Memnopeptides, method for their preparing and their applying

FIELD: organic chemistry, peptides, medicine, pharmacy.

SUBSTANCE: invention relates to peptide derivatives named as memnopeptides that are used as an active component for manufacturing a medicinal preparation used in treatment of bacterial infection. Invention proposes compound of the formula (I): wherein radicals R1, R2, R3, R4, R5, R6, R7, R8 and (A)n have corresponding values, or its salt. Compounds of the formula (I) are prepared by culturing microorganism Memnoniella echinata FH 2272, DSM 13195 under suitable conditions in the nutrient medium containing at least one source of carbon atoms and at least one source of nitrogen atoms and the process is carrying out until the accumulation of at least one compound of the formula (I) in the nutrient medium followed by isolation of indicated compound. The attained technical result involves the development of a pharmaceutical composition eliciting an antibacterial activity. The development of the preparation provides expanding assortment of agents used in treatment of diseases said above.

EFFECT: improved preparing method, valuable medicinal properties of compounds.

10 cl, 2 tbl, 7 ex

 

The invention relates to new derivatives of peptides, called mimepartid, which can be obtained by fermentation Memnoniella echinata FH2272, DSM 13195 in culture medium, method of producing menoitios and menoitios as pharmaceuticals, for example, for the production of a pharmaceutical preparation for the treatment of heart failure.

Cardiovascular disease still ranks first as a cause of death in Western industrial countries. A significant part of them are patients with a diagnosis of heart failure. The meaning of the term "heart failure" implies inadequate functioning of the heart. Heart is not able to produce emission that corresponds to the needs of the mammal. Heart failure is an acute or chronic heart failure in load conditions or even alone to provide the blood needed for metabolism, or to take venous return. It is a condition of the heart in which such compensation mechanisms as heart rate, stroke volume or high blood pressure is no longer sufficient to maintain a normal cardiac output. Heart failure has many causes, for example, inflammatory and degenerative change is of the myocardium (heart muscle), disorders of the coronary circulation and myocardial infarction. Heart failure leads to changes in the peripheral circulation, respiratory disorders, renal function and electrolyte balance, as well as to decrease the strength of the skeletal muscles, and in the end it often leads to death.

Heart failure mostly occurs in old age. Its frequency is 3 disorders in a year per 1000 of the population aged 35 to 64 years and 10/1000/year in the age group from 65 to 94 years. The factor of mortality in persons aged 75 years increased by almost 200 compared with the age group from 35 to 44 years. As shown by studies in the United States, from 1970 to 1983 mortality remained approximately constant. For the Federal Republic of Germany the same figures to be determined. More than 50% of patients die within the first 5 years after diagnosis. This statistical study itself shows the great importance of heart failure for the population, but it also proves inadequate medical treatment that are available to the physician at the present time.

Given the inadequacy of modern methods of treatment have been developed new concepts that should lead to the creation of new cardiac drugs. The ability of cardiac and skeletal muscle juice is to amatice and thus, to perform mechanical work, depends on (1) the contractile structural elements (myofibrils) and (2) chemical energy (ATP)available for myofibrils, which is transformed into mechanical energy in the reduction process. Shortening myofibrils occurs in the reduction process. This can be initiated by motor nerve impulses, under the influence of which calcium ions (CA2+come sarcoplasmatic space from the extracellular space within a few milliseconds, and the depot of calcium emptied. When failure of the myocardium (heart failure) the concentration of CA2+in the myofibrils can be reduced. However, ions of CA2+play a key role in the activation of the contractile apparatus. If there is increased demand, and CA2+mainly injected in sarcoplasmatic reticulum (SR) under the influence of catalysis membrane CA2+dependent Mg2+-ATPase: this enzyme is also called CA2+ATPase Sarko(endo)plasmatic reticulum (SERCA2). In accordance with hydropathicity analysis of CA2+ATPase contains 10 transmembrane helices and a number namebrand loops. With the cytosolic side of the formed domains binding of CA2+and ATP, phosphorylation and interaction with modulatory protein phospholamban (PLB). The last performance is possessing a protein pentamer, which is localized in the membrane of CF and exerts an inhibitory influence on SERCA2 in nefosfaurilirovanna condition. In conditions of physiological stress occurs phosphorylation of PLB, which increases the affinity of SERCA2 and CA2+and, thus, increases the rate of transport of ions of CA2+in CF. Phosphorylation of PLB (protein of 52 amino acids) occurs at two amino acid residues: serine-16 can be phosphorylated by cyclic amp-dependent protein kinase and threonine can be phosphorylated in position 17 kinase-dependent CA2+/calmoduline. Phosphorylation causes conformational changes in PLB, followed by an increased affinity of SERCA2 and CA2+. Antibodies against PLB able to mimic the effect of phosphorylation of PLB and, thus, confirm the key role of PLB as a regulator of contractile activity of the heart (Phospholamban: Protein Structure, Mechanism of Action and Role in Cardiac Function. H.K. Simmermann and L.R. Jones, Physiological Reviews; Vol.78, No. 4,921ff, 1998). Thus, activators SERCA2 should have a beneficial effect in heart failure.

Unexpectedly it was found that the culture of the strain of fungi Memnoniella echinata FH 2272, DSM 13195 contain natural substances that are able to demonstrate a beneficial effect on heart and circulation. Selected active compounds, menapace, are natural substances, VK is chausie specific constituent groups. These compound groups include terpene units, the so-called polyketide part, and a group containing hydrogen.

Terpenes are naturally occurring compounds that can formally be interpreted as the products of polymerization of the hydrocarbon isoprene. In accordance with a variety of isoprene groups can distinguish monoterpene (C10), sesquiterpenes (C15), diterpenes (C20and so a Large number of compounds can be formed from the parent structures by substitution, cyclization, rearrangement, oxidation, etc.; accordingly, in the literature it has been described many thousands of terpenes. It was also reported nitrogen-containing compounds derived from terpenes, but they are listed among alkaloids (e.g., alkaloids Gentiana) [Römpp Chemie Lexikon [Römpp''s Chemical Encyclopedia], 9thEdition, Volume 6, page 4508 ff., Georg Thieme Verlag, Stuttgart/New York, 1992]. However, these terpenes are fundamentally different from menoitios in accordance with the invention, in which the terpenes do not contain polyketide group, through which they can fix nitrogen.

Examples of other known nitrogen-containing terpenes are:

- Sahibiydi [J.Antibiotics, 48:1396 (1995)];

- Stachybotrys [Y.Nozawa et al. J.Antibiotics, 50:635-645 (1997)];

- Spirodihydrofurans the lactam [J.Antibiotics, 49:13 (1996)];

- Nakedgaymen [Tetrahedron, 51:10867-10874 (1995)];

- F1839-A-F1839-J presented Aut a nitrogen-containing terpenes, having polyketide part of [the Japan patent 061864133 and 08283118]. They are inhibitors cholesterylester.

Data terpene derivatives were synthesized from different strains of the genera Memnoniella echinata and Stachybotrys and others. They have been described as antagonists endothelioma receptor, as inhibitors of the protease of HIV-1 and cholesterylester and as tonics for hair. Inhibitor inositoltrifosfata L-671,776 was also isolated from cultures of strain Memnoniella echinata ATCC 20928 [Y...Lam et al. J. Antibiotics, 45, pp.1397-1402, (1992)].

Menopace in accordance with the invention have a different spectrum of activity. Characteristic is their activating effect on SERCA2 and, thus, at the heart of his failure.

Thus, the present invention relates to compounds of formula (I)

in which

R1and R2together represent a double bond, or H2or H and HE, or H and O-C1-C4-alkyl;

R3and R4together represent a double bond, or H2or H and HE, or H and O-C1-C4-alkyl;

R8selected from H, HE, C1-C4-alkyl and O-C1-C4-alkyl, such as O-methyl;

R6represents a group of formula (II)

in which

R9represents the t a N or sugar, linked glycosidic bond, or a group of the formula (III)

in which R10represents H or sugar, linked glycosidic bond;

and in which,

if R6represents a group of formula (II), R5represents a bond with the carbon atom C9 formula (II), and R7represents H, or R7represents a bond with the carbon atom C9 formula (II), a R5represents H and

if R6represents a group of formula (III), R5and R7represent N;

But is an amino acid;

n is an integer selected from 1 to 12, each As the same or different from each other And;

in which the nitrogen atom in isoindoline ring of formula (I) represents the nitrogen of the N-terminal amine of the first amino acids of group (A)n;

or their salts or a derivative thereof;

provided that

But is an amino acid other than Glu, when n is 1 and R1and R2together represent a double bond and R3and R4together represent H2, a R6represents a group of formula (II), a R5represents a bond with the carbon atom C9 group of the formula (II), and R7represent H, a R8represent N and R9represent N.

the formula (I) (A)n can be a, at least one natural amino acid selected from Gly, Ala, Val, Leu, Ile, Pro, Ser, Thr, Phe, Tyr, Trp, Lys, Arg, Asp, His, Glu, Asn, Gin, Cys and Met. A(n) can be a peptide chain having from 2 to 12 amino acids. In one embodiment, the implementation of A(n) consists of 10 amino acids.

With1-C4-alkyl represents remotemachine or branched alkyl having from 1 to 4 carbon atoms, such as methyl, ethyl, ISO-propyl or tert-butyl.

Sugar may be a hexose, such as aldohexose, such as mannose, glucose or galactose, which may be optionally substituted by additional groups, such as C1-C4-alkyl or NH2.

The present invention also applies to all the obvious derivative of compounds of formula I. Derivatives are salts, reduction products, esters, ethers, acetates, as well as amides and products N-alkylation, in addition, all optical antipodes, diastereoisomers and all stereometria form.

In one implementation, the compound of formula (I) has the formula (IV)

in which R1, R2, R3, R4, R9and (A)n have the meanings as indicated above. In the formula (IV) R1and R2taken together, can represent a double bond, R3and R4together you can imagine with the th H 2and R9can represent N.

In one implementation, the connection of the invention is menopace A, C76H108N16O18's formula (IVa)

In this formula declinata structure with 4-methyl and methylene group, is a terpene part; substituted isoindoline ring is getitnow part. Amino acid sequence:

Met His Gln Pro His Gln Pro Leu Pro Pro (SEQ ID NO:1)

is part of the casein sequence. Methionine (Met) can be oxidized to a sulfoxide.

Formula (IVb) shows the preferred spatial form:

where (A)n has the meaning as specified above.

Physico-chemical and spectroscopic properties of preferred compounds in accordance with the invention can be summarized as follows:

Menopace And

Appearance:Colorless substance soluble in polar organic solvents and in water.
HasStability in a neutral, slightly acidic environment
Empirical formula:C76H108N16O18S
Molecular weight:165,87 Yes
The absorption of UV light (λmax):270 nm
These NMR:Cm. table 1

The numbering of the carbon atoms and the associated NMR chemical shifts classified in accordance with the procedure of numbering for cyclic sesquiterpenes.

Figure 1: Numbering of cyclic sesquiterpenes of ketolide.

Table 1

These NMR (chemical shifts) of metropathia And in DMSO - d6when C.
δ-13mδ-1HmnJNNnJCH (10 Hz)C-t
115.42kV0.668d1.8021.802The terpene-8-Me
215.72kV0.978s-2.035, 1.763The terpene-10-IU
320.37t1.409m2.035, 1.5382.035The terpene-6
1.4602.035
421.43kV0.873d1.6781.678, 1.428, 0.890Leu8
522.25kV0.827s0.9180.918, 2.035The terpene-4-Me
622.64t2.182DDT4.892, 2.788, 2.6324.892, 2.788, 2.632Meth1
2.307DDT
22.80t2.19DDT4.869, 2.757, 2.6354.869, 2.752, 2.635
2.31DDT
723.08kV0.890d1.6781.678, 1.428, 0.873Leu6-β'
8At 23.88 of usherd1.678m0.87, 0.88Leu8
923.90t1.764mm 0.962, 1.416, 1.8710.978, 1.416, 3.227The terpene-1
0.962

/tr>
δ-13mδ-1HmnJNNnJCH (10 Hz)C-t
1024.22t1.910m2.144, 4.588, 3.4672.144, 1.771, 4.588,

3.467, 3.689
Pro9
11At 24.32t1.863m2.031, 1.798, 3.6254.356, 3.625, 2.153,

1.792
Pro4
1224.37t1.927m(2.153), (1.814),

3.624
4.388, 3.624, 1.814Pro7
1324.43t1.926m2.144, 1.840,

3.655, 3.538
4.224, 3.655,

3.538
Pro10
14At 24.77t1.840

1.416
m

m
1.416, 1.740, 0.962

1.840, 0.926
-The terpene-2
1526.51t1.940

1.719
m

m
1.719, 4.484, 2.198

1.719, 4.484, 2.198
2.198Gln3
1626.85t1.927

1.722
m

m
1.722, 4.468, 2.166

1.927, 4.468, 2.166
2.166Gln6
1727.04 usert2.976

3.067
DD DD4.598-His5
1827.09t2.937

3.067
DD DD4.570(4.570)His2
1927.50t2.144

1.771
m

m
1.771, 4.578, 1.905,

1.945

2.144, 4.578, 1.905,

1.945
1.945, 3.689, 3.467,

4.578
Pro9
2028.34t2.144

1.840
m

m
1.840, 4.223, 1.926

2.144, 4.223, 1.926
4.223, 3.669,

3.533
Pro10
2128.50KB0.918s0.8270.827The terpene-4-IU
2228.86t.798

2.031
m

m
4.356, 2.031,

(1.863)
Pro4
2328.89t1.814

2.012
m

m
2.012, 4.388, (1.927)

1.814, 4.388, (1.927)
4.388, 3.646Pro7
24At 30.63t1.538

1.430
m

m
1.430, 1.460, (1.409)

(1.802)

1.538, 1.460,

1.802
0.667Terlan-7
2530.64t2.166t1.927, 1.7221.927, 1.722, 4.466,Gln6
26At 30.65t2.192t1.940, 1.7191.940,Gln3
2731.62t3.154

2.792
d

d
2.792

3.154
6.598The terpene-11
2836.43d1.802dqu1.538, 1.430, 0.6670.667, 2.790, 3.156The terpene-8
2937.22S---0.903, 0.824The terpene-4
3037.73

37.84
KB2.548

2.546
s-2.682, 2.758

2.633, 2.783
Meth1

δ-13mδ-1NmnJNNnJCH (10 Hz)C-t
3139.45d2.035DD1.409, 1.4600.903, 0.824, 0.971The terpene-5
3240.07

40.03
t1.428dt4.536, 1.6780.890, 0.873,

4.536
Leu8
3341.71s---3.156, 2.790, 0.977, (1.764), 2.035The terpene-10
3444.25t4.332--4.881The terpene-8'
3546.10t3.669

3.533
1.9344.223, 2.147, 1.934,

1.851
Pro10
3646.53t3.689

3.467
m

m
1.905,1.9364.588, 2.165, 1.781,

1.936
ro 9
37At 46.81t3.624m1.9271.814, 4.388Pro7
3846.87t3.644m1.863-Pro4
3948.56d4.534dt7.918, 1.4287.918, 1.428,1.678Leu8
4049.44

At 49.70
t t2.682

2.758 2.633

2.783
DDT

DDT DDT

DDT
2.758, 2.162, 2.289

2.682, 2.162,2.289 2.783, 2.289 2.162

2.633, 2.289, 2.162
4.892, 2.550

4.869, 2.545
Met1
4150.18d4.466dt8.110, 1.927, 1.7222.166Gln6
42At 50.31d4.484dt8.206, 1.935, 1.7072.182Gln3
43At 51.37d4.570DDD8.135, 2.975, 3.0822.975, 3.082His2
44At 51.68d4.590

4.585
DDD8.496, 2.934,3.067

8.506, 2.934, 3.067
2.934, 3.067His2
4553.30

53.62
d4.892

4.869
2.162, 2.284,

2.162, 2.284
2.162Met1
4657.33d4.588DD2.144, 1.7712.144,

1.771, 1.905,
Pro9
4758.31d4.223DD2.144, 1.8403.669,

3.553,1.926,
Pro10
4859.20d4.388DD2.012, 1.8142.012Pro7
4959.44d4.356DD2.031, 1.7983.635, 1.880Pro4
5073.50

73.51
d3.227DD1.840, 1.4160.903, 0.824The terpene-3
5197.93

97.91
s-3.154, 2.792,

0.972, 0.668
The terpene-9
52100.90

100.88
d6.598

6.596
--The terpene-3'
53112.62

112.55
s---6.593, 4.330The terpene-5'

δ-13mδ-1HMnJHHnJCH (10 Hz)C-t
54116.92

116.90
s---3.154, 2.792, 6.597The terpene-1'
55116.95d7.230s8.655(3.067), 2.937,

8.665
His2
56117.26d7.322s8.7713.092, 3.000,His5
57129.32s---8.783, 7.322, 3.083,

2.976, 4.585
His5
58129.63s---7.230, 3.064, 2.937,

4.592
His2
59133.00

132.96
s-- -4.3301-4'
60133.63d8.655s7.2307.230His2
61133.68d8.771s7.3207.320His5
62153.60

153.62
s---6.597, 3.157,

2.792
The terpene-2'
63155.73s---(6.597), 3.154,

2.792, 4.330
The terpene-6'
64168.11

168.07
s---6.597,4.330, 4.881The terpene-7'
65169.57s---4.588, (4.223)Pro9-CO
66169.59s---8.110, (4.570)His5-CO
67169.69s---4.534, 1.444Leu8-CO
68169.74s--- 8.207, 2.937, 4.585His2-CO
69169.97

169.89
s---8.513, 4.869, 2.190

8.499, 4.892, 2.182
Met1-CO
70170.07s---4.471, 1.733,

1.924, 4.356
Gln3-CO
71170.21s---4.466, 1.722, 1.927,

4.388
Gln6-CO
72170.99s---4.538, 4.388, 2.012,

1.844, 7.918
Pro7-CO
73171.32s---8.138, 4.356, 2.003,

1.805, 4.570
Pro4-CO
74173.10s---4.225, 2.144, 1.840Pro10-CO
75173.80

173.81
s---2.19, 1.93, 1.733-Gln-δ-CO
76173.88

173.86
s---7.26, 2.19, 1.93,

1.73
4-Gln-; -CO
HE9.75UserNOE: 6.598The terpene-2'-HE
NH8.506

8.496
DD4.585 4.590NOE: 4.869/4.692His2-NH

Table 2

δ-13mδ-1HMnJHHnJCH (10 Hz)C-t
NH8.206d4.484NOE: 3.072, 2.948,

4.588
Gln3-NH
NH8.135d4.563His5-NH
NH28.110d4.470NOE:4.570, (4.470),

1.727, (1.927)
Gln6-NH
NH27.918d4.534NOE: 4.388, 1.827,

1.688,1.427
Leu8-NH
NH2 7.260

6.807
s

s
(6.807) (7.260)Gln6-NH2
NH27.230

6.789
s

s
(6.789) (7.230)Gln3-NH2

In one embodiment of the invention the compounds of formula (I) can be obtained by fermentation Memnoniella echinata FH 2272, DSM 13195 or one of its variants or mutants in suitable conditions in a culture medium until at least one menopace formula (I) will not be present in the culture medium, followed by separation of menoitios.

Therefore, the invention also relates to a method for obtaining compounds of formula I, which comprises cultivating a microorganism Memnoniella echinata FH2272, DSM 13195 in culture medium up until at least one menopace formula (I) will not be present in the culture medium, and the subsequent allocation of metropathia from the culture medium.

In one embodiment, the implementation strain FH2272, DSM 13195, its mutants and/or variants are cultivated in nutrient solution (also called the cultural environment), including at least one source of carbon atoms and at least one source of nitrogen atoms and, optionally, include conventional near adicheskie salt, until menopace will not be present in the culture medium. Then menopace can be isolated from the culture medium and, optionally, separated into individual active components and subjected to cleaning. In one implementation, menopace accumulate in the culture medium and separated into individual components.

In another implementation, at least one source of nitrogen atoms used for the culture medium is chosen from amino acids and peptides. Amino acids and peptides that are used as sources of nitrogen atoms that can be the same as the group (A)n as defined above.

The method in accordance with the invention can be used for cultivation in laboratory scale (ranging from ml to l) and on an industrial scale (scale m3).

Was grown colony Memnoniella echinata FH2272, characterized by a strong producing ability. The isolate was deposited on 14 December 1999, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B, 330 Brunswick, Germany, in accordance with the rules of the Budapest Treaty under the following number: Memnoniella echinata FH 2272, DSM 13195.

Memnoniella echinata FH 2272, DSM 13195 has a brown-green mycelium and characterized conidiophore, typical for the genus Memnoniella.

Variants and mutants of strain Memnoniella echinata FH 2272, DSM 13195 can also be used for the synthesis, what about the least one connection menoitios in accordance with the invention. Such mutants can be obtained by per se known method, physical methods, for example, irradiation, such as irradiation with ultraviolet or x-rays, or chemical mutagenesis, using such reagents as ethylmethanesulfonate (EMS), 2-hydroxy-4-methoxybenzophenone (MOB) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Possible options include closely related species of fungi Stachybotrys, such as Stachybotrys atra, Stachybotrys chartarum or Stachybotrys complementi.

Screening for mutants and variants that synthesize at least one connection of menoitios in accordance with the invention may be carried out in accordance with the following scheme:

- Separation of the mycelium after cultivation;

Extraction of the mycelium with an organic solvent;

Extraction of menoitios from the culture filtrate using solid phases;

Analysis by HPLC descending chromatography or testing of biological activity.

The following conditions of cultivation are mushroom Memnoniella echinata FH 2272 deposited to isolate DSM 13195 and its mutants and variants.

In one embodiment, the implementation of the nutrient solution comprising at least one source of carbon atoms, casein peptone as a source of nitrogen atoms and normal is Neorganicheskie salt, Memnoniella echinata FH 2272 produces menopace A. In one embodiment, the implementation of Memnoniella echinata FH 2272 is a DSM 13195.

Possible sources of carbon atoms for aerobic cultivation include assimilated carbohydrates and sugar alcohols such as glucose, lactose, sucrose, starch, dextrin, fructose, molasses, glycerol, galactose and D-mannitol, and carbohydrate natural products, such as malt extract. Possible sources of nitrogen atoms include, for example, amino acids, peptides, proteins, degradation products of amino acids, peptides and proteins, such as casein, gelatin and tryptone; meat extracts; yeast extracts; extracts of peanuts; sprouted seeds, such as corn, wheat, beans, soybeans and cotton; compositions containing seeds; residues of distillation in the production of alcohols; meat flour; yeast extracts; ammonium salts and nitrates. In one implementation, at least one source of nitrogen atoms chosen from synthetically derived peptides and biosynthetic derived peptides. Nutrient solution optionally includes at least one inorganic salt. In one embodiment, the implementation of the inorganic salt is selected from chlorides, carbonates, sulfates and phosphates. Sulfates and phosphates can be selected from sulfates, alkali metal phosphates, alkali metal sulfates saloon the land of metal and alkaline earth metal phosphates, and alkaline earth metals selected from iron, zinc, cobalt and manganese.

The formation of menoitios in accordance with the invention can be carried out in a nutrient solution comprising casein peptone. In one embodiment, the implementation of the nutrient solution includes a concentration of casein peptone in the range from about 0.05 to 5%. Another embodiment includes the concentration of casein peptone in the range from 0.1 to 1%. Another embodiment includes the concentration of casein peptone in the range from 0.2 to 5%. Nutrient solution can include glucose. One embodiment includes a glucose concentration in the range from 0.5 to 3%. Nutrient solution can also include corn hydrolysate. In one embodiment, the implementation of the nutrient solution includes a concentration of corn hydrolysate in the range from 0.05 to 1%. Another embodiment includes a concentration of corn hydrolysate in the range from 0.1 to 0.5%. Nutrient solution can include trace amounts of at least one component selected from potassium chloride, magnesium sulfate and iron sulfate.

In one embodiment, the implementation of the nutrient solution includes casein peptone, glucose, corn hydrolysate and trace amounts of potassium chloride, magnesium sulfate and iron sulfate. In another embodiment, the implementation of nutrient R is the target includes the concentration of casein peptone in the range from about 0.05 to 5%, the glucose concentration in the range from 0.5 to 3%, the concentration of corn hydrolysate in the range from 0.05 to 1%, and trace amounts of potassium chloride, magnesium sulfate and iron sulfate. The percentages in each case are connected with a whole lot of the nutrient solution.

This nutrient solution Memnoniella echinata, which can be Memnoniella echinata FH 2272 DSM 13195, forms a mixture of menoitios. Depending on the composition of the nutrient solution quantitative share one or more menoitios in accordance with the invention can vary. In addition, synthesis of some menoitios can be controlled by the composition of the environments so that menopace may not be produced or may be produced by microorganisms in amounts below the limit of detection.

Cultivation of the microorganism can be carried out in aerobic conditions, ie, for example, by immersion with shaking or stirring in shake flasks or fermenters, not necessarily with the introduction of air or oxygen. It can be carried out in the temperature range from about 18 to 37°S, in a narrower range from about 20 to 32°and in a more narrow range from 25 to 30°C. the pH Range should be from 6 to 8, for example, from 6.5 to 7.5. In General, the microorganism is cultivated under these conditions, during the period from 24 to 300 hours, more common from 36 to 140 hours

P is imushestvenno cultivation can be carried out in several stages, i.e. at least one preculture can be obtained in a liquid nutrient medium and can then be sown in the actual production environment, the main culture, for example, in a volume ratio of 1:10. Preculture can be obtained, for example, the sowing of the mycelium in the nutrient solution and the opportunity to grow within about 36 to 120 hours, for example, within 48 to 72 hours Mycelium can be obtained, for example, by allowing the strain to grow for about 3 to 40 days, for example, from 4 to 10 days, on a solid or liquid nutrient medium, for example, on malt-yeast agar or potato dextrose agar (standard environment for fungi, for example, sold by Difco).

Over the course of cultivation can be monitored by controlling the pH of the cultures, or the amount of mycelium, as well as using chromatographic methods such as thin layer chromatography or high performance liquid chromatography, or testing of biological activity. Connection in accordance with the invention may be in the mycelium and in the culture filtrate, but the biggest part is usually found in the filtrate of the culture.

The following allocation process designed to clean menoitios in accordance with the invention, for example, metropathia A. Separation is s or cleanup metropathia in accordance with the invention from the culture medium can be carried out in accordance with known methods of the chemical, physical and biological properties of natural substances. To test concentrations of menoitios in culture medium at different stages of selection could be applied thin-layer chromatography, for example on silica gel, using as eluent isopropanol/25% NH3or HPLC. Determining when selecting thin-layer chromatography may be performed, for example, by staining reagents, such as chlorosulfonic acid/glacial acetic acid, and the number of formed substances should be compared with a calibration solution.

To highlight metropathia in accordance with the invention mycelium in General first removed from the culture broth using conventional procedures, and then menopace extracted from the cell mass using optional miscible with water and organic solvent. Phase organic solvent contains natural substances in accordance with the invention; it may not necessarily be concentrated in vacuum, and the residue may be subjected to further purification as described below. In one implementation, menopace extracted from the culture by adding a solvent to the mixture of mushrooms and environment.

The culture filtrate may optionally be combined with the concentrate of the extract of the mycelium and extrage is to find a suitable, not miscible with water, an organic solvent, for example n-butanol. Deleted in subsequent organic phase may not necessarily be concentrated in vacuum. For degreasing valuable products, the concentrate can be diluted non-polar solvent in which the compounds according to the invention is not very soluble, for example, hexane, petroleum ether or simple diethyl ether. During this process menopace deposited and lipophilic impurities remain dissolved and can be removed by conventional methods of separation of solid and liquid phase.

The precipitate comprising menapace, can be dissolved in 1/30 of the original volume of water/methanol. The precipitate dissolves completely during this manipulation, and can be lyophilized. The lyophilisate, called in the following the crude product may include from 5 to 50% of menoitios and can be used for further selection.

Further purification of at least one peptide according to the invention can be carried out by chromatography on suitable materials, for example, molecular sieves, silica gel, alumina, ion exchangers or adsorbent resins, or converted phases (RF). Menopace can be separated using this chromatography. Chromatography of menoitios can be used in what Itanium buffer aqueous solutions or mixtures of aqueous and organic solutions.

The term "mixture of water and organic solvents" means all miscible with water and organic solvents, such as methanol, propanol and acetonitrile, at a concentration of from 5 to 80% solvent, more common from 20 to 50% of the solvent or, alternatively, all buffered aqueous solutions which are miscible with organic solvents. Buffers that should be used can be the same as above.

Department of menoitios on the basis of their different polarities may be carried out using chromatography in reversed-phase, for example, MCI® (adsorbing resin from Mitsubishi, Japan) or Amberlite XAD® (Toso Haas), or more hydrophobic materials, such as the phases of the RF-8 RF-18. In addition, the separation can be carried out using normalizes chromatography, for example on silica gel, aluminum oxide and the like.

Chromatography of menoitios can be carried out using a buffer or acidified aqueous solutions or mixtures of aqueous solutions with alcohols or other miscible with water and organic solvents. In one embodiment, the implementation of an organic solvent selected from propanol and acetonitrile.

The term "buffer or acidified aqueous solutions" refers to at least one solution separately or in combination. For example, specified by ENISA least one solution may be selected from water, phosphate buffer, ammonium acetate, citrate buffers and acids. In one embodiment, the implementation of the citrate buffer is in a concentration in the range from 0 to 0.5 M Acid selected from formic acid, acetic acid, triperoxonane acid and from all commercially available acids known to the person skilled in the art. In one embodiment, the implementation of commercially available acid is in a concentration in the range from 0 to 1%. In another implementation, the concentration of the acid is 0.1%.

Chromatography can be carried out using a gradient starting with 100% water and ends with 100% of solvent. Use at least one solvent. May also be a mixture of two or more solvents. In one embodiment, the implementation of a linear gradient is maintained from 20 to 50% of a solvent selected from propanol and acetonitrile.

Alternatively, it may also be a gel-chromatography or chromatography on hydrophobic phases.

Gel chromatography can be carried out on polyacrylamide or copolymer gels, such as Biogel-P 2®(Biorad) or Fractogel TSK HW 40® (Merck, Germany, or Toso Haas, USA).

Another very effective way to clean compounds in accordance with the invention may be the use of ion exchangers. For example, VI is th menopace And can be a great advantage highlighted on cation exchangers, such as Fractogel® EMD SO3. Can be used in buffer solutions with pH from 5 to 8, for example with a pH from 6 to 7.5. Elution can be achieved, for example, by using an increasing salt gradient. In addition to the water it is advisable to use a mixture of aqueous buffer solutions with an organic solvent as a solvent. The proportion of organic solvent can be from 10% to 90%, for example, from 30%to 60%.

The sequence of the above chromatographic processes can be reversible.

Further a very effective treatment stage of menoitios is crystallization. Menopace can be bicrystalline from solutions in organic solvents and mixtures of water with organic solvents. Crystallization can be carried out by a known method per se, for example, by concentration or cooling of saturated solutions of menoitios.

Menopace in accordance with the invention are stable in the solid state and in solutions in the pH range from 3 to 8, for example, from 5 to 7, and, thus, can be included in conventional pharmaceutical preparations.

Education containing nitrogen menoitios can help add the desired amino acids or peptides as a precursor to the culture of Memnoniella echinata. Feature types Memnoniella echinata may be that they are binding the t amino groups of amino acids and peptides, supplied by the synthesis of five-membered ring lactam, usually ring system isoindole. This binding occurs or starting from the aldehyde precursor during oxidation or oxidation steps lactone in the form of an open ring or cyclic lactoovo form. This ability Memnoniella echinata associate amines absolutely amazing and can be used to obtain a secure, converted amine derivatives, such as terpene derivatives of amino acids and peptides.

At least one connection of menoitios in accordance with the invention can be suitable in terms of their valuable pharmacological properties for use as a pharmaceutical in human or veterinary medicine.

Thus, the present invention relates to the use of compounds of formula (I) or its physiologically tolerable salts for the production of a heart drug for the treatment or prevention of heart failure.

Compounds in accordance with the present invention can also be used for the production of a medicinal product for the treatment of diseases in which heart failure is a primary or secondary cause of the disease, such as circulatory failure.

The mechanism of action of menopace is not installed, but it was revealed their significant action.

To identify activators SERCA2 that mimic the effect of phosphorylation of PLB can be carried out by a colorimetric test that determines the activity of CA2+ATPase in the microsomes of the dog's heart in the presence of the tested substances. The test can be performed in 96-cell microtiter plates. In spectrophotometer at a wavelength of 620 nm can be measured enzymatic release of inorganic phosphate from ATP, which forms a blue complex with ammonium molybdate.

Menopace And activates SERCA2 in concentration from 12.5 mol.

Menapace, in addition, possess antimicrobial activity.

Thus, the present invention relates to the use of compounds of formula (I) or its physiologically tolerable salts for the production of a medicinal product for the treatment of microbial, e.g. bacterial infections.

In table 3 shows some of the minimum inhibiting concentration (MS) antimicrobial spectrum of metropathia And against some selected bacteria.

Table 3
StrainResistance against:MIC [g/ml]
Staphylococcus aureus SG 511-16
Staphlococcus aureus 285 Penicillin16
Staphylococcus aureus 503Penicillin16
Staphylococcus aureus FH 1982Methicillin16
Staphylococcus aureus 701EMethicillin16
Staphylococcus aureus 707EMethicillin16
Staphylococcus aureus 9 TübOfloxacin16
Staph. Epidermidis 2c ZH-16
Staph. Epidermidis 763Methicillin>16
Staph. Epidermidis 5747IIWMethicillin32
Staph. Epidermidis 291Ofloxacin8
Staph. Epidermidis 799Ofloxacin8
Enterococcus faecium Md8B-8
Enterococcus faecium VR1Vancomycin>64
Enterococcus faecium VR2Vancomycin>64
Staphylococcus pyogenes VR3Vancomycin>64
Staphylococcus pyogenes 308A-8
Staphylococcus pyogenes 77A-4

In addition to the antimicrobial action of the compounds according to the invention exhibit a weak intimidate the definition, i.e. antifungal properties, for example against Candida albicans.

In addition, the substances according to the invention have certain favorable inhibitory effect on glucose-6-phosphate to translocase (G-6-P-TL), an enzyme that plays an important role in glucose metabolism and, thus, for the treatment of diabetes. For example, the connection menopace And exhibits selective activity against G-6-P-TL, but did not inhibit coenzyme G-6-P phosphatase.

Thus, the present invention relates to the use of compounds of formula (I) or its physiologically tolerable salts for the production of a medicinal product for treatment of diabetes.

The invention also relates to pharmaceutical preparations, at least one connection menoitios in accordance with the invention.

At least one connection of menoitios in accordance with the invention can in General be entered as such in undiluted form. Application in the form of a mixture with suitable excipients or carriers represents one embodiment of the invention. Media that can be used in medicines, can be a normal and pharmacologically tolerated carriers and/or excipients.

Drugs in accordance with the invention can advantage the NGOs be administered orally or parenterally, but rectal administration is also possible. Suitable solid or liquid form of pharmaceutical preparations are, for example, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, aerosols, drops or injectable solutions in this part of the form and drugs, having secured the release of active compounds, and these drugs are commonly used carriers and additives and/or excipients, such as disintegrant, binder, covering tools, agents that cause swelling, glidant or lubricants, flavorings, sweeteners or wetting agents. Commonly used carriers or excipients which may be mentioned are, for example, magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, lactoprotein, gelatin, starch, vitamins, cellulose and its derivatives, animal or vegetable fats/oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols.

Where appropriate, dosage forms can be microencapsulating for oral administration to delay release or to continue it for a longer period of time, for example, coating or pouring active connection is placed in the form of particles in a suitable polymers waxes or the like.

In one embodiment, the implementation of the pharmaceutical preparations can be produced and administered in dosage forms, each dosage form comprises as active ingredient a certain dose of at least one compound of menoitios in accordance with the invention. In the case of solid dosage forms such as tablets, capsules and suppositories, this dose can be up to about 500 mg, but more common it may be approximately from 0.1 to 200 mg, and in the case of injection solutions in this part of the form is about to 200 mg, but is usually from about 0.5 to 100 mg per day.

Daily dose that is to be entered, in General, depends on body weight, age, sex and condition of the mammal. However, under certain conditions, may also be appropriate higher or lower daily doses. The introduction of the daily dose can be carried out as a single introduction in the form of a single dosage unit or as several smaller dosage units, and repeated introduction of divided doses at regular intervals.

Drugs in accordance with the invention can be produced by incorporating at least one compound of menoitios in accordance with the invention suitably is Yu dosage form for administration with conventional carriers and, if appropriate, additives and/or excipients.

The invention is further illustrated by the following examples. Percentages relate to weight. If you were not represented by other items, the mixing ratio, in the case of liquids, refer to the volume.

The following examples are intended to illustrate the invention without limiting the scope of its claims.

Examples

Example 1. Getting glycerol culture Memnoniella echinata FH2272, DSM 13195.

100 ml of nutrient solution (2.0% malt extract, 0.2% of yeast extract, 1.0% glucose, 0.05% of (NH4)2HPO4pH of 6.0) in a sterile Erlenmeyer flask with a capacity of 300 ml inoculated with strain Memnoniella echinata FH 2272, DSM 13195 and incubated on a rotary shaker at 25°and the rotation speed of 140 rpm for 7 days. Then 1.5 ml of this culture is diluted to 2.5 ml of glycerol 80% of the fortress and stored at -20°C.

Example 2. Culture or preculture Memnoniella echinata FH 2272, DSM 13195 in the Erlenmeyer flask.

In a sterile Erlenmeyer flask with a capacity of 300 ml containing 100 ml of the following nutrient solution: 10 g/l glucose, 5 g/l casein peptone, 1.7 g/l liquid corn hydrolysate and 7 ml of trace element solution (10 g/l KCl, 10 g/l MgSO4×7 H2O, 3/6 g/l FeSO4×7 H2O and 6 g/l MgSO4×N2O) seeded with a culture grown in a test tube with a beveled nutrient medium (tacos the same nutrient solution, but with 2% agar) or 1 ml glycerol culture (see example 1) and incubated at 180 rpm and 30°on the shaker. Maximum production of at least one of the compounds of menoitios in accordance with the invention was achieved after approximately 120 hours For seeding fermenters with a capacity of 10 and 200 l was used culture, close to the merger, vynashivalsya in the course lasts for 48-96 h (number of planting about 10%) from the same nutrient solution.

Example 3. Getting menoitios.

The fermenter with a capacity of 30 l worked in the following conditions:

Nutrient medium:

10 g/l glucose

0.5 g/l casein peptone

1.7 g/l liquid corn hydrolysate

7 ml of trace element solution

pH 6.5 (before sterilization)

Trace element solution:KCl 10 g/l, MgSO4×7H2O 10 g/l FeSO4×7H2O 3.6 g/l MgSO4×N2About 6 g/l
Incubation time:45 h
Incubation temperature:28°
Speed stirrer:300 rpm
Aeration:15 l/min

The foam does not necessarily inhibit the re-addition of a solution of ethanol polyhydric alcohol. Maximum production reached approximately 35-70 hours

Example 4. The selection of a mixture of menoitios from the culture solution Memnoniella echinata FH 2272, DSM 13195.

After completion of the cultivation of Memnoniella echinata FH 2272, DSM 13195 culture broth from the fermenter, obtained in accordance with example 3 (200 l) was filtered with the addition of approximately 2% accelerator filtering (e.g., Celite®) and cell mass (22 l) is extracted using 66 l of methanol. A methanol solution containing the target substance is released from the mycelium by filtration and concentrated in vacuo. The concentrate is diluted with water and applied to the prepared 17-liter column of MCI GEL CHP20P together with culture filtrate (180 l). His elute with a gradient of water after 60% propan-2-ol in water. Column flow (25 l/h) collecting the fractions (10 l each) and combine the fractions containing menopace (from 25% to 30% propan-2-ol).

Concentration in vacuo gives 20 l brown solution. 6 l of cation exchanger Fractogel® EMD SO3-, equilibrated at pH 7 phosphate buffer potassium, Packed in a column (125 mm×500 mm). After loading of the ion exchanger 20 liters of the above-described concentrate his elute with a gradient of 10 mm potassium phosphate buffer at pH 7 after 1 M NaCl in 10 mm potassium phosphate buffer, pH 7 in water/methanol (1:1). Column flow, i.e. unbound material, contains a neutral menopace (49 g). Column a flow rate of 12 l/h; portions of l is collected in fractions during gradient elution. Using 0.75 M NaCl (fraction 31 and 32) are menopace A. Fractions 31 and 32 are combined and concentrated to approximately 500 ml in a vacuum.

Example 5: Enrichment metropathia And gel chromatography.

8 g of the product obtained in accordance with example 4, applied to a column with a capacity of 3.9 liters, Packed Fractogel® TSK HW-40 (width (height=10 cm ×50 cm). Eluent: methanol/water (1:1) was pumped through the column at a flow rate of 20 ml/min, and the exit stream from the column is collected in fractions (20 ml). Menopace And find mainly in the fractions 75 to 85. They are combined and freed from methanol in vacuo. This gives 0.9 g of the mixture of the active substance.

Example 6. The HPLC system to identify menoitios.

As described below, the system provided an opportunity for testing the purity and separation and quantification of minoterie, for example, in the raw mixture or in the culture filtrates.

Eluent: 0.1% of triperoxonane acid 32% acetonitrile

Column: Nucleosil (Nucleosil 100C18AB 250/4, Macherey-Nagel)

Flow: 1.0 ml/min

Detection: Absorption of UV light at 210 nm.

In these conditions, menopace And may have the following retention times: Menapace: 7,0 minutes

Example 7. Cleaning metropathia A.

500 ml of metropathia And highlighted and enriched in accordance with approx the rum 5, put on a column of Nucleosil®100-7 C18AB 500 ml and chromatographic with a gradient from 25% to 50% acetonitrile in 0.05% triperoxonane acid in water. The eluent flow 50 ml/min; fraction size 50 ml of Menopace And find in the fractions from 71 to 88. Repeated cleaning of the combined fractions with a constant concentration of solvent 28% acetonitrile in 0.05% triperoxonane acid gives >95% pure metropathia With after lyophilization (100 mg).

Characteristics of metropathia As:

10 g metropathia And hydrolyzing in constantly boiling hydrochloric acid and examined in amino acid analyzer. Find the following common amino acid:

Glutamic acid11 nmol
Proline22 nmol
Histidine10 nmol
Leucine5.6 nmol
Methionine oxide5.5 nmol

The absorption of UV light: λmaxat 269 nm, 305 nm (edge).

Mass spectrum FAB high resolution displays intense MN+at m/z 1565, 7819 Yes, which is in good agreement with the calculated mass (for C76H109T16O18S, monoisotopic), equal 1565, 7827 Yes. Fragmentation MS/MS corresponds to the formula (IVa).

II. The SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified above under item 1, was accompanied by:

( ) research description

(X) the proposed taxonomic designation

(Please mark with a cross)

III. RECEIPT AND ACCEPTANCE

The authority of the International Depositary accepts the microorganism identified above under item I, which was received 12.09.1999 g (date of original Deposit)1

IV. Receipt of the TRANSFER REQUEST

The microorganism identified above under item I, was obtained by this body of International Depositary (the date of the initial Deposit) and a request to transfer the initial Deposit in the Deposit according to the Budapest Treaty was received (the date of receipt of the transfer request).

V. the AUTHORITY of the INTERNATIONAL DEPOSITARY

* If Rule 6.4 (d), this date represents the date on which it was acquired the status of International Depositary authority.

Form DSMZ-BP/4 (one page) 0196.

1. The compound of formula (I)

in which R1and R2together represent a double bond;

R3and R4each independently represents adored;

R8represents hydrogen;

R6represents a group of formula (II)

in which R9represents a hydrogen atom;

thus R5represents a bond with the carbon atom C9 formula (II), a R7represents a hydrogen atom, or

R5represents a hydrogen atom, a R7represents a bond with the carbon atom C9 formula (II);

(A)n is an amino acid sequence

Met-His-Gln-Pro-His-Gln-Pro-Leu-Pro-Pro (SEQ ID NO:1),

or its salt.

2. The compound according to claim 1 of formula (IVb)

where (A)n has the meaning given in claim 1.

3. The compound according to any one of claim 1 or 2, is used as the active substance for the manufacture of a medicinal product for the treatment of microbial infections.

4. Pharmaceutical composition having antimicrobial activity comprising at least one compound of formula (I), in which the specified connection defined in any of the preceding paragraphs, and a pharmaceutically acceptable carrier.

5. The method of obtaining the compounds of formula (I)where the specified connection defined in any of claim 1 or 2, including the cultivation of Memnoniella echinata FH2272, DSM 13195 in suitable conditions in a nutrient the environment, including at least one source of carbon atoms and at least one source of nitrogen atoms, until at least one compound of formula (I) will not be present in the nutrient medium, followed by separation of the compounds.

6. The method according to claim 5, further comprising transforming the compound in a physiologically tolerable salt.

7. The method according to claim 5 or 6, in which the specified cultivation occurs under aerobic conditions.

8. The method according to any of pp.5-7, wherein said at least one source of nitrogen atoms selected from amino acids and peptides.

9. The method according to any of pp.5-8, in which the specified nutrient medium includes casein peptone at a concentration in the range from about 0.05 to 5 wt.% all of the nutrient solution.

10. The method according to any of pp.5-9, in which the specified nutrient medium includes casein peptone, glucose, corn hydrolysate and trace amounts of potassium chloride, magnesium sulfate and iron sulfate.



 

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FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to new derivatives of cyclic amide of the formula (I)

or its salt, or hydrate, or solvate wherein X represents (C1-C6)-alkyl, (C1-C6)-alkyl substituted with phenyl, (C2-C6)-alkenyl substituted with phenyl or halogenphenyl, (C2-C6)-alkynyl substituted with phenyl, phenyl that can be substituted with (C1-C6)-alkyl; one or more halogen atom, nitro-group, phenyl, (C1-C6)-alkoxy-group, halogen-(C1-C6)-alkyl, halogen-(C1-C6)-alkoxy-group, phenyl-(C1-C6)-alkyl, (C1-C6)-alkoxyphenyl-(C1-C6)-alkyl, amino-group, optionally substituted with (C1-C6)-alkyl, acetyl, (C1-C6)-alkoxy-group, substituted with phenyl, phenylcarbonyl, furanyl; 1- or 2-naphthyl, monocyclic (C3-C8)-cycloalkyl, amino-group substituted with one or more substitutes taken among phenyl, halogenphenyl, (C1-C6)-alkoxyphenyl, (C1-C6)-alkyl, halogen-(C1-C6)-alkyl, phenyl-(C1-C6)-alkyl; 5- or 6-membered monocyclic heterocyclic group comprising 1 or 2 heteroatoms, such as nitrogen (N), oxygen (O), sulfur (S) atom optionally substituted with halogenphenyl, halogen atom, benzyl, (C1-C6)-alkyl, phenyl; 8-10-membered bicyclic heteroaryl group comprising 1 or 2 heteroatoms taken among N, O and optionally substituted with halogen atom; 8-10-membered polycyclic cycloalkyl group; Q means -CH2-, -CO-, -O-, -S-, -CH(OR7)- or -C(=NR8)- wherein R7 means hydrogen atom (H), (C1-C6)-alkyl; R8 means OH, (C1-C)-alkoxy-group, acylamino-group, (C1-C6)-alkoxycarbonylamino-group, phenyl-(C1-C6)-alkoxy-group; n = 0-5; B represents group or wherein each among R3, R4, R5 and R6 represents independently substitute taken among group consisting of hydrogen atom (H), halogen atom, NO2 (nitro-group), (C1-C6)-alkoxy-group, CN (cyano-group); m = 1 or 2; ring represents 5- or 6-membered aromatic heterocyclic ring comprising one or two heteroatoms taken among O, S, N. Compound of the formula (I) elicit activity inhibiting binding sigma-receptors that allows their using as component of medicinal agent.

EFFECT: valuable medicinal properties of compounds.

21 cl, 2 sch, 4 tbl, 183 ex

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