WO1994009033A1 - An improved process for cyclohexapeptidyl bisamine compounds - Google Patents
An improved process for cyclohexapeptidyl bisamine compounds Download PDFInfo
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- WO1994009033A1 WO1994009033A1 PCT/US1993/009606 US9309606W WO9409033A1 WO 1994009033 A1 WO1994009033 A1 WO 1994009033A1 US 9309606 W US9309606 W US 9309606W WO 9409033 A1 WO9409033 A1 WO 9409033A1
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- 0 CC(C(C(C(CC1C(NC(*)C(CC(C(NC(C(C)O)C(N(CC(C2)O)C2C(NC2C(C(c(cc3)ccc3O)O)O)=O)=O)=O)NC(CC3=CC4C=CC(**)=CC4C=C3)=O)O)=O)*CCC1O)=O)NC2=O)O Chemical compound CC(C(C(C(CC1C(NC(*)C(CC(C(NC(C(C)O)C(N(CC(C2)O)C2C(NC2C(C(c(cc3)ccc3O)O)O)=O)=O)=O)NC(CC3=CC4C=CC(**)=CC4C=C3)=O)O)=O)*CCC1O)=O)NC2=O)O 0.000 description 3
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
Definitions
- the present invention is directed to an improved process for the preparation of certain cyclohexapeptidyl bisamine compounds.
- Cyclohexapeptidyl bisamine compounds which have one amine group directly on the ring and a second amine group as a substituent or an ether group and which may be represented by the formula
- R 1 is H or OH
- R 2 is H or OH
- R 3 is C n H 2n NR II R III , C n H 2n + NR II R III R IV Y-,
- R 4 is H or OH
- R 5 is H, OH or CH 3
- R 6 is H or CH 3
- R 1 is C 9 -C 21 alkyl, C 9 -C 21 alkenyl,
- R II is H, C 1 -C 4 alkyl or benzyl
- RIII is H, C 1 -C 4 alkyl, benzyl, or R II and
- R III together are -(CH 2 ) 4 - or
- -(CH 2 ) 5 - R IV is H or C 1 -C 4 alkyl
- Y is an anion of a pharmaceutically
- n is an integer of 2 to 4, inclusive.
- bisamine bisamine
- the expression "bisamine” compound is intended to embrace not only the amine (free base) represented by formula (I) but also the acid addition salts.
- the aminoalkyl ethers and the "nitrile compound” which also have an aminoalkyl ether group may be obtained as acid addition salts.
- the acid addition salts are generally trifluoroacetate, acetate or hydrochloride but the products as well as the novel intermediates which are also useful as antibiotics may form salts with any of the pharmaceutically acceptable acids such as those listed in J. Pharm. Soc. 66,
- pneumocandin B O Pneumocandins are names used for certain echinocandin-like structures and the basic structure for pneumocandins are found in the paper by J. M. Balkovec and R. M. Black, in Tetrahedron Letters, 1992, 33, 4529-32.
- sequence ID of the starting materials (X) and the aminoalkyl ether intermediate (Y) would be the same since the change there is the etherification at a hydroxyl and not the replacement of the hydroxyl oxygen.
- sequence IDs are assigned to the nitrile compound (Z) since the glutamine has been modified.
- sequence IDs of (X) and (Y) are as follows: STARTING
- cyclohexapeptide compounds which are starting materials are for most part natural products or modified natural products as hereinafter described.
- the aminoalkanol represented by R 3 OH may be a substituted or unsubstituted amino group and is generally employed as the acid addition salt.
- a protecting group optionally placed on the amino group before the reaction is carried out and the protecting group removed after the etherification is complete as hereinafter more fully described.
- R is a substituted amino group
- a substituted amino alcohol may be the reactant or alternatively an unsubstituted amino alcohol may be employed and the substituent put on the amino group before proceeding to Step (2).
- the amino alcohol is employed in the form of an acid addition salt and is employed in an amount of from about 20 to 200 equivalents.
- the reaction is carried out in the presence of a strong acid.
- a strong organic acid such as camphorsulfonic acid or a mineral acid such as
- hydrochloric acid is preferred. From about 1 to 10 equivalents of the acid are employed.
- a solvent is employed in carrying out the reaction.
- Suitable solvents are aprotic solvents and include dimethylsulfoxide (DMSO), dimethylformamide (DMF), dioxane or combinations thereof.
- the group When the amino alcohol has a primary amino group, the group may be protected before it is used and the protecting group removed before the dehydration step is carried out. Conventional protecting groups are employed.
- the carbobenzyloxy group (CBz) is the preferred group.
- substituted amino groups if the substituent is not already on the amino alcohol, it may be placed on the amino group after the ether is formed by a method appropriate for the particular group and within the knowledge of the skilled in the art. Generally, if the group is a quarternary amine, an alkyl halide would be used, although in the case of methyl the combination of formaldehyde and sodium cyanoborohydride may be the derivatizing agent. If the group is an acid derivative, an activated acid
- derivative may be used as derivatizing agent under basic conditions. Representative reactants which are typical are illustrated in the working examples.
- the preferred reagent is cyanuric chloride.
- Other reagents which may be employed in place of cyanuric chloride are anhydrides such as acetic anhydride, trifluoroacetic anhydride and phosphorus pentoxide; acid chlorides such as oxalyl chloride, phosphorus oxychloride, thionyl chloride, p-toluenesulfonyl chloride and chlorosulfonyl isocyanate; phosphonium reagents such as phosphorus pentachloride, triphenylphosphine/carbon tetrachloride, triphenylphosphonium ditriflate and triphenylphosphonium dichloride; carbodiimides such as dicyclohexylcarbodiimide; other dehydrating agents such as aluminum chloride, titanium tetrachloride, ethyl(carboxysulfamoyl)triethy
- the reaction is carried out in a solvent such as dimethylformamide (DMF).
- a solvent such as dimethylformamide (DMF).
- Other solvents which may be employed include pyridine, collidine and other weakly basic solvents.
- the relative amounts of the aminoalkyl ether compound and the dehydrating agent varies but generally the dehydrating agent is used in excess. From about 1.5 to 15 equivalents of the dehydrating agent are employed .
- the reduction of the nitrile compound to the bisamine compound of the present invention may be carried out employing either chemical or catalytic reduction. When chemical reduction is employed, hydride or hydride combinations have been found useful.
- hydride reducing agents such as sodium cyanoborohydride, aluminum hydride, diborane,
- diisobutyl aluminum hydride and the like also may be used. Frequently these reducing agents are used in combination with a Lewis acid such as cobaltous
- Catalytic hydrogenation also may be carried out over a variety of catalysts including palladium on carbon, platinum oxide, or rhodium on alumina. Low pressure catalytic reduction over Pd/C catalyst is especially preferred.
- Typical solvents depending on the reagent include alcohols, especially methanol and ethanol, dimethylformamide, pyridine, tetrahydrofuran or other ethers.
- the cyclohexapeptide compound, the appropriate aminoalkanol or protected amino-alkanol and strong acid in a dry inert solvent are stirred together at ambient temperature for time sufficient for reaction to take place with the formation of the aminoalkyl ether compound.
- the completion of the reaction may be monitored by HPLC analysis with UV detection at 210 and 277 nm.
- the reaction is usually complete in fifteen to ninety-six hours. The reaction is quenched,
- the aminoalkanol employed is a protected aminoalkanol
- the benzyloxycarbonyl group is removed by low pressure hydrogenation in the presence of 10% Pd/C.
- reaction has occurred, the reaction mixture is filtered to remove the catalyst, the filtrate concentrated in vacuo or lyophilized and the product purified using preparative HPLC.
- the dehydrating agent is added to a solution of the aminoalkyl ether as a salt in an aprotic solvent at 0°C to room temperature.
- the reaction is monitored at 210 and 277 nm using analytical HPLC with solvent system of acetonitrile/water. After several minutes, conversion to product peak is noted.
- About an equal volume of cold sodium acetate is added then further diluted with water, filtered and the filtrate purified using preparative HPLC and monitoring at 220 nm. Appropriate fractions are combined and lyophilized to obtain the nitrile.
- the reaction may be carried out by adding the chemical reducing agent to the nitrile compound in an alcoholic solution under an atmosphere of nitrogen, and stirring until HPLC
- cobaltous chloride is added while stirring to a solution in methanol, or other solvent, of the nitrile, prepared as above described, at ambient temperature, followed by
- the compounds produced by the process of the present invention are active against many fungi and particularly against Candida, Aspergillus and
- Cryptococcus species The antifungal properties may be illustrated with the minimum fungicidal concentration (MFC) determination against certain Candida and
- YNBD Yeast Nitrogen Base
- concentrations ranging from 256 ⁇ g/ml to 0.12 ⁇ g/ml.
- concentrations ranging from 128 ⁇ g/ml to 0.06 drug ⁇ g/ml.
- Each assay includes one row for drug-free control wells and one row for cell-free control wells. After 24 hours of incubation, the microtiter plates were shaken gently on a shaker to resuspend the cells. The MIC-2000 inoculator was used to transfer a 1.5 microliter sample from each well of the 96-well microtiter plate to a single reservoir inoculum plate containing Sabouraud dextrose agar (SDA).
- SDA Sabouraud dextrose agar
- pneumocystis pneumonia from a latent infection. Before drug treatment, two rats were sacrificed to confirm the presence of Pneumocystis carinii pneumonia (PCP); both rats were found to have infections. Five rats
- intermediate nitriles also show good antibiotic potential.
- these compounds may also be employed in compositions for the control of mycotic infections and also for the treatment and control of Pneumocystis carinii infections.
- ether derivative is not only useful as an intermediate but also as an antifungal agent.
- N,N-dimethylformamide was stirred at 25oC.
- the reaction was monitored by analytical HLPC using "ZORBAX" RX-C18 column and a solvent system of 55% CH 3 CN/H 2 O at a flow rate of 1.5 mL/min with UV
- the lyophilizate was dissolved in a minimum volume of H 2 O and the solution was passed through a column of anion exchange resin (Bio-Rad AG2-X8(C1-)) and the eluate was lyophilized to obtain 2.0 grams (95% yield) of the hydrochloride as a white amorphous solid.
- Pentafluorophenyl 6-octyloxy-2-naphthoate for acylating the cyclopeptide was first prepared in the following manner: To a suspension of 6-octyloxy-2-naphthoic acid (3.15 g, 10.5 mmol) and dicyclohexylcarbodiimide in ethyl acetate (25 mL) at 0°C was added pentafluorophenol (2.12 g, 11.5 mmol). The mixture was stirred at 25°C for a period of 18 hours. The precipitate was removed by filtration. The filtrate was washed with water (2 ⁇ 150 mL) and brine and dried with magnesium sulfate.
- Reverse-phase flash chromatography of the triturate eluting with 40-45% CH 3 CN/H 2 O and then lyophilizing the appropriate fractions produced 340 milligrams of 1-[4,5-dihydroxy-N 2 -(6-octyloxy-2-naphthoyl)ornithine]pneumocandin B 0 (80% purity).
- a 40 mg portion was rechromatographed on "ZORBAX" with 43 percent CH 3 CN/H 2 O at 10 ml/min, uv detection at 220 nm to obtain 30 mg of N-acylated compound of >98 percent purity.
- reaction was quenched with 1M NaHCO 3 , the reaction mixture diluted with water and subject to reverse-phase flash chromatography eluting with 40-60 percent CH 3 CN/H 2 O in 10% step gradients to obtain after lyophilization of the 60 percent
- Cyanuric chloride is added to a solution of the trifluoroacetate salt of the aminoethyl ether prepared as above described in anhydrous DMF at 0°C.
- the reaction is monitored by analytical HPLC using a "ZORBAX" RX-C18 column and a solvent system of 35% CH 3 CN/H 2 O at a flow rate of 1.5 mL/min with uv
- Step (3) sodium borohydride is added in portions to a solution of CoCl 2 ⁇ 6H 2 O and the nitrile prepared as described above in methanol under an atmosphere of nitrogen.
- the reaction progress is followed with analytical HPLC and after one hour there is indication of conversion to a more polar product.
- the mixture is acidified to pH 3 by the addition of 1 N HCl and is diluted tenfold with water.
- the solution is subjected to reverse-phase flash chromatography, eluting with 5-10% CH 3 CN/H 2 O, then the appropriate fractions combined and lyophilized to obtain the amine product as the dihydrochloride: C 55 H 81 Cl 2 N 9 O 17
- Example 3 Part B; 150 mg, 0.128 mmol and 1N sodium hydroxide (130 ⁇ L, 0.130 mmol) in water (5 mL) and N,N-dimethylformamide (5 mL) was added ethylacetimidate hydrochloride (160 mg, 1.29 mmol). After a period of 18 hours at pH 8.5, glacial acetic acid was added to pH 7. Reverse-phase (C18) flash column chromatography of the neutralized reaction mixture, eluting with
- the starting materials for the compounds are natural products or derivatives of natural products.
- X-1 may be produced by cultivating Zalerion arboricola ATCC 20868 in a nutrient medium enriched in mannitol as the primary source of carbon as described in U.S. Patent No. 5,021,341, June 4, 1991.
- X-2 may be produced by cultivating Zalerion arboricola ATCC 20868 in nutrient medium as described in U.S. 4,931,352, June 5, 1990 or in nutrient medium enriched in glycerol as described in U.S. 4,968,608, November 6, 1980.
- X-2 nucleus with a different R I may be produced by cultivating Acrophialophora limonispora in nutrient medium as described in U.S. 4,173,629 and thereafter acylating employing R I COCl or an activated ester of R I COOH.
- X-3 and X-7 may be produced by cultivating Cryptosporiopsis ATCC 20594 in nutrient medium as described by Pache et al in 13th ICC (1983), PS 4.8/3, Part 115, Abstract No. 10 and PCT WO 82/00587.
- X-4, X-5 and X-6 may be produced by cultivating Zalerion arboricola ATCC 20868 in nutrient medium.
- X-8 when R 1 is H, R 2 and R 4 are OH, R 5 is H or CH 3 and R 5 is CH 3 , the compound is not a natural product. It may be prepared by reducing a compound in which R 1 is OH and the other R 5 are the same by methods known to the skilled in the art. This may be carried out by adding TFA and triacetoxyborohydride and mixing together until a clear solution is obtained, pouring the solution into water and recovering the product which precipates therein and purifying by HPLC.
- Starting materials in which R I is a different group from that of the natural product may be obtained by deacylating the lipophilic group of the natural product by subjecting the natural product in a nutrient medium to a deacylating enzyme until substantial deacylation occurs, said enzyme having first been obtained by cultivating a microorganism of the family Pseudomondaceae or Actinoplanaceae, as also described in Experentia 34, 1670 (1978) or U.S. 4,293,482, and thereafter recovering the deacylated cyclopeptide, and acylating the deacylated cyclopeptide by mixing together with an appropriate active ester R I COX to obtain Compound E with the desired acyl group using conventional procedures. Methods are also described in U.S. 4,287,120 and 4,293,489.
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Abstract
An improved process for the preparation of cyclohexapeptidyl bisamine compounds represented by formula (I) and which are potent fungicidal and parasiticidal agents is described.
Description
TITLE OF THE INVENTION
AN IMPROVED PROCESS FOR CYCLOHEXAPEPTIDYL BISAMINE COMPOUNDS
The present invention is directed to an improved process for the preparation of certain cyclohexapeptidyl bisamine compounds.
BACKGROUND OF THE INVENTION
Cyclohexapeptidyl bisamine compounds which have one amine group directly on the ring and a second amine group as a substituent or an ether group and which may be represented by the formula
are potent fungicides and parasiticides but because of the complex nature of the compounds it is difficult to obtain the compounds in good yields and further the isolation of the desired compounds free of side products is attended with some difficulty. It is therefore desirable to provide an improved process for the preparation and isolation of the compounds.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention it has been discovered that the bisamine compound represented by the formula
wherein the R groups are hereinafter fully defined, may be obtained in good and more reproducible yields and in good purity by a sequence of reactions comprising:
(a) first preparing an ether derivative (Y) of the cyclohexapeptide compound (X)
(b) dehydrating the aminoalkyl ether derivative so obtained to obtain a nitrile compound (Z) and
(c) reducing the nitrile compound:
In the foregoing and succeeding formulas
R1 is H or OH
R2 is H or OH
R3 is CnH2nNRIIRIII, CnH2n +NRIIRIIIRIVY-,
-(CH2)2-4NHRV, -CH2CH(NH2)CH2NH2
R4 is H or OH
R5 is H, OH or CH3
R6 is H or CH3
R1 is C9-C21 alkyl, C9-C21 alkenyl,
C1-C10 alkoxyphenyl or
C1-C10 alkoxynaphthyl
RII is H, C1-C4 alkyl or benzyl RIII is H, C1-C4 alkyl, benzyl, or RII and
RIII together are -(CH2)4- or
-(CH2)5-
RIV is H or C1-C4 alkyl
RV is (C=NH)NH2, (C=NH)(CH2)0_3H,
(CH2)2-4NH2, CO(CH2)1_3NH2,
(CH2)2-4NH(C=NH)NH2
Y is an anion of a pharmaceutically
acceptable salt
n is an integer of 2 to 4, inclusive.
When the expression "bisamine" compound is employed, it is intended to embrace not only the amine (free base) represented by formula (I) but also the acid addition salts. The aminoalkyl ethers and the "nitrile compound" which also have an aminoalkyl ether group may be obtained as acid addition salts. In the process of the present invention the acid addition salts are generally trifluoroacetate, acetate or hydrochloride but the products as well as the novel intermediates which are also useful as antibiotics may form salts with any of the pharmaceutically acceptable acids such as those listed in J. Pharm. Soc. 66,
2(1977).
In the working examples some of the compounds have been named as pneumocandin BO. Pneumocandins are names used for certain echinocandin-like structures and the basic structure for pneumocandins are found in the paper by J. M. Balkovec and R. M. Black, in Tetrahedron Letters, 1992, 33, 4529-32.
Representative nuclei for the bisamine compounds, Compound I, and the sequence ID for these compounds may be seen in the following table. Since the peptide nucleus would be the same irrespective of the substituent R1 and since the sequence
identification number is assigned for the nuclear variations, both the various R1 and the particular aminoalkyl group which forms the ether at R3 is considered to be the same for the purposes of sequence identifications. Thus, RII, RIII, RIV and RV which are changes within R3 have no effect on sequence
identification.
BISAMINE
COMPOUND R1 R2 R4 R5 R6 SEQ. ID
NUCLEI
I-1 OH OH OH H CH3 1
I-2 OH OH OH CH3 CH3 2
I-3 H OH OH CH3 H 3
I-4 OH H OH CH3 CH3 4
I-5 H H H CH3 CH3 5
I-6 OH OH OH OH CH3 6
I-7 H OH OH H H 7
I-8 H OH OH H CH3 22
The sequence ID of the starting materials (X) and the aminoalkyl ether intermediate (Y) would be the same since the change there is the etherification at a hydroxyl and not the replacement of the hydroxyl oxygen. Different sequence IDs are assigned to the nitrile compound (Z) since the glutamine has been modified.
The sequence IDs of (X) and (Y) are as follows:
STARTING
MATERIAL R1 R2 R4 R5 R6 Seq. ID
(ETHER)
X-1 (Y-1) OH OH OH H CH3 8
X-2 (Y-2) OH OH OH CH3 CH3 9
X-3 (Y-3) H OH OH CH3 H 10
χ-4 (Y-4) OH H OH CH3 CH3 11
X-5 (Y-5) H H H CH3 CH3 12
X-6 (Y-6) OH OH OH OH CH3 13
X-7 (Y-7) H OH OH H H 14
X-8 (Y-8) H OH OH H CH3 23
The sequence IDs of (Z) are as follows
NITRILE
COMPOUND R1 R2 R4 R5 R6 Seq. ID
(Z )
Z-1 OH OH OH H CH3 15
Z-2 OH OH OH CH3 CH3 16
Z-3 H OH OH CH3 H 17
Z-4 OH H OH CH3 CH3 18
Z-5 H H H CH3 CH3 19
Z-6 OH OH OH OH CH3 20
Z-7 H OH OH H H 21
Z-8 H OH OH H CH3 24
The cyclohexapeptide compounds which are starting materials are for most part natural products or modified natural products as hereinafter described.
The aminoalkanol represented by R3OH may be a substituted or unsubstituted amino group and is
generally employed as the acid addition salt. When unsubstituted, a protecting group optionally placed on the amino group before the reaction is carried out and the protecting group removed after the etherification is complete as hereinafter more fully described. When R is a substituted amino group, a substituted amino alcohol may be the reactant or alternatively an unsubstituted amino alcohol may be employed and the substituent put on the amino group before proceeding to Step (2).
The amino alcohol is employed in the form of an acid addition salt and is employed in an amount of from about 20 to 200 equivalents.
The reaction is carried out in the presence of a strong acid. Either a strong organic acid such as camphorsulfonic acid or a mineral acid such as
hydrochloric acid is preferred. From about 1 to 10 equivalents of the acid are employed.
A solvent is employed in carrying out the reaction. Suitable solvents are aprotic solvents and include dimethylsulfoxide (DMSO), dimethylformamide (DMF), dioxane or combinations thereof.
When the amino alcohol has a primary amino group, the group may be protected before it is used and the protecting group removed before the dehydration step is carried out. Conventional protecting groups are employed. The carbobenzyloxy group (CBz) is the preferred group.
With substituted amino groups, if the substituent is not already on the amino alcohol, it may be placed on the amino group after the ether is formed by a method appropriate for the particular group and within the knowledge of the skilled in the art.
Generally, if the group is a quarternary amine, an alkyl halide would be used, although in the case of methyl the combination of formaldehyde and sodium cyanoborohydride may be the derivatizing agent. If the group is an acid derivative, an activated acid
derivative may be used as derivatizing agent under basic conditions. Representative reactants which are typical are illustrated in the working examples.
For dehydration of the carboxamide group, the preferred reagent is cyanuric chloride. Other reagents which may be employed in place of cyanuric chloride are anhydrides such as acetic anhydride, trifluoroacetic anhydride and phosphorus pentoxide; acid chlorides such as oxalyl chloride, phosphorus oxychloride, thionyl chloride, p-toluenesulfonyl chloride and chlorosulfonyl isocyanate; phosphonium reagents such as phosphorus pentachloride, triphenylphosphine/carbon tetrachloride, triphenylphosphonium ditriflate and triphenylphosphonium dichloride; carbodiimides such as dicyclohexylcarbodiimide; other dehydrating agents such as aluminum chloride, titanium tetrachloride, ethyl(carboxysulfamoyl)triethylammonium hydroxide or inner salt.
The reaction is carried out in a solvent such as dimethylformamide (DMF). Other solvents which may be employed include pyridine, collidine and other weakly basic solvents.
The relative amounts of the aminoalkyl ether compound and the dehydrating agent varies but generally the dehydrating agent is used in excess. From about 1.5 to 15 equivalents of the dehydrating agent are employed .
The reduction of the nitrile compound to the bisamine compound of the present invention may be carried out employing either chemical or catalytic reduction. When chemical reduction is employed, hydride or hydride combinations have been found useful.
Sodium borohydride with cobaltous chloride in alcoholic solvent has been found to be particularly useful. When this combination of reagents is used, from about 5 to 50 molar equivalent of sodium
borohydride and from 2 to 10 molar equivalents of cobaltous chloride are used for each molar amount of the nitrile.
Other hydride reducing agents such as sodium cyanoborohydride, aluminum hydride, diborane,
diisobutyl aluminum hydride and the like also may be used. Frequently these reducing agents are used in combination with a Lewis acid such as cobaltous
chloride or aluminum chloride as in the present
combination of sodium borohydride and cobaltous
chloride.
Catalytic hydrogenation also may be carried out over a variety of catalysts including palladium on carbon, platinum oxide, or rhodium on alumina. Low pressure catalytic reduction over Pd/C catalyst is especially preferred.
Typical solvents depending on the reagent include alcohols, especially methanol and ethanol, dimethylformamide, pyridine, tetrahydrofuran or other ethers.
In carrying out the sequence of reactions, as the first step, the cyclohexapeptide compound, the appropriate aminoalkanol or protected amino-alkanol and
strong acid in a dry inert solvent are stirred together at ambient temperature for time sufficient for reaction to take place with the formation of the aminoalkyl ether compound. The completion of the reaction may be monitored by HPLC analysis with UV detection at 210 and 277 nm. The reaction is usually complete in fifteen to ninety-six hours. The reaction is quenched,
conveniently by the addition of dilute aqueous sodium bicarbonate solution. Thereafter, the ether is recovered by diluting with water, filtering and
purifying by appropriate chromatographic procedures. The analytical HPLC is conveniently carried out on a "ZORBAX" (DuPont) RX-C18 column (4.6 × 25 cm) (used in this application) employing acetonitrile/water (each always containing 0.1 percent trifluoroacetic acid (TFA)). For purification there may be used "LICHRO PREP" (E. Merck) RP-18 (40-63 μm), a reverse-phase column, "DELTA-PAK" (Waters) C18 (19 nm × 30 cm), or "ZORBAX" RX-C18 (21.2 nm × 25 cm).
When the aminoalkanol employed is a protected aminoalkanol, after isolation of the ether product, the benzyloxycarbonyl group is removed by low pressure hydrogenation in the presence of 10% Pd/C. The
hydrogenation is monitored by analytical HPLC with acetonitrile/water solvent system, UV detection at 210 and 277 nm. When substantial completion of the
reaction has occurred, the reaction mixture is filtered to remove the catalyst, the filtrate concentrated in vacuo or lyophilized and the product purified using preparative HPLC.
In carrying out the dehydration, the dehydrating agent is added to a solution of the
aminoalkyl ether as a salt in an aprotic solvent at 0°C to room temperature. The reaction is monitored at 210 and 277 nm using analytical HPLC with solvent system of acetonitrile/water. After several minutes, conversion to product peak is noted. About an equal volume of cold sodium acetate is added then further diluted with water, filtered and the filtrate purified using preparative HPLC and monitoring at 220 nm. Appropriate fractions are combined and lyophilized to obtain the nitrile.
In carrying out the reduction using the preferred chemical procedure, the reaction may be carried out by adding the chemical reducing agent to the nitrile compound in an alcoholic solution under an atmosphere of nitrogen, and stirring until HPLC
analysis using detection by ultraviolet absorption at 210 and 277 nm shows substantial completion of the reaction. When sodium borohydride is used in
combination with cobaltous chloride, cobaltous chloride is added while stirring to a solution in methanol, or other solvent, of the nitrile, prepared as above described, at ambient temperature, followed by
portionwise addition of the sodium borohydride which is accompanied by gas evolution. Stirring is continued until substantially complete conversion has been obtained. The mixture may be quenched with acetic or hydrochloric acid at this time. Then the mixture is diluted with water and purified by chromatography, preferably, reverse phase chromatography and the appropriate fractions lyophilized to obtain the desired amine product.
The compounds produced by the process of the present invention are active against many fungi and
particularly against Candida, Aspergillus and
Cryptococcus species. The antifungal properties may be illustrated with the minimum fungicidal concentration (MFC) determination against certain Candida and
Cryptococcus organisms in a microbroth dilution assay carried out in a Yeast Nitrogen Base (Difco) medium with 1 percent dextrose (YNBD).
In a representative assay, Compound IA amine (R1, R2, R4 = OH; R5 = H; R6 = CH3 ; R3 = OCH2CH2NH2 , RI = DMTD)) as dihydrochloride was solubilized in 100 percent dimethyl sulfoxide (DMSO) at an initial concentration of 5 mg/ml. Once dissolved, the drug stock was brought to a concentration of 512 μg/ml by dilution in water such that the final DMSO
concentration was about 10 percent. The solution was then dispensed via a multichannel pipetter into the first column of a 96-well plate (each well containing 0.075 ml of YNBD), resulting in a drug concentration of 256 μg/ml. Compounds in the first column were diluted 2-fold across the rows yielding final drug
concentrations ranging from 256 μg/ml to 0.12 μg/ml.
Four-hour broth cultures of organisms to be tested were adjusted using a spectrophotometer at 600 nm to equal a 0.5 McFarland Standard. This suspension was diluted 1:100 in YNBD to yield a cell concentration of 1-5 × 104 colony forming units (CFU)/ml. Aliquots of the suspension (0.075 ml) were inoculated into each cell of the microtiter plate resulting in a final cell inoculum of 5-25 × 103. CFU/ml and final drug
concentrations ranging from 128 μg/ml to 0.06 drug μg/ml. Each assay includes one row for drug-free control wells and one row for cell-free control wells.
After 24 hours of incubation, the microtiter plates were shaken gently on a shaker to resuspend the cells. The MIC-2000 inoculator was used to transfer a 1.5 microliter sample from each well of the 96-well microtiter plate to a single reservoir inoculum plate containing Sabouraud dextrose agar (SDA). The
inoculated SDA plates were incubated for 24 hours at 35°C. However, for Cryptoccoccus neoformans strains, SDA plates were inoculated at 48 hours and incubated 48 hours after being spotted on SDA before making minimum fungicidal concentration (MFC) readings. The results were as follows:
MFC
Organism μg/mL
C . albicans MY 1028 <0.06
C. albicans MY 1055 0.12
C. albicans MY 1750 0.12
C. guillermondii MY 1019 0.5
C. parapsilosis MY 1010 0.12
C. pseudotropicalis MY 1100 0.12
C. tropicalis MY 1012 <0.06
Cr. neoformans MY 1051 16
Cr. neoformans MY 1146 16
Cr. neoformans MY 2061 16
Cr. neoformans MY 2062 16
Similar operation carried out with compounds (a) R1R2R4=OH, R5=H, R6=CH3, RI = C6H4OC8H17 and (b) R1, R2 and R4=OH, R5=H, R6=CH3, RI=C10H6OC8H7 show good control of fungi.
The compounds may also be useful for
inhibiting or alleviating Pneumocystis carinii
infections in immune-compromised patients. The efficacy of the compounds of the present invention for therapeutic or anti-infective purposes may be
demonstrated in studies on immunosuppressed rats.
In a representative study, the effectiveness of Compound IA was determined. Sprague-Dawley rats (weighing approximately 250 grams) were
immunosuppressed with dexamethasone in the drinking water (2.0 mg/L) and maintained on a low protein diet for seven weeks to induce the development of
pneumocystis pneumonia from a latent infection. Before drug treatment, two rats were sacrificed to confirm the presence of Pneumocystis carinii pneumonia (PCP); both rats were found to have infections. Five rats
(weighing approximately 150 grams) were injected twice daily for four days subcutaneously (sc) with Compound IA in 0.25 ml of vehicle (distilled water). A vehicle control was also carried out. All animals continued to receive dexamethasone in the drinking water and low protein diet during the treatment period. At the completion of the treatment, all animals were
sacrificed, the lungs were removed and processed, and the extent of disease determined by microscopic analysis of stained slides. The results of this study showed Compound IA was 90 percent effective in reducing P. carinii cysts in 5 rats when dosed at 0.019 mg/kg.
In addition, the intermediate nitriles also show good antibiotic potential. Thus, these compounds may also be employed in compositions for the control of
mycotic infections and also for the treatment and control of Pneumocystis carinii infections.
It has also been found that the first intermediate of the process, namely the ether
derivative (Compound Y) has antifungal properties.
Thus, ether derivative is not only useful as an intermediate but also as an antifungal agent.
Especially preferred are derivatives which may be represented by the formula
Illustrative of the antifungal properties of these intermediate compounds are the activities against Candida albicans MY 1055 and Candida tropicalis MY 1012 seen in tests carried out in a manner similar to that
described above and which are presented as illustrative compounds which have I-1 nuclear structure and R3 and R1 as indicated:
MFC
g/mL
R3 RI C . albicans C . tropicalis
MY 1055 MY 1012
-CH2CH2-NH-C(=NH)NH2 DMTD 0 , 123 0.125
-CH2CH2N+(CH3)3 DMTD 0.25 0.25
-(CH2)2NHCOCH2NH2 DMTD 1 .0 0.5
-CH2CH2NH2 DMTD 0. 125 0.25
-CH2CH2NH2 C6H4OC8H17 0.5 0.5
The following examples illustrate the
invention but are not to be construed as limiting:
Example 1
1-[4-Hydroxy-5-aminoethyloxy-N2-(4-octyloxybenzoyl)- ornithine]-5-(3-hydroxyornithine)pneumocandin BO
Dihydrochloride (Seq ID No 1)
N-Benzyloxycarbonylaminoethyl Ether (Seq ID No 8)
A solution of 3.5 grams (3.31 mmol) of
1-[4,5-dihydroxy-N2-(4-octyloxybenzoyl)ornithine]-pneumocandin BO, 16.13 grams (82.7 mmol) 2-(N-benzyloxycarbonylamino)ethanol and 768 milligrams (3.31 mmol) of (lS)-(+)-10-camphorsulfonic acid in 120 mL of anhydrous dioxane and 12 mL of anhydrous
N,N-dimethylformamide was stirred at 25ºC. The reaction was monitored by analytical HLPC using
"ZORBAX" RX-C18 column and a solvent system of 55% CH3CN/H2O at a flow rate of 1.5 mL/min with UV
detection at 210 and 277 nm. After about 20 hours >95 percent conversion to product (tR = 3.76 min) was noted. The reaction mixture was neutralized by the addition of 3.5 mL of 1M NaHCO3 and then diluted with 135 mL of H2O. The resulting solution was filtered and the filtrate pump-injected onto a "DELTA-PAK" C18 cartridge column (47 mm × 30 cm) and eluted using step gradient (45-55% CH3CN/H2O) elution at a flow rate of 50 mL/min. The appropriate fractions were combined, diluted with 250 milliliters of water and the product recovered by solid-phase extraction using the same column in 5% CH3CN/H2O. The extracted material was pump-injected onto the column, then eluted with 95% CH3CN/H2O, the product containing eluates pooled, concentrated and lyophilized to obtain the
N-benzyloxycarbonylaminoethyl ether intermediate in a yield of 2.3 grams (56%) as a white amorphous solid: HPLC assay at 210nm >97% product. Mass spectrum: (FAB) 1242.7 (M+Li)+
Part B
Aminoethyl Ether(R3=HCl●NH2CH2CH2-) (Seq ID No 8)
A solution of 2.30 grams (1.86 mmol) of the N-benzyloxycarbonylaminoethyl ether prepared in Part A in 25 milliliters of acetic acid was hydrogenated under balloon pressure in the presence of 1.80g 10% Pd/C for a period of 1.5 hours. The reaction was monitored by analytical HPLC and a solvent system of 35% CH3CN/H2O at a flow rate of 1.5 mL/min with UV detection 210 and 277 nm. Complete conversion to a product peak at
tR=5.46 min. was observed. The reaction mixture was filtered to remove the catalyst and the filtrate was lyophilized to obtain the aminoethyl ether as the acetate salt. The lyophilizate was dissolved in a minimum volume of H2O and the solution was passed through a column of anion exchange resin (Bio-Rad AG2-X8(C1-)) and the eluate was lyophilized to obtain 2.0 grams (95% yield) of the hydrochloride as a white amorphous solid.
Mass spectrum (FAB) 1109.0 (M+Li)+
Step (2)
Nitrile (Seq ID No 15)
27 milligrams (0.147 mmol) of cyanuric chloride was added to a solution of 100 mg (0.0879 mmol) of the aminoethyl ether prepared in Part B in 0.7 mL of anhydrous DMF at 0°C. The reaction was monitored by analytical HPLC using a solvent system of 35 percent CH3CN/H2O at a flow rate of 1.5 mL/min with UV
detection at 210 and 277nm. After 3.5 hours, a 65% conversion to a product peak (tR=7.97 min) was
observed. 0.672 mL of cold 1M sodium acetate was added to the reaction mixture which was further diluted to 4.5 mL with water. The mixture then was filtered and the filtrate subjected to preparative HPLC on "ZORBAX" RX-C18 with 30% CH3CN/H2O at 10 mL/min and detection at 220 nm. The appropriate fractions were combined and lyophilized to obtain 46 mg (38 percent) of the nitrile compound as the trifluoroacetate salt.
HPLC purity at 210 nm >98%.
Step (3)
Bisamine Product Formula (Seq ID No 1)
49.4 mg (1.31 mmol) of sodium borohydride was added in portions to a solution of 62.2 mg (0.261 mmol) of CoCl2●6H2O and 46 mg (0.0384 mmol) of the nitrile in 3 milliliters of methanol under nitrogen. An
exothermic reaction took place with evolution of hydrogen. HPLC analysis after 1 hour indicated
complete conversion to a more polar product (tR=3.83 min). The reaction mixture was acidified to pH 3 and diluted tenfold with water and then subjected to reverse-phase flash chromatography LICHROPREP eluting with 5-10% CH3CN/H2O, then the appropriate fractions combined and lyophilized to obtain the desired product I-A in a yield of 47 percent (Compound I-la)(Seq ID No 1) ["I-1a "as employed refers to "I-1" nucleus and "a" as the first product hearing said nucleus).
Mass Spectrum: FAB 1094.7 (MLi)+
Example 2
1-[4-Hydroxy-5-aminoethyloxy-N2-(6-octyloxynaphthoyl)-ornithine]-5-(3-hydroxyornithine)pneumocandin BO Dihydrochloride (Seq ID No 1)
Preparation of Starting Cyclopeptide Derivative
Pentafluorophenyl 6-octyloxy-2-naphthoate for acylating the cyclopeptide was first prepared in the following manner: To a suspension of 6-octyloxy-2-naphthoic acid (3.15 g, 10.5 mmol) and dicyclohexylcarbodiimide in ethyl acetate (25 mL) at 0°C was added pentafluorophenol (2.12 g, 11.5 mmol). The mixture was stirred at 25°C for a period of 18 hours. The precipitate was removed by filtration. The filtrate was washed with water (2 × 150 mL) and brine and dried with magnesium sulfate. Removal of the ethyl acetate in vacuo yielded 5.4 g of pentafluorophenyl
6-octyloxy-2-naphthoate as a yellow solid: 1H NMR (400 MHz, CD3OD) δ 0.88 (t, 3, J=6.9 Hz), 4.10 (t, 2, J=6.6 Hz), 7.16 (d, 1), 7.21 (d, 1), 7.80 (d, 1), 7.87 (d, 1), 8.08 (dd, 1), 8.69 (d, 1).
2.0 grams (4.29 mmol) of pentafluorophenyl 6-octyloxy-2-naphthoate, thus prepared, was added to 2.0 grams of 33 percent by weight (0.799 mmol) of
1-(4,5-dihydroxyornithine)pneumocandin B0 in 50 milliliters of anhydrous DMF. HPLC analysis, eluting with 45% CH3CN/H2O at 1.5 mL/min and detection at 210 and 277 nm showed conversion to product peak at tR=5.90 min after 18 hours. The DMF was removed in vacuo and the residue triturated with two 100 milliliter portions of diethyl ether. Reverse-phase flash chromatography of the triturate eluting with 40-45% CH3CN/H2O and then lyophilizing the appropriate fractions produced 340 milligrams of 1-[4,5-dihydroxy-N2-(6-octyloxy-2-naphthoyl)ornithine]pneumocandin B0 (80% purity). A 40 mg portion was rechromatographed on "ZORBAX" with 43 percent CH3CN/H2O at 10 ml/min, uv detection at 220 nm to obtain 30 mg of N-acylated compound of >98 percent purity.
Mass spectrum: FAB 1116.0 (M+Li)+
Step (1)
Part A
N-Benzyloxycarbonylaminoethyl Ether (Seq ID No 8)
To a solution of 300 mg (80% purity, 0.271 mmol) of the 6-octyloxy-2-naphthoyl starting material above prepared, 1.4 grams (7.18 mmol) of 2-(N-benzyloxycarbonylamino)ethanol and 62.9 mg (0.271 mmol)
of (lS)-(+)-10-camphorsulfonic acid in 10 ml anhydrous dioxane and 1 mL anhydrous DMF was stirred at 25°C. HPLC analysis using 65% CH3CN/H2O at 1.5 mL/min with detection at 210 and 277 nm indicated after 18 hours a greater than 95 percent conversion to a less polar product (tR=3.36 min). The reaction was quenched with 1M NaHCO3, the reaction mixture diluted with water and subject to reverse-phase flash chromatography eluting with 40-60 percent CH3CN/H2O in 10% step gradients to obtain after lyophilization of the 60 percent
fractions, ether compound (R1, R2 and R4 = OH, R5 = H, R6 = CH3 and RI = C10H6OC8H17) as a white amorphous solid. HPLC assay: 60% CH3CN/H2O tR=4.30 min. 70% pure
Part B
Aminoethyl Ether (Seq ID No 8)
In a manner similar to that described in Example I, a solution of 180 mg (70% purity, 0.140 mmol) in 10 ml of acetic acid was hydrogenated under balloon pressure in the presence of 160 mg 10% Pd/C for a period of 1.5 hours. The reaction was monitored by analytical HPLC using 40% CH3CN/H2O at a flow rate 1.5 mL/min with detection at 210 and 277 nm. Conversion (>95%) to a more polar product peak (tR=6.43 min) was observed after 1 hour. The reaction mixture was filtered to remove catalyst and the filtrate
concentrated in vacuo. Preparative HPLC of the filtrate using "DELTAPAK" at 10 mL/min, followed by lyophilization of the appropriately combined eluates produced 30 mg of 99% pure and 80 mg of 90% pure aminoethyl ether as the trifluoroacetate salt as a
white amorphous solid. The 90 percent pure material was rechromatographed on "ZORBAX" and eluted with
30-35% CH3CN/H2O with detection at 220 nm to obtain another 35 mg of product of >98% purity.
Total yield 58%.
Mass spectrum: (FAB) 1158.5 (M+Li)+
Step (2)
Nitrile (Seq ID No 15)
Cyanuric chloride is added to a solution of the trifluoroacetate salt of the aminoethyl ether prepared as above described in anhydrous DMF at 0°C. The reaction is monitored by analytical HPLC using a "ZORBAX" RX-C18 column and a solvent system of 35% CH3CN/H2O at a flow rate of 1.5 mL/min with uv
detection 210 and 277 nm. After 3.5 hours conversion to a less polar product is observed. Cold 1M NaOAc (ca. equal volume) is added to the reaction mixture which is further diluted three-fold with H2O. The reaction mixture is filtered. Preparative HPLC of the filtrate "ZORBAX" C-18 (4.6 mm × 25 cm) 30% CH3CN/H2O at 10 mL/min; uv detection at 220 nm) followed by lyophilization of the appropriate fractions produces the nitrile as the trifluoroacetate salt:
C57H76F3N9O19(mw=1248.3).
Step (3)
Bisamine Product (Seq ID No 1)
In a manner similar to that described in Example I, Step (3), sodium borohydride is added in
portions to a solution of CoCl2●6H2O and the nitrile prepared as described above in methanol under an atmosphere of nitrogen. The reaction progress is followed with analytical HPLC and after one hour there is indication of conversion to a more polar product. The mixture is acidified to pH 3 by the addition of 1 N HCl and is diluted tenfold with water. The solution is subjected to reverse-phase flash chromatography, eluting with 5-10% CH3CN/H2O, then the appropriate fractions combined and lyophilized to obtain the amine product as the dihydrochloride: C55H81Cl2N9O17
(mw=1211.2)
Example 3
Part A
In a manner similar to that described in Example I, a solution of 1-[4,5-dihydroxy-N2 (10, 12-dimethylmyristoyl)ornithine]pneumocandin BO,
2-(N-benzyloxycarbonylamino)ethanol and
(IS)-(+)-10-camphorsulfonic acid were stirred at ambient temperature to obtain 1-[4-hydroxy-5-(2-N-benzyloxycarbonylamino)ethyloxy)ornithine-pneumocandin BO (Seq ID No 8) in a yield of 60 percent. 1H NMR (400 MHz, CD3OD) δ 1.15 (d, 3, J=6.2 Hz,
CH3-threo), 2.48 (dd, 1, J=9.5 and 15.4 Hz, H4a-glu), 2.80 (dd, 1, J=3.6 and 15.4 Hz, H4b-glu), 3.49-3.64 (m, 2, OCH2CH2NHZ), 3.78 (m, OCH2CH2NHZ), 4.08 (m, 1, H4-orn), 4.44 (dd, 1, J=6.7 and 10.7 Hz, H2-orn), 4.98 (d, 1, J=3.6 Hz, H2-threo), 5.06 (s, 2, CH2C6H5), 5.08 (d, 1, J=4.0 Hz, H2-glu), 5.16 (d, 2, J=l .8 Hz,
H5-orn), 6.74 (d, 2, J=8.6 Hz, H3 and H5-ArH), 7.13 (d, 2, J=8.6 Hz, H2 and H6-ArH), 7.33 (m, 5, CH2C6H5).
Part B
In a similar manner, a solution of N-benzoylcarbonylaminoethyl ether above prepared is hydrogenated to obtain 1-(4-Hydroxy-5-aminoethyloxy-ornithine)-pneumocandin BO trifluoroacetate (Seq ID No 8) in 90 percent yield.
1H NMR (400 MHz, CD3OD) δ 1.16 (d, 3, J=6.2 Hz,
CH3-threo), 3.12 (m, 2, OCH2CH2NH2●CF3COOH), 3.72 (m, OCH2CH2NH2●CF3COOH), 4.10 (m, 1, H4-orn), 5.24 (d, 1, J=2.3 Hz, H5-orn)
Mass Spectrum: FAB 1108 (MH)+
Anal. Calcd for C52H86ClN9O17: C, 54.56; H, 7.57; N, 11.01; Cl, 3.10. Found: C, 54.28; H, 7.60; N, 10.88; Cl, 3.02.
Step (2)
The aminoethyl ether prepared in Part B of Step 1 is reacted with cyanuric chloride to obtain 1-(4-hydroxy-5-aminoethyloxy-ornithine)-5-(4-cyano-threonine)-pneumocandin BO (Seq ID No 15)
trifluoroacetate in 40% yield.
1H NMR (400 MHz, CD3OD) δ 1.21 (d, 3, J=6.2 Hz,
CH3-threo), 2.73 (dd, 1, J=7.9 and 17.1 Hz,
H4a-(4-cyano-threo)), 2.83 (dd, 1, J=3.8 and 17.1 Hz, H4b-(4-cyano-threo)), 3.13 (m, 2, CH2NH2●CF3COOH) , 4.10 (ddd; 1; J=2.1, 5.5 and 10.3 Hz; H4-orn), 4.46 (dd, 1, J=4.5 and 12.9 Hz, H2-orn), 5.02 (d, 1, J=3.3 Hz, H2-threo), 5.05 (d, 1, J=4.8 Hz, H2-(4-cyano-threo)), 5.32 (d, 1, J=2.1 Hz, H5-orn), 6.76 (d, 2, J=8.6 Hz, H3 and H5-ArH), 7.14 (d, 2, J=8.6 Hz, H2 and H6-ArH) FAB MS (Li) m/z 1097.2 (MLi)+
Step (3)
Under an atmosphere of nitrogen, sodium borohydride is added in portions to a solution in methanol of CoCl2●CH2O and the nitrile prepared in Step 2 to obtain 1-(4-hydroxy-5-aminoethyloxy-ornithine)-5-(3-hydroxy-ornithine)-pneumocandin Bo (Seq ID No 1) dihydrochloride in a yield of 40%
Mass spectrum (FAB): 1101 (M+Li)
lH NMR (400 MHz, CD3OD): δ 7.12 (d, 2H), 6.77 (d, 2H), 5.18 (d, 1H), 3.14 (t, 2H), 3.09 (t, 2H).
Examples 4-10
In operations carried out in a manner similar to those employed in the preceding examples, the following compounds where R1 is C6H4OC8H17 are prepared
SEQ ID NOS
EXAMPLE R1 R2 R4 R 5 R6 ETHER NITRILE PRODUCT
4 OH OH OH H CH3 CH3NHCH2CH2- 8 15 1
5 OH OH OH CH3 CH3 (CH3)2NCH2CH2- 9 16 2
6 H OH OH CH3 H H2NCH2CH2CH2- 10 17 3
7 OH H OH CH3 CH3 H2NCH2CH2- 11 18 4
8 H H H CH3 CH3 C6H5CH2HNCH2CH2- 12 19 5
9 OH OH OH OH CH3 (CH2)5NCH2CH2- 13 20 6
10 H OH OH H H C6H5CH2HN(CH2)3- 14 21 7
Examples 11-17
In still other operations carried out in a manner similar to that described in the preceding examples, the following compounds where RI is
C10H6OC3H17 are prepared.
SEQ ID NOS
EXAMPLE R1 R2 R4 R5 R6 R3 ETHER NITRILE PRODUCT
11 OH OH OH H CH3 CH3NHCH2CH2- 8 15 1
12 OH OH OH CH3 CH3 H2N(CH2)4- 9 16 2
13 H OH OH CH3 H (CH3)2NCH2CH2 10 17 3
14 OH H OH CH3 CH3 (CH2)4NCH2CH2- 11 18 4
15 H H H CH3 CH3 C2H5NHCH2CH2- 12 19 5
16 OH OH OH OH CH3 C6H5CH2NH(CH2)2- 13 20 6
17 H OH OH H H H2N(CH2)3- 14 21 7
Example 18
From the aminoethyl ether obtained in a manner described in Example 3, Step (1), Part B, the following substituted aminoethyl ethers were prepared which then may be used to be dehydrated to the nitrile and thereafter reduced to the bisamine product. All of the following compounds have Seq ID No. 8.
(a) 1-(4-Hydroxy-5-dimethylaminoethyloxy-ornithine)- pneumocandin BO Trifluoroacetate
(R3 = CH2CH2N(CH3)2●CF3COOH) To a stirred solution of 1-(4-hydroxy-5-aminoethyloxy-ornithine)-pneumocandin BO trifluoroacetate (Example 3, Part B; 400 mg, 0.327 mmol) and 530 μL (6.54 mmol) of 37% aqueous formaldehyde in 20 mL of acetonitrile and 10 mL of water was added 62 mg (0.987 mmol) of sodium cyanoborohydride. The reaction mixture was stirred for 15 min and then glacial acetic acid was added to pH 7. The solution was diluted with water (2X) and chromatographed. Reverse-phase (C18) flash column chromatography eluting with 30-507.
acetonitrile/water (1% acetic acid) was followed by lyophilization of the product-containing fractions to provide 355 mg of impure 1-(4-hydroxy-5-dimethylaminoethyloxy-ornithine)-pneumocandin BO acetate.
Preparative reverse-phase HPLC (C18) of this material eluting with 40% acetonitrile/water (0.1% trifluoroacetic acid) provided clean dimethylaminoethyl ether as the trifluoroacetate salt:
FAB-MS (Li), m/z 1143.1 (MLi)+, 1053.9
(b) 1-(4-Hydroxy-5-trimethylaminoethyloxy-ornithine)- pneumocandin BO Iodide
(R3=CH2CH2 +N(CH3)3 I-)
To a stirred solution of 1-(4-hydroxy-5-dimethylaminoethyloxy-ornithine)-pneumocandin BO acetate (Example 3, Part B; 150 mg, 0.125 mmol) in N,
N-dimethylformamide (10 mL) and 1M sodium bicarbonate (2 mL, 2 mmol) was added iodomethane (2 mL, 32.1 mmol). The reaction mixture was stirred for a period
of 18 hours. The mixture was diluted with water (2X) and chromatographed. Reverse-phase (C18) flash column chromatography eluting with 30-50% acetonitrile/water was followed by lyophilization of the
product-containing fractions to provide 83 mg (52%) of
1-(4-hydroxy-5-trimethylaminoethyloxy-ornithine)-pneumocandin BO iodide:
1H NMR (400 MHz, CD3OD) δ 1.18 (d, 3, CH3-threo), 3.19
(m, 11, OCH2CH2N+(CH3)3), 3.58 (m, 2, OCH2), 4.98
(d, 1, H2-threo), 5.08 (d, 1, H2-glu), 5.29 (d, 1,
H5-orn), 6.75 (d, 2, H3 and H5-ArH), 7.13 (d, 2, H2 and
H6-ArH)
FAB-MS (Li), m/z 1150.8 (M+)
(c) 1-(4-Hydroxy-5-guanidinoethyloxy-ornithine)- pneumocandin BO Acetate
(R3=CH2CH2NH-C(=NH)NH2●CH3COCH) To a stirred solution of 1-(4-hydroxy-5-aminoethyloxy-ornithine)-pneumocandin BO acetate
(Example 3, Part B; 160 mg, 0.137 mmol) and 1M sodium bicarbonate (150 μL, 0.150 mmol) in absolute methanol (5 mL) was added aminoiminomethanesulfonic acid (30 mg, 0.242 mmol). After a period of 1.5 hours, the solvent was removed in vacuo. Preparative reverse-phase HPLC (C18) of the residue, eluting with 45%
acetonitrile/water (0.1% acetic acid), was following by lyophilization of the product-containing fractions to give 1-(4-hydroxy-5-guanidinoethyloxy-ornithine)-pneumocandin BO acetate (100 mg, 60%):
FAB MS (Li), m/z 1053.9
(d) 1-(4-Hydroxy-5-acetamidinoethyloxy-ornithine)- pneumocandin BO Acetate
(R3=CH2CH2NH(C=NH)CH3●CH3COOH) To a stirred solution of 0-(4-hydroxy-5-aminoethyloxy-ornithine)-pneumocandin BO acetate
(Example 3, Part B; 150 mg, 0.128 mmol) and 1N sodium hydroxide (130 μL, 0.130 mmol) in water (5 mL) and N,N-dimethylformamide (5 mL) was added ethylacetimidate hydrochloride (160 mg, 1.29 mmol). After a period of 18 hours at pH 8.5, glacial acetic acid was added to pH 7. Reverse-phase (C18) flash column chromatography of the neutralized reaction mixture, eluting with
acetonitrile/water, was followed by lyophilization of the product-containing fractions. Preparative
reverse-phase (C18) HPLC of this material, eluting with acetonitrile/water (0.1% acetic acid), was followed by lyophilization of the product-containing fractions to give 1-(4-hydroxy-5-acetamidinoethyloxy-ornithine)-pneumocandin BO acetate (26 mg, 17%):
1H NMR (400 MHz, CD3OD) δ 1.17 (d, 3), 2.23 (s, 3), 3.42 (m, 2), 3.69 (m, 2), 4.06 (m, 1), 4.98 (dd, 1), 5.08 (dd, 1), 5.22 (d, 1)
FAB MS(Li), m/z 1053.6
(e) 1-(4-Hydroxy-5-(N-benzyloxycarbonylglycyl)-aminoethyloxy-ornithine)-pneumocandin BO
(R3=CH2CH2NHCOCH2NHCOOCH2C6H5) 1-(4-Hydroxy-5-aminoethyloxy-ornithine)-pneumocandin BO acetate (Example 3, Part B; 210 mg; 0.180 mmol) was dissolved in N,N-dimethylformamide (2 mL). To this solution 1M sodium bicarbonate (200 μl, .200
mmol) and pentafluorophenyl N-benzyloxycarbonylglycinate (106 mg, .270 mmol) were added. After 1 h, the reaction mixture was diluted with water (2X).
Isolation by reverse-phase (C18) flash column
chromatography eluting with 50-80% acetonitrile/water gave, after lyophilization of the product-containing fractions, 1-(4-hydroxy-5-(N-benzyloxy-carbonylglycyl)-aminoethyloxy-ornithine)-pneumocandin BO (130 mg, 56%):
FAB-MS (Li), m/z 1306.4 (MLi)+
(g) 1-(4-Hydroxy-5-glycylaminoethyloxy-ornithine)-pneumocandin BO Trifluoroacetate
(R3=CH2CH2NHCOCH2NH3●CF3COOH) This compound was obtained from 1-(4-hydroxy-5-(N-benzyloxycarbonylglycyl)-aminoethyloxy-ornithine)-pneumocandin BO in a manner similar to that described in (Example 3, Part B):
Yield = 49%
FAB-MS (Li), m/z 1171.7 (MLi)+
(g) 1-(4-Hydroxy-5-aminoethyloxy-ornithine)-4-(3- hydroxyhomotyrosine)-pneumocandin BO Trifluoroacetate
(R3=CH2CH2NH2●CF3COOH)
1-(4-Hydroxy-5-aminoethyloxy-ornithine)-pneumocandin BO acetate (250 mg, 0.214 mmol) was dissolved in anhydrous trifluoroacetic acid (5 mL) under an atmosphere of nitrogen. Sodium cyanoborohydride (67 mg, 1.07 mmol) was added immediately resulting in a vigorous gas evolution. After 2 min, the reaction mixture was diluted with water (100 mL) producing a
precipitate. The mixture was filtered. The filter cake was dissolved in methanol and the solvent was removed in vacuo. Preparative reverse-phase (C18) HPLC of the residue, eluting with 40% acetonitrile/water (0.1% CF3COOH), followed by lyophilization of the product-containing fractions to give 1-(4-hydroxy-5-aminoethyloxy-ornithine)-4-(3-hydroxyhomotyrosine)-pneumocandin BO trifluoroacetate (75 mg, 29%):
FAB-MS (Li), m/z 1099.1 (MLi)+
Example 19
In a preparation carried out as described in Examples 1-3, Step 1 and Example 18 the following compounds were prepared.
CBz
(a) R = -CH2CH2NCH2CH2NHCBz
FAB-MS (Li), m/z 1426.1 (MLi)+
(b) R = -CH2CH2NHCH2CH2NH2●2CF3COOH
FAB-MS (Li), m/z 1157.7 (MLi)+
(c) R = -CH2CH2NHCH2CH2NHC(=NH)NH2●2CF3COOH
FAB-MS(Li), m/z 1193.7 (M+)
Example 20
The above compound is obtained in a manner similar to that described in Example I.
Step (1), Part A:
1-[4-Hydroxy-5-(2S,3-(di-N-benzyloxycarbonylamino)propyloxy)ornithine]-pneumocandin BO
C69H100N10O21 mw = 1405.6
Step (1), Part B:
1-[4-Hydroxy-5-(2S,3-diaminopropyloxy)- ornithine]-pneumocandin BO ditrifluoroacetate FAB MS (Li) m/z 1143.7 (MLi)+
Step 2:
1-[4-Hydroxy-5-(2S,3-diaminopropyloxy)- ornithine]-5-(4-cyano-threonine)-pneumocandin BO trifluoroacetate
C53H86N10O16 mw = 1119.3
Step 3:
1-[4-Hydroxy-5-(2S,3-diaminopropyloxy)- ornithine]-5-(3-hydroxy-ornithine)-pneumocandin BO (tris)-trifluoroacetate FAB MS (Li) m/z 1130.0 (MLi)+
EXAMPLE 21
Step (1), Part A:
1-[4-Hydroxy-5-(2R,3-(di-N-benzyloxycarbonyl- amino)propyloxy)ornithine]-pneumocandin BO
C69H100N10O21 mw = 1405.6
Step (1), Part B:
1-[4-Hydroxy-5-(2R,3-diaminopropyloxy)- ornithine]-pneumocandin BO ditrifluoroacetate
FAB MS (Li) m/z 1143.8 (MLi)+
Step 2:
1-[4-Hydroxy-5-(2R,3-diaminopropyloxy)- ornithine]-5-(4-cyano-threonine)-pneumocandin BO trifluoroacetate
C53H86N10O16 mw = 1119.3
Step 3:
1-[4-Hydroxy-5-(2R,3-diaminopropyloxy)- ornithine]-5-(3-hydroxy-ornithine)-pneumocandin BO (tris)-trifluoroacetate
FAB MS (Li) m/z 1130.0 (MLi)+
Preparation of Cyclopeptide Starting Materials:
The starting materials for the compounds are natural products or derivatives of natural products.
The following compounds are natural products produced by cultivating an appropriate organism in nutrient medium as hereinafter described.
X-1 may be produced by cultivating Zalerion arboricola ATCC 20868 in a nutrient medium enriched in mannitol as the primary source of carbon as described in U.S. Patent No. 5,021,341, June 4, 1991.
X-2 may be produced by cultivating Zalerion arboricola ATCC 20868 in nutrient medium as described in U.S. 4,931,352, June 5, 1990 or in nutrient medium enriched in glycerol as described in U.S. 4,968,608, November 6, 1980.
X-2 nucleus with a different RI may be produced by cultivating Acrophialophora limonispora in nutrient medium as described in U.S. 4,173,629 and thereafter acylating employing RICOCl or an activated ester of RICOOH.
X-3 and X-7 may be produced by cultivating Cryptosporiopsis ATCC 20594 in nutrient medium as described by Pache et al in 13th ICC (1983), PS 4.8/3, Part 115, Abstract No. 10 and PCT WO 82/00587.
X-4, X-5 and X-6 may be produced by cultivating Zalerion arboricola ATCC 20868 in nutrient medium.
X-8, when R1 is H, R2 and R4 are OH, R5 is H or CH3 and R5 is CH3, the compound is not a natural product. It may be prepared by reducing a compound in which R1 is OH and the other R5 are the same by methods known to the skilled in the art. This may be carried out by adding TFA and triacetoxyborohydride and mixing together until a clear solution is obtained, pouring the solution into water and recovering the product which precipates therein and purifying by HPLC.
Starting materials in which RI is a different group from that of the natural product may be obtained by deacylating the lipophilic group of the natural
product by subjecting the natural product in a nutrient medium to a deacylating enzyme until substantial deacylation occurs, said enzyme having first been obtained by cultivating a microorganism of the family Pseudomondaceae or Actinoplanaceae, as also described in Experentia 34, 1670 (1978) or U.S. 4,293,482, and thereafter recovering the deacylated cyclopeptide, and acylating the deacylated cyclopeptide by mixing together with an appropriate active ester RICOX to obtain Compound E with the desired acyl group using conventional procedures. Methods are also described in U.S. 4,287,120 and 4,293,489.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: BOUFFARD, FRANCES AILEEN
DROPINSKI, JAMES F.
(ii) TITLE OF INVENTION: AN IMPROVED PROCESS FOR
CYCLOHEXAPEPTIDYL BISAMINE COMPOUNDS
(iii) NUMBER OF SEQUENCES: 24
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MERCK & CO., INC.
(B) STREET: P.O. BOX 200, EAST LINCOLN AVE.
(C) CITY: RAHWAY
(D) STATE: NEW JERSEY
(E) COUNTRY: USA
(F) ZIP: 07065
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette - 5.25 inch, 360Kb
(B) COMPUTER: WANG PC 381
(C) OPERATING SYSTEM: MD-DOS 3.30.10
(D) SOFTWARE: Wang Integrated Word Processing (vi) CURRENT APPLICATION DATE:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA: NONE
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: ALICE O. ROBERTSON
(B) REGISTRATION NUMBER: 18,525
(C) REFERENCE/DOCKET NUMBER: 18850
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908-594-4372
(B) TELEFAX: 908-594-4720
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO : 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS : NA
(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 Xaa Ser Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 Xaa Ser Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9
Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 Xaa Ser Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14 Xaa Ser Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17 Xaa Ser Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO : 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21 Xaa Ser Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23 Xaa Thr Xaa Xaa Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6
(B) TYPE: AMINO ACID
(C) STRANDEDNESS: NA
(D) TOPOLOGY: CIRCULAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24 Xaa Thr Xaa Xaa Xaa Xaa
1 5
Claims
1. An improved process for the preparation of a cyclopeptidyl amine compound having the formula
wherein
R 1 is H or OH
R2 is H or OH
R3 is CnH2nNRllRlII, CnH2n +NRIIRIIIRIVY-,
-(CH2)2-4NHRV, -CH2CH(NH2)CH2NH2
R4 is H or OH
R5 is H, OH or CH3
R6 is H or CH3
RI is C9-C-21 alkyl, C9-C21 alkenyl,
C1-C10 alkoxyphenyl, or
C1-C10 alkoxynaphthyl RII is H, C1-C4 alkyl or benzyl
RIII is H, C1-C4 alkyl, benzyl
or RII and RIII together are -(CH2)4- or -(CH2)5- RIV is H or C1-C4 alkyl
RV is (C=NH)NH2, (C=NH)(CH2)0-3H, (CH2)2-4NH2
CO(CH2)1-3NH2, (CH2)2-4NH(C=NH)NH2
Y is an anion of a pharmaceutically acceptable salt, and n is an integer of from 2 to 4,
inclusive which comprises
(a) preparing an aminoalkyl ether derivative (Y) of the cyclohexapeptide compound (X)
to obtain an aminoalkyl ether derivative of the formula
(b) dehydrating the ether derivative so obtained to obtain a nitrile compound (Z) of the formula
and
(c) reducing the nitrile compound.
2. A process according to Claim 1 wherein the aminoalkyl ether derivative is
(i) an unsubstituted aminoalkyl ether and which is prepared by reacting Compound X with a protected aminoalkanol followed by hydrogenation of the protecting group,
(ii) a substituted or unsubstituted
aminoalkyl ether which is prepared by reacting Compound X directly with the appropriate aminoalkanol (iii) a substituted aminoalkyl ether which is prepared by reacting the
unsubstituted aminoalkyl
ether prepared according to (i) or (ii) above with a derivatizing agent.
3. An improved process for preparing a
cyclohexapeptidyl amine compound having the formula
wherein R5, RI and n are as defined in Claim 1
which comprises
by (b)
(i) reacting said cyclohexapeptide with
an N-protected aminoalkanol, and
hydrogenating in the presence of 10% Pd/C to remove the protecting group or
(ii) directly reacting said cyclohexapepdide
(c) dehydrating the compound so obtained by reacting said compound with cyanuric chloride under anhydrous conditions to obtain a nitrile compound of the formula
(d) reducing the nitrile compound,
4. A compound having the formulm
wherein
R3 is CnH2nNRII RIII, CH2CH(NH2)CH2NH2
RI is C9-C-21 alkyl, C9-C-21alkenyl,
C1-C10 alkoxyphenyl, or
C1-C10 alkoxynaphthyl
RII is H, C1-C4alkyl or benzyl
RIII is H, C1-C4alkyl benzyl
or RII and RIII together -(CH2)4- or -(CH2)5-; and
n is an integer of from 2 to 4, inclusive.
5. A compound according to Claim 4 wherein RI is -C6H4OC8H17.
6. A compound according to Claim 4 wherein RI is -C10H6OC8H17.
7. A compound according to Claim 4 wherein RI is 9, 11-dimethyltridecyl.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU53540/94A AU5354094A (en) | 1992-10-16 | 1993-10-07 | An improved process for cyclohexapeptidyl bisamine compounds |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US96098392A | 1992-10-16 | 1992-10-16 | |
US960,983 | 1992-10-16 |
Publications (1)
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WO1994009033A1 true WO1994009033A1 (en) | 1994-04-28 |
Family
ID=25503915
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1993/009606 WO1994009033A1 (en) | 1992-10-16 | 1993-10-07 | An improved process for cyclohexapeptidyl bisamine compounds |
Country Status (2)
Country | Link |
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AU (1) | AU5354094A (en) |
WO (1) | WO1994009033A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0779895A1 (en) * | 1994-08-23 | 1997-06-25 | Merck & Co. Inc. | An improved process for preparing side chain derivatives of cyclohexapeptidyl lipopeptides |
US5854212A (en) * | 1996-10-23 | 1998-12-29 | Merck & Co., Inc. | Cyclohexapeptidyl bisamine compound, compositions containing said compound and methods of use |
EP1785432A1 (en) * | 2005-11-15 | 2007-05-16 | Sandoz AG | Process and intermediates for the synthesis of caspofungin. |
WO2010008493A2 (en) * | 2008-06-25 | 2010-01-21 | Teva Gyógyszergyár Zártkörüen Müködö Részvénytársaság | Processes for preparing high purity aza cyclohexapeptides |
WO2016201283A1 (en) * | 2015-06-12 | 2016-12-15 | Cidara Therapeutics, Inc. | Antifungal agents |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5874403A (en) * | 1992-10-15 | 1999-02-23 | Merck & Co., Inc. | Amino acid conjugates of cyclohexapeptidyl amines |
AU681119B2 (en) * | 1993-05-17 | 1997-08-21 | Fujisawa Pharmaceutical Co., Ltd. | New polypeptide compound and a process for preparation thereof |
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BE851310A (en) * | 1976-02-12 | 1977-08-10 | Sandoz Sa | NEW DERIVATIVES OF TETRAHYDRO-EQUINOCANDIN B |
BE859067A (en) * | 1976-09-28 | 1978-03-28 | Sandoz Sa | NEW PEPTIDIC AMINO-ETHERS, THEIR PREPARATION AND THEIR APPLICATION AS MEDICINAL PRODUCTS |
GB2065130A (en) * | 1979-12-13 | 1981-06-24 | Lilly Co Eli | Recovery process for a-30912 antibiotics |
US5159059A (en) * | 1990-05-29 | 1992-10-27 | Merck & Co., Inc. | Process for reduction of certain cyclohexapeptide compounds |
US5166135A (en) * | 1988-09-12 | 1992-11-24 | Merck & Company, Inc. | Method for the control of pneumocystis carinii |
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1993
- 1993-10-07 WO PCT/US1993/009606 patent/WO1994009033A1/en active Application Filing
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BE851310A (en) * | 1976-02-12 | 1977-08-10 | Sandoz Sa | NEW DERIVATIVES OF TETRAHYDRO-EQUINOCANDIN B |
BE859067A (en) * | 1976-09-28 | 1978-03-28 | Sandoz Sa | NEW PEPTIDIC AMINO-ETHERS, THEIR PREPARATION AND THEIR APPLICATION AS MEDICINAL PRODUCTS |
GB2065130A (en) * | 1979-12-13 | 1981-06-24 | Lilly Co Eli | Recovery process for a-30912 antibiotics |
US5166135A (en) * | 1988-09-12 | 1992-11-24 | Merck & Company, Inc. | Method for the control of pneumocystis carinii |
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Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0779895A1 (en) * | 1994-08-23 | 1997-06-25 | Merck & Co. Inc. | An improved process for preparing side chain derivatives of cyclohexapeptidyl lipopeptides |
EP0779895A4 (en) * | 1994-08-23 | 1997-10-29 | Merck & Co Inc | An improved process for preparing side chain derivatives of cyclohexapeptidyl lipopeptides |
US5854212A (en) * | 1996-10-23 | 1998-12-29 | Merck & Co., Inc. | Cyclohexapeptidyl bisamine compound, compositions containing said compound and methods of use |
EP1785432A1 (en) * | 2005-11-15 | 2007-05-16 | Sandoz AG | Process and intermediates for the synthesis of caspofungin. |
WO2007057141A1 (en) * | 2005-11-15 | 2007-05-24 | Sandoz Ag | Process and intermediates for the synthesis of caspofungin |
JP2009515917A (en) * | 2005-11-15 | 2009-04-16 | サンド・アクチエンゲゼルシヤフト | Methods and intermediates for the synthesis of caspofungin |
CN101305018B (en) * | 2005-11-15 | 2013-03-27 | 桑多斯股份公司 | Process and intermediates for the synthesis of caspofungin |
KR101342304B1 (en) | 2005-11-15 | 2013-12-16 | 산도즈 아게 | Process and intermediates for the synthesis of caspofungin |
WO2010008493A2 (en) * | 2008-06-25 | 2010-01-21 | Teva Gyógyszergyár Zártkörüen Müködö Részvénytársaság | Processes for preparing high purity aza cyclohexapeptides |
WO2010008493A3 (en) * | 2008-06-25 | 2010-03-11 | Teva Gyógyszergyár Zártkörüen Müködö Részvénytársaság | Processes for preparing high purity aza cyclohexapeptides |
WO2016201283A1 (en) * | 2015-06-12 | 2016-12-15 | Cidara Therapeutics, Inc. | Antifungal agents |
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