Design and synthesis of novel pyrazolo[4,3-d]pyrimidines as potential therapeutic agents for acute lung injury

Abstract

Four series of total 35 new pyrazolo[4,3-d]pyrimidine compounds were designed, synthesized and evaluated for their inhibitory activity against LPS-induced NO production in RAW264.7 macrophages. Among them, compound 4e was found to be the most potent inhibitor, which decreased the production of cytokines in vitro, such as NO, IL-6 and TNF-α, with IC₅₀ values of 2.64, 4.38 and 5.63 μM, respectively. Further studies showed that compound 4e inhibited cytokines secretion of macrophages through suppressing TLR4/p38 signaling pathway. Additionally, compound 4e showed in vivo anti-inflammatory activity in LPS-induced model of acute lung injury. These data suggested that compound 4e may be a promising lead structure for the treatment of ALI.

1. Introduction

Acute lung injury (ALI), characterized by increased permeability of endothelium and epithelium as well as loss of vascular integrity, is an acute inflammatory disease with high morbidity and mortality^1,2. ALI is directly or indirectly caused by pneumonia, inhalation injury, drowning and so forth^2–4, which clinical manifestations include pulmonary edema, dyspnea, hypoxemia^5–7. Many therapies for ALI have been conducted, but effective therapeutic agents were not discovered up to now⁸. Recently, several studies have shown that various inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), play a pivotal role in the development and progrossion of ALI^9–11. Increasing evidences showed that suppressing the over-secretion of inflammatory cytokines have been emeraged as a promising strategy for the treatment of ALI^12,13.

Pyrazolopyrimidine moiety is an important drug-like scaffold¹⁴, which have shown a wide range of clinical applications including bruton's tyrosine kinase inhibitor ibrutinib (I)¹⁵, tumor necrosis factor receptor-associated protein 1 (TRAP1) inhibitor (II)¹⁶, cyclin-dependent kinase (CDK) inhibitors (III, IV)^17,18, anti-inflammation (V)¹⁹ and bumped kinase inhibitors (VI, Figure 1)²⁰.

Figure 1.

The structures of several pyrazolopyrimidines.

In our previous study, a series of pyrazole-pyrimidine derivatives were synthesized for antitumor evalution²¹. Further studies, some analogs exhibited anti-inflammatory activity in RAW 264.7 macrophage cells. In order to find potent anti-inflammatory agents, the scaffold was further modified and evaluated for their inhibitory effect against LPS-induced NO production in RAW264.7 macrophages (Figure 2).

Figure 2.

Summary of target derivatives.

2. Experimental section

2.1. Chemistry

Commercial reagents were used without further purification. Thin layer chromatography (TLC) was carried out on pre-coated silica GF254 plates with visualization by UV light at 254 nm in the appropriate solvents system for all reactions. Unless noted otherwise, the purification of all compounds was processed by silica gel column chromatography. Melting points were determined on a XT4MP apparatus (Taike Corp., Beijing, China) without correction. ¹H and ¹³C NMR spectra were recorded on a Bruker AM-300 (¹H, 300 MHz; ¹³C, 75 MHz) or Agilent DD2 600 MHz (¹H, 600 MHz; ¹³C, 151 MHz). The ¹H and ¹³C spectra were recorded in CDCl₃ or DMSO-d₆ using tetramethylsilane (TMS) as the internal standard. High-resolution electron impact mass spectra (HR-MS) were recorded on a Micro Mass GCT CA 055 instrument under 70 eV electron impact.

2.2. General procedures for the synthesis of title compounds 1a–1j, 2a–2g, 3a–3g and 4a–4k

A mixture of intermediate C1 (1.88 g, 5 mmol) and 4-aminophenol (0.545 g, 5 mmol) were dissolved in isopropanol (IPA) (20 ml). The reaction mixture was refluxed for 6–10 h monitored by TLC. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (gradient elution of PE/EtOAc 85/15 then 80/20 v/v) to obtain compound 1a.

Compounds 1 b–1j, 2a–2g, 3a–3g and 4a–4k were obtained using the same method (Scheme 1).

Scheme 1.

Synthesis of pyrazolo[4,3-d]pyrimidine derivatives 1a∼1j, 2a∼2g, 3a∼3g and 4a∼4k^a. ^aReaction conditions and reagents: (1) substituted carboxylic acid, EDCI, HOBt, TEA, rt, stirred for 18 h; (2) NaOEt, EtOH, reflux, 8–10 h; (3) POCl₃, reflux, 8 h; (4) amine derivatives, IPA, reflux.

4–(1-Methyl-3-propyl-5–(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ylamino)phenol(1a). Title compound 1a was isolated as a white powder in 79.2% yield (1.78 g, 3.96 mmol), mp 241 – 242 °C. ¹HNMR (600 MHz, DMSO-d₆): δ 9.34 (d, J = 3.3 Hz, 1H, NH), 8.70 (brs, 1H, OH), 7.65 (d, J = 2.1 Hz, 2H, ArH), 7.58 (d, J = 6.7 Hz, 2H, ArH), 6.84 (d, J = 6.6 Hz, 2H, ArH), 4.31 (d, J = 2.4 Hz, 3H, OCH₃), 3.84 (d, J = 2.1 Hz, 6H, 2 × OCH₃), 3.72 (d, J = 2.2 Hz, 3H, NCH₃), 2.90 (s, 2H, CH₂), 1.86 – 1.84 (m, 2H, CH₂), 0.99 (t, J = 7.2 Hz, 3H, CH₃). ¹³C NMR (151 MHz, DMSO-d₆): δ 155.0,154.1, 152.6, 147.6, 144.0, 143.1, 138.7, 133.9, 130.1, 125.1, 120.8, 114.6, 104.6, 60.1, 55.6, 39.2, 27.2, 21.4, 13.9. HR-MS (ESI): calcd for C₂₄H₂₈N₅O₄ [M + H]⁺, 450.2136; found 450.2136.

2–(1-Methyl-3-propyl-5–(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ylamino)phenol(1 b). Title compound 1 b was isolated as a white powder in 66.7% yield (1.50 g, 3.34 mmol), mp 207 – 208 °C.¹H NMR (600 MHz, CDCl₃ + DMSO-d₆): δ 7.95 (s, 1H, OH), 7.68 (s, 2H, ArH), 7.12 (t, J = 7.5 Hz, 1H, ArH), 7.07 (d, J = 8.0 Hz, 2H, ArH), 6.90 (t, J = 7.5 Hz, 1H, ArH), 4.43 (s, 3H, OCH₃), 3.90 (s, 6H, 2 × OCH₃), 3.83 (s, 3H, NCH₃), 3.13 (t, J = 7.2 Hz, 2H, CH₂), 1.89 – 1.80 (m, 2H, CH₂), 1.05 (t, J = 7.3 Hz, 3H, CH₃). HR-MS (ESI): calcd for C₂₄H₂₈N₅O₄ [M + H]⁺, 450.2136; found 450.2134.

3–(1-Methyl-3-propyl-5–(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ylamino)phenol(1c). Title compound 1c was isolated as a white powder in 72.0% yield (1.62 g, 3.6 mmol), mp 221 – 222 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 9.76 (s, 1H, OH), 7.74 (s, 2H, ArH), 7.31 (s, 1H, ArH), 7.25 (t, J = 8.0 Hz, 1H, ArH), 7.18 (d, J = 8.0 Hz, 1H, ArH), 6.71 (d, J = 8.0 Hz, 1H, ArH), 4.37 (s, 3H, OCH₃), 3.87(s, 6H, 2 × OCH₃), 3.74 (s, 3H, NCH₃), 3.08 (t, J = 7.5 Hz, 2H, CH₂), 1.84 – 1.75 (m, 2H, CH₂), 0.99 (t, J = 7.3 Hz, 3H, CH₃). ¹³C NMR (151 MHz, DMSO-d₆): δ 157.7, 154.0, 152.8, 148.3, 141.7, 140.3, 138.7, 129.1, 121.3, 114.8, 112.5, 111.3, 107.0, 106.2, 99.3, 60.2, 56.0, 27.4, 25.5, 21.9, 13.8. HR-MS (ESI): calcd for C₂₄H₂₈N₅O₄ [M + H]⁺, 450.2136; found 450.2137.

2–(1-Methyl-3-propyl-5–(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ylamino)benzenethiol(1d). Title compound 1d was isolated as a yellow solid in 47.4% yield (1.10 g, 2.37 mmol), mp 269 – 271 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 11.85 (s, 1H), 10.22 (s, 1H, SH), 9.17 (s, 1H, NH), 8.23 (d, J = 8.1 Hz, 1H, ArH), 8.15 (d, J = 8.2 Hz, 1H, ArH), 7.62 (t, J = 7.7 Hz, 1H, ArH), 7.53 (m, 3H, ArH), 4.34 (s, 3H, OCH₃), 3.96 (s, 6H, 2 × OCH₃), 3.81 (s, 3H, NCH₃), 2.57 (m, 2H, CH₂), 1.71 – 1.67(m, 2H, CH₂), 0.97 (t, J = 7.3 Hz, 3H, CH₃). ¹³C NMR (151 MHz, DMSO-d₆): δ 164.5, 154.1, 153.0, 152.1, 147.8, 142.3, 133.8, 132.0, 127.2, 126.3, 123.3, 122.6, 121.8, 114.7, 106.7, 60.4, 56.5, 40.6, 27.2, 21.4, 14.0. HR-MS (ESI): calcd for C₂₄H₂₈N₅O₃S [M + H]⁺, 466.1907; found 466.1910.

N1-(1-Methyl-3-propyl-5–(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)benzene-1,4-diamine(1e). Title compound 1e was isolated as a yellow solid in 78.4% yield (1.76 g, 3.92 mmol), mp240 – 241 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 10.42 (brs, 2H, NH₂), 9.51 (brs, 1H, NH), 7.92 (d, J = 7.9 Hz, 2H, ArH), 7.66 (s, 2H, ArH), 7.47 (d, J = 7.6 Hz, 2H, ArH), 4.36 (s, 3H, OCH₃), 3.85 (s, 6H, 2 × OCH₃), 3.73 (s, 3H, NCH₃), 2.98 (s, 2H, CH₂), 1.86 – 1.82 (m, 2H, CH₂), 0.99 (t, J = 7.3 Hz, 3H, CH₃). HR-MS (ESI): calcd for C₂₄H₂₉N₆O₃ [M + H]⁺, 449.2296; found 449.2302.

4–(2-(1-Methyl-3-propyl-5–(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-ylamino)ethyl)phenol(1f). Title compound 1f was isolated as a white solid in 64.7% yield (1.54 g, 3.24 mmol), mp 197 – 199 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 9.24 (s, 1H, OH), 7.77 (s, 2H), 7.44 (t, J = 5.6 Hz, 1H, NH), 7.13 (d, J = 8.3 Hz, 2H), 6.72 (d, J = 8.3 Hz, 2H), 4.18 (s, 3H), 3.88 (s, 6H), 3.85 – 3.76 (m, 2H), 3.74 (s, 3H), 2.99 – 2.90 (m, 2H), 2.87 (t, J = 7.4 Hz, 2H), 1.89 – 1.77 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆): δ 155.7, 155.4, 152.6, 149.2, 143.7, 142.1, 138.8, 134.4, 129.6, 129.4, 120.8, 115.1, 104.7, 60.1, 55.7, 42.4, 39.0, 33.9, 27.2, 21.4, 13.9. HR-MS (ESI): calcd for C₂₆H₃₂N₅O₄ [M + H]⁺, 478.2449; found 478.2452.

N-Isobutyl-1-methyl-3-propyl-5–(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(1 g). Title compound 1 g was isolated as a white solid in 88.3% yield (1.83 g, 4.41 mmol), mp 138 – 139 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.79 (s, 2H, ArH), 5.24 (t, J = 5.5 Hz, 1H, NH), 4.25 (s, 3H, OCH₃), 3.99 (s, 6H, 2 × OCH₃), 3.90 (s, 3H, NCH₃), 3.58 (t, J = 6.2 Hz, 2H, NCH₂), 2.98 (t, J = 7.6 Hz, 2H, CH₂), 2.19 (m, 1H, CH), 1.94 – 1.84 (m, 2H, CH₂), 1.07 – 1.02 (m, 9H, CH₃). HR-MS (ESI): calcd for C₂₂H₃₂N₅O₃ [M + H]⁺, 414.2500; found 414.2505.

1-Methyl-N-(3-morpholinopropyl)-3-propyl-5–(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(1 h). Title compound 1 h was isolated as a white solid in 68.0% yield (1.65 g, 3.40 mmol), mp 167 – 168 °C. ¹H NMR (600 MHz, CDCl₃) δ: 7.78 (s, 2H, ArH), 6.20 (s, 1H, NH), 4.27 (s, 3H, OCH₃), 3.99 (s, 6H, 2 × OCH₃), 3.90 (s, 3H, NCH₃), 3.84 (dd, J = 11.6, 5.9 Hz, 2H, CH₂), 3.77 – 3.68 (m, 4H, 2 × CH₂), 2.98 (t, J = 7.6 Hz, 2H, CH₂), 2.59 (dd, J = 12.6, 6.4 Hz, 2H, CH₂), 2.52 (s, 4H, 2 × CH₂), 2.02 – 1.96 (m, 2H, CH₂), 1.93 – 1.88 (m, 2H, CH₂), 1.04 (t, J = 7.4 Hz, 3H, CH₃). ¹³C NMR (151 MHz, CDCl₃): δ 156.9, 153.1, 149.7, 146.0, 143.3, 139.5, 134.9, 121.3, 105.3, 66.7, 61.0, 58.4, 56.2, 54.2, 41.1, 39.4, 27.9, 24.6, 22.2, 14.2. HR-MS (ESI): calcd for C₂₅H₃₇N₆O₄ [M + H]⁺, 485.2871; found 485.2873.

1–(1-Methyl-3-propyl-5–(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperidin-4-ol(1i). Title compound 1i was isolated as a white powder in 55.3% yield (1.22 g, 2.77 mmol), mp 143 – 144 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.73 (s, 2H, ArH), 4.84 (s, 1H, OH), 4.07 (s, 3H, OCH₃), 3.88 (m, 8H, 2 × OCH₃ + CH₂), 3.81 (s, 1H, OCH), 3.74 (s, 3H, NCH₃), 3.30 (s, 2H, CH₂), 2.91 (d, J = 3.1 Hz, 2H, CH₂), 1.95 (s, 2H, CH₂), 1.85 (s, 2H, NCH₂), 1.63 (d, J = 8.2 Hz, 2H, NCH₂), 1.02 – 0.96 (m, 3H, CH₃). ¹³C NMR (151 MHz, DMSO-d₆): δ 154.9, 153.4, 152.8, 145.3, 144.2, 139.1, 133.7, 123.6, 104.8, 65.6, 60.1, 55.8, 46.7, 38.7, 33.7, 27.3, 21.3, 14.0. HR-MS (ESI): calcd for C₂₃H₃₂N₅O₄ [M + H]⁺, 442.2449; found 442.2447.

1-Methyl-7–(4-methylpiperazin-1-yl)-3-propyl-5–(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidine(1j). Title compound 1j was isolated as a white solid in 82.9% yield (1.83 g, 4.15 mmol), mp 116 – 117 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.79 (s, 2H, ArH), 4.11 (s, 3H, OCH₃), 3.99 (s, 6H, 2 × OCH₃), 3.91 (s, 3H, NCH₃), 3.65 (s, 4H, 2 × NCH₂), 3.03 (t, J = 7.6 Hz, 2H, CH₂), 2.69 (s, 4H, 2 × NCH₂), 2.41 (s, 3H, NCH₃), 1.94 – 1.91 (m, 2H, CH₂), 1.06 (t, J = 7.4 Hz, 3H, CH₃). ¹³C NMR (151 MHz, CDCl₃): δ 156.4, 153.9, 153.2, 147.6, 145.6, 139.8, 134.3, 124.5, 105.4, 61.0, 56.3, 54.6, 49.4, 46.3, 38.6, 28.0, 22.2, 14.2. HR-MS (ESI): calcd for C₂₃H₃₃N₆O₃ [M + H]⁺, 441.2609; found 441.2613.

(E)-N1-(1-Methyl-3-propyl-5-styryl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)benzene-1,4-diamine(2a). Title compound 2a was isolated as a white solid in 54.6% yield (1.05 g, 2.73 mmol), mp 163 – 164 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 9.40 (s, 2H), 7.90 – 7.78 (m, 2H, ArH), 7.73 – 7.58 (m, 3H, ArH), 7.46 – 7.26 (m, 6H, ArH), 4.34 (s, 3H, NCH₃), 2.94 – 2.87 (m, 2H, CH₂), 1.86 – 1.69 (m, 2H, CH₂), 1.02 – 0.92 (m, 3H, CH₃). HR-MS (ESI): calcd for C₂₃H₂₅N₆ [M + H]⁺, 385.2135; found 385.2131.

(E)-4–(2-(1-Methyl-3-propyl-5-styryl-1H-pyrazolo[4,3-d]pyrimidin-7-ylamino)ethyl)phenol(2 b). Title compound 2 b was isolated as a white solid in 73.2% yield (1.51 g, 3.66 mmol), mp 163 – 164 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 9.24 (s, 1H), 7.81 (d, J = 15.9 Hz, 1H), 7.67 (d, J = 7.3 Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.0 Hz, 2H), 7.16 (dd, J = 12.1, 3.7 Hz, 3H), 6.75 (d, J = 8.3 Hz, 2H), 4.16 (s, 3H), 3.76 (dd, J = 14.7, 5.8 Hz, 2H), 2.99 – 2.86 (m, 2H), 2.80 (t, J = 7.5 Hz, 2H), 1.83 – 1.70 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 156.6, 155.7, 149.0, 143.8, 141.9, 136.3, 134.2, 129.7, 129.6, 129.3, 128.9, 128.3, 127.1, 120.8, 115.2, 42.6, 39.0, 33.87, 27.3, 21.7, 13.9. HR-MS (ESI): calcd for C₂₅H₂₈N₅O [M + H]⁺, 414.2288; found 414.2291.

(E)-N-(4-Bromophenethyl)-1-methyl-3-propyl-5-styryl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(2c). Title compound 2c was isolated as a yellow solid in 81.1% yield (1.93 g, 4.06 mmol), mp 209 – 210 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.88 (d, J = 15.8 Hz, 1H), 7.62 (d, J = 7.5 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 7.38 (t, J = 7.6 Hz, 2H), 7.30 (t, J = 7.3 Hz, 1H), 7.23 (d, J = 15.8 Hz, 1H), 7.18 (d, J = 8.1 Hz, 2H), 5.05 (s, 1H), 4.07 (s, 3H), 3.98 (dd, J = 12.7, 6.7 Hz, 2H), 3.06 (t, J = 6.9 Hz, 2H), 2.94 (t, J = 7.7 Hz, 2H), 1.90 – 1.82 (m, 2H), 1.03 (t, J = 7.3 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 157.7, 149.0, 145.7, 143.1, 137.9, 136.8, 135.4, 131.9, 130.6, 129.0, 128.7, 128.2, 127.3, 121.0, 120.6, 41.8, 38.9, 34.7, 27.7, 22.3, 14.1. HR-MS (ESI): calcd for C₂₅H₂₇N₅Br [M + H]⁺, 476.1444; found 476.1448.

(E)-1-Methyl-N,3-dipropyl-5-styryl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(2d). Title compound 2d was isolated as a white solid in 84.3% yield (1.41 g, 4.22 mmol), mp 139 – 140 °C. ¹H NMR (600 MHz, CDCl₃ + DMSO-d₆): δ 9.03 (s, 1H, NH), 8.04 (d, J = 15.6 Hz, 1H, ArH), 7.72 (d, J = 15.6 Hz, 1H, ArH), 7.67 (d, J = 3.8 Hz, 2H, ArH), 7.43 (s, 3H, ArH), 4.42 (s, 3H, NCH₃), 3.86 (dd, J = 13.4, 6.6 Hz, 2H, NCH₂), 3.07 (t, J = 7.5 Hz, 2H, CH₂), 1.89 (dt, J = 14.6, 7.3 Hz, 2H, CH₂), 1.86 – 1.77 (m, 2H, CH₂), 1.08 (t, J = 7.4 Hz, 3H, CH₃), 1.04 (t, J = 7.3 Hz, 3H, CH₃). ¹³C NMR (151 MHz, CDCl₃ + DMSO-d₆): δ 152.8, 149.8, 142.4, 139.5, 133.8, 129.8, 128.3, 128.3, 127.7, 120.3, 118.2, 43.1, 39.6, 27.0, 21.7, 21.4, 13.1, 11.0. HR-MS (ESI): calcd for C₂₀H₂₆N₅ [M + H]⁺, 336.2183; found 336.2184.

(E)-N-Butyl-1-methyl-3-propyl-5-styryl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(2e). Title compound 2e was isolated as a white solid in 91.9% yield (1.61 g, 4.60 mmol), mp 156 – 157 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.71 (brs, 1H, NH), 8.06 (d, J = 15.7 Hz, 1H, =CH), 7.70 (d, J = 6.6 Hz, 2H, ArH), 7.59 – 7.42 (m, 4H, ArH), 4.28 (s, 3H, NCH₃), 3.81 (dd, J = 13.2, 6.4 Hz, 2H, CH₂), 2.92 (t, J = 7.5 Hz, 2H, CH₂), 1.76 – 1.67 (m, 4H, 2 × CH₂), 1.50 – 1.42 (m, 2H, CH₂), 1.01 – 0.93 (m, 6H, 2 × CH₃). HR-MS (ESI): calcd for C₂₁H₂₈N₅ [M + H]⁺, 350.2339; found 350.2338.

(E)-1-Methyl-7–(4-methylpiperazin-1-yl)-3-propyl-5-styryl-1H-pyrazolo[4,3-d]pyrimidine(2f). Title compound 2f was isolated as a white solid in 56.8% yield (1.07 g, 2.84 mmol), mp 156 – 157 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 11.86 (brs, 1H), 8.02 (d, J = 15.8 Hz, 1H), 7.74 (d, J = 7.1 Hz, 2H), 7.47 (dt, J = 17.9, 9.1 Hz, 4H), 4.42 (d, J = 13.1 Hz, 2H), 4.14 (s, 3H), 3.79 (t, 2H), 3.55 (d, J = 12.0 Hz, 2H), 3.30 (d, J = 9.7 Hz, 2H), 2.96 (t, J = 7.4 Hz, 2H), 2.83 (s, 3H), 1.80 – 1.73 (m, 2H), 0.98 (t, J = 7.3 Hz, 1H). HR-MS (ESI): calcd for C₂₂H₂₉N₆ [M + H]⁺, 377.2448; found 377.2447.

(E)-N-(2-Fluorobenzyl)-1-methyl-3-propyl-5-styryl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(2 g). Title compound 2 g was isolated as a yellow solid in 77.9% yield (1.56 g, 3.90 mmol), mp 175 – 176 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.88 (d, J = 15.8 Hz, 1H), 7.62 (d, J = 7.7 Hz, 2H), 7.55 (t, J = 7.5 Hz, 1H), 7.38 (t, J = 7.6 Hz, 2H), 7.29 (dd, J = 12.1, 6.3 Hz, 2H), 7.22 (d, J = 15.8 Hz, 1H), 7.16 – 7.08 (m, 2H), 5.52 (s, 1H, NH), 5.01 (d, J = 5.7 Hz, 2H, CH₂), 4.22 (s, 3H), 2.94 (t, J = 7.7 Hz, 2H), 1.87 – 1.83 (m, 2H), 1.02 (t, J = 7.3 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 161.4 (d, J = 245.5 Hz), 157.6, 149.0, 145.7, 143.2, 136.9, 135.5, 130.6 (d, J = 4.4 Hz), 129.5 (d, J = 8.3 Hz), 128.9, 128.6, 128.2, 127.3, 125.5 (d, J = 14.2 Hz), 124.4 (d, J = 3.5 Hz), 121.1, 115.5 (d, J = 21.4 Hz), 39.0, 39.0, 27.7, 22.2, 14.0. HR-MS (ESI): calcd for C₂₄H₂₅N₅F [M + H]⁺, 402.2089; found 402.2084.

(E)-N-(4-Bromophenethyl)-5–(2-(furan-2-yl)vinyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(3a). Title compound 3a was isolated as a yellow solid in 66.6% yield (1.55 g, 3.33 mmol), mp 128 – 129 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.65 (d, J = 15.7 Hz, 1H), 7.47 (d, J = 8.2 Hz, 2H), 7.45 (s, 1H), 7.15 (d, J = 8.2 Hz, 2H), 7.12 (d, J = 15.7 Hz, 1H), 6.50 (d, J = 3.2 Hz, 1H), 6.45 (dd, J = 3.0, 1.7 Hz, 1H), 5.03 (s, 1H), 4.05 (s, 3H), 3.94 (q, J = 6.7 Hz, 2H), 3.03 (t, J = 6.9 Hz, 2H), 2.95 – 2.90 (m, 2H), 1.86 – 1.82(m, 2H), 1.01 (t, J = 7.3 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃); δ 157.5, 153.2, 149.0, 145.7, 143.1, 142.9, 138.0, 131.8, 130.5, 127.3, 122.9, 121.0, 120.6, 111.7, 110.1, 41.8, 38.8, 34.7, 27.7, 22.2, 14.0. HR-MS (ESI): calcd for C₂₃H₂₅BrN₅O [M + H]⁺, 466.1237; found 466.1238.

(E)-N-Butyl-5–(2-(furan-2-yl)vinyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(3 b). Title compound 3 b was isolated as a yellow solid in 87.4% yield (1.48 g, 4.37 mmol), mp 101 – 102 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.61 (d, J = 15.7 Hz, 1H), 7.42 (s, 1H), 7.09 (d, J = 15.7 Hz, 1H), 6.47 (d, J = 3.2 Hz, 1H), 6.42 (dd, J = 3.1, 1.7 Hz, 1H), 5.07 (s, 1H), 4.18 (s, 3H), 3.68 (q, J = 7.0 Hz, 2H), 2.94 – 2.87 (m, 2H), 1.85 – 1.81 (m, 2H), 1.74 – 1.67 (m, 2H), 1.49 – 1.45(m, 2H), 1.00 (t, J = 7.3 Hz, 6H). ¹³C NMR (151 MHz, CDCl₃): δ 157.7, 153.4, 149.5, 145.6, 143.0, 142.9, 127.5, 122.9, 121.2, 111.8, 110.2, 40.7, 39.1, 31.6, 27.8, 22.3, 20.4, 14.2, 14.0. HR-MS (ESI): calcd for C₁₉H₂₆N₅O [M + H]⁺, 340.2132; found 340.2137.

(E)-N-sec-Butyl-5–(2-(furan-2-yl)vinyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(3c). Title compound 3c was isolated as a white solid in 89.4% yield (1.51 g, 4.47 mmol), mp 115 – 116 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.60 (d, J = 15.7 Hz, 1H), 7.43 (s, 1H), 7.10 (d, J = 15.7 Hz, 1H), 6.49 (d, J = 3.1 Hz, 1H), 6.43 (dd, J = 3.2, 1.7 Hz, 1H), 4.80 (d, J = 7.5 Hz, 1H), 4.52 – 4.45 (m, 1H), 4.21 (s, 3H), 2.95 – 2.90 (m, 2H), 1.88 – 1.82 (m, 2H), 1.77 – 1.63 (m, 2H), 1.34 (d, J = 6.5 Hz, 3H), 1.05 – 1.02 (m, 3H), 1.01 (t, J = 5.8 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 157.8, 153.4, 149.1, 145.7, 143.1, 142.9, 127.7, 122.9, 121.1, 111.8, 110.1, 47.8, 39.1, 29.7, 27.8, 22.4, 20.4, 14.2, 10.6. HR-MS (ESI): calcd for C₁₉H₂₆N₅O [M + H]⁺, 340.2132; found 340.2126.

(E)-4–(5–(2-(Furan-2-yl)vinyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)morpholine(3d). Title compound 3d was isolated as a white solid in 73.9% yield (1.31 g, 3.70 mmol), mp 108 – 109 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.63 (d, J = 15.7 Hz, 1H), 7.44 (s, 1H), 7.15 (d, J = 15.7 Hz, 1H), 6.50 (d, J = 3.2 Hz, 1H), 6.44 (dd, J = 3.2, 1.7 Hz, 1H), 4.08 (s, 3H), 3.94 – 3.90 (m, 4H), 3.57 – 3.54 (m, 4H), 2.96 (t, 2H), 1.89 – 1.85 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 157.0, 153.5, 153.0, 147.4, 145.4, 142.9, 126.8, 124.2, 123.1, 111.8, 110.4, 66.4, 49.9, 38.4, 27.8, 22.1, 14.0. HR-MS (ESI): calcd for C₁₉H₂₄N₅O₂ [M + H]⁺, 354.1925; found 354.1925.

(E)-5–(2-(Furan-2-yl)vinyl)-1-methyl-N-(3-morpholinopropyl)-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(3e). Title compound 3e was isolated as a white solid in 56.4% yield (1.16 g, 2.82 mmol), mp 98 – 99 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.62 (d, J = 15.7 Hz, 1H), 7.43 (s, 1H), 7.10 (d, J = 15.7 Hz, 1H), 6.48 (d, J = 3.1 Hz, 1H), 6.43 (dd, J = 3.1, 1.7 Hz, 1H), 6.07 (s, 1H), 4.24 (s, 3H), 3.80 (q, J = 6.1 Hz, 2H), 3.72 (t, J = 4.4 Hz, 4H), 2.94 – 2.91 (m, 2H), 2.57 (t, J = 6.2 Hz, 2H), 2.52 (s, 4H), 1.97 – 1.89 (m, 2H), 1.87 – 1.83 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 157.7, 153.4, 149.5, 145.8, 143.0, 142.9, 127.7, 122.8, 121.3, 111.9, 110.1, 66.8, 58.4, 54.2, 40.9, 39.3, 27.9 24.7 22.4 14.2. HR-MS (ESI): calcd for C₂₂H₃₁N₆O₂ [M + H]⁺, 411.2503; found 411.2510.

(E)-N-(2-Fluorobenzyl)-5–(2-(furan-2-yl)vinyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(3f). Title compound 3f was isolated as a white solid in 79.4% yield (1.55 g, 3.97 mmol), mp132 – 133 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 7.87 (t, J = 5.6 Hz, 1H, ArH), 7.73 (s, 1H, NH), 7.52 (t, J = 7.6 Hz, 1H, ArH), 7.43 (d, J = 15.8 Hz, 1H, =CH), 7.33 – 7.18 (m, 2H, ArH), 7.13 (t, J = 7.2 Hz, 1H, ArH), 6.79 (d, J = 15.8 Hz, 1H, =CH), 6.69 (d, J = 3.3 Hz, 1H, ArH), 6.60 – 6.54 (m, 1H, ArH), 4.84 (d, J = 5.5 Hz, 2H, CH₂), 4.24 (s, 3H, NCH₃), 2.78 (t, J = 7.5 Hz, 2H, CH₂), 1.78 – 1.71 (m, 2H, CH₂), 0.93 (t, J = 7.4 Hz, 3H, CH₃). HR-MS (ESI): calcd for C₂₂H₂₃FN₅O [M + H]⁺, 392.1881; found 392.1885.

(E)-5-(2-(Furan-2-yl)vinyl)-1-methyl-3-propyl-N-(pyridin-4-ylmethyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(3 g). Title compound 3 g was isolated as a yellow solid in 82.2% yield (1.54 g, 4.11 mmol), mp 117 – 118 °C. ¹H NMR (600 MHz, CDCl₃): δ 8.60 – 8.55 (m, 2H), 7.47 (d, J = 15.7 Hz, 1H), 7.41 (d, J = 1.7 Hz, 1H), 7.34 – 7.32 (m, 2H), 7.07 (d, J = 15.7 Hz, 1H), 6.44 (d, J = 3.3 Hz, 1H), 6.42 (dd, J = 3.3, 1.8 Hz, 1H), 5.56 (t, J = 5.8 Hz, 1H), 4.94 (d, J = 5.7 Hz, 2H), 4.24 (s, 3H), 2.93 (t, J = 7.7 Hz, 2H), 1.87 – 1.83 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 157.6, 153.1, 150.2, 149.0, 148.1, 146.0, 143.7, 143.1, 127.0, 123.2, 122.6, 121.0, 111.9, 110.5, 43.8, 39.2, 27.9, 22.4, 14.2. HR-MS (ESI): calcd for C₂₁H₂₃N₆O [M + H]⁺, 375.1928; found 375.1928.

(E)-N-(4-Bromophenethyl)-1-methyl-3-propyl-5–(3,4,5-trimethoxystyryl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(4a). Title compound 4a was isolated as a yellow solid in 87.2% yield (2.47 g, 4.36 mmol), mp 215 – 217 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.79 (d, J = 15.7 Hz, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.21 – 7.17 (m, 2H), 7.15 (d, J = 15.7 Hz, 1H), 6.85 (s, 2H), 5.08 (t, J = 5.7 Hz, 1H), 4.08 (s, 3H), 4.03 – 3.96 (m, 2H), 3.92 (s, 6H), 3.89 (s, 3H), 3.07 (t, J = 6.9 Hz, 2H), 2.96 – 2.87 (m, 2H), 1.88 – 1.84 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 157.8, 153.5, 149.2, 145.7, 143.2, 138.5, 138.1, 135.4, 132.6, 132.0, 130.7, 128.6, 121.2, 120.8, 104.4, 61.1, 56.2, 41.9, 39.0, 34.8, 27.8, 22.4, 14.2. HR-MS (ESI): calcd for C₂₈H₃₂BrN₅O₃ [M + H]⁺, 566.1761; found 566.1760.

(E)-N-(4-Fluorophenethyl)-1-methyl-3-propyl-5–(3,4,5-trimethoxystyryl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(4b). Title compound 4b was isolated as a yellow solid in 67.7% yield (1.71 g, 3.39 mmol), mp 186 – 187 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.80 (d, J = 15.7 Hz, 1H), 7.31 – 7.23 (m, 2H), 7.15 (d, J = 15.7 Hz, 1H), 7.05 (t, J = 8.6 Hz, 2H), 6.86 (s, 2H), 5.07 (t, J = 5.6 Hz, 1H), 4.06 (s, 3H), 3.99 (t, J = 6.4 Hz, 2H), 3.92 (s, 6H), 3.89 (s, 3H), 3.08 (t, J = 6.8 Hz, 2H), 2.94 (t, J = 7.7 Hz, 2H), 1.89 – 1.82 (m, 2H),1.02 (t, J = 7.4 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 161.9 (d, J = 245.4 Hz), 157.8, 153.5, 149.3, 145.8, 143.2, 138.6, 135.4, 134.7 (d, J = 3.4 Hz), 132.7, 130.4 (d, J = 7.9 Hz), 128.7, 121.2, 115.8 (d, J = 21.3 Hz), 104.4, 61.1, 56.3, 42.1, 39.0, 34.6, 27.8, 22.4, 14.2. HR-MS (ESI): calcd for C₂₈H₃₂FN₅O₃ [M + H]⁺, 506.2562; found 506.2560.

(E)-N,N-Diethyl-1-methyl-3-propyl-5–(3,4,5-trimethoxystyryl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(4c). Title compound 4c was isolated as a yellow solid in 81.3% yield (1.79 g, 4.07 mmol), mp 140 – 141 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.75 (d, J = 15.7 Hz, 1H), 7.16 (d, J = 15.7 Hz, 1H), 6.85 (s, 2H), 4.08 (s, 3H), 3.92 (s, 6H), 3.88 (s, 3H), 3.62 (q, J = 7.1 Hz, 4H), 2.99 – 2.96 (m, 2H), 1.91 – 1.85 (m, 2H), 1.26 (t, J = 7.1 Hz, 6H), 1.04 (t, J = 7.4 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 157.0, 153.6, 153.4, 147.2, 145.1, 138.5, 135.3, 132.7, 128.5, 125.1, 104.5, 61.1, 56.3, 43.9, 38.9, 28.0, 22.3, 14.2, 12.6. HR-MS (ESI): calcd for C₂₄H₃₃N₅O₃ [M + H]⁺, 440.2656; found 440.2657.

(E)-N-Isobutyl-1-methyl-3-propyl-5–(3,4,5-trimethoxystyryl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(4d). Title compound 4d was isolated as a yellow solid in 92.3% yield (2.03 g, 4.62 mmol), mp 171 – 172 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.75 (d, J = 15.7 Hz, 1H, =CH), 7.12 (d, J = 15.7 Hz, 1H, =CH), 6.85 (s, 2H, ArH), 5.15 (s, 1H, NH), 4.24 (s, 3H, OCH₃), 3.92 (s, 6H, 2 × OCH₃), 3.88 (s, 3H, NCH₃), 3.59 (t, J = 6.1 Hz, 2H, NCH₂), 2.94 (t, J = 7.7 Hz, 2H, CH₂), 2.12 – 2.08 (m, 1H, CH), 1.91 – 1.82 (m, 2H, CH₂), 1.08 (d, J = 6.7 Hz, 6H, 2 × CH₃), 1.03 (t, J = 7.3 Hz, 3H, CH₃). ¹³C NMR (151 MHz, DMSO-d₆): δ 157.8, 153.4, 149.8, 145.6, 143.0, 138.4, 135.2, 132.7, 128.7, 121.2, 104.4, 61.1, 56.2, 48.3, 39.1, 28.4, 27.8, 22.4, 20.6, 14.1. HR-MS (ESI): calcd for C₂₄H₃₄N₅O₃ [M + H]⁺, 440.2656; found 440.2651.

(E)-1-Methyl-N-(3-morpholinopropyl)-3-propyl-5–(3,4,5-trimethoxystyryl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(4e). Title compound 4e was isolated as a white solid in 62.8% yield (1.74 g, 3.42 mmol), mp 144 – 145 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.75 (d, J = 15.7 Hz, 1H), 7.13 (d, J = 15.6 Hz, 1H), 6.85 (s, 2H), 6.18 (t, J = 5.0 Hz, 1H), 4.28 (s, 3H), 3.92 (s, 6H), 3.88 (s, 3H), 3.87 – 3.84 (m, 2H), 3.73 (t, J = 4.7 Hz, 4H), 2.97 – 2.92 (m, 2H), 2.61 (t, J = 6.2 Hz, 2H), 2.54 (s, 4H), 1.97 (t, J = 6.3 Hz, 2H), 1.88 – 1.84(m, 2H), 1.03 (t, J = 7.3 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 157.8, 153.4, 149.6, 145.8, 142.9, 138.4, 135.2, 132.7, 128.8, 121.4, 104.4, 66.8, 61.1, 58.6, 56.2, 54.3, 41.2, 39.4, 27.9, 24.6, 22.4, 14.2. HR-MS (ESI): calcd for C₂₇H₃₈N₆O₄ [M + H]⁺, 511.3027; found 511.3028.

(E)-2–(1-Methyl-3-propyl-5–(3,4,5-trimethoxystyryl)-1H-pyrazolo[4,3-d]pyrimidin-7-ylamino)ethanol(4f). Title compound 4f was isolated as a white solid in 77.3% yield (1.65 g, 3.87 mmol), mp 127 – 128 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.66 (d, J = 15.8 Hz, 1H), 7.10 (d, J = 15.8 Hz, 1H), 7.06 (s, 1H), 6.97 (s, 2H), 4.83 (t, J = 5.1 Hz, 1H), 4.18 (s, 3H), 3.86 (s, 6H), 3.72 (t, J = 7.3 Hz, 4H), 3.69 (s, 3H), 2.78 (t, J = 7.5 Hz, 2H), 1.77 – 1.73 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃ + DMSO-d): δ 156.6, 153.1, 149.3, 143.6, 141.9, 137.7, 134.3, 132.1, 128.7, 120.8, 104.4, 60.0, 59.3, 55.9, 42.9, 39.0, 27.3, 21.8, 13.9. HR-MS (ESI): calcd for C₂₂H₃₀N₅O₄ [M + H]⁺, 42.2292; found 428.2293.

(E)-4–(2-(4–(1-Methyl-3-propyl-5–(3,4,5-trimethoxystyryl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethyl)morpholine(4 g). Title compound 4 g was isolated as a white solid in 54.7% yield (1.55 g, 2.74 mmol), mp 167 – 169 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.77 (d, J = 15.7 Hz, 1H), 7.16 (d, J = 15.7 Hz, 1H), 6.85 (s, 2H), 4.09 (s, 3H), 3.92 (s, 6H), 3.88 (s, 3H), 3.73 (t, J = 4.7 Hz, 4H), 3.60 (s, 4H), 2.98 (t, J = 7.7 Hz, 2H), 2.74 (s, 4H), 2.64 – 2.56 (m, 4H), 2.52 (t, J = 4.5 Hz, 4H), 1.91 – 1.83 (m, 2H), 1.03 (t, J = 7.3 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 157.2, 153.8, 153.4, 147.3, 145.0, 138.5, 135.7, 132.5, 128.1, 124.5, 104.4, 67.0, 61.1, 56.4, 56.2, 55.8, 54.3, 53.2, 49.5, 38.7, 28.0, 22.3, 14.2. HR-MS (ESI): calcd for C₃₀H₄₃N₇O₄ [M + H]⁺, 566.3449; found 566.3449.

(E)-3–(1-Methyl-3-propyl-5–(3,4,5-trimethoxystyryl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)thiazolidine(4 h). Title compound 4 h was isolated as a yellow solid in 68.2% yield (1.55 g, 3.41 mmol), mp 145 – 146 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.74 (d, J = 15.7 Hz, 1H), 7.16 (d, J = 15.7 Hz, 1H), 6.85 (s, 2H), 4.86 (s, 2H), 4.19 (s, 3H), 4.12 (t, J = 6.3 Hz, 2H), 3.93 (s, 6H), 3.89 (s, 3H), 3.21 (t, J = 6.3 Hz, 2H), 3.02 – 2.97 (m, 2H), 1.90 – 1.87 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 156.8, 153.4, 151.8, 146.9, 145.6, 138.7, 135.7, 132.3, 127.8, 124.0, 104.5, 60.9, 60.9, 56.2, 56.2, 55.1, 52.8, 38.9, 38.9, 30.6, 27.8, 22.1, 14.0. HR-MS (ESI): calcd for C₂₃H₃₀N₅O₃S [M + H]⁺, 456.2064; found 456.2065.

(E)-2–(4-(1-Methyl-3-propyl-5–(3,4,5-trimethoxystyryl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl)piperazin-1-yl)ethanol(4i). Title compound 4i was isolated as a yellow solid in 75.4% yield (1.87 g, 3.77 mmol), mp 141 – 142 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.78 (d, J = 15.7 Hz, 1H), 7.17 (d, J = 15.7 Hz, 1H), 6.86 (s, 2H), 4.11 (s, 3H), 3.93 (s, 6H), 3.89 (s, 3H), 3.70 (t, J = 4.8 Hz, 2H), 3.62 (s, 4H), 2.99 (t, J = 7.7 Hz, 2H), 2.79 (s, 4H), 2.68 (t, J = 4.8 Hz, 2H), 1.90 – 1.86 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 157.2, 153.8, 153.4, 147.3, 145.1, 138.5, 135.7, 132.5, 128.0, 124.4, 104.4, 61.1, 59.5, 57.9, 56.2, 52.5, 49.6, 38.6, 27.9, 22.3, 14.2. HR-MS (ESI): calcd for C₂₆H₃₇N₆O₄ [M + H]⁺, 492.2871; found 492.2871.

(E)-1-Methyl-3-propyl-N-(pyridin-4-ylmethyl)-5–(3,4,5-trimethoxystyryl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(4j). Title compound 4j was isolated as a yellow solid in 86.3% yield (2.05 g, 4.32 mmol), mp 171 – 173 °C. ¹H NMR (600 MHz, CDCl₃): δ 8.62 – 8.56 (m, 2H), 7.56 (d, J = 15.7 Hz, 1H), 7.39 – 7.33 (m, 2H), 7.09 (d, J = 15.7 Hz, 1H), 6.77 (s, 2H), 5.57 (t, J = 5.7 Hz, 1H), 4.98 (d, J = 5.6 Hz, 2H), 4.26 (s, 3H), 3.89 (s, 6H), 3.86 (s, 3H), 2.95 (t, J = 7.7 Hz, 2H), 1.88 – 1.84 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 157.7, 153.4, 150.2, 149.0, 148.1, 145.9, 143.5, 138.5, 135.7, 132.5, 128.2, 122.6, 121.0, 104.3, 61.1, 56.2, 43.9, 39.3, 27.8, 22.4,14.2. HR-MS (ESI): calcd for C₂₆H₃₀N₆O₃ [M + H]⁺, 475.2452; found 475.2452.

(E)-N-(2-Methoxybenzyl)-1-methyl-3-propyl-5–(3,4,5-trimethoxystyryl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(4k). Title compound 4k was isolated as a yellow solid in 79.8% yield (2.01 g, 3.99 mmol), mp 211 – 213 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.83 (d, J = 15.7 Hz, 1H), 7.50 – 7.44 (m, 1H), 7.31 (td, J = 8.0, 1.5 Hz, 1H), 7.14 (d, J = 15.7 Hz, 1H), 6.97 (t, J = 7.9 Hz, 2H), 6.88 (s, 2H), 5.87 (t, J = 5.6 Hz, 1H), 4.96 (d, J = 5.6 Hz, 2H), 4.20 (s, 3H), 3.95 (s, 3H), 3.93 (s, 6H), 3.89 (s, 3H), 2.97 – 2.90 (m, 2H), 1.86 – 1.83 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H). HR-MS (ESI): calcd for C₂₈H₃₄N₅O₄ [M + H]⁺, 504.2065; found 504.2067.

3. Biological evaluation

3.1. Cell culture

Mouse peritoneal RAW264.7 macrophages were obtained from BeNa Culture Collection Company. Cells were cultured in DMEM (Hyclone, USA) supplemented with 10% FBS (Biological Industries, Israel), 100 U/ml penicillin and 100 μg/ml streptomycin (Beyotime, China) at 37 °C in a humidified atmosphere containing 5% CO₂.

3.2. Determination of NO, TNF-α and IL-6

RAW264.7 cells were seeded into 48-well plate with 6 × 10⁴ cells per well and incubated for 24 h. Cells were pretreated with the title compounds (10 μM) for 1 h, followed by exposure to LPS (0.5 μg/ml) for 24 h. The supernatants were collected and examined for NO production using Griess reagent (Beyotime, China). The levels of TNF-α and IL-6 in the supernatant were determined using the ELISA kit, according to the manufacturer's instructions (eBioScience, San Diego, CA).

3.3. Cell viability assay

RAW264.7 cells (6 × 10³ cells/per) were seeded into 96-well plate and incubated for 24 h. Cells were pretreated with the title compounds (20 μM) for 1 h and incubated with LPS (0.5 μg/ml) for 24 h. MTT solution (5 mg/ml in PBS) was added to each well and incubated for 4 h  at 37 °C. The MTT containing media was removed and then 150 μL of DMSO was added. The absorbance was detected at 490 nm by a microplate reader (MQX200, Bio-Tek, USA).

3.4. Western blotting

RAW264.7 cells were seeded into a 6-well plate at a density of 3 × 10⁵ cells per well and then cultured for 24 h. Cells were pretreated with compound 4e (2, 1, 0.5 μM) for 1 h, followed by exposure to LPS (0.5 μg/ml) for 0.5 h.

Cells were lysed with RIPA lysis buffer (Beyotime, China) containing PMSF and phosphatase inhibitors, and then incubated on ice for 30 min. The protein lysates were separated by 12% SDS-PAGE and subsequently transferred onto PVDF membranes (GE Healthcare, UK). The blocked membranes were incubated with the indicated primary antibodies at 4 °C overnight (All of the antibodies were purchased from Cell Signaling Technology, USA). After washing three times with TBST (Beyotime, China), the membranes were incubated with HRP-conjugated secondary antibody (Beyotime, China) for 1 h at room temperature.

3.5. In vivo experiment

Male C57BL/6 mice weighing 18 – 22 g were purchased from Animal Department of Anhui Medical University. Mice were randomly divided into five groups (n = 8): physiological saline as negative control group, LPS (20 mg/kg) stimulated group, compound 4e high dose (20 mg/kg) group, compound 4e low dose (10 mg/kg) group and celecoxib (15 mg/kg) as positive control group. Compound 4e or celecoxib was administrated intraperitoneally (i.p.) 0.5 h before LPS injection via tail vein. Mice were anesthetized and sacrificed 48 h after LPS injection. Lung tissues were collected and fixed in 4% paraformaldehyde, followed by embedded in paraffin. After dehydration, sections were stained with Hematoxylin and Eosin (H&E) staining.

4. Results and discussion

4.1. Chemistry

4-Amino-1-methyl-3-propyl-4,5-dihydro-1H-pyrazole-5-carboxamide (SM) was prepared according to previous protocol²¹. SM was treated with substituted carboxylic acid in the presence of EDCI and HOBt to yield A1–A4. Compounds B1–B4 were synthesized from compounds A1–A4via cyclization in the presence of sodium ethoxide. Key intermediates C1–C4 were carried out under N₂ atmosphere with POCl₃ at 95 °C for 8 h (Scheme 1). (General procedures see Supporting Information).

4.2. Inhibitory activity against LPS-induced NO release

Nitric oxide (NO) is an important pro-inflammatory mediator, relating to several inflammatory diseases, such as rheumatoid arthritis, chronic hepatitis and ALI^22,23. Therefore, inhibition of its overproduction may provide a useful therapy for inflammatory diseases²⁴. Treatment of RAW 264.7 cells with LPS stimulated NF-κB signaling pathway, resulting in the production of cytokines including NO, IL-6 and TNF-α. Briefly, RAW 264.7 cells were pre-incubated with tested compounds, followed by incubated with LPS. The supernatants were collected and then nitrite levels were determined. The results indicated that almost all tested compounds were able to inhibit NO production at 10 µM (Figure 3). Being the most potent, compound 4e reduced NO release more intensely than both celecoxib and resveratrol. Accordingly, the introduction of 3-morpholinopropan-1-amine group at C-7 of pyrazolo[4,3-d]pyrimidine scaffold can improve the inhibitory activity.

Figure 3.

Inhibition of LPS-induced NO releasing by compounds 1a∼4k in RAW264.7 cells^a. ^aRAW264.7 cells were pretreated with compounds (10 μM) for 1 h, and incubated with LPS (0.5 μg/ml) for 24 h. The levels of NO releasing were measured using Griess Reagent assay. (A) Effect of compounds 1a∼2g on NO secretion. (B) Effect of compounds 3a∼4k on NO secretion. Celecoxib (Cel) and resveratrol (Res) were chosen as positive controls. ***p < .001, **p < .01, *p < .05 versus LPS group.

4.3. Assessment of toxicity

To discard that the suppressive effects on NO release was related to cell viability, MTT assay was adopted. As observed in Figure 4, cell viability was not affected by most of compounds at 20 µM, excepting of compounds 1f, 1h, 2b, 4b, 4j and 4k with weak cytotoxicity. The results indicated that their anti-inflammatory activity is not mediated by cytotoxic effect. Therefore, these compounds were worth of further evaluation.

Figure 4.

The cytotoxicity of compounds 1a∼4k in RAW264.7 cells^a. ^aThe cell viability was evaluated by the MTT assay. ***p < .001 compare with control group.

4.4. Inhibition of LPS-induced release of cytokines

Increasing evidences have showed that the two important cytokines IL-6 and TNF-α play important roles in the pathogenesis of ALI through a series of cytokine signaling pathways²⁵. Therefore, the most potent compound 4e was chosen to further evaluate for inhibition of LPS-induced NO, TNF-α and IL-6 releasing. As shown in Figure 5, compound 4e significantly decreased NO, IL-6 and TNF-α secretion in a concentration-dependent manner, with IC₅₀ values of 2.64, 4.38 and 5.63 μM, respectively. On the basis of above findings, the anti-inflammatory mechanisms of compound 4e were further explored.

Figure 5.

Inhibition of the cytokines production^a. ^aRAW264.7 cells were pretreated with compound 4e in a series of concentrations (10, 5, 2.5, 1.25 μM) for 1 h, incubated with LPS (0.5 μg/ml) for 24 h. NO releasing was measured using Griess Reagent assay. The levels of TNF-α and IL-6 in the culture medium were measured by ELISA. *** p< .001, **p < .01, *p < .05 versus LPS group.

4.5. Inhibition of LPS-induced TLR4 expression

As a key factor in LPS-induced inflammation, TLR4 activates series of cellular signaling pathways, including NF-ĸB and mitogen-activated protein kinases (MAPKs), leading to the secretion of pro-inflammatory cytokines, such as NO, IL-6, TNF-α and IL-1β^26–28. Therefore, we investigated whether the expression of TLR4 was down-regulated by compound 4e. As shown in Figure 6, LPS-induced TLR4 overexpression was attenuated by pretreatment of compound 4e in a concentration-dependent manner.

Figure 6.

Inhibit expression of LPS-induced TLR4^a. ^aRAW264.7 cells were pretreated with compound 4e (1 – 4 µM) for 1 h, then stimulated with LPS (0.5 μg/ml) for 24 h. The expression of TLR4 was analyzed by Western blot. The results were showed as means ± SD (n = 3) of at least three independent experiments. TAK-242 was the TLR4 inhibitor. ^###p < .001 compared with LPS un-stimulated cells; *p < .05, **p < .01, ***p < .001 compare with LPS-stimulated cells.

4.6. Inhibition of LPS-induced p38 signaling pathway

MAPKs, including ERK, p38 and JNK, were quite significant in the regulation of inflammation²⁹. Therefore, we detected the effects of compound 4e on LPS-mediated MAPKs signaling activation. As shown in Figure 7, the phosphorylation of p38 but not JNK or ERK was blocked by compound 4e treatment in a concentration-dependent manner. And the total levels of ERK, JNK and p38 were not affected.

Figure 7.

Suppressed LPS-induced p38 activation^a. ^aRAW264.7 cells were pretreated with compound 4e (1–4 µM) for 1 h, then stimulated with LPS (0.5 μg/ml) for 30min. The phosphorylation and total expression of ERK, JNK and p38 were analyzed by Western blot. The results were showed as means ± SD (n = 3) of at least three independent experiments. ^###p < .001 compared with LPS un-stimulated cells; *p < .05, **p < .01, ***p < .001 compare with LPS-stimulated cells.

4.7. In vivo experiment

Next, compound 4e was evaluated in LPS-induced ALI mice model. Mice were pretreated with compound 4e by intraperitoneal injection 0.5 h before LPS challenge. LPS stimulation leads to significant pro-inflammatory alterations, including lung edema, inflammatory cell infiltration and destruction of alveolar structure. Pretreatment of mice with compound 4e effectively reduced airspace inflammation and amended the tissue structure of pulmonary lobules (Figure 8). These results indicated the protective effects of compound 4e against LPS-induced ALI in mice model.

Figure 8.

Compound 4e protected LPS-induced acute lung injury^a. ^aC57/BL6 mice were treated with compound 4e (10 mg/kg, 20 mg/kg), and after 30 min, were challenged with 20 mg/kg LPS by tail vein injection. After Hematoxylin and Eosin staining, histological examination was performed by light microscopy (magnification ×200). Celecoxib (15 mg/kg) was a positive drug. ^###p < .001 compared with control group; *p < .05, **p < .01, ***p < .001 compare with LPS group.

5. Conclusions

In the present studies, 35 novel pyrazolo[4,3-d]pyrimidine derivatives were designed, synthesized and evaluated for their anti-inflammatory activities in RAW264.7 cells. The preliminary SAR studies show that the introduction of 3-morpholinopropan-1-amine group into pyrazolo[4,3-d]pyrimidine could increase anti-inflammatory activity. Specifically, the most potent compound 4e was selected to further study the mechanism. The results showed that compound 4e concentration-dependently inhibited LPS-induced NO, IL-6 and TNF-α secretion through suppressing TLR4/p38 signaling pathway. In vivo studies in LPS-challenged mice showed that compound 4e effectively normalized pulmonary histopathological changes. Taken together, compound 4e could be potential therapeutics for ALI.

Funding Statement

We gratefully acknowledge financial support from Natural Science Foundation of Anhui Provincial Education Department [KJ2017A831] and National Natural Science Funding of China [21572003].

Disclosure statement

No potential conflict of interest was reported by the authors.