Anti-Covid-19 Drug Analogues: Synthesis of Novel Pyrimidine Thioglycosides as Antiviral Agents Against SARS-COV-2 and Avian Influenza H5N1 Viruses.

A class of pyrimidine thioglycoside analogs (6a-h) were synthesized from a reaction of 2-cyano-3,3-dimercapto-N-arylacrylamide (2a-d) and thiourea to produce the corresponding 4-amino-2-mercapto-N-arylpyrimidine-5-carboxamide derivatives (3a-d), and stirring of compounds (3a-d) with peracylated α-d-gluco- and galacto-pyranosyl bromides (4a,b) in DMF-sodium hydride gave the corresponding pyrimidine thioglycosides (5a-h). Deacetylation of the pyrimidine thioglycosides via a reaction with dry NH3/MeOH gave the corresponding free pyrimidine thioglycosides (6a-h). The compounds have been characterized by 13C NMR, 1H NMR, and IR. Pharmacological evaluation of compounds 3a-d, 5a-h, and 6a-h in vitro against SARS-COV-2 and Avian Influenza H5N1 virus strains revealed that some compounds possess interesting activity.

Introduction

Corona viruses n class="Chemical">are a wide group of viruses that infect many different animals, and they have caused severe and dangerous respiratory infections in humans.[1,2] Between 2002 and 2012, two types of corona viruses with the animal origin, severe acute respiratory syndrome coronavirus (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), arose in humans and caused deadly respiratory infection, making emerging corona viruses a source of upcoming and new public health concern in the world during this century.[3] On December 31, 2019, the World Health Organization announced the emergence of a new corona virus called SARS-CoV-2 in the city of Wuhan, China, and this virus has caused an outbreak of viral pneumonia that is rapidly contagious and spreads among humans all over the world leading to death.[4,5] The new corona virus disease is also known as corona virus disease 2019 (COVID-19). It has tremendously surpassed SARS and MERS in terms of the number of infected peoples and the number of areas of the epidemic.[6] The continuing and rapid spread of COVID-19 has now become a serious threat to human health in this world. It has become imperative for the researchers in the field of medicinal chemistry to develop strategic research plans in order to produce medicines to combat these corona viruses. Clearly, no antiviral drugs are approved for treatment of corona viruses. Accordingly, corona viruses have been given importance status by governments for development of avoidance and management strategies due to harshness of these infections and sound epidemic potential.[7−9] Recently, many research and scientific projects focus on trials to development of antiviral nucleoside analogues targeting viral ribonucleic acid synthesis as effective therapeutics against covid-19 virus infections. Nevertheless, the recent development of nucleotide and nucleoside analogue inhibitors with a broad-spectrum activity against multiple covid-19 and a high barrier for resistance holds promise for the treatment of covid-19 disease.[10−13]

Nucleoside analogue inhibitors, currently used to treat vn class="Gene">iral infections, are chemically synthesized analogues of purines and pyrimidines in which the sugar moiety or heterocyclic ring has been altered.[14−16] Nucleoside analogues are managed as prodrugs, which are metabolized by host or viral kinases to their active triphosphate once inside the cell. Nucleoside analogues exert inhibitory effects on viral replication by one or more mechanisms. Through these mechanisms, nucleoside analogues alter the genetic character of the virus, leading to a decrease in viral qualification with every successive replication cycle. Finally, nucleoside analogues could potentially serve as broad-spectrum inhibitors of Covid-19 infection.[17] Several antiviral nucleoside and nucleotide analogues reported in the literature, which are used as antiviral drugs, effective with SARS-CoV-2 are shown in (Figure ). The drugs Remdesivir and Avigan have recently been permitted for use in the treatment of infections from the corona virus pandemic.[18,19]

Figure 1

Nucleoside analogues with demonstrated activity against Covid-19.

The covid-19 and influenza virus H5N1 have matching symptoms. They origin diseases that disturb the breathing system, which may lead to death. Both viruses spread from one person to another through contact.

In our recent work, we have synthesized many antiviral heterocyclic n class="Chemical">thioglycosides, such as purine thioglycosides,[20,21] pyrimidine thioglycosides,[22,23] pyridine thioglycosides,[24,25] quinolone thioglycosides,[26] triazole thioglycosides,[27] thiazole thioglycosides,[28] oxadiazole thioglycosides,[29] imidazole thioglycosides,[30] pyrazole thioglycosides,[31] and thienopyrazole thioglycosides,[32] which exhibited active cytotoxicity, and also we described that dihydropyridine thioglycosides are used as inhibitors in the protein glycosylation process.[33,34]

The main purpose of this work is to synthesize a number of novel n class="Chemical">pyrimidine thioglycosides as pyrimidine nucleoside analogues, which showed an interesting result toward Covid-19 and H5N1 virus strains. This synthesis is accomplished through the reaction of 2-cyano-3,3-dimercapto-N-aryl acrylamide and thiourea, followed by coupling of the resulting pyrimidines with α-halogeno sugars.

Results and Discussion

The synthesis of our desired n class="Chemical">thiopyrimidine derivatives achieved using a mixture of 2-cyano-3,3-dimercapto-N-arylacrylamide (2a–d) and thiourea in absolute ethanol containing drops of piperidine provided thiopyrimidine derivatives (3a–d) in high yields as the sole product (Scheme ). The structures of compounds (3a–d) have been confirmed by using spectral and chemical measurements. Thus, the 1H NMR spectrum of compound 3a showed the absence of a cyano group, which was detected in parent 2, and the emergence of a signal at 6.86 ppm was attributed to an NH2 group. The coupling between the aglycon (3a-d) and activated sugars was achieved in the presence of a basic medium at room temperature to give in a good yield the corresponding pyrimidine S-glycosides (5a–h) (Scheme ). It has been proposed that the cis-(α) sugars interact via a simple SN2 reaction to give the β-glycoside products.[35] Structures of (5a–h) were confirmed based on the spectral data (13C NMR, 1H NMR, and IR). For example, the 1H NMR spectrum of 5a showed the anomeric proton as a doublet at δ = 4.96 ppm with a spin–spin coupling constant (J = 9.8 Hz) demonstrating the β-configuration. The other six protons of glucose were resonated at δ 3.97–4.9 6 ppm, when thioglycosides (5a–h) were reacted with NH3–MeOH at room temperature for 10 min. The deprotected derivatives (6a–h) were obtained in good yields (Scheme ). The structures were confirmed based on the spectral data and elemental analysis. Thus, the 1H NMR spectrum of 6a showed the anomeric proton as a doublet at δ 4.62 ppm with a spin–spin coupling constant (J = 9.4 Hz), signifying only a β-d-configuration and also proved by the 13C NMR spectrum, which exhibited a signal at δ 83.25 ppm corresponding to C-1′. The signals at δ 61.82, 80.14, 69.54, 78.11, and 75.12 ppm correspond to C-6′, C-5′, C-4′, C-3′, and C-2′.

Scheme 1

Synthesis of Pyrimidine Thioglycoside Derivatives 5a–h

Scheme 2

Synthesis of Free Pyrimidine Thioglycoside Derivatives 6a–h

Antiviral Activity

H5N1 Influenza Virus

Antiviral activity of the synthesized compounds was evaluated with respect to n class="Species">H5N1 influenza virus strain A/Egypt/M7217B/2013 utilizing 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxicity (TC50) and Plaque reduction assays exploring inhibition and cytotoxicity percentage values. The accumulated data for inhibition activities and cytotoxicity (see Table and Figures , 3) indicated that most of the compounds demonstrated dose-dependent inhibition behavior.

Table 1

Activity of Tested Compounds (3a–d, 5a–h, and 6a–h) against H5N1 Virus Measured Using the Plaque Reduction Assay

comp. no.	concentration (μM)	initial viral count	viral count PFU/mL	inhibition %	comp. no.	concentration (μM)	initial viral count	viral count PFU/mL	inhibition %
3a	0.125	3.1 × 106	6 × 106	48.33	5g	0.125	3.4 × 106	6 × 106	43.33
	0.25	3.8 × 106	6 × 106	36.67		0.25	2.5 × 106	6 × 106	58.33
3b	0.125	6.0 × 106	6 × 106	0.00	5h	0.125	3.0 × 106	6 × 106	50.00
	0.25	3.0 × 106	6 × 106	50.00		0.25	3.2 × 106	6 × 106	46.67
3c	0.125	3.4 × 106	6 × 106	43.33	6a	0.125	2.3 × 106	6 × 106	61.67
	0.25	4.0 × 106	6 × 106	33.33		0.25	2.5 × 106	6 × 106	58.33
3d	0.125	3.0 × 106	6 × 106	50.00	6b	0.125	3.3 × 106	6 × 106	45.67
	0.25	3.2 × 106	6 × 106	46.67		0.25	3.0 × 106	6 × 106	50.00
5a	0.125	2.5 × 106	6 × 106	58.33	6c	0.125	3.4 × 106	6 × 106	56.33
	0.25	2.3 × 106	6 × 106	61.67		0.25	3.2 × 106	6 × 106	63.67
5b	0.125	3.1 × 106	6 × 106	48.33	6d	0.125	4.0 × 106	6 × 106	61.33
	0.25	1.3 × 106	6 × 106	63.67		0.25	2.4 × 106	6 × 106	60.00
5c	0.125	3.4 × 106	6 × 106	43.33	6e	0.125	2.0 × 106	6 × 106	71.67
	0.25	2.5 × 106	6 × 106	58.33		0.25	2.2 × 106	6 × 106	78.33
5d	0.125	4.0 × 106	6 × 106	33.33	6f	0.125	3.5 × 106	6 × 106	66.67
	0.25	3.40 × 106	6 × 106	43.33		0.25	2.5 × 106	6 × 106	83.33
5e	0.125	3.0 × 106	6 × 106	50.00	6g	0.125	3.6 × 106	6 × 106	65.67
	0.25	2.0 × 106	6 × 106	66.67		0.25	1.3 × 106	6 × 106	63.33
5f	0.125	2.9 × 106	6 × 106	51.67	6h	0.125	3.4 × 106	6 × 106	41.67
	0.25	1.3 × 106	6 × 106	63.33		0.25	1.0 × 106	6 × 106	58.33

Figure 2

Cytotoxicity of compounds 3a, 3b, 3c, 3d, 5a, 5b, 5c, 5d, 5e, 5f, 5g, and 5h at concentrations of 0.25 and 0.125 μM.

Figure 3

Cytotoxicity of compounds 6a, 6b, 6c, 6d, 6e, 6f, 6g, and 6h at concentrations of 0.25 and 0.125 μM.

All tested compounds exhibited the highest to moderate and low potency activity toward n class="Species">H5N1 virus. Attachment of sugar moieties, especially deprotected sugars, to the substituted N-aryl pyrimidine derivatives (compounds 6a–h) demonstrated higher inhibition activity than protected sugar (compounds 5a–h) against H5N1 virus, while pyrimidine derivative-incorporated galacto compounds 5e–h and 6e–h showed higher inhibition activity than its gluco analogues (compounds 5a–d, 6a–d). Most active compounds tested in this study contained N-(phenyl and 4-chlorophenyl moiety pyrimidine), while compounds possessing methyl and methoxyphenyl rings exhibited moderate antiviral activity. On the other hand, pyrimidine 4-merapto derivatives (compounds 3a–d) demonstrated low anti-H5N1 activity.

SARS-COV-2

A series of newly synthesized (twenty) compounds were screened and evaluated for then class="Gene">ir antiviral activity against SARS-COV-2 virus to estimate the half-maximal cytotoxic concentration (CC50) and inhibitory concentration 50(IC50), see (Table and Figures , 5). The results of the MTT assay revealed that some of these compounds have potent activity against SARS-CoV-2 ranging from high, moderate, and low antiviral activity.

Table 2

Determination of the Antiviral CC50 and IC50 and SI of Nontoxic Doses of Compounds (3a–d, 5a–h, and 6a–h) against SARS-COV-2 VIRUS

compound no.	CC50 (μmol)	IC50 (μmol)	SI
3a	510.5	341.3	1.50
3b	503.8	288.7	1.75
3c	204.1	IC50 > CC50
3d	356.0	IC50 > CC50
5a	356.1	126.1	2.80
5b	349.7	151.6	2.30
5c	432.6	287.7	1.50
5d	428.9	168.7	2.54
5e	344.9	86.02	4.00
5f	467.0	77.37	6.04
5g	483.2	106.3	4.55
5h	303.0	92.62	3.27
6a	415.0	47.77	8.69
6b	314.2	45.30	6.90
6c	434.1	73.66	5.89
6d	468.2	49.84	9.39
6e	467.9	18.47	25.33
6f	360.9	15.41	23.40
6g	416.4	34.05	12.22
6h	180.2	30.34	5.90

Figure 4

Graphs of inhibitory concentration 50 (IC50) of tested compounds (3a–d, 5a–h, and 6a–h): antiviral activity against severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) (hCoV19/Egypt/NRC-03/2020, Accession Number on GSAID: EPI_ISL_430820).

Figure 5

Graphs of cytotoxicity concentration 50 (CC50) of tested compounds (3a–d, 5a–h, and 6a–h): on Vero E6 cells.

Graphs of inhibitory concentration 50 (IC50) of tested compounds (3a–d, 5a–h, and n class="Chemical">6a–h): antiviral activity against severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) (hCoV19/Egypt/NRC-03/2020, Accession Number on GSAID: EPI_ISL_430820).

Interestingly, the compounds with n class="Chemical">glycosyl moieties incorporated into the pyrimidine ring system through S-glycosidic linkage (5a–h and 6a–h) demonstrated a marked increase in inhibition activity of SARS-COV-2 virus compared to compounds without these moieties. Moreover, the activity observed for the substituted pyrimidine thioglycosides (6a–h) indicated the importance of the free hydroxyl groups of S-glycopyranosyl moieties for increasing activity compared with protected S-glycopyranosyl moieties (5a–h). In addition, the deprotected galactopyranosyl pyrimidine derivatives (6e–h) showed higher activity than the corresponding glucopyranosyl pyrimidines (6a–d). The inhibition assay results revealed the compounds N-phenyl pyrimidine thiogalactoside (6e), N-P-chlorophenyl pyrimidine thiogalactoside (6f), and N-4-methoxyphenyl pyrimidine thiogalactoside 6h as the moderate potent derivatives with CC50 and IC50 values of 467.9, 360.9, and 180.2 and 18.47, 15.41, 30.34 μM, respectively, and selectivity index values of 25.33, 23.40, and 5.90, respectively, while compounds with methyl and methoxy substitution at the N-p-phenyl ring exhibited a low inhibitory activity against SARS-CoV-2, therefore, the present study confirmed the activities of some of the tested compounds especially (6e) and (6f) as potent inhibitors against COVID-19. These compounds are recommended to be further tested against COVID-19. They may be tested either alone or in combination.

Structure–Activity Relationships

The antiviral activity of compounds 3a–d, n class="Chemical">5a–h, and 6a–h against H5N1 and SARS-COV-2 viruses was investigated and the results are shown in Tables and 2, and compounds 3a–d showed weak activity toward H5N1 and SARS-COV-2 viruses. On the other hand, compounds 5e (X = H) and 5f (X = Cl) shown in Figure exhibited some remarkable activity against H5N1 with inhibition 50 and 51.67% at a concentration of 0.125, also they exhibited inhibition of 66.67% and 63.33 at 0.25 μmol, respectively. Also, similar results were found for compounds 5e and 5f against SARS-COV-2 with IC50 = 86.02 and 77.37 μmol. In a series of compounds 6a–h, the antiviral activity against H5N1virus was improved as noticed in compound 6e (X = H) with inhibition of 71.67 at 0.125 and 78.33 at 0.25 μmol, while compound 6f exhibited good potency among the tested compounds toward H5N1 with 66.67 and 83.33% at 0.125 and 0.25 μmol, respectively. Additionally, the same results were observed for 6e and 6f against SARS-COV-2, with IC50 = 18.47 and 15.41 μmol. Thus, it was concluded that the compounds 6e and 6f showed moderate activity against the two types of tested viruses among the other tested compounds (Figure ).

Figure 6

Antiviral activity of newly prepared compounds against H5N1 and SARA-CoV-2.

Molecular Docking Study

The most potent antiviral compounds n class="Chemical">6e and 6f were docked with the crystal structure of Influenza A Virus H5N1 Nucleoprotein (PDB: 2Q06) and the crystal structure of RNA-dependent RNA polymerase from SARS-CoV-2 (PDB: 7BV2) using molecular docking program soft war 2015.10 to know the exact binding pattern with the receptor. From this study, it was noticed that the docked compounds 6e and 6f with the active site of 2Q06 showed good binding energy in the range between −4.5829 and −4.9411 Kcal mol–1 and displayed good fitness with the active site of 2Q06 protein (Tables and 4). Thus, compound 6e exhibited two hydrogen bonds between two hydroxyl groups and amino acid residues Glu 339 and Ala 387, with bond lengths 2.88 and 2.82 Å, respectively (Figures , 8). Compound 6f has two hydrogen bonds, one with a bond length of 2.97 Å between the hydroxyl group of the sugar moiety and amino acid residue Gly 435 and the other between the NH2 group and Ser 438 with a bond length of 3.08 Å along with bond between chlorine atom and Arg 446 with a bond length of 3.36 Å. Also, it has a cation interaction between the pyrimidine ring and Asp 439 (Figures , 10). On the other hand compounds 6e and 6f have affinity to bind with the active site of RNA-dependent RNA polymerase from SARS-CoV-2 (PDB: 7BV2), thus compound 6e exhibited binding energy S = −5.0696 Kcal mol–1 and exhibited three hydrogen bonds with bond lengths equal to 3.14, 4.06, and 3.73 Å for hydroxyl and thiocarbonyl groups, with the amino acid residues ACH544, Asn 496, and Asn 497, respectively (Figure , 12). Additionally, compound 6f showed the most potent inhibitory activity against SARS-CoV-2 among the other prepared series with three hydrogen bonds, one between the hydroxyl group and ASpA85 with a bond length of 2.91 Å and the other two between the C=S group and Lys A545 and Tyr A546 with bond lengths 3.41 Å and 3.30, along with bond interaction between chlorine and UC12 with S = −5.6214 Kcal mol–1 (Figure , 14).

Table 3

Interactions of Compounds 6e and 6f with the Active Site of 2Q06

compd. no.	B. E. (S)Kcal/mol	interaction groups	interaction amino acids	length of hydrogen bonds Å
6e	–4.5829	OH	Glu 339	2.88 Å
		OH	Ala 387	2.82 Å
		NH2	His 272	cation
6f	–4.9411	OH	Gly 435	2.97 Å
		NH2	Ser 438	3.08 Å
		Cl	Arg 446	3.36 Å
		Pyrimidine	Asp 439	cation

Table 4

Interactions of Compounds 6e and 6f with the Active Site of 7BV2

compd. no.	B. E. (S)Kcal/mol	interaction groups	interaction amino acids	length of hydrogen bonds Å
6e	–5.0696	OH	ACH	3.14
		C=S	Asn A496 Asn A497	4.06
		C=S		3.73
6f	–5.6214	OH	Asp A845 Lys A545	2.91
		C=S	Tyr A546	3.41
		C=S	UC12	3.30
		Cl		3.44

Figure 7

Interaction of 6e (2D) with the active site of 2Q06.

Figure 8

Interaction of 6e (3D) with the active site of 2Q06.

Figure 9

Interaction of 6f (2D) with the active site of 2Q06.

Figure 10

Interaction of 6f (3D) with the active site of 2Q06.

Figure 11

Interaction of 6e (2D) with the active site of 7BV2.

Figure 12

Interaction of 6e (3D) with the active site of 7BV2.

Figure 13

Interaction of 6f (2D) with the active site of 7BV2.

Figure 14

Interaction of 6f (3D) with the active site of 7BV2.

Conclusions

In this paper, a new and innovative method has been successfully developed for synthesizing new and important derivatives of n class="Chemical">pyrimidinethiol compounds and their corresponding thioglycoside derivatives. This method has been proven to be effective in preparing many analogue components of nucleic acids, which are of medical importance in the field of therapeutic chemistry. This method has been proven to be important in the field of industrial chemistry due to the fact that our pyrimidine thioglycosides are prepared at room temperature with high purity and high reaction yield, and therefore it is considered a more efficient method than many methods used to obtain similar compounds. Some novel glycosides such as 6e, 6f, and 6h showed moderate anti-SARS-CoV-2 activity at noncytotoxic micromolar concentrations in vitro with a significant selectivity index (CC50/IC50 = 6–25).

Experimental Part

All melting points were measured on an Electrothermal 9100 digital melting point apparatus. The n class="Chemical">1H NMR and 13C NMR spectra were measured on a JEOL-500MHz spectrometer in DMSO-d6 or CDCl3 using Si(CH3)4 as an internal standard at the National Research Center, Cairo, Egypt. Potential cytotoxicity assay of the newly synthesized compounds was evaluated at the Center of Scientific Excellence for Influenza Viruses, Environmental Research Division, National Research Center (NRC), Dokki, Cairo 12622, Egypt. Elemental analyses were carried out at the Microanalytical Unit, Faculty of Science, Cairo University, Cairo, Egypt.

Progress of the reactions was monitored by thin-layer chromatography (TLC) using aluminum sheets coated with n class="Chemical">silica gel F254 plates with a layer thickness of 0.25 nm (Merck). By viewing under a short-wavelength UV lamp, effective detection can be achieved. Compounds (1a–d and 2a–d) were prepared following our reported procedures.[36,37]

General Procedure for the Synthesis of (3a–d)

To a solution of 2-cyano-3,3-dimercapto-N-phenylacrylamide 2a–d (10 mmol) in absolute ethanol (10 n class="Gene">mL) was added piperidine (2 drops) and stirred, then a solution of thiourea in ethanol (10 mmol) was dropped within 30 min. Stirring was continued under refluxing conditions for 2 h. After completion, the reaction mixture was evaporated under reduced pressure and the residue was poured on ice-water, collected by filtration, dried, and recrystallized from ethanol to give compounds 3a–d.

4-Amino-2-mercapto-N-phenyl-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (3a)

White solid; (EtOH); yield (85%); mp 244 °C; IR (KBr, cm–1) ν: 3365 (NH2), 3269 (NH), 3052 (CH aromatic), 1661 (C=O), 1604 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 6.86 (s, D2O exch., 2H, NH2), 7.33–7.62 (m, 5H, C6H5), 10.54 (s, D2O exch., 1H, NH), 11.12 (s, D2O exch., 1H, NH), 11.63 (s, D2O exch., 1H, SH); 13C NMR: δ 111.31 (C-5), 123.54–139.25 (6C, Ar–C), 161.33 (C-4), 162.21 (C-2), 166.22 (CO), 172.89 (C-6). Anal. Calcd. for. C11H10N4OS2 (278.35): C, 47.46; H, 3.62; N, 20.13; S, 23.04. Found: C, 47.35; H, 3.54; N, 20.15; S, 23.15%.

4-Amino-N-(4-chlorophenyl)-2-mercapto-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (3b)

White solid; (EtOH); yield (80%); mp 239 °C; IR (KBr, cm–1) ν: 3354 (NH2), 3269 (NH), 3244 (NH), 3042 (CH aromatic), 1668 (C=O), 1592 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 6.74 (s, D2O exch., 2H, NH2), 7.42–7.72 (m, 4H, C6H4), 10.68 (s, D2O exch., 1H, NH), 11.26 (s, D2O exch., 1H, NH), 13.26 (s, D2O exch., 1H, SH); 13C NMR: δ 106.96 (C-5), 123.57–141.23 (6C, Ar–C), 163.42 (C-4), 161.53 (C-2), 166.48 (CO), 172.76 (C-6). Anal. Calcd. for. C11H9ClN4OS2 (312.80): C, 42.24; H, 2.90; Cl, 11.33; N, 17.91; S, 20.50. Found: C, 42.16; H, 2.82; Cl, 11.23; N, 17.79; S, 20.43%.

4-Amino-2-mercapto-6-thioxo-N-p-tolyl-1,6-dihydropyrimidine-5-carboxamide (3c)

White solid; (EtOH); yield (85%); mp 234 °C; IR (KBr, cm–1) ν: 3348 (NH2), 3264 (NH), 3241 (NH), 3039 (CH aromatic), 2984 (CH3), 1675 (C=O), 1607 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 2.62 (s, 3H, CH3), 6.72 (s, D2O exch., 2H, NH2), 7.32–7.49 (m, 4H, C6H4), 10.43 (s, D2O exch., 1H, NH), 11.24 (s, D2O exch., 1H, NH), 13.12 (s, D2O exch., 1H, SH); 13C NMR: δ 22.75 (CH3), 113.16 (C-5), 123.45–138.67 (6C, Ar–C), 163.84 (C-4), 165.61 (C-2), 167.29 (C-4), 143.26 (C-6). Anal. Calcd. for. C12H12N4OS2 (292. 38): C, 49.29; H, 4.14; N, 19.16; S, 21.93. found: C, 49.20; H, 4.10; N, 19.11; S, 21.85%.

4-Amino-2-mercapto-N-(4-methoxyphenyl)-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (3d)

White solid; (EtOH); yield (79%); mp 222 °C; IR (KBr, cm–1) ν: 3368 (NH2), 3252 (NH), 3226 (NH), 3039 (CH aromatic), 2986 (CH3), 1669 (C=O), 1609 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 3.91 (s, 3H, CH3), 6.81 (s, D2O exch., 2H, NH2), 7.23–7.66 (m, 4H, C6H4), 10.34 (s, D2O exch., 1H, NH), 11.13 (s, D2O exch., 1H, NH), 13.15 (s, D2O exch., 1H, SH); 13C NMR: δ 59.13 (CH3), 114.16 (C-5), 121.31–134.64 (5C, Ar–C), 161.32 (C, Ar), 164.25 (C-4), 165.88 (C-2), (167.11 CO), 175.76 (C-4), 170.22 (C-6). Anal. Calcd. for C12H12N4O2S2 (308.38): C, 46.74; H, 3.92; N, 18.17; S, 20.80. Found: C, 46.63; H, 3.84; N, 18.11; S, 20.72%.

General Procedure for the Synthesis of (5a–h)

To a solution of 3a–d (10 mmol) in dry DMF (20 mL), NaH (15 mmol) was added portion wise for 15 min and the solution was stirred at room temperature for another 30 min. Then, a solution of 2,3,4,6-tetra-O-acetyl-α-d-gluco- or galactopyronosyl bromide 4a,b in DMF (10 mmol) was dropped within 30 min and the reaction mixture was stirred at room temperature until complete mixing was achieved (TLC, 8 h). After that, the reaction mixture was poured into ice water and the resulting precipitate was collected by filtration, dried, and recrystallized from an appropriate solvent system to give compounds 5a–h.

4-Amino-2-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glucopyranosylthio)-N-phenyl-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (5a)

White solid; (EtOH), yield (89%), mp 242 °C; IR (KBr, cm–1) ν: 3324 (NH2), 3233 (NH), 3038 (CH aromatic), 2952 (CH), 1742 (C=O), 1661 (C=O), 1605 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 1.97, 1.99, 2.01, 2.02 (4s, 12H, 4× OAc), 3.97–4.01 (m, 2H, H-6′, H-6″), 4.23–4.25 (m, 1H, H-5′), 4.52 (t-like, 1H, J = 10.1 Hz, J = 9.9 Hz, H-4′), 4.76 (t-like, 1H, J = 9.6 Hz, J = 9.8 Hz, H-2′), 4.96 (t-like, 1H, J = 9.8 Hz, J = 10.1 Hz, H-3′), 5.38 (d, 1H, J = 9.6 Hz, H-1′), 6.71 (s, D2O exch., 2H, NH2), 7.23–7.65 (m, 5H, C6H5), 8.84 (s, D2O exch., 1H, NH), 10.43 (s, D2O exch., 1H, NH); 13C NMR: δ 22.35 (4× CH3), 62.43 (C-6′), 67.86 (C-4′), 70.19 (C-2′), 73.42 (C-3′), 77.47 (C-5′), 79.68 (C-1′), 89.41 (C-5), 124.26 (2C, Ar–C), 131.37 (Ar–C), 132.54 (2C, Ar–C), 143.65 (Ar–C), 162.28 (C=O), 166.53 (C=O), 168.17 (C-4), 169.79 (C-6), 173.15 (4C=O). Anal. Calcd. for C25H28N4O10S2 (608.64): C, 49.33; H, 4.64; N, 9.21; S, 10.54. Found: C, 49.25; H, 4.52; N, 9.16; S, 10.46.%.

4-Amino-N-(4-chlorophenyl)-2-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glucopyranosylthio)-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (5b)

White solid; (EtOH), yield (86%), mp 239 °C; 1H NMR (500 MHz, DMSO-d6): δ 1.98.1.99, 2.01, 2.02 (4s, 12H, 4× OAc), 3.88–3.89 (m, 2H, H-6′, H6″), 4.24–4.26 (m, 1H, H-5′), 4.45 (t-like, 1H, J = 9.1 Hz, J = 9.2 Hz, H-4′), 4.56 (t-like, 1H, J = 9.4 Hz, J = 9.9 Hz, H-2′), 4.72 (t-like, 1H, J = 9.4 Hz, J = 9.9 Hz, H-3′), 5.41 (d, 1H, J9.4 Hz, H-1′), 6.68 (s, D2O exch., 2H, NH2), 7.43–7.77 (m, 4H, C6H4), 10.62 (s, D2O exch., 1H, NH) 11.53 (s, D2O exch., 1H, NH);13C NMR: δ 21.65 (4× CH3), 62.18 (C-6′), 68.22 (C-4′), 69.52 (C-2′), 73.67 (C-3′), 76.82 (C-5′), 79.11 (C-5), 80.64 (C-1′), 111.23 (C-5), 122.37 (2C, Ar–C), 130.41 (2C, Ar–C), 135.35 (Ar–C), 139.24 (Ar–C), 162.38 (C-4), 165.16 (C-2), 167.42 (C=O), 174.12 (4C=O), 189.29 (C-6). Anal. Calcd. for C25H27ClN4O10S2 (643.09): C, 46.69; H, 4.23; Cl, 5.51; N, 8.71; S, 9.97. Found: C, 46.60; H, 4.15; Cl, 5.43; N, 8.62; S, 9.85%.

4-Amino-2-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glucopyranosylthio)-6-thioxo-N-p-tolyl-1,6-dihydroyrimidine-5-carboxamide (5c)

White solid; (EtOH), yield (85%), mp 252–253 °C; 1H NMR (500 MHz, CDCl3): δ 1.99, 2.01, 2.03, 2.04 (4s, 12H, 4× OAc), 2.36 (s, 3H, CH3), 4.15–4.16 (m, 1H, H-5′), 4.35 (m, 2H, H-6′, H6″), 4.62 (t-like, 1H, J = 9.9 Hz, J = 8.6 Hz, H-4′), 5.34 (t-like, 1H, J = 10.1 Hz, J = 9.9 Hz, H-3′), 5.42 (t-like, 1H, J 9.8 Hz, J 10.1 Hz, H-2′), 5.54 (d, 1H, J = 9.8 Hz H-1′), 6.74 (s, D2O exch., 2H, NH2), 7.31–7.52 (m, 4H, C6H4), 10.35 (s, D2O exch., 1H, NH), 10.65 (s, D2O exch., 1H, NH); 13C NMR: δ 22.15 (4× CH3), 25.62 (CH3),62.41 (C-6′), 69.42 (C-4′), 70.65 (C-2′), 71.11 (C-3′), 72.36 (C-5′), 82.44 (C-1′), 111.24 (C-5), 123.17 (2C, Ar–C), 130.28 (2C, Ar–C), 136.49 (Ar–C), 139.72 (Ar–C), 162.66 (C-4), 153.89 (C-2), 166.68 (C=O), 169.84 (4C=O), 174.89 (C-6). Anal. Calcd. for C26H30N4O10S2 (622.67): CC, 50.15; H, 4.86; N, 9.00; S, 10.30. Found: C, 50.20; H, 4.74; N, 9.12; S, 10.24%.

4-Amino-N-(4-Methoxyphenyl)-2-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glucopyranosylthio)-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (5d)

White solid; (EtOH), yield (87%), mp 247–248 °C; IR (KBr, cm–1) ν: 3334 (NH2), 3239 (NH), 3042 (CH aromatic), 2991 (CH), 1743 (C=O), 1668 (C=O), 1593 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 1.98, 1.99, 2.05, 2.11 (4s, 12H, 4× OAc), 3.86 (s, 3H, OCH3), 3.99–4.02 (m, 2H, 2H-6′), 4.42 (ddd, 1H, J = 2:4 Hz, J = 4:5 Hz, J = 9:1 Hz, H-5′), 4.53 (t-like, 1H, J = 9.2 Hz, J = 9.1 Hz, H-4′), 4.76 (t-like, 1H, J = 9.6 Hz, J = 9.2 Hz, H-3′), 4.88 (t-like, 1H, J = 9.3 Hz, J = 9.6 Hz, H-2′), 5.42 (d, 1H, J9.3 Hz, H-1′), 6.81 (s, D2O exch., 2H, NH2), 7.21–7.72 (m, 4H, C6H4), 10.44 (s, D2O exch., 1H, NH), 11.23 (s, D2O exch., 1H, NH); 13C NMR: δ 22.13 (4× CH3), 58.23 (CH3), 62.13 (C-6′), 69.72 (C-4′), 72.55 (C-2′), 74.26 (C-3′), 76.81 (C-5′), 78.66 (C-1′), 112.46 (C-5), 118.56–162.25 (6C, Ar–C), 166.22 (C-4), 168.16 (C-2), 169.67 (C=O), 172.58 (4C=O), 179.47 (C-6). Anal. Calcd. for C26H30N4O11S2 (638.67): C, 48.90; H, 4.73; N, 8.77; S, 10.04. Found: C, C, 48.82; H, 4.62; N, 8.65; S, 10.15%.

4-Amino-2-(2′,3′,4′,6′-tetra-O-acetyl-β-d-galactopyranosylthio)-N-phenyl-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (5e)

White solid; (EtOH), yield (89%), mp 251–252 °C; IR (KBr, cm–1) ν: 3335 (NH2), 3229 (NH), 3052 (CH aromatic), 2964 (CH), 1744 (C=O), 1669 (C=O), 1608 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 1.98, 2.00, 2.03, 2.05 (4s, 12H, 4× OAc), 3.97–4.01 (m, 2H, H-6′, H-6″), 4.23–4.25 (m, 1H, H-5′), 4.52 (t-like, 1H, J = 10.1 Hz, J = 9.9 Hz, H-4′), 4.76 (t-like, 1H, J = 9.6 Hz, J = 9.8 Hz, H-2′), 4.96 (t-like, 1H, J = 9.8 Hz, J = 10.1 Hz, H-3′), 5.38 (d, 1H, J9.6 Hz, H-1′), 6.71 (s, D2O exch., 2H, NH2), 7.23–7.65 (m, 5H, C6H5), 8.84 (s, D2O exch., 1H, NH), 10.43 (s, D2O exch., 1H, NH); 13C NMR: δ 22.35 (4× CH3), 62.43 (C-6′), 67.86 (C-4′), 70.19 (C-2′), 73.42 (C-3′), 77.47 (C-5′), 79.68 (C-1′), 89.41 (C-5), 124.26 (2C, Ar–C), 131.37 (Ar–C), 132.54 (2C, Ar–C), 143.65 (Ar–C), 162.28 (C=O), 166.53 (C=O), 168.17 (C-4), 169.79 (C-6), 173.15 (4C=O). Anal. Calcd. for C25H28N4O10S2 (608.64): C, 49.33; H, 4.64; N, 9.21; S, 10.54. Found: C, 49.25; H, 4.52; N, 9.16; S, 10.46%.

4-Amino-N-(4-chlorophenyl)-2-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glucopyranosylthio)-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (5f)

White solid; (EtOH), yield (86%), mp 239 °C; 1H NMR (500 MHz, DMSO-d6): δ 1.98.1.99, 2.01, 2.02 (4s, 12H, 4× OAc), 3.88–3.89 (m, 2H, H-6′, H6″), 4.24–4.26 (m, 1H, H-5′), 4.45 (t-like, 1H, J = 9.1 Hz, J = 9.2 Hz, H-4′), 4.56 (t-like, 1H, J = 9.4 Hz, J = 9.9 Hz, H-2′), 4.72 (t-like, 1H, J = 9.4 Hz, J = 9.9 Hz, H-3′), 5.41 (d, 1H, J = 9.4 Hz, H-1′), 6.68 (s, D2O exch., 2H, NH2), 7.43–7.77 (m, 4H, C6H4), 10.62 (s, D2O exch., 1H, NH) 11.53 (s, D2O exch., 1H, NH);13C NMR: δ 21.65 (4× CH3), 62.18 (C-6′), 68.22 (C-4′), 69.52 (C-2′), 73.67 (C-3′), 76.82 (C-5′), 79.11 (C-5), 80.64 (C-1′), 111.23 (C-5), 122.37 (2C, Ar–C), 130.41 (2C, Ar–C), 135.35 (Ar–C), 139.24 (Ar–C), 162.38 (C-4), 165.16 (C-2), 167.42 (C=O), 174.12 (4C=O), 189.29 (C-6). Anal. Calcd. for C25H27ClN4O10S2 (643.09): C, 46.69; H, 4.23; Cl, 5.51; N, 8.71; S, 9.97. Found: C, 46.60; H, 4.15; Cl, 5.43; N, 8.62; S, 9.85%.

4-Amino-2-(2′,3′,4′,6′-tetra-O-acetyl-β-d-galactopyranosylthio)-6-thioxo-N-p-tolyl-1,6-dihydroyrimidine-5-carboxamide (5g)

White solid; (EtOH), yield (85%), mp 252–253 °C; 1H NMR (500 MHz, CDCl3): δ 1.99, 2.01, 2.03, 2.04 (4s, 12H, 4× OAc), 2.36 (s, 3H, CH3), 4.15–4.16 (m, 1H, H-5′), 4.35 (m, 2H, H-6′, H6″), 4.62 (t-like, 1H, J = 9.9 Hz, J = 8.6 Hz, H-4′), 5.34 (t-like, 1H, J = 10.1 Hz, J = 9.9 Hz, H-3′), 5.42 (t-like, 1H, J 9.8 Hz, J 10.1 Hz, H-2′), 5.54 (d, 1H, J = 9.8 Hz H-1′), 6.74 (s, D2O exch., 2H, NH2), 7.31–7.52 (m, 4H, C6H4), 10.35 (s, D2O exch., 1H, NH), 10.65 (s, D2O exch., 1H, NH); 13C NMR: δ 22.15 (4× CH3), 25.62 (CH3),62.41 (C-6′), 69.42 (C-4′), 70.65 (C-2′), 71.11 (C-3′), 72.36 (C-5′), 82.44 (C-1′), 111.24 (C-5), 123.17 (2C, Ar–C), 130.28 (2C, Ar–C), 136.49 (Ar–C), 139.72 (Ar–C), 162.66 (C-4), 153.89 (C-2), 166.68 (C=O), 169.84 (4C=O), 174.89 (C-6). Anal. Calcd. for C26H30N4O10S2 (622.67): CC, 50.15; H, 4.86; N, 9.00; S, 10.30. Found: C, 50.20; H, 4.74; N, 9.12; S, 10.24%.

4-Amino-N-(4-Methoxyphenyl)-2-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glalactopyranosylthio)-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (5h)

White solid; (EtOH), yield (87%), mp 247–248 °C; IR (KBr, cm–1) ν: 3336 (NH2), 3239 (NH), 3042 (CH aromatic), 2991 (CH), 1743 (C=O), 1668 (C=O), 1593 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 1.98, 1.99, 2.05, 2.11 (4s, 12H, 4× OAc), 3.86 (s, 3H, OCH3), 3.99–4.02 (m, 2H, 2H-6′), 4.42 (ddd, 1H, J = 2.4 Hz, J = 4.5 Hz, J = 5.1 Hz, H-5′), 4.53 (t-like, 1H, J = 4.8 Hz, J = 5.1 Hz, H-4′), 4.76 (dd, 1H, J = 9.6 Hz, J = 4.8 Hz, H-3′), 4.88 (t-like, 1H, J = 9.4 Hz, J = 9.6 Hz, H-2′), 5.42 (d, 1H, J9.4 Hz, H-1′), 6.81 (s, D2O exch., 2H, NH2), 7.21–7.66 (m, 4H, C6H4), 10.42 (s, D2O exch., 1H, NH), 11.26 (s, D2O exch., 1H, NH); 13C NMR: δ 22.43 (4× CH3), 57.68 (CH3), 61.41 (C-6′), 69.44 (C-4′), 72.25 (C-2′), 74.57 (C-3′), 75.29 (C-5′), 79.25 (C-1′), 112.36 (C-5), 118.32–161.81 (6C, Ar–C), 164.26 (C-4), 168.28 (C-2), 166.14 (C=O), 172.15 (4C=O), 179.33 (C-6). Anal. Calcd. for C26H30N4O11S2 (638.67): C, 48.90; H, 4.73; N, 8.77; S, 10.04. Found: C, 48.82; H, 4.62; N, 8.65; S, 10.15%.

General Procedure for the Synthesis of (6a–h)

Dry gaseous ammonia was passed through a solution of protected nucleoside 5a–h (10 mmol) in dry methanol (20 mL) for 10 min with cooling at 0 °C and stirring, then the reaction mixture was stirred at room temperature until the reaction was judged complete by TLC using (CHCl3/MeOH 9:1) (Rf, 0.54–0.56). The resulting mixture was concentrated under reduced pressure to afford a solid residue, which was washed several times by boiling chloroform. The residue was dried, purified by column chromatography using chloroform/methanol (9:1), and crystallized using an appropriate solvent to give corresponding compounds 6a-h.

4-Amino-2-(β-d-glucopyranosylthio)-N-phenyl-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (6a)

White solid; (EtOH), yield (66%), mp 192 °C; IR (KBr, cm–1) ν: 3362 (OH), 3284, 3247 (NH2), 3211 (NH), 3042 (CH aromatic), 2928 (CH), 1672 (CO), 1592 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 3.44–3.46 (m, 3H, H-6′, H-6″, H-5′), 3.62 (t-like, 1H, J = 9.6 Hz, H-4′), 3.88 (t-like, 1H, J = 9.4 Hz, H-2′), 4.26 (t-like, 1H, J = 10.3 Hz, H-3′), 4.46 (t-like, 1H, J = 6.1 Hz, D2O exch., 6′-OH), 4.62 (d, 1H, J = 9.4 Hz, H-1′), 4.82 (d, 1H, J = 5.4 Hz, D2O exch., OH), 5.12 (d, 1H, J = 5.1 Hz, D2O exch., OH), 5.41 (d, 1H, J = 6.2 Hz, D2O exch., OH), 6.72 (s, D2O exch., 2H, NH2), 7.23–7.78 (m, 5H, C6H5), 10.45 (s, D2O exch., 1H, NH), 10.96 (s, D2O exch., 1H, NH); 13C NMR: δ 61.82 (C-6′), 69.54 (C-4′), 75.12 (C-2′), 78.11 (C-3′), 80.14 (C-5′), 83.25 (C-1′), 113. 84 (C-5), 123.23 (2C, Ar–C), 132.25 (Ar–C), 134.56 (2C, Ar–C), 139.52 (Ar–C), 163.28 (C-4), 166.11 (C-2), 168.31 (C=O), 179.45 (C-6). Anal. Calcd. for C17H20N4O6S2 (440.49): C, 46.35; H, 4.58; N, 12.72; S, 14.56. Found: C, 46.24; H, 4.49; N, 12.65; S, 14.45%.

4-Amino-N-(4-chlorophenyl)-2-(β-d-glucopyranosylthio)-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (6b)

White solid; (EtOH), yield (63%), mp 186 °C; IR (KBr, cm–1) ν: 3351 (OH), 3322, 3306 (NH2), 3244 (NH), 3032 (CH aromatic), 1673 (C=O), 1605 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 3.35–3.37 (m, 2H, H-6′, H-6″), 3.42 (ddd, 1H, J = 9.2 Hz, 4.3 Hz, 3.4 Hz, H-5′), 3.52–3.54 (t-like, 1H, J = 10.2 Hz, H-4′), 3.74 (t-like, 1H, J = 9.8 Hz, H-2′), 4.01 (t-like, 1H, J = 8.8 Hz, H-3′), 4.81 (t, 1H, J = 6.2 Hz, D2O exch., 6′-OH), 5.24 (d, 1H, = 8.8 Hz, H-1′), 5.43 (d, 1H, J = 5.6 Hz, D2O exch., OH), 5.52 (d, 1H, J = 6.1 Hz,, D2O exch., OH), 5.58 (d, 1H, J = 5.2 Hz, D2O exch., OH), 6.72 (s, D2O exch., 2H, NH2), 7.36–7.82 (m, 4H, C6H4), 10.42 (s, D2O exch., 1H, NH), 11.18 (s, D2O exch., 1H, NH); 13C NMR: δ 62.21 (C-6′), 70.62 (C-4′), 74.52 (C-2′), 76.33 (C-3′), 80.22 (C-5′), 82.56 (C-1′), 108.47 (C-5), 123.41 (2C, Ar–C), 132.45 (2C, Ar–C), 134.88 (Ar–C), 138.62 (Ar–C), 163.19 (C-4), 166.51 (C-2), 168.24 (C=O), 176.38 (C-6). Anal. Calcd. for C17H19ClN4O5S2 (474.94): C, 42.99; H, 4.03; Cl, 7.46; N, 11.80; S, 13.50. Found: C, 42.90; H, 4.12; Cl, 7.35; N, 11.72; S, 13.40%.

4-Amino-2-(β-d-glucopyranosylthio)-6-thioxo-N-p-tolyl-1,6-dihydropyrimidine-5-carboxamide (6c)

White solid; (EtOH), yield (68%), mp 169 °C; IR (KBr, cm–1) ν: 3352 (OH), 3327, 3259 (NH2), 3216 (NH), 3041 (CH aromatic), 1672 (CO), 1598 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 2.36 (s, 3H, CH3), 3.42–3.43 (m, 2H, 2H-6′), 3.56 (ddd, 1H, J = 9.8 Hz, 4.2 Hz, 3.2 Hz, H-5′), 3.62 (t-like, 1H, J = 9.9 Hz, H-4′), 3.78 (t-like, 1H, J = 9.1 Hz, H-2′), 4.02 (t-like, 1H, J = 9.7 Hz, H-3′), 4.46 (t, 1H, J = 5.6 Hz, D2O exch., 6′-OH), 4.49 (d, 1H, J = 9.7 Hz, H-1′), 5.32 (d, 1H, J = 6.1 Hz, D2O exch., OH), 5.46 (d, 1H, J = 6.8 Hz,, D2O exch., OH), 5.51 (d, 1H, J = 6.9, 7.3 Hz,, D2O exch., OH), 6.76 (s, D2O exch., 2H, NH2), 7.23–7.48 (m, 4H, C6H4), 10.86 (s, D2O exch., 1H, NH), 11.29 (s, D2O exch., 1H, NH); 13C NMR: δ 24.62 (CH3), 61.42 (C-6′), 69.36 (C-4′), 70.52 (C-2′), 75.28 (C-3′), 79.16 (C-5′), 83.67 (C-1′), 112.13 (C-5), 123.21 (2C, Ar–C), 132.14 (2C, Ar–C), 133.82 (Ar–C), 138.29 (Ar–C), 163.27 (C-4), 164.62 (C-2), 166.93 (C=O), 179.21 (C-6). Anal. Calcd. for C18H22N4O6S2 (454.52): C, 47.56; H, 4.88; N, 12.33; S, 14.11. Found: C, 47.46; H, 4.76; N, 12.25; S, 14.23%.

4-Amino-N-(4-methoxyphenyl)-2-(β-d-gulcopyranosylthio)-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (6d)

White solid; (EtOH), yield (65%), mp 189 °C; 1H NMR (500 MHz, DMSO-d6): δ 3.89 (s, 3H, OCH3), 3.26–3.29 (m, 2H, H-6′, H-6′), 3.86 (ddd, 1H, J = 9.7 Hz, 4.3 Hz, 3.6 Hz, H-5′), 3.43 (t-like, 1H, J = 10.3 Hz, H-4′), 3.61 (t-like, 1H, J = 9.6 Hz, H-2′), 3.75 (t-like, 1H, J = 10.2 Hz, H-3′), 4.56 (t, 1H, J = 6.8 Hz, D2O exch., 6′-OH), 4.68 (d, 1H, J = 9.8 Hz, H-1′), 4.92 (d, 1H, J = 6.9 Hz, D2O exch., OH), 5.23 (d, 1H, J = 8.2 Hz, D2O exch., OH), 5.43 (d, 1H, J = 8.4 Hz, D2O exch., OH), 6.84 (s, D2O exch., 2H, NH2), 7.23–7.52 (m, 4H, C6H4), 10.26 (s, D2O exch., 1H, NH), 11.41 (s, D2O exch., 1H, NH); 13C NMR: δ 56.44 (CH3), 61.57 (C-6′), 69.36 (C-4′), 73.64 (C-2′), 76.45 (C-3′), 76.65 (C-5′), 83.83 (C-1′), 114.21 (C-5), 123.45 (2C, Ar–C), 126.12 (2C, Ar–C), 134.64 (Ar–C), 153.33 (Ar–C), 164.16 (C-4), 167.35 (C-2), 169.74 (C=O), 178.51 (C-6). Anal. Calcd. for C18H22N4O7S2 (470.52): C, 45.95; H, 4.71; N, 11.91; S, 13.63. Found: C, 45.83; H, 4.65; N, 11.82; S, 13.55%.

4-Amino-2-(β-d-galactopyranosylthio)-N-phenyl-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (6e)

White solid; (EtOH), yield (64%), mp 188 °C; 1H NMR (500 MHz, DMSO-d6): δ 3.39–3.41 (m, 2H, H-6′, H-6″), 3.51 (ddd, 1H, J = 5.2 Hz, 4.6, Hz, 3.4 Hz, H-5′), 3.73 (t-like, 1H, J = 4.6 Hz, H-4′), 3.95 (t-like, 1H, J = 9.8 Hz, H-2′), 4.41 (dd, 1H, J = 10.2 Hz, 5.2 Hz, H-3′), 4.52 (t-like, 1H, J = 8.2 Hz, D2O exch., 6′-OH), 4.68 (d, 1H, J = 9.7 Hz, H-1′), 4.81 (d, 1H, J = 6.2 Hz, D2O exch., OH), 5.04 (d, 1H, J = 5.6 Hz, D2O exch., OH), 5.38 (d, 1H, J = 6.4 Hz, D2O exch., OH), 6.76 (s, D2O exch., 2H, NH2), 7.23–7.76 (m, 5H, C6H5), 10.24 (s, D2O exch., 1H, NH), 10.85 (s, D2O exch., 1H, NH); 13C NMR: δ 61.55 (C-6′), 70.23 (C-4′), 76.14 (C-2′), 79.24 (C-3′), 81.12 (C-5′), 82.11 (C-1′), 112. 42 (C-5), 122.62 (2C, Ar–C), 132.85 (Ar–C), 136.26 (2C, Ar–C), 139.68 (Ar–C), 162.73 (C-4), 167.27 (C-2), 169.68 (C=O), 180.29 (C-6). Anal. Calcd. for C17H20N4O6S2 (440.49): C, 46.35; H, 4.58; N, 12.72; S, 14.56. Found: C, 46.24; H, 4.49; N, 12.65; S, 14.45%.

4-Amino-N-(4-chlorophenyl)-2-(β-d-galactopyranosylthio)-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (6f)

White solid; (EtOH), yield (63%), mp 192 °C; IR (KBr, cm–1) ν: 3358 (OH), 3336, 3314 (NH2), 3258 (NH), 3039 (CH aromatic), 1669 (C=O), 1601 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 3.32–3.33 (m, 2H, H-6′, H-6″), 3.52 (ddd, 1H, J = 5.4 Hz, 4.1 Hz, 3.3 Hz, H-5′), 3.65 (t-like, J = 5.6 Hz, H-4′), 3.75 (t-like, 1H, J = 9.6 Hz, H-2′), 4.12 (dd, 1H, J = 9.9 Hz, 5.1 Hz, H-3′), 4.54 (t, 1H, J = 6.4 Hz, D2O exch., 6′-OH), 5.36 (d, 1H, = 9.7 Hz, H-1′), 5.45 (d, 1H, J = 6.3 Hz, D2O exch., OH), 5.51 (d, 1H, J = 6.4 Hz,, D2O exch., OH), 5.57 (d, 1H, J = 5.7 Hz, D2O exch., OH), 6.76 (s, D2O exch., 2H, NH2), 7.36–7.76 (m, 4H, C6H4), 10.64 (s, D2O exch., 1H, NH), 11.25 (s, D2O exch., 1H, NH); 13C NMR: δ 62.42 (C-6′), 72.18 (C-4′), 74.42 (C-2′), 75.46 (C-3′), 80.66 (C-5′), 83.82 (C-1′), 110.36 (C-5), 123.86 (2C, Ar–C), 133.18 (2C, Ar–C), 136.14 (Ar–C), 138.61 (Ar–C), 164.12 (C-4), 168.01 (C-2), 169.88 (C=O), 179.79 (C-6). Anal. Calcd. for C17H19ClN4O5S2 (474.94): C, 42.99; H, 4.03; Cl, 7.46; N, 11.80; S, 13.50. Found: C, 42.90; H, 4.12; Cl, 7.35; N, 11.72; S, 13.40%.

4-Amino-2-(β-d-galactopyranosylthio)-6-thioxo-N-p-tolyl-1,6-dihydropyrimidine-5-carboxamide (6g)

White solid; (EtOH), yield (61%), mp 187 °C; IR (KBr, cm–1) ν: 3361 (OH), 3312, 3250 (NH2), 3211 (NH), 3039 (CH aromatic), 1676 (CO), 1602 (C=N); 1H NMR (500 MHz, DMSO-d6): δ 2.38 (s, 3H, CH3), 3.46–3.47 (m, 2H, 2H-6′), 3.56 (ddd, 1H, J = 5.1 Hz, 4.3 Hz, 3.2 Hz, H-5′), 3.62 (t-like, 1H, J = 5.4 Hz, H-4′), 3.73 (t-like, 1H, J = 9.8 Hz, H-2′), 4.13 (dd, 1H, J = 5.6 Hz, 4.9 Hz, H-3′), 4.39 (t, 1H, J = 6.8 Hz, D2O exch., 6′-OH), 4.45 (d, 1H, J = 10.3 Hz, H-1′), 5.42 (d, 1H, J = 7.2 Hz, D2O exch., OH), 5.54 (d, 1H, J = 6.4 Hz,, D2O exch., OH), 5.58 (d, 1H, J = 7.3, Hz, D2O exch., OH), 6.75 (s, D2O exch., 2H, NH2), 7.24–7.51 (m, 4H, C6H4), 10.43 (s, D2O exch., 1H, NH), 11.32 (s, D2O exch., 1H, NH); 13C NMR: δ 24.29 (CH3), 62.34 (C-6′), 69.44 (C-4′), 71.41 (C-2′), 74.64 (C-3′), 78.65 (C-5′), 84.51 (C-1′), 111.62 (C-5), 123.36 (2C, Ar–C), 132.88 (2C, Ar–C), 134.39 (Ar–C), 139.23 (Ar–C), 162.67 (C-4), 165.83 (C-2), 168.67 (C=O), 179.76 (C-6). Anal. Calcd. for C18H22N4O6S2 (454.52): C, 47.56; H, 4.88; N, 12.33; S, 14.11. Found: C, 47.46; H, 4.76; N, 12.25; S, 14.23%.

4-Amino-N-(4-methoxyphenyl)-2-(β-d-galacotpyranosylthio)-6-thioxo-1,6-dihydropyrimidine-5-carboxamide (6h)

White solid; (EtOH), yield (65%), mp 196 °C; 1H NMR (500 MHz, DMSO-d6): δ 3.86 (s, 3H, OCH3), 3.36–3.67 (m, 2H, H-6′, H-6′), 3.43 (ddd, 1H, J = 5.4 Hz, 4.2 Hz, 3.5 Hz, H-5′), 3.48 (t-like, 1H, J = 5.2, H-4′), 3.52 (t-like, 1H, J = 10.1 Hz, H-2′), 3.76 (dd, 1H, J = 10.4 Hz, 4.6 Hz, H-3′), 4.57 (t, 1H, J = 7.2 Hz, D2O exch., 6′-OH), 4.62 (d, 1H, J = 9.6 Hz, H-1′), 4.81 (d, 1H, J = 8.1 Hz, D2O exch., OH), 5.05 (d, 1H, J = 6.6 Hz, D2O exch., OH), 5.46 (d, 1H, J = 7.3 Hz, D2O exch., OH), 6.82 (s, D2O exch., 2H, NH2), 7.23–7.56 (m, 4H, C6H4), 10.45 (s, D2O exch., 1H, NH), 11.32 (s, D2O exch., 1H, NH); 13C NMR: δ 58.21 (CH3), 61.89 (C-6′), 68.56 (C-4′), 74.88 (C-2′), 76.92 (C-3′), 79.22 (C-5′), 84.47 (C-1′), 113.43 (C-5), 123.77 (2C, Ar–C), 125.86 (2C, Ar–C), 133.97 (Ar–C), 154.16 (Ar–C), 164.86 (C-4), 168.44 (C-2), 169.95 (C=O), 182.23 (C-6). Anal. Calcd. for C18H22N4O7S2 (470.52): C, 45.95; H, 4.71; N, 11.91; S, 13.63. Found: C, 45.83; H, 4.65; N, 11.82; S, 13.55%.

Cytotoxicity Assay

H5N1 Influenza Virus

Antiviral activity assay and n class="Chemical">MTT cytotoxicity assay (TC50): samples were 10-fold serially diluted with Dulbecco’s Modified Eagle’s Medium (DMEM). Stock solutions of the test compounds were prepared in 10% DMSO in d.d H2O. The cytotoxic activity of the extracts was tested in Madin Darby Canine kidney (MDCK) cells by using the MTT method (Mossman, 1983)[38] with a minor modification. Briefly, the cells were seeded in 96-well plates (100 μL/well at a density of 3 × 105 cells/mL) and incubated for 24 h at 37 °C in 5%CO2. After 24 h, cells were treated with various concentrations of the tested compounds in triplicate. After further 24 h, the supernatant was discarded and cell monolayers were washed with sterile phosphate buffer saline (PBS) three times and MTT solution (20 μL of 5 mg/mL stock solution) was added to each well and incubated at 37 °C for 4 h, followed by medium aspiration. In each well, the formed formazan crystals were dissolved with 200 μL of acidified isopropanol (0.04 M HCl in absolute isopropanol = 0.073 mL HCL in 50 mL isopropanol). Absorbance of formazan solutions was measured at λmax 540 nm with 620 nm as a reference wavelength using a multiwell plate reader. Percentage of cytotoxicity compared to the untreated cells was determined. The plot of % cytotoxicity versus sample concentration was used to calculate the concentration, which was found to be 50% cytotoxicity (LD50).

Plaque Reduction Assay

Assay was carried out according to the method of Hayden et al. 1980[39] in a six-well plate. n class="CellLine">MDCK cells (105 cells/mL) were cultivated for 24 h at 37 °C. A/CHICKEN/7217B/1/2013 (H5N1) virus was diluted to give 105PFU/well and mixed with the safe concentration of the tested compounds and incubated for 30 min at 37 °C before being added to the cells. Growth medium was removed from the cell culture plates and virus-Cpd or virus-extract and Virus-oseltamivir mixtures were inoculated (100 μL/well), after 1 h contact time for virus adsorption, 3 mL of DMEM supplemented with 2% agarose was added onto the cell monolayer, and plates were left to solidify and incubated at 37 °C till formation of viral plaques (3–4 days). Formalin (10%) was added for 2 h and plates were stained with 0.1% crystal violet in distilled water. Control wells were included, where untreated virus was incubated with MDCK cells and finally plaques were counted and percentage reduction in plaque formation in comparison to control wells was recorded.

SARS-COV2

MTT Cytotoxicity Assay

To estimate the half-maximal cytotoxic concentration (CC50), we dissolved stock solutions of the tested compounds in DMSO 10% aqueous solution and diluted further to the working solutions with n class="Chemical">DMEM. We tested the cytotoxic activity by applying the MTT method with minor modifications in VERO-E6 cells. Briefly, we seeded the cells in 96-well plates and the cells were incubated at 37 °C and 5% CO2 for 24 h. After that, the cells were treated with different concentrations of the tested compounds in triplicate. Then, we completed the total methodology as previously mentioned in detail.[38] 50% cytotoxicity (TC50) was calculated by plotting the cytotoxicity percentage versus sample concentration.[39]

Inhibitory Concentration 50 (IC50) Determination

We determined The IC50 concentrations as previously described.[40] Briefly, in 96-well tissue culture plates, we distributed 2.4 × 104 Vero-E6 cells in each well and we incubated the plates overnight at a humidified 37 °C incubator under 5% n class="Chemical">CO2 condition. Then, we washed the cell monolayers once with 1× PBS and we subjected it to virus adsorption for 1 h at room temperature. The cell monolayers were further overlaid with 50 μL of DMEM containing varying concentrations of the test ARBs. After incubation for 72 h, we fixed the cells for 20 min using 100 μL of 4% paraformaldehyde and we stained the cells using 0.1% crystal violet in distilled water for 15 min at room temperature. Then, we dissolved the crystal violet dye using 100 μL absolute methanol per well and the optical density of the color was measured using an Anthos Zenyth 200rt plate reader at 570 nm. The IC50 of the compound is a value that is required to decrease the virus-induced cytopathic effect (CPE) by 50%, compared to the virus control.