Source: http://www.google.com/patents/US4396542?dq=6948823
Timestamp: 2018-01-17 10:19:14
Document Index: 391043128

Matched Legal Cases: ['art 1', 'arts 1', 'art 2', 'art 2', 'art 1', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2', 'art 2']

Patent US4396542 - Method for the total synthesis of cyclosporins, novel cyclosporins and novel ... - Google Patents
A method for the total synthesis of cyclosporins, in particular Cyclosporin A, cyclosporins produced in accordance with the method of the invention and novel intermediates, in particular novel [1S, 2R, 3R]- and [1R, 2S, 3S]-1-nitrilo-1-carbonyl-3-methyl-2-oxy-heptanes and -hept-5-enes, employed in the...http://www.google.com/patents/US4396542?utm_source=gb-gplus-sharePatent US4396542 - Method for the total synthesis of cyclosporins, novel cyclosporins and novel intermediates and methods for their production
Publication number US4396542 A
Application number US 06/299,103
Publication number 06299103, 299103, US 4396542 A, US 4396542A, US-A-4396542, US4396542 A, US4396542A
Inventors Roland Wenger
Patent Citations (5), Non-Patent Citations (2), Referenced by (79), Classifications (9), Legal Events (6)
Method for the total synthesis of cyclosporins, novel cyclosporins and novel intermediates and methods for their production
US 4396542 A
A method for the total synthesis of cyclosporins, in particular Cyclosporin A, cyclosporins produced in accordance with the method of the invention and novel intermediates, in particular novel [1S, 2R, 3R]- and [1R, 2S, 3S]-1-nitrilo-1-carbonyl-3-methyl-2-oxy-heptanes and -hept-5-enes, employed in the method of the invention.
1. Process for the production of a cyclosporin of formula I,
wherein A is a residue of formula Ia, ##STR7## wherein R1 is hydrogen or methyl and -x-y- is -CH2 -CH2 - or -CH═CH- and the positions 2, 3 and 4 have the configuration S,R and R or R,S and S respectively, and B is ethyl, 1-hydroxyethyl, isopropyl or n-propyl, which process comprises
2. A process according to claim 1, in which the straight-chained endecapeptide, in free -N and carboxy-activated form is cyclized to produce the cyclosporin of formula (I).
4. A process according to claim 3, in which the carbonyl azide is cyclized by the addition of a base at -20° to 0° C.
7. A process according to claim 6, in which preparation of the Castro-ester and cyclisation of the ester are carried out concurrently in the presence of a base, at a temperature of from -20° to 30° C. in an inert solvent.
9. A process according to claim 1 for the production of a cyclosporin of formula (I), in which the residue A is an unprotected or O-protected C9 -amino acid of formula (Ia) occupying position l and the remaining amino acid residues of the of the molecule are numbered from 1 clockwise and in sequence, comprising the steps of:
2. Preparation of a dipeptide having the unprotected or O-protected sequence 2-3 and linkage of this dipeptide to the N-terminal of the tetrapeptide obtained in step 1, to obtain a hexapeptide having the sequence 2 through 7;
3. Linkage of the C9 -amino acid to the N-terminal of the hexapeptide obtained in step 2, to obtain a heptapeptide having the sequence 1 through 7;
4. Preparation of a tetrapeptide having the sequence 8 through 11 and linkage of this tetrapeptide to the N-terminal of the heptapeptide obtained in step 3 to obtain a linear endecapeptide having the sequence 8 through 7;
5. Cyclisation of the straight-chain endecapeptide obtained in step 4 in unprotected or O-protected form and, when required removing the O-protecting groups to obtain a cyclosporin wherein -X-Y- is -CH═CH-; and, when desired;
6. Hydrogenation of the cyclosporin obtained in step 5 to obtain the corresponding cyclosporin wherein -X-Y- is -CH2 -CH2 -.
10. A process according to claim 9, in which the cyclosporin obtained is cyclosporin A, B, C, D or G or a corresponding dihydro derivative thereof.
13. A process according to claim 12, in which the carbonyl azide is cyclized by the addition of a base at -20° to 0° C.
16. A process according to claim 11, in which preparation of the Castro-ester and cyclisation of the ester are carried out concurrently in the presence of a base, at a temperature of from -20° to 30° C. in an inert solvent.
I. CYCLOSPORIN TOTAL-SYNTHESIS
In general, peptides may be built up synthetically by any appropriate synthetic pathway and choice of pathway is commonly dictated by considerations of simplicity and economy, e.g. by the number of reaction steps required. The most usual approach is to join together smaller peptide units having the appropriate partial sequences. For cyclosporins, e.g. cyclosporin A, it appears that the choice of synthetic pathway is limited. A primary limitation is of course imposed by the choice of position at which cyclisation is to be effected. It also appears however that there are restrictions in the choice of synthetic sequence for obtaining the cyclosporin in open-chain form. The present invention provides a particular strategy for the synthesis of the required open-chain form, which is employed in conjunction with a choice of highly specific reaction conditions for each synthetic step.
The final cyclisation may be performed by reaction of an obtained, linear endecapeptide having the desired sequence, in free-N and carboxy-activited form (i.e. having an activating group at the C-terminal and a free-amino group at the N-terminal). Suitably the reaction is conducted in the presence of a base. The choice of reaction temperature will of course depend on the particular carboxy-activating group selected but this will generally be in the range of from -20° to 30° C. Suitable bases include e.g. ethyldiisopropylamine and N-methylmorpholine. Suitable carboxy-activated forms include e.g. the carbonylazide and, preferably the activated-ester forming on reaction with benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium-hexafluorophosphate [also known as Castro-reagent], herein referred to as the "Castro-ester".
Carbonylazides may be prepared directly from an obtained linear endecapeptide in free-N and free-carboxy form, e.g. by reaction with diphenylphosphonic acid azide in the presence of dimethyl formamide and a base such as triethylamine. The reaction is conveniently carried out at a temperature of from -10° to 30° C. and proceeds directly with concurrent cyclisation.
(i) reaction of the carboxy-ester-protected endecapeptide in free-N or N-protected form with hydrazine hydrate, optionally dissolved in dimethylformamide, at a temperature of from e.g. 0° to 30° C.,
(ii) removal, when required, of the N-protecting group, e.g. removal of an N-Boc-protecting group by acid treatment, e.g. in the presence of trifluoroacetic acid, at a temperature of e.g. ca. 0° C. and
(iii) oxidation of the obtained hydrazide, e.g. by treatment with t-butylnitrite in the presence of an acid such as HCl (to prevent oxidation of the now unprotected amino group) at a temperature of from e.g. -20° to 0° C.
Final cyclisation of the carbonylazide is thereafter effected by addition of a base, such as triethylamine or ethyldiisopropylamine and at a temperature of e.g. -20° to 0° C.
"Castro-esters" may be prepared from an obtained linear endecapeptide in free-N and free-carboxy form by reaction with benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate in the presence of a base such as triethylamine or N-methylmorpholine, at a temperature of from e.g. -20° to 30° C., in an inert solvent such as chloroform or methylene chloride. The reaction proceeds with concurrent cyclisation. Cyclisation in accordance with this second method is represented in accompanying flow-chart 1B and is exemplified in the following examples 1w through 1y.
For the purposes of the cyclisation reaction the peptide may, if desired, be in O-protected form, i.e. may bear an O-protecting group, such as hereinafter described, at the C9 -amino acid residue and/or, in the case of Cyclosporin C or derivatives thereof, at the 2-threonine residue. Such O-protecting groups are then removed subsequent to ring-closure by methods known in the art, e.g. (i), for the removal of alkyl groups, such as t-butyl, by treatment with an acid such as trifluoroacetic acid or HCl in a solvent medium such as methanol/water at a temperature of from -20° to 30° C., or (ii), for the removal of ester groups, such as acetyl, by hydrolysis in the presence of an alcoholic alkali metal- or alkaline-earth metal-alcoholate, again at a temperature of from 0° to 30° C., or (iii), for the removal of a benzyl group, by hydrogenation in the presence of a metal catalyst such as palladium on charcoal, in an inert solvent such as ethanol, at a temperature of from 0° to 30° C. or by treatment with sodium in liquid ammonia at a temperature of -35° C.
When hydrogenation according to method (iii) above is employed, and the C9 -amino acid moiety of the endecapeptide contains a double-bond, the double-bond is simultaneously reduced to yield the corresponding dihydro-cyclosporin as end-product. Initially obtained cyclosporins containing an unsaturated C9 -amino acid residue may in any case be converted into the corresponding dihydro cyclosporin derivative by reduction, e.g. in accordance with the methods known and described in the art for reducing naturally occurring cyclosporins, e.g. cyclosporins C and D, for example by catalytic hydrogenation, e.g. in accordance with the general methods disclosed in U.K. Patent Specification No. 1,567,201. Reduction is suitably effected under neutral pH conditions at a temperature of from 20° to 30° C. and at atmospheric or slightly elevated pressure, in the presence of a catalyst such as platinum and, preferably, palladium (e.g. palladium on charcoal) in the presence of an inert solvent or diluent, such as ethyl acetate. Flow-charts 1A and 1B represent the total synthetic pathway for producing Cyclosporin A. It will, however, be appreciated that the same basic strategy may be applied to obtain other cyclosporins and cyclosporin derivatives, e.g. by substituting threonine, valine or norvaline residues for the α-amino-butyric acid residue at the 2-position, to obtain Cyclosporins C, D and G or by replacement of the (2S,3R,4R,6E)-3-hydroxy-4-methyl-2-methyl-amino-oct-6-enoic acid residue at the 1-position with derivatives thereof as described herein.
SYNTHESIS OF THE C9 -AMINO ACID
The method of the invention for the synthesis of the C9 -amino acid in stereochemically pure form is represented diagrammatically in the accompanying flow-chart 2 . The individual steps in the synthesis may be carried out as follows:
(a) Etherification to introduce O-protecting C1-4 -alkyl or C7-11 aralkyl groups X' and X" (formula XVII). Preferably X' and X" are the same. The etherfication may be performed by reaction of the compound of formula XVIII with alkyl- or aralkyl-halides, for example a benzyl halide, in particular benzyl bromide, suitably in the presence of a condensation agent, e.g. in the presence of an alkali metal hydroxide, in particular potassium hydroxide. The reaction is preferably performed under anhydrous conditions at a temperature of from e.g. 20° to 100° C. Starting materials of formula XVIII are known (see e.g. J. Chem. Soc., Chem. Commun. 1975, (20) pp. 833-835) and may be produced by known methods.
(b) Reaction with N-chloro- [Hal=Cl] or N-bromo- [Hal=Br] -Succinimide at a temperature of e.g. -10° to 30° C. The reaction is preferably carried out in the dark using carbon tetrachloride as solvent medium Compounds of formula XVII are known (J. Chem. Soc., Supra).
(c) Hydrolysis and epoxidation, e.g. in the presence of an aqueous alkali, in particular an aqueous alkali metal hydroxide, in particular potassium hydroxide. The reaction is suitably conducted at a temperature of from 20° to 60° C.
(d) Methylation to effect opening of the epoxy ring. This step may be effected by reaction of the compound of formula XV with methyl lithium in the presence of a cuprous salt, in particular a cuprous halide, e.g. cuprous iodide, at reduced temperature. Conveniently the methyl lithium and the cuprous salt are added at a temperature of from -20° to 0° C. and the reaction mixture then further cooled to -60° to -20° C.
(e) Ether cleavage to remove protecting groups X' and X". When X' and X" are aralkyl, cleavage may be effected reductively, e.g. by catalytic hydrogenation for example using 10% palladium/charcoal as catalyst, at a temperature of from 0° to 30° C. and at normal or slightly elevated pressure. When X' and X" s.- or t.-alkyl, cleavage is effected hydrolytically, e.g. in the presence of trifluoroacetic or hydrochloric acid, again at a temperature of from e.g. 0° to 30° C.
(f) Introduction of a protecting group (R3)2 C< wherein R3 =methyl or ethyl, e.g. by reaction of a compound of formula XIII with acetone or 2,2-dimethoxypropane (to produce a compound of formula XII wherein R3 =CH3), or with di-ethyl ketone, (to produce a compound of formula XII wherein R3 =C2 H5). The reaction is effected in an acid medium, e.g. in the presence of p-toluenesulfonic acid at elevated temperature, e.g. at 40° C. to reflux.
(g) Tosylation e.g. by reaction with a tolyl-, in particular p-tolyl-sulfonyl halide, e.g. p-toluenesulfonyl chloride, to produce a compound of formula XI wherein R4 =tolyl, in particular p-tolyl. The reaction is carried out in the presence of an organic base such as pyridine, at a temperature of from e.g. -20° to 20° C.
(h) Reaction with an alkali metal cyanide, in particular potassium cyanide, under anhydrous conditions. The reaction is preferably carried out using dimethyl-sulfoxide as solvent, and is suitably performed under an inert atmosphere. The reaction proceeds best at elevated or slightly elevated temperatures of e.g. from 20° to 70° C.
(i) Reduction and hydrolysis, e.g. using diisobutylaluminium hydride in the presence of an aprotic solvent such as tetrahydrofuran, hexane or toluene. The reaction is preferably carried out at a temperature of from -80° to -20° C. under an inert atmosphere.
(j) Condensation with n-butyllithium, e.g. in the presence of ethyltriphenylphosphonium bromide or analogues thereof having 3 to 5 carbon atoms in the alkyl moiety, or in the presence of ethyltriphenylphosphonium bromide in accordance with the method described by Schlosser [Ann. Chem. 708, 1 (1967)]. The reaction is suitably carried out in an aprotic solvent or diluent such as tetrahydrofuran and/or ether. When conducted at a temperature of ca. -90° to -30° C. in the presence of t-butanol, the reaction leads to products of formula VIII in trans-form. When the reaction is carried out at ambient temperatures, e.g. of from 0° to 30° C. in the absence of t-butanol, the corresponding cis-form is obtained.
(k) Removal of the protecting group (R3)2 C< e.g. by hydrolysis e.g. in the presence of a dilute aqueous mineral acid such as hydrochloric acid. The reaction is suitably performed at a temperature of from 0° to 30° C.
(l) Introduction of an O-protecting group X3, e.g. a 1-methoxyethyl, 1-ethoxyethyl or 2-methoxyisopropyl group. The reaction is conveniently carried out by means of a three-step procedure comprising: (i) esterification, in particular benzoylation, of the primary alcohol function, e.g. by reaction with a benzoyl halide, especially benzoyl chloride in the presence of a weak organic base, in particular pyridine, (ii) ketalisation of the secondary alcohol function, e.g. by reaction with a (C1-4 alkoxy)-vinyl ether, for example ethoxy-vinyl ether, in the presence of a catalytic amount of an acid, in particular an organic acid such as trifluoroacetic acid and (iii) hydrolysis, e.g. in the presence of an aqueous base such as sodium hydroxide, with removal of the protecting benzoyl group at the primary alcohol function. The reaction sequence (i)→(ii)→(iii) is conveniently carried out at a temperature of from 0° to 30° C.
(m and m') Oxidation, e.g. in accordance with the method described by Moffat [J.A.C.S. 87, 5661 (1965) and 88, 1762 (1966)] in the presence of dimethylsulfoxide and dicyclohexylcarbodiimide with the addition of pyridine and trifluoroacetic acid. The reaction is suitably performed at a temperature of from 0° to 30° C.
(n and n') The reaction corresponds to the first step of a Strecker synthesis and may be effected by reaction with HCN or, preferably, an alkali metal cyanide, in particular KCN, and ammonia or ammonium chloride (to produce compounds of formula Va or Vb wherein R1 =H) or methylamine (to produce compounds of formula Va or Vb wherein R1 =CH3). The reaction is suitably performed at a temperature of from 0° to 30° C.
(l' and l") Removal of O-protecting group X3 e.g. by deketalisation in the presence of an acid such as trifluoroacetic acid, and dicyclohexylcarbodiimide at a temperature of from 0° to 30° C.
(o) Introduction of a protecting group (X2 +X3), completing a B 6- or, preferably 5-membered ring system, e.g. by reaction of a compound of formula Va with acetone or 2,2-dimethoxy-propane [to produce a compound of formula IV wherein (X2 +X3)=(CH3)2 C<] or with carbonyl or thiocarbonyl-diimidazole or with phosgene [to produce a compound of formula IV wherein (X2 +X3)=-CO- or -CS-]. The reaction is preferably carried out in the presence of methylene chloride or toluene as solvent or diluent and at a temperature of e.g. 0° to 30° C.
(p and p') Selective hydrolysis and isomerisation, e.g. in an aqueous alkali medium such as aqueous sodium hydroxide [to yield the free acids of formula (IIIb1)--process step p] or in the presence of, successively, an alcoholic alkali metal- or alkaline-earth- metal-alcoholate or -carbonate suspension and an acid, e.g. HCl [to yield esters of formula (IIIa1)--process step p']. By variation of the alcoholic component, compounds of formula IIIa1 may be obtained wherein R5 =C1-4 alkyl. Preferably the reaction is carried out employing an ethanolic sodium- or potassium-carbonate suspension to obtain compounds of formula (IIIa1) wherein R5 =C2 H5. Thermodynamically the reaction proceeds such that the carbonyl group assumes the trans-position relative to the adjacent alkyl substituent Z-CH(CH3)-. Conveniently the reaction is carried out at a temperature of from 0° to 20° C.
(q and q') Hydrogenation, e.g. catalytic hydrogenation, for example employing a palladium/charcoal catalyst at a temperature of from e.g. -5° to 40° C. under normal or at slightly elevated pressure. Inclusion of step q or q' leads to product amino acids of formula II wherein -x-y-=-CH2 -CH2 -. Although as shown in flow-chart 2 , hydrogenation is conveniently carried out subsequent to step p or p', it will be appreciated that it is equally possible to effect hydrogenation subsequent to any of steps j through o. Clearly, hydrogenation at an earlier phase in the reaction sequence may be employed to produce the corresponding compounds of formulae VIII through IV wherein the group CH3 -CH═CH-CH2 - (═Z) is replaced by the group CH3 -CH2 -CH2 -CH2 -.
(r) Selective hydrolysis, e.g. in the presence of an aqueous alkali metal- or alkaline-earth metal-hydroxide in particular NaOH or KOH. The reaction is suitably performed at a temperature of from 0° to 20° C. The initially obtained salt may be converted into the free acid of formula IIIb1 or IIIb2, e.g. by addition of a dilute mineral acid such as HCl. At pH 6-7 the free acids precipitate out and may readily be recovered from the reaction medium in pure form.
(s) Removal of protecting group (X2 +X3). When (X2 +X3) is e.g. carbonyl or thiocarbonyl, removal is conveniently effected by alkaline hydrolysis, e.g. in the presence of an aqueous alkali metal or alkaline-earth metal hydroxide, analogously to step r, at a temperature of from e.g. 40° to 70° C. When (X2 +X3) is e.g. a group of formula (CH3)2 C<, removal is effected by acid hydrolysis, e.g. in the presence of a dilute mineral acid such as HCl, at a temperature of from 10° to 40° C.
EXAMPLE 1 Synthesis of cyclosporin A
The synthetic sequence of the following example is that shown in the accompanying flow-chart 1 . The following abbreviations are employed:
______________________________________BOC           =     t.Butyloxy-carbonyl.Bzl           =     Benzyloxy.H--α-Abu--OH         =     α-Aminobutyric acid.H--Sar--OH    =     Sarcosine.H--C.sub.9 A--OH         =     (2S,3R,4R,6E)-3-hydroxy-4-               methyl-2-methylamino-oct-6-               enoic acid (c.f. example 2.1).Oxaz--C.sub.9 A--OH         =     (4R,5S)-4-(hex-2E-en-5R-yl)-               1,2-trimethyl-oxazolin-5-car-               boxylic acid (c.f. example 7).______________________________________
Synthesis of protected Open-Chain form
(a) Boc-α-Abu-Sar-Bzl:
10.3 ml (10.18 g=84.8 mMol) pivaloyl chloride and 16.4 g (162 mMol) N-methyl-morpholine are added to a solution of 15.66 g (17.1 mMol) Boc-α-Abu-OH in 500 ml chloroform and the mixture is stirred for 3 hrs. at room temperature under a nitrogen atmosphere. A solution of 16.5 g (92.5 mMol) H-Sar-Bzl in 500 ml chloroform are then added and the reaction mixture stirred for a further 2 hrs. at room temperature, again under a nitrogen atmosphere. The obtained solution is washed with 300 ml 1-N HCl, the aqueous phase extracted with 200 ml methylene chloride and the combined organic phases washed 2× with 200 ml saturated potassium carbonate solution. The aqueous phases are extracted with 200 ml methylene chloride and the combined organic phases dried over potassium carbonate, filtered and concentrated. The residue is chromatographed on 1 kg silica gel 60 (0.063-0.24 mm) using 1% methanol in methylene chloride as eluant, to yield the title compound: rotation, [α]D 25 =-42° (c=1.0 in chloroform).
10.7 g (29.4 mMol) Boc-α-Abu-Sar-Bzl in 500 ml ethanol containing 2 g 10% palladium/charcoal are debenzylated by hydrogenation for 2 hrs. at room temperature. The product is filtered through talc, evaporated and dried to yield the title compound: Rotation, [α]D 25 =-5,3° (c=1.0 in chloroform).
rotation, [α]D 25 =-59.4° (c=1.0 in chloroform).
46.8 g (115 mMol) BOC-MeLeu-Ala-Bzl are dissolved in 100 ml trifluoroacetic acid pre-cooled to 0° C. and allowed to stand for 1 hr. at 0° C. and 1/2 hr. at room temperature. The obtained solution is concentrated under vacuum, diluted with 500 ml methylene chloride, poured onto ice and washed with 300 ml saturated potassium carbonate solution. The aqueous phase is extracted with 200 ml methylene chloride, the combined organic phases dried over potassium carbonate, filtered and concentrated. The residue is chromatographed on 2 kg silica gel 60 (0.063-0,24 mm) using 5% methanol in methylene chloride as eluant to yield the title compound: rotation, [α]D 25 =-44.5° (c=1.0 in chloroform).
12.9 ml (12.7 g=106 mMol) pivaloyl chloride and 23 g (212 mMol) N-ethyl-diisopropylamine are added to a solution of 23.2 g (106 mMol) BOC-L-Val-OH in 400 ml chloroform and the mixture stirred for 2 hrs. at room temperature under a nitrogen atmosphere. 27.37 g (89 mMol) H-MeLeu-Ala-Bzl dissolved in 100 ml chloroform are then added and the reaction mixture stirred for a further 18 hrs. at 60° C. again under a nitrogen atmosphere. The obtained solution is washed with 300 ml 1 N HCl, the aqueous phase extracted with 200 ml methylene chloride and the combined organic phases washed 2× with 200 ml saturated potassium carbonate solution. The aqueous phases are extracted wit 200 ml methylene chloride, the combined organic phases dried over potassium carbonate, filtered and concentrated. The residue is chromatographed on 2 kg silica gel 60 (0.065-0.2 mm) with 1% methanol in methylene chloride as eluant to yield the title compound as a colourless oil: rotation [α]D 25 =-97.2 (c=1.0 in chloroform).
30.7 g (60.8 mMol) BOC-Val-MeLeu-Ala-Bzl dissolved in 100 ml trifluoroacetic acid are stirred for 2 hrs. at room temperature. The obtained solution is concentrated under vacuum and the residue diluted with 500 ml methylene chloride. The solution is washed 3× with 200 ml saturated potassium carbonate solution, ice being added, the aqueous phases extracted with 200 ml methylene chloride and the combined organic phases dried over potassium carbonate. The title compound is obtained as a pale-yellow oil after further filtration and concentration: rotation, [α]D 25 =-87.1° (c=1.0 in chloroform).
8.5 ml (8.3 g=69.5 mMol) pivaloyl chloride and 12.7 ml (115.8 mMol) N-methylmorpholine are added to a solution of 15.6 g (63.7 mMol) BOC-MeLeu-OH in 500 ml chloroform and the mixture stirred for 2 hrs. at room temperature under a nitrogen atmosphere. 23.45 g (57.9 mMol) H-Val-MeLeu-Ala-Bzl dissolved in 100 ml chloroform are then added and the reaction mixture stirred for a further 2 hrs. at room temperature again under a nitrogen atmosphere. After purification in accordance with the procedure of example 1a the title compound is obtained as a pale yellow oil: rotation, [α]D 25 =-126.7° (c=1.0 in chloroform).
31.27 g (49.5 mMol) BOC-MeLeu-Val-MeLeu-Ala-Bzl dissolved in 100 ml trifluoroacetic acid are stirred for 21/2 hrs. at room temperature and the product solution concentrated under vacuum. The residue is diluted with 500 ml methylene carbonate solution, ice being added and the aqueous phases extracted with 200 ml methylene chloride. The combined organic phases are dried over potassium carbonate, filtered and concentrated to yield the title compound: rotation [α]D 22 =-114.4° (c=1.0 in chloroform), M.P. on re-crystallisation from ether/petrol ether=66°-67°
3.5 ml (3.3 g=27.7 mMol) pivaloyl chloride and 5.4 g (52.9 mMol) N-methylmorpholine are added to a solution of 6.9 g (25.2 mMol) BOC-α-Abu-Sar-OH (example 1b) in 250 ml chloroform and the mixture stirred for 1 hr. at room temperature under a nitrogen atmosphere. 12.0 g (22.7 mMol) H-MeLeu-Val-MeLeu-Ala-Bzl dissolved in 100 ml chloroform are then added and the reaction mixture stirred for a further 15 hrs. at room temperature, again under a nitrogen atmosphere. The obtained product is purified in accordance with the procedure of example 1a to yield the title compound as a colourless foam: rotation, [α]D 25 =-137.9° (c=1.0 in chloroform).
15.75 g (19.9 mMol) BOC-α-Abu-Sar-MeLeu-Val-MeLeu-Ala-Bzl dissolved in 50 ml trifluoroacetic acid are stirred for 4 hrs. at room temperature. The solvent is evaporated under vacuum, the residue dissolved in 300 ml methylene chloride and the obtained solution washed 3× with 200 ml saturated sodium carbonate solution, ice being added. The Aqueous phases are extracted with 200 ml methylene chloride, and the combined organic phases dried over potassium carbonate, filtered and concentrated to yield the title compound: rotation, [α]D 25 =-127.3° (c=1.0 in chloroform).
1.5 g (6.22 mMol) freshly prepared Oxaz-C9 A-OH are dissolved in 50 ml tetrahydrofuran, and 0.7 g (6.93 mMol) N-methyl-morpholine are immediately added. 1.68 g (12.4 mMol) 1-hydroxybenzotriazole are dehydrated by shaking and concentrating 2× with 50 ml toluene and are then added to the solution together with 4.28 g (6.22 mMol) H-α-Abu-Sar-MeLeu-Val-MeLeu-Ala-Bzl.
The whole is cooled to 0° C. and 1.34 g (6.5 mMol) dicyclohexylcarbodiimide are added. The obtained reaction mixture is allowed to warm to room-temperature and is then stirred for 15 hrs. at room-temperature. The obtained solution is diluted with 300 ml methylene chloride and shaken with 200 ml 1 N sodium bicarbonate solution. The aqueous phase is re-extracted with 200 ml methylene chloride, and the combined organic phases are dried over sodium sulfate, filtered and evaporated. The residue is chromatographed on 500 g silica gel (0.063-0.24 mm) using 3% methanol in methylene chloride as eluant, to yield the title compound: rotation [α]D 25 =-117° (c=1.0 in chloroform).
1.5 g (1.6 mMol) Oxaz-C9 A-α-Abu-Sar-MeLeu-Val-MeLeu-Ala-Bzl dissolved in 16 ml methanol are stirred for 15 hrs. at room temperature in the presence of 1.6 ml 1 N HCl. The splitting off of the isopropylidene protecting group may be followed by means of thin-layer chromatography. The acid in the reaction medium is neutralised by the addition of 1 g (12 mMol) sodium bicarbonate and the methanol carefully and fully evaporated off, the temperature being kept at a maximum of 30° C. The residue is taken up in 10 ml methylene chloride containing 5% methanol and chromatographed on 100 g silica gel (0.06-0.24 mm) using 5% methanol in methylene chloride as eluant. The title compound is obtained with a rotation of -138° (c=1.0 in chloroform).
18.9 g (100 m mol) Boc-(D)-Ala-OH are dissolved in 250 ml chloroform and cooled with stirring to -20° C. 23.1 ml (21.2 g=210 m mol) N-methylmorpholine and 12.2 ml (12.0 g=100 m mol) pivaloyl chloride are then added and the whole stirred for 2 hours, still at -20° C. Anhydride formation is followed by means of IR control. When anhydride formation is complete 23.5 g (100 m mol) H-MeLeu-Bzl dissolved in 50 ml chloroform are added dropwise to the obtained reaction mixture at -20° C. and over a period of 5 minutes. Formation of the dipeptide is followed using thin-layer chromatography and IR-spectrometry. After 19 hours no further anhydride can be detected. The obtained reaction solution is poured onto 300 ml water and diluted with 300 ml chloroform. The organic phase is separated, washed with 100 ml water, dried over sodium sulphate, filtered and the solvent carefully evaporated off at max. 40° C. The obtained residue is recrystallised from hexane to yield the title compound: rotation [α]D 25 =-35.3° C. (c=1,05 m chloroform; M.P.=81° C.
32.45 g (79.92 m mol) BOC-(D)-Ala-MeLeu-Bzl in 800 ml ethanol containing 1.6 g 10% palladium/charcoal are debenzylated by hydrogenation for 2 hours at room temperature. After uptake of the calculated quantity of hydrogen the suspension is filtered through talc, the solvent evaporated off and the residue crystallised from hexane to yield the title compound with a rotation of [α]D 25 =-36.7° (c=0.8 in chloroform).
17.9 ml (16.4 g=162.4 m mol) N-methylmorpholine are added to a solution of 24.5 g (77.3 m mol) BOC-(D)-Ala-MeLeu-OH dissolved in 200 ml chloroform and the whole is cooled to -20° C. 9.5 ml (9.27 g=77.3 m mol) pivaloyl chloride are then added over 5 minutes and the reaction mixture stirred for 90 minutes, still at -20° C. Anhydride formation is followed by means of IR control and when complete a solution of 18.2 g (77.3 m mol) H-MeLeu-Bzl in 50 ml chloroform is added dropwise at -20° within 5 minutes. A reaction period of 18 hours at -20° C. is required until no more anhydride is present. The obtained solution is poured onto 300 ml water and is extracted in a separating funnel after first diluting with a further 300 ml chloroform. The aqueous phase is extracted with 300 ml chloroform, the combined organic phases dried over sodium sulphate, filtered and evaporated. The residue is chromatographed on 1 kg silica gel (0.063-0.24 mm) using 2% methanol in methylene chloride as eluant to yield the title compound: rotation, [α]D 25 =-101.3° (c=0.9 in chloroform).
29.3 g (54.97 m mol) BOC-(D)-Ala-MeLeu-MeLeu-Bzl in 800 ml ethanol containing 1.5 g 10% palladium/charcoal are debenzylated by hydrogenation for 2 hours at room temperature. After uptake of the calculated amount of hydrogen, the obtained suspension is filtered through talc, evaporated and the residue crystallised from hexane to yield the title compound: rotation [α]D 25 =-112.6° (c=0.85 in chloroform).
3.48 ml (3.18 g=31.5 m mol) N-methylmorpholine and 1.86 ml (1.8 g=15.0 m mol) pivaloyl chloride are added to a solution of 6.65 g (1.50 m mol) BOC-(D)-Ala-MeLeu-MeLeu-OH in 60 ml chloroform, pre-cooled to -20° C. Conversion to the anhydride is completed after stirring for 2 hours at -20° C. (IR control). 3.35 g (15.0 m mol) H-MeVal-Bzl in 50 ml chloroform are then added dropwise to the reaction mixture at -20° C. The progress of the reaction is followed by means of thin-layer chromatography and IR-spectrometry and is complete after 41/2 days. The obtained solution is poured onto 200 ml 1 N sodium bicarbonate on a shaker, and diluted with 300 ml chloroform. After separation of the organic phase, the aqueous phase is extracted with a further 100 ml chloroform, the combined chloroform phases dried over sodium sulphate, filtered and evaporated. The residue is chromatographed on 600 g silica gel (0.063-0.2 mm) using 2% methanol in methylene chloride as eluant to yield the title compound: rotation, [α]D 25 =-143.7° (c=0.9 in chloroform).
5.53 g (10.0 m mol) of BOC-(D)-Ala-MeLeu-MeLeu-MeVal-Bzl dissolved in 500 ml ethanol containing 0.5 g 10% palladium/charcoal are debenzylated by hydrogenation for 1 hour at room-temperature. The obtained suspension is filtered through talc, evaporated and the residue dried over sodium sulphate to yield the title compound with a rotation of [α]D 25 =-187° (c=0.88 in chloroform).
Cyclisation Steps--Carbonylazide method
(t) BOC-(D)-Ala-MeLeu-MeLeu-MeVal-C9 A-α-Abu-Sar-MeLeu-Val-MeLeu-Ala-NHNH2 :
0.84 g (0.59 m mol) BOC-(D)-Ala-MeLeu-MeLeu-MeVal-C9 A-α-Abu-Sar-MeLeu-Val-MeLeu-Ala-Bzl are stirred for 4 hours at room temperature with 3 ml hydrazine hydrate in 3 ml dimethylformamide, and the dimethylformamide and excess hydrazine hydrate are thereafter evaporated off at room temperature under high vacuum. The residue is dissolved in 250 ml ethylacetate and shaken 2 times with 50 ml saturated NaCl solution. The organic phase is dried over sodium sulphate, filtered and evaporated and the residue chromatographed on 100 g silica gel (0.663-0.24 mm) using 5% methanol in methylene chloride as eluant to yield the title compound: rotation [α]D 20 =-170.2° (c=1.0 in chloroform).
0.2 ml 2 M HCl/dioxane and 22.8 mg (0.22 m mol) t-butylnitrite are added to 136 mg (0.11 m mol) H-(D)-Ala-MeLeu-MeLeu-MeVal-C9 -A-α-Abu-Sar-MeLeu-Val-MeLeu-Ala-NHNH2 dissolved in 5 ml dimethylformamide at a temperature of -20° C. and the whole is stirred for 45 minutes at -20° C. A further 22.8 mg (0.22 m mol) t-butylnitrite are then added and the reaction mixture stirred for a further 2 hours at -20° C., before being diluted with 20 ml dimethyl formamide. 0.051 g (0.4 m mol) ethyldiisopropylamine are then added and the mixture stirred for a further 2 hours at -20° C. The product solution is poured onto 100 ml HCl at pH 3 and shaken with 200 ml diethyl ether. The ether phases are washed with 50 ml water, dried over sodium sulphate, filtered and evaporated, the residue is chromatographed on 50 g silica gel (0.063-0.24 mm) using 2% methanol in methylene chloride as eluant. Fractions identified as Cyclosporin A by thin layer chromatography are collected and recrystallised from acetone. The obtained product has a melting point of 150° C. and a rotation of [α]D 20 =-245° (c=0.8 in chloroform). Identity with the naturally occurring compound Cyclosporin A is confirmed by NMR spectroscopy.
7.1 ml of 0.2 N aqueous NaOH are added to a solution of 2.0 g (1.42 mMol) Boc-(D)-Ala-MeLeu-MeLeu-MeVal-C9 A-α-Abu-Sar-MeLeu-Val-MeLeu-Ala-Bzl [see Example (1 s)] in 50 ml ethanol and the reaction mixture allowed to stand for 24 hours at 0° C. The obtained clear, colourless solution is adjusted to pH 5 by the addition of a few drops of concentrated acetic acid and fully evaporated at 40° C. under vacuum. The remaining amorphous residue is shaken with 100 ml water and 2× with 200 ml methylene chloride, and the organic phase dried over Na2 SO4, filtered and evaporated to yield a white foam. This is then chromatographed on 400 g silica gel 60 and collected in 30 ml fractions using methylene chloride, together with an additional 10% methanol as eluant. From fraction 80 onwards, 30% methanol is used as eluant. The title compound is recovered from fractions 81 onwards by thin-layer chromatography on silica-gel using CH2 Cl2 /30% MeOH as eluant: rotation [α]D 20 =-173° C. (c=1 in chloroform).
(x) H-(D)-Ala-MeLeu-MeLeu-MeVal-C9 A-α-Abu-Sar-MeLeu-Val-MeLeu-Ala-OH:
A 100 ml double-necked flask provided with a stirrer and CaCl-tube and containing 1.2 g (0.91 mMol) BOC-(D)-Ala-MeLeu-MeLeu-MeVal-C9 A-α-Abu-Sar-MeLeu-Val-MeLeu-Ala-OH is cooled in an ice-bath at -20° C. and 20 ml trifluoroacetic acid pre-cooled to -20° C. are added with stirring. The obtained clear and virtually colourless solution is stirred for a further 1 hour at -20° C., and the trifluoroacetic acid evaporated off, still at -20° C., using a water-pump-vacuum. The remaining oil is diluted with 200 ml methylene chloride and shaken in a separating funnel with 100 ml saturated NaHCO3 solution. The organic phase is dried over Na2 SO4, filtered and the solvent fully evaporated. The obtained white foam is rubbed with 25 ml other and filtered through a glass frit. The title compound is obtained after chromatography on silica gel using CH2 Cl2 /20% MeOH as eluant as a white powder: rotation [α]D 20 =-201.8° C. (c= 1 in chloroform).
0.08 ml N-methyl-morpholine and 326 mg (0.738 mMol) benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium-hexafluorophosphate [Castro-reagent] are added to a solution of 900 mg (0.738 mMol) H-(D)-Ala-MeLeu-MeLeu-MeVal-C9 A-α-Abu-Sar-MeLeu-Val-MeLeu-Ala-OH in 60 ml methylene chloride in a double-necked flask and the reaction mixture stirred for 3 days at 20° C. The obtained solution is diluted with 200 ml methylene chloride and shaken with 50 ml water. The organic phase is dried over Na2 SO4, filtered and evaporated. The amorphous residue is then chromatographed on 200 g silica gel 60 using methylene chloride with 4% MeOH as eluant. The eluate is collected in 30 ml fractions. Cyclosporin A is recovered from fractions 28 through 39 and purified by crystallisation from acetone/ether and re-crystallisation from acetone: rotation [α]D 20 =-243.6° C. (c=1 in chloroform). Identity with the naturally occurring compound Cyclosporin A is confirmed by NMR spectroscopy and by x-ray powder diagram according to the method of Guiner and Devolls.
EXAMPLE 2.1 Synthesis of (2S,3R,4R,6E)-3-hydroxy-4-methyl-2-methylamino-oct-6-enoic acid: flow-chart 2 , formula II [R1 ═CH3, -x-y-=trans-CH═CH-], process step s
172 mg (0.76 mMol) (4R,5S)-4-(hex-2E-en-5R-yl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid (product of example 2r) are dissolved in 2.0 ml 2 N KOH. The solution is warmed for 3 hrs. at 80° C., cooled, adjusted to pH 5 by the addition of 1 N HCl and concentrated on a rotary evaporator. The residue is taken up in methanol, filtered through 50 g Sephadex LH 20 and evaporated. The residue is re-crystallised from ethanol to yield the title compound in pure form: M.p. 240°-241° C.; rotation, [α]D 22 =+13.0° (c=0.46 in water).
EXAMPLE 2.2 Synthesis of (2S,3R,4R)-3-hydroxy-4-methyl-2-methylamino-octanoic acid: flow-chart 2 , formula II [R1 ═CH3, -x-y-═-CH2 -CH2 -], process step s
110 mg (0.48 mMol) (4R,5S)-4-(2R-hexyl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid (product of example 2r') are dissolved in 1.4 ml 2 N KOH. The solution is warmed for 2 hrs. at 80° C., cooled, adjusted to pH 4 by the addition of 1 N HCl and filtered through 50 g Sephadex LH 20 using methanol as solvent. The filtrate is evaporated, the residue taken up in 25 ml water, freed from contaminating HCl by passing through 3 g of an anion-exchange resin [Biorad AG 3-×4 (100-200 mesh)], evaporated and re-crystallised from ethanol to yield the title compound in pure form: M.p. 248°-249°; rotation, [α]D 22 =+17.0° (c=0.44 in water).
EXAMPLES 2a (4S,5S)-4,5-dibenzyloxymethyl-2-phenyl-1,3-dioxolan: flow-chart 2 , formula XVII [X' and X"=φ-CH2 -]
15 g (71.4 mMol) (4S,5S)-4,5-dihydroxymethyl-2-phenyl-1,3-dioxolan are rendered completely anhydrous by evaporating 2× with 300 ml toluene and the oil remaining in the reaction vessel is dissolved in 150 ml toluene, 30 g powdered KOH (535 mMol) and 71.5 g (418 mMol) benzyl bromide are added and the reaction mixture stirred for 15 hours at 80° C. The mixture is cooled, the remaining toluene-phase decanted, and the inorganic component removed from the residual phase by stirring 2× with 200 ml toluene and decanting. The combined toluene phases are filtered through talc, evaporated and the remaining oil filtered fraction-wise through 2 kg silica gel 60 using methylene chloride as solvent. Fractions containing benzyl bromide are disposed of. The title compound is obtained as a light yellow fluid with a rotation of [α]D 20 =+10.1 (±1)° (c=1.4 in chloroform).
EXAMPLE 2b (2S,3R)-2-benzoyloxy-1,4-dibenzyloxy-3-bromobutane: flow-chart 2 , formula XVI [X' and X"=φ-CH2 -].
10.9 g (61.2 mMol) N-bromosuccinimide are suspended in 150 ml tetrachloromethane, the suspension cooled to 4° C. and 23.9 g (61.2 mMol) of (4S,5S)-4,5-dibenzyloxymethyl-2-phenyl-1,3-dioxolan dissolved in 250 ml tetrachloromethane added dropwise at the same temperature over a period of 50 minutes. The cooling bath is removed, the reaction flask wrapped in aluminium foil and the reaction mixture stirred for 3 days at room temperature. The obtained orange coloured suspension is diluted with 1 liter methylene chloride and shaken with 200 ml saturated sodium bicarbonate solution. The aqueous phase is extracted with 300 ml methylene chloride and the combined organic phases are dried over sodium sulfate, filtered through a layer of talc and evaporated under vacuum. The residue is chromatographed using 1 kg of silica gel 60 and methylene chloride as eluant. The title compound is obtained as a colourless oil: rotation, [α]D 22 =+18.7°, (c=1.3 in chloroform).
EXAMPLE 2c (2S,3S)-dibenzyloxymethyl-oxiran: flow-chart 2 , formula XV [X' and X"=φ-CH2 -]
16.5 ml aqueous 10 N potassium hydroxide are added to a solution of 25.7 g (54.8 mMol) of (2S,3R)-2-benzoyloxy-1,4-dibenzyloxy-3-bromo-butane in 330 ml ethanol, to give a substantially concommitant precipitation of KBr. The reaction mixture is stirred for 30 minutes at room temperature and then adjusted to pH 5 by the addition of 10 N HCl. The aqueous-ethanolic mixture is evaporated under vacuum, the residue taken up in 500 ml methylene chloride, washed with 200 ml water and the aqueous phase extracted 2× with 200 ml methylene chloride. The combined organic phases are dried over sodium sulfate, filtered through a layer of talc, evaporated and the residue distilled under vacuum to yield the title compound: B.P. 164°-168° C. at 0.2 Torr; rotation, [α]D 20 =-8.7° (c=1,1 in chloroform). The product crystalises in ether/petrolether: M.P. 24° C.
EXAMPLE 2d (2R,3R)-1,4-dibenzyloxy-2-hydroxy-3-methyl-butane: flow-chart 2 , formula XIV [X' and X"=φ-CH2 -]
4.64 g (24.38 mMol) of vacuum dried cuprous iodide are suspended in 100 ml abs. diethylether with dry nitrogen gassing. 23.6 ml (47.52 mMol) of a 4.4% solution of methyllithium in ether are added rapidly at 0° C. and the obtained clear orange-brown solution cooled immediately to -60° using a dry-ice cooling bath. 3.0 g (10.56 mMol) of (2S,3S)-dibenzyloxymethyl-oxiran dissolved in 25 ml abs. ether are then added and the reaction mixture stirred for 1 hour still at -60° C. 5 ml methanol are then added in order to destroy excess methyllithium. The cooling bath is then removed, the reaction mixture warmed to room temperature and 5 ml of water added. The reaction mixture is diluted with 300 ml methylene chloride washed 3× with 200 ml water and the aqueous copper coloured precipitates extracted a further 3× with 200 ml methylene chloride. The combined organic phases are dried over sodium sulfate, filtered through a layer of talc, evaporated under vacuum and the residue purified by chromatographing on 90 g silica gel 60 using methylene chloride as eluant to yield the title compound as a light-beige oil: rotation, [α]D 20 =-4,4° (c=1.5 in chloroform).
EXAMPLE 2e (2R,3R)-1,2,4-trihydroxy-3-methyl-butane: flow-chart 2 , formula XIII
4.5 g (15 mMol) (2R,3R)-1,4-dibenzyloxy-2-hydroxy-3-methyl-butane are de-benzylated by hydrogenation in 120 ml ethanol containing 0.5 g of 10% palladium/charcoal for 2 hours at room-temperature. The product is filtered through talc, evaporated and chromatographed on 120 g silica gel 60, with methylene chloride/methanol (8:2) as eluant. The title compound is obtained as a colourless, viscous oil.
EXAMPLE 2f (4R)-4-(1-hydroxy-2R-propyl)-2-dimethyl-1,3-dioxolan: flow-chart 2 , formula XII
30.5 g (0.254 Mol) (2R,3R)-1,2,4-trihydroxy-3-methyl-butane dissolved in 180 ml benzene are refluxed for 2 hours with 39.8 g (0.381 Mol) 2,2-dimethoxypropane and 180 mg p-toluenesulfonic acid mono-hydrate. The solvent is removed on a rotary evaporator, the remaining oil taken up in 600 ml acetone, 0.6 g p-toluenefulfonic acid mono-hydrate are added and the obtained solution boiled for 16 hours under reflux. The obtained yellow solution is concentrated to 100 ml on a rotary evaporator and chromatographed on 1 kg neutral Alox (activity: II) using 2% methanolic methylene chloride as eluant. The title compound is obtained as an oil: rotation, [α]D 22 =-19.8° (c=1.0 in chloroform; B.P.=56° C. at 0.1 Torr.
EXAMPLE 2g (4R)-4-(1-p-tosyloxy-2R-propyl)-2-dimethyl-1,3-dioxolan: flow-chart 2 , formula XI.
14.3 g (75 mMol) p-toluenesulfonyl-chloride are added at room temperature to a solution of 10.0 g (62.5 mMol) of (4R)-4-(1-hydroxy-2R-propyl)-2-dimethyl-1,3-dioxolan in 65 ml chloroform. 10.1 ml absolute pyridine are added, whereupon the temperature rises to 31° C. The exothermic reaction continues for a further 45 minutes before the temperature begins to sink. The reaction mixture is thereupon stirred for a further 3 hours at 35° C. to complete reaction. The obtained solution is diluted with 300 ml methylene chloride, washed 1× with 150 ml saturated sodium carbonate and 2× with 150 ml saturated cuprous sulfate. The aqueous phases are extracted with 200 ml methylene chloride, the combined organic phases dried over sodium sulfate, filtered through talc and evaporated. The residue is chromatographed on 400 g neutral Alox (activity: II) using methylene chloride as eluant. After crystallisation from petrol ether, the title compound is obtained with a rotation [α]D 20 =+14.2 (c=1.0 in chloroform); M.p.=39°-40° C.
EXAMPLE 2h (4R)-4-(1-cyano-2R-propyl)-2-dimethyl-1,3-dioxolan: flow-chart 2 : formula X
The crystalline (4R)-4-(1-p-tosyloxy-2R-propyl)-2-dimethyl-1,3-dioxolan (17.0 g=54.1 mMol) obtained in accordance with example 2g is hygroscopic. The crystals are accordingly dissolved immediately in 90 ml dimethylsulfoxide, 4.38 g (70.4 mMol) KCN are added and the obtained reaction mixture stirred for 3 days at room temperature under nitrogen. The obtained solution is diluted with 250 ml toluene, shaken with 125 ml water, the aqueous phase extracted 2× with 300 ml methylene chloride, evaporated and the residue taken up in 100 ml water. After shaking 2× with 200 ml toluene the organic phases are combined with the first toluene phase, dried over sodium sulphate, filtered, concentrated and distilled under high vacuum to yield the title compound as a colourless oil: B.P.=60°-63° C. at 0.03 Torr; rotation [α]D 22 =+11.2 (c=1.0 in chloroform).
EXAMPLE 2i (3R)-3 -(4R-2-dimethyl-1,3-dioxolanyl)butyraldehyde: flow-chart 2 : formula IX
41 g (243 mMol) of (4R)-4-(1-cyano-2R-propyl)-2-dimethyl-1,3-dioxolan are dissolved in 1 liter hexane and the solution cooled to -78° C. 205 ml diisobutylaluminium hydride (in the form of a 20% solution in hexane) are added drop-wise under a nitrogen atmosphere over 30 minutes in such a way that the temperature does not rise over -70° C. The reaction mixture is thereafter stirred for a further 11/2 hours at -78° C. Excess reducing agent is then decomposed by the addition of 15 ml water in 60 ml tetrahydrofuran at -70° C. under a nitrogen atmosphere. The obtained reaction mixture is poured onto 1 liter water and shaken. The precipitated Lithiumaluminium sediment is removed with a suction filter. The filtrate is separated from the aqueous phase in a separating funnel and shaken 2× with 300 ml methylene chloride. The combined organic phases are dried over sodium sulfate, filtered, concentrated on a rotary evaporator under reduced prossure and chromatographed on 3 kg silica gel 60 using 2% methanolic methylene chloride as eluant. The title compound is obtained after high-vacuum distillation under a nitrogen atmosphere as a colourless oil: B.P. 45°-50° C. at 0.03 Torr.
EXAMPLE 2j (4R)-4-(hex-2E-en-5R-yl)-2-dimethyl-1,3-dioxolan: flow-chart 2 , formula VIII
19.0 g (51.1 mMol) of ethyltriphenylphosphonium bromide are dried over-night under high-vacuum at 110° C. and made up in solution in 250 ml tetrahydrofuran and 250 ml ether under a nitrogen atmosphere. 1.1 molar equivalents of n-butyl-lithium (31.2 ml of a 15% solution in hexane) are then added with nitrogen gassing within 20 minutes in such a way that the temperature does not rise above 30° C. The reaction mixture takes on a strong red colouration and is stirred for a further 30 minutes at room temperature before colling to -78° C. 8.0 g (46.5 mMol) (3R)-3-(4R-2-dimethyl-1,3-dioxolanyl)-butyraldehyde dissolved in 50 ml absolute diethylether are then added drop-wise so that the temperature does not rise above -73° C. The reaction mixture is then stirred for a further 30 minutes and takes on a light-yellow colouration. A further 31.2 ml (51.1 mMol) n-butyl-lithium dissolved in hexane are then added at -78° C. and the reaction mixture again stirred for 30 minutes. The reaction mixture is warmed to -30° C. and 6.5 ml (69.7 mMol) t.-butanol added drop-wise within 10 minutes. The reaction mixture is stirred for a further 10 minutes at -30° C. and 7.8 g (69.7 mMol) potassium t.-butylate are then added in one go. The reaction solution, which is now yellow, is warmed to room temperature, stirred for 11/2 hours and poured onto 1 liter water. The aqueous phase is removed in a separating funnel and shaken 4× with 200 ml ether. The combined organic phases are dried over sodium sulfate, filtered off and concentrated on a rotary evaporator under reduced pressure to yield the title compound. This is used directly for further reaction without intermediate purification.
EXAMPLE 2k (2R,3R,5E)-2-hydroxy-3-methyl-5-hepten-1-ol: flow-chart 2 , formula VIIa [Z=trans CH3 -CH═CH-CH2 -]
40 ml water and 40 ml 1 N HCl are added to a solution of 6.5 g (35 mMol) (4R)-4-(hex-2E-en-5R-yl)-2-dimethyl-1,3-dioxalan in 300 ml tetrahydrofuran and the reaction mixture allowed to stand for 2 days at room-temperature. The reaction mixture is then adjusted to pH 6-7 by the addition of saturated sodium bicarbonate, the tetrahydrofuran evaporated off in a rotary evaporator and the aqueous solution extracted with methylene chloride until it reacts negatively when tested for the presence of diols. The combined organic phases are dried over sodium sulfate, filtered, concentrated on a rotary evaporator and filtered through 500 g silica gel 60 using 10% methanolic methylene chloride as eluant, to yield the title compound as a colourless oil: rotation, [α]D 22 =-5.7° (c=1.0 in chloroform), and exhibiting the strong IR-absorption at 970 cm-1 characteristic for a trans-double bond.
EXAMPLE 2l (2R, 3R,5E)-2-[(1-ethoxyethyl)-1]oxy-3-methyl-5-hepten-1-ol: flow-chart 2 , formula VIIb [Z=trans CH3 -CH=CH-CH2 -; X3 =C2 H5 O-CH(CH3)-]
6.81 ml (58.62 mMol) benzoyl chloride are added over a period of 10 minutes to a solution of 9.0 g (55.8 mMol) of (2R,3R,5E)-2-hydroxy-3-methyl-5-hepten-1-ol in 90 ml pyridine, pre-cooled to 0° C. The reaction mixture is stirred for 40 minutes at room temperature, diluted with 600 ml methylene chloride and washed 3× with 200 ml cuprous sulfate. The aqueous phases are extracted with 200 ml methylene chloride, the organic phases combined, dried over sodium sulfate, filtered, evaporated and chromatographed on 500 g silica gel 60 using 1% methanolic methylene chloride as eluant. The obtained (2R,3R,5E)-1-benzyloxy-2-hydroxy-3-methyl-5-heptene is dissolved in 200 ml methylene chloride. 14.4 g ethylvinylether and a catalytic amount of trifluoroacetic acid are added and the reaction mixture stirred for 3 days at room temperature. The obtained solution is chromatographed on 800 g basic Alox (activity II) using 1% methanolic methylene chloride as eluant, and the obtained (2R,3R,5E)-2-[(1-ethoxyethyl)-1]oxy-1-benzoyloxy-3-methyl-5-heptene dissolved in 150 ml ethanol. 30 ml 10 N potassium hydroxide solution are then added and the reaction mixture stirred for 90 minutes at room temperature. The obtained solution is diluted with 1 liter methylene chloride, washed with 400 ml water and the aqueous phase extracted with 400 ml methylene chloride. The combined organic phases are dried over sodium sulfate, filtered and evaporated to yield the title compound as a colourless oil, which is used without purification for further reaction.
Example 2m' (2R,3R,5E)-2-[(1-ethoxyethyl)-1]oxy-3-methyl-5-hepten-1-aldehyde: flow-chart 2 , formula VIb [Z=trans CH3 -CH═CH-CH2 -; X3 ═C2 H5 O-CH(CH3)-].
6.3 g (21.3 mMol) of (2R,3R,5E)-2-[(1-ethoxyethyl)-1]oxy-3-methyl-5-hepten-1-ol dissolved in 120 ml dimethylsulfoxide/benzene (1:1) are stirred for 2 hours at room temperature with 0.8 ml (10.65 mMol) trifluoroacetic acid and 18.5 g (89.67 mMol) dicyclohexylcarbodiimide. The obtained suspension is filtered with a suction filter, the filtrate taken up in 500 ml ether and washed with 250 ml water. The aqueous phase is extracted with 300 ml ether, the organic phase dried over sodium sulfate, filtered and evaporated. The residue is chromatographed on silica gel 60 using 0.5% methanolic methylene chloride as eluant to yield the title compound as a colourless oil.
EXAMPLE 2l' (2R,3R,5E)-2-hydroxy-3-methyl-5-hepten-1-aldehyde: flow-chart 2 , formula VIa [Z═trans CH3 -CH═CH-CH2 -]
1.0 ml 1 N HCl are added to a solution of 1.7 g (7.9 mMol) of (2R,3R,5E)-2-[(1-ethoxyethyl)-1]-oxy-3-methyl-5-hepten-1-aldehyde in 20 ml tetrahydrofuran and the reaction mixture allowed to stand for 11/2 hours at room temperature. The obtained solution is shaken with 100 ml water and 200 methylene chloride, the organic phase separated and washed again with 100 ml water. The aqueous phases are extracted with 200 ml methylene chloride, and the combined organic phases dried over sodium sulfate, filtered and evaporated to yield the title compound as a colourless oil. The product is used immediately for further reaction.
EXAMPLE 2n (1RS,2R,3R,5E)-1-cyano-2-hydroxy-3-methyl 1-methylamino-5-heptene: flow-chart 2, formula Va [Z═trans CH3 -CH═CH-CH2 -; R1 ═CH3 ]
0.52 g (7.9 mMol) KCN and 0.54 g (7.9 mMol) methylamine hydrochloride are added with stirring at 20° C. to 1.1 g (7.7 mMol) of (2R,3R,5E)-2-hydroxy-3-methyl-5-hepten-1-aldehyde dissolved in 50 ml methanol. After addition of 7.5 ml water the reaction mixture is stirred for a further two hours at room temperature and then concentrated to 1/2 volume on a rotary evaporator at a water-bath temperature of maximally 40° C. The obtained solution is shaken with 300 ml methylene chloride and 200 ml water and the separated organic phase is shaken with a further 100 ml water. The aqueous phases are extracted one after the other using 2×100 ml methylene chloride, the methylene chloride fractions are combined with the first organic extract, and the whole is dried over sodium sulfate, filtered through a G3 -frit and evaporated to yield the title compound as a diastereomeric mixture. This is used directly without further purification.
EXAMPLE 2n' (1RS,2R,3R,5E)-2-[(1-ethoxyethyl)-1]-oxy-1-cyano-3-methyl-1-methylamino-5-heptene: flow-chart 2 , formula Vb [Z═trans CH3 -CH═CH-CH2 -; X3 ═C2 H5 O-CH-(CH3)-; R1 ═CH3 ]
33.1 mg (4.81 mMol) KCN and 322 g (4.76 mMol) methylamine hydrochloride are added to a solution of 1.0 g (4.67 mMol) of (2R,3R,5E)-2-[(1-ethoxyethyl)-1]oxy-3-methyl-5-hepten-1-aldehyde in 40 ml methanol. After addition of 6 ml water the reaction mixture is stirred for 2 hours at room temperature, evaporated to 20 ml, and the residue shaken with 200 ml methylene chloride and 500 ml water. The aqueous phase is extracted with 50 ml methylene chloride, the combined organic phases dried over sodium sulfate, filtered and evaporated to yield the title compound as a diastereomeric mixture. This is used directly without further purification.
EXAMPLE 2l" (1RS,2R,3R,5E)-1-cyano-2-hydroxy-3-methyl-1-methylamino-5-heptene: flow-chart 2 , formula Va. [R1 ═CH3 ].
1.7 g (6.7 mMol) of (1RS,2R,3R,5E)-2-[(1-ethoxyethyl)-1]-oxy-1-cyano-3-methyl-1-methylamino-5-heptene dissolved in 20 ml dioxan are stirred for 20 hours at room temperature under a nitrogen atmosphere with 3 ml 2 N H2 SO4. The reaction mixture is then poured onto 100 ml water and extracted with 200 ml methylene chloride. The aqueous phase is rendered alkaline by the addition of 6 ml 1 N NaOH, shaken 2× with 200 ml methylene chloride, dried over sodium sulfate, filtered and evaporated to yield the title compound. This is used for further reaction without additional purification.
EXAMPLE 2o (4R,5RS)-5-cyano-4-(hex-2E-en-5R-yl)-1-methyl-2-oxo-oxazoline: flow-chart 2 , formula IV [Z═trans CH3 -CH═CH-CH2 -; (X2 +X3)=>C═O; R1 ═CH3 ].
840 mg (5.2 mMol) carbonyldiimidazole are added to a solution of 630 mg (3.46 mMol) of (1RS,2R,3R,5E)-1-cyano-3-methyl-1-methylamino-2-hydroxy-5-heptene (c.f. examples 2n and 2l") in 30 ml methylene chloride and the reaction mixture is stirred over-night at room temperature. The obtained solution is diluted with 100 ml methylene chloride and shaken with 50 ml water. The aqueous phase is extracted with 100 ml methylene chloride, the organic phase dried over sodium sulfate, filtered and evaporated. The residue is chromatographed on 110 g silica gel 60 using 1% methanolic methylene chloride as eluant to yield the title compound as a diastereomeric mixture.
EXAMPLE 2p' (4R,5S)-4-(hex-2E-en-5R-yl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid ethylester: flow-chart 2 , formula IIIa1 [(X2 +X3)=>C═O; R1 ═CH3 ; R2 ═C2 H5 -]
800 mg (2 mol equivalents) KOH are added to a solution of 600 mg (2.88 mMol) of (4R,5RS)-5-cyano-4-(hex-2E-en-5R-yl)-1-methyl-2-oxo-oxazoline in 95% ethanol and the mixture is stirred for 6 hours at room temperature. The obtained suspension is shaken with 300 ml methylene chloride and 100 ml water and the aqueous phase extracted with 100 ml methylene chloride. The combined organic phases are dried over sodium sulfate, filtered and evaporated. The residue is dissolved in 58 ml 95% ethanol and stirred with 2.9 ml 1 N NCl for 11/2 hours at room temperature. The pH of the obtained solution is adjusted to 7 by the addition of 1 N sodium bicarbonate and extracted with methylene chloride. The combined organic phases are dried over sodium sulfate, filtered and evaporated to yield the title compound: rotation, [α]D 22 =+29.5° (c=1.0 in chloroform).
EXAMPLE 2q (4R,5S)-4-(2R-hexyl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid ethyl ester: flow chart 2 , formula IIIa2 [(X2 +X3)=>C═O; R1 ═CH3 ; R2 ═C2 H5 ]
23 mg 10% palladium/charcoal are added under a nitrogen atmosphere to a solution of 210 mg (0.82 mMol) of (4R,5S)-4-(hex-2-en-5R-yl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid ethyl ester in 10 ml abs. ethanol. Hydrogenation is effected under a hydrogen atmosphere with a 3 hour reaction period. The obtained solution is filtered through talc, evaporated and chromatographed on 60 g silica gel 60 using 2% methanolic methylene chloride as eluant to yield the title compound as a colourless oil: rotation, [α]D 22 =+32.5° (c=0.93 in chloroform).
EXAMPLE 2r.1 (4R,5S)-4-(hex-2E-en-5R-yl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid: flow-chart 2 , formula IIIb1 [(X2 +X3)=>C═O; R1 ═CH3 ].
The (4R,5S)-4-(hex-2E-en-5R-yl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid ethyl ester obtained in accordance with example 2p' is dissolved in 18 ml dioxan and stirred for 1 hour at room temperature with 50 ml 0.1 N NaOH. The obtained solution is adjusted to pH 2 by the addition of 1 N NCl, extracted 2× with 300 ml methylene chloride. The organic phases are dried over sodium sulfate, filtered and evaporated and the obtained residue re-crystallised from ether to yield the title compound in pure enantiomeric form: M.P. 81°-82° C.; rotation, [α]D 22 =+33.5° (c=1.0 in chloroform).
EXAMPLE 2r.2 (4R,5S)-4-(2R-hexyl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid: flow-chart 2 , formula IIIb2 [(X2 +X3)=>C═O; R1 ═CH3 ].
17 ml 0.1 N NaOH are added to a solution of 130 mg (0.50 mMol) of (4R,5S)-4-(2R-hexyl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid ethyl ester in 7 ml dioxan and the reaction mixture stirred for 1 hour at room-temperature. The obtained solution is diluted with 150 ml methylene chloride, adjusted to pH 2 by the addition of 1 N HCl and the aqueous phase extracted with 100 ml methylene chloride. The combined organic phases are dried over sodium sulfate, filtered and evaporated. The title compound is obtained after re-crystallisation from ether: M.P.=109° C.
Proceeding analogously to example 2, but starting from (4R,5R)-4,5-dihydroxymethyl-2-phenyl-1,3-dioxolan in place of the corresponding (4S,5S)-isomer (c.f. example 2a), there are obtained:
(a1) at step p': (4S,5R)-4-(hex-2E-en-5S-yl)-1-methyl 2-oxo-oxazolin-5-carboxylic acid ethyl ester: rotation, [α]D 22 =-29.5° (c=1.0 in chloroform);
(b1) at step q': (4S,5R)-4-(2S-hexyl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid ethyl ester: rotation, [α]D 22 =-32.5° (c=0.93 in chloroform);
(a2) at step r: (4S,5R)-4-(hex-2E-en-5S-yl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid: rotation, [α]D 22 =-33.5° C. (c=1.0 in water);
(b2) at step r: (4S,5R)-4-(2S-hexyl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid: M.P.=110° C.;
(a3) (2R,3S,4S,6E)-3-hydroxy-4-methyl-2-methylaminooct-6-enoic acid: M.P.=240°-241° C.; rotation, [α]D 22 =-13.0° (c=0.46 in water); and
(b3) (2R,3S,4S)-3-hydroxy-4-methyl-2-methylaminooctanoic acid: M.P.=249°; rotation, [α]D 22 =-17° (c=0.44 in water).
Proceeding analogously to examples 2 and 3 but substituting ammonium chloride for methylamine hydrochloride at step n or n', there are obtained:
(a3) (2S,3R,4R,6E)-3-hydroxy-4-methyl-2-amino-oct-6-enoic acid: M.P.=188°-192° C.; rotation, [α]D 20 =+22.6° C. (c=0.265 in 0.1 N HCl); and
(b3) (2R,3S,4S,6E)-3-hydroxy-4-methyl-2-amino-oct-6-enoic acid: M.P. 189°-191° C., rotation, [α]D 20 =22.6° (c=0.265 in 0.1 N HCl).
EXAMPLE 5 Synthesis of (2S,3R,4R,6Z)-3-hydroxy-4-methyl-2-methylamino-oct-6-enoic acid
The above identified compound is produced analogously to example 2, substituting procedures 4j, 4r and 4 below for example 2j, 2r and 2.1 respectively:
40 ml phenyl-lithium are added at room temperature with stirring within 12 mins. to a suspension of 30.0 g triphenylphosphonium bromide in 270 ml abs. tetrahydrofuran and 70 ml diethyl-ether, whereby the temperature rises to 27° C. The obtained red-coloured suspension is stirred for 11/2 hours at room temperature, and a solution of 6.92 g (40.2 mMol) of (3R)-3-[4R-(2-dimethyl)-1,3-dioxolanyl]butyraldehyde in 130 dimethyl-ether is then added at room temperature, whereupon the colour of the reaction medium changes from red to orange. After stirring for a further 5 hours at room temperature the reaction mixture is poured onto 1000 ml water and extracted 2× with 750 ml methylene chloride. The combined organic phases are dried over sodium sulfate, filtered through a glass frit and evaporated under low vacuum. The obtained precipitate is removed by filtration and the concentrated solution chromatographed on 250 g silica gel 60 using 1% methanol in methylene chloride as eluant, to yield the title compound, exhibiting the low IR- absorption at 970 cm-1 characteristic of a cis- double bond.
185 g (0.81 mMol) or (4R,5S)-4-(hex-2Z-en-5R-yl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid ethyl ester are added to 25 ml 0.1 N NaOH in 9 ml dioxane and the reaction mixture stirred for 2 hours at room temperature. The reaction product is poured onto 100 ml water and adjusted to pH 2 by the addition of 1 N HCl. The acid aqueous phase is extracted 2× with 150 ml methylenechloride, and the organic phase dried over sodium sulfate, filtered and evaporated to yield the title compound.
67 mg (0.3 mMol) of (4R,5S)-4-(hex-2Z-en-5R-yl)-1-methyl-2-oxo-oxazolin-5-carboxylic acid are heated at 70° C. for 8 hours in 2.0 ml 2 N KOH. The obtained reaction mixture is cooled, adjusted to pH 6 by the addition of 1 N HCl and extracted 2× with 50 ml dichloromethane to recover non-reacted starting material. The aqueous solution is evaporated to dryness on a rotary evaporator and the crystalline residue taken up in 50 ml methanol. The undissolved portion is removed by filtration, the methanolic solution concentrated to 10 ml and chromatographed on 40 g Sephadex LH 20 using methanol as eluant. The eluate is collected in 8 ml fractions, and the title compound recovered by crystallisation from fractions 9 through 15 by the addition of 3 drops 1 N HCl (pH 5): M.P. 228°-233° C.; rotation, [α] D 22 =+8° C. (c=0.38 in water).
Proceeding analogously to example 4, but starting from (4R,5R)-4,5-dihydroxymethyl-2-phenyl-1,3-dioxolan in place of the corresponding (4S,5S)-isomer of example 2a, there are obtained:
(a3) (2R,3S,4S,6Z)-3-hydroxy-4-methyl-2-methylaminooct-6-enoic acid; rotation, [α]D 22 =-8° (c=0.42 in water).
EXAMPLE 7 Production of (4R,5S)-4-(hex-3E-en-5R-yl)-1,2-trimethyl-oxazolin-5-carboxylic acid [formula III: X1 =OH; (X2 +X3)=>C(CH3)2 ; R1 =CH3 ; x-y=trans -CH=CH-].
201 mg (1 mMol) of (2S,3R,4R,6E)-3-hydroxy-4-methyl-2-methylamino-oct-6-enoic acid, suspended in 80 ml anhydrous acetone are heated under re-flux for 48 hours until a clear solution is obtained. The acetone is completely evaporated under vacuum and the remaining oil taken up in 100 ml methylene chloride, washed with 30 ml water, dried over sodium sulfate and evaporated at room temperature. The obtained title compound may be used directly for synthetic purposes, e.g. for peptide synthesis, without further purification.
EXAMPLE 8 Production of (2S,3R,4R,6E)-2-(N-t.-butyloxy-carbonyl)-methylamino-3-hydroxy-4-methyl-oct-6-enoic acid [formula III: X1 =OH; X2 =(CH3)3 CO-; X3 =H; R1 =CH3 ; -x-y-=trans -CH=CH-].
201 mg (1 mMol) of (2S,3R,4R,6E)-3-hydroxy-4-methyl-2-methylamino-oct-6-enoic acid are dissolved in a solution comprising 2.2 ml 1 N NaOH and 0.33 g (1.5 mMol) bis-(t.-butyl)-dicarbonate in 5 ml dioxan and the reaction mixture stirred for 2 days at room temperature. The obtained solution is taken up in 200 ml methylene chloride, the aqueous phase adjusted to pH 3 by the addition of 2 N H2 SO4 and extracted. The organic phase is washed 2× with 30 ml water, dried over sodium sulfate, filtered and evaporated. The obtained colourless oil is chromatographed on 15 g silica gel 60 using 2% methanolic methylene chloride as eluant to yield the title compound: rotation, [α]D 20 =+7.2° (±2°) (c=1.0 in chloroform). The compound may be employed directly for synthetic purposes, e.g. for peptide synthesis.
Proceeding analogously to examples 7 and 8 but starting from the corresponding (2R,3S,4S)-octenoic acid isomer as starting material, there are obtained:
(a2) (2R,3S,4S,6E)-2-(N-t.-butyloxy-carbonyl)-methylamino-3-hydroxy-4-methyl-oct-6-enoic acid: rotation, [α]D 20 =-7.2° (±2°) (c=1.0 in chloroform).
Proceeding analogously to examples 7 and 8 but starting from (2S,3R,4R,6E)- and (2R,3S,4S,6E)-3-hydroxy-4-methyl-2-amino-oct-6-enoic acid there are obtained:
US4108985 * Oct 27, 1976 Aug 22, 1978 Sandoz Ltd. Dihydrocyclosporin c
US4210581 * Jun 9, 1978 Jul 1, 1980 Sandoz Ltd. Organic compounds
1 * Ko Sawai et al., Agric. Biol. Chem., 45(5), pp. 1223-1228, (1981).
2 * Mori et al., Tetrahedron, vol. 86, pp. 87-90 (1980).
US4554351 * May 5, 1983 Nov 19, 1985 Sandoz Ltd. Method for the total synthesis of cyclosporins, novel cyclosporins and novel intermediates and methods for their production
US4885276 * Oct 24, 1988 Dec 5, 1989 Merck & Co., Inc. Cyclosporin analogs with modified "C-9 amino acids"
US6995139 * Apr 22, 2002 Feb 7, 2006 Debiopharm S.A. Cyclic undecapeptide pro-drugs and uses thereof
US8349312 Jul 8, 2010 Jan 8, 2013 Enanta Pharmaceuticals, Inc. Proline substituted cyclosporin analogues
US8367053 Jul 8, 2010 Feb 5, 2013 Enanta Pharmaceuticals, Inc. Cyclosporin analogues
US8367618 Jan 30, 2010 Feb 5, 2013 Enanta Pharmaceuticals, Inc. Cyclosporin analogues for preventing or treating hepatitis C infection
US8481483 Feb 19, 2010 Jul 9, 2013 Enanta Pharmaceuticals, Inc. Cyclosporin analogues
US8623814 Feb 23, 2011 Jan 7, 2014 Enanta Pharmaceuticals, Inc. Antiviral agents
US8685917 Jul 8, 2010 Apr 1, 2014 Enanta Pharmaceuticals, Inc. Cyclosporin analogues
US8906853 Nov 27, 2013 Dec 9, 2014 Enanta Pharmaceuticals, Inc. [N-Me-4-hydroxyleucine]-9-cyclosporin analogues for treatment and prevention of hepatitis C infection
US9221878 Aug 26, 2014 Dec 29, 2015 Enanta Pharmaceuticals, Inc. Cyclosporin analogues for preventing or treating hepatitis C infection
US9669095 Nov 3, 2015 Jun 6, 2017 Enanta Pharmaceuticals, Inc. Cyclosporin analogues for preventing or treating hepatitis C infection
US20040138108 * Apr 22, 2002 Jul 15, 2004 Roland Wenger Pro-drug and use thereof as a medicament
US20080206287 * Sep 17, 2007 Aug 28, 2008 Reed John C Use of cyclosporin A to sensitize resistant cancer cells to death receptor ligands
US20100196316 * Jan 30, 2010 Aug 5, 2010 Yat Sun Or Cyclosporin analogues for preventing or treating hepatitis c infection
US20100209390 * Feb 19, 2010 Aug 19, 2010 Yat Sun Or Cyclosporin analogues
US20110008284 * Jul 8, 2010 Jan 13, 2011 Xuri Gao Novel clyclosporin analogues
US20110008285 * Jul 8, 2010 Jan 13, 2011 Jiang Long Novel cyclosporin analogues
US20110008286 * Jul 8, 2010 Jan 13, 2011 Guoqiang Wang Novel proline substituted cyclosporin analogues
US20110206637 * Feb 23, 2011 Aug 25, 2011 Yat Sun Or Antiviral agents
EP0414632A2 Jul 23, 1990 Feb 27, 1991 Sandoz Ltd. Cyclosporin derivatives
U.S. Classification 530/321, 930/270, 930/21, 930/20, 930/DIG.546
International Classification C07K7/64
Cooperative Classification Y10S930/27, C07K7/645
Owner name: SANDOZ LTD., 4002 BASLE, SWITZERLAND, A SWISS COMP
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:WENGER, ROLAND;REEL/FRAME:004126/0190
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST. SUBJECT TO CONDITIONS RECITED;ASSIGNOR:SANDOZ, LTD.;REEL/FRAME:004312/0300