HK1140480B

HK1140480B - Cysteine protease inhibitors

Info

Publication number: HK1140480B
Application number: HK10106881.7A
Authority: HK
Inventors: Magnus Nilsson; Lourdes Oden; Pia Kahnberg; Urszula Grabowska
Original assignee: Medivir Ab
Priority date: 2007-06-27
Filing date: 2008-06-26
Publication date: 2013-06-21

Description

Cysteine protease inhibitors

Technical Field

The present invention relates to inhibitors of cysteine proteases, in particular of the papain superfamily. The present invention provides novel compounds useful for preventing or treating diseases caused by an imbalance of physiological proteases, such as cathepsin K.

Background

The papain superfamily of cysteine proteases is widely distributed in a wide variety of species, including mammals, protozoa, plants and bacteria. Many mammalian cathepsins, including cathepsin B, F, H, K, L, O and S, are believed to belong to this superfamily, and dysregulation of their activity has been implicated in a number of metabolic diseases, including arthritis, muscular dystrophy, inflammation, glomerulonephritis and tumour invasion. Pathogenic cathepsin-like enzymes include bacterial gingipain, the plasmodium falciparum cysteine protease falcipainsI, II, III, etc., and cysteine proteases from pneumocystis carinii, trypanosoma cruzi and trypanosoma brucei, brevibacillus plexitus, schistosoma japonicum.

Dysregulation of cathepsin K is associated with a number of diseases including osteoporosis, gum diseases (e.g. gingivitis and periodontitis), paget's disease, hypercalcemia of malignancy, and metabolic bone disease. Due to the elevated concentration of cathepsin K in the broken chondrocytes of osteoarthritic synovium, it is thought to be associated with diseases characterized by cartilage excess or matrix degradation (e.g. osteoarthritis and rheumatoid arthritis).

It is likely that treatment of bone and cartilage diseases (e.g. osteoarticular fire and osteoporosis) requires the lifetime administration of cathepsin K inhibitors, often to a population of patients at or near the elderly stage. This places an unusually high demand on the ease of administration of drugs intended for such diseases. For example, there are attempts to extend the current regimen of osteoporosis drugs in the bisphosphonate class to weekly or longer regimens to improve compliance. However, even with improved dosing regimens, other side effects of bisphosphonates remain. Bisphosphonates block bone turnover rather than attenuate it as cathepsin K inhibitors do. For healthy bones, it is important to maintain this remodeling process, while bisphosphonates block it completely. In addition, bisphosphonates have a long half-life in bone, and thus, if osteonecrosis of the jaw bone itself occurs, it is impossible to remove bisphosphonates from the bone. In contrast, cathepsin K inhibitors generally have a rapid onset and rate of action, meaning that if a problem needs to be identified, dosing can be discontinued without the inhibitor accumulating in the bone matrix.

Accordingly, alternative osteoporosis and osteoarthritis drugs with superior pharmacokinetic and/or pharmacodynamic properties are desired.

International patent application No. wo 02/057270 discloses compounds of formula IA:

wherein UVWXY corresponds approximately to P3 and P2 of dipeptide cysteine protease inhibitors (these are indicated below), Z being in particular O, S, methylene or-NR-, R'₁Is alkyl, alkaryl, etc., P₁And Q is each especially methylene. While the general disclosure in this patent application assumes a wide range of substituents on P1 and Q, none are specified or exemplified and no guidance is provided as to their synthesis. Indeed, the only synthetic proposal provided in WO 02/057270 is completely impermissible at P₁And substitution on Q. The compounds are asserted to be particularly useful in the treatment of protozoal infections such as trypanosoma infections.

Example 9 of international patent application No. wo 2005/066180 discloses inter alia compounds of formula IB:

this compound is an inhibitor of cathepsin K activity, but as shown below, further modifications to this structure lead to improvements in pharmacokinetics and/or pharmacodynamics, significantly improving stability in whole blood and hence better exposure time.

WO 2008/007127, which has not been published between the priority dates of the present application, discloses compounds of formula IC:

the compounds are said to be inhibitors of cysteine proteases, in particular cathepsin K.

Brief description of the invention

According to the present invention there is provided a compound of formula II or a pharmaceutically acceptable salt, prodrug or N-oxide thereof:

wherein

R²Is the side chain of leucine, isoleucine, cyclohexylglycine, O-methylthreonine, 4-fluoroleucine or 3-methoxyvaline;

R³is H, methyl or F;

R_qis a CF having said stereochemistry₃，R_q' is H; or

R_qAnd R_qTogether form a keto group;

q is

Wherein

R⁴Is C₁-C₆An alkyl group;

R⁵is H, methyl or F;

R⁶is C₁-C₆An alkyl group.

According to one aspect of the present invention, there is provided an enantiomeric compound of formula IIa, or a pharmaceutically acceptable salt, N-oxide, or hydrate thereof:

wherein

R³is H, methyl or F;

R⁴is C₁-C₆Alkyl, preferably methyl;

R⁵is H, methyl or F.

Another embodiment of the present invention encompasses compounds of formula lib or a pharmaceutically acceptable salt, N-oxide, or hydrate thereof:

wherein R is²And R³Q is an N-alkylpiperazinoylthiazolyl moiety as defined in formula IIa or 4- (C) as described in WO 07/006716₁-C₄Alkyl) sulfonylphenyl moieties, such as compounds of the formula:

“C_1-6the term "alkyl" denotes an alkyl chain having 1-6 carbon atoms (e.g., C)_1-4Alkyl, or C_1-3Alkyl groups). The alkyl group may be straight or branched. Suitable C_1-6Alkyl groups include, for example, methyl, ethyl, propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl and tert-butyl), pentyl (e.g., n-pentyl), and hexyl (e.g., n-hexyl). Of particular interest is alkyl is methyl.

It will be appreciated that the compounds of the invention may exist in the form of hydrates, for example hydrates of the following partial formulae:

the invention extends to include all such other possible forms.

In a preferred embodiment of the invention, R²Is the side chain of leucine, isoleucine, O-methylthreonine, 4-fluoroleucine or 3-methoxyvaline.

Presently preferred R²Includes those represented by the following partial structural formulae:

in particular R corresponding to the side chain of L-leucine²The meaning of (1).

The embodiment of the preceding paragraph is preferably used wherein R³Or R⁵A compound that is fluorine.

Usually, the variable R is methyl or fluorine³And, if present, is in a meta position relative to the benzylic amide bond, as shown in the following partial structural formula:

representative compounds of this embodiment have the formula:

in certain embodiments of the invention, R⁵Is F, especially when R³Is H and/or R⁴When it is methyl. Preferred compounds in this embodiment have one R as the side chain of 4-fluoroleucine, cyclohexylalanine and preferably leucine². One representative compound of this embodiment has the formula:

preferred embodiments of the invention include those listed below, in each case having the stereochemistry depicted above in formula II:

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [2- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrol-4-yl) -1-cyclohexyl-2-oxoethyl ] -4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methylbutyl ] -4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxypropyl-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxy-2-methylpropyl ] -4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [2- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrol-4-yl) -1-cyclohexyl-2-oxoethyl-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methylbutyl ] -4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxypropyl ] -4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxy-2-methylpropyl ] -4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [2- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrol-4-yl) -1-cyclohexyl-2-oxoethyl ] -4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methylbutyl ] -4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxypropyl-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxy-2-methylpropyl ] -4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -3-methyl-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [2- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrol-4-yl) -1-cyclohexyl-2-oxoethyl ] -3-methyl-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methylbutyl ] -3-methyl-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxypropyl ] -3-methyl-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxy-2-methylpropyl ] -3-methyl-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -3-methyl-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [2- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrol-4-yl) -1-cyclohexyl-2-oxoethyl ] -3-methyl-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methylbutyl ] -3-methyl-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxypropyl ] -3-methyl-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxy-2-methylpropyl ] -3-methyl-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -3-methyl-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [2- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrol-4-yl) -1-cyclohexyl-2-oxoethyl ] -3-methyl-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methylbutyl ] -3-methyl-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxypropyl ] -3-methyl-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxy-2-methylpropyl ] -3-methyl-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [2- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrol-4-yl) -1-cyclohexyl-2-oxoethyl ] -3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methylbutyl-3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxypropyl ] -3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -3-fluoro-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [2- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrol-4-yl) -1-cyclohexyl-2-oxoethyl ] -3-fluoro-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methylbutyl ] -3-fluoro-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxypropyl ] -3-fluoro-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxy-2-methylpropyl ] -3-fluoro-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -3-fluoro-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [2- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrol-4-yl) -1-cyclohexyl-2-oxoethyl ] -3-fluoro-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methylbutyl ] -3-fluoro-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxypropyl ] -3-fluoro-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -2-methoxy-2-methylpropyl-3-fluoro-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

and pharmaceutically acceptable salts, N-oxides and hydrates thereof.

Further preferred embodiments having the stereochemistry described above include:

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-fluoro-3-methylbutyl-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-fluoro-methylbutyl-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-fluoro-3-methylbutyl-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-fluoro-3-methylbutyl ] -3-methyl-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-fluoro-3-methylbutyl ] -3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-fluoro-3-methylbutyl ] -3-methyl-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-fluoro-3-methylbutyl ] -3-methyl-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-fluoro-3-methylbutyl ] -3-fluoro-4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-fluoro-3-methylbutyl ] -3-fluoro-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

and pharmaceutically acceptable salts, N-oxides and hydrates thereof.

Preferred embodiments of the present invention include compounds of formula II below:

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-fluoro-3-methylbutyl ] -4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide;

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide; or a pharmaceutically acceptable salt, hydrate or N-oxide thereof.

Particularly preferred compounds of the present invention include compounds represented by N- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide, formula II, or a pharmaceutically acceptable salt, hydrate or N-oxide thereof.

Particularly preferred compounds of the present invention include compounds represented by N- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide of formula II, or a pharmaceutically acceptable salt, hydrate or N-oxide thereof.

Another aspect of the invention comprises a pharmaceutical composition comprising a compound as defined above and a pharmaceutically acceptable carrier or diluent therefor.

A further aspect of the invention is the use of a compound as defined above for the treatment of diseases mediated by cathepsin K, or for the manufacture of medicaments for the treatment of such diseases, including:

osteoporosis;

gum diseases, such as gingivitis and periodontitis,

the disease of the patient with the perkin's disease,

the hypercalcemia of the malignant tumor is shown,

the bone disease of the metabolic bone is treated,

diseases characterized by cartilage excess or matrix degradation, such as osteoarthritis and rheumatoid arthritis,

bone cancer, including neoplasia,

pain, especially chronic pain.

Also provided is a method for treating or preventing a disorder mediated by cathepsin K, comprising administering to a subject in need thereof a safe and effective amount of a compound according to any one of claims 1 to 17. These subjects are usually mammals, especially humans.

The compounds of the invention may form salts, which form a further aspect of the invention. Suitable pharmaceutically acceptable salts of the compounds of formula II include salts of organic acids, especially carboxylic acids, including but not limited to: acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cypionate, glucoheptonate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, pamoate (palmoate), collooate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactobionate, pivalate, camphorate, undecanoate, and succinate, organic sulfonates such as methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, camphorsulfonate, 2-naphthalenesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, and p-toluenesulfonate; and salts of inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, hemisulfate, thiocyanate, persulfate, phosphate and sulfonate.

The compounds of the invention may in some cases be isolated in the form of hydrates. Hydrates are generally prepared by recrystallization from water/organic solvent mixtures with organic solvents (e.g. dioxane, tetrahydrofuran or methanol). Hydrates can also be generated in situ by administering the corresponding ketone to the patient.

The N-oxides of the compounds of the present invention may be prepared by methods known to those of ordinary skill in the art. For example, the N-oxide may be prepared by treating an unoxidized form of the oxide of the present invention with an oxidizing agent (e.g., trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, m-chloroperbenzoic acid, etc.) in a suitable inert organic solvent (e.g., a halogenated hydrocarbon such as dichloromethane) at about 0 deg.C. Alternatively, the N-oxides of the compounds of the invention may be prepared starting from N-oxides of suitable starting materials.

Examples of the N-oxide of the present invention include N-oxides having the following partial structure:

the compounds of the invention in unoxidized form can be prepared starting from the corresponding N-oxide form of the compound of the invention by treatment with a reducing agent (e.g., sulfur dioxide, triphenylphosphine, lithium borohydride, sodium borohydride, phosphorus dichloride, phosphorus tribromide, etc.) in a suitable inert organic solvent (e.g., acetonitrile, ethanol, aqueous dioxane, etc.) at 0-80 ℃.

It should be noted that the radical position on any molecular moiety used in these definitions may be anywhere on that moiety as long as it is chemically stable.

The groups used in the definition of the variables include all possible isomers unless otherwise indicated.

When any variable occurs more than one time in any constituent, each definition is independent.

Unless otherwise stated or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms which the compound may possess. The mixture may include all diastereomers and/or enantiomers of the basic molecular structure of the compound. All stereochemically isomeric forms of the compounds of the present invention, either pure or in admixture with each other, are intended to be embraced within the scope of the present invention.

Pure stereoisomeric forms of the compounds and intermediates recited herein are defined as those isomers that are substantially free of other enantiomeric or diastereomeric forms of the same basic molecular structure of the compounds or intermediates. In particular, the term "stereoisomerically pure" relates to compounds or intermediates having a stereoisomeric excess of at least 80% (i.e. a minimum of 90% of one isomer and a maximum of 10% of the other possible isomers) up to a stereoisomeric excess of 100% (i.e. 100% of one isomer without the other), in particular compounds or intermediates having a stereoisomeric excess of 90% up to 100%, even a stereoisomeric excess of 94% up to 100%, especially a stereoisomeric excess of 97% up to 100%. The terms "enantiomerically pure" and "diastereomerically pure" are to be understood in a similar manner, but with reference to the enantiomeric and diastereomeric excess, respectively, in the mixture in question.

The compounds of the present invention may be prepared as their individual stereoisomers by reacting a racemic mixture of the compounds with an optically active resolving agent to produce a pair of diastereomeric compounds, separating and recovering the optically pure enantiomers. Resolution of the enantiomers may be carried out using covalent diastereomeric derivatives of the compounds of formula (I), but preferably with dissociable complexes (e.g., crystalline, diastereomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivities, etc.) and can be readily separated by virtue of these differences. Diastereomers can be separated by chromatography (e.g., HPLC) or, preferably, by separation/resolution techniques based on solubility differences. The optically pure enantiomer and the resolving agent are then recovered by any practical means that does not cause racemization. A more detailed description of techniques that can be used to resolve stereoisomers of compounds from their racemic mixtures can be found in Jean Jacques Andre Collet, Samuel H.Wilen, Enantiomers, racemes and solutions, John Wiley & Sons, Inc. (1981).

It will be appreciated that the invention extends to prodrugs, solvates, complexes and other forms which release the compounds of the invention in vivo.

While the active agent may be administered alone, it is preferred that it be part of a pharmaceutical formulation. Such a formulation will contain the active agent as defined above and one or more acceptable carriers/excipients, and optionally other therapeutic ingredients. The carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the user.

Formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, but are preferably oral formulations. The formulations may conveniently be presented in unit dosage form, for example as tablets and sustained release capsules, and may be prepared by any of the methods well known in the art of pharmacy.

These methods include the step of bringing into association the active ingredient defined above with a carrier. In general, the formulations are prepared by bringing into association the active agent with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product. The invention extends to a process for preparing a pharmaceutical composition comprising combining or combining a compound of formula II, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier or excipient. If the manufacture of a pharmaceutical composition involves intimate mixing of the pharmaceutical excipients and the active ingredient in salt form, it is often preferred to use excipients which are non-basic in nature, i.e. acidic or neutral.

The formulation for oral administration in the present invention may be shown in various forms: discrete units such as capsules, cachets or tablets each containing a predetermined amount of active agent; powders or granules; solutions or suspensions of the active agent in aqueous or non-aqueous liquids; or oil-in-water emulsions or water-in-oil solvents, and boluses.

With respect to compositions for oral administration (e.g., tablets and capsules), the term suitable carrier includes vehicles, e.g., commonly used excipients such as binding agents, e.g., syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants, such as magnesium stearate, sodium stearate and stearates of other metals, glyceryl stearate, stearic acid, silicone oil, talc, waxes, oils and colloidal silicon dioxide. Flavoring agents, such as peppermint, oil of wintergreen, cherry green, and the like, may also be used. It may be desirable to add a colorant to make the dosage form easily identifiable. Tablets may also be coated by methods well known in the art.

Tablets may be prepared by compression or moulding, optionally together with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free-flowing form, for example as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to achieve sustained or controlled release of the active agent.

Other formulations suitable for oral administration include lozenges comprising the active agent in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base (e.g. gelatin and glycerin, or sucrose and acacia); and mouthwashes comprising the active agent in a suitable liquid carrier.

The appropriate dosage of the compounds or formulations of the invention will depend on the indication and the patient, and can be readily determined using routine animal testing and confirmed by human clinical trials. It is generally advisable and possible to administer amounts which provide intracellular (physiological proteases for inhibiting the papain superfamily) concentrations in the order of 0.01-100. mu.M, preferably 0.01-10. mu.M, for example 0.1-25. mu.M.

The compounds of the invention are prepared using a wide variety of solution phase and solid phase chemical reactions.

The compounds of the invention are generally prepared as building blocks reflecting the P1, P2 and P3 portions of the terminal production inhibitors. Without wishing to be bound by theory or the binding patterns ascribed to the speculation of particular variables in any way, the abstractions P1, P2 and P3 used herein are provided for convenience only and have essentially their conventional schlecer & Berger meanings, representing those portions of the enzyme believed to fill the S1, S2 and S3 sublines, respectively, of the enzyme, with S1 adjacent to the cleavage site and S3 remote from the cleavage site. Compounds defined by formula II, regardless of their mode of binding, are within the scope of the invention.

In general terms, the P1 building block has the following formula:

wherein

PG is a conventional N-protecting group or a free amine;

two Rb groups define a ketal, such as a bismethyl ketal, or together define a cyclic ketal, such as 1, 3-dioxolane;

rc is a hydroxyl protecting group, or less commonly H, or represents the ketone functionality of the end-product inhibitor in the case where the ketone form of the P1 building block is extended by P2 and P3.

P2 is typically an N-protected L-leucine, L-isoleucine, O-methyl-L-threonine, L-3-hydroxyvaline, 4-fluoroleucine or L-cyclohexylglycine, P3 typically contains a blocking group, such as a benzoic acid derivative, into which has been introduced an N-alkylpiperazinyl-E moiety, or has a synthon in para position for this purpose.

The appropriately protected individual building blocks may be prepared first and subsequently coupled together, preferably in the sequence P2+ P1 → P2-P1, followed by N-alkylpiperazinylthiazolylbenzoic acid^*+ P2-P1 → N-alkylpiperazinothiazolylbenzoate-P2-P2-P1, wherein x represents the activated form, to reduce racemization at P2.

The coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be carried out using standard coupling methods, such as the azide method, the mixed carbonate-carboxylic anhydride (isobutyl chloroformate) method, the carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, the active ester (p-nitrophenyl ester, imino-N-hydroxysuccinate) method, the woodward reagent K method, the carbonyldiimidazole method, the phosphorus reagent, or the redox method. Some of these processes, particularly carbodiimidization, can be enhanced by the addition of 1-hydroxybenzotriazole or 4-DMAP. These coupling reactions can be carried out in solution (liquid phase) or in solid phase.

More specifically, the coupling step involves the dehydrative coupling of a free carboxyl group of one reactant with a free amine group of another reactant in the presence of a coupling agent to form an amide linkage. Descriptions of such coupling agents can be found in general textbooks on Peptide Chemistry, such as m.bodanszky, "Peptide Chemistry", 2^ndrev. ed., Springer-Verlag, Berlin, Germany (1993), and abbreviated as Bodanszky, the contents of which are incorporated herein by reference. Examples of suitable coupling agents are N, N ' -dicyclohexylcarbodiimide, 1-hydroxybenzotriazole (in N, N ' -dicyclohexylcarbodiimide or N-ethyl-N ' - [ (3-dimethylamino) propyl)]In the presence of a carbodiimide). One useful and effective coupling agent is commercially available (benzotriazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate, by itself or in the presence of 1-hydroxybenzotriazole or 4-DMAP. Another useful and effective coupling agent is commercially available 2- (1H-benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium tetrafluoroborate. A further useful and effective coupling agent is commercially available O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate.

The coupling reaction is carried out in an inert solvent such as dichloromethane, acetonitrile or dimethylformamide. An excess of a tertiary amine, such as diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, or 4-DMAP, is added to maintain the reaction mixture at a pH of about 8. The reaction temperature is usually 0 to 50 ℃ and the reaction time is usually 15 minutes to 24 hours.

The functional groups of the unnatural amino acid component typically need to be protected between coupling groups to avoid undesirable bond formation. Protecting Groups which may be used are listed in Greene, "Protective Groups in organic Chemistry", John Wiley & Sons, New York (1981) and "the peptides: analysis, Synthesis, Biology ", vol.3, Academic Press, New York (1981), hereafter simply Greene, the contents of which are incorporated herein by reference.

The alpha-carboxyl group of the C-terminal residue is usually protected in the form of an ester, which can be cleaved to give the carboxylic acid. Protecting groups that may be used include: 1) alkyl esters such as methyl, trimethylsilyl and tert-butyl, 2) aralkyl esters such as benzyl and substituted benzyl, or 3) esters which can be cleaved with mild bases or mild reduction methods, such as trichloroethyl and phenacyl esters.

The alpha-amino ester of each amino acid to be coupled is typically N-protected. Any protecting group known in the art may be used. Examples of such groups include: 1) acyl groups such as formyl, trifluoroacetyl, phthaloyl and p-toluenesulfonyl; 2) aromatic carbamate groups such as benzyloxycarbonyl (Cbz or Z) and substituted benzyloxycarbonyl, and 9-fluorenylmethoxycarbonyl (Fmoc); 3) aliphatic urethane groups such as a tert-butoxycarbonyl group (Boc), an ethoxycarbonyl group, a diisopropylmethoxycarbonyl group and an allyloxycarbonyl group; 4) cyclic alkyl carbamates such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; 5) alkyl groups such as triphenylmethyl and benzyl; 6) trialkylsilyl groups such as trimethylsilyl; and 7) thiol-containing groups such as phenylthiocarbonyl and dithiasuccinyl. Preferred alpha-amino protecting groups are Boc or Fmoc. Many appropriately protected amino acid derivatives are commercially available for peptide synthesis.

The alpha-amino protecting group is usually cleaved off before the next coupling step. When using a Boc group, the method of choice is trifluoroacetic acid neat or in dichloromethane, or HCl in dioxane or ethyl acetate. The resulting ammonium salt is then neutralized with an alkaline solution, such as a water-based buffer, or a solution of a tertiary amine in dichloromethane, acetonitrile or dimethylformamide, either prior to coupling or in situ. When the Fmoc group is used, the reagent of choice is piperidine or substituted piperidine in dimethylformamide, but any secondary amine may be used. Deprotection is carried out at temperatures from 0 ℃ to room temperature, usually at 20-22 ℃.

Once the sequence synthesis of the inhibitor is complete, the remaining protecting groups are removed in any manner, depending on the protecting group chosen. These steps are well known to those skilled in the art.

The first step in the synthesis of compounds of the invention, for example compounds of formula II, is typically to prepare the functionalized P1 building block in solution, for example as shown in the following schematic:

i)MsCl，pyr；ii)NaOAc，Ac₂O，DMF，130℃；iii)BF₃XEt₂O，Et₃SiH，DCM；iv)TBDPS-Cl，Im-H，DMF；v)NaOMe；vi)SO₂Cl₂，Pyr，DCM；vii)pph₃，MeOH，H₂o, then Et₃N，50℃；viii)Boc₂O，Et₃N; ix) TBAF, THF; x) Dess-Martin pentavalent iodine oxidizer; xi) method a: MeOH, TMOF, p-TsOH, then Et₃N，Boc₂O, then chromatographically isolated, finally MeOH, AcCl, method b: MeOH, AcCl, TMOF

Although the above scheme has been illustrated with strategies using different protecting groups such as acetyl, methanesulfonyl, TBDPS and BOC, it is clear that other conventional protecting groups can be modified as described in Greene (supra). In addition, it may be expedient to use the dimethyl hemiacetal of the keto group during the coupling of the P2 and P3 groups and to regenerate the ketone in a later step.

The extension of the building blocks with P2 and P3 building blocks is usually carried out in the presence of a suitable coupling agent, for example benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU), O- (7-azabenzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium Hexafluorophosphate (HATU), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), or 1, 3-Dicyclohexylcarbodiimide (DCC), optionally with 1-Hydroxybenzotriazole (HOBT), and bases such as N, N-diisopropylethylamine, triethylamine, N-methylmorpholine, and the like. The reaction is generally carried out at 20-30 c, preferably at about 25 c, and takes 2-24 hours to complete. Suitable reaction solvents are inert organic solvents such as halogenated organic solvents (e.g., dichloroethane, chloroform, etc.), acetonitrile, N-dimethylformamide, ethereal solvents (e.g., tetrahydrofuran, dioxane), and the like.

Alternatively, the above extended coupling step may be carried out as follows: the P3/P2 building blocks are first converted to an active acid derivative, such as succinimidyl ester, which is then reacted with the P1 amine. This reaction usually takes 2 to 3 hours to complete. The conditions used in this reaction depend on the nature of the reactive acid derivative. For example, if an acid chloride derivative, the reaction is carried out in the presence of a suitable base (e.g., triethylamine, diisopropylethylamine, pyridine, etc.). Suitable reaction solvents are polar organic solvents, such as acetonitrile, N-dimethylformamide, dichloromethane or any suitable mixture thereof.

N-protectedP2 building blocks in the form of L-amino acids are readily available commercially, for example, L-leucine, L-isoleucine, L-cyclohexylglycine, O-methyl-L-threonine and others with various protecting groups such as CBz, Boc or Fomc are available. Other R²Variants are readily prepared from commercially available starting materials. For example, wherein R²is-C (CH)₃)₂OCH₃The compounds of (1) can be prepared by reacting CBz-protected (S) - (+) -2-amino-3-hydroxy-3-methylbutyric acid with 3, 3-dimethoxyhexahydrofuro (3, 2b) pyrrole to form the desired P2-P1 unit. This P2 branched alcohol can now be methylated with methyl iodide under conventional sodium hydride, imidazole, THF conditions to give the desired P2 without significant racemization of the alpha center. This P1-P2 moiety now enables the synthetic reaction to be completed as described herein, i.e., removal of CBz and coupling.

WO 05/565299 describes the preparation of gamma-fluoroleucine P2 building blocks. Truong et al in Synlett 2005(8)1278-1280 describe an alternative method for the synthesis of Fmoc and N-Boc-gamma-fluoroleucine building blocks.

The P3 building block is described in WO 05/66180 or is readily prepared in a similar manner. For example, the following scheme illustrates the preparation of a fluoro-substituted thiazolyl group:

i.HOAc，Br₂RT, 2h, yield 55%; KF, 18-crown-6, CH₃CN, 90 ℃, 16h and 31 percent of yield; HOAc, Br₂The yield is 100 percent at 45 ℃ for 4 h; 4-methylpiperazine-1-carbothioamide, ethanol, 70 ℃, 2h, yield 74%; LiOH, THF, H₂O, RT, 16h, yield 79%.

Synthesis of 4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzoic acid

The starting material methyl 4-acetylbenzoate is commercially available. This ketone was brominated with bromine in acetic acid at the alpha-position to give the desired methyl 4- (2-bromoacetyl) benzoate. Subsequently, methyl 4- (2-bromoacetyl) benzoate is treated with potassium fluoride at 90 ℃ in the presence of 18-crown-6, and the methyl 4- (2-fluoroacetyl) benzoate is obtained after column chromatography. Repeated bromination with bromine in acetic acid at the alpha position of the ketone affords the desired methyl 4- (2-bromo-2-fluoroacetyl) benzoate. Thiazole formation is generally achieved by heating methyl 4- (2-bromo-2-fluoroacetyl) benzoate and 4-methylpiperidinazine-1-carbothioamide at 70 ℃ for 2 hours. On cooling the desired methyl 4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzoate precipitated. The deprotection of the methyl ester is carried out with lithium hydroxide and the desired acid 4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzoic acid is generally obtained in the form of the dihydrochloride salt after work-up with hydrochloric acid and in good yield.

WO 05/066159 and WO 05/065578 describe compounds in which the P2 and P3 units are via C (CF)₃) A process for the preparation of compounds in which moieties are linked together, including compounds in which P3 is diphenyl sulfone. An example of a process for preparing such a P2-P3 building block suitable for preparing a building block wherein R is_qIs trifluoromethyl and Rq' is H.

Schematic diagram 3

The acidic functional group of bromo derivative (3a) prepared according to the method described in bioorg.med.chem.lett.2006, 16, 1985 is protected by reaction with, for example, isopropanol in the presence of an acid (e.g. sulphuric acid) to give the corresponding ester. The desired thiazole derivative can then be coupled to the aromatic group of the resulting ester using, for example, Stille or Suzuki coupling conditions. For example, bromo derivative 3a can be reacted with a borane reagent such as pinacolborane in the presence of [1, 1' -bis (diphenylphosphino) ferrocene ] palladium (II) chloride to give the corresponding dioxaborolane derivative. Subsequent substitution of the boron-containing group with the desired thiazole derivative (3b) in the presence of [1, 1' -bis (diphenylphosphino) ferrocene ] palladium (II) chloride gives the biaryl derivative (3C). The thiazole derivative (3b) can be prepared, for example, as described in J.Med.chem.2005, 48, 7520-7534. Treatment with an acid (e.g., hydrochloric acid) in dioxane removes the acid protecting group to give a P2-P3 building block ready for coupling with a P1 building block to give the compounds of the invention. WO 07/006716 describes methods for the preparation and coupling of such P3 and P3-P2 building blocks.

The term "N-protecting group" or "N-protected" as used herein refers to those groups which are used to protect the N-terminus of an amino acid or peptide or to protect the amino group from adverse reactions during synthetic procedures. A common N-protecting group is described in Greene, "Protective Groups in Organic Synthesis" (John Wiley & Sons, New York, 1981), which is incorporated herein by reference. N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, p-nitrophenoxyacetyl, α -chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; carbamate-forming groups, such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3, 4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4, 5-dimethoxybenzyloxycarbonyl, 3, 4, 5-trimethoxybenzyloxycarbonyl, 1- (p-biphenylyl) -1-methoxybenzyloxycarbonyl, alpha-dimethyl-3, 5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, tert-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2, 2, 2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, Cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, etc.; alkyl groups such as benzyl, trityl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Preferred N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl (bz), t-Butoxycarbonyl (BOC), and benzyloxycarbonyl (Cbz).

Hydroxy and/or carboxy protecting groups are also extensively reviewed in Greene (supra), including ethers such as methyl, substituted methyl ethers such as methoxymethyl, methylthiomethyl, benzyloxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl, and the like; silyl ethers such as Trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), tribenzylsilyl, triphenylsilyl, t-butyldiphenylsilyl, triisopropylsilyl and the like; substituted ethyl ethers such as 1-ethoxymethyl, 1-methyl-1-methoxyethyl, t-butyl, allyl, benzyl, p-methoxybenzyl, diphenylmethyl, triphenylmethyl, and the like; aralkyl radicals, such as the trityl and pixyl (9-hydroxy-9-phenylxanthene derivatives, especially the chloride). Ester hydroxy protecting groups include esters, such as formate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate, pivalate, adamantyl, and the like,Acid esters, benzoates, and the like. Carbonate hydroxy protecting groups include methyl, vinyl, allyl, cinnamyl, benzyl and the like.

Detailed description of the embodiments

Various embodiments of the present invention will now be described, by way of example only, with reference to the following examples.

Example 1

Preparation of P1 building block:

step a)

Compound 1(7.0g, 28.5mmol) (3-azido-3-deoxy-1, 2-O- (1-methylethylidene) - α -D-allofuranose according to the following formula

Tronchet, Jean m.j.; gentile, Bernard; Ojha-Ponce, Joelle; moret, Gilles; schwarzenbach, Dominique; barbalt-Rey, francise; tronchet, Jeannine. carbohydrate Research (1977), 59(1), 87-93) was dissolved in anhydrous pyridine (50ml) and the solution was cooled to 0 ℃. Methanesulfonyl chloride was slowly added to the solution and allowed to warm to room temperature. The reaction mixture was stirred overnight and after 14 h MeOH (10ml) and EtOAc (150ml) were added successively. The solution is mixed with 2M H₂SO₄The aqueous solution was washed 3 times (3X 100ml) with saturated NaHCO₃The aqueous solution was washed 2 times (2X 100ml) and the organic phase was then washed with Na₂SO₄Dried, filtered and concentrated under reduced pressure. The product was placed on a high vacuum pump overnight to remove residual solvent and to give product 2(11.5g) as a pale yellow oil in quantitative yield.

¹H NMR(CDCl₃，400MHz)1.34(s，3H)，1.51(s，3H)，3.07(s，3H)，3.16(s，3H)，4.18(d，1H，J＝3.1)，4.36(dd，1H，J＝8.6，3.2)，4.42(dd，1H，J＝12.0，5.0)，4.67(dd，1H，J＝11.9，2.3)，4.74(d，1H，J＝3.7)，5.11(ddd，1H，J＝8，6，5.0，2.3)，5.89(d，1H，J＝3.6).

Step b)

Compound 2(11.5g, 28.5mmol) was dissolved in DMF (50ml) and NaOAc (234g, 285mmol) and Ac were added to the solution₂O (48.6ml, 0.514mol) and then heated at 125 ℃ for 86 hours. A portion of the solvent was removed on a rotary evaporator and 5000ml EtOAc was added. This very dark solution was filtered through celite. The organic phase was then washed with water (3X 350 ml). Will haveOrganic phase with Na₂SO₄Drying and removing the solvent by a rotary evaporator. The crude product was purified by flash column chromatography (heptane: ethyl acetate 7: 3 → 2: 1) to give the desired compound 3(5.70g), and compound 4(2.34g, 22% yield) in 61% yield.

Compound 3:¹H NMR(CDCl₃，400MHz)1.34(s，3H)，1.53(s，3H)，2.09(s，3H)，2.11(s，3H)，3.94(d，1H，J＝3.4)，4.19(dd，1H，J＝12.2，5.0)，4.32(dd，1H，J＝8.0，3.3)，4.37(dd，1H，J＝12.3，3.5)，4.73(d，1H，J＝3.6)，5.32-5.37(m，1H)，5.94(d，1H，J＝3.8).

compound 4:¹H NMR(CDCl₃，400MHz)1.34(s，3H)，1.51(s，3H)，2.10(s，3H)，3.10(s，3H)，4.11(d，1H，J＝3.6)，4.21(dd，1H，J＝12.8，6.2)，4.32(dd，1H，J＝8.3，3.2)，4.65(dd，1H，J＝12.7，2.2)，4.73(d，1H，J＝3.5)，5.09(ddd，1H，J＝8，3，6.1，2.3)，5.89(d，1H，J＝3.5).

step c)

To compound 3(5.70g, 17.3mmol) dissolved in anhydrous DCM (40ml) was added triethylsilane (27.6ml, 173 mmol). The round bottom flask was placed under an inert atmosphere (N)₂) Placing in ice bath, cooling, and slowly adding BF₃·Et₂O (23.1mL, 173 mmol). The reaction was allowed to proceed slowly and stirred for 3 days, at which time the starting material was still present. NaHCO is slowly added₃Saturated aqueous solution (70mL) followed by gradual addition of solid NaHCO₃Until deflation ceases. The aqueous phase was extracted with DCM (150ml) followed by NaHCO₃Saturated aqueous solution (70mL) and NH₄Washed with saturated aqueous Cl (70 mL). Na for organic phase₂SO₄Drying, filtering and concentrating under reduced pressure. The crude product was purified by flash column chromatography (heptane: ethyl acetate 2: 1) at 41% yieldYield compound 5(1.95 g). 0.69g of unreacted starting material was also isolated.

¹H NMR(CDCl₃，400MHz)2.08(s，3H)，2.11(s，3H)，3.72(dd，1H，J＝10.0，2.6)，3.95(dd，1H，J＝4.6，2.1)，4.17-4.27(m，3H)，4.37(dd，1H，J＝12.1，3.7)，4.52-4.57(m，1H)，5.26-5.31(m，1H).

Step d)

To a solution of compound 5(1.95g, 7.14mmol) in DMF (50mL) was added imidazole (1.46g, 21.4 mmol). After a few minutes TBDPSCI was added and the reaction mixture was stirred at room temperature overnight. Ethyl acetate (200mL) was added and the solution was taken up in 10% aqueous citric acid (3X 50mL) and NaHCO₃Washed with saturated aqueous solution (50 mL). Na for organic phase₂SO₄Dried, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography (heptane: ethyl acetate (4: 1)) to give compound 6 in 92% yield (3.39 g).

¹H NMR(CDCl₃，400MHz)1.09(s，9H)，2.04(s，3H)，2.05(s，3H)，3.71(dd，1H，J＝4.3，2.1)，3.78(dd，1H，J＝9.5，2.2)，3.99(dd，1H，J＝9.6，4.6)，4.12(dd，1H，J＝12.2，5.1)，4.28-4.33(m，2H)，4.36-4.40(m，1H)，5.17-5.22(m，1H)，7.37-7.51(m，6H)，7.58-7.74(m，4H).

Step e)

To a solution of compound 6(3.39g, 6.63mmol) in MeOH (60mL) was added NaOMe (10mL, 0.5M in methanol). The reaction mixture was stirred at room temperature for 2 hours and then addedInto Dowex 50 WX8 (H)⁺Form) the solution was neutralized to neutral pH. The ion exchange resin was filtered off and the solvent was removed by rotary evaporation followed by high vacuum. Product 7(2.66g) was obtained in quantitative yield.

¹H NMR(CDCl₃，400MHz)1.08(s，9H)，3.64(dd，1H，J＝11.5，5.5)，3.71(dd，1H，J＝11.4，3.9)，3.73-3.77(m，2H)，3.85-3.90(m，1H)，3.95(dd，1H，J＝9.6，4.7)，4.15(dd，1H，J＝6.1，4.2)，4.39-4.43(m，1H)，7.37-7.51(m，6H)，7.58-7.74(m，4H).

Step f)

Compound 7(1.4g, 3.27mmol) dissolved in chloroform (10mL) and pyridine (4.77mL, 58.9mmol) was cooled in a dry ice acetone bath and SO was added₂Cl₂(1.56mL, 19.6mmol) and then the cooling bath was removed. The reaction mixture was stirred overnight, which gradually darkened. After 16h the reaction mixture was diluted with DCM (15mL), 10% aqueous citric acid (15mL) and NaHCO₃(saturated aqueous solution) (15 mL). Na for organic phase₂SO₄Dried, filtered and concentrated under reduced pressure. The resulting brown oil was dissolved in MeOH (10mL) and about 0.5mL NaI was added (in MeOH: H)₂0.8% solution in O (1: 1), stirred for 15 minutes. The solvent was then evaporated and the crude product was purified by flash column chromatography (heptane: ethyl acetate (4: 1)) to give compound 8 in 68% yield.

¹H NMR(CDCl₃，400MHz)1.10(s，9H)，3.80-3.85(m，2H)，3.89(dd，1H，J＝12.1，5.8)，3.96(dd，1H，J＝9.7，3.8)，4.00(dd，1H，J＝12.3，2.6)，4.15(ddd，1H，J＝9.7，5.9，2.6)，4.32-4.36(m，2H)，7.35-7.52(m，6H)，7.58-7.75(m，4H)

Step g)

To compound 8(1.08g, 2.24mmol) in MeOH (50ml) and H₂Solution in O (5mL) was added to pph₃(882mg, 3.36 mmol). The reaction mixture was stirred at room temperature overnight. LC-MS showed no starting material, but little cyclized product. To this solution, TEA (9.38mL, 67.2mmol) and H were added₂O (5mL), heated to 50 ℃. After 4 hours LC-MS indicated no uncyclized amine. The solvent was evaporated and the crude product was purified by flash chromatography (heptane: ethyl acetate (3: 2)) to give product 9 in 54% yield (0.49 g). LRMS (M + H) 402.

¹H NMR(CDCl₃，400MHz)1.06(s，9H)，2.71(dd，1H，J＝11.1，10.4)，3.18(dd，1H，J＝11.2，7.0)，3.73(d，1H，J＝4.7)，3.78(dd，1H，J＝9.8，3.5)，3.84(dd，1H，J＝9.8，2.0)，3.95(ddd，1H，J＝10.2，7.1，4.1)，4.16-4.19(m，1H)，4.66(dd，1H，J＝4.4，4.4)，7.35-7.46(m，6H)，7.61-7.67(m，4H).

Step h)

To a solution of compound 9(0.48g, 1.20mmol) in 50mL MeOH/TEA (9: 1) was added BOC anhydride (0.52g, 2.40 mL). The reaction mixture was stirred overnight and then concentrated under reduced pressure to remove the solvent. The crude product was purified by flash column chromatography (heptane: ethyl acetate (4: 1 → 2: 1)) to give product 10(0.6g) in quantitative yield.

¹H NMR(CDCl₃，400MHz)1.07(s，9H)，1.24-1.46(m，9H)^＊，3.05(dd，1H，J＝10.4，10.4)，3.56(d，1H，J＝9.7)，3.70-3.89(m，1H)^＊，3.90-4.15(m，2H)^＊，4.24-4.89(m，3H)^＊，7.34-7.47(m，6H)，7.59-7.78(m，4H).^＊Represents a rotamer.

Step i)

To a solution of compound 10(0.60g, 1.19mmol) in THF (12mL) was added tetrabutylammonium fluoride (1.79mL, 1.79 mmol). The reaction mixture was stirred at room temperature for 3 hours, and then concentrated under reduced pressure to remove the solvent. The crude product was purified by flash column chromatography (heptane: ethyl acetate (1: 1 → 0: 1)) to give compound 11(0.29g) in 94% yield.

¹H NMR(CDCl₃，400MHz)1.45-1.52(m，9H)^＊，3.16-3.32(m，1H)^＊，3.83-4.22(m，5H)^＊，4.41-4.54(m，1H)^＊，4.66-4.71(m，1H)^＊.^＊Represents a rotamer.

Step j)

To a solution of compound 11(0.34g, 1.29mmol) in anhydrous DCM was added Dess-Martin pentavalent iodoxide (0.60g, 1.42 mmol). The reaction mixture is stirred under N₂Stirring was continued for 2 hours, at which point the reaction was deemed complete by TLC. The solution was taken up with 10% Na₂S₂O₃Aqueous solution and NaHCO₃A1: 1 mixture of saturated aqueous solution was washed 3 times (3X 20 mL). Na for organic phase₂SO₄Drying, filtering and concentrating under reduced pressure. The crude product was purified by flash chromatography (heptane: ethyl acetate (3: 1)) to give compound 12 in 84% yield (284 mg).

¹H NMR(CDCl₃，400MHz)1.48(s，9H)，3.45(dd，1H，J＝11.3，9.0)，4.01-4.17(m，2H)，4.19-4.41(m，3H)，4.68-4.87(m，1H).

Step k)

To a solution of compound 12(263mg, 1.01mmol) was added a pre-mixed solution of AcCl (42. mu.L, 0.601mmol) and MeOH (5 mL). After stirring for 2h, AcCl (0.98mL, 14mmol) was added, and after stirring for 16h, AcCl (9.8mL, 140mmol) was added. The reaction was completed shortly, then concentrated under reduced pressure, followed by removal of any residual solvent using high vacuum to give compound 13(253mg) in crude yield of 103%.

¹H NMR(CDCl₃，400MHz)3.34(s，3H)，3.40(s，3H)，3.76(d，1H，J＝10.6)，3.72-3.90(m，1H)，4.15(d，1H，J＝10.4)，4.34(d，1H，J＝4.6)，4.50-4.60(m，1H)，4.69-4.75(m，1H)，4.83(s，1H).

Example 2

Coupling of P-2 with L-Leu

To a solution of crude 13(112mg, 0.460mmol) in DMF (6mL) was added DIEA (304. mu.L, 1.84mmol) and BOC-Leu (117mg, 0.506 mmol). The reaction flask was cooled in an ice bath for 10 min, then HATU (193mg, 0.506mmol) was added. The reaction mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure. The crude product was dissolved in CHCl₃In (15mL), 10% aqueous citric acid (10mL) and NaHCO were added₃Washed with saturated aqueous solution (10 mL). Na for organic phase₂SO₄Drying, filtering and evaporating. Flash chromatography of the crude product (heptane: ethyl acetate (2: 1 → 1: 1)) Purified) to give product 14(121mg) in 62% yield.

Example 3

P3 construction unit

4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl]Benzoic acid

Step a) 4-cyanopropiophenone

4-Bromophenylacetone (5.65g, 26.4mmol), Zn (CN) as described for the preparation of 4-cyanoacetophenone (Synth. Commun 1994, 887-890)₂(1.80g, 15.3mmol) and Pd (pph)₃)₄(2.95g, 2.6mmol) was refluxed at 80 ℃ for 18 h in deoxygenated DMF (35mL, stored on 4A molecular sieves charged with Ar before use). The mixture was partitioned between toluene (100ml) and 2N NH₄OH (100 mL). 2N NH for organic phase₄OH (100mL), washed with saturated aqueous NaCl (2X 100mL), dried, and concentrated under reduced pressure. A10 mmol scale reaction was carried out analogously and the crude products were combined. Flash chromatography (330g silica, 6: 1 petroleum ether/ethyl acetate) afforded the desired compound (5.17g, 89%) as a white solid.

1H NMR(CDCl₃)δppm：1.22(t，3H，J＝7.2Hz)，3.00(q，2H，J＝7.3Hz)，7.75(d，2H，J＝8.8Hz)，8.03(d，2H，J＝8.4Hz)

13C NMR(CDCl₃)δppm：7.8，32.1，116.1，117.9，128.3，132.4，139.7，199.2

Step b) 4-Propionylbenzoic acid

4-Cyanopropiophenone (4.67g, 29.3mmol) was refluxed with 2N NaOH (90mL, 180mmol) and dioxane (90mL) at 95 ℃ overnight. The mixture was diluted with water (150mL), washed with ether (75mL), acidified to pH 2 with concentrated HCl, and extracted with ether (3X 75 mL). The organic phase was washed with saturated aqueous NaCl (3X 75mL), dried, and concentrated to give a yellow solid (5.12g, 98%).

1H NMR(CDCl₃+CD₃OD)δppm：1.18(t，3H，J＝7.2Hz)，2.99，(q，2H，J＝7.1Hz)，7.95(d，2H，J＝8.4Hz)，8.08(d，2H，J＝8.8Hz)

13C NMR(CDCl₃)δppm：7.9，32.1，127.7，130.0，134.0，140.0，168.0，200.8

Step c) methyl 4-propionylbenzoate

Mixing the above benzoic acid (890mg, 5mmol) and NaHCO₃(1.26g, 15mmol) and iodomethane (935. mu.L, 15mmol) in DMF (10mL) were stirred at room temperature overnight. The mixture was diluted with saturated aqueous NaCl solution (50mL) and extracted with ether (3X 50 mL). The organic phase was washed with water (50mL), dried and concentrated. Flash chromatography (90g silica, 2: 1 petroleum ether: ethyl acetate) afforded a white solid (744mg, 77%).

1H NMR(CDCl₃)δppm：1.24(t，3H，J＝7Hz)，3.03(q，2H，J＝7Hz)，3.95(s，3H)，8.0and 8.12(ABq，4H)

Step d) methyl 4- (2-bromopropionyl) benzoate

Methyl 4-propionylbenzoate (744mg, 3.87mmol), pyrrolidone tribromide (1.98g) and 2-pyrrolidone (380mg, 4.5mmol) were heated in THF (38ml) at 50 ℃ under nitrogen for 3 hours. The mixture was cooled, filtered, concentrated under reduced pressure, and then redissolved in ether (50 mL). The ethereal solution was washed successively with water (20mL), Na₂S₂O₅A saturated aqueous solution (20mL), a saturated aqueous solution of NaCl (20mL) and water (20mL) were washed, dried and concentrated under reduced pressure to give a yellow oil (1.025g) which was used directly in the Hantzsch coupling reaction. According to¹This material contained 91% of the desired bromoketone, 5% of the starting ketone, and 4% of 4-bromo-1-butanol, as determined by H NMR.

1H NMR(CDCl₃) δ ppm: 1.92(d, 3H, J ═ 7Hz), 3.96(s, 3H), 5.28(q, 1H, J ═ 7Hz), 8.07 and 8.14(ABq, 4H)

Step e) methyl 4- [2- (4-tert-butoxycarbonylpiperazin-1-yl) -5-methylthiazol-4-yl ] benzoate

All of the above α -bromoketones and tert-butyl 4-thiocarbonylpiperazine-1-carboxylate (J.Med.chem., 1998, 5037-5054, 917mg, 3.73mmol) were dissolved in 36ml THF at 70 ℃ and N₂Reflux for 2 hours. The precipitate was filtered off and the filtrate was concentrated under reduced pressure to a yellow solid. Flash column chromatography (silica gel, 5: 1 petroleum ether: ethyl acetate) afforded 624mg of a pale yellow solid. Chromatography of the precipitate (silica gel, 2: 1 petroleum ether: EtOAc) gave a further 32mg of compound. The total yield is 44%.

1H NMR(CDCl₃) δ ppm: 1.46(s, 9H), 2.43(s, 3H), 3.42, (m, 4H), 3.54(m, 4H), 3.90(s, 3H), 7.68 and 8.04(ABq, 4H).

Step f)4- [2- (4-tert-butoxycarbonylpiperazin-1-yl) -5-methylthiazol-4-yl ] benzoic acid

The above methyl ester (564mg, 1.35mmol) and 1.35mL of 2N NaOH, 5mL of THF, and 3.65mL of water were heated at 60 ℃ for 4 hours. The reaction mixture was evaporated and poured into 20mL of a saturated aqueous solution of NaCl and 20mL of CH in an ice bath₂Cl₂Then acidified with 5% citric acid to pH 3. The layers were separated and the organic phase was further purified with 2X 10mL CH₂Cl₂And (4) extracting. The organic phases were combined, washed with water (10mL), dried, and concentrated under reduced pressure to give a pale yellow solid (537mg, 98%).

1H NMR(CDCl₃) δ ppm: 1.48(s, 9H), 2.47(s, 3H), 3.47(m, 4H), 3.57(m, 4H), 7.74 and 8.12(ABq, 4H).

13C NMR(CDCl₃)δppm：12.6，28.3，42.8，48.1，80.3，119.1，127.8，128.2，130.1，140.5，145.6，154.6，167.2，171.4.

LCMS：(M+H)⁺404，(M-H)^-402.

Step g)4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzoic acid

Reacting 4- [4- (4-carboxyphenyl) -5-methylthiazol-2-yl]Tert-butyl piperazine-1-carboxylate (0.421mmol) was dissolved in 4M HCl/1, 4-dioxane and stirred at room temperature for 1 hour. The solvent was then removed under reduced pressure and the residue, 4- (5-methyl-2-piperazin-1-ylthiazol-4-yl) benzoic acid, was suspended in methanol (10mL) and treated with AcOH/AcONa buffer (pH about 5.5, 5mL) and formaldehyde (0.547 mmol). The reaction mixture was stirred at room temperature1 hour, then NaCNBH₃(0.547mmol) and stirred at room temperature overnight. The solvent was removed under reduced pressure, and the residue was purified by column chromatography to give the title compound (0.403mmol, 95%).

MS(ES)m/z 318(100％，[M+H]⁺).

Example 4

To a solution of compound 14(0.121g, 0.288mmol) in methanol (4mL) was added acetyl chloride (0.4mL) dropwise at 0 ℃. The reaction mixture was then stirred at room temperature overnight and then concentrated under reduced pressure. The residue was dissolved in dry DMF (5mL) and concentrated to dryness, repeated 2 times, then redissolved in DMF (6 mL). Adding 4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl to the solution]Benzoic acid hydrochloride (112mg, 0.316mmol) and DIEA (190. mu.L, 1.15mmol), then cooled to 0 ℃ and HATU (120mg, 0.316mmol) added. The reaction mixture was stirred at room temperature for 3 hours, then the solvent was removed by rotary evaporation. The crude mixture was dissolved in CHCl₃In (15mL), 10% aqueous citric acid (10mL) and NaHCO were added₃Washed with saturated aqueous solution (10 mL). Na for organic phase₂SO₄Drying, filtering and evaporating. The crude product was purified by flash chromatography (ethyl acetate: acetone (1: 1) + 0.2% TEA) to give a colorless oil/solid product in 84% yield (150 mg). LRMS (M + H) 620.

NMR(CDCl₃400MHz) for the major rotamer (13: 1 rotamer mixture): 0.98(d, 3H, J ═ 6.5), 1.04(d, 3H, J ═ 6.4), 1.55-1.89(m, 3H), 2.34(s, 3H), 2.42(s, 3H), 2.49-2.54(m, 2H), 3.25(s, 3H), 3.43(s, 3H), 3.45-3.51(m, 3H), 3.72(d, 1H, J ═ 10.5), 3.91(d, 1H, J ═ 10.5), 4.05-4.15(m, 1H), 4.46-4.53(m, 1 ddh), 4.59 (m, 1H, J ═ 5.2, 5.1), 4.74(d, 1H, J ═ 5.6), 4.96-5.04(m, 1H, 6.83, 1, 8(d, 8, 7.8, d, 8(d, 3H),2H，J＝8.3)，7.79(d，2H，J＝8.5).

example 5

Compound 15(137mg, 0.220mmol) was dissolved in 20mL TFA: H₂O (97.5: 2.5) and stirred for 4 hours. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica gel (EtOAc: acetone (1: 2) containing 0.2% TEA) and lyophilized from dioxane to give the product as a beige solid (90mg) in 71% yield.

NMR(CDCl₃400MHz) for the major rotamer (10: 1 rotamer mixture): 0.98(d, 3H, J ═ 6.5), 1.02(d, 3H, J ═ 6.2), 1.58-1.86(m, 3H), 2.34(s, 3H), 2.42(s, 3H), 2.50-2.54(m, 2H), 3.44-3.53(m, 2H), 3.68(dd, 1H, J ═ 10.4, 8.8), 4.11(d, 1H, J ═ 17.2), 4.29(d, 1H, J ═ 17.3), 4.36-4.44(m, 1H), 4.59(dd, 1H, J ═ 10.5, 7.1), 4.81(d, 1H, J ═ 5.8), 4.87-4.97(m, 2H), 6.83(d, 1H, J ═ 10.5, 7.1), 4.81(d, 1H, J ═ 5.8), 4.87-4.97(m, 2H, 6.83(d, J ═ 8, 7.9, 7.8).

Example 6

N- [ (S) -1- ((3aS, 6R, 6aS) -6-chloro-3-oxohexahydrofuro [3, 2-b)]Pyrrole-4-carbonyl) -3-methylbutyl radical]-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl]Benzamide derivatives

Step a)

To a solution of crude compound 12 (ca. 0.56mmol) in trimethyl orthoformate (0.8mL) and methanol (3mL) was added p-toluenesulfonic acid monohydrate (p-TsOH, 0.007g, 0.037mmol) with stirring, followed by heating at 50 ℃ overnight. The reaction mixture was monitored by TLC (3: 2 hexane: ethyl acetate, ninhydrin stain). The reaction mixture was heated to 60 ℃ and a total of 21mg of p-TsOH was added over 4 hours to give the Boc-deprotected ketal as the main product (as indicated by TLC). The reaction mixture was then cooled to room temperature and triethylamine (0.32mL, 2.24mmol) and di-tert-butyl dicarbonate (0.184g, 0.84mmol) were added sequentially. The reaction mixture was kept at room temperature for 2.5 hours, then concentrated under reduced pressure and pre-absorbed on silica gel. Flash column chromatography of the residue (step gradient elution, ethyl acetate in hexanes, 20-25%) followed by concentration under reduced pressure of the appropriate fractions and further drying on a vacuum line to remove residual solvent afforded product 31 as a beige solid (0.069g, 0.022mmol, 40% over 2 steps).

NMR data (400MHz, 298K, CDCl)₃)：¹H，δ1.47(s，9H)，3.16(m，1H)，3.30(s，3H)，3.37(s，3H)，3.72(d，1H，J 9.3Hz)，3.86(m，1H)，3.98(m，1H)，4.15(m，1H)，4.44(d，1H，J 5.4Hz)，4.61(m，1H).

Step b)

Acetyl chloride (0.3mL) was added dropwise to a solution of compound 31(0.074g, 0.24mmol) in methanol (27mL) over 1 minute at 0 ℃ with stirring. The reaction mixture was monitored by TLC (hexane: ethyl acetate (3: 2) and dichloromethane: methanol (95: 5), ninhydrin staining). The starting material was completely consumed after 4.5 hours. The reaction mixture was then concentrated under reduced pressure, redissolved in dioxane containing a few drops of water and lyophilized. The off-white amorphous solid obtained and N- (tert-butoxycarbonyl) -L-leucine-hydrate (0.066g, 0.26mmol) were dissolved in DMF (3mL) and concentrated under reduced pressure. The residue was redissolved in DMF (3mL) and N-ethyldiisopropylamine (0.13mL, 0.72mmol) was added. To this solution was added N-O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 0.12g, 0.31mmol) at 0 ℃. The reaction mixture was held at 0 ℃ for 40 minutes and at room temperature for 75 minutes. The reaction mixture was then diluted with ethyl acetate (25mL), washed successively with 10% aqueous citric acid (3X 15mL) and saturated aqueous sodium bicarbonate (3X 15mL), then dried (sodium sulfate), filtered, and concentrated under reduced pressure. Flash column chromatography of the residue on silica gel using 2: 1 hexane: ethyl acetate as eluent followed by concentration of the appropriate fractions under reduced pressure gave compound 32b (0.089g, 0.21mmol, 88%) as a colourless syrup which was used directly in the next step.

Step c

Acetyl chloride (0.3mL) was added dropwise to a solution of compound 32b (0.089g, 0.21mmol) in methanol (27mL) at 0 ℃ over a half minute. The reaction mixture was then stirred at room temperature for 5.5 h (monitored by TLC, dichloromethane: methanol (95: 5), stained with a solution of ammonium molybdate-cerium sulfate in 10% aqueous sulfuric acid) and concentrated under reduced pressure. The residue was redissolved in dioxane (5mL), a small amount of water was added, the solution was lyophilized, the resulting colorless amorphous solid and HBr salt of 4- [2- [ 4-methylpiperazin-1-yl ] thiazol-4-yl ] benzoic acid (0.089g, 023mmol) were dissolved in DMF (3mL), N-ethyldiisopropylamine (0.15mL, 0.85mmol) were added, the solution was cooled to 0 ℃ and HATU (0.105g, 0.275mmol) was added. The reaction mixture was stirred at 0 ℃ for 1 hour and at room temperature for an additional 1 hour (monitored by TLC: dichloromethane: methanol (95: 5), visualized with UV light, stained with a solution of ammonium molybdate-cerium sulfate in 10% aqueous sulfuric acid). The reaction mixture was then diluted with ethyl acetate (25mL), washed with 3: 1 saturated aqueous sodium bicarbonate solution/brine (3X 20mL), then dried (sodium sulfate), filtered and concentrated under reduced pressure. The residue was subjected to flash column chromatography with stepwise gradient elution (methanol in dichloromethane, 0-5%) followed by concentration of the appropriate fractions under reduced pressure and freeze drying from dioxane (5mL) and a few drops of water to give product 32c (0.121g, 0.20mmol, 94%) as a colorless amorphous solid.

Step d)

To compound 32c (0.114g, 0.188mmol) was added 97.5: 2.5 TFA: water (6mL) at room temperature, the resulting solution was monitored by LC-MS, and after stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure. The residue was redissolved in ethyl acetate (25mL), washed with a saturated aqueous solution of sodium bicarbonate (3 × 15mL) and brine (1 × 15mL), then dried (sodium sulfate), filtered and concentrated under reduced pressure. The resulting amorphous solid residue was dissolved in DMSO-acetonitrile-water-dioxane (ca. 12mL) and subjected to preparative HPLC-MS (column: Sunfire 19X 100mm (C)₁₈) Eluent A: 10mM aqueous ammonium acetate, eluent B: 10mM ammonium acetate in 9: 1 acetonitrile/water, gradient: 30% B to 80% B in 8 minutes, flow rate: 20 mL/min). The appropriate fractions were concentrated under reduced pressure and the residue redissolved in dioxane containing a few drops of water, frozen and lyophilized to give compound 32d as a beige amorphous solid (0.055g, 0.10mmol, 52%). An aliquot of the product obtained was dissolved in CDCl₃NMR analysis indicated that the product was present as a ketone (no hydrate was detected) as a 9: 1 mixture of rotamers. NMR data represent the major rotamers. The hydrate form is the predominant form when analyzed by HPLC-MS.

NMR data (500MHz, 293K, CDCl)₃)：¹H，δ0.96(d，3H，CH₃-CH)，1.02(d，3H，CH₃-CH)1.62-1.78(m，3H，CH(CH₃)₂ and CH₂CH(CH₃)₂)，2.37(s，3H，CH₃-N)，2.56(m，4H，2×CH₂-N)，3.59(m，4H，2×CH₂-N), 3.70(m, 1H, CHH-CHCl), 4.13(d, 1H, CHH-0), 4.31(d, 1H, CHH-O), 4.42(m, 1H, CHCl), 4.60(m, 1H, CHH-CHCl), 4.82(m, 1H, CHCl-CH), 4.90-4.96(m, 2H, CH-C ═ O and CHNH), 6.85-6.89(m, 2H, NH and thiazole-H), 7.78(d, 2H, Ar-H), 7.89(d, 2H, Ar-H), LR-MS: theoretical value C₂₇H₃₅ClN₅O₄S: 560.2. experimental values: 560.3[ M + H]Theoretical value C₂₇H₃₇ClN₅O₅S: 578.2. experimental values: 578.3.

example 7

N- [2- ((3aS, 6R, 6aS) -6-chloro-3-oxohexahydrofuro [3, 2-b ]]Pyrrole-4-yl) -1-S- Cyclohexyl-2-oxoethyl]-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl]Benzamide derivatives

Step a) [2- ((3aS, 6R, 6aS) -6-chloro-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrol-4-yl) -1-S-cyclohexyl-2-oxoethyl ] carbamic acid tert-butyl ester (7a)

(3aS, 6R, 6aS) -6-chloro-3, 3-dimethoxy hexahydrofuro [3, 2-b]The hydrochloride salt of pyrrole (13) (0.23mmol) and Boc-cyclohexyl-Gly-OH (64.4mg, 0.25mmol) were co-evaporated from DMF, redissolved in 3ml DMF and cooled in an ice bath. DIEA (160. mu.L, 0.92mmol) and HATU (108mg, 0.28mmol) were added sequentially. After 20 minutes, the mixture was stirred at room temperature for 2 hours and 20 minutes, and concentrated under reduced pressure. The residue was redissolved in EtIn OAc (10mL), 10% citric acid (5mL), saturated NaHCO were used in sequence₃The solution (5mL) was washed with saturated NaCl (2X 5 mL). The organic phase was dried (Na)₂SO₄) And concentrated. Flash column chromatography (silica gel, 2: 1 pentane: ethyl acetate) afforded a white solid (86.6mg, 84% yield).

LCMS[M+23]⁺＝269

Step b) N- [2- ((3aS, 6R, 6aS) -6-chloro-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrol-4-yl) -1-S-cyclohexyl-2-oxoethyl ] -4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide (7b)

To an ice-cooled solution of the above tert-butyl carbamate (86.6mg, 0.194mmol) in methanol (2.20mL) was added acetyl chloride (0.25 mL). The mixture was stirred at room temperature for 3 hours 45 minutes and evaporated. Freeze-drying from dioxane/water gave the deprotected amine HCl salt, which was first reacted with 4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl]Benzoic acid hydrobromide (83mg, 0.22mmol) was co-evaporated together from DMF and then redissolved in 2.5mL of DMF. The mixture was cooled in an ice bath and DIEA (140. mu.L, 0.80mmol) was added followed by HATU (83.8mg, 0.22 mmol). After 15 min, the mixture was stirred at room temperature for 2.5 h, concentrated and dissolved in EtOAc (20ml), followed by saturated NaHCO₃(10mL) and saturated NaCl (2X 10mL) solution. The organic phase was dried (Na)₂SO₄) And concentrating. Flash column chromatography (silica gel, CH)₂Cl₂-MeOH-Et₃N) gave a white solid (121.2mg, 99% yield).

LCMS[M+1]⁺＝632

Step c) N- [2- ((3aS, 6R, 6aS) -6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrol-4-yl) -1-S-cyclohexyl-2-oxoethyl ] -4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide (7c)

The above dimethoxy ether (115mg, 0.182mmol) was deprotected in TFA-water (97.5: 2.5v/v, 6.0mL) by stirring at room temperature for 2h 15 min. The mixture was concentrated, dissolved in EtOAc (25mL), and successively saturated NaHCO₃(3X 15mL) and saturated NaCl (15 mL). Na for organic phase₂SO₄Dried and evaporated. The crude product was purified by HPLC-MS (column SunfirePrep C)₁₈OBD 5 μm 19X 100 mm; gradient 60-80% B in a, mobile phase a: 10 nNH₄Aqueous OAc solution, B: 10mM NH₄OAc/90% MeCN) gave the final compound as a white solid (63mg, 59% yield).

LCMS ES⁺604 (hydrate) and ES⁺586 (ketone)

1H NMR(500MHz，CDCl₃) Δ ppm 7.90 and 7.78(ABq, both 2H, 6.89(s, 1H), 6.83(d, 1H, NH), 4.91(m, 1H), 4.86(m, 1H), 4.69(m, 1H), 4.62(dd, 1H), 4.40(m, 1H), 4.32 and 4.14(ABq, both 1H), 3.72(dd, 1H), 3.60(m, 4H), 2.58(m, 4H), 2.38(s, 3H), 2.10-1.04(m, 11H)

Example 8

N- [ (S) -1- ((3aS, 6R, 6aS) -6-chloro-3-oxohexahydrofuro [3, 2-b)]Pyrrole-4-carbonyl) -3-fluoro-3-methylbutyl]-4- [2- (4-methylpiperazin) -1-yl) thiazol-4-yl]Benzamide derivatives

Step a)

Acetyl chloride (0.4mL) was added dropwise to a solution of compound 11(60mg, 0.228mmol) in methanol (4mL) at 0 ℃. The reaction mixture was stirred at room temperature for 6 hours, concentrated, redissolved in 1, 4-dioxane and freeze-dried overnight. The residue was dissolved in 5mL of DMF. To this solution was added gamma-fluoro-Boc-Leu-OH (Truong et al, Synlett 2005, No8, 1279-H1280, 50mg, 0.201mmol) and DIEA (133. mu.L, 0.802mmol), which was then cooled to 0 ℃ and HATU (80mg, 0.211mmol) was added. The reaction mixture was stirred at room temperature for 3 hours, and then the solvent was evaporated under reduced pressure. The product was dissolved in EtOAc (20mL) and washed with NaHCO₃Washed with saturated aqueous solution (10 mL). Na for organic phase₂SO₄Drying, filtering and concentrating under reduced pressure. The product was purified by flash chromatography (ethyl acetate) to afford product 42 in 99% yield (79 mg).

Step b)

Acetyl chloride (0.4ml) was added dropwise to a solution of compound 42(79mg, 0.199mmol) in methanol (4ml) at 0 ℃. The reaction mixture was stirred at room temperature for 6 hours, concentrated, redissolved in 1, 4-dioxane and freeze-dried overnight. The residue was dissolved in 5mL of DMF, and 4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] was added to the solution]Benzoic acid hydrobromide (76mg, 0.198mmol) and DIEA (119. mu.L, 0.721mmol), then cooled to 0 ℃ and HATU (72mg, 0.189mmol) was added. The reaction mixture was stirred at room temperature for 3 hours, and then the solvent was evaporated under reduced pressure. The product was dissolved in CHCl₃In (15mL), NaHCO is used₃Washed with saturated aqueous solution (10 mL). Na for organic phase₂SO₄Drying, filtering and evaporating. The product was purified by flash chromatography (chloroform: ethanol 7: 3+ 0.1% TEA) to afford compound 43 sufficiently pure that it could be used directly in the next step.

Step c)

Compound 43(104mg, 0.198mmol) (impure) was dissolved in 9mL DCM: to DMSO (2: 1), TEA (111. mu.L, 0.797mmol) and SO were added sequentially₃ ^*Pyridine (48mg, 0.299 mmol). The reaction mixture was stirred at room temperature and monitored by LC-MS. After 4 hours, another portion (48mg) of SO was added₃ ^*Pyridine and another portion was added over 4 hours. After 22 hours (overnight), another portion was added and the last portion was added over 2 hours. The solution was poured into a separatory funnel with 40mL DCM and 20mL NaHCO₃Washing with saturated aqueous solution. Na for organic phase₂SO₄Drying, filtering and concentrating under reduced pressure. The product was purified by semi-preparative HPLC on a Sunfire C18 column using mobile phase A (90: 10H)₂O acetonitrile, 10mM NH₄Ac) and B (10: 90H)₂O acetonitrile, 10mM NH₄Ac), from 40% to 75% B. The product was a beige solid in 29% yield (30 mg).

NMR(CDCl₃，400MHz)：1.37-1.52(m，6H)，2.10-2.30(m，2H)，2.37(s，3H)，2.58-2.63(m，2H)，3.57-3.62(m，2H)，3.73(dd，1H，J＝10.5，8.7)，4.10(d，1H，J＝16.9)，4.28(d，1H，J＝16.9)，4.43-4.50(m，1H)，4.71(dd，1H，J＝10.6，6.9)，4.78(d，1H，J＝6.0)，4.87-4.93(m，1H)，4.95-5.03(m，1H)，6.86(s，1H)，7.37(d，1H，J＝7.3)，7.73(d，2H，J＝8.3)，7.82(d，2H，J＝8.6).LRMS(M+H)578.

Example 9

Another P3 construction unit

3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl]Benzoic acid HCl salt

Step a) methyl 4-bromo-3-fluorobenzoate (9a)

4-bromo-3-fluorobenzoic acid (2.46g, 11.2mmol) was dissolved in MeOH (9mL) and toluene (4mL) and cooled in an ice bath. (trimethylsilyl) diazomethane (11mL, 2.0M in hexane, 22mmol) was added dropwise until the yellow color no longer faded. The solution was stirred at room temperature for 40 minutes and then concentrated under reduced pressure. A second crop of carboxylic acid (2.43g) was treated similarly. The two crude products were combined and flash chromatographed (silica gel, 5: 1 pentane: ethyl acetate) to give methyl ester as a white solid (4.92g, 95% yield).

¹H NMR(400MHz，CDCl₃)delta ppm 7.77(m，1H)，7.71(m，1H)，7.64(m，1H)，3.93(s，3H).

Step b) methyl 4-acetoxy-3-fluorobenzoate (9b)

Allyl chloride (105. mu.L, 1.28mmol) and TEA (20. mu.L, 0.26mmol) were added under inert gas to a suspension of zinc powder (480mg, 7.34mmol) and anhydrous cobalt (III) bromide (96.6mg, 0.44mmol) in MeCN (4 mL). After stirring for 10 min at room temperature, the aryl bromide from step a) (1.003g, 4.30mmol, dissolved in 5mL of MeCN) was added followed by acetic anhydride (0.45mL, 4.79mmol) and more MeCN (1 mL). The mixture was stirred overnight, quenched with 1M HCl (20mL), and extracted with EtOAc (3X 20 mL). Organic phase is sequentially treated with NaHCO₃The mixture was washed with a saturated aqueous solution (20mL) and a saturated solution of NaCl (2X 20mL), and dried (Na)₂SO₄) And (4) concentrating. Flash chromatography (silica, 6: 1 to 4: 1 petroleum ether-EtOAc) afforded recovered bromide (161.1mg, 16%) and the desired ketone (white solid, 305.5mg, 36%).

NMR(CDCl₃)δppm：¹H(400MHz)7.94-7.86(m，2H)，7.80(dd，1H，J＝11.2，1.6Hz)，3.95(s，3H)，2.67(d，3H，J＝4.4Hz)；¹⁹F(376MHz)-109.2(m)；¹³C(100MHz)195.4(d，J＝3.7Hz)，165.1(d，J＝2.2Hz)，161.6(d，J＝255Hz)，135.8(d，J＝8.1Hz)，130.7(d，J＝2.9Hz)，129.0(d，J＝14Hz)，125.2(d，J＝3.6Hz)，117.9(d，J＝26Hz)，52.7(s)，31.4(d，J＝7.3Hz).

Step c) methyl 4- (2-bromoacetoxy) -3-fluorobenzoate (9c)

To the mixture of ketone (198mg, 1.01mmol) and pyrrolidone tribromide (532mg, 1.07mmol) of step b) was added THF (10mL) and 2-pyrrolidone (91. mu.L, 1.20 mmol). After heating at 60-65 ℃ for 2h, the mixture was concentrated under reduced pressure and then partitioned between EtOAc (20mL) and Na₂S₂O₃Saturated solution (10 mL). The aqueous phase was extracted with EtOAc (10mL), and the organic phases were combined, washed with a saturated solution of NaCl (2X 10mL), and dried (Na)₂SO₄) And (4) concentrating. Flash chromatography (silica gel, 7: 1 petroleum ether: EtOAc) afforded a white solid (0.2634g) containing 84% of the desired bromide (prepared from sodium chloride)¹⁹F NMR peak integration determination).

NMR(CDCl₃)δppm：¹H(400MHz)7.93(m，1H)，7.88(m，1H)，7.79(dd，1H，J＝11.2，1.6Hz)，4.50(d，2H，J＝2.4Hz)，3.94(s，3H)；¹⁹F(376MHz)-108.4(m).

Step d) methyl 3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzoate

EtOH (5.0mL) was added to the above bromoketone (193mg, 0.70mmol) and 4-methylpiperazine-1-carbothioamide (113mg, 0.71mmol), and the mixture was heated at 70 ℃ for 2 hours and 15 minutes. The precipitate was filtered off, washed with cold EtOH, dried in vacuo and identified. This was repeated on a larger scale for 1.75g of bromoketone (6.36 mmol).

NMR(1/1 CDCl₃-CD₃OD)δppm：¹H(400MHz)8.20(m，1H)，7.86(dd，1H，J＝8.4，1.6Hz)，7.76(dd，1H，J＝11.4，1.8Hz)，7.38(d，1H，J＝2.4Hz)，4.23(br，2H)，3.95，(s，3H)，3.65(br，4H)，3.32(br，2H)，2.98(s，3H)；¹⁹F(376MHz)-114.0(m).LCMS[M+H]⁺＝336.

The precipitates obtained in the two preparations are combined and suspended in NaHCO₃Saturated solution (50 mL). Extraction with EtOAc, washing of the organic phase with water and drying (Na)₂SO₄) Evaporation gave the title compound as a cream solid (1.76 g).

Step e) 3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-1-yl ] benzoic acid hydrochloride

The above methyl ester (1.76g, 5.25mmol) (9d) was heated with 6M hydrochloric acid (40mL) at 80 ℃ for 5.5 hours. More 6M hydrochloric acid (10mL) was added and the mixture was heated at 90 ℃ for 1 hour 15 min. After cooling, the mixture was evaporated under reduced pressure and freeze-dried from water to give the final product as a cream solid in quantitative yield.

NMR(DMSO-d6)δppm：¹H(400MHz)11.60(br，1H)，8.18(t，1H，J＝8.0Hz)，7.82(dd，1H，J＝8.4，1.6Hz)，7.72(dd，1H，J＝12.0，1.6Hz)，7.48(d，1H，J＝2.8Hz)，4.11(m，2H)，3.58(m，2H)，3.49(m，2H)，3.19(m，2H)，2.80(d，3H，J＝4.4Hz)；¹⁹F(376MHz)-113.5(m)；¹³C(100MHz)168.9，166.0，159.0(d，J＝250Hz)，143.4，131.4(d，J＝8Hz)，129.8，125.8(d，J＝11Hz)，125.6，116.6(d，J＝24Hz)，111.1(J＝15Hz)，51.1，45.0，41.9.LCMS[M+H]⁺＝322.

Example 10

N- [ (S) -1- ((3aS, 6R, 6aS) -6-chloro-3-oxo-hexahydrofuro [3, 2-b)]Pyrrole-4-carbonyl) -3-methylbutyl radical]-3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl]Benzamide derivatives

Step a) N- [ (S) -1- ((3S, 3aS, 6R, 6aS) -6-chloro-3-hydroxyhexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide

This compound was prepared by starting from tert-butyl N- [ (S) -1- ((3S, 3aS, 6R, 6aS) -6-chloro-3-hydroxyhexahydrofuro [3, 2-b ] pyrrole-4-carboxylate, a series of deprotection reactions (acetyl chloride, MeOH) and a HATU-mediated coupling reaction first with Boc-leucine and finally with 3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzoic acid hydrochloride prepared aS described in example 9.

LCMS：[M+H]⁺＝580；[M-H]^-＝578.¹⁹F NMR(376MHz，CDCl₃+CD₃OD)δppm-113.3(m).

Step b)

To the alcohol (16.6mg, 0.03mmol) in step a) was added 0.9mL CH₂Cl₂And 0.45ml of a solution in LDMSO Triethylammonium (20. mu.L) and Sulfur trioxide-pyridine complexThe mixture (20mg, 0.125mmol) was stirred at room temperature. After 2 hours, triethylamine (10. mu.L) and sulfur trioxide-pyridine complex (10mg) were added thereto, and the mixture was stirred overnight to complete the oxidation of the ketone. The mixture is treated with CH₂Cl₂Diluting with NaHCO₃The solution was washed with a saturated solution, followed by a saturated solution of NaCl. Na for organic phase₂SO₄Dried and evaporated to give an oil. The crude product was purified by HPLC-MS (column Sunfire Prep C)₁₈μ m 19X 100 mm; an OBD 5; gradient 30-80% B in a, mobile phase a: 10mM NH₄OAc aqueous solution, mobile phase B: 10mM NH₄OAc in 90% MeCN) to give the title compound (4.4mg) as a white solid.

NMR(CDCl₃)δppm：¹H (500MHz, 2 rotamers observed, describing the major rotamer) 8.22(m, 1H, phenyl H5), 7.56-7.54(m, 2H, phenyl H2 and H6), 7.21(m, 1H, thiazole), 6.81(d, 1H, J ═ 8.5Hz, NH), 4.94-4.85(m, 2H, NHCHC ═ O and ClCCHO), 4.84(d, 1H, J ═ 6.0Hz, (O ═ C) NCHC ═ O), 4.56(dd, 1H, J ═ 10.5, 7.0Hz), 4.42(m, 1H, ClCH), 4.32 and 4.14(ABq, all 1H), 3.70(dd, 1H, J ═ 10, 9), 3.59(m, 4H), 2.57(m, 2H, 3.57, 1H, 3.7H, NMe), nmh (m, 1H, 3.6H, 1H, 3.7 Hz), NMe (m, nmh, m, 1H, m-4.6H, 1H₂CHMe₂)，1.03(d，3H，J＝6.0Hz，i-Pr)，0.96(d，3H，J＝6.5Hz，i-Pr)；¹⁹F (376MHz) -112.9(m, 84% of the major rotamer) and-113.2 (m, 16% of the minor rotamer).

LCMS: single isotope molecular weight 577.4 Da; ES + ═ 578.4(M + H)⁺，596.5[M+H₂O+H]⁺.

Example 11

Another P3/P2 building unit

Step a) (S) -2- [ (S) -1- (4-bromophenyl) -2, 2, 2-trifluoroethylamino ] -4-methylpentanoic acid isopropyl ester (11a)

To (S) -2- [ (S) -1- (4-bromophenyl) -2, 2, 2-trifluoroethylamino prepared as described in Li, C, S, et al, Bioorg.Med.chem.Lett, 2006, 16, 1985 was added, with stirring]A solution of-4-methylpentanoic acid (1.80g, 4.9mmol) in isopropanol (100ml) was added to concentrated sulphuric acid (2 ml). The resulting solution was heated at 80 ℃ for 4 hours, then cooled, and then concentrated under reduced pressure. The resulting oil was dispersed in CH₂Cl₂(100mL), with saturated NaHCO₃The solution (2X 50mL) was washed and dried (MgSO)₄) And concentrated under reduced pressure to give the title compound as a brown oil (1.77g, 88%). MS [ M + H ]]412。

Step b) (S) -4-methyl-2- { (S) -2, 2, 2-trifluoro-1- [4- (4, 4, 5, 5-tetramethyl [1, 3, 2] dioxaborolan-2-yl) phenyl ] ethylamino } penta-isopropyl ester (11b)

To a solution of bromo derivative 1K (2.2g, 5.36mmol) in DMF (30mL) was added bis (pinacolato) diboron (2.0g, 8.04mmol), potassium acetate (1.6g, 16.1mmol) and [1, 1' -bis (diphenylphosphino) ferrocene with stirring]Palladium (II) chloride with CH₂Cl₂1: 1 Complex (0.438g, 0.54 mmol). The resulting solution was sealed in a tube and heated in a microwave oven at 160 ℃ for 20 minutes. The reaction mixture was cooled to room temperature and then filtered through a plug of silica gel eluting with ethyl acetate (500 mL). The resulting solution was concentrated under reduced pressure and the crude product was purified by reverse phase C18 column chromatography (H)₂O: MeCN, 50-100% gradient) to give the title compound as a brown oil (0.920g, 38%). MS [ M + H ]]458。

Step C) (S) -4-methyl-2- ((S) -2, 2, 2-trifluoro-1- {4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] phenyl } ethylamino) penta-isopropyl ester (11C)

To a solution of borolane derivative 1K (0.72g, 1.57mmol) in DMF: h₂To a solution in O (1: 1, 20ml) were added 1- (4-bromothiazol-2-yl) -4-methylpiperazine (0.5g, 1.89mmol), sodium carbonate (0.2g, 1.89mmol) and [1, 1' -bis (diphenylphosphino) ferrocene]Palladium (II) chloride with CH₂Cl₂1: 1 Complex (0.129g, 0.16 mmol). The resulting solution was sealed in a tube, heated in a microwave oven at 160 ℃ for 20 minutes, then cooled, and then treated with CH₂Cl₂(100ml) dilution. The organic phase was separated and dried (MgSO)₄) And concentrating under reduced pressure. The crude product was purified by flash column chromatography (ethyl acetate: MeOH, 9: 1) to give the title compound as a dark red solid (0.150g, 13%). MS [ M + H ]]513. Retention time 4.0 min, 50-97% 10mM (NH)₄)₂CO₃: MeCN 6 min gradient C12 reversed phase.

Step d) (S) -4-methyl-2- ((S) -2, 2, 2-trifluoro-1- {4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] phenyl } ethylamino) pentanoic acid, hydrochloride (11d)

To a mixture of 2M hydrochloric acid and dioxane (1: 1, 10ml) was added, with stirring, (S) -4-methyl-2- ((S) -2, 2, 2-trifluoro-1- {4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] phenyl } ethylamino) isopropyl valerate (0.15g, 0.29mmol) prepared as described by Palmer et al in J.Med.chem.2005, 48, 7520-one 7534. The solution was heated at 100 ℃ for 20 h and then concentrated under reduced pressure to give the title compound (0.14g, 98%) as a dark brown solid which was coupled to the P1 building block and the ketone was regenerated by any of the methods described above, usually without further purification. MS M-H469.

Example 11A

Another P3 construction unit

Acoh, bromo, RT, 2h, yield 55%; KF, acetonitrile, 18-crown-6, 90 ℃, 16h, 31% yield; AcOH, bromine, 45 ℃, 4h, 100% yield; 4-methylpiperazine-1-carbothioamide,. DELTA.2 h, yield 74%; lioh, RT, 16h, yield 100%.

Available sources of starting materials

Methyl 4-acetylbenzoate is available from Aldrich; 4-methylpiperazine-1-carbothioamide-11 suppliers (e.g., Chem Pur Products Ltd, Germany) were available to Scifinder.

Step a) methyl 4- (2-bromoacetyl) benzoate

To a solution of methyl 4-acetylbenzoate (8.4mmol) in acetic acid (20ml) was added bromine (8.4 mmol). The reaction mixture was stirred at room temperature for 2 hours, during which time the red color disappeared and a beige precipitate formed. The product was collected by filtration and washed with cold methanol/water (200ml, 1: 1) to give a white powder (55%).

1H NMR(400MHz，CDCl₃)3.98(3H，s)，4.20(2H，s)，8.02(2H，d，J＝8Hz)，8.18(2H，d，J＝8Hz).

Step b) methyl 4- (2-fluoroacetyl) benzoate

To a suspension of potassium fluoride (3.11mmol) in acetonitrile (1mL) was added 18-crown-6 (0.1mmol) and the reaction mixture was heated at 90 ℃ for 30 min. 4- (2-Bromoacetyl) benzoic acid (1.56mmol) was added and the reaction mixture was heated at 90 ℃ for 16h, then diluted with water (10mL) and extracted with ethyl acetate (3X 20 mL). The product was poured onto silica gel, eluted with 5-15% ethyl acetate/isohexane and the desired fractions were concentrated under reduced pressure to give the title compound as a white solid (31%).

1H NMR(400MHz，CDCl₃)3.98(3H，s)，5.55(2H，d，J＝50Hz)，7.95(2H，d，J＝8Hz)，8.18(2H，d，J＝8Hz).

Step c) methyl 4- (2-bromo-2-fluoroacetyl) benzoate

To a suspension of 4- (2-fluoroacetyl) benzoic acid (1.19mmol) in acetic acid (5mL) was added bromine (1.19 mmol). The reaction mixture was heated at 45 ℃ for 4 hours, during which time a green solution was formed. The reaction mixture was concentrated under reduced pressure and azeotroped 2 times with toluene to give the title compound as a green solid (100%). This crude product was used in the next step.

1H NMR(400MHz，CDCl₃)3.98(3H，s)，7.04(1H，s)，8.05-8.10(4H，m).

Step d) methyl 4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzoate

Methyl 4- (2-bromo-2-fluoroacetyl) benzoate (1.18mmol) and 4-methylpiperazine-1-carbothioamide (1.18mmol) were dissolved in ethanol (10 mL). It was heated to reflux for 2 hours and cooled to room temperature causing the product to precipitate. The product was collected by filtration and washed with cold ethanol. The product was treated with aqueous sodium bicarbonate to give the title compound as a colorless oil (74%). MS (ES +)337(M + H, 100%).

Step f 4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzoic acid dihydrochloride

To a solution of methyl 4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzoate (0.43mmol) in tetrahydrofuran/water (2.5ml, 4: 1) was added lithium hydroxide (0.5 mmol). The reaction mixture was stirred at room temperature for 16h and then concentrated under reduced pressure and hydrochloric acid (2N, 3ml) was added to precipitate the product as a white solid. The product was collected by filtration to give the title product as a white solid (79%). MS (ES +)322(M + H, 100%).

Example 12

N- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ]]Pyrrole-4-carbonyl) -3-methylbutyl]-4- [ 5-fluoro -2- (4-methylpiperazin-1-yl) thiazol-4-yl]Benzamide derivatives

Step a) benzyl 6-benzyloxy-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carboxylate (12a)

Dess-Martin pentavalent iodine oxidizer (12.5g, 30mmol) was dissolved in DCM (250 mL). To the stirred oxidant solution was added a solution of compound 10(7.4g, 20mmol) from WO 07/066180 in DCM (50ml) under nitrogen at room temperature over 45 minutes. Once the reaction was indeed complete as judged by TLC, 10% Na was added₂S₂O₃Aqueous solution (200mL) and the mixture was stirred at room temperature for a further 15 minutes. The two phase system was transferred to a separatory funnel and extracted 2 times with EtOAc (200mL and 100mL, respectively). The combined organic phases are treated with NaHCO₃The mixture was washed 1 time with saturated aqueous solution (100mL) and brine (100mL), and Na was added₂SO₄Drying, filtration and evaporation of the solvent under reduced pressure gave crude product 2(7.69g) as a clear oil; ESI⁺，m/z：368(M⁺+1)。

Step b) benzyl 6-benzyloxy-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrole-4-carboxylate (12b)

Compound 12a (7.6g) was dissolved in dry methanol (100 mL). Trimethyl orthoformate (30mL) and pTsOH (0.2g) were added at room temperature under a nitrogen atmosphere. The mixture was heated at 60 ℃ for 8 hours. Once the reaction was judged complete by TLC, it was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel eluting with ethyl acetate/heptane (1: 4) to give the ketal 12b as a clear oil after concentration under reduced pressure (5.9g, 71% over 2 steps); ESI⁺，m/z：382(M⁺-OMe)。

Step c) (3aS, 6R, 6aS) -6-hydroxy-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrole-4-carboxylic acid tert-butyl ester (12c)

At room temperature and H₂A solution of Compound 12b (2.5g, 6.4mmol) in methanol (60mL) and Pd (OH) was stirred under atmosphere₂(0.7g) for 48 hours. The mixture was filtered and concentrated under reduced pressure. The residue (2.8g, 14.8mmol) was dissolved in 75mL of dioxane/water (2: 1) mixture. 10% Na was added dropwise₂CO₃The solution (25mL) was brought to pH 9-9.5. The mixture was cooled to 0 ℃ in an ice-water bath and Boc anhydride was added in one portion. The reaction mixture was stirred at room temperature overnight, if necessary by adding more 10% Na₂CO₃The solution maintains the pH of the mixture at 9-9.5. The mixture was filtered and the solvent was evaporated under reduced pressure. The aqueous mixture was extracted with 3X 100mL EtOAc and the combined organic phases were washed with 100mL water and 100mL brine, Na₂CO₃Drying, filtration and evaporation of the solvent under reduced pressure gave 3.79g of carbamate as a clear oil (89%), ESI⁺，m/z：312(M⁺+Na)。

Step d) (benzyl 3aS, 6R, 6aS) -6-hydroxy-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrole-4-carboxylate (12d)

To compound 12c (3.8g, 13.13mmol) in CH with stirring₂Cl₂(100mL) 2M HCl/MeOH (50mL) was added. The resulting solution was stirred overnight, then concentrated under reduced pressure and azeotroped with toluene (3X 100 mL). The crude residue was dissolved in CH₂Cl₂(100mL), cooled to 0 deg.C, pyridine (1071. mu.L, 13.13mmol) was added followed by dropwise addition of CbzCl (1875. mu.L, 13.13 mmol). The reaction mixture was stirred at room temperature for 2 hours, then treated with 2M HCl (2X 50mL) and NaHCO₃Saturated solution (2X 50mL) was washed and dried (MgSO)₄) And concentrating. The residue was purified by flash column chromatography (5-100% isohexane: EtOAc) to give the title compound as a permeant oil (2510mg, 59%). MS M + H324.

Step e) (3aS, 6R, 6aS) -6-methanesulfonyloxy-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrole-4-carboxylic acid benzyl ester (12e)

To compound 12d (500mg, 1.55mmol) in CH was added under stirring₂Cl₂(20mL) Triethylamine (332. mu.L, 2.32mmol) and methanesulfonyl chloride (266mg, 2.32mmol) were added. After stirring for 30 min, the reaction mixture was washed with NaHCO₃Saturated solution (1X 20mL) and 2M HCl (1X 20mL) were washed and dried (MgSO₄) And concentrated to give the title compound as a yellow oil (655mg, 99%). MS M + H402.

Step f) (3aS, 6S, 6aS) -6-chloro-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrole-4-carboxylic acid benzyl ester (12f)

To a solution of compound 12e) (550mg, 1.37mmol) in DMF (30mL) was added lithium chloride (721mg, 13.7mmol) with stirring. After stirring at 120 ℃ for 120 minutes, the reaction mixture was concentrated under reduced pressure. Residue is CH₂Cl₂Diluted (50mL), washed with water (1X 20mL), MgSO₄Drying, and concentrating under reduced pressure. The residue was purified by flash column chromatography (5-66% isohexane: EtOAc) to give the title compound (330mg, 72%) as a yellow oil. MS M + H342, 344.

Step g) [ tert-butyl 2- (6-chloro-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] carbamate (12g)

6-chloro-3, 3-dimethoxyhexahydrofuro [3, 2-b ] by catalytic hydrogenation using 10% palladium on carbon and hydrogen at atmospheric pressure]Pyrrole-4-carboxylic acid benzyl ester (68mg, 0.20mmol) was deprotected. Stirring deviceAfter stirring for 2 hours, the suspension was filtered through celite and the filtrate was evaporated on a rotary evaporator to give crude 6-chloro-3, 3-dimethoxyhexahydrofuro [3, 2-b ]]Pyrrole, which was coupled with N-Boc-leucine using HATU according to the same method as described in example 2, to give the title compound (78mg, 93%). MS M/z 421.2(M + H)⁺。

Step h) N- [2- (6-chloro-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide (12h)

[2- (6-chloro-3, 3-dimethoxyhexahydrofuro [3, 2-b ] as described in example 4]Pyrrole-4-carbonyl) -3-methylbutyl]Tert-butyl carbamate (78mg, 0.185mmol) was deprotected in acidic conditions (acetyl chloride/methanol) and the crude pyrrole hydrochloride intermediate was then reacted with 4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl using HATU conditions as described in example 2]Benzoate coupling gave the title compound (98mg, 85%). MS M/z 624.2(M + H)⁺。

¹H-NMR(400MHz，CDCl₃)：7.91(d，2H)，7.81(d，2H)，6.85(d，1H)，5.00(m，1H)，4.75(d，1H)，4.60(m，1H)，4.50(dt，1H)，4.12(d，1H)，3.92(d，1H)，3.75(m，1H)，3.49(m，4H)，3.48(m，1H)，3.45(s，3H)，3.28(s，3H)，2.62(m，4H)，2.42(s，3H)，1.85(m，1H)，1.70(m，2H)，1.05(d，3H)，1.00(d，3H).

Step i) N- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide (12i)

Mixing N- [2- (6-chloro-3, 3-dimethoxy-hexahydrofuro [3, 2-b ]]Pyrrole-4-carbonyl) -3-methylbutyl]-4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl]Benzamide (98mg, 0.157mmol) was hydrolyzed under acidic conditions as described in example 5. The residue was purified by preparative HPLC (C8, gradient 10-90% MeCN/H)₂O) to yield the pure title compound 41.8mg (46%) as a ketone (27%) [ MS ]m/z 578.1(M+H)⁺]And hydrate (73%) [ MSm/z 596.1(M + H)₂O+H)⁺]A mixture of two rotamers.

Example 13

6-chloro-4- { 4-methyl-2- {2, 2, 2-trifluoro-1- (4' -methanesulfonylbiphenyl-4-yl) ethylamino } pentanoyl } Tetrahydrofuro [3, 2-b]Pyrrole-3-ones

Step a)1- (6-chloro-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrol-4-yl) -4-methyl-2- [2, 2, 2-trifluoro-1- (4' -methanesulfonylbiphenyl-4-yl) ethylamino ] pentan-1-one (13a)

Benzyl 6-chloro-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrole-4-carboxylate (55mg, 0.16mmol) was deprotected by catalytic hydrogenation using 10% palladium on carbon and atmospheric hydrogen. After stirring for 2 hours, the suspension was filtered through celite and the filtrate was concentrated. The resulting amine was coupled with 4-methyl-2- [2, 2, 2-trifluoro-1- (4' -methanesulfonylbiphenyl-4-yl) ethylamino ] pentanoic acid (76mg, 0.17mmol) prepared as described in WO 07/006716 using the HATU conditions described in example 2 to give the title compound (101mg, 50%).

Step b

Compound 13a (47mg, 0.07mmol) was hydrolyzed under acidic conditions as described in example 5, and the resulting residue was purified by column chromatography (EtOAc-petroleum ether 3: 2) to give the pure title compound (26mg), MS m/z 586.4.

Example 14

6-chloro-4- [ 4-methyl-2- (2, 2, 2-trifluoro-1- {4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl]Benzene and its derivatives Group } ethylamino) pentanoyl]Tetrahydrofuro [3, 2-b]Pyrrole-3-ones

Step a)1- (6-chloro-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrol-4-yl) -4-methyl-2- (2, 2, 2-trifluoro-1- {4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] phenyl } ethylamino) pentan-1-one (14a)

Benzyl group of benzyl 6-chloro-3, 3-dimethoxyhexahydrofuro [3, 2-b ] pyrrole-4-carboxylate (35mg, 0.10mmol) was removed by catalytic hydrogenation using 10% palladium on carbon and atmospheric hydrogen. After stirring for 2 hours, the suspension was filtered through celite and the filtrate was concentrated. The resulting amine was coupled with the acid of example 11 step d (42mg, 0.09mmol) using the HATU conditions described in example 2 to give the title compound (45 mg).

Step b

Compound 14a (47mg, 0.07mmol) was hydrolyzed under acidic conditions as described in example 5. The residue obtained is purified by column Chromatography (CH)₂Cl₂Acetone (2: l) + 0.05% DIEA) to give the pure title compound (20 mg).

Example 15

Alternative synthesis of P1 building blocks

Step i)

Step g) of example 1 was optimized as follows: a mixture of the monocyclic and bicyclic amines (about 1.8mmol) from the first reaction of step g) above was dissolved in ethyl acetate (25ml) and triethylamine (1.5ml) was added. The solution was refluxed for 3 hours, measured by LC-MS salt, then another portion of triethylamine (1.5ml) was added and the reaction was allowed to proceedThe mixture was refluxed for a further 15 hours. It was then cooled to about 0 deg.C, benzyl chloroformate (0.38ml, 2.7mmol) was added in one portion, and then warmed to room temperature. The reaction was monitored by TLC (4: 1 and 3: 2 hexane: ethyl acetate, developed by staining with UV light and AMC), after 4 hours the reaction mixture was diluted with ethyl acetate (15ml), washed successively with 10% aqueous citric acid (3X 25ml) and saturated aqueous sodium bicarbonate (3X 25ml), and dried (Na)₂SO₄) Filtration and concentration. The residue was flash chromatographed (step gradient elution, ethyl acetate/hexanes, 10-20%) and the appropriate fractions concentrated and dried under vacuum overnight to give the title compound as a colorless foam (0.57g, 1.06 mmol).

NMR data (400MHz, 298K, CDCl)₃)：¹H, delta 1.02 and 1.10(2s, 9H, C (CH)₃)₃) 3.13(m, 1H, CHH), 3.59 and 3.80(2m, 2X 1H, CH)₂) 3.99-4.15(m, 2H, CHH and CH), 4.34, 4.42, 4.46 and 4.68(4brs, 2H, major and minor CH), 4.84(m, 1H, CH), 4.92-5.16(m, 2H, CH)₂)，7.11-7.80(m，15H，ArH).

Step ii)

To a solution of the product of step i) (0.56g, 1.05mmol) in THF (6ml) was added 1M tetrabutylammonium fluoride/THF solution (1.26ml) with stirring and stirred at room temperature overnight. The reaction mixture was then concentrated and the residue was subjected to flash column chromatography on silica gel (step gradient elution, ethyl acetate/hexane, 50-100%), followed by concentration of the appropriate fractions and drying under reduced pressure overnight to give the product as a colorless syrup (0.27g, 0.91mmol, 87%).

NMR data (400MHz, 298K, CDCl)₃)：¹H, delta 2.22 and 3.00(2d, 1H, J)_OH，33.5Hz, OH major and minor isomers), 3.30(m, 1H, CHH), 3.89(m, 1H, CHH), 4.00-4.16(m, 3H, 2CHH and CH), 4.24(d, 1H, CH), 4.43 and 4.54(2brs,1H, H-3 major and minor isomers), 4.70(m, 1H, CH), 5.08-5.23(m, 2H, OCH)₂Ph)，7.32-7.40(m，5H，Ar-H).

Step iii)

To a solution of the product of step ii) (0.26g, 0.88mmol) in dichloromethane (6ml) was added Dess-Martin pentavalent iodine oxidizer (0.41g, 0.97mmol) at room temperature with stirring. The reaction was monitored by TLC (3: 2 ethyl acetate: hexane, developed with AMC staining), and after 3.5 hours the reaction mixture was diluted with dichloromethane (20ml), washed with 1: 1 saturated aqueous sodium bicarbonate/10% aqueous sodium thiosulfate (3X 20ml), then dried (sodium sulfate), filtered and concentrated. The residue was redissolved in methanol (5ml) and trimethyl orthoformate (1.25ml) and p-toluenesulfonic acid monohydrate (0.03g, 0.16mmol) was added. The reaction mixture was kept at 60 ℃ overnight, then diisopropylethylamine (0.5ml) was added and the reaction mixture was concentrated. The residue was flash chromatographed (step gradient elution, ethyl acetate/hexanes, 20-40%) then the appropriate fractions were concentrated and dried over the weekend in vacuo to give the product (0.27g, 0.79mmol, 89%) as a colorless hardened slurry.

NMR data (400MHz, 298K, CDCl)₃)：¹H，δ3.08-3.47(m，7H，2×OCH₃Major and minor isomers and CHH), 3.80(m, 2H, CH)₂)，3.98(brs，1H，CH)，4.25(m，1H，CHH)，4.45(m，1H，CH)，4.60(t，1H，CH)，5.04-5.26(m，2H，CH₂)，7.29-7.42(m，5H，Ar-H)

Comparative example 1

N- [ (S) -1- ((3aS, 6R, 6aS) -6-chloro-3-oxo) hexahydrofuro [3, 2-b ] pyrrole-4-carbonyl ] -3-methylbutyl ] -4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide

The 6-S chloro building block is prepared as shown in the following schematic diagram:

compound 10 of WO05/066180

dess-Martin pentavalent iodine oxidizer, DCM, 2h, RT;

trimethyl orthoformate, pTsOH, MeOH, 8h, 60 ℃;

iii.Pd(OH)₂，H₂，MeOH，48h，RT；

iv.Boc₂O，10％Na₂CO₃16h, and the temperature is between 0 ℃ and room temperature;

v.HCl，CH₂CH₂/Py，CB₂Cl；

vi.CH₂CH₂/Et₃N，MsCl；

vii.DMF，LiCl。

this building block was then N-deprotected and the remainder of the synthesis was completed aS described in example 5 to give N- { (S) -1- ((3aS, 6aS) -6R-chloro-3-oxo) hexahydrofuro [3, 2-b ] pyrrole-4-carbonyl } 3-methylbutyl ] -4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide.

Comparative example 2

N- [ (1S) -1- ((3aS, 6aR) -3-oxo) hexahydrofuro [3, 2-b ] pyrrole-4-carbonyl ] -3-methylbutyl ] -4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide

The P1 building block was synthesized as described in WO 02/05720, coupled with N-protected L-leucine and the P3 building block in example 3 above, and oxidized to a ketone as described in the examples.

Comparative example 3

N- [ (1S) -1- ((3aS, 6aR) -3-oxo) hexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide

The synthesis of this comparative example is illustrated in WO 05/66180 as example 9.

Biological examples

Determination of catalytic Activity of cathepsin kappa proteolysis

The experiments for cathepsin k were performed with human recombinase, e.g. the enzyme described in PDB.

ID BC016058 standard; mRNA; HUM; 1699 BP.

DE human cathepsin K (compact osteogenesis imperfecta), mRNA (cDNA clone MGC: 23107)

RX MEDLINE；RX PUBMED；12477932。

DR RZPD；IRALp962G1234。

DR SWISS-PROT；P43235；

The recombinant cathepsin can be expressed in a variety of commercially available expression systems, including the coliform, pichia and baculovirus systems. The purified enzyme is activated by removing the leader sequence by conventional methods.

Standard assay conditions for determining kinetic constants use a fluorogenic peptide substrate, usually H-D-Ala-Leu-Lys-AMC, and are determined in 100mM Mes/Tris, pH 7.0, containing 1mM EDTA and 10mM 2-mercaptoethanol, or 100mM sodium phosphate, 1mM EDTA, 0.1% PEG 4000pH 6.5, or 100mM sodium acetate, pH5, containing 5mM EDTA and 20mM cysteine, optionally containing 1M DTT as a stabilizer in each case. The enzyme concentration used was 5 nM. Substrate stock was prepared as a 10mM DMSO solution. The screening was performed at a fixed substrate concentration of 60. mu.M, and detailed kinetic studies used substrate diluted in multiples from 250. mu.M. The total DMSO concentration in the assay was kept below 3%. All tests were performed at ambient temperature. The product fluorescence (excitation at 390nm, emission at 460 nm) was monitored using a Labsystems Fluoroskan Ascent fluorescence microplate reader. A product progression curve of 15 minutes was formed after production of AMC product.

Ki determination of cathepsin S

This assay uses baculovirus-expressed human cathepsin S and boc-Val-Leu-Lys-AMC fluorogenic substrates available from Bachem in a 384 well plate format, where 7 test compounds can be tested in parallel with a positive control containing a control of known cathepsin S inhibitors.

Substrate diluent

To the B-H row in two rows of 96 deep well polypropylene plates was added 280. mu.l/well of 12.5% DMSO. Add 70. mu.l/well of substrate to row A. Add 2X 250. mu.l/well of assay buffer (100mM sodium phosphate, 100mM NaCl, pH6.5) to row A, mix, and dilute down the plate in multiples to row H.

Inhibitor diluent

To the 96 hole V bottom polypropylene plate in 4 rows of 2-5 and 7-12 add 100 u l/hole test buffer. To rows 1 and 6, 200. mu.l/well of assay buffer was added.

The first test compound, formulated in DMSO, is added to row 1 of the head, typically in a volume that provides K_iThe rough values were calculated from a previous experiment in which 10. mu.l/well of 1mM boc-VLK-AMC (10-fold dilution of a 10mM stock in DMSO into assay buffer) was added to a 96-well Microfluor^TMPlates in rows B to H, 20. mu.l/well were added to row A. To another well on row A of rows 1-10, 2. mu.l of 10mM test compound was added. To each well of row B-H, 90. mu.l of assay buffer containing 1mM DTT and 2nM cathepsin S was added, and 180. mu.l was added to row A. Mix row a with a multi-channel pipettor and dilute in multiples to row G. Row H was mixed and read in a fluorescence photometer. The readings were fitted to the competitive inhibition equation for Prism data, setting S to 100 μ M and K_M100 μ M to obtain K_iEstimates were made up to a maximum of 100. mu.M.

The second test compound is added to row 6 of the first row, the third compound is added to the first row of the second row, and so on. Add 1. mu.l of control to row 6 of the bottom row. Line 1 was mixed and diluted to line 5 by fold. Line 6 was mixed and diluted to line 10 by fold.

An 8-channel multi-channel pipette (set at 5X 10. mu.l) was used to dispense 10. mu.l of substrate per well of the 384-well assay plate. The first row of substrate dilution plates was added to all rows of the assay plates starting from row a. The tips of the multi-channel pipettor are spaced correctly for skipping rows. The second row is added to all rows beginning with row B.

Inhibitors were dispensed into 384 well assay plates at 10. mu.l per well using a 12 channel multi-channel pipette set at 4X 10. mu.l. The first alternate row of inhibitor dilution plates was dispensed into the test plate beginning at a 1. The tip spacing of the multi-channel pipettor is correctly staggered. Similarly, the second, third and fourth rows are added to the alternate rows and rows from A2, B1 and B2, respectively.

20ml of assay buffer was mixed with 20. mu.l of 1M DTT. Sufficient cathepsin S was added to reach a final concentration of 2 nM.

Using a dispenser (e.g., Multidrop 384), 30. mu.l/well is added to all wells of the assay plate and read in a fluorescence photometer (e.g., Ascent).

The fluorescence readings (excitation and emission concentrations of 390nm and 460nm, respectively, set with a pass band filter) reflect the degree of enzymatic cleavage of the fluorogenic substrate, which was fitted to the linear rate of each well regardless of the inhibitor.

For each inhibitor, the fitted rates for all wells were fitted to the competitive inhibition equation using Sigmaplot 2000 to determine V, Km and Ki values.

Ki of cathepsin L

The above procedure was modified as follows to determine Ki for cathepsin L

The enzyme is commercially available human cathepsin L (e.g., Calbiochem). The substrate was H-D-Val-Leu-Lys-AMC sold by Bahcem. The assay buffer was 100mM sodium acetate, 1mM EDTA, pH 5.5. DMSO stock (10mM solution in 100% DMSO) was diluted to 10% in assay buffer. The enzyme was formulated at 5nM concentration in assay buffer plus 1mM dithiothreitol prior to use. Mu.l of 10mM inhibitor formulated in 100% DMSO was added to the A-row wells. 10 μ l of 50 μ M substrate (10mM stock in DMSO diluted 1/200 in assay buffer).

Study of inhibition

Using the above assays, various concentrations of test compounds are used to screen for potential inhibitors. The reaction is initiated by adding the enzyme to a buffer of substrate and inhibitor. K_iValue is calculated according to equation 1

Wherein V₀Is the reaction rate, V is the maximum rate, S is the rate with the Michaelis constant K_MI is the concentration of the inhibitor.

The results are shown in

A: less than 50 nanomolar

B: 50-500 nanomolar

C: 501-1000 nanomole

D: 1001 and 5000 nanomoles

E: 5001-10000 nanomole

F: over 10000 nanomolar

TABLE 1

Examples	Ki cathepsin K	Ki cathepsin S	Ki cathepsin L
				5	A	E	C
6	A	D	C
				7	A	D	D
8	A	F	D
				10	A	D	B
12	A	F	D
				13	A	F	D

Thus, the compounds of formula II are potent inhibitors of cathepsin K and are selective inhibitors compared to the closely related cathepsins S and L.

Representative values for compounds/enzymes/assay cases from a particular batch include:

examples	Ki cathepsin K	Ki cathepsin S	Ki cathepsin L
				5	1.6	7500	880
6	1.5	4000	890
				7	4.2	1700	1200
8	3.3	13000	2900
				10	3.4	4700	490
12	1.8	11000	1600

Metabolic stability

The metabolic stability of the compounds of the present invention and the indicated control examples was tested in a cytosolic assay in which the compounds were incubated with a commercially available human liver cytosol fraction and the disappearance of the compounds was monitored by HPLC or LC/MS. The pooled human liver cytosol fraction is less likely to represent an abnormal individual than blood from a single individual, and unlike whole blood, can be stored frozen. The cytosol assay thus provides a consistent test stand as an indication of the stability of the compound in an in vivo environment (e.g. when exposed to whole blood).

Briefly, test compounds (2. mu.M) were incubated in a mixed human liver cytosol (XenotechLLC Lenexa US, 1mg/mL protein in 0.1M phosphate buffer, pH 4) for 1 hour at 37 ℃. The culture was initiated by adding 1mM NADPH cofactor. Subsamples were removed at 0, 20, 40 and 60 minute timings and "flash precipitated" by the addition of 3 volumes of ice-cold acetonitrile. The samples were centrifuged at low temperature and the supernatant separated and analyzed by LC-MS-MS.

Alternatively, a similar stability assay was performed in human or monkey whole blood.

Comparative example 3 uses the epimer of WO 0566180 with F below the P1 unit. Comparative example 2 the preferred P1 and P2 units of WO 02/057270 and P3 units within the scope of the claims of the present invention (which are not within the scope of WO 02/057270) were used. Comparative example 1 represents the epimer of the compound of example 6 with Cl below.

TABLE 2

As is apparent from the comparative examples, the P1 of prior art WO 02/057270 provides compounds having a cytosolic half-life of slightly more than 1 hour. The widely exemplified F-down P1 in WO 05/66180 is slightly better, with a half-life of more than 1 and a half hour. However, comparing comparative example 1 and comparative example 3, it can be seen that substitution of chlorine for the underlying fluorine in WO 05/66180 significantly reduces stability. In contrast, the chloro overhead epimer of the present invention (example 6) provides a compound with a half-life of more than 5 hours. Similarly, the chlorine-overhead epimer of control example 2 and the invention (example 5) clearly shows a greatly improved half-life for the chlorine-overhead epimer with the same P3 and P2 components.

Permeability of water

This example measures the transport of inhibitors across cells of the human gastrointestinal tract. The well-known Caco-2 cells with channel numbers between 40 and 60 were used in the assay.

Transport of the tip to the outside of the substrate

Typically each compound is tested in 2-4 wells. The outside of the substrate and the apical well were filled with 1.5mL and 0.4mL of Transport Buffer (TB), respectively, and the standard concentration of the assay was 10. mu.M. In addition, all test solutions and buffers contained 1% DMSO. The transfer plate was pre-coated with medium containing 10% serum for 30 minutes prior to testing to avoid non-specific binding to plastic materials. After 21-28 days of culture on the filter scaffolds, the cells were ready for permeability experiments.

Transfer plate No. 1 contains 3 rows of wells, 4 wells per row. Row 1 is indicated by "Wash", row 2 by "30 min" and row 3 by "60 min". Transfer plate No. 2 contains 3 rows of 4 wells, with row 4 being indicated as "90 min", row 5 as "120 min", and the remaining rows not being identified.

The medium in the top wells was removed and the inserts were transferred to a "wash" row (row 1) of transfer plates (plate No. 1) of 2 plates without inserts, which had been prepared with 1.5mL of transfer buffer (HBSS, 25mM HEPES, pH7.4) in rows 1-5. In the a → B screen, TB in the outer pores of the substrate also contained 1% bovine serum albumin.

0.5mL of transport buffer (HBSS, 25mM MES, pH6.5) was added to the insert and the cell monolayer was equilibrated in a Polymix shaker at 37 ℃ for 30 minutes in the transport buffer system. After equilibration with the buffer system, transepithelial resistance values (TEER) were determined using the EVOM chopstick instrument. TEER values are typically between 400 and 1000 Ω per well (depending on the number of channels used).

The transfer buffer (TB, pH6.5) was removed from the apical side, the inserts were transferred to the "30 min" row (row 2), and fresh 425. mu.L TB (pH 6.5) containing the test substance was added to the apical (donor) wells. The plates were incubated at 37 ℃ in a Polymix shaker at low shaking speed (about 150-.

After 30 minutes incubation in row 2, the insert was moved every 30 minutes into a new pre-warmed substrate lateral (receiving) well; row 3 (60 min), row 4 (90 min) and row 5 (120 min).

Samples of 25 μ L were taken from the top solution after about 2 minutes of the experiment and at the end of the experiment. These samples represent donor samples at the beginning and end of the experiment.

300. mu.L of TEER was taken out from the outer side (receiving) well of the substrate on a predetermined schedule, and the post-treatment value of TEER was measured at the end of the experiment. Acetonitrile was added to all samples collected to a total concentration of 50% in the samples. The collected samples will be stored at-20 ℃ until analyzed by HPLC or LC-MS. Substrate lateral to top transport

Typically each compound is tested in 2-4 wells. The outside and top wells of the substrate were filled with 1.55mL and 0.4mL TB, respectively, at a standard concentration of 10. mu.M for the test substance. In addition, all test solutions and buffers contained 1% DMSO. The transfer plate was pre-coated with medium containing 10% serum for 30 minutes prior to the experiment to avoid non-specific binding to plastic material.

After 21 to 28 days of culture on the filter scaffold, the cells were ready for permeability experiments. The media in the top well was removed and the inserts were transferred to a "wash" row (first row) in a new plate (transfer plate) without inserts.

The transfer plate contained 3 rows of 4 wells each. Line 1 is indicated by "wash" and line 3 is labeled "experimental line". The transport plates were prepared beforehand for 1.5mL TB (pH7.4) in the "wash" row (row 1) and for 1.55mL TB (pH7.4) including the test substance in the "test row" (row 3, donor side).

0.5mL of transfer buffer (HBSS, 25mM, pH6.5) was added to the inserts in row 1 and the cell monolayers were equilibrated for 30 min in the transfer buffer system in a Polymix shaker at 37 ℃. After equilibration with the buffer system, TEER values were determined in each well using the EVOM chopper instrument.

The transfer buffer (TB, pH6.5) was removed from the top side, the insert was transferred to row 3 and 400. mu.L of fresh TB, pH6.5, was added to the insert. After 30 minutes 250. mu.L was withdrawn from the top (receiving) well and replaced with fresh transport buffer. Then 250 μ L samples were withdrawn every 30 minutes and replaced with fresh transport buffer until the end of the experiment at 120 minutes and the final TEER post-treatment value was determined at the end of the experiment. Samples of 25 μ L were taken from the outside of the substrate (donor) chamber after about 2 minutes of the experiment and at the end of the experiment. These samples represent donor samples at the beginning and end of the experiment.

Acetonitrile was added to all samples collected to a final concentration of 50% in the samples. The collected samples were stored at-20 ℃ before analysis by HPLC or LC-MS.

Computing

Cumulative absorption fraction FA as a function of time_cumThe measurement of (1). FA_cumCalculated from the following formula:

wherein C is_RiIs to receive the chamber concentration, C, at the end of time interval i_DiIs the donor compartment concentration at the beginning of time interval i. A linear relationship should be obtained.

Permeability coefficient (P)_appCm/s) is calculated from the following formula:

where k is the transport rate (m)^-1) Defined as the cumulative absorption Fraction (FA)_cum) Slope, V, by linear regression as a function of time (min)_RIs the volume in the receiving chamber (mL), A is the area of the filter (cm)²)。

Typical reference compounds:

typical results for the compounds of the invention in this Caco-2 assay include Papp values of 5.2X 10 for the compound of example 10⁶cm/sec, 10X 10 for the compound of example 12⁶cm/sec. Compounds of formula IB containing a trifluoromethyl group, such as example 13, will generally have Papp values 2-5 times higher.

Abbreviations

DMF dimethylformamide DCM dichloromethane

TBDMS tert-butyldimethylsilyl RT Room temperature

THF tetrahydrofuran Ac acetyl

TLC thin layer chromatography DMAP dimethylaminopyridine

EtOAc ethyl acetate

All references, including patents and patent applications, cited in this application are hereby incorporated by reference to the extent possible.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not the exclusion of any other integer, step, group of integers or group of steps.

Claims

1. A compound of formula II:

wherein:

R³is H, methyl or F;

rq is CF having the stereochemistry shown₃Rq' is H; or

Rq and Rq' taken together form a keto group;

q is

Wherein

R⁴Is C₁-C₆An alkyl group;

R⁵is H, methyl or F;

R⁶is C₁-C₆An alkyl group.

2. The compound of claim 1 having formula IIa:

wherein

R³is H, methyl or F;

R⁴is C₁-C₆An alkyl group;

R⁵is H, methyl or F.

3. A compound of formula IIb or a pharmaceutically acceptable salt thereof:

wherein

R³is H, methyl or F;

q is

Wherein

R⁴Is C₁-C₆An alkyl group;

R⁵is H, methyl or F;

R⁶is C₁-C₆An alkyl group.

4. A compound according to any one of claims 1 to 3, wherein R²Is the side chain of leucine, isoleucine, O-methylthreonine, 4-fluoroleucine or 3-methoxyvaline.

5. The compound of claim 4, wherein R²Is the side chain of 4-fluoroleucine.

6. The compound of claim 4, wherein R²Is the side chain of leucine.

7. A compound according to any one of claims 1 to 3, wherein R³Is fluorine or methyl.

8. A compound according to any one of claims 1 to 3, wherein R³Is fluoro or methyl, in the meta position with respect to the benzylic amide.

9. The compound of claim 7, wherein R³Is fluorine.

10. The compound of claim 8, wherein R³Is fluorine.

11. The compound of claim 7, wherein R³Is methyl.

12. The compound of claim 8, wherein R³Is methyl.

13. A compound according to any one of claims 1 to 3, wherein R³Is H.

14. A compound according to any one of claims 1 to 3, wherein R⁵Is fluorine.

15. A compound according to any one of claims 1 to 3, wherein R⁵Is methyl.

16. A compound according to any one of claims 1 to 3, wherein R⁵Is H.

17. A compound according to any one of claims 1 to 3, wherein R⁴Is methyl.

18. The compound of claim 1, wherein R⁶Is methyl, R²Is the side chain of 4-fluoroleucine.

19. The compound of claim 1, wherein R⁶Is methyl, R²Is the side chain of leucine.

20. The compound of claim 1 selected from

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -4- [ 5-methyl-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide; or

Their pharmaceutically acceptable salts.

21. The compound of claim 1, representing:

n- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -3-fluoro-4- [2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide, or a pharmaceutically acceptable salt thereof.

22. The compound of claim 1, representing N- [1- (6-chloro-3-oxohexahydrofuro [3, 2-b ] pyrrole-4-carbonyl) -3-methylbutyl ] -4- [ 5-fluoro-2- (4-methylpiperazin-1-yl) thiazol-4-yl ] benzamide, or a pharmaceutically acceptable salt thereof.

23. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 22 and a pharmaceutically acceptable carrier or diluent therefor.

24. The use of a compound according to any one of claims 1 to 22 in the manufacture of a medicament for the treatment or prevention of diseases mediated by cathepsin K.

25. The use of claim 24, wherein the disease is selected from:

the bone substance is in the form of osteoporosis,

the disease of the gum is caused by the gum disease,

the disease of the patient with the perkin's disease,

the hypercalcemia of the malignant tumor is shown,

the bone disease of the metabolic bone is treated,

diseases characterized by excess cartilage or matrix degradation,

bone cancer, and

pain is caused.

26. The use of claim 24, wherein the disease is selected from:

gingivitis and periodontitis.

27. The use of claim 24, wherein the disease is selected from:

osteoarthritis and rheumatoid arthritis.

28. The use of claim 24, wherein the disease is selected from:

a neoplasia.