Synthesis of Some Novel Compounds of Saccharinyl Acetic Acid Containing Nucleus and Evaluation of Their Biological Activities as Antimicrobial

Introduction

The reported pharmaceutical properties of saccharin and its derivatives^1-4 and in connection^5-9 with our ongoing interest to developing new synthetic strategies for construction of heterocyclic systems involving saccharin due to its significant biological^10-12 and pharmacological activities. Also imidazole derivatives are of interest to medical chemists for many years because of their biological activities such as anticancer, anti tubercular, antibacterial, and antifungal activities^13-22 In view of the above facts we report in the present work the synthesis and antimicrobial activities of Saccharinyl derivatives.

Methods and Experiments

A-Chemistry

Melting points were determined in open capillaries using Gallen Kamp melting point apparatus and are uncorrected F.T.-IR spectra (KBr.disc) were recorded on a Perkin Elmer 1720 F.T. – IR spectrometer. ¹H – NMR spectra were recorded on varin Gemini NMR spectrometer 300 MHz using TMS as internal standard. All reactions were monitored by TLC using aluminum silica gel plates 60 F 245. Elemental analysis and antimicrobial activity were carried out at micro-analytical center, faculty of science, cairo university, Egypt. Elemental analysis of all synthesized compounds is in agreement with the structure elucidated.

Benzylidene-2-Saccharinyl methyl-1,3-Oxazole-5-one.(3)

A mixture of Saccharinyl acetic acid chloride (10mmol) and glycine (10mmol) were refluxed in boiling ethanol for 1hr. The spectral solid was then refluxed for 4 hr. in freshly distilled acetic anhydride (30ml) and anhydrous sodium acetate in the presence of benzaldehyde. The reaction mixture was poured onto water and solid that separated was re-crystallized from ethanol to give (3). Orange (68%) mp 140-142 ⁰C, IR (KBr, Cm^-1) : υ 1700 (10 azlactone), 1610 (CN) , 1337 and 1120 (SO₂). EI – MS: m/z 368 [M⁺]. Analytical calculation for C₁₈H₁₂O₅N₂S; C58.7;H3.3; N7.6 found C58.65; H3.3; N, 7.55.^28,30

5-Benzylidene–3–saccharinyl methyl-1, 2, 4–triazin–6–one (4a,4b)

A mixture of 3 ( 10mmol) and hydrazine hydrate or phenyl hydrazine ( 10 mm ol) in absolute ethanol (30 ml) was refluxed for 3hr. the solid that obtained was recrystallized from ethanol to afford 4a: orange ( 75%), mp 163 -165^oC ; IR (KBr, cm^-1): 1700 (CO) , 1615 ( C=N) , 3350-3450 (br NH – NH) and 1337 and 1138 ( SO₂) MF C₁₈ H₁₄ O₄ N₄.S EI –MS : m/z 382 (M⁺). And 4b : brown ( 80%), mp 95 -97 ; IR (KBr, cm^-1) : 1680 ( CO) , 1610 ( C=2), 3250 (NH), 1330 and 1168 (SO₂) MF C₂₄H₁₈O₄N₄S. EI- MS: m/z 460 (M⁺)

General procedure for the preparation of cinnamamide  derivatives ( 5a-d) and imidazole one derivatives (6a-d).

A mixture of 3(10 mml ol) and aromatic amine oraminobenzoicacid ) was reflexed for 3h. in absolute ethanol (30 ml).

For compounds (5a-d) and in n-nutanol (30ml) for compounds (6a-d).  The solid that obtained was recrystallized from proper solvent to afford (5a-d) and / or (6a-d).³⁰

(α-Saccharinyl- N- acetamido) cinnamamide (5a)

Yellowish (70%) ,mp 210 -212 ; IR( KBr, cm^-1) : 1670 and 1660 (CO), 3300-3200 (NH), 1330 and 1170 (SO₂) . MF C₂₄ H₁₉ O₅ N₃S’ El- MS : m/z 461 (M⁺) , crystallized from benzene  (5b) dark brown (75%) , mp 219-221; IR( KBr cm^-1).

1670 (CO), 3320, 3250 (NH) 1616 (C=N), 1320 and 1170 (SO₂).

MF C₂₅H₂₁O₅N₉S, El- MS: m/z 476 (M+1) recrystallized from toluene.

(5c) yellow (65%) ,mp 90-92 , IR(5Br, cm^-1):1720 , ( CO) , 1680 , (CO amide), 3350-3400 (broud NH, OH) , 1310 and 1120 (SO₂). MF C₂₅H₁₉O₇N₃S, EI –MS:

m/z 505 (M⁺), crystallized from benzene.

(5d) brown ( 70%) , mp 110-112 , IR ( KBr, cm^-1):1710 (CO), 1670 ( CO amide) , 3300-3450(broud NH, OH), 1300 and 1110 (SO₂).

MF C₂₅H₁₉O₇N₃S. EI – MS : m/z 505 (M⁺). recrystallized from benzene.

(6a) brown (75%) ,mp 180-182, IR( KBr, cm^-1):1660 (CO) , 1600 ( CO amidazolone) 1580 (C=N).1330 and 1160 (SO₂). MF C₂₄ H₁₇ O₄ N₃ S EI- MS : m/z 443 (M⁺)  recrystallized from toluene.

(6b). brown (65%) ,mp 202-204 , IR(KBr, cm^-1):

1670 (CO), 1630 ( CO imidazole),  1580( C=N), 1320 and 1170 (SO₂).

M.F. C₂₅H₁₉O₄N₃S. EI- MS: m/z 457 (M⁺).

(6c) brown (70%), mp 150-152 , IR (KBr , cm^-1) , 1680 (coimidazolone) , 1610 (C=N), 1320 and 1110 ( SO₂) , 3450 (OH). MF C₂₅ H₁₇O₆N₃S EI-MS: m/z 487 (M⁺), recrystallized from benzene.

(6d) yellowish (77%) ,mp 155-157, IR (KBr, cm^-1) : 1690 (Coimidazolone) , 1620 (C=N), 1280 and 1120 (SO₂) 3440 (OH).

M.F C₂₅H₁₇O₆N₃S. EI- MS : m/z 487 (M⁺) recrystallized from toluene.

General procedure for the preparation of imidazolone  derivatives ( 6a-d) From ( saccharinyl- N- acetamido ) cinnamide derivatives( 5a-d) by cyclization .

A solution of compounds 5a-d (10 m mole) in acetic anhydride (15ml) was boiled under reflux for 2h. The resulting solution was poured onto crushed ice , and the product that separated out was filtered off, washed with solution of sodium hydrogen carbonate followed by water and then dried. The products were recrystallized from a proper solvent.

Saccharinyl (N’- benzoyl methyl) acetamide(7)

A solution of oxazolonederivative  3 (10 m mole) in dry benzene was treated with anhydrous Alll₃(30m mole) with continuously stirring on water bath for 3h.

The reaction mixture was decomposed with ice- cold hydrochloric acid. Then the ethereal layer was separated and dried over anhydrous Na₂SO₄.

The excess solvent was evaporated then the separated compound was recrystallized from benzene to give (7) : buff (60%) , mp 115-117 , IR (KBr, cm^-1), 1700 (CO ketone), 1680 (CO imide) , 3340 (NH), 1330 and 1140 (SO₂) MF C₁₇H₁₄O₅N₂S.EI-MS: m/z 358 (M⁺)

Oxo -1, 2 ,4 – triazino (4,3-b) (1,2) benzoisothiazole(9)

A mixture of compound 8 ( 10 m mole) in redistilled acetic anhydride 20ml was refluxed for 1h. the reaction mixture was cooled and poured onto ice – cold water. The solid product separated was filtered and recrystallized from ethanol to afford compound (9), yellowish brown (65%) , mp 145-147, IR (KBr, cm^-1) : 1710 (CO) , 3432 (OH) , 3100 (NH) 1634 (C=N) 1363 and 1133 (SO₂) , HNMR (DMSo-d₆) S: 2.5 (s, 2H, cH₂), 4.4 (s,1H, NH) , 5.7 (s. 1H, OH of lactam- lactim dynamic equilibrium) and 7.8-8.1 (m, 4H , ArH), MF C₉H₇N₃O₃S, EI-ms, m/z 251 (M⁺).

General procedure for the preparation of 6 – alkyl–6-hydroxyl– 1,2,4- triazino (4,3-b) (1,2) benzisothiazole (10a-c).

To Mg metal (10m mole) in dry ether (40ml) an alkyl halide namely, ethyl iodide , methyl iodide or benzyl chloride (30m mole) in dry ether (20ml) was added dropwisely. The reaction mixture was refluxed and compound a (10m mole) in dry ether (40ml) added portion wise within 1h.

The reaction mixture was further refluxed for 3h, left over night and then decomposed with dil cold HCl. The ethereal layer was / washed with NaHCO₃ solution then water and dried over analydrous Na2 SO₄ and evaporated to give compound 10a-c which was recrystallized form benzene (10a) : ball yellow (6690) , mp (195-197, IR( KB₆, cm^-1)) 3420 -3390 (OH and NH) , 1610 ( C=N) , 1310 and 1130 (SO₂) , MF C₁₀H₁₁N₃O₃S, EI –MS: m/z    3(M⁺).

(10b) : yellowish (70%) , mp 172-174 ; IR ( KBr, cm^-1)

: 3410 – 3380 ( OH and NH), 1600 (C=N), 1310 and 1110 (SO₂) , ‘HNMR(DMSO-d₆) S: 1.7 (t,3H,CH₃) , 2.5 (q,2H,CH₂) , 4(S,IH,OH), 4.5 (S,2H, CH₂, N-CH₄) , 7.8 (m, 4H , A,H) , 9.1 (S, 1H, NH). MFC₁₁H₁₃N₃O₃S. EI – HS: m/z 267 (M⁺).

(10c) yellow (60%) ,mp 205-207 , IR (KBr, cm^-1) 3400-3370 (OH, and NH) , 1630 (C=N) 1320 and 1120 (SO₂) . MF (C₁₆H₁₃N₃O₃S).

EI – MS : m/z 329 (M⁺)

6- chloro -1,2,4- triazino – (4,3-b) (1,2) benzisolhiazole(11)

A mixture of compound 3(10m mole) , phosphorous oxychloride ( 20m mole) and PCl₅ (1qm) was refluxed on a steam bath for 3h.

Then poured slowly into ice – cold water. The solid that separated was washed several times with water, dried and recrystallized from benzene to give compound (11); brown (70%) ,mp 105-107. IR(KBr, cm^-1), 1630 (C=N), 1330 and 1120 (SO₂) and no absorption for CO and NH.

MF C₉H₆N₃O₂S C₁. EI – MS’ : m/z 255 (M⁺) and 257 (M⁺+2).

Formation of compounds ( 12 a and b)

A mixture of compound 11 ( 10m mole) was refluxed with urea and / or thio- urea ( 10 m mole ) in ( 40ml) sodium ethoxide for 3h, then cooled and poured into water. The solid that separated was dried and recrystallized from benzene – ethanol (1 :1) to give 12a and for 12b.

Compound (12a) : yellow (72%) , mp 72 -74 . IR (KBr, cm^-1)  3350 -3320 (NH), 1610 (C=N) , 1310  and 1120 (SO₂). MF C₁₀H₉N₅O₃S₂ EI- MS: m/2 279 (M⁺ ).

Compound (12b) : yellowish (65%) , mp 235- 237. IR ( KBr, cm^-1) 3370 -3330 (NH), 1630 (C=N), 1240 (C=S) 1310 and 1120 (SO₂). ‘HNMR(DMSO-d₆) :

4.2 (S2H, CH₂, N-CH₂) , 5.9 (2S, 2H, NH₂) 7.5 (m, 4H , ArH), 8.2 (S, IH, NH).

MF C10H9N5O2 S2. EI – MS : m/z 295(M⁺).

6- OXO -5- arylidene – 1,2,4-triazino – (4,3-b) (1,2)- benzisothiazole (13a-c).

A mixture of compound 9 (10m mole) and aromatic aldehyde (10m mole) namely Benz aldehyde , p- chloro- Benz aldehyde and anisaldehyde  in (40 ml).

Acetic anhydride – acetic acid (1: 1) was refluxed for . 3h. after concentrated, the solid that separated was cooled and recrystallized from benzene – ethanol (1: 1) to give (13a –c).

Compound 13a yellow (75%) ,mp 105-107.

IR (KBr, cm^-1) 3400 -3360 (br. NH, OH), 1720 (CO), 1560 , 1480 (C=N, C=C), 1350 and 1150 (SO₂). MF C₁₆H₁₁N₃O₃S. EI –MS : m/z 325 (M⁺).

Compound 13b: yellow (75%) ,mp 200 -202. IR (KBr, cm^-1)  3420-3380(br. OH and NH), 1700 (CO), 1580, 1460 ( C=N, C=C) , 1330 and 1120 (SO₂), MF C₁₆H₁₀N₃O₃SC₁. EI- MS: m/x 359 (M⁺) AND 361 ( 2).

Compound 13c : ball yellow (65%) , mp 188-190. IR (KBR,  cm^-1)  3450- 3400 (br: OH and NH), 1720 (CO) , 1610, 1450 (C=N, C=C), 1300 and 1110 (SO₂). MF C₁₇H₁₀N₃O₄S. EI- MS: m/z 355(M⁺).

Scheme 1  Click here to View scheme

 

Scheme 2  Click here to View scheme

 

Scheme 3 Click here to View scheme

 

Antimicrobial

The newly synthesized compounds (3,4a-b, 5a-b, 5d, 5g, 6c, 7, 9, 10a, 11, 12b, and 13a)were tested for their in vitro growth inhibitory activity against a panel of standard strains of the Institute of fermentation of Osaka (IFO) namely; theGram-positive bacteria (Staphylococcus aureus IFO3060 and Bacillus subtilis IFO 3007), the Gram negative bacteria (Escherichia coli IFO 3301 and Proteus vulgaris IFO 3851.

The primary screening was carried out using the agar disc-diffusion method using Müller-Hinton agar medium.^24-28

Results and Discussion

Chemistry

In the present investigation, saccharinyl acetic acid chloride ⁹reacted with glycine to give N- carboxymethylsaccharinylacetamide(2), which reacted with benzaldehyde in the presence of acetic anhydride and sodium acetate to give 4- benzylidene-2-saccharinyl-methyl-1,3-oxzazole-5-one (3). Treatment of (3) with hydrazine and/or phenylhydrazine in refluxing ethanol afforded 5-benzylidene-3-sacchrinyl-methyl-1, 2, 4 triazine-6-one (4a) and/or 5-benzylidene-3-sacchrinyl-methyl-2-N-phenyl-1, 2, 4- triazine-6-one (4b). Furthermore when compound(3) reacted with aromatic amines (aniline, p-toludine)  and aminobenzoic acids (m, and p-aminobezoic acid) in refluxing ethanol it gave cinnamimide derivatives (5 a-d). But when the same reaction was carried out in n-butanol it gave imidazolone derivatives (6a-d) which were also obtained by refluxing compounds (5a-d) in acetic anhydride. Moreover compound (3) undergoes acid catalyses ring opening reaction with dry benzene in presence of anhydrous AlCl₃ (Fridel – Crafts reaction) to give saccharinyl – (N-benzoyl-methyl) acetamide (7), wherethe reaction product obtained via elimination of arylidene group^{23, 24} On the other hand, when saccharinyl acetic acid hydrazide (8) ⁹ was refluxed in acetic anhydride it cyclized to 6-oxo-1,2,4-triazino-[4,3-b][1,2] benzisothiazole (9).

Also the behavior of compound (9) towards carbon nucleophiles (Grignard reagent) was investigated. Thus when compound (9) submitted to react with ethyl magnesium iodide, methyl magnesium chloride then refluxed in dry benzene, it afforded 6-alkyl-6-hydroxy-1,2,4-triazino-[4,3-b][1,2]benzisothiazole (10). Treatment of compound(9) with mixture of POCl₃/PCl₅ afforded 6-Chloro-1,2,4-triazino[4,3-b][1,2]benzisothiazole (11) which reacted with urea and/or thiourea to give compound (12 a and b) respectively.

Also when compound (9) was allowed to condense with aromatic aldehyde namely bezaldehyde, anisaldehyde, and p-chlorobezaldehde in the presence of pyridine in a mixture of acetic anhydride and acetic acid (1:1) it afforded 5-arylidene-6-oxo-1,2,4-triazino-[4,3-b] benzisothiazole (13a-c).

Antimicrobial Activity

The antimicrobial activity (in vitro) of some synthesized compounds was determined against some bacteria and fungi using tetracycline and amphotericin B as standard antimicrobial agents by using the agar diffusion method²⁸, ²⁹

The obtained zones of inhibition were presented in table 1, which indicated that most of the synthesized derivatives have moderate to good antimicrobial activities.

Table 1: Antimicrobial activity (in vitro) of some synthesized compounds

Compd no.	Zone of inhibition mm/ mg of sample
	Escherickcacoli	Staphylococcus aureus	Candida albicans	Aspergillusflavus
3	0	0	17	15
4a	0	0	15	0
4b	18	12	15	18
5a	0	0	15	0
5b	0	0	16	0
5g	0	0	16	0
5d	12	14	0	0
6c	16	12	15	0
7	15	10	15	0
9	0	0	10	0
10a	19	0	10	0
11	0	0	15	0
12b	0	0	0	0
13a	18	0	0	0
Tetracycline	30	28	0	0
Amphotericin	0	0	16	20

 

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