Heteroatom Chemistry

Synthesis and Anti-osteoclast Activity of Di(1Oxo/Thioxoperhydro-15-[1,3,2] Diazaphospholo [1,5a]Pyridin-1-yl) (4-Substituted Phenyl) Boronates

Journal: Manuscript ID:

Heteroatom Chemistry HC-11-0142 Research Article Borondiphosphonates, Anti-osteoclast, Bone resorption

Wiley - Manuscript type: Keywords:

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Synthesis and Anti-osteoclast Activity of Di(1-Oxo/Thioxoperhydro-15-[1,3,2] Diazaphospholo [1,5-a]Pyridin-1-yl) (4-Substituted Phenyl) Boronates G. Chandra Sekhar Reddy,a M. Veera Narayana Reddy,b C. Radha Rani,a N. Bakthavatchala Reddy,a K. Uma Maheswara Rao,a S.K. Nayakc and C. Suresh Reddya*
a b

Department of Chemistry, Sri Venkateswara University, Tirupati-517502, India Department of Image Science and Engineering, Pukyong National University, Busan, Korea, 608-737 c Bio-Organic Division, BARC, Mumbai, India *Corresponding author. Tel.: + 91-9849694958; Fax: +91-877-2289555; E-mail address: csrsvu@gmail.com (C. Suresh Reddy)
Received

Abstract: A new family of di(1-oxo/thioxoperhydro-15-[1,3,2]diazaphospholo[1,5-a] pyridine-1-yl)(4-substituted phenyl)boronates (4a-j) has been synthesized in a two step process. Reaction of ()-piperidin-2-yl-methanamine (1) phosphoryl/ phosphorothioyl chloride in presence of triethylamine in dry tetrahydrofuran formed the intermediate monochloride (2), which on condensation with p-substituted phenylboronic acids (3a-j) afforded the titled compounds (4a-j). They were characterized by elemental, IR, 1H-,

spectral analyses. All these compounds showed moderate to high anti-osteoclast and

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C-,

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osteoblast activity.

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Synthesis and Anti-osteoclast Activity of Di(1-Oxo/Thioxoperhydro-15-[1,3,2] Diazaphospholo [1,5-a]Pyridin-1-yl) (4-Substituted Phenyl) Boronates

G. Chandra Sekhar Reddy,a M. Veera Narayana Reddy,b C. Radha Rani,a N. Bakthavatchala

Reddy,a K. Uma Maheswara Rao,a S.K. Nayakc and C. Suresh Reddya*
a

Department of Chemistry, Sri Venkateswara University, Tirupati-517502, India Department of Image Science and Engineering, Pukyong National University, Busan, Korea,

608-737
c

Bio-Organic Division, BARC, Mumbai, India

ABSTRACT: A new family of di(1-oxo/thioxoperhydro-15-[1,3,2]diazaphospholo[1,5-a] pyridine-1-yl)(4-substituted phenyl)boronates (4a-j) has been synthesized in a two step process. Reaction of ()-piperidin-2-yl-methanamine (1) phosphoryl/ phosphorothioyl chloride in presence of triethylamine in dry tetrahydrofuran formed the intermediate monochloride (2), which on condensation with p-substituted phenylboronic acids (3a-j) afforded the titled compounds (4a-j). They were characterized by elemental, IR, 1H-,
13

spectral analyses. All these compounds showed moderate to high anti-osteoclast and osteoblast activity.

KEY WORDS: Borondiphosphonates, Anti-osteoclast, Bone resorption, ()-Piperidin-2-ylmethanamine

*Corresponding author. Tel.: + 91-9849694958; Fax: +91-877-2289555; E-mail address: csrsvu@gmail.com (C. Suresh Reddy)

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INTRODUCTION Recently advances in the demands of industry, chemical technology and medicine have opened new opportunities to utilize organoboronphosphorus compounds. 1,3,2-O, N and P heterocycles have attracted great interest due to their valuable pharmacological effects and potential for synthetic applications [1]. They were recently reported to possess matrix metalloproteinase-inhibitory, anticancer [2], pesticidal [3] and antimicrobial [4] activities. The diphosphonates (DPs) have numerous medical applications as drugs for the treatment of bone diseases associated with excessive resorption. Their higher affinity for bone mineral [5] and ability for the prevention of the transition of amorphous calcium phosphate into crystalline hydroxyapatite [6]. They help reduction of hydroxyapatite solubility in vitro [7]. In a number of animal models, they are shown effective in arresting bone loss and in improving the biochemical properties of bone. This led to their successful use as drugs for Paget's disease [8] and osteoporosis [9]. An excellent review appeared recently on bisphosphonates (BPs) as a major class of drugs for the treatm ent of various bone diseases [10].

The organoboron compounds also have important applications in medicine. Recent developments in boron chemistry and pharmacology provided significant progress in designing new organoboron compounds which exhibit hypolipidemic [11], anti-neoplastic [12,13], anti-inflammatory [13,14], anti-osteoporotic [14,15], and analgesic [11] properties. In the past two decades, evidence suggesting that boron is essential for healthy bones and joint function has been mounting, possibly via effects on the balance and absorption of calcium, magnesium and phosphorus [16]. Considering these properties, organoboronphosphates are promising candidates for exploring their synthesis and study of new types of modified DPs analogues.

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The idea that compounds embedded with both the bio-active structural features of organoboron and organophosphonates may have good bioactivity, prompted the author to synthesise several new class of boronodiphosphonates (BDPs) by simple route through the reaction of bioactive groups substituted phosphorochloridates with phenyl boronic acid. Fortunately, these compounds have noticeable anti-osteoporosis properties.

RESULTS AND DISCUSSION Chemistry

Synthesis of the di(1-oxo/thioxoperhydro-15-[1,3,2]diazaphospholo[1,5-a] pyridine1-yl)(4-substituted phenyl) boronates (4a-j) has been accomplished through a two stage process (Scheme-1). Condensation of ()-piperidin-2-yl-methanamine (1) with phosphoryl/ phosphorothioyl chloride in dry tetrahydrofuran (THF) in the presence of triethylamine (TEA) at 0-25 C affords the corresponding monochloride intermediate 1-chlorooctahydro-15[1,3,2]diazaphospholo[1,5-a]pyridine-1-oxide/thione (2). Reaction of 2 with various substituted phenylboronic acids in dry THF in the presence of TEA at 40-45 C for 2-3 h with stirring afforded the title compounds 4a-j in high yields.

All the compounds 4a-j exhibited infra red spectrum (IR) absorption bands for P=O, P=S and P-NH in the regions 1261-1268, 735-756 and 3296-3409 cm-1 respectively [17]. In the proton nuclear magnetic resonance (1H-NMR) spectrum all the aromatic protons resonate as multiplets at H 7.30-8.04. The NH proton gave a broad signal at H 2.68-2.86 due to its coupling with phosphorus and neighboring methylene hydrogens [18]. The NH-CH2 and NCH2 protons resonated as multiplets [18] at H 2.90-3.14 and H 2.58-2.74 respectively. The N-CH proton experienced coupling with the adjacent two methylene protons and resonated as a multiplet at H 3.52-2.21. All the remaining CH2 protons in cyclohexane ring are also

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resonated as multiplets in the region of H 1.10-1.86, due to this reason cyclohexane ring may exist in chair form [19].

NH 2 NH

+ P(X)Cl 3

Et3 N, THF 0-25 C

NH N X P Cl

(1)
OH R B
3 9 4 5 6 6 5 4 7 2 N 1P

(2)
H X O N
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X P O

H N

Et 3N, THF, 35-45 C

Scheme - 1: Synthesis of Di(1-oxo/thioxoperhydro-15-[1,3,2]diazaphospholo [1,5a]pyridin-1-yl) (4-substituted phenyl) boronates (4a-j)

In the carbon nuclear magnetic resonance (13C-NMR) spectra of 4a, 4b, 4e, 4h and 4j all the aromatic carbons resonated at C 120.7-160.8 and the C-9 resonate at C 58.2-59.3. The remaining carbon chemical shifts were observed in the expected regions [18]. The phosphorus nuclear magnetic resonance (31P-NMR) chemical shifts of P=O and P=S were resonated in the region of P 16.72-18.15 and 67.48-72.62 ppm [17] respectively. The atmospheric pressure chemical ionization mass spectra (APCI-MS) of 4a, 4b, 4e, 4h and 4j showed peaks at m/z values corresponding to their protonated molecular ions. In bioactivity the anti-osteoporosis 5
Heteroatom Chemistry

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(3a-j) OH

N B
1 2 3

4a & 4f R H

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(4a-j) 4e & 4j NO2

4b & 4g 4c & 4h 4d & 4i Br F Cl

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activity of compound 4d was exhibited very high osteoblast property and 4b, 4e, 4g and 4i are also showed significant osteoblast activity.

EXPERIMENTAL Chemicals procured from Sigma-Aldrich, Merck and Lancaster were used as such without further purification. All solvents used for spectroscopic and other physical studies were reagent grade and were further purified employing the reported methods. Melting points were determined using a calibrated thermometer by Guna Digital Melting point apparatus. IR spectra were recorded on a PerkinElmer FT-IR 240-C spectro-photometer using KBr optics. 1H-, 13C-,
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P-NMR spectra were recorded on a Bruker-400 and 500 MHz NMR spectrometer operating at

500 MHz for 1H-, 125 MHz for 13C-, 202 MHz for 31P-NMR. Spectra were recorded in DMSOd6 and referenced to TMS (1H & 13C) and 85 % H3PO4 (31P). APCI mass spectra were recorded on a Jeol SX102 DA/600 Mass spectrometer. Elemental analyses were performed on a Thermo Finnigan Insturment at University of Hyderabad, Hyderabad, India. The purity of the compounds were tested by a HPLC-Shimadzu instrument using a C-18 column.

Preparation of di(1-oxo/thioxoperhydro-15-[1,3,2]diazaphospholo[1,5-a] pyridin-1yl)(4-substituted phenyl) boronates (4a-j): A solution of phosphoryl/phosphorothioyl chloride (0.002 mol) in dry THF (10 mL) was added dropwise over a period of 15 min to a stirred solution of ()-piperidin-2-yl-methanamine (1, 0.002 mol) and TEA (0.004 mol) in THF (10 mL) at 0-20 C. After stirring for 1 h at room temperature, formation of intermediate monochloride 2 was ascertained by TLC analysis, TEA hydrochloride was removed from the reaction mixture by filtration. This solution of intermediate is used in the next stage without purification.

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To a stirred solution of various p-substituted phenylboronic acid (3a-j, 0.001 mol) in dry THF (10 mL) and TEA (0.002 mol), the intermediate monochloride 2 solution was added drop wise at 0 C. After the addition, the temperature was slowly raised to 40-45 C and stirred for 2 h. Completion of the reaction was ascertained by TLC conducted on 3:7 mixture of ethylacetate and hexane with an average Rf 0.70. The reaction mixture was filtered to remove TEA hydrochloride and the filtrate on evaporation in a rotaevaporator afforded the crude product. It was further purified by column chromatography on 60-120 mesh silica gel with ethyl acetate: hexane (1:4) as an eluent. The crude product was further purified by crystallization and the title compounds (4a-j) were characterized by IR, 1H-, 13C-, 31P-NMR and mass spectral data.

Spectral data of synthesized compounds Di(1-thioxoperhydro-15-[1,3,2]diazaphospholo[1,5-a]pyridin-1-yl)phenylboronate (4a): Yield 82 %; m.p. 122-124 C; IR(KBr) max [cm-1]: 3395 (-NH), 748 (P=S); 1H NMR (DMSO-d6) : 7.73 (2H, dd, Ar-Hortho), 7.32 (2H, m, Ar-Hpara), 7.36 (1H, m, Ar-Hmeta), 3.413.22 (2H, m, N-CH), 3.01-2.91 (4H, m, NH-CH2), 2.70-2.62 (4H, m, N-CH2), 2.83-2.72 (2H, br, NH), 1.83-1.45 (8H, m, 2 CH2), 1.30-1.14 (4H, m, -CH2); 13C-NMR : 157.7 (C-1 ), 134.1 (C-2 & 6), 130.3 (C-4), 128.7 (C-3 & 5), 58.2 (C-9), 51.5 (C-3), 45.3 (C-7), 39.9 (C-4), 39.6 (C-6), 39.0 (C-5); 31P-NMR : 68.81; APCI-MS m/e: 470 (M+.), 469, 378, 192 (100%); Anal. Calcd for C18H29BN4O2P2S2: C, 45.97; H, 6.21; N, 11.91. Found: C, 45.88; H, 6.19; N, 11.85.

Di(1-thioxoperhydro-15-[1,3,2]diazaphospholo[1,5-a]pyridin-1-yl)(4-fluorophenyl) boronate (4b): Yield 80 %; m.p. 105-107 C; IR(KBr) max [cm-1]: 3409 (-NH), 756 (P=S);
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H NMR (DMSO-d6) [ppm]: 7.93 (2H, dd, Ar-Hortho), 7.79 (2H, m, Ar-Hmeta), 3.50-3.26 (2H,

m, N-CH), 3.13-2.92 (4H, m, NH-CH2), 2.72-2.64 (4H, m, N-CH2), 2.86-2.74 (2H, br, NH), 1.81-1.40 (8H, m, 2 CH2), 1.28-1.11 (4H, m, -CH2); 13C-NMR [ppm]: 160.8 (C-1), 136.1 7
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(C-2 & 6), 147.2 (C-4), 126.0 (C-3 & 5), 59.2 (C-9), 56.8 (C-3), 40.1 (C-7), 40.0 (C-4), 39.8 (C-6), 39.3 (C-5); 31P-NMR [ppm]: 67.48; APCI-MS m/e: 488 (M+.), 484, 483 (100%); Anal. Calcd for C18H28BFN4O2P2S2: C, 44.27; H, 5.78; N, 11.47. Found: C, 44.14; H, 5.66; N, 11.39.

Di(1-thioxoperhydro-15-[1,3,2]diazaphospholo[1,5-a]pyridin-1-yl)(4-chlorophenyl) boronate (4c): Yield 78 %; m.p. 125-127 C; IR(KBr) max [cm-1]: 3401 (-NH), 742 (P=S);
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H NMR (DMSO-d6) [ppm]: 7.75 (2H, dd, Ar-Hortho), 7.49 (2H, m, Ar-Hmeta), 3.46-3.23 (2H,

m, N-CH), 3.04-2.90 (4H, m, NH-CH2), 2.70-2.61 (4H, m, N-CH2), 2.86-2.72 (2H, br, NH), 1.80-1.40 (8H, m, 2 CH2), 1.30-1.10 (4H, m, -CH2); 31P-NMR [ppm]: 72.62; Anal. Calcd for C18H28BClN4O2P2S2: C, 42.83; H, 5.59; N, 11.10. Found: C, 42.80; H; 5.51; N, 11.04.

Di(1-thioxoperhydro-15-[1,3,2]diazaphospholo[1,5-a]pyridin-1-yl)(4-bromophenyl) boronate (4d): Yield 83 %; m.p. 128-130 C; IR(KBr) max [cm-1]: 3392 (-NH), 752 (P=S);

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H NMR (DMSO-d6) : 7.61 (2H, dd, Ar-Hortho), 7.43 (2H, m, Ar-Hmeta), 3.45-3.22 (2H, m, N-

CH), 3.03-2.90 (4H, m, NH-CH2), 2.70-2.61 (4H, m, N-CH2), 2.85-2.71 (2H, br, NH), 1.80-1.40 (8H, m, 2 CH2), 1.25-1.10 (4H, m, -CH2); 13C-NMR [ppm]: 160.1 (C-1), 135.3 (C-2 & 6), 138.4 (C-4), 120.7 (C-3 & 5), 59.0 (C-9), 56.3 (C-3), 40.1 (C-7), 39.9 (C-4), 39.5 (C-6), 39.1

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P-NMR [ppm]: 68.21; Anal. Calcd for C18H28BBrN4O2P2S2: C, 39.36; H, 5.14; N,

10.20. Found: C, 39.25; H, 5.03, N; 10.11.

Di(1-thioxoperhydro-15-[1,3,2]diazaphospholo[1,5-a]pyridin-1-yl)(4-nitrophenyl) boronate (4e): Yield 85 %; m.p. 88-90 C; IR(KBr) max [cm-1]: 3408 (-NH), 735 (P=S); 1H NMR (DMSO-d6) [ppm]: 8.03 (2H, dd, Ar-Hortho), 7.84 (2H, m, Ar-Hmeta), 3.50-3.24 (2H, m, N-CH), 3.12-2.91 (4H, m, NH-CH2), 2.70-2.62 (4H, m, N-CH2), 2.85-2.73 (2H, br, NH), 1.811.41 (8H, m, 2 CH2), 1.30-1.12 (4H, m, -CH2); 13C-NMR [ppm]: 160.7 (C-1), 136.0 (C-2 & 8
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6), 147.5 (C-4), 126.2 (C-3 & 5), 59.0 (C-9), 56.5 (C-3), 40.1 (C-7), 40.0 (C-4), 39.5 (C-6), 39.3 (C-5);
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P-NMR [ppm]: 68.86; APCI-MS m/e: 515 (M+.), 513, 341, 327 (100%), 247,

222, 197; Anal. Calcd for C18H28BN5O4P2S2: C, 41.95; H, 5.48; N, 13.59. Found: C, 41.84; H, 5.39; N, 13.52.

Di(1-oxoperhydro-15-[1,3,2]diazaphospholo[1,5-a]pyridin-1-yl)phenylboronate

(4f):

Yield 76 %; m.p.132-134 C; IR(KBr) max [cm-1]: 3370 (-NH), 1265 (P=O); 1H NMR (DMSO-d6) [ppm]: 7.76 (2H, dd, Ar-Hortho), 7.36 (1H, m, Ar-Hpara), 7.30 (2H, m, Ar-Hmeta), 3.44-3.25 (2H, m, N-CH), 3.02-2.92 (4H, m, NH-CH2), 2.70-2.62 (4H, m, N-CH2), 2.82-2.72 (2H, br, NH), 1.85-1.48 (8H, m, 2 CH2), 1.32-1.15 (4H, m, -CH2); 31P-NMR [ppm]: 16.72; Anal. Calcd for C18H29BN4O4P2: C, 49.34; H, 6.67; N, 12.79. Found: C, 49.01; H, 6.53; N,

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12.59.

Di(1-oxoperhydro-15-[1,3,2]diazaphospholo[1,5-a]pyridin-1-yl)(4-fluorophenyl) boronate (4g): Yield 80 %; m.p. 101-103 C; IR(KBr) max [cm-1]: 3345 (-NH), 1261 (P=O);

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H NMR (DMSO-d6) [ppm]: 7.93 (2H, dd, Ar-Hortho), 7.81 (2H, m, Ar-Hmeta), 3.52-3.28 (2H,

m, N-CH), 3.04-2.91 (4H, m, NH-CH2), 2.72-2.65 (4H, m, N-CH2), 2.86-2.74 (2H, br, NH), 1.80-1.40 (8H, m, 2 CH2), 1.30-1.14 (4H, m, -CH2); 31P-NMR [ppm]: 17.79; Anal. Calcd for C18H28BFN4O4P2: C, 47.39; H, 6.19; N, 12.28. Found: C, 47.27; H, 6.08; N, 12.21.

Di(1-oxoperhydro-15-[1,3,2]diazaphospholo[1,5-a]pyridin-1-yl)(4-chlorophenyl) boronate (4h): Yield 82%; m.p. 95-97 C; IR(KBr) max [cm-1]: 3393 (-NH), 1268 (P=O); 1H NMR (DMSO-d6) [ppm]: 7.79 (2H, dd, Ar-Hortho), 7.51 (2H, m, Ar-Hmeta), 3.48-3.23 (2H, m, N-CH), 3.02-2.90 (4H, m, NH-CH2), 2.70-2.62 (4H, m, N-CH2), 2.85-2.73 (2H, br, NH), 1.811.42 (8H, m, 2 CH2), 1.28-1.14 (4H, m, -CH2); 31P-NMR [ppm]:18.15; APCI-MS m/e: 474 9
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(M+2), 472 (M+.), 445, 444 (100%), 417, 415, 361, 202, 200, 175, 159; Anal. Calcd for C18H28BClN4O4P2: C, 45.74; H, 5.97; N, 11.85. Found: C, 45.66; H,5.91; N, 11.80.

Di(1-oxoperhydro-15-[1,3,2]diazaphospholo[1,5-a]pyridin-1-yl)(4-bromophenyl) boronate (4i): Yield 79 %; m.p. 108-110 C; IR(KBr) max [cm-1]: 3375 (-NH), 1263 (P=O);
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H NMR (DMSO-d6) [ppm]: 7.66 (2H, dd, Ar-Hortho), 7.45 (2H, m, Ar-Hmeta), 3.45-3.20 (2H,

m, N-CH), 3.03-2.90 (4H, m, NH-CH2), 2.71-2.61 (4H, m, N-CH2), 2.85-2.72 (2H, br, NH), 1.81-1.41 (8H, m, 2 CH2), 1.23-1.10 (4H, m, -CH2); 31P-NMR [ppm]: 16.81; Anal. Calcd for C18H28BBrN4O4P2: C, 41.81; H, 5.46; N, 10.83. Found: C, 41.70; H, 5.35; N, 10.75.

Di(1-oxoperhydro-15-[1,3,2]diazaphospholo[1,5-a]pyridin-1-yl)(4-nitrophenyl)boronate (4j): Yield 76 %; m.p. 74-76 C; IR(KBr) max [cm-1]: 3296 (-NH), 1267 (P=O); 1H NMR (DMSO-d6) [ppm]: 8.04 (2H, dd, Ar-Hortho), 7.88 (2H, m, Ar-Hmeta), 3.51-3.26 (2H, m, N-CH), 3.14-2.92 (4H, m, NH-CH2), 2.72-2.63 (4H, m, N-CH2), 2.85-2.74 (2H, br, NH), 1.81-1.42 (8H, m, 2 CH2), 1.30-1.16 (4H, m, -CH2); 13C-NMR [ppm]: 160.8 (C-1), 136.3 (C-2 & 6), 147.8 (C-4), 126.1 (C-3 & 5), 59.3 (C-9), 56.7 (C-3), 40.2 (C-7), 39.9 (C-4), 39.7 (C-6), 39.4 (C-5);
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P-NMR [ppm]: 19.48; APCI-MS m/e: 483 (M+.), 352, 256, 242 (100%), 182, 116; Anal.

Calcd for C18H28BN5O6P2: C, 44.74; H, 5.84; N, 14.49. Found: C, 44.62; H, 5.75; N, 14.41.

Structure - activity relation ship The two phosphonate groups have high affinity for bone mineral with an electronegative atom or groups together and act as a bone hook, allowing rapid and efficient targeting of DPs to bone mineral surfaces. Once localized within bone, the structure and three-dimensional conformation of the side chain as well as substituents in the phosphonate groups in the molecule determine the course of biological activity. 10
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The most potent DPs contain 1 or 2 nitrogen atoms in a heterocyclic moiety with alkyl side chain to a diphosphonate group. The nitrogen-containing diphosphonates (NDPs) are pharmaceutical agents possessing a pyrophosphate-like chemical substructure that confers a strong affinity for calcium. This tridentates structure causes them to chelate circulating calcium and to bind the mineral at bone surfaces.

Biological activity The effect on bone resorption of the new compounds was investigated in terms of the structure activity relationships (SAR) of DPs. Postmenopausal osteoporosis is observed with two important cellular mechanisms that is with reduced osteoblast survival as well as increased osteoclast functions. Hence, for inhibiting osteoclast functions or induces osteoclast cell death, ensuring osteoblast survival becomes essential. Therefore, all the synthesized BDPs (4a-j) were first tested in primary osteoblast cells for their effect on cell viability using MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [20] at concentrations ranging from 10 pM to 1 M. The screening results showed that the most active BDPs were found in the compounds 4d & g that involve the bromo, fluoro substituents respectively shown closer activity to that of standard compound, whereas the moderate activity appeared with compounds 4b,e & i, while 4c and 4h had less effect on osteoblast viability they contain the chloro substituents (Table 1 & 2). Alendronate (Aln) was used as standard and 100 nM of Aln maximally inhibited osteoclastogenesis which was determined by the number of TRAP (tartarate resistant acid phosphatase) positive cells. Biphasic antiosteoclastic action of Aln has been reported in multiple myeloma bone disease [21]. Therefore, the effects of various BDPs were compared at 100 nM concentration along with Aln. Thus, the results obtained are encouraging for further optimization of the anti-resorptive properties of these compounds, and could be used to treat man in the future.

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Materials and methods of bioactivity evaluation: Osteoclasts were prepared from thighbone, shoulder bone, and tibia of 24 h new born rat. The cells were stained with tartrate-resistant acid phosphonates (TRAP) to count the number of positive cells. Primary osteoblast-enriched cultures were prepared from newborn Albino rat calvarias at axenic circumstances. Parietal bones were dissected free from the sutures. The periosteal layers from both sides of the bones were removed carefully. The bones were cut into chips and digested with pancreas for 30 min. After centrifugation, the precipitate cells were diluted with dulbeccos modified eagles medium (DMEM). Then the obtained osteoblast-enriched cell suspension was seeded in culture flask and cultured at 37 C in the presence of 5% CO2. Medium was changed every 3 days. Cell viability was measured using a MTT Cell Proliferation Assay. Absorbance readings of the wells were taken of 492 nm. All the makers of osteoblastic differentiation values were normalized to the relative number of viable cells as determined directly in the 96-well plates using the above mentioned

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proliferation assay.

The osteoclast precursor cells were plated at a density 5104 cells/well in a 96-well plate cultured for 8 days with or without the presence of 10-7 M of BDPs. After culturing (equ. 1), the cells were stained with tartrate-resistant acid phosphonates (TRAP) to count the number of positive cells containing three or more nuclei. The data were inhibitory ratio mean

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Inhibitory ratio (%) =

(Number of control cells) - (Number of treated cells) 100 Number of control cell

(1)

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The cells were plated at a density of 2104 cells/ well in 24-well plates and treated with various concentrations of borondiphosphonates for 72 h. Proliferation was determined using MTT colorimetric assay (equ. 2). The data were proliferative ratio mean SD, n = 3.

Proliferative ratio (%) =

(Number of control cells) - (Number of treated cells) 100 Number of control cell

(2)

In summary, we have reported a series of novel borondiphosphonates that significantly uphold osteoblastic bone formation, besides their roles as intoxicating of osteoclastic bone resorption in cell cultures. This result suggests that these compounds especially compound 4d, might offer discrete advantages over currently available borondiphosphonates, which selectively target osteoclasts. Further investigations of their bioavailability and activities in

vivo are in progress.

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CONCLUSION

The present investigations describe an efficient and simple approach to the synthesis of a variety of the title DPs in satisfactory yields with the use of easily available starting materials. The pharmacological evaluation discusses the anti-resorptive activities of the obtained products. Therefore, study of these results suggest that the compounds have beneficial effects in osteoblast and this holds the hope of developing a new generation of antiresorption drugs after further fine tuning of their chemical structure and detailed pharmacological studies.

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Acknowledgements. The authors thank Prof. C. Devendranath Reddy, Department of Chemistry, S.V. University, Tirupati for his valuable advice and also thank BRNS, BARC, Mumbai for providing financial assistance (2010/37C/26/BRNS/1424).

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REFERENCES [1] Kivel, H.; Zaln, Z.; Thtinen, P. Sillanp, R.; Flp, F.; Pihlaja, K. Eur J Org Chem 2005, 1189-. [2] (a) Srensen, M.D.; Blhr, L.K.A.; Christensen, M.K. Int Pat Appl WO 02/06293; Chem Abstr 2002, 136, 118577. (b) Christensen, M.K.; Blhr, L.K.A. Int Pat Appl WO 03/059921; Chem Abstr 2003, 139, 117538. [3] Shipov, A.E.; Genkina, G.K.; Artyushin, O.I.; Mndzhoyan, Z.O.; Gushchin, B.E.; Chumakova, E.I.; Roslavtseva, S.A.; Eremina, O.Y.; Bakanova, E.I.; Kagan, Y.S.; Ershova, E.A.; Mastryukova, T.A.; Kabachnik, M.I. Russ Chem Bull 1995, 44, 2147. [4] Reddy, P.V.G.; Kiran, Y.B.R.; Reddy, C.S.; Reddy, C.D. Chem Pharm Bull 2004, 52,

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[9] Muehlbauer, R.C.; Russell, R.G.G.; Williams, D.A.; Fleisch, H. Eur J Clin Invest 1971, 1, 336-344.

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[14] Rajendran, K.G.; Burnham, B.S.; Chen, S.Y.; Sood, A.; Spielvogel, B.F.; Shaw, B.R.; Hall, I.H. J Pharm Sci 1994, 83, 1391-1395. [15] Rajendran, K.G.; Chen, S.Y.; Sood, A.; Spielvogel, B.F.; Hall, I.H. Biomed Pharmacother 1995, 49, 131-140. [16] Newnham, R.E. Environ Health Perspect 1994, 102, 83-85. [17] Kishore Kumar Reddy, K.R.; Bhupendra Reddy, C.; Suresh Kumar, K.; Naga Raju, C.; Suresh Reddy, C. Org Commun 2009, 2:2, 28-33. [18] Janardhan Rao, A.; Koteswara Rao, V.; Visweswara Rao, P.; Satheesh Krishna, B.; Naga Raju, C.; Ghosh, S.K. International Journal of Pharma and Bio Sciences 2010, V1(2), 1-5.

[19] Martinek, T. A.; Szolnoki, .; Zaln, Z.; Flp, F. Arkivoc 2007, v, 202-209. [20] Tempone, A.G.; Silva, A.; Brandt, C.A.; Martinez, F.S.; Borborema, S.E.T.; Silveira, M.A.B.; Andrade, H.F. Antimicrob Agents Chemother 2005, 49, 1076-1080. [21] Kitano, M.; Ogata, A.; Sekiguchi, M.; Hamano, T.; Sano, H. J Bone Miner Metab 2005, 23, 48-52.

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Table-1: Compounds and their osteoclast inhibitory activity Compounds 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j Alenb Osteoclast inhibitory ratio mean SDa 36.1 6.5 61.4 2.5 28.4 3.1 68.7 3.1 60.6 5.6 22.1 1.7 66.4 4.3 19.3 4.2 62.7 2.6 48.8 3.4 70.7 6.3

the inhibitory ratio (%) of BDPs of 4a-j were analyzed by MTT assay after 8 days. alendronate (Standard Reference)

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Table-2: Effects of borondiphosphonates on rat calvaria osteoblast proliferation at different concentrations. Compound 4a 4b 4c 4d 4h 10-5 M 26.5 2.4 21.3 2.2 17.1 1.9 32.4 2.9 28.2 2.8 10-6 M 35.2 3.7 28.4 2.8 20.3 2.1 39.8 3.3 32.6 3.8 10-7 M 56.8 5.1 38.3 3.7 23.7 2.2 67.4 7.6 38.5 4.1 10-8 M 20.2 2.1 25. 8 2.5 25.3 2.7 40.6 3.4 27. 7 2.7

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