Biotechnology Reports 5 (2015) 1–6C

Biotechnology Reports 5 (2015) 1–6


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Biotechnology Reports


journal homepage: www.elsevier.com/locate/btre




Acetylcholinesterase enzyme inhibitor activity of some novel pyrazinamide condensed 1,2,3,4-tetrahydropyrimidines

Karthikeyan Elumalaia,c,*, Mohammed Ashraf Alia, Manogaran Elumalaib, Kalpana Elurib, Sivaneswari Srinivasanc

a New Drug Discovery Research, Department of Medicinal Chemistry, Sunrise University, Alwar, Rajasthan 301030, India b Faculty of Pharmaceutical Sciences, UCSI University, Cheras, Kuala Lumpur 56000, Malaysia
c College of Pharmacy, Sree Vidyanikethan Educational Trust, Tirupati 517102, India




A R T I C L E I N F O

Article history: Received 31 July 2014
Received in revised form 19 October 2014 Accepted 20 October 2014
Available online 29 October 2014


Keywords: Pyrazinamide Tetrahydropyrimidines Biginelli reaction
Acetyl cholinesterase inhibitor



1. Introduction

A B S T R A C T

A new series of some novel pyrazinamide condensed 1,2,3,4-tetrahydropyrimidines was prepared by reacting of N-(3-oxobutanoyl)pyrazine-2-carboxamide with urea/thiourea and appropriate aldehyde in the presence of catalytic amount of laboratory made p-toluenesulfonic acid as an efficient catalyst. Confirmation of the chemical structure of the synthesized compounds (4a–l) was substantiated by TLC, different spectral data IR, 1H NMR, mass spectra and elemental analysis. The synthesized compounds were evaluated for acetyl and butyl cholinesterase (AChE and BuChE) inhibitor activity. The titled compounds exhibited weak, moderate or high AChE and BuChE inhibitor activity. Especially, compound
(4l) showed the best AChE and BuChE inhibitory activity of all the 1,2,3,4-tetrahydropyrimidine derivatives, with an IC50 value of 0.11mM and 3.4mM.

ã 2014 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://

creativecommons.org/licenses/by-nc-nd/3.0/).



tangles (NFT), and degeneration or atrophy of the basal forebrain

cholinergic neurons. The loss of basal forebrain cholinergic cells



Acetylcholine (ACH) acts as an excitatory neurotransmitter for voluntary muscles in the somatic nervous system and as a preganglionic and a postganglionic transmitter in the parasympa-thetic nervous system of vertebrates and invertebrates [1,2]. Acetyl cholinesterase (AChE) is a terminator enzyme of nerve impulse transmission at the cholinergic synapses by quick hydrolysis of ACH to choline and acetate. Inhibition of AChE evolves a strategy for the treatment of several diseases as Alzheimer’s disease (AD), senile dementia, ataxia, myasthenia gravis and Parkinson’s disease [3]. AD is one form of senile dementia, which occurs due to various neuropathological conditions such as senile plaques and neurofi-brillary tangles. It is themost common dementias that affect half of the population aged 85 years [4,5] and seventh main cause of life lost affecting 5.3 million people over the world. In AD, growing numbers of nerve cells degenerate and die along with loss in synapse throughwhich information flows fromand tothebrain.As a result, cognitive impairment and dementia occur [6]. The
neuropathologyof AD is generallycharacterized by the presence of numerous amyloidal b-peptide (Ab) plaques, neurofibrillary



* Corresponding author at: College of Pharmacy, Sree Vidyanikethan Educational Trust, Tirupati 517102, India. Tel.: [phone].
E-mail address: [e-mail] (K. Elumalai).

results in an important reduction in ACh level, which plays an important role in the cognitive impairment associated with AD [7]. Both cholinesterase enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are involved in the hydrolysis of acetylcholine; however, studies showed that as the disease progresses, the activity of AChE decreases while the activity of BChE remains unaffected or even increases [8]. In the brain of advancedstagedAD patients,BChE cancompensatefor AChEwhen the activity of AChE is inhibited by AChE inhibitors. Thus, BChE hydrolyses the already depleted levels of ACh in these patients. Furthermore, restoration of ACh levels by BChE inhibition seems to occur without apparent adverse effects [9,10]. It has been also proposed that individuals with low-activity of BChE can sustain cognitive functions better comparing two individuals with normal
BChE activity [11].

Pyrimidine derivatives comprise a diverse and interesting groupofdrugsisextremelyimportantfortheirbiologicalactivities. Dihydropyrimidine and their derivatives have attracted increasing interest owing to their therapeutic and pharmaceutical properties, such as antiviral, antitubercular [12,13], antimicrobial agent [14– 18] antagonists of the human adenosine A2A receptor [19], cyclooxygenase-2 inhibitory activity [20,21], tyrosine kinase inhibitors, antiamoebic activity [22,23], cytotoxicity [24,25] and
acetyl cholinesterase inhibitor activity [26]. The discovery during


http://dx.doi.org/10.1016/j.btre.2014.10.007

2215-017X/ã 2014 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

2 K. Elumalai et al./Biotechnology Reports 5 (2015) 1–6



the 1930s that a dihydropyridine (dihydronicotinamide derivative, NADH), “hydrogen-transferring coenzyme” consequently became importantinbiologicalsystem,hasgeneratednumerousstudieson the biochemical properties of dihydropyridines and their bio-isosteres dihydropyrimidines. The search for more suitable preparation of tetrahydropyrimidinones continues today.
The chemical structure of pyrazinamide provides a most valuable molecular template for the development of agents able to interact with a wide variety of biological activities [27]. Tetrahydropyrimidines are structurally similar to dihydropyrimi-dines. Hence, it was thought worthwhile to synthesize new congeners by incorporating pyrazinamide with 1,2,3,4-tetrahy-dropyrimidinones moieties in a single molecular frameworkand to evaluate their acetyl and butyl cholinesterase inhibitor activity.

2. Experimental

2.1. Materials and methods

All chemicals were supplied by E. Merck (Germany) and SD fine chemicals (India). Melting points were determined by the open tube capillary method and are uncorrected. The purity of the compounds was checked on thin layer chromatography (TLC) plates (silica–gel G) in the solvent system, ethanol, chloroform, ethylacetate(6:2:2);thespotswerelocatedunderiodinevaporsor UV light. IR spectrum was obtained on a PerkinElmer 1720 FT-IR spectrometer (KBr Pellet). 1H NMR spectra were recorded or a Bruker DRX-300 (300MHz FT-NMR) spectrometer using DMSO-d6 as solvent and TMS as internal standard. Mass spectra were obtained using Shimadzu LCMS 2010A under ESI ionization technique. Elemental analyses (C, H, and N) were performed on PerkinElmer model 240C analyzer.

2.2. Preparation of N-(3-oxobutanoyl)pyrazine-2-carboxamide (3)

Pyrazinamide 1 (0.01M) and ethyl acetoacetate 2 (0.01M) were mixed in presence 10ml of glacial acetic acid and refluxed for approximately 3.0h. The colorless liquid formed was then heated on a water bath to remove the alcohol formed during the reaction. After allowing the reaction mixture to cool, crude crystals were obtained. Purification was performed by stirring crude crystals with cold diethyl ether for approximately 20min using a mechanical stirrer. Allowing it to stand for 15min, followed by filtration, resulted in the third compound in a pure form of N-(3-oxobutanoyl)pyrazine-2-carboxamide 3.

2.2.1. General procedure

2.2.1.1. Preparation of 1,2,3,4-tetrahydropyrimidines by microwave

(C¼O, amide),1592 (C¼C),1343 (C—N); 1H NMR (DMSO-d6) d: 2.05 (s, 3H, CH3), 2.87 (s, 2H, CH2), 8.78 (s,1H, Ar—H), 8.93 (s,1H, Ar—H), 9.08 (s, 1H, Ar—H), 9.43 (s, 1H, NH); calculated for C9H9N3O3: C, 52.17; H, 4.38; N, 20.28; found C, 52.12; H, 4.52; N, 20.33.

2.3.2. 6-Methyl-2-oxo-4-phenyl-N-(pyrazin-2-ylcarbonyl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide (4a)
Dark-brownish solid, M.P.: 284–286_C; yield: 70%; IR (KBr,

cm_1): 3246 (N—H), 3152 (Ar—C—H), 2968 (Ali—C—H),1674 (C¼O, amide),1583 (C¼C),1248 (O—C); 1H NMR (DMSO-d6) d: 2.09 (s, 3H, CH3), 5.45 (s, 1H, CH), 7.12–7.23 (m, 5H, Ar—H), 8.78 (s, 1H, Ar—H), 8.93 (s, 1H, Ar—H), 9.08 (s, 1H, Ar—H), 9.41 (s, 1H, NH), 9.76 (s, 1H, NH), 10.11 (s, 1H, NH); MS (m/z): (M+1) calculated 338.12; found

338.07; calculated for C17H15N5O3: C, 60.53; H, 4.48; N, 20.76; found C, 60.48; H, 4.53; N, 20.82.

2.3.3. 6-Methyl-4-phenyl-N-(pyrazin-2-ylcarbonyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (4b)
Ash-colored solid, M.P.: 296–298_C; yield: 77%; IR (KBr, cm_1):

3253 (N—H), 3166 (Ar—C—H), 2948 (Ali—C—H),1677 (C¼O,amide), 1584 (C¼C),1888 (C¼S),1192 (O—C); 1H NMR (DMSO-d6) d: 2.06 (s, 3H, CH3), 5.38 (s, 1H, CH), 7.09–7.25 (m, 5H, Ar—H), 8.78 (s, 1H, Ar— H),8.93 (s,1H,Ar—H),9.08(s,1H,Ar—H),9.39 (s,1H,NH),9.82(s,1H, NH), 10.08 (s, 1H, NH); MS (m/z): (M+1) calculated 354.10; found

354.04. Calculated for C17H15N5O2S: C, 57.78; H, 4.28; N, 19.82; found C, 57.83; H, 4.22; N, 19.87.

2.3.4. 6-Methyl-4-(3-nitrophenyl)-2-oxo-N-(pyrazin-2-ylcarbonyl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide (4c)
Light-yellowish solid, M.P.: 313–315_C; yield: 76%; IR (KBr,

cm_1): 3276 (N—H), 3168 (Ar—C—H), 2984 (Ali—C—H),1678 (C¼O, amide),1558 (C¼C),1162 (O—C); 1H NMR (DMSO-d6) d: 2.07 (s, 3H, CH3),5.49 (s,1H, CH),7.39–7.43 (d,2H,Ar—H), 7.97–8.02(d, 2H, Ar— H), 8.78 (s,1H, Ar—H), 8.93 (s,1H, Ar—H), 9.08 (s,1H, Ar—H), 9.24 (s, 1H, NH), 9.68 (s, 1H, NH), 10.06 (s, 1H, NH); MS (m/z): (M+1)

calculated 383.10; found 383.15; calculated for C17H14N6O5: C, 53.40; H, 3.69; N, 21.98; found C, 53.44; H, 3.75; N, 21.94.

2.3.5. 6-Methyl-4-(3-nitrophenyl)-N-(pyrazin-2-ylcarbonyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (4d)
Light-bluish solid, M.P.: 357–359_C; yield: 71

Kết quả (Việt) 1:

Công nghệ sinh học báo cáo 5 (2015) 1-6Nội dung danh sách có sẵn tại ScienceDirectBáo cáo công nghệ sinh họctạp chí trang chủ: www.elsevier.com/ xác định vị trí/btreHoạt động của chất ức chế enzyme acetylcholinesterase của một số tiểu thuyết pyrazinamide ngưng tụ 1,2,3,4-tetrahydropyrimidinesĐức Vĩnh Elumalaia, c, *, Mohammed Ashraf Alia, Manogaran Elumalaib, Kalpana Elurib, Sivaneswari Srinivasancmột nghiên cứu phát hiện ra loại thuốc mới, các bộ phận của y học hóa học, đại học Sunrise, Alwar, Rajasthan 301030, Ấn Độ b khoa dược phẩm khoa học, đại học UCSI, Cheras, Kuala Lumpur 56000, MalaysiaSân bay Tirupati c College dược, sự tin tưởng giáo dục thanh hiếu Vidyanikethan, 517102, Ấn Độ R T TÔI C L E TÔI N F OBài viết lịch sử: nhận được 31 tháng 7 năm 2014Nhận được trong hình thức sửa đổi ngày 19 tháng 10 năm 2014 chấp nhận 20 tháng 10 năm 2014Có sẵn trực tuyến 29 tháng 10 năm 2014Từ khoá: Pyrazinamide Tetrahydropyrimidines Biginelli phản ứngAxetyl loại chất ức chế1. giới thiệu MỘT B S T R MỘT T CMột loạt mới của một số tiểu thuyết pyrazinamide ngưng tụ 1,2,3,4-tetrahydropyrimidines đã được chuẩn bị bởi phản ứng của N-(3-oxobutanoyl) pyrazine-2-carboxamide với urê/thiourea và thích hợp Anđêhít sự hiện diện của chất xúc tác số tiền của các phòng thí nghiệm thực hiện p-toluenesulfonic acid là một chất xúc tác efficient. Confirmation cấu trúc hóa học của các hợp chất tổng hợp (4a-l) được chứng minh bởi TLC, dữ liệu quang phổ khác nhau IR, 1giờ NMR, quang phổ khối lượng và phân tích nguyên tố. Các hợp chất tổng hợp đã được đánh giá cho axetyl và butyl loại (đau và BuChE) ức chế hoạt động. Các hợp chất có tiêu đề trưng bày yếu, Trung bình hoặc cao đau và BuChE chất ức chế hoạt động. Đặc biệt, hợp chất(4l) cho thấy tốt nhất đau và BuChE ức chế hoạt động của tất cả các dẫn xuất 1,2,3,4-tetrahydropyrimidine, với một giá trị IC50 0,11 mM và 3.4mM.ã 2014 xuất bản bởi Elsevier B.V Đây là một bài viết mở truy cập theo giấy phép CC BY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/3.0/).tangles (NFT), và thoái hóa hoặc teo não trước cơ sởCholin tế bào thần kinh. Sự mất mát của các tế bào não trước cơ sở Cholin Acetylcholine (ACH) acts as an excitatory neurotransmitter for voluntary muscles in the somatic nervous system and as a preganglionic and a postganglionic transmitter in the parasympa-thetic nervous system of vertebrates and invertebrates [1,2]. Acetyl cholinesterase (AChE) is a terminator enzyme of nerve impulse transmission at the cholinergic synapses by quick hydrolysis of ACH to choline and acetate. Inhibition of AChE evolves a strategy for the treatment of several diseases as Alzheimer’s disease (AD), senile dementia, ataxia, myasthenia gravis and Parkinson’s disease [3]. AD is one form of senile dementia, which occurs due to various neuropathological conditions such as senile plaques and neurofi-brillary tangles. It is themost common dementias that affect half of the population aged 85 years [4,5] and seventh main cause of life lost affecting 5.3 million people over the world. In AD, growing numbers of nerve cells degenerate and die along with loss in synapse throughwhich information flows fromand tothebrain.As a result, cognitive impairment and dementia occur [6]. Theneuropathologyof AD is generallycharacterized by the presence of numerous amyloidal b-peptide (Ab) plaques, neurofibrillary* Corresponding author at: College of Pharmacy, Sree Vidyanikethan Educational Trust, Tirupati 517102, India. Tel.: [phone].E-mail address: [e-mail] (K. Elumalai). results in an important reduction in ACh level, which plays an important role in the cognitive impairment associated with AD [7]. Both cholinesterase enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are involved in the hydrolysis of acetylcholine; however, studies showed that as the disease progresses, the activity of AChE decreases while the activity of BChE remains unaffected or even increases [8]. In the brain of advancedstagedAD patients,BChE cancompensatefor AChEwhen the activity of AChE is inhibited by AChE inhibitors. Thus, BChE hydrolyses the already depleted levels of ACh in these patients. Furthermore, restoration of ACh levels by BChE inhibition seems to occur without apparent adverse effects [9,10]. It has been also proposed that individuals with low-activity of BChE can sustain cognitive functions better comparing two individuals with normalBChE activity [11].Pyrimidine derivatives comprise a diverse and interesting groupofdrugsisextremelyimportantfortheirbiologicalactivities. Dihydropyrimidine and their derivatives have attracted increasing interest owing to their therapeutic and pharmaceutical properties, such as antiviral, antitubercular [12,13], antimicrobial agent [14– 18] antagonists of the human adenosine A2A receptor [19], cyclooxygenase-2 inhibitory activity [20,21], tyrosine kinase inhibitors, antiamoebic activity [22,23], cytotoxicity [24,25] andacetyl cholinesterase inhibitor activity [26]. The discovery during http://dx.doi.org/10.1016/j.btre.2014.10.0072215-017X/ã 2014 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). 2 K. Elumalai et al./Biotechnology Reports 5 (2015) 1–6 the 1930s that a dihydropyridine (dihydronicotinamide derivative, NADH), “hydrogen-transferring coenzyme” consequently became importantinbiologicalsystem,hasgeneratednumerousstudieson the biochemical properties of dihydropyridines and their bio-isosteres dihydropyrimidines. The search for more suitable preparation of tetrahydropyrimidinones continues today.The chemical structure of pyrazinamide provides a most valuable molecular template for the development of agents able to interact with a wide variety of biological activities [27]. Tetrahydropyrimidines are structurally similar to dihydropyrimi-dines. Hence, it was thought worthwhile to synthesize new congeners by incorporating pyrazinamide with 1,2,3,4-tetrahy-dropyrimidinones moieties in a single molecular frameworkand to evaluate their acetyl and butyl cholinesterase inhibitor activity.2. Experimental2.1. Materials and methodsAll chemicals were supplied by E. Merck (Germany) and SD fine chemicals (India). Melting points were determined by the open tube capillary method and are uncorrected. The purity of the compounds was checked on thin layer chromatography (TLC) plates (silica–gel G) in the solvent system, ethanol, chloroform, ethylacetate(6:2:2);thespotswerelocatedunderiodinevaporsor UV light. IR spectrum was obtained on a PerkinElmer 1720 FT-IR spectrometer (KBr Pellet). 1H NMR spectra were recorded or a Bruker DRX-300 (300MHz FT-NMR) spectrometer using DMSO-d6 as solvent and TMS as internal standard. Mass spectra were obtained using Shimadzu LCMS 2010A under ESI ionization technique. Elemental analyses (C, H, and N) were performed on PerkinElmer model 240C analyzer.
2.2. Preparation of N-(3-oxobutanoyl)pyrazine-2-carboxamide (3)

Pyrazinamide 1 (0.01M) and ethyl acetoacetate 2 (0.01M) were mixed in presence 10ml of glacial acetic acid and refluxed for approximately 3.0h. The colorless liquid formed was then heated on a water bath to remove the alcohol formed during the reaction. After allowing the reaction mixture to cool, crude crystals were obtained. Purification was performed by stirring crude crystals with cold diethyl ether for approximately 20min using a mechanical stirrer. Allowing it to stand for 15min, followed by filtration, resulted in the third compound in a pure form of N-(3-oxobutanoyl)pyrazine-2-carboxamide 3.

2.2.1. General procedure

2.2.1.1. Preparation of 1,2,3,4-tetrahydropyrimidines by microwave

(C¼O, amide),1592 (C¼C),1343 (C—N); 1H NMR (DMSO-d6) d: 2.05 (s, 3H, CH3), 2.87 (s, 2H, CH2), 8.78 (s,1H, Ar—H), 8.93 (s,1H, Ar—H), 9.08 (s, 1H, Ar—H), 9.43 (s, 1H, NH); calculated for C9H9N3O3: C, 52.17; H, 4.38; N, 20.28; found C, 52.12; H, 4.52; N, 20.33.

2.3.2. 6-Methyl-2-oxo-4-phenyl-N-(pyrazin-2-ylcarbonyl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide (4a)
Dark-brownish solid, M.P.: 284–286_C; yield: 70%; IR (KBr,

cm_1): 3246 (N—H), 3152 (Ar—C—H), 2968 (Ali—C—H),1674 (C¼O, amide),1583 (C¼C),1248 (O—C); 1H NMR (DMSO-d6) d: 2.09 (s, 3H, CH3), 5.45 (s, 1H, CH), 7.12–7.23 (m, 5H, Ar—H), 8.78 (s, 1H, Ar—H), 8.93 (s, 1H, Ar—H), 9.08 (s, 1H, Ar—H), 9.41 (s, 1H, NH), 9.76 (s, 1H, NH), 10.11 (s, 1H, NH); MS (m/z): (M+1) calculated 338.12; found

338.07; calculated for C17H15N5O3: C, 60.53; H, 4.48; N, 20.76; found C, 60.48; H, 4.53; N, 20.82.

2.3.3. 6-Methyl-4-phenyl-N-(pyrazin-2-ylcarbonyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (4b)
Ash-colored solid, M.P.: 296–298_C; yield: 77%; IR (KBr, cm_1):

3253 (N—H), 3166 (Ar—C—H), 2948 (Ali—C—H),1677 (C¼O,amide), 1584 (C¼C),1888 (C¼S),1192 (O—C); 1H NMR (DMSO-d6) d: 2.06 (s, 3H, CH3), 5.38 (s, 1H, CH), 7.09–7.25 (m, 5H, Ar—H), 8.78 (s, 1H, Ar— H),8.93 (s,1H,Ar—H),9.08(s,1H,Ar—H),9.39 (s,1H,NH),9.82(s,1H, NH), 10.08 (s, 1H, NH); MS (m/z): (M+1) calculated 354.10; found

354.04. Calculated for C17H15N5O2S: C, 57.78; H, 4.28; N, 19.82; found C, 57.83; H, 4.22; N, 19.87.

2.3.4. 6-Methyl-4-(3-nitrophenyl)-2-oxo-N-(pyrazin-2-ylcarbonyl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide (4c)
Light-yellowish solid, M.P.: 313–315_C; yield: 76%; IR (KBr,

cm_1): 3276 (N—H), 3168 (Ar—C—H), 2984 (Ali—C—H),1678 (C¼O, amide),1558 (C¼C),1162 (O—C); 1H NMR (DMSO-d6) d: 2.07 (s, 3H, CH3),5.49 (s,1H, CH),7.39–7.43 (d,2H,Ar—H), 7.97–8.02(d, 2H, Ar— H), 8.78 (s,1H, Ar—H), 8.93 (s,1H, Ar—H), 9.08 (s,1H, Ar—H), 9.24 (s, 1H, NH), 9.68 (s, 1H, NH), 10.06 (s, 1H, NH); MS (m/z): (M+1)

calculated 383.10; found 383.15; calculated for C17H14N6O5: C, 53.40; H, 3.69; N, 21.98; found C, 53.44; H, 3.75; N, 21.94.

2.3.5. 6-Methyl-4-(3-nitrophenyl)-N-(pyrazin-2-ylcarbonyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (4d)
Light-bluish solid, M.P.: 357–359_C; yield: 71