Synthetic, Structural, and Anticancer Activity Evaluation Studies on Novel Pyrazolylnucleosides

Abstract

The synthesis of novel pyrazolylnucleosides 3a–e, 4a–e, 5a–e, and 6a–e are described. The structures of the regioisomers were elucidated by using extensive NMR studies. The pyrazolylnucleosides 5a–e and 6a–e were screened for anticancer activities on sixty human tumor cell lines. The compound 6e showed good activity against 39 cancer cell lines. In particular, it showed significant inhibition against the lung cancer cell line Hop-92 (GI₅₀ 9.3 µM) and breast cancer cell line HS 578T (GI₅₀ 3.0 µM).

1. Introduction

The chemistry of nucleosides has been extensively studied and several analogs have been found to exhibit potential as fungicidal, antitumor, and antiviral agents [1,2,3,4,5,6,7]. Modifications in both the heterocyclic bases and the sugar moieties have led to active and safer nucleoside analogues that have found applications as agents effective against human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS), and also against viral infections caused by the herpes simplex virus (HSV types 1 and 2), varicella zoster virus (VZV), hepatitis C virus (HCV), human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV) [8,9]. Nucleoside and nucleotide modifications resulted in an increased interest in the regio- and stereoselective synthesis of nucleosides [10,11]. Moreover, modified nucleosides and nucleotides with a restricted conformation have been used to reach a particular conformation of a rotamer to study the affinity of a biomacromolecule for its natural ligand as well as the molecular recognition in an oligonucleotide chain (RNA/DNA) [12,13,14,15]. Similar studies of anti-sense and anti-gene oligonucleotides (ONs) as potential and selective inhibitors of gene expression [16,17,18,19] and their use as anti-tumor or anti-viral agents [20,21,22,23] have also influenced the developments in the field of nucleic acid-based drugs. Among the nucleoside analogues with significant biological activities, dideoxynucleoside-based compounds such as 2′,3′-dideoxycytidine (ddC) [24], 2′,3′-dideoxyinosine (ddI) [24], and 3′-azidothymidine (AZT) [25] are effective therapeutic agents for the treatment of AIDS, while ribavirin (virazole) [26,27] is an antiviral drug (Figure 1). Similarly, other dideoxynucleosides such as d4T (2′,3′-didehydro-3′-deoxythymidine, stavudine) [28,29] and AZddU (3′-azido-2′,3′-dideoxyuridine) [30] have gone through clinical studies. Different nucleosides isolated from nature such as oxazinomycin [31], pyrazofurin [32,33], showdomycin [34,35], formycin A, and formycin B [36,37] have shown antibiotic properties and have also been found to exhibit anticancer and/or antiviral activities (Figure 1). These examples and new developments in the chemistry and biology of these compounds and their analogs [38,39,40,41,42] have motivated us to work in this area and have led us to investigate the interesting chemical and biological properties of novel nucleoside-based compounds.

Despite the developments in nucleoside chemistry, the clinical use of nucleosides has some drawbacks due to their side-effects and primary or acquired drug resistance [43]. Therefore, the search for the design of new and effective nucleoside-based analogues continues to motivate studies in this field. Our efforts toward this goal have led to the synthesis of twenty novel pyrazolylnucleosides with new structures and potent antiviral and antitumoral behaviors. In this regard, two regioisomers for each pyrazole derivative have been produced and characterized using spectroscopic techniques such as ¹H NMR, ¹³C NMR, NOESY, HMBC, IR, and mass spectroscopy. All compounds were evaluated against the National Cancer Institute (NCI)’s panel of 60 human tumor cell lines for their anticancer activities, and for their antiviral activities against representative viruses.

2. Results and Discussion

2.1. Chemical Synthesis of the Nucleoside Analogues 5 and 6

In our approach, the synthesis of the target compounds 5a–e and 6a–e was achieved in two steps starting from the already reported 3-cyanomethyl-5-aryl-1H-pyrazoles 1a–e [44] and the well-known 2-deoxy-3,5-di-O-p-toluoyl-α-d-ribofuranosyl chloride (2) [45] (Scheme 1). Treatment of pyrazoles 1a–e with sodium hydride in acetonitrile and the subsequent addition of chlorosugar 2 gave two regioisomers of the modified pyrazolyl nucleosides 3a–e (by coupling 2 with the N-1 nitrogen of the pyrazole derivatives) and 4a–e (by coupling chlorosugar 2 with the N-2 nitrogen of the pyrazole derivatives) in 58–65% yields. The products 3a–e and 4a–e were de-toluoylated by using sodium methoxide in methanol, resulting in the formation of the corresponding modified nucleosides 5a–e and 6a–e in 70–80% yields, respectively.

Pyrazoles with no substitution on either of the two N ring atoms can be alkylated to produce two regioisomers under strongly basic conditions. The anion generated produces the two resonance forms that react with chlorosugar 2 in the glycosidation step, leading to mixtures of two regioisomeric pyrazolyl nucleosides (i.e., 3a–e and 4a–e) (Scheme 2).

2.2. Structural Identification of the Isomeric Pyrazolyl Nucleoside Analogues 3–6

The structural identification of the isomeric disubstituted pyrazolyl nucleosides, based on their ¹H NMR spectral data (See Supplementary Materials), has been reported in the literature [46,47,48]. It has been observed that the anomeric protons in the isomeric pyrazolyl nucleosides exhibit different proton chemical shifts. The anomeric proton adjacent to N-1 of the pyrazole compounds 3a–e and 5a–e would appear downfield when compared to the anomeric proton adjacent to N-2 of the pyrazole compounds 4a–e and 6a–e [46,47,48]. In addition to using ¹H NMR chemical shifts, extensive NOE and 2D NMR experiments have been employed to confirm the structures of the isomeric pyrazolyl nucleosides. Using ¹H and ¹³C NMR studies, we confirmed the positional assignments of the isomeric pyrazolyl nucleosides synthesized in the present work (

Table 1. Chemical shift values of the anomeric protons in the ¹H NMR spectra and chemical shift values of the Ar- and CH₂CN bearing carbons of the pyrazole ring in the ¹³C NMR spectra of the isomeric pyrazolyl nucleosides 3a–e, 4a–e, 5a–e, and 6a–e in CDCl₃ on a Bruker Avance 300 spectrometer.

Compound	C-1′ H Shift in the 1H NMR Spectrum (ppm)		Ar- Bearing C Shifts in the 13C NMR Spectrum (ppm)		–CH2CN Bearing C Shifts in the 13C NMR Spectrum (ppm)
	Series 3	Series 5	Series 3	Series 5	Series 3	Series 5
a	6.13	5.99	144.6	145.9	141.7	142.0
b	6.10	6.02	146.2	145.8	144.0	141.8
c	6.05	5.97	145.4	144.9	141.9	141.9
d	6.05	6.00	145.2	144.7	143.7	141.9
e	6.05	6.00	145.6	143.9	142.3	141.1
	Series 4	Series 6	Series 4	Series 6	Series 4	Series 6
a	6.17	6.13	151.0	150.2	137.9	137.7
b	6.16	6.19	150.8	150.7	133.0	133.7
c	6.17	6.16	152.5	149.8	135.8	135.8
d	6.19	6.17	144.1	149.6	134.3	134.6
e	6.17	6.18	149.8	149.2	133.5	134.9

,

Table 2. Chemical shift and coupling constants from the ¹H-NMR of compounds 3d, 4d, 5d, and 6d in acetone-d₆.^a Please refer to structures in Table 4 for skeleton numbering.

Proton	Chemical Shifts (δ)				Coupling Constants (Hz)
	3d	4d	5d	6d	3d	4d	5d	6d
H1′	6.29	6.48	6.09	6.29	dd 5.7, 6.5	dd 6.5, 5.2	t* 6.4	t* 6.2
H2′α	2.72	2.79	2.31	2.42	ddd 14.0, 6.7, 4.1	ddd 14.0, 4.5, 6.6	ddd 13.5, 3.7, 6.8	ddd 13.5, 3.7, 6.7
H2′β	3.54	3.63	3.01	3.00	ddd 14.0, 6.8, 5.6	ddd 14.0, 5.2, 6.7	dt* 13.5, 6.0	dt* 13.5, 5.9
H3′	5.93	5.97	4.66	4.70	m	m	m	m
H4′	4.62	4.65	4.02	4.07	m	m	m	m
H5′α	4.54	4.45	3.61	3.59	m	dd 13.2, 6.4	ddd 11.8, 4.8, 7.7	ddd 11.9, 4.1, 8.1
H5′β	4.63	4.63	3.70	3.71	m	m	dt* 11.9, 4.7	dt* 11.9,4.1
H4	6.50	6.82	6.46	6.83	brs, 1H	t* 0.8	t* 0.5	t 0.8
H6	3.90	4.29	3.96	4.31	brs, 2H	19.0, 0.8	d 0.5	18.9, 0.8
H2″	7.67	7.87	7.60	7.87
H3″			7.57	7.46

^a The chemical shift values presented in Table 2 were all measured in acetone-d₆ (on an Bruker Avance 400 instrument) and may therefore differ from those in Table 1, which were measured in CDCl₃ (on Bruker Avance 300 instrument). * Due to the spatial interactions between different protons as explicitly shown in Table 4, these protons exhibited multiplicities as shown here when the spectra were recorded in acetone-d₆ on a Bruker Avance 400 instrument as against those recorded in CDCl₃, CD₃CN or DMSO-d₆ as given in the Experimental section for the corresponding protons where they appeared as broad singlets or ill resolved multiplets when their spectra were recorded on a Bruker Avance 300 instrument.

and

Table 3. Chemical shift values from the ¹³C-NMR of compounds 3d, 4d, 5d, and 6d in acetone-d₆.^a Please refer to the structures in Table 4 for skeleton numbering.

Chemical Shifts (δ) (Carbon Position)
3d	5d	4d	6d
87.6 (C1′)	87.4 (C1′)	87.6 (C1′)	88.0 (C1′)
37.1 (C2′)	40.5 (C2′)	37.0 (C2′)	41.1 (C2′)
76.6 (C3′)	73.2 (C3′)	76.1 (C3′)	73.0 (C3′)
83.5 (C4′)	89.8 (C4′)	83.7 (C4′)	90.0 (C4′)
65.1 (C5′)	64.2 (C5′)	64.7 (C5′)	63.9 (C5′)
143.8 (C3)	143.6 (C3)	136.3 (C3)	136.1 (C3)
107.2 (C4)	106.7 (C4)	105.4 (C4)	104.9 (C4)
146.0 (C5)	145.8 (C5)	150.6 (C5)	150.5 (C5)
17.6 (C6)	17.6 (C6)	15.3 (C6)	15.2 (C6)
118.1 (CN)	118.0 (CN)	117.0 (CN)	117.1 (CN)
166.4 (C6′′′)		166.5 (C6′′′)
166.5 (C6′′′′)		166.5 (C6′′′′)
*	129.3 (C1′′)	#	134.2 (C1′′)
*	131.6 (C2′′)	#	127.9 (C2′′)
*	129.9 (C3′′)	#	129.7 (C3′′)
*	135.6 (C4′′)	#	132.6 (C4′′)

^a The chemical shift values presented in Table 3 were all measured in acetone-d₆ (on an Bruker Avance 400 instrument) and may therefore differ from those in Table 1, which were measured in CDCl₃ (on Bruker Avance 300 instrument). * Benzene ring Carbons: Quaternary C: 145.0, 144.7, 135.6, 129.3, 128.3, 128.1; CHs: 131.6, 130.5, 130.5, 130.0, 130.0, 129.9. # Benzene ring Carbons: Quaternary C: 145.0, 144.5, 134.1, 132.7, 128.1; CHs: 130.5, 130.4, 130.1, 129.9, 129.5.

). The anomeric proton of the analogues 5a–e generally appeared at 0.14–0.19 ppm upfield when compared to the corresponding proton in the ¹H NMR spectra of its corresponding 6 series isomers. This is in agreement with observations for other 1,5- and 1,3-disubstituted pyrazole nucleosides [46,47,48]. We found that the effect was less pronounced in the 3 series of nucleosides as compared to the 4 series nucleosides where this difference was in the range of 0.04–0.14 ppm. The ¹³C chemical shift of the –CH₂CN bearing pyrazole carbon atom can help distinguish between positional isomers. In the 3 and 5 series of nucleosides, this carbon signal was 4–11 ppm downfield relative to the corresponding signal in the 4 and 6 series nucleosides. In most cases, the aryl bearing pyrazole carbon in the 3 and 5 series was 4–7 ppm upfield relative to the corresponding carbon in the 4 and 6 series. However, in the case of 3d versus 4d, the difference was small and reversed, making the aryl bearing pyrazole carbon less reliable for use in positional assignments.

In order to confirm the above positional assignments, we performed 2D NMR experiments (NOESY and HMBC) on the two pairs of compounds (i.e., one pair consisting of the ditoluoyl protected nucleosides 3d and 4d and another pair consisting of the deprotected nucleosides 5d and 6d) (

Table 4. Results from the NOESY and HMBC spectra of the pairs 3d–4d and 5d–6d *.

Blue and red arrows indicate the NOESY and HMBC cross peaks, respectively.
	3d	4d	5d	6d
NOESY H1′-H6	o	x	o	x
NOESY H1′-H2′′	x	o	x	o
NOESY H4-H2′′	x	x	x	x
HMBC H1′-C5	x	o	x	o
HMBC H1′-C3	o	x	o	x

* The presence of a cross peak is indicated by (x). When it contributes to the positional verification, it is underlined (x). See Table 1, Table 2 and Table 3 for positions of the relevant protons and carbons in the NMR spectra of the compounds 3d, 4d, 5d and 6d.

). The results of the HMBC experiments rely on the fact that the anomeric proton may see the aryl bearing pyrazole carbon in 3d and 5d, but not in 4d and 6d, while the NOESY results would explain the spatial proximity of the anomeric proton to either the protons in the –CH₂CN group or to the ortho protons of the aryl group. Our results verified the positional properties at the pyrazole ring in the four compounds and are summarized in Table 4.

The isomers of the 3 series nucleosides generally had higher R_f values on the TLC than the corresponding isomer of the 4 series of nucleosides. The same was observed for the isomers of the 5 series relative to those in the 6 series of nucleosides.

2.3. Anticancer Activity of the Isomeric Pyrazolyl Nucleoside Analogues 5a–e and 6a–e

The anticancer and toxicity of compounds 5a–e and 6a–e were evaluated by using the National Cancer Institute′s 60 human cancer cell lines. Compounds 5a–e, where the sugar is attached to N-1 of the pyrazole ring, were found to be inactive against all the cell lines irrespective of the substituent on the aromatic ring. In the other series, compounds 6a, 6b, and 6c with 4-methyl, 4-methoxy, and 4-fluoro substituents, respectively, at the aromatic ring were also inactive. However, compounds 6d and 6e with 4-chloro and 4-bromo substituents at the aromatic ring, respectively, showed inhibition against multiple anticancer cell lines. The GI₅₀ (cytostatic parameter) and LC₅₀ (toxicity parameter) values of 6d and 6e for the selected cell lines are given in

Table 5. Antitumor activity (GI₅₀/µM) ^a and toxicity (LC₅₀/µM) ^b data of 6d and 6e with the anticancer drug dasatinib as a positive control.

Panels/Cell Lines	6d		6e		Dasatinib
	GI50	LC50	GI50	LC50	GI50	LC50
Leukemia
CCRF-CEM	>67.5	>67.5	46.3	>57.5	5.3	>100
K-562	>67.5	>67.5	40.0	>57.5	0.01	>100
MOLT-4	>67.5	>67.5	30.9	>57.5	4.1	>100
RPMI-8226	>67.5	>67.5	34.0	>57.5	4.8	99.5
SR	>67.5	>67.5	25.5	>57.5	3.1	89.3
Non-Small Cell Lung Cancer
A549/ATCC	44.8	>67.5	29.3	>57.5	0.05	75.5
HOP-92	>67.5	>67.5	9.3	>57.5	0.01	>100
NCI-H322M	29.8	>67.5	30.2	>57.5	0.04	27.0
NCI-H522	>67.5	>67.5	25.7	>57.5	0.06	55.1
Colon Cancer
HCT-116	>67.5	>67.5	47.7	>57.5	11.8	69.8
HCT-15	>67.5	>67.5	45.1	>57.5	0.6	71.1
CNS Cancer
SF-268	33.5	>67.5	20.5	>57.5	0.07	75.5
SF-295	>67.5	>67.5	24.8	>57.5	1.1	46.8
SNB-19	24.6	>67.5	18.5	>57.5	11.9	76.4
SNB-75	53.5	>67.5	10.5	>57.5	0.01	46.3
U251	>67.5	>67.5	36.4	>57.5	3.2	52.4
Melanoma
LOX IMVI	>67.5	>67.5	49.7	>57.5	0.01	2.8
M14	>67.5	>67.5	57.1	>57.5	3.1	51.5
MDA-MB-435	>67.5	>67.5	53.0	>57.5	4.1	65.5
SK-MEL-2	>67.5	>67.5	39.6	>57.5	1.1	71.8
SK-MEL-5	60.9	>67.5	11.8	>57.5	4.5	44.3
UACC-62	37.8	>67.5	10.5	>57.5	2.8	47.0
Ovarian Cancer
IGROV1	32.6	>67.5	21.5	>57.5	0.01	80.0
OVCAR-3	>67.5	>67.5	39.1	>57.5	0.2	74.6
OVCAR-5	>67.5	>67.5	44.4	>57.5	0.02	86.3
Renal Cancer
A498	22.6	>67.5	18.8	>57.5	0.03	16.0
ACHN	>67.5	>67.5	45.1	>57.5	0.01	520
CAK-1	22.9	>67.5	18.8	>57.5	0.01	5.1
RXF 393	38.0	>67.5	34.6	>57.5	0.03	10.4
SN12C	48.4	>67.5	24.3	>57.5	0.05	44.4
UO-31	19.4	>67.5	16.6	>57.5	0.01	82.0
Prostate Cancer
PC-3	37.4	>67.5	20.0	>57.5	0.2	92.5
DU-145	>67.5	>67.5	45.1	>57.5	0.1	4.9
Breast Cancer
MCF7	48.0	>67.5	32.8	>57.5	12.6	71.4
MDA-MB-231ATCC	>67.5	>67.5	55.8	>57.5	0.01	38.0
HS 578T	16.6	>67.5	3.0	>57.5	0.01	>100
BT-549	49.9	>67.5	30.0	>57.5	5.9	49.8
T-47D	26.6	>67.5	19.4	>57.5	0.2	91.4
MDA-MB-468	31.6	>67.5	13.8	>57.5	0.09	8.4

^a GI₅₀: 50% Growth inhibition, concentration of drug resulting in a 50% reduction in net protein increase when compared with the control cells. ^b LC₅₀: Lethal concentration, concentration of drug lethal to 50% of cells.

. Compound 6d showed inhibition in 19 cell lines, and was most active against the renal cancer cell line UO-31 and breast cancer cell line HS 578T with GI₅₀ < 20 µM in both cases. The most active compound was 6e, which showed moderate inhibition in 39 cell lines. It showed significant inhibition against lung cancer cell line Hop-92 with a GI₅₀ of 9.3 µM and breast cancer cell line HS 578T with a GI₅₀ of 3.0 µM. Most importantly, both compounds (6d and 6e) did not show any toxicity, even at the highest concentration tested, as indicated by their high LC₅₀ values.

The pyrazolyl nucleosides 5a–e and 6a–e were also examined for their antiviral activities against a number of viruses such as Simplex virus type 1 (HSV-1) and type 2 (HSV-2), thymidine kinase-deficient (TK^-) strains of HSV-1, Vaccinia virus, para-influenza-3 virus, Sindbis virus, Coxsackie virus, Punta toro virus, vesicular stomatitis virus (VSV), Coxsackie virus B4 (CV-B4), respiratory syncytial virus (RSV), feline corona virus, and feline herpes virus. However, none of these compounds showed any significant activity against any of these viruses.

3. Experimental

3.1. General Information

Reactions were conducted under an atmosphere of nitrogen in anhydrous solvents. Column chromatography was carried out by using silica gel (100–200 mesh). Melting points were determined in a concentrated H₂SO₄ acid bath and were uncorrected. Analytical TLCs were performed on pre-coated Merck silica gel 60F₂₅₄ plates; the spots were detected either by using UV light or by charring with 4% alcoholic sulfuric acid. The IR spectra were recorded on a Perkin-Elmer 2000 FT-IR spectrophotometer (Waltham, MA, USA). The optical rotations were measured with a Bellingham-Stanley AD 220 polarimeter and the concentrations expressed as g/mL. The ¹H-NMR and ¹³C-NMR spectra were recorded on a Bruker Avance 300 spectrometer (Billerica, MA, USA) at 300 and 75.5 MHz, respectively in CDCl₃, DMSO-d₆, or CD₃CN. All 2D measurements were performed in acetone-d₆ on a Bruker Avance 400 spectrometer (Billerica, MA, USA). Chemical shifts are relative to internal TMS. Assignments were based on COSY, NOESY, HMBC (by using Bruker’s microprogram inv4gslplrnd, which includes the low-pass J-filter to suppress one-bond correlations), HSQC, and JRES spectra. The chemical shift values were reported as δ ppm relative to TMS used as the internal standard and the coupling constants (J) were measured in Hz. The ESI-HRMS spectra of all compounds were recorded on a JEOL JMS-AX505W high-resolution mass spectrometer (Tokyo, Japan) in positive ion mode by using the matrix HEDS (bis-hydroxyethylsulfide) doped with sodium acetate. Acetonitrile was used after distillation over freshly ignited potassium carbonate.

3.2. Synthesis and Characterization

3.2.1. Synthesis of 2′-Deoxy-1′-(3-cyanomethyl-5-aryl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (3a–e) and 2′-deoxy-1′-(3-aryl-5-cyanomethyl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (4a–e)

A solution of 3-cyanomethyl-5-arylpyrazole (1a–e, 15 mmol) in acetonitrile (190 mL) was added into a stirred mixture of sodium hydride (22.5 mmol) in acetonitrile (30 mL) under a nitrogen atmosphere at 30–35 °C, and continuously stirred for 30 min. The reaction mixture was cooled to 0 °C and 1-α-chloro-3,5-di-O-toluoyl-1,2-dideoxyribose (2, 15 mmol) was added and the contents stirred at 0 °C for 2.5 to 3 h. The progress of the reaction was monitored by using silica gel TLC. On completion, the reaction mixture was poured over ice-cold water and extracted with ethyl acetate (3 × 50 mL). The combined organic layer was washed with water (2 × 50 mL), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue thus obtained was column chromatographed over silica gel with ethyl acetate (8–10%) in petroleum ether as the eluent to afford the pyrazolyl nucleosides 3a–e and 4a–e in 31 to 35% and 24 to 29% yields, respectively.

2′-Deoxy-1′-(3-cyanomethyl-5-p-methylphenyl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (3a): Obtained as a white semisolid in a 34% yield. ${[\alpha ]}_{\mathrm{D}}^{18}$ = −115.46° (c 0.25, CHCl₃); R_f = 0.53 (20% ethyl acetate in petroleum ether); IR (Nujol): 2923, 2256 (CN), 1721 (COO), 1611, 1452, 1269, 1177 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.41 (9H, s, 3 × Ar-CH₃), 2.45–2.51(1H, m, C-2′H_α), 3.45–3.52 (1H, m, C-2′H_β), 3.65 (2H, s, CH₂CN), 4.55–4.67 (3H, m, C-4′H and C-5′ H_α+β), 5.86 (1H, brs, C-3′H), 6.13 (1H, t, J = 6.1 Hz, C-1′H), 6.33 (1H, s, C-4H), 7.21–7.24 (4H, brs, 4 × Ar-H), 7.28 (2H, d, J = 7.8 Hz, Ar-H), 7.42 (2H, d, J = 7.8 Hz, Ar-H), 7.90 (2H, d, J = 8.0 Hz, Ar-H), 8.00 (2H, d, J = 8.0, Ar-H); ¹³C NMR (75.5 MHz, CDCl₃): δ 15.6, 19.3, 19.7, 19.7, 34.6, 62.4, 73.9, 80.6, 84.5, 103.8, 115.2, 124.4, 124.7, 125.2, 127.0, 127.1, 127.1, 127.5, 127.7, 127.9, 137.2, 139.9, 142.1, 144.6, 141.7, 164.0, 164.3; HRMS (ESI): Calculated for C₃₃H₃₁N₃O₅ [M + Na]⁺ 572.2156, found [M + Na]⁺ 572.2163.

2′-Deoxy-1′-(3-cyanomethyl-5-p-methoxyphenyl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (3b): Obtained as a semisolid in a 31% yield. ${[\alpha ]}_{\mathrm{D}}^{18}$ = −119.46° (c 0.25, CHCl₃); R_f = 0.31 (20% ethyl acetate in petroleum ether); IR (Nujol): 2926, 2256 (CN), 1724 (CO), 1610, 1505, 1254, 1178 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.40 (6H, s, 2 × Ar-CH₃), 2.46–2.50 (1H, m, C-2′H_α), 3.48–3.53 (1H, m, C-2′H_β), 3.64 (2H, s, CH₂CN), 3.84 (3H, s, OCH₃), 4.54–4.62 (3H, m, C- C-4′H & 5′H_α+β), 5.87 (1H, brs, C-3′H), 6.10 (1H, brs, C-1′H), 6.30 (1H, s, C-4H), 6.99 (2H, d, J = 8.1, Ar-H), 7.21–7.23 (4H, m, Ar-H), 7.46 (2H, d, J = 8.0 Hz, Ar-H), 7.89 (2H, d, J = 7.6 Hz, Ar-H), 7.99 (2H, d, J = 7.6, Ar-H); ¹³C NMR (75.5 MHz, CDCl₃): δ 19.7, 23.7, 23.8, 38.7, 57.5, 66.5, 78.0, 84.7, 88.6, 107.8, 116.4, 119.3, 123.8, 128.9, 129.4, 131.1, 131.2, 131.8, 132.0, 132.6, 144.0, 145.8, 146.2, 148.5, 162.4, 168.1, 168.4; HRMS (ESI): Calculated for C₃₃H₃₁N₃O₆ [M + Na]⁺ 588.2105, found [M + Na]⁺ 588.2127.

2′-Deoxy-1′-(3-cyanomethyl-5-p-flurophenyl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (3c): Obtained as a white semisolid in a 35% yield. ${[\alpha ]}_{\mathrm{D}}^{18}$ = −63.64° (c 0.25, CHCl₃); R_f = 0.54 (20% ethyl acetate in petroleum ether); IR (Nujol): 2925, 2256 (CN), 1723 (COO), 1610, 1506, 1453, 1177, 1104 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.40 (6H, s, 2 × Ar-CH₃), 2.48–2.52 (1H, m, C-2′H_α), 3.50-3.54 (1H, m, C-2′H_β), 3.64 (2H, s, CH₂CN), 4.56–4.66 (3H, m, C-4′H & C-5′H), 5.87 (1H, brs, C-3′H), 6.05 (1H, t, J = 5.8 Hz, C-1′H), 6.33 (1H, s, C-4H), 7.13–7.24 (6H, m, Ar-H), 7.50–7.55 (2H, m, Ar-H), 7.90 (2H, d, J = 7.9 Hz, Ar-H), 7.98 (2H, d, J = 7.9 Hz, Ar-H); ¹³C NMR (75.5 MHz, CDCl₃): δ 17.5, 21.5, 21.6, 36.4, 64.2, 75.7, 82.6, 86.3, 106.1, 115.9 (d, J = 22 Hz), 116.9, 125.4, 126.6, 127.1, 128.9, 129.0, 129.6, 129.8, 131.0 (d, J = 8 Hz), 141.9, 143.7, 144.1, 145.4, 163.1 (d, J = 247 Hz), 165.9, 166.1; HRMS (ESI): Calculated for C₃₂H₂₈N₃O₅F [M + Na]⁺ 576.1905, found [M + Na]⁺ 576.1916.

2′-Deoxy-1′-(3-cyanomethyl-5-p-chlorophenyl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (3d): Obtained as a yellow solid in a 32% yield. ${[\alpha ]}_{\mathrm{D}}^{18}$ = −109.79° (c 0.25, CHCl₃); Mp = 114–115 °C; R_f= 0.52 (20% ethyl acetate in petroleum ether); IR (Nujol): 2924, 2257 (CN), 1728 (COO), 1610, 1494, 1177, 1108 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.40 (6H, s, 2 × Ar-CH₃), 2.46–2.52 (1H, m, C-2′H_α), 3.50–3.57 (1H, m, C-2′H_β), 3.63 (2H, s, CH₂CN), 4.51–4.66 (3H, m, C-4′H & C-5′H), 5.86–5.88 (1H, m, C-3′H), 6.05 (1H, t, J = 6.1 Hz, C-1′H), 6.35 (1H, s, C-4H), 7.21–7.24 (4H, m, Ar-H), 7.43–7.50 (4H, m, Ar-H), 7.90 (2H, d, J = 7.9 Hz, Ar-H), 7.98 (2H, d, J = 7.9 Hz, Ar-H); ¹³C NMR (75.5 MHz, CDCl₃): δ 17.4, 21.5, 21.6, 36.4, 64.1, 75.6, 82.7, 86.4, 106.1, 116.9, 126.4, 126.6, 127.6, 128.9, 129.0, 129.1, 129.6, 129.7, 130.3, 135.3, 141.9, 143.7, 144.1, 145.2, 165.9, 166.1; HRMS (ESI): Calculated for C₃₂H₂₈N₃O₅Cl [M + Na]⁺ 592.1610, found [M + Na]⁺ 592.1625.

2′-Deoxy-1′-(3-cyanomethyl-5-p-bromophenyl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (3e): Obtained as a white solid in a 35% yield. ${[\alpha ]}_{\mathrm{D}}^{18}$ = −96.35° (c 0.25, CHCl₃); Mp = 118–110 °C; R_f = 0.47 (20% ethyl acetate in petroleum ether); IR (Nujol): 2921, 2255 (CN), 1714 (COO), 1610, 1463, 1267, 1176, 1101 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.41 (6H, s, 2 × Ar-CH₃), 2.46–2.54 (1H, m, C-2′H_α), 3.48–3.57 (1H, m, C-2′H_β), 3.64 (2H, s, CH₂CN), 4.51–4.66 (3H, m, C-4′H & C-5′H), 5.85–5.87 (1H, m, C-3′H), 6.05 (1H, t, J = 6.1 Hz, C-1′H), 6.35 (1H, s, C-4H), 7.22 (4H, d, J = 7.9 Hz, Ar-H), 7.42 (2H, d, J = 8.3 Hz, Ar-H), 7.60 (2H, d, J = 8.3 Hz, Ar-H), 7.90 (2H, d, J = 8.0 Hz, Ar-H), 7.98 (2H, d, J = 8.0 Hz, Ar-H); ¹³C NMR (75.5 MHz, CDCl₃): δ 17.8, 21.9, 21.9, 36.8, 64.5, 76.0, 83.1, 86.8, 106.5, 117.3, 123.9, 127.0, 127.4, 128.5, 129.3, 129.4, 130.0, 130.1, 130.9, 132.4, 142.3, 144.0, 144.4, 145.6, 166.2, 166.5; HRMS (ESI): Calculated for C₃₂H₂₈N₃O₅Br [M + Na]⁺ 636.1105, found [M + Na]⁺ 636.1113.

2′-Deoxy-1′-(3-p-methylphenyl-5-cyanomethyl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (4a): Obtained as a white semisolid in a 26% yield. ${[\alpha ]}_{\mathrm{D}}^{18}$ = +25.10° (c 0.25, CHCl₃); R_f = 0.36 (20% ethyl acetate in petroleum ether); IR (Nujol): 2923, 2256 (CN), 1721 (COO), 1611, 1452, 1269, 1177 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.31, 2.38 and 2.42 (9H, 3s, 3H each, 3 × Ar-CH₃), 2.65-2.70 (1H, m, C-2′H_α), 3.74–3.78 (1H, m, C-2′H_β), 3.91 (2H, s, CH₂CN), 4.36 (1H, q, J = 6.3 Hz, C-4′H), 4.54–4.60 (2H, m, C-5′H), 5.85-5.87 (1H, m, C-3′H), 6.17 (1H, t, J = 5.7 Hz, C-1′H), 6.59 (1H, s, C-4H), 7.03 (2H, d, J = 8.0 Hz, Ar-H), 7.18 (2H, d, J = 7.9 Hz, Ar-H), 7.26 (2H, d, J = 8.1 Hz, Ar-H), 7.63 (2H, d, J = 8.0 Hz, Ar-H), 7.83 (2H, d, J = 8.1 Hz, Ar-H), 7.95 (2H, d, J = 8.0 Hz, Ar-H); ¹³C NMR (75.5 MHz, CDCl₃): δ 15.28, 21.2, 21.5, 21.6, 36.0, 63.6, 75.0, 83.2, 87.0, 105.3, 115.3, 125.6, 126.6, 126.7, 128.9, 129.0, 129.1, 129.2, 129.6, 129.7, 133.0, 137.9, 143.5, 144.2, 151.0, 165.9, 166.0; HRMS (ESI): Calculated for C₃₃H₃₁N₃O₅ [M + Na]⁺ 572.2156, found [M + Na]⁺ 572.2155.

2′-Deoxy-1′-(3-p-methoxyphenyl-5-cyanomethyl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (4b): Obtained as a white solid in a 24% yield. ${[\alpha ]}_{\mathrm{D}}^{18}$ = +42.05° (c 0.25, CHCl₃); Mp = 88–90 °C; R_f= 0.24 (20% ethyl acetate in petroleum ether); IR (Nujol): 2924, 2256 (CN), 1724 (COO), 1610, 1456, 1270, 1178 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.32 and 2.42 (6H, 2s, 3H each, 2 × Ar-CH₃), 2.65–2.69 (1H, m, C-2′H_α), 3.73–3.77 (1H, m, C-2′H_β), 3.83 (3H, s, Ar-OCH₃), 3.90 (2H, s, CH₂CN), 4.34–4.37 (1H, m, C-4′H), 4.56–4.59 (2H, m, C-5′H), 5.87 (1H, brs, C-3′H), 6.16 (1H, brs, C-1′H), 6.55 (1H, s, C-4H), 6.91 (2H, d, J = 7.6 Hz, Ar-H), 7.05 (2H, d, J = 7.3 Hz, Ar-H), 7.26 (2H, d, J = 6.9 Hz, Ar-H), 7.67 (2H, d, J = 7.7 Hz, Ar-H), 7.84 (2H, d, J = 7.3 Hz, Ar-H), 7.96 (2H, d, J = 7.4 Hz, Ar-H); ¹³C (75.5 MHz, CDCl₃): δ 15.2, 21.5, 21.6, 36.0, 55.2, 63.6, 75.0, 83.1, 86.9, 105.0, 113.9, 115.4, 125.2, 126.6, 126.9, 128.9, 129.1, 129.5, 129.6, 129.7, 133.0, 143.5, 144.1, 150.8, 159.6, 165.8, 165.9; HRMS (ESI): Calculated for C₃₃H₃₁N₃O₆ [M + Na]⁺ 588.2105, found [M + Na]⁺ 588.2134.

2′-Deoxy-1′-(3-p-flurophenyl-5-cyanomethyl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (4c): Obtained as a white semisolid in a 25% yield. ${[\alpha ]}_{\mathrm{D}}^{18}$ = +14.07° (c 0.25, CHCl₃); R_f = 0.47 (20% ethyl acetate in petroleum ether); IR (Nujol): 2925, 2257 (CN), 1725 (COO), 1610,1522,1443,1272, 1178, 1102 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.31, and 2.42 (6H, 2s, 3H each, 2 × Ar-CH₃), 2.66–2.70 (1H, m, C-2′H_α), 3.73–3.77 (1H, m, C-2′H_β), 3.92 (2H, s, CH₂CN), 4.33–4.35 (1H, m, C-4′H), 4.58–4.63 (2H, m, C-5′H), 5.87 (1H, brs, C-3′H), 6.17 (1H, t, J = 5.1 Hz, C-1′H), 6.56 (1H, s, C-4H), 7.02–7.08 (4H, m, Ar-H), 7.26 (2H, d, J = 7.9 Hz, Ar), 7.67–7.71 (2H, m, Ar-H), 7.83 (2H, d, J = 8.0 Hz, Ar-H), 7.96 (2H, d, J = 8.0 Hz, Ar-H); ¹³C NMR (75.5 MHz, CDCl₃): δ 17.7, 23.9, 24.0, 38.5, 66.0, 77.3, 85.7, 89.5, 107.7, 117.8 (d, J = 22 Hz), 128.0, 129.0 (d, J = 7.5 Hz), 129.7, 129.8, 131.0, 131.4, 131.6, 132.0, 132.1, 135.8, 146.0, 146.7, 152.5, 165.4 (d, J = 250 Hz), 168.2, 168.4; HRMS (ESI): Calculated for C₃₂H₂₈N₃O₅F [M + Na]⁺ 576.1905, found [M + Na]⁺ 576.1915.

2′-Deoxy-1′-(3-p-chlorophenyl-5-cyanomethyl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (4d): Obtained as a yellow solid in a 24% yield. ${[\alpha ]}_{\mathrm{D}}^{18}$ = +34.06° (c 0.25, CHCl₃); Mp = 109–110 °C; R_f = 0.40 (20% ethyl acetate in petroleum ether); IR (nujol): 2924, 2257 (CN), 1716 (COO), 1459, 1377, 1176, cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.33 and 2.45 (6H, 2s, 3H each, 2 × Ar-CH₃), 2.65–2.74 (1H, m, C-2′H_α), 3.74–3.85 (1H, m, C-2′H_β), 3.94 (2H, s, CH₂CN), 4.33–4.38 (1H, m, C-4′H), 4.57–4.65 (2H, m, C-5′H), 5.88–5.90 (1H, m, C-3′H), 6.19 (1H, t, J = 5.8 Hz, C-1′H), 6.59 (1H, s, C-4H), 7.04 (2H, d, J = 7.9 Hz, Ar-H), 7.29 (2H, d, J = 8.0 Hz, Ar-H), 7.35 (2H, d, J = 8.4 Hz, Ar-H), 7.66 (2H, d, J = 8.4 Hz, Ar-H), 7.82 (2H, d, J = 8.1 Hz, Ar-H), 7.98 (2H, d, J = 8.0 Hz, Ar-H); ¹³C NMR (75.5 MHz, CDCl₃): δ 15.7, 21.9, 22.0, 36.5, 63.9, 75.3, 83.8, 87.6, 105.8, 116.5, 127.0, 127.3, 129.1, 129.4, 129.6, 130.0, 130.1, 130.2, 131.3, 133.9, 134.3, 144.1, 144.7, 150.3, 166.2, 166.4; HRMS (ESI): Calculated for C₃₂H₂₈N₃O₅Cl [M + Na]⁺ 592.1610, found [M + Na]⁺ 592.1621.

2′-Deoxy-1′-(3-p-bromophenyl-5-cyanomethyl-)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (4e): Obtained as a white solid in a 29% yield. ${[\alpha ]}_{\mathrm{D}}^{18}$ = +39.82° (c 0.25, CHCl₃); Mp = 118–120 °C; R_f = 0.25 (20% ethyl acetate in petroleum ether); IR (Nujol): 2923, 2255 (CN), 1716 (COO), 1611, 1464, 1270, 1177 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.31 and 2.42 (6H, 2s, 3H each, 2 × Ar-CH₃), 2.62–2.66 (1H, m, C-2′H_α), 3.79–3.82 (1H, m, C-2′H_β), 3.92 (2H, s, CH₂CN), 4.32–4.36 (1H, m, C-4′H), 4.56–4.62 (2H, m, C-5′H), 5.86–5.88 (1H, m, C-3′H), 6.17 (1H, t, J = 5.7 Hz, C-1′H), 6.57 (1H, s, C-4H), 7.03 (2H, d, J = 7.9 Hz, Ar-H), 7.26 (2H, d, J = 7.9 Hz, Ar-H), 7.48 (2H, d, J = 8.3 Hz, Ar-H), 7.57 (2H, d, J = 8.4 Hz, Ar-H), 7.80 (2H, d, J = 8.0 Hz, Ar-H), 7.96 (2H, d, J = 8.0 Hz, Ar-H); ¹³C NMR (75.5 MHz, CDCl₃): δ 15.2, 21.5, 21.6, 36.0, 63.4, 74.8, 83.3, 87.1, 105.3, 115.3, 122.0, 126.5, 126.6, 127.2, 128.9, 129.1, 129.5, 129.7, 131.3, 131.6, 133.5, 143.6, 144.2, 149.8, 166.0, 166.1; HRMS (ESI): Calculated for C₃₂H₂₈N₃O₅Br [M + Na]⁺ 636.1105, found [M + Na]⁺ 636.1116.

3.2.2. Synthesis of 2′-Deoxy-1′-(3-cyanomethyl-5-aryl)pyrazolyl-β-D-ribofuranose (5a–e) and 2′-deoxy-1′-(3-aryl-5-cyanomethyl)pyrazolyl-β-d-ribofuranose (6a–e)

2′-Deoxy-1′-(3-cyanomethyl-5-aryl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (3a–e) or 2′-deoxy-1′-(3-aryl-5-cyanomethyl)pyrazolyl-3′,5′-di-O-toluoyl-β-d-ribofuranose (4a–e, 1.3 mmol) was suspended in dry methanol (18 mL), then a mixture of NaOMe and MeOH (10 mL, 1:3, v/v) was added to the resulting solution, and the contents stirred at room temperature for 3–4 h. The progress of the reaction was monitored by using silica gel TLC. On completion, NH₄Cl was added to the reaction mixture to adjust the pH to 8. One-third of the solvent was removed under reduced pressure and the reaction mixture was poured in water and extracted with ethyl acetate (3 × 30 mL), the organic layer was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue thus obtained was subjected to column chromatography over silica gel with methanol (2–2.5%) in chloroform as the eluent to afford 2′-deoxy-1′-(3-cyanomethyl-5-aryl)pyrazolyl-β-d-ribofuranose (5a–e) and 2′-deoxy-1′-(3-aryl-5-cyanomethyl)pyrazolyl-β-d-ribofuranose (6a–e).

2′-Deoxy-1′-(3-cyanomethyl-5-p-methylphenyl)pyrazolyl-β-d-ribofuranose (5a): Obtained as a white semisolid in a 63% yield. ${[\alpha ]}_{\mathrm{D}}^{30}$ = −87.56° (c 0.25, MeOH); R_f = 0.3 (7% methanol in chloroform); IR (Nujol): 3391 (OH), 2936, 2258 (CN), 1613, 1506, 1456, 1253, 1180, 1055 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.10–2.18 (1H, m, C-2′H_α), 2.39 (3H, s, Ar-CH₃), 2.87–2.91 (1H, m, C-2′H_β), 3.53 (1H, brs, C-5′H_α), 3.61 (1H, brs, C-5′H_β), 3.84 (1H, brs, C-4′H), 3.90 (2H, s, CH₂CN), 4.44 (1H, brs, C-3′H), 4.49 (1H, brs, C-3′OH), 5.13 (1H, brs, C-5′OH), 5.99 (1H, brs, C-1′H), 6.30 (1H, s, C-4H), 7.29 (2H, d, J = 7.3, Ar-H), 7.40 (2H, d, J = 7.7 Hz, Ar-H); ¹³C NMR (75.5 MHz, CDCl₃): δ 17.3, 21.1, 39.9, 63.0, 71.6, 86.0, 88.3, 105.3, 117.7, 126.5, 128.9, 129.5, 138.8, 142.0, 145.9; HRMS (ESI): Calculated for C₁₇H₁₉N₃O₃ [M + Na]⁺ 336.1319, found [M + Na]⁺ 336.1288.

2′-Deoxy-1′-(3-cyanomethyl-5-p-methoxyphenyl)pyrazolyl-β-d-ribofuranose (5b): Obtained as a white semisolid in a 66% yield. ${[\alpha ]}_{\mathrm{D}}^{30}$ = −94.35° (c 0.25, MeOH); R_f = 0.48 (7% methanol in chloroform); IR (Nujol): 3391 (OH), 2932, 2254 (CN), 1612, 1510, 1457, 1253, 1183, 1056 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆): δ 2.17–2.21 (1H, m, C-2′H_α), 2.89–2.93 (1H, m, C-2′H_β), 3.57–3.61 (1H, m, C-5′H_α), 3.65–3.70 (1H, m, C-5′H_β), 3.83-3.85 (5H, m, Ar-OCH₃ & CH₂CN), 3.92-3.94 (1H, m, C-4′H), 4.53 (1H, brs, C-3′H), 4.64 (1H, t, J = 6.6, C-5′OH), 5.09 (1H, d, J = 4.1 Hz, C-3′OH), 6.02 (1H, t, J = 6.3 Hz, C-1′H), 6.26 (1H, s, C-4H), 7.00 (2H, d, J = 8.6 Hz, Ar-H), 7.42 (2H, d, J = 8.6 Hz, Ar-H); ¹³C NMR (75.5 MHz, DMSO-d₆): δ 17.3, 39.9, 55.3, 63.1, 71.8, 86.0, 88.4, 105.1, 114.3, 117.4, 121.5, 130.3, 141.8, 145.8, 160.1; HRMS (ESI): Calculated C₁₇H₁₉N₃O₄ [M + Na]⁺ 352.1268, found [M + Na]⁺ 352.1248.

2′-Deoxy-1′-(3-cyanomethyl-5-p-fluorophenyl)pyrazolyl-β-d-ribofuranose (5c): Obtained as a semisolid in a 63% yield. ${[\alpha ]}_{\mathrm{D}}^{30}$ = −153.52° (c 0.25, MeOH); R_f = 0.41 (7% methanol in chloroform); IR (Nujol): 3393 (OH), 2932, 2256 (CN), 1611, 1508, 1451, 1255, 1182, 1051 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆): δ 2.18–2.22 (1H, m, C-2′H_α), 2.91–2.95 (1H, m, C-2′H_β), 3.61 (1H, brs, C-5′H_α), 3.65 (1H, brs, C-5′H_β), 3.87 (2H, s, CH₂CN), 3.93 (1H, brs, C-4′H), 4.53 (2H, brs, C-3′H and OH), 5.08 (1H, brs, C-5′OH), 5.97 (1H, brs, C-1′H), 6.32 (1H, s, C-4H), 7.22 (2H, brs, Ar-H), 7.53 (2H, brs, Ar-H); ¹³C NMR (75.5 MHz, DMSO-d₆): δ 17.3, 39.9, 63.0, 71.7, 86.1, 88.4, 105.7, 115.9 (d, J = 22 Hz), 117.3, 125.6, 131.1 (d, J = 8 Hz), 141.9, 144.9, 162.8 (d, J = 246 Hz); HRMS (ESI): Calculated for C₁₆H₁₆N₃O₃F [M + Na]⁺ 340.1068, found [M + Na]⁺ 340.1058.

2′-Deoxy-1′-(3-cyanomethyl-5-p-chlorophenyl)pyrazolyl-β-d-ribofuranose (5d): Obtained as a semisolid in a 63% yield. ${[\alpha ]}_{\mathrm{D}}^{30}$ = −124.74° (c 0.25, MeOH); R_f = 0.46 (7% methanol in chloroform); IR (Nujol): 3393 (OH), 2932, 2256 (CN), 1611, 1498, 1451, 1254, 1181, 1048 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆): δ 2.22–2.24 (1H, m, C-2′H_α), 2.92–2.96 (1H, m, C-2′H_β), 3.59–3.61 (1H, m, C-5′H_α), 3.68–3.72 (1H, m, C-5′H_β), 3.85 (2H, brs, CH₂CN), 3.97 (1H, brs, C-4′H), 4.57 (2H, brs, C-3′H and OH), 5.09 (1H, brs, C-5′OH), 6.00 (1H, brs, C-1′H), 6.34 (1H, s, C-4H), 7.24 (2H, d, J = 7.5 Hz, Ar-H), 7.87 (2H, d, J = 8.5 Hz, Ar-H); ¹³C (75.5 MHz, DMSO-d₆): δ 17.3, 39.9, 63.1, 71.8, 86.2, 88.5, 105.7, 117.8, 127.8, 129.3, 130.4, 134.7, 141.9, 144.7; HRMS (ESI): Calculated for C₁₆H₁₆N₃O₃Cl [M + Na]⁺ 356.0772, found [M + Na]⁺ 356.0770.

2′-Deoxy-1′-(3-cyanomethyl-5-p-bromophenyl)pyrazolyl-β-d-ribofuranose (5e): Obtained as a semisolid in a 64% yield. ${[\alpha ]}_{\mathrm{D}}^{30}$ = −105.15° (c 0.25, MeOH); R_f = 0.53 (7% methanol in chloroform); IR (Nujol): 3387 (OH), 2935, 2249 (CN), 1612, 1504, 1454, 1248, 1179, 1048 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆): δ 2.20–2.24 (1H, m, C-2′H_α), 2.90–2.96 (1H, m, C-2′H_β), 3.59–3.61 (1H, m, C-5′H_α), 3.68–3.72 (1H, m, C-5′H_β), 3.85 (2H, brs, CH₂CN), 3.97 (1H, brs, C-4′H), 4.56 (2H, brs, C-3′H & OH ), 5.07 (1H, brs, C-5′OH), 6.00 (1H, t, J = 6.06 Hz, C-1′H), 6.34 (1H, s, C-4H), 7.42 (2H, d, J = 8.1 Hz, Ar-H), 7.61 (2H, d, J = 8.0 Hz, Ar-H); ¹³C (75.5 MHz, DMSO-d₆): δ 16.5, 39.0, 62.2, 71.0, 85.3, 87.6, 104.8, 116.3, 122.2, 128.2, 129.8, 131.0, 141.1, 143.9; HRMS (ESI): Calculated for C₁₆H₁₆N₃O₃Br [M + Na]⁺ 400.0267, found [M + Na]⁺ 400.0232.

2′-Deoxy-1′-(5-cyanomethyl-3-p-methylphenyl)pyrazolyl-β-d-ribofuranose (6a): Obtained as a white semisolid in a 61% yield. ${[\alpha ]}_{\mathrm{D}}^{30}$ = +34.78° (c 0.25, MeOH); R_f = 0.3 (7% methanol in chloroform); IR (Nujol): 3392 (OH), 2934, 2258 (CN), 1611, 1504, 1452, 1254, 1179, 1051 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.23–2.27 (1H, m, C-2′H_α), 2.31 (3H, s, Ph-CH₃), 2.77–2.83 (1H, m, C-2′H_β), 3.36–3.40 (1H, m, C-5′H_α), 3.49–3.51 (1H, m, C5′H_β), 3.82–3.86 (1H, m, C-4′H), 4.32 (2H, s, CH₂CN), 4.46 (1H, brs, C-3′H), 4.86 (1H, t, J = 5.4 Hz, C-5′OH), 5.29 (1H, d, J = 4.0 Hz, C-3′OH), 6.13 (1H, t, J = 5.9 Hz, C-1′H), 6.72 (1H, s, C-4H), 7.22 and 7.69 (4H, 2d, 2H each, J = 7.8 Hz, & 7.9 Hz, ArH); ¹³C NMR (75.5 MHz, DMSO-d₆): δ 14.6, 21.1, 39.8, 62.6, 71.4, 86.5, 88.4, 104.0, 117.5, 125.5, 129.6, 130.1, 135.0, 137.7, 150.2; HRMS (ESI): Calculated for C₁₇H₁₉N₃O₃ [M + Na]⁺ 336.1319, found [M + Na]⁺ 336.1290.

2′-Deoxy-1′-(5-cyanomethyl-3-p-methoxyphenyl)pyrazolyl-β-d-ribofuranose (6b): Obtained as a semisolid in a 62% yield. ${[\alpha ]}_{\mathrm{D}}^{30}$ = +56.37^o (c 0.25, MeOH); R_f = 0.39 (7% methanol in chloroform); IR (Nujol): 3385 (OH), 2937, 2258 (CN), 1610, 1501, 1448, 1251, 1183, 1053 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆): δ 2.40–2.42 (1H, m, C-2′H_α), 2.94–2.98 (1H, m, C-2′H_β), 3.60–3.64 (1H, m, C-5′H_α), 3.72–3.76 (1H, m, C-5′H_β), 3.81 (3H, s, Ph-OCH₃), 3.87 (1H, brs, C-4′H), 4.08 (2H, s, CH₂CN), 4.61 (1H, brs, C-3′H), 5.01–5.05 (2H, m, C-5′OH & C-3′OH), 6.19 (1H, t, J = 6.0 Hz, C-1′H), 6.56 (1H, s, C-4H), 6.91 and 7.66 (4H, 2d, 2H each, J = 8.4 Hz and 8.4 Hz, Ar-H); ¹³C NMR (75.5 MHz, DMSO-d₆): δ 14.9, 39.9, 55.1, 63.2, 71.9, 86.9, 88.9, 103.6, 114.0, 116.1, 126.7, 129.3, 133.7, 150.7, 159.6; HRMS (ESI): Calculated for C₁₇H₁₉N₃O₄ [M + Na]⁺ 352.1268, found [M + Na]⁺ 352.1237.

2′-Deoxy-1′-(5-cyanomethyl-3-p-fluorophenyl)pyrazolyl-β-d-ribofuranose (6c): Obtained as a semisolid in a 59% yield. ${[\alpha ]}_{\mathrm{D}}^{30}$ = +47.97° (c 0.25, MeOH); R_f = 0.36 (7% methanol in chloroform); IR (Nujol): 3391 (OH), 2928, 2249 (CN), 1610, 1503, 1448, 1252, 1179, 1055 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 2.24–2.28 (1H, m, C-2′H_α), 2.85–2.89 (1H, m, C-2′H_β), 3.39–3.41 (1H, m, C-5′H_α), 3.48–3.54 (1H, m, C-5′ H_β), 3.82–3.87 (1H, m, C-4′H), 4.33 (2H, s, CH₂CN), 4.46 (1H, brs, C-3′H), 4.81 (1H, t, J = 5.4 Hz, C-5′OH), 5.27 (1H, d, J = 4.0 Hz, C-3′OH), 6.16 (1H, t, J = 6.0 Hz, C-1′H), 6.76 (1H, s, C-4H), 7.24 (2H, t, J = 7.6 Hz), 7.83 (2H, q, J = 5.3 Hz, Ar-H); ¹³C NMR (75.5 MHz, DMSO-d₆): δ 15.2, 40.3, 63.1, 71.9, 87.0, 89.0, 104.7, 116.5 (d, J = 22 Hz), 117.9, 128.1 (d, J = 8 Hz), 130.0, 135.8, 149.8, 162.9 (d, J = 243 Hz); HRMS (ESI): Calculated for C₁₆H₁₆N₃O₃F [M + Na]⁺ 340.1068, found [M + Na]⁺ 340.1038.

2′-Deoxy-1′-(3-p-chlorophenyl-5-cyanomethyl-)pyrazolyl-β-d-ribofuranose (6d): Obtained as a semisolid in a 61% yield. ${[\alpha ]}_{\mathrm{D}}^{30}$ = +37.58° (c 0.25, MeOH); R_f = 0.36 (7% methanol in chloroform); IR (Nujol): 3387 (OH), 2935, 2249 (CN), 1612, 1504, 1454, 1248, 1179, 1048 cm⁻¹; ¹H NMR (300 MHz, CD₃CN): δ 2.36–2.43 (1H, m, C-2′H_α), 2.88–2.96 (1H, m, C-2′H_β), 3.43 (1H, brs, C-4′H), 3.56–3.63 (1H, m, C-5′H_α), 3.69–3.73 (1H, m, C-5′ H_β), 4.05–4.11 (4H, m, CH₂CN, C-3′H & C-5′OH), 4.62 (1H, brs, C-3′OH), 6.17 (1H, t, J = 6.1 Hz, C-1′H), 6.72 (1H, s, C-4H), 7.44 (2H, d, J = 8.5 Hz, Ar-H), 7.77 (2H, d, J = 8.5 Hz, Ar-H); ¹³C NMR (75.5 MHz, CD₃CN): δ 14.4, 39.9, 62.6, 71.7, 86.6, 88.6, 103.7, 116.9, 126.6, 128.5, 130.9, 133.1, 134.6, 149.6; HRMS (ESI): Calculated for C₁₆H₁₆N₃O₃Cl [M + Na]⁺ 356.0772, found [M + Na]⁺ 356.0757.

2′-Deoxy-1′-(3-p-bromophenyl-5-cyanomethyl)pyrazolyl-β-d-ribofuranose (6e): Obtained as a white solid in a 63% yield. ${[\alpha ]}_{\mathrm{D}}^{30}$ = +50.37° (c 0.25, MeOH); Mp = 74–75 °C; R_f = 0.39 (7% methanol in chloroform); IR (Nujol): 3385 (OH), 2937, 2258 (CN), 1610, 1501, 1448, 1251, 1183, 1053 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆): δ 2.34–2.39 (1H, m, C-2′H_α), 2.92–2.98 (1H, m, C-2′H_β), 3.47 (1H, brs, C-4′H), 3.58 (1H, brs, C-5′H_α), 3.95 (1H, brs, C-5′ H_β), 4.24 (2H, s, CH₂CN), 4.51 (1H, brs, 3′H), 4.77 (1H, brs, C-3′OH), 5.20 (1H, brs, C-5′OH), 6.18 (1H, s, C-1′H), 6.71 (1H, s, C-4H), 7.55 (2H, brs, Ar-H), 7.69 (2H, brs, Ar-H); ¹³C NMR (75.5 MHz, DMSO-d₆): δ 14.9, 39.7, 62.7, 71.4, 86.9, 88.6, 104.3, 116.8, 121.5, 127.3, 129.2, 131.7, 134.9, 149.2; HRMS (ESI): Calculated for C₁₆H₁₆N₃O₃Br [M + Na]⁺ 400.0267, found [M + Na]⁺ 400.0234.

3.3. NCI-60 Human Tumor Cell Line Screen

Details of the methodology are described at http://dtp.nci.nih.gov/branches/btb/ivclsp.html. Briefly, the panel was organized into nine subpanels representing a diverse histology: leukemia, melanoma, and cancers of the lung, colon, kidney, ovary, breast, prostate, and central nervous system. The cells were grown in supplemented RPM1 1640 medium for 24 h. For the five dose study, the test compounds were dissolved in DMSO and incubated with cells at five concentrations with 10-fold dilutions, the highest being 10⁻⁴ M and the others being 10⁻⁵, 10⁻⁶, 10⁻⁷, and 10⁻⁸ M. The assay was terminated by the addition of cold trichloroacetic acid, and the cells were fixed and stained with sulforhodamine B. The bound stain was solubilized, and the absorbance was read on an automated plate reader. The cytostatic parameter, which determines the 50% growth inhibition (GI₅₀) of the tumor cells, was calculated from time zero, control growth, and the absorbance at the five concentration levels. The cytotoxic parameter, lethal concentration (LC₅₀ is the concentration of a drug resulting in a 50% reduction in the measured protein at the end of the drug treatment when compared to that at the beginning), indicating a net loss of cells following the treatment, was calculated as the average of two independent experiments.

4. Conclusions

We successfully achieved the synthesis of twenty novel pyrazolyl nucleosides 3a–e, 4a–e, 5a–e, and 6a–e, which have been characterized in detail by using various NMR spectroscopic techniques such as ¹H NMR, ¹³C NMR, NOESY, HMBC, etc. The pyrazolyl nucleosides 5a–e and 6a–e were screened for anticancer activities on 60 human tumor cell lines, and we identified one compound 6e, which showed good activity against 39 cancer cell lines and showed significant inhibition against lung cancer cell line Hop-92 (GI₅₀ 9.3 µM) and breast cancer cell line HS 578T (GI₅₀ 3.0 µM). Our studies have demonstrated the potential of newly synthesized pyrazolyl-nucleosides that will be pursued further.