Efficient Synthesis of Benzidronate Applying of Phosphorus Trichloride and Phosphorous Acid

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Send Orders for Reprints to [email protected] 78 Letters in Drug Design & Discovery, 2015, 12, 78-84 Efficient Synthesis of Benzidronate Applying of Phosphorus Trichloride and Phosphorous Acid

Alajos Grün1, Rita Kovács1, Dávid Illés Nagy1, Sándor Garadnay2, István Greiner2 and György Keglevich1,*

1Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, 1521 Budapest, Hungary 2Gedeon Richter Plc., 1475 Budapest, Hungary

Abstract: The optimized synthesis of benzidronate involves the reaction of phenylacetic acid with 3 equivalents of phosphorus trichloride and 1 equivalent of phosphorous acid in methanesulfonic acid at 85 °C followed by hydrolysis, pH adjustment and purifications. The positive effect of phosphorous acid on the yield is a novel observation and is probably limited to this model. The heap of our experimental data obtained by varying the molar equivalent quantities of the P-reagents (PCl3 and P(OH)3) and reacting also phenylacetic chloride and ethyl phenylacetate allowed us to draw conclusions on the possible intermediates.

Keywords: Benzidronate, intermediates, optimization, phosphorous acid, phosphorus trichloride.

1. INTRODUCTION alendronate [9], pamidronate [10], fenidronate [11] and etidronate [12]. Our finding was to react the corresponding Dronates/dronic acids are important drugs in the carboxylic acids with 3.2 equivalents of phosphorus treatment of osteoporosis and related illnesses, such as the trichloride in methanesulfonic acid without the addition of Paget-disease, hypercalcemia and they are also effective phosphorous acid. Our recent results have been summarized drugs in bone oncology [1-3]. Bisphosphonates are used as [13, 14]. To broaden the sphere of dronates, we intended to inhibitors of bone resorption. Due to their chemical structure, study also the synthesis of benzidronate. According to the these compounds are able to chelate calcium ions and able to literature, benzidronate was mainly synthesized from - bind to the bone mineral. The P–C–P moiety is responsible oxophosphonates (obtained by the Arbuzov reaction) by the for the strong affinity towards the calcium ions. The hydroxy addition of the P-species (e.g. dimethyl phosphite or group on the central carbon atom increases the affinity to trimethylsilyl phosphite) on the carbonyl group followed by calcium. The side chain determines the biological activity. hydrolysis [15-20]. Not much data can be found on the The first generation of dronates, such as etidronate, synthesis of benzidronate starting from phenylacetic acid and fenidronate and clodronate form non-hydrolysable analogues P-reagents, such as phosphorus trichloride and phosphorous of ATP, while the second and third generations of acid [21, 22]. bisphosphonates containing nitrogen atom in the side chain, act as enzyme inhibitors in osteoclast cells which are responsible for bone resorption. They interfere in several 2. RESULTS AND DISCUSSION processes in these cells, and finally cause apoptosis [1-4]. The first task was the preparation of the benzidronic acid Anti-tumor effects were also described [5]. standard (4). This was done according to a literature The most widespread synthetic method for dronic procedure [23]. Reacting phenylacetyl chloride (1) with acids/dronates involves the reaction of the suitably trimethyl phosphite, -oxophosphonate 2 was obtained that substituted acetic acid (or in the simplest cases acetic acid or was reacted immediately further with dimethyl phosphite benzoic acid) with phosphorus trichloride and phosphorous according to a one-pot protocol. The tetramethyl ester of acid in different solvents [6, 7]. The patent literature on the benzidronic acid (3) so obtained was converted to synthesis of dronic derivates is rather diverging, and the benzidronic acid (4) by acidic hydrolysis (Scheme 1). This experimental data in respect of the excess and molar ratio of standard was utilized in obtaining the titration curve in the potentiometric titration. the P-components (PCl3 and P(OH)3), solvents, work-up and purification are rather contradictory. We clarified the Then, we studied the formation of benzidronate (6, that is situation and elaborated the optimized synthesis of the disodium salt of benzidronic acid) in the reaction of zoledronic acid [8], risedronic acid [8], ibandronate [9], phenylacetic acid (5), phosphorus trichloride and phosphorous acid performed in methanesulfonic acid (MSA) (Scheme 2).

The reaction was carried out applying phosphorus *Address correspondence to this author at the Department of Organic Chemistry and Technology, Budapest University of Technology and trichloride and phosphorous acid in different molar Economics, 1521, Budapest, Hungary; Tel: 36-1-4631111/5883; quantities. After a reaction at 85 °C for 1 day, the mixture Fax: 36-1-4633648; E-mail: [email protected] was hydrolyzed at 105 °C for 4 h. Then the pH was adjusted

0 85 °C 85 °C O O (MeO)3P (MeO)2P(O)H Cl P OMe CHCl3 CHCl3 MeO O 1 2

O OH O O OH O MeO P P OMe HO P P OH 105 °C MeO OMe HO OH cc. HCl

3 4 Scheme (1). Synthesis of the Benzidronic Acid Standard by the Arbuzov Approach. 1) 85 °C/24 h O PCl and/or H PO OH ONa O 3 3 3 P MeSO3H OH OH 2) 105 °C/4 h P 5 H2O OH ONa 3) pH adjustment O 6

Scheme (2). Synthesis of Benzidronate from Phenylacetic Acid Using Phosphorus Trichloride and Phosphorous Acid. to 2.5, and the precipitated crude benzidronate (6) was (Table 1/Entries 6, 7, 9 and 10), as in the case of other purified by newer precipitation from water solution by dronates/dronic acids, such as risedronic acid [8], zoledronic methanol, and by a subsequent digestion by a MeOH–H2O acid [8], ibandronate [9], alendronate [9], pamidronate [10], mixture. The benzidronate samples obtained were analyzed etidronate [12] and fenidronate [11], the outcome of the by potentiometric titration. The purity obtained by 31P NMR reaction of the corresponding carboxylic acid with 2 measurements also bore some information. The experimental equivalents of phosphorus trichloride and 1 equivalent of data are listed in Table 1. The use of 3 equivalents phosphorous acid was in most cases [8–10] lower, or in a phosphorous acid or 1 equivalent of phosphorus trichloride few cases [11, 12] similar as that obtained in the presence of was practically inefficient (Table 1/Entries 1 and 2). 3.2 equivalents phosphorus trichloride. It is important that Measuring 1 equivalent of both phosphorus trichloride and these experiences are related to cases with MSA being the phosphorous acid, benzidronate (6) was obtained in a modest solvent. In only one instance, during the synthesis of yield of 15%, and the purity was only 66% (Table 1/Entry pamidronic acid in sulfolane [10] was phosphorous acid 3). An increase in the quantity of phosphorous acid to 2 involved in the reaction by the formation of a more reactive equivalents led to a significant improvement, the yield of species, (HO)2P-O-PCl2 in reaction with phosphorus dronate 6 was 44%, although the purity was only 77% (Table trichloride. In the present case, we must assume the 1/Entry 4). The use of 2 equivalents of phosphorus formation of this intermediate that may attack the carbonyl trichloride alone allowed a dronate yield of 36% and a purity group of the phenylacetic acid derivatives formed as of 89% (Table 1/Entry 5). It is noteworthy that adding also 1 intermediates. One of these intermediates may be or 2 equivalents of phosphorous acid to the mixture, the yield phenylacetyl chloride (1) that is the result of the interaction of dronate 6 was increased to 74% and 72%, respectively (and of phenylacetic acid with 1 equivalent of phosphorus dronate 6 was obtained in purities of 95%) (Table 1/Entries 6 trichloride. The other intermediate may be the mixed and 7). In the next experiment, only 3.2 equivalents of anhydride of phenylacetic acid (5) and MSA shown as 7 that phosphorus trichloride were applied as the P-reagent. may be formed in two ways, by the reaction of acid chloride Benzidronate (6) was isolated in a yield of 46% and in a purity (1) with MSA, or by the reaction of carboxylic acid (5) with of 95% (Table 1/Entry 8). Adding also 1 or 2 equivalents of methanesulfonyl chloride formed from MSA and phosphorus phosphorous acid to the mixture, a significant increase in the trichloride (Scheme 3). yield to 81% and 79%, respectively, was experienced. The In MSA as the solvent, there is only a slight chance that purities amounted to 98% and 99%, respectively (Table phosphorus trichloride will react with phosphorous acid to 1/Entries 9 and 10). furnish (HO)2P-O-PCl2, as due to the larger excess of MSA, On the basis of our earlier experiences [8-12], the results the chance for the formation of methanesulfonyl chloride is covered by Entries 1, 2, 5 and 8 of Table 1 leading to yields greater. At the same time, phenylacetyl derivatives 1 and 7 of 0%, 2%, 36%, 46%, respectively, are in full agreement may be more capable of reacting with (HO)2P-O-PCl2, that is with our expectations. However, the positive effect of the 1 present only in a low concentration, than the analogous or 2 equivalents of phosphorous acid used beyond the 2 or 3 carboxylic acid derivatives formed from other substituted equivalents of phosphorus trichloride was quite surprising carboxylic acids. 80 Letters in Drug Design & Discovery, 2015, Vol. 12, No. 1 Grün et al.

O Me 1 + MeSO H MeSO2Cl + 5 3 - HCl O - HCl S O O 7

Scheme (3). Possible Routes to the Anhydride Type Intermediate 7.

Table 1. Synthesis of Benzidronate (6).

Reactants Purity (%) Yield of 6a Entry On the Basis of Potentiometric Titrationa (%) PCl3 (equiv.) H3PO3 (equiv.)

1 0 3 0 0

2 1 0 68 2

3 1 1 66 15

4 1 2 77 44

5 2 0 89 (99)b 36

6 2 1 95 (100)b 74

7 2 2 95 (100)b 72

8 3.2 0 95 (100)b 46

9 3 1 98 (100)b 81

10 3 2 99 (100)b 79 aFrom at least two parallel experiments. bOn the basis of 31P NMR

1) 85 °C/20 h PCl3 (2.1 equiv.) 25 °C/6 h MeSO3H PCl3 or SOCl2 2) 105 °C /4 h (1.1 equiv.) 1 H2O 5 and/or 6 MeSO3H 7 3) 50% NaOH/H2O pH 2.5

Scheme (4). Two-Step Preparation of Benzidronate.

Table 2. Synthesis of Benzidronate in Two Steps.

Inorg. Halide P-reactant Purity (%) Yield of 6a Entry (1.1 equiv.) (2.1 equiv.) On the Basis of Potentiometric Titrationa (%)

1 PCl3 PCl3 94 46

2 SOCl2 PCl3 86 24 aFrom at least two parallel experiments.

To justify the intermediacy of acid chloride 1 and mixed The experimental data were collected in Table 2. One can anhydride 7, the synthesis of benzidronate (6) was also see that the reaction with phosphorus trichloride in two steps led performed in two steps. In the first step, phenylacetic acid to the same result obtained by the one-step variation; the yield (5) was reacted with 1.1 equivalents of phosphorus of benzidronate (6) was 46% in both cases, while the purity was trichloride or thionyl chloride in MSA at room temperature 94-95% (Table 2/Entry 1 vs. Table 1/Entry 8). At the same time, for 4 h. Then, in the second step, the mixture of acid chloride when thionyl chloride was used in the first step, dronate 6 was 1 and mixed anhydride 7 was reacted further with 2.1 obtained in only a yield of 24% in a less pure (86%) form. As equivalents of phosphorus trichloride as described above earlier [9-13], these two-step accomplishments justify the (Scheme 4). involvement of intermediates 1 and 7. Efficient Synthesis of Benzidronate Applying Letters in Drug Design & Discovery, 2015, Vol. 12, No. 1 81

1) 85 °C/20 h The last three variations were also performed with ethyl PCl and/or H PO O 3 3 3 phenylacetate (8) as the starting material. Using phosphorus MeSO3H trichloride and phosphorous acid in 1:1, 2:0 and 2:1 6 Y 2) 105 °C/4 h equivalent ratios, the yield of benzidronate (6) was 44%, H2O 55% and 47%, respectively, with purities of 72%, 94% and Y Cl OEt 3) pH adjustment 82%, respectively (Table 3/ Entry 5-7). From among these data, the one obtained with 2 equivalents of phosphorus 18 trichloride is of interest, as shown that the ester (8) is a better Scheme (5). Synthesis of Benzidronate from Phenylacetic Acid starting material than the chloride (1) on the way toward the Derivatives. formation of benzidronate (6). In summary, the formation of benzidronate in the In our last experiments, we attempted to utilize reaction of phenylacetic acid with phosphorus trichloride and phenylacetyl chloride (1) and ethyl phenylacetate (8) in the phosphorous acid in MSA as the solvent was optimized. The synthesis of benzidronate (6). The P-reagents, phosphorus optimum set involved the use of 3 equivalents of phosphorus trichloride and phosphorous acid were applied in different trichloride and 1 equivalent of phosphorous acid at 85 °C. equivalent quantities, and the reactions were carried out as The favorable role of phosphorous acid in this particular above (Scheme 5). case, may be to form (HO)2P–O–PCl2 as an active P-species The experimental data are listed in Table 3. Reacting toward mixed anhydride PhCH2C(O)OSO2Me as a possible phenylacetyl chloride (1) with 2 equivalents of phosphorous intermediate as substantiated by our experimental data. acid, there was no benzidronate (6) formation (Table 3/Entry 1). This confirms that phosphorous acid alone is unreactive 3. EXPERIMENTAL and may be activated by reaction with phosphorus trichloride. The use of 1 equivalent of phosphorus trichloride 3.1. General and the same quantity of phosphorous acid led to a yield of 50% with a purity of 76% (Table 3/Entry 2), while The 31P, 13C and 1H NMR spectra were obtained on a measuring in only phosphorus trichloride in a quantity of 2 Bruker AV-300 spectrometer at 121.5, 75.0 and 300 MHz; equivalents, benzidronate (6) was obtained in a yield of 27%, Chemical shifts are downfield relative to 85% H3PO4 or in a purity of 93% (Table 3/Entry 3). This experiment is the TMS. The couplings are given in Hz. The benzidronate “equivalent” of that starting from phenylacetic acid (5) and content of the samples was determined by potentiometric using 3 equivalents of phosphorus trichloride, still the latter acid-base titrations on a Mettler DL77 potentiometric one is more efficient (yield: 46%, purity: 95%) titrator. (Table 1/Entry 8). This experience may refer to the fact that The titration curve for pure benzidronic acid (4) the predominant intermedier is mixed anhydride 7 and not synthesized by us and for that of the sample obtained from phenylacetyl chloride 1. The reaction applying 2 equivalents the reaction marked by Table 1/Entry 9 are shown in Figs. of phosphorus trichloride together with 1 equivalent of (1 and 2), respectively. phosphorous acid was again the best experiment in the series, furnishing dronate 6 in a yield of 51% and with a purity of 85% (Table 3/Entry 4). This experiment is the 3.2. Preparation of the Benzidronic Acid standard “equivalent” to that marked by Entry 9 of Table 1. However, To 11 mL (0.08 mol) of phenylacetyl chloride (1) in 40 the latter variation was more efficient giving product 6 in a mL of chloroform was added dropwise the solution of 9.8 yield of 81% confirming again the predominant intermediacy mL (0.08 mol) of trimethyl phosphite and 7.6 mL (0.08 mol) of species 7 over 1. dimethyl phosphite in 40 mL of chloroform at 0 °C on

Table 3. Synthesis of Benzidronate from Phenylacetyl Chloride or Ethyl Phenylacetate.

Reactants Purity (%) Yield of 6a Entry Starting Material a On the Basis of Potentiometric Titration (%) PCl3 (equiv.) H3PO3 (equiv.)

1 0 2 – 0

2 1 1 76 50 phenylacetyl chloride 3 2 0 93 27

4 2 1 85 51

5 1 1 72 44 ethyl 6 2 0 94 55 phenylacetate 7 2 1 82 47 aFrom at least two parallel experiments. 82 Letters in Drug Design & Discovery, 2015, Vol. 12, No. 1 Grün et al.

Fig. (1). Titration Curve for Benzidronic Acid (4) Obtained by the Reaction Presented in Scheme (1).

Fig. (2). Titration Curve for the Disodium Salt of Benzidronic Acid (6) Obtained by the Reaction Presented in Table 1/Entry 9. intensive stirring. After the addition was complete, the 54.3 (t, J=10.4, J=3.5, OCH3), 54.2 (t, J=10.4, J=3.5, OCH3), mixture was warmed to 85 °C and then stirred at this 75.7 (t, J=151.7, P-C-P), 127.2 (s, C4), 127.9 (s, C2*), 131.3 * temperature for 18 h. Then the solvent was removed under (s, C3*), 134.4 (t, J = 8.6, C1), may be reversed, [17] reduced pressure to give the crude product as a yellow oil (CDCl3) 39.2 (CH2), 54.2 (bt, J=8.0, J=3.5, OCH3), 75.8 (t, (20 g). Then, the crude product was purified by column J=151.9, P-C-P), 127.2 (Ar), 128.0 (Ar), 131.4 (Ar), 134.5 (t, chromatography (silica gel, 3% methanol in dichlormethane) J = 8.6, Ar). to afford 7.5 g (25%) of benzidronic acid tetramethyl ester 31 (3) as a light yellow solid. P NMR (CDCl3) 21.0, [23] Then, 1.3 g (4.0 mmol) of the tetramethyl ester (3) was 1 20.5; H NMR (CDCl3) 3.36 (t, J=13.7, 2H, CH2), 3.79- hydrolyzed by refluxing it with 15 mL of concentrated HCl 3.72 (m, 12H, OCH3), 7.31-7.24 (m, 3H, ArH), 7.39 (d, for 4 h. The solvent was removed under reduced pressure to J=6.6, 2H, ArH) [23] (CDCl3) 3.4 (t, J=13.5, CH2), 3.8 furnish 0.80 g of the benzidronic acid in a purity of 90% that 13 (OCH3), 7.3 (m, ArH); C NMR (CDCl3) 39.1 (s, CH2), was then suspended in 5 mL of isopropyl alcohol, and the Efficient Synthesis of Benzidronate Applying Letters in Drug Design & Discovery, 2015, Vol. 12, No. 1 83 mixture was stirred at room temperature for 30 min. After (0.026 mol) of thionyl chloride was added dropwise, and the the digestion, the solid product was filtered off. This mixture was stirred at 26 °C for 6 h. Further processing procedure was repeated once to afford 0.60 g (53%) of including the second step involving reaction with benzidronic acid (4) as white solid, in a purity of 100%. 31P phosphorus trichloride (4.6 ml), hydrolysis, pH adjustment, 1 NMR (D2O) 18.6, [23] 19.5, H NMR (D2O) 3.22 filtration of the precipitate, purification by precipitation with MeOH and digestion was performed as described above to (t, J=13.6, 2H, CH2), 7.31-7.18 (m, 5H, ArH); [20] afford 2.3 g (24%) of benzidronate disodium salt (6) in a (CD3COCD3) 3.31 (t, J= 12, 2H, CH2), 7.25–7.03 (m, 3H, 31 13 purity of 86%. P NMR (D2O) 17.8. ArH), 7.50–7.28(m, 2H, ArH), C NMR (D2O) 38.4 (s, CH2), 73.9 (t, J = 144.3, P-C-P), 127.0 (s, C4), 128.0 (s, C2*), * 131.3 (s, C3*), 135.5 (t, J = 8.4, C1), may be reversed, [24] 3.6. Preparation of Benzidronate Disodium Salt (6) from (D2O) 42.0, 78.8 (t, JC,P = 145.7); 130.2, 131.2, 134.4, 138.8. Phenylacetyl Chloride Using Phosphorus Trichloride and Phosphorous Acid (Table 3/Entry 4) 3.3. Preparation of the Disodium Salt of Benzidronate (6) 3.3 mL (0.025 mol) of phenylacetyl chloride (1) and 2.1 Starting from Phenylacetic Acid, Phosphorus Trichloride g (0.025 mol) phosphorous acid were dissolved in 10.5 mL and Phosphorous Acid in a One Step Reaction (Table of methanesulfonic acid on stirring. 4.4 mL (0.050 mol) of 1/Entry 9) phosphorus trichloride was added dropwise and the mixture was stirred at 85 °C for 24 h. Further processing including 3.4 g (0.025 mol) of phenylacetic acid (5) and 2.1 g hydrolysis, pH adjustment, filtration of the precipitate, (0.025 mol) phosphorous acid were added into 10.5 mL of purification by precipitation with MeOH and digestion was MSA on stirring. Then 6.6 mL (0.075 mol) of phosphorus performed as described above to provide 4.9 g (51%) of trichloride was added dropwise in ca. 30 min, and the benzidronate disodium salt (6) in a purity of 85%. 31P NMR contents of the flask were stirred at 85 °C for 24 h. After (D O) 17.8. cooling the mixture to 26 °C, 20 mL (1.1 mol) of water was 2 added and the mixture was stirred further at 105 °C for 4 h. The pH was adjusted to 2.5 by adding ~13 mL of 50% 3.7. Preparation of Benzidronate Disodium Salt (6) from aqueous sodium hydroxide to the mixture. Then, the mixture Ethyl Phenylacetate Using Phosphorus Trichloride was stirred for 12 h and the precipitate was removed by (Table 3/Entry 6) filtration to give ~20 g of the crude product that was 4 mL (0.025 mol) of ethyl phenylacetate (8) was dissolved in 25 mL of hot water, then 100 mL of methanol dissolved in 10.5 mL of methanesulfonic acid on stirring. 4.4 was added, and the mixture was stirred for 1 h. The mL (0.050 mol) of phosphorus trichloride was added precipitate was filtered off and was suspended in 60 mL of dropwise and the mixture was stirred at 85 °C for 24 h. methanol-water 94:6 and the mixture was digested by Further processing including hydrolysis, pH adjustment, stirring at 65 °C for 30 min. The solid product was then filtration of the precipitate, purification by precipitation with filtered off to furnish 6.8 g (81%) of benzidronate disodium 31 MeOH and digestion was performed as described above to salt (6) in a purity of 98%. P NMR (D2O) 17.8, [25] 13 give 4.8 g (55%) of benzidronate disodium salt (6) in a 19.0, C NMR (D2O) 38.4 (s, CH2), 74.3 (t, J = 134.7, P- 31 purity of 94%. P NMR (D2O) 17.8. C-P), 126.7 (s, C4), 128.0 (s, C2*), 131.5 (s, C3*), 136.8 (t, J = 7.9, C1), *may be reversed. CONFLICT OF INTEREST 3.4. Preparation of Benzidronate Disodium Salt (6) from The authors confirm that this article content has no Phenylacetic Acid (5) Applying Phosphorus Trichloride conflict of interest. in a Two Step Manner (Table 2/Entry 1) 3.4 g (0.025 mol) of phenylacetic acid (5) was dissolved ACKNOWLEDGEMENTS in 10.5 mL of methanesulfonic acid on stirring. 2.4 mL (0.028 mol) of phosphorus trichloride was added dropwise, The partial support from Hungarian Scientific Research and the mixture was stirred at 26 °C for 6 h. Then, 4.6 mL Found (OTKA No K83118) is acknowledged. (0.053 mol) of phosphorus trichloride was added dropwise and the mixture was stirred at 85 °C for 18 h. Further processing including hydrolysis, pH adjustment, filtration of REFERENCES the precipitate, purification by precipitation with MeOH and [1] Russell, R.G.G. Bisphosphonates: The first 40 years. Bone, 2011, digestion was performed as described above. 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Received: August 27, 2014 Revised: September 25, 2014 Accepted: September 26, 2014