Synthesis , characterization and anthelmintic activity evaluation of pyrimidine derivatives bearing carboxamide and sulphonamide moieties

Abstract: Pyrimidines, sulphonamides and carboxamides have shown a large number of pharmacological properties against different types of diseases including helminthiasis. Seventeen new pyrimidine derivatives bearing sulphonamide and carboxamide were synthesized and investigated for their in vitro anthelmintic properties. Substituted benzenesulphonyl chlorides 15a–c were treated with various amino acids (16a–h) to obtain benzenesulphonamide derivatives 17a–l. Compounds 17a–f were subsequently treated with benzoyl chloride to obtain the N-benzoylated derivatives 19a–f. Further reactions of compounds 19a–f and 17g–l with 4or 2-aminopyrimidine (20) using boric acid as a catalyst gave the required sulphonamide carboxamide derivatives 21a–q in excellent yields. The compounds were isolated in their analytical grade and characterized using FTIR, 1H-NMR, 13C-NMR and HRMS. The in vitro anthelmintic studies showed that all the synthesized compounds possessed anthelmintic property. Compounds 21a–c, e, g, m and p showed mean paralyzing times of 15, 19, 14, 18, 19, 19 and 18 min, respectively, at 100 mg mL-1 compared to 10 min for albendazole. Compounds 21a–c, g and m had mean death times of 18, 24, 16, 20 and 25 min, respectively, at 100 mg mL-1 compared to 13 min for albendazole.


INTRODUCTION
Helminthiasis is causing untold misery to infected individuals.Anthelmintics are a group of anti-parasitic drugs that expel parasitic worms and other internal parasite from the body by either stunning or killing them without causing significant damage to the host. 1 Resistance to benzimidazoles (1) and closantel (2), Scheme 1, used to treat helminthiasis have been reported.There are genetic features in parasitic helminthes that favour the development of anthelmintic resis- tance. 2 Frequent usage of the same group of anthelmintic drugs, use of anthelmintics in sub-optimal doses, prophylactic mass treatment of domestic animals and continuous use of a single drug have contributed to the widespread development of anthelmintic resistance. 3The spread of resistance to triclabendazole, the main drug used in the treatment of fluke infections, is of global concern. 4YNTHESIS OF NEW ANTHELMINTIC AGENTS 403 Pyrimidines have attracted attention as an important class of heterocyclic compound in chemotherapy.Alloxan (3), a diabetogenic agent, 5 sulfadiazine (4), sulfamerazine (5) and sulfadimidine (6), a potent agent against urinary tract infections and cerebrospinal meningitis 6 all contain the pyrimidine moiety.][12][13][14][15][16][17][18][19] Hunziker 20 in 1967 reported pyrantel pamoate (10) as a depolarizing neuromuscular blocking agent that causes spastic paralysis in helminthes when employed in the treatment of infestations with pinworms and roundworms.This report suggests the possible use of pyrimidine derivatives as anthelmintic agents.
In addition to their traditional uses as antibacterial agents, 21 sulphonamides have also being widely used as diuretics, 22 anticonvulsant, 23 anticancer, 24 antiretroviral, 25 antihypertensive, 26 and antimalarial 27 agents amongst others.Mrozik and Matawan as early as 1976 patented the synthesis of substituted benzenesulphonamides that possessed anthelmintic properties. 28Mohan et al. 29 reported the synthesis of some new methylpyrimidine sulphonamides that possessed anthelmintic activity comparable to that of piperazine citrate.Vijaya et al. 30 reported the synthesis of benzothiazole derived benzenesulphonamides that possessed anthelmintic activity comparable to that of albendazole.Kumar et al. reported some benzenesulphonamide derivatives that possessed mean paralyzing times comparable to that of mebendazole. 31Babu and Selvakumar also reported some isoindole derivatives of benzenesulphonamide that could cause paralysis in 42 min at 10 mg per group. 32These findings point to the fact that sulphonamide derivatives could play a leading role in the fight against helminthiasis.
In view of the need for new anthelmintic agents, 2,4 in this work, the boric acid-catalysed syntheses of pyrimidine-derived carboxamides bearing a sulphonamide functionality are reported.The synergy arising from the successful incorporation of pyrimidine ring, carboxamide and sulphonamide pharmacophore was exploited in this research.It was expected that since the pharmacophores have individual anthelmintic activity, their successful incorporation in one molecule would improve the anthelmintic activity of the compound.________________________________________________________________________________________________________________________ (CC) 2018 SCS.
Available on line at www.shd.org.rs/JSCS/EXPERIMENTAL All reactions requiring inert atmosphere were performed under a nitrogen atmosphere.Drying of solvents was achieved using molecular sieves for 48 h.All reagents were purchased from commercial suppliers, Aldrich, Merck, Fluka, Avra, SD fine and Alfa Aesar.Thin layer chromatography was performed using silica plates purchased from Avra.The plates were visualized under UV light (Popular India, India).The FT-IR spectra of the compounds were run in a Perkin-Elmer Spectrum version 10.03.06 and the bands are presented in wavenumbers.Proton and carbon-13 NMR spectroscopy were run in DMSO-d 6 and CD 3 OD, unless otherwise stated on either Jeol 500 or 400 MHz instruments.The 1 H-NMR and 13 C-NMR spectra were recorded at a frequency of 400 and 100 MHz, respectively, using the 400 MHz instrument and at 500 and 126 MHz, respectively, using the 500 MHz instrument.The chemical shifts are reported in ppm with reference to tetramethylsilane.Mass spectrometry was performed using micro-TOF electrospray time of flight (ESI-TOF) mass spectrometer, sodium formate was used as the calibrant.Some of the mass spectra were recorded in the negative mode and others in the positive mode.All experiments were realised at Prof. Sandeep Verma's Laboratory, Department of Chemistry, Indian Institute of Technology, Kanpur, India.Melting points were determined using a digital melting point apparatus and are uncorrected.

General procedure for the synthesis of substituted benzenesulphonamides 17a-l
Sodium carbonate (Na 2 CO 3 , 1.590 g, 15 mmol) was added to a solution of an amino acid (16a-h, 12.5 mmol) in water (15 mL) with continuous stirring until all the solutes had dissolved.The solution was cooled to -5 °C and the appropriate benzenesulphonyl chloride (15a-c, 15 mmol) was added in four portions over a period of 1 h.The slurry was further stirred at room temperature for about 4 h.The progress of the reaction was monitored using TLC (MeOH/DCM, 1:9).Upon completion of the reaction, the mixture was acidified using 20 % aqueous hydrochloric acid to pH 2. The crystals were filtered via suction and washed with pH 2.2 buffer.The pure products 17a-l were dried over self-indicating fused silica gel in a desiccator.

General procedure for the synthesis of N-benzoyl derivatives of benzenesulphonamides 19a-f
The appropriate benzenesulphonamide (17a-f, 1.0 mmol) was dissolved in NaOH (10 %, 10 mL) in a 50 mL round bottom flask.Benzoyl chloride (18, 1.1 mmol, 0.2 mL) was transferred into the solution of the appropriate benzenesulphonamide and stirred at room temperature.The reaction progress was monitored by TLC (3 % MeOH in DCM) to the disappearance of the benzenesulphonamide spot.Upon completion of the reaction, the solution was transferred into a beaker containing crushed ice and then acidified to a pH of 3 with concentrated hydrochloric acid.The solid was collected via suction filtration and transferred into a beaker containing CCl 4 (10 mL), covered with a watch glass and boiled for 10 min.The mixture was allowed to cool slightly and then filtered.The obtained product 19a-f was washed with 10--20 mL of CCl 4 and dried over fused self-indicating silica gel in a desiccator.

General procedure for the synthesis of the novel pyrimidine derivatives 21a-q
To a suspension of N-benzoyl substituted benzenesulphonamide (19a-f) or substituted benzenesulphonamide (17g-l, 1.0 mmol) in dry toluene (40 mL), in a flask equipped with a Dean-Stark apparatus for azeotropic removal of water, was added 4-or 2-aminopyrimidine (20, 1.0 mmol) and boric acid (0.1 mmol) at room temperature and then refluxed for 8 h.On completion of the reaction, as monitored by TLC, the reaction mixture was precipitated to amides by the addition of about 40 mL n-hexane. 34The pyrimidine derivative was obtained via suction filtration, washed with n-hexane and dried over fused silica gel or concentrated using a rotary evaporator and dried over vacuum in the case of the oily products.The 1 H-NMR and 13 C-NMR spectra were recorded at a frequency of 400 and 100 MHz, respectively, using the 400 MHz instrument and at 500 and 126 MHz, respectively, using the 500 MHz instrument.

Anthelmintics studies
The anthelmintic activity studies were performed against Giardia duodenalis at 2 mg mL -1 concentration using the Garg and Atal method. 35Suspensions of the samples were prepared by triturating the synthesized compounds (100 mg) with Tween 80 (0.5 %) and distilled water and the resulting mixtures were stirred using a mechanical stirrer for 30 min.The suspensions were diluted to contain 0.2 % of the test samples.A suspension of the reference drug, albendazole, was prepared at the same concentration in a similar way.Three sets of G. duodenalis of almost similar sizes (2 inch in length) were placed in Petri plates of 4″ diameter containing 50 mL of a suspension of a test sample or the reference drug at room temperature.Another set of G. duodenalis was kept as control in 50 mL suspension of distilled water and Tween 80 (0.5 %).The paralyzing and death times were noted and their mean was calculated for triplicate sets.The death time was ascertained by placing the earthworms in warm water (50 °C), which stimulated the movement if the worm was alive.

RESULTS AND DISCUSSION
The reaction of substituted benzenesulphonyl chloride (15a-c) with various amino acids (16a-h) in the presence of sodium carbonate gave various benzenesulphonamides (17a-k).Further reactions of compounds 17a-f with benzoyl chloride (18) in the presence of sodium hydroxide gave N-benzoyl derivatives (19a-f).The boric acid catalysed reaction of compounds 19a-f and 17g-l with 4and 2-aminopyrimidine (20) gave new pyrimidine derivatives (21a-q), as shown in Scheme 2.
The analytical and spectral data of the synthesized compounds are given in the Supplementary material to this paper.The spectral characterizations are in agreement with the structures.
In the FTIR spectra, the diagnostic bands at 3412-3217 cm -1 were assigned to NH stretching vibrations.The bands between 1700-1661 cm -1 (two bands per molecule), 1623-1601 cm -1 (two bands per molecule) and 1586-1456 cm -1 (two bands per molecule) were assigned to C=O, C=N and NO 2 stretching, respectively.Compound 21c showed two bands at the NH region which were assigned to the NH of pyrimidine and of the indole ring.Among the hydroxyl derivatives, the bands between 3312-3203 cm -1 were assigned to the OH group.In the proline derivatives 21j-q, the OH band disappeared.In both the 4-hydroxyproline and proline derivatives 21g-q, there was only one C=O band between 1749-1659 cm -1 .
8.25-8.08ppm were assigned to the 2H of the pyrimidine.In the 2-aminopyrimidine derivatives, the pyrimidine rings appeared as a triplet and two doublets between 8.76-8.08ppm.The OH peak appeared as a singlet at 4.31-4.17ppm. 13C-NMR spectra of the derivatives, excepting the proline and 4-hydroxyproline derivatives 21g-q, showed two peaks between 174.99-163.88ppm assigned to the C=O carbons.The peaks at 158.24-152.02ppm were assigned to C=N carbons.The C-OH peak of the 4-hydroxypyrimidine derivatives appeared at 69.31-68.91ppm.
The results of the anthelmintic activity are presented in Table I.The anthelmintic activities increased as the concentration increased.All the synthesized compounds possessed anthelmintic activity with a mean paralyzing time ranging from 25-75, 18-60 and 14-41 min at 25, 50 and 100 mg mL -1 , respectively, compared to 28, 20 and 10 min for albendazole.The mean death time ranged from 30-109, 20-97 and 16-63 min, compared to 35, 25 and 13 min for albendazole at 25, 50 and 100 mg mL -1 , respectively.Compounds 21a and c had comparable anthelmintic activity to that of albendazole.Among the 4-hydroxyproline derivatives 21g-l, the trend of activities showed that 4-hydroxy-1-[(4--nitrophenyl)sulfonyl]-N-(pyrimidin-4-yl)pyrrolidine-2-carboxamide (21g) was the most active.The results showed that with the exception of the 4-aminopyrimidine derivative 21g, which was more active than the 2-aminopyrimidine derivative 21h, the 2-aminopyrimidines were more active than the 4-aminopyrimidine derivatives in the 4-hydroxyproline series.Among the proline derivatives, the 4-aminopyrimidines were more active than the 2-aminopyrimidines.Considering the substitution in the benzene ring, the p-nitro derivatives were the most active.The trend among the 4-aminopyrimidines was NO 2 >CH 3 >H, while that of the 2-aminopyrimidines was H>NO 2 >CH 3 .The trend of the activities imply that substitution at the para position of the benzene ring improved activity among the 4-aminopyrimidine derivatives, whereas the unsubstituted derivatives had better activity among the 2-aminopyrimidine derivatives.Compounds 21a, 21c, 21g and 21m are worthy of further development as anthelmintic agents given their similar anthelmintic activities to that of albendazole.

CONCLUSIONS
In conclusion, novel pyrimidine derivatives bearing sulphonamide and carboxamide moieties were successfully synthesized through boric acid catalysed direct amidation of various benzenesulphonamides and 4-and 2-aminopyrimidine in good to excellent yield.This synthetic approach provided further highlights into the utility of boric acid in direct amidation reactions of unactivated carboxylic acids.The procedure reported herein is simple, economical, efficient and environmentally friendly.Additionally, the derivatives were precipitated in their analytical grade without the necessity for chromatographic purification.Four of the seventeen new derivatives had comparable anthelmintic activity to that of albendazole and as such could be further developed as alternative anthelmintic agents to combat the resistance that will certainly follow the use of monotherapy in helminthiasis.

Scheme 1 .
Scheme 1.The structures of some anthelmintics.
28 ppm was assigned to the 1H of the pyrimidine ring; while the doublet at ________________________________________________________________________________________________________________________ (CC) 2018 SCS.Available on line at www.shd.org.rs/JSCS/

TABLE I .
In vitro anthelmintic activities of the new derivatives 1,2ID I. UGWU1,2, UCHECHUKWU C. OKORO 1 и NARENDRA K. MISHRA 2 1 Department of Pure and Industrial Chemistry, University of Nigeria, Nsukka, 410002, Nigeria and 2 Department of Chemistry, Indian Institute of Technology, Kanpur, 208016, India