FI68243C - FRAMEWORK FOR PHARMACOLOGICAL ACTIVATION OF ACTIVE DIPHOSPHONATERS - Google Patents

Description

ΓΒ1 rt1) MONTHLY CONSUMPTION ££ 30/7

JfltMjA lJ 11 UTLÄGG Nl N GSSKRIFT 0 0 ^ 40 C .... Patent granted 12 08 1935 ^ Patent cedielat (51) Ky.lk. * / Lnt.a. * C 07 F 9/38 FINLAND —FINLAND (21) Pttenttlhakemu * - PstentantBkning 802 ^ 72 (22) Htkemlspilvi - Aniöknlngidag 06.08.80 (O) (23) Starting point— Giltighetsdtg 06.08.80 (41) Has become public - Blivit offentlig 26.03 · 81

National Board of Patents and Registration / 44) Date of publication of the publication

Patent- och registerstyrelsen '' Aniökan utlagd och utl.skrlften publicerad (32) (33) (31) Privilege requested - Begird priority 25.09.79 Iso-Bri Tann ia-Storbri tannien (GB) 7933157 (71) Symphar SA, 6, Chemin des Carpiöres, 1219 Le Lignon, Geneva, Switzerland

Schweiz (CH) (72) Craig L. Bentzen, Onex, Geneva, Mong Lan Nguyen, Nyon, Vaud, Eric Niesor, Gland, Vaud, Switzerland-Schweiz (CH) (7 * 0 Berggren Oy Ab (5 * 0 Method pharmacologically This invention relates to a process for the preparation of novel diphosphonate derivatives and in particular to a process for the preparation of hydroxy-diphosphonates and phosphonophosphates useful in the treatment of cardiac arteriosclerosis.

In the last few years, coronary studies have been performed with common hypolipidemic agents such as clofibrate. More recently, the results of these studies have questioned the therapeutic efficacy of these compounds (see, e.g., New Engl. J. Med. 296, 1185-1190, 1970; Atherosclerosis Rev. 2, 113-153, 1977; The Lancet 8100, 1131-1132 , 1978; and Brit. Med. J. 6152, 1585, 1978).

It would now be desirable to use compounds that rapidly and effectively lower cholesterol directly in tissues and not just in the blood, as is the case with most hypolipidemic agents.

2,6243 Therefore, diphosphono compounds have been studied, in which it has been found that diphosphonates of general formula (I) have a remarkable anti-arterial anti-embrittlement effect and the ability to change the lipoprotein pattern towards very dense lipoproteins and directly purify various tissues of cholesterol.

R03R2 (? V A - C - X (I) PO3R, 2

In the above formula (I), X is OH when m is zero and X is H

when m is 1; R and R 'are the same or different and are H, OL, or C "Hc and A are selected from the group consisting of .ο- · Αΐ; ©? "CX.-Θ '

Y CH II

J o £>, CH2 »n- and ^ Q-O-, CH2» n- 'wherein n is an integer from 1 to 6 and Y is H, CH3, OCH3 or halogen, especially chlorine or fluorine.

The ability of these compounds (I) to remove cholesterol from tissues allows their use in diseases triggered by or due to abnormal cholesterol synthesis, metabolism and precipitation. Examples are cardiovascular diseases in general, which involve the deposition of cholesterol on the arterial walls (Atheromas), high levels of cholesterol in the blood and deposition of cholesterol in subcutaneous tissues (Xanthomatosis), gallstones (cholesterol precipitates), cancerous tissues with cholesterol metabolism ii rich - very sensitive platelets (Shattil, SJ

et al., The Journal of Clinical Investigation 55, 636-634, 1975), etc.

68243

Because cholesterol is the substance that makes up steroid hormones (male and female sex hormones and adrenal cortex hormones), the abnormal synthesis of these hormones can be regulated by the use of such compounds. The potential use of phosphonates in these areas described above is under investigation. In addition, a few compounds of formula (I) have an effect on blood glucose deficiency.

Diphosphonate derivatives of formula (I) can be classified into the two types of compounds below, namely hydroxide phosphonates (Ia) and phosphonophosphates (Ib).

P ° 3R2 P ° 3R2 0 A - C - OH (Ia) A - C -H (Ib) po3r-2 ^ o3r-2 wherein A, R and R1 have the same meaning as above.

It is a primary object of the present invention that compounds (Ia) and (Ib) can be readily prepared from the same starting materials by reacting a dialkyl acyl phosphonate of formula (II) with a dialkyl phosphite of formula (III) in the presence of a base, preferably an amine. The course of the reaction can be controlled by varying the amount of base used, i.e. a catalytic amount of the base (about 5 mol%), for example di-n-butylamine gave the hydroxydiphosphonate compounds (Ia), while the use of the scima base in an amount of about 80 to 100 mol% gave the phosphonophosphates (Ib) according to the following reaction formula: 68243 4 P ° 3R2 5% base A - C - OH (Ia) / R03R2 "/ A - C - PO3R2 + HP (0) (0R ') 2 / (II) (III) \ \ Γ3 * 2 80 - 100 % base A - C - H (1b) PO 3 R 2 The starting material * i.e. dialkyl acyl phosphonates (II) are known compounds and can be synthesized according to a well-known general method, namely by reacting an acid halide with a trialkyl phosphite.

O O

A - C - Cl + P (0R) 3 -> A - C - PO3R2 (II)

The advantage of the process is that although the starting materials (acyl phosphonates, dialkyl phosphites, base) are soluble in cold ether, the obtained compounds (Ia) or (Ib) are not and can therefore be conveniently removed from the reaction mixture by filtration. This allows one-step purification by crystallizing the obtained compounds to the degree of analytical purity. Therefore, the process of the present invention is preferably carried out using ether as a solvent at a temperature of about 0 ° C. In some cases, the ether may be added to a small amount of methylene chloride.

The structure of compounds (Ia) and (Ib) has been confirmed by IR or NMR spectroscopy. All hydroxydiphosphonate compounds (Ia) showed absorption at 3260-300 cm -1 (OH), a peak absent from all books of phosphonophosphates (Ib). Both sets of compounds (Ia) and (Ib) had intense phosphonate absorption at 1260 cm -1 (P = O group) and 1060 cm -1 (P-O-C).

68243 5

The process of the present invention is described in more detail below by means of Examples 1-5, which are directed to the preparation of some diphosphonate derivatives of general formula (I).

Example 1

Tetramethyl 1- (p-chlorophenyl) methane-1-hydroxy-1,1-diphosphonate (Compound 1) (Method from D.A. Nicholson and H. Vaughn, Journal of Organic Chemistry 36, 3843, 1971)

f3 (CH3> 2 Cl - // n-C - OH

\ = / I

P03 (CH 3) 2

Dimethyl phosphite (4.40 g, 40 mmol) and di (n-butyl) amine (0.24 g, 2 μmol) were dissolved in 90 mL of ether and the resulting solution was cooled to 0 ° C. Dimethyl p-chlorobenzoylphosphonate (9.96 g, 40 mmol) (prepared according to Journal of the American Chemical Society 86, 3862, 1964) was added dropwise with vigorous stirring. The white solid separated almost immediately. The mixture was stirred for 1 h at 0 ° C and filtered to give 13.0 g (36 mmol) of the title compound.

Purification was performed by dissolving the crude compound in acetone at room temperature 68243 and adding ether to crystallize it (acetone: ether ratio = 3: 1). 7.9 g (22 mmol) of white crystals were obtained in a yield (pure compound) of 55%.

yield (raw) = 90%

mp = 119-123 ° C

IR (KBr): 3260 cm-1: OH

2880: aliphatic C-H

1500: aromatic C-C

1280 + 1240: p = o

1060: P-O-C

MS: m / e = 360 (M + 2) +: 17% 358 (M) +: 52% 251 (M + 2-PO 3 Me 2) +: 33% 249 (M + PO 3 Me 2) +: 100% NMR (CDCl 3) : δ = 7.90 - 7.20 (multiple dashed line, 4H): phenyl group.

4.50-4.20 (triplet, 1H, J = 7 Hz): H from the hydroxyl group, removed by exchange for deuterium oxide.

3.90 - 3.50 (multiple dashed line, 12H): H of the methyl groups.

Analysis: cnHi7C10yP2

Calculated: C 36.84, H 4.78, P 17.27%

Found: C 36.81, H 4.78, P 17.26%.

To verify the structure of compound 4, it was converted to the corresponding monosodium salt of hydroxydiphosphonic acid (Compound 10) as follows: .V v PCUHNa ci- ^ y— c - oh po3h2

A mixture of 3.59 g (10 ramoles) of compound 4 and 15 g of 37% hydrochloric acid was refluxed for 3 hours. Evaporation of HCl and H 2 O gave 3.2 g (10 mmol) of a white solid, mp: 192-194 ° C (crude) yield: 100% (crude).

it 68243 7

For purification purposes, hydroxy (p-chlorophenyl) methylenediphosphonic acid was converted to its monosodium salt according to the purification method below from P.F. From Pflaumer and J.P. From Filcik, Chemical Abstracts 72, 55656k, 1970:

The solid obtained as described above was dissolved in a mixture of 4.8 g (80 mmol) of glacial acetic acid and 0.7 g (39 mmol) of water at 95 ° C. Sodium acetate titrate hydrate (1.36 g, 10 mmol) was then added gradually. The bulky precipitate was obtained almost immediately. It was filtered and washed thoroughly with ether until the acetic acid odor had disappeared. The purified precipitate was recrystallized from ethanol: water (20:80) to give 1.94 g (6 mmol) of a white powder of the monosodium salt of 1-hydroxy-1- (p-chlorophenyl) methane-1,1-diphosphonic acid (Compound 10). : 60%.

Example 2

Tetramethyl 2,2-dimethyl-2- (p-chlorophenoxy) ethane-1-hydroxy-1,1-diphosphonate (Compound 2) (Method from KD Berlin et al., Journal of Organic Chemistry 30, 1265, 1965 , and an article by DA Nicholson and H. Vaughn in Journal of Organic Chemistry 36, 3843, 1971), -v CH. PO, (CH,) ~

Cl-f V O-C - C - OH

CH3 PO3 (CH3) 2 p-Chlorophenoxy isobutyryl chloride was first prepared by basic hydrolysis of ethyl p-chlorophenoxy isobutyrate and refluxing the acid in thionyl chloride according to the following standard procedure.

10.6 g (86 mmol) of trimethyl phosphite were added dropwise to 20.0 g (86 mmol) of p-chlorophenoxyisobutyryl chloride cooled to 0 ° C. To verify the reaction, it was found that methyl chloride evolved. Distillation under reduced pressure gave 19.0 g (62 mmol) of dimethyl p-chlorophenoxyisobutyrylphosphonate as an almost colorless oil.

8 kp = 115-118 ° / 5 · 10-2 Torr 6 8243 yield = 72%

IR (film): 3000 cm -1: aliphatic C-H

1740: C = 0

1500: aromatic C-C

1250: P = 0

1050: P-O-C

830: 1,4-disubstituted phenyl

A solution of 2.86 g (26 mmol) of dimethyl phosphite and 0.18 g (1.4 mmol) of di (n-butyl) amine in 65 ml of ether was then cooled to 0 ° C and dimethyl-p-chlorophenoxy -isobutyryl phosphonate (7.97 g, 26 mmol) was added slowly with vigorous stirring. A white solid was about to form almost immediately. The mixture was stirred at 0 ° C for 1 hour, then the solid was filtered off. Extraction of benzene: hexane (60:40) gave 7.18 g (17.2 mmol) of white closed crystals of the title compound, m.p. tetramethyl-2,2-dimethyl-2- (p-chlorophenoxy) ethane-1-hydroxy-1,1-diphosphonate.

mp - 137-139 ° C

yield = 66%

IR (KBr): 3360 cm-1: OH

3000: aliphatic C-H

1500: aromatic C-C

1250 + 1220: P = 0

1070: P-O-C

860: 1,4-Disubstituted phenyl NMR (CDCl 3): δ = 7.4-7.0 (multiple dashed line, 4H): phenyl group 4.0 to 3.70 (multiple dashed line, 12H): H of methyl groups attached to phosphonate groups 3.5-3.4 (sharp peak, 1H): H from hydroxyl group, removed by exchange for D 2 O 1.58 (singlet, 6H): H from branched methyl groups.

Analysis: ci4H23C108P2

Calculated: C 40.45, H 5.58, P 14.90%

Found: C 40.29, H 5.94, P 14.93% 9 68243

Example 3

Tetramethyl 1- [4- (4'-chlorobenzoyl) phenyl] methane-1-hydroxy-1,1-diphosphonate (Compound 3) (Method from an article by DA Nicholson and H. Vaughn in Journal of Organic Chemistry 36, 3843 , 1971).

RO3 (CH 3) 2

Cl'-v ^ N γ \ Γ ~ C - OH

EO3 (CH 3) 2

O

The starting compound 4- (4'-chlorobenzoyl) benzoyl chloride was prepared by G.E. Robinson and J.M. According to an article by Vernon in the Journal of the Chemical Society (C), 2586, 1970, and according to an article by E. Wertheim in the Journal of the American Chemical Society 55, 2540, 1933.

Trimethyl phosphite (10.9 g, 88 mmol, 10% excess) was added dropwise to the acid chloride (22.4 g, 80 mmol) heated just below the melting point (about 100 ° C).

The reaction was exothermic and the white crystals of the acid chloride turned to a brown oil, foaming considerably. The reaction mixture was stirred at 100 ° C for 30 min. As it stood and cooled, the oily substance turned to an orange solid. Recrystallization from 60:40 chloroform: petroleum ether gave 20 g (56.7 mmol) of pure dimethyl 4- (4'-chlorobenzoyl) benzoylphosphonate.

mp. = 95-97 ° C (yellow powder) yield = 71%

IR (KBr): 2960 cm -1: aliphatic C-H

1665 + 1650: C-0 (from benzoylphosphonate and benzophenone groups)

1590: aromatic C-C

1250 + 1260: P = O 1050 + 1030: P-O-C

A mixture of 2.20 g (20 nunols) of dimethyl phosphite and 0.144 g (1.10 mmol) of di (n-butyl) amine in 40 ml of ether was cooled to 0 ° C and the filtered solution of 7.04 g ( 20 mmol) of dimethyl 4- (4'-chlorobenzoyl) benzoylphosphonate in 40 ml of dichloromethane were added dropwise. A white precipitate soon separated from the yellow solution. The reaction mixture was stirred for 1 hour at 0 ° C and the precipitate was filtered and washed with ether. Recrystallization from acetone gave white crystals of tetramethyl 1- [4- (4'-chlorobenzoyl) phenyl] methane-1-hydroxy-1,1-diphosphonate (2.4 g, 5.2 mmol).

Mp = 150-153 ° C

yield = 26%

IR (KBr): 3280 cm-1: OH

1670: C = 0

1600: aromatic C-C

1260 + 1240: P = 0

1050 + 1030: P-O-C

MS (m / e): 464 (M + 2) +: 14% 462 (M) +: 42% 355 (M + 2 - PO 3 Me 2) +: 32% 353 (M - PO 3 Me 2) +: 100% NMR (CDCl 3 ): = 8.10-7.30 (multiple dashed line, 3H): H of the two phenyl groups 4.5-4.3 (triplet, 1H, J = 7 Hz): H of the hydroxyl group, removed by exchanging D 2 O: lin 3.95-3.60 (multiline, 12H): H of the methyl group

Example 4

Dimethyl 1- (dimethoxyphosphinyl) -p-chlorobenzyl phosphate (Compound 5) - opo3 (ch3) 9

ex -f \ c - H

'-' po3 (ch3) 2

Dimethyl p-chlorobenzoylphosphonate (9.96 g, 40 mmol) was added dropwise to a solution of equimolar amounts of dimethyl phosphite (4.40 h, 40 mmol) and di (n-butyl) amine (5.16 g, 40 mmol). , which was cooled to 0 ° C before the addition. A white solid began to form almost slightly

II

11 68243 immediately. After stirring for 1 hour at 0 ° C, the solid was filtered off. Recrystallization was performed at room temperature from 1: 3 dichloromethane: ether to give 12.0 g (33 mmol) of white crystals.

mp. = 81-82 ° C

yield = 82%

IR (KBr; = 2980 cm -1: aliphatic OH

1500: aromatic C-C

1290 + 1260: P = 0

1050: P-O-C

NMR (CDCl 3): δ = 7.5-7.3 (multiple dashed line, 4H): phenyl group.

5.85 to 5.40 (double doublet, J-11 and 13 Hz, 1H): H of the methine group (not removable by exchange for deuterium oxide) 3.95 to 3.50 (multiple dashed line, 12H): H of the methyl groups.

Elemental analysis: C 11 H 23 ClO 7 P 2 Calculated: C 36.84 H 4.78 P 17.27% Found: C 36.71 H 4.86 P 17.33%

Example 5

Dimethyl N- (dimethoxyphosphinyl) -2,2-dimethyl-2- (p-chlorophenyl) ethyl phosphate (Compound 7) / 7H-H3O3 (CH3) 2

Cl - (/ / —c - c - H

ch3 po3 (ch3) 2

Dimethylation of p-chlorophenylacetonitrile with sodium amide and methyl iodide in ether gave dimethyl p-chlorophenylacetonitrile. Hydrolysis of this nitrile and refluxing the acid in thionyl chloride gave p-chlorophenyl isobutyryl chloride.

Dimethyl p-chlorophenyl isobutyryl phosphonate was prepared in 90% yield by adding an equimolar amount of trimethyl phosphite to the acid chloride described above cooled to 0 ° C.

12 · 68243

Kp. = 108-110 ° / 5 · 10-2 Torr (white oil) IR (film): 1690 cm -1: C = 0 1270: P = 0

1070 + 1040: P-O-C

A solution of 3.30 g (30 mmol) of dimethyl phosphite and 3.1 g (24 mmol) of di- (n-butyl) amine and dimethyl p-chlorophenyl isobutyrylphosphonate (8.72 g) was then cooled to 0 ° C. g, 30 mmol) was added with rapid stirring. The white solid soon separated. After stirring for 1 hour at 0 ° C, the solid was filtered off. Recrystallization from ether gave 9.0 g (75%) of the title compound, dimethyl (H-dimethoxyphosphinyl) -2,2-dimethyl-2- (p-chlorophenyl) ethyl phosphate as white crystals.

Sp. = 62-63 ° C

IR (KBr) = 2980 cm-1: aliphatic C-H

1500: aromatic C-C

1280 + 1260: P = 0

1080 + 1030: P-O-C

MS (m / e) = 402 (M + 2) +: 0.5% 400 (M) +: 1.5% 248 (M-Cl - ^ ^ Vi 3 + H) +: 100% λmax NMR (CDCl3 ) δ = 7.5 - 7.25 (multiplet, 4H): phenyl group 5.05 - 4.80 (double doublet, J = 9 and 12 Hz, 1H): H of the methine group, (not removable by exchange for D 2 O) 3.85 - 3.50 (multiplet, 12H): H of the four methyl groups bound to the phosphate and phosphonate groups of 1.58 and 1.54 (two partially overlapping singlets, 6H): H of the two branched methyl groups.

Il 1 3 68243

Other compounds of formula (I) were prepared by similar methods as above and the properties of the prepared compounds (I) are shown in Tables I, II and III below.

All NMR spectra of the hydroxydiphosphonate ester compounds (Ia), (compounds 1-3) had the characteristic absorption of the hydroxy group: a sharp peak (for compound 2), or a triplet (for compounds 1 and 3), all of which were exchangeable for deuterium oxide.

The absorption of methine hydrogen atoms was a double doublet (J10 and 12 Hz) characteristic of two different phosphorus atoms and a bound proton: ° -pi

- C - H I

P2

The structure of the hydroxydiphosphonate ester compounds (Ia) was further confirmed by acid hydrolysis of compound 1: the corresponding hydroxydiphosphonic acid was obtained, which was then isolated in its monosodium salt form to facilitate purification (compound 4).

The NMR spectrum of the phosphonophosphate compounds (Ib) also had a characteristic pattern: δ -7.5-7.3 multiplet, phenyl group δ - 5.8 - 5.4 (compound 5) 5.1 - 4.8 (compounds 6, 7 and 8), a double doublet, J ~> 10 and 12 Hz, corresponding to the absorption of the excess hydrogen atom, cannot be removed by exchange for deuterium oxide.

δ ^ 3.9 to 3.5 multiplet, methyl ester groups,

δ = 1.60-1.55 (compounds 6, 7 and 8 only) two singlets, branched methyl group CH

f J

(-c-) ch3 14 68243

The MS spectrum of all didosonate ester compounds (Ia – ib) was characterized by a spectrum: molecular ion (M +) with significant intensity (10–30%) and a fundamental peak (100%), corresponding to the loss of the phosphonate ester group (M -).

The only exception was the mass spectrum of compound 2, which had no molecular ion but the peaks corresponded to the decay of the molecule. The structure of the compound was confirmed without any doubt by microanalysis.

1 5 68243 o Γ »o tC ^ I - U o

Λ G (N

M Φ O T— I

I G X

g · Η II - - O | O

O + * ° θ '»O m' t -P Dl VO II CM I (T> CM U> - Oi

p fö ·· T- - I

o * w u + ~ • H -P ° I * * O: r0 o

Jj EH o O O O CO d) 00

+) Dj O O cm I O MDO> O

dl M T-diin vdcnt— d) * - Ό O **. ^ Ή 3 in + + i 3 Λ * · * ·> (fl Π3 o o o o o o o o ooo • h l in co vo m ^ oo o in vooo

(¾ qj CM CTi CM O CO (N ID O -3 * 0 tN

£ (_ | CO CM t- t— (O CO f— i— CO CO t—

B

e o -p

m 10 H i — I

(0 CO 00 CO

h ro <n co in X U V r- Τ Ο -h 0 1 II o X to o 't "o o X> T- co m co

3 14-1. T- T- T- A

tH Dj 3 - en ro (0

Eh h

G

O

• M

-P

-p to 3 (0

G K S

O

in (¾ ro co co rr rr in * K K \ \ O ». U U U co T- M« -o \ OM co U ro \\ xi u-o-u \ l

It φ 00 ° Φ

<M M M rH

u u u u <u

• P

C/O

•B

o O ^ CM CO Tr Λ ··

tH C

68243 16

S

K CJ CJ> Λ

Il II> 1 o u u cj u

rh "CC CC

I <D O O <L) φ n ro

g CC M CCOKIK

O P -H di P · Η II CJ - CJ - CJ

- P P P P Οι I

P P ·· -p p

4J (0 (0 (0 03 • · M

n (ö (0 u (0 <a cj

• h p E o i p E o o I

ρ ή omo -novo oo O

+ J (¾ rH P CN I γΗΡΓΜτ- | d) ^ nJ n3 '- CP tö ro' - 1— di 'O O.

2 03 ···· - ♦ - # · ·· ·· + H * ·· C Xl

0) 1¾ O OO O OOO O O

H I 00 O O LO 00000 O LO

nj di «JiiniNo σι in cn cm o C) - | rl i r i (N '-' t— r-

♦ H

E

O

P - I ---- '' '' N

CD ^ λ tn

B

rH

ra H rö - h Λί cj m oo n

• rH O 00 ^ VO

O ie ^ I I l Λ! > i r- <n λ; p · ° o -¾1 vo 3 a

rH -. C/O

3 A

ra H

Eh ~

C

0)

• rH

P

P

3 ra p S ps ^ jt> nj K K ffi n * · cj cj o c * o p

O

^ H ΓΟ Π n ro ™ ™, _, T * * T · * T * MrH »JU * I * i» 'MM MH MM MM n - M -

CJ - CJ - CJ CJ - CJ - CJ CJ-CJ-CJ

ä 0 0 0 0

rH O

<rH U ro CJ ffi u 03

P

tn

•B

r ^ J O ^ \ 0 Γ ** CO

··

> <G

II

17 68243

The pharmacological activity of the compounds of formula (I) is described in more detail below in Examples 6-8.

Example 6

Effects of diphosphonates of formula (I) on lipid metabolism in common breeds Method used: Groups of 4 or 5 common male Wistar rats weighing 200 g were orally treated with diphosphonate (200 mg / kg / day) for 4-21 days. Water-soluble compounds were administered in water or dissolved in 24 nmol of bicarbonate buffer. Lipid-soluble compounds were administered in corn oil. Rats were weighed and killed by decapitation (in light ether anesthesia after an overnight fast).

Blood was collected and serum was analyzed. The following changes in blood parameter describing lipid metabolism were reported: free fatty acids were measured by W.G. According to Duncombe (Clin. Acta 9, 122, 1964) triglycerides by the enzymatic method (Boehringer Mannheim Kit 126 012) phospholipids: by the molybdate / vanadate reaction (Boehringer Mannheim Kit 124 974) et al. (La Presse Medicale 43, 974, 1958) and D. Watson (Clin. Chim.

Acta 5, 637, 1960).

Received results:

With the exception of compounds 1 and 3, all diphosphonates (I) tested reduced serum free fatty acids as measured in normal rats or cholesterol-fed rats.

This effect appears to be a relatively common property of these diphosphonates having a p-chlorophenyl group and indicates their involvement in lipid metabolism. Similar features have been described in several; with liposuction agents ("Hypolipidemic Agents", ed. David Krit-chewsky, vol. 41, Handbook of Experimental Phannalogy, Springer-Verlag, 349-408, 1975). Serum triglycerides were significantly reduced by 'A' 18 68243 as measured by compounds 1, 2, 3 and 4 and by the acid form of compound 1. In several cases, an increase in serum phospholipid was observed with compounds 1, 2, 3 and 4, 5, 6 and: 7. In particular, compound 1 was found to be at least twice as active. The cholesterol in the 6-lipc protein fraction (very low density lipoproteins VLDL and low density lipoproteins LDL) decreased while α-lipoprotein (high density lipoproteins HDL) cholesterol increased, leading to a very favorable α-cholesterol / 8-cholesterol. In long-term therapy, this effect was associated with decreased liver and aortic cholesterol levels. Clofibrate was tested in a comparative manner and was found to reduce phospholipids by 33.6% and the α / 8 ratio by 52.2%. The results are consistent with C.E. Dayn et al. (Artery 5, 90-109, 1979) and K.R. Muller and G.G. With results published by Cortes (Artery 4, 564-577, 1978) showing that clofibrate lowers HDL cholesterol in rats.

The above results indicate that these diphosphonates have the property of altering lipid metabolism, in particular by increasing the amount of lipid transported by α-lipoproteins (mainly cholesterol) and decreasing the amount of lipids transported by β-lipoproteins (mainly triglycerides). Because HDL cholesterol levels have been shown to be inversely correlated with cardiovascular risk (see N.E. Miller, Lipids 13, 914-919, 1978), diphosphonates that increase HDL levels may be useful in the potential treatment of atherosclerosis. It is important to note that the acid or salt form of compound 1 and the relatively simple diphosphonate compound 1 do not have these properties. It is also important to note that diphosphonates having a different structure from compounds 1, 2, 3 and tested by others do not have the property of affecting lipid metabolism (see W. Hollander et al., Atherosclerosis 31, 307- 325, 1978 and Mellies et al., Artery 6, 38, 1979).

Example 7

Effect of diphosphonates of formula (I) in cholesterol-fed rats

It 68243 19 Method used:

To increase cholesterol in tissues, especially liver, rats were fed a high fat cholesterol diet for 10 days to 3 months with the following ingredients: casein 20%, butter 37%, cellulose 9.1%, dextrose 18.9%, cholesterol 4.5%, sodium cholate 1.8%, minerals 7.3%, vitamins 1%, choline 0.4%.

The rats were then fed a standard diet and treated for 10 days to 3 months with various compounds (200 mg / kg / day). The serum parameters described above were measured. Liver and aortic lipids were extracted according to J. Folchin et al. (J. Biochem. 226, 497, 1957). Total lipids were determined by the sulfophosphovanil reaction (see N, Zölner and K. Kirsch, Z. Ges. Exp. Med. 135, 545, 1962) and cholesterol was determined by the Liebermann-Burchard reaction.

Received results:

The diet described above increased the total amount of lipids, especially triglycerides and cholesterol in the liver, by 8–10-fold. Treatment with compounds 1, 2, 3, 4 and 7 significantly reduced the total amount of lipids in the liver and / or the amount of cholesterol in the liver. When tested, the same effect was measured in aortic tissue. This indicates that these specific diphosphonates tested have the property of removing cholesterol from tissue. Because cholesterol precipitation has already been well established as an important step in the pathogenesis and / or development of atherosclerosis, these compounds may be useful in preventing or treating injuries caused by atherosclerosis by preventing the deposition of cholesterol in tissues 9 such as the aorta.

Example 8

Effects of the acid and salt forms of the phosphonates of formula (I) d in rats with excessive blood lime Method used:

In recent years, diphosphonic acids have been shown to have an effect on animals with excessive lime in their blood by preventing calcification of the aorta and kidney (see M. Potokar and M.

20 68243

Schmidt-Dunker, Atherosclerosis 30, 313-320, 1978). They have also been shown to prevent vitamin D-induced increases in plasma calcium. This effect may be useful in the recovery of pre-arranged arterial embrittlement disease (see I.Y. Rosenblum et al., Atherosclerosis 22, 411-424, 1975). Because diphosphonates have these effects when administered as acids or sodium salts, the monosodium forms of Compound 1 were also tested using the notes of Potokar (see above). Briefly, groups of four male Wistar rats were administered the acid or salt form of Compound 1 as a 0.05% solution in drinking water, corresponding to about 50 mg / kg per day. Rats were treated with the compounds for 15 days. From day 5 to day 10, blood hypercalcemia was developed by administering 75,000 units of vitamin D 2 / kg / day to control and treated rats. The diphosphonate treatment was continued from the tenth to the fifteenth day. The animals were then sacrificed under light ether anesthesia and serum calcium was determined by B.C. Ray Sarkarin and U.P.S. According to Chanhan (Anal. Biochem. 20, 155, 1967).

Received results:

Calcium deposition has been implicated in the late stages of arterial embrittlement patch development (see W. Hollander, Exp. Mol. Path. 25, 106, 1976), and it has been shown that some diphosphonate acids or salts have an effect on calcium metabolism and may be useful in itself. in the treatment of late stages of atherosclerosis. The fact that none of the esterified forms lowers serum calcium, but the acid and salt forms of Compound 1 lower serum calcium 20 et al.

14% indicate that non-esterified diphosphonates affect calcium metabolism and may reduce calcification of arterial embrittlement.

Some results of the pharmacological activity tests of the diphosphonates of formula (I) described above are shown in Table III below.

li 68243 2 1 • r-4 £ »- * 3 Ai s-iAi to on co o <r co

dl H Z Z Z <N! Z I I

ai rt II

CO Ai

• H

rH

0 μ 0) Pu

du · co σ \ σ * co is O

co w o z - · m z z f ro ι i

u <v II I

μ γη O O <M

C/O

• H · Η | · Μ c tu μ Ρμγ ^ γ ^ οοιλοί u co c Ό: cd · <r <mz ZZI ro il cu μ o · η μ cj iii cu ^ ^ c .: «uo O · η: cd • H <0. id rH £> • 1- ^ μ »id« μ

cc H

0 3 ^

PJ CU

c Cu · vo en co: n co co r · ** ^ • μ ¢ 0 w o uo cm z z z z m i <r 0C O) a) Il li • μ .id * -h

f-t cc O

O £ ^ <0 c μ i co 3 co | j_ * -— co uo co co cm \ c ui ω * μ · co Z cm Z Ζ I ro l en c · η ca oiiiic <0 Ό C: cd a> co * ho μ • μ .id a- ^: cc μ co · μ o: cc • u 22 rrt Ai e rt rt C -ii o —i tn o σ *

M O · CN r · '· «- \ D CM

cndM o cm cm m ^ - oo

O -t-1 a) + rt + + I

* 4-1 g -U

• a d ω -a ia ui tn

rt t — I

- ^ rtoö tococo m mcoa; z | z z z cm c CO C | * f- CO vD <f ρ · μ · μ Pu cm z ro cm c0 S r — t · + + + <0 3 0 U o

Ui cw · μ

0) CO rÖ ro Ό CO

α »ο · μ> vrrsizwcoMr \ o ^" w μ a z + + z Z ro cm «- i> i + +

M

1 · oc <u Pm * - co r--. oo

M * H CO · CM Z ^ - CM

^ g z cj i i + 3 3 r-4 μ r— <μ mC · μ

3 ω · μ * θ ^ tcO'XJONr ^ ocovD

«O <u μ · μ cm co * - cm ·» - Z ro I

H CO μ) μ Ζ ΙΙΙΙΙ + + u U-t o cm Ο · 'νΟ <Γ u ~ i c ο- α ι ι ι • μ «ο g μ χ: 3 cd nj μ co>

ai CU W ΟΟΟΟΟΟϊ ^ Γ ^ O

a »co co <r cm m> *“ cm co LO l l

co> μ z I + I I I Z I

♦ μ μ μ

ΊΟ ^ CO

W ·· O co • με 3, μ Ό Cu i! o) co · μ> ^ μ ^ -ΓνΙΓΟΖΜΤ'-μΐ-Ο'ΜΟΓ ^

^ O

»-Μ 68243 22

Note; - Table IV above gives the results as percentage benchmarks. Except for serum lime, those values that differ from the reference values by less than 15% are considered insignificant (NS).

- Serum free fatty acids, triglycerides and phospholipids were measured in normal rats (N) and rats fed cholesterol for 10 days (C.F).

- Serum α / β-cholesterol, total liver and aortic lipids, and cholesterol were measured in normal and pre-fed high-cholesterol rats.

- Serum lime was measured in rats with dirty lime in their blood.

Pharmacological screening of the diphosphonate derivatives of formula (I) according to the invention has shown that these compounds have certain properties and effects on lipids and Lipi-dimetabolism and can be used for the treatment of cardiovascular disease for the following reasons: increase phospholipids. The latter property can be coupled to HDL lipids, especially HDL cholesterol, which is most dramatically found with compounds 1 and 5.

- They have the important property of significantly reducing and eliminating lipids, especially cholesterol, from the liver and aorta of rats fed a high amount of fat and cholesterol. Experiments not described herein have also shown that compounds such as 1 increase cholesterol excretion in bile and feces, resulting in a net loss of tissue cholesterol.

Thus, it should be noted that the primary effects of said diphosphonates (I) are different and novel compared to classical hypolipidemic compounds. The specificity of these effects (elevated HDL cholesterol and removal of cholesterol from the tissue) strongly suggests a potential pharmaceutical use in arterial embrittlement disease. Rabbit experiments in which experimental atherosclerosis was achieved by feeding cholesterol confirmed the above findings that

II

68243 23 compound 1 having a p-Cl group is the most active.

The fact that the acid and salt forms of Compound 1 affect calcium grain metabolites further suggests that all non-esterified forms of these described diphosphonates may also be used in the treatment of late stages of atherosclerosis.

Example 9

Effect of Diphosphonates of Formula (I) on Blood Glucose 150-200 g of male Wistar rats (5 / group) were treated for 4 days with prescreened phosphonates. They were fasted overnight and killed on the fifth day by severing the head under light ether anesthesia. A blood sample was taken using EDTA as an anticoagulant. Results are given as mean values + sem. The results given in Table IV suggest that p-chlorophenyl diphosphonate was most effective, especially when administered intraperitoneally.

Table IV

Effect on hypoglycemia

Method of administration, dosage Plasma glucose and excipient Comparisons - (m2 / -AQ.Q - Treated____ Cpd 4 Cpd 2 Cpd 21 Cpd 27 ip 50 mg / kg water buffer 87+ 3 36+ 1 po 50 mg / kg water buffer 132+ 8 100+ 12 po 50 mg / kg corn oil 125+ 10 90+ 5 po100 mg / kg water buffer 111+ 7 93 + 12 po 50 mg / kg corn oil 125+ 10 110 + 10 po200 mg / kg water buffer 131+ 4 88+ 5 po200 mg / kg aqueous buffer 131+ 4 102+ 9

Glucose content was determined by W. Werner and H.G. By the Wielinger enzymatic method (Z. Analyt. Chem. 252, 224, 1970), (obtained from Boehringer Mannheim, pack no. 124036).

The scope of the present invention also comprises preparations for reducing hypoglycaemia and acting against arterial embrittlement, which contain as active ingredient a pharmaceutically effective amount of one or more diphosphonate derivatives of formula (I).

2 4 Λ 68243

Safe and effective amounts of the phosphonate compound are prepared to provide the desired effect with a reasonable benefit / risk ratio associated with any drug treatment. Within the limits set by an acceptable and reasonable medical assessment, the dosage of the phosphonate compound will vary depending on the particular condition being treated, the severity of the condition, the duration of treatment, and the particular phosphonate compound used.

Phosphonates are prepared as pharmaceutically acceptable products containing all the ingredients of the mixture used and suitable for use in contact with human and animal tissues without excessive toxicity, irritation or allergy to a reasonable benefit / risk ratio.

The preparation of the pharmaceutical compositions of the present invention for oral dosage unit may be a mixture with a solid excipient containing lactose, sucrose, sorbitol, mannitol, amidone, amylopectin, a cellulose derivative and / or gelatin, which may be prepared together with lubricants such as calcium stearate, magnesium stearate -wax with different forms and / or polyethylene glycol. In some cases, it may be advantageous to use a capsule, in which case the ingredients may consist of a mixture of concentrated sugar, acacia, talc and / or titanium dioxide.

In some cases, certain phosphonates may be mixed with a buffer solution, corn oil, olive oil, commercial glycerol excipients, and administered in a sealed hard gelatin capsule as drops or in the form of a syrup.

Phosphonates can further be prepared with "Imhausen H" as suitable suppositories.

For example, compounds 1-3 were compressed into a tablet form with 10% magnesium stearate and 25% amidone to a final concentration of about 100-300 mg of active ingredient. In addition, the compounds of formula (I) were used in solution in drinking water or corn oil at concentrations of about 2 to 100 mg / ml.

Il V.

Claims (2)

68243 25 Process for the preparation of pharmacologically active diphosphonate derivatives of general formula (I) P ° 3R2 I -3 (°> m A - C - X (I) PO ^ R12 wherein X is OH when m is zero and X is H when m is 1, R and R 'are the same or different and are H, CH 2 or 3a A is selected from the group consisting of: r — v ____ CH- CH_ Qt' Q-O-1-. - - Qh - Cl Y 1 - CH3 υλ - CH3 O in which n is an integer from 1 to 6 and Y is a K, OCH4 or halogen atom, in particular chlorine or fluorine, characterized in that the dialkyl acylphosphonate of the formula (II) O II A - C - pO3R2 (II ) is reacted with an equimolar amount of a dialkyl phosphite of formula (III) in HP (O) (OF?) 2 (III) diethyl ether as a solvent at a temperature of about 0 ° C and in the presence of varying amounts of an organic base such as dialkylamine to give For the preparation of hydroxydiphosphonate derivatives according to I) in which X is OH, m is zero and A, R and R 'are as defined above, the organic base its molar amount corresponds to 5% of the molar amount of each reaction component (II) or (III). To prepare phosphonophosphate derivatives of formula (I) wherein X is H, m is 1 and A, R and R 'are as defined above, the molar amount of organic base corresponds to 80-100% of the molar amount of each of the reaction components (II) or (III). amount. 1. A formulation of a pharmacologically active diphosphonate derivative having the specific formula (I) | ° 3R2 (O) A - C - X (I) PO3Rl2 color X is OH da m is noil och and X is H da m is 1, R och R 'är lika eller oiikä och är H, CH ^ eller C2H5, och A är vald ur den grupp som bestär av D-- Ο-Ρ'Ο-Γ * ci y ch3 y ch3 l | Ij '^^) - (01,) -och (/ 0— (CH ~) -, y 2 n γ> (_ / 2 n 0 color n är ett heltal 1-6 och Y är H, CH3, 0CH3 eller en halogen-atom, preferably chlorine or fluorine, selected from the group consisting of dialkylphosphonates of formula (II) O II A - C - P03R2 (II) having the same equimolar content as dialkylphosphites of formula (III) HP (0) (0R ') ) 2 (III) in diethyl ether at a temperature of about 0 ° C and below