A practical synthesis and the pharmacological potency of gem-diphosphono substituted pyrimidines for treatment of bone resorption

A series of substituted arylidenes was allowed to react with the Wittig-Horner reagent, tetraethyl-methylene-1,1-bisphosphonate to give the corresponding N -containing bisphosphonates (BPs). Acid hydrolysis of one of the BPs gave the corresponding bisphosphonic acid. The in vivo anti-resorptive activities of the products in the rat adjuvant model are discussed in terms of structure-activity relationships (SAR).


Introduction
2][3] The clinical pharmacology of BPs is characterized by low intestinal absorption, but they inhibit bone resorption by being highly selectively taken up and adsorbed to mineral surfaces in bone, where they interfere as osteoclasts.It is likely that BPs are internalized by osteoclasts and interfere with specific biochemical processes and thereby induced programmed cell death or apoptosis. 4,5ecent studies show that BPs can be classified into at least two groups with different modes of action: (a) BPs that most closely resemble pyrophosphates (such as clodronate and etidronate, see Fig. 1) can be metabolically incorporated into non-hydrolysable analogs of ATP that may inhibit ATP-dependant intercellular enzymes.(b) The more potent, N-containing bisphosphonates (such as pamidronate and risedronate, see Fig. 2) are not metabolized in this way but can act on liver enzymes function, which explains the loss of osteoclast activity and induction of apoptosis.

Risedronate Pamidronate
A series of articles 6,7 from our laboratory has reported simple and efficient procedures for the synthesis of several examples of N-heterocycle-substituted-methylene-1,1-bisphosphonic acids 3 derived from the reaction of the parent olefin containing different heterocyclic species 2 with tetraethyl methylene-1,1-bisphosphonate, 1, followed by acid hydrolysis, as shown in Scheme 1.In continuation of our research program [6][7][8][9] on synthesis of a variety of bisphosphonates and their related bisphosphonic acids, it was of interest to introduce another series of these compounds for investigating the structure-activity relationships (SAR).In this work, we have attempted to utilize the high bone-joint-specificity of nitrogen-containing BPs with other

Results and Discussion
The synthesis of the required nitrogen containing bisphosphonates (BPs) was achieved via the Michael addition of a small excess over a molar amount of the Wittig-Horner reagent 1 to acids 4a-f or their N-methyl analogues 4g-j.The reaction was carried out under reflux, with basic catalysis using DMSO as solvent and LiOH (0.5M) as a base.After the reaction was completed (TLC) and the product mixture acid-quenched, , the crude BP product was separated by solvent extraction and purified by column chromatography to give tetraethyl 2-aryl-2-(2,4,6-trioxohexahydropyrimidin-5-yl)ethyl-1,1-bisphosphonate 6a-j as a major product (≈ 65%) together with a very low yield of tetraethyl 2-arylidene-bisphosphonate 8a-f (≤ 7%).Acid hydrolysis (conc.HCl) of the resulting BP product 6h -taken as an example -afforded the corresponding BP-acid 7 (≈ 90%, based on 6h) (Scheme 2).

Scheme 2
The structures of compounds 6a-j were verified by NMR analysis.For instance, the 1 H-NMR (400 MHz, DMSO) spectrum of 6a revealed three types of methine protons at δ = 2.99 (dd, J HH = 9 Hz, 2 J HP = 13.7 Hz, 1H, H a C), 3.87 (ddd, J HH = 9,11 Hz, 1H, H b C), 4.89 (dd, J HH = 11 Hz, 1H, H c C) ppm.The large coupling constant (J HH ) of H b with H a as well as with H c (see Table 3) indicates 10 that H b is trans to H a and H c .On the other hand, IR and NMR analysis established the structure of ethenylidenebisphosphonates 8.The IR (KBr) spectrum of 8a (as an example) showed the absence of stretching bands in the range 1700-1650 cm -1 corresponding to the three C=O groups of the starting arylidene 4a.Furthermore, in the 1 H NMR spectrum, only one methine proton could be indicated at 8.1 (dd, 1H, HC=CP 2 ) ppm.The 13 C NMR as well as MS spectra were in agreement with the proposed structure.
Obviously, the bisphosphosphonate products 6a-j were formed according to a process derived from the Michael addition 11a of arylidine 4 to tetraethyl methylenebisphosphonate (1) to give anion 5.When 5 is quenched by an acid at low temperature, the target bisphosphonate product 6 is obtained.On the other hand, the long time of heating (≈ 2 days) and the presence of a base might be responsible for the formation of the isolated by-product ethenylidenebisphosphonates 8.This could be attributed 11b to the intramolecular proton transfer in anion 5 to form the more stable form 5A; when anion 5A is heated, it splits off to form BP 8 together with a trace amount of acid 9a or its dimethyl analog 9b (mp, TLC, MS and IR spectrum for 9a or 9b were found to be identical with an authentic sample).The low yield of product 8 (≤ 7%) indicates that the reaction seems to be a reversible process.Although Hutchinson et al. 12 reported the inertness of ethenylidenebisphosphonate toward carbanions, Stutz achieved the Michael condensation product using some carbanions in high yields. 13These two contradictory results may best be rationalized by an equilibrium process similar to that shown in Scheme 3. Different results may be obtained with various carbanions or under different experimental conditions.
In order to verify our suggestion, we added p-nitrobenzaldehyde to the reaction solution (1+4a) that underwent Wittig-Horner reaction with anion 5A.As a result, the equilibrium between 5A and 8 was shifted to the left side completely to give the expected Wittig-Horner monophosphonate 10 as a sole product (Scheme 4).This is a strong evidence supporting our postulation.On the other hand, if we could drive the equilibrium completely towards the formation of compound 8, it would be of synthetic importance since only a few methods are available for the preparation of ethenylidenebisphosphonates 8. 14 However our trial on just raising the reaction temperature failed to give the expected result.

+ 4a
LiOH/DMSO/H 2 O p-nitrobenzaldehyde The structures suggested for all the new compounds are in good agreement with their analytical and spectroscopic data (Tables 1-3).

Pharmacological evaluation
The effect on bone resorption of these new compounds was investigated in terms of the structure activity relationships (SAR) of BPs.The screening relied on assessing the potency in an in vivo bone resorption model.Compounds 6a-j and 7 were tested in thyroparathyroidectomized (TPTX) rats with hypercalcemia induced by 1,25-dihydroxy vitamin D 3 , 15 by analogy to the activity of risedronate as a reference.BP-risedronate (cf.Fig 2 ) was selected for its optimal potency and safety in early screening assays. 16ll the tested compounds (Table 4) showed good-to moderate anti-resorptive properties compared to the available BP-drug risedronate.In general, these data indicated only marginal differences for the potency of the tested BP products, due to the different arylidene substituents used.However, screening results showed that the most active BPs were found in the compounds 6d,j that involve the dimethylamino-arylidine substituents, whereas the least activity appeared with compounds 6e,f that contain the chloro-arylidine substituents.
In summary, the inhibition of bone resorption of the new synthesized BPs is significantly high when compared to the available BP-drug, risedronate (standard).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.

Table 1 .
Physical properties and elemental analyses for compounds:

Table 4 .
Anti-resorptive Activity (ARA), Effective Dose (ED 50 ) of the BPs 6a-j and BP-acid 7 All data are generated in the thyroparathyroidectomized (TPTX) acute in vivo rat model.** This result was reproduced experimentally. *