Synthesis of Some <i>N</i>-[(4-Substituted-1-Piperazinyl)-Oxo(Alkyl and Ethylcarbamoyl)]-3-(2-Thiophenyl)Acrylamides as Non-Steroidal Anti-Allergic and Anti-Inflammatory Agents

An interesting approach for the design of anti-allergies is rationally considered. It was proved that current anti-allergic drugs comprise piperazine and acrylic acid segments. In harmony with these findings, new products 5a-u were synthesized starting from conjugated 2-thiopheneacrylic acid with amino acid esters3a-g followed by coupling of their acid derivatives4a-g with some piperazine segments, with the aim to increase their biological activities and decrease side effects. The anti-allergic and anti-inflammatory activities of the products were evaluated and promising results were obtained.

• Thiophene; Amino Acids; Piperazines; Anti-Allergy; Ant-Inflammatory

1. Introduction

The clinical manifestations of the allergic reaction are elicited by the subsequent interaction of the mediators with the end organ smooth muscle or mucous membranes. Traditionally symptomatic treatment of allergies and bronchial asthma has been provided by β-sympathomimetic agents, methylxanthines, corticosteroids, and anticholinergics [1-3]. Japanese authors have synthesized a series of acrylamide derivatives linked with different piperazines via alkyl chains and proved them as promising anti-allergic candidates (Figure 1(A)) [4-7].

It is known that amino acids and peptides are being taxonomically natural, physiologically nontoxic and chemically multifunctional, their rationalized conjugates with biologically active synthetic organic agents, are hypothesized to be more potent, particularly less toxic than their parent biologicals [8]. Further, thiophene derivatives have received interest because of their potential pharmacological utility and their incorporation in synthetic peptides [9-11]. Several thiophene derivatives are known and have been used as antibiotic, anti-allergic, anti-migraine, antioxidant agents [12-14].

In the present work, we have attempted to blend some of the foregoing considerations with our own experience and synthetic techniques, to design compounds [B] with an architectural resemblance to (Figure 1(B)) [15-17]. Thus, some amino acid residues are acting as a bridge between 2-thiopheneacrylic acid and some piperazine derivatives to get the two hypothetical anti-allergic segments i.e. thiophene acrylamide and piperazine. The presence of the amino acid residues introduce an additional hydrogen bond acceptor site (i.e., C=O) that could represent a new structural contribution for the anti-allergic profile of these compounds.

2. Experimental

2.1. Instruments

Melting points were determined in open glass capillaries using an Electrothermal IA 9000 SERIES digital melting point apparatus (Electrothermal, UK) and are uncorrected. Optical rotations were measured by a “Polax-D” po-

larimeter (ATAGO), provided by a SI-Na-1 Sodium lamp. Microanalyses were performed with all final compounds on an Elementar-Vario EL (Elementar-Vario EL, Germany) (Micro-analytical Unit, Central Services Laboratory, National Research Centre, Cairo; Egypt). The ¹H-NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer (Varian, USA). ¹H-NMR spectra were run at 300 MHz in DMSO-d₆ as solvent. Chemical shifts δ are quoted in ppm and were related to that of the solvents. Mass spectra were recorded on a Shimadzu GCMS-QP 1000EX (EI, 70 eV) (Shimadzu, Japan) and Hewlett-Packard (EI, 70 eV) (HewlettPackard, USA). IR spectra were obtained with a BruckerVector 22 (Bruker Rhein-Stetten, Germany). All the reactions were monitored using thin layer chromatography (TLC) using silica gel aluminum sheets 60F₂₅₄ (Merck). While “S” stands for a chromatographic solvent system of chloroform/methanol/acetic acid, 85/10/5 by volume, S₁ and S₂ represent the same solvent system to which petroleum ether (40˚C - 60˚C) was added, in an equal or half ratio volume respectively.

2.2. Organic Preparations

General Procedures

1) Acid Chloride Method [Procedure A]

Triethylamine (2.42 g ≡ 3.4 ml, 24 mmol) was added in portions to a well stirred cold solution (0˚C - 5˚C) of thiophene-2-carbonyl chloride 2 [freshly prepared from thiophene-2-acrylic acid (1.85 g, 12 mmol)] and amino acid ester hydrochloride (12 mmol) in THF/DCM (1:4, 150 ml). The reaction mixture was stirred for 3h at the same temperature with simultaneous adjustment of pH≈ 8. The TEA hydrochloride was filtered off, excess solvent distilled off and residue in, mainly, ethyl acetate (EtOAc) was washed with HCl (1 N, 3 × 20 ml), NaHCO₃ (5%, 3 × 20 ml), H₂O (3 × 20 ml) and dried over anhydrous sodium sulfate, excess solvent was distilled off to get 3a, b and 3 g in good yields (Table 1).

2) In Situ Active Ester Method [Procedure B]

A solution of DCCI (3 g, 15 mmol) in THF (10 ml) was added, over 30 min., to a stirred cold solution (0˚C - 5˚C) of thiophene-2-acrylic acid (2.31 g, 15 mmol) and N-hydroxybenzotiazole (HOBt) (1.7 g, 15 mmol) in THF (50 ml). Stirring was continued for 15 min. at the same temperature, and a mixture of the corresponding amino acid or dipeptide ester hydrochloride (15 mmol) and TEA (1.5 g ≡ 2.13 ml, 15 mmol) in THF (50 ml) was then added. Stirring was continued at the same temperature for 3 h, at pH ≈ 8 (Et₃N) and at 0˚C overnight, then at room temperature for 24 h. Drops of glacial acetic acid were added to the cold (0˚C) suspension and the reaction mixture was filtered off. The filtrate was evaporated, and the residue was taken in acetonitrile (10 ml), then, kept in cold, and the formed urea (DCHU) was separated by filtration. Solvent was distilled off under reduced pressure and the residue was taken up in EtOAc, washed with NaHCO₃ (1 N, 3 × 10 ml), distilled water (3 × 10 ml), KHSO₄ (5%, 3 × 10 ml), distilled water (3 × 10 ml) and dried over anhydrous sodium sulfate. The dried and decolorized EtOAc solution was concentrated (5 ml) and chromatographically purified on manually prepared preparative TLC plates to get the products (3a-g).

The same procedure was undertaken to couple the acid products 4a-g with the appropriate substituted piperazines to afford 5a-u.

3) Mixed Anhydride Method [Procedure C]

Ethyl chloroformate (1.08 g ≡ 0.95 ml, 10 mmol) was added to a stirred and cold (–15˚C) THF solution of 3-(2- thienyl)acrylic acid (1.54 gm, 10 mmol) and N-methylmorpholine (~1 ml, 10 mmol). The reaction mixture was stirred for additional 10 minutes, and then cold solution of free valine methyl ester or glycylglycine ethyl ester (10 mmol) in THF (30 ml) was added. Stirring was maintained for 3 h at (–15˚C) then for 12 h at room temperature. The solvent was evaporated and the residue was taken into EtOAc (30 ml) to be washed with KHSO₄ (3%, 3 × 10), distilled water (3 × 10 ml), NaHCO₃ (3%, 3 × 10), and finally with water (3 × 10 ml) then dried over anhydrous sodium sulfate. The solvent was evaporated

Table 1. Physical data of E-3-(thiophen-2-yl)acrylamido)esters (3a-g).

to dryness and the obtained residue triturated with nhexane. The obtained solid was collected by filtration and crystallized from ethanol/n-hexane to give the products 3d and 3g as identified by melting point and TLC in comparison with samples prepared according to procedure B.

The same procedure was undertaken to couple the acid products 4a, 4d, 4f and 4g with the appropriate substituted piperazines to afford 5a, 5c, 5k, 5p, 5s and 5u.

General Procedure

The KOH solution (30 ml, 0.1 N) was drop-wisely added to a stirred solution cold (–5˚C - 0˚C) of 3a-g (10 mmol) in methanol (20 ml). Then, the reaction mixture was stirred at same temperature for 1 h, at room temperature for 24 h and evaporated under vacuum till dryness. The obtained solid was dissolved in distilled water (50 ml), cooled in an ice bath, stirred, acidified with HCl (0.1 N) to pH ~ 2 - 3 and the precipitate was separated by filtration, washed with distilled water, dried to afford the acids 4a-g (Table 2).

2.2.3. Synthesis of N-[(4-substituted-1-piperazinyl)- oxo-(alkyl and ethylcarbamoyl)]-3-phenyacrylamides (5a-u)

1) (E)-N-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-3-(thiophen-2-yl)acrylamide (5a)

TLC: R_f = 0.35 [S₁], yield: 42% (Procedure B), 53% (Procedure C), m.p. 170˚C - 172˚C. IR ν (cm⁻¹): 3338 (NH), 1657 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 2.13 (s, 3H, NCH₃), 2.27 - 2.39 (m, 4H, 2CH₂, Pip-H), 2.41 - 3.51 (m, 4H, 2CH₂, Pip-H), 4.01 (s, 2H, COCH₂), 6.51 (d, J = 15 Hz, 1H, =CHCO), 6.98 (t, J = 3.5 Hz, 1H, Th-H4), 7.15 (d, J = 5 Hz, 1H, Th-H3), 7.50 (d, J = 15 Hz, 1H, CH=CH), 7.56 (d, J = 5 Hz, 1H, Th-H5), 8.18 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 294 ([M+1]⁺, 12%). Calcd. for C₁₄H₁₉N₃O₂S (293.38). Calcd. C 57.31, H 6.53, N 14.32, S 10.93. Found: C 57.22, H 6.45, N 14.22, S 10.86.

2) (E)-N-(2-(4-ethoxycarbonylpiperazin-1-yl)-2-oxoethyl)-3-(thiophen-2-Yl)acrylamide (5b)

TLC: R_f = 0.55 [S₂)], yield: 47% (Procedure B), m.p. 93˚C - 95˚C. IR ν (cm⁻¹): 3450 (NH), 1694 (C=O ester), 1663 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 1.26 (t, J = 7.5 Hz, 3H, CH₂CH₃), 3.6 - 3.63 (m, 4H, 2CH₂, Pip-H), 3.67-3.7 (m, 4H, 2CH₂, Pip-H), 4.1 (s, 2H, COCH₂), 4.3 (q, J = 7.5 Hz, 2H, CH₂CH₃), 6.44 (d, J = 16 Hz, 1H, =CHCO), 6.98 (t, J = 3.5 Hz, 1H, Th-H4), 7.29 (d, J = 5 Hz, 1H, Th-H3), 7.47 (d, J = 16 Hz, 1H, CH=CH), 7.71 (d, J = 5 Hz, 1H, Th-H5), 8.43 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 352 ([M+1]⁺, 7%). Calcd. for C₁₆H₂₁N₃O₄S (351.42). Calcd. C 54.68, H 6.02, N 11.96, S 9.12. Found: C, 54.56, H 5.96, N 12.96, S 9.00.

3) (E)-N-(2-(4-(4-fluorophenyl)piperazin-1-yl)-2-oxoethyl)- 3-(thiophen-2-yl)acryl-amide (5c)

TLC: R_f = 0.45 [S₁], yield: 53% (Procedure B), 65% (Procedure C); m.p. 120˚C - 122˚C. IR ν (cm⁻¹): 3370 (NH), 1670 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 2.37 - 2.47 (m, 4H, 2CH₂, Pip-H), 3.35 - 3.46 (m, 4H, 2CH₂, Pip-H), 3.96 (s, 2H, COCH₂), 6.23 - 6.33 (m, 2H, Ph-H), 6.84 (d, J = 16 Hz, 1H, =CHCO), 6.99 - 7.05 (m, 2H, Ph-H), 7.06 (t, J = 3.5 Hz, 1H, Th-H4), 7.19 (d, J = 5 Hz, 1H, Th-H3), 7.3 (d, J = 16 Hz, 1H, CH=CH), 7.4 (d, J = 5 Hz, 1H, Th-H5), 8.14 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 374 ([M+1]⁺, 14%). Calcd. For C₁₉H₂₀FN₃O₂S (373.44). Calcd. C 61.11, H 5.40, N 11.25, S 8.59. Found: C 60.98, H 5.32, N 11.39, S 8.67.

4) (E)-N-(3-(4-methylpiperazin-1-yl)-3-oxopropyl)-3-(thiophen-2-yl)acrylamide (5d)

Table 2. Physical data of E-3-(thiophen-2-yl)acrylamido)acids (4a-g).

TLC: R_f = 0.37 [S₂], yield: 39% (Procedure B), m.p. 102˚C - 104˚C. IR ν (cm⁻¹): 3376 (NH), 1669 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 2.23 (s, 3H, NCH₃), 2.27 (t, J = 7.2 Hz, 2H, COCH₂), 2.54 - 2.57 (m, 4H, 2CH₂, Pip-H), 3.1 (t, J = 7.2 Hz, 2H, NHCH₂), 3.28 - 3.31 (m, 4H, 2CH₂, Pip-H), 6.67 (d, J = 15.6 Hz, 1H, =CHCO), 7.05 (t, J = 3.6 Hz, 1H, Th-H4), 7.35 (d, J = 5.1 Hz, 1H, Th-H3), 7.52 (d, J = 15.8 Hz, 1H, CH=CH), 7.63 (d, J = 5.2 Hz, 1H, Th-H5), 8.38 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 308 ([M+1]⁺, 7%). Calcd. for C₁₅H₂₁N₃O₂S (307.41). Calcd. C 58.61, H 6.89, N 13.67, S 10.43. Found: C 58.47, H 6.81, N 13.79, S 10.51.

5) (E)-N-(3-(4-ethoxycarbonylpiperazin-1-yl)-3-oxopropyl)-3-(thiophen-2-Yl)acryl-amide (5e)

TLC: R_f = 0.48 [S₂], yield: 47% (Procedure B), m.p. 113˚C - 115˚C. IR ν (cm⁻¹): 3319 (NH), 1714 (C=O ester), 1657 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 1.17 (t, J = 7.7 Hz, 3H, CH₂CH₃), 2.53 (t, J = 7.1 Hz, 2H, COCH₂), 3.16 (t, J = 7 Hz, 2H, NHCH₂), 3.48 - 3.51 (m, 4H, 2CH₂, Pip-H), 3.71 - 3.73 (m, 4H, 2CH₂, Pip-H), 4.06 (q, J = 7.6 Hz, 2H, CH₂CH₃), 6.36 (d, J = 16.9 Hz, 1H, =CHCO), 7.1 (t, J = 3.5 Hz, 1H, Th-H4), 7.36 (d, J = 5.1 Hz, 1H, Th-H3), 7.62 (d, J = 17 Hz, 1H, CH=CH), 7.68 (d, J = 5.1 Hz, 1H, Th-H5), 8.46 (s, 1H, NHexchangeable with D₂O). Mass: m/z 366 ([M+1]⁺, 11%). Calcd. for C₁₇H₂₃N₃O₄S (365.45). Calc. C 55.87, H 6.34, N 11.50, S 8.77. Found: C 55.73, H 6.27, N 11.64, S 8.86.

6) (E)-N-(3-(4-(4-fluorophenyl)piperazin-1-yl)-3-oxopropyl)-3-(thiophen-2-yl)acryl-amide (5f)

TLC: R_f = 0.42 [S₁], yield: 45% (Procedure B), m.p. 139˚C - 141˚C. IR ν (cm⁻¹): 3317 (NH), 1662 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 2.38 - 2.4 (m, 2H, COCH₂), 2.98 - 3.0 (m, 2H, NHCH₂), 3.11 - 3.14 (m, 4H, 2CH₂, Pip-H), 3.19 - 3.22 (m, 4H, 2CH₂, Pip-H), 6.26 - 6.35 (m, 2H, Ph-H), 6.78 (d, J = 16 Hz, 1H, =CHCO), 6.98 - 7.03 (m, 2H, Ph-H), 7.08 (d, J = 3 Hz, 1H, Th-H4), 7.12 (d, J = 5 Hz, 1H, Th-H3), 7.35 (d, J = 5 Hz, 1H, Th-H5), 7.55 (d, J = 16 Hz, 1H, CH=CH), 8.12 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 387 ([M]⁺, 100%). Calcd. for C₂₀H₂₂FN₃O₂S (387.47). Calcd. C 62.00, H 5.72, N 10.84, S 8.28. Found: C 62.12, H 5.82, N 10.72, S 8.36.

7) (E)-N-(4-(4-methylpiperazin-1-yl)-4-oxobutyl)-3-(thiophen-2-Yl)acrylamide (5g)

TLC: R_f = 0.48 [S₁], yield: 53% (Procedure B), m.p. 182˚C - 184˚C. IR ν (cm⁻¹): 3368 (NH), 1669 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 1.75 - 1.77 (m, 2H, CH₂CH₂CH₂), 2.21 (s, 3H, NCH₃), 2.23 (m, 2H, CH₂CO), 2.52 - 2.54 (m, 4H, 2CH₂, Pip-H), 3.21 (m, 2H, NHCH₂), 3.25 - 3.27 (m, 4H, 2CH₂, Pip-H), 6.42 (d, J = 16 Hz, 1H, =CHCO), 6.99 (t, J = 3.2 Hz, 1H, Th-H4), 7.17 (d, J = 5.1 Hz, 1H, Th-H3), 7.55 (d, J = 5.1 Hz, 1H, Th-H5), 7.62 (d, 1H, J = 16 Hz, 1H, CH=CH), 8.14 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 322 ([M+1]⁺, 12%). Calcd. for C₁₆H₂₃N₃O₂S (321.44). Calcd. C, 59.78; H, 7.21; N, 13.07; S, 9.98. Found: C, 59.75; H, 9.17; N, 13.12; S, 9.94.

8) (E)-N-(4-(4-ethoxycarbonylpiperazin-1-yl)-4-oxobutyl)-3-(thiophen-2-Yl)acrylamide (5h)

TLC: R_f = 0.43 [S₂], yield: 39% (Procedure B), m.p. 121˚C - 123˚C. IR ν (cm⁻¹): 3338 (NH), 1695 (C=O ester), 1660 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 1.17 (t, J = 7.2 Hz, 3H, CH₂CH₃), 1.79 - 1.82 (m, 2H, CH₂CH₂CH₂), 2.53 (m, 2H, CH₂CO), 3.16 (m, 2H, NHCH₂), 3.46 - 3.48 (m, 4H, 2CH₂, Pip-H), 3.79 - 3.82 (m, 4H, 2CH₂, Pip-H), 4.25 (q, 2H, CH₂CH₃), 6.46 (d, J = 16 Hz, 1H, =CHCO), 7.07 (t, J = 3.2 Hz, 1H, Th-H4), 7.4 (d, J = 5 Hz, 1H, Th-H3), 7.54 (d, 1H, J = 16 Hz, 1H, CH=CH), 7.62 (d, J = 5 Hz, 1H, Th-H5), 8.23 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 380 ([M+1]⁺, 2%). Calcd. for C₁₈H₂₅N₃O₄S (379.47). Calcd. C 56.97, H 6.64, N 11.07, S 8.45. Found: C 57.09; H 6.76, N 10.95, S 8.53.

9) (E)-N-(4-(4-(4-fluorophenyl)piperazin-1-yl)-4-oxobutyl)-3-(thiophen-2-yl)acryl-amide (5i)

TLC: R_f = 0.39 [S₁], yield: 55% (Procedure B), m.p. 157˚C - 159˚C. IR ν (cm⁻¹): 3317 (NH), 1662 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 1.85 - 1.87 (m, 2H, CH₂CH₂CH₂), 2.24 - 2.27 (m, 2H, CH₂CO), 2.98-3.0 (m, 2H, NHCH₂), 3.22 - 3.24 (m, 4H, 2CH₂, Pip-H), 3.39-3.42 (m, 4H, 2CH₂, Pip-H), 6.43 (d, J = 17 Hz, 1H, =CHCO), 6.65 (m, 2H, Ph-H), 6.98 (m, 2H, Ph-H), 7.12 (d, J = 3.1 Hz, 1H, Th-H4), 7.17 (J = 5 Hz, 1H, Th-H3), 7.37 (d, J = 5.1 Hz, 1H, Th-H5), 7.57 (d, 1H, J = 17 Hz, 1H, CH=CH), 8.26 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 402 ([M+1]⁺, 4%). Calcd. for C₂₁H₂₄FN₃O₂S (401.50). Calcd. C 62.82, H 6.03, N 10.47, S 7.99. Found: C 62.96; H, 6.11; N, 10.31; S, 7.85.

10) (E)-N-(3-methyl-1-(4-methylpiperazin-1-yl)-1-oxobutan-2-yl)-3-(thiophen-2yl)-acrylamide (5j)

TLC: R_f = 0.37 [S₁], yield: 39% (Procedure B), m.p. 153˚C - 155˚C, [α]²⁵_D (C = 0.5, methanol) = −12. IR ν (cm⁻¹): 3376 (NH), 1669 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 0.98 (s, 6H, 2CH₃),  2.21 (s, 3H, NCH₃), 2.25 - 2.47 (m, 1H, NHCHCH), 2.54 - 2.66 (m, 4H, 2CH₂, Pip-H), 3.21 - 3.32 (m, 4H, 2CH₂, Pip-H), 4.47 - 4.58 (m, 1H, NHCH), 6.42 (d, J = 16 Hz, 1H, =CHCO), 7.1 (t, J = 3 Hz, 1H, Th-H4), 7.35 (d, J = 5 Hz, 1H, Th-H3), 7.48 (d, 1H, J = 16 Hz, 1H, CH=CH), 7.63 (d, J = 5.1 Hz, 1H, Th-H5), 8.11 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 335 ([M]⁺, 38%). Calcd. for C₁₇H₂₅N₃O₂S (335.46). Calcd. C 60.87, H 7.51, N 12.53, S 9.56. Found: C, 60.99, H 7.59, N 12.41, S 9.46.

11) (E)-N-(1-(4-ethoxycarbonylpiperazin-1-yl)-3-methyl- 1-oxobutan-2-yl)-3-(thiophen-2-yl) acrylamide (5k)

TLC: R_f = 0.48 [S₂], yield: 47% (Procedure C), m.p. 109˚C - 111˚C, [α]²⁵_D (C = 0.5, methanol) = +17. IR ν (cm⁻¹): 3319 (NH), 1714 (C=O ester), 1657 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 1.04 (s, 6H, 2CH₃), 1.12 (t, J = 7.1 Hz, 3H, CH₂CH₃), 1.87 - 1.93 (m, 1H, NHCHCH), 2.39 - 2.51 (m, 4H, 2CH₂, Pip-H), 3.32 - 3.43 (m, 4H, 2CH₂, Pip-H), 4.07 (q, J =7.1 Hz, 2H, CH₂CH₃), 4.38 - 4.43 (m, 1H, NHCH), 6.46 (d, J = 16 Hz, 1H, =CHCO), 7.1 (t, J = 3.2 Hz, 1H, Th-H4), 7.36 (d, J = 5 Hz, 1H, Th-H3), 7.54 (d, J = 16 Hz, 1H, CH=CH), 7.87 (d, J = 5 Hz, 1H, Th-H5), 8.09 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 394 ([M+1]⁺, 3%). Calcd. for C₁₉H₂₇N₃O₄S (393.50). Calcd. C 57.99, H 6.92, N 10.68, S 8.15. Found: C 57.83, H 6.85, N 10.75, S, 7.97.

12) (E)-N-(1-(4-(4-fluorophenyl)piperazin-1-yl)-3-methyl- 1-oxobutan-2-yl)-3-(thiophen-2-yl)acrylamide (5l)

TLC: R_f = 0.42 [S₁], yield: 53% (Procedure B), m.p. 139˚C -141˚C, [α]²⁵_D (C = 0.5, methanol) = +5. IR ν (cm⁻¹): 3450 (NH), 1672 (C=O amide). ¹H NMR (DMSOd₆, δ, ppm): 0.98 (s, 6H, 2CH₃), 1.97-2.12 (m, 1H, NHCHCH), 2.26-2.37 (m, 4H, 2CH₂, Pip-H), 3.38 - 3.59 (m, 4H, 2CH₂, Pip-H), 4.45 - 4.56 (m, 1H, NHCH), 6.6 (d, J = 15.6 Hz, 1H, =CHCO), 6.67 (m, 2H, Ph-H), 6.98 (m, 2H, Ph-H), 7.08 (t, J = 3 Hz, 1H, Th-H4), 7.23 (d, J = 4.8 Hz, 1H, Th-H3), 7.54 (d, J = 15 Hz, 1H, CH=CH), 7.65 (d, J = 4.8 Hz, 1H, Th-H5), 8.09 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 416 ([M+1]⁺, 5%). Calcd. for C₂₂H₂₆FN₃O₂S (415.52). Calcd. C 63.59, H 6.31, N 10.11, S 7.72. Found: C 63.45, H 6.25, N 10.23, S 7.79.

13) (E)-N-(1-(4-methylpiperazin-1-yl)-1-oxo-3-phenylpropan-2-yl)-3-(thiophen-2-yl)acrylamide (5m)

TLC: R_f = 0.37 [S₁], yield: 52% (Procedure B); m.p. 127˚C - 129˚C, [α]²⁵_D (C = 0.5, methanol) = +25. IR ν (cm⁻¹): 3376 (NH), 1669 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 2.07 (s, 3H, NCH₃), 2.67 - 2.79 (m, 4H, 2CH₂, Pip-H), 3.15 (brd, J = 5.5 Hz, 2H, CH₂Ph), 3.28 - 3.37 (m, 4H, 2CH₂, PHz ip-H), 5.04 (t, J = 5 Hz, 1H, NHCHCO) 6.42 (d, J = 16, 1H, =CHCO), 7.07 - 7.1 (m, 5H, Ph-H), 7.14 (t, J = 3 Hz, 1H, Th-H4), 7.25 (d, J = 5.1 Hz, 1H, Th-H3), 7.48 (d, J = 16 Hz, 1H, CH=CH), 7.63 (t, J = 5.2 Hz, 1H, Th-H5), 8.56 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 384 ([M+1]⁺, 15%). Calcd. for C₂₁H₂₅N₃O₂S (383.51). Calcd. C 65.77, H 6.57, N 10.96, S 8.36. Found: C 65.89, H 6.65, N 10.78, S 8.26.

14) (E)-N-(1-(4-ethoxycarbonylpiperazin-1-yl)-1-oxo-3- phenylpropan-2-yl)-3-(thiophen-2-yl)acrylamide (5n)

TLC: R_f = 0.48 [S₂], yield: 47% (Procedure B), m.p. 113˚C - 115˚C, [α]²⁵_D (C = 0.5, methanol) = +15. IR ν (cm⁻¹): 3319 (NH), 1714 (C=O ester), 1657 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 1.18 (t, J = 7.15 Hz, 3H, CH₂CH₃), 3.17 (brd, J = 5 Hz, 2H, CH₂Ph), 2.59 - 2.71 (m, 4H, 2CH₂, Pip-H), 3.39 - 3.5 (m, 4H, 2CH₂, Pip-H), 4.03 (q, J =7.1 Hz, 2H, CH₂CH₃), 5.04 (t, J = 5 Hz, 1H, NHCHCO), 6.46 (d, J = 15 Hz, 1H, =CHCO), 7.07 - 7.09 (m, 5H, Ph-H), 7.11 (t, J = 3 Hz, 1H, Th-H4), 7.36 (d, J = 5.1 Hz, 1H, Th-H3), 7.57 (d, J = 15 Hz, 1H, CH=CH), 7.87 (d, J = 5 Hz, 1H, Th-H5), 8.59 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 442 ([M+1]⁺, 13%). Calcd. for C₂₃H₂₇N₃O₄S (441.54). Calcd. C 62.56, H 6.16, N 9.52, S 7.26. Found: C 62.69, H 6.24, N 9.43, S 7.18.

15) (E)-N-(1-(4-(4-fluorophenyl)piperazin-1-yl)-1-oxo-3- phenylpropan-2-yl)-3-(thiophen-2-yl)acrylamide (5o)

TLC: R_f = 0.42 [S₁], yield: 53% (Procedure B); m.p. 139˚C - 141˚C, [α]²⁵_D (C = 0.5, methanol) = −27. IR ν (cm⁻¹): 3370 (NH), 1668 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 2.79-2.90 (m, 4H, 2CH₂, Pip-H), 3.15 (brd, J = 5 Hz, 2H, CH₂Ph), 3.25 - 3.37(m, 4H, 2CH₂, Pip-H), 5.09 (t, J = 5 Hz, 1H, NHCHCO), 6.46 (d, J = 15 Hz, 1H, =CHCO), 6.68 (m, 2H, Ph-H), 6.97 (m, 2H, Ph-H), 7.0 (m, 5H, Ph-H) , 7.08 (t, J = 3.1 Hz, 1H, Th-H4), 7.12 (d, J = 5 Hz, 1H, Th-H3), 7.53 (d, J = 15 Hz, 1H, CH=CH), 7.55 (d, J = 5 Hz, 1H, Th-H5), 8.59 (s, 1H, NH, exchangeable with D₂O). Mass: m/z 464 ((M+1]⁺, 4%). Calcd. for C₂₆H₂₆FN₃O₂S (463.57). Calcd. C 67.36, H 5.65, N 9.06, S 6.92. Found: C 67.42, H 5.69, N 9.00, S 6.88.

16) (E)-N-(3-(4-hydroxyphenyl)-1-(4-methylpiperazin-1- yl)-1-oxo-propan-2-Yl)-3-(thiophen-2-yl)acrylamide (5p)

TLC: R_f = 0.37 [S₁], yield: 39% (Procedure B), 54% (Procedure C); m.p. 127˚C - 129˚C, [α]²⁵_D (C = 0.5, methanol) = -100. IR ν (cm⁻¹): 3376 (NH), 3215 (br OH), 1671 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 2.2 (s, 3H, NCH₃), 2.56-2.67 (m, 4H, 2CH₂, Pip-H), 2.82 (brd, J = 5.5 Hz, 2H, PhCH₂), 3.31 - 3.51 (m, 4H, 2CH₂, Pip-H), 4.7 (t, J = 5.5 Hz, 1H, NHCHCO), 6.47 (d, J = 16 Hz, 1H, =CHCO), 6.84 (d, J = 6.7 Hz, 2H, Ph-H), 6.94 (d, J = 6.7 Hz, 2H, Ph-H), 7.1 (t, J = 3.2 Hz, 1H, Th-H4), 7.35 (d, J = 5.2 Hz, 1H, Th-H3), 7.57 (d, J = 16 Hz, 1H, CH=CH), 7.63 (d, J = 5.2 Hz, 1H, Th-H5), 8.38 (s, 1H, NH, exchangeable with D₂O), 9.15 (s, 1H, OH, exchangeable with D₂O). Mass: m/z 400 ([M+1]⁺, 10%). Calcd. for C₂₁H₂₅N₃O₃S (399.51). Calcd. C 63.13, H 6.31, N 10.52, S 8.03. Found: C 63.25, H 6.35, N 10.40, S 7.95.

17) (E)-N-(1-(4-ethoxycarbonylpiperazin-1-yl)-3-(4- hydroxyphenyl)-1-oxopropan-2-yl)-3-(thiophen-2-yl)- acrylamide (5q)

TLC: R_f = 0.48 [S₂], yield: 47% (Procedure B), m.p. 113˚C - 115˚C, [α]²⁵_D (C = 0.5, methanol) = +18. IR ν (cm⁻¹): 3450 (NH), 3215 (br OH), 1734 (C=O ester), 1678 (C=O amide). ¹H NMR (DMSO-d₆, δ, ppm): 1.14 (t, J = 7.2 Hz, 3H, CH₂CH₃), 2.76 - 2.87 (m, 4H, 2CH₂, Pip-H), 3.15 - 3.27 (m, 6H, Ph-CH₂+ 2CH₂, Pip-H), 4.06 (q, J = 7.2 Hz, 2H, CH₂CH₃), 4.94 (t, J = 5.5 Hz, 1H, NHCHCO), 6.46 (d, J = 15 Hz, 1H, =CHCO), 6.65 (d, J = 6.7 Hz, 2H, Ph-H), 7.03 (d, J = 6.7 Hz, 2H, Ph-H), 7.1 (t, J = 3.1 Hz, 1H, Th-H4), 7.36 (d, J = 5.1 Hz, 1H, Th-H3), 7.57 (d, J = 15.5 Hz, 1H, CH=CH), 7.63 (d, J = 5.1 Hz, 1H, Th-H5), 8.53 (s, 1H, NH, exchangeable with D₂O), 9.25 (s, 1H, OH, exchangeable with D₂O). Mass: m/z 458 ([M+1]⁺, 1%). Calcd. for C₂₃H₂₇N₃O₅S (457.54). Calcd: C 60.38, H 5.95, N 9.18, S 7.01. Found: C 60.23, H 5.87, N 9.34, S 6.93.

18) (E)-N-(1-(4-(4-fluorophenyl)piperazin-1-yl)-3-(4- hydroxyphenyl)-1-oxopropan-2-yl)-3-(thiophen-2-yl)acrylamide (5r)

TLC: R_f = 0.47 [S], yield 56% (Procedure B), m.p. 153˚C - 155˚C, [α]²⁵_D (C = 0.5, methanol) = -15. IR ν (cm⁻¹): 3222 (br, OH), 3317 (NH), 1672 (C=O). ¹H NMR (DMSO-d₆, δ, ppm): 2.74 - 2.85 (m, 4H, 2CH₂, Pip-H), 2.91 - 2.98 (m, 2H, Ph-CH₂), 3.45 - 3.56 (m, 4H, 2CH₂, Pip-H), 4.98 - 5.02 (m, 1H, CH), 6.48 (d, J = 15 Hz, 1H, =CHCO), 6.78 (m, 2H, Ph-H), 6.90 (m, 2H, Ph-H), 6.97 (m, 2H, Ph-H), 7.03 - 7.1 (m, 3H, Ph-H + Th-H4), 7.23 (d, J = 2.5 Hz, 1H, Th-H3), 7.52 (d, J = 15 Hz, 1H, CH=CH), 7.57 (d, J = 5.1 Hz, 1H, Th-H5), 8.55 (s, 1H, NH, exchangeable with D₂O), 9.31 (s, 1H, OH, exchangeable with D₂O). MS: m/z 480 ([M+1]⁺, 45%). Calcd for C₂₆H₂₆FN₃O₃S (479.17). Calcd: C 65.12, H 5.46, N 8.76, S 6.69 %. Found: C 65.26, H 5.52, N 8.65, S 6.61%.

19) (E)-N-[(2-(4-methylpiperazin-1-yl)-2-oxoethylcarbamoyl)-methyl]-3-(thiophen-2-yl)acrylamide (5s)

TLC: R_f = 0.56 [S], yield 58% (Procedure C), m.p. 170˚C - 172˚C. IR ν (cm⁻¹): 3330, 3151 (NH), 1665, 1651 (C=O). ¹H NMR (DMSO-d₆, δ, ppm): 2.2 (s, 3H, CH₃), 3.61 - 3.65 (m, 4H, Pip-H), 3.89 - 3.92 (m, 4H, Pip-H), 4.1 (s, 2H, CH₂), 4.25 (s, 2H, CH₂), 6.64 (d, 1H, J = 15 Hz, =CHCO), 7.13 (t, 1H, J = 5 Hz, Th-H4), 7.14 (d, 1H, J = 2.7 Hz, Th-H3), 7.61 (d, 1H, J = 16 Hz, CH=CH), 7.65 (d, 1H, J = 4.5 Hz, Th-H5), 8.12 (s, br, 2H, 2 NH, D₂O-exchangeable). MS: m/z 351 ([M+1]⁺, 3%). Calcd for C₁₆H₂₂N₄O₃S (350.44). Calcd: C 54.84, H 6.33, N 15.99, S 9.15 %. Found: C 54.92, H 6.39, N 15.86, S 9.23 %.

20) (E)-N-[(2-(4-ethoxycarbonylpiperazin-1-yl)-2-oxoethylcar-bamoyl)-methyl]-3-(thiophen-2-yl)acrylamide (5t)

TLC: R_f = 0.62 [S], yield 42% (Procedure B), m.p. 158˚C - 160˚C. IR ν (cm⁻¹): 3350, 3156 (NH), 1664, 1658 (C=O). ¹H NMR (DMSO-d₆, δ, ppm): 1.2 (t, 3H, J = 7 Hz, CH₂CH₃), 3.6 (m, 4H, Pip-H), 3.9 (m, 4H, Pip-H), 4.1 (q, 2H, J = 7 Hz, CH₂CH₃), 4.19 (s, 2H, CH₂), 4.25 (s, 2H, CH₂), 6.62 (d, 1H, J = 16 Hz, =CHCO), 7.1 (t, 1H, J = 5 Hz, Th-H4), 7.13 (d, 1H, J = 2.5 Hz, Th-H3), 7.51 (d, 1H, J = 16 Hz, CH=CH), 7.7 (d, 1H, J = 5 Hz, Th-H5), 8.21 (s, br, 2H, 2NH, D₂O-exchangeable). MS: m/z 408 ([M+1]⁺, 54%). Calcd for C₁₈H₂₄N₄O₅S (430.5). Calcd: C 52.93, H 5.92, N 13.72, S 7.85 %. Found: C 52.82, H 5.88, N 13.84, S 7.79 %.

21) (E)-N-((2-(4-(4-fluorophenyl)piperazin-1-yl)-2- oxoethylcarbamoyl)-methyl)-3-(thiophen-2-yl)acrylamide (5u)

TLC: R_f = 0.42 [S₁], yield 53% (Procedure C), m.p. 190˚C - 192˚C. IR ν (cm⁻¹): 3377, 3161 (NH), 1662 (C=O). ¹H NMR (DMSO-d₆, δ, ppm): 3.1 - 3.12 (m, 4H, Pip-H), 3.55 - 3.60 (m, 4H, Pip-H), 4.1 (s, 2H, CH₂), 4.25 (s, 2H, CH₂), 6.61 (d, 1H, J = 16 Hz, =CHCO), 6.9 (t, 1H, J = 5 Hz, Th-H4), 7.02 (d, 1H, J = 2.5 Hz, Th-H3), 7.1 (m, 2H, Ph-H), 7.23 (m, 2H, Ph-H), 7.52 (d, 1H, J = 16 Hz, CH=CH), 7.75 (d, 1H, J = 4.5 Hz, Th-H5), 8.32 (s, 2H, 2 NH-D₂O-exchangeable). MS: m/z 430 ([M]⁺, 7%). Calcd for C₂₁H₂₃FN₄O₃S (430.5). Calcd: C 58.59, H 5.39, N 13.01, S 7.45 %. Found: C 58.50, H 5.26, N 13.23, S 7.39 %.

3. Biological Activities

Animals were obtained from the animal house colony of the National Research Center, Cairo, Egypt. All animals were allowed free access to water and were kept on a constant standard diet. All procedures involving animals were carried out in accordance with the guide for the care and use of laboratory animals (National Academy of Science of Egypt) and were approved by the Animals Studies Committee at Washington University. Adult male albino rats, weighing 150 - 180 g, fasted for 12 h and used for determining the anti-inflammatory activity and LD₅₀.

3.1. Anti-Allergic Activity

3.1.1. Evaluation

The percent inhibition of spasmogen induced contraction is calculated. The percentage of inhibition in contraction (due to histamine release) is the anti-allergic potency, which is compared to that of Loratadine (standard reference drug).

3.1.2. Procedure

Albino guinea pigs of either sex weighing 300 - 450 g are sacrificed with an overdose of ether. The chest cavity is opened and the lungs are removed. They were cut into strips of 5 cm and placed into a physiological saline solution. Thereafter, the lung strips are mounted in an organ bath containing a nutritive solution. The bath was bubbled with carbogen and maintained at 37˚C under a preload of 0.5 g - 3 g; the tissue was left to equilibrate for 30 - 60 minutes. Prior to testing carbachol is added to the bath to test the lung strips ability of contraction.Twenty minutes later, two values are obtained by adding the spasmogen.

• Histamine dihydrochloride 10⁻⁶ g/ml for 5 minutes• Ca-ionophore 5 × 10⁻⁶ g/ml for 5 minutes, or

• Leukotriene LTC₄ (10⁻⁹ - 10⁻⁸) g/ml for 10 minutesTo the bath and recording the contractile force at its maximal level. Following 20 minutes, equilibration period resulted, the spasmogen is administrated again, 5 minutes thereafter, and the test compound is added in cumulative dose from 10⁻⁸ - 10⁻⁴ g/ml at 5 or 10 minutes intervals. The contractile response is determined isometriccally [18].

3.2. Evaluation of Anti-Inflammatory Activity

3.2.1. Protection against Edema

The inhibitory activity of the studied compounds on carrageenan-induced rat’s paw edema was carried out according to the method of Winter et al. [19,20]. Groups of adult male albino rats (150 - 180 gm), each of 8 animals were orally dosed with the test compounds at a dose level of 2.5 and 5 mg/kg an hour before carrageenan challenge. Foot paw edema was induced by sub planter injection of 0.05ml of 1% suspension of carrageenin in saline into the planter tissue of one hind paw. An equal volume of saline was injected to the other hind paw and served as control. Four hours after drug administration the animal was decapitated, blood was collected and the paws were rapidly excised.

The average weight of edema was estimated for the treated as well as the control group and the percentage inhibition of weight of edema was also evaluated then percentage protection against edema was estimated (Table 1). Indomethacin (2.5 and 5 mg/kg) was employed as standard reference against which the test compounds were compared.

3.2.2. Estimation of Plasma Prostaglandin E 2 (PGE 2 )

Heparinized blood samples were collected from rats (n = 8), plasma was separated by centrifugation at 12000 g for 2min at 4˚C and immediately stored frozen 20˚C until use.

The designs correlate-EIA prostaglandin in E₂ (PGE₂) kit is a competitive immune assay for the quantitative determination of PGE₂ in biological fluids. The kit uses a monoclonal antibody to PGE₂ to bind, in a competitive manner, the PGE₂ in the sample. After a simultaneous incubation at room temperature the excess reagents were washed away and the substrate was added. After a short incubation time the enzyme reaction was stopped and the yellow color generated was read on a micro plate reader (DYNATCH, MR 5000) at 405 nm. The intensity of the bound yellow color is inversely proportional to the concentration of PGE₂ in either standard or samples. The percentage inhibition of plasma PGE₂ for each compound was estimated [21] (Table 3).

3.3. Evaluation of Acute Toxicity Study

The test compounds were administered orally at different dose levels in separate groups of animals. After 24 h of drug administration percent mortality in each group was observed. From the data obtained, the lethal dose (LD₅₀) was calculated by the method of Smith [22].

4. Results and Discussion

4.1. Chemistry

The starting materials, E-3-(thiophen-2-yl)acrylamido) ester derivatives (3a-g) were obtained by reaction of (E)- 3-(thiophen-2-yl)acrylic acid (1) or (E)-3-(thiophen-2-yl) acryloyl chloride (2) with amino acid ester hydrochlorides of glycine, β-alanine, 4-aminobutyric acid and DValine, L-phenylalanine, L-tyrosine and the dipeptide glycylglycine. The products (3a-g) were prepared by the acid chloride method (procedure A), the 1,3-dicyclohexylcarbodiimide (DCCI) method (procedure B) and/or the mixed anhydride method (procedure C), followed by their alkaline hydrolysis under mild conditions to afford their corresponding acid derivatives (4a-g). The latter compounds were used as intermediates for the preparation of the targeted N-[(4-substituted-1-piperazinyl)-oxo- (alkyl and ethylcarbamoyl)]-3-phenylacrylamides (5a-u) by their reactions with the appropriate piperazines by procedure B and/or procedure C as depicted in Scheme1.

The structures of the synthesized products were confirmed in light of their IR, ¹H NMR and EI-MS spectral data, as well as, their elemental analyses (See experimental section). The infra red spectra indicated the expected absorption of the essential functional groups e.g. amide and C-terminal ester or carboxylic groups. Thus, the secondary amide nature of the compounds was represented by amide band I absorption at 1647 - 1673 cm⁻¹, indicating a resonating N-C=O stretching vibrations. The obtained products were assigned E-configuration on the basis of their proton nuclear magnetic resonance spectra (¹H NMR), showed coupling constants of the olefinic protons (J ≈ 16 - 17 Hz). The electron impact mass spectral data of the products were consistent with the proposed structures and showed molecular ion peaks corresponding to their molecular formulae.

4.2. Pharmacology

All the obtained products (5a-u) have been investigated for their anti-allergic and anti-inflammatory activeties. All tested products manifested highly LD₅₀ values, so they are considered to be of high safety margins (Table 3).

Compounds 5j, 5l, 5p, 5r, 5q, 5m, 5o, 5s, 5u, 5d, 5a and 5c showed to be of high activities relative to that of the reference drug Loratadine (Clorinix^®) (Table 3). It could be in relation with the presence of Valine (5j 98% and 5l 97%) and tyrosine (5p 96%, 5r 94% and 5q 90%) residues, also, the methyl piperazine segment showed to be more active than 4-fluorophenyl and 4-ethoxycarbonyl one (Table 3).

The evaluation of anti-inflammatory activity was carried out according to the method of winter et al, where

Table 3. Anti-allergy and anti-inflammatory activities of compounds (5g-u).

^*NA = no activity.

the inhibitory activity of the studied compounds on carrageenan-induced rat’s paw edema. Most of these compounds have no anti-inflammatory activities except 5g which showed the relative highest activities of 98% (% protection against edema) and 83% (% inhibition of PEG2) in comparison with the reference drug Indomethacine.

5. Conclusion

A series of 21 products from the coupling of three piperazine derivatives with seven 3-(thiophen-2-yl) acrylamide amino acids was designed based on the concept of Nishikawa et al. [Figure 1, structure A]. Our novel design depends on the replacement of the aliphatic chain with the known biologically active amino acid segments. The obtained pharmacological screening results, showed that the presence of the amino acid residues in the products as linkers between 2-thiopheneacrylic acid and piperazine derivatives [Figure 1, structure B], enhance their anti-allergy activity and has no effect on increasing their anti-inflammatory one. All tested products manifested highly LD₅₀ values, so they are considered to be of high safety margins.

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