-phenyl]-acetamide(17) A solution of N-(4-hydroxyphenyl)acetamide (Paracetamol) (1) (1 50 g) and 1,3-dibromobutane (15) (10 0 ml) in acetonitnle (25 0 ml) containing potassium carbonate (5 0g) was refluxed for 8 hrs at room temperature The reaction mixture was filtered and the filtrate was poured into ice cold water The residue was filtered and washed with 5% NaOH solution (3 x 100 ml), water (3 x 50 ml), dried, filtered and crystallized from methanol to get the desired product JV-[4-(4-bromobutoxy)phenyl]acetamide (17) (2 15g ,77 06 ), mp 119-124 ° C Anal IR (KBr) 3289,1660,1552,1409,1236,1046, 825 and 520 cm ' 'H NMR (400 MHz, CDC13) δ 2 04 (s, 3H, -NHCOCH3), δ 2 06 (p, 2H, J=2 13 Hz, (-CH2CH2CH2Br), δ 1 94 (p, 2H, J=3 35 Hz, (-CH2CH2CH2CH2 Br), δ 3 48 (t, 2H, J=6 6 Hz, CH2Br), δ 3 98 (t, 2H, J=6 0 Hz, -OCH2), δ 6 85 (d, 2H, J=3 0 Hz, Ar-H), δ 7 37 (d, 2H, J=3 0 Hz, Ar-H), 8 7 26 (s, 1H, -NH, Exchangeable with D20) 13C NMR (400 MHz, CDC13) δ 24 38 (-NHCOCH3), δ 27 91(-CH2CH2CH2CH2Br), δ 29 48 (-CH2CH2CH2 Br), 8 33 60 (-CH2Br-), 8 67 10 (-OCH2-), 8 114 72 (2 x 2° Ar-C), δ 121 99 (2 x 2° Ar-C), 8 131 07 (1 x 3° Ar-C), 8 155 72 (1 x 3° Ar-C) and 8 168 46(-NHCOCH3) CHN Calculated C, 50 37, H, 5 64, N, 4 89 Found C, 49 96, H, 5 73, N, 4 68 N-[4-(4-Nitrooxy-butoxy)-phenyl]-acetamide (19) A mixture of iV-[4-(4-bromobutoxy)phenyl]acetamide (17) (1 30 g), silver nitrare (5 0 g) and freshly distilled acetomtnle (50 0 ml) was refluxed for 10 hr, cooled, poured into crushed ice and extracted with chloroform (3 x 100 ml) The combined organic layers were dried, filtered, solvent removed under reduced pressure to obtain a residue which was crystallized from methanol to obtain N-[4-(4-Nitrooxy-butoxy)-phenyl]-acetamide (19) (1 lOg ,78%), mp 92-98° C Anal IR (KBr) 3327, 1658, 1519, 1408, 1238, 1040, 832 and 551 cm' lH NMR (400 MHz, CDC13), δ 2 07 (s, 3H, -NHCOCH3), δ1 88 (p, 2H, J=2 7 Hz, -CH2CH2CH2CH2ON02), δ1 79 (p, 2H, J=2 9 Hz,-CH2CH2CH2 ON02), δ 3 91 (t, 2H, 7=5 68 Hz, -CH2ON02), δ4 46 (t, 2H, J= 6 2Hz, -OCH2-), δ 6 76 (d, 2H, J=3 09 Hz, Ar-H), δ 7 32 (d, 2H, J= 3 09Hz, Ar-H) and 6 7 30 (s, 1H, -NH, Exchangeable with D20) 13C NMR (30 MHz, CDC13) δ 23 89 (-NHCOCH3), δ 25 60 (- CH2CH2CH2CH2ON02), δ 24 43 (-CH2CH2CH2ON02), δ 67 22 (- CH2ON02-), δ 73 02 (-OCH2-), δ 114 75 (2 x 2° Ar-C), δ 121 99 (2 x 2° Ar-C), δ 131 16(1 x 3° Ar-C), δ 155 61 (1 x 3° Ar-C) and 5 168 36 (-NHCOCH3) CHN Calculated C, 53 73, H, 6 01, N, 10 44 Found C, 52 94, H, 6 01, N, 9 90 Biological Activity Animals Wistar rats (male 150-200g) of both sex and Laca mice (male, 25-35g) procured from Central Animal House, Panjab University, Chandigarh, India were used Animals were housed under standard laboratory conditions and maintained on rat chow Animals were allowed free access to food and water until used and fasted 24 hr prior to studies Experimental conditions Unless otherwise stated, the following conditions were employed m all experiments The test compounds were suspended in 0 5% carboxymethylcellulose (CMC) and administered per orally (p o) Control animals were given the corresponding amount of vehicle (0 5%, CMC) Anti-inflammatory activity (Table 1) Anti-inflammatory activity was determined by using carrageenan induced rat paw edema model Rats were divided into different groups and the drugs were administered to each group The paracetamol (1) and synthesized NO-releasing derivatives (12 and 13) were administered p o at a dose of 100 mg/kg, p o , emulsified in 0 5% sodium carboxy methyl cellulose (0 5% sodium CMC) Anti-inflammatory activity was determined by using carrageenan induced rat paw edema model Rats were divided into different groups and the drugs/derivatives were administered to each group Acute edema was induced m left hind paw of rats by injecting freshly prepared solution of carrageenan (Type IV, 0 lml, 1%) under plantar region of left hind paw In the right paw, saline (lml, 0 9%) was injected, which served as control for comparison The increase in paw volume was measured by using plethysmometer (water displacement, UGO BASILE, Varese, Italy) at 2 hr and 4 hr after carrageenan challenge Percentage change (increase) in paw volume was calculated and expressed as the amount of inflammation % increase in paw volume at any time = (Vi-Vr)/Vr x 100 Where, V1 = Volume of left paw Vr = Volume of right paw (control) (Table Removed) Table 1 Anti-inflammatory activity of paracetamol (1) and NO-NSAIDs (12 and 13) Results are expressed as mean ± standard error of mean (SEM) * indicates P # indicates P NO-releasing derivatives of paracetamol (1), 12 and 13 showed good anti-inflammatory activity w r t parent drug 1, both at 2 hr and 4 hr interval (shown by less % increase in paw volume) At 2 hr % increase in paw volume for 12 and 13 was 32 68±1 45 and 31 15±1 31 respectively, and at 4 hr % increase in paw volume for 12 and 13 was 26 44±0 10 and 28 45±1 28 respectively No significant activity was observed in case of paracetamol (1) as % increase in paw volume at 2 hr and 4 hr was found to be 38 37±1 26 and 58 92±1 56 respectively Analgesic activity (Table 2 and FIG 1 Sheet No 1 Figure 3) Analgesic activity was determined by using abdominal writhing assay Mice were divided into different groups containing 6 animals in each group Analgesic activity was determined against acetic acid induced writhing assay Writhing was induced by intraperitoneal (lp) injection of freshly prepared acetic acid solution Mice were per orally (p o) administered paracetamol (1) and synthesized NO-releasing derivatives (12 and 13) at a dose of 100 mg/kg, emulsified in 0 5% sodium carboxy methyl cellulose (sodium CMC) vehicle, 30 mm prior to I p acetic acid (1% w/v in saline pH 2 7, 10 ml/kg, I p ) Animals were immediately transferred to individual observation chambers and number of writhes (constriction of abdomen, turning of trunk, and extension of hind limbs) were monitored over the following 20 minutes, beginning 3 min after the injection of acetic acid At the end of observation period the animals were killed by cervical dislocation and exsaguination The average number of writhes in each group of drug treated mice was compared with that of the control group and degree of analgesia was expressed as % inhibition calculated from the equation % Inhibition = (1-Nt/Nc) x 100 Where, Nc = number of writhes in control Nt = number of writhes m drug treated mice COMPOUND % INHIBITION IN WRITHINGS 1 45 36±1 34 10 13 52±1 82* 12 58 35±1 50* 11 17 24±41* 13 61 80±1 69* Table 1 Analgesic of paracetamol (1) and NO-NSAIDs (12 and 13) Results are expressed as mean + standard error of mean (SEM) # indicates P Oalkylated NO-releasing derivatives 12 and 13 of paracetamol (1) exhibited better response than the parent drug indicated by greater % inhibition in writhings The % inhibition in case of 12 and 13 (58 35±1 50, 61 80±1 69 respectively) was found to be better than paracetamol (1) which was 45 36±1 34 The intermediates 10 and 11, having terminal Br (which was later converted to -ONO2 moiety on treatment with AgNOs) showed very negligible % inhibition in writhings indicating that NO release is responsible for better analgesic activity of the compounds 12 and 13 (having NO releasing -ONO2) moiety Biochemical Assays (Table 3 and FIG 2 Sheet No 1 for SGPT(ALT), and Table 4 and FIG 3 Sheet No 2 for ALP) Paracetamol was given at a dose of 1000 mg/Kg and an equivalent dose of 1000 mg/Kg of the derivatives 12 and 13 was given po Before administration blood samples of each animal were taken and after 6 hrs the animals were sacrificed to collect blood samples, and the serum was separated out Measurement of plasma markers of liver Serum Glutamate oxaloacetate transaminase (SGOT) pyruvate transaminase (SGPT) and Alkaline Phosphatase (ALP) were assayed spectrophotometncally using commercially available kits The animals were divided into different groups containing 6-8 animals in each group Animals were lightly anaesthetized with diethyl ether and blood samples were collected which served as the basal reading of the enzyme to be analysed These groups of rats were then treated with paracetamol (1000 mg/Kg, 1 p), and equivalent dose of the test compounds Control group was treated with an equal volume of vehicle (0 9% w/v sodium chloride containing 20% v/v tween-80) Animals were lightly anesthetized with ethyl ether and blood samples were collected and allowed to coagulate at room temperature for 1 hr, serum was then separated by centnfugation (5,000 rpm for 10 mm) at 4°C and stored at -40 0°C till estimation Plasma markers of liver injury were assayed spectrophotometncally using following commercially available kits according to standard laboratory techniques COMPOUND % INCREASE IN SGPT/ALT LEVEL CONTROL 2 92±0 3* Paracetamol (1) 83 11 ±3 3* 12 27 94±2 7# 13 1172±14# Table 3 % increase in SGPT/ALT level Results are expressed as mean ± standard error of mean (SEM) * indicates P # indicates P COMPOUND % INCREASE IN ALP LEVEL CONTROL 1 05±0 49* Paracetamol (1) 15 75±1 07* 12 4 17±0 62* 13 2 85±0 83* Table 4 % increase in ALP level Results are expressed as mean ± standard error of mean (SEM) * indicates P # indicates P Overproduction of this toxic metabolite 2 leads to depletion of glutathione stores in liver and further accumulation of the toxic metabolite causes tissue injury and cell death Enzymes like alkaline phosphatase (ALP, EC 3 13 1), serum glutamate pyruvate transaminase (SGPT) or alanine aminotransferase (ALT) (EC 2 6 12) and the total proteins are considered as plasma markers of liver injury Nitric oxide releasing paracetamol derivatives 12 and 13 were evaluated for their effect on the markers of liver injury like enzymes ALP and SGPT/ALT The plasma level of these enzymes is found to increase in the case of liver toxicity The graph indicates that the rise (with comparison to the basal value) in the case of 1 is much more than in the case of NO releasing derivatives 12 and 13, showing that they cause lesser liver toxicity than the parent drug paracetamol 1 Histopathology (FIG 5 Sheet No 3)For liver histopathology, the livers were removed and portions were fixed in 10% buffered formalin (pH 7 4) They were then processed by standard histopathological techniques, stained with Haematoxyhn and Eosin (H&E), and examineed for morphologic evaluation for liver injury Light microscopic analysis of liver samples obtained from mouse treated with control (0 9% w/v sodium chloride containing 20% v/v tween-80), paracetamol (1), 12 and 13, at a dose of 1000 mg/kg, l p is shown in sections (A), (B), (C), and (D) respectively The interpreted results obtained from histopathological studies are summarized below (l) Control did not cause any damage to the liver Only mild portal triad inflammation was observed (u) Paracetamol (1) a proven liver toxic caused ballooning degeneration, sinusoidal decongestion, portal triad inflammation and spoty necrosis in Zone I (in) Just mild ballooning degeneration was observed in the case of 12 (IV) Compound 13 also didn't cause any significant damage to the liver These results were supportive in summarizing that NO-release counteracts the liver toxic side effects of paracetamol (1) Nitric Oxide Release {Table 5 and FIG 4 Sheet No 2, Fig 5} 100 mg/Kg of the derivatives 12 and 13 was given p o After 6 hours the animals were sacrificed to collect blood samples and the serum was separated out to find out the levels of NO Plasma nitrate/nitrite concentration level was measured by commercially available Cayman Kit COMPOUND NITRATE CONCENTRATION (\i mol/1) CONTROL 182 1 ±0 68 12 354 2±2 17* I 13 ( 265 4±2 47* \| Table 4 % increase m ALP level Results are expressed as mean ± standard error of mean (SEM) * indicates P After a period of 6 hr 12 and 13, NO-releasing derivatives showed increase in % NO serum level w r t control The NO release of these compounds could be the acting force behind their better analgesic and anti-inflammatory activities than the parent drug In vitro cox-2 (ovine) inhibitory assay The prostaglandin product is quantified via enzyme immunoassay (EIA) using a broadly specific antibody that binds to all major prostaglandin compounds Assay kit was purchased from Cayman Chemical Company, USA Celecoxib 79 0% (positive control) PCM 24 0% 12 21 0% 13 32 5% Our compound 13 has shown to have better COX-2 (in vitro) than the parent drug paracetamol The data of analgesic and anti-inflammatory activites is exactly complimenting and supporting it The NO-releasing should have been found to have better peripheral analgesic activity and significant anti-inflammatory activity as compared to the parent drug The derivatives were found to release NO in vivo and are devoid of liver toxicity as indicated by serum lesser increase in serum GPT(ALT) and ALP levels and histopathological studies Moreover, 13 showed better COX-2 (ovine) binding activity than the parent drug On the whole more effective and less toxic derivatives of 1 have been synthesised The advantages of the present investigation are that these NO releasing compounds have shown better analgesic and antiinflammatory activity than the parent drug, and less liver toxicity in biochemical and histopathological studies We claim 1. Nitrate ester derivatives of paracetamol of general formula A, wherein the value of n is selected form 2 to 5. (Formula Removed) 2. A compound as claimed in claim 1 wherein the structural formula comprising (Formula Removed) 3. A compound as claimed in claim 1 wherein the representative compounds of the general formula comprising: (i) N-[4-(2-Nitrooxy-ethoxy)-phenyl]-acetamide (12) (ii) N-[4-(5-Nitrooxy-pentoxy)-phenyl]-acetamide (13) (iii) N-[4-(3-Nitrooxy-propoxy)-phenyl]-acetamide (18) (iv) N-[4-(4-Nitrooxy-butoxy)-phenyl]-acetamide (19) 4. A compound as claimed in claim 1 wherein the compound of formula A is having better peripheral analgesic activity and significant anti-inflammatory activity as compared to paracetamol. 5. A compound as claimed in claim 1 wherein the compounds possess better analgesic and antiinflamatory activity and devoid of toxicity as compared to the parent drug paracetamol (1). 6. A compound as claimed in claim 1 wherein the compounds 12 and 13 showed 32.68±1.45% and 31.15±1.31% at 2 hr, and, 26.44±0.10% and 28.45±1.28% at 4 hr increase respectively, in the paw volume as compared to 38.37±1.26% and 58.92±1.56% (at 2 hr and 4hr respectively) in case of paracetamol at a dose of l00mg/kg body weight. 7. A compound as claimed in claim 1 wherein the compounds 12 and 13 showed increase in NO serum level 354.2±2.17 and 265.4±2.47 respectively w.r.t. control (182.1±0.68) at a dose of l00mg/kg body weight. 8. A compound as claimed in claim 1 wherein the compound 13 showed In vitro cox-2 (ovine) inhibitory activity 32.5% as compared to control 24.0%. 9. A process for preparation of nitrate ester derivative of paracetamol comprising the steps a) reacting paracetamol with dibromo alkane in ketonic solvent in presence of alkali carbonate for a period up to 12 hours, b)filtering the reaction mixture followed by evaporation of solvent to obtain solid residue, c)dissolving the residue in water immisible solvent and washing with sodium hydroxide solution followed by wahing with water,evaprating the solvent to O-alkylated derivative of paracetamol, d)reacting the O-alkylated bromoderivative derivative of paracetamol with sliver nitrate under reflux in an aprotic solvent upto 12 hours,further filtering and evaporating the filterate to obtain the nitrate esters derivative of Paracetamol of general formula A wherein the value of n is selected from 2-5. 4. A compound as claimed in claim 1 wherein the compound of formula A is having better peripheral analgesic activity and significant anti-inflammatory activity as compared to paracetamol. 5. A compound as claimed in claim 1 wherein the compounds possess better analgesic and antiinflamatory activity and devoid of toxicity as compared to the parent drug paracetamol (1). 6. A compound as claimed in claim 1 wherein the compounds 12 and 13 showed 32.68±1.45% and 31.15±1.31% at 2 hr, and, 26.44±0.10% and 28.45±1.28% at 4 hr increase respectively, in the paw volume as compared to 38.37±1.26% and 58.92±1.56% (at 2 hr and 4hr respectively) in case of paracetamol at a dose of l00mg/kg body weight. 7. A compound as claimed in claim 1 wherein the compounds 12 and 13 showed increase in NO serum level 354.2±2.17 and 265.4±2.47 respectively w.r.t. control (182.1±0.68) at a dose of lOOmg/kg body weight. 8. A compound as claimed in claim 1 wherein the compound 13 showed In vitro cox-2 (ovine) inhibitory activity 32.5% as compared to control 24.0%. 9. A process for preparation of nitrate ester derivative of paracetamol comprising the steps a) reacting paracetamol with dibromo alkane in an organic solvent in presence of alkali carbonate for a period up to 12 hours, b)filtering the reaction mixture followed by evaporation of solvent to obtain solid residue, c)dissolving the residue in water immisible solvent and washing with sodium hydroxide solution followed by wahing with water,evaprating the solvent to O-alkylated derivative of paracetamol, d)reacting the O-alkylated bromoderivative derivative of paracetamol with sliver nitrate under reflux in an aprotic solvent upto 12 hours,further filtering and evaporating the filterate to obtain the nitrate esters derivative of Paracetamol of general formula A wherein the value of n is selected from 2-5. 10. A process as claimed in claim 8, wherein dihalo alkane is selected from a group consisting of 1,2-dibromoethane, 1,3-dibromopropane, 1,4-dibromobutane and 1,5- dibromopentane. 11. A process as claimed in claim 8, wherein the organic solvent used is selected from a group consisting of ethyl methyl ketone, acetone, acetonitrile and mixture thereof. 12. A process as claimed in claim 8, wherein the aprotic solvent used is selected from a group consisting of acetonitrile, benzene, hexane and mixture thereof. 13. Nitrate ester derivatives of paracetamol of general formula A, process for preparation thereof is substantially as herein described with reference to the examples and drawings accompanying the specification.

Full Text

FIELD OF THE INVENTION
The present invention relates to nitric oxide releasing derivatives of paracetamol The present invention particularly relates to novel nitrate esters of paracetamol The present invention pertains to process of preparation of nitrate esters of paracetamol More particularly, the invention relates to nitric oxide releasing derivatives of paracetamol which show better analgesic and anti-inflammatory activities and decreased liver toxicities as apparent from their biochemical and histopathologic profile
BACKGROUND OF THE INVENTION
Paracetamol (paracetamol, 1) is among most commonly used analgesics It effectively reduces fever and mild-to moderate pain, and is regarded, in general, as a very safe drug Nevertheless, overdose (deliberate) is a common cause of hepatic injury, accounting for ~ 40% of cases of acute liver failure in the USA [Wu, C C Nitric oxide and inflammation Curr Med Chem - Anti-inflammatory & anti-allergy agents, 2004, 3, 217-222, Joshi, G P NCX-701 NicOx Curr Opin Investig Drugs, 2004, 5, 755-759, and Moore, P K, Marshall, M Nitric oxide releasing paracetamol (nitroparacetamol) Dig Liver Dis, 2003, 35 ] It is an effective analgesic and antipyretic agent and unlike other NSAIDs (Non-steroidal Anti-inflammatory Drugs) has been reported to have little antiinflammatory effects [ Fiorucci, S , Antonelh, E , Mencarelh, A , Palazzetti, B , Alvarez-Miller, L , Muscara, M , del Soldato, P , Sanpaolo, L , Wallace, J L , Morelh, A A NO-releasing derivative of paracetamol spares the liver by acting at several checkpoints in the Fas pathway Br J Pharmacol, 2002, 135, 589-599 ] It is metabolized in liver by three pathways-glucuromdation, sulfonation (both accounting for 95% of metabolism) or via cytochrome P450 enzyme system (5%) in which it is converted to a toxic metabolite (N-acetyl-p-benzoqumone lmine, 2) which further is rendered harmless through an interaction with the endogenous antioxidant glutathione
(Formula Removed)
But overproduction of this toxic metabolite leads to depletion of glutathione stores in liver and further accumulation of the toxic metabolite causes tissue injury and cell death [ Futter, L E, al-Swayeh, O A, Moore, P K A comparison of the effect of nitroparacetamol and paracetamol on liver injury Br J Pharmacol, 2000, 132, 10-12 ] Numerous derivatives of paracetamol have been synthesized to get a safer non-hepatotoxic drug One of the major achievements has been by Nocox in Italy It has designed NCX-701 (3) by adding NO moiety to paracetamol, via an aliphatic spacer to improve the anti-inflammatory activity of paracetamol, based on the well-demonstrated activity of NO on cytokine synthesis and regulation of proinflammatory mediators [Wallace, J L Paracetamol hepatotoxicity NO to the rescue Br J Pharmacol, 2004,143, 1-2 ]
(Formula Removed)
NO may modulate spinal and sensory neuron excitability through multiple mechanisms that may improve the anti-nociceptive activity of paracetamol The pharmacological profile of NCX 701 has been reviewed [ Marshall, M , Moore, P K Effect of nitric oxide releasing paracetamol and flurbiprofen on cytokine production in human blood Eur J Pharmacol, 2004, 483, 317-322, and al-Swayeh, O A, Futter, L E, Clifford, R H, Moore, P K Nitroparacetamol exhibits anti-inflammatory and anti-nociceptive activity Br J Pharmacol, 2000, 130, 1453-1456] Compared with NSAIDs, paracetamol possesses little anti-inflammatory activity in humans and it does not induce damage of
the gastric mucosa, but it could cause liver damage (or even poisoning) when used in high dosage The aim of coupling paracetamol with NO has, therefore, been to improve its anti-inflammatory activity, and also to exploit the cytoprotective properties of NO to reduce potential liver damage Concerning safety, several studies have highlighted the liver sparing profile of NCX 701 compared to paracetamol The beneficial protective effect on the liver has been supposed to be related to the fact that NO can reduce the synthesis of several pro-inflammatory cytokines [ Wallace, J L Paracetamol hepatotoxicity NO to the rescue Br J Pharmacol, 2004, 143, 1-2] The antiinflammatory activity related to NO release has been demonstrated in vitro and in vivo, [Marshall, M , Moore, P K Effect of nitric oxide releasing paracetamol and flurbiprofen on cytokine production in human blood Eur J Pharmacol, 2004, 483, 317-322 , al-Swayeh, O A, Futter, L E, Clifford, R H, Moore, P K Nitroparacetamol exhibits anti-inflammatory and anti-nociceptive activity Br J Pharmacol, 2000, 130, 1453-1456, AND, Paul Clark, M J, Howat, D , Flower, R J, Moore, P K , Perretti, M Nitroparacetamol (NCX-701) exhibits anti-inflammatory activity in the zymosan air pouch Inflamm Res, 2001, 50, S158]demonstrating that NCX 701 is not only a safer drug than paracetamol, but that it also possesses a wider action due to additive mechanisms Several papers have compared the anti-nociceptive activity of NCX 701 versus paracetamol, [Romero-Sandoval, E A , Mazano, J, Howat, D , Herrero, J F NCX-701 (nitroparacetamol) is an effective antinociceptive agent in rat withdrawal reflexes and wind-up Br J Pharmacol, 2002, 135, 1556-1562 , Romero-Sandoval, A E, Del Soldato, P , Herrero, J F The effects of sham and full spinahzation on the antinociceptive effects of NCX-701 (nitroparacetamol) in monoarthritic rats Neuropharmacology, 2003, 45, 412-419 , and Gaitan, G , Del Soldato, P , Herrero, J F Low doses of nitroparacetamol or dexketoprofen trometamol enhance fentanyl antinociceptive activity Eur J Pharmacol, 2003, 481, 181-188 ] regularly showing the superiority of NCX 701 Moreover, sub-effective doses of NCX 701 have enhanced the anti-nociceptive activity of the u-opioid receptor agonist fentanyl A Phase II study clinical trial has demonstrated that lg NCX 701 provides similar analgesic efficacy to paracetamol lg in post-operative dental pain, therefore with a reduced exposure to
paracetamol, confirming a contribution of NO to the anti-nociceptive mechanisms of NCX 701
O-Dealkylation of a homologous series of alkoxy acetanihdes [p-methoxy, p-ethoxy phenacetin, p-(n)-propoxy- and p-(n)-butoxy acetanihdes, 4-7] have shown to release paracetamol
The compounds (6 and 7) were structurally related to phenacetin (5), and were synthesized to check whether they release paracetamol (1) upon O-dealkylation, which they did The chain length in the synthesized compounds was varied to study the effect of chain lengthening on the rate of release of paracetamol
[Chul-Ho, Y , Miller, G P , Guengench, F P Oxidations of p-Alkoxyacylanihdes
Catalyzed by Human Cytochrome P450 1A2 Structure-Activity Relationships and
Simulation of Rate Constants of Individual Steps m Catalysisf Biochemistry 2001, 40,
4521-4530]
The compounds (4, 6 and 7) are structurally related to phenacetin (5) therefore, on
metabolism they will liberate paracetamol (1) (Formula Removed)
Release of paracetamol (1) by alkoxy acetamhdes (4-7)
There was a need to develop the novel molecules which are having better analgesic and antnnflamatory activity The advantages of the present invention are that these NO releasing compounds have shown better analgesic and antiinflammatory activity than the parent drug, and less liver toxicity in biochemical and histopathological studies
OBJECTS OF THE INVENTION
The main object of the present invention is to provide novel nitrate esters of paracetamol
Another object of the present invention is to provide process for preparation of novel nitrate esters of paracetamol
Yet another object of the present invention is to develop nitric oxide releasing derivative of paracetamol that shows better analgesic, anti-inflammatory, biochemical (SGOT, ALP levels) and histopathologic profile
Further object of the present invention is to develop a pharmaceutical composition comprising nitric oxide releasing derivatives of paracetamol
Still another object of the present invention is to inhibit COX-2 in vitro and release NO in \i\o
Compound (13) has shown to have better COX-2 (in vitro) inhibition activity l e 32 5% than the parent drug paracetamol (1) which had 24 0% The compounds 12 and 13 also released NO in vivo
SUMMARY OF THE INVENTION
The present invention relates to the synthesis of NO-releasing derivatives of paracetamol and their evaluation The compounds showed better analgesic, anti-inflammatory, biochemical and histopathologic profile The present invention particularly relates to novel nitrate esters of paracetamol The nitrate esters of paracetamol are prepared by reacting the paracetamol with dihaloalkyl compound and followed by reaction with silver nitrate to obtain the corresponding nitrate ester derivatives
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 Analgesic activity of paracetamol (1) and NO-NSAIDs (12 and 13)
10 and 11 are intermediates only which were later converted to -ONO2 moiety on treatment with AgN03
Figure 2 % increase in SGOT levels after 6 hours
Figure 3 % increase in ALP levels after 6 hours
(Explaination for Figure 2 and Figure 3)
Overproduction of toxic metabolite 2 leads to depletion of glutathione stores in liver and further accumulation of the toxic metabolite causes tissue injury and cell death Enzymes like alkaline phosphatase (ALP, EC 3 13 1), serum glutamate pyruvate transaminase (SGPT) or alanine aminotransferase (ALT) (EC 2 6 12) and the total proteins are considered as plasma markers of liver injury Nitric oxide releasing paracetamol derivatives (12 and 13) were evaluated for their effect on the markers of liver injury like enzymes ALP and SGPT/ALT The plasma levels of these enzymes was found to increase in the case of liver toxicity The graph indicates that the rise (with comparison to the basal value) in the case of 1 is much more than in the case of NO releasing derivatives (12 and 13), showing that they cause lesser liver toxicity than the parent drug paracetamol (1)
Figure 4 Nitrite ion concentration (umol/L) in serum after 6 hours
The graph indicates that the serum NO concentration 6 hours after administration of 12 and 13 was greater than the control (0 9% w/v sodium chloride containing 20% v/v tween-80) confirming that these compounds release NO in vivo
Figure 5 Histopathology (hacmatoxylm and eosin) of liver samples
Light microscopic analysis of liver samples obtained from mouse treated with control (0 9% w/v sodium chloride containing 20% v/v tween-80), paracetamol (1), (12) and (13), at a dose of 1000 mg/kg, lp is shown in sections (A), (B), (C), and (D),
respectively The interpreted results obtained from histopathological studies are summarized below
(1) Control did not cause any damage to the liver Only mild portal triad inflammation was observed
(n) Paracetamol (1) a proven liver toxic caused ballooning degeneration, sinusoidal decongestion, portal triad inflammation and spoty necrosis in Zone I
(in) Just mild ballooning degeneration was observed in the case of 12
(IV) Compound 13 also didn't cause any significant damage to the liver
These results were supportive m summarizing that NO-release counteracts the liver toxic side effects of paracetamol (1)
DETAILED DESCRIPTION OF THE INVENTION
Accordingly the present invention provides nitrate ester derivatives of paracetamol of general formula A, wherein the value of n is selected from 2 to 5
(Formula Removed)
In an embodiment of the invention wherein the structural formula A comprising
(Formula Removed)
In an embodiment of the invention wherein the representative compounds of the general formula A comprising
(1) N-[4-(2-Nitrooxy-ethoxy)-phenyl]-acetamide (12)
(n) N-[4-(5-Nitrooxy-pentoxy)-phenyl]-acetamide (13)
(in) N-[4-(3-Nitrooxy-propoxy)-phenyl]-acetamide (18)
(IV) N-[4-(4-Nitrooxy-butoxy)-phenyl]-acetamide (19)
In another embodiment of the invention wherein the compound of formula A is having better peripheral analgesic activity and significant anti-inflammatory activity as compared to paracetamol
In yet another embodiment of the invention wherein the compounds possess better analgesic and antiinflamatory activity and devoid of toxicity as compared to the parent drug paracetamol (1)
In still another embodiment of the invention wherein the compounds 12 and 13 showed 32 68±1 45% and 31 15±1 31% at 2 hr, and, 26 44±0 10% and 28 45±1 28% at 4 hr increase respectively, in the paw volume as compared to 38 37±1 26% and 58 92±1 56% (at 2 hr and 4hr respectively) in case of paracetamol at a dose of l00mg/kg body weight
In a further embodiment of the invention wherein the compounds 12 and 13 showed increase in NO serum level 354 2±2 17 and 265 4±2 47 respectively wrt control (182 1±0 68) at a dose of 100mg/kg body weight
In an embodiment of the invention wherein the compound 13 showed In vitro cox-2 (ovine) inhibitory activity 32 5% as compared to control 24 0%
Accordingly the present invention rovides a process for preparation of nitrate ester derivative of paracetamol comprising the steps
a) reacting paracetamol with dibromo alkane in organic solvent in presence of alkali carbonate for a period up to 12 hours,
b)filtenng the reaction mixture followed by evaporation of solvent to obtain solid residue,
c)dissolving the residue in water immisible solvent and washing with sodium hydroxide solution followed by washing with water,evaprating the solvent to O-alkylated derivative of paracetamol,
d)reacting the O-alkylated bromodenvative derivative of paracetamol with sliver nitrate under reflux in an aprotic solvent upto 12 hours,further filtering and evaporating the filterate to obtain the nitrate esters derivative of Paracetamol of general formula A wherein the value of n is selected from 2-5
In an embodiment of the invention wherein dihalo alkane may be selected from a group consisting of 1,2-dibromoethane, 1,3-dibromopropane, 1,4-dibromobutane and 1,5-dibromopentane
In an embodiment of the invention wherein the organic solvent used may be selected from a group consisting of ethyl methyl ketone, acetone, acetomtnle and mixture thereof
In an embodiment of the invention wherein the aprotic solvent used may be selected from a group consisting of acetomtnle, benzene, hexane and mixture thereof
The pharmaceutical composition may be prepared using the compounds of the general formula A along with the pharmaceutical^ acceptable excepients
Compounds 12 and 13 are novel and have not been reported earlier Compound (13) has shown to have better COX-2 inhibition activity (in vitro) than the parent drug paracetamol The data of analgesic and anti-inflammatory activites is exactly complimenting and supporting it Therefore, it can be concluded that the NO releasing compound is showing better activity than the parent drug candidate, and moreover, the biochemical and histopathological studies carried out, indicate that the compound is causing less liver toxicity than paracetamol
NO-releasing derivatives of paracetamol, (12) and (13) showed good anti-inflammatory activity wrt parent drug, both at 2 hr and 4 hr interval No significant activity was observed in case of paracetamol (1) Oalkylated NO-releasing derivatives (12 and 13) of paracetamol (1) exhibited better response than the parent drug indicated by greater % inhibition in writhings The intermediates 10 and 11, having terminal Br (which was later converted to -ONO2 moiety on treatment with AgNOs) showed very negligible % inhibition in writhings indicating that NO release is responsible for better analgesic activity of the compounds 12 and 13 (having NO releasing -ONO2) moiety In NO releasing activity they also showed significant increase in level of NO The NO release of these compounds could be the acting force behind their better analgesic and antiinflammatory activities than the parent drug Our compound (13) has shown to have better COX-2 (in vitro) than the parent drug paracetamol In the case of biochemical studies paracetamol (1) caused a significant increase in the serum ALP levels as compared to the NO-releasing derivatives (12 and 13), indicating its liver toxicity Both the derivatives were found to increase the levels of ALP but not more than the drug Increase in the levels of SGPT (ALT) δ hr after administration of paracetamol (1) justified its liver toxicity, with % increase more than 80% Increase m the case of both the derivatives (12 and 13) was not that significant In histopathological studies, also the damage caused by 12 and 13, to liver was less m comparison to paracetamol (1) Therefore, compounds 12 and 13 showed better activity and lesser toxicity than the parent drug 1
Paracetamol (1), a proven liver toxic and having no anti-inflammatory activity of its own has been converted to more active and lesser toxic NO releasing compounds 12 and 13
Nitric oxide releasing derivatives of paracetamol are developed (12 and 13) (Figure 2), which have shown better analgesic, anti-inflammatory, biochemical (SGOT, ALP levels) and histopathologic profile Besides these derivatives have shown to inhibit COX-2 in vitro and release NO in vivo The liver toxic drug paracetamol (1) having no antiinflammatory activity has been converted to less liver toxic and more active derivatives Briefly, Paracetamol was stirred in a solution of 1, 2-dibromoethane (8) and 1, 5-dibromopentane (9), in ethyl methyl ketone and K2CO3 for 8-10 hours
The resulting solution was filtered, dried and evaporated to get a solid residue The residue was dissolved in CHC13 and washed with 5% NaOH (3 X 100ml) and water (3 X 100ml), dried over anhydrous Na2SO4, filtered and solution evaporated to get O-alkyl derivatives 10 and 11, respectively These were refluxed in a solution of AgN03 and acetomtnle overnight, filtered, and evaporated to get nitrate esters 12 and 13, respectively
(Formula Removed)
Synthesis of NO releasing derivatives (12 and 13) of paracetamol (1)
Following examples are given by way of illustration and should not construed to limit the scope of the invention
Example 1
N-[4-(2-Bromo-ethoxy)-phenyl]-acetamide (10)
A solution of N-(4-hydroxyphenyl)-acetamide (paracetamol) (1) (1 50 g, 0 01 mol) and 1,2-dibromoethane (8) (10 0 ml) in ethyl methyl ketone (25 0 ml) containing potassium carbonate (5 0 g) was stirred for 8 hr at room temperature The reaction mixture was filtered, residue washed with ethyl methyl ketone and solvent evaporated under reduced pressure to get the residue The residue was dissolved m chloroform (200 0 ml), washed with 5% NaOH solution (3 x 100 ml), water (3 x 50 ml), dried, filtered and crystallised from methanol to get the desired product N-[4-(2-bromo-ethoxy)-phenyl]-acetamide (10) (2 01 g, 78 51 %), mp 126-127° C
Anal
IR (KBr) 3302, 3024, 1662, 1551,1242, 1029 and 828 cm l
1H NMR (CDC13) δ 2 10 (s, 3H, -NHCOCHc), 3 65 (t, 2H, J = 5 9 Hz, CH2Br), 4 26 (t, 2H, J = 5 9 Hz, -OCH2), δ 83 (d, 2H, Ar-H), 7 49 (d, 2H, Ar-H), 9 42 (s, 1H, -NH Exchangeable with D2O)
13C NMR (CDCI3) δ 23 49 (-NHCOCH3), 29 03 (-CH2Br-), δ7 66 (-OCH2-), 114 40 (2 x 2° Ar-Q, 121 06 (2 x 2° Ar-Q, 132 20 (1 x 3° Ar-Q, 153 72 (1 x 3° Ar-Q and 168 26 (-NHCOCH3)
Calcd for Ci0Hi2BrNO2 C, 46 53, H, 4 69, N, 5 43 Found C, 46 77, H, 4 17, N,
5 24
Nr-[4-(2-Nitrooxy-ethoxy)-phenyl]-acetamide(12)
A mixture of Ar-[4-(2-bromo-ethoxy)-phenyl]-acetamide (10) (1 30 g, 0 005 mol), silver nitrate (5 0 g) and freshly distilled acetomtnle (50 0 ml) was refluxed for 10 hr, cooled, poured into crushed ice and extracted with chloroform (3 x 100 ml) The combined organic layers were dried, filtered, solvent removed under reduced pressure to obtain a residue which was crystallised from methanol to obtain N-[4-(2-mtrooxy-ethoxy)-phenylj-acetamide (12) (0 91 g, 75 83 %), mp 106-107° C
Anal
IR (KBr) 3324, 3060, 2920, 1667, 1634, 1522, 1238, 1046 and 861 cm '
1H NMR (CDC13) δ 2 15 (s, 3H, -NHCOCH3), 4 21 (t, 2H, J = 4 6 Hz, -OCH2-% 4 80 (t, 2H, J = 4 6 Hz, -CH2ONO2), δ 83 (d, 2H, Ar-H), 7 49 (d, 2H, Ar-H) and 7 43 (s, 1H, -NH, Exchangeable with D2O)
13C NMR (CDCI3) δ 24 32 (-NHCOCH3), δ4 46 (-CH2ON02-), 71 09 (-OCH2-), 115 00 (2 x 2° Ar-Q, 122 02 (2 x 2° Ar-Q, 132 03 (1 x 30 Ar-Q, 154 74 (1 x 3° Ar-Q and 168 62 (-NHCOCH3)
Calcd for C10H12N2O5 C, 50 00, H, 5 04, N, 11 66 Found C, 49 76, H, 4 60, N, 11 19
Example 2 Nr-[4-(5-Bromo-pentoxy)-phenyl]-acetamide (11)
A solution of NL(4-hydroxyphenyl)-acetamide (paracetamol) (1) (1 50 g, 001 mol) and 1,5-dibromopentane (9) (10 0 ml) in ethyl methyl ketone (25 0 ml) containing potassium carbonate (5 0 g) was stirred for 8 hr at room temperature The reaction mixture was filtered, residue washed with ethyl methyl ketone and solvent evaporated under reduced pressure to get the residue The residue was dissolved m chloroform (200 0 ml), washed with 5% sodium hydroxide solution (3 x 100 ml), water (3 * 50 ml), dried, filtered and crystallised from methanol to get the desired product JV-[4-(5-bromo-pentoxy)-phenyl]-acetamide (11) (2 15 g, 72 15 %), mp 110-111° C
Anal
IR (KBr) 3303, 3031, 2944, 1660, 1549, 1410, 1239, 1045 and 735 cm '
1H NMR (CDCI3) δ 1 62 (m, 2H, -CH2CH2CH2-), 1 81 (p, 2H, J = 7 4 Hz, - OCH2CH2-), 1 92 (m, 2H, -CH2CH2Br-), 2 11 (s, 3H, -NHCOCH3), 3 43 (t, 2H, J= 6 3 Hz, -CH2Bx), 3 92 (t, 2H, J = 6 3Hz, -OCH2-), δ 81 (d, 2H, Ax-H), 7 37 (d, 2H, Ar-H) and 9 42 (s, 1H, -NH Exchangeable with D20)
13C NMR (CDCI3) δ 24 20 (-NHCOCH3), 24 83 (-CH2CH2CH2-), 28 45 (-OCH2CH2-), 32 49 (-CH2CH2Br-), 33 68 (-CH2Br-), δ7 87 (-OCH2-), 114 72 (2 x 2° Ar-Q, 122 06 (2 x 2° Ar-Q, 131 05 (1 x 3° Ar-Q, 155 84 (1 x 30 Ar-Q and 168 70 (-NHCOCH3)
Calcd for Ci3Hi8BrN02 C, 52 01, H, 6 04, N, 4 67 Found C, 52 67, H, 5 54, N,
4 74
N-[4-(5-Nitrooxy-pentoxy)-phenyI]-acetamide(13)
A mixture of N-[4-(5-bromo-pentoxy)-phenyl]-acetamide (11) (150 g, 0 005 mol)), silver nitrate (5 0 g) and freshly distilled acetonitnle (50 0 ml) was refluxed for 10 hr, cooled, poured into crushed ice and extracted with chloroform (3 x 100 ml) The combined organic layers were dried, filtered, solvent removed under reduced pressure to obtain a residue which was crystallised from methanol to obtain N-[4-(5-mtrooxy-pentoxy)-phenyl]-acetamide (13) (1 02 g, 72 34 %), mp 96-98° C
Anal
IR (KBr) 3322, 3025, 2951, 1663, 1547,1236, 1048, 878 and 739 cm '
1H NMR (CDCI3) δ 1 59 (m, 2H, -CH2CH2CH2-), 1 84 (m, 4H, -CH2CH2CH2-), 2 10 (s, 3H, -NHCOCH3), 3 93 (t, 2H, J = 6 6 Hz, -OCH2-), 4 49 (t, 2H, J = 6 6 Hz, -CH2ON02),
δ 80 (d, 2H, Ar-H), 7 45 (d, 2H, Ar-H), 9 10 (s, 1H, -NH, Exchangeable with D20)
13C NMR (CDCI3) δ 22 52 (-NHCOCH3), 24 36 (-CH2CH2CH2-), 26 64 (-OCH2CH2-), 28 83 (-CH2CH2ON02-), δ7 74 (-CH2ON02-), 73 23 (-OCH2-), 114 80 (2 x 2° Ar-C), 122 01 (2 x 2° Ar-Q, 131 11 (1 x 3° Ar-Q, 155 83 (1 x 3° Ar-Q and 168 40 (-NHCOCH3)
Calcd for Ci3Hi8N205 C, 55 31, H, 6 43, N, 9 92 Found C, 54 89, H, 6 19, N, 9 47
Compounds 10 and 11 are the intermediates and are synthesized as per literature references
(10) A Multi-Mode-Driven Molecular Shuttle Photochemically and Thermally Reactive Azobenzene Rotaxanes Murakami, Hiroto, Kawabuchi, Atsushi, Matsumoto, Rika, Ido, Takeshi, Nakashima, Naotoshi Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Nagasaki, Japan Journal of the American Chemical Society (2005), 127(45), 15891-15899
(11) Antagonists of slow reacting substance of anaphylaxis Synthesis of a series of chromone-2-carboxyhc acids Appleton, R A , Bantick, J R , Chamberlain, T R , Hardern, D N, Lee, T B, Pratt, A D Pharm Div, Fisons Ltd, Loughborough/Leicestershire, UK Journal of Medicinal Chemistry (1977), 20(3), 371-9
Examples of compounds with C3 and C4 chains are given below,
(Formula Removed)
Example 3 N-[4-(3-Bromo-propoxy)-phenyll-acetamide(16)
A solution of N-(4-hydroxyphenyl)acetamide (paracetamol) (1) (1 50 g) and 1,3-dibromobutane (14) (10 0 ml) in ethyl methyl ketone (25 0 ml) containing potassium

carbonate (5 0 g) was stirred overnight at room temperature The reaction mixture was filtered, residue washed with ethyl methyl ketone and sovent evaporated under reduced pressure to get the residue The residue was dissolved in chloroform (200 0 ml), washed with 5% NaOH solution (3 x 100 ml), water (3 x 50 ml), dried, filtered and crystallized from methanol to get the desired product N-[4-(3-Bromo-propoxy)-phenylj-acetamide (16) (2 0lg ,75 8%), mp 115-118 C
Anal
IR (KBr) 3284,1658, 1559, 1410,1239,1032, 832 and 521 cm '
1H NMR (400 MHz, CDC13) δ 2 14 (s, 3H, -NHCOCH3), δ 2 30 (p, 2H,7=6 1, -CH2CH2Br-), δ 3 59 (t, 2H, J=6 4 Hz, -CH2Br), δ 4 07 (t, 2H, J=5 8 Hz, -OCH2-), δ 6 86 (d, 2H, J=3 0 Hz, Ar-H), δ 7 37 (d, 2H, J=3 0 Hz, Ar-H) and 8 7 27(s, 1H, -NH, Exchangeable with D20)
I3C NMR (400 MHz, CDC13) δ 24 40 (-NHCOCH3), δ 30 14 (-CH2CH2CH2Br-), δ 32 36 (-CH2Br-), δ 65 58 (-OCH2-), δ 114 84 (2 x 2° Ar-C), δ 122 (2 x 2° Ar-C), δ 131 23 (1 x 3° Ar-C), δ 155 55 (1 x 30 Ar-C) and 8 168 43 (-NHCOCH3)
CHN Calculated C, 48 55, H, 5 19, N, 5 15 Found C, 48 76, H, 5 10, N, 4 36
Nr-[4-(3-Nitrooxy-propoxy)-phenyl]-acetamide(18)
A mixture of N-[4-(3-Bromo-propoxy)-phenyl]-acetamide (16) (1 50 g), silver nitrare (5 0 g) and freshly distilled acetomtnle (50 0 ml) was refluxed for 10 hr, cooled, poured into crushed ice and extracted with chloroform (3 x 100 ml) The combined organic layers were dried, filtered, solvent removed under reduced pressure to obtain a residue which was crystallized from methanol to obtain N-[4-(3-Nitrooxy-propoxy)-phenyl]-acetamide (18) (1 09g ,77 30%), mp 89-92 C
Anal
IR(KBr 3333, 1664, 1548, 1411, 1238, 1057, 839 and 704 cm '
1H NMR (400 MHz, CDCI3) δ 2 14 (s, 3H, -NHCOCH3), δ 2 19 (p, 2H,J =6 1 -CH2CH2CH2-), δ 4 66 (t, 2H, J =6 3 Hz, -OCH2-), δ 4 03 (t, 2H, J =5 9Hz, -CH2ON02), δ 6 84 (d, 2H, J=3 1 Hz, Ar-H), δ 7 41 (d, 2H, J=3 8 Hz, Ar-H) and 6 7 26(s, 1H, -NH, Exchangeable with D20)
13C NMR (400 MHz, CDC13) δ 24 40 (-NHCOCH3), δ 27 0 (-CH2CH2CH2-), δ 63 80 (-CH2ONOr), δ 70 00 (-OCH2), δ 77 11 (-OCHr), δ 114 76 (2 x 2° Ar-C), δ 121 98 (2 x 2° Ar-C), δ 131 46 (1 x 3° Ar-C), δ 155 27 (1 x 3° Ar-C) and 5 168 46 (-NHCOCH3)
CHN Calculated C, 51 97, H, 5 55, N, 11 02 Found C, 48 63, H, 5 37, N, 10 79
Example 4 N-[4-(4-Bromo-butoxy)-phenyl]-acetamide(17)
A solution of N-(4-hydroxyphenyl)acetamide (Paracetamol) (1) (1 50 g) and 1,3-dibromobutane (15) (10 0 ml) in acetonitnle (25 0 ml) containing potassium carbonate (5 0g) was refluxed for 8 hrs at room temperature The reaction mixture was filtered and the filtrate was poured into ice cold water The residue was filtered and washed with 5% NaOH solution (3 x 100 ml), water (3 x 50 ml), dried, filtered and crystallized from methanol to get the desired product JV-[4-(4-bromobutoxy)phenyl]acetamide (17) (2 15g ,77 06 ), mp 119-124 ° C
Anal
IR (KBr) 3289,1660,1552,1409,1236,1046, 825 and 520 cm '
'H NMR (400 MHz, CDC13) δ 2 04 (s, 3H, -NHCOCH3), δ 2 06 (p, 2H, J=2 13 Hz, (-CH2CH2CH2Br), δ 1 94 (p, 2H, J=3 35 Hz, (-CH2CH2CH2CH2 Br), δ 3 48 (t, 2H, J=6 6 Hz, CH2Br), δ 3 98 (t, 2H, J=6 0 Hz, -OCH2), δ 6 85 (d, 2H, J=3 0 Hz, Ar-H),
δ 7 37 (d, 2H, J=3 0 Hz, Ar-H), 8 7 26 (s, 1H, -NH, Exchangeable with D20)
13C NMR (400 MHz, CDC13) δ 24 38 (-NHCOCH3), δ 27 91(-CH2CH2CH2CH2Br), δ 29 48 (-CH2CH2CH2 Br), 8 33 60 (-CH2Br-), 8 67 10 (-OCH2-), 8 114 72 (2 x 2° Ar-C), δ 121 99 (2 x 2° Ar-C), 8 131 07 (1 x 3° Ar-C), 8 155 72 (1 x 3° Ar-C) and 8 168 46(-NHCOCH3)
CHN Calculated C, 50 37, H, 5 64, N, 4 89 Found C, 49 96, H, 5 73, N, 4 68
N-[4-(4-Nitrooxy-butoxy)-phenyl]-acetamide (19)
A mixture of iV-[4-(4-bromobutoxy)phenyl]acetamide (17) (1 30 g), silver nitrare (5 0 g) and freshly distilled acetomtnle (50 0 ml) was refluxed for 10 hr, cooled, poured into crushed ice and extracted with chloroform (3 x 100 ml) The combined organic layers were dried, filtered, solvent removed under reduced pressure to obtain a residue which was crystallized from methanol to obtain N-[4-(4-Nitrooxy-butoxy)-phenyl]-acetamide (19) (1 lOg ,78%), mp 92-98° C
Anal
IR (KBr) 3327, 1658, 1519, 1408, 1238, 1040, 832 and 551 cm'
lH NMR (400 MHz, CDC13), δ 2 07 (s, 3H, -NHCOCH3), δ1 88 (p, 2H, J=2 7 Hz, -CH2CH2CH2CH2ON02), δ1 79 (p, 2H, J=2 9 Hz,-CH2CH2CH2 ON02), δ 3 91 (t, 2H, 7=5 68 Hz, -CH2ON02), δ4 46 (t, 2H, J= 6 2Hz, -OCH2-), δ 6 76 (d, 2H, J=3 09 Hz, Ar-H), δ 7 32 (d, 2H, J= 3 09Hz, Ar-H) and 6 7 30 (s, 1H, -NH, Exchangeable with D20)
13C NMR (30 MHz, CDC13) δ 23 89 (-NHCOCH3), δ 25 60 (-
CH2CH2CH2CH2ON02), δ 24 43 (-CH2CH2CH2ON02), δ 67 22 (-
CH2ON02-), δ 73 02 (-OCH2-), δ 114 75 (2 x 2° Ar-C), δ 121 99 (2 x 2° Ar-C), δ 131 16(1 x 3° Ar-C), δ 155 61 (1 x 3° Ar-C) and 5 168 36 (-NHCOCH3)
CHN Calculated C, 53 73, H, 6 01, N, 10 44 Found C, 52 94, H, 6 01, N, 9 90 Biological Activity
Animals Wistar rats (male 150-200g) of both sex and Laca mice (male, 25-35g) procured from Central Animal House, Panjab University, Chandigarh, India were used Animals were housed under standard laboratory conditions and maintained on rat chow Animals were allowed free access to food and water until used and fasted 24 hr prior to studies
Experimental conditions Unless otherwise stated, the following conditions were employed m all experiments The test compounds were suspended in 0 5% carboxymethylcellulose (CMC) and administered per orally (p o) Control animals were given the corresponding amount of vehicle (0 5%, CMC)
Anti-inflammatory activity (Table 1) Anti-inflammatory activity was determined by using carrageenan induced rat paw edema model Rats were divided into different groups and the drugs were administered to each group The paracetamol (1) and synthesized NO-releasing derivatives (12 and 13) were administered p o at a dose of 100 mg/kg, p o , emulsified in 0 5% sodium carboxy methyl cellulose (0 5% sodium CMC) Anti-inflammatory activity was determined by using carrageenan induced rat paw edema model Rats were divided into different groups and the drugs/derivatives were administered to each group Acute edema was induced m left hind paw of rats by injecting freshly prepared solution of carrageenan (Type IV, 0 lml, 1%) under plantar region of left hind paw In the right paw, saline (lml, 0 9%) was injected, which served as control for comparison The increase in paw volume was measured by using plethysmometer (water displacement, UGO BASILE, Varese, Italy) at 2 hr and 4 hr after carrageenan challenge Percentage change (increase) in paw volume was calculated and expressed as the amount of inflammation
% increase in paw volume at any time = (Vi-Vr)/Vr x 100 Where, V1 = Volume of left paw
Vr = Volume of right paw (control)
(Table Removed)
Table 1 Anti-inflammatory activity of paracetamol (1) and NO-NSAIDs (12 and 13)
Results are expressed as mean ± standard error of mean (SEM)
* indicates P # indicates P NO-releasing derivatives of paracetamol (1), 12 and 13 showed good anti-inflammatory activity w r t parent drug 1, both at 2 hr and 4 hr interval (shown by less % increase in paw volume) At 2 hr % increase in paw volume for 12 and 13 was 32 68±1 45 and 31 15±1 31 respectively, and at 4 hr % increase in paw volume for 12 and 13 was 26 44±0 10 and 28 45±1 28 respectively No significant activity was observed in case of paracetamol (1) as % increase in paw volume at 2 hr and 4 hr was found to be 38 37±1 26 and 58 92±1 56 respectively
Analgesic activity (Table 2 and FIG 1 Sheet No 1 Figure 3) Analgesic activity was determined by using abdominal writhing assay Mice were divided into different groups containing 6 animals in each group Analgesic activity was determined against acetic acid induced writhing assay Writhing was induced by intraperitoneal (lp) injection of freshly prepared acetic acid solution Mice were per orally (p o) administered paracetamol (1) and synthesized NO-releasing derivatives (12 and 13) at a dose of 100 mg/kg, emulsified in 0 5% sodium carboxy methyl cellulose (sodium CMC) vehicle, 30 mm prior to I p acetic acid (1% w/v in saline pH 2 7, 10 ml/kg, I p ) Animals were immediately transferred to individual observation chambers and number of writhes (constriction of abdomen, turning of trunk, and extension of hind limbs) were monitored over the following 20 minutes, beginning 3 min after the injection of acetic acid At the end of observation period the animals were killed by cervical dislocation and exsaguination The average number of writhes in each group of drug treated mice was compared with that of the control group and degree of analgesia was expressed as % inhibition calculated from the equation
% Inhibition = (1-Nt/Nc) x 100
Where, Nc = number of writhes in control
Nt = number of writhes m drug treated mice
COMPOUND % INHIBITION IN WRITHINGS
1 45 36±1 34
10 13 52±1 82*
12 58 35±1 50*
11 17 24±41*
13 61 80±1 69*
Table 1 Analgesic of paracetamol (1) and NO-NSAIDs (12 and 13) Results are expressed as mean + standard error of mean (SEM) # indicates P Oalkylated NO-releasing derivatives 12 and 13 of paracetamol (1) exhibited better response than the parent drug indicated by greater % inhibition in writhings The % inhibition in case of 12 and 13 (58 35±1 50, 61 80±1 69 respectively) was found to be better than paracetamol (1) which was 45 36±1 34 The intermediates 10 and 11, having terminal Br (which was later converted to -ONO2 moiety on treatment with AgNOs) showed very negligible % inhibition in writhings indicating that NO release is responsible for better analgesic activity of the compounds 12 and 13 (having NO releasing -ONO2) moiety
Biochemical Assays (Table 3 and FIG 2 Sheet No 1 for SGPT(ALT), and Table 4 and FIG 3 Sheet No 2 for ALP) Paracetamol was given at a dose of 1000 mg/Kg and an equivalent dose of 1000 mg/Kg of the derivatives 12 and 13 was given po Before administration blood samples of each animal were taken and after 6 hrs the animals were sacrificed to collect blood samples, and the serum was separated out Measurement of plasma markers of liver Serum Glutamate oxaloacetate transaminase (SGOT) pyruvate transaminase (SGPT) and Alkaline Phosphatase (ALP) were assayed spectrophotometncally using commercially available kits
The animals were divided into different groups containing 6-8 animals in each group Animals were lightly anaesthetized with diethyl ether and blood samples were
collected which served as the basal reading of the enzyme to be analysed These groups of rats were then treated with paracetamol (1000 mg/Kg, 1 p), and equivalent dose of the test compounds Control group was treated with an equal volume of vehicle (0 9% w/v sodium chloride containing 20% v/v tween-80) Animals were lightly anesthetized with ethyl ether and blood samples were collected and allowed to coagulate at room temperature for 1 hr, serum was then separated by centnfugation (5,000 rpm for 10 mm) at 4°C and stored at -40 0°C till estimation Plasma markers of liver injury were assayed spectrophotometncally using following commercially available kits according to standard laboratory techniques
COMPOUND % INCREASE IN SGPT/ALT LEVEL
CONTROL 2 92±0 3*
Paracetamol (1) 83 11 ±3 3*
12 27 94±2 7*#
13 1172±14*# Table 3 % increase in SGPT/ALT level
Results are expressed as mean ± standard error of mean (SEM)
* indicates P # indicates P COMPOUND % INCREASE IN ALP LEVEL
CONTROL 1 05±0 49*
Paracetamol (1) 15 75±1 07*
12 4 17±0 62*
13 2 85±0 83* Table 4 % increase in ALP level
Results are expressed as mean ± standard error of mean (SEM)
* indicates P # indicates P Overproduction of this toxic metabolite 2 leads to depletion of glutathione stores in liver
and further accumulation of the toxic metabolite causes tissue injury and cell death Enzymes like alkaline phosphatase (ALP, EC 3 13 1), serum glutamate pyruvate transaminase (SGPT) or alanine aminotransferase (ALT) (EC 2 6 12) and the total proteins are considered as plasma markers of liver injury Nitric oxide releasing paracetamol derivatives 12 and 13 were evaluated for their effect on the markers of liver injury like enzymes ALP and SGPT/ALT The plasma level of these enzymes is found to
increase in the case of liver toxicity The graph indicates that the rise (with comparison to the basal value) in the case of 1 is much more than in the case of NO releasing derivatives 12 and 13, showing that they cause lesser liver toxicity than the parent drug paracetamol 1
Histopathology (FIG 5 Sheet No 3)For liver histopathology, the livers were removed and portions were fixed in 10% buffered formalin (pH 7 4) They were then processed by standard histopathological techniques, stained with Haematoxyhn and Eosin (H&E), and examineed for morphologic evaluation for liver injury
Light microscopic analysis of liver samples obtained from mouse treated with control (0 9% w/v sodium chloride containing 20% v/v tween-80), paracetamol (1), 12 and 13, at a dose of 1000 mg/kg, l p is shown in sections (A), (B), (C), and (D) respectively The interpreted results obtained from histopathological studies are summarized below
(l) Control did not cause any damage to the liver Only mild portal triad inflammation was observed
(u) Paracetamol (1) a proven liver toxic caused ballooning degeneration, sinusoidal decongestion, portal triad inflammation and spoty necrosis in Zone I
(in) Just mild ballooning degeneration was observed in the case of 12
(IV) Compound 13 also didn't cause any significant damage to the liver
These results were supportive in summarizing that NO-release counteracts the liver toxic side effects of paracetamol (1)
Nitric Oxide Release {Table 5 and FIG 4 Sheet No 2, Fig 5}
100 mg/Kg of the derivatives 12 and 13 was given p o After 6 hours the animals were
sacrificed to collect blood samples and the serum was separated out to find out the levels
of NO Plasma nitrate/nitrite concentration level was measured by commercially
available Cayman Kit
COMPOUND NITRATE CONCENTRATION (\i mol/1)
CONTROL 182 1 ±0 68
12 354 2±2 17*

I 13 ( 265 4±2 47* |
Table 4 % increase m ALP level
Results are expressed as mean ± standard error of mean (SEM) * indicates P After a period of 6 hr 12 and 13, NO-releasing derivatives showed increase in % NO serum level w r t control The NO release of these compounds could be the acting force behind their better analgesic and anti-inflammatory activities than the parent drug
In vitro cox-2 (ovine) inhibitory assay The prostaglandin product is quantified via enzyme immunoassay (EIA) using a broadly specific antibody that binds to all major prostaglandin compounds Assay kit was purchased from Cayman Chemical Company, USA
Celecoxib 79 0% (positive control) PCM 24 0%
12 21 0%
13 32 5%
Our compound 13 has shown to have better COX-2 (in vitro) than the parent drug paracetamol The data of analgesic and anti-inflammatory activites is exactly complimenting and supporting it
The NO-releasing should have been found to have better peripheral analgesic activity and significant anti-inflammatory activity as compared to the parent drug The derivatives were found to release NO in vivo and are devoid of liver toxicity as indicated by serum lesser increase in serum GPT(ALT) and ALP levels and histopathological studies Moreover, 13 showed better COX-2 (ovine) binding activity than the parent drug On the whole more effective and less toxic derivatives of 1 have been synthesised
The advantages of the present investigation are that these NO releasing compounds have shown better analgesic and antiinflammatory activity than the parent drug, and less liver toxicity in biochemical and histopathological studies

We claim
1. Nitrate ester derivatives of paracetamol of general formula A, wherein the value of n
is selected form 2 to 5.
(Formula Removed)
2. A compound as claimed in claim 1 wherein the structural formula comprising
(Formula Removed)
3. A compound as claimed in claim 1 wherein the representative compounds of the
general formula comprising:
(i) N-[4-(2-Nitrooxy-ethoxy)-phenyl]-acetamide (12) (ii) N-[4-(5-Nitrooxy-pentoxy)-phenyl]-acetamide (13) (iii) N-[4-(3-Nitrooxy-propoxy)-phenyl]-acetamide (18) (iv) N-[4-(4-Nitrooxy-butoxy)-phenyl]-acetamide (19)
4. A compound as claimed in claim 1 wherein the compound of formula A is having better peripheral analgesic activity and significant anti-inflammatory activity as compared to paracetamol.
5. A compound as claimed in claim 1 wherein the compounds possess better analgesic and antiinflamatory activity and devoid of toxicity as compared to the parent drug paracetamol (1).
6. A compound as claimed in claim 1 wherein the compounds 12 and 13 showed
32.68±1.45% and 31.15±1.31% at 2 hr, and, 26.44±0.10% and 28.45±1.28% at 4 hr
increase respectively, in the paw volume as compared to 38.37±1.26% and 58.92±1.56%
(at 2 hr and 4hr respectively) in case of paracetamol at a dose of l00mg/kg body weight.
7. A compound as claimed in claim 1 wherein the compounds 12 and 13 showed increase in NO serum level 354.2±2.17 and 265.4±2.47 respectively w.r.t. control (182.1±0.68) at a dose of l00mg/kg body weight.
8. A compound as claimed in claim 1 wherein the compound 13 showed In vitro cox-2 (ovine) inhibitory activity 32.5% as compared to control 24.0%.
9. A process for preparation of nitrate ester derivative of paracetamol comprising the
steps
a) reacting paracetamol with dibromo alkane in ketonic solvent in presence of alkali carbonate for a period up to 12 hours,
b)filtering the reaction mixture followed by evaporation of solvent to obtain solid residue,
c)dissolving the residue in water immisible solvent and washing with sodium hydroxide solution followed by wahing with water,evaprating the solvent to O-alkylated derivative of paracetamol,
d)reacting the O-alkylated bromoderivative derivative of paracetamol with sliver nitrate under reflux in an aprotic solvent upto 12 hours,further filtering and evaporating the filterate to obtain the nitrate esters derivative of Paracetamol of general formula A wherein the value of n is selected from 2-5.
4. A compound as claimed in claim 1 wherein the compound of formula A is having better peripheral analgesic activity and significant anti-inflammatory activity as compared to paracetamol.
5. A compound as claimed in claim 1 wherein the compounds possess better analgesic and antiinflamatory activity and devoid of toxicity as compared to the parent drug paracetamol (1).
6. A compound as claimed in claim 1 wherein the compounds 12 and 13 showed
32.68±1.45% and 31.15±1.31% at 2 hr, and, 26.44±0.10% and 28.45±1.28% at 4 hr
increase respectively, in the paw volume as compared to 38.37±1.26% and 58.92±1.56%
(at 2 hr and 4hr respectively) in case of paracetamol at a dose of l00mg/kg body weight.
7. A compound as claimed in claim 1 wherein the compounds 12 and 13 showed increase in NO serum level 354.2±2.17 and 265.4±2.47 respectively w.r.t. control (182.1±0.68) at a dose of lOOmg/kg body weight.
8. A compound as claimed in claim 1 wherein the compound 13 showed In vitro cox-2 (ovine) inhibitory activity 32.5% as compared to control 24.0%.
9. A process for preparation of nitrate ester derivative of paracetamol comprising the
steps
a) reacting paracetamol with dibromo alkane in an organic solvent in presence of alkali carbonate for a period up to 12 hours,
b)filtering the reaction mixture followed by evaporation of solvent to obtain solid residue,
c)dissolving the residue in water immisible solvent and washing with sodium hydroxide solution followed by wahing with water,evaprating the solvent to O-alkylated derivative of paracetamol,
d)reacting the O-alkylated bromoderivative derivative of paracetamol with sliver nitrate under reflux in an aprotic solvent upto 12 hours,further filtering and evaporating the filterate to obtain the nitrate esters derivative of Paracetamol of general formula A wherein the value of n is selected from 2-5.
10. A process as claimed in claim 8, wherein dihalo alkane is selected from a group
consisting of 1,2-dibromoethane, 1,3-dibromopropane, 1,4-dibromobutane and 1,5-
dibromopentane.
11. A process as claimed in claim 8, wherein the organic solvent used is selected from a group consisting of ethyl methyl ketone, acetone, acetonitrile and mixture thereof.
12. A process as claimed in claim 8, wherein the aprotic solvent used is selected from a group consisting of acetonitrile, benzene, hexane and mixture thereof.
13. Nitrate ester derivatives of paracetamol of general formula A, process for preparation thereof is substantially as herein described with reference to the examples and drawings accompanying the specification.

Documents:

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Patent Number

279922

Indian Patent Application Number

1753/DEL/2007

PG Journal Number

06/2017

Publication Date

10-Feb-2017

Grant Date

03-Feb-2017

Date of Filing

17-Aug-2007

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110 001,INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	TILAK RAJ BHARDWAJ	UNIVERSITY INSTT OF PHARMACEUTICAL SCIENCES, PANJAB UNIV, CHANDIGARH-160101
2	MANOJ KUMAR	UNIVERSITY INSTT OF PHARMACEUTICAL SCIENCES, PANJAB UNIV, CHANDIGARH-160101
3	NEERAJ MEHTA	UNIVERSITY INSTT OF PHARMACEUTICAL SCIENCES, PANJAB UNIV, CHANDIGARH-160101
4	NEELIMA DHINGRA	UNIVERSITY INSTT OF PHARMACEUTICAL SCIENCES, PANJAB UNIV, CHANDIGARH-160101

PCT International Classification Number

N/A

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA