PT86793B - PROCESS FOR THE PRODUCTION OF CYTOLIC ANTIGONIST PEPTIDE OF OCITOCIN AND VASOPRESSIN - Google Patents

Abstract

Cyclic peptides of formula (I) and oxidn. prods. of synthetic analogues of formulae (IIA-C) and pharmaceutically acceptable salts are new: where, for component (1) S R1 = CHMe -Et; R2 = CH2pH; R3 = OH; for (1a) S R1 = CHM2-Et; R2 = CH2Ph; R3 = H; for (A) S R1 = iPr; R2 = CH2Ph; R3 = OH; for (B) S R1 = CH2-iPs; R2 = iPr; R3 = OH; for (C) S R1 = CH2-iPr, R2 = CH2Ph, R3 = OH, and for (D) S R1 = CH2-iPr, R2 = CHMe-Et, and R3 = OH. Also new are cyclic peptides with empirical formulae C33H56N8O7 (component (E)), C33 H56 N8 O8 (Compnent (F)) and C34 H58 N8 O8 (Component (G)) and given NMR spectra. Prodn. of oxytocin or vasopressin antagonist cpds. comprises aerobic cultivation of a Streptomyces silvensis strain in aq. nutrient medium. Biologically pure culture of Streptomyces silvensis is new, specifically strains ATCC 53525 and ATCC 53562.

Description

PROCESS FOR THE PRODUCTION OF CYCLIC PEPTIDS ANTAGONISTS OF Oxytocin and VASOPRESSIN

thus being useful in the treatment and prevention of pathological situations in which vasopressin may be involved, such as, for example, congestive heart failure, hypertension, edema and hyponatremia.

The aforementioned compounds have, for example, the formula

on what

R ¹ , R ² and R ³ are, for example, -CH (CH ₃ ) (CH ₂ ) (CH ₃ ),

-CH ^ CgH ^ and -OH, respectively, or their pharmaceutically acceptable salts.

Fundamentals of the Invention

This invention belongs to the field of oxytocin and vasopressin antagonists. In the field of obstetrics, one of the most important problems is the treatment (control) of premature labor (ahead of time). A significant number of pregnancies that progress beyond 20 weeks of pregnancy experience (present) premature labor and delivery that are a major cause of neonatal morbidity and mortality.

It has recently been proposed that a selective oxytocin antagonist would be an ideal tocolytic agent. In recent years, evidence has accumulated that strongly suggests that oxytocin constitutes the physiologic initiator of labor in various species of mammals including humans. Oxytocin is thought to exert this effect in part by causing the uterine myometrium to contract and in part by increasing the synthesis and release of contractile prostaginandins formed by the uterine / p1acenta endometrium.

These prostaginins may, moreover, be important in the process of cervical dilation (of the cervix). Through these mechanisms, the labor process (term and preterm) is initiated through an increased sensitivity of the uterus to oxytocin, which results in part in a well-documented increase in the number of oxytocin receptors in this tecj, do. This upregulation of oxytocin receptors and increased uterine sensitivity appears to be due to the trophic effects of the rising plasma levels of estrogens in relation to the end of pregnancy. By blocking the effects of oxytocin on the uterus, both direct (contractile) and indirect (increased synthesis of prostaglandins), a selective oxytocin antagonist would likely be more effective for the treatment of premature (pre-term) labor than current regimes. Furthermore, as oxytocin at the end of pregnancy has more important effects on the uterus alone, it is expected that such a compound will have few, if any, side effects.

Vasopressin (also known as Antidiuretic Hormone, ADH (HAD) is a pituitary peptide hormone that is vitally important for maintaining an appropriate water balance in animals and humans. The diuretic effect of vasopressin, concentrates urine by increasing the reabsorption of water from the kidney filtrate, so even under mild conditions of dehydration the increased blood levels of vasopressin act to concentrate water and control the appropriate osmorality of blood and extracellular fluids. contracting the vascularized smooth muscle 1 and may normally be involved in maintaining peripheral vascular resistance (McNeil, JR, Can J. Physio 1 .Pharmaco1 61: 1226-1235 (1983); Cowley, AW, Quillen, EW and Skelton, MM, Federation Proceedings, 42: 3170-3176 (1983).

Recent studies have led to the development of specific relatively potent and specific antagonists of vasopressin's antidiuretic or pressor effects (Sawyer, WH, and Manning, M, Federation Proceedings 43: 87-90 (1984). Due to the prominent actions of vasopressin on renal functions and cardiovascular, vasopressin antagonists are useful in the treatment of various conditions including congestive heart failure, hypertension, and edema situations.The diuretic activity of water that saves salts of vasopressin antagonists will be particularly useful in the treatment of hyponatremia that can arise from a number of situations (Zerbe, R. Stropes, L. Robertson, G., Ann. Rev. Med. 31: 315-327 (1980). The commonly known vasopressin antagonists are vasopressin analog peptides ( Huffman, WH Ali, FE, Bryan, WM et al, J. Med. Chem .28; 1759-1760 (1985), and are thus likely to be rapidly metabolized in vi v and have little, if any, oral activity (Lynn, R.K. Straub, K. M., Landvat ter S.W. and Garvey, C.T. Federation Proceeding 45: 205 (1986); Kinter L.B. Mann, W.A. Woodward, P. DePalma, D. and Brennan F., Federation Proceedings 45: 649 (1986).

It is therefore an object of the present invention to provide cyclic and synthetic peptide analogs which are antagonists of oxytocin and vasopressin and are useful as pharmaceutical agents. It is also an object of the present invention to provide processes for producing these cyclic peptides. It is another objective of the present event to provide a mixture of cyclic peptides with formula I and its secondary related components A, B, C, D, E, F and G, produced by aerobic fermentation of the organism Streptomyces silvensis. Another objective is to provide cultures of the organism Streptomyces silvensis ATCC No.53525 or ATCC No.53526 that are capable of producing cyclic peptides and mixtures thereof. Yet another objective is to provide synthetic analogue products of these cyclic peptides.

SHORT DESCRIPTION OF THE DRAWINGS

Fig. 1 is a proton spectrum of nuclear magnetic resonance (H-NMR) of the compound with formula I.

Fig. 2 is a ^ -RMN spectrum of secondary component A.

Fig. 3 is a ^ H-NMR spectrum of secondary component B.

Fig.4 is an H-NMR spectrum of secondary component C.

Fig.5 is a ¹ H-NMR spectrum of secondary component D.

Fig.6 is a spectrum

H-NMR of secondary component E.

Fig.7 is a spectrum

H-NMR of secondary component F.

Fig.8 is a spectrum

H-NMR of secondary component G.

Summary of the Invention

It has now been found that the compound with Formula I and several related secondary compounds are produced by means of controlled aerobic fermentation of Streptomyces silvensis ATCC No.53525 or ATCC No.53526

(I)

The compounds of formula I, their synthetic analogs and the related secondary compounds are antagonists of oxytocin and vasopressin. Oxytocin antagonists are useful for the treatment of premature labor (before term). Vasopressin antagonists are useful in the treatment and prevention of disorders of the renal and cardiovascular system of animals, including humans. The preparation and isolation of the compound of formula I is described. The in vitro activity of the compound of formula I, its analogs and related secondary compounds as oxytocin and vasopressin antagonists is also described.

DETAILED DESCRIPTION

The compounds of formula I and several secondary related compounds are produced by means of controlled aerobic fermentation of a new strain of Streptomyces silvensis, ATCC No. 53525 or ATCC No.53526.

microorganism ATCC No. 53525 or ATCC No. 53526 was isolated from the garbage of the forest floor in Pimpri, India. A deposit under the Budapest Treaty of a biologically pure culture of this microorganism was made at the American Type Culture Collection, Rockville, Maryland, on July 29, 1986, under no. ATCC Accessibility 53525. The microorganism ATCC No.53526 is a naturally occurring subisolate of ATCC No.53525.

The culture characteristics of this organism were compared with the culture descriptions of Streptomyces species given in Bergey's Manual of Determinative Bacteriology, Eighth Edition, 1974, Williams, & Wilkens, Baltimore, MD and the reports of the Inter National Streptomyces Project, Shirling, EB & D. Gottlieb: Cooperative description of type cultures of Streptomyces

II. Species description from first Study Intern. J. Syst. Bacteriol. 18: 69 189, 1968;

Shirling, E.B. & D. Gottlieb Cooperative description of type cultures of Streptomyces.

III. Additional Species descriptions from first and second studies, Intern. J. Syst. Bacteriol. 18: 279 392, 1968;

Shirling, E.B. & Gottlieb: Cooperative description of type cultures of Streptomyces.

IV. Species descriptions from the second, Third, and fourty studies. Inter. J. Syst. Bacteriol. 19: 391 512, 1969;

V. Add i t iona 1.

Descriptions, Inter J. Syst. Bacteriol. 22: 265 394, 1972.

organism has characteristics in common with Streptomyces fradiae but there are significant differences, such as the color of the vegetative mycelium and the. development of green color in specific environments and at certain temperatures.

As a result of the comparison, it was concluded that the organism represents a new species of Streptomyces, and has thus been designated as Streptomyces si 1 vensis sp. nov.

The cultural characteristics of Streptomyces silvensis, ATCC No.53525 or ATCC No.53526, are shown in Table I, below:

TABLE I

Cultural Characteristics of ATCC No.53526 (V = vegetative growth; A = aerial mycelium;

PS = soluble pigment)

Morphology: Sporophores of tufts of spore chains revealing loose, open hooks, loops and coiled ropes. The spore surface is soft (TEM) ..

Malt extract yeast extract agar (ISP Medium 2)

V: Reverse -Yellow color edged by light orange and purple

A: Mix of cream, coral and purple color

PS: None

Oatmeal Agar (ISP 3 Medium)

V: Reverse - reddish orange color

A: Light purple and cream colored becoming dark pink as the culture ages

BS: None

Inorganic salts-starch agar (ISP Medium 4)

V: Reverse - Coral mixed with purple

A: Pale purple mixed with cream color, becoming

darker pink as the culture ages

PS: None

Glycerol asparagine agar (ISP 5 Medium)

V: reverse -coral mixed with orange and purple color

A: Coral mixed with orange, purple and cream color

PS: None

Peptone-iron agar-yeast extract (ISP 6 medium)

V: Brownish-yellow1

A: None

PS: None

Melanin: None

Tyrosine Agar (ISP 7 Medium)

V: Reverse - greenish tint

A: Light greenish white edged with white and purple tones

PS: none

Egg albumin agar

V · 'Reverse-bright greenish yellow

THE: Yellow with a green matrix PS: none Agar Czapek Dox V: Reverse - cream and light orange THE: Cream color with yellow tones PS: none

USE OF CARBON

Basal Pridham-Gott1ieb medium + 1% carbon source + = growth;

+ = little or doubtful growth;

- = no growth compared to the control (the control) (negative) (no carbon source)

Glucose +

Arabinose +

Cellulose

Fructose +

Inositol +

Lactose

Maltose +

Mannitol +

Manose +

Rafinose

Ramnose

Sucrose +

Xylose +

Temperature Variation (yeast-dextrose extract agar + salts)

28 ° C

- Good vegetative and aerial growth with spore

37 ° C - Good vegetative and aerial growth with sporulation

42 ° C - No growth

50 ° C - No growth

Oxygen requirements (Culture carried out by deep inoculation in a culture medium solidified in a vertical column in a tube to minimize the surface, on yeast-dextrose extract agar + salts)

Aerobics

All readings were taken after three weeks and 28 ° C unless otherwise indicated. pH of all media approximately neutral (6.8-7.2).

Designations of the color numbers collected by Color Harmony Manual, 1958, 4th Edition, Container Corporation of America, Chicago, Illino i s.

Cultural Characteristics of ATCC No.53525 (V = vegetative growth;

A = air mycelium:

PS = soluble pigment)

Morphology: Sporophores of tufts of spore chains revealing loose, open hooks, loops and coiled strings.

The spore surface is smooth.

Yeast extract-malt extract agar (ISP 2 Medium)

V: Reverse - dark reddish orange edged with red.

mycelium of the substrate turns yellow with the addition of NaOH and turns red when acidified

A: A mixture of coral and cream color, developing shades of purple as the culture ages. The center appears yellowish green when the culture is incubated at 37 ° C.

PS: none

Oatmeal agar (ISP 3 Medium)

V: Dark reddish orange color

A: Purple and coral becoming dark pink as the culture ages

PS: none

Inorganic salts-starch agar (ISP 4 Medium)

V: Reverse - reddish orange color edged with red

A: Light coral becoming dark pink as the culture ages

PS: none

Glycerol asparagine agar (ISP 5 Medium)

V: reverse - orange

A: light coral and orange color edged with dark orange color

PS: none

Peptone-iron agar-yeast extract (ISP 6 medium)

V: Yellow-brown color

A: sparse, whitish

PS: None

Melanin: None

Tyrosine agar (ISP 7 medium)

V: Reverse - orange color mixed with olive green

A: The center is yellowish green edged with the color of orange and coral

PS: None

Egg albumin agar

V: Reverse-colorless mixed with green and purple edge THE: Lime green edged with a mixture of and cream color purple PS : none Agar of Czapek DOX V: Reverse - purple mixed with light and coral color orange THE: Light purple with light orange color and coral PS : none Use of carbon

Basal Pridham-Gottlieb medium + 1% carbon source;

+ = growth + = little or doubtful growth;

- = no growth compared to negative control (a testimony) (no carbon source)

Glucose + Arabinose + Cellulose Fructose + Inositol + Lactose + Maltose + Mannitol + Mannose + Rafinose + Ramnose + Sucrose + Xylose +

Temperature variation (yeast extract dextro agar + salts)

28 ° C - Good vegetative and aerial growth with spores

37 ° C - Good vegetative and aerial growth with spores

42 ° C - No growth

50 ° C - No growth

Oxygen requirements (Culture carried out by deep inoculation in a culture medium solidified in a vertical column in a tube to minimize the surface, in a yeast-dextrose extract agar + salts)

Aerobi ca

All readings were taken after three weeks at 28 ° C unless otherwise indicated, pH of all media approximately neutral (6.8-72)

Designation of the numbers of the colors collected from the Color Harmony Manual, 1958, 4th

Edition, Container Coproration of America, Chicago, Illinois

Fermentation Conditions compound of formula I and the related secondary compounds are produced by aerobic fermentation of appropriate aqueous nutrient media under controlled conditions by inoculation with a culture of the organism Streptomyces si1 vensis ATCC No.53526. The media contains sources of carbon, nitrogen, and assimilable inorganic salts. In general, carbohydrates (eg glucose, fructose, maltose, sucrose, xylose, etc.) can be used either alone or in combination with

sources of assimilable carbon in the nutrient medium. These carbon sources can be used individually or combined in between.

Generally, many protonaceous materials can be used as nitrogen sources for the fermentation process. Suitable sources of nitrogen include, for example, yeast hydrolysates, primary yeast, soy flour, cottonseed meal flour, casein hydrolysates, corn liqueur, soluble distillers or tomato paste, etc,

Organic salts of the nutrient that can be incorporated into the culture medium include usual salts capable of providing sodium, potassium, ammonium, calcium, phosphate, sulfate, chloride, carbonate, and the like ions. Trace metals such as cobalt, manganese, iron, magnesium, etc. are also included.

It must be taken into account that the nutrient media described here are merely illustrative of the wide range of media that can be used, and are not intended to be limiting.

Fermentation is carried out at temperatures ranging between about 20 ° C and 37 ° C; however, for optimal results it is preferable to conduct the fermentation at temperatures of about 28 ° C. The pH of the nutrient medium for the growth of Streptomyces silvensis, ATCC No.53525 or ATCC No. 53526, cultures and production of the vasopressin antagonist with formula I and various secondary related compounds can vary between about 6.8 and 7, 4.

It should be taken into account that for the production of fermentation of the compound of formula I and several related secondary compounds, the present invention is not limited to the use of Streptomyces s i1 vens is ATCC No. 53525 or ATCC No.53526, and especially It is desired and intended to include within the scope of this invention the use of other natural or artificial mutants produced or derived from the described cultures, or other variants or species of the genus Streptomyces to the extent that they can produce the compound of formula I or any of the various secondary related compounds. Artificial production of septic strains or mutant strains of Streptomyces from ATCC No. 53525 or ATCC No. 53526 can be achieved by conventional physical or chemical mutagens, for example, ultraviolet irradiation of the described cultures, or nitrosoguanidine tantamination , etc. Recent recombinant DNA techniques such as proto-plastic fusion, incorporation of plasmid, incorporation of chromosome fragments, etc., may also prove useful.

In a preferred embodiment of the present invention, the compound of formula I and its various secondary related compounds are produced by means of controlled aerobic fermentation of the microorganism Streptomyces silvensis, ATCC No.53525 or ATCC No. 53526. Fermentation should be carried out at a temperature ranging from about 20 ° C to 37 ° C, preferably at about 28 ° C. Generally the composition of the assimilable nutrient medium can vary widely. The essential ingredients of the nutrient are a carbon source and a nitrogen source. Other essential nutrients are provided by means of mineral salts such as chlorides, nitrates, sulfates, carbonates and phosphates of sodium, potassium, ammonium and calcium. The nutrient medium may also contain sources of inorganic elements such as magnesium, iron, copper, manganese

ganesium, zinc, cobalt, etc.

Typical carbon sources include: glucose, oils, organic acids, dextrin, starches, glycerol, etc. Typical nitrogen sources include: amino acids, cotton seed, figs, tomatoes, corn, etc.), animal offal, various hydrolysates (eg casein, yeast, etc.) and derivative products such as lard water and soluble distillation.

Maximum production of the compound with formula I can be achieved within 24 to 200 hours, usually within 72 to 96 hours, of fermentation under optimal conditions. The inoculum for fermentation can be provided from vegetative growth in a medium that supports rapid growth of the microorganism, nisno, or directly from spores.

Following fermentation, the compound accumulated with formula I can be separated from its secondary related compounds and recovered from the broth using conventional chromatographic techniques.

fermentation broth is filtered to separate the mycelium from the floating liquid (supernatant). These are extracted as follows:

A. The floating product is stirred with an equal volume of a moderately polar solvent, not miscible with water such as: chloroform, ethyl acetate, methyl methyl ketone, etc. The layers are allowed to settle; the organic phase contains all the compound with formula I initially locates

floating product.

B. Mycelia are shaken vigorously (homogenized) with various volumes of acetone, ethyl acetate, methyl methyl ketone, etc. These solvents will dissolve most of the compound with formula I located inside the mycelia.

The combined mycelial and floating organic extractors are then concentrated until a small volume is obtained under reduced pressure. The resulting mass is subjected to a series of dividing steps, by solvents and washing. The solvents chosen include petroleum ether, hexane, methylene chloride ether, methanol and similar solvents.

They can be adsorption and fractionation chromatographies, gel filtration, reverse phase liquid chromatography, etc., in conjunction with eluents with appropriate polarity and solubilization characteristics to provide the compound with formula I in the form of an almost powder. White.

The various secondary components of the fermentation broth can be recovered through a similar application of a series of chromatographic techniques. Seven secondary components, here referred to as A to G, were thus isolated from the fermentation broth of Streptomyces silvensis, ATCC No. 53525 or ATCC No. 53526. By weight of these secondary components, combined, are equivalent to about five percent by weight of the

isolated stand with formula I.

A number of different nutrient media can be used in the fermentation of Streptomyces s i1 vens i s, ATCC No. 53525 or ATCC No.53526. Variation of the medium or microorganism will vary the yield of the production of the compound with formula I and / or its rate of production. Variation of the medium or microorganism can also increase or decrease the type and quantity of various secondary related compounds present. in the broth. The compositions of the preferred media are shown in Table II.

TABLE II

MEANS

KE

Glucose 1, 0 g / 1 Dextrin 10.0 g / i Meat extract 3.0 g / 1 Ardamina 5.0 g / i Nzami na 5.0 g / 1 MgS0 ₄ , 7H ₂ 0 0.05 g / 1 Phosphate buffer 2.0 ml C to C 0g 0.5 g / i

pH 7.0-7.2

Phosphate cap

KH ₂ P0 ₄

On ₂ HP0 ₄

PH 7.0

YME

Yeast extract (Difco)

Malt Extract (Difco)

Glucose

Agar pH 7.0-7.4, 0 g / 1

95.0 g / 1

4.0 g / 1

10.0 g / 1

4.0 g / 1

20.0 g / 1

KL

Glucose 10.0 g / 1 L- Asparagine 1.0 g / 1 Yeast extract 1.0 g / 1

MOPS 25 mM p-2,000 - 1 drop / 50 ml pH 7.2-7.4

ΝΡΑ-4

Asparag i na 1.0 g / 1 Edam i na 2.5 g / 1 Pr i matona 2.5 g / 1 Yeast extract 5.0 g / 1 Malt extract 10.0 g / 1 Sucrose 5.0 g / 1 CaCO ^ 5.0 g / 1 pH 7.2-7.4 NPA-6 Asparagine 1.0 g / 1 Primaton 2.5 g / 1 Yeast extract 5.0 g / 1 Glucose 10.0 g / 1 C to C Οβ 5.0 g / 1 Mineral Salts 250.0 ml

ρΗ 7.2-7.4

-26 MINERAL SALTS

KC 1 0.74 g / 1 C to C12 0.02 g / 1 NaH ₂ P0 ₄ 1.4 g / 1 Citric acid 0.38 g / 1 MgCl ₂ 0.25 g / 1 Na ₂ S0 ₄ 0.36 g / 1

trace elements

VESTIGINAL ELEMENTS

HC1 Cone.

ml

FeC1 2 · 6H20 5.4 g / i MnCl ₂ .2H ₂ 0 2.0 g / i CuCl ₂ .2H ₂ 0 0.17 g / i CoCl ₂ .6H ₂ 0 0.48 g / i ^H 3 ^B0 4 0.06 g / i Na ₂ Mo0 ₄ 0.2 g / i

The expressions means of sowing and production are used as expressions of the technique. Generally, a seed medium supports rapid growth of the microorganism and an aliquot (seed) portion of this medium is used to inoculate a production medium for large-scale fermentation.

The following Examples describe the production of fermentation and isolation of the compound of formula I and the various secondary related compounds. These Examples are merely illustrative and are not intended to limit the scope of the invention.

Example 1

A frozen container (-80 ° C) with ATCC No. 53525 culture was thawed and the entire contents (2.0 ml) were inoculated into 54 ml KE seed medium in a 250 ml Erlenmeyer flask with divider. This seed was incubated at 28 ° C, 220 rpm for 4 days. A 4% inoculum was then used to inoculate each of the 4 2L Erlenmeyer flasks with divider containing 250 ml of NPA-6 medium. These flasks were incubated at 28 ° C, 220 rpm for 4 days. After harvesting, the vials were collected.

Example 2

A frozen container (-80 ° C) with ATCC No.53525 culture was thawed and the entire contents (2.0 ml) were inoculated into 54 ml KE seed medium in a 250 ml Erlenmeyer flask with divider. This seed was incubated at 28 ° C, 220 rpm for 1 day. A 4% inoculum was used to inoculate another KE seed vial, which was incubated at 28 ° C, 220 rpm for 3 days. A 4% inoculum of this seed was then used in 2 L Erlenmeyer flasks with a partition containing 250 ml of NPA-4 medium. After incubation at 28 ° C, 220 rpm for 4 days, these flasks were harvested and collected.

Example 3

A frozen container (~ 80 ° C) with ATCC No. 53525 culture was thawed and the entire contents (2.0 ml) were inoculated into 54 ml KE seed medium in an Erlenmeyer flask with 250 ml divider. This seed was incubated at 28 ° C, 220 rpm for 2 days. A 4% inoculum was used to inoculate another KE seed vial, which was incubated at 28 ° C, 220 rpm for 2 days. A 4% seed seed inoculum was used to inoculate 4 2 L Erlenmeyer flasks containing 250 ml of NPA-4 medium. After incubation

at 28 ° C, 220 rpm for 4 days these vials were collected and collected.

Example 4

The fermentation charge of Example 1 was stirred with ethyl methyl ketones (1.2 volumes) for half an hour. After allowing the layers to settle and separate, the organic phase was evaporated to dryness. The residue was redissolved in a small amount of methanol and fractionated by gel filtration on a 70 cc column packed with Sephadex LH-20. Elution with methanol provided a compound if mi-purified with formula I with an elution volume of 0.51-0.63 column volumes.

The fractions containing the compound with formula I were separated by drying, dissolved again in 0.5 ml of methanol and subsequently fractionated by RP-HPLC (CLEP-FR) (high performance liquid chromatography of the reverse phase) in a column Whatman Magnum-9 ODS-3- eluted at 8 ml / min. and 40 ° C with a 30 minute gradient of acetonitrile (from 35% to 100%).

Purified compound with formula I was obtained after 8.5 column volumes of the eluted product. The appropriate fractions were separated by drying under vacuum and examined spectroscopically.

Example 5 The fermentation broth of Example 2 was filtered through a Celite filter aid. The cells and the filtrate broth were extracted separately with vigorous stirring with successive portions of ethyl acetate. The two strata were combined, evaporated to dryness, reconstituted in a small volume of methylene chloride-methanol (3: 1) and fractionated at high speed through a 100 cc layer of Merck 60 silica gel surrounded by methylene chloride- methanol (95: 5).

Elution with a grade of methanol in methylene chloride from 5% to 100% proposed, added the components, secondary A and B semi-purified in 10% 10% methanol eluate. This solution was evaporated, the residue was redissolved in 0.5 ml of methanol and the final purification was achieved by HPLC (CLEP) (Whatman Magnum-9 ODS-3 column, eluted at 8 ml / min. And 40 ° C for 15 minutes with 35% aqueous acetonitrile, followed by a linear gradient of 100% acetonitrile over the next 30 minutes). Component Pure A (6 mg) was obtained at 14 column volumes of the eluate and Component B (35 mg) at 14.5 column volumes.

The presence of other members of this family of cyclic hexapeptides was recognized during the realization of this fermentation, however, its low concentration made isolation impossible. The isolation was carried out in later loads.

Example 6

The fermentation charges of Example 2 and Example 3 were filtered through a filter aid. The cells, containing over 85% of the components of interest, were extracted in a homogenizer twice with ethyl acetate. The combined extracts were separated by evaporation in vacuo. The solid residue was redissolved in 10 cc of methylene methanol chloride (92: 8) and passed slowly through a 100 cc column surrounded by silica gel. E. Merck 60 balanced with 8% methanol in methylene chloride. The column was eluted with the same solvent mixture. Components E, F and G were obtained together in Fraction I; Fraction II contained raw Component D.

Fraction I was evaporated to dryness, reconstituted in 0.5 ml of methanol, and this solution was fractionated at 42 ° C on a Whatman ODS-3 Magnum-9 column eluted at 8 ml / min. for 15 minutes with acetonitrile-water (4: 6) followed by a linear gradient of 20 minutes until a 50:50 mixture. The pure component F was obtained after 10 column volumes of eluent; The pure Component G (12 mg) was obtained after 13 column volumes; and pure Component E (8 mg) was obtained after 17 column volumes. Each fraction was separated by concentration under vacuum and freeze-dried in preparation for spectroscopic analysis.

The aforementioned fraction II was evaporated, redissolved in 0.5 ml of methanol and partial purification was obtained by passing this solution through a Magnum-9 ODS-3- column using 60% acetate

aqueous nitrile at 42 ° C administered at 8 ml / min. The final purification of Component D was performed by HPLC (CLEP) on the same column, eluting with acetonitrile: water 1: 1 at 8 ml / min. and 42 ° C.

compound was obtained as a large well by eluting to approximately 16-17 column volumes.

Example 7

Although its presence was detected by HPLC (CLEP) in previous fermentations, the isolation of Component C was carried out only during the purification of gram gram amounts of compound with formula I from large-scale charges.

_; Approximately 110 liters of fermentation broth / 14 L + 70 L loads of 7 were centrifuged using a continuous supply Sharples centrifuge. The compactly formed cells, which contained about 95% of the compounds of interest, were extracted as follows: twice successively with 5 liters of ethyl acetate, twice with 50% methanol in ethyl acetate, once with methanol and finally three times in succession with ethyl acetate-water (1: 1).

The last three extracts were combined and the layers were allowed to settle. The ethyl acetate phase was collected and concentrated until a thick oil was obtained. This was triturated with 50 cc of methylene methane chloride and left to stand at -20 ° C. The resulting heavy suspension was filtered to remove the soft (velvety) solids that had formed (containing only impurities) and the filtrate was evaporated off. The gummy residue was redissolved in 80 cc of methanol and passed through a 4.2 liter column of Sephedex LH-20, (in methanol). The loading of compound.com with Formula I mixed with Component C was eluted with between 0.66 and 0.77 column volumes.

The fractions were kept at 4 ° C overnight. More impurities were removed by filtration.

The soluble portion was dried to a solid under vacuum. The residue, mixed with 10 cc of 19: 1 ethylene methane chloride, was fractionated on 450 cc of silica gel. E. Merck 60 previously equilibrated with 3% methanol in methylene chloride. The elution of the doluna was performed at 10 ml / min. with the same solvent mixture. The compound of Formula I and Component C were obtained together in a large fraction (elution volume 1-1.5 column volumes). The appropriate fractions were separated by drying.

The separation of the two components was performed by HPLC (CLEP); a series of aliquots of methanolic solution of the solids obtained after the silica gel step was fractionated on an ODS-3 column (0 cm x 9 mm) eluting at 40 ° C with 50%

of aqueous acetonitrile administered at 8 ml / min. Component C was obtained as a very large peak centered around the 40 minute retention time (Retention volume 16 column volumes). These fractions were extracted with ethyl acetate, concentrated, on a rotary evaporator and the resulting suspension was freeze-dried to give the purified Component C.

Example 8

Characterization of Compounds with Formula I and

Secondary components solid material obtained in the aforementioned Example 4 was characterized by high resolution mass spectrometry and by nuclear magnetic resonance spectroscopy, as will be discussed below. Based on these data, the structure of Formula I was attributed:

(I)

Structure I as mentioned above is intended to include all possible stereoisomers. The structures of the analogue and secondary components are also intended to include all possible stereoisomers.

1. Mass Spectral Data

All mass spectra were recorded in an instant on a FinniganMAT 212 instrument by means of electron impact at 90 eV. Accelerated hydrolyses were carried out in 6N hydrochloric acid in airtight containers at 100 ° C for 18 hours. The amino acids were identified by GC-MS as

the derivatives being trimethylsi1i1. Trimethylsilyl derivatives were prepared with a mixture of BSTFA-pyridone at room temperature. Accurate mass measurements were made on the same instrument with high resolution using the peak matching method using perfluoro kerosene (PFK) as an internal standard

2. Spectra ^ H-NMR

The spectra were recorded at room temperature in Οθϋθ on a Varian XL-400 spectrometer. Chemical shifts are reported in ppm relative to tetramethylsi1 year (TMS) with zero ppm using the solvent peak at 7.15 ppm with internal reference. The spectrum for the compound of formula I is shown in Fig. 1.

3. ¹³ C-NMR spectra

Spectra were recorded on CD2Cl2 or CgDg at room temperature on a Varian XL-400 NMR spectrophotometer. Chemical shifts are indicated in ppm with respect to tetramethylsi1 year with zero ppm using the solvent peak as the internal reference at 53.8 ppm for CD2CI2 and 128.0 ppm for C ^ Dg.

Compound with Formula I: (CD ₂ C1 ₂ ) 12.1 15.7 20.521.3

24.4 24.9 25.5 26.4 31.7 34.2 35.1 37.8 46.65,

46.74 47.3 47.5 48.6 50.4 56.1 60.2 60.9126.8

127.0 128.54 (2χ) 128.6 (2χ) 129.6 (2χ) 129.7 (2χ)

137.56 137.63 171.0 (2χ) 171.3 171.8 173.5 and 174.7 ppm.

Component C (Οθϋθ): 20.7 20.9 (2χ) 23.7 24.224.4

24.7 24.8 26.2 32.1 35.4 36.2 36.9 45.4 46.1 46.6

47.0 49.5 50.5 58.1 58.5 59.6 126.7 126.8 127.5-129.5 (obsc), 129.7 (2χ) 130.4 (2χ) 137.5 138.7169,0

170.2 171.7 (2χ) 173.3 and 174.2 ppm.

Component D (CgDg) 10.8 15.4 20.7 21.0 21.07 23.8

24.2 24.4 24.8 25.0 26.5 (2χ) 32.1 35.1 35.5 36.3 45, 4 46, 2 46.7 47.0 49.5 54.4 58.1 58.7 59.7 126.7 128.6 (2χ) 130.4 (2χ) 138, .8 169.0 172.2 172, 6 173.3

and 174.3 ppm.

-38Component G (C ₆ D ₅ ) 11.0 15.1 20.4 20.7 22.9 23.1

23.7 24.4 25, 1 (2χ) 25.3 26.2 26.4 29.9 31.0 36.2 36.4 38.4 44, 1 46, 8 48.2 51.4 52.7 53.4 54.3 57.4 58.7 60.1 170.5 171.7 172.0 172.3 173.9 and 174.1 ppm.

Definitions

The following abbreviations will be used for amino acid residues:

ILE ................................ isoleucine

NOH-FE ............................. N-hydroxy-phenyl to 1anine

NOH-ILE ............................ N-h idroxi -i soleucina

NOH-LEU ............................ N-hydroxy-leucine

NOH-VAL ............................ N-hydroxy-va 1 ina

N-MeLEU ............................ N-met i 1 -1 eucí na

N-MeFe .............................. N-metyl-phenylalanine

Fe ................................. fen i I to 1 an i na

PIP

HN-CH-CO-H / \ ²

............ HN ÇH _O

I / ²

CH ₂ CH ₂

PRO ........................ proline

VAL ........................ valine

The structure of the component with formula I was investigated in detail and the structures of the other components were attributed by comparing the critical data.

COMPOUND WITH FORMULA I

HR ^ MS Found Calculated

Formu1 a

758.4135 758.4115 ^C 40 ^H 54 ^N 8 ° 7 740,4007 740,4010 ^C 40 ^H 52 ^N 8 ° 6 628.3380 628,3373 ^C 35 ^H 44 ^N 6 ° 5 544.2681 544.2686 ^C 31 ^H 36 ^N 4 ° 5 517.2792 517.2815 ^C 30 ^H 37 ^N 4 ° 4 487,2467 487.2471 ^C 29 ^H 33 ^N 3 ° 4 376,1759 376,1787 ^C 23 ^H 24 ^N 2 ° 3 257.1287 257.1290 ^C 15 ^H 17 ^N 2 ° 2 229.1312 229.1341 ^C 14 ^H 17 ^N 2 ° 229.1103 229.1103 ^C 14 ^H 15 ^N ° 2 134.0970 134.0970 ^C 9 ^H 12 ^N

compound of formula I contain an equivalent of each of Fe, N-MeFe, and PRO by hydrolysis. NMR additionally indicates two equivalents of PIP and one NOH-ILE. These six amino acids joined by peptide bonds in a cyclic structure are taken into account for the empirical formula. The complete peptide sequence is as follows:

PRO -> FE NOH-ILE> PIP PIP -> N-MeFe

T _____________________________

Data for Components A, B, C and D (DATA FOR THE COMPOUND WITH FORMULA I FOR COMPARISON)

HR-MS

Compounds Found Calculated Formu1 a Formula I ^C 40 ^H 54 ^N 8 ° 7 THE 744.3971 744,3959 ^C 39 ^H 52 ^N 8 ° 7 B 710.4117 710.4115 ^C 36 ^H 54 ^N 8 ° 7 Ç 758.4113 758.4115 ^C 40 ^H 54 ^N 8 ° 7 D 724,4267 724,4272 ^C 37 ^H 56 ^N 8 ° 7

.-41-

AMINO ACID COMPOSITION AA-3 AA-4 AA-5 AA-6 Compound AA-1 AA-2 Formula I PIP PIP N-MeFe PRO FAITH NOH_ILE THE PIP PIP N-MeFE PRO FAITH NOH-VAL B PIP PIP N-MeFE PRO VAL NOH-LEU Ç PIP PIP N-MeFE PRO FAITH NOH-LEU D PIP PIP N-MeFE PRO ILE NOH-LEU

Residues ΑΑ-3, ΑΑ-4, and ΑΑ-5 are found in the acid hydrolyzate. Residues AA-1, AA-2 and AA-6 are assigned by NMR.

CRITICAL FRAGMENTARY IOES

Compound M ⁺

Formula I 758 628 544 487 376 229 THE 744 614 530 473 376 229 B 710 580 496 439 328 229 Ç 758 628 544 487 376 229 D 724 594 510 453 342 229

STRUCTURES OF SECONDARY COMPONENTS A,

N-ÇH— CO

N — CH— CO / \

HN CH_ \ / ² ch ₂ ch ₂ - N —CH— CO - / \

HN CH_ \ / ² ch ₂ ch ₂

CH _O \ ² C = CH / \

HC CH W // HC —CH

Component ^R 1 Rn THE -CH (CH ₃ ) ₂ ~ ^CH 2 ^C 6 ^H 5 B -CH ₂ CH (CH ₃ ) ₂ -ch (ch ₃ ) ₂ Ç -CH ₂ CH (CH ₂ ) ₂ - ^CH 2 ^C 6 ^H 5 D -CH ₂ CH (CH ₃ ) ₂ -CH (CH ₃ ) CH ₂ CH ₃

DATA FOR COMPONENTS E, F and G

HR-MS

Component 1 Found ( ( AND 676.4275 F 692.4179 G 706.4392

Formu1 a

Calculated Molecular 676,4272 ^C 33 ^H 56 ^N 8 ° 7 692.4221 ^C 33 ^H 56 ^N 8 ° 8 706.4378 ^C 34 ^H 58 ^N 8 ° 8

Composition of AMINO ACIDS

Component E: Contains an equivalent of each from among

VAL, PRO, and N-MeLEU by hydrolysis. Two equivalents of PIP plus an equivalent of NOH-LEU (or NOH-ILE) suggested by MS (EM) are taken into account for the empirical formula.

Component F: Contains an equivalent of each of VAL, PRO, and N-MeLEU by hydrolysis. Component F contains more oxygen than Component E. An equivalent of each of PIP, hydroxy-PIP, and NOH-LEU (or NOH-ILE) is suggested by MS (EM) and is taken into account for the empirical formula.

Component G: Contains an equivalent of each from ILE PRO, and N-MeLEU by hydrolysis. An equivalent of each of PIP, a hydroxy-PIP, and NOH-LEU (or NOH-ILE) are suggested by MS (EM) and are taken into account for the empirical formula.

C-NMR suggests NOH-LEU.

The structures of components E, F and G have not been established but are not considered to be analogous to structures containing N-MeFe.

Example 9

Synthetic Analog of the Compound with Formula I

To a magnetically stirred mixture of the compound with Formula I (2.0 grams) in methanol (46 ml) and water (19 ml) with sodium acetate (2.68 grams) under nitrogen was added TiCl2 (20% aqueous) 1 ml)

The reaction mixture turned blue. After about 35 minutes the reaction mixture became colorless and a percipitate formed. A second 1 ml of TiCl _{3 was added} and after 35 minutes the solution became colorless again. Thin plate chromatography using plates of Analtech silica and methylene chloride (100 ml) stirred with 10 ml of concentrated aqueous ammonium hydroxide (95%) and methanol (5%) revealed the presence of starting material.

A third 1ml portion of TiCl2 was added and even though a light blue color appears after 1-1 / 2 hours, a fourth 1ml portion of TiCl2 is added. Thin plate chromatography as previously described indicated that the starting material was gone. The reaction mixture was filtered through Supercell, washed with methanol, slurried in methanol and filtered, and the methanol was evaporated in vacuo. The material was purified by means of high pressure liquid chromatography and identified as the dehydroxy analogue of the compound of formula I having the following structure:

(Ia) amino acid analysis: PRO, 0.99; ILe 1.01; Fe 1.00 Mass spectrum 742 (M +). ^ H-NMR was consistent.

Example 10

Synthetic Analog Oxidation Products

After synthesis of the compound of Example 9 and after standing at room temperature, oxidation products formed having the following structures:

Example 11

/ ^ Da h7 Oxytocin binding assay

Oxytocin receptors have been identified in uterine tissue of mammals through the use of radioligand binding assays in vitro.

These receptor binding sites mediate the uterine myometrial contractile response to oxytocin.

= 480 The present radioligand binding process was adapted from the methods of Soloff, MS and Grzonka, Z., Endocrinology, 1 19: 1564-1569 (1986) and Fuchs, AR., Fuchs, F., and Soloff, MS

J. Clinicai Endocrinology and Metabolism 60: 37-41 (1985) and is used here to measure the relative affinity of test compounds to the / H7 oxytocin-labeled oxytocin receptor. The assay uses a raw rat uterine membrane preparation as a source of oxytocin receptors. IC ^ q values (test compound concentration to inhibit 50% of / ~ ³ H7ocytocin binding) are determined by incubating uterine tissue with / H7 oxytocin in the presence of / or in the absence of varying concentrations of the compound, and as power estimates, they are inversely related to affinities.

TABLE III

POWERS OF COMPOUNDS IN CONNECTION TESTS

Oxytocin

Compound ^IC 50 / ' ³ H7 ot (nM) (Dtero) Formula I 370 + 70 Example 9 170 + 15 Example 10 42 + 29

I

Example 12

Antagonism to Vasopressin In Vitro

The biological activity of the compound with Formula I and the related secondary components is shown in Table IV, below. The table summarizes the in vitro potencies of the various components to compete with H-vasopressin binding to the two proposed vasopressin receptor subtypes (V ^ V ₂ ), as well as to inhibitors of vasopressin-stimulated adenylate cyclase activity, another measurement of antagonism to vasopressin.

/ H7Arginine Vasopressin Binding Assay, / ~ 3H binding assay 7Arginine Vasopressin is an in vitro process that has been adapted from the method of Guillon, et a, Eur. J. Pharmacol 85 219-304 (1982). This assay measures the relative affinity of the various test compounds to the vasopressin receptor (s) labeled / H7vasopressin.

Vasoprésine receptors have been pharmacologically divided into two subtypes: the V (depressant) receptor found on the liver and vascular smooth muscle and the V ₂ (antidiuretic) receptor found on the kidney medulla. The binding assay uses in a raw membrane preparation of the rat liver (V. receptor) or kidney marrow (V ₂ receptor) that is incubated with / ”' ^{* 3} H / vasopressin in the presence of / or in the absence of test compound. The IC _5Q values (concentrations to inhibit the binding of / “ ^B H7vasopressin to the various compounds by 50% (see Table III) are measures of their potencies and are inversely related to their affinities.

50Vasopressin exerts its antidiuretic effects on the kidney by stimulating the activity of adenylate cyclase. Thus, vasopressin-stimulated adenylate cyclase in the kidney marrow is used as a functional assay to determine the potencies of the various test compounds as antagonists of V ₂ -vasopresine. In this assay, the ΙΟ ^ θ values obtained for the various compounds (see Table III) are measures of their potencies to inhibit the conversion of ATP to cAMP by vasopressin-stimulated adenylate cyclase. This method was adapted from Seamon et al, PNAS 78: 3363-3367 (1981).

CL ίο OΌ

-Ρ

GO

Φ

Φ co

TABLE IV

CO

O * rcs o05

CD

The ’i— <

Ç-)

Ό «X

Ç

LO Φ ίο, GO

* - u X c x X d σ> X d d d r * X m d only · - O CD. +1 +1 · - 44 r * cn CD «. O * uo CD m O

Έ Έ X *S d CD CD CD d d un r * r * only Λ zs

(J X ε X d r * X d d ú0 O v σ · _Λ d χ X d E O X Έ Έ > s. o *> CT CO d σ » CD 44 CO 44 d d d • 44 O r * <o m - · M3 - ' CO r * · r * ♦ CM lO r- O Λ Z \ ^ 7 CM

H < PQ O Q M cold 0 <ϋ φ Φ d) d) d) d) d) '-1 P 4-> 4-> -P 4-> -P 4-1 5 ç Ç Ç ç Ç ç ç ε Φ d) d) d) φ d) d) ρ ç Ç G ç ç ç ç 0 0 0 0 O 0 0 0 you Qi Qi Qj Cb & Qj Cb ε ε ε AND ε ε ε O 0 0 s 0 s 0 0 0 O u O X u x u O u

O ο Η ο II Γ 'φ • Η η3 Ε 0 s • Η T3 Ρ Φ Ε —- Ή φ P s Qj 0 X O Τ3 P Φ ο α Φ φ ω Φ • ^ r 0 Ε Ό Φ 0 Ρ - ' υ Φ 0 ^ 4 Τ3 ·. Ο Φ 0 ε εη <Φ • Η Ρ ο 44 ΙΓ) <0 υ α Η X ω ·· 0 0 σχ Ρ τ3 ·. Ρ Η , - φ υ φ Φ ε Λ τι 0 Ç υ 0 • Η ω ο χ; 0 0 φ • Η ΙΦ TS Ç φ 'd) Ό Ε 0 Ç 4- * 0 ω Ο Λ d) Φ ω Ρ X Φ 0 φ Ρ γΗ Ρ (ύ 3 0 > s υ Φ Λ ο

The ability of the compound with formula I, its synthetic analogs and its related secondary compounds to antagonize oxytocin and vasopressin makes these compounds useful as pharmaceutical agents. These compounds will be especially useful in the treatment and prevention of premature labor (before term) or of pathological situations in which vasopressin may be involved, for example, congestive heart failure, hypertension, edema and hyponatremia.

compound with formula I, its analogs and its related secondary compounds or a pharmaceutically acceptable salt thereof, can be administered to a human being either alone or preferably, in combination with pharmaceutically acceptable vehicles or diluents, in a pharmaceutical composition, in accordance with with standard pharmaceutical technology. The compound can be administered orally or parenterally. Parenteral administration includes intravenous, intramuscular, intraperitoneal, subcutaneous and topical administration.

For oral use of an oxytocin or vasopressin antagonist of this invention, the selected compound can be administered, for example, in the form of tablets or capsules, or in the form of an aqueous solution or suspension. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch, and lubricating agents, such as magnesium stearate, are commonly used. For oral administration in the form of capsules the useful diluents are lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying agents

-53ci

fication and suspension. If desired, certain sweetening and / or flavoring agents can be added. For intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the active ingredient are usually prepared and the pH of the solution must be adjusted and buffered accordingly. For intravenous use, the total concentration of the solutes must be controlled to make the preparation isotonic.

When the compound of formula I, its synthetic analogs and its related secondary compounds or a salt thereof are used as an oxytocin or vasopressin antagonist in a human, the daily dosage will normally be determined by doctor's prescription. In addition, the dosage will vary according to the age, weight and response of each patient, and also according to the severity of the patient's symptoms. However, in most cases, a daily dose will vary between about 1 mg and about 1500 mg and preferably between 10 mg and 500 mg in a single dose or in divided doses. On the other hand, it may be necessary in certain cases to use dosages beyond these limits.

Claims (9)

1 - Process for the production of an oxytocin or vasopressin antagonist compound characterized by growing a strain of Streptomyces isolates in an aqueous nutrient medium by aerobic fermentation from an assimilable carbon source and recovering said compound. 2. The method of claim 1, wherein the strain of Streptomyces is isolated from ATCC No. 53525 or ATCC NO. 53526. 3®.- Process according to claim 1 or 2, characterized in that cyclic peptides are produced with the formula: N- CH - CO - N - CH - CO - N - CH - CO where R have the following meanings: Cot.pos to R ¹ R ² R ³ I -Cri (CH ^) Cri ₂ (CH ₃ ) - ^CH 2 ^C 6 ^H 5 -OH Ia -Cri (CH j) CH ₂ (CH ₃ ) - ^CH 2 ^C 6 ^H 5 -H THE -ch (ch ₃ ) ₂ -CH_C _r H _c z O 2> -OH B -Ch ₂ CH (CH ₃ ) ₂ -CH (Cri ₃ ) ₂ -OH ç -ch ₂ ch (ch ₃ ) ₂ -CHCH. 2 b o -OH D -ch ₂ ch (ch _{3> 2} -CH (Cri ₃ ) CH ₂ CH ₃ . -OH or their pharmaceutically acceptable salts. 4. A process according to claim 1 or 2, characterized in that cyclic peptides are produced with the formula: or their pharmaceutically acceptable salts: 5. ^A process according to claim 1 or 2, characterized in that cyclic peptides are produced with the empirical formula ^C 33 ^H 56 ^N 3 ° 7 and the NMR spectrum of Fig. 6; PPM the empirical formula ^C 33 ^H 56 ^N 8 ° 8 ^{and 0 is} P ^{ectr0 de} of Fig-7; the empirical formula ^C 34 ^H θΝθΟθ and the NMR spectrum of Fig.8; 6 ^? Method for antagonizing vasopressin in animals, characterized in that a compound obtained according to claim 1, 2, 3, 4 or 5 is administered to a patient, the daily dosage range being from 1 mg to 1500 mg. 7 ^? Method for antagonizing oxytocin in animals, characterized in that a compound obtained according to claim 1, 2,3, 4 or 5 is administered to a patient, the daily dosage range being from 1 mg to 1500 mg. 8 ^â .- Biologically pure culture characterized by being from the organism Streptomyces si1vens is. 9 ^? Culture according to claim 8, characterized in that the microorganism is ATCC No.53525 or ATCC No.53526.