INTRODUCTION:

Cyclic imides are well known important organic compounds that exhibit diverse biological activities like anti-inflammatory, antibacterial, analgesic, hypoglycemic and antifungal activities^1-11. Besides these compounds are useful building blocks in the synthesis of many drugs and pharmaceuticals^12-14. On the other hand trimethoprim is a well-known antibiotic that is used in combination with Sulfamethoxazole in treating urinary tract infections, bacterial infections and acute invasive diarrhea^15-21. Moreover Schiff bases represent the most active intermediates that exhibit wide spectrum of biological activities and play a vital role in the production of different pharmaceutical and bioactive heterocycles^22-27. Based on all these facts, it seems worthwhile to design and synthesize new molecules that contain these three active moieties (cyclic imide, trimethoprim and Schiff base) together in the same molecule since this may exhibit the new compounds high biological activity and may open possibilities for fighting bacterial infections.

EXPERIMENTAL:

Chemicals used in performing this work were purchesed from Aldrich, BDH and Fluka companies. Melting points of the synthesized compounds were determined on Gallenkamp capillary melting point apparatus and are uncorrected. FTIR spectra were recorded on Shimadzu FTIR-8400 Fourier Transform Infrared spectrophotometer in College of science, University of Baghdad. ¹H-NMR and ¹³CNMR spectra were recorded on near magnetic resonance Bruker, Bruker, Ultrashield 300 MHz in Iran.Antibacterial activity study was performed in Biology Department, College of Science, University of Baghdad.

METHOD:

1- Synthesis of Trimethoprim Bis Schiff base [1]

The titled Compound was synthesized by following the literature procedure²⁸ some modifications. A mixture of trimethoprim (0.01mol, 2.78g) and (0.02 mo l, 2.79g) of 4-amino acetophenone in (40mL) absolute ethanol with a few drops of glacial acetic acid was heated under reflux for (8 hrs.). After completion of reflux time the mixture was cooled and the obtained precipitate was collected by filtration, washed with ether and dried. Recrystallization of the product from acetone afforded pale yellow crystals in (88%) yield and have me melting point (110-112)⁰C.

2-Synthesis of Bis Schiff base Trimethoprim Bis Amic Acids [2-7]

The titled bis amic acids [2-7] were synthesized via reaction of (0.005mol, 2.62g) of compound [1] with (0.01mol) of different cyclic anhydrides in dry acetone with stirring for two hrs. at room temperature²⁹. After completion of stirring time the obtained precipitate was filtered, washed with acetone dried and finally recrystallized from a suitable solvent. Melting points, colours and percent yields of compounds [2-7] are shown in Table (1).

3-Synthesis of Bis Schiff base Trimethoprim Bis cyclic imides [8-13]

The titled bis imides [8-13] were Synthesized by dehydration of the corresponding bis amic acids [2-7].

Dehydration was performed via fusion (2g) of bis amic acids [2-7] in oil bath for four hrs. at temperatures higher than bis amic acid melting point by ten degrees²⁹. After completion of fusion the resulted solid was, recrystallized from a suitable Solvent. Melting points, colours and percent yields of compounds [8-13] are shown in Table (2).

RESULTS AND DISCUSSION:

Since cyclic imides are very important compounds with wide spectrum of activities and applications the core of this work is synthesis a series of novel bis cyclic imides based on the known drug trimethoprim and the links between imide cycles and trimethoprim drug molecule is done through Schiff base moiety. That means this work involved designing of new molecules contain the three biologically active components (cyclic imide, trimethoprim drug and Schiff base) together in the same fram and this may exhibit the new molecules high biological activity leading to new possibilities for fighting bacterial infections. Synthesis of the new bis cyclic imides was performed by many steps as shown in scheme (1) in the first step trimethoprim was introduced in condensation reaction with 4-amino acetophenone producing trimethoprim bis Schiff base [1].

Compound [1] was afforded as pale yellow crystals in (88%) percent yield and melting point (110-112)⁰C.

FTIR spectrum of compound [1] showed characteristic absorption bands at (3425-3450) cm^-1 and (3332- 3398) cm^-1 due to asym. and sym. ν(NH₂). The spectrum showed also absorption bands at (3062) cm^-1, (2962, 2833) cm^-1 (1654, 1641) cm^-1, (1591) cm^-1 and (1280, 1126) cm^-1which are due to ν(C-H) aromatic, asym. And sym. ν(C-H) aliphatic, ν(C=N), ν(C=C) and ν(C-O) ether respectively³⁰.

¹H-NMR spectrum of compound [1] showed signals at (δ= 1.92) ppm, (δ = 2.4) ppm, (3.58) ppm and at (3.65-3.75) ppm, which are belong to NH₂ protons, (two CH₃) protons, (CH₂) protons and three OCH₃) protons respectively³⁰. Other signals appeared at (δ = 6.06-7.69) ppm and (δ =7.71) ppm belong to aromatic protons and proton in pyrimidine ring.

¹³C-NMR spectrum of compound [1] showed signals at (δ = 22.04, 26.25) ppm, (33.39) ppm and (56.22-60.42) ppm, which are belong to methyl groups carbons, (CH₂) carbon and (three OCH₃) carbons respectively. The spectrum showed also signals at δ = (106.29- 136.27) ppm, (153.22-I54.07) ppm, (161.86, 162.98) ppm and (195.5) ppm belong to aromatic carbons, (C=N) imine carbons, carbons in pyrimidine ring and (C=N) in pyrimidine ring respectively³⁰.

Compound [1] represents the important key compound from which the target bis cycles imides were synthesized through two steps, in the first one compound [1] was introduced in reaction with different cyclic anhydrides producing the corresponding bis amic acids [2-7]. Melting points, colours and percent yields of the prepared bis amic acids are shown in Table (1).

FTIR spectra of bis amic acids [2-7] showed clear absorption bands at (3400-3463) cm^-1and (3184- 3388) cm^-1due to ν (O-H) carboxyl and ν(N-H) amide and other absorption bands at (1685-1722) cm^-1and (1656-1685) cm^-1due to ν(C=O) carboxyl and ν(C=O) amide respectively³⁰.

The spectra showed also absorption bands at (1631-1664) cm^-1, (1591-1595) cm^-1, (1263-1280) cm^-1and (1122 - 1128) cm^-1which are attributed to ν(C=N), ν(C = C), asym. ν(C-O) ether and sym. ν(C-O) ether respectively³⁰.

All details of FTIR spectral data of compounds [2-7] are listed in Table (3).

¹H-NMR spectrum of bis amic acid [3] showed signals at (δ= 2.1- 2.66) ppm, (3.58) ppm and (3.65-3.77) ppm belong to (two CH₃) protons, (CH₂) protons and (three OCH₃) protons respectively. The spectrum showed other signals at (δ= 6.64-7.72) ppm, (7.84-7.87) ppm, (7.96-8.16) ppm and (11.2) ppm, which are belong to aromatic protons in pyrimidine ring, (2 NH) amide protons and (2OH) carboxyl protons.

¹³C-NMR spectrum of compound [3] showed signals at (δ= 26.86,31.14) ppm, (32.63) ppm and (56.31-60.41) ppm, which are belong to (two CH₃) carbons, (CH₂) carbon and (three OCH₃)carbons. Other signals appeared at (δ= 106.64- 143.21) ppm, (I53.35-155.30) ppm, (162.69-166.65) ppm, (196.87) ppm and (197.8)ppm which are belong to aromatic carbons, (C=N) imine carbons, pyrimidine ring carbons, (C=N) in pyrimidine ring and carbonyl carbons.

¹H-NMR spectrum of bis amic acid [4] showed signals at (δ= 2.39-2.53) ppm, (3.65-3.76) ppm and (6.13)ppm which are belong to (2CH₃) protons, (CH₂, 3OCH₃) protons and vinylic protons.

Other signals appeared at (δ=6.31-7.70) ppm, (7.77-7.80) ppm, (7.93-7.96) ppm and at (δ= 11.50) ppm, which belong to aromatic protons, proton in pyrimidine ring, (2 NH) amide protons and (2OH) carboxyl protons respectively³⁰.

¹³C-NMR spectrum of compound [4] showed signals at (δ= 26.23,26.80) ppm, (32.66) ppm and (56.29-60.41) ppm, which are belong to (two CH₃ Carbons), (CH₂) carbon and (three OCH₃) carbons. signals belong to aromatic and vinylic carbons appeared at (δ= 106.57-143.64) ppm, Signals belong to (C=N) imine appeared at (δ= 153.37- 155.12) ppm, signals belong to pyrimidine ring carbons appeared at (δ= 164.24-168.36) ppm and signals belong to carbonyl carbons and (C=N) in pyrimidine ring appeared at (δ=195.49-196.99)ppm.

In the second step the prepared bis amic acids [2-7] were introduced in dehydration reaction producing the target bis cyclic imides [8-13]. Dehydration reaction was performed by fusion. process and involved elimination of water molecules and ring closure, producing the target bis imides. Physical properties of bis imides [8-13] are shown in Table (2).

FTIR Spectra of bis cyclic imides [8-13] showed disappearance of absorption bands due to ν(O-H) carboxyl and ν(N-H) amide and appearance of sholder absorption band at (1743-1778) cm^-1 and clear bands at (1703-1724) cm^-1 due to asym. and sym. ν(C=O) imide. These two points are good proofs for success of bis imide formation.

The spectra showed also other absorption bands at (1622- 1683) cm^-1, (1591-1602) cm^-1, (1382-1394) cm^-1, (1236-1269) cm^-1 and (1122-1124) cm^-1 which are attributed to ν (C=N), ν(C=C), ν(C-N) imide, asym. ν(C-O) ether and sym. ν (C-O) etherrespectively³⁰.

Details of FTIR spectral data of bis cyclic imides [8-13] are listed in table (4).

¹H-NMR spectrum of bis imide [9] showed signals at (δ= 2.53-2.66) ppm and (3.43-3.78) ppm belong to (two CH₃) protons, (CH₂) protons and (three OCH₃) protons. Signals belong to aromatic protons appeared at (δ= 6.25-7.66) ppm while the signal which belong to proton in pyrimidine ring appeared at (δ=8.02-8.17) ppm. It is noticeable that the spectrum showed disappearance of (OH) carboxyl signal proving success of imide formation.

¹³C-NMR spectrum of compound [9] showed signals at (δ= 27.35) ppm, (32.94) ppm and (56.31-60.43) ppm belong to (2CH₃) carbons, (CH₂) carbon and (three OCH₃) carbons.

Other signals appeared at (δ= 106.53-139.22) ppm, (153.28-153.39) ppm, (162.09-163.79) ppm and (197.78) ppm, which belong to aromatic carbons, (C=N) imine carbons, pyrimidine ring carbons and carbonyl and (C=N) in pyrimidine ring carbons respectively.

¹H-NMR spectrum of bis imide [10] showed signals at (δ= 2.4-2.64) ppm, and (3.56-3.76) ppm belong to (two CH₃) protons, (CH₂) protons and (three OCH₃) protons.

Other signals appeared at (δ=6.05-6.23) ppm, (6.54-7.82) ppm and (7.96-8.11) ppm belong to vinylic protons, aromatic protons and pyrimidine ring proton.

The spectrum also showed disappearance of (OH) carboxyl proton signal proving success of imide formation.

¹³C-NMR spectrum of compound [10] showed signals at (δ= 26.30, 27.29) ppm. (33.28) ppm and (56.27-60.43) ppm belong to (2CH₃) carbons, CH₂ carbon and (3 OCH₃) carbons. The spectrum showed also signals at (δ= 106.37-136.29) ppm belong to aromatic and vinylic carbons, signal at. (δ=153.21)ppm belong to (C=N)imine carbons and signal at (δ=161.04-163.09)ppm belong to pyrimidine ring carbons while signal belong to (C =O) imide carbons and (C =N) carbon in pyrimidine ring appeared at(δ= 197) ppm.

Scheme 1:

Table 1: Physical properties of compounds [2-7]

Comp. No.	Compound structure	Melting point ⁰C	Yield %	Colour	Recrystallization solvent
2		220-222	90	White	Ethanol
3		217-218	88	Off white	Acetone
4		166-168	91	Off white	Ethanol
5		125-127	92	White	Acetone
6		144-146	90	Off white	Acetone
7		102-104	86	Pale yellow	Acetone

Table 2: physical properties of compounds [8-13]

Comp. No.	Compound structure	Melting point	Yield %	Colour	Recrystallization solvent
8		266-268	82	Dark Brown	Acetone
9		303-304	80	Black	Dioxane
10		293-295	77	Brown	Cyclohexane
11		287-288	81	Brown	Dioxane
12		240-242	84	Brown	Cyclohexane
13		311-313	78	Black	Acetone

Table 3: FTIR spectral data (cm^-1) of bis amic acids [2-7]

Comp. No.	ν(O-H) ν(N-H)	ν(C-H) Aromatic	ν(C-H) Aliphatic	ν(C=O) Carboxyl	ν(C=O) Amide	ν(C=N)	ν(C=C)	ν(C-O) Ether
2	3419 3334 3226	3002	2939 2839	1685	1685 (overlap)	1656	1593	1280 1128
3	3334 3191	3001	2972 2839	1722	1660	1631	1595	1265 1126
4	3400 3344 3184	3099	2939 2837	1712	1676	1633	1593	1271 1124
5	3463 3388 3334 3224	3058	2939 2840	1714	1656	1643	1595	1280 1128
6	3406 3284 3199	3002	2937 2837	1706	1679	1662	1591	1267 1128
7	3400 3346 3184	3002	2964 2833	1718	1685	1664	1593	1263 1122

Table 4: FTIR spectral data (cm^-1) of bis imides [8-13]

Comp. No.	ν(C-H) Aromatic	ν(C-H) Aliphatic	ν(C=O) Imide	ν(C=N)	ν(C=C)	ν(C-N) Imide	ν(C-O) Ether
8	3006	2939 2837	1743(sh) 1716	1679	1598	1382	1269 1124
9	3001	2939 2833	1778(sh) 1724	1681 1660	1602	1390	1265 1124
10	3080	2939 2837	1714	1660 1629	1595	1379	1269 1124
11	3075	2937 2835	1747(sh) 1708	1650	1595	1394	1236 1122
12	3010	2941 2837	1776(sh) 1716	1683 1622	1600	1394	1267 1124
13	3099	2941 2833	1751(sh) 1703	1650 1629	1591	1394	1236 1122

(Sh=shoulder)

Antibactrial Activity Study:

Antibacterial activity study for the synthesized bis cyclic imides [8-13] was performed against many types of gram-positive and gram-negative bacteria including staphylococcus aurus, Pseudomonas auroginosa, Escherichia Coli, Klebsiella pneumonia and Bacillus subtilis using Muller Hinton agar and incubation was performed at (37) Cͦ for 24 hrs. Results of inhibition Zones caused by the tested compounds against the above mentioned types of bacteria are listed in Table (5).

The results in Table (5) indicated that all the tested compounds [8-13] showed very high activity against Pseudomonas auroginosa. The tested compounds [8-12] showed very high or high activity against Staphylococcus aurus, while compounds [8, 1l, 12,13] showed high activity against Escherichia coli. Compounds [8,12,13] showed high activity against Klebsiella pneumonia, and compounds [11, 12] showed high activity against Bacillus subtilis.

CONCLUSION:

In this work new development was made on trimethoprim drug molecule through intraducing biscyclic imides and Schiff base moieties in original drug molecule. Introducing of these moieties increased antibacterial activity of the resulted molecules, thus most of them showed very high antibacterial activity against various types of bacteria. These promising results can lead to find new drug which may fight different bacterial infections.

ACKNOWLEDGEMENT:

We thank like the biological Dr. Hiba for her help in study in performing the biological activity study of prepare compounds.

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Received on 26.12.2020 Modified on 02.03.2021

Research J. Pharm.and Tech 2021; 14(11):5874-5880.

DOI: 10.52711/0974-360X.2021.01049

Comp. No.	*Escherichia Coli*	*Pseudomonas auroginosa*	*staphylococcus aurus*	*Klebsiella pneumonia*	*Bacillus subtilis*
8	31	35	26	27	13
9	13	30	33	13	18
10	14	34	35	10	12
11	24	37	27	14	24
12	23	39	24	24	20
13	25	38	15	20	14
Drug	16	33	33	11	18