Accepted Manuscript Design, synthesis, characterization, quantum-chemical calculations and anti-inflammatory activity of novel series of thiophene derivatives M.H. Helal, M.A. Salem, M.A. Gouda, N.S. Ahmed, A.A. El-Sherif PII: DOI: Reference:

S1386-1425(15)00367-4 http://dx.doi.org/10.1016/j.saa.2015.03.070 SAA 13485

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date:

12 November 2014 22 February 2015

Please cite this article as: M.H. Helal, M.A. Salem, M.A. Gouda, N.S. Ahmed, A.A. El-Sherif, Design, synthesis, characterization, quantum-chemical calculations and anti-inflammatory activity of novel series of thiophene derivatives, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/ 10.1016/j.saa.2015.03.070

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Design, synthesis, characterization, quantum-chemical calculations and antiinflammatory activity of novel series of thiophene derivatives M. H. Helala,b , M. A. Salemb,c, M. A. Goudad,e, N. S. Ahmed a,f, A. A. El-Sherifg Department of Chemistry, Faculty of Arts and Science, Northern Border University, Rafha, KSA.

a b

Department of Chemistry, Faculty of Science, Al-Azhar University, 11284 Nasr City, Cairo, Egypt.

c

Department of Chemistry, Faculty of Science and Arts, King Khalid University, Mohail Assir, KSA.

d

Chemistry Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt.

e f

Department of Chemistry, Faculty of Science and Arts, Taibah University, Ulla, 41411, KSA.

Department of Chemistry, National Institute of Oceanography and Fisheres, Alex, Egypt.

g

Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt.

Abstract Interaction of 1-(4-morpholinophenyl)ethanone 1 with either malononitrile or ethyl cyanoacetate 2 afforded Knoevenagel-Cope product 3. In subsequent treatment of 3 with sulfur, the 2-aminothiophene derivatives (4a, 4b) are formed under basic conditions. The solvent-free reaction of thiophene derivative 4a with ethyl cyanoacetate afforded thieno[2,3-d][1,3]oxazine derivative 6. The base catalyzed condensation of 2aminothiophene derivative (4a) with ethyl cyanoacetate afforded N-(thieno-2-yl) cyanoacetamide derivative 7. The latter was used to synthesize different heterocyclic derivatives comprising, pyridine and coumarin rings. Also, several substituted thieno[2,3d]pyrimidines have been prepared from reaction of 2-aminothiophene-3- carbonitrile 4b with some electrophilic reagents. The structure of the newly compounds were confirmed on the basis of elemental analysis and spectral data. The molecular modeling of the synthesized compounds has been drawn and their molecular parameters were calculated. Also, valuable information is obtained from calculation of the molecular parameters including electronegativity, net dipole moment of the compounds, total energy, electronic energy, binding energy, HOMO and LUMO energy. Evaluation of anti-inflammatory activity of the tested compounds was performed in albino rats by producing carrageenan induced paw oedema and measuring the zone of inflammation at different time intervals 1

i.e. 1, 2, 3 and 4 h after carrageenan injection. Results indicated that most of the tested compounds showed moderate to good activity comparable to indomethacin. Also, compound 16 with additional morpholine ring beside the thiophene ring inhibits carrageenan induced paw oedema more than the standard indomethacin drug at all the time scales studied. Thus, compound 16 is considered as a promising compound for further modification to obtain clinically useful anti-inflammatory agent. Keywords: Thiophene, morpholine, Synthesis, Spectra, Modeling, Anti-inflammatory.

1. Introduct ion As it is known, the wide occurrence of the heterocycles in bioactive natural products and pharmaceuticals has made them as important synthetic targets and had attracted the attention of pharmaceutical chemists to synthesize a large number of novel chemotherapeutic agents of heterocyclic nature. Among these heterocyclic compounds, multisubstituted 2-aminothiophenes are privileged structures as they have attracted considerable attention in designing of bioactive molecules and found in several biologically active and natural compounds[1-4] due to their broad spectrum of biological activities, such as antioxidant [5], antibacterial[6], antifungal [7], antitumor [8], antiinflammatory [9], antianxiety [10] and antitubercular activities. Additionally, Nfunctionalized morpholines have found to possess diverse pharmacological activities. They are reported to exert a number of important physiological activities such as antidiabetic [11], antihyperlipo-proteinemics [12], antiemetic [13], platelet aggregation inhibitors, bronchodilators and growth stimulates [14] and anti-inflammatory [15]. These were also used in treatment of inflammatory diseases, pain, migraine and asthma [16]. The experimental studies have been accompanied by computational studies, especially in recent years [17] due to their important role in understanding of the probably behavior of the compound during reactions and identification of the important information about the compounds under investigations, like total energy, binding energy, electronic energy, dipole moment, bond lengths, HOMO, LUMO [18]. The applicability of the semi2

empirical methods PM3 for the calculation of novel synthesized compounds has been evaluated [19]. In a continuance of our research program directed towards synthesis of medicinally potent new chemical entities and their biological screening, the present study is aimed to study synthesis, characterization, molecular modeling and biological evaluation of various novel bioactive compounds heterocyclic compounds [20-25]. It seems therefore to be of considerable interest to synthesize newly heterocyclic compounds containing both the thiophene and morpholine moieties. Additionally, our objective is also to study the anti-inflammatory activities of the synthesized compounds.

2. Experimental All melting points are uncorrected. IR spectra (KBr) were measured on Shimadzu 440 spectrometer, 1HNMR and

13

CNMR spectra were obtained in DMSO on a Varian

Gemini 600 MHz spectrometer using TMS as internal standard; chemical shifts are reported as (ppm). Mass spectra were obtained on GCMS\QP 1000 Ex mass spectrometer at 70 ev. Elemental analyses were carried out at the Department of Chemistry, Faculty of Science, Cairo University, Egypt. Microbiology screening was carried out in Microbiology department, National Research Center, Cairo, Egypt.

2.1. Chemistry 2.1.1. Preparation of compounds (3a & 3b): General procedure A mixture of acetophenone 1 (0.05 mol), malononitrile (or ethyl cyanoacetate) (0.051 mol), acetic acid (2 mol) and ammonium acetate (0.05 mol) was added to 50 ml benzene with a Dean-Stark trap. The reaction mixture was stirred under reflux for 3 h for malononitrile (5 h for ethyl cyanoacetate) with removal of the condensed water. The excess benzene was evaporated and the resulting product was cooled to room temperature. The separated solid was then filtered, washed with water, and subjected to air drier and recrystallized from ethanol to give intermediate ethylidene 3.

3

Ethyl 2-cyano-3-(4-morpholinophenyl)but-2-enoate(3a) , cm-1):3010 (arom. CH), 2910 (aliph. CH),

Yield (70%); m.p. 57-58 ºC; IR (KBr,

2235(C≡N) and 1660 (C=O). 1HNMR (300 MHz, DMSO-d6): δ = 1.27 (t, 3H, CH3), 2.49 (s, 3H, CH3), 3.26, 3.79 (2t, 8H, morphonyl-H), 4.27 (q, 2H, CH2) and 7.03-7.53 (2d, 4H, Ar-H) ppm; 13CNMR (300 MHz, CDCl3): δ = 13.93 (CH3), 22.53 (CH3), 47.21 (C3, C5 of morpholine), 61.35 (CH2), 65.86 (C2, C6 of morpholine), 113.33, 117.26, 128.70, 129.46 (phenyl-C), 100,22, 152.56 (C=C) ppm. Elemental analysis for C17H20N2O3. Calcd. C, 67.98; H, 6.71; N, 9.33; Found: C, 67.80; H, 6.20; N, 9.40. 2-(1-(4-morpholinophenyl)ethylidene)malononitrile(3b) Yield (70%); m.p. 98-90ºC; IR (KBr,

, cm-1): 3001 (arom. CH), 2871(aliph. CH),

,2215 (C≡N) and 1609 (C=N). 1HNMR (300 MHz, DMSO-d6): δ = 2.49 (s, 3H, CH3), 3.27, 3.74 (2t, 8H, morphonyl-H), and 6.96-7.83 (2d, 4H, Ar-H) ppm; Elemental analysis for C15H15N3O. Calcd. C, 71.13; H, 5.97; N, 16.59; Found: C, 70.90; H, 5.60; N, 16.10. 2.1.2. Preparation of compounds (4a & 4b): General procedure In reaction flask both ethylidene 3 (0.05 mol) and sulphur ( 0.05 mol) were mixed in 50 ml ethanol. After the mixture was cooled to 15 °C, a solution of diethylamine (0.05 mol) was added dropwise at 15 °C, and stirred for 3 h at 65 °C. Left to cool, the separated solid was filtered, dried and recrystallized from ethanol to give 4.

Ethyl 2-amino-4-(4-morpholinophenyl)thiophene-3-carboxylate(4a) , cm-1): 3404, 3300, 3165 (NH2), 3090

Yield (70%); m.p. 209-210 ºC; IR (KBr,

(arom. CH), 2869 (aliph. CH) and 1666 (C=O). MS: 332(M+1, 55.0%), 286(38.8%), 228 (100.0%), 172(30.2%), 114 ( 25.5%), 77(14.5%).1HNMR (300 MHz, DMSO-d6): δ = 0.98 (t, 3H, CH3), 3.10, 3.76 (2t, 8H, morphonyl-H), 4.01 (q, 2H, CH2), 6.06 (s, 1H, thiophene-H5), and 6.89-7.29 (m, 6H, Ar-H + NH2) ppm; 13CNMR (300 MHz, CDCl3): δ = 13.84 (CH3), 48.59 (C3, C5 of morpholine), 58.53 (CH2), 66.05 (C2, C6 of morpholine), 103.01, 103.95 (thiophene- C5, C3),113.86, 129.16, 129.21( phenyl-C),

4

140.47, 149.78 (thiophene- C4, C2), 164.92 (C=O) ppm. Elemental analysis for C17H20N2O3S. Calcd. C, 61.42; H, 6.06; N, 8.43; Found: C, 61.30; H, 5.80; N, 8.10. 2-amino-4-(4-morpholinophenyl)thiophene-3-carbonitrile(4b) , cm-1): 3382, 3315, 3205 (NH2), 2825

Yield (70%); m.p. 185-186 ºC; IR (KBr,

(aliph. CH), 2197 (C≡N) and 1613 (C=N). MS: 385 (M+1,60.4%), 217 (100.0%). 199 (15.5%), 172(12.5%), 113(32.0%), 99(12.5%), 77(10.0%). 1HNMR (300 MHz, DMSOd6): δ = 3.13, 3.73 (2t, 8H, morphonyl-H), 6.37 (s, 1H, thiophene-H5), and 6.96-7.49 (m, 6H, Ar-H + NH2) ppm; 13CNMR (300 MHz, CDCl3): δ = 48.01 (C3, C5 of morpholine), 65.99 (C2, C6 of morpholine), 83.32, 102.85 (thiophene- C5, C3),114.73, 116.83, 125.20, 127.45 ( phenyl-C), 183.37, 150.46 (thiophene- C4, C2), 166.20 (C=O) ppm. Elemental analysis for C15H15N3OS. Calcd. C, 63.13; H, 5.30; N, 14.73; Found: C, 62.90; H, 5.00; N, 14.90. 2-(5-(4-morpholinophenyl)-4-oxo-4H-thieno[2,3-d][1,3]oxazin-2-yl)acetonitrile(6) 2-Aminothiophene 3a, (0.01mol) was fused with excess ethyl cyanoacetate at ~210 ºC in an oil bath for 40 min. Excess ethyl cyanoacetate was evaporated under vacuum. The solid product remained was treated with ethanol (20 mL) and then filtered. The ethanolic filtrate was poured on to crushed ice. The solid product obtained was filtered and crystallized from toluene: Yield (70%); m.p. 250 ºC; IR (KBr,

, cm-1): 2923 (aliph.

CH), 2210 (C≡N) and 1650 (C=O). MS: 353 (M+1, 58%),197 (19.5%), 166(10.0%), 115(23.0%), 218 (100%). 1HNMR (300 MHz, DMSO-d6): δ = 3.13, 3.75 (2t, 8H, morphonyl-H), 4.24 (s, 2H, CH2), and 6.99-7.83 (m, 5H, Ar-H + thiophene-H5) ppm; Elemental analysis for C18H15N3O3S. Calcd. C, 61.18; H, 4.28; N, 11.89; Found: C, 60.80; H, 4.10; N, 11.80. Ethyl 2-(2-cyanoacetamido)-4-(4-morpholinophenyl)thiophene-3-carboxylate(7) A mixture of compound 3a (0.01mol), ethyl cyanoacetate (0.01 mol) and sodium metal (0.5 gm) in ethanol (30 ml) was refluxed for 4h. The reaction mixture was poured into ice/water and acidified with 0.1 N HCl at pH 3-4 and then the resulting precipitate was

5

filtered off, dried, and recrystallized from acetic acid. Yield (70%); m.p. 149-150 ºC; IR (KBr,

, cm-1): 2890 (aliph. CH), 2205 (C≡N), 1670(C=O, ester) and 1660(C=O,

amide) cm-1. 1HNMR (300 MHz, DMSO-d6): δ = 1.01 (t, 3H, CH3), 3.13, 3.76 (2t, 8H, morphonyl-H), 4.07 (q, 2H, CH2), 4.25 (s, 2H, CH2), ,6.91-7.17 (m, 5H, Ar-H + thiophene-H5) and 11.15(s, 1H, NH) ppm;

13

CNMR (300 MHz, CDCl3): δ = 13.53

(CH3), 26.11 (CH2CN), 60.37 (CH2), 48.41 (C3, C5 of morpholine), 66.01 (C2, C6 of morpholine), 94.32, 139.04, 146.01, 150.15 (thiophene - C), 114.13, 115.17, 120.51, 127.38, 129.25 ( phenyl-C), 161.01, 164.36 (2C=O) ppm. Elemental analysis for C20H21N3O4S. Calcd. C, 60.13; H, 5.30; N, 10.52; Found: C, 60.00; H, 5.15; N, 10.30.

2.1.3. Preparation of compounds (8a & 8b) Equ imolar amounts of compound 4a (0.01 mol) and aromatic aldehyde (0.01 mol) in ethanol (30 mL) were treated with 0.5 ml of piperidine and refluxed for 3 h. The solid product which produced on heating was collected and recrystallized from the proper solvent. (E)-ethyl-2-(3-(4-chlorophenyl)-2-cyanoacrylamido)-4-(4-morpholinopheny-l)thiophene3-carboxylate(8a) Yield (60%); m.p. 255 ºC; IR (KBr,

, cm-1): 3160 (NH), 2900 (aliph. CH), 2190

(C≡N), 1677(C=O, ester) and 1662(C=O, amide) cm-1. 1HNMR (300 MHz, DMSO-d6): δ = 1.02 (t, 3H, CH3), 3.11, 3.70 (2t, 8H, morphonyl-H), 4.10 (q, 2H, CH2), 6.85(s, 1H, thiophene-H5), 7.10-8.60 (m, 5H, Ar-H+ olephenic- CH) and 10.90 (s, 1H, NH) ppm; Elemental analysis for C27H24ClN3O4S. Calcd. C, 62.12; H, 4.63; N, 8.05; Found: C, 62.00; H, 4.50; N, 8.00.

(E)-ethyl-2-(2-cyano-3-(4-methoxyphenyl)acrylamido)-4-(4-morpholinophenyl)thiophene-3-carboxylate(8b) , cm-1): 3130 (NH), 2925 (aliph. CH), 2220

Yield (70%); m.p. 245-247 ºC; IR (KBr,

(C≡N), 1675(C=O, ester) and 1662(C=O, amide) cm-1. 1HNMR (300 MHz, DMSO-d6): δ = 1.03 (t, 3H, CH3), 3.12, 3.74 (2t, 8H, morphonyl-H), 3.89 (s, 3H, OCH3), 4.16 (q, 2H,

6

CH2), 6.91(s, 1H, thiophene-H5), 7.18-8.42 (m, 5H, Ar-H+ olephenic- CH) and 11.81 (s, 1H, NH) ppm;

13

CNMR (300 MHz, CDCl3): δ = 13.54 (CH3), 55.75 (OCH3), 60.51

(CH2), 48.45 (C3, C5 of morpholine),

66.02 (C2, C6 of morpholine), 108.10,

150.20(C=C), 94.10, 139.90, 148.01, 154.30 (thiophene - C), 113.99, 115.05, 115.90, 120.51, 124.19, 127.58, 129.58, 133.39 ( phenyl-C), 163.57,

164.98 (2C=O) ppm.

Elemental analysis for C28H27N3O5S. Calcd. C, 64.97; H, 5.26; N, 8.12; Found: C, 64.50; H, 5.10; N, 8.00. 2.1.4. Preparation of compounds (10a & 10b) Compounds (10a & 10b) can be prepared by two different methods as shown below

2.1.4.1.

Method A

Equimolar amounts of compound 4a (0.01 mol) and aryldine malononitrile (0.01 mol) in ethanol (30 ml) were treated with 0.5 mL of piperidine and refluxed for 3 h. The solid product which produced on heating was collected and recrystallized from the proper solvent.

2.1.4.2.

Method B

A mixture of compound 8 (0.01 mol), malononitrile (0.01 mol) and 0.5 ml of piperidine in 1,4-dioxan (30 ml) was refluxed for 3 h. The solid product which produced on heating was collected and recrystallized from the proper solvent. Ethyl-2-(6-amino-4-(4-chlorophenyl)-3,5-dicyano-2-oxopyridin-1(2H)-yl)-4-(4morpholinophenyl)thiophene-3-carboxylate(10a) Yield (55%); m.p. 227-228 ºC; IR (KBr,

, cm-1): 3172, 3091 (NH2), 2863 (aliph. CH),

2216 (C≡N) and 1668 (C=O). 1HNMR (300 MHz, DMSO-d6): δ = 1.05 (t, 3H, CH3), 3.12, 3.75 (2t, 8H, morphonyl-H), 4.16 (q, 2H, CH2), 6.97-8.51 (m, 9H, Ar-H+ thiophene-H5), and 11.88 (s, 2H, NH2) ppm;

13

CNMR (300 MHz, CDCl3): δ = 13.53

(CH3), 60.57 (CH2), 48.43 (C3, C5 of morpholine), 66.01 (C2, C6 of morpholine), 94.20,

7

132.93, 137.90, 139.10 (thiophene-C), 102.20, 147.66, 152.33, 158.20 (pyridine-C), 113.98, 115.52, 116.16, 120.57, 127.47, 129.58, 132.27 (phenyl-C), 152.33 (C≡N), 164.95 (C=O) ppm. Elemental analysis for C30H24ClN5O4S. Calcd. C, 61.48; H, 4.13; N, 11.95; Found: C, 61.40; H, 4.00; N, 11.70. Ethyl-2-(6-amino-3,5-dicyano-4-(4-methoxyphenyl)-2-oxopyridin-1(2H)-yl)-4-(4morpholinophenyl)thiophene-3-carboxylate(10b) Yield (50%); m.p. 260-262 ºC; IR (KBr,

, cm-1): 3396, 3165 (NH2), 3077 (arom. CH),

2927 (aliph. CH), 2210 (C≡N) and 1671 (C=O). 1HNMR (300 MHz, DMSO-d6): δ = 1.08 (t, 3H, CH3), 3.12, 3.76 (2t, 8H, morphonyl-H), 3.85 (s, 3H, OCH3), 4.16 (q, 2H, CH2), 6.97-8.42 (m, 9H, Ar-H, thiophene-H5), and 11.90 (s, 2H, NH2) ppm; Elemental analysis for C31H27N5O5S. Calcd. C, 64.01; H, 4.68; N, 12.04; Found: C, 63.80; H, 4.40; N, 11.95. 2.1.5. Preparation of compounds 13 A mixture of 4a (0.01 mol), 1, 3-dicarbonyl compound (0.01 mol) and piperidine (0.5 ml) was fused in an oil bath at 150 ºC for 30 min. Ethanol was then added and the precipitate filtered out to give 13. Ethyl-2-(3-cyano-4,6-dimethyl-2-oxopyridin-1(2H)-yl)-4-(4-morpholinophenyl)thiophene-3-carboxylate(13) Yield (53%); m.p. 210-212 ºC; IR (KBr,

, cm-1): 3078 (arom. CH), 2825 (aliph. CH),

2219 (C≡N) and 1721 (C=O). 1HNMR (300 MHz, DMSO-d6): δ = 1.06 (t, 3H, CH3), 2.21, 2.41 (2s, 6H, 2CH3), 3.13, 3.75 (2t, 8H, morphonyl-H), 4.22 (q, 2H, CH2), 6.52 (s, 1H, thiophene-H5), 6.95, 7.25 (2d, 4H, Ar-H), and 7.65 (s, 1H, pyridine-H5) ppm; 13

CNMR (300 MHz, CDCl3): δ = 13.32 (CH3), 20.75 (2CH3), 60.54 (CH2), 48.12 (C3, C5

of morpholine), 66.01 (C2, C6 of morpholine), 99.84, 130.94, 140.99, 141.04 (thiophene - C), 109.26, 153.09, 160.22, 160.86 (pyridine - C), 114.36, 115.27, 123.23, 125.84, 128.93 (phenyl-C), 150.42, (C≡N), 161.60 (2C=O) ppm. Elemental analysis for C25H25N3O4S. Calcd. C, 64.78; H, 5.44; N, 9.06; Found: C, 64.80; H, 5.20; N, 8.85. 2.1.6. Preparation of compound 14

8

Ethyl-2-(2-imino-2H-chromene-3-carboxamido)-4-(4-morpholinophenyl)thio-phene-3carboxylate(14) Equimolar amounts of compound 4a (0.01 mol) and salisaldehyde (0.01 mol) in ethanol (30 mL) were treated with and ammonium acetate (0.5 g) and refluxed for 3 h. The solid product which produced on heating was collected and recrystallized from 1,4-dioxan as white crystals. Yield (70%); m.p. 229-230 ºC; IR (KBr,

, cm-1): 3318, 3243 (NH),

3100 (arom. CH), 2815 (aliph. CH), 1715 (C=O, ester) and 1675 (C=O, amid). 1HNMR (300 MHz, DMSO-d6): δ = 1.06 (t, 3H, CH3), 3.14, 3.75 (2t, 8H, morphonyl-H), 4.19 (q, 2H, CH2), 6.90 (s, 1H, thiophene-H5), 7.15- 7.84 (m, 8H, Ar-H), 8.61 (s, 1H, chromene H4), 9.70 and 14.26 (2s, 2H, 2NH) ppm; Elemental analysis for C27H25N3O5S. Calcd. C, 64.40; H, 5.00; N, 8.34; Found: C, 64.20; H, 5.10; N, 8.13.

2.1.7. Preparation of compound 15 2-Chloro-N-(3-cyano-4-(4-morpholinophenyl)thiophen-2-yl)acetamide (15) To a solution of 4b (0.01 mol) in DMF (20 ml), chloro acetylchloride (0.01 mol) was added and the reaction was allwed to stirred for 1 h. All contents were then poured on ice, the product was collected, dried and recrystallized from ethanol, to yield 15 colorless crystals. Yield (60%); m.p. 199-200 ºC; IR (KBr,

, cm-1): 3365 (NH), 3091 (Arom.

CH), 2855 (aliph. CH), 2218 (C≡N) and 1681 (C=O). MS: 361(M+, 65%), 303 (15.0%), 227 (100%),198 (20.2%), 171(18.0%), 77(66.0%). 1HNMR (300 MHz, DMSO-d6): δ = 3.15, 3.75 (2t, 8H, morphonyl-H), 4.52 (s, 2H, CH2), 7.15- 7.84 (m, 5H, Ar-H + thiophene-H5) and 11.80 (s, 1H, NH) ppm; Elemental analysis for C17H16ClN3O2S. Calcd. C, 56.43; H, 4.46; N, 11.61; Found: C, 56.10; H, 4.20; N, 11.50. 2.1.8. Preparation of compound 16 N-(3-cyano-4-(4-morpholinophenyl)thiophen-2-yl)-2-morpholinoacetamide (16) A mixture of 15 (0.01 mol), morpholine (0.01 mol) and potassium carbonate (0.01 mol) was dissolved in DMF (7 mL) and refluxed under stirring for 2 h. After cooling, the suspension was filtered and the solution was extracted with chloroform and washed with

9

water. The organic layers were dried over anhydrous sodium sulfate and evaporated. Recrystallization from ethanol gave white crystals. Yield (70%); m.p. > 300 ºC; IR (KBr, , cm-1): 3240 (NH), 3110 (arom. CH), 2825 (aliph. CH) and 1668 (C=O,). 1HNMR (300 MHz, DMSO-d6): δ = 3.16, 3.19, 3.75, 3.78 (4t, 16H, 2 morphonyl-H), 4.11 (s, 2H, CH2), 6.98 -7.48 (m, 5H, Ar-H + thiophene-H5) and 12.58 (s, 1H, NH) ppm;

13

CNMR

(300 MHz, CDCl3): δ = 52.23 (CH2), 46.84, 48.04 (2 morpholine- C3, C5), 65.66, 66.32 (2 morpholine- C2, C6), 94.10 (thiophene - C3), 138.37 (thiophene – C4), 149.37 (thiophene – C5), 150.61 (thiophene – C2), 114.61, 114.94, 120.57, 124.14, 128.15 (phenyl-C), 162.42, (C≡N), 188.65 (C=O) ppm. Elemental analysis for C21H24N4O3S. Calcd. C, 61.14; H, 5.86; N, 13.58; Found: C, 61.00; H, 5.50; N, 13.10.

2.1.9. Preparation of compound 17 2-(4-Hydroxy-2-iminothiazol-3(2H)-yl)-4-(4-morpholinophenyl)thiophene-3-carbonitrile(17) To a solution of 15 (0.01mol) in EtOH (60 ml) KSCN (0.01mol) was added. The reaction mixture was refluxed for 2 h. The mixture was then poured on ice-cold water, the solid collected by filtration and recrystallized from EtOH to give yellow crystals. Yield (70%); m.p. > 300 ºC; IR (KBr,

, cm-1): 3432 (br OH), 2974 (arom. CH), 2825 (aliph. CH),

2210 (C≡N) and 1609 (C=N). 1HNMR (300 MHz, DMSO-d6): δ = 3.15, 3.75 (2t, 8H, morphonyl-H), 6.68 (s, 1H, thiazole-H5), 6.98- 7.51 (m, 5H, Ar-H + thiophene-H5), 9.90 (s, 1H, NH) and 11.90 (s, 1H, OH) ppm; Elemental analysis for C18H16N4O2S2. Calcd. C, 56.23; H, 4.19; N, 14.57; Found: C, 56.00; H, 3.90; N, 14.15.

2.1.10. Preparation of compound 19 5-(4-Morpholinophenyl)thieno[2,3-d]pyrimidin-4-amine(19) A solution of 4b in formamide (10ml) was refluxed for 2 hr and the obtained product after cooling and filtration was recrystallized from acetic acid to give brown crystals. Yield (70%); m.p. > 300 ºC; IR (KBr,

, cm-1): 3473, 3240 (NH2), 3087 (Arom. CH),

10

2840 (Aliph. CH) and 1620 (C=N). 1HNMR (300 MHz, DMSO-d6): δ = 3.21, 3.75 (2t, 8H, morphonyl-H), 7.06- 7.35 (m, 5H, Ar-H + thiophene-H5), 8.10 (s, 1H, pyrimidineH2), and 8.35 (s, 2H, NH2) ppm; Elemental analysis for C16H16N4OS. Calcd. C, 61.52; H, 5.16; N, 17.93;Found: C, 61.20; H, 4.95; N, 18.00. 2.1.11. Preparation of compound 20 5-(4-Morpholinophenyl)thieno[2,3-d]pyrimidin-4(3H)-one(20) A solution of 4b in formic acid (10ml) was refluxed for 4 hr and the obtained product after cooling and filtration was recrystallized from acetic acid to give white crystals. Yield (70%); m.p. > 300 ºC; IR (KBr,

, cm-1): 3140 (NH), 3055 (arom. CH), 2812

(aliph. CH) and 1666 (C=O). 1HNMR (300 MHz, DMSO-d6): δ = 3.15, 3.76 (2t, 8H, morphonyl-H), 6.86- 7.44 (m, 5H, Ar-H + thiophene-H5), 8.12 (s, 1H, pyrimidine-H2), and 12.20 (s, 1H, NH) ppm; Elemental analysis for C16H15N3O2S. Calcd. C, 61.32; H, 4.82; N, 13.41; Found: C, 61.00; H, 4.55; N, 13.15. 2.1.12. Preparation of compound 21 N-(3-cyano-4-(4-morpholinophenyl)thiophen-2-yl)acetamide(21) A suspension of 4b (0.01 mol) in acetic anhydride (15 ml) was refluxed for 6 hr, cooled and the resulting precipitate was filtered off and recrystallized from acetic acid as white crystals. Yield (70%); m.p. 274-275 ºC; IR (KBr,

, cm-1): 3140 (NH), 3010 (arom.

CH), 2815 (aliph. CH) and 1668 (C=O,). 1HNMR (300 MHz, DMSO-d6): δ = 2.23 (s, 3H, CH3), 3.17, 3.77 (2t, 8H, morphonyl-H), 7.00- 7.48 (m, 5H, Ar-H + thiophene-H5) and 11.60 (s, 1H, NH) ppm;

13

CNMR (300 MHz, CDCl3): δ = 22.49 (CH3), 47.92,

(morpholine- C3, C5), 65.98 (morpholine- C2, C6), 91.73 (thiophene - C3), 137.68 (thiophene – C4), 150.71 (thiophene – C5), 150.82 (thiophene – C2), 113.25, 114.76, 120.62, 124.19, 128.04 (phenyl-C), 148.42, (C≡N), 168.66 (C=O) ppm. Elemental analysis for C17H17N3O2S. Calcd. C, 62.36; H, 5.23; N, 12.83;Found: C, 62.00; H, 5.15; N, 12.60. 2.1.13. Preparation of compound 23

11

1-(3-Cyano-4-(4-morpholinophenyl)thiophen-2-yl)-3-phenylthiourea(23) To solution of 4b (0.01 mol) in ethanol (20 ml), phenyl isothiocyanate (0.01 mol) and triethylamine (0.5 ml) were added. The reaction mixture was refluxed for 3 hr, cooled and the resulting solid was filtered off and recrystallized from methanol as yellow crystals. Yield (70%); m.p. 240 ºC; IR (KBr,

, cm-1): 3432 (NH), 2840 (aliph. CH),

2202 (C≡N), and 1631 (C=N). 1HNMR (300 MHz, DMSO-d6): δ = 3.17, 3.75 (2t, 8H, morphonyl-H), 4.49 (s, 1H, NH), 6.91- 7.33 (m, 5H, Ar-H + thiophene-H5) and 11.03 (s, 1H, NH) ppm; Elemental analysis for C22H20N4OS2. Calcd. C, 62.83; H, 4.79; N, 13.32; Found: C, 63.00; H, 4.80; N, 13.10.

2.3. Molecular modeling studies An attempt to gain a better insight on the molecular structure of the synthesized compounds, geometric optimization and conformation analysis has performed using semi-empirical method PM3 as implemented in HyperChem 7.5 [26]. The structures of synthesized reported compounds were optimized with semi-empirical method PM3. A gradient of 0.01 kcal/Å was set as a convergence criterion in all the molecular mechanics and quantum calculations. The lowest energy structure was used for each molecule to calculate physicochemical properties.

2.3. Anti-inflammatory activity In order to screen the anti-inflammatory profile of the synthesized compounds, carrageenan-induced paw edema assay was employed as a model for acute inflammation [27]. Albino Wistar rats of either sex (130-150 g) were divided into various groups. Each group contained six mice fasted for 12 h. First group was used as a control group and received 1 ml of 20% v/v DMSO solution. The second group received indomethacin orally (10 mg/kg) dissolved in 20% v/v DMSO solution. Other groups received DMSO solution of test compounds at a dose of 50 mg/kg orally. One hour after 12

the administration of the tested compounds, carrageenan suspension (0.1 ml of 1% w/v suspension in 0.9% saline solution) was injected into the sub-planter region of left hind paw of animals. Carrageenan caused visible redness and pronounced swelling that was well developed by 4 h and persisted for more than 48 h. immediately, the initial paw volume was measured using plethysmometer (UGO Basile 21025 Comerio, Italy) before carrageenan injection. Also, the paw volume was measured after 1, 2, 3 and 4 h after carrageenan administration. The difference between initial and subsequent readings gave the change in edema volume for the corresponding time. Indomethacin (10 mg/kg) was administrated in second group of animals and served as reference drug for comparison. Anti-inflammatory activities of the reported compounds and indomethacin were determined by comparing their results with the ones obtained in the control group. Edema volume of control (Vc) and volume of treated (Vt) were used to calculate (%) edema volume by using following formula: % Edema volume=100 × (Edema volume after drug treatment/Initial volume) 2.4. Statistical analysis Values were expressed as means ± S.E. Comparisons between means were carried out using one-way ANOVA followed by least significant difference (LSD) and Turkey multiple comparisons test.

3. Results and discussion

In this investigation, the authors aimed to synthesize the substituted 2aminothiophenes 4 via a two-step Gewald synthesis [28] In a reaction of 1-(4morpholinophenyl)ethanone 1 [29] with either malononitrile or ethyl cyanoacetate 2 afforded Knoevenagel-Cope product 3. The structure of compound 3 was confirmed on

13

the basis of its elemental and spectral data. The IR spectrum of 3b revealed absorption bands at 2871, 2215 cm-1 assignable to aliphatic CH and nitrile group. The 1H-NMR spectrum of 3a displayed singlet signals at 2.45 characteristic for CH3, two triplet signals at 3.27, 3.73 ppm for morpholine protons. In subsequent treatment of 3 with sulfur, the 2aminothiophenes 4 is formed under basic conditions with 70% yield (Scheme 1). The infrared spectrum of compound 4a showed the presence of intense absorption bands at 3404, 3300, 3165 and 1666 cm-1 assignable for NH2 and carbonyl ester groups while its 1

HNMR spectrum (DMSO-d6) indicated a triplet at 0.98 and quartet at 4.01 ppm for ester

group protons, singlet signals at 6.06 and 7.29 ppm for CH-thiophene and NH2 protons. Also, mass spectrum of 4a showed a molecular ion peak at m/z 332 (55%) with base peak at m/z = 228.

Scheme 1 The solvent-free reaction [30] of thiophene derivative 4a with ethyl cyanoacetate afforded

2-(5-(4-morpholinophenyl)-4-oxo-4H-thieno

[2,3-d][1,3]-oxazin-2-

yl)acetonitrile (6). The IR spectrum revealed absorption bands at 2210 and 1650 cm-1 assignable to C≡N and carbonyl groups, respectively. The 1H-NMR spectrum displayed a 14

lack of ethoxy, NH signals and the appearance of singlet signals at δ 4.24 ppm for methylene group. The formation of 6 is assumed to take place via the formation of nonisolable acyclic intermediate 5, followed by loss of ethanol to afford thieno[2,3-d] oxazine 6. On the other hand, the base catalyzed condensation [31] of 2-aminothiophene derivative 4a with ethyl cyanoacetate afforded N-(thieno-2-yl) cyanoacetamide derivative 7. The spectroscopic data of the isolated product was in complete agreement with structure 7 as its IR spectrum exhibited nitrile, carbonyl ester, and amidic carbonyl stretching frequencies at 2205, 1670 and 1660 cm-1 respectively. The1H-NMR spectrum of compound 7 displayed signals at δ 1.01, 4.07, 4.25, 6.91 and 11.15 ppm for ester group, methylene group, thiophene-H5 and NH proton, respectively. O Ar

OEt

O Ar

S

Fusion Ar

O S

Ar

- EtOH

CN

N

S 6

5

CN

NH2

COOEt

EtOH

4a Ar =

CN O

O

COOEt

O

NH

EtONa

O

NH

S

N

CN O 7

Scheme 2 The Knoevenagel condensation of cyanoacetamide 7 with aromatic aldehydes such as pchlorobenzaldehyde and p-methoxy- benzaldehyde in ethanolic piperidine solution under reflux conditions afforded the corresponding benzylidene derivatives 8a and 8b. The 1HNMR spectrum of 8b revealed four singlet signals at 3.89, 6.91, 8.42 and 11.81 ppm characteristic for methoxy protons, thiophene-H5, an olefinic proton (CH=) and NH proton respectively. When compounds 8a and 8b were reacted with malononitrile in

15

boiling ethanol containing a catalytic amount of piperidine, aminopyridone derivatives 10a and 10b were obtained as the sole products via intermediate 9. The 1 H-NMR spectra of 10a and 10b displayed triplet at 1.05 and quartet at 4.16 ppm for ester group protons and singlet at 11.88 ppm for NH2 protons. The formation of 10 is assumed to proceed via the initial Michael addition of the active methylene group of malononitrile to the activated double bond of 8 to form Michael adduct 9 which cyclized and auto-oxidation under the reaction conditions yielded 10. Additionally, the structures of 10a and 10b were established chemically through reaction of compound 7 with benzylidene derivatives 11 (1:1 M ratio) by refluxing in ethanol and in presence of piperidine (Scheme 3). Cyclocondensation of compound 7 with acetylacetone furnished 4-methyl-2oxopyridine derivative 13 via intramolecular hetero-cyclization of the non-isolable intermediate 12 by loss of water molecule. The

1

H-NMR spectrum of 13 revealed four

singlet signals at δ 2.21, 2.41, 6.52 and 7.65 ppm for 2 CH3, thiophene-H5 and pyridineH5,

respectively.

Also,

condensation

of

cyanoacetanilide

derivative

7

with

salicylaldehyde using ammonium acetate as a catalyst in refluxing ethanol gave 2-imino2H-chromene 14 ( Scheme 3). IR spectrum showed the absence of C≡N absorption band. 1

HNMR spectrum revealed three singlet signals at 8.61, 9.70 and 14.26 ppm

characteristic to CH-chromene and 2NH.

16

Ar

COOEt NH

S

CN

i

O Ar

8a,b

ii Ar

COOEt O

CN

iii Ar

Ar

COOEt O S

N

S

N C

COOEt

CN

NH

H2N

Ar

Ar

CN NH

S

CN

9 Ar

CN

10a,b

COOEt O

O

Ar

7

S

v

N H CH3 O

CH3

Ar

iv O N

S

- H2O

CN

N

H3C

CH3 13

12

Ar =

COOEt O

CN

COOEt O S

N H HN

O

14

i = p-chlorobenzaldehyde and/or p-anisaldehyde, ii = malononitrile, iii = arylidine v = acetylacetone, iv = salisaldehyde

Scheme 3 Chloroacetylchloride was reacted with 2-aminothiophene 4b in recently distilled dimethylformamide (DMF) to afford chloroacetamide derivative 15 in good yield. The mass spectrum of compound 15 revealed a molecular ion peak at m/z = 361(M+, 65%), and a base peak was observed in the spectrum at m/z = 227 (100%), which is compatible with its molecular formula of C17H16ClN3O2S. Chloroacetamide derivative 15 acts as a key intermediate for introduce the thiazolidinone and morpholine moieties on the 17

monamide of thiophene derivative 4b. Thus, the nucleophilic substitution reaction between compound 15 and morpholine afforded 2-morpholinoacetamide derivative 16. Also, the nucleophilic attack of potassium thiocyanate on N-substituted chloroacetamide derivative 15 afforded the 4-hydroxy thiazoline derivative 17 and the other possible ketoform 18 was excluded on the basis of infrared spectra which revealed charactrestic absorption broad band at 3423 cm-1 assignable to OH with absence of carbonyl absorption band. Also, 1HNMR spectrum of 18 revealed four singlet signals at 6.68, 6.98, 9.90 and 11.90 ppm characteristic for CH-thiazole, thiophene-H5, NH and OH protons respectively, (Scheme 4).

Scheme 4 The interaction of 4b with formamide via cyclization reaction afforded pyrido[2,3d]pyrimidine derivative 19. Both elemental analysis and spectroscopic data are in accord with the assigned structure. The infrared spectrum exhibited absorption bands at 3473, 3240 (NH2), 1620 cm-1(C=N) with absence of C≡N characteristic absorption band. 1

HNMR spectrum of 19 revealed two singlet signals at 8.10 and 8.35 ppm assignable to

18

CH-pyrimidine and NH2 protons respectively. Also, pyrido[2,3-d]pyrimidine derivative 20 was obtained upon treatment of 4b with formic acid. 1HNMR spectrum for compound 20 revealed two singlet signals at 8.12, 12.20 ppm assignable to CH-pyrimidine and NH protons respectively. Additionally, treatment of 4b with acetic anhydride gave acyclic product 21 and the other possible pyrido[2,3-d]pyrimidine structure 22 was ruled out on the basis of the presence of C≡N characterisƟc absorpƟon band at 2210 cm-1. Similarly, reaction of 2-aminothiophene derivative 4b with phenyl isothiocyanate afforded 3-cyano4-(4-morpholinophenyl)thiophen-2-yl)-3-phenylthiourea (23) and the other possible pyrido[2,3-d]pyrimidine structure 24 was excluded on the basis of infrared spectrum which revealed charactrestic absorption band at 3432 and 2202 cm-1 assignable to NH and C≡N group respecƟvely. Also, 1HNMR spectrum of 23 revealed two singlet signals at 4.48,11.07 ppm (2NH) and 6.91 ppm for CH- thiophene-H5 proton (Scheme 5).

19

CN

Ar

N H

S

NH2

Ar

S N H

Ph

N

i S

23

Ar

CN

iv NH

Ar

N S

N H

Ph

S 4b

S

NH

ii S

24 Ar

O

N 20

CN O

NH S Ar =

O

Ar

NH2

iii

Ar

N 19

O

N H

S

N 22

21

N i = Formamide, ii = formic acid, iii = acetic anhydride, iv = phenyl isithiocyanate

Scheme 5 3.2. Molecular modeling and computational study In the absence of a crystal structure, to obtain the molecular conformation of a compound, energy minimization studies were carried out on the basis of the semiempirical PM3 level provided by HyperChem 7.5 software. The most stable structures obtained were subsequently optimized to the closest local minimum at the semiempirical level using PM3 parameterizations. The calculated dipole moment (µ), total energy (ET), binding energy (EB), and electronic energy (EE) after geometrical optimization of the structures of compounds (Table 1). The values of the following parameters: the highest occupied molecular orbital energy (EHOMO), the lowest unoccupied molecular orbital energy (ELUMO), the difference

20

between HOMO and LUMO energy levels (ΔE), Mulliken electronegativity (χ), chemical potential (Pi), global hardness (η), global softness (S), global electrophilicity (ω) [32-35] and additional electronic charge (ΔNmax) have been calculated [36] using semi-empirical PM3 method as implemented in HyperChem [26]. In a first step, the molecular geometries of all compounds were fully optimized in the gas phase to gradients of 0.01 kcal·mol-1Å-1 and afterwards the molecular descriptors were determined. The molecular parameters were calculated as reported in literature [37-39]. Recently, quantumchemical calculation methods have become available to provide a powerful approach for crystal structure prediction [37-39].

3.2.1. Bond length and bond angle calculations Theoretical

calculations

have

paid

a

considerable

attention

to

the

characterization and inferences of geometrical optimization of the prepared compounds; therefore we could obtain the optimized structure for the prepared compounds by computing the theoretical physical parameters, such as, bond lengths and bond angles using the HyperChem 7.5 software. The optimized structure for the 4a, 15, 17 and 20 with the atomic numbering scheme is shown in the Figs. 1-4. The bond angles and bond lengths (Ǻ) obtained from the energy minimum optimized structures of 20 as a representative example of the synthesized compounds are given in the Tables 2 and 3. In most of the cases, the actual bond lengths and bond angles are close to the optimal values, and thus the proposed structure of the compound is acceptable. 3.2.2. Molecular parameters From the obtained data (Table 4) we can deduce each of the following: i) The molecular stability and reactivity can be measured by important properties like hardness (η) and softness (σ). Soft molecules have small energy gap and more reactive 21

than hard molecules with a large energy gap. This can be attributed to the fact that soft molecules could easily offer electrons to an acceptor. Also, the reactivity of soft molecules increases than hard molecules if electron transfer or rearrangement is necessary for the reaction. ii) The reactivity index measures the energy stabilization when the system acquires an additional electronic charge (ΔNmax) from the environment. The electrophilicity index is positive quantity and the direction of the charge transfer is completely determined by the electronic chemical potential (Pi) of the molecule because an electrophile is a chemical species capable of accepting electrons from the environment and its energy must decrease upon accepting electronic charge. Therefore, the electronic chemical potential must be negative, exactly as supported by the values in Table 4. iii)The HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) called the frontier molecular orbitals (FMOs) used to determine the way of interaction for the molecule with other species. The negative values of the HOMO and LUMO (Table 4) indicate the title thiophene compounds are stable molecules [40-42]. iv) The concept hard and soft nucleophiles and electrophiles has also been directly related to the relative energies of the HOMO and LUMO orbitals. Hard nucleophiles have a low energy HOMO, soft nucleophiles have a high energy HOMO, hard electrophiles have a high energy LUMO and soft electrophiles have a low energy LUMO. Electron-donating atoms possess high HOMO with a loose holding of valence electron, thereby being susceptible to oxidation. Substances with low ionization energy give up electrons easily and hence are likely to participate in chemical reactions. v) The correlation between the highest molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the synthesized compounds was investigated.

22

The energies of HOMO of (4a), (4b), (6), (7), (14), (15), (16), (20) and (21) are closely spaced (EHOMO, (4a) -8.23 eV; EHOMO, (4b) -8.51 eV; EHOMO, (6) -8.47 eV, EHOMO, (7) -8.90 eV, EHOMO, (14) -8.84 eV, EHOMO, (15) -8.76 eV, EHOMO, (16) -8.64, EHOMO, (20) -8.23 eV and EHOMO, (21) -8.64 eV whereas (10a), (13) and (17) are substantially higher (EHOMO, (10a) 5.40 eV, EHOMO, (13) -6.04 eV and EHOMO, (17) -5.36 eV. vi) The HOMO–LUMO energy separation has been used as an important stability index and an indicator for the chemical reactivity of the molecule. A molecule with a small energy gap is more polarized and is known as soft molecule [43,44]. The values of the energy separation between the HOMO and LUMO for the synthesized compounds lie in the range 3.97-7.70. This large HOMO–LUMO gap automatically means high excitation energies for many of the excited states and good stability for the title compounds. vii) It was observed that, the energy difference between the HOMO and LUMO in 10a, 17 and (13) significantly decreases compared to that of the other compounds, indicating a lower kinetic stability of (10a), (17) and (13) compared to the others. The low value of energy gap may be due to the groups that enter into conjugation [37]. viii) The electrophilicity index (ω), which shows the ability of the molecule to accept electrons follow the trend; 6 (ω = 3.74) > 7 (ω = 3.58) > 15 (ω = 3.43) > 14 (ω = 3.43) > 16 (ω = 3.23) > 21 (ω = 3.22) > 20 (ω = 3.12) > 10a (ω = 2.94) > 4b (ω = 2.82) > 17 (ω = 2.81) > 13 (ω = 2.62) > 4a (ω = 2.58). Compound (6) exhibits the highest value of electrophilicity (Table 4) which confirms its highest capacity to accept electrons. 3.3. Anti-inflammatory activity Inflammation is the integral part of the body's defense mechanism. All the newly synthesized compounds and indomethacin, as a reference drug, were subjected to in vivo anti-inflammatory studies using carrageenan-induced rat paw edema model. Edema, which develops after carrageenan inflammation, is a biphasic event. In the

23

present investigation, the results of the anti-inflammatory studies are given in Table 5 as percent edema at the right hind paw. The right hind paw volume was measured immediately before carrageenan injection and at selected times (1, 2, 3 and 4 hours) thereafter by water displacement plethysmometer. All the drugs were administrated one hour before carrageenan injection. Carrageenan-induced edema is a non-specific inflammation maintained by the release of histamine, 5-hydroxytryptamine, kinins and later by prostaglandins [45,46]. Since edemas of this type are highly sensitive to nonsteroidal anti-inflammatory drugs (NSAIDs), carrageenan has been accepted as a useful agent for studying new anti-inflammatory drugs.. The anti-inflammatory properties were recorded at successive intervals of 1, 2, 3 and 4 h and compared with that of standard indomethacin. The inhibitory effect of acid NSAIDs, such as indomethacin, is usually weak in the first phase (1–2 h), in contrast with their strong inhibition in the second phase (3–4 h) [47]. Good inhibition of the second phase of carrageenan induced edema was observed for the compounds tested, suggesting that they interfere with prostaglandin synthesis (Table 5). The following points could be deduced after analyzing of the anti-inflammatory results: 1- The data obtained (Fig. 5) revealed that, paw edema was inhibited by the oral administration of the most of the test compounds at a dose level of 50 mg/kg dose. 2- The anti-inflammatory activity data (Table 5) indicated that all the synthesized thiophene compounds exhibited significant activity by decreasing the paw volume that was produced by carrageenan. 3- Compounds 4a, 4b, 6, 7, 10a, 13a, 14, 15, 16, 17, 20 and 21, at 50 mg/kg p.o. exhibited 34.45%, 37.03%, 54.01%, 43.39%, 56.61%, 49.17%, 45.16%, 50.85%, 58.46%, 39.85, 31.35% and 36.87% activity, respectively whereas indomethacin exhibited 47.73% anti-inflammatory activity at 4h.

24

4- The anti-inflammatory activity data (Table 5) indicated that all the tested compounds protected rats from carrageenan-induced inflammation moderately at 1h of reaction time; the activity increased and reached to the peak level at 4 h. 5- Most of the tested compounds showed carrageenan activity nearest to indomethacin. 6- The anti-inflammatory activity of the synthesized thiophene derivatives obeyed this order 16 > 6 >10a > 15 > 14 > 13 > 7 > 21 > 20 > 17 > 4b > 4a 7- Compounds 17, 20, 21, 4a and 4b have lower anti-inflammatory activity than indomethacin. 8- Among all the test compounds, it is interesting to note that compounds 16, 6 and 10a showed best anti-inflammatory activity. 3.4. SAR studies Structure-activity relationships for the synthesized thiophene derivatives showed that compound 16 containing extra morpholine ring appeared more anti-inflammatory potent than indomethacin standard drug at all investigated time scales from the start of the first hour to the fourth hour. Thus, compound 16 is a promising compound. Thiophene compounds with additional oxazinone heterocyclic ring like 6, showed carrageenan activity in the same range of indomethacin reference drug. Thiophene compounds with additional chromene ring as 14 or pyridone moieties like 10a and 13, showed equipotent anti-inflammatory activity when compared to the reference standard indomethacin reference drug. Since our findings are preliminary results; further studies need to be carried out to investigate the other specifications, such as toxicological studies or side effect-activity profiles of the most active compounds.

25

4. Conclusions In summary, the objective of the present study was to synthesize and investigate the anti-inflammatory activities of some new thiophenes with the hope of discovering new compounds possessing an anti-inflammatory activity. The novel series of thiophene derivatives were characterized by elemental analysis and 1H-NMR,

13

C-NMR, mass

spectrometry, IR studies and elemental analyses. The reported compounds were evaluated for their anti-inflammatory activities. The in vivo anti-inflammatory properties were investigated by standard methodologies. As regards the relationships between the structure of the heterocyclic scaffold and the detected biological activities, it showed varied biological activity. Probably in this case the presence of different substituent’s caused a certain change of activity. SAR indicated that the thiophene compounds having and additional heterocyclic rings like pyridone, or oxazinone, chromene and extra morpholine moieties have increased activity compared to other compounds. In conclusion, compound having additional morpholine ring emerged as lead compound in the anti-inflammatory studies among all the synthesized thiophene compounds. To support the solid state structure, the molecular modeling optimization and electronic parameters have been calculated using PM3 method. Acknowledgments The authors express their sincere thanks to the Northern Border University for financial support of the project number (433-038).

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29

[46] K. Tsurumi, K. Kyuki, M. Niwa, S. Kokuba, H. Fujimura, Arzneim. Forsch. Drug Res. 36 (1986) 1796–1800. [47] A. Gavalas, L. Kourounakis, D. Litina, P. Kourounakis, Arzneim. Forsch. Drug Res. 41 (1991) 423–426.

30

Fig. 1. The molecular structure of compound 4a along with the atom numbering scheme.

31

Fig. 2. The molecular structure of compound 15 along with the atom numbering scheme.

32

Fig. 3. The molecular structure of compound 17 along with the atom numbering Scheme.

33

Fig. 4. The molecular structure of compound 20 along with the atom numbering scheme.

34

80 70

50 40 30 20 4h 3h

10 0 Indomethacin4a

2h 1h

4b

6

7

10a

13a

14

15

16

17

20

21

Compounds

Fig. 5. Anti-inflammatory activity of the synthesized compounds.

35

Time

% Inhibition

60

Table 1. Some energetic properties of synthesized compounds calculated by PM3 method: Compound

4a

Total energy kcal/mol -85636.07

Binding energy kcal/mol -4482.6

Electronic energy kcal/mol -642009.5

Nuclear energy kcal/mol 556373.5

Dipole moment Debyes 2.93

2.41

4b

-68562.3

-3756.7

-480203.3

411640.9

3.03

2.38

6

-90292.6

-4446.7

-668187.2

577894.6

3.70

2.85

7

-104545.6

-5088.2

-844900.2

740354.5

6.25

2.24

10a

-150410.8

-7294.9

-14464589.8

1314179.0

7.28

4.39

13a

-119613.9

-6125.3

-1077953.1

958613.1

4.86

3.71

14

-131845.6

-6554.3

-1218429.1

1086583.5

2.56

3.49

15

-88457.1

-4278.7

-642808.9

554351.7

5.22

2.60

16

-105444.4

-5555.3

-880545.0

775100.5

4.67

1.53

17

-91469.9

-4431.2

-703195.9

611725.9

4.53

4.63

20

-78084.1

-4040.8

-563246.9

485162.8

1.78

1.80

21

-81514.2

-4302.9

-594536.8

513022.6

4.79

2.01

Table 2. Various bond lengths (Å) of 20 compound Atoms Actual bond length (Å)

Optimal bond length (Å)

C(22)-H(37)

1.11

1.11

C(22)-H(36)

1.11

1.11

C(21)-H(35)

1.11

1.11

C(21)-H(34)

1.10

1.111

C(21)-C(22)

1.53

1.52

O(20)-C(21)

1.41

1.39

C(19)-H(33)

1.11

1.11

C(19)-H(32)

1.10

1.11

C(19)-O(20)

1.415

1.39

C(18)-H(31)

1.11

1.11

C(18)-H(30)

1.11

1.11

C(18)-C(19)

1.53

1.52

log P

N(17)-C(22)

1.48

1.47

N(17)-C(18)

1.48

1.47

C(16)-H(29)

1.10

1.10

C(15)-H(28)

1.10

1.10

C(15)-C(16)

1.38

1.42

C(14)-H(27)

1.10

1.10

C(13)-H(26)

1.10

1.10

C(13)-C(14)

1.39

1.42

C(12)-N(17)

1.44

1.46

C(12)-C(16)

1.41

1.42

C(12)-C(13)

1.40

1.42

C(11)-C(15)

1.40

1.42

C(11)-C(14)

1.40

1.42

C(9)-O(10)

1.22

1.21

N(8)-H(25)

1.00

1.01

N(8)-C(9)

1.43

1.37

C(7)-H(24)

1.10

1.10

C(7)-N(8)

1.39

1.35

N(6)-C(7)

1.31

1.26

C(4)-C(5)

1.40

1.42

C(2)-H(23)

1.10

1.10

C(2)-S(3)

1.71

1.66

C(1)-C(5)

1.45

1.42

Table 3. Various bond Angles (o) of 20 compound Atoms

Actual bond length (Å)

Optimal bond length (Å)

H(37)-C(22)-H(36)

106.63

109.40

H(37)-C(22)-C(21)

110.03

109.41

H(36)-C(22)-C(21)

110.87

109.41

H(35)-C(21)-H(34)

108.30

109.4

H(35)-C(21)-C(22)

111.54

109.41

H(35)-C(21)-O(20)

109.77

106.70

H(34)-C(21)-C(22)

111.77

109.41

C(22)-C(21)-O(20)

112.54

107.40

H(33)-C(19)-H(32)

108.29

109.40

H(33)-C(19)-O(20)

109.75

106.70

H(33)-C(19)-C(18)

111.57

109.41

H(32)-C(19)-C(18)

111.75

109.41

H(31)-C(18)-H(30)

106.57

109.40

H(31)-C(18)-C(19)

110.01

109.41

H(31)-C(18)-N(17)

110.24

109.80

H(30)-C(18)-C(19)

110.84

109.41

C(22)-N(17)-C(18)

110.47

109.41

H(29)-C(16)-C(15)

119.182

120.00

H(29)-C(16)-C(12)

120.28

120.00

H(28)-C(15)-C(16)

118.54

120.00

H(28)-C(15)-C(11)

119.77

120.00

H(27)-C(14)-C(13)

118.72

120.00

H(27)-C(14)-C(11)

120.05

120.00

C(13)-C(14)-C(11)

121.22

121.00

H(26)-C(13)-C(14)

118.67

120.00

H(26)-C(13)-C(12)

120.26

120.00

C(14)-C(13)-C(12)

121.05

121.00

N(17)-C(12)-C(16)

120.68

120.00

N(17)-C(12)-C(13)

121.017

120.00

C(16)-C(12)-C(13)

117.996

120.00

C(15)-C(11)-C(14)

117.507

120.00

C(15)-C(11)-C(1)

118.771

120.00

C(14)-C(11)-C(1)

123.72

120.00

N(8)-C(9)-C(5)

116.049

112.74

H(25)-N(8)-C(9)

119.403

117.40

H(24)-C(7)-N(8)

119.979

116.56

H(24)-C(7)-N(6)

118.409

116.50

C(7)-N(6)-C(4)

119.196

115.00

C(9)-C(5)-C(4)

117.067

117.6

N(6)-C(4)-C(5)

123.94

120.00

N(6)-C(4)-S(3)

123.099

126.00

H(23)-C(2)-S(3)

121.04

120.00

H(23)-C(2)-C(1)

123.466

120.00

C(11)-C(1)-C(2)

119.879

120.00

Table 4. Calculated EHOMO (EH), ELUMO (EL), energy band gap (EH-EL), chemical potential (Pi), electronegativity (χ), global softness (S), global hardness (η), electrophilicity index (ω) and absolute softness (σ) for the synthesized compounds Compound

EH (eV)

4a 4b 6 7 10a 13a 14 15 16

-8.23 -8.51 -8.47

17

-5.36 -8.23

20 21

-8.9 -5.40 -6.04 -8.84 -8.76 -8.64

-8.64

EL(eV) ∆E(eV) χ (eV) -0.63 7.60 4.43 -0.81 7.70 4.66 -1.64 6.83 5.06 7.46 -1.44 5.17 -1.43 3.97 3.42 -1.13 4.91 3.59 -1.32 7.52 5.08 -1.37 7.39 5.07 -1.18 7.46 4.91 -1.35 4.01 3.355 -1.16 7.07 4.70 -1.17

7.47

4.91

η(eV)

σ (eV-1)

Pi(eV)

ω(eV)

S (eV-1)

∆Nmax

3.8 3.85 3.42 3.73 1.98 2.46 3.76 3.70

0.26 0.26 0.29 0.27 0.50 0.41 0.27 0.27

-4.43 -4.66 -5.06 -5.17 -3.42 -3.59 -5.08 -5.07

2.58 2.82 3.74 3.58 2.94 2.62 3.43 3.47

0.13 0.13 0.15 0.13 0.25 0.20 0.13 0.14

1.17 1.21 1.48 1.39 1.72 1.46 1.35 1.37

3.73

0.27

-4.91

3.23

0.13

1.32

2.01 3.54 3.74

0.50 0.28 0.27

-3.365 -4.70 -4.91

2.81 3.12 3.22

0.25 0.14 0.13

1.67 1.33 1.31

Table 5. Anti-inflammatory activity of the synthesized thiophene compounds. Compd. No.

% Oedema volume (% Inhibition of test compounds relative to carrageenan group control) 1h

2h

3h

4h

Control

76.87± 5.83

101.6±10.41

125.3±6.75

154.7±5.27

Indomethacin (St.)

24.92± 2.51*(67.58)

46.57±4.08*(54.16)

73.54±6.23*(41.31)

80.86±5.91*(47.73)

4a

40.24± 3.61*(67.58)

55.91±3.58*(44.97)

86.95±7.87*(30.61)

101.4±1.4*(34.45)

4b

28.70± 1.32*(62.66)

51.40±4.85*(49.41)

74.79±5.01*(40.31)

97.42±2.98*(37.03)

6

27.54± 2.02*(64.17)

41.68±2.59*(58.98)

60.44±3.62*(51.76)

71.15±3.68*(54.01)

7

29.10±2.15*(62.14)

40.85±2.81*(59.97)

65.28±3.38*(47.90)

87.58±3.10*(43.39)

10a

31.85± 2.10*(58.57)

46.11±4.48*(54.62)

54.65±4.95*(56.38)

67.04±5.94*(56.66)

13a

32.22± 1.50*(58.09)

53.45±3.19*(47.39)

69.13±3.36*(44.83)

78.63±4.68*(49.17)

14

31.43± 2.69*(59.11)

45.54±2.23*(55.18)

68.73±5.96*(45.15)

84.84±7.84*(45.16)

15

26.23± 2.95*(65.88)

39.64±3.10*(60.98)

56.97±3.43*(54.53)

76.03±4.75*(50.85)

16

21.23± 2.14*(72.38)

33.14±2.99*(67.38)

48.39±4.80*(61.38)

64.26±6.41*(58.46)

17

34.33± 2.59*(55.34)

54.49±4.42*(46.37)

75.55±6.48*(39.70)

93.05±6.85*(39.85)

20

31.38± 1.35*(59.18)

49.63±4.26*(51.51)

81.23±7.94*(35.17)

106.2±6.56*(31.35)

21

31.85± 2.35*(58.57)

50.16±5.28*(50.63)

76.04±6.95*(39.31)

97.66±6.71*(36.87)

-Values are expressed as means ± SEM (n = 6). * Significantly different from control group at P < 0.05. ª Significantly different from indomethacin group at P < 0.05. Statistical analysis was done using one way ANOVA followed Turkey for multiple comparisons respectively.

Graphical Abstract Design, synthesis, characterization, quantumchemical calculations and anti-inflammatory activity of novel series of thiophene derivatives M. H. Helala,b , M. A. Salemb,c, M. A. Goudad,e, N. S.

80 70

Ahmed a,f, A. A. El-Sherifg

50 40 30 20 4h 3h

10 2h

0 Indomethacin4a

1h

4b

6

7

10a

13a

14

Compounds

15

16

17

20

21

T im e

% In h ib it io n

60

Novel series of thiophene heterocyclic compounds have been synthesized and characterized by elemental analyses and spectral like IR, 1H NMR, 13C NMR and MS studies. The molecular modeling of the synthesized compounds has been drawn and their molecular parameters were calculated. The antiinflammatory activity was investigated.

Research Highlights Design, synthesis, characterization, quantum-chemical calculations and antiinflammatory activity of novel series of thiophene derivatives M. H. Helala,b , M. A. Salemb,c, M. A. Goudad,e, N. S. Ahmed a,f, A. A. El-Sherifg

● Novel series of thiophene heterocyclic compounds were synthesized and characterized ● Optimization of the geometric shape was carried out. ● Molecular parameters were calculated. ● The anti-inflammatory activities of the synthesized compounds were investigated.

37