INOSITOL PHOSPHATE ANALOGUES

Abstract: Novel inositol phosphorothioates have the general formula (I) C6H6(OH)n[(OPO2S)<2->]m[(OPO2O)<2->]6-m-n in which n is zero or an integer from 1 to 5 and, m is an integer from 1 to 6. They may be prepared by a method comprising reacting an inositol derivative having the general formula (II) C6H6(OB)n[OP(OCH2CH2CN)2]6-n in which n is zero or an integer from 1 to 5 and B represents a hydroxyl-protecting group, with a solution of elemental sulphur, which may or may not be radioactive, in a solvent therefor, and removing the hydroxyl-protecting groups from the product of the reaction. Phosphate esters may be prepared by oxidising the compound of formula (II) with an organic peroxide.

Description: INOSITOL PHOSPHATE ANALOGUES This invention relates to analogues of inositol phosphate esters and to a method for the preparation thereo . Inositol [C g H g (OH)g] is hexahydroxycyclohe-icane of which there are nine isomeric forms. Myo- inositol is cis-1,2,3,5-trans-4,6-hexahydro ycyclohexane. Certain of the inositol phosphates are known, One well-known derivative of commercial importance is phytic acid (inositol hexaphosphoric acid) . Also known are D-myo-inositol-1,3,4-trisρhosphate , the 1,4.,5-isomer and the 1,4,5,6- and 1,3,4,5-tetrakisphosphates. These molecules are of biological importance although their precise biological roles have not yet been fully- elucidated. They may be isolated with some difficulty and in low yield from natural sources. Synthesis of the 1,4,5-trisρhosphate has been achieved, but not entirely satisfactorily, by phosphoryl- ation using P(V) agents which methods often result in the formation of undesirable five-membered phosphate rings. The biological significance of inositol phosphates is the subject of much current interest and research. One area in which the inositol phosphates have been found to be involved is in calcium ion transport mechanisms in cells. A need exists, then, for materials for conducting scientific research into the biochemistry of inositol phosphate esters and, of course, such materials may possess pharmacological utility for therapy of clinical conditions arising from malfunction of the inositol phosphate biochemistry. According to the present invention there is provided an inositol phosphorothioate having the general formula I; C 6I6, (OH)tL [(OPO2.S) 2" ] [-(0P02,0) 2" ] ή o— m m— n n (I) in which n is zero or an integer from 1 to 5 and, m is an integer from 1 to 6. Thus, the present invention provides an inositol molecule in which one or more of its six hydro yl groups is esterified by a phosphorothioate group, the remaining positions being occupied by hydroxyl or phosphate ester groups, The sulphur atom, of the thioate group may be a radioisotope of sulphur to provide radiolabelled compounds for use as research reagents. The present invention also provides a method of preparing an inositol phosphorothioate having the general formula I; C6H6 (OH)n [(0P02S) 2" ]m [(0P020) 2~ ]6—m—n (I) in which n is zero or an integer from 1 to 5 and, m is an integer from 1 to 6, said method comprising reacting an -3- inositol derivative having the general formula II ; C 6 H 6 (0B) n [0P(0CH 2 CH 2 CN ) 2 ] 6 _ n (II) in which n is zero * or an integer from 1 to 5 and B represents a hydroxyl-protecting group, with a solution of elemental sulphur in a solvent therefor, and removing the protecting groups from the product of the reaction. Preferably the hydroxyl-protecting group represented by B in formula II is a benzyl group. Alternatively a divalent group such as isopropylidene may be utilised when n is an even number and the hydroxyl groups are thereby protected in pairs.. Thus the compound of general formula II may have the alternative formulae IIA or IIB: C 6 H 6 <0Bz) n [0P<0CH 2 CH 2 C N 2 ] 6 _ ιι (IIA) C 6 H 6 (0-iPr'-0) m [0P(0GH 2 CH 2 CN) 2 ] p (IIB) in which Bz is a benzyl group, iPr' is an isopropylidenyl group, n is zero or an integer from 1 to 5, m is zero or 1 or 2 and p is 2 or 4 or 6. It is to be noted that the compound of general formula II above may be oxidised with, for example a hydroperoxide such as tert- butylhydroperoxide, to form a compound having the formula VI; G 6 H 6 (0B Z ) n [0 P 0 (0CH 2 CH 2 CrI) 2 ] 6 _ n (VI) which may then be reduced to deprotect the hydroxy groups and form phosphate ester groups. The starting material, that is, the inositol derivative having the ' general formula II defined above, may be prepared by a method comprising reacting a compound having the general formula III: G 6 H 6 (0B) n (0H) 6 _ a (III) in which n and B are as defined hereinabove, with chloro (2-cyanoethoxy) -N, N-diisoρropylamino phosphine, which has the formula IV: (iPr) 2 N P (Cl) 0CH 2 CH 2 CT (IV) in which iPr represents an isopropyl group, to obtain a compound having the general formula V: G 6 H 6 (0B) a [0P(0CH 2 GH 2 GIf) H iPr 2 3 6 _ a (V) in which n, B and iPr are as defined hereinabove; and reacting the compound of formula V with terazole and 2-cyanoethanol to form the said compound of general formula I The benzylated inositol starting material (formula III above) may be prepared from inositol, via the isopropylidene derivatives, according to the method described in J. Gigg, R. Gigg, S. Payne & R. Conant; Carb. Res,, 140, cl, (1985); J. Chem. Soc. Perk. Trans., I, (1987), 423. ' The isopropylidene-containing starting materials and their preparation are described in J. Gigg, R. Gigg, S. Payne & R. Conant; Carb. Res., 142, 132 (1985) Concerning the reagent having the formula IV, reference is directed to N.D. Sinha, J.Biernat, J. McManus & H. oester; Nucl. Acids Res.,12, 4539 (1984). The principal intended use of this reagent is for the synthesis of polynucleotides: see, for example, European Patent Application Number 35,719 and United States Patent Numbers 3,534,017; 4,415,732 and 4,458,066. The present invention also provides a method of preparing an inositol phosphate having the general formula I; C 6,H,6 (OH)n [(0P02.S) 2" ]m [(0P00) 2~ ]o,—m—n (I) in which n is zero or an integer from 1 to 5 and, m is zero, said method comprising reacting an inositol derivative having the general formula II; ' C 6 H 6 (0B) n [0P 0CH 2 CH 2 CN 2 ] 6 _ n (II) in which n is zero or an integer from 1 to 5 and B represents a hydroxyl-protecting group, with an organic peroxide, and removing the protecting groups from the product of the reaction. The organic preroxide is preferably tert-butyl . hydroperoxide. Reference is made to the accompanying drawing which shows the reaction scheme of the invention using the tetrabenzylated derivative of my__-inositol as an example.'' The reagents involved in the steps identified in the drawing as (i) to (iv) were as follows: (i) (iPr 2 N P (Cl) 0CH 2 CH 2 CN, C 2 H 5 N(iPr 2 in dichloromethane) ; (ii) Tetrazole, HOCr j CH-CN in dichloromethane; (iii) tert-BuOOH(for X=0) : sulphur in pyridine (for X=S);and, (iv) NH.OH at 60°C followed by sodium in liquid NH 3 . The invention will now be described, by way of illustration, in the following Examples. EXAMPLE 1 (+,-)- 1,2,4-tri-O-benzyl-myo-inositol (0.20 mmole) was treated with 0.66 mmole of chloro (2-cyano- ethoxy)- N,N diisopropylamino phosphine and diisopropyl- ethylamine ((0.60 mmole) in dichloromethane to yield the trisphosphoramidite. Reaction of the trisphosphor- amidite with tetrazole and 2-cyanoethanol yielded the trisphosphite triester m about 90% yield. 31P NMR spectroscopy showed 5Jpp spm-spm coupling m the form of an AB system for two of the phosphite triesters confirming the phosphitylation of the vicinal 4,5-diol system. Oxidation with anyhydrous tert-butyl hydro- peroxide afforded quantitatively the trisphosphate ester and finally deblocking using sodium in liquid ammonia effected reductive removal of the benzyl groups and removal of the 2-cyanoethyl groups by beta-elimination to give the trisphosphate product which was purified by ion-exchange chromatography. In order to remove contaminating traces of other inositol phosphates the product was further purified by HPLC. DL-mvo-inositol- 1,4,5-trisphosphate was obtained in about 50% yield. The 31P and 1H NMR spectra and the SAB mass spectrum of the purified product corresponded with that reported for the same compound isolated from natural sources. EXAMPLE 2 (+,-)-l,2,3,4-tetra-0-benzyl myo-inositol was treated with 2 equivalents of chloro(2-cyanoethoxy)- N,N diisopropylamino phosphine in dichloromethane to yield the bisphosphoramidite. Treatment of the bisphosphor- amidite with tetrazole- and 2-cyanoethanol gave the bisphosphite tπester. P NMR spectroscopy showed that about 90% of the phosphorus could be accounted for by an AB system assignable to the 4,5-bisρhosphite triester system. Oxidation of the bisphosphite triester with tert- butyl hydroperoxide gave the bisphosphate triester. Ammonolysis removed the 2-cyanoethyl groups (heating to 60°C for 3 hours was necessary as mild ammonolysis only removed one of the 2-cyanoethyl groups from each phosphotriester) and treatment with sodium in liquid ammonia quantitatively removed the benzyl groups to give (+,-) yo-inositol 4,5-bisphosρhate. EXAMPLE 3 The 4,5-bisphosphite triester, prepared as described in Example 2 above, was reacted with a solution of sulphur in pyridine to yield the bis- phosphoro-thioate triester and subsequent removal of the protecting groups using sodium in liquid ammonia yielded (+,-)myo-inositol 4,5-bisphosρhorothioate. For particular uses, the sulphur may be radioactive. EXAMPLE 4 (+,-)-1.2. ,5-Bisisopropylidene-mvo-inositol (0.325 mmole) was reacted with chloro(2-cyanoethoxy)- N,N diisopropylamino phosphine (0.715 mmole) and diisopropylethylamine (0.715 mmole) in dichloromethane (1 ml) for one hour to yield the bisphosphoramidite. Reaction of the phosphoramidite with tetrazole and 2-cγanoethanol (0.715 mmole of each) gave the 1,4-bis- phosphite triester. Oxidation with tert-butyl hydroperoxide (1 ml) gave the bisphosphate triester. Ammonolysis (2 hours at 65 C) removed the 2-cyanoethyl groups and treatment of the ammonium salt in water with excess Dowex (Trade Mark) H ion-exchange resin for one hour gave myo-inositol 1,4-bisphosρhate (80%) which was converted into the solid cyclohexylammonium salt. EXAMPLE 5 The bisphosphite triester, obtained as described in Example 4 above, (0.325 mmole) was oxidised using sulphur (200 mg) in pyridine (2 ml) . The mixture was filtered, the solvent evaporated and the residue dissolved in methanol. Ammonolysis (3 hours at 65°C) gave the bisphosphorothioate and treatment with Dowex (Trade Mark) H ion-exchange resin for 3 hours followed by evaporation and addition of ammonia gave the crude ammonium salt as a gum which, on trituration with methanol, gave the ammonium salt of (+,-)-inositol 1,4- bisphosphorothioate as a white solid (51%) . EXAMPLE 6 In analogous manner, (+)-1,2,4-tri-O-benzyl-myo- inositol was converted to the corresponding tri[di(2-cyanoethyl)]phosphite via the phosphoramidite as previously described and then oxidised using sulphur in pyridine. Complete deprotection of all the protecting groups was accomplished using sodium in liquid ammonia, the 2-cyanoethyl groups being removed by beta-elimination and the benzyl groups cleaved to yield a crude product. Purification of the crude product on a column of DEAE Sephadex A-25 (Trade Mark) using a linear gradient of triethylammonium bicarbonate gave DL-myo- inositol- 1,4,5-trisphosphorothioate eluting at around 800mM buffer in a yield of about 25%. [ 31 P NMR (D 2 0, pH9) proton coupled α ' p 3d, 42.0 (J pH =8.0 Hz); 44.9 (J pH =9.3 Hz); 45.1 (J pH =9.3 Hz)] (shown in Fig.2). . The proton-coupled P NMR spectrum was similar to that reported for authentic D-mγo-inositol-1,4.5- trisphosphate and its synthetic DL-isomer except that the resonances were shifted about 40ρpm downfield as would be expected for the phosphorothioates. In biological evaluation of trisphosphorothioate, using rat cerebellum membrane receptor-binding assay specific for myo-inositol-l,4,5-trisρhosphate, the tris¬ phosphorothioate was found to have a high affinity for such receptors, indicating the potential of the sulphur analogues in studies on receptor-linked phosphoinositide metabolism. Additionally it"was found to release calcium from permeablised cells as a full agonist with an EC-o.u Λ of only three times higher than the 1,4,5- trisphosphate. EXAMPLE 7 (+)-1 r 2:4 ,5-Diisopropylidene-mvo-inositol (0.325 mmole) was reacted with chloro(2-cyanoethoxy)- N,N diisopropylamino phosphine (0.715 mmole) and diisoproplyamine (0.715 mmole) in dichloromethane for one hour at ambient temperature to yield the corresponding bisphophoramidite. [d p (CH 2 Cl 2 ) 4s, 152.24, 152.30, 152.67, 152.71]. 3 P NMR resonances corresponding to unequal pairs of epimers were observed, on account of the two chiral phosphorus centres (intensity ratios, downfield pair: upfield pair, approximately 2:1) Reaction of the bisphosphoramidite with tetrazole and 2-cyanoethanol (0.715 mmole of each) gave the bisphosphite triester. [S p (CH 2 Cl 2 ) 2s, 138.8, 139.61]. The bisphosphite triester (0.325 mmole) was oxidised to the bisphosphorothioate ester using sulphur (200 mg) in pyridine (2 ml) . The reaction mixture was filtered, the liquid evaporated and the residue dissolved in methanol [c , (MeOH) 2s, 65.85, 67.41]. Ammonolysis (3 hours at 65 C) gave a bisphosphoro¬ thioate possessing two isopropylidene ketals [d_ (D 2 0, pH 12) proton coupled, S, 43.16, J=10.96 Hz], and treatment with excess Dowex H (Trade Mark) ion- exchange resin in water for 3 hours followed by evaporation and addition of ammonia gave a crude gum, which on trituration with methanol gave the ammonium salt of (±)-my_o_-inositol-l,4-bisphosphorothioate (51% yield) [d p (D_0, pH 12) proton coupled, c , 44.82, J=8.52 Hz; d, 46.34, J=9.74 Hz] contaminated with only minor amounts of -impurities. Final purification was effected on a column of DEAE Sephadex (Trade Mark) as described above. To my_o-inositol-l,4-bisρhosphorothioate (25 micromoles) dissolved in 100 mM glycine buffer at pH 10.1 containing 1 mM MgCl- an 0.1 mM ZnCl- (0.5 ml, ca. 50% D O) in a precision NMR tube was added alkaline phosphatase (30 microlitres, 25 units) and the 31 P spectrum was monitored. No change in the NMR spectrum was observed when monitored over a few hours and overnight incubation (23 hours) showed only the two peaks assignable to the bisphosphorothioate. In comparison, using the bisphosphate ester in a similar experiment, clear evidence was observed for the degradation of the bisphosphate to the monophosphates and inorganic phosphate by the phosphatase. Additionally it was found to be resistant to the action of the inositol 1,4,5-trisphosphate-specific 5-phosphatase enzyme, thus demonstrating the phosphatase resistance of the phosphorothioate analogues. EXAMPLES 8 to 15 By methods analogous to those described in the preceding Examples, the following inositol phosphoro- thioates may be prepared: 8. mvo-inositol-1-phosphorothioate. 9. myo-inositol-1,4,-bisphosphorothioate. 10. mvo-inositol-1,4,5-triρhosρhorothioate. 11. myo-inositol-1,4,5-triphosρhorothioate. 12. myo-inositol-1,3,4,5-tetrakisphosphorothioate. 13. myo-inositol-1,4,5, 6-tetrakisphosphorothioate. 14. myo-inositol-1,3,4,5, 6-pen akisphosphorothioate. 15. myo-inositol-1,2,3,4,5, 6-hexakisphosphorothioate The optical configuration of these products is determined by the optical configuration of the starting materials. If the precursors of the products are optically resolved, the resolution will be reflected in the configuration of the product'. EXAMPLE 16 (±)-2,3, 6-tri-O-benzyl-l,4-(2,2,2-trichloroethyl phosphate)-myo-inositol (0.091 gram, 80 micromole) was dissolved in dry acetonitrile (1 ml) . To this solution were added N,N-diisoρroρylamine (0.011 gram, 80 micromole) and chloro(2-cyanoethyl)-N,N-diisopropylamino phosphine (0.024 gram, 100 micromole). The mixture was allowed to stand at room temperature for one hour. The reaction mixture was then treated with tetrazole (0.014 gram, 200 micromole) and 2-cyanoethanol (0.014 gram, 200 micromole) . After a further hour at ambient temperature the resulting phosphite triester was oxidised with sulphur (0.5 gram) in pyridine (2 ml) at room temperature for 16 hours. All volatile products were evaporated and the residue dissolved in dry dioxan (1ml) . This solution was added to a solution of sodium (0.5 gram) in liquid ammonia (50 ml) . After 10 minutes the reaction was quenched with methanol and the ammonia allowed to evaporate. The residue was taken up in water (100 ml) and treated with. Dowex (Trade Mark) H ion-exchange resin until the solution was acidic. The resin was removed by filtration and the solution rendered basic with triethylamine. The solvent was removed by evaporation and the resulting residue, after dissolution in water, was chromatographed on a column of DEAE Sephadex (Trade Mark) A-25 resin (12 x 2.5 cm) using a gradient of triethylammonium bicarbonate (600 ml of each of 150 mM to 1M) . DL-rrry_o_-inositol-l,4-bisphosphate-5-ρhosphorothioate was eluted around 550 mM buffer as the triethylammonium salt. Evaporation of the pooled fractions gave the product as a glass (35 micromole, 44% , 31P NMR, Cd (H 2 0) 2.6, 4.6, PO3 " ), 45.0 (PSO^ " ) •

Claims: CLAIMS An inositol phosphorothioate having the general formula I ; C 6 H 6 (0H) n [ <0P0 2 S 2 -] m [ o*° 2 ° 2 - ] 6 _ m _ n (I) in which n is zero or an integer from 1 to 5 and, m is an integer from 1 to 6. mvo-inositol-1-phosphorothioate. mvo-inositol-1,4,-bisphosphorothioate. my_o-inosi ol-1,4,5-trisρhosphorothioate 5. myo-inositol-1,4,5-trisρhosρhorothioate. 6. myo-inositol-1,3,4,5-tetrakisρhosρhorothioate. • myo-inositol-1,4,5, 6-tetrakisphosphorothioate. myo-inositol-1,3,4,5,6-pentakisphosρhorothioate. mvo-inositol-1,2,3,4,5,6-hexakisρhosphorothioate 10. An isositol phosphorothioate as claimed in any of the preceding claims in which a suplhur atom is a radioisotope. 11. A method of preparing an inositol phosphorothioate having the general formula I; C fi 6H6, (OH) TL [(OPO2-S) 2- ] [(OPO2-O) 2- ],6—m—n (I) in which n is zero or an integer from 1 to 5 and, m is an integer from 1 to 6, said method comprising reacting an inositol derivative having the general formula II; C 6 H 6 (0B) n [0 0CH 2 CH 2 CN 2 ] 6 _ u (II) in which n is zero or an integer from 1 to 5 and B represents a hydroxyl-protecting group, with a solution of elemental sulphur in a solvent therefor, and removing the hydroxyl-protecting groups from the product of the reaction. 12. A method as claimed in claim 11, in which the hydroxyl-protecting group represented by B in formula II is a benzyl group, whereby the compound of formula II has the formula IIA: . C 6.Ho, (OBz n [OP(OCH Δ-CH Δ-GH)]o,—n (IIA) in which Bz is a benzyl group, and n is zero or an integer from 1 to 5. 13. A method as claimed in claim 11 or 12, in which n is an even number and the group represented by B in formula II is an isopropylidene group, whereby the compound of formula II has the formula IIB:. C D,HO, (O-iPr'-O HI [0P(0CH Δ„CH Δ,CN)]p π (IIB) in which iPr' is an isopropylidenyl group, m is- zero or 1 or 2 and p is 2 or 4 or 6. 14. A method as claimed in claim 11 or 12 or 13, in which the reaction with sulphur is effected by treatment of the compound of formula II with a solution of sulphur in pyridine. 15. A method as claimed in any of claims 11 to 14, in which the protecting groups are removed by treatment with sodium and liquid ammonia. 16. A method as claimed in any of claims 11 to 15, in which the inositol derivative having the general formula II defined in claim 10, is prepared by a method comprising reacting a compound having the general formula III: • . in which n and B are as defined hereinabove, with chloro(2-cyanoethoxy)-N,N-diisoproρylamino phosphine, which has the formula IV: (iPr) 2 N P (Cl) 0CH 2 CH 2 C (IV) in which iPr represents an isopropyl group, to obtain a compound having the general formula V: C 6 H 6 (0B) n [0P(0CH 2 CH 2 CN ) N i^^ 2 6 -τι (V) in which n, B and iPr are as defined hereinabove; and reacting the compound of formula V with terazole and 2-cyanoethanol to form the said compound of general formula II 17. A method as claimed in any of claims 11 to 16, in which the compound having the general formula II is reacted with an organic oxidising agent followed by removal of the hydroxyl-protecting groups from the product of the reaction, to give a product of general formula I in which one or more of the substituents is "a phosphate ester group. 18. A method as claimed in claim 17, in which the oxidising agent is tert-butylhydroperoxide. 19. Pharmaceutical preparations containing as active principle an inositol phosphorothioate having the general formula I defined in claim 1. 20. A method of preparing an inositol phosphate having the general formula I; C 6H6, (OH)n. [(0P0 ΔS) 2" ]m [(0P020) 2" ]o,—πi—n (I) in which n is zero or an integer from 1 to 5 and, m is zero, said method comprising reacting an inositol derivative having the general formula II; G H (0B CO COC CH N l ( II ) in which n is zero or an integer from 1 to 5 and B represents a hydroxyl-protecting group, with an organic peroxide, and removing the protecting groups from the product of the reaction. 21. A method as claimed in claim 20 in which the organic peroxide is text-butyl hydroperoxide.

Inventors: POTTER BARRY VICTOR LLOYD, POTTER, BARRY, VICTOR, LLOYD
Applicants: RESEARCH CORP, RESEARCH CORPORATION LIMITED
Application year: 1988
Main classification: C07F9
IPC codes: A61K31, A61P3, C07F9