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 have revealed that moderate to remarkable antimicrobial action is present in several compounds, belonging to various pharmacological categories, such as antihistamines^1-3, tranquilizers ⁴, antihypertensive ⁵, antipsychotics ^6-10 and anti-inflammatory agents ¹¹. Such compounds, having antimicrobial properties in addition to their predesignated pharmacological actions, are termed as non-antibiotics ¹².

Since many of these compounds possess two or three benzene rings and nitrogen in the secondary or tertiary state, the present study was designed to determine antimicrobial action of an antispasmodic drug 3,4-dimethoxybenzoic acid 4-[ethyl{2-(4-methoxyphenyl)-1-methylethyl}amino]butyl ester hydrochloride having two benzene rings. It is a potential musculotropic antispasmodic drug without atropic side-effects whose major therapeutic role is in the treatment of irritable bowel syndrome. It is also indicated for treatment of gastrointestinal spasm secondary to organic disorder ¹³.

The synthetic process study for preparation of the drug in laboratory was attempted and the developed process was further standardized to achieve consistent quality and yield of the product. The synthesized drug was well characterized using different spectroscopic, elemental and chromatographic techniques to establish structure and confirm purity.

In this research Paper we report a simple three-stage synthesis process for the said drug, its characterization data to establish structure and chromatographic purity. The research paper further describe results of the study to evaluate antimicrobial potential, toxicity and protective efficacy of the drug using agar and broth dilution methods in vitro and in vivo respectively.

MATERIALS AND METHODS:

Drug: The synthetic process study and subsequent synthesis of target drug product was carried out in Laboratory at Chemistry Department, Ruparel College, Mumbai 400016. The synthesized drug substance was well characterized with the help of “IR” using Perkin Elmer FTIR , MS (m/z) using Shimadzu Qp-2010 Mass spectrometer, “H¹ NMR” using NMR Bruker, 300 MHz spectrophotometer at “NPIL Research Centre”, Mumbai. The compound which was obtained in white dry powder form was analyzed by “HPLC” technique using Agilent 1100 HPLC system equipped with Quaternary gradient pump and Photodiode array detector for purity confirmation at RPG LS Research Centre, Mumbai.

For activity testing drug samples were dissolved in sterile distilled water, and kept at 4^oC.

Bacteria: A total of 414 fully characterized bacterial isolates belonging to 15 genera comprising 100 gram positive and 314 gram negative types were tested. Of these, 46 were “National collection of type culture” (NCTC) and “American type culture collection” (ATCC) strains and preserved in freeze-dried state.

Media: Peptone water (PW; Oxoid brand, UK), nutrient broth (NB; Oxiod), and Mueller Hinton broth (MHB; Difco, USA) were obtained, and peptone agar (PA), nutrient agar (NA), and Mueller Hinton agar (MHA) were prepared by adding agar to the respective liquid media, according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines¹⁴, and used for determining minimum inhibitory concentration (MIC) of MEB HCl. Streptococcus pyogenes was grown in MHB supplemented with blood.

The Anti-microbial testing was carried out at “Department of Toxicology, NPIL Research Centre, Mumbai.

Brief Synthetic Process for preparation of drug substance:

The proposed novel method for the synthesis as in Figure 1 involves condensation of 4'-bromobutyl-3, 4-dimethoxy benzoate (Stage I Intermediate) with 2-ethylamino-1-(4’-methoxyphenyl) propane (Stage II Intermediate) in ethyl methyl ketone (EMK) solvent medium at 75-80 ^0C for 32 hours. The stage I intermediate is prepared by simple condensation of sodium salt of 4-bromobutyl-3, 4-dimethoxybenzoic acid and 1, 4 -dibromobutane in two step process. Preparation of Stage II Intermediate is carried out in Laboratory Autoclave reactor by catalytic reduction of 4-methoxyphenyl acetone with hydrogen gas and 5 % platinum on charcoal as catalyst followed by in-situ condensation with ethylamine.

Detailed Laboratory process:

Stage I-Step 1: Method of preparation of sodium- 3, 4-dimethoxy benzoate [2]:

To a solution of 3, 4-dimethoxy benzoic acid (200 gm, 1.09 moles) in methanol (3.0 L), sodium methoxide solution (205 mL, 29 %) was added. The mixture was then warmed to 45^oC and maintained for one hour. The methanol is recovered by distillation till thick product slurry is obtained (~60 %). The solid product was filtered and slurry washed with methanol. Yield: 204 g, 91.7%,

Stage I-Step 2: Method of preparation of 4’-bromobutyl-3, 4-dimethoxy benzoate [3]:

A mixture of 3, 4-dimethoxy benzoate (204 gm, 1.0 mole) and 1,4-dibromobutane (432 gm, 2.0 moles) was heated in an oil bath at 150-155° for 3h.The progress of the reaction was monitored by thin layer chromatography (TLC) to ensure formation of product and complete conversion. On reaction completion excess 1,4-dibromobutane was recovered (~194.4 gm, 0.9 moles) by distillation under reduced pressure (1mm, 62-68 ^oC) followed by isolation of product (1mm, 72-78 ^oC) as next fraction. Yield: 190 gm, 60%,

Stage II: Method of preparation of 2-ethylamino-1-(4’-methoxyphenyl) propane [4]:

To the mixture of 4-methoxyphenylacetone (175 gm, 1.06 moles) in methanol (1.05 L) was added platinum on carbon catalyst (Pt/C-5%, 5 gm), and cooled to 15^oC. To this pre-cooled mixture ethylamine solution (135 gm, 70 %, 2.1 moles) was added. The mixture was transferred to autoclave reaction vassal and purged hydrogen after inertisation with Nitrogen. The reaction continued maintaining the temperature 50^oC and Hydrogen pressure 6 Kg/cm² for 15 hours. The progress of the reaction was monitored by TLC to ensure complete conversion of starting. The reaction mass was cooled to 30^oC and catalyst was filtered off. The mixture of methanol and unreacted ethyl amine was recovered by simple distillation followed by isolation of product by distillation under reduced pressure (1 mm, 89^oC). Yield: 173 gm, 84.5%;

Stage III: Method of preparation of 3, 4-dimethoxybenzoic acid 4-[ethyl {2-(4-methoxyphenyl)-1-methylethyl} amino] butyl ester hydrochloride [5].

A mixture of compound [3] (150 gm, 0.473 moles) and Compound [4] (95.2 gm, 0.493 moles) in ethyl methyl ketone (300 mL) was refluxed for a period of 32 hours at 75-80^oC. The progress of the reaction was monitored by TLC to ensure formation of product and complete conversion of starting. On reaction completion solvent was distilled off completely followed by addition of Water (750 mL) and Toluene (300 mL). The solution cooled to 30^oC and stirred for 30 minutes and settled before layer separation.

Table 1:Physical data and Elemental Analysis of the drug and its intermediates

Sr.

No.

Compound

Melting

Range

Molecular Formula

Molecular Weight

Elemental Analysis

Found % and (calculated) %

Sodium 3, 4-dimethoxy benzoate

(StageI-Step1)

C₉H₉O₄Na

204

52.9448

(52.9411)

4.4079

(4.4117)

4^l-bromobutyl-3, 4-dimethoxy benzoate

(StageI-Step2)

C₁₃H₁₇BrO₄

317

49.2148

(49.1135)

5.3595

(5.3627)

N-ethyl-n,n^l-methoxyphenyl isopropylamine (Stage II)

C₂₅H₃₅NO₅

193

74.6152

(74.6113)

9.8412

(9.8445)

7.2572

(7.2538)

3, 4-dimethoxybenzoic acid 4-[ethyl {2-(4-methoxyphenyl)-1-methylethyl} amino] butyl ester hydrochloride Drug substance

(Stage III)

105-107⁰C

C₂₅H₃₅NO₅.HCl

466

64.3739

(64.3776)

7.7215

(7.7253)

3.0008

(3.0042)

Table 2: Spectral analysis data of drug and Intermediates

Sr. No

Compound

Spectroscopic Technique

Analysis Results

Sodium 3, 4-dimethoxy benzoate

(StageI-Step1)

MS (m/z)/Intensity

(w. r. to base peak)

183 [M+1]⁺/100%(base peak), 165 [M-CH₃]⁺/ 5%

IR (KBr) (cm^-1)

-C=O stretching (1673), -O-CH₃ stretching (1466),

Benzene stretching (787)

¹H NMR

Solvent: CDCl₃

Chemical Shift ð 3.74 ppm (s, 6H, -CH₃),

6.91 ppm (d, 1H, Ar-H, j = 8.4),

7.41-7.36 ppm (q, 2H, Ar-H, j = 4.7)

HPLC Purity

99.66 %

4^l-bromobutyl-3, 4-dimethoxy benzoate

(StageI-Step2)

MS (m/z)/Intensity

(w. r. to base peak)

317 M⁺/ 99%, 182 [M-{(CH₂)₄Br}]⁺/ 18%, 165 [M-{O(CH₂)₄Br}]⁺/100% (base peak),

137 [M-{-CO-O-(CH₂)₄Br}]⁺/92%

IR (KBr) (cm^-1)

-CH₂ stretching (2958), -OCH₃ stretching (2838),

-C=O stretching (1704),

-C-O stretching (1133), Benzene stretching (764)

¹H NMR

Solvent: CDCl₃

Chemical Shift ð 2.05 ppm (m, 4H, -CH₂),

3.50 ppm (t, 2H, CH₂,j = 10.5), 3.93 ppm (s, 6H, CH₃),4.35 ppm (t, 2H, CH₂, j = 6.0), 6.89 ppm (d, 1H, Ar-H, j = 8.4), 7.53 ppm (d, 1H, Ar-H, j = 1.5), 7.68 ppm (q, 1H, Ar-H, j = 2.9),

HPLC Purity

97.07%

N-ethyl-n,n^l-methoxyphenyl isopropylamine

(Stage II)

MS (m/z)/Intensity

(w. r. to base peak)

194 [M + 1]⁺/100% (base peak) , 149 [M-{CH₃-CH₂-NH}]⁺/ 4%, 121 [M-{CH₃-CH₂}]⁺/ 3%

IR (KBr) (cm^-1)

-CH3 stretching (2962), -OCH3 stretching (2834),

-CH3 stretching (1464), -C-N stretching (1300),

-C-O stretching (1035), Benzene stretching (806)

¹H NMR

Solvent: CDCl₃

Chemical Shift ð 1.0 ppm (m, 6H, -CH₃),

2.87 ppm (m, 5H, -CH₂,-CH),

3.75 ppm (s, 3H, -CH₃), 6.85 ppm (d, 2H, Ar-H, j = 8.4), 7.10 ppm (d, 2H, Ar-H, j = 8.4)

HPLC Purity

98.59 %

Final drug molecule

(Stage III)

MS (m/z)/Intensity (w. r. to base peak)

308 [ M- {CH₂-(C₆H₄)-OCH₃}]⁺/ 100% (base peak)

165 [ M- {O-(CH₂)₄-N(C₂H₅)-CH₂(CH₃)-CH₂-(C₆H₄)-OCH₃}]⁺/ 22%

IR (KBr) (cm^-1)

-C-H stretching (2959.57-2840.22), -C=O stretching (1717.43),

-C=C stretching (1605, 1514, 1459),

Asymmetrical -C-O-C and --C-O stretching (1265-1130),

Symmetrical -C-O-C stretching (1023.04)

¹H NMR

Solvent: CDCl₃

Chemical Shift ð 2.54-1.10 ppm (m, 10H, -CH₃,-CH₂), 3.24-3.05 (m, 5H,-CH₂, -CH), 3.58 (t, 2H,-CH₂, j= 11.75), 3.77 (d, 3H, -CH₃, j = 3.0), 3.91 (d, 6H, -CH₃, j = 1.5), 4.37-4.36 (q, 2H, -CH₂, j = 7.16), 6.87-6.79 (m, 2H, Ar-H, j = 8.4), 7.14 (d, 1H, Ar-H, j = 11.0), 7.26 (s, 2H, Ar-H), 7.52 (d, 1H, Ar-H, j = 6.5), 7.86-7.65 (t, 1H, Ar-H, j = 8.25), 12.14 (s, 1H, -NH)

HPLC Purity

99.95 %

Figure 1: Reaction Scheme of proposed synthetic route for target compound

Table 3: In vitro activity of the drug on Gram positive and Gram negative bacteria

Sr. No.	Bacteria	Number tested	No. inhibited by Mebeverine Hydrochloride µg/ml
			5	10	25	50	100	200	>200
1	Bacillus spp.	7	2	2	2	1
2	Stephylococus aureus	89	2	9	31	22	15	3	7
3	Streptococcus spp.	4			1		1	1	1
4	Escherichia coli	47		1	1	3			42
5	Salmonella spp.	29		3	15	4	3	2	2
6	Shigella spp.	48		1	9	2	1	15	20
7	Klebsiella spp.	8						1	7
8	Arizona spp.	1			1
9	Providencia spp.	1			1
10	Proteus spp.	4							4
11	Pseudomonas spp.	10					1	1	8
12	Pasturella septica	1							1
13	Bordetella bronchiseptica	1			1
14	Hafnia spp.	1			1
15	Vibrio cholerae	133	3	7	56	27	29	10	1
16	Vibrio parahemolyticus	30		5	6	7	7	5
Total		414	7	28	125	66	57	38	93

Table 4: Reduction in cfu/ml of S. typhimurium NCTC 74 in organ homogenates of mice treated with drug substance.

Time of Sampling (h)	Group	Mouse No.	Drug/Mouse	Cfu/ml counts in
Time of Sampling (h)	Group	Mouse No.	Drug/Mouse	Heart blood	Liver	Spleen
2	I	3	Mebeverine Hydrochloride 60 µg	1.9 x10³ ^to3.0 x10^4*	1.3 x10³ ^to6.8 x10^4*	1.2 x10³ ^to2.7 x10^4*
2	II	3	Saline (Control)	4.2 x10⁵ ^to6.9 x10⁶	3.0 x10⁶ ^to9.1 x10⁶	1.3 x10⁵ ^to8.9 x10⁶
18	III	3	Mebeverine Hydrochloride 60 µg	1.0 x10³ ^to4.1 x10^4**	4.9 x10³ ^to6.9 x10^4**	3.1 x10³ ^to6.8 x10^5**
18	IV	3	Saline (Control)	5.8 x10⁸ ^to8.2 x10⁹	9.1 x10³ ^to7.1 x10⁹	2.5 x10⁸ ^to7.3 x10⁹

P*<0.05, **<0.01 compared to control group

The Toluene layer was washed further with Water (2x100 mL) and dried over anhydrous sodium sulfate. To the toluene layer IPA-HCl (72 gm, 20 %) was added till pH is acidic (2-2.5). The solid precipitated as hydrochloride salt was isolated by filtration as crude product (196.1 gm, 89 %) that was further purified by crystallization from methanol. Yield: 180.7 gm, 82%.

Pharmacology Test Protocol for evaluation of antimicrobial potential, toxicity and protective efficacy of the drug:

Determination of MIC of the drug: Both broth and agar dilution methods were used to determine the MIC of the drug with respect to different test bacteria^{15, 16}.For these methods, MEB HCl was added to each tube or plate at concentrations of 0 (control), 5, 10, 25, 50, 100 and 200 µg/ml. Since one solid agar medium containing the drug could be used for inoculation of a large number of bacteria at a time, this was done at least three times for every test bacterium and the results of agar dilution method only were presented.

Determination of bacteriostatic / bactericidal action of the drug¹⁷: Bacterial isolates sensitive to drug molecule were chosen, viz. Shigella dysenteriae 7 and Staphytococcus aureus NTTC 6571, 8530, 8531. The drug was added at a concentration higher than the respective MIC level (50µg/ml) at the logarithmic growth phase of the cultures and colony forming units/ml (cfu/ml) counts were determined at 2 hourly intervals up to 18 hours.

In vivo tests: Swiss male white mice weighing 18-20g were maintained at the animal house at standard conditions of temperature (21±1°C) and relative humidity (50-60%) with a photoperiod of 14:10h of light-darkness. Water and a dry pellet diet were given ad libitum. The virulence of the test strain Salmonella typhimurium NCTC 74 was exalted by repeated mouse passages and the median lethal dose (MLD or LD₅₀) of the passaged strain corresponding to 1.85x10⁹cfu/mouse suspended in 0.5 ml NB served as the challenge dose¹⁸ for the test groups of animals.

Toxicity of the drug: To determine the toxicity of the drug molecule, 10 mice were studied. Of these 5 were injected intraperitoneally 60 µg of the drug, and the rest received 30

µg doze. The animals were kept under observation up to 100 hours.

Protective efficacy of the drug: Of the two groups of 5 mice each (18-20g) group I was intraperitoneally administrated 30 µg of drug per mouse (0.1 ml from 300

µg/ml solution of drug), and group II was given 60 µg of the drug per mouse (0.1 ml from 600 µg/ml solution of drug). After 3 h, mice in both the groups were challenged with 50 MLD of S. typhimurium 74. A control group of 5 mice were also injected similarly with the same bacterial strain, and 0.1 ml sterile saline instead of drug. The protective capacity of the drug was determined by recording the mortality of the mice in different groups up to 100 hours.

In another experiment, 4 groups of three mice each were made. Animals in groups I and III received 60 µg of drug, while animals in groups II and IV received 0.1 ml sterile saline. After 3 h, all groups were given a 50 MLD challenge of S. typhimurium74. Two hours after the challenge, animals of group I and II were sacrificed. The heart blood was collected aseptically; livers and spleens were removed aseptically and homogenized in tissue homogenizers. The cfu/ml homogenate counts of the individual organs were determined separately. The same procedure was applied on groups III and IV, 18h after the challenge. The concentration of drug in mouse blood was assayed by measuring the diameter of the inhibition zones by serum soaked filter paper discs (6mm diameter, 3mm thick, Millipore, USA absorbing 0.03ml volume) on a lawn flooded with 10⁶ bacteria from an 18h broth culture of S. typhimurium 74 on peptone agar. The drug concentrations in serum samples were determined by referring these values to a standard calibration curve prepared with known concentrations of the drugs¹⁹.

Statistical analysis: The data were analyzed using Student’s ‘t’-test and Chi square test.

RESULTS:

Synthetic Process Development Study:

A simple three stage synthetic method was proposed for preparation of 3, 4-dimethoxybenzoic acid 4-[ethyl {2-(4-methoxyphenyl)-1-methylethyl} amino] butyl ester hydrochloride. The target drug substance was synthesized in the Laboratory using the proposed method and was obtained in 82.0 % yield. The product isolated as a white crystalline powder was established using different characterization and structure elucidation techniques like physical data, elemental analysis MS (m/z), ¹H NMR and FTIR presented in Table 2. The purity of the compound was confirmed by HPLC analysis stated in Table 1 and 2.

Pharmacology for evaluation of antimicrobial potential, toxicity and protective efficacy of the drug:

Bacterial inhibitory spectrum:

Of the 414 bacterial isolates tested, were inhibited by the drug at 5-25 µg/ml concentrations presented in Table 3. The staphylococci, vibrios and some enterbacteria like Arizona, Bordetella and Hafnia were sensitive to this drug; 79 of 89 isolates of S. Aureus and 122 of 133 isolates of V. cholerae were inhibited within 100µg/ml concentration. Bacilli and salmonellae were also found to be sensitive to Mebeverine Hydrochloride. Resistant isolates mostly belonged to Escherichia coli, Pseudomonas and Klebsiella spp.

Bacteriostatic and bactericidal action:

The MIC of Mebeverine Hydrochloride against S. dysenteriae 7 was found to be 25µg/ml. At the logarithmic growth phase of the culture, when the cfu/ml count of the strain was 5.5 x10⁸, 50 µg/ml of Mebeverine Hydrochloride was added. Subsequently, the cfu/ml counts of the culture were determined after 2, 4, and 6 and at the end of 18h. The counts were 1.0x10⁶, 2.0x10⁴ and 1.5x10⁴/ml respectively. The drug was bacteriostatic on some other Gram negative bacteria like Salmonella typhimurium 74 and Shigella boydii 8. However, Mebeverine Hydrochloride proved to be highly bactericidal when tested against S. aureus NCTC 6571, 8530, 8531.

In vivo toxicity:

No mortality was recorded in the two groups of mice injected with 30 and 60 µg Mebeverine Hydrochloride during the observation period of 100h.

In vivo protection:

Of the 5 mice in the control group, 4 (80%) died within 100h of the challenge while 60 and 20 percent mortality was recorded in the two test groups of mice that received 30 and 60 µg of Mebeverine Hydrochloride respectively. The difference in the mortality was found to be significant (p<0.001) between the drug treated and control groups. Mebeverine Hydrochloride significantly reduced the number of viable bacteria in heart blood, liver and spleen of mice in groups I and III both at 2 (p<0.05) and 18h (p<0.01) after challenge, compared with the control (saline treated) mice as presented in Table 4.

DISCUSSION:

The proposed novel synthetic process for drug substance is well established in the Laboratory to achieve the expected yield and quality of the product. The process was observed to be short, simple, robust, reproducible and with minimal by-product formation.

The synthesized drug substance is confirmed by characterization and structure elucidation techniques as described in the write up which supports the validity of proposed synthetic route.

The process can be further scaled up to pilot plant scale to evaluate commercial feasibility.

The test drug is a potential musculotropic antispasmodic drug without atropic side-effects whose major therapeutic role is in the treatment of irritable bowel syndrome. It is used to relieve cramps or spasms of the stomach and intestines (gut). It is also indicated for treatment of gastrointestinal spasm secondary to organic disorder and irritable bowel syndrom (IBS).

The test drug showed significant antibacterial potential against many Gram negative and Gram positive bacteria in the present evaluation study. The drug was found to be more active against generic pathogens. Though it was bacteriostatic against Gram negative bacteria, it was bactericidal against Gram positive strains. The animal experiments were undertaken to determine its relevance to the human therapeutic application. The results of in vivo experiments showed that the drug was non toxic and significantly protected the mice against the bacterial challenge.

Of the various classes of pharmacological agents the Phenothiazine, which contain tricyclic benzene rings, possess moderate to powerful antimicrobial action¹⁰. In the tested drug substance, the presence of two benzene rings and nitrogen in the secondary or tertiary state may be plying a key role in conferring antimicrobial activity to this compound. Since this drug already is in routine therapeutic usage, it has the potential of being developed as a second or even first line antibacterial agent in many infections, especially those of the gastrointestinal tract. Structural modifications and clinical or chemotherapeutic synergistic combinations of the drug with conventional antimicrobial and/or non-antibiotics could enhance the antibacterial activity of this drug.

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