AU616367B2 - Ginkgolide derivatives - Google Patents

Description

11 Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION (OR 1(1 INAL) 616367 tnt. Class Class Application Number: Lodged: Comrplute Specification Lodged: 4 Accepted: 0 Published: Related Art .N,-mq,3f Applicant: Add ress of Applicant Actual Inventor: Address for Service SCIENTIFIQUJES 51/53 ruie clu Docteur Blanche 75016 Paris, France PIERRE BRAQUET and ANDRE ESANU EJ)WD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled: The following statement is a full description of this invention, including the best method of performing it known to us~ 4 ?A

LI

S 1.

~ti 4. The basic application referred to in paragraph 2 of this Declaration first application made in a Convention country in respect of the invention the subject of the application.

D ECLA R ED th d ay o a 19 SOCIETE DE CONSEILS DE RECHERCHES 6 ET D'APPLICATIONS SCIENTIFIQUES GMA E QnR- a..Me..

t Signature.

:i II i nr CII~~lrrr~3 I N -_II -la- The invention relates to Ginkgolide derivatives, to methods for their preparation, and to pharmaceutical compositions containing them.

ano The invention provides l-alkoxy, driti-v ginkgolides A,B,C,J and M and further provides sefrakyP/ 0 t- Od a&itr3o a a F e in, Ah0^ z 1".

Sderivativesof th said inkgclids. The preferred alkoxy groups are methoxy and ethoxy groups.

Q 00 0oo The invention also provides a method for the o000 0 0. preparation of 1-alkoxy derivatives of Ginkgolides A,B,C,J 00 a 0° 0 10 and M and of 10-alkoxy derivatives of the said Ginkgolides the method comprising reacting, in solution, Ginkgolide A,B,C, J or M with an excess of diazoalkane and separating 0 0 0 the resultant mixture of 1-alkoxy-Ginkgolide and 0 00 00 0 6 15 In a preferred procedure, the selected Ginkgolide 000000 0 0 is dissolved in dioxan, suitably at a concentration of Ig per 100 ml, and the selected diazoalkane is dissolved 0 0 in diethyl ether. The solution containi .g the diazoalkane is slowly added to that containing the Ginkgolide, allowing ten equivalents of diazoalkane per equivalent of Ginkgolide. The mixed solution is stood at ambient temperature for from 3 to 8 hours, yielding a mixture of 1-alkoxy-Ginkgolide and 10-alkoxy-Ginkgolide. The separation of the two products from the remaining nonreacted Ginkgolide may suitably be achieved by evaporating off the solvents and eluting the residue through a silica asAL gel column using ethyl acetate hexane 1:1 by volume 4as eluent. The resulting solution is evaporated off 4

I

V- -2and treated by chloroform which dissolved the lO-alkoxy derivatives. The said 10-alkoxy derivative is recovered from this chloroformic solution and the remaining solution ~is then treateu' with diethyl ether which gives the 1-alkoxy derivative.

The alkoxy-Ginkgolides of the invention are of interest in the treatment of PAF-Acether induced maladies, and the invention accordingly also provides a pharmaceutical composition comprising a 1-alkoxy derixratzixwo or 1O-aIko><i derivci-H-i c-P on-c -rhe. (-ikpolides AG, *T or 11 Or mo fu' 0 -r~e I 411/0 Yj 6i1 O 0-aj.9 dervn-fe.s c- one e- -the saed 0&-,kjolde-s i7n adritufe- 0000c p~raLf7~/ ~~epfbe dtih~ent or- arrler, 0000 0 0 0 1 150 The invention is illustrated by the following 0000 00 0 examples: 0 0 CExample 1 1-Methoxy-Ginkgolide B and 10-Methoxy-Ginkgolide

B

To a solution of Ginkgolide B in dioxan (l0g/1) was 0*0 slowly added 10 equivalents of a solution of diazomethane 00 in diethyl ether. The mixture was stood at ambient a 00 0 0 00- 0temperature for 4 hours, and then separated following o the preferred separation procedure described above. 1- 25 methoxy-Ginkgolide B, the structure of which was confirmed o by HPLC, was obtained in 66.1% yield and lO-methoxy -Ginkgolide B was obtained in 24.4%/ yield.

t 1-methoxy lO0-methoxy Ginkgolide A 56.3% 13.2% Ginkgolide C 49.1 16.7% 00lt 0 C0 0 0*60 0 0 0, oO 0 0 009 0 0 0 00 00 0 0 00 00 0 O 4.9 000006 o 0 Example 2 l-Ethoxy-Ginkgolide B and 10-Ethoxy-Ginkgolide B Following the procedure described in Example 1, but using diazoethane in place of diazomethane (duration 6 hours), 1-ethoxy-Ginkgolide B was obtained in 63.2% yield and 10-ethoxy-Ginkgolide B was obtained in 25.7% yield.

Proceeding as above, but with Ginkgolides A and C, the following yields were obtained 1-ethoxy Ginkgolide A 72.8% 20.1% Ginkgolide C 59.2% 30.4%

TOXICITY

The toxicity of the compounds of the invention has been 15 measured on mice by oral route.

No death was noticed at maximum administration does t-or mice.

PHARMACOLOGY

A proof of th armaceutical interest of the compounds he invention has been established by the fjowing pharmaceutical experimentations S?-A D/ v rl It- 11- IIj 3a No death was noticed at the maximum administration dose.

PHARMACOLOGY

The pharmaceutical interest of the compounds of the invention is shown by the following pharmaceutical 1i experiments.

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etee 0 00* *j oe on ca e a eB O 4 1) Inhibition of the platelets aggregation on New Zealand rabbits.

The experimentation was conducted on platelets with plasma of New Zealand rabbits.

Blood samples were taken from auricular artery and placed in a citrate buffer (3.8 pH 7.4) blood was further centrifugated for 15 mn at 1200 RPM.

The tested sample was prepared in DMSO, then poured on platelets rich plasma for 1 mn, then a dose of 2.5 nM of 10 PAF was added.

000 0ooS The determination is made on a Cronolog Coultronics 000 ooo apparatus which determine the transmission percentage 0000 1o1° corresponding to the maximum height of the peak before the .O desaggregation.

0OO 0000 o o 15 The percentage of variation of the inhibition with respect to the transmission percentage is calculated (control 0 000 pure DMSO).

0 o 0 0 This method was described in detail in LABORATORY O 0 0 0 INVESTIGATIONS, Vol. 41, No. 3, p. 275, 1979, JEAN-PIERRE CAZENAVE, Dr. MED., JACQUES BENVENISTE, Dr. MED., AND J. FRASER MUSTARD, M. "Aggregation of Rabbits Platelets 0° by Platelet-Activating Factor is independent of the Release Reaction and the Arachidonate Pathway and inhibited by Membrane-Active Drugs".

The results demonstrate that the compounds inhibit the aggregation induced by 2.5 nM of PAF. Five tests made Son 5 different rabbits allowed us to calculate the IC 0 of the various compounds using the linear regression test.

The values for IC 50 on platelets have been found as follows Ii! L 5 0000 0000 o 00 0 oo 000 000 oo oo

QOOO

00 0 0 0 0 oa O 0 00 0 0 00 000000 0 00 0 Q0 0 0 Ginkgolide type and substitution

OCH

3 OCzIl s position B 1- 6.6 10 7 1.1 10 6 B 10- 2.9 10 7 7.2 10-6 C 1- 4.2 10 6 8.5 10 6 C 10- 3.0 10 6 9.3 10- 6 A 1- 4.6 10 6 8.7 10 6 A 10- 1.3 10 5 6.2 10 4 10 2) Anaphylactic bronchoconstriction of a passively sensitized guinea-pig Passive heterolog sensitizing Male Hartley guinea-pigs (400-500g) were sensitized by an intravenous injection (IV) of an antiovalbumin immune-serum rabbit (Cooper Biomedical, To obtain a satisfactory anaphylactic response, 24 hours later, the following conditions of use were fixed injection into the penis of a diluted serum (to half concentration 0.05 ml/100 g).

Bronchoconstriction measure Guinea-pigs were anesthetized with urethan (2 c/kg IP) then tracheotomized and ventilated by mean of a respiratory pump (UGO BASILE) stroke volume 1 ml/100 g, strokes/mn.

4 ,0 -6- A pneumothorax was done to abolish spontaneous respiration.

The initial resistance was kept constant at 10 cm water pressure according to the method of Konzett and Rossler and the excess of air volume was measured with a bronchospasm tranducer (UGO BASILE) connected to a UGO BASILE recorder "Gemini". The jugular vein was catheterized for intravenous injections. The anaphylactic shock was induced by an intravenous injection of 0.75 mg/kg of heterolog passive of ovalbumine. Products were given by oral route, 1 hour before the antigenic stimulation in the form of a gummy water suspension, at the dose of 25 mg/kg.

00*" 00 0 0 Results 0 0 0004 The bronchoconstriction induced by ovalbumin was expressed in percentage of maximal bronchoconstriction given by clamping of the trachea. The results are reported in the following table 0 00 0 00 0oooo0 o 0 o0 Ginkgolide type and substitution OCH, OCzaH position B 1- 49.7 38.3 B 10- 54.9 36.2 C 1- 40.1 39.8 C 10- 30.6 23.1 A 1- 32.0 15.1 NS A 10- 25.2 12.2 NS NS Non Significative Significative Very Significative Highly Significative I 1 -7-

POSOLOGY

In human therapy, usual1 doses for per as administration are 0. 5 to 1 g per diem, in tablets or gelatine capsules for one month.

In L.V. administration, three weekly injections at 0.05 to 0.2 g in isotonic solution, for one month are recommended.

00 Oc.

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a w 0 0000 0000 00 ci 0 00 0 00 0 0 0 00 Pci 0 0 ccci 6 *0*000 0 0 a 000000 o 0 00 00 00 0 ci

Claims (9)

1. A 1-alkoxy or 10-alkoxy derivative of one of the Ginkgolides A,B,C,J or M or a mixture of the 1-alkoxy and derivaties of one of the said Ginkgolides.

2. A Ginkgolide derivative according to claim 1 or mixture of Ginkgolide derivatives according to claim 1 in which the or each alkoxy group is a methoxy or ethoxy group.

3. A process for the preparation of a Ginkgolide derivative according to claim 1 or a mixture of Ginkgolide derivaties according to claim 1, the process comprising o, reacting one of the Ginkgolides A,B,C,J or M with an excess of a diazoalkane in a solvent, and optionally separating the resultant mixture of l-alkoxy-Ginkgolide and o 0'.

4. A process according to claim 3 in which the diazoalkane is diazomethane or diazoethane.

A process according to claim 3 or claim 4 in which approximately 10 equivalents of the diazoalkane are employed per equivalent of Ginkgolide. ,,o 0

6. A process according to any of claims 3 to 5 in 00.0 which a diethyl ether solution of the diazoalkane is mixed f,4 with a dioxan solution of the Ginkgolide and the mixture is allowed to react for from 3 to 8 hours at ambient temperature.

7. A process according to any of claims 3 to 6 in which the mixture is separated by evaporating off the solvent, eluting the residue through a silica gel column using ethyl acetate: hexane 1:1 by volume as eluent, 0 9 evaporating off the solvent from the eluate, taking up the residue in chloroform, recrystallising the from solution and adding diethyl ether to the remaining solution to obtain the 1-alkoxy-Ginkgolide.

8. A process for the preparation of a Ginkgolide derivative according to claim 1 or a mixture of Ginkgolide derivatives according to claim 1, the process being substantially as described herein with reference to either of the Examples.

9. A pharmaceutical composition comprising a l-aloxy or 10-alkoxy derivative of one of the Ginkgolides A,B,C,J and M or a mixture of the 1-alkoxy and 10-alkoxy derivatives of one of the said Ginkgolides in admixture with a pharmaceutically acceptable diluent or carrier. DATED this 15th day of Febuary, 1991 I 0" 0I 0 0 0 0'Q 4 SOCIETE DE CONSEILS DE RECHERCHES ET D'APPLICATIONS SCIENTIFIQUES WATERMARK PATENT TRADEMARK ATTORNEYS 2ND FLOOR, 'THE ATRIUM' 290 BURWOOD ROAD, HAWTHORN VIC. 3122 AUSTRALIA 01 0 6 0 0q 0.0i aL:lcg:(125) Ui