Piperidine- and piperazine substituted n-hydroxyformamides as inhibitors of metalloproteinases

专利摘要:
The present invention provides compounds of formula (I) which are useful as metalloproteinase inhibitors, in particular as inhibitors of MMP13. Formula I
公开号:KR20020073594A
申请号:KR1020027010762
申请日:2001-02-15
公开日:2002-09-27
发明作者:발람베르나르크리스토프；도웰로버트이언；핀레이마우리스레이몬드버스코일；뉴콤니콜라스존；터커하워드；워터슨데이비드
申请人:아스트라제네카 아베；
IPC主号:

专利说明:

PIPERIDINE- AND PIPERAZINE SUBSTITUTED N-HYDROXYFORMAMIDES AS INHIBITORS OF METALLOPROTEINASES As Inhibitors of Metalloproteinases
[2] The compounds of the present invention are inhibitors of one or more metalloproteinase enzymes. Metalloproteinases are the superfamily of proteinases (enzymes) that have recently increased significantly in number. Based on the structural and functional considerations, such enzymes are classified into the family and subfamily described in NM Hooper (1994) FEBS Letters 354 : 1-6. Examples of metalloproteinases are matrix metalloproteinases (MMPs) such as collagenase (MMP1, MMP8, MMP13), gelatinases (MMP2, MMP9), stromelysin (MMP3, MMP10, MMP11), Matrylicin (MMP7), metalloelase (MMP12), enamelin (MMP19), MT-MMP (MMP14, MMP15, MMP16, MMP17); Leprolysine or adamalisine or MDC, including secretases and cedases, such as TNF converting enzymes (ADAM 10 and TACE); The astaxin family comprising enzymes such as procollagen processing proteinases (PCPs); And other metalloproteinases such as agrecanase, endothelin converting enzyme family and angiotensin converting enzyme family.
[3] Metalloproteinases are believed to be critical for multiplegia of physiological disease processes involving tissue reconstitution such as embryonic development, bone formation and uterine reconstruction during menstruation. This is based on the ability of metalloproteinases to cleave a wide range of matrix substrates such as collagen, proteoglycans and fibronectin. Metalloproteinases also include the processing or secretion of biologically important mediators such as tumor necrosis factor (TNF); And post-translational proteolytic processing or shedding of biologically important membrane proteins such as low-affinity IgE receptor CD23. For a more complete list, see NM Hooper et al. (1997) Biochem. J. 321 : 265-279.
[4] Metalloproteinases are associated with many disease states. Inhibition of the activity of one or more metalloproteinases can affect these disease states, for example: various inflammatory and allergic diseases such as inflammation of the joints (especially rheumatoid arthritis, osteoarthritis and gout), inflammation of the gastrointestinal tract (especially inflammatory Intestinal diseases, ulcerative appendicitis and gastritis), inflammation of the skin (especially psoriasis, eczema, dermatitis); Tumor metastasis or invasion; Diseases associated with unregulated degeneration of the extracellular matrix such as osteoarthritis; Bone resorption diseases (eg, osteoporosis and Paget's disease); Diseases associated with modified angiogenesis; Enhanced collagen reconstitution associated with diabetes mellitus, periodontal disease (eg, gingivitis), corneal ulcers, skin ulcers, postoperative conditions (eg colon anastomosis) and skin wound healing; Dehydration diseases of the central and peripheral nervous systems (eg, multiple sclerosis); Alzheimer's disease; Extracellular matrix reconstitutions observed in cardiovascular diseases such as restenosis and atherosclerosis; And the role of MMPs such as chronic obstructive pulmonary disease, COPD (eg, MMP12, are discussed in Anderson and Shinagawa, 1999, Current Opinion in Anti-inflammatory and Immunomodulatory Investigational Drugs , 1 (1) : 29-38 ) . } Can have many advantages.
[5] Many metalloproteinase inhibitors are known and different classes of compounds can have different degrees of efficacy and selectivity in inhibiting various metalloproteinases. We have discovered a new class of compounds that are inhibitors of metalloproteinases and are of particular interest in inhibiting MMP-13, as well as MMP-9. Compounds of the invention have beneficial efficacy and / or pharmacokinetic properties.
[6] Initially, MMP13 or collagenase 3 was cloned from a cDNA library derived from breast tumors (JMP Freije et al. (1994) Journal of Biological Chemistry 269 (24) : 16766-16773). PCR-RNA analysis of RNA from a wide range of tissues indicates that MMP13 expression is breast fibroadenoma, normal or resting breast gland, placenta, liver, ovary, uterus, prostate or parotid gland, or breast cancer cell lines (T47-D, MCF-7 and ZR75). -1), which is not found, indicating that it is limited to breast carcinoma. According to this observation, MMP13 was transformed into epidermal keratinocytes [N. Johansson et al. (1997) Cell Growth Differ . 8 (2) : 243-250], platelet cell sarcoma [N. Johansson et al., (1997) Am. J. Pathol. 151 (2) : 499-508] and epidermal tumors [K. Airola et al., (1997) J. Invest. Dermatol. 109 (2) : 225-231. These results suggest that MMP13 is secreted by transformed epithelial cells and may involve extracellular matrix degradation and cell-matrix interactions associated with metastasis as observed in malignant epithelial growth, particularly in invasive breast cancer lesions and skin carcinogenesis. Suggests that.
[7] Recent distributed data suggest that MMP13 plays a role in the replacement of other connective tissues. For example, consistent with the substrate specificity and preference of MMP13 for degradation of type II collagen [PG Mitchell et al. , (1996) J. Clin. Invest. 97 (3) : 761-768; V. Knauper et al., (1996) The Biochemical Journal 271 : 1544-1550], MMP13 was found during primary ossification and bone reconstitution [M. Stahle-Backdahl et al., (1997) Lab. Invest. 76 (5) : 717-728; N. Johansson et al. (1997) Dev. Dyn. 208 (3) : 387-397], in degenerative joint diseases such as rheumatoid arthritis and osteoarthritis [D. Wernicke et al., (1996) J. Rheumatol. 23 : 590-595; PG Mitchell et al. (1996) J. Clin. Invest. 97 (3) : 761-768; O. Lindy et al., (1997) Arthritis Rheum 40 (8) : 1391-1399; And during aseptic loosening of the hip abdomen [S. Imari et al. (1998) J. Bone Joint Surg. Br. 80 (4) : 701-710]. MMP13 is also associated with chronic adult periodontitis because it is localized to the epithelium of chronically inflamed mucosal human gingival tissue [VJ Uitto et al., (1998) Am. J. Pathol 152 (6) : 1489-1499], involved in the reconstitution of the collagen matrix in chronic wound sites [M. Vaalamo et al., (1997) J. Invest. Dermatol. 109 (1) : 96-101].
[8] MMP9 (gelatinase B; 92 kDa type IV collagenase; 92 kDa gelatinase) is a cloned, sequenced secreted protein that was purified for the first time in 1989 [SM Wilhelm et al., (1989) J. Biol. Chem. 264 (29) : 17213-17221; J. Biol. Chem. (1990) 265 (36) : issued corrected to 22570]. Recent studies of MMP9 provide an excellent source for details and references to this protease: TH Vu and Z. Werb (1998) (In: Matrix Metalloproteinases. 1998. Edited by WC Parks and RP Mecham. Pp. 115-148 Academic Press.ISBN 0-12-545090-7). The following section is taken from TH Vu and Z. Werb (1998), supra.
[9] Usually, expression of MMP9 is limited to several cell types, including feeder cells, osteoclasts, neutrophils and macrophages. However, its expression can be induced in these same cells and other cell types by several mediators, including exposing the cells to growth factors or cytokines. These are often the same mediators involved in initiating an inflammatory response. As with other secreted MMPs, MMP9 is released as an inactive proenzyme that is later cleaved to form enzymatically active enzymes. The proteases required for this activity in vivo are unknown. The balance of active MMP9 versus inactive enzymes is further regulated in vivo by interaction with the native protein TIMP-1 (tissue inhibitor of metalloprotease-1). TIMP-1 binds to the C-terminal region of MMP9 resulting in inhibition of the catalytic domain of MMP9. The balance of induced expression of proMMP9, cleavage from proMMP to active MMP9, and the presence of TIMP-1 are determined to determine the amount of catalytically active MMP9 present at the local site. Proteolytically active MMP9 attacks gelatin, elastin and substrates comprising native type IV and type V collagen, which has no activity against native type I collagen, proteoglycans or laminin.
[10] There is a growing body of data suggesting a role for MMP9 in various physiological and pathological processes. Physiological roles include invasion of embryonic feeders through the uterine epithelium in the early stages of embryo implantation; Role in bone growth and development; And migration of inflammatory cells from pulses to tissues. Since increased MMP9 expression is observed in certain pathological conditions, MMP9 is involved in advanced diseases such as arthritis, tumor metastasis, Alzheimer's disease, multiple sclerosis, and plaque rupture in atherosclerosis resulting in acute coronary conditions such as myocardial infarction. Suggests that there is.
[11] WO 99/38843 relates to compounds of the formulas for use in the manufacture of a medicament for the treatment or prevention of conditions associated with matrix metalloproteinases:
[12] BX- (CH ₂ ) _m- (CR ¹ R ² ) _n -W-COY
[13] In particular, the compound N- {1S- [4- (4-chlorophenyl) piperazine-1-sulfonylmethyl] -2-methylpropyl} -N-hydroxyformamide is disclosed.
[1] The present invention relates to compounds useful for inhibitors of metalloproteinases, in particular pharmaceutical compositions comprising them, as well as their use.
[14] We have now found compounds that are potent MMP13 inhibitors and possess desirable activity profiles.
[15] In a first aspect of the invention, we provide a compound of formula (I)
[16]
[17] Wherein B represents a phenyl group monosubstituted with halogen or trifluoromethyl at position 3 or 4 or a phenyl group disubstituted with halogen (which may be the same or different) at position 3 or 4; Or B represents a 2-pyridyl group or 2-pyridyloxy group monosubstituted with halogen, trifluoromethyl, cyano or C _1-4 alkyl at positions 4, 5 or 6; Or B represents a 4-pyrimidinyl group, optionally substituted at position 6 with halogen or C _1-4 alkyl;
[18] X represents a carbon or nitrogen atom;
[19] R 1 is a trimethyl-1-hydantoin C _2-4 alkyl group or a trimethyl-3-hydantoin C _2-4 alkyl group; Phenyl or C _2-4 alkylphenyl monosubstituted with halogen, trifluoromethyl, thio or C _1-3 alkyl or C _1-3 alkoxy at position 3 or 4; Phenyl-SO ₂ NHC _2-4 alkyl; 2-pyridyl or 2-pyridyl C _2-4 alkyl; 3-pyridyl or 3-pyridyl C _2-4 alkyl; 2-pyrimidine-SCH ₂ CH ₂ ; Optionally monosubstituted with one of halogen, trifluoromethyl, C _1-3 alkyl, C _1-3 alkyloxy, 2-pyrazinyl optionally substituted with halogen or 2-pyrazinyl C _2-4 alkyl optionally substituted with halogen 2- or 4-pyrimidinyl C _2-4 alkyl;
[20] Any of the alkyl groups described above may be straight or branched chain.
[21] Preferred compounds of the invention are those to which any one or more of the following applies:
[22] B is 4-chlorophenyl, 4-fluorophenyl, 4-bromophenyl or 4-trifluorophenyl; 2-pyridyl or 2-pyridyloxy monosubstituted at the 4 or 5 position, such as 5-chloro-2-pyridyl, 5-bromo-2-pyridyl, 5-fluoro-2-pyri Dill, 5-trifluoromethyl-2-pyridyl, 5-cyano-2-pyridyl, 5-methyl-2-pyridyl; In particular, 4-fluorophenyl, 5-chloro-2-pyridyl or 5-trifluoromethyl-2-pyridyl;
[23] X represents a nitrogen atom;
[24] R 1 is 3-chlorophenyl, 4-chlorophenyl, 3-pyridyl, 2-pyridylpropyl, 2- or 4-pyrimidinylethyl (optionally monosubstituted with fluorine), 2- or 4-pyrimidinylpropyl , 2- (2-pyrimidinyl) propyl (optionally monosubstituted with fluorine); In particular, 2-pyrimidinylpropyl, 2- (2-pyrimidinyl) propyl (optionally monosubstituted with fluorine) or 5-fluoro-2-pyrimidinylethyl.
[25] For the compounds of formula (I), certain subgroups may be phenyl groups in which B is monosubstituted with halogen or trifluoromethyl at position 3 or 4, or halogen (same or different) at positions 3 and 4 Phenyl group disubstituted; Or B is a 2-pyridyl group or a 2-pyridyloxy group monosubstituted with halogen, trifluoromethyl or cyano at the 5th or 6th position; Or a 4-pyrimidinyl group optionally substituted with halogen or C _1-4 alkyl at position 6; X is a carbon or nitrogen atom; R 1 is C _2-4 alkyl or trimethyl-3-hydantoin C _2-4 alkyl group; Or R 1 is phenyl or C _2-4 alkylphenyl monosubstituted with halogen, trifluoromethyl, thio or C _1-3 alkyl or C _1-3 alkoxy at the 3 or 4 position; Or R 1 is phenyl-SO ₂ NHC _2-4 alkyl; Or R 1 is 2-pyridyl or 2-pyridyl C _2-4 alkyl; Or R 1 is 3-pyridyl or 3-pyridyl C _2-4 alkyl; Or R 1 is 2-pyrimidine-SCH ₂ CH ₂ ; Or 2- or 4-pyrimi, wherein R ₁ is optionally monosubstituted with one of halogen, trifluoromethyl, C _1-3 alkyl, C _1-3 alkyloxy, 2-pyrazinyl or 2-pyrazinyl C _2-4 alkyl Dinyl C _2-4 alkyl; Any alkyl group is represented by a compound which may be straight or branched.
[26] It should be appreciated that the specific substituents on B and / or R1 and the number of substituents are chosen to avoid steric undesirable combinations.
[27] Each illustrated compound represents a specific and independent aspect of the invention.
[28] When optically active centers are present in the compounds of formula (I), the inventors disclose all individual optically active forms and combinations thereof, as well as their corresponding racemates, as individual specific embodiments of the invention. Racemates are known procedures, including, for example, formation of diastereomeric derivatives with easy optically active co-species, followed by separation and cleavage of the co-species (Advanced Organic Chemistry: Third Edition). : Author J. March, p 104-107), can be isolated into individual optically active forms.
[29] It should be appreciated that the compounds according to the invention may contain one or more asymmetric substituted carbon atoms. The presence of one or more such asymmetric centers (chiral centers) in the compounds of formula (I) results in stereoisomers, in which case the invention includes mixtures including enantiomers and diastereomers, and racemic mixtures thereof It should be understood that it extends to all such stereoisomers.
[30] In the examples, we disclose the isolation and characterization of certain enantiomers. Enantiomers can be prepared by reacting a racemic material with a chiral aid, separating the diastereomers formed using chromatography, and then cleaving the chiral aid. After eluting second from the column (using the conditions described herein), the cleaved diastereomers provide more enantiomeric isomers than tested. In each case, we believe that the active enantiomers have S stereochemistry, but do not wish to be limited to these initial considerations. The active enantiomer is characterized in that the derivative is eluted second from the separation column. The use of different compounds of formula (I), alternative columns and / or different solvents can affect the elution order of the most active enantiomers.
[31] In the examples, we disclose the isolation and characterization of certain diastereomers. Chromatographic separation and subsequent testing revealed that the more active diastereomer elutes first from the separation column (ie, the more active diastereomer is eluted first from the separation column). Different compounds of formula (I), alternative columns and / or different solvents can affect the elution order of the most active diastereomers.
[32] In the case of compounds of formula (I) having two chiral centers, we believe that active enantiomers have S, S stereochemistry, but do not wish to be limited to this initial consideration.
[33] When tautomers are present in a compound of formula (I), we disclose all individual tautomer forms and combinations thereof as individual specific embodiments of the invention.
[34] As mentioned above, the compounds of the present invention are metalloproteinase inhibitors, in particular they are inhibitors of MMP13. Each of the above symbols for the compounds of formula (I) represent independent and specific embodiments of the invention. While not wishing to be bound by theoretical considerations, it is believed that the compounds of the present invention exhibit selective inhibition of any of the above symbols relative to any MMP1 inhibitory activity, which, by way of non-limiting example, is intended to provide any MMP1 inhibitory activity. Compared to 100 to 1000 times selectivity can be shown.
[35] Certain compounds of the invention are particularly useful as agrecanases, i.e. inhibitors of aggrecan degradation. Certain compounds of the invention are particularly useful as inhibitors of MMP9 and / or MMP12.
[36] The compounds of the present invention may be provided as pharmaceutically acceptable salts. These include acid addition salts such as hydrochloride, bromate, citrate and maleate and salts formed with phosphoric acid and sulfuric acid. In other embodiments, suitable salts are alkali metal salts such as sodium or potassium, alkaline earth metal salts such as calcium or magnesium, or base salts such as organic amines such as triethylamine.
[37] They may also be provided as hydrolyzable esters in vivo. These are pharmaceutically acceptable esters that hydrolyze in the human body to produce the parent compound. Such esters can be identified by examining the fluid of the test animal after intravenous administration of the compound under test, for example, to the test animal. Suitable in vivo hydrolyzable esters for carboxy include methoxymethyl and for hydroxy formyl and acetyl, in particular acetyl.
[38] For the use of a compound of formula (I) or a pharmaceutically acceptable salt or in vivo hydrolyzable ester for therapeutic treatment (including prophylactic treatment) of mammals, including humans, this is usually standard pharmaceutical as a pharmaceutical composition. It is prepared according to the implementation.
[39] Therefore, in another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt or hydrolyzable ester in vivo and a pharmaceutically acceptable carrier.
[40] The pharmaceutical compositions of the invention may be administered in a standard manner for the disease state to be treated, such as oral, topical, parenteral, buccal, intranasal, intravaginal or enteral or aeration. For this purpose, the compounds of the invention can be prepared by means known in the art, for example, tablets, capsules, aqueous solutions, oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, fine powders or kegs For airborne aerosols and parenteral use (including intravenous, intramuscular or infusion), it may be formulated in the form of sterile aqueous or oily solutions or suspensions or sterile emulsions.
[41] In addition to the compounds of the present invention, the pharmaceutical compositions of the present invention may contain or co-administer (simultaneously or sequentially) one or more pharmaceutical agents useful for treating one or more of the aforementioned disease states.
[42] Usually, the pharmaceutical composition of the present invention is administered to a human to receive 0.5 to 75 mg / kg body weight (preferably 0.5 to 30 mg / kg body weight) per day. Such daily dosages can be given in divided doses as needed, the exact amount of compound received and the route of administration depend on the weight, age and gender of the patient to be treated, and according to principles known in the art. Depends on the specific disease state.
[43] Typically, unit dosage forms contain about 1 mg to 500 mg of a compound of the present invention.
[44] Therefore, in another aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof for use in a therapeutic treatment method of a human or animal body. In particular, the inventors disclose the use in the treatment of diseases or conditions mediated by MMP13 and / or agrecanase and / or MMP9 and / or MMP12.
[45] In another aspect, the invention provides a method for treating a metalloproteinase mediated disease comprising administering to a warm blooded animal a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof or a hydrolyzable ester in vivo. Provide a method of treatment. Metalloproteinase mediated diseases include arthritis (eg, osteoarthritis), atherosclerosis, chronic obstructive pulmonary disease (COPD).
[46] In another aspect, the present invention provides a process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof or a hydrolyzable ester in vivo, wherein the process comprises a compound of formula (II) By reacting with an appropriate compound to obtain an alkene of formula III and then converting it to a compound of formula IV, which is a precursor of formula I, optionally followed by a pharmaceutically acceptable salt or in vivo hydrolyzable ester of a compound of formula I Forming.
[47]
[48] Compounds of formula (II) are readily prepared by reacting a compound of formula (V) with a compound of formula (VI), wherein B 'is a precursor of B, and X' is an X or a precursor of X or X's suitable for reaction with B ' Indicates an activated form. Compound II may also be prepared from Compound VII, as shown below.
[49]
[50] It should be recognized that most of the relevant starting materials are commercially available. In addition, the following table shows the details of the aldehyde intermediates and their corresponding Chemical Abstracts registration numbers.
[51] RCHO Chemical abstract 3- (2-pyrimidinylthio) propionaldehyde 155957-56-5 3- (2-pyrazinyl) butyraldehyde 177615-94-0 3-phenylsulfonylamidopropanal 57483-28-0 4- (4-methoxyphenyl) butyraldehyde 160093-24-3 4- (3-methoxyphenyl) butyraldehyde 113504-55-5
[52] Aldehyde without chemical abstract registration number
[53] 3- (2-pyrimidyl) propionaldehyde. To a solution of 2-bromopyrimidine (7.95 g, 0.05 M) in acetonitrile (150 mL) propargyl alcohol (4.2 g, 0.075 M), bis (triphenylphosphine) palladium (II) chloride (750 mg , 1 mM), copper iodide (100 mg, 0.5 mM) and triethylamine (25 mL, 0.25 M) were added and the mixture was stirred at 70 ° C. for 2 hours and heated. An additional amount of propargyl alcohol (2.1 g, 0.038 M), bis (triphenylphosphine) palladium (II) chloride (375 mg, 0.5 mil) and copper iodide (50 mg, 0.25 mM) were then added to the reaction mixture. Added, stirred at 70 ° C. for 1 hour, and heated.
[54] The reaction mixture was evaporated to dryness and the residue which was preabsorbed on silica was chromatographed. Elution with ethyl acetate gave 4.45 g (66%) of 3- (2-pyrimidyl) prop-2-yn-3-ol as a yellow solid. NMR (CDCl ₃ ) 2.9 (1H, t), 4.5 (2H, d), 7.3 (1H, d), 8.8 (2H, t), MS found MH ⁺ 135.
[55] 3- (2-pyrimidyl) prop-2-in-1-ol (4.45 g. 0.033 M) was dissolved in ethyl acetate (140 mL) and 10% Pd / C (890 mg) was added and the mixture Was stirred for 6 h under hydrogen atmosphere. The reaction mixture was passed through celite and the filtrate was evaporated to give 4.15 g (91%) of 3- (2-pyrimidyl) propan-1-ol as a yellow oil. NMR (CDCl ₃ ) 2.1 (2H, m), 3.2 (2H, t), 3.8 (2H, t), 7.2 (1H, t), 8.7 (2H, d) MS found MH ⁺ 139.
[56] 3- (2-pyrimidyl) propan-1-ol was oxidized using the following resource conditions to obtain 3- (2-pyrimidyl) propionaldehyde. DMSO (21.3 mL) was added to oxalyl chloride (14.3 mL) dissolved in dichloromethane (700 mL), keeping the temperature below -60 ° C. After 15 minutes, alcohol (20.8 g) dissolved in dichloromethane (20 mL) was slowly added and after 30 minutes triethylamine (125 mL) was added. After 15 minutes, the reaction mixture was warmed to room temperature with addition of water (100 mL). The solvent was separated and the organic layer was washed with water (3 × 150 mL), dried (MgSO ₄ ), evaporated to an oil, purified by flash column chromatography eluting with ethyl acetate / methanol (5%). To give the product (8.71 g, 43%) as an oil. NMR (CDCl ₃ ) 3.0 (2H, t), 3.4 (2H, t), 7.1 (1H, t), 8.7 (2H, d), 9.9 (1H, s).
[57] The following aldehydes were prepared using the procedure described above:
[58] 3-butyn-1-ol was used in place of 4- (2-pyrimidyl) butyraldehyde propargyl alcohol. NMR CDCl ₃ 9.8 (1H, s), 8.6 (2H, m), 7.15 (1H, m), 3.0 (2H, m), 2.5 (2H, m), 2.2 (2H, m).
[59] 2-bromopyrazine was used instead of 3- (2-pyrazinyl) propionaldehyde 2-bromopyrimidine. NMR (d ₆ -DMSO) 9.77 (s, 1H), 8.61 (d, 1H), 8.54 (dd, 1H), 8.46 (d, 1H), 3.10 (t, 2H), 2.92 (t, 2H).
[60] 2-bromopyrazine was used in place of 4- (2-pyrazinyl) butyraldehyde 2-bromopyrimidine and 3-butyn-1-ol was used in place of propargyl alcohol. NMR (d ₆ -DMSO) 9.68 (s, 1H), 8.56 (m, 2H), 8.49 (m, 1H), 2.80 (t, 2H), 2.5 (m, 2H), 1.96 (m, 2H).
[61] 2-chloro-4-trifluoropyrimidine [CAS Registry No. 33034-67-2] was used instead of 4- (4-trifluoromethylpyrimidin-2-yl) butanal 2-bromopyrimidine 3-butyno-1-ol was used instead of propargyl alcohol. ¹ H NMR (CDCl ₃ ): 9.80 (s, 1 H), 8.92 (d, 1 H, J = 5.0 Hz), 7.47 (d, 1H, J = 5.0 Hz), 3.11 (dd, 2H, J = 7.5, 7.5 Hz), 2.60 (dd, 2H, J = 6.1, 6.1 Hz), 2.21 (m, 3H).
[62] 2-chloro-5-fluoro-pyrimidine [CAS Registry No. 62802-42-0] was used instead of 4- (5-fluoropyrimidin-2-yl) butanal 2-bromopyrimidine, 3-butynio-1-ol was used in place of pargill alcohol. ¹ H NMR (CDCl ₃ ): 9.90 (s, 1 H), 8.52 (s, 2H, J = 5.0 Hz), 7.47, 3.47 (m, 2H), 3.33 (dd, 2H, J = 6.8, 6.8 Hz), 3.02 (m, 2 H).
[63] 2-chloro-4-methoxypyrimidine [CAS Registry No. 22536-63-6] was used instead of 4- (4-methoxypyrimidin-2-yl) butanal 2-bromopyrimidine and propar 3-butynio-1-ol was used instead of gil alcohol. ¹ H NMR (CDCl ₃ ): 9.80 (s, 1 H), 8.34 (d, 1 H, J = 5.0 Hz), 6.55 (d, 1H, J = 5.0 Hz), 3.97 (s, 3H), 2.91 (dd, 2H, J = 6.8, 6.8 Hz), 2.58 (m, 2H), 2.20 (m, 2H).
[64] 2-chloro-5-ethyl-pyrimidine [CAS Registry No. l11196-81-7] was used instead of 4- (5-ethylpyrimidin-2-yl) butanal 2-bromopyrimidine and propargyl 3-butyno-1-ol was used instead of alcohol. ¹ H NNR (CDCl ₃ ): 9.79 (s, 1H), 8.51 (s, 2H), 2.99 (dd, 2H, J = 7.4, 7.4 Hz), 2.54 (m, 4H), 2.17 (p, 1H, J = 7.4 Hz), 1.04 (t, 2H, J = 7.2 Hz).
[65] 5- (2-pyrimidyl) pentanal 2-bromopyrimidine and 4-pentyn-1-ol were used in place of propargyl alcohol: NMR (CDCl ₃ ) 9.8 (1H, s), 8.65 (2H , m), 7.1 (1H, m), 3.0 (2H, m), 2.5 (2H, m), 1.9 (2H, m), 1.7 (2H, m).
[66] 2-iodo-5-bromopyrimidine was used in place of 3- (5-bromopyrimidin-2-yl) propanal 2-bromopyrimidine. ¹ H NMR (CDCl ₃ ): 9.90 (s, 1 H), 8.70 (s, 2H), 3.30 (dd, 2H), 3.0 (dd, 2H).
[67] 4- (4-pyrimidyl) butan-1-al. 2,4-dichloropyrimidine (4.47 g, 0.03 M) was dissolved in triethylamine (250 mL) under argon. (Ph ₃ P) ₂ PdCl ₂ (420 mg, 0.006 M), CuI (28 mg, 0.00015 M) and 3-butyn-1-ol (2.36 mL, 0.03 M) were added and the mixture was stirred at ambient temperature for 18 hours. It was. After evaporation to dryness, water (250 mL) was added and extracted with dichloromethane. The combined organic phases were dried and evaporated to dryness. The residual oil was chromatographed eluting with isohexane / ethyl acetate 1: 1 to afford 4- (2-chloro-4-pyrimidyl) -3-butyn-1-ol (3.3 g) as an oil. NMR (CDCl ₃ ) d 8.5, (d 1 H); 7.3, (d 1 H); 3.9, (t 2 H); 2.8, (m 2 H); 1.6, (s 1 H). Mass spectrum found MH + 183. This material was hydrogenated as described above, but in the presence of 1 equivalent of triethylamine to afford the required saturated alcohol, which was oxidized using the abovementioned one-way oxidation to yield the required 4- (4-pyrimidyl). Butane-1-al was obtained. NMR CDCl ₃ d 9.8, (s 1H); 9.1; (s 1H); 8.5, (d 1 H); 7.1, (d 1 H); 2.8, (t 2 H); 2.5, (t 2 H); 2.1, (m 2 H). Mass spectrum found MH-149.
[68] 3-5-fluoropyrimidin-2-yl) propanal. (E) -1-Ethoxy-3- (5-fluoropyrimidin-2-yl) prop-2-enyl ethyl ether and (Z) -1- in anhydrous ethanol (100 mL) at argon atmosphere at room temperature To a stirred solution of ethoxy-3- (5-fluoropyrimidin-2-yl) prop-2-enyl ethyl ether (9.7 g, 43 mmol) was added 10% palladium (1.O g) on activated charcoal. It was. The reaction flask was then emptied and filled with hydrogen gas. Then the mixture was stirred for 18 hours at room temperature. The reaction was then filtered through a pad of celite and evaporated under reduced pressure to yield a yellow oil (8.7 g, 89%). To a solution of this oil (15 g, 66 mmol) in THF (200 mL) at room temperature was added an aqueous hydrochloric acid solution (36 mL of 2 M solution, 72 mmol) and the reaction was stirred at room temperature for 3 hours. Then diluted with ethyl acetate (100 mL) and aqueous sodium bicarbonate solution (saturated, 100 mL) was added to bring the pH of the mixture to pH = 9. The layers were then separated and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The combined organic extracts were then dried (Na ₂ SO ₄ ), filtered and evaporated under reduced pressure to afford 3- (5-fluoropyrimidin-2-yl) propanal (16 g) which was no longer purified. Used without. ¹ H NMR (CDCl ₃ ): 9.90 (s, 1 H), 8.50 (s, 2 H), 3.33 (dd, 2H, J = 6.9, 6.9 Hz), 3.00 (dd, 2H, J = 6.9, 6.9 Hz).
[69] The starting material was obtained by the following method: 2-chloro-5-fluoropyrimidine [CAS Registry No. 62802-42-0] (9.0 g, 68 mmol) and 1-tree in anhydrous DMF (140 mL) under argon atmosphere. Solid potassium carbonate (9.4 g, 68 mmol), tetraethylammonium chloride in a solution of butylstanyl-3,3-diethoxyprop-1-ene (42.8 g, 102 mmol, E: Z isomer 5: 1 mixture) (11.2 g, 68 mmol) and bis (triphenylphosphine) palladium (II) chloride (2.4 g, 3.4 mmol) were added sequentially. The resulting mixture was then heated to 120 ° C. for 3 hours. The reaction was then cooled to room temperature and diluted with water (100 mL) and diethyl ether (150 mL). This mixture was then filtered through a pad of celite. The layers were separated and the aqueous phase was extracted with diethyl ether (3 x 100 mL). The combined organic extracts were then dried (MgSO ₄ ), filtered and evaporated under reduced pressure. Flash chromatography (silica gel, 10% ethyl acetate in hexanes) then gave the product (9.7 g, 63%) as a pale yellow oil and E: Z isomer 3: 1 mixture.
[70] E-isomer: ¹ H NMR (CDCl ₃ ): 8.53 (s, 2H), 6.99 (dd, 1H, J = 15.4, 4.1 Hz), 6.86 (d, 1H, J = 15.4 Hz), 5.14 (d, 1H , J = 4.1 Hz), 3.56 (m, 4H), 1.24 (t, 6H, J = 7.1 Hz)
[71] Z-isomer: ¹ H NMR (CDCl ₃ ): 8.57 (s, 2H), 6.65 (d, 1H, J = 12.1 Hz), 6.49 (d, 1H, J = 7.5 Hz), 6.09 (dd, 1H, J = 12.1, 7.5 Hz), 3.70 (m, 4H), 1.21 (t, 6H, J = 7.1 Hz)
[72] The following aldehydes were prepared using appropriately substituted 2-chloropyrimidine using a similar method:
[73] 3- (4-methoxyoxypyrimidin-2-yl) propanal ¹ H NMR (CDCl ₃ ): 9.82 (s, 1 H), 8.34 (d, 1 H, J = 8.4 Hz), 6.55 (d, 1H, J = 7,4 Hz), 3.91 (s, 3H), 3.28 (dd, 2H, J = 7.4, 7.4 Hz). 2.99 (dd, 2H, J = 7.4, 7.4 HZ).
[74] 3- (4-trifluoromethylpyrimidin-2-yl) propanal ¹ H NMR (CDCl ₃ ): 9.92 (s, 1 H), 8.90 (d, 1 H, J = 5.0 Hz), 7.47 (d, 1 H) , J = 5.0 Hz), 3.43 (dd, 2H, J = 6.8, 6.8 Hz), 3.07 (dd, 2H, J = 6.8, 6.8 Hz).
[75] 3- (5-ethylpyrimidin-2-yl) propanal ¹ H NMR (CDCl ₃ ): 9.91 (s, 1 H), 8.49 (s, 2H), 3.31 (dd, 2H, J = 6.9, 6.9 Hz) , 2.98 (dd, 2H, J = 6.9, 6.9 Hz), 2.61 (q, 2H, J = 7.6 Hz), 1.26 (t, 3H, J = 7.6 Hz).
[76] 3,5,5-trimethyl-1-propane hydantoin
[77]
[78] 3,5,5-trimethyl hydantoin [CAS (6345-19-3)] (3.5 g, 0.025 mol), 2- (2-bromoethyl) -1,3-dioxolane in MeCN (100 mL) 4.8 mL: 0.041 mol), K ₂ CO ₃ (8.5 g, 0.062 mol), benzyltrimethylammonium chloride (2.23 g, 0.012 mol) were refluxed together for 24 hours. The reaction was cooled to rt, filtered and the filtrate was evaporated in vacuo. The residue was dissolved in DCM and then washed with water (X3) and then evaporated in vacuo. The residue was azeotropic with toluene (X3) to give a yellow oil (5.4 g). The oil was then stirred in THF (30 mL) with concentrated HCl (4 mL) at room temperature for 20 hours. Neutralized with aqueous NaHCO ₃ and extracted with DCM (X8). The combined organics were dried over Na ₂ SO ₄ and evaporated in vacuo to give a yellow oil (4.3 g). ¹ H NMR (CDCl ₃ ): 9.82 (s, 1 H), 3.62 (t, 2H), 3.04 (s, 3H), 2.90 (m, 2H), 1.37 (s, 6H).
[79] 1,5,5-trimethyl-3-propanal hydantoin
[80]
[81] 1,5,5-trimethylhydantoin [CAS (685l-81-6)] (5.0 g, 35.0 mol) was added to a mixture of NaOEt (0.02 g, 0.298 mmol, catalytic amount) and EtOH (8 mL) and under argon Stirred. After the mixture was warmed to 300 ° C., acrolein (2.35 mL) was added slowly and the reaction exothermed to 45 ° C. The reaction was cooled to rt and stirred for 2 h more. AcOH (0.136 mL, 2.4 mmol) and silica gel (3.5 g) were added to the mixture and then evaporated in vacuo. The product on silica was chromatographed on a silica column (eluent: 5% acetone / DCM) to give a clear oil (6.2 g). The residue was further purified on alumina (eluent: DCM) to give a clear oil (2.7 g). ¹ H NMR (CDCl ₃ ): 9.78 (s, 1 H), 3.88 (t, 2H), 2.86 (s, 3H), 2.82 (m, 2H), 1.37 (s, 6H).
[82] 1,5,5-trimethyl-3-butanal hydantoin was prepared in a similar manner. [M + H 213].
[83] 3- (3-chlorophenyl) butyraldehyde. 3-chloroiodobenzene (2.38 g), palladium acetate (20 mg), sodium bicarbonate (1.01 g) and crotyl alcohol (1.28 mL) in N-methylpyrrolidone (4 mL) were stirred and at 130 ° C Heated for 2 hours. The reaction mixture was cooled, water (50 mL) was added, and the mixed radish was extracted with diethyl ether (2 X 50 mL). The combined organic extracts were dried and the residue obtained upon removal of solvent was purified by chromatography through silica, eluting with a mixture of ethyl acetate and methylene chloride (1:20) to afford the title compound as an oil. Yield 519 mg, MH = 181
[84] 3- (2-pyridyl) butyraldehyde. Prepared by the one-way oxidation of the alcohol (CAS 90642-86-7).
[85] 3- (5-fluoropyrimidin) -2-yl-butanal
[86]
[87] Concentrated hydrochloric acid (1 mL) was added to 2- [2- (1,3-dioxolan-2-yl) -1-methylethyl] -5-fluoropyrimidine (1.1 g) in tetrahydrofuran (10 mL) at room temperature. ) Was stirred, stirred for 3 hours, and then sodium hydrogen carbonate was added to neutral pH. The mixture was poured into a Chemelute cartridge, washed with ethyl acetate (3 x 20 mL), the combined organics were dried over NaSO ₄ and vacuum dried to 3- (5-fluoropyrimidin-2-yl) butane Eggs (300 mg, 35%) were obtained and used without further purification.
[88] Starting materials were prepared as follows:
[89] 2- [2- (1,3-dioxolan-2-yl) -1-methylethyl] -5-fluoropyrimidine
[90]
[91] To a stirred suspension of activated "Rieke" zinc in tetrahydrofuran (21 mg, 1.53 M), 2- (2-bromopropyl) -1,3-dioxolane (6.6) in tetrahydrofuran (50 mL) g) was added and a temperature rise from 21 ° C. to 40 ° C. was observed, heated to 40 ° C. for 1 h, then cooled to room temperature, followed by 2-chloro-5-fluoropyrimidine (3 g) and [1 , 2-bis (diphenylphosphino) propane] dichloronickel (II) chloride (368 mg) was added. The mixture was stirred at room temperature for 4 hours, then filtered through a pad of celite and the lysate was evaporated under reduced pressure. Then flash chromatography (silica gel, 25% ethyl acetate in hexanes-hexanes) gave the product (1.1 g) as a pale yellow oil; ¹ H NMR (d ₆ -DMSO): 8.81 (s, 2H), 4.73 (dd, 1H), 3.66-3.87 (m, 4H), 3.21-3.30 (m, 1H), 2.19 (ddd, 1H), 1.83 (ddd, 1 H), 1.27 (d, 3 H); m / z 213 (M + l).
[92] 2- (2-bromopropyl) -1,3-dioxolane
[93]
[94] Crotonaldehyde (9.18 g, 108 mmol) was added dropwise to a stirred solution of bromotriethylsilane (24 g, 156 mmol) at 0 ° C., stirred at 0 ° C., then warmed to room temperature, and further 1 hour Stirred. Ethylene glycol (9.5 g, 156 mmol) and p-toluenesulfonic acid (100 mg) were added, the solution was heated to reflux and water was removed using a Dean Stark apparatus. Upon completion, the mixture was cooled to room temperature and washed with aqueous sodium hydrogen carbonate solution (saturated, 2 x 50 mL). The residue was purified by vacuum distillation to give 2- (2-bromopropyl) -1,3-dioxolane (18.8 g, 40-42 ° C., 89% at 1 mmHg).
[95] ¹ H NMR (CDCl ₃ ): 5.05 (dd, 1H), 4.18-4.33 (m, 1H), 3.84-4.0 (m, 4H), 2.25 (ddd, 1H), 2.03 (ddd, 1H), 1.75 (d , 3H).
[96] Using a similar method, the following aldehydes were prepared using suitably substituted 2-chloropyrimidine and 1,3-dioxolane:
[97] 3- (5-chloropyrimidin-2-yl) propanal
[98]
[99] ¹ H NMR (CDCl ₃ ): 9.90 (s, 1 H), 8.60 (s, 2H), 3.32 (dd, 2H), 3.04 (dd, 2H).
[100] 3- (5-chloropyrimidin-2 -yl) butanal
[101]
[102] ¹ H NMR (CDCl ₃ ): 9.85 (s, 1H), 8.60 (s, 2H), 3.65 (m, 1H), 3.14 (dd, 1H), 2.75 (dd, 1H), 1.39 (d, 3H).
[103] 3- [2- (6-chloropyrazinyl)] propanal
[104]
[105] 3- [2- (6-chloropyrazinyl)] propanal diethyl acetal (200 mg, 0.82 mmol) was treated with 2 N hydrochloric acid (450 μl) in tetrahydrofuran (2.5 mL) at room temperature for 18 hours. After adjusting the pH to 8 with saturated aqueous sodium bicarbonate, the reaction was extracted with ethyl acetate (x3) and the organics dried (sodium sulphate anhydrous), filtered and concentrated in vacuo to give the title compound (137 mg) as a dark brown oil. , 98%). This material was used without further purification.
[106] ¹ H NMR (CDCl ₃ ) δ9.85 (1H. S); 8.4 (2H, 2xs); 3.5 (2H, t); 3.0 (2H, t).
[107] Starting material was obtained by the following method:
[108] 3- [2- (6-chloropyrazinyl)] propanal diethyl acetal
[109]
[110] 3- [2- (6-chloropyrazinyl)] propynal diethyl acetal (5.5 g, 22.9 mmol) in ethanol (55 mL) was degassed with argon and palladium (IV) oxide (52 mg, 0.23 mmol) Was added. The reaction vessel was emptied and a hydrogen atmosphere was introduced. After 2 days, the reaction mixture was concentrated in vacuo and purified by flash chromatography eluting with a gradient of 0-50% ethyl acetate in isohexane to give 3- [2- (6-chloropyrazinyl)] propanal as a pale yellow oil. Diethyl acetal (1.17 g, 21%) was obtained.
[111] ¹ H NMR (CDCl ₃ ) δ8.4 (1H, s); 8.35 (1 H, s); 4.5 (1 H, t); 3.75-3.55 (2H, m); 3.55-3.4 (2H, m); 2.9 (2H, doublet); 2.1 (2H, doublet); 1.2 (6 H, t).
[112] 3- [2- (6-chloropyrazinyl)] propynal diethyl acetal
[113]
[114] To a solution of 2,6-dichloropyrazine (1 g, 6.7 mmol) and propionaldehyde diethyl acetal (1.1 mL, 7.4 mmol) in acetonitrile (10 mL) at room temperature under argon atmosphere bis (triphenylphosphine) palladium ( II) Dichloride (94 mg, 0.13 mmol) and copper iodide (I) (51 mg, 0.27 mmol) were added followed by triethylamine (4.7 mL, 33.6 mmol). The reaction was stirred at rt overnight. The solvent was removed in vacuo and the residue was purified by flash chromatography, eluting with 10-20% ethyl acetate in isohexane to give 3- [2- (6-chloropyrazinyl)] propanal diethyl acetal (as yellow oil). 660 mg, 41%).
[115] ¹ H NMR (CDCl ₃ ) δ 8.6 (1H, s); 8.55 (1 H, s); 5.5 (1 H, s); 3.9-3.75 (2H, m); 3.7-3.4 (2H, m); 1.25 (6 H, t)
[116] m / s (EI ⁺ ) 241/243 (MH ⁺ ).
[117] Another method for preparing Formula I or its pharmaceutically acceptable salts or in vivo hydrolysable esters is to react a compound of Formula II with a compound of Formula R1COOR to obtain a compound of Formula VIII, as described below: Converting it to a compound of formula (IX), converting the compound of formula (IX) to an alkene of formula (III), and then to a compound of formula (IV) which is a precursor of compound (I), and then optionally a compound of formula (I) Forming a pharmaceutically acceptable salt or hydrolyzable ester in vivo.
[118] Suitable esters of formula R 1 COOR may be purchased commercially or prepared, eg, using procedures similar to those described in Example 10. It is to be appreciated that it is possible to use any ester of the formula R 1 COOR, wherein R 1 is as defined above, wherein R may be any group, including, for example, alkyl, aralkyl, heteroaryl, and the like.
[119]
[120] Compounds of the invention can be assessed, for example, in the following assays:
[121] Isolated Enzyme Assay
[122] Matrix metalloproteinase classes, including for example MMP13
[123] Recombinant human proMMP13 is described in V. Knauper et al., (1996) The Biochemical Journal 271 : 1544-1550 (1996), can be expressed and purified. Purified enzymes can be used to monitor inhibitors of activity as follows: Purified proMMP13 was activated at 21 ° C. for 20 hours using 1 mM amino phenyl mercury acid (APMA) and activated MMP13 (analyzed sugar). 11.25 ng) in assay buffer (0.1 M Tris-HCl, pH 7.5) containing 0.1 M NaCl, 20 mM CaCl ₂ , 0.02 mM ZnCl and 0.05% (w / v) Brij 35 at 35 ° C. for 4-5 hours. Synthetic substrate 7-methoxycoumarin-4-yl) acetyl. Pro.Leu.Gly.N-3- (2,4-dinitrophenyl) -L-2,3-diaminopropionyl.Ala.Arg.NH ₂ is used to incubate in the presence or absence of the inhibitor. Activity is determined by measuring fluorescence at λex 328 nm and λem 393 nm. % Inhibition is calculated as follows:% inhibition is [fluorescence _{plus inhibitor} -fluorescence _background ] divided by [fluorescence _{minus inhibitor} -fluorescence _background ].
[124] See, eg, C. Graham Knight et al., (1992) FEBS Lett. 296 (3) : 263-266, similar protocols can be used for other expressed and purified proMMPs using substrate and buffer conditions that are optimal for a particular MMP.
[125] Adamadamin class, including, for example, TNF converting enzyme
[126] The ability of compounds to inhibit proTNFa converting enzymes can be assessed using partially purified isolated enzyme assays, which are described in THM-1 as described by KM Mohler et al. (1994) Nature 370 : 218-220. Is obtained from the membrane. Purified enzyme activity and its inhibition rate was determined by substrate 4 ′ in assay buffer (50 mM Tris HCl, pH 7.4, containing 0.1% (w / v) Triton X-100 and 2 mM CaCl ₂ , for 18 hours at 26 ° C.). , 5'-dimethoxyfluororesinyl Ser.Pro.Leu.Ala.Gln.Ala.Val.Arg.Ser.Ser.Ser.Arg.Cys (4- (3-succinimide-1-yl) fluore New) -NH ₂ is used to incubate partially purified enzymes in the presence or absence of test compounds. Inhibition was measured as for MMP13 except that λex 490 nm and λem 530 nm were used. Substrates were synthesized as follows. The peptide portion of the substrate is either passively on the Fmoc-NH-Rink-MBHA-polystyrene resin or O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl as a coupling agent with Fmoc-amino acid. Four or five or more excess Fmoc-amino acids and HBTU were collected on an automated peptide synthesizer by standard methods involving the use of uronium hexafluorophosphate (HBTU). Ser ¹ and Pro ² were double coupled. The following side chain protection strategy was used; Ser ¹ (But), Gln ⁵ (Trityl), Arg ^8,12 (Pmc or Pbf), Ser ^9,10,11 (Trityl), Cys ¹³ (Trity). After aggregation, the N-terminal Fmoc-protecting group was removed by treating Fmoc-peptidyl-resin with DMF. The amino-peptidyl-resin thus obtained was 1.5 to 2 equivalents of 4 ', 5'-dimethoxyfluorescein-4 (5) -carboxylic acid at 70 ° C. for 1.5 to 2 hours [Khanna & Ullman, (1980). ) Anal Biochem. 108 : 156-161, preactivated with diisopropylcarbodiimide and 1-hydroxybenzotriazole in DMF]. The dimethoxyfluororesinyl-peptide was then deprotected simultaneously and separated from the resin by treatment with trifluoroacetic acid containing 5% each of water and triethylsilane. The isolated peptide was reacted with 4- (N-maleimido) fluorescein in DMF containing diisopropylethylamine, the product was purified by RP-HPLC and finally isolated by freeze drying from aqueous acetic acid. The product was characterized by MALDI-TOF MS and amino acid analysis.
[127] Natural substrate
[128] The activity of the compounds of the invention as inhibitors of aggrecan degradation is described, for example, in EC Arner et al. (1998) Osteoarthritis and Cartilage 6 : 214-228; (1999) Journal of Biological Chemistry , 274 (10) , 6594-6601, and assays using the antibodies described therein. The efficacy of compounds acting as inhibitors to collagenase is described in T. Cawston and A. Barrett (1979) Anal. Biochem. 99 : 340-345].
[129] Inhibition of metalloproteinase activity in cell / tissue activity
[130] Test as an Agent to Inhibit Membrane Shadase, such as TNF Converting Enzyme
[131] Ability of the compounds of the present invention to inhibit the cellular processing of TNFα produced was essentially the literature [Mohler KM, etc., (1994) Nature 370: 218-220 ] using an ELISA to detect the released TNF as described in THP- It can evaluate within 1 cell. In a similar fashion, NM Hooper et al. (1997) Biochem. J. 321 : 265-279 processing or shedding of other membrane molecules, such as those described, can be tested using appropriate cell lines and appropriate antibodies that detect shed proteins.
[132] Test as an Agent to Inhibit Cell Invasion
[133] The ability of the compounds of the invention to inhibit cell migration in invasive assays is described in A. Albini et al., (1987) Cancer Research 47 : 3239-3245.
[134] Test as an Agent That Inhibits Whole Blood TNF Shadase Activity
[135] The ability of the compounds of the present invention to inhibit TNFα production is assessed in human whole blood assays that stimulate the release of TNFα using LPS. Heparinized (10 units / ml) human whole blood obtained from volunteers was diluted 1: 5 in medium (RPMI1640 + bicarbonate, penicillin, streptomycin and glutamine) and humidified at 37 ° C. (5% CO ₂ /95% for 30 minutes). Air) incubator (160 μl) with 20 μl of test compound (3 sets) in DMSO or the appropriate vehicle, then add 20 μl LPS (E. coli. 0111: B4; final concentration 10 μg / ml) do. Each assay includes conventional TNFα inhibitors as a control or standard of diluted blood incubated with medium alone (6 wells / plate). The plates were then incubated for 6 hours at 37 ° C. (humidified incubator), centrifuged (2000 rpm for 10 minutes; 4 ° C.), plasma was harvested (50-100 μl) and 96 wells at −70 ° C. After storage on plates, subsequent analysis of TNFα concentrations is performed by ELISA.
[136] Test as an agent that inhibits in vitro cartilage degradation
[137] The ability of the compounds of the present invention to inhibit degradation of the aggrecan or collagen component of cartilage is essentially described in KM Bottomley et al. (1997) Biochem. J. 323 : 483-488.
[138] Pharmacokinetic Test
[139] In order to assess the clearance properties and bioavailability of the compounds of the present invention, ex vivo pharmacokinetic tests using HPLC or mass spectrometry analysis are used as synthetic substrate assays or alternatively. This is a general test that can be used to assess the clearance rate of compounds across various species. Animals (eg, rats, silk monkeys, etc.) are administered intravenously or orally with water-soluble preparations of compounds (eg, 20% w / v DMSO, 60% w / v PEG400) and continuous time points (eg, 5, 15 (30, 60, 120, 240, 480, 720, 1220 min), blood samples are taken as 10 U heparin from the appropriate vessels. Plasma fractions are obtained after centrifugation and plasma proteins are precipitated with acetonitrile (80% w / v final concentration). After 30 minutes plasma proteins are precipitated by centrifugation at -20 ° C and the supernatant fractions are evaporated to dryness using a Savant rapid dry machine. The precipitate is reconstituted with assay buffer and then analyzed for compound components using synthetic substrate analysis. In short, the compound concentration-response curve is constructed for the compound being evaluated. Serial dilution of the reconstituted plasma extract is assessed for activity, and the amount of compound present in the original plasma sample is calculated using the concentration-response curve taking into account the total plasma dilution factor.
[140] In vivo evaluation
[141] Test as anti-TNF agent
[142] The ability of the compounds of the invention as ex vivo TNFα inhibitors is evaluated for rats. In brief, the group of male Wistar Alderry Park (AP) rats (180-210 g) is administered with a compound (6 rats) or drug vehicle (10 rats) by the appropriate route, such as oral, intraperitoneal, subcutaneous. do. After 90 minutes, rats are sacrificed using high concentrations of CO ₂ and blood drawn through the posterior ventral vein with 5 units sodium heparin / ml blood. Blood samples are immediately placed on ice, centrifuged at 4 ° C. for 10 minutes at 2000 rpm and harvested plasma is later frozen to −20 ° C. to analyze their effect on TNFα production by LPS stimulated human blood. . Rat plasma samples are thawed and 175 μl of each sample is added to a set format pattern of 96 U well plates. Then 50 μl of heparinized human blood are added to each well, mixed and the plate incubated at 37 ° C. for 30 minutes (humidity incubator). LPS (25 μl; final concentration 10 μg / ml) is added to the wells and the incubation is continued for another 5.5 hours. Control wells are incubated with 25 μl of medium alone. The plate is then centrifuged at 2000 rpm for 10 minutes, 200 μl of supernatant is transferred to a 96 well plate and frozen at −20 ° C. for subsequent analysis of TNF concentration by ELISA.
[143] Data analysis by dedicated software is calculated for each compound / dose:
[144]
[145] Test as an antiarthritis
[146] The activity of compounds as anti-arthritis agents is described in DE Trentham et al., (1977) J. Exp. Med. 146 : 857 as tested in collagen induced arthritis (CIA). In this model, acid soluble native type II collagen causes multiple arthritis in rats when administered with Freund's incomplete adjuvant. Similar conditions can be used to cause arthritis in mice and primates.
[147] Test as anticancer agent
[148] The activity of the compound as an anticancer agent is, for example, B16 cell line {B. Hibner et al., Abstract 283 p75, described in the 10th NCI-EORTC Symposium, Amsterdam June 16-19, 1998], essentially using IJ Fidler (1978) Methods in Cancer Research 15 : 399-439. ] Can be evaluated as described.
[149] The present invention is now illustrated in the following examples, but is not limited to these.
[150] Example 1
[151] N- [1-([4- (4-bromophenyl) piperazino] sulfonylmethyl) -4-pyrimidin-2-ylbutyl] -N-hydroxyformamide
[152]
[153] N- [1-([4- (4-bromophenyl) piperazino] sulfonylmethyl) -4-pyrimidin-2-yl in THF (5.0 mL) and formic acid (2.5 mL) cooled to 0 ° C. To a stirred solution of butyl] hydroxyamine (497 mg, 1.0 mmol) was added to the resulting mixture of acetic anhydride (566 μl, 6.0 mmol) and formic acid (2.0 mL). The mixture was stirred at 0 ° C. for 1 h and allowed to come to room temperature. The solvent was removed by rotary evaporation and the residue was chromatographed (50 g silica combined eluent, eluent 0 → 15% methanol / dichloromethane). Purified by evaporation and crystallized from hot ethyl acetate to give N- [1-([4- (4-bromophenyl) piperazino] sulfonylmethyl) -4-pyrimidine- as a white crystalline powder. 2-ylbutyl] -N-hydroxyformamide (262 mg, 51%) was obtained.
[154] NMR (300 MHz DMSO-d ₆ ) δ / ppm: 9.87 (s, 1H *), 9.55 (s, 1H *), 8.70 (m, 2H), 8.29 (s, 1H *), 7.98 (s, 1H * ), 7.33 (m, 3H), 6.92 (dd, 2H), 4.68 (m, 1H *), 4.13 (m, 1H *), 3.55-3.31 (m, 5H, partially ambiguous), 3.25-3.09 (m , 7H, partially obscure), 1.80-1.50 (m, 4H).
[155] Rotational Isomer Signal
[156] MS: ES ⁺ , (M + H) ⁺ = 512, 514 (Br isotope pattern)
[157] Starting materials were prepared as follows:
[158] i) Methanesulfonyl chloride (2.83 mL, 36.3 mmol) in a solution of 1- (4-bromophenyl) piperazine hydrochloride (5.09 g, 18.3 mmol) and triethylamine (7.67 mL) in dichloromethane (100 mL) Was added dropwise. The mixture was stirred at rt for 1 h and then dichloromethane (100 mL) was added. The organics were washed with water (2 ×), brine, dried (Na ₂ SO ₄ ) and evaporated in vacuo to give a yellow solid which was crystallized from ethanol and washed with diethyl ether to give 1- (as a white downy powder. 4-Bromophenyl) -4- (methanesulfonyl) piperazine (4.74 g, 81% yield) was obtained.
[159] ¹ H NMR (300 MHz CDCl ₃ ) δ / ppm: 7.38 (d, 2H), 6.91 (d, 2H), 3.21 (m, 8H), 2.89 (s, 3H) MS: ES ⁺ , (M + H) ⁺ = 318, 320 (Br isotope pattern)
[160] ii) in 1- (4-bromophenyl) -4- (methanesulfonyl) piperazine (902 mg, 2.0 mmol) suspended in dry THF (15 mL) cooled to -20 ° C to -30 ° C under nitrogen. Lithium bis (trimethylsilyl) amide (1.0 M in THF, 4.0 mL), chloromethylsilane (217 mg, 2.0 mmol, 253 μL) and 4-pyrimidin-2-ylbutanal (300 mg, 2.0 mmol) in succession Was added. The mixture was stirred at −20 ° C. for 1 hour, quenched with saturated ammonium chloride solution and left overnight at room temperature. The solvent was removed in vacuo, the residue was partitioned between dichloromethane (15 mL) and water (5 mL), the organics were separated and chromatographed (50 g silica bound eluent, 0 → 100% ethyl acetate / hexanes gradient). ) To give 2- (5- [4- (4-bromophenyl) piperazino] sulfonylpent-4-enyl) pyrimidine (759 mg, 84% yield) as a white crystalline material.
[161] MS: ES ⁺ , (M + H) ⁺ = 451, 453 (Br isotope pattern)
[162] iii) stirring 2-((E) -5- [4- (4-bromophenyl) piperazino] sulfonylpent-4-enyl) pyrimidine (451 mg, 1.0 mmol) in THF (10 mL) To the solution hydroxyamine (50% solution in water, 500 μl) was added and the mixture was stirred overnight. The solvent was removed in vacuo and azeotropic with toluene (3x) to yield N- [1-([4- (4-bromophenyl) piperazino] sulfonylmethyl) -4-pyrimidin-2-ylbutyl] hydroxy Amine (497 mg, quant.) Was obtained.
[163] MS: ES ⁺ , (M + H) ⁺ = 484, 486 (Br isotope pattern)
[164] Example 2
[165] N- [1-([4- (5-chloropyridin-2-yl) piperazino] sulfonylmethyl) -3- (5-fluoropyrimidin-2-yl) propyl] -N-hydroxyform amides
[166]
[167] Acetic anhydride (0.51 mL) was added directly to formic acid (2.0 mL) cooled to 0 ° C., followed by 2- [4- [4- (5-chloropyridin-2-yl) pipera in tetrahydrofuran (11 mL). A solution of Zino] sulfonyl-3- (hydroxyamino) butyl] -5-fluoropyrimidine (0.485 g) was added. The solution was stirred at rt for 3 h, then evaporated in vacuo, the resulting residue was azeotropic with toluene and then dissolved in methanol and heated to 40 ° C. for 30 min. The solution was evaporated to dryness, then diethyl ether was added and stirred at room temperature for 10 minutes, the solid was filtered and dried in vacuo to N- [1-([4- (5-chloropyrimidin-2-yl) Piperazino] sulfonylmethyl) -3- (5-fluoropyrimidin-2-yl) propyl] -N-hydroxyformamide (0.218 g) was obtained. mp 154-155 ° C.
[168] NMR (d ₆ -DMSO 373 ° K): 2.20 (m, 2H), 2.95 (m, 2H), 3.23 (dd, 1H), 3.30 (m, 4H), 3.49 (dd, 1H), 3.60 (m, 4H), 4.42 (vbs, 1H), 6.88 (d, 1H), 7.59 (dd, 1H), 8.05 (vbs, 1H), 8.12 (dd, 1H), 8.71 (s, 2H), 9.40 (vbs, 1H) ); m / z 473 (M + l).
[169] Starting materials were prepared as follows:
[170] (i) 1- (5-chloropyridin-2-yl) -4- (methylsulfonyl) piperazine (0.600 g) was stirred in anhydrous tetrahydrofuran (22 mL) under argon and then cooled to -10 ° C. After the addition, lithium bis (triethylsilyl) amide (4.8 mL of 1.0 M solution in tetrahydrofuran) was added. The mixture was stirred at −10 ° C. for 30 minutes and a solution of diethylchlorophosphate (0.345 mL) was added. The mixture was stirred at −10 ° C. for 15 minutes, then 3- (5-fluoropyrimidin-2-yl) propanal (0.334 g) was added and stirred at −10 ° C. for 30 minutes. The mixture was allowed to warm to rt, then washed with aqueous ammonium chloride and extracted with ethyl acetate. The organic layer was dried over Na ₂ SO ₄ .
[171] Purify the residue on silica, eluting with 70% ethyl acetate 30% hexane to give 2-((E) -4- [4- (5-chloropyridin-2-yl) piperazino] sulfonylbut-3-enyl ) -5-fluoropyrimidine and 2-((Z) -4- [4- (5-chloropyrimidin-2-yl) piperazino] sulfonylbut-3-enyl) -5-fluoropyripy A 6: 4 mixture (0.44 g) of midine was obtained.
[172] ¹ H NMR (CDCl ₃ ): 8.55 (d, 1H), 8.48 (s, 1H), 7.46 (dd, 1H), 6.85 (m, 1H), 6.60 (d, 1H), * 6.45 (m, 1H) , 6.15 (d, 1H), * 6.03 (d, 1H), 3.61 (m, 4H), 3.28 (m, 2H), 3.15 (m, 4H), * 2.81 (m, 2H); MS (ES &^lt; ⁺ & gt ^; ): 412.3 (MH ^< ⁺ & gt ^; ).
[173] The symbol * denotes an isomer.
[174] (ii) 2-((E) -4- [4- (5-chloropyridin-2-yl) piperazino] sulfonylbut-3-enyl) -5-fluoro in tetrahydrofuran (5 mL) A solution of pyrimidine and 2-((Z) -4- [4- (5-chloropyridin-2-yl) piperazino] sulfonylbut-3-enyl) -5-fluoropyrimidine (0.44 g) To hydroxylamine (1.0 mL, 50% aqueous solution) was added. The mixture was stirred for 18 hours, then diluted with EtOAc (10 mL) and washed with saturated ammonium chloride solution (10 mL). The organic layer was dried over Na ₂ SO ₄ and evaporated in vacuo to afford 2- [4- [4- (5-chloropyridin-2-yl) piperazino] sulfonyl-3- (hydroxyamino) butyl] -5- Fluoropyrimidine (0.483 g) was obtained.
[175] ¹ H NMR (CDCl ₃ ): 8.45 (s, 2H), 8.08 (d, 1H), 7.39 (dd, 1H), 6.55 (d, 1H), 5.76 (bs, 2H), 3.59 (m, 4H), 3.46 (m, 1H), 3.42 (m, 2H), 3.33 (m, 4H), 3.10 (m, 4H), 2.82 (m, 1H), 2.15 (m, 1H), 2.01 (m, 1H); MS (ES &^lt; ⁺ & gt ^; ): 445.3 (MH ^< ⁺ & gt ^; ).
[176] Example 3
[177] The following compounds were prepared.
[178]
[179] BXR1mptM + HExamples of Methods Used in Manufacturing 5-Cl-2-pyridylN1,5,5-trimethyl-3-hydantoinCH ₂ CH ₂ 517.32 (5-Cl-2-pyridyl) oxyC4-Cl-phenyl 474.3One 5-Cl-2-pyridylN3,5,5-trimethyl-1-hydantoinCH ₂ CH ₂ 517.3One (5-Cl-2-pyridyl) oxyC2-pyrimidinylCH ₂ CH ₂ 470.3One 5-Cl-2-pyridylN2-pyrimidinyl-SCH ₂ CH ₂ 487One (5-Br-2-pyridyl) oxyC2-pyrimidinylCH ₂ CH ₂ CH ₂ 528.2One
[180] 5-Cl-2-pyridylN3- (OCH ₂ Ph) -Ph 531One 3,4-diCl-phenylN2-pyrimidinylCH ₂ CH ₂ CH ₂ 502One 4-Cl-phenylN2-pyrimidinylCH ₂ CH ₂ CH ₂ 468One 5-Cl-2-pyridylN3-CF ₃ -Ph 493One 4-Cl-phenylN3-pyridyl 397.42 5-Cl-2-pyridylN4-CF ₃ -Ph 493One 5-Cl-2-pyridylN3-thiophenyl 431One 5-Cl-2-pyridylN2-pyrazinylCH ₂ CH ₂ CH ₂ 4692 5-Cl-2-pyridylN2-pyrazinylCH ₂ CH ₂ 455.42 3-Cl-phenylN2-pyrimidinylCH ₂ CH ₂ CH ₂ 468.42 6-Me-4-pyrimidylN2-pyrimidinylCH ₂ CH ₂ CH ₂ 450.52 5-cyano-2-pyridylN2-pyrazinylCH ₂ CH ₂ CH ₂ 460.52 5-cyano-2-pyridylN2-pyrazinylCH ₂ CH ₂ 446.52 4-F-PhN2-pyrimidinylCH ₂ CH ₂ 438One 5-CF ₃ -2- pyridylN2-pyrimidinylCH ₂ CH ₂ 489One 5-cyano-2-pyridylN2-pyrimidinylCH ₂ CH ₂ 446One 5-CF ₃ -2- pyridylN2-pyrimidinylCH ₂ CH ₂ CH ₂ 503One 5-Cl-2-pyridylN4-pyrimidinylCH ₂ CH ₂ CH ₂ 469One 4-F-PhC2-pyrimidinylCH ₂ CH ₂ CH ₂ 4512 4-F-PhC2-pyrimidinylCH ₂ CH ₂ 4372 5-Cl-2-pyridylN2- (4-MeO-pyrimidinyl) CH ₂ CH ₂ 4852 5-Cl-2-pyridylC2-pyrimidinylCH ₂ CH ₂ CH ₂ 4682 5-Cl-2-pyridylC2-pyrimidinylCH ₂ CH ₂ 4542 5-Cl-2-pyridylN2- (4-CF ₃ -pyrimidinyl) CH ₂ CH ₂ 5232 5-Cl-2-pyridylN2- (5-ethylpyrimidinyl) CH ₂ CH ₂ 4832 5-Cl-2-pyridylN2- (4-MeO-pyrimidinyl) CH ₂ CH ₂ CH ₂ 4992 5-cyano-2-pyridylN2- (4-MeO-pyrimidinyl) CH ₂ CH ₂ CH ₂ 4902 5-Cl-2-pyridylN2- (5-F-pyrimidinyl) CH ₂ CH ₂ CH ₂ 4872 5-Br-2-pyridylN2- (4-CF ₃ -pyrimidinyl) CH ₂ CH ₂ CH ₂ 5832 5-Cl-2-pyridylN2- (4-CF ₃ -pyrimidinyl) CH ₂ CH ₂ CH ₂ 5372 5-cyano-2-pyridylN2- (4-CF ₃ -pyrimidinyl) CH ₂ CH ₂ CH ₂ 5282 5-Cl-2-pyridylN2- (5-ethylpyrimidinyl) CH ₂ CH ₂ CH ₂ 4972
[181] 5-Br-2-pyridylN2- (5-ethylpyrimidinyl) CH ₂ CH ₂ CH ₂ 541/5432 5-cyano-2-pyridylN2- (5-ethylpyrimidinyl) CH ₂ CH ₂ CH ₂ 4882 4-F-PhNPhSO ₂ NHCH ₂ CH ₂ 515One 5-Cl-2-pyridylCPhCH (Me) CH ₂ 64-65 2 4-F-PhN1,5,5-trimethyl-3-hydantoin CH (Me) CH ₂ 85 One 4-F-PhN4-MeO-PhCH (Me) CH ₂ 480One 4-F-PhN3-MeO-PhCH (Me) CH ₂ 480One 4-F-PhC1,5,5-trimethyl-3-hydantoin CH (Me) CH ₂ 77-79 One 4-Cl-PhN3-Cl-PhCH (Me) CH ₂ 500, 502One 6-Cl-2-pyrimidinylN2-pyrazinylCH (Me) CH ₂ 79-81470One 5-Cl-2-pyridylN2-pyridylCH (Me) CH ₂ 4682 5-cyano-2-pyridylN2-pyridylCH (Me) CH ₂ 4592 5-cyano-2-pyridylN2-pyrazinylCH (Me) CH ₂ 804602 5-CN-2-pyridylN2-pyrimidinylCH ₂ CH ₂ CH ₂ CH ₂ 474.5One 4-Cl-2-phenylN2-pyrimidinylCH ₂ CH ₂ CH ₂ CH ₂ 482.45One 5-Cl-2-pyridylN2-pyrimidinylCH ₂ CH ₂ CH ₂ CH ₂ 483.4One 5-Cl-2-pyridylN4-Cl-phenyl 459.3One 5-F-2-pyridylN2-pyrimidinylCH ₂ CH ₂ CH ₂ 453.22 5-F-2-pyridylN2- (5-F-pyrimidinyl) CH ₂ CH ₂ 457.12 5-Br-2-pyridylN2- (5-F-pyrimidinyl) CH ₂ CH ₂ 517/5192 4-Cl-phenylN2- (5-F-pyrimidinyl) CH ₂ CH ₂ 472.12 5-CN-2-pyridylN2- (5-F-pyrimidinyl) CH ₂ CH ₂ 464.182 5-CF ₃ -pyridylN2- (5-F-pyrimidinyl) CH ₂ CH ₂ 507.142 5-Cl-2-pyridylN2- (5-Br-pyrimidinyl) CH ₂ CH ₂ 533/5352 5-F-2-pyridylN2- (5-Br-pyrimidinyl) CH ₂ CH ₂ 517/5192 4-F-phenylN2- (5-Br-pyrimidinyl) CH ₂ CH ₂ 516/5182 5-F-2-pyridylN2- (5-Me-pyrimidinyl) CH ₂ CH ₂ 453.42 4-Cl-phenylN2- (5-Me-pyrimidinyl) CH ₂ CH ₂ 468.4One 5-Br-2-pyridylN2- (5-Me-pyrimidinyl) CH ₂ CH ₂ 513/5152
[182] 5-CF ₃ -2- pyridylN2- (5-Me-pyrimidinyl) CH ₂ CH ₂ 503.42 5-F-2-pyridylN2- (4-CF ₃ -pyrimidinyl) CH ₂ CH ₂ 507.062 4-Cl-phenylN2- (4-CF ₃ -pyrimidinyl) CH ₂ CH ₂ 521.92 5-CF ₃ -2- pyridylN2- (4-CF ₃ -pyrimidinyl) CH ₂ CH ₂ 556.952 5-Br-2-pyridylN2- (4-CF ₃ -pyrimidinyl) CH ₂ CH ₂ 566/5682 5-Cl-2-pyridylN2- (5-Cl-pyrimidinyl) CH ₂ CH ₂ 489/4912 5-Br-2-pyridylN2- (5-Cl-pyrimidinyl) CH ₂ CH ₂ 532/5342 5-F-2-pyridylN2- (5-Cl-pyrimidinyl) CH ₂ CH ₂ 4732 4-F-phenylN2- (5-Cl-pyrimidinyl) CH ₂ CH ₂ 4722 4-Cl-phenylN2- (5-Cl-pyrimidinyl) CH ₂ CH ₂ 488/4902 5-Br-2-pyridylN2- (5-Br-pyrimidinyl) CH ₂ CH ₂ 576/578/5802 4-Cl-phenylN2- (5-Br-pyrimidylyl) CH ₂ CH ₂ 531/533/5352 5-CN-2-pyridylN3- (5-pyridyl) CH ₂ CH ₂ 479/4812 4-CF ₃ -phenylN2-pyrimidinylCH ₂ CH ₂ CH ₂ 5022 4-Br-phenylN2- (5-F-pyrimidinyl) CH ₂ CH ₂ 518.32 3,4-diCl-phenylN2- (5-F-pyrimidinyl) CH ₂ CH ₂ 506.342 3-Cl-phenylN2- (5-F-pyrimidinyl) CH ₂ CH ₂ 472.382 4-CF ₃ -phenylN2- (5-F-pyrimidinyl) CH ₂ CH ₂ 506.42 4-F-PhN2-pyrimidinylCH ₂ CH ₂ 87-89 One 3,4-diCl-PhN2-pyrimidinylCH ₂ CH ₂ 489One 4-Cl-PhN2-pyrimidinylCH ₂ CH ₂ 455One 5-Me-2-pyridylN2-pyrimidinylCH ₂ CH ₂ CH ₂ 449One 5-Me-2-pyridylN2-pyrimidinylCH ₂ CH ₂ 435One 4-F-PhN2-pyrazinylCH ₂ CH ₂ CH ₂ 452One 4-F-PhN(6-Cl-2-pyrazinyl) CH ₂ CH ₂ 91-92 2
[183] 4-F-PhN5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ 143-4 2 4-Cl-PhN2-pyrazinylCH (CH ₃ ) CH ₂ 468One 4-F-PhN5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ 469One
[184] Starting piperazine sulfonamides and piperidine sulfonamides required for the synthesis of the compounds were either commercially available or prepared as follows:
[185] 1- (4-fluorophenyl) -4- (methanesulfonyl) piperazine
[186]
[187] Methanesulfonyl chloride (20 mL, 258 mmol) was added to a solution of 1- (4-fluorophenyl) piperazine (35 g, 194 mmol) and pyridine (17.5 mL) in anhydrous dichloromethane (200 mL) at 0 ° C. Added dropwise. The mixture was stirred for 3 hours at room temperature. The mixture was washed with water and extracted with dichloromethane (2 x 100 mL). The organic layer was dried over MgSO ₄ and evaporated in vacuo. The residue was triturated and washed with methanol to give 1- (4-fluorophenyl) -4- (methanesulfonyl) piperazine (39.35 g) as white crystals.
[188] ¹ H NMR (CDCl ₃ ): 7.00 (m, 2H), 6.90 (m, 2H), 3.40 (m, 4H), 3.20 (m, 4H), 2.83 (s, 3H).
[189] Aryl / heteroarylpiperazine and piperidine used as starting materials are either commercially available or described in the scientific literature.
[190] 1- (6-chloropyrimidin-4-yl) -4-mesylpiperazine
[191] A mixture of 4,6-dichloropyrimidine (39.4 g), 1-mesylpiperazine hydrochloride (55.7 g) and triethylamine (116 mL) in ethanol (500 mL) was stirred at reflux for 4 h. The mixture was then stirred at rt for 12 h. The separated solid was collected by filtration, the slurry was washed with ethanol (2 x 80 mL, 160 mL), then diethyl ether (150 mL) and dried to give 1- (6-chloropyrimidine-4- as a cream solid. I) -4-mesyl piperazine (71.9 g) was obtained. mp 200-202 ℃
[192] NMR (d ₆ -DMSO): 2.88 (s, 3H), 3.18 (m, 4H), 3.80 (m, 4H), 7.04 (s, 1H), 8.38 (m, 1H); m / z 277.3 (M + l).
[193] Using a similar procedure, 1-mesylpiperazine hydrochloride, CAS (161357-89-7), was reacted with the appropriate chloropyridine to afford the following compounds:
[194]
[195] Rm / z (M + 1) 5-Cl-2-pyridyl276 5-CF ₃ -2- pyridyl310 5-CN-2-pyridyl267 5-Br-2-pyridyl320/322
[196] 2- (4-piperidinyloxy) -5-chloropyridine
[197] i) NaH (2.88 g, 66 mmol, 55% dispersion in mineral oil) was stirred in anhydrous DME (200 mL) under argon. A mixture of 2,5-dichloropyridine (8.87 g, 60 mmol) and 4-hydroxypiperidine (6.67 g, 66 mmol) dissolved in anhydrous DME (200 mL) was added dropwise to the NaH suspension over 30 minutes. After the addition was complete, the reaction was heated at 82 ° C. for 48 hours while maintaining the argon blanket. The reaction was quenched slowly with water and then most of the THF was removed. The aqueous phase was washed with DCM (x3). The organic layer was dried over Na ₂ SO ₄ and evaporated in vacuo to afford 2- (4-piperidinyloxy) -5-chloropyridine (12.7 g, quantitative) as a yellow oil.
[198] ¹ H NMR (DMSO): 8.17 (d, 1H), 7.76 (dd, 1H), 6.81 (d, 1H), 4.96 (m, 1H), 2.93 (m, 2H), 2.53 (m, 2H), 1.91 (m, 2H), 1.46 (m, 2H); MS (ES < ⁺ & gt ^; ): 213.3 (MH ⁺ ), 225.3 (MNa ⁺ ).
[199] In a similar manner, 2- (4-piperidinyloxy) -5-bromopiperidine was prepared. MH + 257.3
[200] Example 4-Disassembly
[201] N-[(1S) -1-({[4- (5-chloropyridin-2-yl) piperazino] sulfonyl} methyl) -4- (pyrimidin-2-yl) butyl] -N-hydrate Oxyformamide
[202]
[203] To methanol (76 mL) was added to carbamate 1 (3.8 g, 5.66 mmol) dissolved in THF (76 mL), followed by water (38 mL). Lithium hydroxide monohydrate (2.37 g, 56.6 mmol) was added to the solution. ) Was added. After stirring for 2 hours at room temperature, the solvent was removed under reduced pressure, and the residue was dissolved in water (250 mL) and washed with ethyl acetate (200 mL) and diethyl ether (2 x 250 mL). Saturated aqueous ammonium chloride was added until the aqueous layer was approximately pH 8 and extracted with dichloromethane (3 x 250 mL). The combined dichloromethane extracts were dried (MgSO ₄ ) and evaporated to afford the product (2.2 g, 83%) as a white powder. Chiral HPLC using a Chiralpak AS column showed that the product was isolated at 96% ee (believed to have S stereochemistry). Mpt (from EtOH) 124.5-126.5 ° C .; [a] _D ²⁵ = -l7.2 (MeOH); NMR CDCl ₃ d 9.9 (br s, 1H) *; 8.7 (m, 2 H); 8.5 (s, 1 H) *; 8.1 (brs, 1 H); 8.0 (s, 1 H) *; 7.5 (dd, 1 H); 7.2 (m, 1 H); 6.6 (d, 1 H); 4.9 (m, 1 H) *; 4.2 (m, 1 H) *; 3.7-3.5 (m, 4 H); 3.5 (m, 1 H) *; 3.4-3.2 (m, 4 H); 3.3 (m, 1 H) *; 3.1-2.9 (m, 3 H); 2.0-1.6 (m, 4 H). MS (M + H) calc'd for C ₁₉ H ₂₅ ClN ₆ O ₄ S, 469. found 469.
[204] * Rotational Isomer Signal
[205] Step A
[206]
[207] Triethylamine (10.4 mL, 75 mmol) was added to reversed phase hydroxysamate 2 (18.76 g, 40 mmol) dissolved in dichloromethane (300 mL) and cooled to 0 ° C., followed by (4S) -4-benzyl- 2-oxalidinone-3-carbonyl chloride (10.55 g, 44 mmol) [CAS No. 139149-49-8] was added. After stirring at −3 to 0 ° C. for 3 hours, the mixture was washed with water (250 mL), dried (MgSO ₄ ) and evaporated to give a beige foam (27.1 g). The diastereomers were separated using preparative hplc eluting with ethyl acetate / EtOH (5%). The more polar diastereomers were isolated in 35% yield. MS (M + H) calcd for C ₃₀ H ₃₄ ClN ₇ O ₇ S 672. found 672.
[208] Compound 2 was prepared using the method described in Example 2: (M + H 469), mpt 131-134 ° C .; NMR (DMSO) 9.8 (1H, br), 8.7 (2H, m), 8.3 and 7.9 (1H, s), 8.1 (2H, s), 7.6 (1H, m), 6.9 (1H, m), 4.1 ( 1H, br m), 3.6 (4H, m), 3.2 (6H, m), 2.8 (2H, m), 1.8 (4H, m)
[209] Example 5
[210] The following compounds were produced in a manner similar to that described in Example 4:
[211] N-[(1S) -1-({[4- (5-bromopyridin-2-yl) piperazino] sulfonyl} methyl) -4- (pyridin-2-yl) butyl] -N-hydrate Oxyformamide
[212]
[213] NMR CDCl ₃ d 11.9 (br s, 1H) *; 8.5 (s, 1 H) *; 8.5-8.4 (m, 1 H); 8.2 (m, 1 H); 8.1 (s, 1 H) *; 7.8-7.7 (m, 1 H); 7.6 (m, 1 H); 7.3-7.2 (m, 2 H); 6.6 (m, 1 H); 5.0-4.9 (m, 1 H) *; 4.3-4.2 (m, 1 H) *; 3.7-3.6 (m, 4 H); 3.6 (m, 1 H) *; 3.4-3.3 (m, 4 H); 3.3 (m, 1 H) *; 3.1 (dd, 1H) *, 2.9 (m, 1H) *, 2.9-2.8 (m, 2H); 2.1-1.6 (m, 4H). MS (M + H) calc'd for C ₂₀ H ₂₆ BrN ₅ O ₄ S 514, found 514.
[214] * Rotational Isomer Signal
[215] [a] _D ²⁵ = -14 (c = 2.3, MeOH)
[216] The racemic starting material was prepared using the method described in Example 2. M + H = 512/514.
[217] N-[(1S) -1-({[4- (5-chloropyridin-2-yl) piperazino] sulfonyl} methyl) -3- (5-fluoropiperidin-2-yl) Propyl] -N-hydroxyformamide
[218]
[219] ¹ H NMR (DMSO, 373K): 9.44 (br s, 1H), 8.70 (s, 2H), 8.10 (d, 1H, J = 2.6 Hz), 8.05 (br s, 1H), 7.57 (dd, 1H) , J = 9.1, 2.6 Hz), 6.86 (d, 1H, J = 9.1 Hz), 4.40 (br s, 1H), 3.59 (dd, 4H, J = 5.3, 5.0 Hz), 3.47 (dd, 1H, J = 14.6, 7.4 Hz), 3.28 (dd, 4H, J = 5.3, 5.0 Hz), 3.24 (dd, 1H, J = 14.6, 4.3 Hz), 2.93 (m, 2H), 2.16 (m, 2H).
[220] MS (ESI): 473 (MH < ⁺ & gt ^; )
[221] a _d = -11.03 (MeOH, c = 1.242).
[222] The racemic starting material was prepared in Example 2.
[223] N-[(1S) -1-({[4- (4-fluorophenyl) piperazino] sulfonyl} methyl) -4- (pyrimidin-2-yl) butyl] -N-hydroxyformamide
[224]
[225] M + H 452.44; NMR CDCl ₃ d 9.9 (br s, 1H) *; 8.7 (m, 2 H); 8.5 (s, 1 H) *; 8.05 (s, 1 H) *; 7.2 (m, 1 H); 7.0-6.9 (m, 4 H); 4.9 (m, 1 H) *; 4.2 (m, 1 H) *; 3.5-3.4 (m, 4 H); 3.5 (m, 1 H) *; 3.2-3.1 (m, 4 H); 3.3 (m, 1 H) *; 3.1-2.9 (m, 3 H); 2.0-1.6 (m, 4 H).
[226] * Rotational Isomer Signal
[227] The racemic starting material was prepared using the method described in Example 3. NMR (DMSO) 10.0 (1H, br s), 8.6 (2H, m), 8.2 (1H, d), 7.2 (1H, m), 6.9 (4H, m), 4.9 and 4,2 (1H, br) , 3.4 (6H, m), 3.0 (6H, m), 1.9 (4H, m).
[228] Example 6 Chromatography Decomposition
[229] N-{(1S) -1-({[4- (5-chloropyridin-2-yl) piperazino] sulfonyl} methyl) -3- (pyrimidin-2-yl) propyl] -N-hydrate Roxyformamide and N-[(lR) -1-({[4- (5-chloropyridin-2-yl) piperazino] sulfonyl} methyl) -3- (pyrimidin-2-yl) propyl] -N-hydroxyformamide
[230]
[231] N- [1-({[4- (5-chloropyridin-2-yl) piperazino] sulfonyl} methyl) -3- (pyrimidin-2-yl) propyl] -N prepared in racemic form Hydroxyformamide was converted into chiralpak AD No. Charged with AD00CJ-HK002 and separated into single enantiomers by chromatographic separation on a column eluted with ethanol. The biological activity is in the second eluted compound from the column and is assumed to have S stereochemistry.
[232] Eluted with first enantiomeric isomer MH + 455.
[233] Eluted with the second enantiomeric isomer MH + 455.
[234] The racemic starting material was prepared using the method described in Example 2.
[235] NH + = 455. 1H, d), 4.7 and 4.2 (1H, br m), 3.6 (4H, m), 3.4-3.2 (6H, m), 2.8 (2H, m), 2.1 (2H, m).
[236] Example 7-Another Example of Chromatographic Decomposition
[237] The following compounds were decomposed using the conditions described in Example 6.
[238] N-[(1S) -1-({[4- (5-trifluoromethylpyridin-2-yl) piperazino] sulfonyl} methyl) -3- (pyrimidin-2-yl) propyl]- N-hydroxyformamide and N-[(1R) -1-({[4- (5-trifluoromethylpyridin-2-yl) piperazino] sulfonyl} methyl) -3- (pyrimidine- 2-yl) propyl] -N-hydroxyformamide
[239]
[240] Eluted with first enantiomeric isomer M + H 489.5.
[241] Eluted with the second enantiomer, M + H 489.5.
[242] The racemic starting material was prepared in Example 3.
[243] N-{(1S)-({[4- (5-bromopyridin-2-yl) piperazino] sulfonyl} methyl) -4- (pyrimidin-2-yl) butyl] -N-hydroxy Formamide and N-{(1R)-({[4- (5-bromopyridin-2-yl) piperazino] sulfonyl} methyl) -4- (pyrimidin-2-yl) butyl] -N Hydroxyformamide
[244]
[245] First enantiomeric isomer M + H 513/515
[246] Second enantiomeric isomer M + H 513/515
[247] Racemic starting materials were prepared using the method outlined in Example 2: M + H 513/515.
[248] Example 8
[249] The following compounds were prepared.
[250]
[251] BXR1mptM + HExamples of Methods Used in Manufacturing (5-Cl-2-pyridyl) oxyC2-pyrimidinylCH ₂ CH ₂ CH ₂ (S enantiomer) 4844 5-CF ₃ -2- pyridylN2-pyrimidinylCH ₂ CH ₂ CH ₂ (S enantiomer)141-1425034 4-F-phenylN2- (5-F-pyrimidinyl) CH ₂ CH ₂ (S enantiomer) 456.246 ** 4-F-phenylN2- (5-F-pyrimidinyl) CH ₂ CH ₂ 456.22 4-Br-PhN2-pyrazinylCH (CH ₃ ) CH ₂ mixed diastereomer 3: 1 (A: B) 512One 4-Cl-PhC2-pyrazinylCH (CH ₃ ) CH ₂ diastereomer A 467One 4-Cl-PhC2-pyrazinylCH (CH ₃ ) CH ₂ mixed diastereomer 1: 2 (A: B) 467One 4-Br-PhC2-pyrazinylCH (CH ₃ ) CH ₂ mixed diastereomer 3: 1 (A: B) 511One 5-Cl-2-pyridylN5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ mixed diastereomer 3: 1 (A: B) 487One 4-Cl-PhN5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ Diastereomer A157-9 One
[252] 4-Cl-PhN5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ diastereomer B164-7 One 4-Br-PhN5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ Diastereomer A167-9 One 4-Br-PhN5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ diastereomer B183-5 One 4-Cl-PhC5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ Diastereomer A195-8 One 4-Cl-PhC5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ diastereomer B155-8 One 3,4-diCl-PhN5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ Diastereomer A172-3 One 3,4-diCl-PhN5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ diastereomer B172-3 One 5-CN-2-pyridylN5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ Diastereomer A 478One 4-F-PhN5-F-2-pyrimidinylCH (CH ₃ ) CH ₂ (S enantiomer) 4707 4-F-PhN(R, S) -pyrazinylCH (CH ₃ ) CH ₂ (S enantiomer) 4524
[253] In the table above,
[254] ** is column chiralpak AD (250 mm x 4.6 mm) No. Compound (S enantiomeric isomer) prepared by the method of Example 6 using ADooCE-JJ122 and eluent MeOH / MeCN 15/85,
[255] Enantiomers A and B relate to the elution sequence from a silica column eluted with 3-5% ethanol in dichloromethane (diastereomer A is the eluted first fraction and diastereomer B is the second fraction). .
[256] Example 9
[257] We provide NMR data for the following compounds listed in Example 8:
[258] N-[(1S) -1-({[4- (5-trifluuromethylpyridin-2-yl) piperazino] sulfonyl} methyl) -4- (pyrimidin-2-yl) butyl]- N-hydroxyformamide
[259]
[260] NMR CDCl ₃ δ 10.1 (br s, 1H) *; 8.7 (m, 2 H); 8.5 (s, 1 H) *; 8.4 (br s, 1 H); 8.1 (s, 1 H) *; 7.7 (dd, 1 H); 7.2 (m, 1 H); 6.7 (d, 1 H); 4.9 (m, 1 H) *; 4.2 (m, 1 H) *; 3.9-3.7 (m, 4 H); 3.6 (m, 1 H) *; 3.4-3.2 (m, 4 H); 3.3 (m, 1 H) *; 3.1-2.9 (m, 3 H); 2.0-1.6 (m, 4 H). * Rotational Isomer Signal
[261] N-({[4-fluorophenylpiperazino] sulfonyl} methyl) -3-[(5-fluoropyrimidin-2-yl) propyl] -N-hydroxyformamide
[262]
[263] ¹ H NMR (DMSO, 373K): 9.46 (br s, 1 H), 8.73 (s, 2 H), 7.08-6.96 (m, 4 H), 4.42 (br s, 1 H), 3.50 (dd, J = 14.8, 7.5 Hz, 1H), 3.35 (m, 4H), 3.28 (dd, J = 14.8, 4.4 Hz, 1H), 3.18 (m, 4H), 2.97 (m, 2H), 2.21 (m, 2H).
[264] N-[(1R or 1S)-({[4-chlorophenylpiperazino] sulfonyl} methyl) -3-[(3R or 3S)-(5-fluoropyrimidin-2-yl) butyl]- N-hydroxyformamide (single diastereomer A)
[265]
[266] ¹ H NMR (CDCl ₃ ) (approximate proportions of 2 rotamers): 8.72 (s, 0.5H), 8.57 (d, 2H), 8.25 (s, 0.5H), 7.89 (s, 0.5H), 7.23 ( dd, 2H), 6.83 (dd, 2H), 4.94 (sext, 0.5H), 4.30 (m, 0.5H), 3.57 (dd, 0.5H), 3.44 (m, 2H), 3.37 (m, 2.5H) , 3.16 (m, 5.5H), 3.02 (dd, 0.5H), 2.52 (ddd, 0.5H), 2.35 (ddd, 0.5H), 2.02 (dt, 0.5H), 1.89 (ddd, 0.5H), 1.40 (dd, 3H);
[267] N-[(1R or 1S)-({[4-bromophenylpiperazino] sulfonyl} methyl) -3-[(3R or 3S)-(5-fluoropyrimidin-2-yl) butyl] -N-hydroxyformamide (single diastereomer A)
[268]
[269] ¹ H NMR (CDCl ₃ ) (approximate proportions of 2 rotamers): 8.72 (s, 0.5H), 8.57 (d, 2H), 8.25 (s, 0.5H), 7.89 (s, 0.5H), 7.38 ( dd, 2H), 6.80 (dd, 2H), 4.94 (sext, 0.5H), 4.30 (m, 0.5H), 3.57 (dd, 0.5H), 3.44 (m, 2H), 3.37 (m, 2.5H) , 3.16 (m, 5.5H), 3.02 (dd, 0.5H), 2.52 (ddd, 0.5H), 2.35 (ddd, 0.5H), 2.02 (dt, 0.5H), 1.89 (dt, 0.5H), 1.40 (dd, 3H);
[270] N-[(1R or 1S)-({[4-chlorophenylpiperidino] sulfonyl} methyl) -3-[(3R or 3S)-(5-fluoropyrimidin-2-yl) butyl]- N-hydroxyformamide (single diastereomer A)
[271]
[272] ¹ H NMR (CDCl ₃ ) (approximate proportions of two isomers): 8.69 (s, 0.5H), 8.57 (d, 2H), 8.25 (s, 0.5H), 7.89 (s, 0.5H), 7.27 ( Ambiguous), 7.13 (dd, 2H), 4.91 (sext, 0.5H), 4.30 (m, 0.5H), 3.87 (m, 2H), 3.57 (dd, 0.5H), 3.35 (dd, 0.5H), 3.18 (m, 1.5H), 3.00 (dd, 0.5H), 2.85 (m, 2H), 2.55 (m, 1.5H), 2.35 (ddd, 0.5H), 2.06 (dt, 0.5H), 1.88 (m , 2.5H), 1.7 (unclear), 1.40 (dd, 3H);
[273] N-[(1R or 1S)-({[3,4-dichlorophenylpiperazino} methyl) -3-[(3R or 3S)-(5-fluoropyrimidin-2-yl) butyl] -N Hydroxyformamide (single diastereomer A)
[274]
[275] ¹ H NMR (CDCl ₃ ) (approximate proportions of two isomers): 8.62 (s, 0.5H), 8.55 (d, 2H), 8.22 (s, 0.5H), 7.86 (s, 0.5H), 7.28 ( m, 1H), 6.95 (m, 1H), 6.73 (m, 1H), 4.92 (sext, 0.5H), 4.30 (m, 0.5H), 3.57 (dd, 0.5H), 3.44 (m, 2H), 3.37 (m, 2.5H), 3.16 (m, 5.5H), 3.02 (dd, 0.5H), 2.52 (ddd, 0.5H), 2.37 (ddd, 0.5H), 2.04 (dt, 0.5H), 1.89 ( dt, 0.5 H), 1.40 (dd, 3H);
[276] N-[(1R or 1S)-({[4- (5-cyanopyridin-2-yl) piperazino] sulfonyl} methyl) -3-[(3R or 3S)-(5-fluoropyripy Midin-2-yl) butyl] -N-hydroxyformamide (single diastereomer A)
[277]
[278] ¹ H NMR (CDCl ₃ ) (approximately proportional dimer): 8.72 (s, 0.5H), 8.55 (s, 2H), 8.41 (s, 1H), 8.22 (s, 0.5H), 7.86 (s , 0.5H), 7.65 (m, 1H), 6.61 (dd, 1H), 4.92 (m, 0.5H), 4.30 (m, 0.5H), 3.78 (m, 4H), 3.57 (dd, 0.5H), 3.38 (m, 2H), 3.30 (m, 2.5H), 3.16 (m, 1.5H), 3.02 (dd, 0.5H), 2.52 (m, 0.5H), 2.37 (m, 0.5H), 2.04 (dt , 0.5H), 1.84 (dt, 0.5H), 1.40 (dd, 3H);
[279] N-[(1S)-({[4- (4-fluorophenylpiperazino] sulfonyl} methyl) -3-[(3S)-(5-fluoropyrimidin-2-yl) butyl]- N-hydroxyformamide
[280]
[281] ¹ H NMR (DMSO-d ₆ ): 9.9, 9.53 (2s, 1H), 8.78 (s, 2H), 7.98 (d, 1H), 7.12-6.91 (m, 4H), 4.8, 4.17 (2s, 1H) , 3.13 (m, 4H), 3.0 (m, 1H), 1.86 (m, 1H), 1.22 (m, 3H).
[282] Example 10
[283] 1-({[4- (4-chlorophenyl) piperazin-1-yl] sulfonyl} methyl) -3- (5-chloropyridin-3-yl) propyl (hydroxy) formamide
[284]
[285] Acetic anhydride (102 μl, 1.1 mmol) was added to formic acid (400 μl, 10.8 mmol) at 0 ° C., followed by stirring at room temperature for 15 minutes. The mixture is then cooled to 0 ° C. and 1- (4-chlorophenyl) -4-{[4- (5-chloropyridin-3-yl) -2- (hydroxyamino) butyl] sulfonyl in THF } A solution of piperazine (100 mg, 0.22 mmol) was added dropwise by syringe. After stirring at room temperature for 1.5 hours, the volatiles were removed in vacuo and the residue was azeotropic with toluene (2 mL). The residue was then dissolved in methanol (5 mL). Then diethyl ether (5 mL) was added and the cloudy suspension was stirred at rt for 1 h. The precipitated solid was filtered off, washed with diethyl ether and dried in vacuo to yield the title compound (48 mg, 0.099 mmol) as an off white solid.
[286] ¹ H NMR (DMSO, 373K): 9.55 (br s, 1 H), 8.43 (d, 1 H), 8.41 (d, 1 H), 8.17 (br s, 1 H), 7.76 (dd, 1 H), 7.25 (m, 2H), 6.96 (m, 2H), 4.35 (br s, 1H), 3.49 (dd, 1H), 3.34 (m, 4H), 3.25 (m, 5H), 2.67 (m, 2H), 2.02 (m, 2H).
[287] MS (ESI): 487.06, 489.04, 490.08 (MH ⁺ 2 x Cl)
[288] Standard materials were prepared as follows:
[289] (i) ethyl-3- (5-chloropyridin-3-yl) propanoate
[290]
[291] Ethyl (2E) -3- (5-chloropyridin-3-yl) prop-2-enoate (338 mg, 1.6 mmol) in anhydrous ethanol (10 mL) at 0 ° C. [CAS No. 163083-45 -2] was added solid sodium borohydride (67 mg, 1.75 mmol). The reaction mixture was allowed to warm to rt and stirred for 4 h, during which more sodium borohydride (67 mg, 1.75 mmol) was added. After further stirring for 18 hours, saturated aqueous ammonium chloride solution (5 ml) was added. The volatiles were removed in vacuo and the residue was partitioned between water (10 mL) and acetate (10 mL). The layers were separated and the aqueous phase was extracted with ethyl acetate (3 x 10 mL). The combined organic extracts were then dried (MgSO ₄ ), filtered and concentrated in vacuo. Flash chromatography (silica gel, 20% to 100% ethyl acetate in hexanes) gave the title compound (132 mg, 0.62 mmol) and saturated alcohol (70 mg).
[292] ¹ H NMR (CDCl ₃ ): 8.43 (m, 1H), 8.34 (m, 1H), 7.55 (m, 1H), 4.16 (q, 2H), 2.96 (dd, 2H), 2.63 (dd, 2H).
[293] (ii) 1-{[4- (4-chlorophenyl) piperazin-1-yl] sulfonyl} -4- (5-chloropyridin-3-yl) butan-2-one
[294]
[295] 1.71 mL of LiHMDS (1.0 M solution in THF over 4 minutes in a stirred solution of 1- (4-chlorophenyl) -4- (methylsulfonyl) piperazine (235 mg, 0.85 mmol) at -10 ° C. over 4 minutes, 1.71 mmol) was added dropwise. The solution was then stirred at this temperature for 40 minutes. Then ethyl 3- (5-chloropyridin-3-yl) propanoate (201 mg, 0.94 mmol) in THF (1 mL) was added dropwise by cannula over 5 minutes. The reaction was further stirred at −10 ° C. for 30 minutes and then quenched with saturated aqueous ammonium chloride solution (5 mL). The volatiles were removed in vacuo and the residue was extracted with CH ₂ Cl ₂ (3 × 5 mL). The combined organic extracts were washed with water (10 mL) and brine (10 mL), then dried (MgSO ₄ ), filtered and concentrated in vacuo. Flash chromatography (silica gel, 50% ethyl acetate in hexanes) afforded the title compound (228 mg, 0.52 mmol) and ethyl 3- (5-chloropyridin-3-yl) propanoate (74 mg, 0.35 mmol). ) Was recovered.
[296] ¹ H NMR (CDCl ₃ ): 8.46 (m, 1H), 8.38 (m, 1H), 7.58 (m, 1H), 7.21 (m, 2H), 6.83 (m, 2H), 3.96 (s, 2H), 3.37 (m, 4H), 3.17 (m, 6H), 2.95 (dd, 2H),
[297] MS (ESI): 442.07, 444.06, 445.1 (MH ⁺ 2 x Cl).
[298] (iii) 1-{[4- (4-chlorophenyl) piperazin-1-yl] sulfonyl} -4- (5-chloropyridin-3-yl) butan-2-ol
[299]
[300] 1-{[4- (4-chlorophenyl) piperazin-1-yl] sulfonyl} -4- (5-chloro in a mixed solvent system of CH ₂ Cl ₂ / MeOH (1: 1, 5 mL) at room temperature To a stirred solution of pyridin-3-yl) butan-2-one (228 mg, 0.51 mmol) was added solid sodium borohydride in one portion. The reaction was stirred for 40 minutes and then quenched with aqueous hydrochloric acid (1 M, 2 mL). The layers were then separated and the aqueous phase was extracted with CH ₂ Cl ₂ (3 × 5 mL). The combined organic extracts were dried (MgSO ₄ ), filtered and concentrated in vacuo. The crude product was then filtered through a plug of silica gel eluting with 50% ethyl acetate in hexanes to afford the title compound (111 mg, 0.25 mmol).
[301] ¹ H NMR (CDCl ₃ ): 8.47 (m, 1 H), 8.40 (m, 1 H), 7.59 (m, 1 H), 7.21 (m, 2 H), 6.86 (m, 2H), 4.21 (m, 1 H), 3.45 (m, 4H), 3.24 (m, 4H), 3.11 (m, 2H), 2.88 (m, 2H), 1.89 (m, 2H).
[302] (iv) 1- (4-chlorophenyl) -4-{[(1E) -4- (5-chloropyridin-3-yl) but-1-enyl] sulfonyl} piperazine
[303]
[304] 1-{[4- (4-chlorophenyl) piperazin-1-yl] sulfonyl} -4- (5-chloropyridin-3-yl) butane-2 in anhydrous CH ₂ Cl ₂ (2.5 mL) at room temperature To a stirred solution of -ol (111 mg, 0.25 mmol) trimethylamine hydrochloride (2 mg, 0.02 mmol), triethylamine (52 μL, 0.25 mmol), followed by methanesulfonyl chloride (21 μL, 0.25) under argon atmosphere. mmol) was added. The reaction was stirred at rt for 30 min and then quenched by addition of saturated aqueous sodium bicarbonate solution (5 mL). The layers were separated and the aqueous phase was extracted with ethyl acetate (3 x 6 mL). The combined organics were then dried (MgSO ₄ ), filtered and concentrated in vacuo. The residue was then dissolved in CH ₂ Cl ₂ (2.5 mL) and treated with triethylamine (100 μl, 1.36 mmol). After 30 minutes, the reaction was quenched by the addition of saturated aqueous sodium bicarbonate solution (5 mL). The layers were separated and the aqueous phase was extracted with ethyl acetate (3 x 6 mL). The combined organic extracts were then dried (MgSO ₄ ), filtered and concentrated in vacuo. The crude material was used in the next step.
[305] MS (ESI): 446.06, 428.06, 430.07 (MH ⁺ 2 x Cl)
[306] (v) 1- (4-chlorophenyl) -4-{[4- (5-chloropyridin-3-yl) -2- (hydroxyamino) butyl] sulfonyl} piperazine
[307]
[308] 1- (4-chlorophenyl) -4-{[(1E) -4- (5-chloropyridin-3-yl) but-1-enyl] sulfonyl} piperazine (formerly in THF (10 mL) at room temperature) To a stirred solution of crude from step) was added a solution of hydroxylamine (2 mL, 50% aqueous solution in water). The reaction was stirred at rt for 3 h and then quenched with saturated aqueous ammonium bicarbonate solution (5 mL). The layers were separated and the aqueous phase was extracted with ethyl acetate (3 x 10 mL). The combined organic extracts were then dried (MgSO ₄ ), filtered and concentrated in vacuo. The residue was then purified by flash chromatography (silica, 100% ethyl acetate) to give the title compound (100 mg, 0.22 mmol).

权利要求:
Claims (22)
[1" claim-type="Currently amended] A compound of formula (I) or a pharmaceutically acceptable salt or in vivo hydrolyzable ester thereof:
Formula I

Where
B represents a phenyl group monosubstituted with halogen or trifluoromethyl at position 3 or 4 or a phenyl group disubstituted with halogen (which may be the same or different) at position 3 or 4; Or B represents a 2-pyridyl group or 2-pyridyloxy group monosubstituted with halogen, trifluoromethyl, cyano or C _1-4 alkyl at positions 4, 5 or 6; Or B represents a 4-pyrimidinyl group, optionally substituted at position 6 with halogen or C _1-4 alkyl;
X represents a carbon or nitrogen atom;
R 1 is a trimethyl-1-hydantoin C _2-4 alkyl group or a trimethyl-3-hydantoin C _2-4 alkyl group; Or R 1 is phenyl or C _2-4 alkylphenyl monosubstituted with halogen, trifluoromethyl, thio, C _1-3 alkyl or C _1-3 alkoxy at the 3 or 4 position; Or R 1 is phenyl-SO ₂ NHC _2-4 alkyl; Or R 1 is 2-pyridyl or 2-pyridyl C _2-4 alkyl; Or R 1 is 3-pyridyl or 3-pyridyl C _2-4 alkyl; Or R 1 is 2-pyrimidine-SCH ₂ CH ₂ ; Or R 1 is one of halogen, trifluoromethyl, C _1-3 alkyl, C _1-3 alkyloxy, 2-pyrazinyl optionally substituted with halogen or 2-pyrazinyl C _2-4 alkyl optionally substituted with halogen Monosubstituted 2- or 4-pyrimidinyl C _2-4 alkyl.
[2" claim-type="Currently amended] The method of claim 1,
B represents a phenyl group monosubstituted with halogen or trifluoromethyl at position 3 or 4 or a phenyl group disubstituted with halogen (which may be the same or different) at position 3 or 4; Or B represents a 2-pyridyl group or 2-pyridyloxy group monosubstituted with halogen, trifluoromethyl or cyano at the 5 or 6 position; Or B represents a 4-pyrimidinyl group, optionally substituted at position 6 with halogen or C _1-4 alkyl;
X represents a carbon or nitrogen atom;
R 1 is a trimethyl-1-hydantoin C _2-4 alkyl group or a trimethyl-3-hydantoin C _2-4 alkyl group; Or R 1 is phenyl or C _2-4 alkylphenyl monosubstituted with halogen, trifluoromethyl, thio, C _1-3 alkyl or C _1-3 alkoxy at the 3 or 4 position; Or R 1 is phenyl-SO ₂ NHC _2-4 alkyl; Or R 1 is 2-pyridyl or 2-pyridyl C _2-4 alkyl; Or R 1 is 3-pyridyl or 3-pyridyl C _2-4 alkyl; Or R 1 is 2-pyrimidine-SCH ₂ CH ₂ ; Or R 1 is 2- or 4-pyrimi optionally monosubstituted with one of halogen, trifluoromethyl, C _1-3 alkyl, C _1-3 alkyloxy, 2-pyrazinyl or 2-pyrazinyl C _2-4 alkyl A compound representing a dinyl C _2-4 alkyl or a pharmaceutically acceptable salt or in vivo hydrolyzable ester thereof.
[3" claim-type="Currently amended] The compound of claim 1, wherein B is 4-chlorophenyl, 4-fluorophenyl, 4-bromophenyl, 4-trifluorophenyl, 5-chloro-2-pyridyl, 5-bromo-2-pyridyl , 5-fluoro-2-pyridyl, 5-trifluoromethyl-2-pyridyl, 5-cyano-2-pyridyl, 5-methyl-2-pyridyl, or a compound thereof Pharmaceutically acceptable salts or in vivo hydrolyzable esters.
[4" claim-type="Currently amended] The compound of claim 3, or a pharmaceutically acceptable salt or in vivo thereof, wherein B is 4-fluorophenyl, 5-chloro-2-pyridyl or 5-trifluoromethyl-2-pyridyl. Hydrolyzable esters.
[5" claim-type="Currently amended] The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt or in vivo hydrolyzable ester thereof, wherein X is a nitrogen atom.
[6" claim-type="Currently amended] 6. The compound of claim 1, wherein R 1 is 3-chlorophenyl, 4-chlorophenyl, 3-pyridyl, 2-pyridylpropyl, 2- or 4-pyrimidinylethyl (optionally fluorine). Mono-substituted), 2- or 4-pyrimidinylpropyl, 2- (2-pyrimidinyl) propyl (optionally monosubstituted with fluorine) or a pharmaceutically acceptable salt or biological thereof Hydrolyzable esters.
[7" claim-type="Currently amended] The compound of claim 6, wherein R 1 is 2-pyrimidinylpropyl, 2- (2-pyrimidinyl) propyl (optionally monosubstituted with fluorine) or 5-fluoro-2-pyrimidinylethyl Its pharmaceutically acceptable salts or in vivo hydrolysable esters.
[8" claim-type="Currently amended] The compound of claim 1, or a pharmaceutically acceptable salt or in vivo hydrolyzable ester thereof, wherein the compound of formula I is as exemplified herein.
[9" claim-type="Currently amended] The compound of claim 8, wherein the compound is N- [1-([4- (4-bromophenyl) piperazino] sulfonylmethyl) -4-pyrimidin-2-ylbutyl] -N-hydroxyform Amide, N- [1-([4- (5-chloropyridin-2-yl) piperazino] sulfonylmethyl) -3- (5-fluoropyrimidin-2-yl) propyl] -N-hydride Oxyformamide, N-[(1S) -1-({[4- (5-chloropyridin-2-yl) piperazino] sulfonyl} methyl) -4- (pyrimidin-2-yl) butyl] -N-hydroxyformamide, N-[(1S) -1-({[4- (5-bromopyridin-2-yl) piperazino] sulfonyl} methyl) -4- (pyridine-2- Yl) butyl] -N-hydroxyformamide, N-[(1S) -1-({[4- (5-chloropyridin-2-yl) piperazino] sulfonyl} methyl) -3- (5 -Fluoropyrimidin-2-yl) propyl] -N-hydroxyformamide, N-[(1S) -1-({[4- (4-fluorophenyl) piperazino] sulfonyl} methyl) 4- (pyrimidin-2-yl) butyl] -N-hydroxyformamide, N-[(1S) -1-({[4- (5-chloropyridin-2-yl) piperazino] sul Ponyl} methyl) -3- (pyrimidin-2-yl) propyl] -N-hydroxyform Amide, N-[(1R) -1-({[4- (5-chloropyridin-2-yl) piperazino] sulfonyl} methyl) -3- (pyrimidin-2-yl) propyl] -N -Hydroxyformamide, N-[(1S) -1-({[4- (5-trifluoromethylpyridin-2-yl) piperazino] sulfonyl} methyl) -3- (pyrimidine-2 -Yl) propyl] -N-hydroxyformamide, N-[(1R) -1-({[4- (5-trifluoromethylpyridin-2-yl) piperazino] sulfonyl} methyl)- 3- (pyrimidin-2-yl) propyl] -N-hydroxyformamide, N-[(1S) -1-({[4- (5-bromopyridin-2-yl) piperazino] sul Ponyl} methyl) -4- (pyrimidin-2-yl) butyl] -N-hydroxyformamide, N-[(1R) -1-({[4- (5-bromopyridin-2-yl) Piperazino] sulfonyl} methyl) -4- (pyrimidin-2-yl) butyl] -N-hydroxyformamide, N-[(1S) -1-({[4- (5-trifluoro Methylpyridin-2-yl) piperazino] sulfonyl} methyl-4- (pyrimidin-2-yl) butyl] -N-hydroxyformamide, N-({[4-fluorophenylpiperazino] Sulfonyl} methyl) -3-[(5-fluoropyrimidin-2-yl) propyl] -N -Hydroxyformamide, N-[(1R or 1S)-({[4-chlorophenylpiperazino] sulfonyl} methyl) -3-[(3R or 3S)-(5-fluoropyrimidine-2 -Yl) butyl] -N-hydroxyformamide, N-[(1R or 1S)-({[4-bromophenylpiperazino] sulfonyl} methyl) -3-[(3R or 3S)-( 5-fluoropyrimidin-2-yl) butyl] -N-hydroxyformamide, N-[(1R or 1S)-({[4-chlorophenylpiperidino] sulfonyl} methyl) -3- [ (3R or 3S)-(5-fluoropyrimidin-2-yl) butyl] -N-hydroxyformamide, N-[(1R or 1S)-({[3,4-dichlorophenylpiperazino] Sulfonyl} methyl) -3-[(3R or 3S)-(5-fluoropyrimidin-2-yl) butyl] -N-hydroxyformamide, N-[(1R or 1S)-({[4 -(5-cyanopyridin-2-yl) piperazino] sulfonyl} methyl) -3-[(3R or 3S)-(5-fluoropyrimidin-2-yl) butyl] -N-hydroxy Formamide, N-[(1S)-({[4- (4-fluorophenylpiperazino] sulfonyl} methyl) -3-[(3S)-(5-fluoropyrimidin-2-yl) Butyl] -N-hydroxypo Amide, 1-({[4- (4-chlorophenyl) piperazin-1-yl] sulfonyl} methyl) -3- (5-chloropyridin-3-yl) propyl (hydroxy) formamide Compound or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof.
[10" claim-type="Currently amended] The compound or pharmaceutically acceptable salt or in vivo hydrolyzable ester thereof according to any one of claims 1 to 9, wherein the compound of formula (I) is the most active enantiomer.
[11" claim-type="Currently amended] The compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt or in vivo hydrolyzable ester thereof, wherein the compound of formula (I) is an S enantiomer or an S, S enantiomer. .
[12" claim-type="Currently amended] A pharmaceutical composition comprising a compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt or hydrolyzable ester thereof in vivo, and a pharmaceutically acceptable carrier.
[13" claim-type="Currently amended] A compound of formula (I) or a pharmaceutically acceptable salt or in vivo hydrolyzable ester thereof according to claim 1 for use in a therapeutic treatment method of a human or animal body.
[14" claim-type="Currently amended] A compound of formula (I) or a pharmaceutically acceptable salt or in vivo hydrolyzable ester thereof according to claim 1 for use as a therapeutic agent.
[15" claim-type="Currently amended] A method of treating a metalloproteinase mediated disease condition comprising administering to a warm blooded animal a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or hydrolyzable ester thereof in vivo.
[16" claim-type="Currently amended] The method of claim 15, comprising treating the disease state mediated by at least one of MMP13, agrecanase, MMP9, MMP12.
[17" claim-type="Currently amended] Use of a compound of formula (I) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof in the manufacture of a medicament for treating a disease state mediated by one or more metalloproteinase enzymes.
[18" claim-type="Currently amended] Use of a compound of formula (I) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof in the manufacture of a medicament for the treatment of arthritis.
[19" claim-type="Currently amended] Use of a compound of formula (I) or a pharmaceutically acceptable salt or in vivo hydrolyzable ester thereof in the manufacture of a medicament for the treatment of atherosclerosis.
[20" claim-type="Currently amended] Use of a compound of formula (I) or a pharmaceutically acceptable salt or in vivo hydrolyzable ester thereof in the manufacture of a medicament for the treatment of chronic obstructive pulmonary disease.
[21" claim-type="Currently amended] As in the following scheme, reacting a compound of formula II with a compound of formula R1CHO to obtain an alkene of formula III, converting the alkene to a compound of formula IV, then converting a compound of formula IV A compound of formula (I) or a pharmaceutically acceptable thereof, comprising the step of converting to a compound of formula (I), and then optionally forming a pharmaceutically acceptable salt or in vivo hydrolyzable ester of the compound of formula (I) Processes for preparing possible salts or hydrolyzable esters in vivo:

[22" claim-type="Currently amended] As in the following scheme, reacting a compound of formula II with a compound of formula R1COOR to obtain a compound of formula VIII, converting a compound of formula VIII to a compound of formula IX, a compound of formula IX Converting the alkene of III, converting the alkene to a compound of formula IV, then converting a compound of formula IV to a compound of formula I, and then optionally a pharmaceutical of the compound of formula I A method for preparing a compound of formula (I) or a pharmaceutically acceptable salt or in vivo hydrolyzable ester thereof, comprising the step of forming an acceptable salt or in vivo hydrolyzable ester:

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-02-21|Priority to EP00400467.7

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2000-02-21|Priority to EP00400467

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2001-02-15|Application filed by 아스트라제네카 아베

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2001-02-15|Priority to PCT/GB2001/000624

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2002-09-27|Publication of KR20020073594A

优先权:

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申请号 | 申请日 | 专利标题

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EP00400467.7|2000-02-21|

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EP00400467|2000-02-21|

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PCT/GB2001/000624|WO2001062742A1|2000-02-21|2001-02-15|Piperidine- and piperazine substituted n-hydroxyformamides as inhibitors of metalloproteinases|