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JS403 is a carbamoylphosphonate (CPO) molecule that showed anti-metastatic properties in mice. Since JS403 is intended to be a chronic prophylactic drug, the preferred route of administration should be oral. However, it exhibits poor oral bioavailability of less than 1%. The poor intestinal permeability and high solubility implies its classification as BCS class III drug. The aim of this study was to overcome the limited intestinal permeation of JS403 that is regarded as an unmet need in the pharmaceutical industry for this class of drugs. Therefore, the impact of acceptable absorption enhancers on the intestinal permeability of JS403 were examined using established experimental models. The absorption enhancers were: I) sodium caprate (C10), II) sodium deoxycholate (SDC) and III) mono-carboxymethylated chitosan (MCC). The effect of each enhancer was examined alone and also in combinations. In-vitro permeability through enterocytes monolayer was studied using the Caco-2 model, while the oral bioavailability was determined by using the freely moving rat model. The results of this investigation showed that while the use of a single absorption enhancer had no effect on JS403 permeability, the combination of C10 and sodium deoxycholate increased the permeability of JS403 by 10-fold in the in-vitro model. In addition, this blend showed a 2-fold elevation in JS403 oral bioavailability. Both in-vitro and in-vivo results highlight the synergistic potential of the combined enhancers C10 and sodium deoxycholate in enhancing oral bioavailability of BCS class III medications.

Carbamoylphosphonates (CPOs) have recently been identified as extracellular in vivo active inhibitors of the cancer and metastasis-promoting extracellular enzymes, carbonic anhydrases (CAs) IX and XII, and matrix metalloproteinase-2 (MMP-2). This article describes the stereoselective synthesis and the evaluation of the biological properties of a pair of enantiomeric aminoalkylcarbamoylphosphonates, which have been enantioselectively synthesized from l-serine, using functional group-transformations. The structures of the enantiomeric products have been determined by X-ray crystallography. The enantiomeric purities of compounds have been confirmed by chiral shift reagent NMR experiments and by circular dichroism. In vitro evaluation of the CPOs synthesized revealed that they possess micromolar IC50 inhibitory action against CAIX and CAXII. The two enantiomers as well as the racemic or optically inactive ones do not differ by any significant extent from previously reported CPOs’ CA inhibitory values. On the other hand, MMPs inhibitory activities are rather weak; only MMP-2 showed a notable IC50 value.

Carbamoylphosphonates (CPOs) have been identified as inhibitors of matrix metalloproteinases (MMPs) and as orally active, bioavailable, and safe antimetastatic agents. In this article, we focus on the direct antitumor activity of the CPOs. We discovered that CPOs also inhibit carbonic anhydrases (CAs), especially the IX and XII isoforms identified as cancer promoting factors. Thus, CPOs can be regarded as novel nontoxic drug candidates for tumor microenvironment targeted chemotherapy acting by two synergistic mechanisms, namely, inhibiting CAs and MMPs simultaneously. We have also demonstrated that the ionized CPO acid is unable to cross the cell membrane and thus limited to interact with the extracellular domains of isozymes CAIX and CAXII. Finally, applying CPOs against cancer cells in hypoxic conditions resulted in the dose dependent release of lactate dehydrogenase, confirming the direct interaction of the CPOs with the cancer related isozymes CAIX and XII and thereby promoting cellular damage.

Seven 4-phenoxybenzenesulfonamidopolymethylene carbamoylphosphonates (CPOs) bearing two to eight methylene units in the polymethylene chain were synthesized and evaluated as matrix metalloproteinase (MMP) inhibitors. The five lowest homologues [(CH₂)₂-₆] are selective MMP-2 inhibitors, whereas the two with the longest linkers [(CH₂)₇,₈] lack inhibitory activity. The most potent homologues are those with (CH₂)₅,₆; these two were evaluated for antimetastatic activity in a murine melanoma model and showed good potency both by oral and intraperitoneal administration without any toxic--including musculoskeletal--side effects. In contrast to the previously reported cis-ACCP, which was shown to inhibit MMP-2 for ∼30 min, the new compounds inhibit MMP activity for the duration of measurement, lasting several hours. Pharmacokinetic evaluation revealed, on the one hand, low oral bioavailability; on the other hand, a relatively large calculated volume of distribution, consistent with the observed reversible absorption of CPO 5 to hydroxyapatite, as a model for bone.

Some carbamoylphosphonates (CPOs) inhibit matrix metalloproteinases (MMPs). Although MMPs are involved in inflammatory processes, the anti-inflammatory activity of CPOs has not been reported. In this context we compared the biological activity of the three aminoCPOs, PYR-CPO, PIP-CPO and cis-ACCP. We were particularly interested in their capability to modulate the secretion of tumor necrosis factor alpha (TNFα). LPS-activated mouse peritoneal macrophages and LPS-activated mouse splenocytes were used to explore this question. It was found that the aminoCPOs were able to reduce TNFα secretion to a level equivalent to the reduction caused by the steroid drug budesonide (BUD). The reduction in TNFα levels was neither accompanied by cytotoxicity, nor did it inhibit cell proliferation. To explicate whether the aminoCPOs affect TNFα processing by TNFα-converting enzyme (TACE), TACE inhibitory properties of the three molecules was tested in vitro. Only PIP-CPO exerted TACE inhibitory activity at therapeutic (non-cytotoxic) concentrations, indicating on its potential to serve as an anti-inflammatory agent by reducing TNFα secretion.

This article reviews the current state of phosphorus-based matrix metalloproteinase (MMP) inhibitors. MMPs are a potentially harmful group of enzymes and their successful inhibition can be expected to alleviate a large diversity of severe pathologies that are listed in the beginning of the review. More than 20 years of worldwide search by the scientific and industrial community for a clinically useful inhibitor have resulted in failure because of toxic side effects or lack of in vivo efficacy of the tested molecules. In the majority of inhibitors reviewed in this article, the phosphorus plays the role of zinc bonding, as most MMP inhibitors are designed to do, since the catalytic zinc ion is crucial for the functioning of these enzymes. The main classes of phosphorus based MMP inhibitors are: bisphosphonates, phosphonamidates, phosphinamidates, phosphinates, phosphonates and carbamoylphosphonates.

This Glossary of Terms in Pharmaceutics is needed by practitioners in the field of pharmaceutics as this field fulfills an important and crucial role, different from the roles of other scientific disciplines involved in the drug-making process. The glossary contains 168 definitions used in pharmaceutics. These are related to various aspects of this discipline such as: (1) physicochemical characterization of pharmaceutical preparations and the active ingredients they contain; (2) unit operations used in the practice of pharmaceutics; (3) terms related to the various dosage forms; (4) terms related to the various modes and routes of drug delivery; (5) terms used in pharmacokinetics and biopharmaceutics in general, and additional miscellaneous terms. The field of pharmaceutics itself is of a multidisciplinary nature as its practitioners come from a variety of disciplines, such as chemistry or various biological sciences, thus a glossary containing authoritative definitions would be useful for them. The terms used in pharmaceutics are rarely covered by existing glossaries, and in the cases they are, their definitions are often inappropriate for the field of pharmaceutics and require new or modified definitions to better fit the new context.

cis-2-Aminocyclohexylcarbamoylphosphonic acid ( cis-ACCP) was evaluated in vitro and in two in vivo cancer metastasis models. It reduced metastasis formation in mice by approximately 90% when administered by a repetitive once daily dosing regimen of 50 mg/kg via oral or intraperitoneal routes and was nontoxic up to 500 mg/kg, following intraperitoneal administration daily for two weeks. Pharmacokinetic investigation of cis-ACCP in rats revealed distribution restricted into the extracellular fluid, which is the site of action for the antimetastatic activity and rapid elimination ( t 1/2 approximately 19 min) from blood. Sustained and prolonged absorption ( t 1/2 approximately 126 min) occurred via paracellular mechanism along the small and large intestine with overall bioavailability of 0.3%. The in vivo concentrations of cis-ACCP in the blood in rats was above the minimal concentration for antimetastatic/MMP-inhibitory activity, thus explaining the prolonged action following once daily administration. Finally, 84% of the intravenously administered cis-ACCP to rats was excreted intact in the urine.

Low molecular weight compounds were isolated by high-performance liquid chromatography from the maggot or haemolymph extracts of Lucilia sericata (Meigen) (Diptera: Calliphoridae). Using gas chromatography-mass spectrometry analysis, three compounds were obtained: p-hydroxybenzoic acid (molecular weight 138 Da), p-hydroxyphenylacetic acid (molecular weight 152 Da) and octahydro-dipyrrolo[1,2-a;1',2'-d] pyrazine-5,10-dione (molecular weight 194 Da), also known as the cyclic dimer of proline (or proline diketopiperazine or cyclo[Pro,Pro]). All three molecules revealed antibacterial activity when tested against Micrococcus luteus and/or Pseudomonas aeruginosa, and the effect was even more pronounced when these molecules were tested in combination and caused lysis of these bacteria.

Autotaxin is an extracellular, two zinc-centered enzyme that hydrolyzes lysophosphatidyl choline to lysophosphatidic acid, involved in various cancerous processes, e.g. migration, proliferation and tumor progression. We examined the autotaxin inhibitory properties of extended structure carbamoylphosphonates (CPOs) PhOC(6)H(4)SO(2)NH(CH(2))nNHCOPO(3)H(2), with increasing lengths of methylene chains, (CH(2))(n), n = 4-8. Carbamoylphosphonates having n = 6, 7, 8 inhibited autotaxin in vitro with IC(50) ≈ 1.5 µM. Using an imaging probe we demonstrated that compound n = 6 inhibits recombinant autotaxin activity in vitro and in vivo, following oral CPO administration. Additionally, daily oral administration of compound n = 7 inhibited over 90% of lung metastases in a murine melanoma metastasis model. Both the carbamoylphosphonates and the enzymes reside and interact in the extracellular space expecting minimal toxic side effects, and presenting a novel approach for inhibiting tumor proliferation and metastasis dissemination.

This article reviews the unique reactivities of � -hydroxyiminoalkylphosphonates, which result from the proximity and the interaction between the hydroxyimino and the phosphonic groups. The reaction course of � -hydroxy- iminoalkylphosphonates depends markedly on the type of groups linked to the phosphorus and on the stereochemistry of the oxime function. (Z)-� -Hydroxyiminoalkylphosphonates and phosphinates undergo, upon heating, fragmentation to a nitrile and a phosphate or phosphonate, while (E)-� -hydroxyiminoalkylphosphonates and -phosphinates undergo Beckmann rearrangement to N-acylphosphoramidates or N-acylphosphonamidates, respectively. In contrast to the diesters, � -hydroxyiminoalkylphosphonic acids (and monoesters) are unstable and fragment to metaphosphates, which can perform in situ phosphorylation of appropriate groups. Kinetic and mechanistic studies of the fragmentations of � - hydroxyiminobenzylphosphonates to metaphosphoric acid and esters are reviewed. The fragmentation of � -hydroxyimino- benzylphosphonic acid monoesters to metaphosphate esters requires acid catalysis and slows down at about pH 3. In contrast, � -hydroxyiminobenzylphosphonic acid undergoes fragmentation in aqueous solutions at a wide range of pH, from strongly acidic up to about pH 9. The � -hydroxyiminophosphonate fragmentation methodology was applied to phosphorylation of silica gel, which can potentially be useful as chromatographic stationary phase. A variety of stable precursors of � - hydroxyiminophosphonates that can be induced to fragment thermally, photochemically or by base catalysis is described. (E)-� -Hydroxyiminobenzylphosphonamidates of various types have been found to undergo Beckmann rearrangement to yield N-acylphosphordiamidates upon heating in nonpolar solvents such as toluene, or fragmentation to metaphosphonamidate by heating in polar solvents. The fragmentation of (E)-� -hydroxyiminobenzyl- phosphonates to metaphosphates is considered a special case of the Beckmann fragmentation.

Three pyrimidine derivatives (8a–c), differing from isouramil and divicine at C2, have been synthesized and their autoxidation rates measured spectrophotometrically. The autoxidation rates of all five pyrimidines (8a–c, isouramil and divicine) were correlated with σ p + values (rho=−1.28, r2=0.949).

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endo-peptidases that have the capacity to degrade all elements of the extracellular matrix (ECM) and are required for homeostatic maintenance of the ECM. Interest in MMPs arose from the accumulating evidence implying that over-activity of MMPs plays a role in mediating or accompanying a diverse array of pathologies. Because of this, there has been an ongoing, and alas unsuccessful, effort for nearly two decades to develop clinically applicable MMP inhibitors (MMPIs) as drugs. The largest family of the inhibitor candidates that failed in clinical trials is that of hydroxamic acids. This review i) attempts to rationalise the failure of hydroxamates as MMPIs, ii) critically reviews publications and patents of the last few years, which report new non-hydroxamate based MMPIs, and iii) summarises factors that are considered important for success in developing clinically useful MMPIs.

Matrix metalloproteinases (MMPs) are a family of over twenty zinc-dependent enzymes that hydrolyze connective tissue and are involved in a variety of diseases, which are associated with undesired tissue breakdown. Previously we described the synthesis of a series of achiral alkyl and cycloalkylcarbamoylphosphonic acids and their biological evaluation. Herein we report the effect of chirality and geometrical isomerism on the potency and selectivity of inhibition. The inhibitory potencies of pairs of enantiomeric and stereoisomeric alkyl and cycloalkylcarbamoylphosphonic acids were evaluated on recombinant MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 enzymes. The results show that the enantiomers and stereoisomers studied differ considerably in their inhibitory potencies and selectivities on the enzyme subtypes studied. Such a result is consistent with the assumption that the carbamoylphosphonates interact with a chiral environment such as an enzyme.

Matrix metalloproteinases (MMPs) are a family of over 20 zinc-dependent enzymes that hydrolyze connective tissue and are involved in a variety of diseases, which are associated with undesired tissue breakdown. This paper reports the synthesis, characterization, and biological evaluation of a novel class of MMP inhibitors based on the carbamoylphosphonic acid function. We report a series of 10 open chain N-alkylcarbamoylphosphonic acids (ranging from R = C(1) to C(6) groups), eight N-cycloalkylcarbamoylphosphonic acids (ranging from cyclopropyl to cyclooctyl rings), and four N,N-dialkylcarbamoylphosphonic acids. The compounds were evaluated in three in vitro models, which consisted of (a) the in vitro invasion across a reconstituted basement membrane, (b) determination of the IC(50) values on recombinant MMP-1, MMP-2 MMP-3, MMP-8, and MMP-9 enzymes, and (c) an in vitro capillary formation model, which is a model of angiogenesis. Several of the compounds were also tested in an in vivo murine melanoma model. The following general conclusions have been reached: Most compounds show selectivity for MMP-2 over the other MMP subtypes examined. Cycloalkylcarbamoylphosphonic acids are more potent than comparable open-chain alkyl compounds. Optimal activity against MMP-2 among the cycloalkyl derivatives was shown by N-cyclopentylcarbamoylphosphonic acid (3m). N,N-Dialkylcarbamoylphosphonic acids that were examined showed weak or no activity. The compounds examined showed toxic effects neither in vitro nor in vivo in the concentrations used. Carbamoylphosphonic acids are water soluble at physiological pH and are stable indefinitely.

The role of the microenvironment is of critical importance during the initiation and progression of carcinogenesis, both for understanding cancer biology and exploiting new knowledge for improved diagnostics and therapeutics at the cellular and molecular level. This summary emphasizes the most dynamic aspects of the microenvironment, namely tumor promoting enzymes and their inhibition. The enzymes mentioned are matrix metalloproteinases (MMPs) degrading connective tissue and carbonic anhydrases regulating pH level in the extracellular space. Additional enzymes reviewed are ectonucleotidases, such as autotaxin (ATX), CD73, and CD39. There is a short introduction to carbamoylphosphonates, inhibitors that regulate in vivo three kinds of zinc enzymes, MMP2, CAIX, and ATX, incapacitating the cancer.

Epoxy crosslinking agents have been investigated for use in the fabrication of bioprosthetic devices, such as heterograft heart valve prostheses. It has been generally assumed that epoxy crosslinking takes place via amino-epoxy reactions. The present study investigated the hypothesis that the reactions of methionine residues with epoxides also can occur in biomaterial crosslinking. A series of model reactions were studied in which a mono-epoxide was combined with individual alkyl sulfides. In the present studies epoxides rapidly alkylate aliphatic sulfides, including methionine derivatives, in buffered aqueous solutions at room temperature and pH close to neutral, forming sulfonium compounds, which are stable at pH 5-7 at temperatures up to 50 degrees C, except for cases in which methionine derivatives with non-protected carboxy groups are used. The rate of reaction remains practically unchanged within the range of pH from 5 to 12, whereas in strongly alkaline media the reverse reaction occurs. This discovery can provide a better understanding of processes occurring in the fixation of bioprosthetic tissues with polyepoxides. It can also develop into a site-specific method to label methionine residues in proteins. The carboxy group-containing sulfonium betaines derived from N-protected methionines undergo cyclization in unexpectedly mild conditions, which can be used as an efficient method for preparation of N-protected 2-amino-4-butyrolactones with sensitive protective groups.

This work was aimed at improving the absorption of bisphosphonates by targeting carrier systems in the intestine and the intestinal peptide carrier system (hPEPT1), in particular. (14)C-Labeled pamidronate and alendronate as well as radiolabeled and "cold" peptidyl-bisphosphonates, Pro-[(3)H]Phe-[(14)C]pamidronate, and Pro-[(3)H]Phe-[(14)C]alendronate were synthesized. In situ single-pass perfusion studies revealed competitive inhibition of transport by Pro-Phe, suggesting peptide carrier-mediated transport. Prodrug transport in the Caco-2 cell line was significantly better than that of the parent drugs, and the prodrugs exhibited high affinity to the intestinal tissue. Oral administration of the dipeptidyl prodrugs resulted in a 3-fold increase in drug absorption following oral administration in rats, and the bioavailability of Pro-Phe-alendronate was 3.3 (F(TIBIA)) and 1.9 (F(URINE)) times higher than that of the parent drug. The results indicate that the oral absorption of bisphosphonates can be improved by peptidyl prodrugs via the hPEPT1; however, other transporters may also be involved.

Bisacylphosphonates have previously been shown to possess in vitro activity with regard to hydroxyapatite (HAP) formation and dissolution, and in vivo on tissue calcification and bone resorption. This work was aimed at elucidating the role of the keto groups in the biological activity of these compounds. For this purpose, we have synthesized a series of long-chain bisphosphonates possessing different functional groups adjacent to the phosphonic functions and examined them in some in vitro and in vivo models. The functional groups introduced into the newly synthesized bisphosphonates (hydroxyl, amide, and difluoromethylene) were chosen as to represent certain isolated aspects of the keto groups in the original compounds. None of the functional groups introduced into the long chain bisphosphonates bestowed higher activity on the molecules than the carbonyl groups. The unique effect of the α-keto groups in rendering long-chain bisphosphonates active is presumably attained through a combination of chelating ability with sufficiently low pKa values to enable the bisacylphosphonate molecules to be fully ionized at physiological pH. © 2000 John Wiley & Sons, Inc. Heteroatom Chem 11:470–479, 2000

The thermodynamics, pH dependency and solvent effects of the fragmentation reaction of a series of α-oxyiminobenzylphosphonate monomethyl esters [(E)-1a–f] were examined in water and other hydroxylic solvents by UV and by 31P NMR spectroscopy at pH 0–3.1. The fragmentation of compounds (E)-1a–f was found to be a first-order reaction in substrate over the acidity range studied, while the dependence on the acidity is more complex, with rate constants k1 and k2. The ρ values corresponding to the first and second order rate constants were −1.12 and −0.835, respectively, indicating that the reaction is facilitated by electron-donating substituents, which probably enhance the protonation of the oxime OH group. Activation parameters for k1 and k2 reactions were also calculated. The near- zero values of the entropies of activation obtained are consistent with a dissociative transition state with almost no bonding to a nucleophilic solvent. Monitoring the fragmentation reaction of (E)-1a in several binary alcohol–water mixtures at different acidities showed that the reaction rate is enhanced by the alcohol’s acidity and not hampered by the steric requirements of the alcohol molecule. This rules out in our opinion, the likelihood for nucleophilic solvent assistance in the rate-determining step. On the other hand, product studies show that both the nucleophilicity and the steric requirements of the alcohol are of importance in determining the product formed in the fragmentation of (E)-1a. The highest selectivity (S) value was found for MeOH, while S values of

Solid-state 31P and 13C magic angle spinning (MAS) NMR spectra were used to characterize pure bisphosphonates and also bisphosphonates adsorbed on hydroxyapatite. Four geminal bisphosphonates, including the clinically used compounds ethane-1-hydroxy-1,1-diphosphonic acid, 3-amino-1-hydroxypropane-1,1-diphosphonic acid and 4-amino-1-hydroxybutane-1,1-diphosphonic acid, five α,ω-bisphosphonates and phosphonoacetic acid were investigated. NMR spectra of pure and adsorbed bisphosphonates differ in the observed linewidths and in the isotropic chemical shifts. The broad lines reflect mainly the poor crystallinity of the adsorbed compounds. Shifts of δiso in both directions do not reveal a correlation with the molecular structures. The molar ratio of phosphonates adsorbed on hydroxyapatite determined by 31P spectra without cross-polarization (CP) is approximately two times larger for geminal bisphosphonates than for α,ω-bisphosphonates and phosphonoacetic acid. 13C CP/MAS spectra of pure and adsorbed bisphosphonates recorded in two cases for identification of adsorbed compounds give additional information about the state of adsorbed compounds. Disodium α,ω-dihydroxypolymethylene-α,ω-bisphosphonates in the solid state show characteristic 13C chemical shifts which are indicative of either odd or even numbers of CH2 groups. Copyright © 2000 John Wiley & Sons, Ltd.

Bisphosphonates are clinically used mainly to reduce bone resorption. We studied the transplacental effects of two bisphosphonates on the fetal skeleton in rats. Pregnant rats were treated during days 11-20 of pregnancy with daily subcutaneous injections of 0.1 mg/kg of alendronate or a newly synthesized bisphosphonate, VS-b6. This period of pregnancy was chosen because the active development of bones from mesenchyme through cartilaginous models occurs during that time. Histological examination of midlongitudinal sections of the 21-day-old fetuses showed an increase in the amount of diaphyseal bone trabeculae with slight shortening of the diaphysis in the experimental fetuses, in comparison to controls. Computerized histomorphometric studies similarly showed an increase in the amount of diaphyseal bone trabeculae with a concomitant decrease in bone marrow volume, but no change in cartilage volume. In addition, chemical analysis of the fetal bones showed an increase in calcium content in the treated fetuses. 14C-alendronate was shown to pass through the rat placenta and accumulate in the fetuses, most probably in their bones. This is presumed because bisphosphonates are known to accumulate in bone, being stored there for long periods of time. It is important, in light of our results, to give careful consideration to the treatment of women with bisphosphonates at childbearing age, whenever this is needed.

Purpose. To validate our hypothesis that a bisphosphonate (BP) having a nitrogen-containing heterocyclic ring on the side chain, and with no hydroxyl on the geminal carbon would possess increased activity, and better oral bioavailability due to enhanced solubility of its calcium complexes/salts and weaker Ca chelating properties.

Hindered primary amines that resist other reagents can be converted to carbamoylphosphonates by means of 4-nitrophenoxycarbonylphosphonate diesters in high yields and under mild conditions.

The new type of phosphoniminoxy free radicals was generated electrochemically or by chemical oxidation of oximes. It was established the strong difference of ESR parameters for isomeric Z and E forms of the radicals. The spin distribution and its correlation with electronic and spatial characteristics of molecules as well as with intramolecular interactions are discussed.

Purpose. This work was aimed at synthesizing novel bisphosphonates (BPs) and examining them in comparison to clinically used BPs such as pamidronate and alendronate, and to tetracycline, in order to evaluate their potential as anticalcification and antiresorption agents. The correlation between the various models was examined in order to establish facile experimental models for pre-screening of potential compounds.

(i-PrO)2P(O)COSEt (1) reacted with NH2OH in pyridine at room temperature to give mainly (i-PrO)2P(O)NH2 (4). The formation of 4 was interpreted in terms of a spontaneous Lossen rearrangement of (i-PrO)2P(O)CONHOH (2a) formed in the reaction. A transient 31P NMR signal appearing in the reaction mixture at δ −1.8 was assigned to 2a. Trapping of (i-PrO)2P(O)NCO (5), formed in the reaction of 1 and NH2OH, by cyclohexylamine gave (i-PrO)2P(O)NHCONHC6H11 (6). Attempted isolation of 6 gave the hydrolyzed product N-cyclohexylurea (7). The reaction of 1 with NH2OMe proceeded slower than that with NH2OH and gave the expected (i-PrO)2P(O)CONHOMe (2b), which was isolated and identified. 2b converts slowly to 4 in pyridine at room temperature. In contrast, MeNHOH reacted rapidly with 1 and gave the stable crystalline (i-PrO)2P(O)CON(Me)OH (2c). The structure of hydroxamates 2 were assigned on the basis of 1H, 13C, and 31P NMR spectral data. This facile Lossen rearrangement is discussed in terms of the unusually high m...

Examination of alcoholic solutions of dimethyl acetylphosphonate (1) and dimethyl benzoylphosphonate (2) by 31P NMR spectroscopy reveals the presence of considerable amounts of hemiketals. Because of the great difference between the 31P chemical shifts of acylphosphonates (ca. 0 ppm) and their hemiketals (17–21 ppm), 31P NMR spectroscopy is a uniquely suitable method for studying the rates and equilibria of hemiketal formation of acylphosphonates with different alcohols. The equilibrium constants Kf, K′f (K′f = Kf [ROH]), pseudo-first-order rate constants k′f, the second order rate constants, kf for hemiketal formation from dimethyl acetylphosphonate with various alcohols, as well as the reverse reaction rate constants, kr to starting materials, were determined. The kinetic isotope effect of 2.8 for the forward reaction kf (EtOH addition) and the backward reaction kr indicates a general catalysis pathway. On the other hand, the calculated values of the enthalpies of activation ΔH‡ = 10.37 kcal mol-1 (forward), ΔH‡ = 13.66 kcal mol-1 (backward) and the entropies of activation, ΔS‡ = -17.25 cal mol-1 K-1 (forward), ΔS‡ = -9.82 cal mol-1 K-1 (backward) are not in accord with high molecularity of the reaction (1 cal = 4.184 J). Our analysis led to the conclusion that this is probably due to the fact that the transition state is mainly reactant-like with the development of only limited extent of bond formation. Various plausible reaction pathways for hemiketal formation are discussed. In addition, we have calculated the value of 2.65 σ* for the P(O)(OMe)2 group based on proton affinity obtained from heats of formation (ΔHf) of applying the MNDO techniques. The following linear correlation between pKa values and PA values of hemiketals of the form (Me)(R)C(OH)(OCH2X) was developed: pKa = PA - 356.58 + 9.18 [σ*(Me) + σ*(R) 0.2σ*(X)].

The title compound, C8H16NO4P, is the first carbamoylphospho­nic acid for which the crystal structure has been solved. The –PO3H2 group has three distinct P—O bond distances of 1.483 (1), 1.506 (1) and 1.545 (2) A. Hydrogen bonds of types P—O—H⋯O=C and N—H⋯O=P [2.416 (2) and 2.954 (2) A, respectively] connect the mol­ecules to form a chain of condensed ten-membered hydrogen-bonded rings. The second P—O—H group of each phospho­nic acid is hydrogen-bonded to an O=P group of the next chain, so serving to link adjacent chains together.

Geminal bisphosphonates R~R2C(PO3H2)2, such as etidronate (R 1 = Me, R 2= OH), pamidronate (R l = C H z C H z N H 2 , R 2 = OH), clodrohate (R 1 = R 2 = C1), and alendronate (R 1 = C H z ( C H 2 ) z N H 2 , R 2 = OH), characterized by a P-C-P type structure are used clinically as antiresorption [1] and anticalcification [2] agents. The introduction of fluorine or perfluoroalkyl groups into molecules is a versatile tool for modifying their physicochemical properties and physiological behavior [3, 4] by increasing lipophilicity [3] and, due to the strong electron-withdrawing inductive effect of fluorine, influencing the reactivity of neighboring groups, for example, acidity [4]. Recently monoand difluoromethylene bisphosphonates have been shown to be potential candidates for bisphosphate analogues inhibiting bone lysis ([5, 6] and references cited in [6]). Since a correlation between increasing acidity and improved antiresorption activity of bisphosphonates has been suggested [7], we considered it of interest to synthesize and to examine a perfluorinated analogue of clinically used bisphosphonate. In this paper we describe the synthesis and evaluation of the trifluoro derivative of etidronate. Trimethylsilylated phosphites are wellknown precursors for phosphonates and phosphonic acids [8-13]. Trifluoroacetyl and acetyl chloride were reported to react with tris(trimethylsilyl)phosphite [9] to yield the corresponding a-ketophosphonates [14-16] (for a recent comprehensive review on acylphosphonates a-ketophosphonates see [16]). We were encouraged to reinvestigate this reaction as a synthetic route to our target compound, trifluoro analogue of l-hydroxyethane-l,l-bisphosphonate [17]. The reaction of trifluoroacetyl chloride and tris(trimethylsilyl) phosphite (1) at 7 0 ° C furnished chlorotrimethylsilane and 1-trimethylsiloxy-2,2,2-trifluoroethane-tetrakis(trimethylsilyl) bisphosphonate (3) [F3 pentakis(trimethylsilyl) etidronate] (Fig. 1), as a water-sensitive, colorless, low melting point solid. The expected a-ketophosphonate [141 CF3C(O)P(O)(OSiM%) 2 (2) was not observed but probably formed via intermediate A, regardless of the starting materials' stoichiometry (Fig. 1). However, 2-(trifluoroacetyloxy)pyridine (TFAP) [18] reacted with phosphite 1 and furnished monophosphonate 4, which was characterized by nuclear magnetic resonance (NMR) spectroscopy. Presumably the reaction proceeded via the 1,3 dipolar intermediate B, which underwent a 1,4-trimethylsilyl group. Warming up to ambient temperature, compound 4 decomposed to give the a-ketophosphonate 2 and 2-trimethylsiloxypyridine (Fig. 1). Further addition of phosphite 1 to compound 2 again gave bisphosphonate 3, via intermediate C. A stabilizing effect for compound 4 could possibly be a Lewis acid base interaction of the phosphonate phosphorus and the pyridine nitrogen atom since the analogous reaction carried out with phenyltrifluoroacetate directly gave bisphosphonate 3 exclusively. The bisphosphonate was then easily converted using a methanol/ water mixture to form the waxy free acid 5 (F3 etidronic acid), for which two pKa values (1.55 and 5.56) were measured. No thermal rearrangement of the PC(CF3)(OH)P skeleton to give a POCH(CFflP framework was observed [17]. To obtain more information about the molecular structure of compounds 3 or 5 we tried unsuccessfully to grow single crystals for X-ray diffraction; however, after partial hydrolysis appropriate crystals of l-hydroxy-2,2,2-trifluoroethylidene-tris(trimethylsilyl)bisphosphonate (6) were obtained. Molecules of compound 6 were found to arrange pairwise in the unit cell taking advantage of two P = O. • .HO-P (251.5 pro) and two P = O . . . H O C (264.1pm) hydrogen bridges (Fig. 1). The molecular structure showed a slightly distorted tetrahedral geometry at carbon C1, P1, and P2 (Fig. 2) presumably caused by the involvement of the oxygen intermolecular hydrogen bonding comparable to other bisphosphonate structures [19, 20]. The P1-C1-P2 angle was found to be 110.3 ° correlating [20] to the relatively long P1-C1 and P2-C1 distances (185.6 pro); for the mono hydrate of eftdronic acid the corresponding parameters were 115.1° and 183.2 and 184.0 pm [19]. The calcification of bioprosthetic tissue implanted subdermally in rats is a wellestablished model for the study of ectopic calcification and for the evaluation of drugs potentially affecting it [21-26]. It has been shown [23-25] that there is a good correlation between anticalcification effect of bisphosphonates in vivo and antimineralization effect in vitro, at least in part as a consequence of their action on crystal growth [23-25]. In addition, the subdermal model of bioprosthetic tissue calcification is not mediated by osteogenic cellular factors [24, 27]. Thus, novel bisphosphonates can be screened conveniently by examining their effect

E,Z-isomeric forms of phosphorylated oximes were established by NMR 1H, 13C, 31P spectroscopy. Stereospecifity of phosphorus-carbon coupling is shown to be suitable criteria for distinction of spatial isomers. The new type of phosphoniminoxy radicals was generated from these oximes and spin distribution dependencies upon phosphorus environment and spatial structure of the molecules were established.

Reactions of 2,2,2-trihaloethyl or 1,1,1,3,3,3-hexafluoro-2-propyl bensoylphosphonate anions 7 or 10 with hydroxylamine gave the corresponding (α-hydroxyiminobensyl)phosphonates, 3 or 11, respectively, as predominantly (E)-isomers. Refluxing (E)-3a or (E)-3b in EtOH or 2-PrOH caused them to fragment to bensonitrile and mixed phosphodiesters: ethyl trihaloethylphosphate (12) or 2-propyl trihaloethyl phosphate (13), respectively. (E)-3a did not undergo any fragmentation in boiling water or MeOH. Refluxing (E)-3a in aprotic solvents led to the formation of bensonitrile and P,P'-bis-(2,2,2-trifluoroethyl)pyrophosphate (4a). The rate of fragmentation increased with solvent polarity. Similar behavior was exhibited by 11. The fragmentation of anions 3 and 11 is interpreted in terms of a dissociative mechanism leading to the formation of metaphosphate in the first step. The lack of reactivity in water and methanol is rationalized by assuming stabilization of the starting material by H-bond formation, while the rate enhancement by polar solvent is attributed to stabilization of the transition state and solvation of the departing OH - by the solvent. The metaphosphate formed in the thermal fragmentation of 3a in MeCN was trapped by styrene oxide, as evidenced by the isolation and identification of 2-oxo-2-(2',2',2'-trifluoroethyl)-4-phenyl-1,3,2-dioxaphospholane as a mixture of diastereoisomers 18 and 19

Certain irregularities in calcium metabolism such as Paget's disease, osteoporosis, as well as osteolysis in bone cancer, are characterized by excessive destruction of the bone by resorption. In contrast, a number of clinically important diseases, for example, atherosclerosis, kidney and renal calculu8, arthritis, and bioprosthetic heart valve calcification are characterized by the deposition of calcium phosphate. nis~hosphonates are a relct3vely new class of drugs, that have been used in these diseases to inhibit both mineralization and bone resorption, following the recognition, that they are stable analogs of endogenous pyrophosphate, which is a physiological inhibitor of calcification and bone resorption. A number of geminal bisphosphonates, namely, Pamidronate (ABP), Etidronate (HEBP), and Clodronate (ClMBP), have been approved for clinical use.

We describe results of our initial experiments regarding the use of various protecting groups for the preparation of some N-protected aminoacylphosphonates and amino hydroxyimino phosphonates derived from them. The protecting groups that have been examined are benzyloxycarbonyl (Cbz), [(9-fluorenylmethyl)oxy]carbonyl (Fmoc), and phthaloyl (Pht)

Modified amino acids and peptides are in the focus of medicinal chemistry as potential mimics of natural compounds that may act as inhibitors and modulators of natural processes. Along with many other types of analogs, considerable effort was invested into the synthesis of phospha amino acids. Presently phospha analogs for all amino acids and for many peptides are known and some of these are of considerable practical importance.

The reaction of benzoylphosphonic acid (3) with thionyl chloride leads to the formation of benzoylphosphonic dichloride (5) and P,P′-dibenzoylpyrophosphonic dichloride (6). These products were identified by 31P nmr spectra and through their products of alcoholysis with 2-propanol.

Arbuzov reactions of diethyl 2,2,2-trihaloethyl phosphites (3) with benzoyl chloride afforded ethyl 2,2,2-trihaloethyl benzoylphosphonates (4). The reactions of 4 with NH2OH.HCl led to the formation of methyl benzoate and ethyl methyl H-phosphonate as a result of alcoholysis of 4, followed by alkoxy group exchange. Methanol solutions of benzoylphosphonates 4 were found by 31P NMR spectroscopy to contain considerable proportions of hemiacetals, 7, which undergo base-catalyzed CP bond cleavage. The formation of hemiacetals from benzoylphosphonates 4 is suppressed in 2-propanol, and in this solvent the corresponding oximes 2 could be obtained in good yields. Reactions of methyl benzoylphosphonochloridate (10) with 2,2,2-trihaloethanols, in CH2Cl2, gave methyl 2,2,2-trihaloethyl benzoylphosphonates (11) which could be converted directly to oximes 12 by NH2OH.HCl in a one-pot procedure. In contrast to the previously studied dimethyl (E)-α-hydroxyiminobenzylphosphonate, which underwent thermal Beckmann rearrangement to afford N-benzoylphosphoramidates, both (E) and (Z)-trihaloethyl esters 2 and 12 underwent fragmentation to benzonitrile and to the corresponding dialkyl hydrogen phosphate, reflecting the increased electrophilicity of the phosphorus in these compounds. Demethylation of methyl esters 12 was effected smoothly by iodide or bromide ions to yield benzoylphosphonate salts 15, which in turn were converted to oxime salts 14 by treatment with hydroxylamine. In contrast, attempted deethylation of ethylesters 2 in refluxing acetonitrile led to benzonitrile and pyrophosphate type product as indicated by 31P spectroscopic examination of the reaction mixture. Oxime salts 14 behaved similarly when heated. Acidification of lithium 2,2,2-trifluoroethyl α-hydroxyiminobenzylphosphonate (14a) gave the corresponding hydrogen trifluoroethyl phosphonate (19a). The fragmentation of 19a in 0.6 N ethanolic hydrogen chloride to ethyl trifluoroethyl hydrogen phosphate and benzonitrile at room temperature had a T1/2 value of approx 18 hours, which is greater by a factor of 2 than that of the corresponding methyl ester. When the fragmentation of 19 was carried out in solvent mixtures of either water with methanol or 2-propanol, or methanol with tbutanol, the composition of the solvents was reflected in the products, indicating a dissociative type mechanism, involving metaphosphate as reactive intermediates.

Methyl benzoylphosphonochloridate 3 reacted with 2-cyanoethanol or p-nitrophenethyl alcohol to yield methyl 2-cyanoethyl benzoylphosphonate 4a or methyl p-nitrophenethyl benzoylphosphonate 4b. Ketones 4a and 4b were converted into the corresponding oximes, 5a and 5b as a mixture of E and Z isomers, by hydroxylamine. Oximes 5a and 5b were demethylated by treatment with sodium iodide or lithium bromide to give the corresponding monester monosalts 6a and 6b. Reaction of lithium 2-cyanoethyl or p-nitrophenethyl benzoylphosphonate with methoxylamine afforded the respective oxime methyl ether salts 8a and 8b as predominantly E isomers. Base treatment of 6a and 6b caused their dealkylation to the anion of α-hydroxyiminobenzylphosphonic acid 9 which did not decompose further. In contrast, 8a and 8b in aqueous base gave the dianion of α-methoxyaminobenzylphosphonic acid 10, which slowly fragmented to benzonitrile and phosporic acid. The fragmentation is interpreted in terms of a dissociative mechanism involving the formation of metaphosphate anion.