Synthesis and biological evaluation of novel N3-substituted dihydropyrimidine derivatives as T-type calcium channel blockers and their efficacy as analgesics in mouse models of inflammatory pain

Accepted Manuscript Synthesis and Biological Evaluation of Novel N3-substituted Dihydropyrimidine Derivatives as T-Type Calcium Channel Blockers and Their Efficacy as Analgesics in Mouse Models of Inflammatory Pain Mohamed Teleb, Fang-Xiong Zhang, Junting Huang, Vinicius M Gadotti, Ahmed M. Farghaly, Omaima M. AboulWafa, Gerald W. Zamponi, Hesham Fahmy PII: DOI: Reference:

S0968-0896(16)31121-X http://dx.doi.org/10.1016/j.bmc.2017.02.015 BMC 13543

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

3 November 2016 2 February 2017 8 February 2017

Please cite this article as: Teleb, M., Zhang, F-X., Huang, J., Gadotti, V.M., Farghaly, A.M., AboulWafa, O.M., Zamponi, G.W., Fahmy, H., Synthesis and Biological Evaluation of Novel N3-substituted Dihydropyrimidine Derivatives as T-Type Calcium Channel Blockers and Their Efficacy as Analgesics in Mouse Models of Inflammatory Pain, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.02.015

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Synthesis and Biological Evaluation of Novel N3-substituted Dihydropyrimidine Derivatives as T-Type Calcium Channel Blockers and Their Efficacy as Analgesics in Mouse Models of Inflammatory Pain Mohamed Teleb a, b, Fang-Xiong Zhang c, Junting Huang c, Vinicius M Gadotti c, Ahmed M. Farghaly b, Omaima M. AboulWafa b, Gerald W. Zamponi c and Hesham Fahmy a, * a

Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, SD 57007, USA b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt c Department of Physiology & Pharmacology, Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Drive NW, Calgary T2N 4N1, Canada

ABSTRACT Low-voltage-activated calcium channels are important regulators of neurotransmission and membrane ion conductance. A plethora of intracellular events rely on their modulation. Accordingly, they are implicated in many disorders including epilepsy, Parkinson's disease, pain and other neurological diseases. Among different subfamilies, T-type calcium channels, and in particular the CaV3.2 isoform, were shown to be involved in nociceptive neurotransmission. The role of CaV3.2 in pain modulation was supported by demonstrating selective antisense oligonucleotide-mediated CaV3.2 knockdown, in vivo antinociceptive effects of T-type blockers, and pain attenuation in CaV3.2 knockout formalin-induced pain model. These Emerging investigations have provided new insights into targeting T-type calcium channels for pain management. Within this scope, various T-type calcium channel blockers have been developed such as mibefradil and ethosuximide. Although being active, most of these molecules interact with other receptors as well. This addresses the need for T- selectivity. Few selective T-type channel blockers of diverse chemical classes were developed such as ABT-639 and TTA-P2. Interestingly, R(-) efonidipine which is a dihydropyridine (DHP) showed T-channel selectivity. Systematic modification of 1,4-dihydropyridine scaffold introduced novel derivatives with 40 fold T-type selectivity over L-type calcium channels. Along these lines, substitution of the DHP core with various analogues favored T-selectivity and may serve as novel pharmacophores. Several dihydropyrimidine (DHPM) mimics were introduced by Squibb as potential candidates. As a continuation of this approach, the current study describes the synthesis of Novel N3 substituted DHPMs with structure similarities to the active DHPs. Different functional groups were introduced to the N3 position through a spacer to gain more information about activity and selectivity. Furthermore, the spacer aims at improving the metabolic stability of the molecules. Initial screening data by whole patch clamp technique showed a robust inhibition of Cav3.2 T-type channels by eleven compounds. Interestingly, four compounds of these were efficient selective T-type blockers. Based on selectivity and efficiency, two compounds were selected for in vivo evaluation in mouse models of inflammatory pain. Results showed effective attenuation of nociception and mechanical hypersensitivity. ∗

Corresponding author: E-mail: [email protected] (Hesham Fahmy) 1

Keywords: Pain, Calcium channels; T-type calcium channel blockers; Whole patch clamp technique; Dihydropyrimidines; 1,4-Dihydropyridines. 1. Introduction. The mechanisms of pain have been subject to extensive investigation. However, only few novel analgesic classes have been introduced to the clinic. The drug discovery sector is thus focusing on identifying new targets and pharmacophores for pain management. Within this approach, designing specific channel and receptor modulators is currently in progress. Over the last few years, a number of calcium channel blockers with varying channel isoform selectivity have been reported to show robust antinociception effects in different pain models 1,2. Among different calcium channel subfamilies, T-type calcium channels are well recognized as important pain transmission regulators 3-6. These are classified into three isoforms: Cav3.1, Cav3.2 and Cav3.3 7,8. Cav3.2 is the predominant isoform expressed in spinal cord and primary sensory afferent neurons 9. Studies showed an increased level of Cav3.2 channel expression in diabetic neuropathic animal models 10 and neuropathic pain models following sciatic nerve injury 11. Further evidence of T-type Cav3.2 channel implication in nociceptive neurotransmission was supported by reduced acute nociceptive sensitivity to noxious mechanical, thermal, and chemical stimuli in transgenic mice lacking Cav3.2 channels compared to wild-type mice 12. Other studies showed that intrathecal delivery of a specific Cav3.2 antisense oligonucleotide produces a significant robust antihyperalgesic effects in neuropathic pain mode 13. Along these lines, there are considerable efforts to identify and develop T-type calcium channel blockers. Several molecules have been shown to target Ttype calcium channel such as mibefradil and ethosuximide (Fig. 1) but also interact with other ion channels or receptors 14,15. Reviewing literature for selective T-blockers revealed some chemically diverse molecules such as ABT-639 2, TTA-P2 16 and interestingly R(−) efonidipine 17 (Fig. 1) which belongs to the class of dihydropyridines (DHPs). Although DHPs are best known to be selective inhibitors of L-type calcium channels, a number of reports showed that T-type calcium channels may also be sensitive to some DHPs 18,19. Within this context, some structure activity relationship studies were carried out to gain more information about T-type over L-type selectivity. The DHP scaffold was modified at almost every position. Modification of ester groups at the C3-and C5- positions of the typical DHP scaffold have been shown to be important for modulating tissue selectivity 20-22. Aromatic ring substitutions also allowed promising T-selectivity of some derivatives 19. Studies were extended to evaluate the effect of modifying the DHP core itself. Various analogues were introduced such as hexahydroquinoline (condensed DHP ring system) (Fig. 1) which conferred 30-40 fold selectivity for T- over L-type 23,24. Interestingly, BristolMyers Squibb Co. synthesized various N3-substitued Dihydropyrimidines (DHPMs) as aza-analogues of DHPs (Fig. 1) 25. Unlike DHPs which face metabolic inactivation by aromatization 26, DHPMs mimics are metabolically stable owing to their structural characteristics, particularly the presence of N3-substituents 27 . However, some potent DHPMs showed only in vitro activity due to first pass effect through hydrolysis and removal of the N3-substitutions 28. In this paper, a newly synthesized series of N3-substituted DHPMs was designed as T-type calcium channel blockers. Lead compounds showed considerable T-type selectivity. The Basic scaffold of the novel compounds was designed to mimic the structural requirements for calcium channel blocking activity in clinically used DHPs and active DHPM CCBs 29 (Fig. 1). N3-substitutions were studied as a function of activity and selectivity. These were designed to include various functionalities reported as 2

pharmacophoric framework of agents with potent antinociceptive activities such as acyl hydrazones 30-34, arylsulfonylhydrazides 35, semicarbazides 36 and their thio-isosters. The study was extended to include different acyl hydrazides and structurally related esters. Heterocycles that were reported to confer antiinflammatory activities to different pharmacophores were also introduced to the DHPM core. These are substituted oxadiazoles 37, their thio-isosteres; thiadiazoles and thiazolones 38. The designed functionalities were introduced to the N3 of the DHPM core via two-carbon spacer. The introduction of this linker was designed to avoid common metabolic inactivation mechanisms of both DHPs and DHPMs. Representative derivatives were evaluated as antagonists for Cav3.2 and Cav1.2 by applying the wholecell patch clamp technique in an attempt to identify the most active and selective compounds. Following the initial screening, the promising T-type calcium channel blockers were further assessed for in vivo evaluation in different mouse models of nociception and hypersensitivity.

Fig. 1. T-Type calcium channel blockers and designed target compounds 3

2. Results and discussion 2.1. Chemistry The general synthetic schemes for synthesis of the target compounds are described in Schemes 1 and 2. The N3-substituted 3,4-dihydropyrimidinone (1) was synthesized according to aza-Michael addition reactions catalyzed by KF/Al2O3 39, from the parent DHPM; Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo1,2,3,4-tetrahydropyrimidine-5-carboxylate 40. Nucleophilic substitution of the ethyl ester (1) with hydrazine hydrate afforded the key intermediate hydrazide (2) 41. Acetylation of Hydrazide (2) by refluxing in acetic anhydride gave the tri-acetylated product (3) 42. However, stirring a solution of hydrazide (2) in pyridine with 1.2 equivalent acetylchloride at room temperature afforded the monoacetylated derivative (4a) 43. The carbamate derivatives (4b & 4c) were obtained utilizing 1.2 equivalent of the appropriate alkyl chloroformate under similar conditions. Direct sulfonylation of hydrazide (2) with different substituted sulfonyl chloride derivatives in pyridine afforded the corresponding aryl and aralkylsulfono-hydrazides (5a & 5b) 44. Hydrazones (6a-l) were readily synthesized in high yields and purity by condensing hydrazide (2) with the appropriate aldehydes, ketones 45, isatins 42 or β-ketoester 46 in boiling alcohol. The utilized carbonyl precursors include various aryl and aralkyl aldehydes and ketones, substituted isatins and representative β-ketoester; ethyl acetoacetate. Interestingly, the open chain condensation product with ethyl acetoacetate was isolated without subsequent cyclization, while condensing hydrazide (2) with acetyl acetone under similar conditions afforded the cyclized pyrazole derivative (7) 47. Moreover, classical Michael addition with α,β-unsaturated systems provided as easy route for hydrazide (2) alkylation 48. Unexpectedly, hydrazide (2) was dialkylated to the N’,N’dialkoxycarbonylethyl derivatives (8a & 8b) utilizing the appropriate alkyl acrylate under basic catalysis. Heating the hydrazide (2) with different isocyanates and isothiocyanates in ethanol at reflux temerature furnished the corresponding semicarbazide and thiosemicarbazide derivatives (9a-e) (Scheme2). These were utilized as starting compounds for further cyclization reactions. The acid catalyzed dehydrative intramolecular cyclization of thiosemicarbazide (9a) afforded thiadiazole derivative (10) 49. Desulphurization of thiosemicarbazide (9a & 9b) by reaction with yellow mercuric oxide in boiling ethanol yielded the corresponding oxadiazole derivatives (11a & 11b) 50. Cyclization of thiosemicarbazides (9a & 9b) into the corresponding thiazolines (12a-d) was achieved by reaction with the appropriate 2-bromoacetophenone derivatives 51. Moreover, treatment of thiosemicarbazide precursors (9a & 9b) with ethyl bromoacetate furnished the corresponding thiazolidinone derivatives (13a & 13b) 50. Structures of synthesized compounds were elucidated using 1H NMR, 13C NMR, IR, elemental analysis and High resolution mass spectroscopy.

4

Scheme 1. Synthesis of the target compounds (2-8)

5

NO2 O

O

O N H

NH2

N H

N O

2 RNCX

NO2

S

N

O N H

O

H2SO4

N N

O

NO2

NO2

O

N H

N

N H

H N

N O H 9(a-e)

O

10

NO2

O

N

Br

O

R N

O N

O

R

N H

X

N H

N

O

S

13a,b 13a: R= C6H5 13b: R= p-CH3C6H4

O Br

R2 NO2

N N

O

H N

9a: X=S, R= C6H5 9b: X=S, R= p-CH3C6H4 9c: X=S, R= CH(C6H5)2 9d: X=S, R= CH2CH=CH2 9e: X=O, R= C6H5

HgO

O

O

O

O

N H

R

O

R1 N

O N

O

N O H 11a,b

N

N H

R2

S

N O H 12(a-d)

11a: R= C6H5 11b: R= p-CH3C6H4

12a: R1= 12b: R1= 12c: R 1= 12d: R1=

C6H5 , R2= F C6H5 , R2= NO2 p-CH3C6H4, R2= F p-CH3C6H4, R2= NO2

Scheme 2. Synthesis of the target compounds (9-13) 2.2. Whole patch clamp assay Representative compounds from different series were selected for evaluation by whole-cell patch clamp recording assay with Cav1.2 and Cav3.2 channels (Fig. 2). 2.2.1. Cav1.2 Antagonism (L-type Block) L-type currents from tsA-201 cells transiently transfected with rat Cav1.2 and ancillary calcium channel subunit cDNA were recorded. Data were represented as percent inhibition of the current after applying test compound at a 10 µM concentration to measure the tonic (resting state) block. Fig. 2a shows a robust current inhibition (60-85%) of Cav1.2 by compounds 6(a-f) & 6h and a moderate inhibition (around 50%) by compounds 5a and 6j. Other derivatives were less active. 2.2.1. Cav3.2 Antagonism (T-type Block) Fig. 2b shows data recorded from similar experiments utilizing cells transfected with the Cav3.2 α1 subunit. Compounds 6(a-f), 6h, 9c, 10, 11a and 13b showed efficient Cav3.2 block (70-95%) with most of them showing almost complete T-type current inhibition. Moderate inhibition (around 50%) was recorded by compounds 5a and 6j, while the remainder of test compounds did not show considerable activity. 6

O

Thus, compounds 6(a-f) and 6h were found to be effective blockers of both Cav1.2 and Cav3.2 channels with compounds 6(d-f) showing a slightly higher degree of Cav3.2 block. Interestingly, compounds 9c, 10, 11a and 13a preferentially showed high affinity Cav3.2 block suggesting selective antagonism of Cav3.2 over Cav1.2. Furthermore, it is notable that compound 9c conferred the best T-type selectivity.

Fig. 2 (a) Tonic block of rat Cav1.2 (L-type) induced by a 10 micromolar application of test compounds (n = 4-5 per channel). (b) Tonic block of human Cav3.2 (T-type) with the same compounds (n = 4-5 per channel, at 10 µM). Error bars reflect standard errors Next we conducted a more detailed analysis of the blocking properties of the most efficient (compound 6a) and most selective (compound 9c) T-type channel blockers on transiently expressed Cav3.2 channels. Fig. 3a depicts the dose response relation for block of Cav3.2 by compound 6a along with a representative current trace obtained before and after application of the compound. The IC50 obtained from the fit to the dose response curve was 3.39 ± 0.10 µM. Figs. 3b and 3c illustrate the effects of this compound on the current-voltage and steady state inactivation curves, respectively. At a concentration of 3µM, compound 6a inhibited Cav3.2 currents at all test potentials, but did not affect half-activation or halfinactivation potentials. Compound 9c inhibited Cav3.2 channels with ten-fold greater potency (IC50=0.34 ± 0.10 µM, Fig. 3d), however block appeared to saturate at 80% inhibition. There was no effect on the half-activation potential (Fig. 3e). This compound did produce a ~5 mV hyperpolarizing shift in the halfinactivation potential (Fig. 3f), which is indicative of inactivation-state dependent block and suggest the possibility of use-dependent action. The time course of inhibition by both compounds was slow, requiring 8-10 minutes to reach steady state when applied at 3 and 1µM respectively. The blocking effects of compounds 6a and 9c were tested on transiently expressed the Cav2.2 using whole-cell patch clamp recording. At a concentration of 10 µM, compound 6a blocked Cav2.2 by 31.90 ± 2.39% and compound 9c blocked Cav2.2 by 7.64 ± 3.18%, (n = 3 for each compound), altogether indicating a poor ability to block N-type calcium channels. 7

Fig. 3(a) Dose–response relation for compound 6a inhibition of Cav3.2. The IC50 obtained from the fit curve was 3.39 ± 0.10 µM (n = 3 per dose). The inset shows a representative T-type current in the presence and the absence of 3 µM of compound 6a. (b) Effects of compound 6a on the current–voltage relation of Cav3.2. The half-activation potentials obtained from the fitted curves were -32.07 ± 0.80 mV and -29.29 ± 0.94 mV before and after the application of compound 6a, respectively (3 µM, n = 3), p = 0.56 (paired t test). (c) Steady-state inactivation curve for Cav3.2 before and after application of compound 6a. The half-inactivation potentials from the fitted curves were -47.60 ± 0.87 and -48.77 ± 1.83 mV before and after treatment with compound 6a (3 µM, n = 3), p = 0.31 (paired t test). (d) Dose– response relation for compound 9c inhibition of Cav3.2. The IC50 from the fitted curve was 0.34 ± 0.10 µM (n = 3 per dose). The inset shows a representative T-type current in the presence and the absence of 3 µM of compound 9c. (e) Effect of compound 9c on the current–voltage relation of Cav3.2. The halfactivation potentials from the fitted curves were -30.84 ± 0.46 mV and -30.57 ± 1.00 mV before and after the application of compound 9c (1 µM, n = 3), p = 0.98 (paired t test). (f) The steady-state inactivation curves for Cav3.2 before and after application of compound 9c. The half-inactivation potentials obtained from the fitted curves were -46.27 ± 0.57 and -51.20 ± 1.03 mV before and after the application of compound 9c (1 µM, n = 3), p = 0.02 (paired t test). 2.3. In vivo Studies Compounds 6a and 9c were selected for in vivo assessment in different mouse models of nociception. 2.3.1. Formalin-induced nociception Fig. 4 illustrates the effects of compound 6a and 9c in the formalin model 1. In this model, formalin is injected into the hindpaw, triggering two phases of spontaneous nocifensive behavior that is characterized by paw biting and licking - an early phase (phase I) that occurs in the first five minutes before subsiding, and a second phase (phase II) that reappears approximately 30 minutes after formalin injection. The 8

response is expressed as the total time spent licking and biting (termed “nocifensive response”) in each of the phases, and these parameters are then plotted separately. The compounds were delivered intrathecally 20 minutes before the formalin test to circumvent issues with blood-brain barrier penetration, and to control the site of action. Such intrathecal treatment of the mice with compound 6a significantly reduced the nocifensive response in a dose dependent manner in both; the first phase (Fig. 4a) and the second phase (Fig. 4b) of the formalin test. Maximum efficacy was achieved at a dose of 3µg/10µl. Compound 9c showed significant pain attenuation in both phases with maximum efficacy at 1µg/10µl (Fig. 4c & 4d). Its effect on nocifensive response when delivered at 1µg/10µl is comparable to that of 6a at the 3µg/10µl dose. However, its analgesic effects abated when delivered at 3µg/10µl (Fig. 4c & 4d). This may be due to activation of other pronociceptive targets at such dose 1. Accordingly, both 6a and 9c efficiently alleviate acute inflammatory hypersensitivity with compound 9c exhibiting more potency.

Fig. 4 Intrathecal injection of 6a and 9c attenuated formalin-induced nocifensive behaviour. a-b: Pretreatment with 6a reduced formalin-induced nocifensive response at 1µg and 3µg for phase I (a) and 3µg for phase II (b) and with maximal inhibitory effects at 3µg/10µl. c-d: Pre-treatment with 9c reduced formalin-induced nocifensive response at 1µg and 3µg for phase I (c) and all concentrations for phase II (d) and the peak effects is at 1µg/10µl. Data are mean ± S.E.M (n= 4-6 mice per group), analyzed with one-way ANOVA with Bonferroni’s post-hoc test. *p<0.05, **p<0.01, ***p<0.001 vs vehicle group and #p<0.05 vs 0.3µg group.

9

2.3.2. CFA-induced persistent inflammatory nociception Intraplantar injection of CFA induces hypersensitivity due to the development of persistent paw inflammation. Unlike in the formalin test where nocifensive behavior is scored, CFA-induced hypersensitivity can be assessed by mechanical paw withdrawal thresholds (in grams) in response to mechanical stimulation of the hind paw with a digital plantar aesthesiometer. Mechanical hypersensitivity in CFA-injected animals was evaluated after intrathecal injection of compounds 6a and 9c to assess their ability to modulate nociceptive transmission under chronic inflammatory conditions 52. As shown in Fig 5, CFA injected mice developed mechanical hyperalgesia as indicated by a decrease in paw withdrawal thresholds when compared to the pre-CFA baseline levels of the vehicle control group. Spinal treatment of mice with compound 6a (3 µg/10µl) significantly attenuated the CFA –induced mechanical hyperalgesia when compared with the control group, at 90 min (P ≤ 0.001) up to 150 min (P ≤ 0.001) (Fig. 5a). Interestingly compound 9c significantly reversed mechanical hyperalgesia from 20 min (P ≤ 0.05) through 45 min (P ≤ 0.001), 90 min (P ≤ 0.001) and up to 150 min (P ≤ 0.001) relative to vehicle-treated controls (Fig. 5b). These data indicate that compounds 6a and 9c are regulators of chronic inflammatory hypersensitivity when injected intrathecally with compound 9c showing higher potency.

Fig. 5. Intrathecal injection of compounds 6a and 9c partially reduced CFA-induced mechanical hyperalgesia. Compound 6a (a) reversed paw withdrawal threshold at 90 and 150 min and compound 9c (b) reversed the threshold at 45, 90 and 150 min in the CFA-treated mice compared to vehicle controls. Data are mean ± S.E.M (n= 6 mice per group), analyzed by a two-way ANOVA with Bonferroni’s posthoc test. *p<0.05, ***p<0.001 vs PBS+vehicle group. 2.4. Structure Activity Relationship Studies Generally, the two-carbon spacer at N3 of the DHPM core retained the intrinsic calcium channel activity of the N3 substituted DHPMs 53-55. Efficacy was a function of the terminal functionalities. Hydrazones as a functional group conferred the highest calcium channel blocking activity. Within this series, hydrazones derived from aromatic aldehydes (6a-c) showed almost complete L- & T-type channel block. Diphenyl hydrazone derivatives and those derived from aromatic ketones exhibited slightly lower L-type current 10

block while failing to inhibit T-type currents altogether. It is notable that slight structural modifications on 6d to 6g derivative abolished either current block. In order to gain more SAR information, the effect of the hydrazone side chain size on activity was investigated. Interestingly, a one carbon increase in the side chain length of the hydrazone (6g) to give 6h, while retaining the other substituents restored the relative moderate L- and T-block activity. The SAR within this series was then extended to test the effect of introducing different moieties to the DHPM core through hydrazone ligation as well. As a representative example, the ester derivative (6i) was used in this study. Fig 2 shows that such modification was detrimental to both L- and T-type channel blocking activities. Moreover, Hydrazones derived from isatins represented by 6j showed moderate L- and T-type channel block. Other modifications were also studied. Hydrazide acetylation (3) or cyclization to a pyrazole ring (7) diminished both L- and T-type channel activities, while the sulfonohydrazide derivative (5a) showed moderate L-type block with slightly higher T-type block. The thiosemicarbazide and its cyclized heterocyclic derivatives favor T-type selectivity. The benzhydryl derivative (9c) showed outstanding T-current block with a negligible L-type current blocking activity. Hence, 9c is the most promising T-type blocker identified in this study, although its selectivity on other targets beyond the L-type and N-type channels tested here will need to be determined. Interestingly, modifying the thiosemicarbazide terminal group to simple phenyl group as in 9a dramatically abolished activity. However, the cyclization of 9a into thiadiazole (10), an oxadiazole (11a) or a thiazolidinone (13a) conferred promising T-type selectivity while the corresponding thiazoline (12a) was inactive.

3. Conclusion Novel N3 substituted Dihydropyrimidnes were synthesized as T-type calcium channel blockers. The compounds were designed to mimic the structural requirements for calcium channel blocking activity of DHPs. Various moieties were introduced at the N3 position through a spacer aiming at improving the pharmacodynamics and the pharmacokinetic profiles. Representatives of different series were evaluated for T- and L-type calcium channel blocking activity. Eleven compounds showed promising T-type block. Four lead compounds (9c, 10, 11a & 13b) of these were selective T-blockers. Compounds with highest Ttype blocking activity and selectivity over L-type channels (6a & 9c) were assessed for in vivo biological evaluation in formalin and CFA-induced models of nociception. Interestingly, both 6a & 9c attenuate hypersensitivity. 9c was more potent and was selected as a lead for further structure optimization studies. 4. Experimental Procedures 4.1. Chemistry 4.1.1. General information All chemicals were purchased from commercial sources. Flash column chromatography separation was performed using Acros organics silica gel 40-60 µm, 60 Å using combination of ethyl acetate and hexanes. Preparative thin layer chromatography was performed using UNIPLATE TM1500 µm silica gel plates with UV 254 preparative layer. Whatman and sigma TLC plates were utilized for thin layer chromatography and visualization was done using UV fluorescence at 254 nm. Melting points were recorded on a Mel-Temp, Laboratory devices, Inc and are uncorrected. %CHN Analyzer by combustion/ 11

TCD and %S by O flask combustion/IC were used for elemental analysis of final compounds and performed by Micro Analysis Inc., Wilmington DE, USA and are within 0.4%. 1 H and 13C NMR spectra were obtained on a Bruker Avance 400 MHz & 600 MHz instrument using DMSO-d6 as solvent unless otherwise stated. 1H NMR Spectra are reported in order; multiplicity, number of protons and signals were characterized as s (singlet), d (doublet), dd (doublet of doublet), t (triplet), m (multiplet), br s (broad signal), q (quartet), quin (quintet), tquin (triplet of quintet), sxt (sextet), spt (septet). Chemical shifts are relative to TMS as an internal standard. Mass spectra were recorded on Thermo Finnigan MAT95XL high resolution magnetic sector mass spectrometer, using electrospray ionization method. The IR spectra were recorded on ZnSe crystal at 8 cm-1 resolution in Nicolet 380 ATR-FTIR spectrophotometer (Thermo electron Corporation, Madison, WI). 4.1.2. Ethyl 3-[2-(ethoxycarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4tetrahydropyrimidine-5-carboxylate (1) To a solution of DHPM 40 (15 mmol) and the appropriate ethyl acrylate (15 mmol) in anhydrous DMF (30 mL), KF/neutral alumina (10 mol %) was added in one portion. The mixture was stirred at room temperature for 1-5 days. The completion of the reaction was monitored by TLC using 1:1 toluene: acetone. The catalyst was removed by filtration. The filtrate was then poured into ice-cold water (500 mL). The obtained product was filtered, washed with water, dried and purified by recrystallization from ethanol ; Yield: 4.6 g (76.3%); MP: 121 oC; IR: 3214, 1729, 1695, 1645, 1526, 1249, 1204, 1118, 1086 cm-1; 1H NMR (400 MHz, DMSO) δ 1.17 (2t, J = 8, 6 Hz, 6H, 2CH3CH2), 2.26 (s, 3H, C6-CH3), 2.392.45 (m, 1H, CH), 2.60-2.66 (m, 1H, CH), 3.05-3.13 (m, 1H, CH), 3.68-3.74 (m, 1H, CH), 3.99- 4.09 (m, 4H, 2CH3CH2), 5.54 (s, 1H, C4-H), 7.68-8.19 (m, 4H, ArHs), 9.64 (s, 1H, NH, D2O-exchangeable); 13C NMR (400 MHz, DMSO) δ 13.89, 13.91, 17.57, 32.49, 41.34, 59.33, 59.48, 60.00, 99.28, 121.61, 122.77, 130.39, 133.31, 144.98, 147.69, 148.38, 151.44, 164.65, 171.1; Elemental Analysis Calcd for C19H23N3O7: C 56.29, H 5.72, N 10.37; Found: C 56.07; H 5.93; N 10.32. 4.1.3. Ethyl 3-[2-(hydrazocarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4tetrahydropyrimidine-5-carboxylate (2) To an ethanolic solution of 1 (50 mL, 1 mmol), hydrazine-hydrate (99%) (3 mL) was added and refluxed for 7 h. The reaction was then cooled, concentrated, diluted with water. The precipitate was washed with water, dried and recrystallized from ethanol; Yield: 3 g (80%); MP: 184 oC; IR: 3363, 3224, 3215, 1693, 1643, 1520, 1240, 1085 cm-1; 1H NMR (400 MHz, DMSO) δ 1.16 (t, J = 8 Hz, 3H, CH3CH2), 2.15-2.22 (m, 1H, CH), 2.29 (s, 3H, C6-CH3), 2.68-2.72 (m, 1H, CH), 3.03-3.10 (m, 1H, CH), 3.70-3.77 (m, 1H, CH), 3.99- 4.11 (m, 2H, CH3CH2), 4.21 (s, 2H, NH2, D2O-exchangeable), 5.55 (s, 1H, C4-H), 7.69-8.20 (m, 4H, ArHs), 9.10 (s, 1H, CONH, D2O-exchangeable), 9.63 (s, 1H, NH, D2O-exchangeable); 13C NMR (400 MHz, DMSO) δ 13.88, 17.59, 32.13, 41.66, 59.23, 59.47, 99.23, 121.61, 122.72, 130.30, 133.35, 145.07, 147.69, 148.39, 151.42, 164.72, 169.64, Elemental Analysis Calcd for C17H21N5O6: C 52.17, H 5.41, N 17.89; Found: C 52.21, H 5.34, N 17.85. 4.1.4. Acetic N',N'-diacetyl-3-[5-(ethoxycarbonyl)-4-methyl-6-(3-nitrophenyl)-2-oxo-2,3dihydropyrimidin-1(6H)-yl]propanehydrazonic anhydride (3)

12

A mixture of acid hydrazide (2) (2mmol) in acetic anhydride (3 mL) was heated under reflux for 8 h. After completion, the reaction was concentrated under reduced pressure, and then poured into ice-cold water (500 mL). The obtained product was filtered, washed with water, dried and purified by preparative TLC using gradient acetone in toluene; Yield: 0.86 g (84%); MP: 157 oC; IR: 3325, 1744, 1720, 1690, 1642, 1530, 1211, 1092 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.16 (t, J = 6 Hz, 3H, CH3CH2), 2.27 (s, 3H, C6-CH3), 2.33 (d, J = 1.6 Hz, 9H, 3COCH3), 2.69-2.75 (m, 1H, CH), 3.04-3.10 (m, 1H, CH), 3.183.25 (m, 1H, CH), 3.64-3.71 (m, 1H, CH), 3.98-4.08 (m, 2H, CH3CH2) 5.58 (s, 1H, C4-H), 7.67-8.20 (m, 4H, ArHs), 9.65 (s, 1H, NH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.91, 17.59, 24.24, 24.31, 24.41, 34.61, 41.15, 59.48, 59.59, 99.35, 121.63, 122.79, 130.39, 133.34, 145.08, 147.68, 148.35, 151.48, 164.67, 170.78, 170.81, 170.94, 172.00; HRMS (ESI) Calcd. for C23H28N5O9 [M+1] +: 518.1882; Found: 518.1889. 4.1.5. Ethyl 3-[2-(2-substituitedhydrazinylcarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4tetrahydropyrimidine-5-carboxylate (4a-c) A mixture of acid hydrazide (2) (1 mmol) and acetyl chloride (1.2 mmol) (for 4a) or the proper alkyl chloroformate (1.2 mmol) (for 4b & 4c) in anhydrous pyridine (3mL) was stirred overnight at room temperature under nitrogen. The separated product was then filtered washed with water, dried and purified as mentioned. 4.1.5.1. Ethyl 3-[2-(2-acetylhydrazinylcarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4tetrahydropyrimidine-5-carboxylate (4a) Yield: 0.25 g (57%); Purified by preparative TLC using gradient acetone in toluene; MP: 118 oC; IR: 3221, 3183, 3122, 1659, 1646, 1606, 1530, 1241, 1028 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.12-1.19 (m, 3H, CH3CH2), 1.84 (s, 3H, NCOCH3), 1.99, 2.09 (2s, 1H, CH), 2.18-2.28 (m mixed with s, 4H, CH & C6-CH3), 2.96-3.03 (m, 1H, CH), 3.60-3.67 (m, 1H, CH), 3.94-4.06 (m, 2H, CH3CH2), 5.53 (s, 1H, C4H,), 7.68 (t, J = 8 Hz, 1H, ArHs), 7.75 (m, 1H, ArH), 8.15 (m, 2H, ArHs), 9.58 (s, 1H, NH, D2Oexchangeable), 9.75 (s, 1H, CONH, D2O-exchangeable), 9.79 (s, 1H, CONH, D2O-exchangeable); 13CNMR (DMSO, 400 MHz) δ ppm: 13.90, 17.58, 20.37, 31.78, 41.46, 59.30, 59.44, 99.28, 121.66, 122.77, 130.37, 133.42, 145.17, 147.67, 148.21, 151.37, 164.67, 167.76, 168.92; HRMS (ESI) Calcd. for C19H23N5O7Na [M+Na] +: 456.1490; Found: 456.1503. 4.1.5.2. Ethyl 3-[2-(2-{methoxycarbonyl}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2-oxo1,2,3,4-tetrahydropyrimidine-5-carboxylate (4b) Yield: 0.31 g (68.8%); the product was used as such without further purification; MP: 125 oC; IR: 3321, 3223, 3105, 1732, 1696, 1682, 1661, 1630, 1530, 1239, 1220, 1088, 1021 cm-1; 1H-NMR (CDCl3, 400 MHz) δ 1.23 (t, J = 8 Hz, 3H, CH3CH2), 2.37 (s, 3H, C6-CH3), 2.41-2.45 (m, 1H, CH), 2.66-2.74 (m, 1H, CH), 3.27-3.34 (m, 1H, CH), 3.71-3.82 (m mixed with s, 4H, CH &NCO2CH3), 4.04- 4.18 (m, 2H, CH3CH2), 5.52 (s, 1H, C4-H), 7.51 (t, J = 6 Hz, 1H, ArH), 7.69-7.74 (m, 2H, ArH & NH, D2Oexchangeable), 8.14 (d,J= 4 Hz, 1H, ArH), 8.21 (s, 1H, ArH), 8.62,8.68, 10.39 (3br s, 2H, 2NH , D2Oexchangeable); HRMS (ESI) Calcd. for C19H23N5O8Na [M+Na] +: 472.1439; Found: 472.1439.

13

4.1.5.3. Ethyl 3-[2-(2-{ethoxycarbonyl}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2-oxo1,2,3,4-tetrahydropyrimidine-5-carboxylate (4c) Yield: 0.4 g (87%); the product was used as such without further purification; MP: 116 oC;IR: 3371, 3334, 3222, 1725, 1700, 1678, 1659, 1631, 1530, 1239, 1222, 1087, 1021 cm-1; 1H-NMR (CDCl3, 400 MHz) δ 1.23 (t, J = 8 Hz, 3H, CH3CH2), 1.30 (t, J = 6 Hz , 3H, NCO2CH2CH3), 2.36 (s, 3H, C6-CH3), 2.402.43(m, 1H, CH), 2.65-2.67 (m, 1H, CH), 3.28-3.35 (m, 1H, CH), 3.69-3.75 (m, 1H, CH), 4.04- 4.16 (m, 2H, CH3CH2) 4.18- 4.24 (m, 2H, NCO2CH2CH3), 5.50 (s, 1H, C4-H), 7.20 (d, J = 4 Hz, 1H, NH, D2Oexchangeable), 7.51 (t, J = 4 Hz, 1H, ArH), 7.72 (d, J = 8 Hz, 1H, ArH), 8.14 (dd, J = 4 Hz, 1H, ArH), 8.21 (t, J = 4 Hz, 1H, ArH), 8.63 (br s, 1H, NH, D2O-exchangeable), 10.39 (br s, 1H, NH , D2Oexchangeable); 13C-NMR (CDCl3, 400 MHz) δ 14.17, 14.38, 18.22, 32.73, 43.01, 60.36, 61.24, 62.53, 100.83, 122.33, 123.15, 129.67, 133.62, 144.38, 147.27, 148.46, 153.03, 157.10, 165.16, 170.13; HRMS (ESI) Calcd. for C20H25N5O8Na [M+Na] +: 486.1595; Found: 486.1597. 4.1.6. Ethyl 3-[2-(2-{substitutedsulfonyl}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2-oxo1,2,3,4-tetrahydropyrimidine-5-carboxylate (5a & b) A mixture of acid hydrazide (2) (1 mmol) and the proper sulfonyl chloride (1.5 mmol) in anhydrous pyridine (2mL) was stirred for 4 days at room temperature. Excess pyridine was removed under reduced pressure. The reaction mixture was then poured into ice-cold water (500 mL). The separated product was filtered, washed with water, dried and purified by recrystallization from methanol/dioxane (9:1) 4.1.6.1. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(2-{phenylsulfonyl}hydrazinylcarbonyl)ethyl]1,2,3,4-tetrahydropyrimidine-5-carboxylate (5a) Yield: 0.2 g (38%); MP: 207 oC; IR: 3296, 3243, 1710, 1681, 1626, 1529, 1327, 1159, 1240, 1079 cm-1; H NMR (DMSO, 400 MHz) δ 1.12 (t, J = 8 Hz, 3H, CH3CH2), 2.07-2.14 (m, 1H, CH), 2.25 (s, 3H, C6CH3), 2.38-2.48 (m, 1H, CH), 2.77-2.84 (m, 1H, CH), 3.41-3.51 (m, 1H, CH), 3.91- 4.05 (m, 2H, CH3CH2), 5.44 (s, 1H, C4-H), 7.48-8.17 (m, 9H, ArHs), 9.58 (s, 1H, NH, D2O-exchangeable), 9.85 (s, 1H, NH, D2O-exchangeable), 10.09 (s, 1H, SO2NH, D2O-exchangeable); Elemental Analysis Calcd for C23H25N5O8S: C 51.97, H 4.74, N 13.18; Found: C 52.04, H 4.69, N 12.69. 1

4.1.6.2. Ethyl 3-[2-(2-{benzylsulfonyl}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2-oxo1,2,3,4-tetrahydropyrimidine-5-carboxylate (5b) Yield: 0.2 g (37%); MP: 199 oC; IR: 3301, 3233, 1685, 1643, 1530, 1346, 1160, 1247, 1087 cm-1; 1H NMR (DMSO, 400 MHz) δ 11.07, 1.14 (2t, J= 6, 8 Hz, 3H, CH3CH2), 2.23 (s, 3H, C6-CH3), 2.28-2.37 (m, 1H, CH), 2.56-2.64 (m, 1H, CH), 3.04-3.11 (m, 1H, CH), 3.68-3.37 (m, 1H, CH), 3.82-3.91 (m, 2H, CH3CH2), 4.47- 4.34 (m, 2H, SO2CH2), 5.59 (s, 1H, C4-H), 7.33-7. 46 (m, 5H, ArHs), 7.66-7.77 (m, 2H, ArHs), 8.14-8.17 (m, 2H, ArHs), 9.53-9.58 (m, 1H, NH, D2O-exchangeable), 9.63, 9.70 (2s, 1H, NH, D2O-exchangeable), 10.25(s, 1H, SO2NH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.84, 17.63, 31.91, 41.39, 57.51, 59.44, 59.56, 99.30, 121.57, 122.81, 128.05, 128.21, 129.28, 130.44, 130.98, 133.32, 142.91, 144.99, 147.71, 148.23, 151.54, 164.63, 170.20; Elemental Analysis Calcd for C24H27N5O8S: C 52.84, H 4.99, N 12.84; Found: C 52.69, H 5.04, N 12.71. 14

4.1.7. General procedure for preparation of hydrazones (6a-l) To a stirred solution of hydrazide (2) (1.0 mmol) in absolute ethanol, the appropriate aldehyde (1eq.) , ketone (1eq.), isatin (1eq.) or ethylacetoacetate (6 eq.) was added, the mixture was refluxed for 4-30 h, then allowed to cool to room temperature and the obtained precipitate was recrystallized from proper solvent (s). 4.1.7.1. Ethyl 3-[2-(2-benzylidenehydrazinylcarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4tetrahydropyrimidine-5-carboxylate (6a) Yield: 0.4 g (83%); purified by recrystallization from ethanol/dioxane (9:1); MP: 199 oC; IR: 3205, 1707, 1665, 1638, 1528, 1240, 1086 cm-1; 1H NMR (DMSO, 400 MHz) δ: 1.10, 1.12 (2t, J = 8, 6 Hz, 6H, 2CH3CH2), 2.20, 2.25 (2s, 6H, 2C6-CH3), 2.37-2.40 (m, 1H, CH), 2.61-2.75 (m, 2H, 2CH), 2.96-3.03 (m, 1H, CH), 3.16- 3.28 (m, 2H, 2CH), 3.74- 3.81 (m, 2H, 2CH), 3.90- 4.06 (m, 4H, 2CH3CH2), 5.57, 5.63 (2s, 2H, 2C4-H), 7.35-8.20 (m, 20H, ArHs & 2CH=N), 9.60, 9.64 (2s, 2H, 2NH, D2O-exchangeable), 11.36, 11.43 (2s, 2H, 2CONH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ : 13.86, 13.88, 17.57, 17.63, 31.17, 31.18, 41.37, 41.39, 59.43, 59.52, 66.29, 99.29, 99.30, 121.57, 121.61, 122.65, 122.71, 126.53, 126.94, 128.63, 129.60, 129.84, 130.26, 133.36, 134.12, 134.21, 142.87, 145.04, 145.36, 146.10, 146.12, 147.67, 147.69, 148.36, 148.39, 151.49, 151.54, 164.65, 164.68, 166.83, 172.59; Elemental Analysis Calcd for C24H25N5O6: C 60.12, H 5.26, N 14.61; Found: C 60.13, H 5.31, N 14.45. 4.1.7.2. Ethyl 3-[2-(2-{4-fluorobenzylidene}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6b) Yield: 0.4 g (84%);%); purified by recrystallization from ethanol/dioxane (9:1); MP: 217 oC; IR: 3200, 1705, 1675, 1636, 1525, 1234, 1071 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.15, 1.17 (2t, J = 8, 8 Hz, 6H, 2CH3CH2), 2.23, 2.28 (2s, 6H, 2C6-CH3), 2.37-2.43 (m, 1H, CH), 2.62-2.76 (m, 2H, 2CH), 2.95-3.03 (m, 1H, CH), 3.17-3.30 (m, 2H, 2CH), 3.74-3.82 (m, 2H, 2CH), 3.94-4.10 (m, 4H, 2CH3CH2), 5.58, 5.64 (2s, 2H, 2C4-H), 7.24-7.31 (m, 4H, ArHs), 7.66-7.82 (m, 8H, ArHs), 7.97 (s, 1H, CH=N), 8.16-8.21 (m, 5H, 4ArHs &CH=N), 9.62, 9.66 (2s, 2H, 2NH, D2O-exchangeable), 11.40, 11.46 (2s, 2H, 2CONH, D2Oexchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.88, 13.89, 17.55, 17.61, 31.12, 33.06, 41.35, 41.60, 59.44, 59.49, 66.30, 99.27, 99.30, 115.60, 115.61, 115.82, 115.83, 121.58,122.70, 122.74, 128.63, 128.71, 129.04, 129.12, 130.33, 130.72, 130.80, 130.83, 133.33, 133.37, 141.69, 144.95, 144.97, 145.02, 145.34, 147.65, 147.69, 148.34, 151.43, 151.50, 161.60, 161.77, 164.06, 164.65, 164.68, 166.78, 172.57; Elemental Analysis Calcd for C24H24FN5O6: C 57.94, H 4.86, N 14.08; Found: C 57.93, H 4.70, N 14.08. 4.1.7.3. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(2-{3-phenylallylidene}hydrazinylcarbonyl)ethyl]1,2,3,4-tetrahydropyrimidine-5-carboxylate (6c) Yield: 0.4 g (82%); purified by recrystallization from ethanol/dimethylformamide (9:1); MP: 232 oC; IR: 3217,1669,1651, 1524,1243,1090 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.17 (t, J = 8 Hz, 6H, 2CH3CH2), 2.23,2.25 (2s, 6H, 2C6-CH3), 2.29-2.36 (m, 1H, CH), 2.55-2.69 (m, 2H, 2CH), 2.91-2.97 (m, 1H, CH), 3.06-3.16 (m, 2H, 2CH), 3.69-3.79 (m, 2H, 2CH), 3.95-4.08 (m, 4H, 2CH3CH2), 5.54, 5.61 (2s, 2H, 2C4H), 6.86-7.06 (m, 4H,2CH=CH), 7.33 (t, J = 6 Hz, 2H, ArHs), 7.39 (t, J = 8 Hz, 4H, ArHs), 7.60 (d, J = 8 15

Hz, 4H, ArHs), 7.69 (t, J = 8 Hz, 2H, 2CH=N), 7.77 (t, J = 4 Hz, 3H, ArHs), 7.90-7.79 (m, 1H, ArH), 8.17 (d, J = 8 Hz, 4H, 2ArHs & 2CH=N), 9.59, 9.62 (2s, 1H, NH, D2O-exchangeable), 9.63, 9.66 (2s, 1H, NH, D2O-exchangeable), 11.26, 11.31 (2s, 2H, 2CONH, D2O-exchangeable); 13C-NMR(DMSO, 400 MHz) δ 13.93, 17.61, 30.65, 40.98, 59.32, 59.48, 99.18, 121.63, 122.75, 125.12, 126.94, 128.75, 130.39, 133.39, 135.86, 138.33, 138.68, 145.17, 145.43, 147.69, 148.37, 151.39, 151.48, 164.66, 164.74, 172.35; HRMS (ESI) Calcd for C26H28N5O6 [M+1] +:506.2044; Found: 506.2034. 4.1.7.4. Ethyl 3-[2-(2-{2,2-diphenylethylidene}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6d) Yield: 0.52 g (91%); purified by recrystallization from ethanol/dioxane (9:1); MP: 216 oC; IR: 3200, 1705, 1675, 1636, 1534, 1238, 1083 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.13, 1.15 (2t, J = 8, 6 Hz, 6H, 2CH3CH2), 2.25 (s, 6H, 2C6-CH3), 2.28-2.32 (m, 1H, CH), 2.49-2.61 (m, 2H, 2CH), 2.84-2.92 (m, 1H, CH), 3.05-3.16 (m, 2H, 2CH), 3.68-3.76 (m, 2H, 2CH), 3.92-4.08 (m, 4H, 2CH3CH2), 4.91, 4.96 (2d, J = 4, 8 Hz, 2H, 2CH(C6H5)2), 5.55, 5.60 (2s, 2H, 2C4-H), 7.23-7.38 (m, 20H, ArHs), 7.63-7.68 (m, 2H, ArHs), 7.74-7.79 (m, 3H, ArHs), 7.96 (d, J = 8 Hz, 1H, ArHs), 8.12-8.18 (m, 4H, 2ArHs & 2CH=N), 9.61, 9.63 (2s, 2H, 2NH, D2O-exchangeable), 11.26, 11.31 (2s, 2H, 2CONH, D2O-exchangeable); 13CNMR (DMSO, 400 MHz) δ 13.93, 17.61, 30.91, 32.85, 40.99, 41.46, 52.86, 52.93, 59.30, 59.46, 66.32, 99.19, 99.27, 121.58, 122.74, 126.77, 128.14, 128.15, 128.66, 128.69, 130.31, 133.33, 141.08,141.11,141.18, 145.05, 145.18, 147.25, 147.67, 148.40, 151.40, 151.42, 151.46, 164.65, 164.72, 166.56, 172.25; HRMS (ESI) Calcd for C31H31N5O6Na [M+Na] +: 592.2167, Found: 592.2157. 4.1.7.5. Ethyl 3-[2-(2-{1-phenylethylidene}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6e) Yield: 0.44 g (89%); purified by recrystallization from ethanol/dioxane (9:1); MP: 210 oC; IR: 3206, 1674, 1640, 1526, 1240, 1082 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.10, 1.13 (2t, J = 6, 8 Hz, 6H, 2CH3CH2), 2.21, 2.24, 2.26 (3s, 12H, 2C6-CH3 & 2CH3C=N), 2.70-2.75 (m, 2H, 2CH), 3.02-3.08 (m, 2H, 2CH), 3.27-3.33 (m, 2H, 2CH), 3.75-3.94 (m, 2H, 2CH), 3.95-4.04 (m, 4H, 2CH3CH2), 5.58, 5.65 (2s, 2H, 2C4-H), 7.37-8.18 (m, 18H, ArHs), 9.60, 9.65 (2s, 2H, 2NH, D2O-exchangeable), 10.41, 10.57 (2s, 2H, 2CONH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.37, 13.88,17.57, 17.62, 31.70, 41.50, 59.43, 59.62, 66.30, 99.29, 121.56, 122.63, 125.77, 126.21, 128.13, 128.21, 128.85, 129.04, 130.26, 133.32, 137.99, 138.17, 144.99, 145.38, 147.31, 147.66, 148.38, 151.11 151.52, 164.68, 173.48; HRMS (ESI) Calcd for C25H28N5O6 [M+1] +: 494.2034; Found: 494.2016. 4.1.7.6. Ethyl 3-[2-(2-{1-(4-fluorophenyl)ethylidene}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6f) Yield: 0.3 g (61.5%); purified by recrystallization from dioxane; MP: 191 oC; IR: 3200, 1719, 1673, 1642, 1530, 1243, 1080 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.10-1.17 (m, 3H, CH3CH2), 2.21, 2.24, 2.25 (3s, 6H, C6-CH3 & CH3C=N), 2.65-2.80 (m, 1H, CH), 2.96-3.12 (m, 1H, CH), 3.21-3.31 (m, 1H, CH), 3.673.84 (m, 1H, CH), 3.91- 4.07 (m, 2H, CH3CH2), 5.55, 5.62 (2s, 1H, C4-H), 7.21-7.26 (m, 2H, ArHs), 7.667.85 (m, 4H, ArHs), 8.16, 8.17 (dist. d, 2H, ArHs), 9.57, 9.63 (2s, 1H, NH, D2O-exchangeable), 10.40, 10.57 (2s, 1H, CONH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.45, 13.91, 17.57, 31.65, 16

41.48, 59.44, 66.32, 99.29, 121.56, 122.70, 127.94, 128.03, 130.37, 133.35, 134.52, 145.36, 146.40, 147.64, 148.35, 149.74, 151.45, 151.48, 164.68, 173.43; Elemental Analysis Calcd for C25H26FN5O6: C 58.70, H 5.12, N 13.69; Found: C 58.71, H 5.03, N 13.49. 4.1.7.7. Ethyl 3-[2-(2-{1,1-diphenylpropan-2-ylidene}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6g) Yield: 0.4 g (61.5%); purified by recrystallization from ethanol/dioxane (9:1); MP: 233 oC; IR: 3192, 1705, 1675, 1632, 1531, 1238, 1076 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.11-1.18 (m, 6H, 2CH3CH2), 1. 86 (s, 6H, 2CH3C=N), 2.26,2.27 (2s, 6H, 2C6-CH3), 2.37-2.45 (m, 2H, 2CH), 2.65-2.75 (m, 2H, 2CH), 2.89-2.96 (m, 1.5H, CH), 3.04-3.11 (m, 0.5H, CH), 3.66- 3.79 (m, 2H, 2CH), 3.90- 4.10 (m, 4H, 2CH3CH2), 5.02,5.12 (2s, 2H, 2CH(C6H5)2), 5.51, 5.56 (2s, 2H, 2C4-H), 7.17-7.35 (m, 22H, ArHs), 7.657.78 (m, 4H, ArHs), 8.12-8.18 (m, 4H, 2ArHs& 2CH=N), 9.63, 9.67 (2s, 2H, 2NH, D2O-exchangeable), 10.20, 10.41 (2s, 2H, 2CONH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.94, 16.39, 17.64, 31.78, 40.83, 58.94, 59.47, 66.32, 99.13, 121.57, 122.67, 126.37, 126.43, 126.55, 128.07, 128.15, 128.30, 128.84, 130.32, 133.26, 140.75, 140.86, 140.90, 145.09, 147.66, 147.71, 148.47, 151.37, 151.49, 152.13, 164.73, 166.75, 173.42; HRMS (ESI) calcd for C32H33N5O6Na [M+Na] +: 606.2323, Found: 606.2315. 4.1.7.8. Ethyl 3-[2-(2-{4,4-diphenylbutan-2-ylidene}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6h) Yield: 0.35 g (58.6%); purified by recrystallization from ethanol/dioxane (9:1); MP: 202 oC; IR: 3207, 1689, 1654, 1639, 1528, 1244, 1079 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.13-1.17 (m, 3H, CH3CH2), 1.75,1.79 (2s, 3H, CH3C=N), 2.26, 2.29 (2s, 3H, C6-CH3), 2.38-2.45 (m, 1H, CH), 2.57-2.72 (m, 1H, CH), 3.29-3.11 (m, 3H, CH2CH), 3.63-3.75 (m, 1H, CH), 3.95-4.09 (m, 2H, CH3CH2), 4.37-4.45 (m, 1H, CH(C6H5)2), 5.54-5.60 (m, 1H, C4-H), 7.14 (d, J = 8 Hz, 2H, ArHs), 7.22-7.36 (m, 8H, ArHs), 7.64-7.69 (m, 1H, ArHs), 7.76 (t, J = 8 Hz, 1H, ArHs), 8.15-8.19 (m, 2H, 2ArHs), 9.62, 9.67 (2s, 1H, NH, D2Oexchangeable), 9.94, 10.20 (2s, 1H, CONH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.94, 16.12, 17.66, 31.55, 43.45, 47.32, 59.35, 59.48, 66.32, 99.19, 121.58, 122.68, 122.70, 125.92, 125.93, 127.58, 127.59, 127.69, 128.19, 128.20, 128.30, 130.34, 130.36, 133.32, 144.47, 144.54, 145.25, 147.67, 148.48, 150.80, 151.38, 164.76,172.83; HRMS (ESI) Calcd for C33H36N5O6 [M+1] +: 598.2660; Found: 598.2637. 4.1.7.9. Ethyl 3-[2-(2-{4-ethoxy-4-oxobutan-2-ylidene}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6i) Yield: 0.29 g (57.7%); purified by recrystallization from ethanol; MP: 148 oC; IR: 3230, 1728, 1694, 1643, 1528, 1233, 1081 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.13-1.23 (m, 6H, 2CH3CH2), 1.89, 1.91, (2s, 3H, CH3C=N), 2.25 (s, 3H, C6-CH3), 2.35-2.42 (m, 0.5H, CH), 2.55-2.70 (m, 1H, CH), 2.85-2.93 (m, 0.5H, CH), 3.03-3.14 (m,1H, CH), 3.27, 3.32, 3.38 (3s, 2H, CH2), 3.67-3.78 (m, 1H, CH), 3.94-4.14 (m, 4H, 2CH3CH2), 5.54, 5.61 (2s, 1H, C4-H), 7.69 (t, J = 8 Hz, 1H, ArH), 7.77 (d, J = 8 Hz, 1H, ArH), 8.148.18 (m, 2H, ArHs), 9.58, 9.62 (2s, 1H, NH, D2O-exchangeable), 1017, 10.34 (2s, 1H, NH, D2Oexchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.91, 13.94, 16.15, 16.54, 17.57, 17.59, 31.34, 31.42, 32.63, 32.64, 41.02, 41.39, 43.84, 43.91, 59.40, 59.44, 60.35, 60.40, 99.16, 99.24, 121.60, 122.67, 122.70, 17

122.74, 130.34, 130.39, 133.34, 144.98, 145.21, 146.67, 147.66, 147.69, 148.37, 148.39, 148.42, 151.39, 151.46, 164.65, 164.68, 167.02, 167.05, 169.43, 169.57, 172.98; Elemental Analysis Calcd for C23H29N5O8: C 54.86, H 5.81, N 13.91; Found: C 54.60, H 5.64, N 13.77. 4.1.7.10. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(2-{2-oxoindolin-3-ylidene}hydrazinylcarbonyl) ethyl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6j) Yield: 0.29 g (55%); purified by recrystallization from acetone/dioxane (9:1); MP: 249 oC; IR: 3220, 1710, 1690, 1639, 1529, 1243, 1083 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.17 (t, J = 6 Hz, 3H, CH3CH2), 2.16 (s, 3H, C6-CH3), 2.58-2.89 (m, 1H, CH), 3.14-3.28 (m, 2H, CH2), 3.84-3.91 (m, 1H, CH), 3.99- 4.05 (m, 2H, CH3CH2), 5.62 (s, 1H, C4-H), 6.95-8.21 (m, 8H, ArHs), 9.64 (s, 1H, NH, D2O-exchangeable), 11.27 (s, 1H, NH, D2O-exchangeable), 12.51 (s, 1H, NH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.88, 17.49, 30.18, 41.36, 59.48, , 66.30, 99.23, 111.00, 119.63, 120.32, 121.56, 122.42, 122.73, 130.36, 131.31, 133.37, 134.04, 142.31, 145.15, 147.67, 148.36, 151.49, 162.35, 164.65, 173.68; HRMS (ESI) Calcd for C25H25N6O7 [M+1] +: 521.1779; Found: 521.1785. 4.1.7.11. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(2-{2-oxo-1-phenylindolin-3-ylidene} hydrazinylcarbonyl)ethyl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6k) Yield: 0.24 g (40%); purified by recrystallization from dioxane (9:1); MP: 152 oC; IR: 3208, 1699, 1677, 1629, 1531, 1228, 1077 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.15 (t, J = 6 Hz, 3H, CH3CH2), 2.15 (s, 3H, C6-CH3), 2.88-2.91 (m, 1H, CH), 3.15-3.26 (m, 2H, CH2), 3.76-3.88 (m, 1H, CH), 3.98-4.10 (m, 2H, CH3CH2), 5.61 (s, 1H, C4-H), 6.84 (d, J = 8 Hz, 1H, ArH), 7.21 (t, J = 8 Hz, 1H, ArH), 7.41 (t, J = 8 Hz, 1H, ArH), 7.48-7.54 (m, 3H, ArHs), 7.59-7.65 (m, 3H, ArHs), 7.69 (t, J = 8 Hz, 1H, ArH), 7.78 (d, J = 4 Hz, 1H, ArH), 8.15-8.18 (m, 2H, ArHs), 9.63 (s, 1H, NH, D2O-exchangeable), 12.36, 12.79 (s &br s, 1H, NH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.91, 17.49, 30.32, 41.40, 59.49, 99.22, 110.03, 119.22, 120.86, 121.58, 122.77, 123.56, 123.57, 126.57, 128.59, 128.60, 129.70, 129.72, 130.42, 131.34 132.78, 133.37, 133.40, 143.40, 147.63, 147.67, 148.42, 151.46, 159.83, 164.65, 172.13; Elemental Analysis Calcd for C31H28N6O7: C 62.41, H 4.73, N 14.09; Found: C 62.20, H 4.37, N 14.06. 4.1.7.12. Ethyl 3-[2-(2-{5-fluoro-2-oxoindolin-3-ylidene}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6l) Yield: 0.49 g (91%); purified by recrystallization from acetone; MP: 276 oC; IR: 3231, 1711, 1694, 1642, 1530, 1244, 1086 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.13 (t, J = 8 Hz, 3H, CH3CH2), 2.11 (s, 3H, C6CH3), 2.85-2.88 (m, 1H, CH), 3.07-3.19 (m, 2H, CH2), 3.80-3.86 (m, 1H, CH), 3.96-3.99 (m, 2H, CH3CH2), 5.56 (s, 1H, C4-H), 6.90,6.92 (2d, J = 4, 4 Hz, 1H, ArH), 7.18,7.19 (2t, J = 8, 8 Hz, 1H, ArH), 7.26 (d, J = 8 Hz, 1H, ArH), 7.67 (t, J = 8 Hz, 1H, ArH), 7.75-7.77 (m, 1H, ArH), 8.11-8.15 (m, 2H, ArHs), 9.60 (s, 1H, NH, D2O-exchangeable), 11.25 (s, 1H, NH , D2O-exchangeable), 12.45, 1285 (s &br s, 1H, NH, D2O-exchangeable); Elemental Analysis Calcd for C25H23FN6O7: C 55.76, H 4.31, N 15.61; Found: C 55.49, H 4.27, N 15.24. 4.1.8. Ethyl 3-[2-({3,5-dimethyl-1H-pyrazol-1-yl}carbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2-oxo1,2,3,4-tetrahydropyrimidine-5-carboxylate (7) 18

An equimolar mixture of hydrazide(2) (1 mmol) and acetyl acetone (1mmol) in absolute methanol (4 mL) was refluxed for 12 h. The reaction was then allowed to cool and the separated product was filtered, washed with methanol, dried and recrystallized from ethanol; Yield: 0.42 g (91.5%); MP: 195 oC; IR: 3204, 1728, 1700, 1684, 1635, 1527, 1242, 1080 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.16 (t, J = 8 Hz, 3H, CH3CH2), 2.16 (s, 3H, CH3 pyrazole), 2.27 (s, 3H, C6-CH3), 2.43 (s, 3H, CH3 pyrazole), 3.19-3.41 (m, 3H, CH2CH), 3.83-3.88 (m, 1H, CH), 3.98-4.10 (m, 2H, CH3CH2), 5.56 (s, 1H, C4-H), 6.15 (s, 1H, pyrazole C4’H), 7.67-8.18 (m, 4H, ArHs), 9.65 (s, 1H, NH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.25, 13.88, 13.91, 17.57, 33.78, 41.15, 59.46, 66.31, 99.18, 111.06, 121.66, 122.65, 130.28, 133.40, 143.06, 145.04, 147.56, 148.39, 151.35, 151.39, 164.65, 171.67; Elemental Analysis Calcd for C22H25N5O6: C 58.01, H 5.53, N 15.38; Found: C 57.66, H 5.51, N 15.56. 4.1.9. Dialkyl 3,3'-[2-(3-{5-(ethoxycarbonyl)-4-methyl-6-(3-nitrophenyl)-2-oxo-2,3-dihydropyrimidin1(6H)-yl}propanoyl)hydrazine-1,1-diyl]dipropanoate (8a & b) A mixture of hydrazide (2) (1 mmol), the appropriate acrylate (1.2 mmol) and 4Dimethylaminopyridine (DMAP) (3 mmol) in DMF (20 mL) was stirred at 70oC for 3 days. The solvent was removed under reduced pressure. The resulting residue was purified by gradient column chromatographyusing ethyl acetate in hexanes to afford the purified products. 4.1.9.1. Dimethyl 3,3'-[2-(3-{5-(ethoxycarbonyl)-4-methyl-6-(3-nitrophenyl)-2-oxo-2,3dihydropyrimidin-1(6H)-yl}propanoyl)hydrazine-1,1-diyl]dipropanoate (8a) Yield: 0.3 g (55.6%); Oil; IR: 3224, 3097, 1735, 1675, 1639, 1530, 1235, 1198, 1085, 1028 cm-1;1H-NMR (CDCl3, 400 MHz) δ 1.13-1.19 (m, 3H, CH3CH2), 2.01 (s, 3H, CO2CH3), 2.28 (s, 3H, C6-CH3), 2.35-2.64 (m, 4H, CH2CH2), 2.82-3.17 ( m, 5H, CH2CH2 &CH), 3.58 (t,J =6 Hz, 3H, CO2CH3), 3.64-3.86 (m, 3H, CH2CH), 3.93-4.20 (m, 2H, CH3CH2), 5.49, 5.53, 5.59 (3s, 1H, C4-H), 7.44-7.50 (m, 2H, ArH & NH, D2O-exchangeable), 7.67(d, J = 4 Hz,1H, ArH), 8.08 (d, J = 8 Hz,1H, ArH), 8.17 (d, J = 12 Hz, 1H, ArH), 8.81, 8.99 (2s, 1H, NH, D2O-exchangeable);HRMS (ESI) Calcd for C25H33N5O10Na [M+Na] +: 586.2120; Found: 586.2125. 4.1.9.2. Diethyl 3,3'-[2-(3-{5-(ethoxycarbonyl)-4-methyl-6-(3-nitrophenyl)-2-oxo-2,3-dihydropyrimidin1(6H)-yl}propanoyl)hydrazine-1,1-diyl]dipropanoate (8b) Yield: 0.3 g (52.8%); MP: 129 oC; 3217, 3092, 1729, 1666, 1644, 1532, 1249, 1180, 1089, 1027 cm-1;1HNMR (CDCl3, 400 MHz) δ 1.17-1.29 (m, 9H, 3CH3CH2), 2.27-2.34 (m, 1H, CH), 2.36 (s, 3H, C6-CH3), 2.41-2.45 (m, 1H, CH), 2.47-2.62 (m, 4H, CH2CH2), 2.84-2.95 (m, 1H, CH), 3.04-3.26 (m, 4H, CH2CH2), 3.67-3.84 (m, 1H, CH), 4.05-4.17 (m, 6H, 3CH3CH2), 5.56, 5.65 (2s, 1H, C4-H), 6.48 (s, 1H, NH, D2Oexchangeable), 7.48-7.54 (m, 1H, ArH), 7.75(t, J = 8 Hz, 1H, ArH) 8.00 (s, 1H, NH, D2Oexchangeable), 8.15 (m, 1H, ArH) 8.21, 8.23, 8.26 (m, 1H, ArH); 13C-NMR (Acetone-d6, 400 MHz) δ 14.55, 14.56, 14.61, 18.34, 18.38, 31.16, 32.94, 33.57, 33.80, 42.21, 42.79, 52.91, 55.03, 60.49, 60.52, 60.71, 60.97, 61.04, 61.09, 61.22, 61.23, 101.15, 101.25, 122.99, 123.14, 123.55, 123.60, 130.46, 130.99, 134.44, 134.47, 146.27, 146.47, 148.78, 148.94, 149.23, 149.25, 152.97, 165.68, 165.78, 170.61, 172.30, 172.33, 172.46, 175.58; HRMS (ESI) Calcd for C27H37N5O10Na [M+Na] +: 614.2433; Found: 614.2427. 19

4.1.10. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(2-{substitutedcarbamothioyl or carbamoyl}hydrazinylcarbonyl)ethyl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (9a-e) An equimolar mixture of hydrazide (2) (1 mmol) and the appropriate substituted isothiocyanate or isocyanate (1mmol) in ethanol (20 ml) was allowed to reflux for 8-48 h. On cooling, the separated product was filtered, washed with cold ethanol, dried and recrystallized from the appropriate solvent (s). 4.1.10.1. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(2-{phenylcarbamothioyl}hydrazinylcarbonyl) ethyl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (9a) Yield: 0.48 g (91.6%); purified by recrystallization from ethanol/acetone (7:3); MP: 121 oC; 3251, 1687, 1645, 1529, 1529, 1348, 1236, 1022, 1236, 1086 cm-1;1H-NMR (DMSO, 400 MHz) δ 1.16 (t, J = 8 Hz, 3H, CH3CH2), 2.29 (s, 3H, C6-CH3), 2.37-2.42 (m, 1H, CH), 2.57-2.64 (m, 1H, CH), 3.12-3.19 (m, 1H, CH), 3.71- 3.79 (m, 1H, CH), 3.95-4.10 (m, 2H, CH3CH2), 5.62 (s, 1H, C4-H), 7.17-8.22 (m, 9H, ArHs), 9.59, 9.63 (2s, 3H, 3NH, D2O-exchangeable), 10.00 (s, 1H, NH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.94, 17.65, 32.22, 41.47, 59.51, 69.66, 99.35, 121.64, 122.78, 123.34, 125.01, 126.01, 127.99, 128.02, 130.40, 133.41, 139.07, 145.17, 147.69, 148.28, 151.53, 164.72; HRMS (ESI) Calcd for C24H27N6O6S [M+1] +: 527.1707; Found:527.1703. 4.1.10.2. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(2-{p-tolylcarbamothioyl}hydrazinylcarbonyl) ethyl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (9b) Yield: 0.43 g (80%); purified by column chromatography using gradient ethyl acetate in hexanes; MP: 118 oC; 3232, 1679, 1641, 1527, 1527, 1348, 1236, 1022, 1236, 1083 cm-1;1H-NMR (DMSO, 400 MHz) δ 1.13 (t, J = 8 Hz, 3H, CH3CH2), 2.09 (s, 1H, CH), 2.23 (s, 3H, C6H4CH3), 2.28 (s, 3H, C6-CH3), 2.612.70 (m, 1H, CH), 3.01-3.12 (m, 1H, CH), 3.62- 3.71 (m, 1H, CH), 3.92-4.07(m, 2H, CH3CH2), 5.55 (s, 1H, C4-H), 7.12 (d, J = 8 Hz, 2H, ArHs), 7.27 (d, J = 8 Hz, 2H, ArHs), 7.69 (t, J = 8 Hz, 1H, ArH), 7.76 (d, J = 8 Hz, 1H, ArH), 8.16 (t, J = 10 Hz, 1H, ArH), 9.29, 9.46, 9.58 (3s, 3H, 3NH, D2Oexchangeable), 9.90 (s, 1H, NH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.94, 17.63, 20.50, 32.17, 41.44, 59.49, 59.51, 99.32, 121.61, 122.79, 123.41, 125.65, 125.88, 126.04, 126.06, 128.45, 128.48, 130.41, 133.41, 136.47, 145.16, 147.69, 148.28, 151.50, 164.72; HRMS (ESI) Calcd for C25H29N6O6S [M+1] +: 541.1864; Found: 541.1870. 4.1.10.3. Ethyl 3-[2-(2-{benzhydrylcarbamothioyl}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl) -2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (9c) Yield: 0.48 g (79%); purified by recrystallization from ethanol/dioxane (8:2); MP: 140 oC; 3250, 1693, 1646, 1528, 1528, 1349, 1235, 1024, 1235, 1085 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.08-1.16 (m, 3H, CH3CH2), 2.82, 2.92 (2s, 3H, C6-CH3), 2.33-2.39 (m, 1H, CH), 2.56-2.63 (m, 1H, CH), 3.06-3.13 (m, 1H, CH), 3.41- 3.53 (m, 1H, CH), 3.68-3.76 (m, 1H, CH(C6H5)2), 3.94-4.10 (m, 2H, CH3CH2), 5.61 (s, 1H, C4-H), 7.27-7.42 (m, 10H, ArHs), 7.66-7.81 (m, 2H, ArHs), 8.18-8.21 (m, 2H, ArHs), 8.68, 8.69 (d, J = 8 Hz, 1H, NH, D2O-exchangeable), 9.14, 9.27, 9.39 (2s, 1H, NH, D2O-exchangeable), 9.61, 9.67 (2s, 1H, NH, D2O-exchangeable), 9.83, 9.99 (s &br s, 1H, NH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) 20

δ 13.95, 17.66, 32.17, 41.58, 59.51, 60.58, 66.33, 99.33, 121.61, 122.79,126.98, 127.60, 127.66, 127.68, 128.19, 130.41, 133.37, 141.62, 141.65, 141.69, 145.16, 147.70, 148.29, 151.49, 164.73; HRMS (ESI) Calcd for C31H33N6O6S [M+1] +: 617.2177; Found: 617.2190. 4.1.10.4. Ethyl 3-[2-(2-{allylcarbamothioyl}hydrazinylcarbonyl)ethyl]-6-methyl-4-(3-nitrophenyl)-2oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (9d) Yield: 0.43 g (87.8%); purified by recrystallization from ethanol; MP: 135 oC; 3284, 3238, 1709, 1689, 1640, 1527, 1527, 1347, 1248, 1021, 1221, 1081 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.07, 1.16 (2t, J= 6, 8 Hz, 3H, CH3CH2), 2.26 (s, 3H, C6-CH3), 2.29-2.35 (m, 1H, CH), 2.48-2.52 (m, 1H, CH), 3.04-3.11 (m, 1H, CH), 3.66-3.71 (m, 1H, CH), 3.95-4.08 (m, 2H, CH3CH2), 4.11 (s, 2H, allyl CH2), 5.04-5.15(m, 2H, allyl HA & HM), 5.57 (s, 1H, C4-H), 5.79-5.88 (group of s, 1H, allyl HX), 7.71 (t, J = 8 Hz, 1H, ArH ), 7.77 (t, J = 8 Hz, 1H, ArH), 8.09 (s, 1H, ArH), 8.15-8.19 (m, 2H, ArHs), 9.21 (s, 1H, NH, D2Oexchangeable), 9.60, 9.62 (2s, 1H, NH, D2O-exchangeable), 9.78 (s, 1H, NH, D2O-exchangeable); 13CNMR (DMSO, 400 MHz) δ 13.94, 17.63, 32.08, 41.45, 45.74, 59.39, 59.51, 99.32, 115.09, 121.57, 122.76,122.79, 130.41, 133.37, 134.88, 145.11, 147.69, 148.26, 151.52, 164.70, 170.15; HRMS (ESI) Calcd for C21H27N6O6S [M+1] +: 491.1707, Found: 491.1702. 4.1.10.5. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(2-{phenylcarbamoyl}hydrazinylcarbonyl)ethyl]1,2,3,4-tetrahydropyrimidine-5-carboxylate (9e) Yield: 0.32 g (82.6%); purified by recrystallization from ethanol; MP: 131oC; 3239, 1667, 1640, 1601, 1528, 1348, 1236, 1025, 1236, 1083 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.08-1.18, 1.25 (m &t, J = 6 Hz, 3H, CH3CH2), 2.27 (s, 3H, C6-CH3), 2.53-2.58 (m, 1H, CH), 3.05-3.12 (m, 1H, CH), 3.36-3.40 (m, 1H, CH), 3.67- 3.73 (m, 1H, CH), 3.91-4.14 (m, 2H, CH3CH2), 5.57, 5.59 (s, 1H, C4-H), 6.96 (t, J = 8 Hz, 1H, ArH), 7.26 (t, J = 8 Hz, 2H, ArHs), 7.47 (d, J = 8 Hz, 2H, ArHs), 7.70 (t, J = 8 Hz, 1H, ArH), 7.79 (d, J = 8 Hz, 1H, ArH), 8.04 (s, 1H, ArH), 8.18 (s, 1H, ArH), 8.20 (s, 1H, NH, D2O-exchangeable), 8.74 (s, 1H, NH, D2O-exchangeable), 9.61 (s, 1H, NH, D2O-exchangeable), 9.75 (s, 1H, NH, D2Oexchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.86, 17.58, 32.02, 41.49, 59.43, 59.48, 99.36, 118.48, 121.69, 121.92, 122.79, 128.58, 130.36, 133.43, 139.46, 145.14, 147.66, 148.18, 151.46, 155.26, 164.72, 170.47; HRMS (ESI) Calcd for C24H27N6O7[M+1] +: 511.1936; Found: 511.1942. 4.1.11. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(5-{phenylamino}-1,3,4-thiadiazol-2-yl)ethyl]1,2,3,4-tetrahydropyrimidine-5-carboxylate (10) A mixture of the thiosemicarbazide (9a) (1mmol) and concentrated H2SO4 (1 mL) was stirred at 0-5oC for 1 h then stirring was continued for overnight at room temperature. After reaction completion, the mixture was poured onto saturated cold sodium carbonate solution. The precipitated solid was filtered, washed with water and recrystallized from aqueous methanol; Yield: 0.4 g (80%); MP: 206oC; 3213, 1705, 1684, 1636, 1526, 1232, 1080, 750 cm-1;1H-NMR (DMSO, 400 MHz) δ 1.09 (t, J = 8 Hz, 3H, CH3CH2), 2.25 (s, 3H, C6-CH3), 3.06-3.21 (m, 3H, CH2CH), 3.89-4.00 (m, 3H, CH3CH2& CH), 5.45 (s, 1H, C4-H), 6.958.15 (m, 9H, ArHs), 9.64 (s, 1H, NH, D2O-exchangeable), 10.27 (s, 1H, NH, D2O-exchangeable); 13CNMR (DMSO, 400 MHz) δ 13.82, 17.55, 27.89, 44.24, 59.08, 59.53, 99.34, 117.14, 121.60, 121.66,

21

122.79, 128.98, 130.38, 133.28, 140.66, 144.71, 147.67, 148.40, 151.62, 156.39, 164.41, 164.62; Elemental Analysis Calcd for C24H24N6O5S: C 56.68, H 4.76, N 16.53; Found: C 56.61, H 4.70, N 16.10. 4.1.12.1. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(5-{arylamine})-1,3,4-oxadiazol-2-yl)ethyl]1,2,3,4-tetrahydropyrimidine-5-carboxylate (11a & b) A mixture of the thiosemicarbazide (9a) or (9b) (1mmol) and yellow HgO (2 mmol) in methanol was stirred under reflux for 12-16 h. The reaction mixture was allowed to cool to room temperature and the black mercuric sulfide was filtered and washed with ethanol. The filtrate and alcoholic washing were combined and evaporated under reduced pressure and the residue was purified by recrystallization from acetone/ ethanol (1:1). 4.1.12.1. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(5-{phenylamino}-1,3,4-oxadiazol-2-yl)ethyl]1,2,3,4-tetrahydropyrimidine-5-carboxylate (11a) Yield: 0.2 g (41%); MP: 206oC; 3286, 3209, 1685, 1630, 1530, 1239, 1091 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.08, 1.13 (2t, J= 8, 6 Hz, 3H, CH3CH2), 2.10, 2.26 (2s, 3H, C6-CH3), 2.90-3.05 (m, 2H, CH2), 3.16-3.23 (m, 1H, CH), 3.90-4.03 (m, 3H, CH3CH2& CH), 5.54, 5.56 (2s, 1H, C4-H), 6.99 (t, J = 8 Hz, 1H, ArH), 7.34 (t, J = 8 Hz, 2H, ArHs), 7.58 (d, J = 8 Hz, 2H, ArHs), 7.69 (t, J= 6Hz, 1H, ArH), 7.80 (d, J= 8Hz, 1H, ArH), 8.18 (d, J = 8 Hz, 1H, ArH), 8.21 (t, J= 4Hz, 1H, ArH), 9.66, 9.68 (2s, 1H, NH, D2Oexchangeable), 10.39 (s, 1H, NH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.86, 17.62, 23.62, 42.34, 59.21, 59.48, 99.25, 116.74, 121.47, 121.77, 122.81, 128.93, 130.37, 133.40, 138.83, 144.98, 147.67, 148.34, 151.43, 157.74, 159.78, 164.60; HRMS (EI) Calcd for C24H24N6O6 [M] +: 492.1752; Found: 492.1748. 4.1.12.2. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(5-{p-tolylamino}-1,3,4-oxadiazol-2-yl)ethyl]1,2,3,4-tetrahydropyrimidine-5-carboxylate (11b) Yield: 0.37 g (70%); MP: 145oC; 3211, 1676, 1632, 1528, 1233, 1082 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.13 (t, J= 8 Hz, 3H, CH3CH2), 2.21 (s, 3H, C6-CH3), 2.39 (s, 3H, C6H4CH3), 2.46-2.54 (m, 1H, CH), 2.66-2.75 (m, 1H, CH), 2.99-3.09 (m, 1H, CH), 3.61-3.69 (m, 1H, CH), 3.90-4.04 (m, 2H, CH3CH2), 5.31 (s, 1H, C4-H), 7.22 (d, J= 8Hz, 2H, ArHs), 7.33 (d, J= 8Hz, 2H, ArHs), 7.61 (d, J= 4Hz, 2H, ArHs), 8.02 (dist.s, 1H, ArH), 8.10-8.14 (m, 1H, ArH), 9.55 (s, 1H, NH, D2O-exchangeable); HRMS (ESI) Calcd for C25H26N6O6 [M] +: 506.1908; Found: 506.1914. 4.1.13.1. Ethyl 3-[2-(2-{4-(4-sunstituted phenyl)-3-arylthiazol-2(3H)-ylidene}hydrazinylcarbonyl)ethyl]6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (12a-d) A suspension of thiosemicarbazide (9a) or (9b) (1 mmol) in absolute methanol (20 mL) and the appropriate substituted 2-bromoacetophenone derivative (1 mmol) was stirred under reflux for 4-23 h. The reaction mixture was then concentrated under reduced pressure and allowed to cool overnight. The separated precipitate was filtered and purified by recrystallization from the proper solvent(s).

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4.1.13. Ethyl 3-[2-(2-{4-(4-fluorophenyl)-3-phenylthiazol-2(3H)-ylidene}hydrazinylcarbonyl)ethyl]-6methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (12a) Yield: 0.4 g (63%); purified by recrystallization from dichloromethane/petroleum ether (3:2); MP: 263oC; IR: 3150, 1705, 1666, 1636, 1529, 1237, 1074, 1165, 1017 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.12 (t, J = 8 Hz, 3H, CH3CH2), 2.17-2.23 (m, 1H, CH), 2.25 (s, 3H, C6-CH3), under DMSO (m, 1H, CH), 2.943.01 (m, 1H, CH), 3.66-3.73 (m, 1H, CH), 3.91-4.06 (m, 2H, CH3CH2), 5.52 (s, 1H, C4-H), 6.56 (s, 1H, CHThiazole), 7.07-8.18 (m, 13H, ArHs), 9.60 (s, 1H, NH, D2O-exchangeable), 10.12 (s, 1H, NH, D2Oexchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.98, 17.60, 32.38, 41.66, 59.46, 59.52, 99.20, 115.64, 121.49, 122.78, 123.47, 123.56, 123.68, 126.86, 130.25, 130.34, 133.42, 139.54,140.46, 144.94, 145.07, 147.67, 148.24, 151.41, 153.23, 154.32, 161.25, 164.64, 169.78, 169.85; HRMS (ESI) Calcd for C32H29N6O6FS [M] +: 644.1848; Found: 644.1852. 4.1.13.2. Ethyl 3-[2-(2-{4-(4-nitrophenyl)-3-phenylthiazol-2(3H)-ylidene}hydrazinylcarbonyl)ethy]-6methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (12b) Yield: 0.29 g (42.5%); purified by recrystallization from ethanol/acetone (9:1); MP: 262oC; IR: 3156, 1703, 1635, 1524, 1242, 1077, 1199, 1016 cm-1;1H-NMR (DMSO, 400 MHz) δ 1.04-1.18 (m, 3H, CH3CH2), 2.03-2.18 (m, 1H, CH), 2.23, 2.24 (2s, 3H, C6-CH3), 2.31-2.50(m, 0.5H, CH), 2.58-2.70(m, 1H, CH), 2.74-2.90 (m, 1H, CH), 2.93-3.09 (m, 0.5H, CH), 3.40-3.56 (m, 1H, CH), 3.85-4.10 (m, 2H, CH3CH2), 5.41-5.62 (m, 1H, C4-H), 6.83-7.02, 7.13-7.21 (m, 1H, CH Thiazole), 7.37-7.45 (m, 3H, ArHs), 7.58 (q, J= 8 Hz, 2H, ArHs,), 7.63-7.86 (m, 4H, ArHs), 8.08-8.34 (m, 4H, ArHs), 9.55-9.63 (m, 1H, NH, D2O-exchangeable), 11.03-11.28 (m, 0.5H, NH, D2O-exchangeable), 12.00-12.07 (2 br, 0.5H, NH, D2Oexchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.94,17.46, 32.08, 40.76, 59.45, 66.31, 99.11, 101.78, 121.49, 122.76, 123.57, 123.78, 127.78, 127.80,129.60, 130.25, 130.33, 133.40,138.37, 140.84, 144.89, 147.65, 151.39, 155.08, 160.61, 164.64, 169.72, 169.78; HRMS (ESI) Calcd for C32H29N7O8S [M] +: 671.1774; Found: 671.1793. 4.1.13.3. Ethyl 3-[2-(2-{4-(4-fluorophenyl)-3-(p-tolyl)thiazol-2(3H)-ylidene}hydrazinylcarbonyl)ethyl]6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (12c) Yield: 0.33 g (50.7%); purified by recrystallization from ethanol/DMF (9:1); MP: 258oC; IR: 3150, 1706, 1667, 1636, 1529, 1241, 1076, 1207, 1017 cm-1;1H-NMR (DMSO, 400 MHz) δ 1.03-1.15 (m, 3H, CH3CH2), 2.06-2.17 (m, 0.5H, CH), 2.22, 2.23 (2s, 3H, C6-CH3), 2.35 (s, 3H, C6H4CH3), 2.57-2.61(m, 0.5H, CH), 2.68-2.79 (m, 1H, CH), 2.89-3.07 (m, 1H, CH), 3.41-3.50(m, 1H, CH), 3.89-4.09 (m, 2H, CH3CH2), 5.45, 5.51 (2s, 1H, C4-H), 7.04 (m, 1H, CHThiazole), 7.15-7.77 (m, 10H, ArHs), 8.09-8.19 (m, 2H, ArHs), 9.60,9.61 (dist. d, 1H, NH, D2O-exchangeable), 11.84 (br s, 1H, NH, D2O-exchangeable); HRMS (ESI) Calcd for C33H31N6O6FS [M] +: 658.2004; Found: 658.2022. 4.1.13.4. Ethyl 6-methyl-4-(3-nitrophenyl)-3-[2-(2-{4-(4-nitrophenyl)-3-(p-tolyl)thiazol-2(3H)ylidene}hydrazinylcarbonyl)ethyl]-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (12d) Yield: 0.25 g (38.7%); purified by recrystallization from ethanol/DMF; MP: 228oC; IR: 3217, 3151, 1702, 1682, 1636, 1528, 1244, 1079, 1200, 1011 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.04-1.18 (m, 3H, 23

CH3CH2), 2.20, 2.21 (2s, 3H, C6-CH3), 2.27, 2.29, 2.3 (3s, 3H, C6H4CH3), 2.56-2.73 (m, 1H, CH), 2.842.92, 2.98-3.05 (m, 1H, CH), 3.53-3.58 (m, 1H, CH), 3.69-3.79 (m, 1H, CH), 3.88-4.07 (m, 2H, CH3CH2), 5.41, 5.45, 5.52, 5.55, 5.58 (group of singlets, 1H, C4-H), 6.85-6.90 (m, 1H, CHThiazole), 6.97-6.99 (m, 1H, ArH), 7.12-7.22 (m, 2.5H, ArHs), 7.47 (d, J = 8 Hz, 0.5H, ArH), 7.63-7.78 (m, 4H, ArHs), 8.10-8.32 (m, 4H, ArHs), 9.58-9.64 (m, 1H, NH, D2O-exchangeable), 10.38, 11.33 (2br s, 1H, NH, D2Oexchangeable); HRMS (ESI) Calcd for C33H31N7O8S [M] +: 658.1949; Found: 658.1935. 4.1.14.1. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(2-{4-oxo-3-arylthiazolidin-2ylidene}hydrazinylcarbonyl)ethyl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (13a &b) An equimolar mixture of the appropriate thiosemicarbazide derivatives (9a) or (9b) (1 mmol) and ethyl bromoacetate (1 mmol) in absolute methanol (20 mL) was refluxed for 10-23 h. The reaction mixture was concentrated under reduced pressure, diluted with water after cooling and allowed to stand overnight; the separated product was filtered off, dried and crystallized from the proper solvent(s). 4.1.14.1. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(2-{4-oxo-3-phenylthiazolidin-2-ylidene} hydrazinylcarbonyl)ethyl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (13a) Yield: 0.48 g (84.8%); purified by recrystallization from aqueous ethanol; MP: 122oC; IR: 3211, 1735, 1690, 1638, 1528, 1235, 1083, 770 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.16 (t, J = 8 Hz, 3H, CH3CH2), 2.24 (s, 3H, C6-CH3), 2.61-2.66 (m, 1H, CH), 2.81-2.86 (m, 1H, CH), 3.10-3.14 (m, 1H, CH), 3.69- 3.79 (m, 1H, CH), 3.97-4.02 (m, 2H, CH3CH2), 4.05 (s, 2H, thiazolidone CH2), 5.37 (s, 1H, C4-H), 7.41-8.15 (m, 9H, ArHs), 9.59 (s, 1H, NH, D2O-exchangeable); 13C-NMR (DMSO, 400 MHz) δ 13.91, 17.60, 23.46, 33.71, 52.41, 59.34, 59.48, 99.13, 121.61, 122.77, 128.94, 129.87, 129.97, 130.33, 132.56, 133.31, 145.00, 147.55, 148.28, 149.11, 151.23, 153.42, 164.54, 168.58; Elemental Analysis Calcd for C26H27N6O7.5S: C 54.25, H 4.73, N 14.60; Found: C 54.64, H 5.02, N 14.20. 4.1.14.2. Ethyl 6-methyl-4-(3-nitrophenyl)-2-oxo-3-[2-(2-{4-oxo-3-(p-tolyl)thiazolidin-2-ylidene} hydrazinylcarbonyl)ethyl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (13b) Yield: 0.3 g (51.7%); purified by recrystallization from ethanol/acetone (9:1); MP: 193oC; IR: 3221, 1752, 1714, 1672, 1634, 1525, 1224, 1077, 777 cm-1; 1H-NMR (DMSO, 400 MHz) δ 1.15, 1.18 (2t, J = 8, 8 Hz, 3H, CH3CH2), 2.23 (s, 3H, C6-CH3), 2.42 (s, 3H, C6H4CH3), 2.54-2.62 (m, 1H, CH), 2.77-2.84(m, 1H, CH), 3.08-3.15 (m, 1H, CH), 3.68- 3.75 (m, 1H, CH), 3.92-4.12 (m, 2H, CH3CH2), 4.04 (s, 2H, thiazolidinone CH2), 5.35 (s, 1H, C4-H), 7.27 (d, J = 8 Hz, 2H, ArHs), 7.38 (d, J = 12 Hz, 2H, ArHs), 7.61- 7.67 (m, 2H, ArHs), 8.06-8.14 (m, 2H, ArHs), 9.59 (s, 1H, NH, D2O-exchangeable); Elemental Analysis Calcd for C27H28N6O7S: C 55.85, H 4.86, N 14.47; Found: C 56.49, H 5.08, N 13.97. 4.2. Whole patch clamp assay 4.2.1. Chemicals

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Test compounds were dissolved in DMSO to prepare stock solutions of 10 or 30 mM concentrations. They were diluted prior to the experiments so that the final concentration of DMSO was 0.1 % or less. Calcium channel currents were not affected by a concentration of 0.1 % DMSO.

4.2.2. Cell Culture and Transient Transfection Human embryonic kidney cells (HEK) tsA-201 cells were grown to 80~90% confluence at 37°C (5% CO2) in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY), supplemented with 10% (vol/vol) fetal bovine serum (HyClone, Thermo Scientific, Pittsburgh, PA), 200 U/ml penicillin, and 0.2 mg/ml streptomycin (Life Technologies, Grand Island, NY). Cells were suspended with 0.25% trypsin/ethylenediaminetetraacetic acid and plated onto glass coverslips in 10-cm culture dishes (Corning, Corning, NY) at 10% confluence 6 h before transfection. Calcium channel (5 µg) and green fluorescent protein marker (0.5 µg) DNAs were transfected into cells with calcium phosphate. For Cav1.2, the additional Cavβ1b (5 µg) and Cavα2δ1 (5 µg) subunits were coexpressed. Cells were transferred to 30°C 16-18 h later following transfection and stored for two days before recording. 4.2.3. Electrophysiology Cells on a glass coverslip were transferred into an external bath solution of 20 mM BaCl2, 1 mM MgCl2, 40 mM TEACl, 65 mM CsCl, 10 mM HEPES, 10 mM glucose, pH 7.4. Borosilicate glass pipettes (Sutter Instrument Co., Novato, CA, 3–5 MΩ) were filled with internal solution containing 140 mM CsCl, 2.5 mM CaCl2, 1 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 2 mM Na-ATP and 0.3 mM Na-GTP, pH 7.3. Whole-cell patch clamp recordings were performed by using an EPC 10 amplifier (HEKA Elektronik, Bellmore, NY) linked to a personal computer equipped with Pulse (V8.65) software (HEKA Elektronik, Bellmore, NY). After seal formation, the membrane beneath the pipette was ruptured and the pipette solution was allowed to dialyze into the cell for 2–5 min before recording. Voltage-dependent currents were leak corrected with an online P/4 subtraction paradigm. Data were recorded at 10 kHz and filtered at 2.9 kHz. T-type calcium currents were elicited by depolarization from a holding potential of -110 mV to a test potential of -20 mV and L-type calcium currents were elicited by depolarization from a holding potential of -90 mV to a test potential of +20 mV, with an interpulse interval of 20 s. The duration of the test pulse was typically 100 ms. 4.2.4. Data Analysis and Statistics. Data analysis was performed by using online analysis built in Pulse software (HEKA Elektronik, Bellmore, NY), and all graphs were prepared by using GraphPad Prism 5 (GraphPad Software, La Jolla, CA). All data were given as mean values ± standard errors. 4.3. In vivo experiments 4.3.1. Animals Experiments were approved by the Institutional Animal Care and Use Committee and conform to the guidelines of the International Association for the Study of Pain. Adult male C57BL/6 J wild-type (n=62, 7-9 weeks) mice were used (The Jackson Laboratory, CAN). Mice were kept on a 12-hr light/dark cycle

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(lights on at 7 am) and at a maximum of five per cage in a holding room kept at a temperature of 23 ± 1°C; food and water were available ad libitum. 4.3.2. Pretreatment with 6a/9c in the formalin-induced model In the formalin test, animals were pre-treated with sequential concentrations (0.3 µg, 1 µg and 3 µg in 10 µl) of 6a and 9c or vehicle (1% DMSO in PBS, 10 µl) via intrathecal (i.t) injection. After 20 minutes, animals were intraplantarly (i.pl.) injected with 20 µl of 1.25% formalin solution into the right hind paw as previously described 56,57. Briefly, two phases of nocifensive behavior (Phase I: 0-5 min, Phase II: 1530 min) were immediately recorded by counting the time spent on licking or biting the injected paw. 4.3.3. CFA-induced mechanical hypersensitivity and drug intervention Inflammatory chronic hypersensitivity was induced by Complete Freud’s Adjuvant (CFA) as previously described 46,58. Briefly, baseline of mechanical paw withdrawal threshold was measured for the animals and then 30 µl of CFA or PBS was intraplantarly injected into the right hind paw. Animals were treated with either 6a (3 µg/10 µl/i.t), 9c (1 µg/10 µg i.t) or vehicle (1% DMSO in PBS, 30 µl) based on the maximal effects in the formalin test, 2days following CFA injection. Mechanical paw withdrawal thresholds were measured in a blinded fashion at 0, 20, 45, 90 and 150 min post drug administration. All the animals were habituated in the chambers for at least 1 hour before measuring the baseline and time 0. 4.3.4. Measurement of mechanical hyperalgesia in the CFA model Mechanical hyperalgesia was measured using the digital plantar aesthesiometer (DPA,UgoBasile, Varese, Italy) as previously described [58]. Mice were individually placed in a plexiglass chamber (20 cm× 18.5 cm × 13 cm, length × width × height) over a wire mesh floor. The aesthesiometer was placed under the animal with the filament directly stimulating the central plantar surface of the ipsilateral hind paw. Each paw was tested three times with intervals between each stimulation and the force which elicited a paw withdrawal was recorded as a threshold. 4.3.5. Statistical analysis Data were analyzed with GraphPad Prism 5 and are presented as mean ± S.E.M. Behaviour data were analyzed by one-way ANOVA with Bonferroni post-hoc test for the formalin test and two-way ANOVA with Bonferroni post-hoc test for the CFA-induced model. Acknowledgments This research project was funded by The Egyptian Ministry of Higher Education and Scientific Research and the Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University. Gerald W. Zamponi is supported by a Foundation Grant from the Canadian Institutes of Health Research and holds a Canada Research Chair. References 1. C. Bladen, V. M. Gadotti, M. G. Gündüz, N. D. Berger, R. Şimşek, C. Şafak, G.W. Zamponi, Pflugers Arch – Eur. J. Physiol. 467 (2015)1237-1247.

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Highlights:    

Novel N3-substituted dihydropyrimidine derivatives as selective T-type calcium channel blockers. Evaluation of L-/T- calcium channel antagonism by whole patch clamp technique. Novel N3-substituted dihydropyrimidines reduce the formalin-induced nocifensive response Novel N3-substituted dihydropyrimidines reduce the CFA-induced mechanical hyperalgesia

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