Iraqi J Pharm Sci, Vol.18(2) 2009 Cyclic amines derivatives of metronidazole 1 Synthesis of New Cyclic Amines-Linked Metronidazole Derivatives as Possible Prodrugs Maadh Q. Abdul-Kadir *,1 , Nadhum E. AL-Ani * and Shakir M. Alwan * * Department of Pharmaceutical Chemistry, College of Pharmacy ,University of Baghdad, Baghdad, Iraq Abstract Certain cyclic amine derivatives of metronidazole via acetate spacer were prepared. Cyclic amines used are piperidine and piperazine to improve the physicochemical properties and reduce some of metronidazole side effects. This is believed to be done by modification of its structural features to get prodrugs with improved properties over that of metronidazole. The present work includes esterification of metronidazole with choroacetic acid, N-alkylation of the cyclic amines by the halogenated ester and characterization of their structures by spectral(UV and IR) and elemental(CHN)analysis.The melting points, degree of solubilities and partition coefficients were also determined. Both metronidazole-piperazine and metronidazole-piperidine derivatives have different structural and physicochemical properties that may be excellent to diminish problems associated with metronidazole. Key words: Metronidazole, chloroacetate, prodrug. الخالصة ميّ نهًخشَٔيذاصٔل يٍ خالل جسش انخالث. اٌ االييُاث انحهميت في ْزا انبحث َمٕو بخحضيش بعغ انًشخماث االييُيّ انحه انًسخعًهتْي انببشاصيٍ ٔانببشيذيٍ ٔانغشع يٍ ْزا انبحث ْٕ ححسيٍ انخٕاص انفيضيأيت ٔانكيًيأيت ٔحمهيم بعغ انخأثيشاث يمذياث ادٔيّ راث خظائض افضم يٍ انجاَبيت نهًخشَٔيذاصٔل .اٌ ْزا يخى بأجشاء ححٕيش في حشكيبت انًخشَٔيذاصٔل نهحظٕل عهى كهٕسٔ حايغ انخهيك ثى حعٕيغ انًشكب اعالِ عهى رسة َخشٔجيٍ -انًخشَٔيذاصٔل .يشًم انجاَب انعًهي اسخشة انًخشَٔيذاصٔل يع انفا حهيم انذليك االييُاث انحهميت ٔيٍ ثى حشخيض ْزِ انًشكباث بٕاسطت انخحهيالث انطيفيّ ) ححج انحًشاء ٔفٕق انبُفسجيت (ٔكزنك انخ َخشٔجيٍ(.كزنك فأٌ دسجاث االَظٓاس,لابهياث انزٔباٌ ٔانخٕصع بيٍ سائهيٍ غيش يًخضجيٍ لذ -ْيذسٔجيٍ-نهعُاطش انًكَّٕ نٓا)كاسبٌٕ حى ححذيذْا ايضا ٔكهٓا اثبخج حظٕنُا عهى يشخميٍ نهًخشَٔيذاصٔل يًخهكاٌ خٕاص فيضيأيت ٔكيًيأيت ٔحشكيبيّ حخخهف عٍ ٔلذ حكٌٕ افضم يٍ حيث حمهيم انجٕاَب انسهبيت نّ.انًخشَٔيذاصٔل Introduction Metronidazole is one of the nitroimidazole derivatives that are mostly used in treating many infections nowadays. It is effective against wide range bacterial, protozoal and parasitic infections (1a) .It has little more effect over mebendazole against Giardia intestinalis as well as intestinal nematodes (2) .Intravaginal metronidazole is effective in the treatment of bacterial vaginosis (3) .Metronidazole,as benzoyl form,could be used as supportive , suppressive and/or synergistic /additive drug in treatment of african trypanosomiasis (4) . Metronidazole can be considered as a prodrug in the sense that it requires metabolic activation by sensitive microorganisms in which the nitro group is reduced to hydroxylamine and covalently bind the DNA of the microorganism so that triggering the lethal effect (1a,5) . Metronidazole is rapidly and completely absorbed when given orally. It has a limited plasma protein binding, but can attain very favorable and rapid tissue distribution, including CNS and CSF (6) . Resistance to metronidazole can be attributed to decreased activity of pyruvate: ferredoxin oxidoreductase which reduces metronidazole, induction of oxidative stress mechanism including superoxide dismutase and peroxiredoxin (7) ,mutation of genes rdxA (8) or lower activities of oxidases and reductases (9) .Increasing the dose and duration of treatment with acid suppression can overcome H.pylori resistance to metronidazole (10,11) . In therapeutic dose, the side effects of metronidazole include metallic bitter taste and CNS symptoms (dizziness, headache and sensory neuropathies). It also has a strong reported teratogenic effect (6) . In case of piperazine, it has the ability to dissolve uric acid, and so might be useful in gout and other rheumatic diseases. It is used as an effective anthelmentic in citrate form for the treatment of pinworms as well as round worms (1b) . Piperazine moiety was introduced to certain antibacterial drugs to increase activities of norfloxacin and ciprofloxacin against some bacteria (1c) . 1 Corresponding author E- mail : maadhqm@yahoo.com Received : 14 / 9 / 2008 Accepted : 9 / 2 / 2009 Iraqi J Pharm Sci, Vol.18(2) 2009 Cyclic amines derivatives of metronidazole 2 It was found that parenteral combination of ciprofloxacin with metronidazole enhances the antimicrobial spectrum of quinolone derivatives against anaerobic organisms and gram-positive bacteria (12) . In cetrizine (H1- antihistaminic drug) the piperazine moiety participate in formation of zwitterions of the drug to prevent penetration into the brain and in turn minimizes CNS effects (13,14) . On the other hand piperidine is a cyclic amine and can be used as a moiety for the synthesis of many drugs such as the antihistamine, diphenylpyraline, which is structurally related to cetrizine (15, 1d) . Several of the functionalized piperidines have been shown to exhibit diverse biological activities through some enzyme inhibition such as glucosidase (16,17) , tryptase (18) and some serine protease (19) . Both piperidine and piperazine moieties in fibrates structures were found to have superior activities in lowering serum trigiycerides, cholesterol and sugar in mice and rats (20) .Also many opioid receptors ligands are piperazine or piperidine-based agents (21) . Prodrugs of metronidazole; to reduce or abolish some of its side effects as well as improving its activities; like some amino acids (22,23,24) , organic acids (25) and others (26,27,28) were synthesized. This field requires more investigations to get the most effective and suitable derivatives. Accordingly, we synthesized two derivatives of metronidazole using piperazine and piperidine linked via acetate bridging in order possibly to get a new suitable mutual ester prodrug with piperazine - diacetate or piperidine-acetate moiety; and these prodrugs (especially compound III) were supposed to be hydrolyzed to generate the parent drugs that may have gastrointestinal or systemic anthelmentic effects as in using diethyl carbamazine for ascaris and filareasis. This will occur in parallel to metronidazole release exhibiting its anti-protozoal and anti-anaerobic action; or a new derivative of possible better physicochemical and antibacterial properties since the presence of piperazine moiety might contribute in improving activity, as shown in ciprofloxacin antipseudomonal activity (1c) . Materials and Methods Metronidazole was supplied by SDI, Iraq; Piperazine hexahydrate and Methylene chloride (E. Merck AG, Germany); Piperidine, Chloroacetic acid and Pyridine (Fluka, Switzerland); Absolute ethanol 99% and Thionyl chloride (BDH, England), Dioxan (Riedel-Dehean AG "Seelze-Hannover", Germany); Methanol and Chloroform (GCC, UK);while Cyclohexane by (Chem supply, South Australia). Methods of synthesis used were the conventional procedures (scheme 1) (22) , melting points and TLC (Rf) values of intermediates and products were used to follow up the reactions, spectral analysis (UV and IR spectra) and elemental analysis (CHN analysis) to characterize the products were conducted. Partition coefficients and water solubilities were also determined. The synthesis started by activation of the carboxyl group of chloroacetic acid(15 gm,158.73 mmole) with 15 ml thionyl chloride (SOCl2) in 50 ml dried chloroform to produce chloroacetyl chloride, which(11.37 gm,120 mmole) was reacted with metronidazole(15 gm,87.66 mmole) in pyridine(7.95 gm,100.63 mmole) and methylene chloride(125 ml) to give metronidazole chloroacetate ester as an intermediate (compound II). Rf values and CHN analysis were given in tables (2) and (3) respectively. Metronidazole - piperazine derivative (compound III) was prepared by reaction of compound II (9.9 gm , 40 mmole) dissolved in 125 ml dry dioxin with piperazine hexahydrate(1.942 gm,10 mmole) dissolved in dry methanol. Compound III was then purified by washing with saturated sodium chloride solution, dried over anhydrous sodium sulphate and recrystalized from chloroform- cyclohexan mixture. The percent yield, physical description and melting point are given in table (1). Rf values and CHN analysis are given in tables (2) and (3) respectively. Metronidazole-piperidine (compound IV) was synthesized by the same method as for compound III except piperidine was used instead of piperazine solution and its properties were also shown in tables (1), (2)and(3). The partition coefficients of both compounds (III) and (IV) were determined by shaking 20 ml of 0.00012 M chloroform(organic solvent) solution of either compound with equal volume of distilled water for 30 min and their concentrations were measured spectrophotometrically at  max 310nm. A standard curve using series of concentrations of each compound in chloroform was constructed. The partition coefficients of the prepared compounds were calculated and shown in table (4) (29) . Iraqi J Pharm Sci, Vol.18(2) 2009 Cyclic amines derivatives of metronidazole 3 CH2 CCl OH O SOCl2 CH2 CCl Cl O N N O2N CH3 CH2CH2OH CH2 CCl Cl O N HN NH N N N N O2N CH3 CH2CH2 H2CC Cl O Chloroacetic acid Chloroacetyl chloride Metronidazole + Metronidazole chloroacetate ester Compound II Piprazine 1 mole Compound III Compound II 2 moles + NH N Piperidine 1 mole Compound IV Compound II 1 moles + O N N O2N CH3 CH2CH2 H2CC O O N N NO2H3C H2CH2CCH2 C O O N N O2N CH3 CH2CH2 H2CC O O N N O2N CH3 CH2CH2 H2CC Cl O O N N O2N CH3 CH2CH2 H2CC Cl O O Scheme 1: Synthesis of compounds III and IV. Table 1: Physical description, melting points and percent yield of metronidazole and compounds I, II, Ill and IV. Compound Empirical formula Molecular weight (gm/mole) Description % yield M.P. OC (observed) Metronidazole C6H9O3N3 171.1 Yellow crystals --- 159-161 Compound I C2H2OCl2 113.0 Colorless-faint yellow oily liquid 93 --- Compound II C8H10O4N3Cl 247.5 Yellowish brown crystals 76 70-72 Compound III C20H28O8N8 508.5 Off white powder 61 201-203 (dec.) Compound IV C13H20O4N4 296.0 Deep yellow crystals 78 106-109 Iraqi J Pharm Sci, Vol.18(2) 2009 Cyclic amines derivatives of metronidazole 4 Table2: Rf values of metronidazole, compound II, III and IV. Solvent system: A = chloroform : ethanol (4:1), B = chloroform : methanol (6:1), C = methanol alone. Table 3: Elemental microanalysis of metronidazole and compounds II, III and IV. Compound Molecular weight (g/mole) Empirical formula Element Elemental analysis % Calculated % Observed Metronidazole 171,1 C6H9O3N3 C H N 42.10 5.26 24.56 41.96 5.190 24.43 Compound II 247.5 C8H10O4N3Cl C H N 38.78 4.040 16.96 38.65 3.980 16.84 Compound III 508.5 C20H28O8N8 C H N 47.24 5.510 22.04 46.98 5.330 21.86 Compound IV 296.0 C13H20O4N4 C H N 52.70 6.750 18.91 52.51 6.580 18.63 Table 4: Partition coefficients and water solubilities of metronidazole and compounds III and IV. Compound (P) Log (P) Water solubility (mg/ml) Metronidazole 0.677 ~ -0.169 10 Compound III 98.26 ~ 1.99 0.13 Compound IV 6.800 ~ 0.832 0.59 Compound Rf values A B C Metronidazole 0.662 0.851 0.611 Compound II 0.822 0.864 0.641 Compound III 0.39 0.54 0.269 Compound IV 0.794 0.826 0.723 Iraqi J Pharm Sci, Vol.18(2) 2009 Cyclic amines derivatives of metronidazole 5 Results and Discussion The products obtained were analyzed by measurement of physical properties like, melting points, Rf values, partition coefficients, solubilities, as well as spectral methods, including UV and IR and were supported by significant analysis of the empirical formula of metronidazole, compounds II, III and IV as shown in tables 1, 2 and 3 respectively. The UV spectra shown in figures (1 and 2) showed that compounds II, III and IV differ from metronidazole in their chemical constitutions. The higher partition coefficient and lower water solubility of products III and IV indicate a higher lipophilicity and in turn might enhance the absorption through biological compartments, even the blood-brain barrier, since log (P) values occur within the optimum value of (0- 3) (30) . With respect to ultraviolet spectra using equal molar concentrations of these compounds in chloroform show the same  max with just small variations within 5nm ( max 313-317 nm). Changing metronidazole to compounds II, III and IV has no effect on the extent of conjugation as shown in figures (1 and 2) (31) . This follows the principle stating that, when two or more chromophoric groups are present in a molecule and they are separated by two or more single bond, the effect on spectrum is usually additive and there is little electronic interaction between isolated chromophoric groups (32,33) . Figure 1: shows nearly equal max (313 – 317 ) nm for Metronidazole (b),Compound II(C) and Compound III(D) using equal conc.(0.0008M) of the three compounds (A) for solvent . Figure 2 : shows nearly equal max (313 – 317) nm for Metronidazole (b),Compound II(C) and Compound III(D) using equal conc.(0.00012M) of the three compounds (A) for solvent . This is also seen in the extent of absorption intensity. The intensity of compound III(having molar absorbitivity  =2.72 X 10 4 mole -1 .L.cm -1 ) is nearly twice that of metronidazole and compound II(=1.61 X 10 4 mole -1 .L.cm -1 ), since compound III has double concentration of chromophore of metronidazole and compound II as shown in figure (1); whereas in figure (2), the intensities of absorption of metronidazole, compound II and IV(=1.03 X 10 4 mole -1 .L.cm -1 ) are nearly equal, since they have the same concentration of chromophore. The comparison of the IR spectra for piperazine, piperidine, metronidazole and compound II with those for compounds III and IV showed the following differences :  Strong bands at (1715-1720) cm -1 and (1750) cm -1 due to ester (C=O) stretching vibration for compounds II , III and IV respectively.  Two asymmetrical coupled bands at (1265 and 1180) cm -1 , (1245 and 1155) cm -1 and (1225 and 1193) cm -1 for (C-C(=O)-O) and (O-C-C) groups were appeared in compounds II, III and IV respectively.  Two bands at (2870 and 2780) cm -1 due to asymmetrical and symmetrical stretching vibrations and two bands within fingerprint region at (1440 and 1380) cm -1 due to (C-H) bending vibrations of methyl group.  The disappearance of a single weak-band in lower than (3550-3200) cm -1 region which is due to (N-H) stretching of secondary amine in spectrum of compounds III and IV. Iraqi J Pharm Sci, Vol.18(2) 2009 Cyclic amines derivatives of metronidazole 6  Disappearance of the strong-broad band (3550-3200) cm -1 ; which corresponds to hydroxyl (OH) stretching vibrations ; in compounds II, Ill and IV spectra.  A strong broad band between(910-712)cm -1 and medium broad band between (820-710) cm -1 in piperazine and piperidine spectra respectively arisen from secondary amine (N-H) wagging. Compounds III and IV spectra have no this band.  In the lowest frequency region of the spectra, which is sensitive region, there is a prominent variation in spectra of compounds III and IV from that of compound II which shows (C-C1) stretching (850-550) cm -1 , and the (CH2) wagging band of (CH2Cl) group at (1300-1150) cm -1 . Conclusion Cross-linking metronidazole with either piperazine or piperidine via an ester spacer was done resulting in new compounds with different and possibly better physicochemical and biological properties. 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