Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
  • C07C271/06—Esters of carbamic acids
  • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
  • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
  • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0207—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)4-C(=0), e.g. 'isosters', replacing two amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Retroviruses that is, viruses within the family of Retroviridae, are a class of viruses which transport their genetic material as ribonucleic acid rather than
• RNA-tumor viruses also known as RNA-tumor viruses, their presence has been associated with a wide range of diseases in humans and animals. They are believed to be the causative agents in pathological states associated with infection by Rous sarcoma virus (RSV), murine leukemia virus (MLV), mouse mammary tumor virus (MMTV), feline leukemia virus (FeLV), bovine leukemia virus (BLV), Mason-Pfizer monkey virus (MPMV) , simian sarcoma virus (SSV), simian acquired immunodeficiency syndrome (SAIDS), human T-lymphotropic virus (HTLV-I, -II) and human immunodeficiency virus (HIV-1, HIV-2), which is the etiologic agent of AIDS (acquired immunodeficiency syndrome) and AIDS related
• the extracellular virus particle is composed of an outer membrane studded with viral glycoproteins, a core of structural proteins, and a genome of single stranded ribonucleic acid.
• the retroviral genome has a distinctive regional organization, referred to as the 5'- gag-pol-env-3' structure, wherein the gag region encodes the core structural proteins, the pol region encodes certain critical viral enzymes such as reverse transcriptase, integrase and protease, and the env region encodes the envelope glycoproteins.
• Viral replication occurs only within host cells and is dependent upon host cellular functions.
• the method of isosteric replacement has been disclosedas a strategy for the development of protease inhibitors for HIV-1.
• Published European Patent applications EP-A 337 714, EP-A 352 000 and EP-A 357 332, EP-A 346 847, EP-A 342 541 and EP-A 393 445 are representative. Similar strategies have also been reported for inhibition of renin in U.S. Patents 4,713,445 and 4,661,473.
• protease- inhibiting compounds which have a favorable balance of potency and pharmacokinetics properties.
• the compounds of this invention have a unique 2,4 dihydroxy isostere wherein the hydroxyl group in the 2 position is further substituted to create a tertiary alcohol.
• the compounds are potent inhibitors of retroviral protease and are able to penetrate affected cells.
• This invention comprises compounds, hereinafter
• This invention is also a pharmaceutical composition, which comprises a compound of formula (I) and a
• This invention further constitutes a method for treating retroviral disease, which comprises administering to a mammal in need thereof an effective amount of a compound of formula
• R 1 is A, A-B or A-B-D
• A is R 11 , R 11 CO, R 11 - (CHR 11 ' ) n -CO, R 1 1 OCO, R 1 1 OCH (R 11 ' ) CO, R 11 NHCH ( R 11 ' ) CO, R 11 SCH ( R 11 ' ) CO, R 1 1 SO 2 , R 1 1 SO or R 1 1 C (O) CO;
• R 2 and R 4 are C 1-6 alkyl, optionally substituted with 1-5 fluorine atoms, C 3-6 alkenyl, C 1-6 alkyl-O-CH 2 or (CH 2 ) n -T, wherein T is phenyl, naphthyl, C 5-6 cycloalkyl or indolyl, optionally substituted with nitro, halogen, C 1-4 alkyl,
• R 5 is H, C 1-6 alkyl, C 1-6 alkyl-O-CH 2 or J-CH 2 (CH 2 )n, wherein J is CONHR', CO 2 R', NHR', SR', or phenyl, naphthyl, C 5-6 cycloalkyl or indolyl, optionally substituted with nitro, halogen, hydroxy, C 1-4 alkyl, C 1-4 alkoxy, C 1-4 alkylthio or trifluoromethyl;
• R 3 is G, X-G or X-Y-G; X and Y are
• G is R 10 , NR'R 10 , NR'NR'-A, OR 10 or SR 10 ;
• R 10 is (CR 11 R 11 ') n -W;
• R 11 and R 11 ' are H, Alk, Ar, Het, Ar-C 1-5 alkyl,
• W is H, CH 2 OR", COR", OR 11 , OCOR 11 , NR'R 11 , NR'COR 11 , SR 11 , Ar or Het;
• Alk is C 1-6 alkyl or C ⁇ -6alkyl substituted by one or two hydroxy, nitro or C 1-4 alkoxy groups, or one to five fluoro atoms;
• R' is H, C 1-6 alkyl or (CH 2 ) n -Ar;
• R" is H, C 1-6 alkyl or (CH 2 ) n -Ph;
• R 1 is A or A-B.
• R 2 is C 1-6 alkyl or (CH 2 ) n -phenyl.
• R 3 is G.
• R 3 is NHCH(R 11 )-W, wherein R 11 is H or Alk and W is CH 2 OH, CONH 2 or phenyl.
• R 1 is A. More suitably R 1 is C 1-6 alkoxycarbonyl. Preferably R 2 is benzyl.
• R 3 is NHCH(i-pr)CH 2 OH.
• R 4 is benzyl
• Prodrugs are considered to be any covalently bonded carriers which release the active parent drug according to formula (I) in vivo.
• Ar indicates aryl, Ar is phenyl or naphthyl, or phenyl or naphthyl substituted by one to three C 1-4 alkyl, C 1-4 alkoxy, C 1-4 alkylthio, trifluoromethyl, nitro, mercapto, hydroxy, halogen, cyano, CO 2 R" or CON(R") 2 .
• Het indicates an
• heterocyclic ring is a saturated or unsaturated, five or six membered ring or nine or ten-membered bicyclic ring, containing one to three heteroatoms chosen from the group of nitrogen, oxygen and sulfur, which are stable and available by conventional chemical synthesis.
• Illustrative heterocycles are pyridyl, furyl, thienyl, morpholinyl, pyrrolinyl, pyrrolidinyl, pyrazolyl,
• Boc refers to the t-butyloxycarbonyl radical
• Z and Cbz refer to the carbobenzyloxy radical
• BrZ refers to the o-bromobenzyloxy-carbonyl radical
• C1Z is the
• C12Z refers to the 2,4-dichlorocarbobenzyloxy radical
• Bn refers to the benzyl radical
• Ac refers to acetyl
• Ph refers to phenyl
• BrMgDA refers to bromomagnesium diisopropylamide
• DCC refers to dicyclohexyl-carbodiimide
• DMAP refers to dimethylaminopyridine
• HOBT 1-hydroxybenzotriazole
• OSU is
• 1-hydroxysuccinimide NMM is N-methylmorpholine
• DTT is dithiothreitol
• EDTA is ethylenediamine tetraacetic acid
• DIEA is diisopropyl ethylamine
• DBU is 1,8 diazobicyclo [5.4.0]undec-7-ene
• DMSO is dimethylsulfoxide
• DMF is dimethyl formamide
• HMPA hexamethylphosphoramide
• DMAPEC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
• DCC dicyclohexylcarbodiimide
• EDC N-ethyl-N'(dimethylaminopropyl)-carbodiimide
• PPA 1-propanephosphonic acid cyclic anhydride
• DPPA diphenylphosphoryl azide
• BOP refers to
• TMS-Imid N-trimethysilylimidazole
• valinol (2S)-2-amino-3-methyl-butanol
• THF is
• HF refers to hydrofluoric acid
• TFA refers to trifluoroacetic acid.
• C 1-6 alkyl as applied herein is meant to include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tertiary butyl, n-pentyl, isopentyl arid hexyl. As used herein in the compounds of this invention.
• Representative compounds of this invention are:
• Preferred compounds of this invention are:
• the chiral centers of the compounds of the invention may be racemic or nonracemic. Racemic mixtures, mixtures of diastereomers, as well as single diastereomers and
• Pr 1 is an amino protecting group
• Pr 2 is a carboxy protecting group
• Pr 3 and Pr 5 are H or a hydroxy
• Pr2 is generally OH, O-alkyl, O-benzyl or O-phenyl.
• Methods of general synthetic utility are found in U.S. Patents 4,661,473 and 4,713,445, published European Patent applications EP-A 352 000 and 337 714,
• Pr 3 is H or a hydroxy-protecting group
• Pr 5 is H or a hydroxy-protecting group
• A, B, D, R 4 and R 2 are as defined for formula (I), with any reactive groups protected
• Pr 3 is H or a hydroxy-protecting group
• X, Y, G, R 2 and R 4 are as defined in formula (I), with any reactive groups protected
• G, X, Y, A and R 10 are as defined for formula (I), with any reactive groups protected, and
• Typical coupling agents include carbodiimides, such as DCC, EDC, DMAPEC, DPPA, PPA, and the BOP reagent, and
• coupling reagents would additionally include organometallic reagents, such as lithium dimethyl copper which could be reacted with an acyl halide to form a ketone.
• Typical bases include organic nitrogenous bases, such as triethylamine or diisopropylamine, or an alkali metal
• Typical reactive groups which may require protection during synthesis are the hydroxyl, amino, carboxyl, carbonyl and mercapto groups. Conventional methods for protection and deprotection of reactive groups are disclosed in Greene,
• R 4 may be introduced into the isosteres (II) or (III) as any desired amino acid side chain, with any reactive groups suitably protected.
• R 2 may be introduced into the isostere and varied as
• An ⁇ -substituted, ⁇ -amino-aldehyde, such as (IV), is prepared by conventional means.
• a suitable ⁇ - amino acid may be reduced to its corresponding carboxaldehyde to initiate the preparation.
• Suitable methods are disclosed Coppola et al . , Asymmetric Synthesis, Construction of Chiral Molecules Using Amino Acids, Wiley Interscience, New York, 1987.
• the aldehyde (IV) is condensed with the enolate derived from methyl pyruvate (V) and a suitable base, such as
• the enol lactone 1 is reduced with an appropriate reducing agent, such as hydrogen with a palladium catalyst to yield an ⁇ -hydroxy lactone as mixture of epimers at the hydroxyl position.
• an appropriate reducing agent such as hydrogen with a palladium catalyst to yield an ⁇ -hydroxy lactone as mixture of epimers at the hydroxyl position.
• the hydroxyl group is then protected with an appropriate protective group which is stable to strong base, such as a silyl ether.
• Subsequent treatment of the lactone with a strong base such as lithium alkyl or
• hydroxyl group at the 4 position can be protected, or the hydroxyl group at the 4 position can be selectively protected.
• a silyl ether is the most useful group for this purpose.
• the protected compound allows further modification of the carboxy terminus via amidation, esterification or coupling of an activated acyl intermediate, such as an acyl halide, with an organometallic reagent.
• deprotecting the amino terminus allows further modification of the amine group by alkylation, acylation or sulfonylation.
• Solution synthesis of the peptide bonds is accomplished using conventional methods for coupling the appropriate amino acid residues and optionally removing any protective groups. Suitable methods are disclosed, for instance, in Bodansky et al . , The Practice of Peptide Synthesis, Springer-Verlag, Berlin, 1984. Typically, a protected Boc-amino acid which has a free carboxyl group is coupled to a protected amino acid which has a free amino group using a suitable
• carbodiimide coupling agent such as N, N' dicyclohexyl carbodiimide (DCC) or 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (DMAPEC), optionally in the presence of catalysts such as 1-hydroxybenzotriazole (HOBT) and dimethylamino pyridine (DMAP).
• DCC N, N' dicyclohexyl carbodiimide
• DMAPEC 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride
• catalysts such as 1-hydroxybenzotriazole (HOBT) and dimethylamino pyridine (DMAP).
• HOBT 1-hydroxybenzotriazole
• DMAP dimethylamino pyridine
• a protected Boc-amino acid or peptide is treated in an anhydrous solvent, such as methylene chloride or tetrahydrofuran (THF), in the presence of a base, such as N-methyl morpholine, DMAP or a trialkyl amine, with isobutyl chloroformate to form the "activated anhydride", which is subsequently reacted with the free amine of a second protected amino acid or peptide.
• anhydrous solvent such as methylene chloride or tetrahydrofuran (THF)
• a base such as N-methyl morpholine, DMAP or a trialkyl amine
• Esters are often used to protect the terminal carboxyl group of peptides in solution synthesis. They may be
• the acids may be converted to other esters via an activated acyl intermediate as previously described.
• the amides and substituted amides of this invention are prepared from carboxylic acids of the peptides in much the same manner.
• ammonia or a substituted amine may be reacted with an activated acyl intermediate to produce the amide.
• Use of coupling reagents, such as DCC or DMAPEC, is convenient for forming substituted amides from the carboxylic acid itself and a suitable amine.
• methyl esters of this invention may be converted to the amides, or substituted-amides, directly by treatment with ammonia, or a substituted amine, in methanol solution.
• a methanol solution of the methyl ester of the peptide is saturated with ammonia and stirred in a
• an acid addition salt may be
• Acid addition salts of the peptides are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic or methanesulfonic .
• the acetate salt form is especially useful.
• cationic salts may be prepared.
• the parent compound is treated with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation.
• Cations such as Na + , K + , Ca ++ and NH 4 + are examples of cations present in pharmaceutically
• Certain of the compounds form inner salts or zwitterions which may also be acceptable.
• the compounds of formula (I) are used to induce antiviral activity in patients which are infected with
• the this invention is a method of treating disease resulting from infection by retroviruses by
• this invention is a method of treating Acquired Immune Deficiency Syndrome (AIDS).
• the method of treatment comprises the administration orally, parenterally, buccally, trans-dermally, rectally or by insufflation, of an effective quantity of the chosen
• Dosage units of the active ingredient are generally selected from the range of 0.01 to 25 mg/kg, but will be readily determined by one skilled in the art depending upon the route of administration, age and condition of the patient. These dosage units may be administered one to ten times daily for acute or chronic infection.
• protease inhibiting properties of the peptides of this invention are demonstrated by their ability to inhibit the hydrolysis of a peptide substrate by rHIV protease in the range of about 20 nM to about 60 ⁇ M.
• the following table is representative of the inhibition constants of the compounds of this invention.
• compositions of the peptides of this invention, or derivatives thereof, may be formulated as solutions or lyophilized powders for parenteral
• Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use.
• the liquid formulation is generally a buffered, isotonic, aqueous solution.
• suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution.
• Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
• these peptides may be encapsulated, tableted or prepared in a emulsion or syrup for oral
• compositions may be added to enhance or stabilize the
• Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline and water.
• Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
• the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl
• the amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit.
• the pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms .
• a liquid carrier When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
• a pulverized powder of the peptides of this invention may be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
• excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols
• the pulverized powders may also be compounded with an oily preparation, gel, cream or emulsion, buffered or unbuffered, and administered through a transdermal patch.
• Beneficial effects may be realized by co-administering, individually or in combination, other anti-viral agents with the protease inhibiting compounds of this invention.
• anti-viral agents examples include nucleoside analogues, phosphonoformate, rifabutin, ribaviran, phosphonothioate oligodeoxynucleotides, castanospermine, dextran sulfate, alpha interferon and ampligen.
• Nucleoside analogues which include 2',3'-dideoxycytidine(ddC), 2',3'-dideoxyadenine(ddA) and 3'-azido-2',3'-dideoxythymide(AZT), are especially useful.
• AZT is one preferred agent.
• pharmaceutical compositions comprise an anti-viral agent, a protease
• the enzyme used to assay the peptide of this invention was produced in this manner and purified from the cell pellet as follows.
• the E. coli cell pellet was resuspended in a buffer consisting of 50 mM Tris- HCl, pH 7.5; 1.0 mM each DTT, EDTA and PMSF
• the column was equilibrated in the same buffer at a flow rate of 4 ml/min.
• the effluent of the column was monitored at 280 nm and 1 min. fractions collected.
• the rHIVPRT recombinant HIV protease
• the protease was 85-95% pure.
• By immunoblot analysis >90% of the immunoreactive material was precipitated at the ammonium sulfate step.
• activity assay the highest peak of activity was found in the fractions collected at 45 and 46 minutes.
• the overall yield of rHIVPRT was N1 mg from a 50 gm E. coli cell pellet.
• MENDT buffer 50 mM Mes (pH 6.0; 2-(N-morpholino)ethanesulfonic acid), 1 mM
• reaction was initiated with purified 0.01-1 mg HIV protease.
• Reaction mixtures 37°C
• reaction mixtures 37°C
• Ultrasphere ® and Ultrasphere ® ODS are silica gel and octadecylsilane chromatographic supports
• Microsorb ® is a silica gel chromatographic support manufactured by Rainin Instruments Co., Woburn, Mass. Bakerbond ® is a silica gel chromatographic support
• Celite ® is filter aid composed of acid washed diatomaceous silica manufactured by Mansville Corp., Denver, Colorado.
• steps 1(b) and 1(c) except substituting the enantiomerically pure (5S)-5-((1S)-1-t-butyloxycarbonylamino-2-phenyl)ethyl-4H,5H-dihydrofuran-2,3-dione 1a in step 1(b), (5S)-5-((1S)-1-t-butyloxycarbonylamino-2-phenyl)ethyl-3-trimethysilyloxy-tetrahydrofuran-2-one (3a) was prepared.
• reaction mixture was poured into 0.05 M HCl, and separated.
• the aqueous layer was extracted ethyl acetate (3X), the combined organic layers were washed with 0.05 M HCl, brine, dried over MgSO 4 , and concentrated to a yellow oil.
• steps 1 (b) through 1(d) except using the enantiomerically pure (5S)-5-((1S)-1-t-butyloxycarbonylamino- 2-phenyl)ethyl-4H,5H-dihydrofuran-2,3-dione, 1a, in step 1(b), (3S,5S)-5-((1S)-1-t-butyloxycarbonylamino-2-phenyl)ethyl-3-trimethysilyloxy-3-benzyl-tetrahydrofuran-2- one (4a) was prepared.
• n-butyllithium (1.6M solution in hexane, 1.25 mL, 1.98 mmol). The reaction mixture was allowed to stir for 20 min. To this mixture was added a solution of 4 (240 mg, 0.495 mmol) in THF (10 mL) via cannula. The reaction mixture was allowed to stir for 1 h, poured into 0.05 M HCl, and extracted with ethyl acetate (3X). The organic layers were washed with 0.05 M HCl, brine, dried over MgSO 4 , and concentrated to a white solid. The solid was recrystallized from ethyl acetate, to afford the product (220 mg,86%):
• reaction mixture was cooled to 0°C and Cbz-D-Ala (34 mg, 0.15 mmol), 1-hydroxybenzotriazole (HOBT) (21 mg, 0.15 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
• reaction mixture was cooled to 0°C and Cbz-L-Ala (40 mg, 0.18 mmol), HOBT (24 mg, 0.18 mmol), and DMAPEC (35 mg, 0.18 mmol) were added.
• the reaction mixture was allowed to stir at 0°C for 1 h, and the ice bath was removed.
• the mixture was allowed to warm and stir to room temperature for 16 h.
• the mixture was diluted with methylene chloride, washed with 10% NaHCO 3 , 0.05 M HCl, brine, dried over MgSO 4 , and
• reaction mixture was poured into 0.05 M HCl, and separated.
• the aqueous layer was extracted ethyl acetate (3X), the combined organic layers were washed with 0.05 M HCl, brine, dried over MgS ⁇ 4 , and concentrated to a yellow oil. This was
• Phosphatidylcholine 1.4 g
• phosphatidylglycerol 0.6g
• polycarbonate filter five times to produce a uniform size distribution. If necessary the suspension can be dialysed or ultracentrifuged to remove non-encapsulated peptide.
• a preparation which contains 250 mg of the compound of Example 5 is prepared as follows:
• Example 15 distilled water. The solution is filtered under sterile conditions in to a multi-dose ampoule and lyophilized. The powder is reconstituted by addition of 200 mL of 5% dextrose in water (D5W) for intravenous injection. The dosage is thereby determined by the injection volume. This solution is also suitable for use in other methods for administration, such as in a bottle or bag for IV drip infusion.
• Example 15
• a capsule for oral administration is prepared by mixing and milling 350 mg of the compound of Example 5 with 750 mg of lactose and 50 mg of magnesium stearate. The resulting powder is screened and filled into a hard gelatin capsule.

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