Synthesis, Characterization of 2-Methylthio-1, 4-dihydropyrimidines for its Antibacterial potential

 

Varsha I. Sarode1*, Ritesh P. Bhole1

Dr. D. Y. Patil Unitech Society’s, Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research,

Pimpri, Pune-411018

*Corresponding Author E-mail: varsha.isarode@gmail.com

 

ABSTRACT:

Some novel 2-methylthio-1, 4-dihydropyrimidine derivatives (II) have been prepared in good yields by alkylation of 1,2,3,4-tetrahydropyrimidines (I) with methyl iodide in the presence of pyridine. All the synthesized compounds were characterized by, IR and 1H NMR spectra, Mass spectra and by elemental analysis. The title compounds were screened for their antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Proteus vulgaris. Ampicillin was used as a standard drug for evaluating antibacterial activity. Compounds with chlorine (IIe, IIf), nitro (IIi) and 3, 4, 5-trimethoxy groups (IIj) at para position in the aryl moiety were found to have potent activity amongst other compounds. Other compounds shows moderate activity.

 

KEYWORDS: 2-Methylthio-1, 4-dihydropyrimidine, Biginelli reaction, synthesis, antibacterial activity.

 

 

INTRODUCTION:

In recent years acid-catalyzed cyclocondensation of acetoacetate with aldehydes and (thio)ureas, known as the Biginelli reaction, has attracted significant attention1-9. The resulting dihydropyrimidines (DHPMs) have been reported to have antibacterial10, antiviral11, anti-inflammatory12, analgesic13, antihypertensive as well as calcium channel blocker14,15, cardioprotective16 and antioxidant17.

 

An antimicrobial agent is anything that can kill or inhibit the growth of bacteria, such as high heat or radiation or a chemical. Antibacterial agents are used in relatively low concentrations to treat specific bacterial diseases without harming the host organism. Antimicrobial chemotherapy plays an important role in the treatment of many infectious diseases.

 

However, repeated use of some antibiotics results in resistance i.e. ineffectiveness of drug against the microorganisms.

 

The emergence of drug resistance against bacteria is posing a major problem in antimicrobial therapy18. This situation stimulated us to prepare new series of antimicrobial compounds with greater potency.19-21

 

Minimum inhibitory concentration (MIC) may be defined as the lowest concentration of antimicrobial agent required to inhibit the growth of the microorganism. MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against a microorganisms20.

 

MATERIAL AND METHODS:

Melting points were determined in open capillaries and are uncorrected. All compounds were characterized by elemental analysis, IR and 1H NMR spectra. The IR spectra were recorded on a JASCO FT-IR 4100 spectrometer, using KBr discs. The 1H NMR spectra were obtained on a Varian-NMR-mercury 300 spectrometer in DMSO-d6 as solvent and TMS as an internal standard, chemical shifts are given in ppm.

 

General Procedure for the synthesis of compounds (IIa-IIl):

A mixture of appropriate aldehyde (0.02 mol), acetoacetate (0.02 mol), thiourea (0.03 mol), catalyst aluminum chloride (0.01 mol) in methanol (10 mL) and concentrated hydrochoric acid (2 drops) was placed in the round bottom flask. The mixture was stirred well and then refluxed. The reaction was monitored by thin layer chromatography. After cooling, the precipitate was formed which was filtered and washed with cold methanol (I).

 

Compound I (0.01 mol), methyl iodide (0.011 mol) in methanol (20 mL) was placed in a round bottom flask and refluxed for 2 h. Pyridine (0.037 mol) was then added and refluxed again for 10 min After cooling, the reaction mixture was poured onto crushed ice (approx. 200 g) and stirred for 5 min. Compound II obtained was filtered.

 

Scheme 1: Synthesis of 2-methylthio-1, 4-dihydropyrimidines

 

 

Ethyl 6-methyl-2-(methylthio)-4-phenyl-1,4-dihydropyrimidine-5-carboxylate (IIa):

yield: 82.10 %; m.p. 160-162ºC;  IR-(KBr) cm-1: 3318.89 (NH), 1659.45 (C=O), 1573.63 (C=N); 1H NMR (300 MHz, DMSO): 14.18 (s, 1H, NH), 7.75-7.20 (m,  5H, C6H5), 5.988 (S, 1H, CH), 3.98 (q, 2H, OCH2CH3 ),  2.35 (s, 1H, S-CH3), 2.193 (s, 3H, CH3), 1.11 (t, 3H, OCH2CH3), EIMS (M+1): 290

 

Methyl 6-methyl-2-(methylthio)-4-phenyl-1,4-dihydropyrimidine-5-carboxylate (IIb):

yield: 67.51 %; m.p. 100-102ºC;  IR-(KBr) cm-1: 3316.96 (NH), 1708.62 (C=O), 1646.91 (C=N);  1H NMR (300 MHz, DMSO):  14.876 (s, 1H, NH), 7.751-7.201 (m,   5H, C6H5), 5.988 (s, 1H, CH), 3.71 (s, 3H, OCH3),  2.351 (s, 1H, S-CH3), 2.193 (s, 3H, CH3). EIMS (M+1): 276

 

 

Ethyl 4-(4-methoxyphenyl)-6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIc):

yield: 78.94 %; m.p. 116-117ºC; IR-(KBr) cm-1: 3318.89 (NH), 1649.8 (C=0), 1150.33(C-O); 1H NMR (300 MHz, DMSO): 14.876 (s, 1H, NH), 7.68-7.069 (m, 4H, C6H4), 5.988 (s, 1H, CH), 3.98 (q, 2H, OCH2CH3), 3.605 (s, 3H, OCH3), 2.351 (s, 3H, S-CH3), 2.193 (s, 3H, CH3), 1.11 (t, 3H, OCH2CH3). EIMS (M+1): 320

 

Methyl 4-(4-methoxyphenyl)-6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IId):

yield: 73.68 %; m.p. 146-147ºC; IR-(KBr) cm-1: 3318.89 (NH), 1654.62 (C=O), 1249.65 (C-O); 1H NMR (300 MHz, DMSO):14.87 (s, 1H, NH), 7.68-7.069 (m, 4H, C6H4), 5.988 (s, 1H, CH), 3.708 (s, 3H, OCH3), 3.605 (s, 3H, OCH3), 2.351 (s, 3H, S-CH3), 2.193 (s, 3H, CH3). EIMS (M+1): 306

 

Ethyl 4-(4-chlorophenyl)-6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIe):

yield: 92.98 %; m.p. 138-139ºC; IR-(KBr) cm-1:  3341.07 (NH), 1674.87 (C=O), 1574.59 (C=N), 745.35 (C-Cl); 1H NMR (300 MHz, DMSO): 14.876 (s, 1H, NH), 7.778-7.396 (m, 4H, C6H4), 5.988 (s, 1H, CH), 3.98 (q, 2H, OCH2CH3), 2.351 (s, 3H, S-CH3), 2.193 (s, 3H, CH3), 1.11 (t, 3H, OCH2CH3). EIMS (M+1): 324

 

Methyl 4-(4-chlorophenyl)-6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIf):

yield: 57.89 %; m.p. 130-132ºC; IR-(KBr) cm-1:  3308.29 (NH), 1659.45 (C=O), 1561.09 (C=N), 782.95 (C-Cl); 1H NMR (300 MHz, DMSO):14.876 (s, 1H, NH), 7.778-7.396 (m, 4H, C6H4), 5.988 (s, 1H, CH), 3.708 (s, 3H, OCH3), 2.351 (s, 3H, S-CH3), 2.193 (s, 3H, CH3). EIMS (M+1): 310

 

Ethyl 4-[4-(dimethylamino) phenyl]-6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIg):

yield: 62.93 %; m.p.124-126ºC; IR-(KBr) cm-1 3334.32 (NH), 1654.62 (C=O), 1514.81 (C=N); 1H NMR (300 MHz, DMSO):14.876 (s, 1H, NH), 7.532-6.577 (m, 4H, C6H4),  5.988 (s, 1H, CH), 3.98 (q, 2H, OCH2CH3), 2.831 (s, 6H, N(CH3)2), 2.351 (s, 3H, S-CH3), 2.193 (s, 3H, CH3), 1.11 (t, 3H, OCH2CH3),. EIMS (M+1): 333

 

Methyl 4-[4-(dimethylamino) phenyl]-6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIh):

yield: 75.75 %;  m.p. 214-216ºC; IR-(KBr) cm-1: 3327.57 (NH), 1668.12 (C=O), 1555.31 (C=N); 1H NMR (300 MHz, DMSO): 14.876 (s, 1H, NH), 7.532-6.577 (m, 4H, C6H4), 5.988 (s, 1H, CH),  3.708(s, 3H, OCH3), 2.831 (s, 6H, N(CH3) 2), 2.351 (s, 3H, S-CH3), 2.193 (s, 3H, CH3),. EIMS (M+1): 319

 

Ethyl 6-methyl-2-(methylthio)-4-(4-nitrophenyl)-1,4-dihydropyrimidine-5-carboxylate (IIi):

yield: 83.68 %; m.p.180-182ºC; IR-(KBr) cm-1: 3235.97 (NH), 1693.19 (C=O), 1528.31 (Asy Ar-NO2), 1349 (Sym Ar-NO2), 855.27 (C-N); 1H NMR (300 MHz, DMSO): 14.876 (s, 1H, NH), 7.89-8.10 (m, 4H, C6H4), 5.988 (s, 1H, CH),  3.98 (q, 2H, OCH2CH3), 2.351 (s, 3H, S-CH3), 2.193 (s, 3H, CH3), 1.11 (t, 3H, OCH2CH3). EIMS (M+1): 335

 

Ethyl 6-methyl-2-(methylthio)-4-(3,4,5-trimethoxyphenyl)-1,4-dihydropyrimidine-5-carboxylate (IIj):

yield: 75.14 %; m.p.206-208ºC; IR-(KBr) cm-1: 3288.04 (NH), 1669.09 (C=O), 1573.63 (C=N),1184, 1123 (C-O); 1H NMR (300 MHz, DMSO): 14.87 (s, 1H, NH), 5.874 (s, 1H, CH), 3.98 (q, 2H, OCH2CH3), 3.83 (s,  9H, OCH3), 2.351 (s, 3H, S-CH3), 2.193 (s, 3H, CH3), 1.11 (t, 3H, OCH2CH3). EIMS (M+1): 380

 

Ethyl 6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIk):

yield: 65.47 %; m.p. 210-211ºC; IR-(KBr) cm-1: 3209.93 (NH), 1659.45 (C=O), 1501.31 (C=N); 1H NMR (300 MHz, DMSO): 15.179 (s, 1H, NH), 4.723 (S, 2H, CH2), 4.089 (q, 2H, OCH2CH3), 2.402 (s, 3H, S-CH3), 2.173 (s, 3H, CH3),  1.222 (t, 3H, OCH2CH3). EIMS (M+1): 214

 

Methyl 6-methyl-2- (methylthio)-1, 4-dihydropyrimidine-5-carboxylate (IIl):

yield: 75.14 % ; m.p. 198-200ºC; IR-KBr (cm-1): 3318.07 (NH), 1668.12 (C=O), 1594.84 (C=N); 1H NMR (300 MHz, DMSO): 15.17 (s, 1H, NH), 4.723 (S, 2H, CH2), 3.705 (s, 3H, OCH3), 2.402 (s, 3H, S-CH3), 2.173 (s, 3H, CH3). EIMS (M+1): 200

 

Antibacterial Activity:

Procedure:

1)       Into test tube rack, place a one set of 8 sterile 13×100 mm test tubes labeled 1 through 8. Label one rack set I- Control (blank), Gram positive Set II- Staphylococcus aureus, Set III- Bacillus Subtilis and gram negative Set IV-Escherichia coli, Set V-Proteus vulgaris.

 

Table 1.1 Microorganisms used for antibacterial activity

Sr. No.

Standard  Micro-organism

NCIM Code No.

Gram + Ve

1

Staphylococcus aureus

2079

2

Bacillus Subtilis

2063

Gram – Ve

3

Escherichia coli

2089

4

Proteus vulgaris

2027

 

2)    Using a sterile 1 mL glass syringe, add 0.8 mL of DMSO in labeled 1 and 0.5 mL of DMSO labeled 2 through labeled 8 in Set I-V.

3)    With a sterile 1 mL glass syringe, add 0.2 mL test compounds solution in labeled 1 test tube and mix it.

4)    Using a sterile 1 mL glass syringe, transfer test compounds solution 0.5 mL from tube 1 to tube 2. Mix well and transfer 0.5 mL from tube 2 to tube 3. Continue this procedure through tube 8. Discard 0.5 mL from test tube 8.

5)    Using a sterile 5 mL glass syringe, add 4.4 mL of nutrient broth labeled 1 to labeled 8 in set I-V.

6)    Using a sterile 1 mL glass syringe, add 0.1 mL of the 1:1000 dilution of the microorganism in set I-V.

7)    At the same time, two control tubes were prepared.

a)     One containing the inoculated media control.

b)    One containing the inoculated culture media control.

8)    Incubate all sets of the tube for 42-48 h at 370C.

9)    The growth of the test organism was measured by determining the extinction at 600 nm of each of the solution in the tube against the blank (Set I).

 

RESULTS AND DISCUSSION:

The 1,2,3,4-tetrahydropyrimidine-2-thione derivatives were prepared by Biginelli three-component reaction of appropriate aldehyde, acetoacetate, and thiourea. C-2 modified dihydropyrimidine derivatives of type II was synthesized by alkylation reaction. Thus 2-methylthio-1, 4-dihydropyrimidine derivatives were synthesized from the respective tetrahydropyrimidine-2-thiones by reaction with methyl iodide in the presence of pyridine (Scheme 1).

 

The purity of the compounds was checked by thin layer chromatography. All the synthesized compounds were characterized by elemental analysis, IR and 1H NMR spectra. In the IR spectra, compounds (IIa-IIl) displayed a strong absorption band for NH at 3200-3350 cm-1.

 

Table1.2 Test compounds serial dilution-broth medium setup

Test Tube Number

Additions (mL) to:

1

2

3

4

5

6

7

8

Media Control

Culture Control

DMSO

0.8

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

Compound

0.2

Serial Dilutions

-

-

Medium

4.4

4.4

4.4

4.4

4.4

4.4

4.4

4.4

4.4

4.4

Test Culture

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

-

0.1

Compound (µg/mL)

500

250

125

62.5

31.25

15.625

7.8125

3.9

-

-

 

 

 

The ester carbonyl stretching frequency was observed in the range of 1600-1700 cm-1. Absorption band for C=N was observed in the range of 1500-1600 cm-1. The presence of the nitro group in compound IIi can be identified from the two intense absorption bands at 1528.31 and 1349.93 cm−1 for asymmetric and symmetric stretching vibrations, respectively. These stretching vibrations are due to highly polar nitrogen–oxygen bonds.

 

All the title compounds were screened for their antibacterial activity against Staphylococcus aureus, Bacillus Subtilis, Escherichia coli, and Proteus vulgaris. For evaluating antibacterial activity Ampicillin was used as standard drug. The observed minimum inhibitory concentrations (MIC) are given in Table 1.3 & figure 1.1.

 

Table 1.3. Antibacterial activity of title compounds (IIa-IIl)

Compound

Minimum inhibitory concentration (µg/ml)

Staphylococcus aureus

Bacillus Subtilis

Escherichia coli

Proteus vulgaris

IIa

125

62.5

62.5

31.2

IIb

62.5

125

125

62.5

IIc

125

62.5

250

125

IId

31.2

125

62.5

62.5

IIe

125

15.62

31.2

31.2

IIf

250

62.5

31.2

15.62

IIg

125

31.2

31.2

62.5

IIh

62.5

62.5

125

62.5

IIi

31.2

15.62

62.5

31.2

IIj

15.62

31.2

31.2

62.5

IIk

125

250

125

62.5

IIl

125

62.5

250

125

Ampicillin

25

12.5

12.5

25

 

Figure 1.1. Graphical representation of the antibacterial activity of title compounds (IIa-IIl).

 

Compounds with chlorine (IIe, IIf), nitro (IIi) and 3, 4, 5-trimethoxy group (IIj) at para position in the aryl moiety are the most potent compounds. Compounds with no aryl moiety at C-4 position are poor acting drugs i.e. compound IIk and IIl.  The electron withdrawing groups may impart its lipophilicity and results in more penetration inside the bacterial cell wall.

 

Compound IIj were found to possess potent activity against Staphylococcus aureus with MIC value 15.62 μg/mL, compounds IIe and IIi were found to possess potent activity against Bacillus subtilis with MIC value 15.62 μg/mL, compounds IIe, IIf, IIg and IIj have good activity against Escherichia coli with MIC value 31.2 μg/mL and compound IIf were found to possess potent activity against Proteus vulgaris with MIC value 15.62 μg/mL.

 

CONCLUSION:

Various 2-methylthio-1, 4-dihydropyrimidines are synthesized in reasonable yield by a simple, efficient and one-pot reaction. The result suggests that the antibacterial activities are markedly influenced by the aromatic substituent. Thus compounds IIe, IIf, IIi, and IIj with electron withdrawing substituents in the aromatic ring, show greater antibacterial activity. This could be taken as a lead compound for the development of some new heterocyclic derivatives for antimicrobial activity. Moreover, these compounds may be further exploited for other pharmacological activities.

 

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Received on 13.12.2018         Modified on 31.12.2018

Accepted on 21.01.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(4):1585-1589.

DOI: 10.5958/0974-360X.2019.00263.4