GB2266890A - Antihypertensive compounds by dechlorination of cyclic depsipeptides - Google Patents
Antihypertensive compounds by dechlorination of cyclic depsipeptides Download PDFInfo
- Publication number
- GB2266890A GB2266890A GB9307909A GB9307909A GB2266890A GB 2266890 A GB2266890 A GB 2266890A GB 9307909 A GB9307909 A GB 9307909A GB 9307909 A GB9307909 A GB 9307909A GB 2266890 A GB2266890 A GB 2266890A
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- United Kingdom
- Prior art keywords
- dhpg
- endothelin
- iii
- dechlorination
- cochinmycin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- XNOPRXBHLZRZKH-DSZYJQQASA-N oxytocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 XNOPRXBHLZRZKH-DSZYJQQASA-N 0.000 description 1
- 229960001723 oxytocin Drugs 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000013618 particulate matter Substances 0.000 description 1
- 230000004963 pathophysiological condition Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108010091212 pepstatin Proteins 0.000 description 1
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 210000005059 placental tissue Anatomy 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000029865 regulation of blood pressure Effects 0.000 description 1
- 230000008327 renal blood flow Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000012134 supernatant fraction Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 230000002620 ureteric effect Effects 0.000 description 1
- 230000025033 vasoconstriction Effects 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K11/00—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K11/02—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof cyclic, e.g. valinomycins ; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Chemical dechlorination of the natural fermentation products, cyclic depsipeptides II and III derived from a culture of Microbispora, produces cyclic depsipeptides I and V, active as endotholin antagonists and useful for treating hypertension and cardiovascular disorders.
Description
TITLE OF THE INVENTION
PRODUCTION OF ANTIHYPERTENSIVE COMPOUNDS
BACKGROUND OF THE INVENTION 1. Field of the Invention
The present invention belongs to the field of antagonists of the endothelin receptor.
Endothelin (ET-1) itself is an endothelium-derived potent vasoconstrictor peptide consisting of 21 amino acids. The unusually prolonged vasoconstriction induced by endothelin in the presence or absence of extracellular Ca2+ suggests that the action of this peptide may profoundly influence blood pressure regulation under normal and pathophysiological conditions. As described further below, endothelin also has a number of other physiological effects, and antagonists which bind to its receptor are thus expected to have advantageous pharmacological properties and corresponding therapeutic benefits.
Endothelin Activity and the Effects of Antagonizing
Its Receptor Binding Endothelin (ET-1), and two closely related bioactive peptides, ET-2 and ET-3, are widely distributed in mammalian tissues, and they can induce numerous biological responses in non-vascular as well as vascular tissues by binding to at least two distinct endothelin receptor subtypes. In addition to smooth muscle, neural and atrial sites, endothelin receptors may be found in gastrointestinal, urogenital, uteral and placental tissues.
Endothelin is a potent vasoconstrictor peptide and thus plays a role in vivo in arterial pressure-volume homeostasis. Not only peripheral, but coronary vascular resistance as well, is increased by endothelin. Cardiac output is decreased, while plasma renin activity is increased.
There is a reduction in renal blood flow and glomerular filtration rate, while levels of atrial natriuretic factor, vasopressin, and aldosterone become elevated.
It is also considered, in accordance with the present invention, that antagonists for the endothelin receptor may be useful in preventing or reducing denudation following angioplasty Such denudation results from myointimal thickening following angioplasty, which is caused by increased endothelin release. Endothelin acts as a growth factor with resepct to smooth muscle and fibroblastic cells, and possibly other types of cells, as well.
Endothelin is also a neuropeptide, acting on the posterior pituitary, where it modulates the release of the neurosecretory hormones vasopressin and oxytocin. Endothelin released from the posterior pituitary also acts as a circulating hormone, having a wide range of actions as discussed further above.
This includes effects on the endocrine system, especially the adrenal glands. Endothelin increases plasma levels of epinephrine.
Consequently, cochinmycins I and V of the present invention, which are antagonists for the endothelin receptor, have therapeutic usefulness in preventing, decreasing or modulating the various physiological effects of endothelin discussed above, by wholly or partially blocking access of endothein to its receptor.
2. Brief Description of the Prior Art
Endothelin was initially purified from the culture medium of porcine aortic endothelial cells;
Yanagisawa et al. (1988) Nature 332, 411-415.
Further investigations of the activity of endothelin have been described in publications such as the following:
- Takagi et al. (1988) 3iochem. Biophys. Res.
Commun. 157, 1164-1168.
- Sugiura et al. (1989) Biochem. Biophys. Res.
Commun. 158, 170-176.
- Miller et al. (1989) J. Clin. Invest. 83, 317-320.
- Du Pont Biotech Update (1990).
- Warner.et al. (1989) J. Cardiovasc. Pharmacol.
13(Suppl. 5), S85-S88.
- Yoshizawa et al. (1990) Science 247, 462-464.
- Bousso-Mittler et al. (1989) Biochem. Biophys.
Res. Commun. 162, 952-957.
- Saito et al. (1989) Hypertension 14, 335-336.
- Tomita et al. (1989) New Engl. J. Med. 321, 1127.
- Kurihara et al. (1989) J. Cardiovasc.
Pharmacol. 13(suppl. 5) S13-S17.
- Sugiura et al. (1989) Biochem. Biophys. Res.
Commun. 161, 1220-1227.
U.S. Pat. No. 4,810,692 discloses two immunosuppressant cyclodepsipeptides designated 55185
RP and 59451 RP, although represented by a more general formula which includes many stereoisomers not specified in the formula. While species 55185 RP and 59451 RP are characterized by various chemical and physical data, they are nevertheless unspecified stereoisomers that have been found to be different from cyclic depsipeptides I and V of the present invention.
The present invention relates to two stereoisomers that, even though falling within the general formula of the '692 patent, are neither isolated nor suggested in said patent, and are produced by a different microorganism than that in the '692 patent. Moreover, these two stereoisomers have no immunosuppressant activity. Since the characterization data in the '692 patent is not consistent with the structures proposed therein for the isolated species 55185 RP and 59451 RP, the present invention also relates to the two species described but not enabled in the '692 patent.
U.S. Patent Application Serial No. 07/645 535 filed January 24, 1991, describes the isolation of the cyclic depsipeptides I, II, and III (now referred to as cochinmycins I, II, and III) from a fermentation culture of Nicrobispora and the use of cochinmycins I, II and III as endothelin antagonists to treat hypertension. A patent application with
Attorney Docket No. 18694, filed concurrently with this application, describes the isolation of cochinmycins IV and V from a culture of Microbispora, and their use as endothelin antagonists.
SUMMARY OF THE INVENTION
The present invention relates to two novel processes for the production of peptidolactones (I and V) through chemical dechlorination of natural fermentation products III and II, respectively.
Cochinmycins I, V, II, and III are active as endothelin anatagnists.
The process of the present invention further relates to a method of preparing two novel cyclodepsipeptides (II and III) by culturing
Microbispora sp. MA6857 (ATCC 55140) and isolating the compounds from the fermentation broth.
Dechlorination is accomplished by dissolving the starting material, either cochinmycin II or cochinmycin III, in an alcoholic solvent such as methanol, isopropanol, ethanol, preferably methanol and hydrogenating at (1-5) kg/cm2 H2 in the presence of 5%-10% Palladium/Charcoal and MgO 5 to 30% for 1 to 16 hours.
The reaction mixture is filtered to remove catalyst, and the filtrate is then evaporated to dryness, preferably by flash evaporation at under 40"C. The product may be purified by chromatography in a suitable solvent. The fractions are monitored by HPLC at 215nm and those containing product are evaporated to dryness to yield the purified final product.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention there is provided an endothelin receptor antagonist cyclic depsipeptide of the formula:
Cochinmycin PyrX Y-DHPG *
I X = H D-DHPG
II X = C1 L-DHPG S
III X = C1 D-DHPG
V X = H L-DHPG S
PROTON NUCLEAR MAGNETIC RESONANCE (1H NMR) OF
COCHINMYCIN I AND V
The proton nuclear magnetic resonance (1H
NMR) spectra for cochinmycins I and V of the formula shown above were recorded at 300 MHz in DMSO-d6 on a
VARIAN SC300 NMR spectrometer. Chemical shifts were recorded in ppm relative to tetramethylsilane (TMS) at zero ppm using the solvent peak at 2.49 ppm as internal standard. For convenience, the data is presented in tabular form immediately below.
TABLE I 1H NMR Assignments of Cyclic Depsipeptide I in DMSO-d6 at 300 C
Recorded at 300 MHza,b Residue Hα H# NH Aromatic D-Ala 4.22 dq 1.29 d (3H) --- 7.81 d
J = 7.5 J = 7.5 J = 7.5
D-allo-Thr 4.62 t 5.18 dq 0.84 d (3H) 8.81 d
J = 9.5 J = 10,6.5 J = 6.5 J = 9
L-Phe 4.71 ddd 3.06 dd --- 8.10 d 7.1-7.34 (1OH)
J = 5, 8.5, J = 5, 13.5 J = 8.5
10.5 2.92 dd
J = -10.5,-14
D-Phe 4.33 dt 2.99 dd --- 7.80 d 7.1-7.34 (1OH) J = 5.5, 8 J = -8,-14 J = 7.5
2.90 dd
J = 5.5,-14
L-DHPG 5.22 d --- --- 7.94 d 6.07 d (2H)c J = 8 J = 8 J = 2 6.11 tc J = 2 D-DHPG 5.22 d --- --- 8.39 d 6.02 d (2H)c
J = 10 J = 9.5 J = 2
6.09 tc J = 2 Pyrrole --- --- --- 11.36 br.d 6.02 d (1H)
J = 1.8
J = 1.5 6.80 m (1H)
6.88 m (1H) a,b,c See after Table II.
1H NMR Assignments of Cochinmycin V in DMSO-d6 at 200 C Recorded at 300
MHza,b Residue Hα Hss NH Aromatic D-Ala 3.92 dq 1.32 d (3H) --- 8.22 d
J = 5, 7.5 J = 7.0 J = 4.5
D-allo-Thr 4.45 t 4.96 dq 1.11 d (3H) 8.66 d
J = 9.5 J = 10, 6. J = 6 J = 9
L-Phe 4.80 ddd 3.02 dd --- 8.12 d 7.14m(5H)C J = 5, 8.5, J = 5, 13.5 J = 8.5 9.5 2.89 dd
J = -10.5, 13.5
D-Phe 4.49 dt 3.04 dd --- 8.67 7.32m(2H)C
J = 4,8,9 J = -6, 13.5 J = 9.5 7.22m(2H)C
2.93 dd 7.02m(lH)C
J = 9, 13.5
L-DHPG 5.44 d --- --- 7.80 d 6.22 d (2H)
J = 8 J=9 J = 2 6.15 t J = 2 D-DHPG 5.24 d --- --- 8.31 d 6.18 d (2H) J = 8 J = 8 J = 2
6.19 t
J = 2
Pyrrole --- --- --- 11.40 br.d 6.02 dd,
J = 2.5, 6
6.82 m
6.89 m a,b,c, See next page.
a Coupling constants + 0.2 Hz.
b Except where otherwise noted, all chemical shift
resonances correspond to one-proton intensity and
are given in ppm using the solvent peak at o 2.49 as interval standard.
c Assignments may be interchanged.
ABBREVIATIONS: In Tables I and II above, the following abbreviations have been used: s = singlet; d = doublet; t = triplet; m = multiplet; br = broad
CARBON 13 NUCLEAR MAGNETIC RESONANCE (12C NMR)
In order to further characterize the semi synthetic cyclic depsipeptides I and V, 13C NMR spectra were also recorded in DMSO-d6 at 75 MHz on a
VARIAN SC300 spectrometer at 20 C, with chemical shifts being given in ppm relative to tetramethylsilane (TMS) at zero ppm using the solvent peak at 39.5 ppm as internal standard.The spectral peaks are indicated below:
Compound I 13C NMR Chemical Shifts:
CH3's - 6 15.7, 17.8; CH21s - 6 37.3, (2x); CH's - 6 48.6*, 50.6*, 54.2, 54.4, 55.3*, 56.8, 69.1, 101.7, 102.2, 104.2 (2x), 104.7 (2x), 108.6*, 110.7*, 121.6*, 126.3, 126.6, 128.1 (2x), 128.4 (2x), 129.2 (2x), 129.25 (2x); quarternarys - 8 125.8, 136.9, 138.1*, 138.9, 139.5, 158.4 (4x), 160.6*, 167.1, 168.5, 168.7, 170.5, 171.8 and 172.3* ppm.
*13C Chemical shifts with > 0.2 ppm difference from starting material.
Compound V 13C NMR Chemical Shifts:
CH3's - 6 16.6, 16.9 CH2,s - 6 36.7, 38.0
CH's - 6 51.9, 53.9, 55.0, 55.8, 56.1, 56.2, 70.7, 101.9, 102.1, 106.6 (2x), 106.9 (2x), 108.6*, 110.7*, 121.5*, 126.2, 126.3, 128.0 (2x), 128.2 (2x), 129.1 (2x), 129.2 (2x); quarternary's - 6 125.8, 137.0, 138.0, 138.3, 139.7, 158.0 (2x), 158.2 (2x), 160.2* 168.4, 168.7, 168.9, 171.2, 171.7 and 172.3 ppm.
*13C Chemical shifts with > 0.2 ppm difference from starting material.
Stereochemistry
Cochinmycins I and V have the stereochemistries shown in Formula I below:
Cochinmvcin Y-DHPG *
I D-DHPG t
V L-DHPG S
TABLE III
Physico-chemical properties of Cochinmycins I and V.
Cochinmycin I Cochinmycin V
Molecular Formula C46H47N7012 C46H46N7012 HR-FAB-MSa((M+H)+m/z) Found: 890.3380 890.3360
Calcd: 890.3361 890.3361
23b [a]D -10.0 (c 0.1, MeOH) +20.10(c 0.1, MeOH)
MeOH UVc#max nm(E%) 212 (446), 230 (sh,172), 214 (489), 230 (sh,237),
269 (175) 270 (224) FTIRd(ZnSe)Vmaxcml 3299,1733,1661,1623 3302,1739,1661,
1603,1555,1516 1555,1516
HPLCetR(minutes) 4.8 3.3 a JOEL HX110 mass spectrometer at lOkV using ULTRAMARK 1621 as the
internal standard.
b PERKIN-ELMER 241 polarimeter c Beckman DU-70 ultraviolet spectrophotometer.
d PERKIN-ELMER 1750 FOURIER transform infrared spectrophotometer; sample
deposited on a zinc selenide crystal at ambient temperture.
e Whatman PARTISIL 5 ODS-3, 4.6 x 100 mm; MeOH-H20(45:55); flow rate, 1
ml/minute; 400C; detection - UV 215nm.
sh=shoulder
Both semi synthetic cochinmycins I and V show physicochemical characteristics identical to their "natural" counterparts. The absolute stereochemistry of cochinmycin I was as described in U.S. Patent Application Serial
No. 07/645535 filed January, 1991. The absolute stereochemistry of cochinmycin V was determined by comparison of its NMR spectra to those of'its starting material, cochinmycin II. A conservation of stereochemistry was indicated.
Fermentation of Microbispora sp. MA6857
Starting materials, cochinmycins II and III were isolated from a culture of a strain of
Microbispora which is novel and which has been designated sp. MA6857. A sample of this microorganism was deposited under the Budapest Treaty at the American Type Culture Collection (ATCC), 12301
Parklawn Drive, Rockville, Maryland 20852, on January 17, 1991, where it has been assigned the accession number ATCC 55140. Any restrictions relating to public access to the microorganism shall be irrevocably removed upon a patent grant. Based on the macro and micromorphology, the microorganism is a member of the genus Microbispora which is characterized by the formation of paired spores borne on an aerial mycelium. However, the microorganism has taxonomic and other features which distinguish it from the known strains of the genus Microbispora, and on that basis it is believed to be novel. It has, consequently, been designated herein Microbispora sp.
MA6857.
The microorganism Microbispora sp. MA6857 was isolated from a culture prepared by well known methods from a soil sample originating in Cochin,
India. In order to produce scaled-up culture batches for supplying additional quantities of cochinmycins
II and III, a seed culture was produced by inoculating 50 ml of aqueous nutrient medium, ATCC, in a 250 ml triple baffled erlenmeyer flask with 2 ml of refrigerated or thawed frozen vegatative mycelia.
The nutrient medium ATCC has the following composition:
ATCC
Glucose 10;0g
Soluble starch 20.0g
Yeast extract 5.0g pH adjusted to 7.0 with
N-Z amine E 5.0g NaOH prior to CaCO3
CaCO3 1.0g addition
Beef extract 3.og
BACTO-PEPTONE 5.0g
The culture vessel was incubated at 28 C and shaken at 220 rpm for 96 hrs in order to obtain sufficient biomass for use as an inoculum for production medium. The production medium employed, SAM-4, contained sources of assimilable organic nutrients for growth of the culture in the form of dextrin, soybean flour and peptone.Its composition is set out below:
SAM-4
Dextrin 50.0g
Soybean flour 30.0g pH adjusted to 7.0 with
DIFCO PEPTONE 1.0g NaOH prior to CaCO3
CaCO3 5.0g addition
Distilled H2O 1000ml The production medium, 44 ml per 250 ml non-baffled erlenmeyer flask, was inoculated with 2 ml of seed culture and incubated at 28 C and shaken at 220 rpm for 6 to 10 days.
From the seed culture described above, three different production media were prepared, as described below: 2E - 2 ml of cell suspension from a 4 day seed culture (obtained by inoculating ATCC medium, 50 ml per 250 ml baffled erlenmeyer, with thawed frozen mycelium and incubating at 28 C and 220 rpm) was used to inoculate SAM-4 production medium which was incubated for 10 days at 28 C and 220 rpm prior to harvest.
3G - 2 ml of cell suspension from a 4 day seed culture (obtained by inoculating ATCC medium with cells scraped from a BAM-2 agar slant) was used to inoculate SAM-4 production medium which was incubated for 6 days prior to harvest. Both seed and production media were incubated at 28 C and 220 rpm.
5L - 2 ml of cell suspension from a 4 day seed culture (obtained by inoculating ATCC medium with thawed frozen mycelia preserved in 10% glycerol) was used to inoculate SAM-4 production medium which was incubated for 10 days prior to harvest. Both seed and production medium were incubated at 28 C and 220 rpm.
Isolation of Cyclic Depsipeptides II and III from
Cultures 2E and 3G
The 2E and 3G cultures were pooled (115 ml whole broth) and extracted with methyl ethyl ketone.
The organic layer (dry weight = 89 mg) was dissolved in 0.2 ml of DMSO solution and purified as one shot on a Whatman PARTISIL 10 ODS-3 column (0.94 x 50 cm) at room temperature at 5 ml/min using a gradient of 0-100% acetonitrile/water (0-10 min, at 0%; 10-60 min, 0 to 100%, linear). Eighty one-minute fractions were collected, followed by a 200 ml acetonitrile wash. Fractions 38-44 were active in 125I-ET-l/bovine aorta binding assay. (Reverse phase and normal phase TLC analyses of these fractions revealed heterogeneity, without a consistent -pattern. Also, fractions 40 and 41 were titrated and a zig-zag dose response curve was noticed for each.)
Fractions 38-44 were then pooled (dry weight = 16.6 mg), dissolved in 0.25 ml solution of methanol and dimethylsulfoxide (2:3) and rechromoatographed on the same column, at room temperature.This time, a gradient of 20-75% methanol/water was employed (0-10 min, at 20%; 10-65 min, 20-75%, linear; 65-75 min, 75-100%, linear). Fractions 57-64 were active.
Three active components were realized by HPLC analyses (analytical HPLC: column - Whatman PARTISIL 5 ODS-3 0.46 x 10 cm; mobile phase - MeOH-H20, 50:50, e lml/min, 40 C; detection - UV 215 nm) of these fractions: 58-59 (1.5 mg), 60-61 (4.5 mg), and 63-64 (1.6 mg). Further purification in E. Merck silica gel 60F TLC plates (10 x 20 cm, 0.2 mm thickness) using trifluoro acetic acid - methanol dichloromethane, 1:5:95, revealed activity at the origin in each case.Subsequent purification on
Whatman PARTISIL 10 ODS-3 (0.46 x 25 cm) at 40 C using isocratic elutions of 45%, 50% and 50% methanol/water at 1 ml/min for the respective fractions, collecting 1 minute fractions, gave 10-13 (3.2 mg, Cochinmycin II) and (0.64 mg, Cochinmycin
III), as revealed by binding data and homogeneity indicated by analytical HPLC (analytical HPLC: column - Whatman PARTISIL 5 ODS-3 0.46 x 10 cm; mobile phase - MeOH - H20, 45:55, @ 1 ml/min, 40"C; detection - W 215 nm). W, FT-IR, and 1H-NMR (CD30D) data showed the two components to be structurally related.
Isolation of Cyclic Depsipeptides II and III from
Culture 5L
Culture 5L (2700 ml whole broth, pH 8.4) was adjusted to pH 7.2 with dilute HC1 and extracted with methyl ethyl ketone (3800 ml). The inactive aqueous layer was discarded. The active organic layer (1.34 g dry weight; EC50 = 25 > 1 WBE/ml) was purified on a column of E. Merck silica gel 60 (50g, 40-63 Fm) in
CH2C12 with stepwise elution of CH2C12 (180 ml), 5% MeOH/CH2Cl2 (5 x 100 ml), 10% MeOH/CH2C12 (2 x 100 ml and 200 ml) and 200 ml each of 20, 30, 50 and 100% MeOH/CH2C12. HPLC analyses revealed desired components in factions 8 and 9 (i.e., the second and third 10% MeOH/CH2C12 eluates) and also confirmed by 125I-ET-l/bovine aorta binding data (91 and 74% inhibitions respectively at 900 > 1 WBE/ml). These fractions were pooled (dry weight = 264 mg) and purified on a Whatman PARTISIL 10 ODS-3 column (2.21 x 25 cm) at 40 C and 15 ml/min using a 40 - 55 100% MeOH/H2O gradient elution (0-5 min, at 40%; 5-65 min, from 40-55%, linear; 65-120 min, from 55 to 100%, linear). One minute fractions were collected.
Analytical HPLC (column - Whatman PARTISIL 5 ODS-3 0.46 x 10 cm; mobile phase - MeOH - H2O, 45:55, fl 1 ml/min, 40O C; detection - W 215 nm) suggested pooling fractions 41-46, 51-55, and 64-72. Thus flash evaporation of solvents gave 88 mg of 51-55 (Cochinmycin II) and 33 mg of 64-72 (Cochinmycin
III). The analytical data is shown in the table of values below:
TABLE IV
Physico-chemical properties of Cochinmycins II and
III.
cochinmycin II cochinmycin III
Molecular Formula C46H46N7012C1 C46H46N7012C1 HR-FAB-MSa((M+H)+m/z) Found: 924.3005 924.2949
Calcd: 924.2971 924.2971
23b [a]D +20.00(c 0.1, MeOH) -20.00(c 0.1, MeOH)
MeOH UVc#max nm(E%) 213 (483), 230 (sh,208), 212 (471), 230 (sh,185),
275 (219) 274 (207)
FT-IRd(ZnSe)vmaxcm-1 3304,1734,1657, 3304,1733,1662,
1607,1521 1558,1519 HPLCt(minutes) 6.1 9.4 a JOEL HXllO mass spectrometer at lOkV using ULTRAMARK 1621 as the
internal standard.
b PERKIN-ELMER 241 polarimeter c Beckman DU-70 ultraviolet spectrophotometer.
d PERKIN-ELMER 1750 FOURIER transform infrared spectrophotometer; sample
deposited on a zinc selenide crystal at ambient temperture.
e Whatman PARTISIL 5 ODS-3, 4.6 x 100 mm; MeOH-H20(45:55); flow rate, 1
ml/minute; 400C; detection - UV 215nm.
sh=shoulder
PROTON NUCLEAR MAGNETIC RESONANCE (1H NMR) OF STARTING MATERIALS
The proton nuclear magnetic resonance (1H
NMR) spectra for the two starting materials cochinmycins II and III were recorded at 400 MHz in
DMSO-d6 on a VARIAN XL400 NMR spectrometer. Chemical shifts were recorded in ppm relative to tetramethylsilane (TMS) at zero ppm using the solvent peak at 2.49 ppm as internal standard. For convenience, the data is presented in tabular form immediately below.
TABLE V 1H NMR Assignments of Cyclic Depsipeptide II in DMSO-d6 at 300 C
Recorded at 400 MHza,b
Residue Hg Hss H# H Aromatic
D-Ala 3.94 dq 1.32 d (3H) --- 8.17 d
J = 5, 7.5 J = 7.5 J = 5
D-allo-Thr 4.45 t 4.96 dq 1.11 d (3H) 8.60 d
J = 9.5 J = 10,6.5 J = 6.5 J = 8
L-Phe 4.79 ddd 3.02 dd --- 8.12 d 7.15 m (5H)C
J = 5, 9, J = 5, 13.5 J = 9
10 2.88 dd
J = 10.5, 13.5
D-Phe 4.50 dt 3.04 dd --- 8.62 d 7.29 m (2H)C
J = 6, 9 J = 6, 13.5 J = 8.5 7.22 m (2H)c
2.94 dd 7.04 m (1H)c
J = 9, 13.5
L-DHPG 5.44 d --- --- 7.78 d 6.23 d (2H)
J=9 J=9 J = 2 6.15 t J = 2 D-DHPG 5.25 d --- --- 8.27 d 6.17 d (211) J = 8 J = 8 J = 2
6.20 t
J = 2
Cl-Pyrrole --- --- --- 12.16 br.s 6.04 d
6.87 d
J = 4 a,b,c See after Table VI.
TABLE VI 1H NMR Assignments of Cyclic Depsipeptide III in DMSO-d6 at 300 C
Recorded at 400 MHza,b
Residue Hα Hss H# NH Aromatic
D-Ala 4.22 dq 1.31 d (3H) --- 7.81 d
J = 7.5 J = 7.5 J = 7.5
D-allo-Thr 4.62 t 5.18 dq 0.85 d (3H) 8.82 d
J = 9.5 J = 10,6.5 J = 6.5 J = 9.5
L-Phe 4.72 ddd 3.07 dd --- 8.15 d 7.1-7.33 (5H)
J = 4.5, 8.5,J = 4.5, 13.5 J = 8.5
10.5 2.89 dd
J = 10.5, 13.5
D-Phe 4.33 dt 2.98 dd --- 7.79 d 7.1-7.33 (10H)
J = 5.5, 8 J = 8, 13.5 J = 8
2.89 dd
J = 5.5, 13.5
D-DHPG 5.23 d --- --- 7.96 d 6.08 d (2H)C J=8 J = 10 J = 2
6.12 tc J = 2 D-DHPG 5.23 d --- --- 8.39 d 6.03 d (2H)C
J = 10 J = 10 J = 2
6.10 tc J = 2 Pyrrole --- -- -- 12.15 br.s6.87 d
6.02 d
J = 4 a,b,c See next page.
a Coupling constants + 0.2 Hz.
b Except where otherwise noted, all chemical shift
resonances correspond to one-proton intensity and
are given in ppm using the solvent peak at 6
2.49 as interval standard.
c Assignments may be interchanged.
ABBREVIATIONS: In Tables V and VI above, the following abbreviations have been used: s = singlet; d = doublet; t = triplet; m = multiplet; br = broad
CARBON 13 NUCLEAR MAGNETIC RESONANCE (13C NMR) OF
STARTING MATERIALS
In order to further characterize the cyclopeptides II and III, 13C NMR spectra were also recorded in DMSO-d6 at 100 MHz on a VARIAN XL400 spectrometer at 30 C, with chemical shifts being given in ppm relative to tetramethylsilane (TMS) at zero ppm using the solvent peak at 39.5 ppm as internal standard.The spectral peaks are indicated below:
Compound II 13C NMR Chemical Shifts: 16.6, 16.9, 36.8, 38.0, 51.8, 53.8, 54.9, 55.8, 56.16, 56.24, 70.6, 101.9, 102.2, 106.6 (2x), 106.8 (2x), 107.0, 111.8, 117.2, 125.7, 126.2, 126.3, 128.0 (2x), 128.1 (2x), 129.0 (2x), 129.2 (2x), 136.9, 137.8, 138.3, 139.6, 158.0 (2x), 158.2 (2x), 159.2, 168.4, 168.6, 168.9, 171.1, 171.5, 172.2 ppm.
Compound III 13C NMR Chemical Shifts: 15.7, 17.7, 37.26, 37.31, 50.5, 54.2 (2x), 55.2, 56.75, 56.81, 69.0, 101.7, 102.2, 104.2 (2x), 104.7 (2x), 107.0, 111.7, 117.3, 125.6, 126.3, 126.4, 128.0 (2x), 128.3 (2x), 129.07 (2x), 129.12 (2x), 136.8, 137.8, 138.8, 139.4, 158.3 (4x), 159.5, 167.0, 168.4, 168.6, 170.4, 171.7, 172.0 ppm.
Dechlorination of component III [conversion of component III to component I
Component III (0.80 g, 0.87 mmole) was dissolved in methanol (6 mL) and kept under H2 (2.8 kg/cm2) in the presence of 5% Pd/C (0.20 g) and MgO (0.20 g) for 15 h. HPLC analysis revealed 98% completion of conversion. The reaction mixture was filtered over CELITE and Whatman NO. 3 filter paper.
Flash evaporation, under vacuo at < 40 C, to dryness afforded 0.79 g of material. Purification on a
Whatman PARTISIL 10 ODS-3 column (2.21 x 50 cm) was performed using 50% methanol/water as the isocratic mobile phase for elution at 10 mL/min at room temperature. The effluent was monitored at 215 nm.
1.5 min fractions were collected. HPLC analyses suggested pooling frs. 34 - 40 (Ve = 495 - 600 mL), which was flash evaporated to dryness in vacuo at < 400C, to afford 0.40 g of component I, 16 mg of starting component III was recovered from fractions 61 - 80.
Dechlorination of component II to produce component V
Component II (1.0 g, 1.08 mmole) was dissolved in methanol (12 mL) and kept under H2 (2.81 kg/cm2) in the presence of 5% Pd/C (0.25 g) and MgO (0.25 g) for 6 h. HPLC analysis revealed -80% completion of conversion. The reaction mixture was filtered over CELITE and Whatman NO. 3 filter paper.
Flash evaporation, under vacuo at < 40 C, to dryness afforded 0.96 g of material. Purification on a
Whatman PARTISIL 10 ODS-3 column (2.21 x 50 cm) was performed using 45% methanol/water as the isocratic mobile phase for elution at 10 mL/min at room temperature. The effluent was monitored at 215 nm.
1.5-min fractions were collected. HPLC analyses suggested pooling frs. 38-45 (Ve = 555 - 675 mL), which was flash evaporated to dryness in vacuo at < 400C, to afford 0.40 g of component V (see above).
Endothelin Receptor Binding Assav Results
The binding of semi synthetic cyclicdepsipeptides I and V to the endothelin receptor was determined in accordance with the assay described in detail immediately below. It is similar to the assay described in Ambar et al. (1989) Biochem. Biophys.
Res. Commun. 158, 195-201; and Khoog et al. (1989)
Febs Letters, 253, 199-202.
Endothelin-l (ET-1) was purchased from Peptides
International (Louisville, KY ). 125I-ET-1 (2000
Ci/mMol) was purchased from Amersham (Arlington
Heights, IL).
Membranes were prepared from rat hippocampus, and rat or cow aorta. Dissected tissue was homogenized twice for 30 seconds with a Brinkman POLYTRON (setting 10, Generator PTA 20 TS (Westbury, NY)] in ice cold 250 mM sucrose, 50 mM Tris-HCl pH 7.4 with 7 Fg/ml pepstatin A and 0.5 Fg/ml leupeptin. The crude particulate matter was removed by centrifugation at 750 x g for 10 min. The membranes were sedimented from the supernatant fraction by centrifugation at 48,000 g for 30 min. Membrane pellets were resuspended in the above buffer with protease inhibitors. Aliquots of these suspensions were stored at -70" C.
Binding studies with 125I-ET-l were conducted in 50 mM potassium phosphate pH 7.5 with 0.1% bovine serum albumin (BSA) using 12-well SKATRON (Lier,
Norway) cell harvester tube strips. 125I-ET-1 concentrations were 25 pM for hippocampus, 150 pM for aorta. Samples were dissolved in dimethylsulfoxide (DMSO). Upon addition of the sample, the final DMSO concentration was 3%. Membranes were added last to start the binding reaction. The reaction mixture was incubated at 37C C for 30 or 60 minutes. Binding reactions were terminated using a SKATRON cell harvester by filtration through glass fiber filter pads presoaked with 2% BSA. The samples on the pads were immediately washed with 150 mM NaCl 0.1% BSA.
The pads were punched out and radioactivity was evaluated in a Beckman GAMMA 5500 gamma counter (Fullerton, CA). Nonspecific binding was determined in the presence of 100 nM ET-1. The results of this
The results of this binding assay are set out in the table of values below:
TABLE VII Affinitv for ET-A and ET-B Receptors Ki (uM) vs.[125I]-ET-1
Aorta Hippocampusa
(ET-A) (ET-B)
Cochinmycin I O.lOå 0.20
Cochinmycin II 3.0a 3.0
Cochinmycin III 0.20a 0.5
Cochinmycin V 90.0b 25.0 a Rat b Cow
In accordane with the present invention, cyclic depsipeptides I and V are useful in human therapy for treating asthma, hypertension, renal failure particularly post-ischemic renal failure, cyclosporin nephrotoxicity, vasospasm, cerebral and cardiac ischemia, myocardial infarction, or endotoxin shock caused by or associated with endothelin, comprising administering to a human patient in need of such treatment, a therapeutically effective amount of cyclic depsipeptides I or V.
The particular dosage to be administered in the course of such treatment is the result of a number of factors, such as the particular condition being treated, the route of administration, the age, sex, weight and general condition of the patient being treated, and whether acute or chronic treatment is envisioned. With those considerations in mind, it can be stated that, as a general matter, cochinmycins
I and V of the present invention will be administered orally to a patient in dosage amounts between 1 and 200 mg/kg/day, and will be administered parenterally to a patient in dosage amounts between 0.5 and 100 mg/kg/day.
The present invention also relates to pharmaceutical compositions for treating asthma, hypertension, renal failure particularly post-ischemic renal failure, cyclosporin nephrotoxicity, vasospasm, cerebral and cardiac ischemia, myocardial infarction, or endotoxin shock caused by or associated with endothelin, comprising a therapeutically effective amount of cyclic depsipeptides I, or V together with a pharmaceutically acceptable carrier therefor.
Where the pharmaceutical compositions of the present invention are for oral administration, e.g., as tablets, the active ingredient, i.e., cochinmycins
I or V, will be used in combination with other compounding ingredients such as talc, vegetable oils, polyols, benzyl alcohols, gums, gelatin, starches and other carriers, all of which are well known in the art. Where the pharmaceutical compositions of the present invention are for parenteral administration, the active ingredient will be dissolved or dispersed in a suitable liquid carrier, or emulsions may be formed using suitable emulsifying agents.
Claims (4)
1. A process for preparing a cyclic depsipeptide of the formula below wherein X = H and the stereochemistry at Y-DHPG is L-DHPG or D-DHPG from a compound of Formula I wherein X is C1 and the stereochemistry at Y-DHPG is L-DHPG or D-DHPG by dechlorination, wherein Formula I is:
* = An asymmetric carbon.
2. The process of. Claim 1 wherein dechlorination comprises dissolving the starting material in an alcoholic solvent selected from the group consisting of methanol, ethanol, and isopropanol, hydrogenating for 1 to 16 hours in the presence of a catalyst, filtering, isolating and recovering the product.
3. The process of Claim 2 wherein the alcoholic solvent is methanol and the catalyst is 1:1 MgO:5% Pd/C.
4. The process of Claim 3 wherein the hydrogenation is for 14-16 hours at 2-4 kg/cm2 H2.
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| US87037192A | 1992-04-17 | 1992-04-17 |
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| GB2266890A true GB2266890A (en) | 1993-11-17 |
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ID=25355241
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4810692A (en) * | 1986-05-22 | 1989-03-07 | Rhone-Poulenc Sante | Immunosuppressant substances, process for their preparation and pharmaceutical compositions containing them |
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1993
- 1993-04-16 GB GB9307909A patent/GB2266890A/en not_active Withdrawn
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4810692A (en) * | 1986-05-22 | 1989-03-07 | Rhone-Poulenc Sante | Immunosuppressant substances, process for their preparation and pharmaceutical compositions containing them |
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