JP2008534538A - Cyclic iminopeptide derivatives - Google Patents

Abstract

  The present invention relates to antibacterial cyclic iminopeptide derivatives and methods for their production, their use for the treatment and / or prevention of diseases, their use for the manufacture of medicaments for the treatment and / or prevention of diseases, in particular Relates to those for bacterial infections.

Description

  The present invention relates to antibacterial cyclic iminopeptide derivatives and methods for their production, their use for the treatment and / or prevention of diseases, their use for the manufacture of medicaments for the treatment and / or prevention of diseases, in particular Relates to those for bacterial infections.

  The fermentation of bottromycin is described in JP43010998, JP47010036, US3650904, JP491116297 and HU52165. US 33808885 describes botromycin as an antibiotic for the treatment of chickens and turkeys, and JP 6165332 describes for the treatment of livestock mycoplasma disease and swine dysentery. The structure described for botromycin in the current state of the art indicates incorrect stereochemistry.

  The amide and hydrazine derivatives of botromomycin have the wrong basic structure of botromycin in JP 420040505, DE 1620027, J. Antibiotics 19, 1966, 149, J. Antibiotics 20, 1967, 162 and J. Med. Chem. 11, 1968, 746. Presumably, it has been published as an antibiotic.

  These natural products do not meet the requirements of antibacterial drugs in terms of their properties. Although structurally different substances with antibacterial activity are available on the market, there is always a development of resistance. Therefore, new substances are desirable for better and more effective treatment.

  Accordingly, one object of the present invention is to provide new and alternative compounds with equivalent or improved antimicrobial activity for the treatment of bacterial diseases in humans and animals.

  Surprisingly, certain derivatives of natural products in which the ester groups of these natural products are replaced with substituted hydroxamates have antibacterial activity and improved pharmacokinetic properties compared to natural products It was found.

［式中、
Ｒ^１およびＲ^２は、水素を表すか、
または、
Ｒ^１およびＲ^２は、メチルを表すか、
または、
Ｒ^１はメチルを表し、そして、
Ｒ^２は水素を表し、
そして、 The present invention has the formula

[Where:
R ¹ and R ² represent hydrogen or
Or
R ¹ and R ² represent methyl or
Or
R ¹ represents methyl and
R ² represents hydrogen,
And

｛ここで、＊は、カルボニル基への結合部位である｝
の基を形成している］
の化合物、並びにそれらの塩、それらの溶媒和物およびそれらの塩の溶媒和物に関する。 R ³ and R ⁴ , independently of one another, represent C ₁ -C ₄ -alkyl {which is cyano, hydroxycarbonyl, aminocarbonyl, C ₁ -C ₄ -alkoxycarbonyl, C ₁ -C _6- alkylaminocarbonyl, C 3 _-C 6 _- cycloalkyl, C 6 _-C 10 _- aryl and 5- or may be substituted with one substituent selected from the group consisting of heteroaryl 6-membered, C _{When 1} -C ₄ -alkyl is not methyl, alternatively or additionally, they are independently of each other from the group consisting of halogen, hydroxy, amino, C ₁ -C ₄ -alkoxy and C ₁ -C ₆ -alkylamino. Optionally substituted with 1 to 3 substituents selected from
_Or, C 3 -C ₆ - or {it represents a cycloalkyl, halogen, hydroxy, _amino, C 1 -C ₄ - _alkyl, C 1 -C ₄ - _alkoxy, C 1 -C ₆ - alkylamino, cyano, hydroxy Optionally substituted by 1 to 3 substituents independently selected from the group consisting of carbonyl, aminocarbonyl, C ₁ -C ₄ -alkoxycarbonyl and C ₁ -C ₆ -alkylaminocarbonyl },
Or
R ³ and R ⁴ together represent (CH ₂ ) ₃ or (CH ₂ ) ₄ , and together with the nitrogen or oxygen atom to which they are respectively attached, are 5 or 6 membered Forming a ring, wherein the ring is halogen, hydroxy, amino, C ₁ -C ₄ -alkyl, C ₁ -C ₄ -alkoxy, C ₁ -C ₆ -alkylamino, cyano, hydroxycarbonyl, May be substituted with 1 to 2 substituents independently selected from the group consisting of aminocarbonyl, C ₁ -C ₄ -alkoxycarbonyl and C ₁ -C ₆ -alkylaminocarbonyl,
Or
R ³ and R ⁴ are combined with the nitrogen or oxygen atom to which they are each attached to form a formula

{Where * is the binding site to the carbonyl group}
Is forming a group]
And their salts, solvates and solvates of these salts.

  The compounds of the present invention are encompassed by the compounds of formula (I) and (Ia) and their salts, solvates and solvates of salts, and formulas (I) and (Ia), as exemplary embodiments Compounds described below and salts, solvates thereof and solvates of those salts (compounds included in formulas (I) and (Ia) described below are still salts, solvates and solvent of salts) In the case of non-Japanese).

Depending on their structure, the compounds of the invention can exist in stereoisomeric forms (enantiomers, diastereomers). The invention therefore relates to the enantiomers or diastereomers and their respective mixtures. From a mixture of such enantiomers and / or diastereomers, a stereoisomerically homogeneous component can be isolated by known methods such as chromatography on chiral phases or crystallization using chiral amines or chiral acids.
The invention also relates to tautomers of the compounds, depending on the structure of the compounds.

For the purposes of the present invention, preferred salts are physiologically acceptable salts of the compounds of the present invention.
Physiologically acceptable salts of compound (I) include acid addition salts of mineral acids, carboxylic acids and sulfonic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, Examples include toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid, trifluoroacetic acid, and benzoic acid salts.

  Physiologically acceptable salts of compound (I) include common base salts such as, and preferably, alkali metal salts (eg, sodium and potassium salts), alkaline earth metal salts (eg, calcium and magnesium). Salt) and ammonia or organic amines having 1 to 16 carbon atoms (eg and preferably ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylamino) Also included are the ammonium salts derived from ethanol, procaine, dibenzylamine, N-methylmorpholine, dihydroabiethylamine, arginine, lysine, ethylenediamine and methylpiperidine).

For the purposes of the present invention, a solvate represents the form of a compound that forms a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a special form of solvates where coordination occurs with water.

For the purposes of the present invention, unless otherwise indicated, the substituents have the following meanings:
Alkyl itself, as well as “alk” and “alkyl” in alkoxy, alkylamino, alkoxycarbonyl and alkylaminocarbonyl are generally 1 to 6, preferably 1 to 4 carbon atoms. Represents a straight-chain or branched alkyl radical having, for example and preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, tert.-butyl, n-pentyl and n-hexyl.

Alkoxy by way of example and preferably represents methoxy, ethoxy, n-propoxy, isopropoxy and tert.-butoxy.

Alkylamino is an alkylamino radical having one or two alkyl substituents (selected independently of each other), for example and preferably methylamino, ethylamino, n-propylamino, isopropylamino, tert .-Butylamino, n-pentylamino, n-hexylamino, N, N-dimethylamino, N, N-diethylamino, N-ethyl-N-methylamino, N-methyl-Nn-propylamino, N- Represents isopropyl-Nn-propylamino, N-tert.-butyl-N-methylamino, N-ethyl-Nn-pentylamino and Nn-hexyl-N-methylamino. C ₁ -C ₃ -alkylamino represents, for example, a monoalkylamino radical having 1 to 3 carbon atoms or a dialkylamino radical having 1 to 3 carbon atoms per alkyl substituent.

Alkoxycarbonyl by way of example and preferably represents methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, tert.-butoxycarbonyl, n-pentoxycarbonyl and n-hexoxycarbonyl.

Alkylaminocarbonyl is an alkylaminocarbonyl radical having one or two alkyl substituents (selected independently of each other), wherein the alkyl substituents are independently of each other generally 1 1 to 6, preferably 1 to 4, particularly preferably 1 to 3 carbon atoms), for example and preferably methylaminocarbonyl, ethylaminocarbonyl, n-propylaminocarbonyl, isopropylamino Carbonyl, tert.-butylaminocarbonyl, n-pentylaminocarbonyl, n-hexylaminocarbonyl, N, N-dimethylaminocarbonyl, N, N-diethylaminocarbonyl, N-ethyl-N-methylaminocarbonyl, N-methyl- Nn-propylaminocarbonyl, N-isopropyl N-n-propylamino carbonyl, represents an N-tert.-butyl -N- methylaminocarbonyl, N- ethyl -N-n-pentyl-amino carbonyl and N-n-hexyl--N- methylaminocarbonyl. C ₁ -C ₃ -alkylaminocarbonyl is, for example, a monoalkylaminocarbonyl radical having 1 to 3 carbon atoms or a dialkylaminocarbonyl radical having 1 to 3 carbon atoms per alkyl substituent. Represents.

Cycloalkyl generally represents a cycloalkyl group having 3 to 6 carbon atoms, for example and preferably, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl are mentioned as cycloalkyl.

Aryl generally represents a monocyclic or bicyclic aromatic radical having 6 to 10 carbon atoms, for example and preferably phenyl and naphthyl are mentioned as aryl.

Heteroaryl typically has 5 to 6 ring atoms and has up to 4 heteroatoms from the series S, O and N, where the nitrogen atom forms an N-oxide. (Optionally) represents an aromatic monocyclic radical, for example and preferably thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, oxadiazolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl.

Halogen represents fluorine, chlorine, bromine and iodine, preferably fluorine and chlorine.

If a radical in a compound of the invention is substituted , the radical is substituted one or several times, identical or different, unless otherwise specified. Substitution with up to 3 identical or different substituents is preferred. Very particular preference is given to substitution with one substituent.

｛ここで、＊は、カルボニル基への結合部位である｝
の基を形成している、
式（Ｉ）の化合物並びにそれらの塩、それらの溶媒和物およびそれらの塩の溶媒和物である。 For the purposes of the present invention, preferred compounds are:
R ¹ and R ² represent hydrogen,
Or
R ¹ and R ² represent methyl,
Or
R ¹ represents methyl and
R ² represents hydrogen,
And
R ³ and R ⁴ independently of one another represent methyl or ethyl,
Or
R ³ and R ⁴ together represent (CH ₂ ) ₃ or (CH ₂ ) ₄ , and together with the nitrogen or oxygen atom to which they are respectively attached, are 5 or 6 membered Forming a ring, wherein the ring may be substituted with one substituent selected from the group consisting of hydroxy and ethoxycarbonyl;
Or
R ³ and R ⁴ are combined with the nitrogen or oxygen atom to which they are each attached to form the formula

{Where * is the binding site to the carbonyl group}
Forming the basis of
Compounds of formula (I) and their salts, solvates thereof and solvates of their salts.

For the purposes of the present invention, preferred compounds are also:
R ¹ represents methyl and
R ² represents hydrogen,
And
R ³ and R ⁴ independently of one another represent methyl or ethyl,
Or
R ³ and R ⁴ together represent (CH ₂ ) ₃ or (CH ₂ ) ₄ , and together with the nitrogen or oxygen atom to which they are respectively attached, are 5 or 6 membered Forming a ring, wherein the ring may be substituted with one substituent selected from the group consisting of hydroxy and ethoxycarbonyl;
Compounds of formula (I) and their salts, solvates thereof and solvates of their salts.

（式中、Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４は、上記の意味を有する）
に相当する式（Ｉ）の化合物並びにそれらの塩、それらの溶媒和物およびそれらの塩の溶媒和物である。 For the purposes of the present invention, preferred compounds are also of the formula

(Wherein R ¹ , R ² , R ³ and R ⁴ have the above meanings)
And the salts thereof, the solvates thereof and the solvates of the salts thereof.

（式中、Ｒ^１およびＲ^２は、上記の意味を有する）
の化合物を、１種またはそれ以上の脱水剤の存在下、式

（式中、Ｒ^３およびＲ^４は、上記の意味を有する）
の化合物と反応させる、
または、 The present invention further relates to a process for the preparation of the compounds of formula (I) or their salts, their solvates or their salts,
[A] Formula

(Wherein R ¹ and R ² have the above meanings)
In the presence of one or more dehydrating agents.

(Wherein R ³ and R ⁴ have the above meanings)
React with a compound of
Or

（式中、Ｒ^３は、上記の意味を有する）
の化合物と反応させ、第２段階で、式

（式中、Ｒ^４は上記の意味を有し、Ｘ^１は、ハロゲン、好ましくは臭素またはヨウ素を表す）
の化合物と、塩基の存在下で反応させる。 [B] A compound of formula (II) is reacted in the first step in the presence of one or more dehydrating agents.

(Wherein R ³ has the above meaning)
In the second step, the compound is reacted with

(Wherein R ⁴ has the above meaning and X ¹ represents halogen, preferably bromine or iodine)
Is reacted with a compound in the presence of a base.

  The reaction according to method [A] and the first step according to method [B] are generally carried out in an inert solvent, optionally in the presence of a base, preferably in the temperature range of 0 ° C. to 40 ° C., at atmospheric pressure. To do.

  Suitable dehydrating agents here are, for example, carbodiimides, such as N, N′-diethyl-, N, N′-dipropyl-, N, N′-diisopropyl-, N, N′-dicyclohexylcarbodiimide, N- (3 -Dimethylaminoisopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), N-cyclohexylcarbodiimide-N'-propyloxymethylpolystyrene (PS-carbodiimide) or carbonyl compounds such as carbonyldiimidazole or 1,2-oxazolium Compounds such as 2-ethyl-5-phenyl-1,2-oxazolium-3-sulfate, or 2-tert.-butyl-5-methylisoxazolium perchlorate, or acylamino compounds such as 2-ethoxy- 1-ethoxycarbonyl-1,2-dihydro Norin, or propanephosphonic anhydride, or isobutyl chloroformate, or bis- (2-oxo-3-oxazolidinyl) phosphoryl chloride, or benzotriazolyloxytri (dimethylamino) phosphonium hexafluorophosphate or O- (benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HBTU), 2- (2-oxo-1- (2H) -pyridyl) -1, 1,3,3-tetramethyluronium tetrafluoroborate (TPTU) or O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HATU) ) Or 1-hydroxybenzotriazole (HOBt) or ben Triazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP), or a mixture thereof, or a mixture thereof with a base.

  Bases are, for example, alkali carbonates such as sodium or potassium carbonate, or bicarbonates, or organic bases such as trialkylamines such as triethylamine, N-methylmorpholine, N-methylpiperidine, 4-dimethylaminopyridine. Or diisopropylethylamine.

  Preferably, the condensation is carried out with 1-hydroxybenzotriazole in the presence of a base, in particular diisopropylethylamine.

  Inert solvents are, for example, halohydrocarbons such as dichloromethane or trichloromethane, hydrocarbons such as benzene, or nitromethane, dioxane, dimethylformamide or acetonitrile. It is also possible to use a mixture of solvents. Dimethylformamide is particularly preferred.

  The second stage reaction according to method [B] is generally carried out in an inert solvent, preferably in the temperature range of 0 ° C. to 40 ° C., under atmospheric pressure.

  The base is, for example, an alkali carbonate such as cesium carbonate, sodium or potassium, or bicarbonate; cesium carbonate is preferred.

  Inert solvents include, for example, halohydrocarbons such as methylene chloride, trichloromethane or 1,2-dichloroethane, or ethers such as dioxane, tetrahydrofuran or 1,2-dimethoxyethane, or acetone, dimethylformamide, dimethyl Other solvents such as acetamide, 2-butanone or acetonitrile, or mixtures of these solvents with water; dioxane, dioxane / water or dimethylformamide are preferred.

  Compounds of formula (III), (IIIa) and (IIIb) are known or can be prepared analogously to known methods.

（式中、Ｒ^１およびＲ^２は、上記の意味を有する）
の化合物を、塩基と反応させることにより製造できる。 Compounds of formula (II) are known or have the formula

(Wherein R ¹ and R ² have the above meanings)
This compound can be produced by reacting with a base.

  This reaction is generally carried out in an inert solvent, preferably in the temperature range of 0 ° C. to 40 ° C., at atmospheric pressure.

  The base is, for example, an alkali hydroxide such as sodium hydroxide, lithium or potassium, or an alkali carbonate such as cesium carbonate, sodium carbonate or potassium, preferably lithium hydroxide.

  Inert solvents include, for example, methylene chloride, trichloromethane, tetrachloromethane, trichloroethane, tetrachloroethane, halohydrocarbons such as 1,2-dichloroethane or trichloroethylene, diethyl ether, methyl tert.-butyl ether, 1,2-dimethoxy. Ethers such as ethane, dioxane, tetrahydrofuran, glycol dimethyl ether or diethylene glycol dimethyl ether, alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol or tert-butanol, or dimethylformamide, dimethylacetamide, dimethyl sulfoxide, acetonitrile Or other solvents such as pyridine, or mixtures of solvents, the preferred solvent being A hydrofuran and / or methanol.

  The compound of formula (IV) can be produced by fermentation. They ferment, for example, Streptomyces bottropensis (DSM16814) or Streptomyces spec. (DSM17025) strains in an aqueous culture medium under aerobic conditions. Sometimes produced. Typically, these microorganisms are fermented in a culture medium containing a carbon source and optionally proteinaceous material. Preferred carbon sources are, for example, glucose, brown sugar, sucrose, glycerol, starch, corn starch, lactose, dextrin and molasses. Preferred nitrogen sources include, for example, cottonseed flour, yeast, autolysis baker's yeast, milk solids, soy flour, corn flour, casein pancreas or papain digestion degradation products, solid distillation products, animal peptone broth And meat and bone meal. Preferably, a combination of these carbon and nitrogen sources is used. If tap water and unpurified components are used as components of the medium, it is not necessary to add trace elements such as zinc, magnesium, manganese, cobalt and iron to the fermentation medium.

  Manufacturing can include any temperature that ensures sufficient growth of the microorganism. This temperature is preferably 21 ° C. to 32 ° C., particularly preferably about 28 ° C.

  Optimal manufacturing is generally achieved within 4 to 8 days, preferably about 7 days. The fermentation broth usually remains slightly basic (pH 7.2 to pH 8.4) during fermentation. The final pH depends in part on any buffer used and in part on the initial pH of the culture medium. Preferably, the pH is adjusted to about 6.5 to 7.5, particularly preferably pH 7.2 before sterilization.

  Production takes place both in shake flasks and in stirred fermenters. When culturing is carried out in shake flasks or large bats and tanks, preferably in the spore form of the microorganism, in order to avoid an obvious lag phase in the production of metabolites and thus avoid inefficient utilization of the equipment Use nutritional form for sowing. Therefore, it is advantageous to prepare a seed form of nutrient form in an aqueous culture medium by seeding this medium with an aliquot of soil or slope culture. Once a fresh, vigorous nutrient form of inoculum is prepared in this manner, it is aseptically transferred to another shake flask or other suitable facility for microbial fermentation. The medium that produces the vegetative form of the inoculum may be the same or different from the medium used to produce the compounds of the present invention, as long as it ensures sufficient growth of the microorganism.

  In general, the production is carried out in a stirred fermentor under aerobic conditions using the microorganisms described above. However, the production is independent of the fermentor used and the starting culture. These precursors can also be obtained using shaking culture. For large-volume fermentation, it is preferred to use a vegetative inoculum. Nutritional inoculum is produced by inoculating a small amount of culture medium with the spore form, mycelium fragment or lyophilized pellet of the microorganism. The vegetative form of the inoculum is then transferred to the fermentation vat. Thus, after a suitable culture time, the compound of formula (IV) is produced in optimal yield. Usually, in aerobic submerged fermentation, sterile air is passed through the culture medium. For efficient growth of microorganisms, the volume of air used is in the range of about 0.25 to about 0.5 times the volume of air / volume of culture medium / minute (vvm). The optimal rate in a 30 liter vat is about 0.3 vvm, with agitation, which is effected at about 200-500 rev / min, preferably 240 rev / min with a conventional propeller revolution. If foam formation is a problem, it is necessary to add a small amount of antifoam such as 1 ml / l of silicon to the fermentation medium.

  Production generally begins after about 96 hours and occurs for at least 3 days during the fermentation period. Peak production is reached with a fermentation time of about 6 to 7 days.

The compound of formula (IV) can be isolated from the fermentation medium by conventional methods.
The compounds of formula (IV) are mainly contained in the fermentation filtrate of fermented microorganisms, but they can also be present in small amounts in the mycelium of the microorganism. The culture broth can be obtained by separation using a separator in a simple manner.

  To separate the compounds of formula (IV) from the fermentation broth and purify them, chromatographic adsorption methods (eg by column chromatography, liquid-liquid separation chromatography, gel permeation chromatography) followed by suitable Various methods can be used, such as elution with solvents, crystallization from solvents, and combinations thereof. In a preferred purification method, the compound of formula (IV) is extracted from the mycelium or from the supernatant extract. The latter can be prepared using an adsorption resin such as XAD, HP20 or Lewapol. A column chromatography method, preferably Biotage C18 KP, is used to perform the initial purification. Final purification of the compound is preferably performed by preparative high performance liquid chromatography (HPLC) using C18 column material.

  Preferred fermentation conditions and media are described in Examples 1A-3A.

  The above-mentioned strains Streptomyces botropensis DSM 16814 and Streptomyces genus DSM 17025 have been deposited in accordance with the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Brunswick, Germany.

The compounds of the present invention exhibit valuable antibacterial and pharmacokinetic properties that could not have been predicted.
They are therefore suitable for use as medicaments for the treatment and / or prevention of human and animal diseases.

  The compounds according to the invention can be used for the treatment and / or prevention of infectious diseases, in particular bacterial infections, alone or in combination with other active compounds because of their pharmacological properties.

For example, it is possible to treat and / or prevent local and / or systemic diseases resulting from the following pathogens or mixtures of the following pathogens:
Gram-positive cocci such as staphylococci (Staphylococcus aureus, Staphylococcus epidermidis) and streptococci (Streptococcus agalactia, faecalis, Streptococcus pneumoniae, Streptococcus pyogenes); Internal bacteria such as Escherichia coli, Haemophilus influenzae, Citrobacter (Citrobacter Freundi, Citrobacter gibernis), Salmonella and Shigella; , Enterobacter (Enterobacter aerogenes, Enterobacter agglomerans), Hafnia, Serratia (Seratia marcescens), Proteus (Proteus mirabilis, Proteus rechgeri), Prote Us Bulgaris), Providencia, Yersinia and Acinetobacter. Furthermore, the antibacterial spectrum includes representative examples of the genus Pseudomonas (Pseudomonas aeruginosa, Pseudomonas maltophilia) and strictly anaerobic bacteria such as Bacteroides fragilis, Peptococcus, Peptostreptococcus and Clostridium; And mycoplasma (pneumonia mycoplasma, mycoplasma hominis, mycoplasma urealyticum), and mycobacteria such as tuberculosis.
The above list of pathogens is exemplary only and should not be considered limiting in any way.

Examples that may be referred to as diseases resulting from the above mentioned pathogens or mixed infections and that can be prevented, ameliorated or cured by the preparations that can be used according to the invention are
Infectious diseases in humans, eg septic infection, bone and joint infection, skin infection, postoperative wound infection, abscess, cellulitis, wound infection, infection burn, burn, oral area infection, infection after dental surgery Septic arthritis, mastitis, tonsillitis, genital infection and eye infection.

In addition to humans, other species can treat bacterial infections. Examples that may be mentioned include:
Pig: E. coli diarrhea, enterotoxemia, sepsis, dysentery, salmonellosis, uteritis-mastitis-agalactia syndrome, mastitis;
Ruminants (cow, sheep, goat): diarrhea, sepsis, bronchopneumonia, salmonellosis, pasteurellosis, mycoplasma disease, genital infection;
Horses: bronchial pneumonia, joint disease, postpartum and postpartum infection, salmonellosis;
Dogs and cats: bronchial pneumonia, diarrhea, dermatitis, otitis, urinary tract infection, prostatitis;
Poultry (chicken, turkey, quail, pigeons, ornamental birds, etc.): Mycoplasma disease, Escherichia coli infection, chronic respiratory disease, salmonellosis, pasturellosis, parrot disease.

  Treatment of bacterial diseases in the breeding and management of production and ornamental fish is possible as well, where the antibacterial spectrum goes beyond the pathogens described above, for example, Pasteurella, Brucella, Campylobacter, Listeria, It covers additional pathogens such as Erysipelothris, Corynebacterium, Borellia, Treponema, Nocardia, Rickettsia, and Yersinia.

The invention further relates to the use of the compounds according to the invention for the treatment and / or prevention of diseases, preferably bacterial diseases, in particular bacterial infections.
The invention further relates to the use of the compounds according to the invention for the treatment and / or prevention of diseases, in particular the diseases mentioned above.
The invention further relates to the use of the compounds according to the invention for the manufacture of a medicament for the treatment and / or prevention of diseases, in particular the aforementioned diseases.
The invention further relates to a method for the treatment and / or prophylaxis of diseases, in particular the aforementioned diseases, using an antibacterial effective amount of the compounds of the invention.

  The compounds of the invention can act systemically and / or locally. They can be used for this purpose, for example, orally, parenterally, pulmonary, nasal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival, or Can be administered in a suitable manner as an ear or as an implant or stent.

The compounds of the present invention can be administered in dosage forms suitable for these administration routes.
Suitable for oral administration functions in accordance with the current state of the art, releasing the compounds of the invention in a rapid and / or modified manner and the compounds of the invention in crystalline form and / or Dosage forms containing in amorphous and / or dissolved form, eg tablets (uncoated or coated tablets, eg enteric coated, or delayed dissolving or insoluble, controlling the release of the compounds of the invention Tablets or films / oblates that disintegrate rapidly in the oral cavity, film / lyophilizers, capsules (eg hard or soft gelatin capsules), dragees, granules, pellets, powders, emulsions, Suspension, aerosol or liquid.

  Parenteral administration avoids the absorption phase (eg intravenous, intraarterial, intracardiac, spinal or lumbar) or includes absorption (eg intramuscular, subcutaneous, intradermal, transdermal or Intraperitoneal). Dosage forms suitable for parenteral administration include, inter alia, injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilizates or sterile powders.

  Suitable for other administration routes are, for example, pharmaceutical forms for inhalation (among others, powder inhalers, nebulizers), nasal drops, liquids, sprays; tablets for administration to the tongue, sublingually or buccal; Film / oblate or capsules, suppositories, ear or ophthalmic preparations, vaginal capsules, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (eg patches etc.) ), Milk, paste, foam, dusting powder, implant or stent.

  The compounds of the invention can be converted into the stated administration forms. This can be done in a manner known per se by mixing with inert, non-toxic, pharmaceutically acceptable auxiliaries. These adjuvants include, inter alia, carriers (eg microcrystalline cellulose, lactose, mannitol), solvents (eg liquid polyethylene glycols), emulsifiers and dispersants or wetting agents (eg sodium dodecyl sulfate, polyoxysorbitan oleate), Binders (eg polyvinylpyrrolidone), synthetic and natural polymers (eg albumin), stabilizers (eg antioxidants such as ascorbic acid), colorants (eg inorganic pigments eg iron oxide etc.) and taste and / or odor Concealer.

  The present invention further comprises medicaments comprising at least one compound of the invention, usually together with one or more inert, non-toxic, pharmaceutically acceptable auxiliaries, and their use for the purposes mentioned above About.

  It has generally been found that administering parenteral doses of about 5 to 250 mg / kg body weight per 24 hours is advantageous to achieve effective results. For oral administration, the amount is about 5 to 100 mg / kg body weight per 24 hours.

  Nevertheless, it may be necessary to deviate from the above-mentioned amounts, in particular depending on body weight, route of administration, individual behavior with respect to the active compound, type of formulation and time or interval at which the administration takes place. Thus, it may be sufficient to make less than the above-mentioned minimum amount, while in other cases the upper limit mentioned must be exceeded. For large doses it may be desirable to divide these into several individual doses over the day.

  Unless otherwise noted, percentage data in the following tests and examples are percentages by weight; parts are parts by weight. The solvent ratio, dilution ratio and concentration of the liquid / liquid solution are in each case based on volume.

A. Examples
Abbreviations used:

LC-MS and HPLC methods:
Method 1 (standard preparative HPLC conditions to purify the botomycin derivative): Column: VP 250/21 Nucleodur 100-5, C18 ec, Macherey & Nagel: 762002; Eluent A: 0.01% trifluoroacetic acid in water Eluent B: acetonitrile / 0.01% trifluoroacetic acid; gradient: 0 min 0% B, 20 min 20% B, 40 min 20% B, 60 min 30% B, 100 min 30% B, 110 min 100 % B, 132 min 100% B; flow rate: 5 ml / min; oven: 30 ° C .; UV detection: 210 nm.

Method 2 (analytical HPLC standard conditions for botomycin derivatives): column: XTerra 3.9 x 150 WAT 186000478; temperature: 40 ° C; flow rate: 1 ml / min; eluent A: 0.01% trifluoroacetic acid in water, elution Agent B: acetonitrile + 0.01% trifluoroacetic acid, gradient: 0.0 min 20% B → 1 min 20% B → 4 min 90% B → 6 min 90% B → 6.2 min 20% B.

Method 3 (Standard Conditions for Analytical LC-MS Measurement of Bothromycin Derivatives): Instrument: HPLC Micromass LCT with Agilent Series 1100; Column: Waters Symmetry C18; 3.5 μm; 50 mm x 2.1 mm; Eluent A: Water 1 l + 98 -100% formic acid 1 ml; eluent B: acetonitrile 1 l + 98-100% formic acid 1 ml; gradient: 0 min 100% A → 1 min 100% A → 6 min 10% A → 8 min 0% A → 10 min 0% A → 10.1 min 100% A → 12 min 100% A; flow rate from 0 min to 10 min 0.5 ml / min → 10.1 min 1 ml / min → 12 min 0.5 ml / min; oven: 40 ° C .; UV detection DAD : 208-500 nm.

NMR measurement: NMR measurement was performed with Bruker DMX500 having a proton frequency of 500.13 MHz. The solvent was DMSO-d ₆ and the temperature was 302K. Calibration was based on a DMSO signal of 2.5 ppm.

The structure of the starting compound botromycin A2 (Example 1A) was confirmed by X-ray structural analysis. The other botomycins listed (Examples 2A-5A) are assumed to have the same stereochemistry as they show similar NMR spectra. Therefore, these structures have different stereochemistry from the current state of the art.

  Compounds 1A-5A formed during fermentation are used for further reactions (see Example 1). If the stereochemical attributes of compounds 1A to 5A are actually different, then the stereochemistry of the secondary product is correspondingly different.

Example 1A
N-[(3S, 6S, 14R, 14aS) -6-tert-butyl-3-isopropyl-14-methyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3-methoxy-3-oxo-1- (1 , 3-thiazol-2-yl) propyl] -beta-methyl-L-phenylalaninamide (bothromycin A2)

Example 2A
N-[(3S, 6S, 14aS) -6-tert-butyl-3-isopropyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4,7,10] tetraaza Cyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3-methoxy-3-oxo-1- (1,3-thiazole-2 -Yl) propyl] -beta-methyl-L-phenylalaninamide (bothromycin B2)

Example 3A
N-[(3S, 6S, 14aS) -6-tert-butyl-3-isopropyl-14,14-dimethyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3-methoxy-3-oxo-1- (1 , 3-thiazol-2-yl) propyl] -beta-methyl-L-phenylalaninamide (bothromycin C2)

Example 4A
N-[(3S, 6S, 14R, 14aS) -6-tert-butyl-3-isopropyl-14-methyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -2-carboxy-1- (1,3-thiazole -2-yl) ethyl] -beta-methyl-L-phenylalaninamide (botromycin A2 as acid)

Example 5A
N-[(3S, 6S, 14aS) -6-tert-butyl-3-isopropyl-14,14-dimethyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -2-carboxy-1- (1,3-thiazole -2-yl) ethyl] -beta-methyl-L-phenylalaninamide (botromomycin C2 as acid)

ATCC = American Type Culture Collection, Manassas, Virginia, U.S.A.
DSM = Deutsche Sammlung fuer Mikroorganismen und Zellkuluren, Brunswick, Germany.
CBS = Centraalbureau voor Schimmelcultures, Baarn, The Netherlands Synthesis of compounds 1A, 2A, 3A, 4A and 5A Strains used Table 1: Strains capable of producing the precursors botomycins A and B:

ATCC = American Type Culture Collection, Manassas, Virginia, USA
DSM = Deutsche Sammlung fuer Mikroorganismen und Zellkuluren, Brunswick, Germany.
CBS = Centraalbureau voor Schimmelcultures, Baarn, The Netherlands

  Streptomyces botropensis was isolated and identified by Koninklijke Nederlandsche Gisten Spiritusfabriek, N.V. and deposited at CBS, Baarn, The Netherlands (BP 762736, 1954) under strain deposit number CBS 163.64. To confirm this patent, the strain was deposited again at Deutsche Sammlung fuer Mikroorganismen und Zellkuluren, Brunswick, Germany under the deposit number DSM 16814.

  Streptomyces genus DSM 17025 was isolated from a soil sample from Eifel / Germany and included in the Bayer Healthcare AG stock collection with strain number BC16019.

Example 1A Production of 2A and 3A Preparation of Preculture of S. botropensis DSM16814 For the preparation of the preculture, the strain S. botropensis DSM16814 was cultured in the following medium: Medium 1: Yeast-Mort Medium: D-glucose 0.4%, yeast extract 0.4%, malt extract 1.0%, tap water up to 1 liter. The pH value of the medium was adjusted to 7.2 using an aqueous sodium hydroxide solution. In each case, the medium was sterilized for 20 minutes at 121 ° C. and 1.1 bar overpressure.

  In each case, 2 ml of mycelial suspension in 50% glycerol was prepared as a seed for 15 × 150 ml of medium 1 in a 1000 ml Erlenmeyer flask, 96 hours, 240 revolutions per minute (rev / min), 28 ° C. And incubated on a rotary shaker. These cultures were used to prepare fresh glycerol stock samples stored at -20 ° C or they served as seeds for production cultures.

Culture for production of S. botropensis DSM 16814 (30 l scale)
For the preparation of the production culture, the above 15 × 150 ml preculture was prepared by adding 22 l of the following medium 2: glucose (1%), starch (1.5%), yeast extract (0.5%), The defatted soybean flour (1.0%), sodium chloride (0.5%) and calcium carbonate (0.3%) were used as inoculum. Prior to seeding, 1 ml / l of antifoam SAG 5693 (Union Carbide, USA) was added to the medium and the medium was sterilized with steam under 1.1 bar for 60 minutes. The production culture was incubated in a 30 l Bioengineering (Switzerland) stirred fermenter (blade stirrer) for 120-160 hours at 28 ° C., agitation speed of 240 rpm, with an aeration rate of 0.3 vvm. A 20 ml daily sample obtained under aseptic conditions was tested using analytical HPLC and the manufacturing process was monitored. Under these conditions, a yield of botromycin A (Example 1A) of 20 mg / l was obtained.

Preparation of a preculture of S. genus DSM 17025 For the preparation of the preculture, the strain S. genus DSM 17025 was cultured in the following medium: medium 1: yeast-malt medium: 0.4% D-glucose, Up to 0.4% yeast extract, 1.0% malt extract and 1 liter of tap water. The pH value of the medium was adjusted to 7.2 using an aqueous sodium hydroxide solution. In each case, the medium was sterilized for 20 minutes at 121 ° C. and 1.1 bar overpressure.

  In each case, 2 ml of mycelium suspension in 50% glycerol was prepared as 2 × 150 ml of medium 1 in a 1000 ml Erlenmeyer flask as inoculum, 96 hours, 240 revolutions per minute (rev / min), 28 ° C. And incubated on a rotary shaker. These cultures were used to prepare fresh glycerol stock samples stored at -20 ° C or they served as seeds for production cultures.

Culture for production of S. genus DSM 17025 (30 l scale)
For the preparation of the production culture, the above 2 × 150 ml preculture was added to 30 l of the following medium 2: glucose (1%), starch (1.5%), yeast extract (0.5%), The defatted soybean flour (1.0%), sodium chloride (0.5%) and calcium carbonate (0.3%) were used as inoculum. Prior to seeding, 1 ml / l of antifoam SAG 5693 (Union Carbide, USA) was added to the medium and the medium was sterilized with steam under 1.1 bar for 60 minutes. The production culture was incubated in a 40 l Bioengineering (Switzerland) stirred fermentor (blade stirrer) for 90-140 hours at 28 ° C., agitation speed of 240 rev / min and aeration rate of 0.3 vvm. A 20 ml daily sample obtained under aseptic conditions was tested using analytical HPLC and the manufacturing process was monitored. Under these conditions, a yield of botromycin A (Example 1A) of 20 mg / l was obtained.

Analytical HPLC
An analytical HPLC method with UV / visible (HPLC-UV / Vis) detection and detection by mass spectrometry was used for detection during biosynthesis.
Prior to HPLC analysis, the aqueous fermentation sample was divided into mycelium and culture supernatant and methanol was added to both products obtained: the culture filtrate was diluted 1: 4 with methanol and centrifuged mycelium Was extracted with an initial amount of methanol in an ultrasonic bath for 15 minutes. Methanolic samples were filtered through 0.45 μm filters and injected into analytical HPLC or LC-MS, respectively.

Analysis by HPLC-MS was performed in ESI ⁺ and ESI ⁻ modes on an HP 1100 HPLC system combined with a Micromass-LCT mass spectrometer (Micromass, Manchester, Great Britain). Mass spectra were recorded in the range of m / z = 100-1650. Individual botomycins have the following retention times:
Bothromycin A2 (Example 1A): R _t = 4.54 min Bothromycin B2 (Example 2A): R _t = 4.51 min Bothromycin C2 (Example 3A): R _t = 4.61 min Bothromycin A2 free acid ( Example 4A): R _t = 4.51 min Bothromycin C2 free acid (Example 5A): R _t = 4.48 min

The HPLC-MS system used was operated with the following parameters: stationary phase: Waters Symmetry C18, 3.5 μm 2.1 × 50 mm; mobile phase: water with gradient 0.1% formic acid (A) and 0.1. Acetonitrile with% formic acid (B): 0-1 min 100% A; 1-6 min linear gradient to 90% B; 6-8 min 90% B-100% B; 8-10 min 100% B; flow rate 0.5 ml / min; column oven 40 ° C., UV detection: DAD 208-700 nm. The detection of botomycin was performed based on protonated molecular ion (M + H) ⁺ and deprotonated molecular ion (M−H) ⁻ , doubly charged molecule (M + 2H) ⁺⁺ and formic acid adduct (M + AS) ⁻ .

Bothromycin A2 (Example 1A): (M + H) ⁺ = 823.8; (M + 2H) ⁺⁺ = 412.5; (M−H) ⁻ = 821.6; (M + AS) ⁻ = 867.7
Bothromycin B2 (Example 2A): (M + H) ⁺ = 809.8; (M + 2H) ⁺⁺ = 405.5; (M−H) ⁻ = 807.6; (M + AS) ⁻ = 853.6
Bothromycin C2 (Example 3A): (M + H) ⁺ = 837.7; (M + 2H) ⁺⁺ = 419.5; (M−H) ⁻ = 835.6; (M + AS) ⁻ = 881.6
Bothromycin A2 free acid (Example 4A): (M + H) ⁺ = 809.6; (M + 2H) ⁺⁺ = 405.5; (M−H) ⁻ = 807.5
Bothromycin C2 free acid (Example 5A): (M + H) ⁺ = 823.7; (M + 2H) ⁺⁺ = 412.5; (M−H) ⁻ = 821.5; (M + AS) ⁻ = 867.5

  Using a Hewlett Packard Series 1100 Analytical HPLC System (HP, Waldbronn, Germany) consisting of a G 1312A binary pump system, G 1315A diode array detector, G 1316A column temperature control system, G 1322A degassing system and G 1313A autoinjector. HPLC-UV / Vis analysis was performed. Waters Symmetry C18 with a 10 mm precolumn using water (A) containing 0.05% trifluoroacetic acid and acetonitrile (B) containing 0.05% trifluoroacetic acid as the mobile phase at a flow rate of 0.4 ml / min. A 3.5 μm 2.1 × 50 mm column served as the stationary phase. Gradual gradient 0-100% B (25-25-45-45-65-100-25% in 0-1-2-3.5-6.7-6.8-7.8-8.4 min) The sample was separated in B). HPLC-UV chromatograms were recorded at 210 nm (detection of impurities), 225 nm and 254 nm. Diode array detection in the 200-600 nm range resulted in HPLC-UV / Vis spectra. The column oven was set at 45 ° C.

The compounds described have the following UV spectra: UV _{max at} 206-207 nm, weak shoulder at 230 nm, the following retention times:
Bothromycin A2 (Example 1A): R _t = 4.68 min Bothromycin B2 (Example 2A): R _t = 4.54 min Bothromycin C2 (Example 3A): R _t = 4.88 min Bothromycin A2 free acid ( Example 4A): R _t = 3.44 min botomycin C2 free acid (Example 5A): R _t = 4.22 min

Isolation of the compounds of Examples 1A, 2A, 3A, 4A and 5A from a stirred fermentor culture (30 l scale) Mycelium from a 30 l scale Streptomyces botropensis DSM16814 fermentation is centrifuged from the supernatant (15 minutes at 2300 rpm).
The culture supernatant obtained after centrifugation was loaded onto a 2500 ml Lewapol column (adsorption resin, OC 1064, Bayer AG, Leverkusen, Germany). The flow through and wash water (about 30 l) were discarded. A subsequent wash was then performed with 6 column volumes of 60% methanol and 3 column volumes of 80% methanol. The obtained fraction was discarded, and botromycin bound to the adsorption resin was eluted with 100% methanol at least 10 times the column volume. The eluate was then dried in vacuo.

  The mycelium obtained after centrifugation was treated twice with 5 l each of methanol in an Ultra-Turrax P50 DPX (Janke & Kunkel Labortechnik), then separated and discarded. The methanolic extract obtained from the mycelia digestion was concentrated on a rotary evaporator and the aqueous residue (about 2.6 l) was extracted 4 times with 2 l each of ethyl acetate. The ethyl acetate phase was dried over sodium sulfate, filtered and dried in vacuo.

  The mycelium (about 11 g, content of botomycin A2 → major metabolite, about 3%) and culture supernatant (about 5 g, content of botromomycin A2 content of about 10%) were combined and partially methanol And further purified with a Biotage chromatography column (Flash 75, 75 × 300 mm, Biotage C18 KP-WP, 15-20 μM). Eluent water (A) containing 0.05% trifluoroacetic acid and acetonitrile (B) containing 0.05% trifluoroacetic acid at a flow rate of 80 ml / min at 12, 16, 20, 25, 28, 31, 31, Step gradients were used at 35 and 50% B steps. Bothromycin, eluting at 31% B, was concentrated to dryness in vacuo (residue about 0.9 g, about 61% yield of botomycin A2) and dissolved in methanol. The total volume was divided into three parts and then separated in three separate runs using a preparative HPLC system: hardware Gilson Abimed HPLC (UV detector 210 nm, binary pump system, fraction collector); solid Phase: Inertsil ODS-3 C18, 5 μm with precolumn Nucleosil C18, 7 μm, 20 × 30 mm; 30 × 250 mm; flow rate: 41.2 ml / min; fraction size 20.6 ml / 0.5 min, 50 Stepwise gradient from water (A) containing 0.05% trifluoroacetic acid to acetonitrile (B) containing 0.05% trifluoroacetic acid: 0-15-75-105-115-125 minutes 10-10-30-30-100-100% B.

  Bothromycin eluted with a gradient region of 31-35% B. After HPLC and HPLC-MS analysis, the individual fractions were combined according to the target material and dried in vacuo.

  A yield of 320 mg with> 97% purity was achieved for the major metabolite botromycin A2 (Example 1A). For the second metabolites botromomycin B2 (Example 2A) and botomycin C2 (Example 3A),> 98% pure 12 mg and> 90% pure ˜15 mg were achieved. The free acid of botomycin A2 (Example 4A) and botomycin C2 (Example 5A) is present as a minor component in an amount of <5 mg. The second metabolite (Example 3A) and minor components (Example 4A), (Example 5A) were further purified to> 98% purity in a further purification step with the same preparative HPLC method.

  After purification, botromycin exists as a salt of trifluoroacetic acid and salt exchange to mesyl and formate is completely possible.

Example 4A
Alternative synthesis:
265 mg (322 μmol) of botromomycin A2 is dissolved in 125 ml of methanol and 2.4 ml of 2M aqueous lithium hydroxide solution is added. The solution is heated under reflux for 1 hour, then cooled to room temperature and adjusted to pH 1-2 with 5.4 ml of 1M hydrochloric acid. The mixture was concentrated and the residue was partitioned between 300 ml ethyl acetate and 100 ml water. The aqueous phase is separated, saturated with ammonium chloride and extracted again with 100 ml of ethyl acetate. The combined ethyl acetate phases are concentrated and the residue is taken up in dichloromethane / methanol 1: 1. The target product is cooled and precipitated by the addition of diethyl ether and petroleum ether. After recovery by suction filtration and drying, 199 mg (76% of theory) of the acid are obtained.
HPLC (Method 2): R _t = 4.14 min;
LC-MS (Method 3): R _t = 4.34 min; m / z = 809 (M + H) ⁺

Example 5A
Alternative synthesis:
Similar to the alternative synthesis of Example 4A, the title compound is prepared from 28 mg (33.45 μM) of botomycin C2.
Yield: 20 mg (73% of theory)
HPLC (Method 2): R _t = 4.16 min;
LC-MS (Method 3): R _t = 4.47 min; m / z = 821 (M−H) ⁻

実施例４Ａの代替的合成と同様に、ボトロマイシンＢ２ ２２ｍｇ（２７μＭ）から表題化合物を製造する。
収量：２１ｍｇ（理論値の９７％）
ＨＰＬＣ（方法２）：Ｒ_ｔ＝４.０１分；
ＬＣ−ＭＳ（方法３）：Ｒ_ｔ＝４.３４分；ｍ／ｚ＝７９３（Ｍ−Ｈ）⁻ Example 6A
N-[(3S, 6S, 14aS) -6-tert-butyl-3-isopropyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4,7,10] tetraaza- Cyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -2-carboxy-1- (1,3-thiazol-2-yl) ethyl]- Beta-methyl-L-phenylalaninamide

Analogous to the alternative synthesis of Example 4A, the title compound is prepared from 22 mg (27 μM) of botomycin B2.
Yield: 21 mg (97% of theory)
HPLC (Method 2): R _t = 4.01 min;
LC-MS (Method 3): R _t = 4.34 min; m / z = 793 (M−H) ⁻

Exemplary Embodiment
Example 1
N-[(3S, 6S, 14R, 14aS) -6-tert-butyl-3-isopropyl-14-methyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3- [methoxy (methyl) amino] -3- Oxo-1- (1,3-thiazol-2-yl) propyl] -beta-methyl-L-phenylalaninamide

75 mg (92.7 μM) of the compound derived from Example 4A was dissolved in 3 ml of DMF, 18.8 mg (139 μmol) of 1-hydroxy-1H-benzotriazole, N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride 21.3 mg (111.2 μmol) of salt and then 28.3 mg (463.5 μmol) of N, O-dimethylhydroxylamine are added. The mixture is stirred at room temperature for 16 hours. The mixture is then concentrated. The remaining residue is taken up in 300 ml of dichloromethane and extracted by shaking with 50 ml of 5% citric acid. The aqueous phase is separated, saturated with ammonium chloride and extracted again with dichloromethane. The combined organic phases are concentrated and the residue is purified by preparative HPLC (Method 1). Corresponding fractions are combined, the solvent is evaporated and the remaining residue is lyophilized from dioxane / water 1: 1. 55 mg (70% of theory) of the target compound are obtained.
HPLC (Method 2): R _t = 4.31 min;
LC-MS (Method 3): R _t = 4.59 min; m / z = 852 (M + H) ^+
1 H-NMR (500 MHz, DMSO-d ₆ ): δ = 0.8 and 0.89 (2d, 6H), 0.84 (s, 9H), 0.9 (s, 9H), 0.97 (d, 3H), 1.12 (d, 3H ), 1.68 (m, 1H), 1.88 (m, 1H), 2.2 (m, 1H), 2.55 (m, 1H), 2.9-3.3 (m, 6H), 3.65-3.8 (m, 5H), 3.4 and 3.9 (2m, 2H), 3.95 (d, 1H), 4.12 (d, 1H), 4.2 (dd, 1H), 4.7 (t, 1H), 5.0 (d, 1H), 5.63 (m, 1H), 6.7 (d, 1H), 7.1-7.3 (m, 5H), 7.45 (m, 1H), 7.63 and 7.73 (2d, 2H), 7.8-8.0 (m, 2H), 8.95 (d, 1H), 9.22 (d , 1H).

実施例１の合成と同様に、実施例４Ａ由来の化合物５ｍｇ（６.２μＭ）、イソオキサゾリジン塩酸塩 (Perkin 2, 2000, 1435 or J. Chem. Soc. 1942, 432 と同様に製造）１.３５ｍｇ（１２.４μＭ）、１−ヒドロキシ−１Ｈ−ベンゾトリアゾール１.２５ｍｇ、Ｎ−（３−ジメチルアミノプロピル）−Ｎ'−エチルカルボジイミド塩酸塩１.４ｍｇおよびエチル−ジイソプロピルアミン３μｌから、表題化合物を製造する。
収量：２.１ｍｇ（理論値の４０％）
ＨＰＬＣ（方法２）：Ｒ_ｔ＝４.２６分；
ＬＣ−ＭＳ（方法３）：Ｒ_ｔ＝４.０分；ｍ／ｚ＝８６４（Ｍ＋Ｈ）^＋ Example 2
N-[(3S, 6S, 14R, 14aS) -6-tert-butyl-3-isopropyl-14-methyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3-isoxazolidin-2-yl-3-oxo -1- (1,3-thiazol-2-yl) propyl] -beta-methyl-L-phenylalaninamide

Similar to the synthesis of Example 1, 5 mg (6.2 μM) of the compound derived from Example 4A, isoxazolidine hydrochloride (produced in the same manner as Perkin 2, 2000, 1435 or J. Chem. Soc. 1942, 432) From 35 mg (12.4 μM), 1-hydroxy-1H-benzotriazole 1.25 mg, N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride 1.4 mg and ethyl-diisopropylamine 3 μl To manufacture.
Yield: 2.1 mg (40% of theory)
HPLC (Method 2): R _t = 4.26 min;
LC-MS (Method 3): R _t = 4.0 min; m / z = 864 (M + H) ⁺

実施例１の合成と同様に、実施例４Ａ由来の化合物１０ｍｇ（１２.４μＭ）、１,２−オキサジナン塩酸塩（Perkin 2, 2000, 1435 または J. Chem. Soc. 1942, 432 と同様に製造）３.１ｍｇ（２４.７μＭ）、１−ヒドロキシ−１Ｈ−ベンゾトリアゾール２.５ｍｇ、Ｎ−（３−ジメチルアミノプロピル）−Ｎ'−エチルカルボジイミド塩酸塩２.９ｍｇおよびエチル−ジイソプロピルアミン６.５μｌから、表題化合物を製造する。
収量：３.８ｍｇ（理論値の３５％）
ＨＰＬＣ（方法２）：Ｒ_ｔ＝４.３６分；
ＬＣ−ＭＳ（方法３）：Ｒ_ｔ＝４.６分；ｍ／ｚ＝８７８（Ｍ＋Ｈ）^＋ Example 3
N-[(3S, 6S, 14R, 14aS) -6-tert-butyl-3-isopropyl-14-methyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -beta-methyl-N-[(1R) -3- (1,2-oxazinane -2-yl) -3-oxo-1- (1,3-thiazol-2-yl) propyl] -L-phenylalaninamide

Analogously to the synthesis of Example 1, 10 mg (12.4 μM) of the compound from Example 4A, 1,2-oxazinane hydrochloride (Perkin 2, 2000, 1435 or J. Chem. Soc. 1942, 432 ) 3.1 mg (24.7 μM), 1-hydroxy-1H-benzotriazole 2.5 mg, N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride 2.9 mg and ethyl-diisopropylamine 6.5 μl The title compound is prepared from
Yield: 3.8 mg (35% of theory)
HPLC (Method 2): R _t = 4.36 min;
LC-MS (Method 3): R _t = 4.6 min; m / z = 878 (M + H) ⁺

実施例１の合成と同様に、実施例６Ａ由来の化合物１１ｍｇ（１３.８５μＭ）、１,２−オキサジナン塩酸塩（Perkin 2, 2000, 1435 または J. Chem. Soc. 1942, 432と同様に製造) ５.１ｍｇ（４１.５μＭ）、１−ヒドロキシ−１Ｈ−ベンゾトリアゾール２.８ｍｇ、Ｎ−（３−ジメチルアミノプロピル）−Ｎ'−エチルカルボジイミド塩酸塩３.２ｍｇおよびエチル−ジイソプロピルアミン７μｌから、表題化合物を製造する。
収量：１.７ｍｇ（理論値の１４％）
ＨＰＬＣ（方法２）：Ｒ_ｔ＝４.２６分；
ＬＣ−ＭＳ（方法３）：Ｒ_ｔ＝４.６０分；ｍ／ｚ＝８６４（Ｍ＋Ｈ）^＋ Example 4
N-[(3S, 6S, 14aS) -6-tert-butyl-3-isopropyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4,7,10] tetraaza- Cyclododecine-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -beta-methyl-N-[(1R) -3- (1,2-oxazinan-2-yl) -3- Oxo-1- (1,3-thiazol-2-yl) propyl] -L-phenylalaninamide

Analogously to the synthesis of Example 1, 11 mg (13.85 μM) of the compound from Example 6A, 1,2-oxazinane hydrochloride (Perkin 2, 2000, 1435 or J. Chem. Soc. 1942, 432 ) 5.1 mg (41.5 μM), 1-hydroxy-1H-benzotriazole 2.8 mg, N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride 3.2 mg and ethyl-diisopropylamine 7 μl The title compound is prepared.
Yield: 1.7 mg (14% of theory)
HPLC (Method 2): R _t = 4.26 min;
LC-MS (Method 3): R _t = 4.60 min; m / z = 864 (M + H) ⁺

実施例１の合成と同様に、実施例６Ａ由来の化合物１１ｍｇ（１３.９μＭ）、Ｎ,Ｏ−ジメチルヒドロキシルアミン４.２ｍｇ（６９μＭ）、１−ヒドロキシ−１Ｈ−ベンゾトリアゾール２.８ｍｇおよびＮ−（３−ジメチルアミノプロピル）−Ｎ'−エチルカルボジイミド塩酸塩３.２ｍｇから、表題化合物を製造する。
収量：８.８ｍｇ（理論値の７６％）
ＨＰＬＣ（方法２）：Ｒ_ｔ＝４.１７分；
ＬＣ−ＭＳ（方法３）：Ｒ_ｔ＝４.４９分；ｍ／ｚ＝８３８（Ｍ＋Ｈ）^＋ Example 5
N-[(3S, 6S, 14aS) -6-tert-butyl-3-isopropyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4,7,10] tetraaza- Cyclododecine-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3- [methoxy (methyl) amino] -3-oxo-1- (1,3 -Thiazol-2-yl) propyl] -beta-methyl-L-phenylalaninamide

Similar to the synthesis of Example 1, 11 mg (13.9 μM) of the compound from Example 6A, 4.2 mg (69 μM) of N, O-dimethylhydroxylamine, 2.8 mg of 1-hydroxy-1H-benzotriazole and N- The title compound is prepared from 3.2 mg of (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride.
Yield: 8.8 mg (76% of theory)
HPLC (Method 2): R _t = 4.17 min;
LC-MS (Method 3): R _t = 4.49 min; m / z = 838 (M + H) ⁺

実施例１の合成と同様に、実施例５Ａ由来の化合物１０ｍｇ（１２μＭ）、Ｎ,Ｏ−ジメチルヒドロキシルアミン３.７ｍｇ（６１μＭ）、１−ヒドロキシ−１Ｈ−ベンゾトリアゾール２.５ｍｇおよびＮ−（３−ジメチルアミノプロピル）−Ｎ'−エチルカルボジイミド塩酸塩２.８ｍｇから表題化合物を製造する。
収量：１.７ｍｇ（理論値の１６％）
ＨＰＬＣ（方法２）：Ｒ_ｔ＝４.３１分；
ＬＣ−ＭＳ（方法３）：Ｒ_ｔ＝４.５４分；ｍ／ｚ＝８６６（Ｍ＋Ｈ）^＋ Example 6
N-[(3S, 6S, 14aS) -6-tert-butyl-3-isopropyl-14,14-dimethyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3- [methoxy (methyl) amino] -3- Oxo-1- (1,3-thiazol-2-yl) propyl] -beta-methyl-L-phenylalaninamide

Analogously to the synthesis of Example 1, 10 mg (12 μM) of the compound from Example 5A, 3.7 mg (61 μM) of N, O-dimethylhydroxylamine, 2.5 mg of 1-hydroxy-1H-benzotriazole and N- (3 The title compound is prepared from 2.8 mg of -dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride.
Yield: 1.7 mg (16% of theory)
HPLC (Method 2): R _t = 4.31 min;
LC-MS (Method 3): R _t = 4.54 min; m / z = 866 (M + H) ⁺

実施例１の合成と同様に、実施例４Ａ由来の化合物１０ｍｇ（１２.４μＭ）、Ｎ,Ｏ−ジエチルヒドロキシルアミン（ＤＥ２１１３３５５（１９７１）と同様に製造）５.５ｍｇ（６２μＭ）、１−ヒドロキシ−１Ｈ−ベンゾトリアゾール３.３ｍｇおよびＮ−（３−ジメチルアミノプロピル）−Ｎ'−エチルカルボジイミド塩酸塩３.６ｍｇから、表題化合物を製造する。
収量：５.４ｍｇ（理論値の５０％）
ＨＰＬＣ（方法２）：Ｒ_ｔ＝４.３５分；
ＬＣ−ＭＳ（方法３）：Ｒ_ｔ＝４.７８分；ｍ／ｚ＝８８０（Ｍ＋Ｈ）^＋ Example 7
N-[(3S, 6S, 14R, 14aS) -6-tert-butyl-3-isopropyl-14-methyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3- [ethoxy (ethyl) amino] -3- Oxo-1- (1,3-thiazol-2-yl) propyl] -beta-methyl-L-phenylalaninamide

Similar to the synthesis of Example 1, 10 mg (12.4 μM) of the compound from Example 4A, N, O-diethylhydroxylamine (prepared in the same way as DE211355 (1971)) 5.5 mg (62 μM), 1-hydroxy- The title compound is prepared from 3.3 mg of 1H-benzotriazole and 3.6 mg of N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride.
Yield: 5.4 mg (50% of theory)
HPLC (Method 2): R _t = 4.35 min;
LC-MS (Method 3): R _t = 4.78 min; m / z = 880 (M + H) ⁺

実施例１の合成と同様に、実施例４Ａ由来の化合物１０ｍｇ（１２.４μＭ）、Ｎ−メチル−Ｏ−エチルヒドロキシルアミン（ＤＥ２１１３３５５（１９７１）と同様に製造）４.６ｍｇ（６２μＭ）、１−ヒドロキシ−１Ｈ−ベンゾトリアゾール３.３ｍｇおよびＮ−（３−ジメチルアミノプロピル）−Ｎ'−エチルカルボジイミド塩酸塩３.６ｍｇから、表題化合物を製造する。
収量：５ｍｇ（理論値の４７％）
ＨＰＬＣ（方法２）：Ｒ_ｔ＝４.２３分；
ＬＣ−ＭＳ（方法３）：Ｒ_ｔ＝４.６３分；ｍ／ｚ＝８６６（Ｍ＋Ｈ）^＋ Example 8
N-[(3S, 6S, 14R, 14aS) -6-tert-butyl-3-isopropyl-14-methyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3- [ethoxy (methyl) amino] -3- Oxo-1- (1,3-thiazol-2-yl) propyl] -beta-methyl-L-phenylalaninamide

Similar to the synthesis of Example 1, 10 mg (12.4 μM) of the compound derived from Example 4A, N-methyl-O-ethylhydroxylamine (produced in the same manner as DE211355 (1971)), 4.6 mg (62 μM), 1- The title compound is prepared from 3.3 mg of hydroxy-1H-benzotriazole and 3.6 mg of N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride.
Yield: 5 mg (47% of theory)
HPLC (Method 2): R _t = 4.23 min;
LC-MS (Method 3): R _t = 4.63 min; m / z = 866 (M + H) ⁺

実施例１の合成と同様に、実施例４Ａ由来の化合物１０ｍｇ（１２.４μＭ）およびイソオキサゾリジン−４−オール（Synthesis 1982, 5, 426と同様に製造）２.２ｍｇ（２５μＭ）から、表題化合物を製造する。ここで、Ｎ−（３−ジメチルアミノプロピル）−Ｎ'−エチルカルボジイミド塩酸塩／１−ヒドロキシ−１Ｈ−ベンゾトリアゾールの代わりに、［Ｏ−（７−アザベンゾトリアゾール−１−イル）−１,１,３,３−テトラメチルウロニウムヘキサフルオロホスフェート］（ＨＡＴＵ）７.１ｍｇ（１８.６μＭ）を、エチルジイソプロピルアミン３.２ｍｇの存在下でカップリング剤として使用する。１６時間室温で撹拌した後、混合物を濃縮し、分取用ＨＰＬＣ（方法１）により残渣を直接精製し、表題化合物を実施例１と同様に単離する。
収量：６.８ｍｇ（理論値の６３％）
ＨＰＬＣ（方法２）：Ｒ_ｔ＝３.８６分；
ＬＣ−ＭＳ（方法３）：Ｒ_ｔ＝４.７分；ｍ／ｚ＝８８０（Ｍ＋Ｈ）^＋ Example 9
N-[(3S, 6S, 14R, 14aS) -6-tert-butyl-3-isopropyl-14-methyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3- (4-hydroxyisoxazolidin-2-yl ) -3-Oxo-1- (1,3-thiazol-2-yl) propyl] -beta-methyl-L-phenylalaninamide

Similar to the synthesis of Example 1, the title compound was obtained from 10 mg (12.4 μM) of the compound from Example 4A and 2.2 mg (25 μM) of isoxazolidin-4-ol (prepared as Synthesis 1982, 5, 426). Manufacturing. Here, instead of N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride / 1-hydroxy-1H-benzotriazole, [O- (7-azabenzotriazol-1-yl) -1, 7.1 mg (18.6 μM) of 1,3,3-tetramethyluronium hexafluorophosphate] (HATU) is used as coupling agent in the presence of 3.2 mg of ethyldiisopropylamine. After stirring for 16 hours at room temperature, the mixture is concentrated and the residue is purified directly by preparative HPLC (Method 1) and the title compound is isolated as in Example 1.
Yield: 6.8 mg (63% of theory)
HPLC (Method 2): R _t = 3.86 min;
LC-MS (Method 3): R _t = 4.7 min; m / z = 880 (M + H) ⁺

実施例１の合成と同様に、実施例４Ａ由来の化合物１０ｍｇ（１２.４μＭ）およびエチルイソオキサゾリジン−５−カルボキシレート（J. Chem. Soc. Chem. Commun. 1984, 9, 606と同様に製造）３.６ｍｇ（２５μＭ）から、表題化合物を製造する。ここで、Ｎ−（３−ジメチルアミノプロピル）−Ｎ'−エチルカルボジイミド塩酸塩／１−ヒドロキシ−１Ｈ−ベンゾトリアゾールの代わりに、［Ｏ−（７−アザベンゾトリアゾール−１−イル）−１,１,３,３−テトラメチルウロニウムヘキサフルオロホスフェート］（ＨＡＴＵ）９.４ｍｇ（２４.７μＭ）を、エチルジイソプロピルアミン３.２ｍｇの存在下で、カップリング剤として使用する。室温で１６時間撹拌した後、混合物を濃縮し、分取用ＨＰＬＣ（方法１）により残渣を直接精製し、実施例１と同様に、表題化合物を単離する。
収量：６ｍｇ（理論値の５２％）
ＨＰＬＣ（方法２）：Ｒ_ｔ＝４.３分；
ＬＣ−ＭＳ（方法３）：Ｒ_ｔ＝５.１分；ｍ／ｚ＝９３６（Ｍ＋Ｈ）^＋ Example 10
N-[(3S, 6S, 14R, 14aS) -6-tert-butyl-3-isopropyl-14-methyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3- [5- (ethoxycarbonyl) isoxazolidine- 2-yl] -3-oxo-1- (1,3-thiazol-2-yl) propyl] -beta-methyl-L-phenylalaninamide

Analogously to the synthesis of Example 1, 10 mg (12.4 μM) of the compound from Example 4A and ethyl isoxazolidine-5-carboxylate (J. Chem. Soc. Chem. Commun. 1984, 9, 606) ) The title compound is prepared from 3.6 mg (25 μM). Here, instead of N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride / 1-hydroxy-1H-benzotriazole, [O- (7-azabenzotriazol-1-yl) -1, 9.4 mg (24.7 μM) of 1,3,3-tetramethyluronium hexafluorophosphate] (HATU) is used as a coupling agent in the presence of 3.2 mg of ethyldiisopropylamine. After stirring for 16 hours at room temperature, the mixture is concentrated and the residue is directly purified by preparative HPLC (Method 1), and the title compound is isolated as in Example 1.
Yield: 6 mg (52% of theory)
HPLC (Method 2): R _t = 4.3 min;
LC-MS (Method 3): R _t = 5.1 min; m / z = 936 (M + H) ⁺

実施例１の合成と同様に、実施例４Ａ由来の化合物１７ｍｇ（２１μＭ）およびエチル−１,２−オキサジナン−６−カルボキシレート（J. Org. Chem. 2000, 65, 7667から製造）６.７ｍｇ（４２μＭ）から、表題化合物を製造する。ここで、Ｎ−（３−ジメチルアミノプロピル）−Ｎ'−エチルカルボジイミド塩酸塩／１−ヒドロキシ−１Ｈ−ベンゾトリアゾールの代わりに、［Ｏ−（７−アザベンゾトリアゾール−１−イル）−１,１,３,３−テトラメチルウロニウムヘキサフルオロホスフェート］（ＨＡＴＵ）１６ｍｇ（４２μＭ）を、エチルジイソプロピルアミン５.４ｍｇの存在下で、カップリング剤として使用する。１６時間室温で撹拌した後、混合物を濃縮し、残渣を分取用ＨＰＬＣ（方法１）により直接精製し、表題化合物を実施例１と同様に単離する。
収量：１６.７ｍｇ（理論値の８４％）
ＨＰＬＣ（方法２）：Ｒ_ｔ＝４.５分；
ＬＣ−ＭＳ（方法３）：Ｒ_ｔ＝５.３分；ｍ／ｚ＝９４８（Ｍ−Ｈ）⁻ Example 11
N-[(3S, 6S, 14R, 14aS) -6-tert-butyl-3-isopropyl-14-methyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3- [6- (ethoxycarbonyl) -1, 2-Oxazinan-2-yl] -3-oxo-1- (1,3-thiazol-2-yl) propyl] -beta-methyl-L-phenylalaninamide

Analogously to the synthesis of Example 1, 17 mg (21 μM) of the compound from Example 4A and 6.7 mg of ethyl-1,2-oxazinane-6-carboxylate (prepared from J. Org. Chem. 2000, 65, 7667) The title compound is prepared from (42 μM). Here, instead of N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride / 1-hydroxy-1H-benzotriazole, [O- (7-azabenzotriazol-1-yl) -1, 16 mg (42 μM) of 1,3,3-tetramethyluronium hexafluorophosphate] (HATU) is used as a coupling agent in the presence of 5.4 mg of ethyldiisopropylamine. After stirring for 16 hours at room temperature, the mixture is concentrated, the residue is purified directly by preparative HPLC (Method 1) and the title compound is isolated as in Example 1.
Yield: 16.7 mg (84% of theory)
HPLC (Method 2): R _t = 4.5 min;
LC-MS (Method 3): R _t = 5.3 min; m / z = 948 (M−H) ⁻

実施例１の合成と同様に、実施例４Ａ由来の化合物５ｍｇ（６.２μＭ）および２−オキサ−３−アザビシクロ［２.２.２］オクタ−５−エントリフルオロアセテート（Organic Letters 2004, 11, 1805 から製造）４.２ｍｇ（１９μＭ）から、表題化合物を製造する。ここで、Ｎ−（３−ジメチルアミノプロピル）−Ｎ'−エチルカルボジイミド塩酸塩／１−ヒドロキシ−１Ｈ−ベンゾトリアゾールの代わりに、［Ｏ−（７−アザベンゾトリアゾール−１−イル）−１,１,３,３−テトラメチルウロニウムヘキサフルオロホスフェート］（ＨＡＴＵ）３.５ｍｇ（９.５μＭ）を、エチルジイソプロピルアミン１.６ｍｇの存在下で、カップリング剤として使用する。室温で１６時間撹拌した後、混合物を濃縮し、残渣を分取用ＨＰＬＣ（方法１）により直接精製し、表題化合物を実施例１と同様に単離する。
収量：５ｍｇ（理論値の９０％）
ＨＰＬＣ（方法２）：Ｒ_ｔ＝４.３分；
ＬＣ−ＭＳ（方法３）：Ｒ_ｔ＝５.０および５.１分；ｍ／ｚ＝９０２（Ｍ＋Ｈ）^＋ Example 12
N-[(3S, 6S, 14R, 14aS) -6-tert-butyl-3-isopropyl-14-methyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -beta-methyl-N-[(1R) -3- (2-oxa-3 -Azabicyclo [2.2.2] oct-5-en-3-yl) -3-oxo-1- (1,3-thiazol-2-yl) propyl] -L-phenylalaninamide

Similar to the synthesis of Example 1, 5 mg (6.2 μM) of the compound from Example 4A and 2-oxa-3-azabicyclo [2.2.2] oct-5-entryfluoroacetate (Organic Letters 2004, 11, The title compound is prepared from 4.2 mg (19 μM). Here, instead of N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride / 1-hydroxy-1H-benzotriazole, [O- (7-azabenzotriazol-1-yl) -1, 1,3,3-tetramethyluronium hexafluorophosphate] (HATU) 3.5 mg (9.5 μM) is used as coupling agent in the presence of 1.6 mg ethyldiisopropylamine. After stirring at room temperature for 16 hours, the mixture is concentrated, the residue is purified directly by preparative HPLC (Method 1) and the title compound is isolated as in Example 1.
Yield: 5 mg (90% of theory)
HPLC (Method 2): R _t = 4.3 min;
LC-MS (Method 3): R _t = 5.0 and 5.1 min; m / z = 902 (M + H) ⁺

実施例４Ａ由来の化合物４０ｍｇ（４９.４μｍｏｌ）を、ＤＭＦ２０ｍｌに溶解し、Ｎ−ベンジルヒドロキシルアミン塩酸塩１１.８ｍｇ（７４.２μｍｏｌ）、［Ｏ−（７−アザベンゾトリアゾール−１−イル）−１,１,３,３−テトラメチルウロニウムヘキサフルオロホスフェート］（ＨＡＴＵ）３７.６ｍｇ（９８.８μｍｏｌ）およびエチルジイソプロピルアミン１２.８ｍｇを添加する。１６時間室温で撹拌した後、混合物を濃縮し、残渣をアセトニトリル／水１：２に取り、分取用ＨＰＬＣ（方法１）により精製する。対応する画分を合わせ、溶媒を蒸発させ、残っている残渣をジオキサンから凍結乾燥する。標的化合物４４.８ｍｇ（理論値の９９％）を得る。
ＨＰＬＣ（方法２）：Ｒ_ｔ＝４.３８分 Example 13
N-[(3S, 6S, 14R, 14aS) -6-tert-butyl-3-isopropyl-14-methyl-1,4,10-trioxododecahydropyrrolo [1,2-a] [1,4, 7,10] tetraazacyclododecin-7 (8H) -ylidene] -3-methyl-L-valyl- (betaS) -N-[(1R) -3-benzyl (methoxy) amino] -3-oxo -1- (1,3-thiazol-2-yl) propyl] -beta-methyl-L-phenylalaninamide

40 mg (49.4 μmol) of the compound derived from Example 4A was dissolved in 20 ml of DMF, and 11.8 mg (74.2 μmol) of N-benzylhydroxylamine hydrochloride, [O- (7-azabenzotriazol-1-yl)- 1,3,3,3-Tetramethyluronium hexafluorophosphate] (HATU) 37.6 mg (98.8 μmol) and ethyldiisopropylamine 12.8 mg are added. After stirring for 16 hours at room temperature, the mixture is concentrated and the residue is taken up in acetonitrile / water 1: 2 and purified by preparative HPLC (Method 1). Corresponding fractions are combined, the solvent is evaporated and the remaining residue is lyophilized from dioxane. 44.8 mg (99% of theory) of the target compound are obtained.
HPLC (Method 2): R _t = 4.38 min

25 mg (27.3 μmol) of this intermediate is dissolved in 10 ml of dioxane / water 1: 1 and 26.7 mg (82 μmol) of cesium carbonate is added. After stirring for 10 minutes at room temperature, the mixture is lyophilized. The residue is taken up in 10 ml of DMF and 85 μl (1.4 mmol) of methyl iodide are added at room temperature. The reaction is complete after 30 minutes. The mixture is concentrated and the residue is purified by preparative HPLC (Method 1). 8.4 mg (33% of theory) of the title compound are obtained.
HPLC (Method 2): R _t = 4.3 min;
LC-MS (Method 3): R _t = 5.65 min; m / z = 928 (M + H) ⁺

B. Evaluation of physiological activity
Abbreviations used:

The in vitro and in vivo activity of the compounds of the invention can be demonstrated in the following assays:
Determination of Minimum Inhibitory Concentration (MIC) The minimum inhibitory concentration (MIC) is the lowest concentration of antibiotic at which growth of the test microorganism is inhibited over 18-24 hours. In these cases, the inhibitor concentration can be determined by standard microbiological methods (eg, The National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-fifth edition. NCCLS document M7-A5 [ISBN 1-56238-394-9]. See NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2000). The MIC of the compounds of the invention is determined in a liquid dilution test on a 96-well microtiter plate scale. Adjust fresh bacterial streaks on Colombian blood agar plates (Becton Dickinson) with 5% sheep blood [Chocolate for H. influenzae-Becton Dickinson] to OD ₅₇₈ 0.1 in saline. . Test substances are dissolved in 10 mM DMSO and further diluted with DMSO in a 1: 2 dilution step. In each case, 1 μl of diluted test substance is added to a 96-well microtiter plate and 100 μl of a microbial suspension diluted 1: 300 with Mueller-Hinton broth (HTM for H. influenzae) is added. For Streptococcus pneumoniae and Streptococcus pyogenes, the microbial dilution is 1: 100 in Mueller-Hinton broth supplemented with 2% hemolyzed horse blood. Microtiter plates are incubated aerobically for 18-24 hours at 37 ° C .; streptococci and Haemophilus influenzae are microaerobically incubated in the presence of 5% CO ₂ .
In each case, the lowest substance concentration at which no visible bacterial growth occurs is defined as the MIC.

濃度のデータ：μＭ表記のＭＩＣ Table A

Concentration data: MIC in μM

Systemic infection with S. aureus 133:
The suitability of the compounds of the present invention for the treatment of bacterial infections can be demonstrated in various animal models. For this purpose, animals are generally infected with a suitable pathogenic microorganism and then treated with the compound to be tested present in a formulation adapted to the particular therapeutic model. In particular, the suitability of the compounds of the invention for the treatment of bacterial infections can be demonstrated in a mouse sepsis model after infection with S. aureus.

For this purpose, S. aureus 133 cells are cultured overnight in BH broth (Oxoid, Germany). The overnight culture was diluted 1: 100 in fresh BH broth and grown for 3 hours. Bacteria in logarithmic growth phase are centrifuged and washed twice with buffered saline. The cell suspension in 50 units of saline is then adjusted with a photometer (Dr Lange LP 2W). After the dilution step (1:15), this suspension is mixed 1: 1 with a 10% mucine suspension. 0.2 ml of this infection solution is administered ip per 20 g of mouse. This corresponds to a cell number of about 1-2 × 10 ⁶ microorganisms / mouse. iv treatment is given 30 minutes after infection. Female CFW1 mice are used for this infection experiment. Animal survival is recorded for 5 days. The animal model is adjusted so that untreated animals die within 24 hours after infection. The ED ₁₀₀ represents the lowest dose at which all animals survive from infection during the observation period.

Pulmonary infection with Streptococcus pneumoniae L3TV The suitability of the compounds of the invention for the treatment of bacterial infections can also be demonstrated in a model of pulmonary infection in mice after infection with Streptococcus pneumoniae.
In this case, female CFW1 mice are infected intranasally with 8 × 10 ⁵ microbial pneumococcus L3TV. The inoculum is adjusted by dilution of the cryopreservation culture. On days 1 and 2 of infection, iv treatment is applied twice a day in each case. Three days after infection, the lungs are removed, the tissue is homogenized, and a microbial count is performed.

Determination of in vivo pharmacokinetic properties In general, the pharmacokinetic properties (eg, clearance, distribution, etc.) of a compound are determined by measuring the plasma concentration of the compound in a particular species. To this end, blood is collected at various times (eg 2, 5, 10, 20, 40 minutes, 1, 2, 4, 6, 8, 24 hours), for example after intravenous administration of the compound, and centrifuged. . The plasma concentration of the compound is then measured by an appropriate analytical method by LC / MSMS.

In mice, one animal is required for each sampling time. Blood is collected from the tail vein. In rats, blood is obtained from catheterized animals: a silicone catheter is advanced through the right external jugular vein to the heart or secured in the tail vein, secured in the cervical muscle and brought subcutaneously to the back Guide through the skin and close with plastic obstruction. This catheter provides access to a constant vein so as to make it possible to obtain complete blood dynamics in one animal.
For dogs, blood is collected from the jugular vein. Again, complete blood dynamics in one animal is obtained.

Exemplary Embodiments of Pharmaceutical Compositions Compounds of the present invention can be converted to pharmaceutical formulations in the following manner:
Solutions that can be administered intravenously:
composition:
1 mg of the compound of Example 1, 15 g of polyethylene glycol 400 and 250 g of water for injection.
Manufacturing:
The compound of the present invention is dissolved in water with stirring with polyethylene glycol 400. The solution is sterilized by filtration (pore diameter 0.22 μm) and dispensed under aseptic conditions into a heat-sterilized infusion bottle. The latter is closed with an infusion stopper and a crimp cap.

Claims (11)

formula

[Where:
R ¹ and R ² represent hydrogen or
Or
R ¹ and R ² represent methyl or
Or
R ¹ represents methyl and
R ² represents hydrogen,
And
R ³ and R ⁴ , independently of one another, represent C ₁ -C ₄ -alkyl {which is cyano, hydroxycarbonyl, aminocarbonyl, C ₁ -C ₄ -alkoxycarbonyl, C ₁ -C _6- alkylaminocarbonyl, C 3 _-C 6 _- cycloalkyl, C 6 _-C 10 _- aryl and 5- or may be substituted with one substituent selected from the group consisting of heteroaryl 6-membered, C _{When 1} -C ₄ -alkyl is not methyl, alternatively or additionally, they are independently of each other from the group consisting of halogen, hydroxy, amino, C ₁ -C ₄ -alkoxy and C ₁ -C ₆ -alkylamino. Optionally substituted with 1 to 3 substituents selected from
_Or, C 3 -C ₆ - or {it represents a cycloalkyl, halogen, hydroxy, _amino, C 1 -C ₄ - _alkyl, C 1 -C ₄ - _alkoxy, C 1 -C ₆ - alkylamino, cyano, hydroxy Optionally substituted by 1 to 3 substituents independently selected from the group consisting of carbonyl, aminocarbonyl, C ₁ -C ₄ -alkoxycarbonyl and C ₁ -C ₆ -alkylaminocarbonyl },
Or
R ³ and R ⁴ together represent (CH ₂ ) ₃ or (CH ₂ ) ₄ , and together with the nitrogen or oxygen atom to which they are respectively attached, are 5 or 6 membered Forming a ring, wherein the ring is halogen, hydroxy, amino, C ₁ -C ₄ -alkyl, C ₁ -C ₄ -alkoxy, C ₁ -C ₆ -alkylamino, cyano, hydroxycarbonyl, May be substituted with 1 to 2 substituents independently selected from the group consisting of aminocarbonyl, C ₁ -C ₄ -alkoxycarbonyl and C ₁ -C ₆ -alkylaminocarbonyl,
Or
R ³ and R ⁴ are combined with the nitrogen or oxygen atom to which they are each attached to form a formula

{Where * is the binding site to the carbonyl group}
Is forming a group]
Or a salt thereof, a solvate thereof or a solvate of a salt thereof. Where
R ¹ and R ² represent hydrogen,
Or
R ¹ and R ² represent methyl,
Or
R ¹ represents methyl and
R ² represents hydrogen,
And
R ³ and R ⁴ independently of one another represent methyl or ethyl,
Or
R ³ and R ⁴ together represent (CH ₂ ) ₃ or (CH ₂ ) ₄ , and together with the nitrogen or oxygen atom to which they are respectively attached, are 5 or 6 membered Forming a ring, wherein the ring may be substituted with one substituent selected from the group consisting of hydroxy and ethoxycarbonyl;
Or
R ³ and R ⁴ are combined with the nitrogen or oxygen atom to which they are each attached to form the formula

{Where * is the binding site to the carbonyl group}
Forming the basis of
One of the compounds according to claim 1, or a salt, a solvate thereof or a solvate of the salt thereof. Where
R ¹ represents methyl and
R ² represents hydrogen,
And
R ³ and R ⁴ independently of one another represent methyl or ethyl,
Or
R ³ and R ⁴ together represent (CH ₂ ) ₃ or (CH ₂ ) ₄ , and together with the nitrogen or oxygen atom to which they are respectively attached, are 5 or 6 membered Forming a ring, wherein the ring may be substituted with one substituent selected from the group consisting of hydroxy and ethoxycarbonyl;
A compound according to claim 1 or claim 2, or a salt thereof, a solvate thereof or a solvate of a salt thereof. formula

(Wherein R ¹ , R ² , R ³ and R ⁴ have the meanings of claim 1, claim 2 or claim 3).
The compound according to any one of claims 1 to 3, or a salt thereof, a solvate thereof, or one of solvates of the salt thereof, wherein A process for the preparation of a compound of formula (I) according to claim 1 or a salt thereof, a solvate thereof or a solvate of a salt thereof,
[A] Formula

(Wherein R ¹ and R ² have the meaning of claim 1)
In the presence of one or more dehydrating agents.

(Wherein R ³ and R ⁴ have the meaning of claim 1)
React with a compound of
Or
[B] A compound of formula (II) is reacted in the first stage in the presence of one or more dehydrating agents.

(Wherein R ³ has the meaning of claim 1)
In the second step, the compound is reacted with

(Wherein R ⁴ has the meaning of claim 1 and X ¹ represents halogen, preferably bromine or iodine)
In the presence of a base,
A method characterized by.   5. A compound according to any one of claims 1 to 4 for the treatment and / or prevention of disease.   Use of a compound according to any of claims 1 to 4 for the manufacture of a medicament for the treatment and / or prevention of diseases.   Use of a compound according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment and / or prevention of bacterial diseases.   A medicament comprising at least one compound according to any of claims 1 to 4 in combination with at least one inert, non-toxic, pharmaceutically acceptable adjuvant.   The medicament according to claim 9, for the treatment and / or prevention of bacterial infections.   A method for controlling bacterial infections in humans and animals by administering an antibacterial effective amount of at least one compound according to any one of claims 1 to 4 or a medicament according to claim 9 or claim 10. .