Pharmacological Evaluation of Substituted Benzeneacetic Acid Ester Derivatives for Their Sedative, Antibacterial and Antifungal Potential

 

Nandini R Pai* and Deepnandan S Dubhashi

Department of Organic Chemistry, D.G. Ruparel College, Senapati Bapat Marg, Mahim, Mumbai 400016, India

*Corresponding Author E-mail: nandini_pai@hotmail.com or deepdubhashi@hotmail.com

ABSTRACT:

Antispasmodic medication is used to relieve urinary and bladder difficulties by relieving muscle spasms of the bladder. Several compounds of this family are known to possess antimicrobial potency in addition to their predesignated pharmacological actions. Based on this rationale present study is intended towards evaluating sedative, antibacterial and antifungal potency of an established antispasmodic drug to establish it as effective sedative and antimicrobial agent. This is based on the theory that the compound is expected to exhibit antimicrobial potency may be due to two benzene rings and secondary or tertiary nitrogen in its core molecule structure. For pursuing this objective, novel and structurally diverse analogs of the lead compound α-cyclohexyl-α-hydroxy-benzeneacetic acid-4-(diethylamino)-2-butynyl ester hydrochloride, a potential antispasmodic drug having two benzene rings and secondary or tertiary nitrogen in its structural framework were synthesized. All the synthesized compounds were confirmed by using physical data and different structure elucidation tools. The novel compounds were screened for their sedative, antibacterial and antifungal potential. The sedative potency of the compounds was evaluated using Thiopental sodium (Thiosol ®) as standard drug following one-way ANOVA followed by Scheffe’s post hoc analysis to find out the significance. The compounds were evaluated in vitro, for their antibacterial activity using acetone as a solvent and MIC was done by broth dilution method. The fungal susceptibility testing on different fungi was done by cup-diffusion method using Clotrimazole (100mcg/mL) as standard using acetone as a solvent. The compounds have shown promising results and definite structure-activity relationship could be established.

 

INTRODUCTION:

Antibiotics are known to be the major protective agents against bacterial infections.  However, the usage of antibiotics and antibacterial chemotherapeutics is becoming more and more restricted in the present age, despite the fact that there exist a large number of antibiotics.  This is largely attributed to the emergence of drug-resistant bacteria, which render even some of the most broad spectrum antibiotics ineffective.  In addition, most antibiotics have side effects.  Thus, it becomes essential to investigate newer drugs with less resistance.  Different studies on search of newer antimicrobials and antibacterial have revealed that moderate to remarkable antimicrobial or antibacterial action is present in several compounds, belonging to various pharmacological categories, such as antihistamines^1-3, tranquilizers⁴, antihypertensive⁵, anti-psychotics^6-10 anti-spasmodic¹¹ and anti-inflammatory agents¹². Such compounds, having antibacterial properties in addition to their predesignated pharmacological actions, are termed as non-antibiotics¹¹.

 

Many of these compounds possess two or three benzene rings and nitrogen in the secondary or tertiary state in their molecular structure which is expected to be one of the bases for exhibiting antimicrobial potency. Based on this rationale the present study was designed to synthesize structurally diverse novel analogs of lead compound having two benzene rings and secondary nitrogen in its structural framework, estimate their sedative, antibacterial and antifungal activity.

 

The incorporation of structural modifications in the main framework of the lead compound was thought to enhance the pharmacological potency. In the quest of novel antispasmodic agents with antimicrobial and sedative properties, we envisioned to synthesize novel analogs of the α-cyclohexyl-α-hydroxy-benzeneacetic acid-4-(diethylamino)-2-butynyl ester hydrochloride (Lead Compound), with diverse structural modifications. The novel analogs of lead compound were confirmed by structure elucidation techniques and tested for their antispasmodic and anticholinergic potency to establish structure activity relationship. The study then extended further to evaluate their sedative, antibacterial and antifungal potential.

The desired structural modifications in the lead compound were achieved by introducing respective modifications in the structural framework of its process intermediates. This was achieved by using respective starting materials like different homologs of α-oxo-benzeneacetic acid and various secondary amines.

 

The diverse analogs of process intermediate, 4-diethylamino-2-butynyl acetate (Intermediate A) were synthesized in a two step process by preparation of propargyl acetate in the first step followed by its simple condensation with various amines. Their reaction with different analogs of α-cyclohexyl-α-hydroxybenzeneacetic acid methyl ester (Intermediate B) synthesized by Grignard reaction involving in situ preparation of cyclohexyl magnesium bromide and condensation with different homologs of α-oxo-benzeneacetic acid methyl ester, achieved the targeted structurally diverse analogs of lead compound, α-cyclohexyl-α-hydroxy-benzeneaceticacid-4-(diethylamino)-2-butynylester hydrochloride (Table I). 

 

The structures of all the novel analogs were established by physical data, Mass spectra (Table 2), FTIR (Table 3) and 1H NMR (Table 4) spectroscopy. Synthesized compounds were then screened over different pharmacological potential like sedative, antibacterial and antifungal activities. The results are presented in respective tables (Table 5, 6 and 7).

This research paper describes the synthesis, characterization and pharmacological testing of novel analogs of lead compound. Based on these results obtained definite structure-activity relationship established was discussed further.

 

MATERIALS AND METHODS:

For Synthesis and Characterization of compounds

Synthesis of lead molecule, α-cyclohexyl-α-hydroxy-benzeneacetic acid-4-(diethylamino)-2-butynyl ester hydrochloride involves simple condensation of 4-diethylamino-2-butynyl acetate (Intermediate A) and α-cyclohexyl-α-hydroxybenzeneacetic acid methyl ester (Intermediate B) as shown in Figure 1. The standard laboratory synthetic process is presented in following chapters.

 

Melting points of the synthesized compounds were determined on a melting point apparatus MP II and are uncorrected. IR spectra of the synthesized compounds were determined on Perkin Elmer FTIR Spectrum. 1H NMR, spectra were taken on Bruker 300MHz NMR spectrometer. The mass spectra were taken on Shimadzu Qp-2010 mass spectrometer. Progress of the reaction was monitored using Perkin Elmer Clarus 500 GC system and Agilent 1100 HPLC system. The required chemicals and solvents were procured from Sigma-Aldrich and Rankem. The sample of reference standard Thiopental sodium, Penicillin and Clotrimazole was obtained from “Aarti Drugs” and “Reliance life sciences” research centre Mumbai.

 

Standard process for preparation of propargyl acetate (2) and its analogs.

To a pre-cooled solution (<10oC) of propargyl alcohol (200 gm, 3.57 moles) and triethylamine (430.5 gm, 4.26 mol)  in dichloromethane (1.0 L), was added acetyl chloride (311.0 gm, 3.96 mol) maintaining temperature less than 15oC. The reaction mass was slowly warmed to 20-25oC and stirred for 0.5 hour. Completion of reaction was confirmed by GC and water was added to the reaction mass. The reaction mass was then stirred for 15 minutes, layers settled and organic layer containing product was separated. The product was isolated by distillation after recovery of dichloromethane solvent. Yield: 318.5 g, 91%.

 

Standard process for the preparation of 4-diethylamino-2-butynyl acetate (3) and its analogs.

To the mixture of para-formaldehyde (90 gm, 3.0 mole), diethylamine (210 gm, 2.87 moles) and cuprous chloride (5 gm) in 1,4-dioxane (1050 mL) was added propargyl acetate (250 gm, 2.55 mol) under stirring at 30-35oC. The reaction mass was then heated using an oil bath to 90-95°C and maintained for 1 hour. Completion of reaction was confirmed by GC, cooled to 15oC and filtered on hyflo bed. On recovery of solvent 1,4-dioxane (70 %) crude product was isolated (415.5 gm, 89 %) which was purified by distillation under reduced pressure (1mm, 75-80oC) to get pure product as brownish yellow oil. Yield: 373.5 gm, 80%.

   

Standard process for the preparation of α-oxo-benzeneacetic acid methyl ester (5) and its analogs.

To the solution of α-oxo-benzeneacetic acid (150 gm, 1.0 moles) and dimethylformamide (1.0 mL) in toluene (450 mL) was added thionyl chloride (178.5gm, 1.5 mol). The solution was heated to 45-50oC and continued for 1 hour. The reaction mass was then subjected to distillation when toluene was recovered (~390 mL) followed by distillation of product under reduced pressure (1 mm, 60oC). The isolated acid chloride product (155 gm, 92 %) was poured in methanol (300 mL) and methyl ester of was isolated by distillation under reduced pressure (1 mm, 98oC) after initial recovery of methanol. Yield: 143.5 gm, 87%.

 

Standard process for the preparation of α-cyclohexyl-α-hydroxybenzeneacetic acid methyl ester (6) and its analogs.

Magnesium turnings (15.8 gm) and iodine (200 mg.) were added to tetrahydrofuran (73.0 gm) under nitrogen atmosphere and the mixture was stirred at about 25oC for 0.5 hour. Initially first lot of cycloxexyl bromide (4.1 gm, 0.025 mol) was added drop wise followed by tetrahydrofuran (292.0 gm) was added and then remaining cyclohexyl bromide (86 gm, 0.53 mol) was added drop wise at 60-70oC. The mixture was stirred at 60-70oC and reaction completion was confirmed by GC. The mixture was cooled to 20-30oC and this Grignard solution was added drop wise to a mixture of α-oxo-benzeneacetic acid methyl ester (82.1 gm, 0.5 mol) and tetrahydrofurane (82.0 mL) at 5-15oC. The reaction mass further stirred for 1 hour and  completion of the reaction was confirmed by HPLC for absence of α-oxo-benzeneacetic acid methyl ester.

Figure 1: Reaction scheme for preparation of lead compound and its analogs (7-7l).

 

 

Table 1: Analogs of lead molecule (7-7l)

Product	R1	R2	R3	R4	R5	n	R*	R**
7	H	H	H	H	H	0	-C2H5	-C2H5
7a	H	H	H	H	H	2	-C2H5	-C2H5
7b	H	H	H	H	H	3	-C2H5	-C2H5
7c	H	CH3	H	H	H	2	-C2H5	-C2H5
7d	CH3	CH3	H	H	H	2	-C2H5	-C2H5
7e	H	H	H	H	H	0	-CH(CH3)2	-CH(CH3)2
7f	H	H	H	H	H	0	-(CH2)3-CH3	-(CH2)3-CH3
7g	H	H	H	H	H	2	-CH(CH3)2	-CH(CH3)2
7h	H	H	H	H	H	3	-CH(CH3)2	-CH(CH3)2
7i	H	H	H	H	H	2	-CH3	-CH3
7j	H	H	H	H	H	3	-CH3	-CH3
7k	H	H	H	H	H	2	1H-pyrrole
7l	H	H	H	H	H	2	3-methylpyrrole

 

Table 2: Physical, Yield and Mass spectral data of synthesized compound (7-7l)

Compound	Mol.  Formula	Mol. Weight	M.P  0C	Yield  %	MS (M/Z)
7	C22H31NO3 .HCl	393.5	129-130	86.4 %	358 (M+1)+
7a	C24H35NO3.HCl	421.5	143-144.5	72.3 %	386 (M+1)+
7b	C25H37NO3.HCl	435.5	161-162	71.9 %	400 (M+1)+
7c	C25H37NO3.HCl	435.5	157-158.5	76.8 %	400 (M+1)+
7d	C26H39NO3.HCl	449.5	177-178.6	84.3 %	413 (M)+
7e	C24H35NO3.HCl	421.5	121-123	79.5 %	386 (M+1)+
7f	C26H39NO3.HCl	449.5	183-184.7	80.5 %	414 (M+1)+
7g	C26H39NO3.HCl	449.5	191-193	69.4 %	414 (M+1)+
7h	C27H41NO3.HCl	463.5	173-175	65.4 %	427 (M)+
7i	C22H31NO3.HCl	393.5	163-165.3	71.5 %	358 (M+1)+
7j	C23H33NO3.HCl	407.5	153-153.4	80.3 %	372 (M+1)+
7k	C24H29NO3.HCl	415.5	194-195.7	59.4 %	380 (M+1)+
7l	C25H31NO3.HCl	429.5	142-143.5	60.3 %	394 (M+1)+

Tetrahydrofurane was evaporated under reduced pressure at 65-80oC and toluene (150.0 mL) was added. This mixture was then added drop wise at < 35oC to 7N hydrochloric acid (215.0 mL) and allowed to stir for 0.5 hour. The layers were settled, organic layer containing product was separated, cooled to 0-5oC and product isolated by filtration. The product was further purified by crystallization from ethyl acetate. Yield: 80.7 gm, 65%.

 

Standard process for the preparation of α-cyclohexyl-α-hydroxy-benzeneacetic acid-4-(diethylamino)-2-butynyl ester hydrochloride (7) and its analogs (7a-7l).

To the solution of α-cyclohexyl-α-hydroxybenzeneacetic acid methyl ester (160 gm, 0.65 mol) in n-heptane (800 mL) was added solution of 4-diethylamino-2-butynyl acetate (130 gm, 0.71 mol) in n-heptane (800 mL) followed by sodium methoxide (8.0 gm) at 25-30oC. Heat the reaction mass to 90-95oC when distillation of methyl acetate and n-heptane mixture starts. This is continued further for 3 hours. Reaction completion was confirmed by HPLC and added n-heptane (400 mL) followed by water (400 mL). The reaction mass stirred for 10 minutes, layers settled and organic layer containing product was separated and washed further with water (100 mL). The organic layer was then extracted with 10 % hydrochloric acid solution (4 x 100 mL) and combined acidic aqueous layer was subjected to chilling (0 ± 5oC) when product is precipitated as hydrochloride salt. The product slurry stirred for 1 hour at same temperature, filtered and product dried at 40-45oC under reduced pressure (120 mm/Hg). The crude product is

 

purified by crystallization from ethyl acetate to isolate pure product as off white crystalline powder. Yield 218.5 gm, 86.4 %.

 

Biological activity of synthesized compounds

Animals Used for Biological activity studies

Study was performed using healthy Wistar rats of average weight of either sex. Wister rats weighing in the range of 300-500 gm were procured and sedative activity was carried out on approval of Institutional Animal Ethics Committee constituted for the purpose.

 

Sedative activity

Male Wister rats weighing in the range of 200-400 gm were selected from an inbred strain colony.  They were maintained at constant temperature and relative humidity.  Acute toxicity was done by following the proposed test method from literature13.  Thiopental sodium (Thiosol ®) was used as standard drug, 2% CMC suspension was used as control and suspensions of the synthesized compounds were used.  The mean sleeping times of compounds were compared with the standard, using one-way ANOVA followed by Scheffe’s post hoc analysis to find out the significance. The results are presented in Table 5.

 

Antibacterial activity 

Antibacterial activity of the synthesized compounds (7, 7a-7p) was evaluated using acetone as a solvent and MIC was done by broth dilution method14. The bacterial stains used for the assay include, against gram-positive organisms B.Subtilis (MTCC 441), B.sphaericus (MTCC 511) and

 

Table 3: FTIR spectral data of synthesized compound (7-7l)

Product	IR Data
7	-OH stretching (3316 cm-1), aromatic C-H stretching (3096 cm-1), aliphatic C-H stretching (2959.57-2840.22 cm-1), -C≡C- stretching (2150 cm-1), -C=O stretching (1760 cm-1), asymmetrical -C-O-C and --C-O stretching (1265-1130 cm-1), monosubstituted benzene stretching (two bands at760 cm-1 and 690 cm-1)
7a	-OH stretching (3316 cm-1), aromatic C-H stretching (3096 cm-1), aliphatic C-H stretching (2928 cm-1), -C≡C- stretching (2070 cm-1), -C=O stretching (1744 cm-1), asymmetrical -C-O-C and --C-O stretching (1246-1161 cm-1), monosubstituted benzene stretching (two bands at770 cm-1 and 696 cm-1)
7b	-OH stretching (3318 cm-1), aromatic C-H stretching (3096 cm-1), aliphatic C-H stretching (2992-2845 cm-1), -C≡C- stretching (2069 cm-1), -C=O stretching (1756 cm-1), asymmetrical -C-O-C and --C-O stretching (1246-1133 cm-1), monosubstituted benzene stretching (two bands at770 cm-1 and 695 cm-1)
7c	-OH stretching (3317 cm-1), aromatic C-H stretching (3096 cm-1), aliphatic C-H stretching (2992-2845 cm-1), -C≡C- stretching (2070 cm-1), -C=O stretching (1745 cm-1), asymmetrical -C-O-C and --C-O stretching (1273-1161 cm-1), monosubstituted benzene stretching (two bands at770 cm-1 and 695 cm-1), mata substituted benzene stretching (two bands at704 cm-1 and 781 cm-1)
7d	-OH stretching (3316 cm-1), aromatic C-H stretching (3096 cm-1), aliphatic C-H stretching (2992-2860 cm-1), -C≡C- stretching (2070 cm-1), -C=O stretching (1744 cm-1), asymmetrical -C-O-C and --C-O stretching (1274-1161 cm-1), monosubstituted benzene stretching (two bands at770 cm-1 and 696 cm-1), mata disubstituted benzene stretching (two bands at704 cm-1 and 781 cm-1)
7e	-OH stretching (3316 cm-1), aromatic C-H stretching (3096 cm-1), aliphatic C-H stretching (2992-2861 cm-1), -C≡C- stretching (2069 cm-1), -C=O stretching (1745 cm-1), asymmetrical -C-O-C and --C-O stretching (1274-1161 cm-1), monosubstituted benzene stretching (two bands at770 cm-1 and 695 cm-1)
7f	-OH stretching (3317 cm-1), aromatic C-H stretching (3096 cm-1), aliphatic C-H stretching (2992-2861 cm-1), -C≡C- stretching (2070 cm-1), -C=O stretching (1745 cm-1), asymmetrical -C-O-C and --C-O stretching (1273-1161 cm-1), monosubstituted benzene stretching (two bands at770 cm-1 and 695 cm-1)
7g	-OH stretching (3316 cm-1), aromatic C-H stretching (3096 cm-1), aliphatic C-H stretching (2992-2861cm-1), -C≡C- stretching (2142 cm-1), -C=O stretching (1746 cm-1), asymmetrical -C-O-C and --C-O stretching (1274-1161 cm-1), monosubstituted benzene stretching (two bands at770 cm-1 and 695 cm-1)
7h	-OH stretching (3316 cm-1), aromatic C-H stretching (3096 cm-1), aliphatic C-H stretching (2992-2861cm-1), -C≡C- stretching (2070 cm-1), -C=O stretching (1744 cm-1), asymmetrical -C-O-C and --C-O stretching (1274-1161 cm-1), monosubstituted benzene stretching (two bands at770 cm-1 and 695 cm-1)
7i	- OH stretching (3316 cm-1), aromatic C-H stretching (3096 cm-1), aliphatic C-H stretching (2992-2861cm-1), -C≡C- stretching (2142 cm-1), -C=O stretching (1745 cm-1), asymmetrical -C-O-C and --C-O stretching (1274-1161 cm-1), monosubstituted benzene stretching (two bands at770 cm-1 and 695 cm-1)
7j	- OH stretching (3317 cm-1), aromatic C-H stretching (3095 cm-1), aliphatic C-H stretching (2992-2860cm-1), -C≡C- stretching (2069 cm-1), -C=O stretching (1744 cm-1), asymmetrical -C-O-C and --C-O stretching (1274-1161 cm-1), monosubstituted benzene stretching (two bands at770 cm-1 and 695 cm-1)
7k	- OH stretching (3317 cm-1), aromatic C-H stretching (3096 cm-1), aliphatic C-H stretching (2992-2861cm-1), -C≡C- stretching (2070 cm-1), -C=O stretching (1745 cm-1), asymmetrical -C-O-C and --C-O stretching (1274-1161 cm-1), monosubstituted benzene stretching (two bands at770 cm-1 and 695 cm-1)
7l	- OH stretching (3317 cm-1), aromatic C-H stretching (3095 cm-1), aliphatic C-H stretching (2992-2860cm-1), -C≡C- stretching (2069 cm-1), -C=O stretching (1744 cm-1), asymmetrical -C-O-C and --C-O stretching (1274-1161 cm-1), monosubstituted benzene stretching (two bands at770 cm-1 and 695 cm-1)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

S. aureus (MTCC 96) and Gram-negative organisms P. aeruginosa (MTCC 741) K. aerogenes (MTCC 39) and C. violaceum (MTCC 2656) at 100µg/ml concentration. Standard antibacterial drugs were also screened under similar conditions for comparison.  Penicillin (100mcg/mL) from stock solution of 1000mg/mL was used as standard for B. Subtilis, B. sphaericus and S. aureus and Gentamycin (100mcg/mL) from stock solution of 1000mg/mL was used as a standard for other organisms. The results are presented in Table 6.

 

Antifungal activity

The antifungal activity of compounds (7,7a-7p) was evaluated against A. niger (MTCC 282), C. tropicum (MTCC 2821), R. oryzae (MTCC 262), F. moliliforme (MTCC 1848) and C. lunata (MTCC 2030) using Acetone as a solvent by cup diffusion method14 at 100mcg/mL concentrations. The fungal susceptibility testing was done

 

 

 

by cup-diffusion method using Clotrimazole (100mcg/mL) from stock solution of 1000mg/mL as standard. The results are presented in Table 7. The zone of inhibition was measured after 24 hr of incubation at 37oC. The zone of inhibition developed if any, was then accurately measured and recorded.

 

RESULTS AND DISCUSSIONS:

The structurally diverse analogs of the lead compound were synthesized by standard process as described in Figure 1. The structures of the novel compounds have been confirmed by structure elucidation techniques and results are described for ¹H NMR (Table 4), FTIR (Table 3) and Mass spectral (Table 2) analysis and also supported by physical data (Table 2) such as melting point differences.

 

 

 

Table 4: ¹H NMR spectral data of synthesized compound (7-7l)

Product	1H NMR (ð)
7	Chemical Shift ð, 1.10 ppm (t, 6H, -CH3), 1.27 ppm (m, 2H, -CH of cyclohexyl), 1.35-1.57 (m, 8H, -CH of cyclohexyl), 2.40 (q, 4H, -CH2), 2.53 (m, 1H, -CH of cyclohexyl), 3.05 (s, 2H, -CH2), 4.77 (s, 2H, -CH2), 6.91 (bs, 1H, -OH), 7.19 (d, 4H, CH-Ar), 7.37 (m, 1H, CH-Ar)
7a	Chemical Shift ð, 1.00 ppm (t, 6H, -CH3), 1.30 (m, 2H, -CH of cyclohexyl), 1.40-1.52 (m, 8H, -CH of cycloxexyl), 2.00 (m, 1H, -CH of cyclohexyl), 2.12 (t, 2H, -CH2), 2.25 (t, 2H, -CH2), 2.40 (q, 4H, -CH2), 3.05 (s, 2H, -CH2), 4.77 (s, 2H, -CH2), 5.52 (bs, 1H, -OH), 7.30-7.37 (d, 3H, CH-Ar), 7.52 (m, 2H, CH-Ar)
7b	Chemical Shift ð, 1.05 ppm (t, 6H, -CH3), 1.20 (m, 2H, -CH of cyclohexyl), 1.32-1.50 (m, 8H, -CH of cycloxexyl), 1.68-1.73 (m, 4H, -CH2), 1.89 (m, 1H, -CH of cyclohexyl), 2.30 (t, 2H, -CH2), 2.41 (q, 4H, -CH2), 3.15 (s, 2H, -CH2), 4.69 (s, 2H, -CH2), 6.00 (bs 1H, -OH), 7.14 (d, 4H, CH-Ar), 7.26 (m, 1H, CH-Ar)
7c	Chemical Shift ð, 1.10 ppm (t, 6H, -CH3), 1.21 ppm (m, 2H, -CH of cyclohexyl), 1.30-1.51 (m, 8H, -CH of cyclohexyl), 1.89 (m, 1H, -CH of cyclohexyl), 2.12 (t, 2H, -CH2), 2.25 (t, 2H, -CH2), 2.35 (s, 3H, -CH3-Ar), 2.43 (q, 4H, -CH2), 3.05 (s, 2H, -CH2), 4.76 (s, 2H, -CH2), 5.90 (bs, 1H, -OH), 6.99 (d, 2H, CH-Ar)7.07 (t, 1H, CH-Ar), 7.35 (m, 1H, CH-Ar)
7d	Chemical Shift ð, 1.00 ppm (t, 6H, -CH3), 1.23 ppm (m, 2H, -CH of cyclohexyl), 1.36-1.50 (m, 8H, -CH of cyclohexyl), 1.95 (m, 1H, -CH of cycloxexyl), 2.17 (t, 2H, -CH2), 2.24 (t, 2H, -CH2), 2.39 (s, 6H, -CH3-Ar), 2.45 (q, 4H, -CH2), 3.10 (s, 2H, -CH2), 4.73 (s, 2H, -CH2), 5.85 (bs, 1H, -OH), 6.83 (s, 3H, -CH-Ar),
7e	Chemical Shift ð, 1.05 (s, 12H, -CH3), 1.19 (s, 2H, -CH of cycloxexyl), 1.40-1.51 (m, 8H, -CH of cyclohexyl), 2.12 (m, 1H, -CH of cycloxexyl), 2.91 (m, 2H, -CH), 3.15 (s, 2H, -CH2), 4.71 (s, 2H, -CH2), 6.85 (bs, 1H, -OH), 7.21 (d, 4H, CH-Ar), 7.37 (m, 1H, CH-Ar)
7f	Chemical Shift ð, 0.96 ppm (s, 6H, -CH3), 1.25 (s, 2H, -CH of cycloxexyl), 1.36-1.50 (m, 16H, -CH of cyclohexyl, CH2), 2.36 (t, 4H, -CH2), 2.55 (m, 1H, -CH of cyclohexyl), 3.05 (s, 2H, -CH2), 4.73 (s, 2H, -CH2), 6.91 (bs 1H, -OH), 7.22 (d, 4H, CH-Ar), 7.35 (m, 1H, CH-Ar)
7g	Chemical Shift ð, 1.08 (s, 12H, -CH3), 1.24 (sm, 2H, -CH of cycloxexyl), 1.31-1.49 (m, 8H, -CH of cyclohexyl), 1.90 (s, 1H, -CH of cycloxexyl), 2.12 (t, 2H, -CH2), 2.30 (t, 2H, -CH2), 3.01 (m, 2H, -CH), 3.10 (s, 2H, -CH2), 4.72 (s, 2H, -CH2), 5.90 (bs, 1H, -OH), 7.37 (m, 3H, CH-Ar), 7.57 (t, 2H, CH-Ar),
7h	Chemical Shift ð, 1.05 (d, 12H, -CH3), 1.26 (m, 2H, -CH of cycloxexyl), 1.36-1.52 (m, 8H, -CH of cycloxexyl), 1.68-1.73 (m, 4H, -CH2), 1.89 (m, 1H, -CH of cyclohexyl), 2.25 (t, 2H, -CH2), 2.97 (s, 2H, -CH), 3.10 (s, 2H, -CH2), 4.75 (s, 2H,-CH2), 5.91 (bs, 1H, -OH), 7.36 (d, 3H, CH-Ar), 7.50 (m, 2H, CH-Ar)
7i	Chemical Shift ð, 1.27 ppm (m, 2H, -CH of cyclohexyl), 1.37-1.50 (m, 8H, -CH of cycloxexyl), 1.9 (m, 1H, -CH of cyclohexyl), 2.12 (m, 2H, -CH2), 2.25 (t, 2H, --CH2), 2.27 (s, 6H, -CH3), 3.05 (s, 2H, -CH2), 4.77 (s, 2H, -CH2), 5.52 (bs, 1H, -OH), 7.36 (m, 3H, -CH-Ar), 7.57 (m, 2H, -CH-Ar)
7j	Chemical Shift ð, 1.30 (m, 2H, -CH of cyclohexyl), 1.39-1.56 (m, 8H, -CH of cycloxexyl), 1.68-1.75 (m, 4H, -CH2), 2.0 (m, 1H, -CH of cyclohexyl), 2.25 (t, 2H, -CH2) 2.27 ppm (s, 6H, -CH3), 3.05 (s, 2H, -CH2), 4.71 (s, 2H, -CH2), 6.81 (bs, 1H, -OH), 7.10 (m, 3H, -CH-Ar), 7.31 (m, 2H, -CH-Ar)
7k	Chemical Shift ð, 1.22 (m, 2H, -CH of cyclohexyl), 1.31-1.46 (m, 8H, -CH of cyclohexyl), 1.90 (m, 1H, -CH of cycloxexyl), 2.18 (m, 2H, -CH2), 2.30 (m, 2H, -CH2), 4.54 (s, 2H, -CH2), 4.75 (s, 2H,-CH2), 5.40 (bs, 1H, -OH), 5.90 (m, 2H, CH-Ar), 6.37 (m, 2H, CH-Ar), 7.30-7.37 (m, 3H, CH-Ar), 7.40 (t, 2H, CH-Ar)
7l	Chemical Shift ð, 1.22 (m, 2H, -CH of cyclohexyl), 1.31-1.46 (m, 8H, -CH of cyclohexyl), 1.98 (m, 1H, -CH of cycloxexyl), 2.05 (s, 3H, -CH3), 2.12 (m, 2H, -CH2), 2.25 (m, 2H, -CH2), 4.54 (s, 2H, -CH2), 4.75 (s, 2H,-CH2), 5.52 (bs, 1H, -OH), 5.90 (m, 1H, CH-Ar), 6.20 (m, 2H, CH-Ar), 7.30-7.37 (m, 3H, CH-Ar), 7.40 (t, 2H, CH-Ar)

 

Structure activity relationships

Sedative activity   

The six compounds viz. “7a”, “7b”, “7g”, “7h”, “7i” and “7j” with n= 2 or 3, were found to be statistically significant against standard Thiopental sodium at P<0.05 by applying Scheffe’s Post Hoc method at 100 mg/kg (Table 5). The lead compound “7” and its analogs “7e”, “7f” were not significant where n= 0. This can be attributed to presence of respective ethyl or propyl linker (n = 2 or 3) prior to carbonyl group in the structural framework of the compound. But in compounds “7c” and “7d” with methyl and dimethyl substitution on phenyl ring has led them insignificant even with n=2.

 

No added benefit was observed in compounds “7k” and “7l” where diethyl amine in the lead compound is replaced with 1 H-pyrrole and with n=2,  were found insignificant like lead compound.

 

Table 5: Sedative Activity of the Synthesized Compounds (7-7l)

 

Sr. No.	Compound	Mean Sleeping Time min ± S.E.
1	Control	-
2	Standard Thiopental sodium	13.16 ± 1.66
3	7	11.36 ± 0.76
4	7a	22.10 ± 0.55.
5	7b	21.33 ± 0.37.
6	7c	10.36 ± 0.76
7	7d	13.26 ± 0.65
8	7e	12.25 ± 0.37
9	7f	12.05 ± 0.65
10	7g	21.10 ± 0.66
11	7h	21.53 ± 0.57.
12	7i	22.77 ± 0.54.
13	7j	20.30 ± 0.45
14	7k	11.45 ± 0.61
15	7l	13.10 ± 0.43

Dose: 100 mg/Kg; The results are significant (^.) at p<0.0

 

Table 6: Antibacterial Activity of the Synthesized Compounds (7-7l)

Sr. No.	Compound	Microorganisms
		Gram-positive			Gram-negative
		B. subtilis	B. sphaericus	S. aureus	P. aeruginosa	K. aerogenes	C. violaceum
Reference Standard	Penicillin	30	29	22	23	18	19
1	7	25	25	15	18	12	13
2	7a	14	15	10	10	09	07
3	7b	16	14	06	06	04	07
4	7c	19	17	11	11	09	08
5	7d	20	16	10	12	10	08
6	7e	24	22	14	17	10	11
7	7f	22	20	13	12	11	10
8	7g	11	11	05	08	05	07
9	7h	13	17	04	09	08	09
10	7i	13	10	06	10	07	07
11	7j	12	15	05	09	07	08
12	7k	18	14	10	11	07	08
13	7l	19	13	09	11	08	10

 

Table 7: Antifungal Activity of Synthesized Compounds (7-7l)

 

Sr. No.	Compound	Zone of inhibition in mm
		A. niger	C. tropicum	R. oryzae	F. moniliforme	C. lunata
Reference Standard	Clotrimazole	31	30	27	29	32
1	7	24	26	21	24	25
2	7a	19	18	19	17	17
3	7b	18	16	19	16	18
4	7c	17	17	18	16	17
5	7d	17	19	17	16	15
6	7e	23	26	23	22	27
7	7f	25	25	22	24	21
8	7g	10	09	10	08	09
9	7h	10	07	09	06	08
10	7i	14	15	12	11	08
11	7j	13	14	10	09	09
12	7k	20	23	21	22	20
13	7l	22	22	23	20	21

In conclusion all the novel analogs with diverse structural modifications especially with introduction of ethyl or propyl linker prior to carbonyl carbon have demonstrated significant sedative activity and are recommended for further evaluation. Introduction of electron donating group on phenyl ring have diminished the activity of respective compounds.

 

Antibacterial and antifungal activity   

From Table 6, it is clearly evident that the compounds are active against the bacterial and fungal stains. Among thirteen compounds (“7-7l”), the compounds “7”, “7e” and “7f” has shown excellent antibacterial activity well comparable to that of reference standard Penicillin.

 

The compounds “7a” and “7b” exhibited reduced antibacterial potential than the lead compound “7”, where respective linker is introduced in the structural framework of molecule prior to carbonyl carbon with n=2 and 3 respectively. The activity is slightly enhanced with introduction of methyl and dimethyl group on phenyl ring as in compounds “7c” and “7d”. Similar reduced antibacterial potency was shown by compounds “7g”, “7h”, “7i” and “7j” with n=2 or 3 in respective compounds.

 

The data indicated that only the lead compound “7” and analogs “7e” and “7f” with n= 0, have shown promising results but surprisingly compounds “7k” and “7l” with n=2 and with pyrrole or substituted pyrrole replacement respectively have shown moderate antibacterial activity equivalent to compounds“7c” and “7d”.

 

From the above study it is concluded that introduction of ethyl or propyl linker prior to carbonyl carbon in the structural framework (n>0, n=2 or 3) has adverse impact on potency leading to diminished antibacterial activity of the compounds. None of the compounds tested have exhibited MIC higher than that of the reference standard Penicillin. The antibacterial potency of compounds “7”, “7i” and “7j” against the standard drug is promising and can be evaluated further for their formulation potentials.

 

The antifungal activity studies as presented in Table 7 indicated that the compounds “7”, “7e” and “7f” exhibited an excellent antifungal activity (21-27) close to reference standard Clotrimazole (23-29) and can be exploited for formulation of fungicide. The compounds “7a”, “7b”, “7c” and “7d” have shown moderate activity (< 20) whereas compounds “7i” and “7j” with respective structural substitutions have shown reduced antifungal activity (<15). Surprisingly the compounds “7k” and “7l” which have shown moderate antibacterial activity have exhibited promising antifungal potency (21-24). Based on results other compounds “7g” and “7h” can be claimed as inactive (< 15) based on data.

 

It is clearly evident from the test result that introducing linker prior to carbonyl carbon and replacing ethyl groups on nitrogen with higher alkane homologs in structural framework of lead compound led to diminished antifungal potency of respective compounds as similarly observed during antibacterial testing.

 

In conclusion, the tested novel compounds have moderate to excellent activity towards the bacteria and fungi under investigation. The lead compound “7” and analogs “7e” and “7f” can be exploited for formulations of bactericidal and fungicide following further detailed study.

 

 CONCLUSION:

The novel analogs of the lead compound α-cyclohexyl-α-hydroxy-benzeneacetic acid-4-(diethylamino)-2-butynyl ester hydrochloride with diverse structural modifications have exhibited promising results as desired when screened over pharmacological activities like sedative, antibacterial and antifungal potentials. The definite structure-activity relationship could be established based on acquired test results of different compounds against induced structural modifications. The potentially active compounds, in respective areas of pharmacology, identified with preliminary screening studies are recommended for further evaluation of their formulation potential that can replace some of the existing drugs in the family as a better alternative with minimal side effects characteristics.

 

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Received on 08.12.2009       Modified on 14.01.2010

Accepted on 17.02.2010      © RJPT All right reserved