Synthesis, Antiproliferative Activity, and ADME Profiling of Novel Racemic and Optically Pure Aryl-Substituted Purines and Purine Bioisosteres

Abstract

The aim of this study was to synthesize new racemic and optically pure aryl-substituted purine bioisosteres using ultrasound-assisted Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition. Regioselective synthesis of α-azido alcohols was applied to afford heterocycles with a 2-hydroxyeth-1-yl linker. Catalytic asymmetric synthesis using halohydrin dehalogenase in the ring-opening of epoxides gave enantioenriched azido alcohols, which subsequently afforded R- and S-enantiomers of purine and pyrrolo[2,3-d]pyrimidines with a 1-hydroxyeth-2-yl linker. The newly synthesized compounds were evaluated in vitro for their antiproliferative activity against four malignant tumor cell lines. The influence of regioisomerism and the stereochemistry of the hydroxyethyl group, as well as a N-heterocyclic scaffold linked to the aryl moiety on cytostatic activity was evaluated. Of all the compounds tested, purine 40a and pyrrolo[2,3-d]pyrimidine 45a derivatives with p-trifluoromethyl-substituted aryl connected to 1,2,3-triazole via a 2-hydroxyeth-1-yl spacer showed promising submicromolar antiproliferative activity. In addition, compound 45a exhibited selectivity towards the tumor cell line, with a selectivity index (SI) of 40, moderate clearance, and good membrane permeability.

1. Introduction

Due to the increasing incidence of cancer and the problems of multiple side effects and resistance to classical chemotherapeutic agents, the search for new antitumor agents is becoming more urgent [1]. It was estimated that in 2020, more than 19.3 million new cancer cases were diagnosed and approximately 10.0 million deaths were recorded from cancer worldwide [2]. The growing proportion of chiral drug molecules on the market takes advantage of the chiral switching of already marketed racemates and develops de novo enantiomerically pure compounds [3,4,5,6]. As a consequence, enantioselective synthesis has gained considerable attention in medicinal chemistry because the different enantiomers or diastereomers of a molecule often exhibit different biological activities [7,8,9]. These differences between enantiomers may arise not only from drug interactions at the receptors, but also from their absorption, distribution, metabolism, and excretion [10]. The synthesis of enantiomerically pure compounds is one of the most demanding challenges in organic chemistry. Among successful applications of transition metal catalysis, organocatalysis, and biocatalysis, enzymes have emerged as environmentally friendly catalysts for high regioselective, chemoselective, and stereoselective transformations [11,12]. Halohydrin dehalogenases (HHDHs) are biotechnologically interesting enzymes that catalyze a wide range of bond formations, such as carbon–carbon, carbon–nitrogen, carbon–oxygen, carbon–chlorine, and carbon–bromine. HHDHs have been used for the synthesis of highly optically active epoxides and 1,2-azido alcohols [13]. Nucleobase-derived compounds have appeared as important pharmacophores interacting with the synthesis and functions of nucleic acids and enzymes [14,15,16,17,18,19]. These N-heterocycles have gained importance in recent years in combating different types of cancer [20,21]. Some purine-based drugs are well known antimetabolites that interfere with DNA replication and cell division by causing cell death when incorporated into DNA or RNA [22,23]. Purine-based compounds have emerged as potent kinase inhibitors that play a pivotal role in the proliferation, migration, and survival of human tumor cells [16,24,25,26]. First-generation purine and second-generation deazapurine inhibitors of heat shock protein 90 (HSP90) have also been developed [27]. Recently, some purine analogs have been identified as inhibitors targeting the microtubule-severing enzyme katanin, which plays an essential role in various carcinomas [28]. Others have found application as antagonists of the Smoothened (SMO) receptor, which is the most druggable target in the Hedgehog signaling pathway for anticancer agents [29].

Among the increasing number of small-molecule targeted drugs that have been developed for the treatment of malignancies, some chiral purine and purine isostere derivatives are associated with antitumor activities given their ability to interfere with carcinogenesis processes, and have been approved by the FDA for the treatment of various types of cancer (Figure 1) [30,31,32,33,34].

Figure 1.

Chiral purine and purine isostere derivatives as antitumor drugs.

Targeted therapies of idelalisib and duvelisib include the inhibition of phosphatidylinositol 3-kinase (PI3K), while acalabrutinib and ibrutinib emerged as Bruton’s tyrosine kinase (BTK) inhibitors [35] and ruxolitinib as Janus kinase (JAK) inhibitors [36]. Futibatinib is a highly selective irreversible fibroblast growth factor receptor 1 (FGFR) inhibitor in patients with advanced solid tumors [37] and glasdegib acts as a SMO inhibitor [38].

In continuation of our previous study on the antiproliferative activity of purine and pseudopurine derivatives [39,40,41,42,43], herein we propose the synthesis of selected racemic N-heterocyclic analogs with hydroxyethyl linker containing primary and secondary hydroxyl groups, instead of directly connected aromatic and 1,2,3-triazole scaffolds (Figure 2) [43]. The introduction of a spacer between the heterocyclic moieties enables additional flexibility in structural preorganization, while the introduction of the primary and secondary hydroxyl groups affects both the biological and physicochemical properties of the prepared molecules [44]. The physicochemical properties can be significantly altered by the introduction of a hydroxyl group, mainly by increasing the polarity and thus the solubility in aqueous media.

Figure 2.

Design of novel racemic α- and β-regioisomers and optically pure aryl-substituted purine bioisosteres.

Since aliphatic alcohols have pKa values above 14, they do not alter the charge of a ligand, but have the potential for a moderate increase in hydrophilicity without impairing membrane permeation. In addition to the regioisomeric effect, the impact of the chirality of enantiomerically enriched N-aryl-substituted 6-chloropurine and 4-chloropyrrolo[2,3-d]pyrimidine derivatives on antiproliferative activity was evaluated.

2. Materials and Methods

2.1. General

Solvents and chemicals, including starting purines and purine bioisosters 1–5, as well as para-substituted 2-phenyloxiranes 11–15 and phenacyl bromide 21–25, were purchased from Sigma-Aldrich (St. Louis, MO, USA), Alfa Aesar (Haverhill, MA, USA), and Fisher Scientific International (Pittsburgh, PA, USA). The progress of all reactions was monitored by thin layer chromatography on pre-coated Merck (Darmstadt, Germany) silica gel plates 60F254. The synthesized compounds were purified via column chromatography using Fluka (Buchs, Switzerland) silica gel (0.063−0.2 mm) and the appropriate solvent. All NMR spectra were recorded using a Bruker 300, 400, and 600 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) in deuterated DMSO solutions, with TMS as the internal standard. The melting points of all newly synthesized compounds were determined using a Koffler (Reichert, Vienna) hot stage microscope. The ¹H (δ/ppm) and ¹³C NMR (δ/ppm) spectra of compounds 26a,b–49a,b can be found in the Supporting Information (Figures S1–S48). The purity of all newly synthesized compounds was determined by elemental analysis and the elemental analysis results are within 0.5% of the theoretical values. The UV/Vis absorption spectra of compounds 40a, 45a, and 49b were measured at a concentration of 1 × 10⁻⁴ mol dm⁻³ in phosphate buffer and DMSO (99:1, v/v) at pH 7.4 using a Varian Cary 50 spectrophotometer in double-beam mode at 25 °C.

2.2. Experimental Procedures for the Synthesis of Compounds

Moreover, 1-(Prop-2-yn-1-yl)-1H-indole 6 [45], 1-(prop-2-yn-1-yl)-1H-benzimidazole 7 [43], 6-chloro-9-(prop-2-yn-1-yl)-9H-purine 8 [43,46], 4-chloro-7-(prop-2-yn-1-yl)-7H-pyrrolo[2,3-d]pyrimidine 9 [47], 4-chloro-1-(prop-2-yn-1-yl)-1H-imidazo[4,5-c]pyridine 10 [43], 2-azido-2-phenylethan-1-ol 16a [48], 2-azido-2-(4-fluorophenyl)ethan-1-ol 17a [49], 2-azido-2-(4-chlorophenyl)ethan-1-ol 18a [50], 2-azido-2-(4-bromophenyl)ethan-1-ol 19a [50], 2-azido-2-(4-(trifluoromethyl)phenyl)ethan-1-ol 20a [49], 2-azido-1-phenylethan-1-ol 16b [51], 2-azido-1-(4-fluorophenyl)ethan-1-ol 17b [51], 2-azido-1-(4-chlorophenyl)ethan-1-ol 18b [52], 2-azido-1-(4-bromophenyl)ethan-1-ol 19b [51], and 2-azido-1-(4-(trifluoromethyl)phenyl)ethan-1-ol 20b [49] were synthesized according to a known procedure. Enantioenriched (ee = 89−99%) ß-azido alcohols (R), (S)-16b-(R), (S)-18b and (R), and (S)-20b were also synthesized according to a well-known procedure given in the literature [49].

2.2.1. General Procedure for the Synthesis of Racemic Aryl-Substituted Purine Bioisosteres with Primary Hydroxyl (26a−49a) and Secondary Hydroxyl (26b−49b) Groups

The corresponding N-propargylated heterocyclic base 6−10 (1 eq.) was dissolved in methanol, and the corresponding racemic 1,2-azido alcohol with primary hydroxyl group 16a,b−20a,b (1.2 eq.) and Cu(OAc)₂ (0.05 eq.) were added. The reaction mixture was irradiated under ultrasound for 2 h in a laboratory ultrasonic cleaning bath. The solvent was removed under reduced pressure, and the residue was purified using column chromatography.

1-((1-(2-Hydroxy-1-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indole (26a)

Compound 26a was prepared using the procedure mentioned above from compound 6 (50 mg, 0.32 mmol) and 2-azido-2-phenylethanol 16a (62 mg, 0.39 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 26a was isolated as a white powder (43 mg, 42%, m.p. = 167–169 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.23 (s, 1H, H-triazole), 7.58 (d, J = 7.8 Hz, 1H, H-7), 7.53 (d, J = 7.8 Hz, 1H, H-4), 7.43 (d, J = 3.1 Hz, 1H, H-2), 7.36–7.29 (m, 5H, H-Ph), 7.14–7.09 (m, 1H, H-6), 7.05–6.98 (m, 1H, H-5), 6.44 (d, J = 3.1 Hz, 1H, H-3), 5.82–5.65 (m, 1H, CHCH₂), 5.46 (s, 2H, CH₂), 5.26 (t, J = 5.3 Hz, 1H, OH), 4.27–4.20 (m, 1H, CH_A-OH), 3.98–3.93 (m, 1H, CH_B-OH). ¹³C NMR (151 MHz, DMSO) δ/ppm: 143.78, 137.83, 136.03, 129.08, 129.06, 128.68, 128.67, 127.61, 123.54, 121.55, 120.87, 119.58, 110.56, 101.41, 66.54, 63.55, 41.30. Anal. calcd. For C₁₉H₁₈N₄O (Mr = 318.38): C, 71.68; H, 5.70; N, 17.60; found: C 71.61, H 5.66, N 17.57.

1-((1-(2-Hydroxy-1-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indole (27a)

Compound 27a was prepared using the procedure mentioned above from compound 6 (50 mg, 0.32 mmol) and 2-azido-2-(4-fluorophenyl)ethanol 17a (69 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 27a was isolated as a yellow oil (101 mg, 94%). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.20 (s, 1H, H-triazole), 7.57 (d, J = 7.8 Hz, 1H, H-7), 7.53 (d, J = 7.8 Hz, 1H, H-4), 7.42 (d, J = 3.1 Hz, 1H, H-2), 7.39–7.36 (m, 2H, H-Ph), 7.18–7.15 (m, 2H, H-Ph), 7.12 (t, J = 7.2 Hz, 1H, H-6), 7.01 (t, J = 7.2 Hz, 1H, H-5), 6.43 (d, J = 3.1 Hz, 1H, H-3), 5.78–5.76 (m, 1H, CHCH₂), 5.45 (s, 2H, CH₂), 5.26 (t, J = 5.0 Hz, 1H, OH), 4.22–4.18 (m, 1H, CH_A-OH), 3.96–3.92 (m, 1H, CH_B-OH).¹³C NMR (151 MHz, DMSO) δ/ppm: 162.48 (d, J_CF = 244.6 Hz), 143.97, 136.19, 134.22 (d, J_CF = 3.0 Hz), 130.08 (d, J_CF = 8.4 Hz), 129.20, 128.82, 123.69, 121.72, 121.02, 119.74, 116.04 (d, J_CF = 21.5 Hz), 110.69, 101.59, 65.79, 63.66, 41.43. Anal. calcd. For C₁₉H₁₇FN₄O (Mr = 336.37): C, 67.84; H, 5.09; N, 16.66; found: C, 67.91; H, 5.02; N, 16.60.

1-((1-(2-Hydroxy-1-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indole (28a)

Compound 28a was prepared using the procedure mentioned above from compound 6 (50 mg, 0.32 mmol) and 2-azido-2-(4-chlorophenyl)ethanol 18a (76 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 28a was isolated as a colorless oil (58 mg, 51%). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.21 (s, 1H, H-triazole), 7.58 (d, J = 8.3 Hz, 1H, H-7), 7.53 (d, J = 7.9 Hz, 1H, H-4), 7.43 (d, J = 3.4 Hz, 2H, H-Ph), 7.39–7.41 (m, 1H, H-2), 7.34 (d, J = 8.6 Hz, 2H, H-Ph), 7.13 (t, J = 7.4 Hz, 1H, H-6), 7.02 (t, J = 7.4 Hz, 1H, H-5), 6.44 (d, J = 3.1 Hz, 1H, H-3), 5.80–5.76 (m, 1H, CHCH₂), 5.46 (s, 2H, CH₂), 5.29 (t, J = 5.3 Hz, 1H, OH), 4.23–4.17 (m, 1H, CH_A-OH), 3.98–3.92 (m, 1H, CH_B-OH).¹³C NMR (101 MHz, DMSO) δ/ppm: 143.86, 136.80, 136.03, 133.40, 129.67, 129.06, 128.67, 123.65, 121.57, 120.87, 119.59, 110.55, 101.44, 65.60, 63.38, 41.27. Anal. calcd. For C₁₉H₁₇ClN₄O (Mr = 352.82): C, 64.68; H, 4.86; N, 15.88; found: C, 64.61; H, 4.91; N, 15.81.

1-((1-(2-Hydroxy-1-(4-bromophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indole (29a)

Compound 29a was prepared using the procedure mentioned above from compound 6 (50 mg, 0.32 mmol) and 2-azido-2-(4-bromophenyl)ethanol 19a (93 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 29a was isolated as a yellow oil (31 mg, 24%). ¹H NMR (600 MHz, DMSO) ¹H NMR (300 MHz, DMSO) δ/ppm: 8.14 (s, 1H, H-triazole), 7.54–7.44 (m, 4H, H-7, H-4, H-Ph), 7.36 (d, J = 3.1 Hz, 1H, H-2), 7.21 (d, J = 8.4 Hz, 2H, H-Ph), 7.10–7.02 (t, J = 7.0 Hz, 1H, H-6), 6.95 (t, J = 7.0 Hz, 1H, H-5), 6.37 (dd, J = 3.1, 0.7 Hz, 1H, H-3), 5.70 (t, J = 5.8 Hz, 1H, CHCH₂), 5.39 (s, 2H, CH₂), 5.23 (t, J = 5.3 Hz, 1H, OH), 4.18–4.08 (m, 1H, CH_A-OH), 3.92–3.85 (m, 1H, CH_B-OH).¹³C NMR (101 MHz, DMSO) δ/ppm: 143.86, 137.21, 136.04, 132.00, 129.99, 129.07, 128.67, 123.66, 121.99, 121.58, 120.88, 119.60, 110.55, 101.52, 65.54, 63.34, 41.28. Anal. calcd. For C₁₉H₁₇BrN₄O (Mr = 397.28): C, 57.44; H, 4.31; N, 14.10; found: C, 57.38; H, 4.37; N, 14.17.

1-((1-(2-Hydroxy-1-(4-(trifluoromethyl)phenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indole (30a)

Compound 30a was prepared using the procedure mentioned above from compound 6 (50 mg, 0.32 mmol) and 2-azido-2-(4-(trifluoromethyl)phenyl)ethanol 20a (88 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 30a was isolated as a yellow oil (93 mg, 76%). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.24 (s, 1H, H-triazole), 7.71 (d, J = 8.2 Hz, 2H, H-Ph, H-7), 7.58 (dd, J = 8.2, 0.8 Hz, 1H, H-4), 7.54–7.51 (m, 3H, H-Ph), 7.43 (d, J = 3.2 Hz, 1H, H-2), 7.12 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H, H-6), 7.01 (ddd, J = 8.0, 7.0, 0.9 Hz, 1H, H-5), 6.44 (dd, J = 3.2, 0.8 Hz, 1H, H-3), 5.90 (dd, J = 8.3, 5.0 Hz, 1H, CHCH₂), 5.47 (s, 2H, CH₂), 5.34 (t, J = 5.3 Hz, 1H, OH), 4.25–4.21 (m, 1H, CH_A-OH), 4.03–3.99 (m, 1H, CH_B-OH). ¹³C NMR (151 MHz, DMSO) δ/ppm: 143.44, 141.87, 135.54, 128.82, 128.57, 128.40, 128.17, 128.14, 125.86 (q, J = 273.8 Hz), 125.49 (q, J = 3.6 Hz), 123.36, 121.07, 120.37, 119.08, 110.04, 100.95, 65.19, 62.81. Anal. calcd. For C₂₀H₁₇F₃N₄O (Mr = 386.38) C, 62.17; H, 4.44; N, 14.50; found: C, 62.23; H, 4.54; N, 14.59.

1-((1-(2-Hydroxy-1-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-benzimidazole (31a)

Compound 31a was prepared using the procedure mentioned above from compound 7 (50 mg, 0.32 mmol) and 2-azido-2-phenylethanol 16a (62 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 31a was isolated as a white powder (77 mg, 75%, m.p = 214–215 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.35 (s, 1H, H-2), 8.32 (s, 1H, H-triazole), 7.67–7.64 (m, 1H, H-4/H-7), 7.64–7.62 (m, 1H, H-4/H-7), 7.34–7.29 (m, 5H, H-Ph), 7.25–7.17 (m, 2H, H-5, H-6), 5.79–5.76 (m, 1H, CHCH₂), 5.57 (s, 2H, CH₂), 5.28 (t, J = 5.4 Hz, 1H, OH), 4.28–4.21 (m, 1H, CH_A-OH), 3.99–3.94 (m, 1H, CH_B-OH). ¹³C NMR (101 MHz, DMSO) δ/ppm: 144.40, 143.93, 142.77, 137.74, 134.05, 129.09, 128.65, 127.60, 123.88, 122.79, 122.06, 119.90, 111.18, 66.62, 63.53, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₈H₁₇N₅O (Mr = 319.37) C, 67.70; H, 5.37; N, 21.93; found: C, 67.79; H, 5.33; N, 21.84.

1-((1-(2-Hydroxy-1-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-benzimidazole (32a)

Compound 32a was prepared using the procedure mentioned above from compound 7 (50 mg, 0.32 mmol) and 2-azido-2-(4-fluorophenyl)ethanol 17a (69 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 32a was isolated as a white powder (20 mg, 19%, m.p. = 190–192 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.32 (s, 1H, H-2/H-triazole), 8.31 (s, 1H, H-2/H-triazole), 7.65–7.62 (m, 2H, H-4, H-7), 7.40–7.37 (m, 2H, H-Ph), 7.23 (d, J = 8.2 Hz, 1H, H-5/H-6), 7.18 (dd, J = 14.1, 5.2 Hz, 3H, H-5/H-6, H-Ph), 5.79 (dd, J = 8.7, 4.9 Hz, 1H, CHCH₂), 5.56 (s, 2H, CH₂), 5.28 (t, J = 5.3 Hz, 1H, OH), 4.24–4.19 (m, 1H, CH_A-OH), 3.97–3.93 (m, 1H, CH_B-OH). ¹³C NMR (151 MHz, DMSO) δ/ppm: 161.86 (d, J = 244.5 Hz), 144.93, 143.95, 143.46, 142.33, 133.59, 133.51 (d, J = 3.2 Hz), 129.46 (d, J = 8.5 Hz), 123.41, 122.34, 121.59, 119.43, 115.44 (d, J = 21.7 Hz), 110.70, 65.24, 62.99. Anal. calcd. For C₁₈H₁₆FN₅O (Mr = 337.36) C, 64.09; H, 4.78; N, 20.76; found: C, 64.12; H, 4.82; N, 20.80.

1-((1-(2-Hydroxy-1-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-benzimidazole (33a)

Compound 33a was prepared using the procedure mentioned above from compound 7 (50 mg, 0.32 mmol) and 2-azido-2-(4-chlorophenyl)ethanol 18a (75 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 33a was isolated as a white powder (28 mg, 25%, m.p. = 174–176 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.33 (s, 1H, H-triazole/H-2), 8.32 (s, 1H, H-triazole/H-2), 7.65 (d, J = 4.0 Hz, 1H, H-4/H-7), 7.63 (d, J = 4.1 Hz, 1H, H-4/H-7), 7.43–7.40 (m, 2H, H-Ph), 7.36–7.33 (m, 2H, H-Ph), 7.27–7.22 (m, 1H, H-5/H-6), 7.22–7.17 (m, 1H, H-5/H-6), 5.80–5.78 (m, 1H, CHCH₂), 5.57 (s, 2H, CH₂), 5.31 (t, J = 5.3 Hz, 1H, OH), 4.24–4.18 (m, 1H, CH_A-OH), 3.99–3.93 (m, 1H, CH_B-OH). ¹³C NMR (101 MHz, DMSO) δ 144.21, 143.95, 142.83, 142.70, 136.70, 133.43, 129.67, 129.07, 124.00, 122.82, 122.07, 119.92, 111.18, 65.67, 63.36, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₈H₁₆ClN₅O (Mr = 353.81) C, 61.11; H, 4.56; N, 19.79; found: C, 61.03; H, 4.63; N, 19.87.

1-((1-(2-Hydroxy-1-(4-bromophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-benzimidazole (34a)

Compound 34a was prepared using the procedure mentioned above from compound 7 (50 mg, 0.32 mmol) and 2-azido-2-(4-bromophenyl)ethanol 19a (93 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 34a was isolated as a white powder (80 mg, 63%, m.p. = 203–205 °C). ¹H NMR (300 MHz, DMSO) δ/ppm: 8.26 (s, 1H, H-triazole/H-2), 8.25 (s, 1H, H-triazole/H-2), 7.60–7.55 (m, 2H, H-4, H-7), 7.48 (d, J = 8.4 Hz, 2H, H-Ph), 7.23–7.11 (m, 4H, H-Ph, H-5, H-6), 5.72 (dd, J = 8.6, 5.1 Hz, 1H, CHCH₂), 5.51 (s, 2H, CH₂), 5.25 (t, J = 5.3 Hz, 1H, OH), 4.14 (ddd, J = 11.3, 8.4, 5.6 Hz, 1H, CH_A-OH), 3.90 (dt, J = 11.0, 4.7 Hz, 1H, CH_B-OH). ¹³C NMR (151 MHz, DMSO) δ/ppm: 144.68, 144.21, 143.11, 137.39, 134.35, 132.27, 130.25, 124.28, 123.06, 122.34, 122.29, 120.18, 111.44, 66.00, 63.58, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₈H₁₆BrN₅O (Mr = 398.26) C, 54.29; H, 4.05; N, 17.59; found: C, 54.34; H, 4.01; N, 17.66.

1-((1-(2-Hydroxy-1-(4-(trifluoromethyl)phenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-benzimidazole (35a)

Compound 35a was prepared using the procedure mentioned above from compound 7 (50 mg, 0.32 mmol) and 2-azido-2-(4-(trifluoromethyl)phenyl)ethanol 20a (88 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 35a was isolated as a white powder (45 mg, 36%, m.p. = 71–73 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.36 (s, 1H, H-triazole), 8.33 (s, 1H, H-2), 7.72 (d, J = 8.2 Hz, 2H, H-Ph), 7.64 (d, J = 8.0 Hz, 2H, H-4, H-7), 7.52 (d, J = 8.2 Hz, 2H, H-Ph), 7.24 (t, J = 7.5 Hz, 1H, H-5/H-6), 7.19 (dd, J = 10.9, 4.1 Hz, 1H, H-5/H-6), 5.91 (dd, J = 8.2, 5.0 Hz, 1H, CHCH₂), 5.58 (s, 2H, CH₂), 5.36 (t, J = 5.3 Hz, 1H, OH), 4.26–4.22 (m, 1H, CH_A-OH), 3.93–3.86 (m, J = 11.5, 5.0 Hz, 1H, CH_B-OH). ¹³C NMR (151 MHz, DMSO) δ/ppm: 142.39, 141.77, 128.83, 128.51 (q, J_CF3 = 34.2 Hz), 128.41, 128.26, 128.13, 125.49 (q, J_CF3 = 3.7 Hz), 125.47 (q, J_CF3 = 277.8 Hz), 122.32, 123.10 (q, J_CF3 = 267.1 Hz), 121.57, 119.40, 110.68, 65.25, 62.77, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₉H₁₆F₃N₅O (Mr = 387.37) C, 58.91; H, 4.16; N, 18.08; found: C, 58.88; H, 4.09; N, 18.11.

6-Chloro-9-((1-(2-hydroxy-1-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine (36a)

Compound 36a was prepared using the abovementioned procedure from compound 8 (50 mg, 0.26 mmol) and 2-azido-2-phenylethanol 16a (51 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 36a was isolated as a white powder (70 mg, 76%, m.p. = 151−153 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.80 (s, 1H, H-2), 8.79 (s, 1H, H-8), 8.35 (s, 1H, H-triazole), 7.34–7.31 (m, 5H, H-Ph), 5.78–5.73 (m, 1H, CHCH₂), 5.63 (s, 2H, CH₂), 5.26 (t, J = 5.4 Hz, 1H, OH), 4.26–4.21 (m, 1H, CH_A-OH), 3.98–3.94 (m, 1H, CH_B-OH). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.70, 151.64, 149.05, 141.65, 141.50, 137.14, 130.72, 128.59, 128.22, 127.12, 123.32, 66.24, 63.02, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₄ClN₇O (Mr = 355.79) C, 54.01; H, 3.97; N, 27.56; found: C, 54.11; H, 3.89; N, 27.49.

6-Chloro-9-((1-(2-hydroxy-1-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine (37a)

Compound 37a was prepared using the procedure mentioned above from compound 8 (50 mg, 0.26 mmol) and 2-azido-2-(4-fluorophenyl)ethanol 17a (56 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 37a was isolated as a white powder (77 mg, 79%, m.p. = 90–91 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.80 (s, 2H, H-2, H-8), 8.34 (s, 1H, H-triazole), 7.42–7.38 (m, 2H, H-Ph), 7.21–7.17 (m, 2H, H-Ph), 5.78–5.81 (m, 1H, CHCH₂), 5.64 (s, 2H, CH₂), 5.28 (t, J = 5.4 Hz, 1H, OH), 4.25–4.18 (m, 1H, CH_A-OH), 3.98–3.92 (m, 1H, CH_B-OH). ¹³C NMR (101 MHz, DMSO) δ/ppm: 161.47 (d, J_CF = 242.9 Hz), 151.67, 149.08, 147.35, 141.28, 138.04, 130.74, 127.98 (d, J_CF = 8.1 Hz), 127.43, 124.46, 114.82 (d, J = 21.3 Hz), 70.55, 56.47, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₃ClFN₇O (Mr = 373.78) C, 51.41; H, 3.51; N, 26.23; found: C, 54.38; H, 3.48; N, 26.18.

6-Chloro-9-((1-(2-hydroxy-1-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine (38a)

Compound 38a was prepared using the procedure mentioned above from compound 8 (50 mg, 0.26 mmol) and 2-azido-2-(4-chlorophenyl)ethanol 18a (61 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 38a was isolated as a white powder (52 mg, 51%, m.p. = 98−100 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.80 (s, 2H, H-2, H-8), 8.34 (s, 1H, H-triazole), 7.44–7.41 (m, 2H, H-Ph), 7.36–7.34 (m, 2H, H-Ph), 5.82–5.78 (m, 1H, CHCH₂), 5.64 (s, 2H, CH₂), 5.30 (t, J = 5.3 Hz, 1H, OH), 4.24–4.17 (m, 1H, CH_A-OH), 3.99–3.93 (m, 1H, CH_B-OH). ¹³C NMR (101 MHz, DMSO) δ/ppm: 152.22, 152.17, 149.57, 147.95, 142.06, 136.62, 133.46, 131.23, 129.68, 129.09, 123.98, 65.80, 63.36, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₃Cl₂N₇O (Mr = 390.23) C, 49.25; H, 3.36; N, 25.13; found: C, 49.19; H, 3.42; N, 25.08.

6-Chloro-9-((1-(2-hydroxy-1-(4-bromophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine (39a)

Compound 39a was prepared using the procedure mentioned above from compound 8 (50 mg, 0.26 mmol) and 2-azido-2-(4-bromophenyl)ethanol 19a (75 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 39a was isolated as a white powder (32 mg, 28%, m.p. = 98−101 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.80 (s, 2H, H-2, H-8), 8.34 (s, 1H, H-triazole), 7.56 (d, J = 8.5 Hz, 2H, H-Ph), 7.29 (d, J = 8.5 Hz, 2H, H-Ph), 5.78 (m, 1H, CHCH₂), 5.64 (s, 2H, CH₂), 5.30 (t, J = 5.4 Hz, 1H, OH), 4.24–4.16 (m, 1H, CH_A-OH), 3.99–3.93 (m, 1H, CH_B-OH). ¹³C NMR (101 MHz, DMSO) δ/ppm: 152.21, 152.17, 149.57, 148.03, 142.05, 137.03, 132.01, 131.22, 129.99, 123.98, 122.04, 65.85, 63.30, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₃BrClN₇O (Mr = 434.68) C, 44.21; H, 3.01; N, 22.56; found: C, 44.16; H, 3.07; N, 22.62.

6-Chloro-9-((1-(2-hydroxy-1-(4-(trifluoromethyl)phenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine (40a)

Compound 40a was prepared using the procedure mentioned above from compound 8 (50 mg, 0.26 mmol) and 2-azido-2-(4-(trifluoromethyl)phenyl)ethanol 20a (72 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 30a was isolated as a colorless oil (40 mg, 36%). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.79 (s, 2H, H-2, H-8), 8.37 (s, 1H, H-triazole), 7.73 (d, J = 8.1 Hz, 2H, H-Ph), 7.53 (d, J = 8.1 Hz, 2H, H-Ph), 5.91 (dd, J = 8.1, 5.0 Hz, 1H, CHCH₂), 5.64 (d, J = 6.3 Hz, 2H, CH₂), 5.35 (t, J = 5.3 Hz, 1H, OH), 4.25–4.21 (m, 1H, CH_A-OH), 4.03–4.00 (m, 1H, CH_B-OH). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.71, 151.66, 149.06, 141.68, 141.61, 130.72, 128.75 (q, J = 32.1 Hz), 128.14, 126.70, 125.50 (q, J = 3.2 Hz), 124.00 (q, J = 272.4 Hz), 123.70, 65.37, 62.77, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₃ClF₃N₇O (Mr = 423.78) C, 48.18; H, 3.09; N, 23.14; found: C, 48.26; H, 3.15; N, 23.17.

4-Chloro-7-((1-(2-hydroxy-1-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine (41a)

Compound 41a was prepared using the procedure mentioned above from compound 9 (50 mg, 0.26 mmol) and 2-azido-2-phenylethanol 16a (51 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 41a was isolated as a white powder (75 mg, 81%, m.p. = 139–141 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.66 (s, 1H, H-2), 8.28 (s, 1H, H-triazole), 7.81 (d, J = 3.6 Hz, 1H, H-6), 7.31–7.35 (m, 5H, H-Ph), 6.68 (d, J = 3.6 Hz, 1H, d, H-5), 5.75 (m, 1H, CHCH₂), 5.59 (s, 2H, CH₂), 5.26 (t, J = 5.2 Hz, 1H, OH), 4.26–4.22 (m, 1H, CH_A-OH), 3.97–3.92 (m, 1H, CH_B-OH) ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.19, 150.90, 150.90, 142.83, 137.72, 131.80, 129.13, 128.76, 127.66, 123.72, 117.25, 99.39, 66.72, 63.54, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₅ClN₆O (Mr = 354.80) C, 57.55; H, 4.26; N, 23.69; found C, 57.61; H, 4.19; N, 23.76.

4-Chloro-7-((1-(2-hydroxy-1-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine (42a)

Compound 42a was prepared using the procedure mentioned above from compound 9 (50 mg, 0.26 mmol) and 2-azido-2-(4-fluorophenyl)ethanol 17a (56 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 42a was isolated as a white powder (73 mg, 75%, m.p. = 90–91 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.67 (s, 1H, H-2), 8.26 (s, 1H, H-triazole), 7.81 (d, J = 3.6 Hz, 1H, H-6), 7.39 (m, 2H, H-Ph), 7.17 (d, J = 8.9 Hz, 2H, H-Ph), 6.68 (d, J = 3.6 Hz, 1H, H-5), 5.76–5.78 (m, 1H, CHCH₂), 5.58 (s, 2H, CH₂), 5.27 (t, J = 5.2 Hz, 1H, OH), 4.26–4.21 (m, 1H, CH_A-OH), 3.96–3.92 (m, 1H, CH_B-OH). ¹³C NMR (151 MHz, DMSO) δ/ppm: 162.06 (d, J_CF = 244.6 Hz), 150.87, 150.62, 150.59, 142.55, 133.69 (d, J_CF = 2.9 Hz), 131.49, 129.67 (d, J_CF = 8.4 Hz), 123.41, 116.94, 115.63 (d, J_CF = 21.4 Hz), 99.07, 65.49, 63.18, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₄ClFN₆O (Mr = 372.79) C, 54.77; H, 3.79; N, 22.54; found C, 54.72; H, 3.83; N, 22.61.

4-Chloro-7-((1-(2-hydroxy-1-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine (43a)

Compound 43a was prepared using the procedure mentioned above from compound 9 (50 mg, 0.26 mmol) and 2-azido-2-(4-chlorophenyl)ethanol 18a (62 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 43a was isolated as a white powder (83 mg, 82%, m.p. = 149–150 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.65 (s, 1H, H-2), 8.25 (s, 1H, H-triazole), 7.80 (d, J = 3.6 Hz, 1H, H-6), 7.39 (s, 2H, H-Ph), 7.34 (d, 2H, H-Ph), 6.67 (d, J = 3.6 Hz, 1H, H-5), 5.79–5.75 (m, 1H, CHCH₂), 5.58 (s, 2H, CH₂), 5.28 (t, J = 5.2 Hz, 1H, OH), 4.23–4.16 (m, 1H, CH_A-OH), 3.97–3.93 (m, 1H, CH_B-OH). ¹³C NMR (101 MHz, DMSO) δ/ppm: 151.16, 150.91, 150.87, 142.88, 136.68, 133.45, 131.78, 129.69, 129.09, 123.82, 117.23, 99.37, 65.74, 63.35, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₄Cl₂N₆O (Mr = 389.24) C, 52.46; H, 3.63; N, 21.59; found C, 52.51; H, 3.71; N, 21.67.

4-Chloro-7-((1-(2-hydroxy-1-(4-bromophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine (44a)

Compound 44a was prepared using the procedure mentioned above from compound 9 (50 mg, 0.26 mmol) and 2-azido-2-(4-bromophenyl)ethanol 19a (75 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 44a was isolated as a white powder (39 mg, 35%, m.p. = 211–213 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.66 (s, 1H, H-2), 8.26 (s, 1H, H-triazole), 7.81 (d, J = 3.6 Hz, 1H, H-6), 7.55 (d, J = 8.5 Hz, 2H, H-Ph), 7.28 (d, J = 8.5 Hz, 2H, H-Ph), 6.68 (d, J = 3.6 Hz, 1H, H-5), 5.78–5.74 (m, 1H, CHCH₂), 5.58 (s, 2H, CH₂), 5.29 (t, J = 5.3 Hz, 1H, OH), 4.23–4.17 (m, 1H, CH_A-OH), 3.97–3.92 (m, 1H, CH_B-OH). ¹³C NMR (151 MHz, DMSO) δ/ppm: 150.67, 150.44, 150.39, 142.40, 136.61, 131.54, 131.32, 129.52, 123.34, 121.55, 116.74, 98.88, 65.30, 62.80, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₄BrClN₆O (Mr = 433.69) C, 47.08; H, 3.25; N, 19.38; found C, 47.13; H, 3.29; N, 19.44.

4-Chloro-7-((1-(2-hydroxy-1 -(4-(trifluoromethyl)phenyl) ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine (45a)

Compound 45a was prepared using the procedure mentioned above from compound 9 (50 mg, 0.26 mmol) and 2-azido-2-(4-(trifluoromethyl)phenyl)ethanol 20a (72 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 45a was isolated as a colorless oil (38 mg, 35%). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.66 (s, 1H, H-2), 8.26 (s, 1H, H-triazole), 7.81 (d, J = 3.6 Hz, 1H, H-6), 7.40 (d, J = 8.6 Hz, 2H, H-Ph), 7.35 (d, J = 8.6 Hz, 2H, H-Ph), 6.68 (d, J = 3.6 Hz, 1H, H-5), 5.78 (dd, J = 8.7, 5.0 Hz, 1H, CHCH₂), 5.59 (s, 2H, CH₂), 5.29 (t, J = 5.6 Hz, 1H, OH), 4.24–4.19 (m, CH_A-OH, 1H), 3.98–3.92 (m, 1H, CH_B-OH). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.14, 150.88, 150.81, 147.02, 142.72, 131.67, 128.55 (q, J_CF3 = 31.5 Hz), 127.29, 125.41 (q, J_CF3 = 3.6 Hz), 124.77, 124.69 (q, J_CF3 = 272.2 Hz), 117.22, 99.26, 71.10, 56.61, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₈H₁₄ClF₃N₆O (Mr = 422.80) C, 51.14; H, 3.34; N, 19.88; found C, 51.09; H, 3.39; N, 19.81.

4-Chloro-1-((1-(2-hydroxy-1-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-imidazo[4,5-c]pyridine (46a)

Compound 46a was prepared using the procedure mentioned above from compound 10 (50 mg, 0.26 mmol) and 2-azido-2-phenylethanol 16a (51 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 46a was isolated as a white powder (59 mg, 64%, m.p. = 92–94 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.58 (s, 1H, H-2), 8.37 (s, 1H, H-triazole), 8.15 (d, J = 5.6 Hz, 1H, H-6), 7.74 (d, J = 5.6 Hz, 1H, H-7), 7.35–7.29 (m, 5H, H-Ph), 5.80–5.74 (m, 1H, CHCH₂), 5.65 (s, 2H, CH₂), 5.27 (t, J = 5.7 Hz, 1H, OH), 4.25–4.21 (m, 1H, CH_A-OH), 3.98–3.94 (m, 1H, CH_B-OH). ¹³C NMR (75 MHz, DMSO) δ/ppm: 143.59, 142.33, 141.66, 140.74, 137.75, 129.47, 129.15, 127.81, 126.69, 124.30, 107.74, 63.75, a signal assigned to the CH₂ group is obscured by the DMSO signal, and signals of two quaternary C atoms were not observed. Anal. calcd. For C₁₇H₁₅ClN₆O (Mr = 354.80) C, 57.55; H, 4.26; N, 23.69; found C, 57.61; H, 4.32; N, 23.61.

4-Chloro-1-((1-(2-hydroxy-1-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-imidazo[4,5-c]pyridine (47a)

Compound 47a was prepared using the procedure mentioned above from compound 10 (50 mg, 0.26 mmol) and 2-azido-2-(4-fluorophenyl)ethanol 17a (56 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 47a was isolated as a white powder (60 mg, 62%, m.p. = 100–101 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 8.31 (s, 1H, H-triazole), 8.15 (d, J = 5.5 Hz, 1H, H-6), 7.73 (d, J = 5.5 Hz, 1H, H-7), 7.41–7.37 (m, 2H, H-Ph), 7.21–7.16 (m, 2H, H-Ph), 5.85 (s, 2H, CH₂), 5.81–5.76 (m, 1H, CHCH₂), 5.28 (t, J = 5.3 Hz, 1H, OH), 4.29–4.26 (m, 1H, CH_A-OH), 3.98–3.92 (m, 1H, CH_B-OH). ¹³C NMR (75 MHz, DMSO) δ/ppm: 162.34 (d, J_CF = 244.8 Hz), 151.42, 145.10, 143.32, 141.16, 134.00 (d, J_CF = 2.9 Hz), 129.89 (d, J_CF = 8.3 Hz), 123.38, 116.04, 115.75 (d, J_CF = 21.5 Hz), 65.80, 63.47, 41.75, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₄ClFN₆O (Mr = 372.79) C, 54.77; H, 3.79; N, 22.54; found C, 54.71; H, 3.72; N, 22.49.

4-Chloro-1-((1-(2-hydroxy-1-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-imidazo[4,5-c]pyridine (48a)

Compound 48a was prepared using the procedure mentioned above from compound 10 (50 mg, 0.26 mmol) and 2-azido-2-(4-chlorophenyl)ethanol 18a (62 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 48a was isolated as a white powder (47 mg, 46%, m.p. = 108–110 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.58 (s, 1H, H-2), 8.36 (s, 1H, H-triazole), 8.16 (d, J = 5.6 Hz, 1H, H-6), 7.75 (d, J = 5.6 Hz, 1H, H-7), 7.42 (d, J = 8.5 Hz, 2H, H-Ph), 7.34 (d, J = 8.6 Hz, 2H, H-Ph), 5.81 (dd, J = 8.5, 4.9 Hz, 1H, CHCH₂), 5.66 (s, 2H, CH₂), 5.31 (t, J = 5.3 Hz, 1H, OH), 4.24–4.17 (m, 1H CH_A-OH), 3.99–3.93 (m, 1H, CH_B-OH). ¹³C NMR (101 MHz, DMSO) δ/ppm: 146.71, 142.15, 141.42, 141.32, 140.50, 137.55, 136.61, 133.46, 129.67, 129.09, 124.11, 107.44, 65.74, 63.34. Anal. calcd. For C₁₇H₁₄Cl₂N₆O (Mr = 389.24) C, 52.46; H, 3.63; N, 21.59; found C, 52.41; H, 3.59; N, 21.52.

4-Chloro-1-((1-(2-hydroxy-1-(4-bromophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-imidazo[4,5-c]pyridine (49a)

Compound 49a was prepared using the procedure mentioned above from compound 10 (50 mg, 0.26 mmol) and 2-azido-2-(4-bromophenyl)ethanol 19a (75 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1) compound 49a was isolated as a white powder (41 mg, 36%, m.p. = 99–102 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.58 (s, 1H, H-2), 8.36 (s, 1H, H-triazole), 8.16 (d, J = 5.6 Hz, 1H, H-6), 7.75 (d, J = 5.6 Hz, 1H, H-7), 7.42 (d, J = 8.5 Hz, 2H, H-5), 7.34 (d, J = 8.6 Hz, 2H, H-Ph), 5.81 (d, J = 8.5 Hz, 1H, H-Ph), 5.81–5.78 (m, CHCH₂), 5.66 (s, 2H, CH₂), 5.31 (t, J = 5.3 Hz, 1H, OH), 4.24–4.17 (m, 1H, CH_A-OH), 3.99–3.94 (m, 1H, CH_B-OH). ¹³C NMR (101 MHz, DMSO) δ/ppm: 146.73, 142.16, 141.44, 141.34, 140.51, 137.56, 137.04, 132.04, 130.00, 124.15, 122.07, 107.46, 65.36, 63.30, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₄BrClN₆O (Mr = 433.69) C, 47.08; H, 3.25; N, 19.38; found C, 47.11; H, 3.31; N, 19.31.

1-((1-(2-Hydroxy-2-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indole (26b)

Compound 26b was prepared using the abovementioned procedure from compound 6 (50 mg, 0.32 mmol) and 2-azido-1-phenylethanol 16b (62 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 26b was isolated as a white powder (65 mg, 64%, m.p. = 119–120 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 7.93 (s, 1H, H-triazole), 7.59–7.52 (m, 2H, H-4, H-7), 7.41 (d, J = 3.2 Hz, 1H, H-2), 7.33–7.24 (m, 5H, H-Ph), 7.16–7.14 (m, 1H, H-6), 7.05–7.01 (m, 1H, H-5), 6.44 (d, J = 3.1, 1H, H-3), 5.76 (d, J = 4.7 Hz, 1H, OH), 5.45 (s, 2H, CH₂), 4.95–4.91 (m, 1H, CHCH₂), 4.48–4.38 (m, 2H, CHCH₂). ¹³C NMR (101 MHz, DMSO) δ/ppm: 143.64, 142.46, 136.02, 129.05, 128.70, 128.61, 127.98, 126.46, 124.42, 121.54, 120.83, 119.56, 110.59, 101.34, 71.79, 56.97, 41.28. Anal. calcd. For C₁₉H₁₈N₄O (Mr = 318.38) C, 71.68; H, 5.70; N, 17.60; found C, 71.72; H, 5.68; N, 17.68.

1-((1-(2-Hydroxy-2-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indole (27b)

Compound 27b was prepared using the procedure mentioned above from compound 6 (50 mg, 0.32 mmol) and 2-azido-1-(4-fluorophenyl)ethanol 17b (69 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 27b was isolated as a white powder (53 mg, 49%, m.p. = 165–167 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 7.90 (s, 1H, H-triazole), 7.56–7.54 (m, 2H, H-4, H-7), 7.41 (d, J = 3.2 Hz, 1H, H-2), 7.32–7.30 (m, 2H, H-Ph), 7.16–7.10 (m, 1H, H-6), 7.10–7.06 (m, 2H, H-Ph), 7.05–7.00 (m, 1H, H-5), 6.46–6.43 (m, 1H, H-3), 5.81 (d, J = 4.7 Hz, 1H, OH), 5.44 (s, 2H, CH₂), 4.96–4.93 (m, 1H, CHCH₂), 4.50–4.39 (m, 2H, CHCH₂). ¹³C NMR (75 MHz, CDCl₃) δ/ppm: 157.91 (d, J_CF = 246.13 Hz), 143.20, 138.34 (d, J_CF = 2.9 Hz), 135.52, 128.57, 128.23, 127.96 (d, J_CF = 8.3 Hz), 123.90, 121.04, 120.35, 119.08 (d, J_CF = 21.3 Hz), 114.69, 110.10, 100.85, 70.59, 56.36, 40.80. Anal. calcd. For C₁₉H₁₇FN₄O (Mr = 336.37) C, 67.84; H, 5.09; N, 16.66; found C, 67.79; H, 5.13; N, 16.70.

1-((1-(2-Hydroxy-2-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol (28b)

Compound 28b was prepared using the procedure mentioned above from compound 6 (50 mg, 0.32 mmol) and 2-azido-1-(4-chlorophenyl)ethanol 18b (76 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 28b was isolated as a white powder (47 mg, 42%, m.p. = 154–156 °C). ¹H NMR (300 MHz, DMSO) δ/ppm: 7.90 (s, 1H, H-triazole), 7.56 (d, J = 3.5 Hz, 1H, H-7), 7.55 (d, J = 3.2 Hz, 1H, H-4), 7.40 (d, J = 3.1 Hz, 1H, H-2), 7.26–7.32 (m, 4H, H-Ph), 7.14 (dd, J = 11.2, 4.2 Hz, 1H, H-6), 7.03 (dd, J = 11.0, 4.1 Hz, 1H, H-5), 6.44 (d, J = 2.6 Hz, 1H, H-3), 5.85 (d, J = 4.7 Hz, 1H, OH), 5.44 (s, 2H, CH₂), 4.96–4.92 (m, 1H, CHCH₂), 4.45–4.42 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 143.70, 141.37, 136.00, 132.45, 129.05, 128.72, 128.53, 128.34, 124.41, 121.53, 120.84, 119.56, 110.58, 101.34, 71.03, 56.69, 41.29. Anal. calcd. For C₁₉H₁₇ClN₄O (Mr = 352.82) C, 64.68; H, 4.86; N, 15.88; found C, 64.62; H, 4.71; N, 15.80.

1-((1-(2-Hydroxy-2-(4-bromophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-indol (29b)

Compound 29b was prepared using the procedure mentioned above from compound 6 (76 mg, 0.32 mmol) and 2-azido-1-(4-bromophenyl)ethanol 19b (93 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 29b was isolated as a white powder (62 mg, 49%, m.p. = 65–67 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 7.91 (s, 1H, H-triazole), 7.54–7.56 (m, 2H, H-4, H-7), 7.45–7.43 (m, 2H, H-Ph), 7.40 (d, J = 3.1 Hz, 1H, H-2), 7.22 (d, J = 8.4 Hz, 2H, H-Ph), 7.15–7.12 (m, 1H, H-6), 7.04–7.01 (m, 1H, H-5), 6.44 (d, J = 3.1, 1H, H-3), 5.86 (d, J = 4.7 Hz, 1H, OH), 5.44 (s, 2H, CHCH₂), 4.95–4.92 (m, 1H, CHCH₂), 4.47–4.41 (m, 2H, CH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 143.72, 141.80, 136.01, 131.46, 129.07, 128.73, 128.71, 124.43, 121.54, 121.02, 120.85, 119.58, 110.60, 101.35, 71.08, 56.64, 41.30. Anal. calcd. For C₁₉H₁₇BrN₄O (Mr = 397.28) C, 57.44; H, 4.31; N, 14.10; found C, 57.51; H, 4.27; N, 14.18.

1-((1-(2-Hydroxy-2-(4-(trifluoromethyl)phenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H- indol (30b)

Compound 30b was prepared using the procedure mentioned above from compound 6 (76 mg, 0.32 mmol) and 2-azido-1-(4-(trifluoromethyl)phenyl)ethanol 19b (88 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 30b was isolated as a white powder (53 mg, 43%, m.p. = 73–75 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.09 (s, 1H, H-triazoli), 7.62 (m, 3H, H-7, H-4, H-2), 7.61 (d, J = 8.2 Hz, 2H, H-Ph), 7.50 (d, J = 8.3 Hz, 2H, H-Ph), 7.24 (ddd, J = 24.5, 13.0, 7.6 Hz, 3H, H-6, H-5, H-3), 6.01 (d, J = 4.7 Hz, 1H, OH), 5.56 (s, 2H, CH₂), 5.10–5.04 (m, 1H, CHCH₂), 4.56–4.43 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 147.04, 145.46, 142.70, 128.53 (q, J = 31.5 Hz), 127.27, 126.39 (q, J = 271.4 Hz), 125.44 (q, J = 3.7 Hz), 124.88, 123.81, 123.81, 123.24, 123.24, 122.77, 122.03, 119.91, 111.23, 71.08, 56.62. Anal. calcd. For C₂₀H₁₇F₃N₄O (Mr = 386.38) C, 62.17; H, 4.44; N, 14.50; found C, 62.21; H, 4.40; N, 14.56.

1-((1-(2-Hydroxy-2-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-benzimidazole (31b)

Compound 31b was prepared using the procedure mentioned above from compound 7 (50 mg, 0.32 mmol) and 2-azido-1-phenylethanol 16b (62 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 31b was isolated as a white powder (87 mg, 85%, m.p. = 178–179 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.30 (s, 1H, H-2), 8.06 (s, 1H, H-triazole), 7.64 (m, 2H, H-4, H-7), 7.32–7.19 (m, 7H, H-Ph, H-5, H-6, H-3), 5.78 (d, J = 4.7 Hz, 1H, OH), 5.56 (s, 2H, CH₂), 4.97–4.92 (m, 1H, CHCH₂), 4.50–4.43 (m, 2H, CHCH₂). ¹³C NMR (101 MHz, DMSO) δ/ppm: 144.36, 143.97, 142.57, 142.37, 134.05, 128.74, 127.99, 126.45, 124.84, 122.79, 122.19, 119.87, 111.23, 71.62, 57.03, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₈H₁₇N₅O (Mr = 319.37) C, 67.70; H, 5.37; N, 21.93; found C, 67.67; H, 5.41; N, 21.81.

1-((1-(2-Hydroxy-2-(4-fluorphenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-benzimidazole (32b)

Compound 32b was prepared using the procedure mentioned above from compound 7 (50 mg, 0.32 mmol) and 2-azido-1-(4-fluorophenyl)ethanol 17b (69 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 32b was isolated as a white powder (51 mg, 47%, m.p. = 173–174 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.30 (s, 1H, H-2), 8.03 (s, 1H, H-triazole), 7.66–7.64 (m, 1H, H-4), 7.62–7.60 (d, J = 7.9 Hz, 1H, H-7), 7.32–7.29 (m, 2H, H-Ph), 7.26–7.23 (m, 1H, H-5), 7.21–7.19 (m, 1H, H-6), 7.07–7.04 (m, 2H, H-Ph), 5.83 (d, J = 4.7 Hz, 1H, OH), 5.54 (s, 2H, CH₂), 4.94–4.97 (m, 1H, CHCH₂), 4.47–4.42 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 161.61 (d, J_CF = 242.8 Hz), 144.02, 143.62, 142.26, 138.18 (d, J_CF = 2.3 Hz), 133.66, 128.09 (J_CF = 7.8 Hz), 124.46, 122.43, 121.70, 119.52, 114.96, 110.87, 70.69, 56.56, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₈H₁₆FN₅O (Mr = 337.36) C, 64.09; H, 4.78; N, 20.76; found C, 64.14; H, 4.82; N, 20.69.

1-((1-(2-Hydroxy-2-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-benzimidazole (33b)

Compound 33b was prepared using the procedure mentioned above from compound 7 (50 mg, 0.32 mmol) and 2-azido-1-(4-chlorophenyl)ethanol 18b (75 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 33b was isolated as a white powder (37 mg, 33%, m.p. = 218–220 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.32 (s, 1H, H-2), 8.05 (s, 1H, H-triazole), 7.66 (d, J = 7.9 Hz, 1H, H-4), 7.62 (d, J = 7.9 Hz, 1H, H-7), 7.29 (s, 4H, H-Ph), 7.23 (m, 1H, H-5), 7.21 (m, 1H, H-6), 5.89 (d, J = 4.4 Hz, 1H, OH), 5.55 (s, 2H, CH₂), 4.97 (m, 1H, CHCH₂), 4.47 (t, J = 6.0 Hz, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 144.55, 144.35, 142.92, 141.62, 134.18, 132.75, 128.82, 128.62, 125.12, 123.06, 122.33, 120.19, 111.52, 71.27, 57.04, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₈H₁₆ClN₅O (Mr = 353.81) C, 61.11; H, 4.56; N, 19.79; found C, 61.06; H, 4.61; N, 19.70.

1-((1-(2-Hydroxy-2-(4-bromophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-benzimidazole (34b)

Compound 34b was prepared using the procedure mentioned above from compound 7 (50 mg, 0.32 mmol) and 2-azido-1-(4-bromophenyl)ethanol 19b (93 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 34b was isolated as a white powder (25 mg, 20%, m.p. = 224–226 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.31 (s, 1H, H-2), 8.06 (s, 1H, H-triazole), 7.66 (d, J = 7.7 Hz, 1H, H-4), 7.62 (d, J = 7.8 Hz, 1H, H-7), 7.44 (d, J = 8.4 Hz, 4H, H-Ph), 7.26–7.19 (m, 4H, H-Ph, H-5, H-6), 5.89 (d, J = 4.7 Hz, 1H, OH), 5.56 (s, 2H, CH₂), 4.95 (dt, J = 9.1, 4.6 Hz, 1H, CHCH₂), 4.52–4.41 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 144.54, 144.15, 142.79, 141.89, 134.17, 131.59, 128.84, 124.98, 122.92, 122.19, 121.17, 120.04, 111.36, 71.18, 56.84, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₈H₁₆BrN₅O (Mr = 398.26) C, 54.29; H, 4.05; N, 17.59; found C, 54.21; H, 4.12; N, 17.64.

1-((1-(2-Hydroxy-2-(4-(trifluoromethyl)phenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-benzimidazole (35b)

Compound 35b was prepared using the procedure mentioned above from compound 7 (50 mg, 0.32 mmol) and 2-azido-1-(4-(trifluoromethyl)phenyl)ethanol 20b (88 mg, 0.38 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 35b was isolated as a white powder (25 mg, 20%, m.p. = 208–210 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.30 (s, 1H, H-2), 8.05 (s, 1H, H-triazole), 7.65 (d, J = 7.8 Hz, 1H, H-4), 7.60 (m, 1H, H-7), 7.44–7.42 (m, 2H, H-Ph), 7.21 (ddd, J = 9.0, 3.8, 2.0 Hz, 3H, H-Ph, H-5, H-6), 5.88 (d, J = 4.7 Hz, 1H, OH), 5.55 (s, 2H, CH₂), 4.94 (dt, J = 7.4, 4.5 Hz, 1H, CHCH₂), 4.46 (qd, J = 13.9, 6.0 Hz, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 142.90, 142.28, 129.23 (q, J = 31.8 Hz), 128.64, 127.27, 126.01, 124.51 (q, J = 271.8 Hz), 124.89, 124.23, 122.81, 122.06, 121.81, 119.96, 111.25, 65.76, 63.27, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₉H₁₆F₃N₅O (Mr = 387.37) C, 58.91; H, 4.16; N, 18.08; found C, 58.86; H, 4.09; N, 18.13.

6-Chloro -9 -((1-(2-hydroxy-2-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine (36b)

Compound 36b was prepared using the abovementioned procedure from compound 8 (50 mg, 0.26 mmol) and 2-azido-1-phenylethanol 16b (51 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 36b was isolated as a white powder (55 mg, 60%, m.p = 129–131 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.81 (s, 1H, H-2), 8.77 (s, 1H, H-8), 8.07 (s, 1H, H-triazole), 7.33–7.23 (m, 5H, H-Ph), 5.77 (d, J = 4.7 Hz, 1H, OH), 5.62 (s, 2H, CH₂), 4.95 (dt, J = 8.6, 4.5 Hz, 1H, CHCH₂), 4.46 (qd, J = 13.7, 6.2 Hz, 2H, CHCH₂). ¹³C NMR (101 MHz, DMSO) δ/ppm: 152.18, 152.13, 149.55, 147.83, 142.34, 141.74, 131.24, 128.57, 127.97, 126.45, 124.97, 71.75, 57.07, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₄ClN₇O (Mr = 355.79) C, 54.01; H, 3.97; N, 27.56; found C, 54.09; H, 3.91; N, 27.49.

6-Chloro-9-((1-(2-hydroxy-2-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine (37b)

Compound 37b was prepared using the procedure mentioned above from compound 8 (50 mg, 0.26 mmol) and 2-azido-1-(4-fluorophenyl)ethanol 17b (56 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 37b was isolated as a white powder (28 mg, 29%, m.p. = 131–134 °C). ¹H NMR (400 MHz, DMSO) δ 8.81 (s, 1H, H-2), 8.77 (s, 1H, H-8), 8.06 (s, 1H, H-triazole), 7.34 (d, J = 2.9 Hz, 2H, H-Ph), 7.09 (m, 2H, H-Ph), 5.82 (d, J = 4.7 Hz, 1H, OH), 5.61 (s, 2H, CH₂), 4.98–4.94 (m, 1H, CHCH₂), 4.46 (t, J = 6.6 Hz, 2H, CHCH₂). ¹³C NMR (101 MHz, DMSO) δ 161.97 (d, J_CF = 242.9 Hz), 152.17, 149.58, 147.85, 141.78, 138.54, 131.24, 128.48 (d, J_CF = 8.1 Hz), 127.93, 124.96, 115.32 (d, J_CF = 21.3 Hz), 71.05, 56.97, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₃FClN₇O (Mr = 373.78) C, 51.41; H, 3.51; N, 26.23; found C, 51.49; H, 3.47; N, 26.19.

6-Chloro-9-((1-(2-hydroxy-2-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine (38b)

Compound 38b was prepared using the procedure mentioned above from compound 8 (50 mg, 0.26 mmol) and 2-azido-1-(4-chlorophenyl)ethanol 18b (61 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 38b was isolated as a white powder (81 mg, 80%, m.p. = 89–92 °C). ¹H NMR (300 MHz, DMSO) δ/ppm: 8.81 (s, 1H, H-2), 8.77 (s, 1H, H-8), 8.07 (s, 1H, H-triazole), 7.32 (m, 4H, H-Ph), 5.87 (d, J = 4.7 Hz, 1H, OH), 5.61 (s, 2H, CH₂), 4.96 (dt, J = 9.2, 4.6 Hz, 1H, CHCH₂), 4.54–4.37 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.55, 151.51, 148.96, 147.24, 141.18, 140.69, 131.86, 130.61, 127.92, 127.75, 124.36, 70.37, 56.19, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₃Cl₂N₇O (Mr = 390.23) C, 49.25; H, 3.36; N, 25.13; found C, 49.19; H, 3.41; N, 25.09.

6-Chloro-9-((1-(2-hydroxy-2-(4-bromophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine (39b)

Compound 39b was prepared using the procedure mentioned above from compound 8 (50 mg, 0.26 mmol) and 2-azido-1-(4-bromophenyl)ethanol 19b (75 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 39b was isolated as a white powder (66 mg, 58%, m.p. = 86–89 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.79 (s, 1H, H-2), 8.74 (s, 1H, H-8), 8.05 (s, 1H, H-triazole), 7.45–7.43 (m, 2H, H-Ph), 7.24 (d, J = 1.6 Hz, 2H, H-Ph), 5.82 (d, J = 4.8 Hz, 1H, OH), 5.60 (s, 2H, CH₂), 4.96–4.93 (m, 1H, CHCH₂), 4.46 (dd, J = 15.9, 6.0 Hz, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.69, 151.65, 149.09, 147.38, 141.32, 141.25, 130.97, 130.75, 128.25, 124.49, 120.55, 70.55, 56.27, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₃BrClN₇O (Mr = 434.68) C, 44.21; H, 3.01 N, 22.56; found C, 44.27; H, 3.08; N, 22.50.

6-Chloro-9-((1-(2-hydroxy-2-(4-(trifluoromethyl)phenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine (40b)

Compound 40b was prepared using the procedure mentioned above from compound 8 (50 mg, 0.26 mmol) and 2-azido-1-(4-(trifluoromethyl)phenyl)ethanol 20b (72 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound 40b was isolated as a white powder (68 mg, 60%, m.p. = 84–86 °C). ¹H NMR (300 MHz, DMSO) δ/ppm: 8.81 (s, 1H, H-2), 8.76 (d, J = 6.0 Hz, 1H, H-8), 8.11 (s, 1H, H-triazole), 7.64 (d, J = 8.3 Hz, 2H, H-Ph), 7.53 (d, J = 8.1 Hz, 2H, H-Ph), 5.98 (d, J = 4.7 Hz, 1H, OH), 5.62 (s, 2H, CH₂), 5.13–4.93 (m, 1H, CHCH₂), 4.51 (qd, J = 13.8, 6.0 Hz, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.65, 151.63, 149.07, 147.35, 146.50, 141.34, 130.72, 128.06 (q, J_CF3 = 31.7 Hz), 126.89 (q, J_CF3 = 271.8 Hz), 124.93 (q, J_CF3 = 3.6 Hz), 124.50, 121.45, 70.59, 56.18, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₃ClF₃N₇O (Mr = 434.68) C, 48.18; H, 3.09; N, 23.14; found C, 48.23; H, 3.02; N, 23.11.

4-Chloro-7-((1-(2-hydroxy-2-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine (41b)

Compound 41b was prepared using the procedure mentioned above from compound 9 (50 mg, 0.26 mmol) and 2-azido-2-phenylethanol 16b (51 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 41b was isolated as a white powder (57 mg, 62%, m.p. = 114–117 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 7.97 (s, 1H, H-triazole), 7.78 (d, J = 3.6 Hz, 1H, H-6), 7.29 (dd, J = 12.4, 4.5 Hz, 5H, H-Ph), 6.69 (d, J = 3.6 Hz, 1H, H-5), 5.76 (d, J = 4.7 Hz, 1H, OH), 5.57 (s, 2H, CH₂), 4.94 (dt, J = 8.7, 4.6 Hz, 1H, CHCH₂), 4.50–4.40 (m, 2H, CHCH₂). ¹³C NMR (101 MHz, DMSO) δ/ppm: 151.13, 150.90, 150.82, 142.61, 142.39, 131.71, 128.58, 127.96, 126.46, 124.74, 117.25, 99.28, 71.64, 57.01, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₅ClN₆O (Mr = 354.80) C, 57.55; H, 4.26; N, 23.69; found C, 57.49; H, 4.31; N, 23.73.

4-Chloro-7-((1-(2-hydroxy-2-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine (42b)

Compound 42b was prepared using the procedure mentioned above from compound 9 (50 mg, 0.26 mmol) and 2-azido-1-(4-fluorophenyl)ethanol 17b (56 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 42b was isolated as a white powder (49 mg, 51%, m.p. = 154–156 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 7.95 (s, 1H, H-triazole), 7.77 (d, J = 3.6 Hz, 1H, H-6), 7.33 (dd, J = 8.5, 5.7 Hz, 2H, H-Ph), 7.09 (t, J = 8.9 Hz, 2H, H-Ph), 6.69 (d, J = 3.6 Hz, 1H, H-5), 5.81 (d, J = 4.7 Hz, 1H, OH), 5.57 (s, 2H, CH₂), 4.96 (dt, J = 9.4, 4.8 Hz, 1H, CHCH₂), 4.44 (dd, J = 12.1, 4.9 Hz, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 161.45 (d, J_CF = 243.0 Hz), 150.63, 150.38, 150.30, 142.13, 138.04 (d, J = 2.7 Hz), 131.18, 127.94 (d, J_CF = 8.2 Hz), 124.21, 116.72, 114.79 (d, J_CF = 21.2 Hz), 98.76, 70.54, 56.38, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₄ClFN₆O (Mr = 372.79) C, 54.77; H, 3.79; N, 22.54; found C, 54.83; H, 3.74; N, 22.59.

4-Chloro-7-((1-(2-hydroxy-2-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine (43b)

Compound 43b was prepared using the procedure mentioned above from compound 9 (50 mg, 0.26 mmol) and 2-azido-1-(4-chlorophenyl)ethanol 18b (61 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 43b was isolated as a white powder (52 mg, 51%, m.p. = 152–154 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 7.96 (s, 1H, H-triazole), 7.77 (d, J = 3.6 Hz, 1H, H-6), 7.33–7.29 (m, 4H, H-Ph), 6.68 (d, J = 3.6 Hz, 1H, H-5), 5.86 (d, J = 4.7 Hz, 1H, OH), 5.56 (s, 2H, CH₂), 4.98–4.94 (m, 1H, CHCH₂), 4.49–4.41 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.16, 150.91, 150.83, 142.69, 141.36, 132.48, 131.71, 128.47, 128.29, 124.75, 117.24, 99.29, 71.01, 56.75, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₄Cl₂N₆O (Mr = 389.24) C, 52.46; H, 3.63; N, 21.59; found C, 52.40; H, 3.71; N, 21.65.

4-Chloro-7-((1-(2-hydroxy-2-(4-bromophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine (44b)

Compound 44b was prepared using the procedure mentioned above from compound 9 (50 mg, 0.26 mmol) and 2-azido-1-(4-bromophenyl)ethanol 19b (75 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 44b was isolated as a white powder (50 mg, 44%, m.p. = 161–164 °C). ¹H NMR (300 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 7.97 (s, 1H, H-triazole), 7.77 (d, J = 3.6 Hz, 1H, H-6), 7.46 (d, J = 8.4 Hz, 2H, H-Ph), 7.25 (d, J = 8.4 Hz, 2H, H-Ph), 6.69 (d, J = 3.6 Hz, 1H, H-5), 5.86 (d, J = 4.7 Hz, 1H, OH), 5.57 (s, 2H, CH₂), 4.94 (dd, J = 8.4, 3.7 Hz, 1H, CHCH₂), 4.45 (dd, J = 5.9, 4.2 Hz, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.16, 150.90, 150.82, 142.67, 141.76, 131.69, 131.45, 128.72, 124.75, 121.02, 117.24, 99.28, 71.05, 56.69, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₄BrClN₆O (Mr = 433.69) C, 47.08; H, 3.25; N, 19.38; found C, 47.15; H, 3.17; N, 19.43.

4-Chloro-7-((1-(2-hydroxy-2-(4-(trifluoromethyl)phenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine (45b)

Compound 45b was prepared using the procedure mentioned above from compound 9 (50 mg, 0.26 mmol) and 2-azido-1-(4-(trifluoromethyl)phenyl)ethanol 20b (72 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 45b was isolated as a white powder (23 mg, 21%, m.p. = 74–75 °C). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 8.00 (s, 1H, H-triazole), 7.77 (d, J = 3.6 Hz, 1H, H-6), 7.63 (d, J = 8.2 Hz, 2H, H-Ph), 7.52 (d, J = 8.1 Hz, 2H, H-Ph), 6.67 (d, J = 3.6 Hz, 1H, H-5), 5.98 (d, J = 4.8 Hz, 1H, OH), 5.57 (s, 2H, CH₂), 5.08–5.06 (m, 1H, CHCH₂), 4.55–4.45 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.14, 150.88, 150.81, 147.03, 142.72, 131.67, 128.55 (q, J_CF3 = 31.5 Hz), 125.50, 125.42 (q, J_CF3 = 3.5 Hz), 124.77, 124.69 (d, J_CF3 = 272.2 Hz), 117.22, 99.26, 71.10, 56.61, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₈H₁₄F₃N₆O (Mr = 422.80) C, 51.14; H, 3.34; N, 19.88; found C, 51.09; H, 3.39; N, 19.81.

4-Chloro-1-((1-(2-hydroxy-2-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-imidazo[4,5-c]pyridine (46b)

Compound 46b was prepared using the procedure mentioned above from compound 10 (50 mg, 0.26 mmol) and 2-azido-1-phenylethanol 16b (51 mg, 0.31mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 46b was isolated as a white powder (71 mg, 77%, m.p. = 92–94 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.57 (s, 1H, H-2), 8.17 (d, J = 5.6 Hz, 1H, H-6), 8.10 (s, 1H, H-triazole), 7.73 (d, J = 5.6 Hz, 1H, H-7), 7.28 (m, 5H, H-Ph), 5.81 (d, J = 4.6 Hz, 1H, OH), 5.65 (s, 2H, CH₂), 4.99–4.93 (m, 1H, CHCH₂), 4.54–4.42 (m, 2H, CHCH₂,). ¹³C NMR (101 MHz, DMSO) δ/ppm: 146.64, 142.31, 141.82, 141.37, 141.30, 140.41, 137.59, 128.58, 128.00, 126.44, 125.06, 107.47, 71.73, 57.06, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₅ClN₆O (Mr = 354.80) C, 57.55; H, 4.26; N, 23.69; found C, 57.41; H, 4.39; N, 23.73.

4-Chloro-1-((1-(2-hydroxy-2-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-imidazo[4,5-c]pyridine (47b)

Compound 47b was prepared using the procedure mentioned above from compound 10 (50 mg, 0.26 mmol) and 2-azido-1-(4-fluorophenyl)ethanol 17b (56 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 47b was isolated as a white powder (49 mg, 51%, m.p. = 98–100 °C). ¹H NMR (300 MHz, DMSO) δ/ppm: 8.69 (s, 1H, H-2), 8.16 (d, J = 4.5 Hz, 1H, H-6), 7.98 (s, 1H, H-triazole), 7.74 (d, J = 4.5 Hz, 1H, H-7), 7.31 (dd, J = 8.6, 5.6 Hz, 2H, H-Ph), 7.06 (t, J = 8.9 Hz, 2H, H-Ph), 5.85–5.79 (m, 3H, OH, CH₂), 4.96 (dd, J = 11.7, 4.8 Hz, 1H, CHCH₂), 4.49–4.43 (m, 2H, CHCH₂). ¹³C NMR (75 MHz, DMSO) δ/ppm: 160.33, 154.70 (d, J_CF = 234.4 Hz), 152.26, 143.04, 141.16, 138.51 (d, J_CF = 2.9 Hz), 136.68, 133.61, 128.43 (d, J_CF = 8.2 Hz), 124.29, 115.27 (d, J_CF = 21.3 Hz), 105.46, 71.02, 56.90, 41.86. Anal. calcd. For C₁₇H₁₄ClFN₆O (Mr = 372.79) C, 54.77; H, 3.79; N, 22.54; found C, 54.81; H, 3.74; N, 22.48.

4-Chloro-1-((1-(2-hydroxy-2-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-imidazo[4,5-c]pyridine (48b)

Compound 48b was prepared using the procedure mentioned above from compound 10 (50 mg, 0.26 mmol) and 2-azido-1-(4-chlorophenyl)ethanol 18b (61 mg, 0.31mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 48b was isolated as a white powder (39 mg, 39%, m.p. = 95–98 °C). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.57 (s, 1H, H-2), 8.17 (d, J = 5.6 Hz, 1H, H-6), 8.09 (s, 1H, H-triazole), 7.72 (d, J = 5.6 Hz, 1H, H-7), 7.30 (s, 4H, H-Ph), 5.89 (d, J = 4.6 Hz, 1H, OH), 5.64 (s, 2H, CH₂), 4.97 (dt, J = 7.4, 4.6 Hz, 1H, CHCH₂), 4.52–4.42 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 146.66, 141.89, 141.42, 141.29, 140.40, 137.59, 132.48, 128.53, 128.35, 125.05, 107.46, 70.96, 56.79, a signal assigned to the CH₂ group is obscured by the DMSO signal, and the signal of the one quaternary C atom was not observed. Anal. calcd. For C₁₇H₁₄Cl₂N₆O (Mr = 389.24) C, 52.46; H, 3.63; N, 21.59; found C, 52.51; H, 3.70; N, 21.51.

4-Chloro-1-((1-(2-hydroxy-2-(4-bromophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-imidazo[4,5-c]pyridine (49b)

Compound 49b was prepared using the procedure mentioned above from compound 10 (50 mg, 0.26 mmol) and 2-azido-1-(4-bromophenyl)ethanol 19b (75 mg, 0.31 mmol). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound 49b was isolated as a white powder (45 mg, 40%, m.p. = 107–109 °C). ¹H NMR (300 MHz, DMSO) δ/ppm: 8.56 (s, 1H, H-2), 8.16 (d, J = 5.6 Hz, 1H, H-6), 8.09 (s, 1H, H-triazole), 7.71 (d, J = 5.6 Hz, 1H, H-7), 7.30 (s, 4H, H-Ph), 5.89 (d, J = 4.6 Hz, 1H, H-Ph), 5.63 (s, 2H, CH₂), 4.96 (dt, J = 7.2, 4.7 Hz, 1H, CHCH₂), 4.53–4.40 (m, 2H, CHCH₂). ¹³C NMR (75 MHz, DMSO) δ/ppm: 146.66, 141.89, 141.42, 141.28, 140.40, 137.59, 132.48, 128.53, 128.35, 125.05, 107.46, 70.96, 56.79, a signal assigned to the CH₂ group is obscured by the DMSO signal, and the signal of the one quaternary C atom was not observed. Anal. calcd. For C₁₇H₁₄BrClN₆O (Mr = 433.69) C, 47.08; H, 3.25; N, 19.38; found C, 47.00; H, 3.16; N, 19.31.

2.2.2. General Procedure for the Synthesis of Optically Enriched (R)- and (S)-N-Aryl-Substituted Derivatives of 6-Chloropurine and Pyrrolo[2,3-d]Pyrimidine with a Secondary Hydroxyl Group

The corresponding N-propargylated heterocyclic base 8, 9 (1 eq.) was dissolved in methanol, and the corresponding optically enriched azido alcohols with a secondary hydroxyl group (R), (S)-16b-(R), (S)-18b and (R), (S)-20b (1.2 eq.), and Cu(OAc)₂ (0.05 eq.) were added. The reaction mixture was irradiated under ultrasound for 2 h in a laboratory ultrasonic cleaning bath. The solvent was removed under reduced pressure, and the residue was purified via column chromatography.

(R)-6-Chloro -9 -((1-(2-hydroxy-2-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine ((R)-36b)

Compound (R)-36b was prepared using the procedure mentioned above from compound 3 (45 mg, 0.23 mmol) and (R)-2-azido-1-phenylethanol (R)-16b (46 mg, 0.28 mmol, ee = 95%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1) compound (R)-36b was isolated as a white powder (30 mg, 37%, m.p. = 129–131 °C, ee = 95%, [α]_D²⁴ − 30.0 (c = 0.0010)). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.81 (s, 1H, H-2), 8.77 (s, 1H, H-8), 8.07 (s, 1H, H-triazole), 7.33–7.23 (m, 5H, H-Ph), 5.77 (d, J = 4.7 Hz, 1H, OH), 5.62 (s, 2H, CH₂), 4.95 (dt, J = 8.6, 4.5 Hz, 1H, CHCH₂), 4.46 (qd, J = 13.7, 6.2 Hz, 2H, CHCH₂). ¹³C NMR (101 MHz, DMSO) δ/ppm: 152.18, 152.13, 149.55, 147.83, 142.34, 141.74, 131.24, 128.57, 127.97, 126.45, 124.97, 71.75, 57.07, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₄ClN₇O (Mr = 355.79) C, 54.01; H, 3.97; N, 27.56; found C, 54.11; H, 3.89; N, 27.64.

(S)-6-Chloro -9 -((1-(2-hydroxy-2-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine ((S)-36b)

Compound (S)-36b was prepared using the procedure mentioned above from compound 3 (45 mg, 0.23 mmol) and (S)-2-azido-1-phenylethanol (S)-16b (45 mg, 0.28 mmol, ee = 99%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound (S)-36b was isolated as a white powder (48 mg, 59%, m.p. = 129–131 °C, ee = 99%, [α]_D²⁴ + 30.0 (c = 0.0010)). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.81 (s, 1H, H-2), 8.77 (s, 1H, H-8), 8.07 (s, 1H, H-triazole), 7.33–7.23 (m, 5H, H-Ph), 5.77 (d, J = 4.7 Hz, 1H, OH), 5.62 (s, 2H, CH₂), 4.95 (dt, J = 8.6, 4.5 Hz, 1H, CHCH₂), 4.46 (qd, J = 13.7, 6.2 Hz, 2H, CHCH₂). ¹³C NMR (101 MHz, DMSO) δ/ppm: 152.18, 152.13, 149.55, 147.83, 142.34, 141.74, 131.24, 128.57, 127.97, 126.45, 124.97, 71.75, 57.07, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₄ClN₇O (Mr = 355.79) C, 54.01; H, 3.97; N, 27.56; found C, 54.07; H, 4.03; N, 27.50.

(R)-6-Chloro-9-((1-(2-hydroxy-2-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine ((R)-37b)

Compound (R)-37b was prepared using the procedure mentioned above from compound 3 (30 mg, 0.16 mmol) and 2-azido-1-(4-fluorophenyl)ethanol (R)-17b (34 mg, 0.19 mmol, ee = 99%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound (R)-37b was isolated as a white powder (26 mg, 27%, m.p. = 131–134 °C, ee = 99%, [α]_D²⁴ −20.0 (c = 0.0010)). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.81 (s, 1H, H-2), 8.77 (s, 1H, H-8), 8.06 (s, 1H, H-triazole), 7.34 (d, J = 2.9 Hz, 2H, H-Ph), 7.09 (m, 2H, H-Ph), 5.82 (d, J = 4.7 Hz, 1H, OH), 5.61 (s, 2H, CH₂), 4.98–4.94 (m, 1H, CHCH₂), 4.46 (t, J = 6.6 Hz, 2H, CHCH₂). ¹³C NMR (101 MHz, DMSO) δ/ppm: 161.97 (d, J_CF = 242.9 Hz), 152.17, 149.58, 147.85, 141.78, 138.54, 131.24, 128.48 (d, J_CF = 8.1 Hz), 127.93, 124.96, 115.32 (d, J_CF = 21.3 Hz), 71.05, 56.97, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₃FClN₇O (Mr = 373.78) C, 51.41; H, 3.51; N, 26.23; found C, 51.46; H, 3.58; N, 26.19.

(S)-6-Chloro-9-((1-(2-hydroxy-2-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine ((S)-37b)

Compound (S)-37b was prepared using the procedure mentioned above from compound 3 (30 mg, 0.16 mmol) and 2-azido-1-(4-fluorophenyl)ethanol (S)-17b (34 mg, 0.19 mmol, ee = 97%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 30:1), compound (S)-37b was isolated as a white powder (20 mg, 33%, m.p. = 131–134 °C, ee = 97%, [α]_D²⁴ + 18.2 (c = 0.0011)). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.81 (s, 1H, H-2), 8.77 (s, 1H, H-8), 8.06 (s, 1H, H-triazole), 7.34 (d, J = 2.9 Hz, 2H, H-Ph), 7.09 (m, 2H, H-Ph), 5.82 (d, J = 4.7 Hz, 1H, OH), 5.61 (s, 2H, CH₂), 4.98–4.94 (m, 1H, CHCH₂), 4.46 (t, J = 6.6 Hz, 2H, CHCH₂). ¹³C NMR (101 MHz, DMSO) δ/ppm: 161.97 (d, J_CF = 242.9 Hz), 152.17, 149.58, 147.85, 141.78, 138.54, 131.24, 128.48 (d, J_CF = 8.1 Hz), 127.93, 124.96, 115.32 (d, J_CF = 21.3 Hz), 71.05, 56.97, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₃FClN₇O (Mr = 373.78) C, 51.41; H, 3.51; N, 26.23; found C, 51.38; H, 3.55; N, 26.18.

(R)-6-Chloro-9-((1-(2-hydroxy-2-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine) ((R)-38b)

Compound (R)-38b was prepared using the procedure mentioned above from compound 5 (50 mg, 0.26 mmol) and 2-azido-1-(4-chlorophenyl)ethanol (R)-18b (61 mg, 0.31 mmol, ee = 89%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound (R)-38b was isolated as a white powder (43 mg, 44%, m.p. = 89–92 °C, ee = 89%, [α]_D²⁴ − 36.4 (c =0.0011)). ¹H NMR (300 MHz, DMSO) δ/ppm: 8.81 (s, 1H, H-2), 8.77 (s, 1H, H-8), 8.07 (s, 1H, H-triazole), 7.32 (m, 4H, H-Ph), 5.87 (d, J = 4.7 Hz, 1H, OH), 5.61 (s, 2H, CH₂), 4.96 (dt, J = 9.2, 4.6 Hz, 1H, CHCH₂), 4.54–4.37 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.55, 151.51, 148.96, 147.24, 141.18, 140.69, 131.86, 130.61, 127.92, 127.75, 124.36, 70.37, 56.19, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₃Cl₂N₇O (Mr = 390.23) C, 49.25; H, 3.36; N, 25.13; found C, 49.33; H, 3.31; N, 25.19.

(S)-6-Chloro-9-((1-(2-hydroxy-2-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine) ((S)-38b)

Compound (S)-38b was prepared using the procedure mentioned above from compound 5 (58 mg, 0.30 mmol) and 2-azido-1-(4-chlorophenyl)ethanol (S)-18b (72 mg, 0.36 mmol, ee = 95%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound (S)-38b was isolated as a white powder (85 mg, 73%, m.p. = 89–92 °C, ee = 95%, [α]_D²⁴ + 33.3 (c = 0.0012)). ¹H NMR (300 MHz, DMSO) δ/ppm: 8.81 (s, 1H, H-2), 8.77 (s, 1H, H-8), 8.07 (s, 1H, H-triazole), 7.32 (m, 4H, H-Ph), 5.87 (d, J = 4.7 Hz, 1H, OH), 5.61 (s, 2H, CH₂), 4.96 (dt, J = 9.2, 4.6 Hz, 1H, CHCH₂), 4.54–4.37 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ 151.55, 151.51, 148.96, 147.24, 141.18, 140.69, 131.86, 130.61, 127.92, 127.75, 124.36, 70.37, 56.19, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₆H₁₃C_l2N₇O (Mr = 390.23) C, 49.25; H, 3.36; N, 25.13; found C, 49.32; H, 3.289; N, 25.08.

(R)-6-Chloro-9-((1-(2-hydroxy-2-(4-(trifluoromethyl)phenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine ((R)-40b)

Compound (R)-40b was prepared using the procedure mentioned above from compound 5 (50 mg, 0.26 mmol) and (R)-2-azido-1-(4-(trifluoromethyl)phenyl)ethanol (R)-40b (72 mg, 0.31 mmol, ee = 97%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound (R)-40b was isolated as a white powder (85 mg, 75%, m.p. = 84–86 °C, ee = 97%, [α]_D²⁴ − 45.5 (c = 0.0011). ¹H NMR (300 MHz, DMSO) δ/ppm: 8.81 (s, 1H, H-2), 8.76 (d, J = 6.0 Hz, 1H, H-8), 8.11 (s, 1H, H-triazole), 7.64 (d, J = 8.3 Hz, 2H, H-Ph), 7.53 (d, J = 8.1 Hz, 2H, H-Ph), 5.98 (d, J = 4.7 Hz, 1H, OH), 5.62 (s, 2H, CH₂), 5.13–4.93 (m, 1H, CHCH₂), 4.51 (qd, J = 13.8, 6.0 Hz, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.65, 151.63, 149.07, 147.35, 146.50, 141.34, 130.72, 128.06 (q, J_CF3 = 31.7 Hz), 126.89 (q, J_CF3 = 271.8 Hz), 124.93 (q, J_CF3 = 3.6 Hz), 124.50, 121.45, 70.59, 56.18, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₃ClF₃N₇O (Mr = 434.68) C, 48.18; H, 3.09; N, 23.14; found C, 48.23; H, 3.14; N, 23.20.

(S)-6-Chloro-9-((1-(2-hydroxy-2-(4-(trifluoromethyl)phenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-purine ((S)-40b)

Compound (S)-40b was prepared using the procedure mentioned above from compound 5 (50 mg, 0.26 mmol) and (R)-2-azido-1-(4-(trifluoromethyl)phenyl)ethanol (S)-40b (72 mg, 0.31 mmol, ee = 97%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound (S)-40b was isolated as a white powder (91 mg, 81%, m.p. = 84–86 °C, ee = 97%, [α]_D²⁴ + 50 (c =0.0010)). ¹H NMR (300 MHz, DMSO) δ/ppm: 8.81 (s, 1H, H-2), 8.76 (d, J = 6.0 Hz, 1H, H-8), 8.11 (s, 1H, H-triazole), 7.64 (d, J = 8.3 Hz, 2H, H-Ph), 7.53 (d, J = 8.1 Hz, 2H, H-Ph), 5.98 (d, J = 4.7 Hz, 1H, OH), 5.62 (s, 2H, CH₂), 5.13–4.93 (m, 1H, CHCH₂), 4.51 (qd, J = 13.8, 6.0 Hz, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.65, 151.63, 149.07, 147.35, 146.50, 141.34, 130.72, 128.06 (q, J_CF3 = 31.7 Hz), 126.89 (q, J_CF3 = 271.8 Hz), 124.93 (q, J_CF3 = 3.6 Hz), 124.50, 121.45, 70.59, 56.18, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₃ClF₃N₇O (Mr = 434.68) C, 48.18; H, 3.09; N, 23.14; found C, 48.11; H, 3.13; N, 23.09.

(R)-4-Chloro-7-((1-(2-hydroxy-2-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine ((R)-41b)

Compound (R)-41b was prepared using the procedure mentioned above from compound 9 (25 mg, 0.13 mmol) and 2-azido-2-phenylethanol (R)-16b (21 mg, 0.16 mmol, ee = 95%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound (R)-41b was isolated as a white powder (16 mg, 35%, m.p. = 114–117, ee = 95%, [α]_D²⁴ − 30.0 (c = 0.0010)). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 7.97 (s, 1H, H-triazole), 7.78 (d, J = 3.6 Hz, 1H, H-6), 7.29 (dd, J = 12.4, 4.5 Hz, 5H, H-Ph), 6.69 (d, J = 3.6 Hz, 1H, H-5), 5.76 (d, J = 4.7 Hz, 1H, OH), 5.57 (s, 2H, CH₂), 4.94 (dt, J = 8.7, 4.6 Hz, 1H, CHCH₂), 4.50–4.40 (m, 2H, CHCH₂). ¹³C NMR (101 MHz, DMSO) δ/ppm: 151.13, 150.90, 150.82, 142.61, 142.39, 131.71, 128.58, 127.96, 126.46, 124.74, 117.25, 99.28, 71.64, 57.01. Anal. calcd. For C₁₇H₁₅ClN₆O (Mr = 354.80) C, 57.55; H, 4.26; N, 23.69; found C, 57.61; H, 4.19; N, 23.61.

(S)-4-Chloro-7-((1-(2-hydroxy-2-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine ((S)-41b)

Compound (S)-41b was prepared using the procedure mentioned above from compound 9 (54 mg, 0.28 mmol) and 2-azido-2-phenylethanol (S)-16b (55 mg, 0.34 mmol, ee = 99%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound (S)-41b was isolated as white powder (88 mg, 76%, m.p. = 114–117, ee = 99%, [α]_D²⁴ + 30.0 (c = 0.0010)). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 7.97 (s, 1H, H-triazole), 7.78 (d, J = 3.6 Hz, 1H, H-6), 7.29 (dd, J = 12.4, 4.5 Hz, 5H, H-Ph), 6.69 (d, J = 3.6 Hz, 1H, H-5), 5.76 (d, J = 4.7 Hz, 1H, OH), 5.57 (s, 2H, CH₂), 4.94 (dt, J = 8.7, 4.6 Hz, 1H, CHCH₂), 4.50–4.40 (m, 2H, CHCH₂). ¹³C NMR (101 MHz, DMSO) δ/ppm: 151.13, 150.90, 150.82, 142.61, 142.39, 131.71, 128.58, 127.96, 126.46, 124.74, 117.25, 99.28, 71.64, 57.01. Anal. calcd. For C₁₇H₁₅ClN₆O (Mr = 354.80) C, 57.55; H, 4.26; N, 23.69; found C, 57.48; H, 4.19; N, 23.62.

(R)-4-Chloro-7-((1-(2-hydroxy-2-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine ((R)-42b)

Compound (R)-42b was prepared using the procedure mentioned above from compound 9 (43 mg, 0.22 mmol) and 2-azido-1-(4-fluorophenyl)ethanol (R)-17b (50 mg, 0.27 mmol, ee = 99%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound (R)-42b was isolated as a white powder (44 mg, 54%, m.p. = 154–156 °C, ee = 99%, [α]_D²⁴ − 18.2 (c = 0.0011)). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 7.95 (s, 1H, H-triazole), 7.77 (d, J = 3.6 Hz, 1H, H-6), 7.33 (dd, J = 8.5, 5.7 Hz, 2H, H-Ph), 7.09 (t, J = 8.9 Hz, 2H, H-Ph), 6.69 (d, J = 3.6 Hz, 1H, H-5), 5.81 (d, J = 4.7 Hz, 1H, OH), 5.57 (s, 2H, CH₂), 4.96 (dt, J = 9.4, 4.8 Hz, 1H, CHCH₂), 4.44 (dd, J = 12.1, 4.9 Hz, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ 161.45 (d, J_CF = 243.0 Hz), 150.63, 150.38, 150.30, 142.13, 138.04 (d, J = 2.7 Hz), 131.18, 127.94 (d, J_CF = 8.2 Hz), 124.21, 116.72, 114.79 (d, J_CF = 21.2 Hz), 98.76, 70.54, 56.38, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₄ClFN₆O (Mr = 372.79) C, 54.77; H, 3.79; N, 22.54; found C, 54.71; H, 3.73; N, 22.49.

(S)-4-Chloro-7-((1-(2-hydroxy-2-(4-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine ((S)-42b)

Compound (S)-42b was prepared using the procedure mentioned above from compound 9 (30 mg, 0.15 mmol) and 2-azido-1-(4-fluorophenyl)ethanol (S)-17b (36 mg, 0.19 mmol, ee = 97%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound (S)-42b was isolated as a white powder (30 mg, 54%, m.p. = 154–156 °C, ee = 97%, [α]_D²⁴ + 16.7 (c = 0.0012)). ¹H NMR (400 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 7.95 (s, 1H, H-triazole), 7.77 (d, J = 3.6 Hz, 1H, H-6), 7.33 (dd, J = 8.5, 5.7 Hz, 2H, H-Ph), 7.09 (t, J = 8.9 Hz, 2H, H-Ph), 6.69 (d, J = 3.6 Hz, 1H, H-5), 5.81 (d, J = 4.7 Hz, 1H, OH), 5.57 (s, 2H, CH₂), 4.96 (dt, J = 9.4, 4.8 Hz, 1H, CHCH₂), 4.44 (dd, J = 12.1, 4.9 Hz, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 161.45 (d, J_CF = 243.0 Hz), 150.63, 150.38, 150.30, 142.13, 138.04 (d, J = 2.7 Hz), 131.18, 127.94 (d, J_CF = 8.2 Hz), 124.21, 116.72, 114.79 (d, J_CF = 21.2 Hz), 98.76, 70.54, 56.38, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₄ClFN₆O (Mr = 372.79) C, 54.77; H, 3.79; N, 22.54; found C, 54.82; H, 3.83; N, 22.49.

(R)-4-Chloro-7-((1-(2-hydroxy-2-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine ((R)-43b)

Compound (R)-43b was prepared using the procedure mentioned above from compound 9 (52 mg, 0.27 mmol) and 2-azido-1-(4-chlorophenyl)ethanol (R)-18b (63 mg, 0.32 mmol, ee = 89%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound (R)-43b was isolated as a white powder (74 mg, 71%, m.p. = 152–154 °C, ee = 89%, [α]_D²⁴ − 40.0 (c = 0.0010)). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 7.96 (s, 1H, H-triazole), 7.77 (d, J = 3.6 Hz, 1H, H-6), 7.33–7.29 (m, 4H, H-Ph), 6.68 (d, J = 3.6 Hz, 1H, H-5), 5.86 (d, J = 4.7 Hz, 1H, OH), 5.56 (s, 2H, CH₂), 4.98–4.94 (m, 1H, CHCH₂), 4.49–4.41 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.16, 150.91, 150.83, 142.69, 141.36, 132.48, 131.71, 128.47, 128.29, 124.75, 117.24, 99.29, 71.01, 56.75, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₄Cl₂N₆O (Mr = 389.24) C, 52.46; H, 3.63; N, 21.59; found C, 52.40; H, 3.69; N, 21.63.

(S)-4-Chloro-7-((1-(2-hydroxy-2-(4-chlorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidine ((S)-43b)

Compound (S)-43b was prepared using the procedure mentioned above from compound 9 (52 mg, 0.27 mmol) and 2-azido-1-(4-chlorophenyl)ethanol (S)-18b (63 mg, 0.32 mmol, ee = 95%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound (S)-43b was isolated as a white powder (94 mg, 89%, m.p. = 152–154 °C, ee = 95%, [α]_D²⁴ + 40.0 (c = 0.0010)). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 7.96 (s, 1H, H-triazole), 7.77 (d, J = 3.6 Hz, 1H, H-6), 7.33–7.29 (m, 4H, H-Ph), 6.68 (d, J = 3.6 Hz, 1H, H-5), 5.86 (d, J = 4.7 Hz, 1H, OH), 5.56 (s, 2H, CH₂), 4.98–4.94 (m, 1H, CHCH₂), 4.49–4.41 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.16, 150.91, 150.83, 142.69, 141.36, 132.48, 131.71, 128.47, 128.29, 124.75, 117.24, 99.29, 71.01, 56.75, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₇H₁₄Cl₂N₆O (Mr = 389.24) C, 52.46; H, 3.63; N, 21.59; found C, 52.51; H, 3.58; N, 21.51.

(R)-4-Chloro-7-((1-(2-hydroxy-2-(4-(trifluoromethyl)phenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin ((R)-45b)

Compound (R)-45b was prepared using the procedure mentioned above from compound 9 (76 mg, 0.40 mmol) and (R)-2-azido-2-(4-(trifluoromethyl)phenyl)ethanol (R)-20a (110 mg, 0.47 mmol, ee = 97%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound (R)-45b was isolated as a white powder (101 mg, 60%, m.p. = 74–75 °C, ee = 97%, [α]_D²⁴ − 44.4 (c = 0.0009)). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 8.00 (s, 1H, H-triazole), 7.77 (d, J = 3.6 Hz, 1H, H-6), 7.63 (d, J = 8.2 Hz, 2H, H-Ph), 7.52 (d, J = 8.1 Hz, 2H, H-Ph), 6.67 (d, J = 3.6 Hz, 1H, H-5), 5.98 (d, J = 4.8 Hz, 1H, OH), 5.57 (s, 2H, CH₂), 5.08–5.06 (m, 1H, CHCH₂), 4.55–4.45 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.14, 150.88, 150.81, 147.03, 142.72, 131.67, 128.55 (q, J_CF3 = 31.5 Hz), 125.50, 125.42 (q, J_CF3 = 3.5 Hz), 124.77, 124.69 (d, J_CF3 = 272.2 Hz), 117.22, 99.26, 71.10, 56.61, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₈H₁₄F₃N₆O (Mr = 422.80) C, 51.14; H, 3.34; N, 19.88; found C, 51.21; H, 3.28; N, 19.95.

(S)-4-Chloro-7-((1-(2-hydroxy-2-(4-(trifluoromethyl)phenyl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin ((S)-45b)

Compound (S)-45b was prepared using the procedure mentioned above from compound 9 (76 mg, 0.40 mmol) and (S)-2-azido-2-(4-(trifluoromethyl)phenyl)ethanol (S)-20a (110 mg, 0.47 mmol, ee = 97%). After purification via column chromatography (CH₂Cl₂/CH₃OH = 50:1), compound (S)-45b was isolated as a white powder (112 mg, 66%, m.p. = 74–75 °C, ee = 97%, [α]_D²⁴ + 50.0 (c = 0.0010)). ¹H NMR (600 MHz, DMSO) δ/ppm: 8.68 (s, 1H, H-2), 8.00 (s, 1H, H-triazole), 7.77 (d, J = 3.6 Hz, 1H, H-6), 7.63 (d, J = 8.2 Hz, 2H, H-Ph), 7.52 (d, J = 8.1 Hz, 2H, H-Ph), 6.67 (d, J = 3.6 Hz, 1H, H-5), 5.98 (d, J = 4.8 Hz, 1H, OH), 5.57 (s, 2H, CH₂), 5.08–5.06 (m, 1H, CHCH₂), 4.55–4.45 (m, 2H, CHCH₂). ¹³C NMR (151 MHz, DMSO) δ/ppm: 151.14, 150.88, 150.81, 147.03, 142.72, 131.67, 128.55 (q, J_CF3 = 31.5 Hz), 125.50, 125.42 (q, J_CF3 = 3.5 Hz), 124.77, 124.69 (d, J_CF3 = 272.2 Hz), 117.22, 99.26, 71.10, 56.61, a signal assigned to the CH₂ group is obscured by the DMSO signal. Anal. calcd. For C₁₈H₁₄F₃N₆O (Mr = 422.80) C, 51.14; H, 3.34; N, 19.88; found C, 51.21; H, 3.29; N, 19.82.

2.3. Examination of Antiproliferative Activity In Vitro

Cell Culturing and Proliferation Assays

The human ATCC tumor cell lines breast adenocarcinoma (MCF7), colorectal carcinoma (HT-29), pancreatic ductal adenocarcinoma (CFPAC-1), hepatocellular carcinoma (HepG2), and normal skin fibroblasts (HFF) derived from ATCC (American Type Culture Collection) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) or Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Lonza, Basel, Switzerland) in a humidified atmosphere with 5% CO₂ at 37 °C. For the experiment, carcinoma cell lines and normal human cell lines were seeded in 96-well microtiter plates at a density of 3000 cells per well and 5000 cells per well, respectively, depending on the doubling time of the specific cell line. The test substances were then added at fivefold, tenfold dilutions (0.01 to 100 µM), freshly prepared in the growth medium on the day of testing, and incubated for a further 72 h. After 72 h of incubation, the growth rate of the cells was determined using the MTT assay according to the manufacturer’s guidelines. The percentage of growth was calculated by transforming the experimentally determined absorbance values using the formulas suggested by the National Institutes of Health (NIH). The IC₅₀ values were calculated from the dose–response curves using linear regression analysis. The experiment was performed in tetraplicates in two individual experiments.

2.4. In Vitro ADME Profiling

2.4.1. MDCKII-MDR1 Permeability Assay

MDCKII-hMDR1 cells were obtained from Solvo Biotechnology, Hungary. DMEM, fetal bovine serum, Glutamax-100, Antibiotic/Antimycotic, DMSO, Dulbecco’s phosphate-buffered saline, and MEM Non-essential amino acids were purchased from Sigma (St. Louis, MO, USA). Bi-directional permeability and P-glycoprotein substrate assessments were carried out in Madin-Darby canine epithelial cells with the over-expressed human MDR1 gene (MDCKII-MDR1), coding for P-glycoprotein. Experimental procedures, as well as cell culture conditions, were the same as previously described [53]. Briefly, compounds (10 µM, 1% DMSO v/v) in duplicate were incubated at 37 °C for 60 min with cell monolayer on 24-well Millicell inserts (Millipore, Burlington, MA, USA) with and without the P-glycoprotein inhibitor Elacridar (2 µM, International Laboratory, San Francisco, CA, USA). The inhibition of P-glycoprotein was verified by amprenavir (Moravek Biochemicals Inc, Brea, CA, USA) and monolayer integrity using Lucifer yellow (Sigma, St. Louis, MO, USA). LC-MS/MS measured compound concentrations and Lucifer yellow was measured on an Infinite F500 (Tecan, Männedorf, CH, Switzerland) using excitation of 485 nm and emission of 530 nm.

2.4.2. Metabolic Stability

Mouse liver microsomes were obtained from Corning Life Sciences (Corning, NY, USA). DMSO, nicotinamide adenine dinucleotide phosphate (NADP), glucose-6-phosphate, glucose-6-phosphate dehydrogenase, magnesium chloride, propranolol, caffeine, diclofenac, and phosphate-buffered saline (PBS) were purchased from Sigma (St. Louis, MO, USA). Acetonitrile (ACN) and methanol (MeOH) were obtained from Merck (Darmstadt, Germany). Testosterone was purchased from Steraloids (Newport, RI, USA). Metabolic stability was assessed in mouse liver microsomes. Compounds (final concentration of 1 µM, 0.03% DMSO v/v) were incubated in duplicate in phosphate buffer (50 mM, pH 7.4) at 37 °C together with mouse liver microsomes in the absence and presence of the NADPH cofactor (0.5 mM nicotinamide adenine dinucleotide phosphate, 5 mM glucose-6-phosphate, 1.5 U/mL glucose-6-phosphate dehydrogenase, and 0.5 mM magnesium chloride). Incubation and sampling were performed on a Freedom EVO 200 (Tecan, Männedorf, CH, Switzerland) at 0.3, 10, 20, 30, 45, and 60 min. The reaction was quenched using 3 volumes of a mixture of ACN/MeOH (2:1) containing internal standard (diclofenac), centrifuged, and the supernatants were analyzed using LC-MS/MS. The metabolic activity of the microsomes was verified via simultaneous analysis of several controls including testosterone, propranolol, and caffeine. The in vitro half-life (t_1/2) was calculated using GraphPad Prism (v9.0 software) non-linear regression of % of parent compound remaining versus time. In vitro clearance, expressed as µL/min/mg, was estimated from the in vitro half-life (t_1/2) and normalized for the protein amount in the incubation mixture, assuming 52.5 mg of protein per gram of liver and using constant values for mouse liver weight/body weight [87.5 g/kg] and mouse liver blood flow (LBF) [131 mL/min/kg].

2.4.3. LC-MS/MS Analysis

All samples were quantified using tandem mass spectrometry coupled to liquid chromatography. Samples were analyzed on a Sciex API4000 Triple Quadrupole Mass Spectrometer (Sciex, Division of MDS Inc., Toronto, Canada) coupled to a Shimadzu Nexera X2 UHPLC frontend (Kyoto, Japan). Samples were injected onto a UHPLC column (HALO2 C18, 2.1 × 20 mm, 2 µm or Luna Omega 1.6 µm Polar C18 100A, 30 × 2.1 mm) and eluted with a gradient at 50 °C. The mobile phase was composed of acetonitrile/water mixture (9/1, with 0.1% formic acid) and 0.1% formic acid in deionized water. The flow rate was 0.7 mL/min under gradient conditions, leading to a total run time of 1.5–2 min. A positive ion mode with turbo spray, an ion source temperature of 550 °C, and a dwell time of 150 ms were utilized for mass spectrometric detection. Quantitation was performed using multiple reaction monitoring (MRM) at the specific transitions for each compound.

2.4.4. In Silico Profiling

The in silico ADME properties as well as the structural parameters were calculated using ACD Percepta software (ACD/Percepta, v2021.1.3) [54]. Data analysis and visualization were performed using DataWarrior (DataWarrior v06.01.01, 2024). Cresset suite software (Flare™, v7.0) was used to perform field based alignment and the conformational search [55].

3. Results

3.1. Chemistry

The targeted racemic 26a–49a, 26b–49b, and enantiomeric-enriched N-heterocycles (R)-36b-(R)-38b, (R)-40b, (R)-41b-(R)-43b, (R)-45b, and (S)-36b-(S)-38b, (S)-40b, (S)-41b-(S)-43b, and (S)-45b were synthesized via the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of terminal alkynyl derivatives of purine and purine isosteres 6–10 and 1,2-azido alcohols 16a–20a and 16b–20b using an environmentally benign synthetic protocol [56] with ultrasonic irradiation (Scheme 1).

Scheme 1.

Cu(I)-catalyzed synthesis of novel racemic aryl-substituted derivatives of purines and purine isosteres. Reagents and conditions: (i) propargyl bromide, NaH, DMF, r.t., overnight; (ii) NaN₃, NH₄Cl, MeOH/H₂O (8:1), 75 °C, overnight; (iii) NaN₃, DMF, r.t, 0.5 h; (iv) NaBH_4, MeOH, r.t., 1 h; and (v) Cu(OAc)₂, MeOH, 2 h, under ultrasound irradiation.

The key intermediates N-propargylated indole (6) [45], benzimidazole (7) [43], 6-chloro-9H-purine (8) [43], 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (9) [47], and 4-chloro-1H-imidazo[4,5-c]pyridine (10) [43] were obtained via N-alkylation of the corresponding heterocyclic base 1–5. N-Alkylation of 6-chloropurine (3) and 4-chloro-1H-imidazo[4,5-c]pyridine (5) afforded N-9 regioisomers 8 and 10, which was confirmed by 1D and 2D NMR techniques [43]. Racemic 1,2-azido alcohols with primary hydroxyl groups 16a–20a were synthesized via a regioselective ring-opening reaction of various para-substituted 2-phenyloxiranes 11–15 with sodium azide under basic conditions. The regioselectivity of the 2-phenyloxirane derivatives can be explained by the electronic effects of the aromatic substituent that direct the azide nucleophile towards the benzylic α-position due to the stabilization of the positive charge via resonance with the aromatic moiety [57]. However, the strong electron-withdrawing trifluoromethyl substituent at the phenyl ring in 15 significantly decreases the regioselectivity of 20a/20b from 15.6 (α/β) to 1.5 (α/β) (Table 1). Consequently, α-azido alcohols 16a–20a with the primary hydroxyl group were isolated as major products in very good yields (51–88%) [48,49,50], while β-azido alcohols 16b–20b with the secondary hydroxyl group were obtained in yields of 6–34% (Table 1).

Table 1.

Regioselectivity and conversion of ring-opening reaction of 2-phenyloxirane derivatives via NaN₃ under basic conditions ^a.

R2	Regioisomers	Conversion (%)	Regioselectivity (α/β)
H	16a/16b	96%	15.6
F	17a/17b	71%	4.5
Cl	18a/18b	68%	5.2
Br	19a/19b	87%	2.1
CF3	20a/20b	85%	1.5

^a Reaction conditions: NaN₃ (1.2 eq) and NH₄Cl (1.0 eq) in MeOH/H₂O (8:1), 75 °C, overnight.

Therefore, a new synthetic pathway for β-azido alcohols was used in which p-substituted phenacyl bromides were treated with sodium azide, followed by fast reduction to afford corresponding β-azido alcohols 16b–20b [49,51,52]. Enantioenriched (ee = 89–99%) β-azido alcohols 16b–18b and 20b, as R- and S-enantiomers, were obtained via biocatalytic azidolysis of various para-substituted 2-phenyloxiranes with sodium azide as a nucleophile and halohydrin dehalogenase, which were found to be enantioselective (Scheme 2) [58].

Scheme 2.

Cu(I)-catalyzed synthesis of novel optically enriched (R)- and (S)-aryl-substituted derivatives of 6-chloropurine and 4-chloropyrrolo[2,3-d]pyrimidine. Reagents and conditions: (i) MeOH, Cu(OAc)₂, 2 h, under ultrasound irradiation.

The application of halohydrin dehalogenases and biocatalysis in general, particularly in the synthesis of active pharmaceutical ingredients, has evolved into an environmentally friendly method [59]. For the synthesis of enantiopure (R)-1,2-azido alcohols, the HheC-W249P variant was used, while for (S)-1,2-azido alcohols, HheA-N178A was applied [60]. Absolute configurations of 1,2-azido alcohols were assigned based on the previously reported data [60,61]. Novel enantioenriched (R)- and (S)-N-aryl-substituted derivatives of 6-chloropurine ((R)-36b-(R)-38b, (R)-40b, (S)-36b-(S)-38b, and (S)-40b) and pyrrolo[2,3-d]pyrimidine ((R)-41b-(R)-43b, (R)-45b, (S)-41b-(S)-43b, and (S)-45b) were subsequently synthesized using the copper(I)-catalyzed 1,3-dipolar cycloaddition of propargylated N-heterocycles 8 or 9 and optically pure ß-substituted azido alcohols 16b–18b and 20b under ultrasound irradiation (Scheme 2).

3.2. Biological Profiling

3.2.1. Evaluation of Antiproliferative Activity

Newly synthesized compounds were evaluated for their cytotoxic activity in vitro against four human cancer cell lines, including human breast adenocarcinoma (MCF-7), ductal pancreatic adenocarcinoma (CFPAC-1), colorectal carcinoma (HT-29), and hepatocellular carcinoma (HepG2), as well as on non-cancerous skin fibroblasts (HFF-1), to check for non-specific toxicity. Furthermore, 5-Fluorouracil (5-FU) was used as a reference drug. The results are expressed as 50% inhibitory concentration (IC₅₀) values and are listed in Table 2, Table 3, Table 4 and Table 5. It can be observed that purine (36a–40a and 36b–40b) (Table 3) and pyrrolo[2,3-d]pyrimidine (41a–45a and 41b–45b) (Table 4) derivatives exhibited better antiproliferative activity than indole (26a–30a and 26b–30b) (Table 2), benzimidazole (31a–35a and 31b–35b) (Table 2), and imidazo[4,5-c]pyridine (46a–49a and 46b–49b) (Table 5) derivatives.

Table 2.

The growth inhibition effects of racemic indole (26a,b–30a,b) and benzimidazole derivatives (31a,b–35a,b) on MCF-7, CFPAC-1, HT-29, and HepG2, as well as on normal skin fibroblasts (HFF-1).

				IC50 a (µM)
Compd	X	Linker	R2	MCF-7	CFPAC-1 b	HT-29	HepG2	HFF-1	SI (CFPAC-1) c
26a	CH		H	>100	>100	>100	>100	>100	-
26b	CH		H	>100	>100	>100	>100	>100	-
27a	CH		F	51 ± 6.7	68 ± 12.7	47 ± 16.9	49 ± 2.5	42 ± 10.5	0.6
27b	CH		F	>100	>100	>100	>100	>100	-
28a	CH		Cl	32 ± 0.0	30 ± 0.6	31 ± 0.9	32 ± 0.2	42 ± 4.6	1.4
28b	CH		Cl	35 ± 16.2	35 ± 19.8	46 ± 18.4	66 ± 2.7	>100	>2.9
29a	CH		Br	24 ± 1.0	39 ± 2.8	34 ± 0.3	29 ± 0.8	>100	>2.6
29b	CH		Br	29 ± 14	43 ± 9.1	36 ± 1.4	35 ± 1.8	25 ± 4.1	0.6
30a	CH		CF3	21 ± 1.6	9 ± 3.3	28 ± 1.9	32 ± 3.6	34 ± 1.6	3.7
30b	CH		CF3	22 ± 3.4	27 ± 0.7	51 ± 15.3	65 ± 2.4	18 ± 0.6	0.7
31a	N		H	>100	>100	>100	>100	>100	-
31b	N		H	91 ± 7.2	>100	>100	>100	>100	-
32a	N		F	98 ± 2.8	>100	>100	74 ± 0.6	>100	-
32b	N		F	>100	>100	>100	>100	>100	-
33a	N		Cl	40 ± 1.8	34 ± 2.3	63 ± 9.8	50 ± 12.8	>100	>2.9
33b	N		Cl	70 ± 20.5	69 ± 12.1	>100	>100	64 ± 6.17	0.9
34a	N		Br	22 ± 5.1	15 ± 8.9	39 ± 4.3	35 ± 14.2	42 ± 8.3	2.8
34b	N		Br	39 ± 10.1	8 ± 3.2	70 ± 14.5	64 ± 19.5	32 ± 14.1	4.0
35a	N		CF3	30 ± 0.9	27 ± 0.7	61 ± 0.7	59 ± 4.6	>100	>3.7
35b	N		CF3	31 ± 13.7	36 ± 1.3	67 ± 2.9	67 ± 3.5	22 ± 18.1	0.6
5-FU				1 ± 0.8	0.14 ± 0.1	0.23 ± 0.0	9 ± 1.3	0.94 ± 0.0	6.7

^a IC₅₀—compound concentration that inhibited cell growth by 50%. Data represent mean IC₅₀ (μM) values ± standard deviation (SD) of three independent experiments. ^b Indole analogs of 27, 28, and 30 without linker exhibited IC₅₀ > 100 μM (CFPAC-1), while benzimidazole analogs of 32, 33, and 35 exhibited IC₅₀ values of 86.52 μM, 52.55 μM, and 56.43 μM, respectively (CFPAC-1). ^c Selectivity index towards ductal pancreatic adenocarcinoma cells (CFPAC-1), SI = [IC₅₀ HFF]/[IC₅₀ CFPAC-1].

Table 3.

The growth inhibition effects of racemic and enantiomeric-enriched 6-chloropurine derivatives (36a,b–40a,b, (R),(S)-36b-(R),(S)-38b and (R),(S)-40b) on MCF-7, CFPAC-1, HT-29, and HepG2, as well as on normal skin fibroblasts (HFF-1).

			IC50 a (µM)
Compd	Linker	R2	MCF-7	CFPAC-1 b	HT-29	HepG2	HFF-1	SI (HT-29) c	SI (HepG2) c
36a		H	53 ± 5.5	60 ± 0.9	54 ± 7.4	55 ± 2.4	>100	>1.9	>1.8
36b		H	69 ± 10.6	65 ± 18.9	61 ± 2.4	41 ± 10.6	80 ± 14.0	1.3	1.9
(R)-36b		H	61 ± 3.7	66 ± 3.2	86 ± 11.6	57 ± 9.7	>100	>1.2	>1.8
(S)-36b		H	33 ± 5.8	32 ± 5.3	48 ± 4.8	21 ± 0.8	>100	>2.1	>4.8
37a		F	38 ± 15.7	50 ± 12.4	41 ± 2.3	37 ± 12.9	64 ± 6.3	1.6	1.7
37b		F	49 ± 9.9	56 ± 5.5	46 ± 9.9	54 ± 6.0	59 ± 9.2	1.3	1.1
(R)-37b		F	>100	>100	>100	64 ± 3.1	35 ± 5.5	0.4	0.6
(S)-37b		F	73 ± 2.9	65 ± 5.4	24 ± 2.9	45 ± 1.9	70 ± 19.5	2.9	1.6
38a		Cl	24 ± 8.8	48 ± 4.9	33 ± 6.06	34 ± 4.2	41 ± 16.3	1.2	1.2
38b		Cl	51 ± 8.9	36 ± 8.2	56 ± 2.8	49 ± 13.3	31 ± 5.3	0.6	0.6
(R)-38b		Cl	64 ± 4.8	60 ± 10.3	51 ± 17.8	39 ± 10.5	31 ± 13.4	0.6	0.8
(S)-38b		Cl	58 ± 1.9	51 ± 11.7	47 ± 16.8	28 ± 7.5	81 ± 1.8	1.7	2.9
39a		Br	14 ± 4.8	46 ± 3.8	31 ± 2.6	16 ± 5.6	14 ± 4.7	0.5	0.9
39b		Br	21 ± 14.0	15 ± 1.0	50 ± 0.6	44 ± 8.1	30 ± 2.7	0.6	0.7
40a		CF3	19 ± 4.3	18 ± 3.5	0.69 ± 0.01	0.64 ± 0.1	18 ± 0.6	26.1	28.1
40b		CF3	28 ± 6.7	25 ± 6.4	14 ± 8.1	15 ± 9.0	8 ± 4.8	0.6	0.5
(R)-40b		CF3	36 ± 23.1	16 ± 2.6	6 ± 0.6	8 ± 0.6	6 ± 4.8	1.0	0.7
(S)-40b		CF3	10 ± 0.4	19 ± 2.4	5 ± 0.1	7 ± 1.5	12 ± 3.4	2.4	1.7
5-FU			1 ± 0.8	0.1 ± 0.1	0.2 ± 0.0	9 ± 1.3	0.9 ± 0.0	4.5	0.1

^a IC₅₀—compound concentration that inhibited cell growth by 50%. Data represent mean IC₅₀ (μM) values ± standard deviation (SD) of three independent experiments, ^b 6-Chloropurine analogs of 37 and 40 without linker exhibited IC₅₀ values of 33.39 μM and 7.90 μM, respectively (CFPAC-1). ^c Selectivity index towards colorectal carcinoma (HT-29) SI = [IC₅₀ HFF]/[IC₅₀ HT-29] and hepatocellular carcinoma (HepG2), SI = [IC₅₀ HFF]/[IC₅₀ HepG2].

Table 4.

The growth inhibition effects of racemic and enantiomeric-enriched 4-chloropyrrolo[2,3-d]pyrimidine derivatives (41a,b–45a,b, ((R),(S)-41b-(R),(S)-43b and (R),(S)-45b)) on MCF-7, CFPAC-1, HT-29, and HepG2, as well as on normal skin fibroblasts (HFF-1).

			IC50 a (µM)
Compd	Linker	R2	MCF-7	CFPAC-1 b	HT-29	HepG2	HFF-1	SI (MCF-7) c	SI (CFPAC-1) c
41a		H	39 ± 7.1	33 ± 4.5	33 ± 3.2	38 ± 1.8	62 ± 23.3	1.6	1.9
41b		H	44 ± 17.4	49 ± 11.3	50 ± 1.3	50 ± 10.2	44 ± 14.1	1.0	0.9
(R)-41b		H	46 ± 2.7	58 ± 12.1	46 ± 2.3	52 ± 2.7	23 ± 5.8	0.5	0.4
(S)-41b		H	45 ± 7.2	49 ± 18.5	41 ± 5.6	41 ± 12.4	58 ± 3.1	1.3	1.2
42a		F	39 ± 16.9	36 ± 2.2	39 ± 1.9	47 ± 13.5	51 ± 16.5	1.3	1.4
42b		F	45 ± 5.7	34 ± 3.2	49 ± 14.6	38 ± 11.0	51 ± 3.6	1.1	1.5
(R)-42b		F	60 ± 1.2	20 ± 0.9	27 ± 1.4	32 ± 6.04	24 ± 2.4	0.4	1.2
(S)-42b		F	41 ± 0.0	13 ± 2.7	33 ± 0.2	49 ± 2.0	73 ± 2.4	1.8	5.6
43a		Cl	24 ± 8.8	8 ± 3.2	33 ± 6.1	33 ± 4.2	41 ± 16.3	1.7	5.1
43b		Cl	39 ± 10.1	48 ± 4.9	28 ± 3.8	46 ± 2.2	32 ± 14.1	0.8	0.7
(R)-43b		Cl	35 ± 1.9	32 ± 16.1	48 ± 4.8	26 ± 8.2	20 ± 6.5	0.6	0.6
(S)-43b		Cl	33 ± 5.8	31 ± 5.3	33 ± 7.8	21 ± 0.8	41 ± 1.1	1.2	1.3
44a		Br	79 ± 17.6	69 ± 2.0	87 ± 9.9	83 ± 28.3	79 ± 17.6	1.0	1.1
44b		Br	25 ± 6.4	15 ± 0.2	30 ± 0.7	32 ± 3.2	27 ± 11.9	1.1	1.8
45a		CF3	0.5 ± 0.1	1 ± 0.7	26 ± 1.2	28 ± 0.5	20 ± 4.7	40.0	20.0
45b		CF3	21 ± 1.6	13 ± 2.2	19 ± 7.5	32 ± 16.1	12 ± 3.1	0.6	0.9
(R)-45b		CF3	43 ± 4.6	49 ± 8.7	45 ± 4.0	38 ± 7.6	21 ± 3.8	0.5	0.4
(S)-45b		CF3	26 ± 8.5	43 ± 10.8	31 ± 2.2	23 ± 2.3	43 ± 3.9	1.7	1.0
5-FU			1 ± 0.8	0.1 ± 0.1	0.2 ± 0.0	9 ± 1.3	0.9 ± 0.0	0.9	9.0

^a IC₅₀—compound concentration that inhibited cell growth by 50%. Data represent mean IC₅₀ (μM) values ± standard deviation (SD) of three independent experiments, ^b 4-chloropyrrolo[2,3-d]pyrimidine analogs of 42, 43, and 45 without linker exhibited IC₅₀ values of 48.37 μM, 47.69 μM, and 46.55 μM, respectively (CFPAC-1). ^c Selectivity index towards human breast adenocarcinoma cells (MCF-7), SI = [IC₅₀ HFF]/[IC₅₀ MCF-7] and towards ductal pancreatic adenocarcinoma cells (CFPAC-1), SI = [IC₅₀ HFF]/[IC₅₀ CFPAC-1].

Table 5.

The growth inhibition effects of racemic 4-chloro-1H-imidazo[4,5-c]pyridine derivatives (46a,b–49a,b) on MCF-7, CFPAC-1, HT-29, and HepG2, as well as on normal skin fibroblasts (HFF-1).

			IC50 a (µM)
Compd	Linker	R2	MCF-7	CFPAC-1	HT-29	HepG2	HFF-1	SI (HepG2) b
46a		H	>100	>100	>100	>100	>100	-
46b		H	>100	>100	>100	>100	>100	-
47a		F	63 ± 8.4	51 ± 6.5	>100	>100	75 ± 14.2	0.8
47b		F	21 ± 2.6	18 ± 0.8	65 ± 12.6	55 ± 8.8	18 ± 2.9	0.3
48a		Cl	96 ± 1.8	>100	>100	>100	>100	-
48b		Cl	>100	>100	>100	>100	>100	-
49a		Br	77 ± 3.0	77 ± 0.5	77 ± 1.1	66 ± 22.8	90 ± 4.04	1.4
49b		Br	46 ± 0.0	21 ± 0.7	10 ± 3.2	4 ± 0.0	21 ± 5.8	5.3
5-FU			1 ± 0.8	0.1 ± 0.1	0.2 ± 0.0	9 ± 1.3	0.9 ± 0.0	0.1

^a IC₅₀—compound concentration that inhibited cell growth by 50%. Data represent mean IC₅₀ (μM) values ± standard deviation (SD) of three independent experiments. ^b Selectivity index towards hepatocellular carcinoma (HepG2), SI = [IC₅₀ HFF]/[IC₅₀ HepG2].

From indoles and benzimidazoles, the best growth inhibition was observed in CFPAC-1 cells, while from 6-chloropurines and 4-chloro-1H-imidazo[4,5-c]pyridines, in HepG2 cells. Among 4-chloropyrrolo[2,3-d]pyrimidines, the most pronounced inhibition was found in MCF-7 and CFPAC-1 cells.

In addition, N-heterocyclic derivatives with the halogen-substituted aromatic unit had better inhibitory activity than their unsubstituted analogs.

From indoles, purines, and pyrrolo[2,3-d]pyrimidines, activities decreased in the following order: CF₃ > Br > Cl > F. For instance, indole 30a, purine 40a, and pyrrolo[2,3-d]pyrimidine 45a with 4-(trifluoromethyl)phenyl moiety showed the highest inhibitory effect (30a: IC₅₀ = 9 µM on CFPAC-1; 40a: IC₅₀ = 0.69 µM on HT-29, IC₅₀ = 0.64 µM on HepG2; and 45a: IC₅₀ = 0.5 µM on MCF-7 cell line). These compounds also contain the primary hydroxyl group in a 2-hydroxyeth-1-yl spacer.

They exhibited toxicity to normal skin fibroblasts (HFF-1) with selectivity indexes (SI) ranging from 4 to 40. The best selectivity compared to a non-tumor cell line was observed for pyrrolo[2,3-d]pyrimidine 45a, with a SI of 40. Pyrrolo[2,3-d]pyrimidine derivatives (43a and 45a) showed the best inhibitory activity on the CFPAC-1 cell line, with IC₅₀ values ranging from 1 to 8 µM. Non-substituted aryl indoles (26a and 26b), benzimidazoles (31a and 31b), and imidazo[4,5-c]pyridines (46a and 46b) were deprived of any activity.

Comparing the influence of the hydroxyethyl linker and enantiomers of 6-chloropurines and 4-chloropyrrolo[2,3-d]pyrimidines on activity, we may conclude that the linker with the primary hydroxyl group in regioisomers of the 26a–45a series had a generally higher growth-inhibiting effect on the tested cell lines than the linker with the secondary hydroxyl group in the 26b–45b series, and that the S-enantiomers were more active than the corresponding R-enantiomers. Although the difference in antitumor activity was not pronounced, the S-enantiomers were twofold less cytotoxic to normal fibroblasts (HFF-1) than the corresponding R-enantiomers.

To better understand the observed SAR, the p-bromophenyl-substituted derivatives with the primary hydroxyl group—29a, 34a, 39a, 44a, and 49a—were aligned on the minimum energy conformation of compound 40a, and the molecular fields calculated by the Cresset software [55] were compared (Figure 3). All compounds were well aligned on the minimum energy conformation of compound 40a, indicating that the observed potency differences came from differences in the distribution of the electrostatic potential. The combination of the lower lipophilicity and larger negative electrostatic potential of the 6-chloropurine scaffold compared to the other heterocycles resulted in its higher potency. In addition, the increased lipophilic potential of the trifluoromethyl-phenyl substitution also contributed to the better potency profile when compared to the bromo analogs, and resulted in the most active compound 40a.

Figure 3.

Electrostatic potential calculated from molecular fields for p-bromophenyl-substituted derivatives from explored subseries aligned on the minimum energy conformation of the p-(trifluoromethyl)phenyl-substituted 6-chloropurine 40a.

Due to a larger number of rotational bonds, conformational space is large, with over 100 conformations within 3 kcal/mol, as demonstrated in Figure 4 for the most active compound 40a on HT-29 and HepG2 cells. The most stable is trans-conformation, while the least stable conformations adopt a cis-geometry of left- and right-hand side aromatic moieties with respect to the central triazole.

Figure 4.

Conformational analysis of compound 40a: left—conformational distribution, middle—energy histogram of generated conformations, and right—minimum energy conformation.

3.2.2. Physico-Chemical and ADME Properties

The calculated structural (logP, TPSA, HBD, and HBA) and PhysChem parameters (solubility, %PPB, and probability of CYP3A4 inhibition), as well as the measured permeability and metabolic stability (clearance values, CL) are shown in Table 6. All compounds are in the good lipophilicity range and fit within the Lipinski rule of 5. The predicted solubility varies from very soluble to insoluble, as a balance between lipophilicity and polarity, additionally influenced by the number of heteroatoms in the bicyclic moiety, the primary or secondary alcohol, and the substituent on the right-hand side phenyl. The predicted plasma protein binding is in the range of 91.5–99.5, and although not necessarily very accurate in absolute value predictions, gives quite a good understanding of the relative free fraction within the studied series. Most significant cytochrome P450 (CYP3A4) inhibition is flagged for pyrrolo[2,3-d]pyrimidine derivatives 44a,b and 45a,b, while compounds 39a,b, 40a,b,c, and 49a,b are predicted as possible CYP3A4 inhibitors (Table 6). This should be further de-risked for future, more advanced compounds.

Table 6.

Calculated structural and PhysChem parameters, as well as measured permeability and metabolic stability of representative compounds.

Compd	LogP	TPSA a	HBD b	HBA c	Solubility pH = 7.4 Water	%PPB d	CYP3A4 Inhibition e	Clearance Values (CL) f	Clearance Class.	Papp (AB) [×10−6 cm/s]	Papp (BA) [×10−6 cm/s]	Efflux Ratio g	Permeability
29a	3.11	55.87	1	5	highly insoluble	98.66	0.35	43	MODERATE	10.0	25.5	2.6	HIGH
29b	3.16	55.87	1	5	insoluble	97.32	0.41	92	HIGH	18.3	28.6	1.7	HIGH
34a	2.25	68.76	1	6	soluble	98.7	0.40	<30	LOW	2.0	65.7	32.8	LOW
34b	2.44	68.76	1	6	soluble	95.59	0.42	48	MODERATE	4.3	69.4	17.2	MODERATE
39a	1.85	94.54	1	8	very soluble	95.74	0.59	<30	LOW	0.9	58.0	68.1	LOW
39b	1.78	94.54	1	8	soluble	91.26	0.74	<30	LOW	3.1	69.6	22.5	MODERATE
40a	1.7	94.54	1	8	insoluble	98.4	0.72	<30	LOW	2.1	60.6	29.5	MODERATE
(R)-40b	1.79	94.54	1	8	soluble	96.98	0.68	<30	LOW	2.2	45.2	21.0	MODERATE
(S)-40b	1.79	94.54	1	8	soluble	96.98	0.68	<30	LOW	2.2	42.9	19.5	MODERATE
44a	2.4	81.65	1	7	insoluble	98.46	0.77	56	MODERATE	10.9	67.4	6.2	HIGH
44b	2.53	81.65	1	7	insoluble	97.35	0.81	84	HIGH	25.3	62.7	2.5	HIGH
45a	2.26	81.65	1	7	insoluble	99.4	0.76	54	MODERATE	7.6	66.4	8.8	MODERATE
45b	2.53	81.65	1	7	insoluble	98.84	0.75	90	HIGH	26.5	90.7	3.4	HIGH
49a	2.55	81.65	1	7	very soluble	98.04	0.58	<30	LOW	1.0	41.1	41.4	LOW
49b	2.54	81.65	1	7	soluble	96.6	0.66	<30	LOW	1.6	36.9	23.3	LOW

^a TPSA—total polar surface area; ^b HBD—hydrogen bond donor; ^c HBA—hydrogen bond acceptor; ^d PPB—plasma protein binding; ^e probability of CYP3A4 inhibition less than IC₅₀ < 10 µM; ^f predicted in vivo hepatic clearance; CL: <30% low, 30–70% moderate, and >70% high. ^g Efflux ratio: <2 no efflux, >2 active efflux.

Purine and imidazo[4,5-c]pyridine analogs showed high metabolic stability due to decreased potential for aromatic hydroxylation, while indole and pyrrolo[2,3-d]pyrimidine derivatives exhibited low metabolic stability. Compounds with secondary alcohols were metabolically less stable than compounds with primary alcohols, indicating a higher contribution of N-dealkylation in comparison with O-dealkylation. Indole and pyrrolo[2,3-d]pyrimidine derivatives had good permeability, while benzimidazole, 6-chloropurine, and imidazo[4,5-c]pyridine analogs had low permeability, due to decreased lipophilicity.

Selected 6-chloropurine (40a), 4-chloropyrrolo[2,3-d]pyrimidine (45a), and 4-chloroimidazo[4,5-c]pyridine (49b) with significant antiproliferative activity were examined for hydrolytic stability in an aqueous buffer solution. No significant changes were observed in their UV/Vis spectra over 24 h, indicating that these compounds remain hydrolytically stable under the tested conditions (Supporting Information S49).

Permeability is also influenced by a high efflux ratio for the majority of compounds. Compounds 29b, 44b, and 45b had the highest permeability and low efflux, but also had high metabolic clearance, while compounds 39a, 39b, 40a, and 40b with low clearance demonstrated low permeability and higher efflux values, as shown in Figure 5.

Figure 5.

Graphical presentation of measured ADME parameters.

4. Discussion

Enantiomers of 6-chloropurine and 4-chloropyrrolo[2,3-d]pyrimidine with hydroxyethyl linker were obtained via the biocatalytic ring-opening of epoxides with halohydrin dehalogenase to afford optically pure ß-substituted azido alcohols that subsequently, with N-propargylated 6-chloropurine and 4-chloropyrrolo[2,3-d]pyrimidine, gave the corresponding (R)- and (S)-N-aryl-substituted derivatives (R)-36b–(R)-38b, (R)-40b (R)-41b–(R)-43b, and (R)-45b and (S)-36b–(S)-38b, (S)-40b, (S)-41b–(S)-43b, and (S)-45b, respectively. Ultrasound-assisted synthesis and the catalytic asymmetric route using halohydrin dehalogenase contribute to a more sustainable synthetic approach to obtaining the target compounds.

Comparing the influence of the hydroxyethyl linker between 1,2,3-triazole and the aromatic moieties on antiproliferative activity, we may conclude that the linker with the primary hydroxyl group in the regioisomers of the 26a–45a series had a generally higher growth-inhibiting effect on the tested cell lines than the linker with the secondary hydroxyl group in the 26b–45b series (Figure 6).

Figure 6.

Insight into structure–antiproliferative activity relationship of the racemic and enantioenriched aryl-substituted N-heterocycles.

The only exceptions are imidazo[4,5-c]pyridine derivatives, among which compounds 47b–49b, with a 2-hydroxyeth-2-yl spacer, showed better activity than their analogs 47a–49a, with a 2-hydroxyeth-1-yl spacer. Overall, regioisomers 26a–45a, with the primary hydroxyl group, also exhibited a somewhat lower toxicity towards normal fibroblasts (HHF-1) than the regioisomers of the 26b–45b series. As mentioned before, from a series of regioisomers with a 2-hydroxyeth-2-yl spacer, benzimidazole 34b and imidazo[4,5-c]pyridine 49b derivatives with 4-bromophenyl moiety displayed the highest inhibitory effect. A comparison of the antiproliferative activity of enantiomeric-enriched 6-chloropurines and 4-chloropyrrolo[2,3-d]pyrimidines showed that S-enantiomers were more active than the corresponding R-enantiomers.

Based on the previous antiproliferative results on purine isosteres with an aryl moiety directly linked to 1,2,3-triazole [43], it can be observed that the introduction of a spacer, especially 2-hydroxyeth-1-yl, between aryl and 1,2,3-triazole, generally improves the activity and reduces the cytotoxic effect on non-cancerous cells. The difference in growth inhibition is more pronounced for indoles, benzimidazoles, and 4-chloropyrrolo[2,3-d]pyrimidines than for 6-chloropurines. Similarly to the 6-chloropurine 40a and the 4-chloropyrrolo[2,3-d]pyrimidine 45a with a 2-hydroxyeth-1-yl spacer, which showed the best antiproliferative activity (40a: IC₅₀ = 0.69 µM on HT-29, IC₅₀ = 7.9 µM on HepG2; 45a: IC₅₀ = 0.5 µM on MCF-7; and 40a: IC₅₀ = 1 µM on CFPAC-1), a structural analog with p-(trifluoromethyl)phenyl directly connected to 1,2,3-triazole exhibited the best activity (IC₅₀ = 7.9 µM on CFPAC-1), albeit high toxicity to normal cells (IC₅₀ = 0.75 µM) [43]. Conversely, 40a and 45b were found to be less toxic to normal cells than analogs, with aryl directly connected to 1,2,3-trazole, with SIs between 20 and 40.

Evaluation of the ADME properties showed that metabolic stability ranges from high for purine and imidazo[4,5-c]pyridine analogs to low for indole and pyrrolo[2,3-d]pyrimidine derivatives. Compounds with secondary alcohols are metabolically less stable than compounds with primary alcohols. Indole and pyrrolo[2,3-d]pyrimidine derivatives have good permeability, while benzimidazole 34a, 6-chloropurine 39a, and imidazo[4,5-c]pyridines 49a and 49b have low permeability (Papp (AB) ≤ 2 × 10⁻⁶ cm/s). All compounds are P-glycoprotein (Pgp) substrates, with the exception of compound 29b.

5. Conclusions

A new series of aryl-substituted purine bioisosteres containing a linker with the primary (26a–49a) and secondary hydroxyl groups (26b–49b) was synthesized via the ultrasound-assisted Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of terminal alkynyl N-heterocyclic derivatives and aryl-substituted 1,2-azido alcohols. The results of antiproliferative profiling showed that among all purine and purine isosteres, purine and 4-chloropyrrolo[2,3-d]pyrimidine derivatives with halogen-substituted aromatic residue had better inhibitory activity compared to indole, benzimidazole, and 4-chloroimidazo[4,5-c]pyridine derivatives and their unsubstituted analogs. In addition, the introduction of a spacer between aryl and 1,2,3-triazole generally improved the growth-inhibiting activity, showing that the linker with the primary hydroxyl group had a higher cytostatic effect and lower toxicity on normal fibroblasts (HHF-1) than the linker with the secondary hydroxyl group. S-enantiomers of 6-chloropurines and 4-chloropyrrolo[2,3-d]pyrimidines were more active than the corresponding R-enantiomers. We can conclude that purine 40a and 4-chloropyrrolo[2,3-d]pyrimidine 45a, both with 4-(trifluoromethyl)phenyl moiety and the primary hydroxyl group, showed the most significant inhibitory effect (40a: IC₅₀ = 0.69 µM on HT-29, IC₅₀ = 0.64 µM on HepG2; 45a: IC₅₀ = 0.5 µM on MCF-7 cell line). The best selectivity compared to the non-tumor cell line was observed for 4-chloropyrrolo[2,3-d]pyrimidine 45a. Compound 45a has moderate clearance and good permeability, which makes it quite interesting in combination with promising bioactivity and the selectivity profile. Further studies are required to elucidate the mechanism of action of 45a as a promising candidate. However, the active efflux has a negative influence on the cellular concentration, indicating a further need for the multi-parametric optimization of the PhysChem and ADME properties.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biom15030351/s1. Figures S1–S49: ¹H, ¹³C NMR, and UV-Vis spectra of novel compounds.

Author Contributions

Formal analysis, M.P., A.M., S.K., M.M.E., P.G., S.K.P., and K.P.; investigation, A.M., M.M.E., P.G., S.K.P., and K.P.; methodology, M.P., S.K., and M.M.E.; project administration, S.R.-M.; supervision, S.R.-M.; writing—original draft, M.P., S.K., and S.R.-M.; writing—review and editing, S.R.-M. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data that support the findings of this study are available in the Supporting Information of this article.

Conflicts of Interest

Although the authors Sanja Koštrun and Astrid Milić are employees of Selvita d.o.o., they declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. The remaining authors also declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Funding Statement

This work was supported by the Croatian Science Foundation under the project number (HRZZ-IP-2022-10-9420).