Developing efficient protocols for synthesis, antiosteoarthritic, antiinflammatory assessments and docking studies of nitrogen-containing bisphosphonate derivatives

Wafaa M. Abdou; Neven A. Ganoub; Mohamed A.H. Ismail; Eman Sabry; Reham F. Barghash; Athina Geronikaki

doi:10.1016/j.arabjc.2016.02.019

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Original article

10 (

); 1084-1097

doi:

10.1016/j.arabjc.2016.02.019

Developing efficient protocols for synthesis, antiosteoarthritic, antiinflammatory assessments and docking studies of nitrogen-containing bisphosphonate derivatives

Wafaa M. Abdou^{^a,⁎}, Neven A. Ganoub^{^a}, Mohamed A.H. Ismail^{^b}, Eman Sabry^{^a}, Reham F. Barghash^{^a}, Athina Geronikaki^{^c}

a Chemical Industries Division, National Research Centre, Elbehouth St., D-12311, Dokki, Cairo, Egypt

b Pharmaceutical Chemistry, Faculty of Pharmacy, Ain-Shams University, Cairo, Egypt

c School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece

⁎Corresponding author. wabdou@link.net (Wafaa M. Abdou)

Received: 2015-11-16, Accepted: 2016-2-29, Published: 2016-12-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Abstract

We report herein, two synthetic approaches to three types of nitrogen-containing bisphosphonates (N-BPs) in moderate to high yields (58–88%). Ester cleavage of selected bisphosphonates was undertaken to obtain their N-BP-acid analogs. Based on the reported bisphosphonate properties and the prospective biological prediction using computer-assisted molecular modeling (CAMM), new compounds were evaluated in a mouse model of antigen-induced arthritis (AIA) and the delayed-type hypersensitivity granuloma reaction (DTH-GRA) for chronic inflammation. Pharmacological results showed that N-BP-acids are more favorable for antiarthritis activity than N-BP-esters. On the other hand, the majority of N-BPs revealed good antiinflammatory potency compared to their acid analogs. The results also showed that the presence of a free thiol group in a molecule enhances the anti-inflammatory activity. Furthermore, bioscreening results were in good agreement with the prediction investigation. A hypothesis of molecular modeling study, including fitting of the synthesized compounds into 3D-pharmacophore using Discovery Studio 2.5 software and their docking into the human farnesyl pyrophosphate synthase (hFPPS, PDB code: 2F8C protein) showed good results consistent with the observed pharmacological properties.

Keywords

N-bisphosphonates

N-bisphosphonic acids

Antichronic inflammation

CAMM-drug research

Docking studies

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1 Introduction

Bisphosphonates (BPs) are synthetic pyrophosphate mimics, which have been developed and used for the treatment of bone diseases (Fig. 1, Emesto et al., 2007). The pronounced selective activity of BPs for bone tissues gives them a key advantage in clinical practice and the mainstay of antiresorptive therapy for osteoporosis (Balakrishna et al., 2011; Emesto et al., 2007; Fleisch, 1988, 1995 ). BPs inhibit bone resorption by reducing osteoclast activity and inducing osteoclast apoptosis (Balakrishna et al., 2011; Fleisch, 1995, 1988 ). They are therefore, used for treatment post-menopausal osteoporosis in women and reducing the associated bone fracture risk (Cheng and Oldfield, 2004).

Structures of bisphosphonates and pyrophosphate.

Nitrogen-containing bisphosphonates are known as the second generation of BPs and are proven to be more effective. These drugs are used in diverse therapeutic areas such as microbial (Wang et al., 2001), parasitic (Docampo and Moreno, 2001), herbicidal (Cromartie et al., 1999), viral (HIV) (Mckenna et al., 1999), cancer diseases (Fleisch, 2002; Senaratne and Colston, 2002) as well as chronic inflammation and arthritis (Francis et al., 1989; Nugent et al., 1993; Österman et al., 1997 ). The activity of N-BPs has accounted to their effect on human farnesyl (hFPP)-and human geranyl geranyl pyrophosphate synthase (hGGPP), which are enzymes in the mevalonate pathway, decreasing the prenylation of essential GTP-binding proteins (Dunford et al., 2006). Furthermore, it is well established that N-BPs bearing free amine group (or alkylated amine, known as the third generation of BPs) at one of the side chains are usually preferable in clinical use because of their higher potency (Bijvoet et al., 1995).

In human rheumatoid arthritis (RA), osteoclasts are found at sites of tissues invasion and emerge from synovial inflammatory tissues (Hannig et al., 2007; Österman et al., 1997), therefore, inhibition of osteoclastogenesis is a powerful approach to prevent arthritic bone destruction (Hannig et al., 2007). As a part of our ongoing research program on the applications of BPs in pharmacology (Abdou et al., 2014, 2012b,c,d; Kamel et al., 2012; Shaddy et al., 2013 ), we herein, accomplished a synthesis of a variety of β-aminobisphosphonates and their relevant bisphosphonic acids. New compounds were examined vs RA in animal antigen-induced arthritis (AIA) and for the chronic inflammation in animal model of the delayed-type hypersensitivity granuloma reaction (DTH-GRA). The optimized antiarthritis/antiinflammatory activities of synthesized N-BPs and N-BP-acids are also based on the prospective predicted potency, using the computer-assisted molecular modeling (CAMM) (Da Silva et al., 2010; Lagunin et al., 2003). Later, a hypothesis of molecular modeling study, including fitting of the synthesized compounds into 3D-pharmacophore using Discovery Studio 2.5 software (Girgis et al., 2011; Shahin et al., 2014) and their docking into the human farnesyl pyrophosphate synthase (hFPPS, PDB code: 2F8C protein) showed good results consistent with the observed pharmacological properties.

2 Material and methods

2.1

2.1 General data

Melting points were determined with open capillary tube on an Electrothermal (variable heater) melting point apparatus and were not corrected. IR spectra were recorded on a JASCO FT-IR 6100 using KBr disk (JASCO, Japan). NMR spectra were measured with a JEOL E.C.A-500 MHz (¹³C: 125.7 MHz, ¹H: 500 MHz, ³¹P: 202.4 MHz) spectrometer (JEOL, Japan). ³¹P NMR spectra were recorded with H₃PO₄ (85%) as external reference. ¹H and ¹³C NMR spectra were recorded with trimethylsilane as internal standard in CDCl₃. Chemical shifts (δ) are given in ppm. The mass spectra were performed at 70 eV on an MS-50 Kratos (A.E.I.) spectrometer provided with a data system spectrometer (Kratos, UK). Elemental analyses were carried out at the Microanalysis Laboratory, Cairo University, Cairo, Egypt, using elementary Analysen-systeme GmbH-vario EL III Element Analyzer, Germany. The appropriate precautions in handling moisture-sensitive compounds were observed. The purity of all new samples was verified by microchemical analysis (C/H/N/S) and spectroscopy. Solvents were dried by standard techniques. Thin-layer chromatography (TLC): Merck 0.2 mm silica gel 60 F254 analytic aluminum plates. All international principles and local regulations concerning the care and the use of laboratory animals were considered during the pharmacological screening. The azide 2 was prepared according to the reported method (Pokhodylo et al., 2010).

2.2

2.2 Preparation of tetraethyl (3-amino-4,5,6,7-tetrahydro-2H[1]benzothieno[2,3-b]pyrrole-2,2-diyl)bisphosphonate (4)

A solution of tetraethyl methylenebisphosphonate (0.9 g, 3.2 mmol) of (1) in absolute ethanol (10 mL) containing sodium (80 mg, 3.5 mmol) was stirred at 0 °C for 0.5 h. A solution of 2-azido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (2) (0.6 g, 3 mmol) in EtOH (10 mL) was then added in one portion, and the reaction mixture was stirred under reflux for 6 h (TLC). The product mixture was cooled, poured into iced water, and acidified with conc HCl, followed by extraction with AcOEt, dried, and the volatile materials were removed under reduced pressure until dryness. Crystallization of the resulting residue from MeCN afforded the bisphosphonate 4 as strew yellow crystals. Yield: 1.2 g (87.6%); mp 162–164 °C; υ_max/cm⁻¹ 3441–3333 (NH₂), 1254 (P⚌O, free), 1234 (P⚌O, H-bonded), 1075, 1029 (2P—O—C); δ_H (500 MHz, CDCl₃) 1.24, 1.35 (2dt, ³J_HH = 6.7, ⁴J_PH = 4.4 Hz, 12H, 4H₃CH₂COP), 1.91, 2.45 (2 m, 8H, 4H₂C-hexyl), 4.04–4.27 (m, 8H, 4H₂COP), 6.36, 8.73 (2br, 2H, H₂N, D₂O, exchanged); δ_C (125.7 MHz, CDCl₃) 167.8 (t, ³J_PC = 7.4 Hz, C⚌N), 141.6, 134.6 (C⚌C-hexyl), 120.6 (t, ²J_PC = 33.6 Hz, C—NH₂), 102.7 (t, ³J_PC = 7.1 Hz, C⚌C—NH₂), 77.4 (t, ¹J_PC = 179.4 Hz, C-P₂), 62.1 (d, ²J_PC = 13.7 Hz, 4CH₂OP), 26.4, 25.1, 23.6, 22.3 (CH₂-hexyl), 16.5 (d, ³J_PC = 7.5 Hz, 4CH₃H₂COP); δ_P (202.4 MHz, CDCl₃) 21.5, 24.2 (2d, ²J_PP = 24.2 Hz, C-P₂); m/z (%) 462 [M⁺−2, 65%], 188 [100, M⁺−276 (2H + 2[P(O)(OEt)₂])]. Anal. calcd. for C₁₈H₃₀N₂O₆P₂S (464.45): C, 46.55; H, 6.51; N, 6.03; S, 6.90. Found: C, 46.62; H, 6.45; N, 5.96; S, 6.84.

2.3

2.3 Preparation of ethyl 4-azido-5-cyano-1-phenyl-1H-pyrazole-3-carboxylate (6)

A solution of NaNO₂ (2.76 g, 40 mmol) in water (20 mL) was added to a solution of ethyl 4-amino-5-cyano-1-phenyl-1H-pyrazole-3-carboxylate (7.68 g, 30 mmol) in 10% aqueous HCl (120 mL) at 0–5 °C. The reaction mixture was stirred at this temperature for 30 min, any resinous sediment that has formed should be filtered, followed by adding a solution of NaN₃ (3.25 g, 50 mmol) in water (50 mL). The mixture was further stirred for 1 h and the precipitate was filtered off, washed with water, and dried. The resulting residue was crystallized from cyclohexane to give 6 as pale yellow crystals. Yield: 6.5 g (76.5%); mp 158–160 °C; υ_max/cm⁻¹ 2219 (CN), 2152 (N₃), 1704 (C⚌O); δ_H (500 MHz, CDCl₃) 1.23 (t, ³J_HH = 6.5 Hz, 3H, H₃CH₂CO), 4.35 (q, ³J_HH = 6.5 Hz, 2H, H₂CO), 7.55–7.62 (m, 5H, H-Ph); δ_C (125.7 MHz, CDCl₃) 160.2 (C⚌O), 142.1, 136.3, 128.1, 126.4, 125.5, 111.5, 101.4 (C-pyrazole, C-Ph), 122.1 (CN), 61.3 (CH₂O), 13.6 (CH₃H₂CO); m/z (%) 282 [M⁺, 74%], 254 [100, M⁺−28]. Anal. calcd. for C₁₃H₁₀N₆O₂ (282.26): C, 55.32; H, 3.57; N, 29.77. Found: C, 55.39; H, 3.43; N, 29.64.

2.4

2.4 Preparation of ethyl 6-amino-5,5-bis(diethoxyphosphoryl)-1-phenyl-1,5-dihydropyrrolo-[3,2-c]pyrazole-3-carboxylate (7)

Following the previous procedure described for the synthesis of compound 4 and using the same amounts, the azide 6 (0.8 g, 3 mmol) reacted with 1 (0.9 g, 3.2 mmol) in EtONa (80 mg, 3.5 mmol) solution at r.t. for 6 h. The product mixture was cooled, poured into iced water, and acidified with conc HCl, followed by extraction with AcOEt, dried, and the volatile materials were removed under reduced pressure until dryness. Crystallization of the resulting residue from CHCl₃ afforded the bisphosphonate 7 as pale yellow crystals. Yield: 1.4 g (84.8%); mp 186–188 °C; υ_max/cm⁻¹ 3429–3334 (NH₂), 1259 (P⚌O, free), 1229 (P⚌O, H-bonded), 1130, 1072 (2P—O—C); δ_H (500 MHz, CDCl₃) 0.97 (t, ³J_HH = 6.5 Hz, 3H, H₃CH₂CO), 1.26, 1.38 (2dt, ³J_HH = 5.3, ⁴J_PH = 4.2 Hz, 12H, 4H₃CCH₂OP), 4.18 (q, ³J_HH = 6.5 Hz, 2H, H₂CO), 4.32–4.45 (m, 8H, 4H₂COP), 6.43, 8.94 (2br, 2H, H₂N, D₂O, exchanged), 7.52–7.69 (m, 5H, H-Ph); δ_C (125.7 MHz, CDCl₃) 158.7 (t, ³J_PC = 7.2 Hz, C⚌N), 157.6 (C⚌O), 148.2, 141.3, 129.5, 124.5, 115.2 (C-Ph, C-pyrazole), 116.2 (t, ³J_PC = 9.5 Hz, C⚌C—NH₂), 106.3 (t, ²J_PC = 34.5 Hz, C—NH₂), 73.6 (t, ¹J_PC = 175.3 Hz, C-P₂), 62.4 (d, ²J_PC = 10.7 Hz, 4CH₂OP), 61.2 (CH₂O), 16.5 (d, ³J_PC = 6.9 Hz, 4CH₃H₂COP), 14.3 (CH₃CH₂O); δ_P (202.4 MHz, CDCl₃) 27.6, 28.3 (2d, ²J_PP = 21.5 Hz, C-P₂); m/z (%) 540 [M⁺−2, 65%], 221 [100, M⁺−321 (2H + OEt + 2[P(O)(OEt)₂])]; Anal. calcd. for C₂₂H₃₂N₄O₈P₂ (542.46): C, 48.71; H, 5.95; N, 10.33. Found: C, 48.79; H, 5.81; N, 10.27.

2.5

2.5 Preparation of nitrogen-bisphosphonates (N-BPs, 11a–d)

Step 1: to a solution of azide 6 (2.82 g, 10 mmol) in CH₂Cl₂ (10 mL), triphenylphosphine (PPh₃) (3.27 g, 12.5 mmol) in CH₂Cl₂ (10 mL) was added. The reaction mixture was stirred at r.t. for 6 h and the volatile materials were removed under vacuum. The precipitated material was collected (4.65 g, 90.2%), washed with ligroin to give the iminophosphorane (A) as colorless material in 90.2% yield (Abdou et al., 2012a). Compound A is pure enough and stable for few days at −10 °C; Step 2: to a solution of A (1.29 g, 2.5 mmol) in THF (15 mL), methyl-, ethyl-, phenyl-, and/or cyclohexaneisocyanate (9a–d, 2.5 mmol) were added at r.t. After the mixture was stirred for ≈8 h (TLC) at 0 to −5 °C, the solvent was distilled off in vacuum, and the residue was washed thoroughly with ether/petroleum ether (1:2, 20 mL) to get rid of Ph₃PO, yielding the carbodiimides 10. Compounds 10a–d (≈80%) were generally used directly without further purification; Step 3: to the in situ carbodiimides 10a–d (2 mmol), phosphoryl carbanion 1 (0.69 g, 2.4 mmol) in EtONa solution (64 mg Na, 2.8 mmol/20 mL EtOH) was added in one portion, and the mixture was further stirred at r.t. for ≈8–10 h (TLC). The product mixture was cooled-up, poured into iced water, and acidified with conc HCl, followed by extraction with AcOEt, dried, and the volatile materials were removed under reduced pressure until dryness. The resulting residue was crystallized from the proper solvent to give the bisphosphonates 11a–d.

2.5.1

2.5.1 Ethyl 7-amino-6,6-bis(diethoxyphosphoryl)-5-(methylimino)-1-phenyl-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-3-carboxylate (11a) was obtained as pale yellow crystals

Yield: 0.86 g (74.4%); mp 162–164 °C (CH₂Cl₂); υ_max/cm⁻¹ 3435–3342 (NH₂), 1702 (C⚌O), 1256, 1226 (2P⚌O), 1132, 1079 (2P—O—C); δ_H (500 MHz, CDCl₃) 1.17 (t, ³J_HH = 6.7 Hz, 3H, H₃CH₂CO), 1.24, 1.28 (2dt, ³J_HH = 6.8, ⁴J_PH = 4.8 Hz, 12H, 4H₃CH₂COP), 3.3 (s, 3H, H₃CN), 3.78 (q, ³J_HH = 6.7 Hz, 2H, H₂CO), 4.21–4.25 (m, 8H, 4H₂COP), 6.31, 8.72 (2br, 2H, H₂N, D₂O exchanged), 7.42–7.63 (m, 5H, H-Ph); δ_C (125.7 MHz, CDCl₃) 178.3 (t, ²J_PC = 25.2 Hz, C⚌NMe), 162.6 (C⚌O), 158.2, 147.5, 145.9, 129.4, 128.5, 123.7 (C-Ph, C-pyrazole), 118.8 (t, ²J_PC = 28.4 Hz, C—NH₂), 112.4 (t, ³J_PC = 8.7 Hz, C⚌C—NH₂), 61.7 (d, ²J_PC = 12.7 Hz, 4CH₂OP), 60.2 (CH₂O), 47.8 (t, ¹J_PC = 168.7 Hz, C-P₂), 41.5 (CH₃N), 16.4 (d, ³J_PC = 6.8 Hz, 4CH₃CH₂OP), 14.5 (CH₃CH₂O); δ_P (202.4 MHz, CDCl₃) 24.4, 25.6 (2d, ²J_PP = 22.6 Hz, C-P₂); m/z (%) 581 [M⁺−2, 53%], 307 [100, M⁺−276 (2H + 2[P(O)-(OEt)₂])]. Anal. calcd. for C₂₄H₃₅N₅O₈P₂ (583.51): C, 49.40; H, 6.05; N, 12.00. Found: C, 49.31; H, 5.93; N, 11.93.

2.5.2

2.5.2 Ethyl 7-amino-6,6-bis(diethoxyphosphoryl)-5-(ethylimino)-1-phenyl-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-3-carboxylate (11b) was obtained as yellow crystals

Yield: 0.74 g (62.2%); mp 155–157 °C (MeCN); υ_max/cm⁻¹ 3440–3346 (NH₂), 1706 (C⚌O), 1258, 1229 (2P⚌O), 1136, 1076 (2P—O—C); δ_H (500 MHz, CDCl₃): 1.11 (t, ³J_HH = 6.3 Hz, 3H, H₃CH₂CN), 1.23 (t, ³J_HH = 6.4 Hz, 3H, H₃CH₂CO), 1.28, 1.31 (2dt, ³J_HH = 6.2, ⁴J_PH = 5.1 Hz, 12H, 4H₃CCH₂OP), 3.51 (q, ³J_HH = 6.3 Hz, 2H, H₂C·N), 3.75 (q, ³J_HH = 6.4 Hz, 2H, H₂CO), 4.11–4.26 (m, 8H, 4H₂COP), 6.35, 8.69 (2br, 2H, H₂N, D₂O, exchanged), 7.45–7.61 (m, 5H, H-Ph); δ_C (125.7 MHz, CDCl₃) 176.7 (t, ²J_PC = 28.4 Hz, C⚌NEt), 163.6 (C⚌O), 158.6, 146.9, 144.2, 129.5, 128.2, 123.5 (C-pyrazole, C-Ph), 117.5 (t, ²J_PC = 27.8 Hz, C—NH₂), 113.5 (t, ³J_PC = 8.2 Hz, C⚌C—NH₂), 62.5 (d, ²J_PC = 12.6 Hz, 4CH₂OP), 61.8 (CH₂O), 55.6 (CH₂N), 45.6 (t, ¹J_PC = 167.3 Hz, C-P₂), 16.3 (d, ³J_PC = 7.3 Hz, 4CH₃CH₂OP), 15.6 (CH₃CH₂O), 14.8 (CH₃CH₂N); δ_P (202.4 MHz, CDCl₃) 26.1, 27.5 (2d, ²J_PP = 23.8 Hz, C-P₂); m/z (%) 595 [M⁺−2, 63%], 321 [100, M⁺−276 (2H + 2[P(O)(OEt)₂])]; Anal. calcd. for C₂₅H₃₇N₅O₈P₂ (597.54): C, 50.25; H, 6.24; N, 11.72. Found: C, 50.38; H, 6.14; N, 11.64.

2.5.3

2.5.3 Ethyl 7-amino-6,6-bis(diethoxyphosphoryl)-1-phenyl-5-(phenylimino)-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-3-carboxylate (11c) was obtained as yellow crystals

Yield: 0.86 g (66.7%); mp 176–178 °C (EtOH); υ_max/cm⁻¹ 3435–3343 (NH₂), 1712 (C⚌O), 1254, 1232 (2P⚌O), 1132, 1078 (2P—O—C); δ_H (500 MHz, CDCl₃) 1.21 (t, ³J_HH = 6.8 Hz, 3H, H₃CCH₂O), 1.25, 1.34 (2dt, ³J_HH = 7.3, ⁴J_PH = 4.8 Hz, 12H, 4H₃CCH₂OP), 4.12 (q, ³J_HH = 6.8 Hz, 2H, H₂CO), 4.16–4.37 (m, 8H, 4H₂COP), 6.33, 8.61 (2br, 2H, H₂N, D₂O exchanged), 7.36–7.63 (m, 10H, H-Ph); δ_C (125.7 MHz, CDCl₃) 177.4 (t, ²J_PC = 27.3 Hz, C⚌NPh), 162.4 (C⚌O), 158.9, 156.3, 147.5 145.3, 129.6, 128.7, 126.2, 123.6, 123.1 (C-pyrazole, C-Ph), 117.5 (t, ²J_PC = 29.4 Hz, C—NH₂), 112.7 (t, ³J_PC = 9.2 Hz, C⚌C—NH₂), 61.7 (d, ²J_PC = 11.6 Hz, 4CH₂OP), 61.3 (CH₂O), 46.5 (t, ¹J_PC = 181.3 Hz, C-P₂), 16.4 (d, ³J_PC = 8.7 Hz, 4CH₃CH₂OP), 14.3 (CH₃CH₂O); δ_P (202.4 MHz, CDCl₃) 25.7, 27.1 (2d, ²J_PP = 24.1 Hz, C-P₂) ppm; m/z (%) 643 [M⁺−2, 63%], 369 [100, M⁺−276 (2H + 2[P(O)(OEt)₂])]; Anal. calcd. for C₂₉H₃₇N₅O₈P₂ (645.58): C, 53.95; H, 5.78; N, 10.85. Found: C, 54.04; H, 5.69; N, 10.71.

2.5.4

2.5.4 (Ethyl 7-amino-5-(cyclohexylimino)-6,6-bis(diethoxyphosphoryl)-1-phenyl-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-3-carboxylate (11d) was obtained as yellow crystals

Yield: 0.94 g (72.3%); mp 165–166 °C (diethyl ether); υ_max/cm⁻¹ 3439–3333 (NH₂), 1705 (C⚌O), 1259, 1230 (2P⚌O), 1130, 1072 (2P—O—C); δ_H (500 MHz, CDCl₃) 0.9, 1.19 (2 m, 8H, 4H₂C), 1.47 (t, ³J_HH = 6.9 Hz, 3H, H₃CH₂CO), 1.61, 1.73 (2dt, ³J_HH = 6.2, ⁴J_PH = 4.7 Hz, 12H, 4H₃CCH₂OP), 1.82 (m, 2H, H₂C), 2.6 (m, 1H, HC), 4.32 (q, ³J_HH = 6.9 Hz, 2H, H₂CO), 4.41–4.56 (m, 8H, 4H₂COP), 6.34, 8.74 (2br, 2H, H₂N, D₂O exchanged), 7.67–7.89 (m, 5H, H-Ph); δ_C (125.7 MHz, CDCl₃) 166.5 (t, ²J_PC = 29.5 Hz, C⚌NC₆H₁₁), 162.7 (C⚌O), 158.6, 147.8, 145.7, 129.4, 128.3, 123.6 (C-pyrazole, C-Ph), 118.2 (t, ²J_PC = 27.8 Hz, C—NH₂), 112.4 (t, ³J_PC = 8.9 Hz, C⚌C—NH₂), 61.9 (d, ²J_PC = 11.2 Hz, 4CH₂OP), 60.8 (CH₂O), 56.3 (CH-cyclohexane), 45.6 (t, ¹J_PC = 178.5 Hz, C-P₂), 29.4, 25.6, 23.2 (CH₂-cyclohexane), 16.5 (d, ³J_PC = 8.6 Hz, 4CH₃CH₂OP), 14.6 (6CH₃CH₂O); δ_P (202.7 MHz, CDCl₃) 26.2, 27.6 (2d, ²J_PP = 36 Hz, C-P₂); m/z (%) 649 [M⁺−2, 64%], 375 [100, M⁺−276 (2H + 2[P(O)(OEt)₂])]. Anal. calcd. for C₂₉H₄₃N₅O₈P₂ (651.63): C, 53.45; H, 6.65; N, 10.75. Found: C, 53.31; H, 6.56; N, 10.62.

2.6

2.6 Preparation of nitrogen-bisphosphonates 16a–d

A mixture of 2-aminobenzenethiol (13) (1.25 g, 10 mmol) and aldehyde: 4-(dimethylamino)-, 4-hydroxy-, 2-hydroxy-, or 4-methoxy-benzaldehyde 14a–d (10 mmol) in EtOH (20 mL) containing AcOH (1 mL) was stirred under reflux for ≈3 h (TLC). The resulting Schiff-bases 15a–d were precipitated and allowed to react in situ with 1. Phosphoryl carbanion 1 (1.2 g, 4.2 mmol) in DMF (20 mL) containing LiH (0.1 g, 12.6 mmol) was added under stirring to the substrates 15a–d (3.5 mmol). The suspension was stirred at r.t. for further 30 min and then heated under reflux for the appropriate time ≈18 h (TLC). The product mixture was cooled-up, poured into iced water, and acidified with conc HCl, followed by extraction with AcOEt, dried, and the volatile materials were removed under reduced pressure until dryness. The resulting residue was crystallized from the proper solvent to give the bisphosphonates 16a–d.

2.6.1

2.6.1 Tetraethyl {2-(4-(dimethylamino)phenyl)-2-[(2-mercaptophenyl)amino]ethane-1,1-diyl}bisphosphonate (16a) was obtained as buff crystals

Yield: 1.3 g (68.3%); mp 170–172 °C (MeCN); υ_max/cm⁻¹ 3429–3357 (NH), 2565 (SH, free), 1254, 1228 (2P⚌O), 1090, 1028 (2P—O—C); δ_H (500 MHz, CDCl₃) 1.14, 1.18 (2dt, ³J_HH = 6.3, ⁴J_PH = 4.4 Hz, 12H, 4H₃CCH₂OP), 2.88 (br, 1H, HS, D₂O exchanged), 2.94 (s, 6H, Me₂N), 3.84 (td, ³J_HH = 9.8, ²J_PH = 17.7 Hz, 1H, HC-P₂), 4.13–4.26 (m, 8H, 4H₂COP), 4.45 (td, ³J_HH = 9.8, ³J_PH = 6.8 Hz, 1H, HC-Ar), 6.64–7.42 (m, 8H, H-Ar), 9.12 (br, 1H, HN, D₂O exchanged); δ_C (125.7 MHz, CDCl₃) 149.8, 148.5, 131.4, 129.3, 125.6, 122.5, 115.7, 114.2 (C-Ar), 139.3 (t, ³J_PC = 8.9 Hz, C-Ar), 64.5 (t, ²J_PC = 26.7 Hz, CH—NH), 60.5 (d, ²J_PC = 12.8 Hz, 4CH₂OP), 44.7 (t, ¹J_PC = 182.4 Hz, C-P₂), 40.1 (Me₂C), 15.7 (d, ³J_PC = 8.4 Hz, 4CH₃CH₂OP); δ_P (202.4 MHz, CDCl₃) 24.3, 25.6 (2d, ²J_PP = 23.9 Hz, C-P₂); m/z (%) 542 [M⁺−2, 65%], 254 [100, M⁺−290 (2H + CH₂[P(O)(OEt)₂]₂)]. Anal. calcd. for C₂₄H₃₈N₂O₆P₂S (544.58): C, 52.93; H, 7.03; N, 5.14; S, 5.89. Found: C, 53.01; H, 6.96; N, 5.06; S, 6.02.

2.6.2

2.6.2 Tetraethyl {2-(4-hydroxyphenyl)-2-[(2-mercaptophenyl)amino]ethane-1,1-diyl}bisphosphonate (16b) was obtained as buff crystals

Yield: 1.12 g (62.2%); mp 144–146 °C (CH₂Cl₂); υ_max/cm⁻¹ 3421–3373 (OH & NH), 2555 (SH), 1260, 1242 (2P⚌O), 1087, 1030 (2P—O—C); δ_H (500 MHz, CDCl₃) 1.16, 1.19 (2dt, ³J_HH = 5.6, ⁴J_PH = 4.6 Hz, 12H, 4H₃CCH₂OP), 2.91 (br, 1H, HS, D₂O exchanged), 3.87 (td, ³J_HH = 10.1, ²J_PH = 18.4 Hz, 1H, HC-P₂), 4.11–4.31 (m, 8H, 4H₂COP), 4.51 (td, ³J_HH = 10.1, ³J_PH = 7.1 Hz, 1H, HC-Ar), 6.62–7.35 (m, 8H, H-Ar), 9.34 (br, 1H, HN, D₂O exchanged), 10.43 (br, 1H, HO, D₂O exchanged); δ_C (125.7 MHz, CDCl₃) 157.5, 149.8, 130.3, 129.3, 125.6, 122.7, 119.5, 115.6 (C-Ar), 141.6 (t, ³J_PC = 9.1 Hz, C-Ar), 64.7 (t, ²J_PC = 27.4 Hz, CH—NH), 60.4 (d, ²J_PC = 11.7 Hz, 4CH₂OP), 44.5 (t, ¹J_PC = 185.4 Hz, C-P₂), 15.4 (d, ³J_PC = 7.6 Hz, 4CH₃CH₂OP); δ_P (202.4 MHz, CDCl₃) 23.6, 25.8 (2d, ²J_PP = 23.6 Hz, C-P₂); m/z (%) 515 [M⁺−2, 57%], 227 [100, M⁺−290 (2H + CH₂[P(O)(OEt)₂]₂)]. Anal. calcd. for C₂₂H₃₃NO₇P₂S (517.51): C, 51.06; H, 6.43; N, 2.71; S, 6.20. Found: C, 50.99; H, 6.37; N, 2.61; S, 6.31.

2.6.3

2.6.3 Tetraethyl {2-(2-hydroxyphenyl)-2-[(2-mercaptophenyl)amino]ethane-1,1-diyl}bis-phosphonate (16c) was obtained as buff crystals

Yield: 1.0 g (58.2%); mp 140–141 °C (cyclohexane); υ_max/cm⁻¹ 3427–3393 (OH & NH), 2543 (SH), 1262, 1228 (2P⚌O), 1085, 1033 (2P—O—C); δ_H (500 MHz, CDCl₃) 1.14, 1.28 (2dt, ³J_HH = 5.4, ⁴J_PH = 4.6 Hz, 12H, 4H₃CCH₂OP), 2.95 (br, 1H, HS, D₂O exchanged), 3.85 (td, ³J_HH = 10.5, ²J_PH = 16.8 Hz, 1H, HC-P₂), 4.13–4.29 (m, 8H, 4H₂COP), 4.46 (td, ³J_HH = 10.5, ³J_PH = 6.8 Hz, 1H, HC-Ar), 6.45–7.54 (m, 8H, H-Ar), 9.36 (br, 1H, HN, D₂O exchanged), 10.47 (br, 1H, HO, D₂O exchanged); δ_C (125.7 MHz, CDCl₃) 161.3, 151.4, 131.3, 129.8, 125.6, 123.1, 122.4, 115.7 (C-Ar), 135.7 (t, ³J_PC = 9.4 Hz, C-Ar), 60.6 (d, ²J_PC = 12.5 Hz, 4CH₂OP), 57.4 (t, ²J_PC = 26.3 Hz, CH—NH), 45.3 (t, ¹J_PC = 179.5 Hz, C-P₂), 15.1 (d, ³J_PC = 7.4 Hz, 4CH₃CH₂OP); δ_P (202.4 MHz, CDCl₃) 23.8, 25.6 (2d, ²J_PP = 23.5 Hz, C-P₂); m/z (%) 515 [M⁺−2, 62%], 227 [100, M⁺−290 (2H + CH₂[P(O)(OEt)₂]₂)]. Anal. calcd. for C₂₂H₃₃NO₇P₂S (517.51): C, 51.06; H, 6.43; N, 2.71; S, 6.20. Found: C, 50.98; H, 6.38; N, 2.65; S, 6.29.

2.6.4

2.6.4 Tetraethyl 2-(2-mercaptophenylamino)-2-(4-methoxyphenyl)ethane-1,1-diyl-bisphosphonate (16d) was obtained as buff crystals

Yield: 1.2 g (64.6%); mp 155–157 °C (CH₂Cl₂); υ_max/cm⁻¹ 3433–3365 (NH), 2552 (SH), 1261, 1240 (2P⚌O), 1083, 1035 (2P—O—C); δ_H (500 MHz, CDCl₃) 1.15, 1.23 (2dt, ³J_HH = 5.9, ⁴J_PH = 4.6 Hz, 12H, 4H₃CCH₂OP), 3.01 (br, 1H, HS, D₂O exchanged), 3.67 (td, ³J_HH = 10.9, ²J_PH = 19.4 Hz, 1H, HC-P₂), 3.95 (s, 3H, H₃CO), 4.12–4.29 (m, 8H, 4H₂COP), 4.51 (td, ³J_HH = 10.9, ³J_PH = 6.5 Hz, 1H, HC-Ar), 6.62–7.35 (m, 8H, H-Ar), 9.34 (br, 1H, HN, D₂O exchanged); δ_C (125.7 MHz, CDCl₃) 158.3, 149.6, 130.6, 129.5, 125.4, 122.5, 118.4, 115.7 (C-Ar), 142.4 (t, ³J_PC = 9.4 Hz, C-Ar), 64.5 (t, ²J_PC = 29.4 Hz, CH—NH), 60.7 (d, ²J_PC = 11.2 Hz, 4CH₂OP), 55.7 (CH₃O), 44.8 (t, ¹J_PC = 178.4 Hz, C-P₂), 15.7 (d, ³J_PC = 7.3 Hz, 4CH₃CH₂OP); δ_P (202.4 MHz, CDCl₃) 23.6, 25.7 (2d, ²J_PP = 23.8 Hz, C-P₂); m/z (%): 529 [M⁺−2, 57%], 241 [100, M⁺−290 (2H + CH₂[P(O)(OEt)₂]₂)]. Anal. calcd. for C₂₃H₃₅NO₇P₂S (531.54): C, 51.97; H, 6.64; N, 2.64; S, 6.03. Found: C, 51.91; H, 6.52; N, 2.58; S, 6.12.

2.7

2.7 General procedure for N-BP-acid-salts 5A, 8A, 12aA,bA, 17aA,bA

0.63 mmol of bisphosphonates 4, 7, 11a, 11b, 16a or 16b were dissolved in CHCl₃ (10 mL) and treated with trimethylsilyl bromide (3.8 mmol) and was heated at 40 °C for ≈6 h. After concentrating the product mixture in vacuum, the crude material was diluted with water and stirred for 30 min. The layers were separated, and the aqueous layer was evaporated to dryness. The precipitate was collected and dried to give the crude acids 5, 8, 12a, 12b, 17a or 17b. The resulted acids are hygroscopic and can be kept only under vacuum for identification purposes. The acids were later stirred in a solution of aqueous NaOH (10%/Methanol, 20 mL (1:3 v/v) for 1 h, followed by concentration. The precipitate 5A, 8A, 12aA, 12bA, 17aA or 17bA was filtered-off and air-dried.

2.7.1

2.7.1 (3-Amino-4,5,6,7-tetrahydro-2H-[1]benzothieno[2,3-b]pyrrole-2,2-diyl)bisphosphonic acid (5) was obtained as white substance

Yield: 0.34 g (71.6%); mp > 300 °C; υ_max/cm⁻¹ 1252, 1227 (2P⚌O); δ_H (500 MHz, D₂O) 1.93, 2.46 (2 m, 8H, 4CH₂-hexyl); δ_C (125.7 MHz, D₂O) 167.6 (t, ³J_PC = 7.3 Hz, C⚌N), 141.4, 134.8 (C⚌C-hexyl), 120.1 (t, ²J_PC = 33.9 Hz, C—NH₂), 102.9 (t, ³J_PC = 7.5 Hz, C⚌C—NH₂), 77.1 (t, ¹J_PC = 178.4 Hz, C-P₂), 26.1, 24.9, 23.2, 21.7 (CH₂-hexyl); δ_P (202.4 MHz, D₂O) 18.8, 19.4 (2d, ²J_PP = 16.3 Hz, C-P₂); m/z (%) 350 [M⁺−2, 33%]. Anal. calcd. for C₁₀H₁₄N₂O₆P₂S (352.24): C, 34.10; H, 4.01; N, 7.95; S, 9.10. Found: C, 34.21; H, 3.93; N, 7.86; S, 9.19.

2.7.2

2.7.2 6-Amino-3-(ethoxycarbonyl)-1-phenyl-1,5-dihydropyrrolo[3,2-c]pyrazole-5,5-diyldiphosphonic acid (8) was obtained as white substance

Yield: 0.32 g (65.5%); mp > 300 °C; υ_max/cm⁻¹ 1259, 1235 (2P⚌O); δ_H (500 MHz, D₂O) 1.35 (t, ³J_HH = 7.5 Hz, 3H, H₃CCH₂O), 4.45 (q, ³J_HH = 7.5 Hz, 2H, H₂CO), 7.51–7.67 (m, 5H, H-Ph); δ_C (125.7 MHz, D₂O) 158.4 (t, ³J_PC = 7.4 Hz, C⚌N), 156.8 (C⚌O), 147.8, 140.9, 129.2, 124.2, 116.1 (C-Ph, C-pyrazole), 117.1 (t, ³J_PC = 9.3 Hz, C⚌C—NH₂), 106.6 (t, ²J_PC = 33.9 Hz, C—NH₂), 72.9 (t, ¹J_PC = 175.6 Hz, C-P₂), 61.7 (CH₂O), 14.6 (CH₃CH₂O); δ_P (202.4 MHz, D₂O) 19.4, 19.6 (2d, ²J_PP = 20.6 Hz, C-P₂); m/z (%) 428 [M⁺−2, 21%]. Anal. calcd. for C₁₄H₁₆N₄O₈P₂ (430.25): C, 39.08; H, 3.75; N, 13.02. Found: C, 39.17; H, 3.67; N, 13.13.

2.7.3

2.7.3 7-Amino-3-(ethoxycarbonyl)-5-(methylimino)-1-phenyl-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-6,6-diylbis-phosphonic acid (12a) was obtained as white solid

Yield: 0.32 g (63.2%); mp > 300 °C; υ_max/cm⁻¹ 1706 (C⚌O), 1254, 1239 (2P⚌O); δ_H (500 MHz, D₂O) 1.17 (t, ³J_HH = 7.6 Hz, 3H, H₃CCH₂O), 3.5 (s, 3H, H₃CN), 4.14 (q, ³J_HH = 7.6 Hz, 2H, H₂CO), 7.41–7.64 (m, 5H, H-Ph); δ_C (125.7 MHz, D₂O) 177.6 (t, ²J_PC = 24.9 Hz, C⚌NMe), 162.3 (C⚌O), 157.9, 148.1, 145.9, 128.9, 128.1, 123.2 (C-Ph, C-pyrazole), 118.5 (t, ²J_PC = 28.1 Hz, C—NH₂), 113.1 (t, ³J_PC = 8.9 Hz, C⚌C—NH₂), 60.6 (CH₂O), 47.2 (t, ¹J_PC = 168.2 Hz, C-P₂), 42.1 (CH₃N), 14.9 (CH₃CH₂O); δ_P (202.4 MHz, D₂O) 18.5, 19.2 (2d, ²J_PP = 15.6 Hz, CP₂); m/z (%) 469 [M⁺−2, 24%]. Anal. calcd. for C₁₆H₁₉N₅O₈P₂ (471.30): C, 40.77; H, 4.06; N, 14.86. Found: C, 40.86; H, 3.99; N, 14.92.

2.7.4

2.7.4 7-Amino-3-(ethoxycarbonyl)-5-(ethylimino)-1-phenyl-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-6,6-diylbisphosphonic acid-salt (12b) was obtained as white substance

Yield: 0.33 g (65.3%); mp > 300 °C; υ_max/cm⁻¹ 1710 (C⚌O), 1256, 1234 (P⚌O); δ_H (500 MHz, D₂O) 1.18 (t, ³J_HH = 7.8 Hz, 3H, H₃CCH₂N), 1.24 (t, ³J_HH = 7.1 Hz, 3H, H₃CCH₂O), 4.13 (q, ³J_HH = 7.8 Hz, 2H, H₂CN), 4.16 (q, ³J_HH = 7.1 Hz, 2H, H₂CO), 7.46–7.74 (m, 5H, H-Ph); δ_C (125.7 MHz, D₂O) 177.1 (t, ²J_PC = 27.8 Hz, C⚌NEt), 163.2 (C⚌O), 157.9, 146.2, 143.8, 128.1, 128.9, 124.2 (C-pyrazole, C-Ph), 116.3 (t, ²J_PC = 29.8 Hz, C—NH₂), 113.6 (t, ³J_PC = 7.9 Hz, C⚌C—NH₂), 62.3 (CH₂O), 56.1 (CH₂N), 44.9 (t, ¹J_PC = 169.5 Hz, C-P₂), 15.9 (CH₃CH₂O), 15.2 (CH₃CH₂N); δ_P (202.4 MHz, D₂O) 18.6, 19.3 (2d, ²J_PP = 15.5 Hz, C-P₂); m/z (%) 483 [M⁺−2, 28%]. Anal. calcd. for C₁₆H₁₉N₅O₈P₂ (485.32): C, 42.07; H, 4.36; N, 14.43. Found: C, 42.16; H, 4.29; N, 14.34.

2.7.5

2.7.5 2-(4-(Dimethylamino)phenyl)-2-(2-mercaptophenylamino)ethane-1,1-diylbisphosphonic acid-salt (17a) was obtained as yellow crystals

Yield: 0.34 g (68.3%); mp > 300 °C; υ_max/cm⁻¹ 1251, 1229 (2P⚌O); δ_H (500 MHz, D₂O) 2.96 (s, 6H, NMe₂), 3.87 (td, ³J_HH = 10.2, ²J_PH = 17.4 Hz, 1H, HC-P₂), 4.45 (td, ³J_HH = 10.2, ³J_PH = 6.9 Hz, 1H, HC-Ar), 6.65–7.22 (m, 8H, H-Ar); δ_C (125.7 MHz, D₂O) 148.5, 147.2, 132.4, 129.9, 125.6, 123.5, 116.8, 114.7 (C-Ar), 139.6 (t, ³J_PC = 8.2 Hz, C-Ar), 65.1 (t, ²J_PC = 27.1 Hz, CH—NH), 45.4 (t, ¹J_PC = 184.4 Hz, C-P₂), 40.9 (Me₂C); δ_P (202.4 MHz, D₂O) 19.7, 20.1 (2d, ²J_PP = 21.9 Hz, C-P₂); m/z (%) 430 [M⁺−2, 17%]. Anal. calcd. for C₁₆H₂₂N₂O₆P₂S (432.37): C, 44.45; H, 5.13; N, 6.48; S, 7.42. Found: C, 44.52; H, 5.05; N, 6.39; S, 7.32.

2.7.6

2.7.6 2-(4-Hydroxyphenyl)-2-(2-mercaptophenylamino)ethane-1,1-diylbisphosphonic acid-salt (17b) was obtained as yellow crystals

Yield: 0.3 g (62.4%); mp > 300 °C; υ_max/cm⁻¹ 1253, 1232 (2P⚌O); δ_H (500 MHz, D₂O) 3.81 (td, ³J_HH = 9.7, ²J_PH = 18.4 Hz, 1H, HC-P₂), 4.41 (td, ³J_HH = 9.7, ³J_PH = 6.7 Hz, 1H, HC-Ar), 6.62–7.27 (m, 8H, H-Ar); δ_C (125.7 MHz, D₂O) 156.9, 149.1, 131.3, 130.2, 124.9, 122.3, 120.4, 115.9 (C-Ar), 140.8 (t, ³J_PC = 9.4 Hz, C-Ar), 64.6 (t, ²J_PC = 28.3 Hz, CH—NH), 44.9 (t, ¹J_PC = 186.8 Hz, C-P₂); δ_P (202.4 MHz, D₂O) 18.9, 19.3 (2d, ²J_PP = 23.4 Hz, C-P₂); m/z (%) 403 [M⁺−2, 27%]. Anal. calcd. for C₁₄H₁₇NO₇P₂S (405.30): C, 41.49; H, 4.23; N, 3.46; S, 7.91. Found: C, 41.57; H, 4.14; N, 3.39; S, 8.01.

2.8

2.8 Biological procedures

2.8.1

2.8.1 Antigen-induced arthritis

According to the reported method by Nugent et al. (1993), groups of 6 female albino mice, 6–8 weeks of age, were immunized subcutaneously (sc) with an emulsion of methylated bovine serum albumin (mBSA) and Freund’s complete adjuvant supplemented with extra heat killed M-tuberculosis. Secondary immunizations were performed after 7 days, and after a further 14 days the animals were challenged intraarticularly with 200 mg of mBSA in saline into the left rear stifle joint. The tested compounds 4, 5A, 7, 8A, 11a,b, 12aA, 16a,b, and 17aA,bA as well as the positive control, N-BP-drug (zoledronic acid, Zol) 18 were dissolved, suspended, or emulsified in sterile saline and sonicated where appropriate to homogeneous doses. All compounds were then adjusted to pH 7.0 with 0.1 M NaOH and stored frozen in aliquots. Fresh aliquots were used for each day of dosing. Mice were dosed sc in the scruff of the neck from the day of intraarticular mBSA challenge (day 0) using a 5 of 7 day dosing regimen until the conclusion of the study on day 28. The mBSA-injected stifle joint was then skimmed, removed, and fixed in phosphate-buffered formaldehyde solution prior to decalcification and histological preparation. The assessment of arthritis was performed on sagittal joint sections stained with hematoxylin and eosin. Sections were graded 1 (mild) to 5 (severe) for soft tissue inflammation, pannus formation, and extent to cartilage and bone erosion. The changes (ΔPV) which occurred in the arthritic control and treated rats (injected and noninjected-mBSA) were quantitated on day 28 by mercury displacement plethysmography. The component scores were summed to give arthritis score. Statistical comparisons were performed by one-way analysis of variance (ANOVA), compared with vehicle-treated control. Resulted data are displayed in Table 1.

Table 1 Antiarthritis evaluation results of N-BPs 4, 7, 11a,b, 16a,b & N-BP-acid-salts 5A, 8A, 12aA, 17aA,bA.

Antigen-induced arthritis
No.	Dose sc mg/kg	%Inhibition^a	No.	Dose sc^b mg/kg	%Inhibition
18	200	54^∗∗∗	11b	200	23^∗∗
	100	53^∗∗∗		100	51^∗∗
	50	44^∗∗∗		60	35^∗∗
	30	44^∗∗∗

4	200	38^∗	12aA	200	40^∗∗
	100	28^∗		100	36^∗∗∗
	50	–		60	52^∗∗∗
	30	–		40	44^∗

5A	200	13^∗∗	16a	200	50^∗∗∗
	100	38^∗∗		100	45^∗∗∗
	60	42^∗∗		60	48^∗∗∗
	40	40^∗		50	44^∗∗∗

7	200	53^∗∗∗	16b	200	51^∗∗∗
	100	46^∗∗∗		100	46^∗∗∗
	60	40^∗∗∗		60	42^∗∗
	40	38^∗∗∗		50	40^∗∗∗

8A	200	56^∗∗∗	17aA	200	58^∗∗∗
	100	52^∗∗∗		100	55^∗∗∗
	50	40^∗∗∗		60	48^∗∗∗
	30	38^∗∗∗		40	46^∗∗∗

11a	200	40^∗∗	17bA	200	60^∗∗∗
	100	48^∗∗		100	56^∗∗∗
	60	24^∗		60	44^∗∗∗
				40	36^∗∗∗

a (^∗∗∗) p < 0.001, (^∗∗) p < 0.01, (^∗) p < 0.05.

b sc: subcutaneous.

2.8.2

2.8.2 Delayed-type hypersensitivity granuloma

According to the reported methods by Nugent et al. (1993) and Österman et al. (1997), groups of 6 female albino mice (25–30 g) were sensitized with an emulsion of methylated bovine serum albumin (mBSA) and Freund’s incomplete adjuvant and dextran by sc injection over the inguinal lymph node. Three weeks later, hydroxyapatite (HA) disks (6 mm diameter) soaked in mBSA solution (30 mg/mL saline) were implanted sc in dorsum of the mice (two disks, bilaterally). All tested compounds (4, 5A, 7, 8A, 11a–d, 12aA, 12bA, 16a–d, 17aA, 17bA, and Zometa®) were prepared as solutions, suspensions, or emulsion in sterile saline and sonicated where appropriate to homogeneous doses, and the pH was adjusted to 7.4 with 0.1 M NaOH. Each mouse received compound in a volume of 0.1 mL/10 g body weight sc in the scruff of the neck. Dosing commenced on the day of implantation of the mBSA soaked disks and was continued thereafter on a daily basis until day nine, when the mice were euthanized. The granulomatous lesions were then excised and both wet and dry tissue weights measured. Results were analyzed by students paired test. Resulted data are displayed in Table 2. Potency of the tested compounds was calculated regarding zoledronic acid, reference standard according to the following equation: $% Potency = \frac{Group % edema inhibition of tested compound}{Group % edema inhibition of zoledronic acid}$

Table 2 DTH-GRA results of synthesized N-BPs and N-BP-acid salts.^a

No.	Dose sc mg/kg	% Inhibition of granuloma		% Potency (wet)	No.	Dose sc mg/kg	% Inhibition of granuloma		% Potency (wet)
No.	Dose sc mg/kg	Dry wt	Wet wt	% Potency (wet)	No.	Dose sc mg/kg	Dry wt	Wet wt	% Potency (wet)
18	100	47^∗∗∗	44^∗∗∗	100	16a	100	68^∗∗∗	65^∗∗∗	144.7
4	100	31^∗	33^∗	65.9		50	54^∗∗∗	62^∗∗∗
5A	100	14^∗	18^∗∗	29.8		25	43^∗∗∗	55^∗∗∗
7	100	64^∗∗∗	66^∗∗∗	136.2	16b	100	66^∗∗∗	65^∗∗∗	140.4
	50	48^∗∗∗	44^∗∗∗			50	63^∗∗∗	60^∗∗∗
	25	38^∗∗∗	35^∗∗∗			25	50^∗∗∗	52^∗∗∗

8A	100	20^∗	26^∗	42.6	16c	100	48^∗∗∗	49^∗∗∗	102.1
11a	100	44^∗∗	46^∗∗	93.6		50	38^∗∗∗	45^∗∗∗
11b	100	41^∗∗	42^∗∗	87.2	16d	100	50^∗∗	48^∗∗	106.4
11c	100	40^∗∗∗	38^∗∗∗	85.1		50	52^∗∗	47^∗∗
11d	100	35^∗∗∗	38^∗∗∗	74.5

12aA	100	16^∗	20^∗	34.1	17aA	100	19^∗	22^∗	40.4
12bA	100	18^∗	24^∗	38.3	17bA	100	18^∗	25^∗	38.3

a (^∗∗∗) p < 0.001, (^∗∗) p < 0.01, (^∗) p < 0.05.

2.8.3

2.8.3 Toxicity evaluation of promised BP-derivatives 7, 16a, and 17bA

The LD₅₀ determination of the most promising synthesized and antiinflammatory active agents (7, 16a, and 17bA) was carried out by the standard known LD₅₀ method in mice (Kärber, 1931). Albino mice weighing 20–25 g were divided into six groups of six mice each. Administrations of the tested compounds dissolved in the same vehicle solution in 500, 750, and 1000 mg/kg (body weight) were given intraperitoneally. Control groups were given in buffer solution only. Toxic symptoms, mortality rates, and postmortem findings in each group were recorded 24 h post administration.

LD₅₀ of the tested compounds were calculated according to the following formula: ${LD}_{50} = D_{m} - Σ (z \times d) / n$ where D_m is the largest dose, which kills all animals, z is the mean of dead animals between two successive doses, d = the constant factor between two successive doses, n is the number of animals in each group, Σ is the sum of (z × d).

3 Chemistry and results of biological studies

3.1

3.1 Chemistry

We adopted two different protocols for synthesis of our targets, N-BPs: (i) consisted of coupling azido-substrates with the phosphorus reagent, tetraethyl methylenebisphosphonate (1)/catalyzed by sodium ethanolate (EtONa); (ii) involved one-pot three-component synthesis, by applying 1 in an alkaline solution to carbodiimide- or Schiff-base derivatives, initially generated in situ.

Schemes 1 and 2 describe synthesis of β-amino-BPs 4, 7 and their acid analogs 5 and 8 from the coupling condensation of 1 with the azides 2 and 6. When the azide 2 (Pokhodylo et al., 2010) was caused to react with slight excess of phosphonyl carbanion 1 in EtONa solution, tetraethyl (3-amino-4,5,6,7-tetrahydro-2H-[1]benzothieno-[2,3-b]pyrrole-2,2-diyl)bisphosphonate (4) was isolated in 87.6% yield. As the pharma laboratories proved remarkable therapeutic value of BP-acids than BP-esters (Fleisch, 1995, 1988), hydrolysis of 4 to the corresponding acid 5 was undertaken using trimethylsilyl bromide (TMS-Br) in chloroform, followed by quenching with water. The resulted acid 5 is hygroscopic and can be kept only under vacuum for identification purposes. Later on, it was stirred in a solution of aqueous NaOH (10%/Methanol, 20 mL (1:3 v/v) for 1 h, followed by concentration to give its disodium salt 5A (Nugent et al., 1993; Zhou et al., 2014) (Scheme 1).

Synthesis of N-BP 4, N-BP-acid 5 & its salt 5A.

Synthesis of N-BP 7, N-BP-acid 8 & its salt 8A.

The IR (υ_max/cm⁻¹) spectrum of N-BP 4 revealed the lack of the azido- and the nitrile groups and revealed the presence of stretching frequencies at 3441–3333 (NH₂), P⚌O (1254, free), 1234 (P⚌O, H-bonded, due to its H-bonding with one proton of the amino group), and at 1075, 1029 (P—O—C). In this context, the NH₂-protons were displayed in the ¹H NMR (CDCl₃, δ_H) spectrum at 6.36 and 8.73 (2br, 2 × 1H) ppm. Furthermore, the ¹³C NMR (CDCl₃, δ_C) spectrum of 4 revealed among others, two triplets at 77.4 (t, ¹J_PC = 179.4 Hz, C-P₂), and 120.6 (t, ²J_PC = 33.6 Hz, C—NH₂) and its ³¹P NMR (CDCl₃, δ_P) showed two doublets peaks at 21.5, 24.2 (2d, J_PP = 24.2 Hz). For the N-BP-acid 5, the ³¹P NMR (D₂O, δ_P) chemical shifts were displayed at 18.8, 19.4 ppm (2d, J_PP = 16.3 Hz) (Du et al., 2002; Turhanen and Vepsaelaeinen, 2008). As displayed in Scheme 1, the azides smoothly react with the reagent 1 in refluxing ethanol with the evolution of nitrogen to yield the intermediate 3 (Deep et al., 1991; Cadogan et al., 1978). Further reduction of the cyano group and intramolecular cyclization led to the product 4. The hydrolysis of the nitrile group was observed due to the presence of the alkaline medium and the heating.

Similarly, the fused pyrrole-2,2-diylbisphosphonate 7 was synthesized in 84.8% yield by treating ethyl 4-azido-5-cyano-1-phenyl-1H-pyrazole-3-carboxylate (6) with the phosphorus reagent 1 in EtONa solution at r.t. under the same reaction conditions. It is notable to mention that the azide 6 was prepared and identified for the first time. Compound 7 underwent ester cleavage as previously mentioned to give 8, followed by its conversion to its disodium salt 8A. The ¹H-, ¹³P-, and ¹³C NMR data of 7 and 8 are consistent with the assigned structures (see the experimental section) (Scheme 2).

Next, one-pot three-component reactions were applied for the synthesis of N-BPs 11a–d and relevant N-BP-acids 12a,b (Scheme 3). The first step of the process involved the iminophosphorane (A) formation (90%) from the reaction of the azide 6 with Ph₃P in CH₂Cl₂. The precipitated phosphorane (A) was treated with a proper isocyanate 9a–d in tetrahydrofuran (THF) to give the required carbodiimides 10a–d in >80% yields via the aza-Wittig reaction. N-Heterocumulenes 10a–d were highly unstable, and should be quickly freed from Ph₃PO, and could be kept at −5 to 0 °C for few days (Liu et al., 2008; Abdou et al., 2013). Further addition of ethanolate solution of phosphonyl carbanion 1 to 10a–d afforded the target fused pyridine-3,3-diyl-bisphosphonates 11a–d in 62–74% yields. 11a,b underwent ester cleavage as previously described to afford the respective acids 12a, 12b followed by their disodium salts 12aA and 12bA (Scheme 3).

Synthesis of N-BPs 11a–d, N-BP-acids 12a,b & salts 12aA, 12bA.

The IR absorptions (υ_max/cm⁻¹) for 11a–d showed bands within the ranges 1259–1254 (P⚌O, free) and 1234–1226 (P⚌O, H-bonded). The NH₂ protons in 11a–d were observed at ≈6.31 and ≈8.72 ppm in their ¹H NMR (CDCl₃, δ_H). The carbon chemical shifts for CP₂, CNH₂, and C⚌NMe in 11a (as a representative example) were observed in their expected region at δ_C 47.8 (t, ¹J_PC = 168.7 Hz, CP₂), 118.8 (t, ²J_PC = 28.4 Hz, C—NH₂), and at 178.3 (t, ²J_PC = 25.2 Hz, C⚌NMe). The ³¹P NMR (CDCl₃, δ_P) chemical shifts of 11a were observed at 25.6 and 24.4 (2d, J_PP = 22.6 Hz). Chemical ionization mass spectra of 11a–d gave molecular ions and diagnostic N-BP-daughter ion peaks at their expected m/z values. For the N-BP-acids 12a,b, the ³¹P NMR (D₂O, δ_P) chemical shifts were displayed at δ ≈ 18–19 ppm.

In another type of multi component reaction (MCR), the Schiff-base intermediates 15 generated in situ from 2-aminobenzenethiol (13) and the proper aldehyde 14a–d were trapped by phosphoryl carbanion 1 in DMF solution containing lithium hydride (LiH) (Keglevich and Bálint, 2012; Li et al., 2014). After the working up, N-BPs 16a–d were obtained in 58–68% yields, via the addition reaction at the exocyclic imines. N-BPs 16a,b underwent ester cleavage using TMSBr, as previously mentioned to afford the respective acids 17a, 17b followed by conversion to their disodium salts 17aA and 17bA (Scheme 4).

Synthesis of N-BPs 16a–d, N-BP-acids 17a,b & salts 17aA, 17bA.

In the IR (υ_max, cm⁻¹) spectra of 16a–d showed the NH band the range 3433–3357 and the SH moiety at ≈2558 (free thiol), the two P⚌O bands within the range 1262–1254 (free) and 1242–1228 (H-bonded). The ¹H NMR (CDCl₃, δ_H) spectrum of 16a, as a represented examples exhibited two types of methine protons with different chemical shifts at 3.84 (td, J_HH = 9.8, ²J_PH = 17.7 Hz, H^aC-P₂) and at δ_H 4.45 ppm (td, J_HH = 9.8, ³J_PH = 6.8 Hz) ascribed to the proton at the exocyclic asymmetric carbon (H^b—C^∗—Ar). The ³¹P NMR (CDCl₃, δ_P) chemical shifts of 16a–d were observed at δ_P ≈ 26, 24 (2d, J_PP ≈ 24 Hz). The presence of the free thiol moiety in the IR and ¹H NMR spectra as well as the δ_H data of the exocyclic methine-hydrogens confirms the assigned structure and rule out any kind of SH-interaction in the reaction to form a heterocycle.

3.2

3.2 Pharmacology

Bisphosphonates (BPs) have been proven to be an important asset in the treatment and prevention of bone resorption and osteoporosis (Fleisch, 1995, 1988; Balakrishna et al., 2011 ). However, they seem to have different effect on hypocalcemia resulted from diseases such as tumor-induced osteolytic bone diseases, chronic inflammation, and human arthritis (Balakrishna et al., 2011).

3.2.1

3.2.1 Biological activity spectra prediction

Elaborating a new entity in drug discovery needs optimization: Data Base, permeability, and Computer Assisted Programs are used for this purpose. PASS program is an important tool to be used for designing in drug discovery. Prospective biological activity spectra of the molecular structures of synthesized N-BPs 4, 7, 11a–d, 16a–d, and N-BP-acids 5, 8, 12a,b, 17a,b, starting materials 2 and 6 as well as the reference drug zol-18 were predicted in the early stage of the investigation. The computer-assisted molecular modeling (CAMM) program: PASS program 2012.1 version (IBMC, Moscow, Russia) was adopted for designing – in silico – the potential structures (Da Silva et al., 2010; Lagunin et al., 2003). Data of the PASS results were presented as a list of activities (supplementary materials, appendix 1). The study indicated that the main expected biological activity of the designed compounds was the antiosteoarthritic and antiinflammation potencies as common properties among these compounds. Furthermore, the results demonstrated that structures 7, 8, 16a, 16b, 16d, and 17b are the most molecules of potential efficacy while compounds 4, 11a, 11b, 12b, and 16c showed moderate activity. On the other hand, BP-derivatives 2, 5, 6, 11c, 11d, 12a, and 17a displayed only weak potency.

3.2.2

3.2.2 Bioassays

The articular pathology of antigen-induced arthritis (AIA) (Nugent et al., 1993; Österman et al., 1997) involves an initial intense inflammatory synovitis followed by chronic inflammation and severe erosion of articular cartilage and subchondral bone resembling human rheumatoid arthritis (RA) (Nugent et al., 1993; Brackertz et al., 1977). Both the synovitis and joint destruction are unaffected by non-steroidal antiinflammatory drugs (NSAID) (Nugent et al., 1993) but can be suppressed by corticosteroids such as dexamethasone.

The delayed type hypersensitivity granuloma assay is a model of chronic inflammation, in which mice were previously sensitized to methylated bovine serum albumin (mBSA) and surgically implanted with hydroxyapatite disks (two per mouse) soaked in mBSA in order to generate granulomas. This model is not affected by tradition NSAID such as indomethancin (Nugent et al., 1993). However, the antiarthritic properties of bisphosphonic acids (BPs) are poorly understood and have not been demonstrated in any other models of arthritis (Markusse et al., 1990). Furthermore, the antiinflammatory potential of BP-acids has been largely ignored (Daoud et al., 1987; Hyvonen and Kowolik, 1992).

Another article reported that may be BPs and BP-acids take different pathways (Nugent et al., 1993).

3.2.3

3.2.3 Antiarthritis

The antiarthritic assay result was displayed in Table 1. Data reflected the effects of sc administered selective six out of 10 new N-BPs, 4, 7, 11a,b, 16a,b and five out 6 new N-BP-acid salts, 5A, 8A, 12aA, 17aA and 17bA on AIA in a mouse model over 28 days. Zometa® (18, zoledronic acid, Fig. 2), which is N-BP-acid drug was used as a positive control since it revealed an activity against RA (Dunn et al., 1990). In our experiments, 18 (dose: 30–200 mg/kg) exerted significant inhibitory effects on the arthritis, and in general the tested compounds displayed a moderate to good activity in the same dose range as 18. These exceptions were 17aA, 17bA (N-BP-acid salts), which showed higher inhibition (p < 0.001) at 40–200 mg/kg. Compound 8 also displayed a significant inhibition (p < 0.001) at 30–200 at a slightly higher range (100–200 mg/kg). The N-BPs 7, 16a, 16b, and 11a revealed an excellent inhibition (p < 0.001) at 60–200 mg/kg whereas they showed only a marginal activity at a higher dose (100 mg/kg). Finally, compound 11b displayed a good activity at 100 mg/kg, while 4, 5A showed only a marginal one at 100–200 mg/kg, which is not statistically remarkable.

3.2.4

3.2.4 Antiinflammatory, DTH-GRA biological evaluation

The previous eleven compounds and others (all sixteen new compounds) were profiled in a delayed-type hypersensitivity granuloma model (Nugent et al., 1993). Thus, the tested compounds 4, 5A, 7, 8A, 11a–d, 12aA,bA, 16a–d, and 17aA,bA as well as the positive control Zometa® 18 administered sc, and the results were displayed in Table 2. Compound 18 reproducibly inhibited granuloma wet and dry weights. Bisphosphonate esters 7, 16a and 16b significantly inhibited (higher than the positive control) the granuloma in a dose-dependent manner at the doses examined (25–100 mg/kg), while the esters 11a,b and 16c,d exerted significant inhibition, which were almost equivalent to that of 18 over the same dose, at 100 mg/kg. Others 4, 11c,d displayed moderate activity against dry/wet granuloma and were not dose related. In contrast, the acid salts 5A, 8A, 12aA,bA and 17aA,bA, all showed dramatic drop in the activity in relative to their phosphonate ester analogs against the dry/wet weight granuloma.

The structure activity correlation based on the obtained results in Tables 1 and 2 indicates that bisphosphonate esters 7, 16a, 16b showed an excellent efficacy in both models. However, the inhibition of DTH-GRA was not always predictive of the activity of the BPs in the AIA models, suggesting that antiarthritis/antiinflammatory activity may involve more than one mechanism that the effect of BPs as anti-inflammatory is better than the parallel BP-acids (Nugent et al., 1993). In favor of this suggestion speaks the fact that while some BP-acid salts 8A, 17aA and 17bA demonstrated excellent antiarthritic properties, these acids showed only moderate antiinflammatory potential. The fact that S,N-BPs 16a–d induce significant antiinflammatory property implies that the presence of a free thiol moiety enhances the antiinflammatory efficacy of these compounds (Abdou et al., 2006; Almstead et al., 1999; Cawston, 1996; Siestema and Ebetino, 1994 ). Furthermore, this result probably (suggested by the referee) should be due to the different lipophilicity, and permeability, of the esters with respect to the acids. Probably the esters were hydrolyzed in vivo with different time, and in the arthritic test that was performed in 28 days with somministration after 5 or 7 days the difference between the acids and the esters is reduced with respect to the anti-inflammatory test, that was performed in 9 days with a daily somministration. At this point, the comparison of the activity of NBPS with NBP-acids is not completely correct.

Furthermore, the analysis of the biological activity spectra prediction of the new compounds made in this publication is a good example of in silico study of chemical compounds before their experimental investigations. The analysis was performed using the free available web site with the internet version of PASS and PharmaExpert: http://www.ibmc.msk.ru/PASS (CAMM) (Da Silva et al., 2010; Lagunin et al., 2003). The spectrum for a substance is a list of the biological activity types for which the probability to be revealed (Pa) and the probability not to be revealed (Pi) are calculated. Pa and Pi values are independent and their values vary from 0 to 1 (supplementary material, appendix 1). By default, in PASS Pa = Pi value is chosen as a threshold, therefore all compounds with Pa > Pi are suggested to be active. Correlation between the results of the computer-assisted prediction and the in vivo pharmacological results is demonstrated in Table 3.

Table 3 Correlation between the results of the computer-assisted prediction and the in vivo pharmacological results.^a

Comp.	Prediction^b Pa^∗/Pa^∗∗	P/E^c (Anti-arthritis)	P/E (Anti-inflammation)	Comp.	Prediction Pa∗/Pa∗∗	P/E (Anti-arthritis)	P/E (Anti-inflammatn)
18	0.847	+/+	+/+	12a	0.675	+/+	+/+
	0.980				0.980
4	0.702	+/−	+/+	12b	0.742	NT	+/+
	0.612				0.672
5	0.675	+/−	+/+	16a	0.762	+/+	+/+
	0.669				0.684
7	0.782	++	+/+	16b	0.741	+/+	−/+
	0.962				0.124
8	0.442	+/+	+/+	16c	0.770	NT	+/+
	0.512				0.672
11a	0.570	+/+	+/+	16d	0.607	NT	+/+
	0.764				0.776
11b	0.577	+/+	+/+	17a	0.536	+/+	+/+
	0.964				0.904
11c	0.706	NT	+/+	17b	0.589	+/+	+/−
	0.770				0.564
11d	0.526	NT	+/+
	0.967

a AAP, antiarthritis: 74.5%; AAP, antiinflammation: 99.5%.

b Pa^∗/Pa^∗∗: Propable activity (Pa) as antiarthritis/propable activity (Pa) as an antiinflammatoric agent.

c P/E: prediction result/evaluation of bioscreening result.

3.2.5

3.2.5 Toxicity of the promised products

Toxicological study of the most promising synthesized active compounds 7, 16a and 17bA was performed using medium lethal dose 50% (LD₅₀) standard method (Kärber, 1931) in mice in 500, 750, and 1000 mg/kg (body weight), i.e. ∼5 folds of the used antiarthritis and antiinflammatory effective doses. However, no toxic symptoms or mortality rates were observed after 24 h post administrations explaining the safe behavior of the used doses.

3.2.6

3.2.6 Molecular modeling docking studies

Molecular docking of the new bisphosphonate esters (N-BPs) 4, 7, 11a–d, 16a–d, relevant N-BP-acids 5, 8, 12a,b, 17a,b, and the reference drug zoledronic acid (Zol, 18) into the human farnesyl pyrophosphate synthase (hFPPS, PDB code: 2F8C protein) (Rondeau et al., 2006) was performed using the Accelrys Discovery Studio 2.5 modules (Girgis et al., 2011; Shahin et al., 2014). The results are displayed in Table 4.

Table 4 Molecular modeling docking results.

No.	Docking score (kcal/mol)	HB & lipophilic interactions	No.	Docking score (kcal/mol)	HB & lipophilic interactions
18	−49.6774	2 HB (ARG 112)	12a	−48.649	2 HB (ASP 103)
		3 HB (Asp 103)			3 HB (ARG 112)
		1 HB (ASP 107)			1 (+)-π–π-Interaction (ARG 60)

4	−36.1248	1 HB (ASP 103)	12b	−49.8194	2 HB (ASP 103)
					1 HB (ASP 107)
					2 HB (ARG 112)
					1 (+)-π–π-Interaction (ARG60)

5	−39.5462	2 HB (ARG 112)	16a	−43.6762	2 HB (ARG 112)
		2 HB (ASP 103)			1 (+)-π–π-Interaction (ARG112)
		2 HB (ASP 107)

7	−49.2206	1 HB (ARG 112)	16b	−39.6825	1 HB (ASP 103)
		2 (+)-π–π-Interaction (ARG 112)			1 HB (ARG 112)

8	−50.0093	2 HB (ASP 103)	16c	−39.1039	3 HB (ASP 103)
		1 HB (ASP 107)			2 HB (ASP 107)
		1 (+)-π–π-Interaction (ARG 112)			1 (+)-π–π-Interaction (ARG112)

11a	−44.2512	1 HB (GLN 96)	16d	−38.1832	2 HB (ARG 112)
		1 (+)-π–π-Interaction (ARG 60)			1 HB (ASP 103)

11b	−38.5265	2 HB (ARG 112)	17a	−50.8548	1 HB (ASP 174)
					2 HB (ASP 107)
					2 HB (ASP 103)
					1 HB (ARG 112)
					1 HB (GLN 96)

11c	−42.156	1 HB (ARG 112)	17b	−53.4131	2 HB (ASP 107)
		2 (+)-π–π-Interaction (ARG 112)			3 HB (ASP 103)
		1 (+)-π–π-Interaction (ARG 60)			2 HB (ARG 112)
					1 HB (GLN 96)

11d	−43.3475	1 (+)-π–π-Interaction (ARG 60)

Considering N-BP-acids, except 5, BP-acids 8, 17a,b showed markedly higher docking scores than the reference drug 18 while 12a,b showed close docking scores to 18. On the other hand, all N-BPs indicated lower scores than the same reference. Description of the binding sites of interactions between these most active molecules 8, 17a, 17b and the enzyme in comparison with the reference drug 18 is recorded in Fig. 3. The binding interaction forces of the lead compound 18 at the binding sites showed 6H-bonds with the amino acids # Arg 112 (2 HB), Asp 103 (3 HB), and Asp 107 (1 HB), but didn’t show π–π interactions; the Cdock score = −49.67 kcal/mol. This assumes that the interaction of the reference 18 with these three amino acids is essential for the activity and that they represent the catalytic triad of such enzyme. Concerning our tested molecules, compound 17b showed docking score = −53.41 kcal/mol, and 8 HB with Arg 112 (2 HB), Asp 103 (3 HB), Asp 107 (2 HB) and Gln 96 (1 HB). Compound 17a showed docking score = −50.85 kcal/mol and 7 HB with Arg 112 (1 HB), Asp 103 (2 HB), Asp 107 (2 HB), Asp 174 (1 HB) and Gln 96 (1 HB). In addition, compound 8 showed docking score (−50.00 kcal/mol) with 3 HB at the same triad [Asp 103 (2 HB), and Asp 107 (1 HB) and one π–π interaction with Arg 112]. These data are predicting that N-BP-acids have the same mode of action like the reference drug 18, but with higher efficacy as inhibitor for FPPS enzyme and slightly higher activity. While compound 12b showed nearly similar docking score (−49.81 kcal/mol) in comparison with 18, with 5 HB at the same catalytic triad [Arg 112 (2 HB), Asp 103 (2 HB), and Asp 107 (1 HB), and one π–π interaction with Arg 60].

Binding sites of interactions between the most active molecules 8, 17a, 17b and the enzyme in comparison with the reference drug 18. (A1) The ionization state of Zometa (18) after running prepared ligand by adjusting ionization method by selecting the parameter (Rule base and not the pH base). (A2) 3D diagram of 18 with the binding site after rotating to the opposite side-view. It showed one OH of PO3H2 engaged with HB with (Asp103) and (Asp107), while the other OH is free. (A) Docking of 18 with FPPS enzyme (Cdock score = −49.6774); (B) Docking of 8 with FPPS enzyme (Cdock score = −50.0093); (C) Docking of 17a with FPPS enzyme (Cdock score = −50.8548); (D) Docking of 17b with FPPS enzyme (Cdock score = −53.4131).

On the other hand, the prepared ester bisphosphonates 4, 6, 11a–d, 16a–d showed lower docking scores and fewer number of H-bonding than the reference drug 18, as shown in Table 4. The data also indicate that these compounds have less inhibitory activity for FPPS enzyme. Fig. 3 presents the binding sites of interactions between the most active molecules 8, 17a, 17b and the enzyme in comparison with the reference drug 18. In addition, a list of binding sites of interactions between all new molecules 4, 5, 7, 8, 11a–d, 12a,b, 16a–d, and 17a,b as well as the enzyme in comparison with the reference drug 18 are displayed as supplementary material (Appendix 2).

3.2.7

3.2.7 Correlation between molecular docking studies and antiarithritic-DTH-GRA biological evaluation

Since the inhibition of FPPS enzyme is an important step in the inhibition of osteoclastic activity, it is now widely used as an approach for preventing the bone loss associated with osteoporosis and antiarthritis activity.

In this study, we found that the acids 17a,b, 12a,b, and 8 that showed significant docking scores and high numbers of binding-site interactions at the hFPPS enzyme have the highest antiarithritic activity among all the tested compounds and the reference drug 18. The bisphosphonate molecules (4, 7, 11a–d, 16a–d) that showed less docking scores and fewer number of binding-site interactions with hFPPS, also showed lower antiarithritic activity (Tables 1 and 4). The above conclusion indicated that the correlation between the virtual docking scores and the biological activities are closely matching to each other.

4 Conclusion

In summary, efficient and useful two synthetic protocols were applied to construct under mild conditions, various β-aminomethylene-1,1-bisphosphonates (N-BPs) in good to excellent yields (58–88%). We attempted in this research work to utilize the high bone specificity of the third generation of N-BPs that contain primary amine group as one of the side chain (Bijvoet et al., 1995) to elaborate new entities for treating rheumatoid arthritis and chronic inflammation. Other elements such as chemical moieties of potential anticatabolic activities, e.g., the pyrrol and/or pyridine nucleus as well as the biological activity spectra prediction using CAMM programs were also considered. The obtained results indicated that three new BPs have antiarthritic/antiinflammation activities higher than the positive control (BP-drug) at the same doses. These three BP-acids have either free thiol and/or free amino side chain groups. These groups may facilitate the binding to the metal atom in the active site of the matrix metalloproteinases (MMPs) (Almstead et al., 1999; Balakrishna et al., 2011; Cawston, 1996; Cheng and Oldfield, 2004 ). Finally, from previous (Barbier et al., 1986; Hannig et al., 2007; Nugent et al., 1993 ) and present studies, it is concluded that osteoclast activity plays an important role in bone loss in rheumatoid arthritis (RA). This suggests that osteoclast-targeted therapies may effectively prevent skeletal damage in patients with RA. Furthermore, docking studies for new compounds were performed in order to explore their possible binding mode at hFPPS, PDB code: 2F8C.

Acknowledgement

Authors thank the National Research Centre, Dokki, Cairo, Egypt (project # 10010101) for the financial support of the present work.

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Appendix A

Supplementary material

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.arabjc.2016.02.019.

Appendix A

Supplementary material

Supplementary Appendix 1

Supplementary Appendix 1 Computer prediction list of biological activity of new compounds and positive standards.

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Supplementary Appendix 2

Supplementary Appendix 2 Description of binding sites of interactions between all new molecules 4, 5, 7, 8, 11a–d, 12a,b, 16a–d, and 17a,b, 18 to the hFPPS enzyme.

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Show Sections

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Developing efficient protocols for synthesis, antiosteoarthritic, antiinflammatory assessments and docking studies of nitrogen-containing bisphosphonate derivatives

Abstract

Abstract

Keywords

N-bisphosphonates

N-bisphosphonic acids

Antichronic inflammation

CAMM-drug research

Docking studies

1 Introduction

2 Material and methods

2.1 General data

2.2 Preparation of tetraethyl (3-amino-4,5,6,7-tetrahydro-2H[1]benzothieno[2,3-b]pyrrole-2,2-diyl)bisphosphonate (4)

2.3 Preparation of ethyl 4-azido-5-cyano-1-phenyl-1H-pyrazole-3-carboxylate (6)

2.4 Preparation of ethyl 6-amino-5,5-bis(diethoxyphosphoryl)-1-phenyl-1,5-dihydropyrrolo-[3,2-c]pyrazole-3-carboxylate (7)

2.5 Preparation of nitrogen-bisphosphonates (N-BPs, 11a–d)

2.5.1 Ethyl 7-amino-6,6-bis(diethoxyphosphoryl)-5-(methylimino)-1-phenyl-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-3-carboxylate (11a) was obtained as pale yellow crystals

2.5.2 Ethyl 7-amino-6,6-bis(diethoxyphosphoryl)-5-(ethylimino)-1-phenyl-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-3-carboxylate (11b) was obtained as yellow crystals

2.5.3 Ethyl 7-amino-6,6-bis(diethoxyphosphoryl)-1-phenyl-5-(phenylimino)-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-3-carboxylate (11c) was obtained as yellow crystals

2.5.4 (Ethyl 7-amino-5-(cyclohexylimino)-6,6-bis(diethoxyphosphoryl)-1-phenyl-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-3-carboxylate (11d) was obtained as yellow crystals

2.6 Preparation of nitrogen-bisphosphonates 16a–d

2.6.1 Tetraethyl {2-(4-(dimethylamino)phenyl)-2-[(2-mercaptophenyl)amino]ethane-1,1-diyl}bisphosphonate (16a) was obtained as buff crystals

2.6.2 Tetraethyl {2-(4-hydroxyphenyl)-2-[(2-mercaptophenyl)amino]ethane-1,1-diyl}bisphosphonate (16b) was obtained as buff crystals

2.6.3 Tetraethyl {2-(2-hydroxyphenyl)-2-[(2-mercaptophenyl)amino]ethane-1,1-diyl}bis-phosphonate (16c) was obtained as buff crystals

2.6.4 Tetraethyl 2-(2-mercaptophenylamino)-2-(4-methoxyphenyl)ethane-1,1-diyl-bisphosphonate (16d) was obtained as buff crystals

2.7 General procedure for N-BP-acid-salts 5A, 8A, 12aA,bA, 17aA,bA

2.7.1 (3-Amino-4,5,6,7-tetrahydro-2H-[1]benzothieno[2,3-b]pyrrole-2,2-diyl)bisphosphonic acid (5) was obtained as white substance

2.7.2 6-Amino-3-(ethoxycarbonyl)-1-phenyl-1,5-dihydropyrrolo[3,2-c]pyrazole-5,5-diyldiphosphonic acid (8) was obtained as white substance

2.7.3 7-Amino-3-(ethoxycarbonyl)-5-(methylimino)-1-phenyl-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-6,6-diylbis-phosphonic acid (12a) was obtained as white solid

2.7.4 7-Amino-3-(ethoxycarbonyl)-5-(ethylimino)-1-phenyl-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-6,6-diylbisphosphonic acid-salt (12b) was obtained as white substance

2.7.5 2-(4-(Dimethylamino)phenyl)-2-(2-mercaptophenylamino)ethane-1,1-diylbisphosphonic acid-salt (17a) was obtained as yellow crystals

2.7.6 2-(4-Hydroxyphenyl)-2-(2-mercaptophenylamino)ethane-1,1-diylbisphosphonic acid-salt (17b) was obtained as yellow crystals

2.8 Biological procedures

2.8.1 Antigen-induced arthritis

2.8.2 Delayed-type hypersensitivity granuloma

2.8.3 Toxicity evaluation of promised BP-derivatives 7, 16a, and 17bA

3 Chemistry and results of biological studies

3.1 Chemistry

3.2 Pharmacology

3.2.1 Biological activity spectra prediction

3.2.2 Bioassays

3.2.3 Antiarthritis

3.2.4 Antiinflammatory, DTH-GRA biological evaluation

3.2.5 Toxicity of the promised products

3.2.6 Molecular modeling docking studies

3.2.7 Correlation between molecular docking studies and antiarithritic-DTH-GRA biological evaluation

4 Conclusion

Acknowledgement

References

Appendix A

Supplementary material

Appendix A

Supplementary material

Supplementary Appendix 1

Supplementary Appendix 2

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