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Keywords:

  • bisphosphonates;
  • cytogenetic activity;
  • cytotoxic/cytostatic properties;
  • insertion reaction;
  • oxazinones;
  • Schiff bases

Abstract

  1. Top of page
  2. Abstract
  3. Experimental
  4. Results and Discussion
  5. Cytogenetic Activity of Selected Newly Synthesized Bisphosphonates
  6. Conclusion
  7. Acknowledgments
  8. References

Methods for the preparation of various aminomethylene bisphosphonates were developed. The required bisphosphonates were obtained by applying tetraethyl methylenebisphosphonate reagent to different types of oxazinones and the relevant Schiff base derivatives. Based on the prediction results (Pass program), we further estimated the sister chromatid exchange frequency and proliferation rate index values of human lymphocyte cultures after the administration of four newly synthesized bisphosphonates in order to evaluate their cytotoxic/cytostatic and possible antineoplastic potency. The results showed that all four bisphosphonates cause a dose-dependent increase in sister chromatid exchange frequency, followed by a decrease in proliferation rate index in both experiments compared to the control.

Although at least 25% of patients with breast cancer develop skeletal metastases, with bone, the site of disease producing the greatest morbidity, little new research has been performed in the last decade in the setting of bone metastatic breast cancer (1–4). Skeletal complication is often associated with bone pain and includes hypocalcemia and pathological fracture. In addition to inhibiting bone resorption, bisphosphonates (BPs) have also been shown to exhibit antitumor effects such as the inhibition of proliferation and induction of apoptosis in cultured human breast cancer cells. Recent studies reported that BP drug treatment interferes with breast cancer cell adhesion to bone matrix and inhibits cell migration and invasion (2). Diel et al. (5) studied a group of 302 patients with primary breast cancer who had no overt evidence of metastatic disease but had tumor cells in the bone marrow, a risk factor for the development of distant metastases. Patients were randomly assigned to receive oral bisphosphonate drug, clodronate for 2 years (157 patients), and a standard group follow-up (145 patients). The median observation period was over 36 months (p < 0.002), whereby an unexpected reduction in the incidence of osseous metastases was significantly lower in the clodronate group. On the other hand, a randomized, prospective, double-blind, placebo-controlled study (n = 378) with i.v. pamidronate has been evaluated with disappointing results (6). Consequently, identifying a definite adjuvant role of BPs will therefore require a further large number of BPs and randomized studies to resolve these and other issues.

In the pursuance of our interests for the synthesis and investigating the reactivity of BPs against the bone resorption and inflammatory diseases (7–15) as well as elaborating N-BP acids as efficient antiproliferation agents of cancer cells (15), we herein intended to present a synthetic strategy of new series of substituted 5- and 6-N-heterocyclic bisphosphonate esters and our initial biological studies of antitumor potency. The computer-assisted approach, pass program (16–18), was adopted for designing in silico the structures of potentially active molecules for future synthesis. It is based on a robust analysis of structure–activity relationships (18) in a heterogeneous training set currently including about two hundred thousand of biologically active compounds from different chemical series with about 4500 types of biological activity. There are many examples of successful use of pass approach for finding new pharmacological agents (19–22). It is based on a robust analysis of structure–activity relationships (18) in a heterogeneous training set currently including about two hundred thousand of biologically active compounds from different chemical series with about 4500 types of biological activity. Because only the structural formula of chemical compound is necessary to obtain pass predictions, this approach was used to estimate theoretically the cytotoxicity of the speculated BP products and was compared with the prediction of known drugs zoledronic acid, pamidronate, and clodronate (Table 3). Later on, we further estimated the sister chromatid exchange (SCE) frequency and proliferation rate index (PRI) values of human lymphocyte cultures after the administration of four newly synthesized BPs, in order to evaluate their cytotoxic/cytostatic and possible antineoplastic potential. The prospective cytogenetic activity of newly synthesized BPs was based on the results of the prediction that had been carried out, in the earlier stage.

Experimental

  1. Top of page
  2. Abstract
  3. Experimental
  4. Results and Discussion
  5. Cytogenetic Activity of Selected Newly Synthesized Bisphosphonates
  6. Conclusion
  7. Acknowledgments
  8. References

General

Melting points (uncorrected) were determined using open capillary tube on an Electrothermal (variable heater) melting point apparatus. IR spectra were recorded on a JASCO FT-IR 6100 using KBr disk. NMR spectra were measured using a JEOL E.C.A-500 MHz (13C: 125.8 MHz, 1H: 500.6 MHz, 31P: 200.7 MHz) spectrometer. 31P NMR spectra were recorded using H3PO4 (85%) as external reference. 1H and 13C NMR spectra were recorded using trimethylsilane as internal standard in CDCl3 or DMSO-d6. Chemical shifts (δ) are given in p.p.m. The mass spectra were performed at 70 eV on an MS-50 Kratos (A.E.I.) spectrometer provided with a data system. The appropriate precautions in handling moisture-sensitive compounds were observed. The purity of all new samples was verified by microchemical analysis (H/C/N) and spectroscopy.

Synthesis

Reaction of benzooxazines 2a, 2b, 7a, and 7b, with methylenebisphosphonate (1)

Synthesis of 5a, 5b, 8a, and 8b.  General method: To slurry of 9.36 mmol of lithium hydride (LiH) dispersion (80% in mineral oil) in 20 mL anhydrous DMF was added dropwise 3.8 mmol of 1 in 5 mL dry DMF at 0 °C. The reaction mixture was further stirred at r.t. for 1 h, followed by the addition of 15 mL DMF containing 3.6 mmol of 4-(4-methylphenyl)-2,3-benzoxazin-1-one (2a), 4-(4-methoxyphenyl)-2,3-benzoxazin-1-one (2b), 3-phenyl-2,4-benzoxazin-1-one (7a), and 3-methyl-2,4-benzoxazin-1-one (7b), and the mixture was then heated under reflux for 21–30 h (TLC). The reaction mixture was poured into 200 mL of distilled water and HCl (1 N) was added (at −5 °C) until the pH became acidic, and extracted with AcOEt (3 × 50 mL). The combined organic phase was washed, dried over anhydrous sodium sulfate, followed by the removal of the solvent under reduced pressure. The resulting residue was collected, washed several times with cyclohexane, and crystallized from the proper solvent to give the respective BPs 5a, 5b, 8a, and 8b.

Tetraethyl 4-oxo-1-p-tolyl-3,4-dihydroisoquinoline-3,3-diyldiphosphonate (5a).  Yellow crystals; mp 223–225 °C (CH2Cl2); yield: 65%; IR (KBr): νmax 1758 (C=O), 1562 (C=N), 1258, 1244 (P=O), 1148, 1079 (P-O-C)/cm; 1H NMR (CDCl3): δ 1.22, 1.35 (2dt, JH-H =6.8, 4JP-H =4.6 Hz, 2 × 6H, 4H3CC.OP), 2.32 (s, 3H, H3C-tolyl), 3.99, 4.18 (2qt, 3JP-H = 12.6 Hz, 2 × 4 H, 4H2COP) 7.36, 7.56, 7.79, 8.39 (4m, 8H, H-Ar) p.p.m.; 13C NMR (CDCl3): δ 176.95 (d, 2JP-C =14.4 Hz, C=O), 170.6 (d, 3JP-C =6.8 Hz, C=N), 138.8, 137.7, 134.2, 131.3, 129.9, 127.3, 126.8, 124.2 (C-Ar), 77.6 (t, 1JP-C =168.7 Hz, C-P2), 61.7 (d, 2JP-C = 8.3 Hz, CH2OP), 21.5 (CH3-tolyl), 16.7 (d, 3JP-C =5.3 Hz, H3C.COP) p.p.m.; 31P NMR (CDCl3): δP 20.7, 24.7 (2d, 2JP-P =32 Hz, P-C-P) p.p.m.; EI-MS: m/z (%): 508 (18) [M++1], 492 (28) [M+-15], 355 (20) [M+-(15 + 137) (Me + C4H10O3P)], 218 (100) [M+-(15 + 274) {Me + (C4H10O3P)2}], 137 (77), 77 (84); Anal. Calcd for C24H31NO7P2 (507.45): C, 56.80; H, 6.16; N, 2.76; P, 12.21. Found: C, 56.87; H, 6.21; N, 2.64; P, 12.14.

Tetraethyl 1-(4-methoxyphenyl)-4-oxo-3,4-dihydroisoquinoline-3,3-diyldiphosphonate (5b).  Yellow crystals; mp 223–225 °C (CHCl3); yield: 63%; IR (KBr): νmax 1763 (C=O), 1561 (C=N), 1252, 1240 (P=O), 1139, 1071 (P-O-C)/cm; 1H NMR (CDCl3): δ 1.28, 1.33 (2dt, JH-H =7.6, 4JP-H =4.3 Hz, 2 × 6H, 4H3CC.O), 3.75 (s, 3H, H3CO.C6H4), 4.19, 4.38 (2qt, 3JP-H =12.8 Hz, 8H, 4H2COP), 7.22, 7.34, 7.66, 8.36 (4m, 8H, H-Ar) p.p.m.; 13C NMR (CDCl3): δ 175.3 (d, 2JP-C =12.6 Hz, C=O), 159.6 (d, 3JP-C =6.8 Hz, C=N), 138.4, 137.3, 134.6, 131.7, 130.9, 129.8, 127.2, 113.3 (C-Ar), 76.8 (t, 1JP-C =166.7 Hz, C-P2), 62.5 (d, 2JP-C =133 Hz, CH2OP), 56.5 (CH3O), 16.3 (d, 3JP-C =6.4 Hz, H3C.COP) p.p.m.; 31P NMR (CDCl3): δP 24.7, 26.8 (2d, 2JP-P =34.4 Hz, P-C-P) p.p.m.; EI-MS: m/z (%): 524 (11) [M++1], 492 (31) [M+-31, OMe], 355 (24) [M+-(31 + 137) (OMe+ C4H10O3P)], 218 (100) [M+-(31 + 274) {OMe+ (C4H10O3P)2}], 137 (77), 77 (88); Anal. Calcd for C24H31NO8P2 (523.45): C, 55.07; H, 5.97; N, 2.68; P, 11.83. Found: C, 55.15; H, 6.01; N, 2.64; P, 11.71.

Tetraethyl 4-oxo-2-phenyl-3,4-dihydroquinoline-3,3-diyldiphosphonate (8a).  Pale yellow crystals; mp 177–179 °C (CH2Cl2); yield: 68%; IR (KBr): νmax 1758 (C=O), 1565 (C=N), 1260, 1248 (P=O), 1101, 1046 (P-O-C)/cm; 1H NMR (CDCl3): δ 1.24, 1.31 (2dt, JH-H =7.3, 4JP-H =4.3 Hz, 2 × 6H, 4H3CC.OP), 4.07, 4.28 (2qt, 3JP-H =12.8 Hz, 8H, 4H2COP), 7.38, 7.56, 7.68 (3m, 7H, H-Ar), 8.39, 8.42 (2d, JH-H =6.8 Hz, 2H, H-Ar) p.p.m.; 13C NMR (CDCl3): δ 179.6 (d, 2JP-C =12.6 Hz, C=O), 168.6 (d, 2JP-C =11.7 Hz, C=N), 138.6, 135.7, 134.2, 133.1, 130.7, 129.1, 127.2, 126.3 (C-Ar), 67.8 (t, 1JP-C =144.6 Hz, C-P2), 62.2 (d, 2JP-C =9.7 Hz, CH2OP), 16.1 (d, 3JP-C =4.8 Hz, H3C.COP) p.p.m.; 31P NMR (CDCl3): δP 27.4, 28.3 (2d, 2JP-P =32 Hz, P-C-P) p.p.m.; EI-MS: m/z (%): 494 (17) [M++1], 355 (33) [M+-137, C4H10O3P)], 218 (100) [M+-274), (C4H10O3P)2], 137 (78), 77 (92); Anal. Calcd for C23H29NO7P2 (493.4): C, 55.99; H, 5.92; N, 2.84; P, 12.55. Found: C, 56.04; H, 5.98; N, 2.73; P, 12.46.

Tetraethyl 2-methyl-4-oxo-3,4-dihydroquinoline-3,3-diyldiphosphonate (8b).  Yellow crystals; mp 118–120 °C (hexane); yield: 66%; IR (KBr): νmax 1765 (C=O), 1556 (C=N), 1256, 1248 (P=O), 1110, 1050 (P-O-C)/cm; 1H NMR (CDCl3): δ 1.25, 1.34 (2dt, JH-H =7.6, 4JP-H =4.3 Hz, 2 × 6H, 4H3CC.OP), 2.32 (s, 3H, H3C), 3.95, 4.19 (2qt, 3JP-H =12.8 Hz, 8H, 4H2COP), 7.53, 7.93 (2d, JH-H =7.4 Hz, 4H, H-Ar) p.p.m.; 13C NMR (CDCl3): δ 181.5 (d, 2JP-C =12.4 Hz, C=O), 167.6 (d, 2JP-C =10.8 Hz, C=N), 143.8, 133.7, 130.4, 129.3, 128.7, 126.3 (C-Ar), 62.2 (d, 2JP-C =9.7 Hz, CH2OP), 57.8 (t, 1JP-C =148.4 Hz, C-P2), 24.3 (d, 3JP-C =4.4 Hz, CH3), 16.9 (d, 3JP-C =4.8 Hz, H3C.COP) p.p.m.; 31P NMR (CDCl3): δP 23.4, 28.7 (2d, 2JP-P =34 Hz, P-C-P) p.p.m.; EI-MS: m/z (%): 431 (15) [M+], 416 (20) [M+-15], 279 (41) [M+-(15 + 137) (Me+ C4H10O3P)], 142 (100) [M+-(15 + 274) {Me+ (C4H10O3P)2}], 137 (54), 77 (84); Anal. Calcd for C18H27NO7P2 (431.4): C, 50.12; H, 6.31; N, 3.25; P, 14.36. Found: C, 50.24; H, 6.42; N, 3.18; P, 14.21.

Reaction of benzooxazines 9a and 9b with 1

Synthesis of 10a, 10b, 11a, and 11b.  Following the general procedure, a mixture of 9.2 mmol of LiH, 4.2 mmol of the bisphosphonate reagent 1, and 3.6 mmol of 1H benzo[d][1,3]-oxazine-2,4-dione (9a) or 1-methyl-1H-benzo[d][1,3]oxazine-2,4-dione (9b) in 30 mL DMF was heated under reflux for 15–20 h (TLC). After the usual workup, the resulting residue was purified by chromatography on silica gel, gradient eluting using CHCl3–EtOAc to yield compounds 10a and 10b, respectively. Anthranilic acid (11a, 17% yield, mp 146–147 °C) and N-methyl anthranilic acid (11b, 14% yield, mp 170–172 °C) were obtained and identified. Mp and mixed mps, as well as IR and mass spectral data, were comparable to those of an available sample (23).

Tetraethyl 3-oxoindoline-2,2-diyldiphosphonate (10a).  Yellow crystals; mp 189–190 °C (EtOH); yield: 58%; IR (KBr): νmax 3356w (NH), 1755 (C=O), 1535 (C=N), 1254, 1236 (P=O), 1148, 1124 (P-O-C)/cm; 1H NMR (CDCl3): δ 1.25, 1.3 (2dt, JH-H =6.6, 4JP-H =4.7 Hz, 2 × 6H, 4H3CC.OP), 4.15, 4.29 (2qt, 3JP-H =13.3 Hz, 8H, 4H2COP), 7.42, 7.78 (2d, JH-H =7.4 Hz, 4H, H-Ar), 11.05 (s, 1H, HN) p.p.m.; 13C NMR (CDCl3): δ 182.9 (d, 2JP-C =11.4 Hz, C=O), 161.8, 137.3, 127.3, 123.5, 112.3 (C-Ar), 75.5 (t, 1JP-C =137.4 Hz, C-P2), 61.6 (d, 2JP-C =6.7 Hz, CH2OP), 16.4 (d, 3JP-C =4.8 Hz, H3C.COP) p.p.m.; 31P NMR (CDCl3): δP 23.4, 26.7 (2d, 2JP-P =34.6 Hz, P-C-P) p.p.m.; EI-MS: m/z(%): 404 (15) [M+-1], 131 (100) [M+-274, (C4H10O3P)2], 137 (74); Anal. Calcd for C16H25NO7P2 (405.3): C, 47.41; H, 6.22; N, 3.46; P, 15.28. Found: C, 47.50; H, 6.30; N, 3.33; P, 15.12.

Synthesis of the Schiff bases 13a, 13b, 15a, and 15b

A mixture of 10 mmol of 4-(4-methylphenyl)-2,3-benzoxazin-1-one (2a) or 3-phenyl-2,4-benzoxazin-1-one (7a) and hydrazine hydrate (15 mmol) was refluxed in 20 mL boiling ethanol for 4 h. After cooling, the resulting precipitate was filtered, washed with ether, and dried to afford the corresponding hydrazide 12 (84% yield) or 14 (87% yield) (24). Compounds 12 and 14 (10 mmol) were allowed to react with 10 mmol p-cholorobenzaldehyde or p-dimethylamino-benzaldehyde in 20 mL boiling ethanol for 4–6 h (TLC). The resulting mixture was cooled, filtered, washed with 10 mL chloroform, and dried. Crystallization of the collected residue from ethanol afforded the corresponding new Schiff bases 13a and 13b or the known (25) Schiff bases E-3-(7-chlorohepta-2,4,6-triynylideneamino)-2-phenylquinazolin-4(3H)-one (15a, 74% yield) and E-3-(4(dimethylamino) benzylideneamino)-2-phenylquinazolin-4(3H)-one (15b, 76% yield).

E-2-(4-Chlorobenzylideneamino)-4-p-tolylphthalazin-1(2H)-one (13a).  Pale yellow crystals, mp 210–212 °C (MeOH); yield: 72%; IR (KBr): νmax 1679 (C=O), 1610, 1554 (2C=N)/cm; 1H NMR (DMSO-d6): δ 2.34 (s, 3H, H3C-tolyl), 7.42–8.02 (m, 12H, H-Ar), 8.13 (s, 1H, HC=N) p.p.m.; 13C NMR (DMSO-d6): δ 159.8 (C=O), 154.40 (HC=N, exocycl.), 151.2, 149.2, 147.3, 140.6, 138.4, 134.7, 134.3, 131.3, 130.0, 129.2, 129.7, 127.9, 125.4, 123.2 (C-Ar), 20.7 (CH3-tolyl), p.p.m.; EI-MS: in m/z (%): 373 (100) [M+], 374 (20) [M++1], 375 (34) [M++2], 376 (7)[M++3)]; Anal. Calcd for C22H16ClN3O (373.8): C, 70.68; H, 4.31; Cl, 9.48; N, 11.24; Found: C, 70.71; H, 4.40; Cl, 9.34; N, 11.14.

E-2-(4-(Dimethylamino)benzylideneamino)-4-p-tolylphthalazin-1(2H)-one (13b).  Yellow crystals; mp 222–224 °C; yield: 75%; IR (KBr): νmax 1672 (C=O), 1612, 1550 (2C=N)/cm; 1H NMR (DMSO-d6): δ 2.37 (s, 3H, H3C-tolyl), 2.79 (s, 6H, (H3C)2N),7.44–8.05 (m, 12H, H-Ar), 8.21 (s, 1H, HC=N) p.p.m.; 13C NMR (DMSO-d6): δ 160.8 (C=O), 154.4 (H-C=N, exocycl.), 151.7,148.6, 145.1, 142.4, 138. 6, 134.8, 131.3, 130.8, 130.00, 129. 2, 127.9, 127.3, 125.3, 124.1, 122.7 (C-Ar), 112.4 (C-N(CH3)2), 40.4 (CH3)2N), 20.9 (CH3-tolyl) p.p.m.; EI-MS: m/z (%): 384 (18) [M++2], 368 (74) [M+-15], 323 (100) [M+-(15 + 44)]; Anal. Calcd for C24H22N4O (382.46): C, 75.37; H, 5.80; N, 14.65; Found: C, 75.45; H, 5.71; N, 14.57.

Reaction of Schiff bases 13a, 13b, 15a, and 15b with methylenebisphosphonate 1

Synthesis of 16a, 16b, 17a, and 17b.  General method: To a slurry of 9.36 mmol of LiH (80% in mineral oil) in 20 mL of dry DMF was added dropwise of 4.68 mmol of 1 in 5 mL dry DMF at 0 °C. The reaction mixture was further stirred at r.t. for 1 h, and a solution of 3.6 mmol of 13a, 13b, 15a, or 15b in 10 mL DMF was introduced all at once, and the mixture was then heated under reflux for 18–30 h (TLC). After the usual workup, the resulting residue was collected, washed several times with pentane, and crystallized from the proper solvent to give the respective BPs 16a, 16b, 17a, and 17b, respectively.

E -Tetraethyl 2-(4-chlorophenyl)-2-(1-oxo-4-p-tolylphthalazin-2(1H)-ylamino)ethane-1,1-diyldiphosphonate (16a).  Yellow crystals; mp 230–232 °C (EtOH); yield: 68%; IR (KBr): νmax 3364w (NH), 1763 (C=O), 1565 (C=N), 1254, 1250 (P=O), 1148, 1079 (P-O-C)/cm; 1H NMR (DMSO-d6/D2O): δ 1.21, 1.36 (2dt, JH-H =7.6, 4JP-H =4.3 Hz, 2 × 6H, 4H3CC.OP), 2.17 (dd, JH-H =9.5, 2JP-H =18.4 Hz, 1H, HC-P2), 2.24 (s, 3H, H3C-tolyl), 4.19, 4.33 (2 qt, 3JP-H =11.8 Hz, 8H, 4H2COP), 5.98 (dd, JH-H =9.5, 3JP-H =5.8 Hz, 1H, HCN-CH),), 7.36–8.13 (m, 12H, H-Ar) p.p.m.; 13C NMR (DMSO-d6): δ 163.9 (C=O), 149.4, 147.6, 139.6, 136.8, 135.3, 134.8, 132.6, 130.0, 129.2, 127.7, 125.4, 123.2 (C-Ar), 64.6 (d, 2JP-C =13.2 Hz, CH-N), 60.6 (d, 2JP-C =9.7 Hz, CH2OP), 40.2 (t, 1JP-C =124.6 Hz, C-P2), 20.9 (CH3-Ar), 15.8 (d, 3JP-C =5.2 Hz, H3C.COP) p.p.m.; 31P NMR [DMSO-d6]: δP 24.3, 28.6 (2d, 2JP-P =34 Hz, P-C-P) p.p.m.; EI-MS: m/z (%): 662 (17) [M+], 663 (<5) [M++1], 664 (7)[M++2], 665 (<5)[M++3]; Anal. Calcd for C31H38ClN3O7P2 (662.05): C, 56.24; H, 5.79; Cl, 5.36; N, 6.35; P, 9.36; Found: C, 56.41; H, 5.86; Cl, 5.18; N, 6.29; P, 9.16.

E -Tetraethyl 2-(4-(dimethylamino) phenyl)-2-(1-oxo-4-p-tolylphthalazin-2(1H)-ylamino) ethane-1,1-diyldiphosphonate (16b).  Orange crystals; mp 239–241 °C (Ethanol); yield: 59%; IR (KBr): νmax 3364w (NH), 1766 (C=O), 1555 (C=N), 1253, 1250 (P=O), 1148, 1079 (P-O-C)/cm; 1H NMR (DMSO.d6/D2O): 1.17, 1.35 (2dt, JH-H =6.8, 4JP-H =4.7 Hz, 2 × 6H, 4CH3CC.OP), 2.43 (s, 3H, CH3-Ar), 2.72 (dd, JH-H =9.8, 2JP-H = 17.5 Hz, 1H, HC-P2), 2.84 (s, 6H, (H3C)2N), 4.07, 4.21 (2qt, 3JP-H =13.2 Hz, 8H, 4H2COP), 5.87 (dd, JH-H =9.8, 3JP-H =5.4 Hz, 1H, HCN-CH), 7.36–8.13 (m, 12H, H-Ar) p.p.m.; 13C NMR (DMSO.d6): δ 163.9 (C=O), 149.4, 147.6, 139.6, 136.8, 135.6, 133.8, 131.2, 129.4, 126.4, 125.4, 123.6, 114.3 (C-Ar), 64.2 (d, 2JP-C =13.6 Hz, CH-N), 61.2 (d, 2JP-C =9.8 Hz, 4CH2OP), 40.3 ((CH3)2N), 38.6 (t, 1JP-C =129.8 Hz, C-P2), 20.9 (CH3-Ar), 15.8 (d, 3JP-C =4.2 Hz, H3C.COP) p.p.m.; 31P NMR (DMSO.d6): δP 23.1, 27.5 (2d, 2JP-P =34 Hz, P-C-P) p.p.m.; EI-MS: m/z (%): 672 (9) [M++2], 655 (13 [M+-15, Me], 611 (21) [M+-(15 + 44)], 474 (55), 337 (100), 137 (57); Anal. Calcd for C33H44N4O7P2 (670.67): C, 59.10; H, 6.61; N, 8.35; P, 9.24; Found: C, 59.21; H, 6.76; N, 8.19; P, 9.09.

E -Tetraethyl 2-(4-chlorophenyl)-2-(4-oxo-2-phenylquinazolin-3(4H)-ylamino)ethane-1,1-diyldiphosphonate (17a).  Reddish brown crystals; mp 187–190 °C (cyclohexane); yield: 72%; IR (KBr): νmax 3364w (NH), 1772 (C=O), 1553 (C=N), 1262, 1258 (P=O), 1150, 1079 (P-O-C)/cm; 1H NMR (DMSO.d6/D2O): δ 1.09, 1.30 (2dt, JH-H =7.2, 4JP-H =4.3 Hz, 2 × 6H, 4H3CC.OP), 2.24 (dd, JH-H =11.4, 2JP-H =18.8 Hz, 1H, HC-P2), 4.12, 4.28 (2qt, 3JP-H =12.5 Hz, 8H, 4H2COP), 5.78 (dd, JH-H =11.4, 3JP-H =5.5 Hz, 1H, HCN-CH),), 7.36–8.13 (m, 13H, H-Ar) p.p.m.; 13C NMR (DMSO-d6): δ 162.5 (C=O), 158.9, 146.7, 145.6, 134.6, 133.8, 132.4, 129.5, 127.6, 126.2, 125.4, 123.6, 120.3 (C-Ar), 64.0 (d, 2JP-C =14.2 Hz, CH-N), 61.5 (d, 2JP-C =8.8 Hz, 4CH2OP), 42.3 (t, 1JP-C =135.8 Hz, C-P2), 15.8 (d, 3JP-C =4.8 Hz, H3C.COP) p.p.m.; 31P NMR (DMSO-d6): δP 24.3, 28.2 (2d, 2JP-P =34 Hz, P-C-P) p.p.m.; EI-MS: m/z(%): 648 (32) [M+], 649 (7)[M++1], 650 (11) [M++2], 651 (<5) [M++3]; Anal. Calcd for C30H36ClN3O7P2 (648.02): C, 55.60; H, 5.60; Cl, 5.47; N, 5.47; P, 9.56; Found: C, 55.71; H, 5.65; Cl, 5.34; N, 5.31; P, 9.46.

E -Tetraethyl 2-(4-dimethylamino)-2-(4-oxo-2-phenylquinazolin-3(4H)-ylamino)ethane-1,1-diyldiphosphonate (17b).  Yellow crystals; mp 140–142 °C (CH2CL2); yield (69%); IR (KBr): νmax 3364w (NH), 1769 (C=O), 1556 (C=N), 1261, 1255 (P=O), 1148, 1079 (P-O-C)/cm; 1H NMR (DMSO.d6/D2O): δ 1.15, 1.38 (2dt, JH-H =7.4, 4JP-H =4.4 Hz, 2 × 6H, 4H3CC-OP), 2.24 (dd, JH-H =11.6, 2JP-H =17.7 Hz, 1H, HC-P2), 2.81 (s, 6H, (H3C)2N), 4.09, 4.32 (2qt, 3JP-H =12.6 Hz, 8H, 4H2COP), 5.87 (ddd, JH-H =11.6, 3JP-H =4.9 Hz, 1H, HC-CH-P2), 7.35–8.36 (m, 13H, H-Ar) p.p.m.; 13C NMR (DMSO-d6): δ 162.8 (C=O), 152.8, 147.6, 135.6, 134.3, 131.8, 130.6, 128.9, 127.9, 124.4, 120.3, 114.4 (C-Ar), 64.7 (d, 2JP-C =12.2 Hz, CH-N), 61.7 (d, 2JP-C =8.8 Hz, 4CH2OP), 42.4 (t, 1JP-C =137.8 Hz, C-P2), 40.31 (N(CH3)2), 15.3 (d, 3JP-C =4.8 Hz, H3C.COP) p.p.m.; 31P NMR [DMSO-d6]: δP 22.3, 26.2 (2d, 2JP-P =34 Hz, P-C-P) p.p.m.; EI-MS: m/z (%): 657 (14) [M + 1], 612 (25) [M+-44], 474 (61), 337 (100), 137 (57); Anal. Calcd for C32H42N4O7P2 (656.64): C, 58.53; H, 6.45; N, 8.53; P, 9.43; Found: C, 58.62; H, 6.54; N, 8.39; P, 9.31.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Experimental
  4. Results and Discussion
  5. Cytogenetic Activity of Selected Newly Synthesized Bisphosphonates
  6. Conclusion
  7. Acknowledgments
  8. References

During our earlier work, we have concerned with the insertion reactions of different types of benzoxazines with phosphorus (III) and (V) reagents (26–30). In parallel, 4-substituted 2,3-benzoxazin-1-one (2a and 2b) were treated with a slight excess (1.3 equiv.) of methylenebisphosphonate reagent (1) in boiling dimethylformamide (DMF) containing excess LiH (2 equiv of 1) to afford 3,4-dihydroisoquinoline-3,3-diphosphonates 5a and 5b in 65 and 63% yields, respectively. A reasonable mechanism (Scheme 1) of the condensation of 2 with phosphonyl carbanion 1 involves an initial nucleophilic attack of the carbanion center in 1 onto the C(1)=O group, with subsequent ring opening to give 4via the intermediate 3. Under basic conditions, the carbanion, alpha to the diphosphonates, attacked the nitrogen in a SN type reaction to give the substituted isoquinoline-BPs 5a and 5b with concomitant loss of H2O molecule (26–32). Acid hydrolysis of 5a and 5b was undertaken to obtain the corresponding BP acids 6a (69%) and 6b (62%) for potency comparability.

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Figure Scheme 1:.  Synthesis of BPs 5a, 5b, and BP-acids 6a, and 6b.

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On the same basis, 2-substituted 4-oxo-3,4-dihydroquinoline-3,3-diyldiphosphonates 8a (68%) and 8b (66%) were obtained when 3-phenyl-2,4-benzoxazin-1-one (7a) and its 3-methyl analog 7b were allowed to react with methylenebisphosphonate 1 under the same reaction conditions as described in Scheme 2 (24,26–28).

image

Figure Scheme 2:.  Synthesis of BPs 8a and 8b .

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On the other hand, when 1H-benzo[d][1,3]oxazine-2,4-dione (9a) and its N-methyl derivative 9b were allowed to react with methylenebisphosphonate 3-oxoindoline-2,2-diyldiphosphonates, 10a (58%) and 10b (54%) were obtained along with anthranilic acids 11a (or its N-methyl derivative 11b, approximately 15%) as described in Scheme 3 (30–33).

image

Figure Scheme 3:.  Synthesis of BPs 10a and 10b.

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Postulated structures of the newly synthesized compounds 5a, 5b, 6a, 6b, 8a, 8b, 10a, and 10b are in agreement with their IR, 1H-, 13C-, 31P-NMR, molecular weight measurements (MS), and elemental analytical data. The 31P NMR spectroscopy of these compounds showed the presence of two separate doublets with equal 2JP-P coupling constants (34–39).

Expecting an enhancement of biological activity, synthesis of substituted pyrimidine–based BPs was undertaken by applying the reagent 1 to the Schiff bases 13a, 13b, 15a, and 15b. The required substrates derived from the condensation of oxazinones 2a and 7a with hydrazine hydrate to give the corresponding hydrazides 12 and 14, followed by the condensation of the latter with p-chloro- and p-dimethylaminobenzaldehyde to yield the new imines 13a and 13b or the known oxazolones 15a and 15b, respectively.

The configuration of the substitution on the methine-carbon atom at the exocyclic double bond in 15a (as a representative example of Schiff bases) was examined by selective heteronuclear nOe experiments, which were also useful for the assignment of the 13C NMR signals (40). The irradiation of =HC-Ar proton (9.1 p.p.m.) resulted only in the enhancement of the intensity of 13C signals of the carbonyl carbon [C(4), 159.4 p.p.m.] and the carbon of C-Ar-doublet (164.4 p.p.m.), whereas there was no effect on C-2′ signal(127.3 p.p.m.). The lack of nOe between the irradiated methine proton and C-2′ (Structure 15a) indicates the E-configuration of the exocyclic H and the C(2)-phenyl substituent; therefore, =HC-Ar proton is too far from the phenyl moiety.

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Further treatment of these imines with the BP reagent 1 in the usual manner afforded the respective BPs 16a, 16b, 17a, and 17b in reasonable yields (Scheme 4). The configuration of compounds 16 and 17 was assigned as (E) based on the 1H NMR (DMSO-d6/D2O) spectrum of 16a (as a representative example) that revealed two types of methine-protons with different chemical shifts at δ 2.17 (dd, JH-H =9.5, 2JP-H = 18.4 Hz, 1H, HC-P2) and a doublet of doublet centered at δ = 5.98 p.p.m. (JH-H =9.5, 3JP-H =5.8 Hz) ascribed to the proton at the exocyclic asymmetric (chiral) carbon (HCN-CH), which confirmed the presence of the HCN and the HC-P2 in a staggered antiarrangement (E) for the adduct 16a and by extension 16b, 17a, and 17b.

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Figure Scheme 4:.  Synthesis of BPs 16a, 16b, 17a, and 17b.

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Cytogenetic Activity of Selected Newly Synthesized Bisphosphonates

  1. Top of page
  2. Abstract
  3. Experimental
  4. Results and Discussion
  5. Cytogenetic Activity of Selected Newly Synthesized Bisphosphonates
  6. Conclusion
  7. Acknowledgments
  8. References

Sister chromatid exchange is a rapid, sensitive, and simple method for the detection of chromosomal DNA damage and/or subsequent DNA repair. The alteration in cell cycle kinetics as indicated by the suppression of PRI in normal lymphocyte cultures has been proved to be a very useful and sensitive marker of the cytostatic action of various environmental hazards or therapeutic agents (41). In vitro studies have shown that antitumor-alkylating drugs cause an induction of SCE frequency, followed by a decrease in PRI values in normal human lymphocyte cultures (42). In this respect, estimation of SCEs and PRI can provide an initial assessment of the possible antineoplastic activity of newly synthesized drugs. In this study, we estimated the SCE frequency and PRI values of human lymphocyte cultures after the administration of four newly synthesized BPs 5a, 8a, 16a, and 17a (as representative examples), in order to evaluate their cytotoxic/cytostatic and possible antineoplastic potential.

Materials and methods

In vitro SCE and PRI assays

Blood from two different healthy donors, who are of 25–35 years old, non-smokers, not receiving any drugs, not consuming considerable quantities of alcohol, or not having suffered from any kind of infection for the last 15 days, was used for two different experiments, in order to investigate the cytogenetic activities of the new BPs 5a, 8a, 16a, and 17a. Human lymphocyte cultures were prepared by adding in 5 mL chromosome medium consisted of RPMI-1640 (Biochrome, Berlin, Germany) supplemented with 20% fetal calf serum (FCS), 0.63%l-glutamine, 0.63% penicillin/streptomycin, and 2% phytohemagglutinin, at the beginning of culture life, with 11–12 drops of normal human heparinized whole blood, 5 μg/mL 5-Bromo-deoxyuridine (BrdU), and the solutions of the evaluated BPs at final concentrations/culture 12.5, 25, and 50 μm (the solvent was ethanol/H2O:10/90).

The concentrations of the BPs used were equivalent to the doses of BPs already used as drugs such as zoledronic acid, pamidronate, and clodronate (43). The cultures were incubated at 37 °C for 72 h in the dark to minimize the photolysis of BrdU. Colchicine (0.3 μg/mL) was added 2 h before the collection of the cultures. The cells were then collected by centrifugation and exposed to 0.075 MKCl for 10 min. The hypotonic solution spreads the chromosomes and hemolyses the red blood cells. The pellet was fixed three times with methanol/acetic acid (3:1). Drops of concentrated suspension of cells were placed on microslides that allowed to air dry. For SCE and PRI analysis, the slides were stained by a modification of the Fluorescence plus Giemsa procedure to obtain harlequin chromosomes (44).

Statistical analysis

For SCEs, 30 suitably spread 2nd division metaphases from each culture were blindly scored. Each SCE value represents the mean ± standard error (SE). For PRI, at least 100 cells in the 1st, 2nd, 3rd, and higher divisions from each culture were blindly scored. PRI = M+ 2M+ 3M3/100, where M1, M2, and M3 are the per cent values of cells in the 1st, 2nd, 3rd, and higher divisions, respectively. Student’s t-test was performed to determine whether any of the SCE values calculated differed significantly from the controls, and the χ2 test was used for the cell kinetic comparisons (PRI). Simple linear correlation between SCEs frequencies and PRI values was also calculated using Pearson’s product–moment correlation coefficient r. Then, a criterion for testing whether r differs significantly from zero was applied, whose sampling distribution is Student’s t-test with n − 2 degrees of freedom.

Results

We have studied the cytogenetic effect of the four newly synthesized BPs, by estimating the SCE frequency (Table 1, Figure 1) and PRI values (Table 2, Figure 2) in normal human lymphocyte cultures from two different healthy donors. Each compound was tested at three doses (12.5, 25, and 50 μm final concentrations per culture) equivalent to the doses of BPs already used as drugs. The results showed that the four BPs cause a dose-dependent increase in SCE frequency, followed by a decrease in PRI in both experiments compared with the control. Among the four BPs, the heterocyclic methylenebisphosphonates 8a and 5a are more active than the BPs 16a and 17a that are derived from the Schiff bases.

Table 1.   Effect of bisphosphonates (BPs) 5a, 8a, 16a, and 17a on SCE frequency in normal human lymphocyte cultures
Dose (μm)BPS (SCEs ± SE/cell)
5a8a16a17a
1st exp.2nd exp.1st exp.2nd exp.1st exp.2nd exp.1st exp.2nd exp.
  1. *Statistically significant (p < 0.001) increase over the corresponding control (t-test).

  2. **Statistically significant (p < 0.01) increase over the corresponding control (t-test).

  3. ***Statistically significant (p < 0.05) increase over the corresponding control (t-test).

  4. SE, standard error. A minimum of 30 cells were scored for sister chromatid exchange (SCEs) from each culture.

0 (control)7.68 ± 1.637.59 ± 0.827.68 ± 1.637.59 ± 0.827.68 ± 1.637.59 ± 0.827.68 ± 1.637.59 ± 0.82
12.510.50 ± 2.09*10.07 ± 0.81*11.14 ± 2.29*12.03 ± 0.96*8.09 ± 1.588.16 ± 1.428.22 ± 1.458.10 ± 0.59
2511.63 ± 1.74*11.60 ± 0.43*12.18 ± 2.39*13.01 ± 0.71*8.86 ± 1.83***8.90 ± 0.59***8.73 ± 1.818.69 ± 1.59
5013.18 ± 2.16*12.96 ± 0.78*14.04 ± 1.93*14.67 ± 0.93*10.09 ± 2.19**10.36 ± 1.50**9.14 ± 1.83**9.96 ± 0.86**
image

Figure 1.  Sister chromatid exchanges of normal human lymphocyte cultures after (A) 5a, (B) 8a, (C) 16a, and (D) 17a administration.

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Table 2.   Effect of bisphosphonates (BPs) 5a, 8a, 16a, and 17a on proliferation rate index (PRI) in normal human lymphocyte cultures
Dose (μm)BPs (PRI)
5a8a16a17a
1st exp.2nd exp.1st exp.2nd exp.1st exp.2nd exp.1st exp.2nd exp.
  1. *Statistically significant (p < 0.001) decrease over the corresponding control (χ2 test).

0 (control)2.302.192.302.192.302.192.302.19
12.52.172.112.03*1.98*2.182.122.222.15
252.02*1.96*1.90*1.85*2.162.062.242.06
501.86*1.83*1.68*1.71*1.91*1.85*2.112.01
image

Figure 2.  PRI of normal human lymphocyte cultures after (A) 5a, (B) 8a, (C) 16a, and (D) 17a administration.

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Bisphosphonates 5a caused a dose-dependent, statistically significant increase in SCE frequency at all three concentrations tested (p < 0.001). Proliferation rate index values were statistically significantly diminished at 25 and 50 μm. Bisphosphonates 8a, the most cytogenetically active one, causing a statistically significant increase in SCE frequency at all concentrations tested (p < 0.001), followed by an equally significant decrease in PRI (p < 0.001) in both experiments. Bisphosphonates 16a imposed a dose-dependent increase in SCE frequency at all three concentrations tested, which became statistically significant at 25 μm (p < 0.05). Proliferation rate index values were statistically significantly decreased at 50 μm (p < 0.001). Bisphosphonates 17a was the least active compound, causing a statistically significant increase in SCE frequency only at 50 μm (p < 0.01). Proliferation rate index values were decreased compared with the control, but this decrease did not reach statistically significant level at any of the concentrations tested.

The Pearson’s correlation coefficient (r) was used as a simple linear correlation between SCEs frequencies and PRI values. Statistically significant correlation was observed between the magnitude of the SCE induction and the PRI depression for BP 5a (r = −0.9973, p < 0.05) and BP 8a (r = −0.9999, p < 0.01), showing that there is a higher correlation for BP 8a. A statistically significant negative (r) coefficient is indicative of antineoplastic potential of the compound.

Discussion

In the present study, we evaluated the cytogenetic activity of four newly synthesized BPs in normal human lymphocyte cultures by estimating SCEs and PRI, in order to explore their antineoplastic potential. All four compounds were cytogenetically active, causing dose-dependent increase in SCE frequency and decrease in PRI values, which became statistically significant at different concentrations. The order of activity is as follows: 8a 5a 16a 17a. The observed different activities can be either due to the different active groups of the molecules or to their different sizes, which could affect their uptake from the cells. Among the four compounds, only 8a and 5a had a statistically significant negative Pearson’s coefficient r, suggesting possible antineoplastic activity. Finally, because in the present study a strong correlation (p < 0.01) between SCE enhancement and PRI depression was established in lymphocyte cultures (in vitro), further studies in experimental tumors in vivo for evaluating the possible antineoplastic potential by these and the other synthesized compounds are warranted.

Furthermore, the analysis of biological activity spectra prediction for the synthesized compounds made in this publication is a good example of in silico study of chemical compounds before their experimental investigations. Anyone can do the same analysis using the free available website with the internet version of pass and PharmaExpert: http://www.ibmc.msk.ru/PASS/. A biological activity 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. Biological activity spectra were predicted for representatives of new BPs and the known drugs: zoledronic acid, pamidronate, and clodronate with pass 2009.1 version (Table 3). As it can be observed from the pass results, only for four of the eight compounds (5a, 8a, 16a, and 17a), cytotoxicity was predicted. Some common types of activities for new class of BPs and the known BP drugs (zoledronic acid, pamidronate, and clodronate) were observed. Thus, such activities as bone and lung cancer were predicted almost for all tested new compounds as well as for zoledronic acid, pamidronate, and clodronate. By default, in pass, Pa = Pi value is chosen as a threshold; therefore, all compounds with Pa > Pi are suggested to be active.

Table 3.   Computer prediction of biological activity
CompoundsPredictive activityPaCompoundsPredictive activityPa
5aAntineoplastic (lung cancer)0.42216bAntineoplastic (lung cancer)0.843
Antineoplastic (non-Hodgkin’s lymphoma)0.370Antineoplastic (ovarian cancer)0.758
Antineoplastic (bone cancer)0.344Antineoplastic (melanoma)0.690
Antineoplastic (ovarian cancer)0.427Bone formation stimulant0.527
Cytotoxic0.268Antiosteoporotic0.514
Bone disease treatment0.502
Antileukemic0.431
Antineoplastic (bone cancer)0.408
Antineoplastic (brain cancer0.453
5bAntineoplastic (lung cancer)0.45417aAntineoplastic (lung cancer)0.490
Antineoplastic (ovarian cancer)0.430Antineoplastic (ovarian cancer)0.594
Antineoplastic (brain cancer)0.396Antineoplastic (gastric cancer)0.535
Antineoplastic (bone cancer)0.341Antineoplastic (melanoma)0.475
Antineoplastic0.330Cytotoxic0.270
Antiosteoporotic0.21017bAntineoplastic (lung cancer)0.842
8aAntineoplastic (bone cancer)0.356Antiarthritic0.780
Cytotoxic0.218Antineoplastic (ovarian cancer)0.751
Antineoplastic (melanoma)0.694
Antineoplastic (gastric cancer)0.510
Bone formation stimulant0.503
Antiosteoporotic0.484
Antileukemic0.473
PamidronateBone disease treatment0.995
8bBone formation stimulant0.671Antiosteoporotic0.994
Bone disease treatment0.509Bone formation stimulant0.985
Antineoplastic (bone cancer)0.975
Osteoclast antagonist0.912
Antineoplastic (multiple myeloma)0.718
Antineoplastic (sarcoma)0.573
Antineoplastic (breast cancer)0.542
Antileukemic0.451
 16aAntineoplastic (lung cancer)0.672Paget’s disease treatment0.423
Antineoplastic (ovarian cancer)0.585Prostate cancer treatment0.380
Antineoplastic (melanoma)0.415Antineoplastic0.348
Cytotoxic0.255(hematological cancer)Miotic0.350
Zoledonic acidBone disease treatment0.9228
Antiosteoporotic0.916
Antineoplastic (bone cancer)0.874ClodronateBone disease treatment0.996
Bone formation stimulant0.857Bone formation stimulant0.995
Antineoplastic (bone cancer)0.770
Antineoplastic (sarcoma)0.596
Osteoclast antagonist0.708Antineoplastic (multiple myeloma)0.499
Antineoplastic (multiple myeloma)0.666Antiosteoporotic0.488
Antineoplastic (lung cancer)0.486Antineoplastic (lung cancer)0.435
Antineoplastic (sarcoma)0.466Antineoplastic (hematological cancer)0.412
Antileukemic0.403Miotic0.415
Antineoplastic (hematological cancer)0.351Paget’s disease treatment0.266
Antineoplastic (brain cancer)0.345
Antineoplastic (breast cancer)0.277

Conclusion

  1. Top of page
  2. Abstract
  3. Experimental
  4. Results and Discussion
  5. Cytogenetic Activity of Selected Newly Synthesized Bisphosphonates
  6. Conclusion
  7. Acknowledgments
  8. References

In conclusion, reactions between oxazinones and Horner reagent, methylenebisphosphonate, are of significant value from the synthetic and applied points of view. We described convenient routes to a new class of bisphosphonate ester derivatives. The protocol demonstrated an efficient site selective attack for insertion reactions providing an easy route to isoquinolines and pyridazines-based BPs, in high yields. The recognized actions of the newly synthesized BPs on lymphatic cell adhesion, invasion, and cell viability indicate that these agents may have a wider role to play in the treatment of patients suffering from cancers with a propensity to metastize to breast and bone.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Experimental
  4. Results and Discussion
  5. Cytogenetic Activity of Selected Newly Synthesized Bisphosphonates
  6. Conclusion
  7. Acknowledgments
  8. References

Financial support from the Egyptian Academy of Scientific Research and Technology is gratefully acknowledged.

References

  1. Top of page
  2. Abstract
  3. Experimental
  4. Results and Discussion
  5. Cytogenetic Activity of Selected Newly Synthesized Bisphosphonates
  6. Conclusion
  7. Acknowledgments
  8. References
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