Iraqi J Pharm Sci, Vol.29(1) 2020 Indole based N-acyl hydrazones with antibacterial activity DOI: https://doi.org/10.31351/vol29iss1pp207-2015 207 Synthesis, Characterization and Antibacterial Activity Evaluation of New Indole-Based Derivatives Ahmed T. Sulaiman* and Susan W. Sarsam*, 1 *Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Baghdad, Iraq. Abstract Indole is widely distributed heterocycle found in natural biologically active molecules, drugs, and other substances. Starting from Indole-3-propionic acid (IPA) a metabolite produced by gut’s bacteria, new series of N- acyl hydrazones (4a-g) was synthesized. These N-acyl hydrazones were prepared by the reaction of 3-(1H-indol- 3-yl) propane hydrazide and aldehyde in the presence of glacial acetic acid as a catalyst. 1HNMR and FT-IR analyses were used to identify the synthesized compounds. In vitro study was performed to evaluate the antibacterial activity of the synthesized compounds against six different types of microorganisms by using well diffusion method. All the tested N-acyl hydrazones (4a-g) displayed moderate activity against the Gram-negative E.coli which was comparable to Amoxicillin, except compound (4e), which showed high activity. Also, selective moderate activities against other Gram-negative bacteria were shown by compounds (4a, 4c, 4e, 4f and 4g), while, compounds (4b) and (4d) exhibited intermediate activity against Gram-positive B.subtilis. All the synthesized compounds exhibited selective lower antibacterial activity compared to Ciprofloxacin. Additionally, no activity was exhibited by any of the examined compounds against the Gram-positive S. aureus. Keywords: N-acyl hydrazone, Indole-3-propionic acid, Antibacterial. تحضير وتشخيص وتقييم الفعالية المضادة للبكتريا لمشتقات اندول جديدة 1*،و سوزان وديع سرسم *أحمد طالل سليمان *فرع الكيمياء الصيدالنية، كلية الصيدلة، جامعة بغداد، بغداد، العراق . الخالصة الطبيعية واألدوية. في هذا البحث تم تحضير سلسلة يعتبر اإلندول من اوسع الحلقات الغير متجانسة إنتشاراً في المركبات البايولوجية . هذه المركبات حضرت عن طريق بروبيونيك -3 - مض اإلندولاتحتوي على مجموعة االسيل مشتقة من ح( 4a-g)زونات جديدة من الهيدرو بروبيونك مع عدة الديهايدات بوجود حامض الخليك الثلجي كعامل مساعد. تم تشخيص جميع -3 -الهايدرازايد المشتق من حامض اإلندول مفاعلة أُجريت دراسة مختبرية لتقييم المركبات المحضرة .مطياف األشعة تحت الحمراء وجهاز الرنين النووي المغناطيسي المركبات المحضرة عن طريق ( أظهرت فعالية متوسطة ضد 4a-gبكتيرية لستة انواع مختلفة من البكتريا.جميع المركبات المحضرة )كمضادات ,4a, 4c, 4eحيث كان له فعالية عالية. كما أظهرت المركبات ) (4e)والتي كانت مماثلة لألموكسيسلين،عدا المركب اإلشريكية القولونيةبكتريا 4f, 4g) فعاليات متوسطة ضد االنوا( 4ع البكترية االخرى السالبة لصبغة الغرام. بينما المركباتb, 4d ) كان لها فعالية متوسطة ضد البكتريا سين. إضافة العصوية الرقيقة الموجبة لصبغة الغرام. جميع المركبات المحضرة كان لها فعالية منتقاة ضد البكتريا أقل عند مقارنتها بالسيبروفلوكسا الذهبية الموجبة لصبغة الغرام. المكورات العنقوديةمن المركبات فعالية ضد بكتريا إلى انه لم تظهر اٌي بروبيونيك، مضاد بكتيري. -3-مض اإلندولاوزونات المرتبطة بمجموعة االسيل، حراكلمات مفتاحية: الهيد Introduction Indole (benzo[b]pyrrole) is widely distributed heterocycle found in natural biologically active molecules, drugs, and other substances. In the biological system, several indole-based biomolecules occur with different effects such as serotonin (5-hydroxytryptamine) a neurotransmitter, melatonin the sleep hormone, tryptamine and related amino acid tryptophan. Also, natural alkaloids with an important pharmacological activity contain indole base had been isolated, among them; vinblastine and vincristine (vinca alkaloids) with anti-cancer activity (1). Also, many synthetic drugs carrying indole pharmacophore are now available such as Sunitinib an anticancer drug, Delavirdine an antiviral clinically used for HIV, Indomethacin, Etodolac NSAIDs, and many other drugs with various pharmacological activities (2). Indole propionic acid (IPA) is a type of plant auxin (plant hormone) which is involved in plenty of developmental ways through the growth period of the plant (3). In human, IPA is detected in serum and cerebrospinal fluid (4), it originates from Clostridium sporogenes an intestinal flora, its presence in plasma depends on this type of bacteria (5), and recently it was discovered other types of gut bacteria are capable of the production of IPA, which are Peptostreptococcus anaerobius and three strains of Clostridium cadaveris (6). 1Corresponding author E-mail: sarsam14@yahoo.com Received:28 / 9 /2019 Accepted: 14/ 1/ 2020 Iraqi Journal of Pharmaceutical Sciences https://doi.org/10.31351/vol29iss1pp207-2015 Iraqi J Pharm Sci, Vol.29(1) 2020 Indole based N-acyl hydrazones with antibacterial activity 208 These types of bacteria produce IPA through deamination of tryptophan by bacterial enzyme tryptophan deaminase (7). IPA has antioxidant activity and acts as a free radical scavenger which completely protects nerve cells against the beta-amyloid’s toxic effect, so it perhaps applied in the treatment of Alzheimer’s disease (8, 9). Also, IPA is considered a ligand for pregnane X receptor (PXR), and together regulates mucosal homeostasis and integrity of GIT by decreasing tumour necrosis factor-alpha and increases junctional protein-coding mRNAs (10, 11). Recently, it was observed that IPA has anti- tuberculosis activity in vitro and in vivo by a mechanism which involves inhibition of bacterial tryptophan biosynthesis through a negative feedback mechanism (12, 13). Hydrazones constitute a significant group of organic compounds contain (-NH-N=C-) moiety. They are synthesized by the reaction of hydrazine derivatives with carbonyl-containing compound (aldehyde or ketone) (14). Hydrazones have received considerable interest by many medicinal chemistry researchers owing to the ease of their synthesis as well as the exhibition of wide range of pharmacological effects. They have been notified to have antibacterial, analgesic, anti- anticonvulsant, antiplatelet, anti-tubercular, inflammatory and antitumor activities (15). N-acyl hydrazone (NAH) is considered a privileged structure, which resembles the smallest essential subunit, shared in many drugs or lead- compounds, capable of interacting with a single receptor or more than one class of receptors. Also, due to its ease of production, the stability to hydrolysis, changing H-bonding (donating ↔ accepting) and altering conformation which results in different molecular property with diverse pharmacological activities (16). Several drugs containing NAH pharmacophore are now available for clinical use such as Dantrolene the only drug approved for malignant hyperthermia treatment (17), Nitrofurantoin, an antibacterial and Carbazochrome which has been used for the treatment of capillary and parenchymal haemorrhage (18). The aim of the present investigation was to synthesize new indole derivatives containing N-acyl hydrazone starting from indole-3-propionic acid then to evaluate their antibacterial activity. Materials and Methods Starting material 3-indole propionic acid (IPA) and aldehydes were purchased from Himedia (India), Hydrazine hydrate from Merck (Germany), methanol from Thomas Baker (India) and ethanol from Scharlau (Spain). The progress of reactions as well as the purity of newly synthesized compounds was checked out by thin-layer chromatography (TLC) by using silica gel GF (type 60) pre-coated aluminium sheet from Merck (Germany). UV-254 lamp was used to visualize the spots, and the elution system used was (ethyl acetate: toluene: methanol (2:2:1)). Stuart SMP3 melting points device was used to measure the melting points and were incorrected. Infrared spectra were made at the College of Pharmacy/ University of Baghdad using FT-IR (IR Affinity-1) spectrometer (Shimadzu, Japan). 1HNMR spectra were obtained using Bruker (400 MHz) device and DMSO-d6 as a solvent and were performed at the Central instrumental laboratory/ College of Science/ University of Tehran / Iran. All the synthesized compounds were identified and their structures were determined by FTIR and 1HNMR spectral analyses (19) Synthesis of ester (methyl 3-(1H-indol-3-yl) propionate) (2) (20) Ten gm (0.052mole) of indole propionic acid (1) was dissolved in (50 mL) of methanol and chilled to 0 oC using an ice bath. Then 3 mL of concentrated H2SO4 was added to the cold solution drop by drop with vigorous stirring, and then the temperature of the solution was raised gradually to 70 Co and started the reflux. The reaction was monitored by TLC, after 4 hours of reflux the reaction completed and the solution was poured into a crushed ice containing beaker, a precipitate was formed, and then the solution was neutralized with 5% (W/V) NaHCO3. The final precipitate was obtained by filtration and recrystallized from (methanol/water) to yield off-white fine crystals. Yield 94%; m.p. 75-76 oC; Rf = 0.86; IR (v, cm-1): 3390: (N-H) str. of Indole. 3086: Ar (C-H) str., 2951, 2916: (C-H) asym. Str. of CH3 & CH2, 2897, 2854: (C-H) sym. str. of CH3& CH2, 1716: (C=O) str. of ester, 1165: (C-O-C) Str.; 1HNMR (δ, ppm) : 2.66 (2H, t, -CH2-CH2-COOCH3), 2.95 (2H, t, - CH2-CH2-COOCH3 ), 3.58 (3H, s, -COOCH3), 6.96,7.05 (2H,2t, Ar-H), 7.10 (1H, s ,Ar-H), 7.32,7.50 (2H,2 d, Ar-H), 10.79 (1H,s, Indole N-H). Synthesis of acid hydrazide (3-(1H-indol-3-yl) propanehydrazide) (3) (21) 5gm (0.024 mole) of compound (2) was dissolved by heating and stirring in 30 mL ethanol, then (20mL, 0.41 mole) of hydrazine hydrate was added gradually to the hot solution with continuous agitation and started the reflux. After 5 hours, TLC showed hydrazide spot only, the reaction was stopped and left to cool, then poured into a beaker contains (50 mL) of distilled water. Excess ethanol was evaporated and the formed precipitate was obtained by filtration, dried, rinsed with ether then recrystallized from water to obtain white needle shape crystals. Yield =88%; m.p 143 oC; Rf = 0.48; IR (v, cm-1): 3309, 3278: (N-H) str. of Indole and hydrazide, 3082: Ar. (C-H) str., 2897, 2854: (C-H) str. (asym.& sym.) of CH2, 1666: (C=O) str. of amide, 1508: Ar. (C=C) str.; 1H NMR (δ, ppm): 2.38 (2H, t, -CH2- CH2-CONHNH2), 2.90 (2H, t, -CH2-CH2- CONHNH2 ), 4.16 (2H, s, -NH-NH2), 6.96, 7.04 Iraqi J Pharm Sci, Vol.29(1) 2020 Indole based N-acyl hydrazones with antibacterial activity 209 (2H,2 t, Ar-H), 7.08 (1H, s ,Ar-H), 7.31, 7.50 (2H, 2d, Ar-H), 8.99 (1H,s, -NH-NH2), 10.75 (1H,s, Indole N-H) (22). Synthesis of N-acyl Hydrazones compounds (4a-g) (23). Equimolar (1mmole) of acid hydrazide (3) and appropriate aldehyde were dissolved in suitable solvent (ethanol or water) and reacted, using glacial acetic acid as a catalyst then refluxed for 1-2 hour. Ethanol was used as a solvent except for the reaction with vanillin where water was used instead (24). When the reaction was finished, the residue was filtered and dried. The quantities of each reactant are listed in table 1. Table 1. Quantity of reactants that equal to 1 mmole. Compound Quantity Acid hydrazide (3) 203.1 mg p-OH benzaldehyde 122.1 mg p-Cl benzaldehyde 140.5 mg p-Br benzaldehyde 185 mg Benzaldehyde 0.1 mL p-NO2 benzaldehyde 151.1mg p-N(CH3)2 benzaldehyde 149.2 mg Vanillin 152.1 mg N'-(4-hydroxybenzylidene)-3-(1H-indol-3-yl) propanehydrazide (4a) White powder, yield 73%; m.p. 208-209 oC; Rf = 0.7; IR (v, cm-1) : 3429: (O-H) str. of phenol, 3302, 3186 : (N-H) str. of indole and hydrazone, 3059: Ar (C-H) str., 2900, 2862: (C-H) str.(asym.& sym.) of CH2, 1624 : ( C=O) str. of amide, 1604: (C=N) str., 1566: Ar. (C=C) str.; 1HNMR (δ, ppm): 2.53 (2H, t, -CH2-CH2-CO-NH- ), 2.97 (2H, t, -CH2-CH2-CO-NH-), 6.79 (2H, t, Ar- H ), 6.96, 7.05 (2H,2 t, Ar-H), 7.12 (1H, d ,Ar-H), 7.32 (1H, d, Ar-H), 7.46 (2H, dd, Ar-H), 7.55 (1H, d, Ar-H), 7.87, 8.02 (1H, 2s, -N=CH-), 9.83, 9.87 (1H, s, -OH) , 10.77 (1H,s, Indole N-H), 11.07, 11.16 (1H,2s, -NH-N=C-). N'-(4-chlorobenzylidene)-3-(1H-indol-3-yl) propanehydrazide (4b) White grain-like crystals, yield 69%; m.p. 197-198 oC; Rf = 0.77; IR (v, cm-1 ): 3383, 3170: (N-H) str. of indole and hydrazone, 3055: Ar. (C-H) str., 2958, 2843: (C-H) str. (asym. & sym.) of CH2, 1654: (C=O) str. of amide, 1612: (C=N) str., 1558: Ar. (C=C) str.; 1HNMR (δ, ppm): 2.58 (2H, t, -CH2- CH2-CO-NH-), 3.00 (2H, t, -CH2-CH2-CO-NH-), 6.97, 7.06 (2H,2t, Ar-H), 7.13 (1H,d,Ar-H), 7.32 (1H, d, Ar-H), 7.46 (2H, dd, Ar-H), 7.55 (1H,d, Ar- H), 7.66 (2H,dd, Ar-H), 7.96, 8.13 (1H, 2s, -N=CH- ), 10.78 (1H,s, Indole N-H), 11.35, 11.45 (1H,2s, - NH-N=C-). N'-(4-bromobenzylidene)-3-(1H-indol-3-yl) propanehydrazide (4c) White fine crystals, yield 72%; m.p. 175- 176 oC; Rf = 0.78; IR (v, cm-1 ): 3433, 3174 (N-H) str. of indole and hydrazone, 3062: Ar. (C-H) str., 2970, 2862 : (C-H) str. (asym.& sym.) of CH2 , 1654 : (C=O) str. of amide, 1608: (C=N)str., 1558: Ar. (C=C) str.; 1HNMR (δ, ppm): 2.57 (2H, t, -CH2- CH2-CO-NH-), 2.97 (2H, t, -CH2-CH2-CO-NH-), 6.97, 7.06 (2H,2 t, Ar-H), 7.12 (1H,d ,Ar-H), 7.32 (1H, d, Ar-H), 7.54 (2H,dd, Ar-H), 7.56 (1H,d, Ar- H), 7.61 (2H,d, Ar-H), 7.94, 8.11 (1H, 2s, -N=CH-), 10.77 (1H,s, Indole N-H), 11.33,11.44 (1H,2s, -NH- N=C-). N'- benzylidene- 3-(1H-indol-3-yl) propanehydrazide (4d) White fine needle-like crystals, yield 63%; m.p. 173-174 oC; Rf = 0.74; IR (v, cm-1): 3294, 3182 (N-H) str. of indole and hydrazone, 3020: Ar (C-H) str., 2900, 2846: (C-H) str. of CH2 (asym. & sym.), 1620: (C=O) str. of amide, 1600: (C=N) str., 1566: Ar. (C=C) str. 1HNMR (δ, ppm): 2.57 (2H, t, -CH2- CH2-CO-NH-), 3.00 (2H, t, -CH2-CH2-CO-NH-), 6.97, 7.06 (2H,2t, Ar-H), 7.13 (1H,d ,Ar-H), 7.32 (1H, d, Ar-H), 7.37-7.45(3H, m, Ar-H), 7.55 (1H,d, Ar-H), 7.64 (2H,dd, Ar-H), 7.98, 8.14 (1H, 2s, - N=CH-), 10.78 (1H,s, Indole N-H), 11.28, 11.38 (1H,2s, -NH-N=C-). 3-(1H-indol-3-yl)-N'-(4-nitrobenzylidene) propanehydrazide (4e) Yellow fine crystals, yield 98%; m.p. 238- 239 oC; Rf = 0.76; IR (v, cm-1 ): 3387, 3190 ( N-H) str. of indole and hydrazone, 3055: Ar (C-H) str., 2954, 2835: (C-H) str. of CH2 (asym & sym.), 1678 : (C=O)str. of amide, 1581: (C=N) str., 1512: Ar. (C=C) str. & asym (NO2) str., 1334: (NO2) sym. str.; 1HNMR (δ, ppm): 2.61 (2H, t, -CH2-CH2-CO-NH- ), 3.02 (2H, t, -CH2-CH2-CO-NH-), 6.97, 7.05 (2H, 2t, Ar-H), 7.13 (1H,d ,Ar-H), 7.32, 7.55 (2H,2d, Ar- H), 7.89 (2H,dd, Ar-H), 8.06, 8.22 (1H, 2s, -N=CH- ), 8.26 (2H,dd, Ar-H), 10.77 (1H,s, Indole N-H), 11.57, 11.67 (1H,2s, -NH-N=C-). N'-(4-(dimethylamino) benzylidene)-3-(1H-indol- 3-yl) propanehydrazide (4f) White fluffy crystals, yield 83.8%; m.p. 206-207 oC; Rf = 0.74; IR (v, cm-1): 3163 ( N-H) str. of indole and hydrazone overlap, 3043: Ar (C-H) str., 2974, 2908, 2850: (C-H) str. of CH3 & CH2 (asym. & sym.), 1643 : (C=O) str. of amide, 1597: (C=N) str., 1527 : Ar.(C=C) str.; 1HNMR (δ, ppm): 2.52 (2H, t, -CH2-CH2-CO-NH-), 2.93, 2.95 (6H,2s, -N(CH3)2 ), 2.97 (2H, t, -CH2-CH2-CO-NH-), 6.71 (2H, t, Ar-H), 6.97, 7.05 (2H,2t, Ar-H), 7.12 (1H,d ,Ar-H), 7.32 (1H, d, Ar-H), 7.45 (2H,dd, Ar-H), 7.55 (1H,dd, Ar-H), 7.84,7.98 (1H, 2s, -N=CH-), 10.77 (1H,s, Indole N-H), 10.98, 11.06 (1H, 2s, -NH-N=C- ). Iraqi J Pharm Sci, Vol.29(1) 2020 Indole based N-acyl hydrazones with antibacterial activity 210 N’-(4-hydroxy-3-methoxybenzylidene)-3-(1H- indol-3-yl) propanehydrazide (4g) White powder, yield 65%; m.p. 160-165 oC; Rf = 0.68; IR (v, cm-1 ): 3406 : (O-H) str. of phenol, 3383, 3217 ( N-H) str. of indole and hydrazone, 3032: Ar. (C-H) str., 2939, 2846: (C-H) str. of CH2 (asym. & sym.), 1635 : (C=O) str. of amide, 1581 (C=N) str., 1516 Ar.(C=C) str., 1265 : Ar (C-O-C) str.; 1HNMR (δ, ppm): 2.54 (2H, t, - CH2-CH2-CO-NH-), 2.98 (2H, t, -CH2-CH2-CO- NH-), 3.75, 3.80 (3H, s, -OCH3), 6.79 (1H, dd, Ar- H), 6.94 (1H, t, Ar-H), 7.00 (1H,s, Ar-H), 7.06 (1H,t, Ar-H), 7.13 (1H,d, Ar-H), 7.21 (1H, d, Ar-H), 7.32, 7.55 (2H, 2d, Ar-H), 7.86, 8.01 (1H,2s, -N=CH-), 9.45, 9.49 (1H, s, -OH) , 10.77 (1H,s, Indole N-H), 11.11, 11.18 (1H, 2s, -NH-N=C-) (25,26). Antibacterial activity The antibacterial activity was tested by well diffusion method, by using bacterial suspension obtained from McFarland turbidity standard (number 0.5). Mueller-Hinton agar MHA plates were inoculated by this suspension. In each agar plate of examined bacteria, four wells were made, and 80μl from the solution of the synthesized compound was added to them. The plates were incubated at 37 °C for 24 hours, and then the antibacterial activity was evaluated by measuring the diameter of the inhibition zone around the well in (mm) (27). Four types of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia and Proteus mirabilis) and two types of Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) were tested in vitro for antibacterial activity. All types of bacteria have already been isolated and identified in the College of the Medicine/ University of Al- Nahrain. Ciprofloxacin and Amoxicillin were used as standards for antibacterial activity. The concentrations of standers were 1mg/mL in Dimethyl sulfoxide (DMSO). Results and Discussion Chemistry The pathway for the synthesis of the targeted N-acyl hydrazones (4a-g) is depicted in scheme 1. Compound 4a 4b 4c 4d 4e 4f 4g R1 OH Cl Br H NO2 N(CH3)2 OH R2 H H H H H H OCH3 Scheme 1. General synthetic pathway of the titled compounds Iraqi J Pharm Sci, Vol.29(1) 2020 Indole based N-acyl hydrazones with antibacterial activity 211 The synthetic pathway started by the preparation of methyl ester derivative (2) of IPA by Fischer esterification method using H2SO4 as a catalyst. The acid protonates oxygen of the carbonyl group and makes carbon more electrophile, subjected to nucleophilic attack by the lone pair of electrons of alcohol. The Nucleophilic attack breaks carbonyl group and forms the tetrahedral intermediate oxonium ion. At this point, the excess of alcohol will deprotonate the tetrahedral intermediate oxonium ion to form neutral molecule. Then, transfer of the proton to hydroxyl group which is eliminated as water and the carbonyl bond is regenerated (28) (scheme 2).This method showed considerable yield with ease of production. . Scheme 2. Mechanism of carboxylic acid esterification (Fischer Esterification) (28) Hydrazine hydrate (N2H4.H2O) was used to prepare the acid hydrazide (3) from compound (2). The reaction is base catalyzed ester aminolysis. The mechanism involves two hydrazine molecules either occurred in single step (concert pathway) in which aminolysis occurs through carbonyl attack by amine associate with proton abstraction from the amine, while departure of leaving group associated with proton transfer from ammonium ion, this process happen in one step. The second possibility occurs through multi steps pathway in which the first hydrazine molecule attacks the electrophilic carbonyl group to form tetrahedral intermediate and the second hydrazine molecule will transfer the proton to methoxy group resulting in dissociation of the methoxy group(29-32) (scheme 3). Iraqi J Pharm Sci, Vol.29(1) 2020 Indole based N-acyl hydrazones with antibacterial activity 212 Scheme 3. Mechanism of hydrazide formation from ester (Ester aminolysis) (29, 30) Subsequently, the final N-acyl hydrazones (4a-g) were synthesized by condensing 3-(1H-indol- 3-yl) propanehydrazide compound (3) with the corresponding aldehyde in acidic media as a catalyst. In this reaction, one water molecule had been eliminated to form a carbon-nitrogen double bond (imine or Schiff base) (33) (scheme 4). The target compounds (4a-g) were obtained in good to excellent yields (63-98%). Scheme 4. Mechanism of imine formation (Schiff base) (34) Iraqi J Pharm Sci, Vol.29(1) 2020 Indole based N-acyl hydrazones with antibacterial activity 213 The IR spectra demonstrated shifting of the absorption band of C=O from 1716 cm-1 in ester to 1666 in hydrazide (amide formation), in addition, two bands appeared at 3309, 3278 attributed to primary amine N-H stretching overlapping with N- H stretching of indole. The infrared spectra of the synthesized N-acyl hydrazones (4a-g) had a characteristic absorption band at 1581-1612 cm-1 of carbon-nitrogen double bond (C=N) stretching of imine. Additionally, compounds (4a-g) had recognized bands at 1620-1678 cm-1, attributed to the carbonyl group (C=O) stretching of amide. The 1HNMR spectra of the ester, acid hydrazide and NAHs were consistent with the assigned structures. Two sets of two separated singlets were displayed in the 1HNMR of N-acyl hydrazones (4a-g), attributed to both the –N=CH- and -CONH- protons. These protons appeared as two singlets resonating in the regions (7.84 – 8.22) and (10.98 – 11.67) ppm, respectively. The presence of these paired signals can be explained on the basis of the existence of hydrazones as E/Z geometric isomers around the C=N double bonds as well as the cis/trans amide conformers (35, 36). Antibacterial activity Table 2 shows the activity of the synthesized compounds (NAHs) against the selected bacteria at a concentration of 1mg/mL. Table 2. In vitro antibacterial activity of the synthesized N-acyl hydrazones at 1mg/mL . Zone of inhibition (mm) Compounds P. mirabilis K. pneumoniae E. coli P. aeruginosa B. subtilis S. aureus 12 14 14 - - - 4a - - 12 - 11 - 4b - - 14 12 - - 4c - - 11 - 11 - 4d - 14 18 - - - 4e 13 - 12 - - - 4f - - 12 12 - - 4g - 10 15 38 - 35 Amoxicillin 45 18 30 50 29 52 Ciprofloxacin - - - - - - DMSO (-) = No activity, (zone of inhibition between 5-10 mm) = slightly active, (zone of inhibition between 10-15 mm) = moderately active, (zone of inhibition more than 15 mm) = highly active (37). The data illustrated in the table 2 reveals that all the synthesized tested compounds showed moderate activity against E.coli comparable to that of Amoxicillin, except compound 4e, which showed high activity. Also, compounds 4a and 4e showed moderate activity against Klebsiella pneumonia, which was greater than Amoxicillin. In addition, moderate activity was viewed by compounds 4a and 4f against Proteus mirabilis. Also, moderate anti- Pseudomonas activity was seen with compounds 4c and 4g, which was lower compared to Amoxicillin. All the tested compounds demonstrated various antibacterial activities against selected bacteria, which were lower compared to ciprofloxacin. Generally, all the tested compounds had no activity against the selected Gram-positive bacteria, except compounds 4b and 4d, which showed moderate activity against Bacillus subtilis. The using of local isolates and not the control strains (as American Type Collection Culture strains ATCC), may be the cause of low activity of the synthesized compounds due to mutation and resistance (38). Conclusion New N-acyl hydrazone derivatives containing indole base were successfully synthesized and the structures of newly synthesized compounds were characterized using IR and HNMR spectral analyses methods. The synthesized compounds had moderate activity against Escherichia coli and selective activity against other types of bacteria. Antituberculosis activity evaluation of the synthesized compounds and using the control strains of bacteria instead of local isolates, are recommended for a future study. Acknowledgement The authors are grateful to the College of Pharmacy/ University of Baghdad for all the facilities to perform the research. References 1. Mekky H, Al-Sabahi J, Abdel-Kreem MF. Potentiating biosynthesis of the anticancer alkaloids vincristine and vinblastine in callus cultures of Catharanthus roseus. South African Journal of Botany. 2018 Jan 1; 114:29-31. 2. Chadha, N. Indoles: As Multitarget Directed Ligands in Medicinal Chemistry. In: Silakari O, editor. Key heterocycle cores for designing multitargeting molecules. Elsevier, Amsterdam: Netherlands; 2018: pp. 289-305. Iraqi J Pharm Sci, Vol.29(1) 2020 Indole based N-acyl hydrazones with antibacterial activity 214 3. Ludwig-Müller J. Auxin conjugates: their role for plant development and in the evolution of land plants. Journal of experimental botany. 2011; 62 (6):1757-1773. 4. Wall, R., Cryan, J. F., Ross, R. P. et al. Bacterial Neuroactive Compounds Produced by Psychobiotics.In: Mark Lyte John, F. Cryan, editors. Microbial Endocrinology: The Microbiota-Gut-Brain Axis in Health and Disease. Springer, New York; 2014. P.P. 221– 239. Doi: 10.1007/978-1-4939-0897-4_10. 5. Wikoff WR, Anfora AT, Liu J. et al. Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proceedings of the national academy of sciences. 2009; 106 (10):3698- 3703. 6. Dodd D, Spitzer MH, Van Treuren W. et al. A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites. Nature. 2017; 551 (7682):648-652. 7. Zhang LS, Davies SS. Microbial metabolism of dietary components to bioactive metabolites: opportunities for new therapeutic interventions. Genome medicine. 2016; 8 (1):46-64. 8. Chyan YJ, Poeggeler B, Omar RA. et al Potent neuroprotective properties against the Alzheimer β-amyloid by an endogenous melatonin-related indole structure, indole-3- propionic acid. Journal of Biological Chemistry. 1999; 274(31):21937-21942. 9. Hwang IK, Yoo KY, Li H. et al. Indole‐3‐ propionic acid attenuates neuronal damage and oxidative stress in the ischemic hippocampus. Journal of neuroscience research. 2009; 87(9):2126-2137. 10. Venkatesh M, Mukherjee S, Wang H. et al. Symbiotic bacterial metabolites regulate gastrointestinal barrier function via the xenobiotic sensor PXR and Toll-like receptor 4. Immunity. 2014; 41(2):296-310. 11. Mani S, Li H. Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4. The FASEB Journal. 2015; 29(1_supplement):575-617. 12. Negatu DA, Liu JJ, Zimmerman M. et al. Whole-cell screen of fragment library identifies gut microbiota metabolite indole propionic acid as antitubercular. Antimicrobial agents and chemotherapy. 2018; 62(3):1571-1579. 13. Negatu DA, Yamada Y, Xi Y. et al. Gut microbiota metabolite indole propionic acid targets tryptophan biosynthesis in Mycobacterium tuberculosis. MBio. 2019; 10(2): 2781-2796. 14. Larsen D, Kietrys AM, Clark SA. et al. Exceptionally rapid oxime and hydrazone formation promoted by catalytic amine buffers with low toxicity. Chemical science. 2018; 9(23):5252-5259. 15. Hussain I, Ali A. Exploring the Pharmacological Activities of Hydrazone Derivatives: A Review. J. Phytochem. Biochem. 2017; 1:1-5. 16. Maia RD, Tesch R, Fraga CA. Acylhydrazone derivatives: a patent review. Expert opinion on therapeutic patents. 2014 Nov 1;24(11):1161- 1170. 17. Rosenberg H, Pollock N, Schiemann A. et al. Malignant hyperthermia: a review. Orphanet journal of rare diseases. 2015;10 (1):93-112. 18. Thota S, Rodrigues DA, Pinheiro PD. et al. N- Acylhydrazones as drugs. Bioorganic & medicinal chemistry letters. 2018; 28(17):2797- 2806 19. Silverstein R. M., Webster F. X., Kiemle D. K., Spectrometric Identification of Organic Compounds, seventh edition, Chapter 2 and 3 John Wily and sons, Inc., 111River Street, Hoboken. USA 2005;pp. 72-137. 20. Abbas AH, Elias AN, Fadhil AA. Synthesis, characterization and biological evaluation of new potentially active hydrazones of naproxen hydrazide. Der Pharma Chemica, 2015, 7(10):93-101. 21. Bhatia R, Rathore P, Nayak S. Synthesis, Characterization and Anti-inflammatory Activity of 1, 2, 4 Triazole Derivatives. Current Research in Pharmaceutical Sciences. 2012 Oct 5:160-165. 22. Amir M, Kumar S. Synthesis of some new 2-(2- fluoro-4-biphenylyl) propionic acid derivatives as potential anti-inflammatory agents. Die Pharmazie-An International Journal of Pharmaceutical Sciences. 2005; 1;60(3):175- 80. 23. Kumar P, Kadyan K, Duhan M, et al. Design, synthesis, conformational and molecular docking study of some novel acyl hydrazone based molecular hybrids as antimalarial and antimicrobial agents. Chemistry Central Journal. 2017 Dec; 11(1):115-129. 24. Rao VK, Reddy SS, Krishna BS. et al. Synthesis of Schiff's bases in aqueous medium: a green alternative approach with effective mass yield and high reaction rates. Green Chemistry Letters and Reviews. 2010; 3(3):217-223. 25. Han MI, Gurol G, Yildirim T. et al. Synthesis and antibacterial activity of new hydrazide- hydrazones derived from Benzocaine. Marmara Pharmaceutical Journal. 2017; 1;21(4):961-6. Iraqi J Pharm Sci, Vol.29(1) 2020 Indole based N-acyl hydrazones with antibacterial activity 215 26. Gürkök G, Coban T, Suzen S. Melatonin analogue new indole hydrazide/hydrazone derivatives with antioxidant behavior: synthesis and structure–activity relationships. Journal of enzyme inhibition and medicinal chemistry. 2009;1;24(2):506-15. 27. Bauer AW, Kirby WMM, Sherris JC, Truck M. Antibiotic susceptibility testing by standardized single disk method. Am J Clin Pathol. 1966;45:493–496. 28. McMurry J. Ch. 21: Carboxylic acid derivatives: Nucleophilic acyl substitution, In: Organic Chemistry, 7th edition, Brooks by Thomson Learning, Inc., USA, 2008; pp. 785– 841. 29. Bunnett JF, Davis GT. The mechanism of aminolysis of esters1, 2. Journal of the American Chemical Society. 1960;82(3):665- 674. 30. Bruice TC, Mayahi MF. The influence of the leaving tendency of the phenoxy group on the ammonolysis and hydrolysis of substituted phenyl acetates. Journal of the American Chemical Society. 1960; 82(12):3067-3071. 31. Galabov B, Atanasov Y, Ilieva S. et al. Mechanism of the aminolysis of methyl benzoate: a computational study. The Journal of Physical Chemistry A. 2005 Dec 22;109(50):11470-11474. 32. Yi GQ, Zeng Y, Xia XF. et al. The substituent effects of the leaving groups on the aminolysis of phenyl acetates: DFT studies. Chemical Physics. 2008 3;345(1):73-81. 33. Carey and Sandburg RJ. Ch. 7: Addition, condensation and substitution reactions of carbonyl compounds; Advanced Organic Chemistry, Part A: Structure and Mechanisms, 5th edition, Springer Science, New York, USA, 2007. pp. 629-714. 34. Klein D. Organic Chemistry 3rd ed. Hoboken: John Wiley and Sons, Inc.; 2017. Chapter 19, Aldehydes and Ketones; pp. 844-897. 35. Lopes A, Miguez E, Kümmerle A, Rumjanek V, Fraga C, Barreiro E. Characterization of amide bond conformers for a novel heterocyclic template of N-acylhydrazone derivatives. Molecules. 2013;18(10):11683-11704. 36. Hassan OM, Sarsam SW. Synthesis, Characterization and Preliminary Anti- inflammatory Evaluation of New Etodolac Derivatives. Iraqi Journal of Pharmaceutical Sciences. 2019; 28(1):107-113. 37. Dabholkar VV,Gavande RP.synthesis and antimicrobial activities of novel 1, 4- benzothiazine derivatives. Arab. J. Chem. 2016; 9:225-229. 38. Landecker H. Antibiotic resistance and the biology of history. Body & Society. 2016;22(4):19-52. Baghdad Iraqi Journal Pharmaceutical Sciences by bijps is licensed under a Creative Commons Attribution 4.0 International License. Copyrights© 2015 College of Pharmacy - University of Baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/