Patent Grant
10087144
The present invention relates to a series of quinolone derivatives which act as C-type natriuretic peptide receptor (NPRC) agonists.
C-type natriuretic peptide (CNP) is a vasoactive peptide that has vasodilatory and diuretic properties. CNP is a potent relaxant of vascular smooth muscle, particularly in the coronary circulation. CNP also inhibits smooth muscle cell proliferation and aldosterone production. CNP is also an atherogenic agent, i.e. has antiaggregatory and anticoagulant properties, and is an anti-inflammatory. The biological actions of CNP are mediated via activation of specific natriuretic peptide receptors (NPR5), particularly the NPR-B and -C subtypes. NPR-C is the predominant subtype involved in the vasorelaxant response to CNP (Chauhan, S. D. et al, (2003) Proceedings of the National Academy of Sciences, 100, 3, 1426-1431).
CNP has been found to be useful in the prevention and treatment of ischemic vascular disease, e.g. myocardial infarction or stroke, and other cardiovascular disorders, e.g. hypertension, atherosclerosis, restenosis, and myocardial ischaemia/reperfusion injury (Hobbs, A., et al, (2004), Circulation, 110, 1231-1235). CNP has also been found to exert a strong anti-atherogenic influence on blood vessel walls (Ahluwali, A., et al, (2005) Trends in Pharmacological Sciences, 26, 3, 162-167).
CNP is therefore potentially useful in treating a wide range of vascular disorders. There are, of course, disadvantages associated with using a protein-based drug in a therapeutic context. In particular, oral administration results in the rapid hydrolysis of amide bonds in the protein, leading to the destruction of the protein and loss of therapeutic efficacy in vivo. There is therefore a need to provide small molecules that mimic CNP and which do not degrade easily when administered in a therapeutic context.
It has now been found that the quinolone derivatives of formula I mimic CNP in binding to NPRC receptors. They will therefore be therapeutically useful, but are unlikely to suffer from the disadvantages outlined above.
Quinolone antibacterial agents are disclosed in Koga, et al, J. Med. Chem. 1980, 23, 1358-1363.
The present invention therefore provides compounds of formula I, tautomers thereof, or pharmaceutically acceptable salts or N-oxides thereof for use in the treatment or prevention of cardiovascular disease:
Some of the compounds of formula I are new, and accordingly, the present invention also provides compounds of formula (1), tautomers thereof, or pharmaceutically acceptable salts or N-oxides thereof:
wherein
and/or
and/or
and/or
The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Compounds of formula I containing one or more chiral centres may be used in enantiomerically or diastereoisomerically pure form, or in the form of a mixture of isomers. For the avoidance of doubt, the compounds of formula I can, if desired, be used in the form of solvates. Further, for the avoidance of doubt, the compounds of the invention may be used in any tautomeric form.
As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p-toluenesulphonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines and heterocyclic amines.
A compound of formula I can be salified by known methods, by contacting the compound with an appropriate acid or base.
As used herein, a said C1-C6 alkylene group or moiety is a linear or branched alkylene group or moiety. Typically, a C1-C6 alkylene group or moiety is a C1-C4 alkylene group or moiety, preferably a C1-C2 alkylene group or moiety. Examples of a C1-C6 alkylene group or moiety include methylene, ethylene, n-propylene and n-butylene groups and moieties. Methylene and ethylene are preferred.
As used herein, a C2-C6 alkenylene group or moiety is a linear or branched alkylene group or moiety. Typically, a C2-C6 alkenylene group or moiety is a C2-C4 alkenylene group or moiety. Examples include —CH═CH—, —CH2—CH═CH—, —CH═CH—CH2—, —CH═CH—CH2—CH2—, —CH2—CH═CH—CH2—, —CH2—CH2—CH═CH—, and —CH═CH—CH═CH—. —CH═CH— is preferred.
As used herein, a C2-C6 alkynylene group or moiety is a linear or branched alkylene group or moiety. Typically, a C2-C6 alkynylene group or moiety is a C2-C4 alkynylene group or moiety. Examples include —C≡C—, —CH2—C≡C—, —C≡C—CH2—, —C≡C—CH2—CH2—, CH2—C≡C—CH2—, —CH2—CH2—C≡C—, and —C≡C—C≡C—. —C≡C— is preferred.
As used herein, a halogen is typically chlorine, fluorine, bromine or iodine and is preferably chlorine, bromine or fluorine, more preferably chlorine or bromine, most preferably bromine. A halogen atom may be chosen from chlorine, bromine and iodine.
As used herein, a C1-C8 alkyl group or moiety is a linear or branched alkyl group or moiety containing from 1 to 8 carbon atoms. Typically, a C1-C8 alkyl group or moiety is a C1-C6 alkyl group or moiety. Examples of C1-C6 alkyl groups and moieties include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl and hexyl. More typically, a C1-C8 alkyl group or moiety is a C1-C4 alkyl group or moiety. Examples of C1-C4 alkyl groups and moieties include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl and t-butyl. For the avoidance of doubt, where two alkyl moieties are present in a group or compound of formula (I), the alkyl moieties may be the same or different.
As used herein, a C1-C6 alkoxy group is typically a said C1-C6 alkyl group attached to an oxygen atom. Typically, a C1-C6 alkoxy group or moiety is a C1-C4 alkoxy group or moiety, preferably a C1-C2 alkoxy group. Methoxy is preferred.
A haloalkyl or haloalkoxy group is typically a said alkyl or alkoxy group substituted by one or more said halogen atoms. Typically, it is substituted by 1, 2 or 3 said halogen atoms. Preferred haloalkyl and haloalkoxy groups include perhaloalkyl and perhaloalkoxy groups such as —CX3 and —OCX3 wherein X is a said halogen atom, for example chlorine and fluorine. Particularly preferred haloalkyl groups are —CF3 and —CCl3, —CF3 is especially preferred. Particularly preferred haloalkoxy groups are —OCF3 and —OCCl3. —OCF3 is especially preferred.
A C1-C4 hydroxyalkyl group is typically a said alkyl group substituted by one or more hydroxyl (—OH) groups. Typically, it is substituted by 1, 2 or 3 hydroxyl groups, preferably 1 or 2, more preferably one hydroxyl group. Preferred hydroxyalkyl groups are —CH2OH, —CH2CH2OH and —CH(OH)CH3 groups. —CH2OH is especially preferred.
As used herein, a C6-C10 aryl group or moiety is a monocyclic or polycyclic, aromatic ring containing from 6 to 10 carbon atoms. Examples of such aryl groups include phenyl, naphthalene and azulene. Phenyl and naphthylene are preferred.
As used herein, a 5- to 10-membered heteroaryl group or moiety is typically a monocyclic 5- to 10-membered aromatic group or moiety, such as a 5- or 6-membered ring containing at least one heteroatom, for example 1, 2 or 3 heteroatoms, selected from O, S and N. Examples include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, furanyl, thienyl, pyrazolidinyl, pyrrolyl, oxadiazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, imidazolyl, pyridazolyl, and pyrazolyl, groups. Preferred examples include furanyl, pyridyl, and thienyl groups. Thienyl and pyridyl groups are particularly preferred.
As used herein, a C3-C10 cycloalkyl group or moiety is typically a monocyclic or bicyclic, preferably monocyclic, non-aromatic, saturated or unsaturated C5-C10 carbocyclic ring. Saturated rings are preferred. Typically, a C3-C10 cycloalkyl group or moiety is a C3-C6 cycloalkyl group or moiety, preferably a C3-C4 cycloalkyl group or moiety. Examples of C3-C10 cycloalkyl groups and moieties include cyclopropyl and cyclobutyl groups. Cyclopropyl groups are preferred.
As used herein, a 5- to 10-membered heterocyclyl group or moiety or heterocyclic ring is typically a monocyclic or bicyclic, non-aromatic, saturated or unsaturated C5-C10 carbocyclic ring in which one or more, for example 1, 2 or 3, of the carbon atoms are replaced with a moiety selected from N, O, S, S(O) and S(O)2, preferably N or O. One or more, for example 1, 2 or 3, of the carbon atoms may be replaced with a moiety selected from C═O and C═NR, where R is hydrogen or C1-C4 alkyl, preferably hydrogen. Typically, it is a 5- to 9-membered ring. Suitable heterocyclyl groups, moieties and rings include pyrazolidinyl, piperidyl, piperazinyl, morpholinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, 1,3-dioxolanyl, 1,4-dioxolyl, pyrazolinyl and octahydro-1H-pyrrolo[3,4-b]pyridine groups and moieties and the group
Piperidyl, piperazinyl, morpholinyl, 1,3-dioxolanyl, 1H-pyrrolo[3,4-b]pyridine and
groups, moieties and rings are preferred.
The aryl, heteroaryl, cycloalkyl and heterocyclyl groups in the compounds of formula (I) are unsubstituted or substituted with one or more substituents, for example 1, 2 or 3 substituents, selected from halogen, hydroxy, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, —SOR, —SO2R, —NR′R″, —NR′(C═O)R″, —COOR, nitro and cyano substituents, wherein R, R′ and R″ are the same or different and each represents a hydrogen atom or C1-C4 alkyl group.
Typically, said aryl, heteroaryl, cycloalkyl and heterocyclyl groups in the compounds of formula (I) are unsubstituted or substituted with one or more, for example 1, 2 or 3, substituents selected from halogen, hydroxy, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, —SOR, —SO2R, —NR′R″, —NR′(C═O)R″, and —COOR substituents, wherein R, R′ and R″ are the same or different and each represents a hydrogen atom or C1-C4 alkyl group. For the avoidance of doubt, the substituents on said aryl, heteroaryl, cycloalkyl and heterocyclyl groups in the compound of formula (I) are themselves unsubstituted.
Typically, the present invention provides a compound of formula I as defined herein, or a pharmaceutically acceptable salt or N-oxide thereof for use in the treatment or prevention of cardiovascular disease. Preferably, the present invention provides a compound of formula I as defined herein, or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of cardiovascular disease.
Typically, the compound of formula (I) is other than 4-hydroxyquinoline-3-carboxylic acid, 5-phenylethyl-4-hydroxyquinoline-3-carboxylic acid, 7-benzyloxy-4-hydroxyquinoline-3-carboxylic acid, 7-phenethyloxy-4-hydroxyquinoline-3-carboxylic acid, 7-phenoxyethoxy-4-hydroxyquinoline-3-carboxylic acid, or tautomers thereof, for example
and/or when R is —OH, then R2, R3, R4, R5, R6 and R7 are not all hydrogen atoms; and/or when R is —OR′ wherein R′ is a hydrogen atom, a C1-C6 alkyl group or a C1-C6 haloalkyl group, then R2, R3, R4, R5, R6 and R7 are not all hydrogen atoms; and/or when R is —OR′ wherein R′ is a hydrogen atom, a C1-C6 alkyl group or a C1-C6 haloalkyl group, then R4 and R5 are not both isobutoxy groups; and/or when R is —OR′ wherein R′ is a hydrogen atom, a C1-C6 alkyl group or a C1-C6 haloalkyl group, then R6 is hydrogen.
More typically, when R is —OH, then R2, R3, R4, R5, and R7 are not all hydrogen atoms; and/or when R is —OR′ wherein R′ is a hydrogen atom, a C1-C6 alkyl group or a C1-C6 haloalkyl group, then R2, R3, R4, R5, and R7 are not all hydrogen atoms.
Generally, when R is —OR′ wherein R′ is a hydrogen atom, a C1-C6 alkyl group or a C1-C6 haloalkyl group, then R2, R3, R4, R5, and R7 are not all hydrogen atoms; when R is —OR′ wherein R′ is a hydrogen atom, a C1-C6 alkyl group or a C1-C6 haloalkyl group, then R4 and R5 are not both isobutoxy groups; when R is —OR′ wherein R′ is a hydrogen atom, a C1-C6 alkyl group or a C1-C6 haloalkyl group, then R6 is hydrogen; and the compound of formula (I) is other than 5-phenylethyl-4-hydroxyquinoline-3-carboxylic acid, 7-benzyloxy-4-hydroxyquinoline-3-carboxylic acid, 7-phenethyloxy-4-hydroxyquinoline-3-carboxylic acid, 7-phenoxyethoxy-4-hydroxyquinoline-3-carboxylic acid, or tautomers thereof.
Typically, the compound of formula (I) is other than flumequine, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, grepafloxacin, levofloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gatifloxacin, gemifloxacin, moxifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, delafloxacin, danofloxacin, difloxacin, enrofloxacin, ibafloxacin, marbofloxacin, orbifloxacin, and sarafloxacin. Preferably, the compound of formula (I) is other than a compound of formula (If) or (Ig), as defined herein, where R4 and/or R5 is a fluorine atom. More preferably in the compound of formula (I), R4 and/or R5 is other than a fluorine atom. Thus, in certain embodiments, the compound of formula (I) is other than a fluoroquinolone. Fluoroquinolones are a well-known class of antiobiotic compound which would be familiar to the skilled person.
Typically, V is CR3.
Typically, B is —(C═O)R1.
Typically, the 5- to 10-membered heteroaryl groups which may be present at the B position are 5- to 6-membered, preferably 5-membered. Preferred groups include oxadiazole and thiazole groups. Those groups are typically unsubstituted or substituted with 1, 2 or 3, preferably 1 or 2, more preferably 1 substituent chosen from halogen, hydroxy, C1-C4 alkyl and C1-C4 haloalkyl substituents.
Typically, in the group -L′″-NRR′, which may be present at the B position, R is a hydrogen atom. Typically, R′ is a hydrogen atom or a C1-C4 alkyl or C haloalkyl group. The group L′″ in the group -L′″-NRR′, which may be present at the B position, is typically a C1-C6 alkylene group, preferably a C alkylene group, more preferably a C alkylene group, most preferably a methylene group.
Typically, the 5- to 10-membered heterocyclyl groups which may be present at the R1 position are 5- to 6-membered, preferably 5-membered. Typically, the heterocyclyl group is a monocyclic carbocyclic ring in which 1, 2 or 3, preferably 2 of the carbon atoms are replaced with a moiety selected from N, O, or S, preferably N. One or more, for example 1, 2 or 3, preferably 2 of the carbon atoms may be replaced with a moiety selected from C═O and C═NR, where R is hydrogen or C1-C4 alkyl, preferably hydrogen. Suitable groups include the group
Those groups are typically unsubstituted or substituted with 1, 2 or 3, preferably 1 or 2, more preferably 1 substituent chosen from halogen, hydroxy, C1-C4 alkyl and C1-C4 haloalkyl substituents. Those groups are preferably unsubstituted.
Typically, R1 is —OR′, wherein R′ is a hydrogen atom, a C1-C6 alkyl group or a C1-C6 haloalkyl group, or R1 is a proteinogenic α amino acid, which is linked to the carbonyl moiety in the compound of formula (I) via the α amino group, which amino acid is optionally esterified at the α carboxylic acid group with a C1-C6 alkyl group or a C6-C10 aryl group.
Typically, in the group —NR″R′″, which may be present at the R1 position, R″ is a hydrogen atom. R′″ is typically a hydrogen atom, a C1-C4 alkyl or C1-C4 haloalkyl group.
Typically, in the group —NRIV-L′″-(C═O)NR″R′″ which may be present at the R1 position, RIV is a hydrogen atom. L′″ is typically a a C1-C6 alkylene group, preferably a C1-C4 alkylene group, more preferably a C1-C2 alkylene group, most preferably a methylene group. R″ is typically a hydrogen atom. R′″ is typically a hydrogen atom, a C1-C4 alkyl or C1-C4 haloalkyl group. The group —NRIV-L′″-(C═O)NR″R′″ is preferably —NH—CH2—(C═O)NHR′″.
Typically, in the group —NRIV-L′″-(C═O)OR which may be present at the R1 position, RIV is a hydrogen atom. L′″ is typically a a C1-C6 alkylene group, preferably a C1-C4 alkylene group, more preferably a C1-C2 alkylene group, most preferably an ethylene group. R is typically a hydrogen atom or C1-C4 alkyl group, preferably a hydrogen atom or methyl group. The group —NRIV-L′″-(C═O)OR is preferably —NH—(CH2)2—(C═O)OR, more preferably —NH—(CH2)2—(C═O)OH or —NH—(CH2)2—(C═O)OCH3.
Typically, the compound of formula I is of formula I′:
wherein W, X, Y, Z, R1, R2, R3, R7 and R9 are as defined above.
As mentioned above, either (a) W is N and R9 and R2 together form a bond, or (b) W is CR8, and R8 and R9 together form a bond. Thus, the compound of formula (I) is either a compound of formula (Ia) or (Ib):
wherein R2, R7, B, V, X, Y and Z are as defined herein.
Typically, A2, represents a C6-C10 aryl group or a 5-6-membered heteroaryl group, preferably a phenyl, naphthyl or 5- to 6-membered heteroaryl group, more preferably a phenyl group.
R1 may be a proteinogenic α amino acid, which is linked to the carbonyl moiety in the compound of formula (I) via the α amino group, which amino acid is optionally esterified at the α carboxylic acid group with a C1-C6 alkyl group or a C6-C10 aryl group. Proteinogenic α amino acids are well known to the person skilled in the art. For the avoidance of doubt, however, the proteinogenic α amino acids are those amino acids that are found in proteins. These are alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan and tyrosine.
When R1 is a proteinogenic α amino acid, then R1 is typically a group of formula —NH—CHR—CO2R′, wherein R′ is a hydrogen atom, C1-C6 alkyl group or C6-C10 aryl group and R is a hydrogen atom, or a methyl, —(CH2)3—NH—(C═NH)—NH2, —CH2CONH2, —CH2CO2H, —CH2SH, —(CH2)2CO2H, —(CH2)2CONH2, —CH(CH3)CH2CH3, CH2CH(CH3)2, —(CH2)4NH2, —(CH2)2SCH3, —CH2Ph, —CH2OH, —CH(CH3)OH, —CH2-p-hydroxy-Ph, —CH(CH3)2, —CH2SeH group, or a
group.
R′ in the moiety —NH—CHR—CO2R′ is typically a hydrogen atom or C1-C6 alkyl group, preferably a hydrogen atom or C1-C4 alkyl group, more preferably a hydrogen atom, or a methyl or t-butyl group.
R in the moiety —NH—CHR—CO2R′ is preferably a hydrogen atom, or a methyl, —CH2CH(CH3)2, —CH2-Ph or —CH(CH3)2 group.
Thus, the proteinogenic α amino acid is preferably glycine, alanine, leucine, phenylalanine or valine. Alanine is typically L-alanine. Leucine is typically L-leucine. Phenylalanine is typically L-phenylalanine. Valine is typically L-valine.
The amino acid is typically optionally esterified at the α carboxylic acid group with a C1-C6 alkyl group, preferably a C1-C4 alkyl group, more preferably a methyl or t-butyl group.
Typically, when R is a moiety OR′, it is a hydroxyl group or C1-C6 alkoxy group, preferably a hydroxyl group or C1-C4 alkoxy group, more preferably a hydroxyl group or C1-C2 alkoxy group.
When R1 is a group —NRIV-L′″-COOR, L′″ is typically a C2-C6 alkylene group. Typically, the group —NRIV-L′″-COOR is other than a proteinogenic α amino acid which is linked to the carbonyl moiety in the compound of formula (I) via the α amino group as defined herein.
Typically, R1 is a 5- to 10-membered heterocyclyl group, or R1 is a proteinogenic α amino acid, which is linked to the carbonyl moiety in the compound of formula (I) via the α amino group, which amino acid is optionally esterified at the a carboxylic acid group with a C1-C6 alkyl group or a C6-C10 aryl group, or R1 is —NR″R′″, —NRIV-L′″-CONR″R′″, or —NRIV-L′″-COOR, wherein L′″, R, R″, R′″ and RIV are the same or different and each represents a hydrogen atom, a C1-C6 alkyl group or a C1-C6 haloalkyl group;
More typically, R1 is a 5- to 10-membered heterocyclyl group, or —OR′, wherein R′ represents a hydrogen atom or C1-C4 alkyl group, or R1 is a group of formula —NH—CHRIV—CO2RV, wherein RV is a hydrogen atom or C1-C6 alkyl group, and RIV is a hydrogen atom, or a methyl, —(CH2)3—NH—(C═NH)—NH2, —CH2CONH2, —CH2CO2H, —CH2SH, —(CH2)2CO2H, —(CH2)2CONH2, —CH(CH3)CH2CH3, CH2CH(CH3)2, —(CH2)4NH2, —(CH2)2SCH3, —CH2Ph, —CH2OH, —CH(CH3)OH, —CH2-p-hydroxy-Ph, —CH(CH3)2, —CH2SeH group, or a
group; or R1 is a group of formula —NRIV-L′″-COOR wherein RIV is a hydrogen atom, L′″ is a C2-C4 alkylene group, and R is a hydrogen atom or C1-C4 alkyl group.
Even more typically, R1 is a 5- to 10-membered heterocyclyl group, or R1 is a group of formula —NH—CHRIV—CO2RV, wherein RV is a hydrogen atom or C1-C6 alkyl group, and RIV is a hydrogen atom, or a methyl, —(CH2)3—NH—(C═NH)—NH2, —CH2CONH2, —CH2CO2H, —CH2SH, —(CH2)2CO2H, —(CH2)2CONH2, —CH(CH3)CH2CH3, CH2CH(CH3)2, —(CH2)4NH2, —(CH2)2SCH3, —CH2Ph, —CH2OH, —CH(CH3)OH, —CH2-p-hydroxy-Ph, —CH(CH3)2, —CH2SeH group, or a
group; or R1 is a group of formula —NRIV-L′″-COOR wherein RIV is a hydrogen atom, L′″ is a C2-C4 alkylene group, and R is a hydrogen atom or C1-C4 alkyl group.
Preferably, R1 is a 5-membered heterocyclyl group, or —OR′, wherein R′ is a hydrogen atom or C1-C4 alkyl group, or R1 is a group of formula —NH—CHRIV—CO2RV, wherein RV is a hydrogen atom or C1-C6 alkyl group, and RIV is a hydrogen atom, or a methyl, —CH2CH(CH3)2, —CH2-Ph or —CH(CH3)2 group; or R1 is a group —NH—(CH2)2—COOR, where R is hydrogen or C1-C4 alkyl.
More preferably, R1 is a 5-membered heterocyclyl group, or R1 is a group of formula —NH—CHRIV—CO2RV, wherein RV is a hydrogen atom or C1-C6 alkyl group, and RIV is a hydrogen atom, or a methyl, —CH2CH(CH3)2, —CH2-Ph or —CH(CH3)2 group; or R1 is a group of —NH—(CH2)2—COOR, where R is hydrogen or C1-C4 alkyl.
Even more preferably, R1 is
or R1 is —OR′ wherein R′ is a hydrogen atom or C1-C2 alkyl group,
or R1 is a group of formula —NH—CHRIV—CO2RV, wherein RV is a hydrogen atom or C1-C4 alkyl group, and RIV is a hydrogen atom, or a methyl, —CH2CH(CH3)2, —CH2-Ph or —CH(CH3)2 group; or R1 is —NH—(CH2)2—COOH or —NH—(CH2)2—COOCH3.
Most preferably, R1 is
or R1 is a group of formula —NH—CHRIV—CO2RV, wherein RV is a hydrogen atom or C1-C4 alkyl group, and RIV is a hydrogen atom, or a methyl, —CH2CH(CH3)2, —CH2-Ph or —CH(CH3)2 group; or R1 is —NH—(CH2)2—COOH or —NH—(CH2)2—COOCH3.
Typically, L′ is a C1-C6 alkylene group, preferably a C1-C4 alkylene group, more preferably a C1-C2 alkylene group, most preferably a —CH2— group.
Typically, A2 is a C6-C10 aryl group, preferably a phenyl group.
Typically, the moiety A2 is unsubstituted or substituted with one, two or three, preferably one or two, substituents selected from halogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, —SO2R, and —NR′(C═O)R″ substituents, wherein R, R′ and R″ are the same or different and each represents a hydrogen atom or C1-C4 alkyl group. Preferably, these substituents are selected from halogen, C1-C2 alkyl, C1-C2 alkoxy, C1-C2 haloalkyl, C1-C2 haloalkoxy —SO2R, and —NH(C═O)R″ substituents wherein R and R″ are the same or different and each represents a C1-C2 alkyl group.
More preferably, A2 is a phenyl group which is unsubstituted or substituted by one or two substituents selected from chlorine, fluorine, C1-C2 alkyl, C1-C2 alkoxy, CF3O—, —SO2Me, and —NHAc substituents. Most preferably, A2 is unsubstituted phenyl, chlorophenyl, fluorophenyl, methoxyphenyl, trifluoromethoxyphenyl, dimethylphenyl, methylsulphonylphenyl and acetaminophenyl.
As mentioned above, when W is CR8 and Z is CR6, R2 may form, together with R6 and the carbon and nitrogen atoms which connect R2 and R6 in the formula (I), a 5- to 6-membered heterocyclic ring. In this embodiment, the compound of formula (I) is of formula (Ic):
wherein B, V, X, Y, R1, and R7 are as defined herein and the ring R represents a 5- to 6-membered heterocyclic ring.
In addition to the nitrogen atom to which R2 is bonded, said heterocyclic ring typically contains zero, one or two, preferably one, further heteroatoms selected from N and O, preferably O. Said heterocyclic ring is typically a 6-membered ring. Said heterocyclic ring is more preferably a piperidine or morpholine ring. Said heterocyclic ring is typically unsubstituted or substituted substituted with one or two substituents selected from halogen, C1-C4 alkyl and C1-C4 alkoxy substituents. Said heterocyclic ring is preferably substituted with one C1-C4 alkyl substituent, more preferably one C1-C2 alkyl substituent, most preferably a methyl substituent.
In a preferred embodiment, R2 forms, together with R6 and the carbon and nitrogen atoms which connect R2 and R6 in the formula (I), a morpholine or piperidine group, said morpholine and piperidine groups being unsubstituted or substituted with a C1-C2 alkyl group.
In one particularly preferred embodiment, said heterocyclic ring is a piperidine ring substituted with a methyl group. In this embodiment, said compound of formula (I) is preferably a compound of formula (Ic′):
wherein B, V, X, Y, R1, and R7 are as defined herein.
In another particularly preferred embodiment, said heterocyclic ring is a morpholine ring substituted with a methyl group. In this embodiment, said compound of formula (I) is preferably a compound of formula (Id):
wherein B, V, X, Y, R1, and R7 are as defined herein.
Typically, when W is CR8, R2 is a hydrogen atom, or a C1-C6 alkyl, phenyl, -L′-A2, C3-C6 cycloalkyl, or —COOR′ group, wherein R′ is a hydrogen atom or C1-C4 alkyl group, or when Z is a moiety CR6, R2 may form, together with R6 and the carbon and nitrogen atoms which connect R2 and R6 in the formula (I), a 5- to 6-membered heterocyclic ring, said cycloalkyl, phenyl and heterocyclic groups being typically unsubstituted or substituted with one or two substituents selected from halogen, C1-C4 alkyl and C1-C4 alkoxy substituents, L′ is a C1-C6 alkylene group, and A2 is a phenyl group, which is unsubstituted or substituted with one, two or three substituents selected from halogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, —SO2R, and —NR′(C═O)R″ substituents, wherein R, R′ and R″ are the same or different and each represents a hydrogen atom or C1-C4 alkyl group.
Preferably, when W is CR8, R2 is a hydrogen atom, or a C1-C4 alkyl, phenyl, A2, C3-C4 cycloalkyl, or —COOR′ group, wherein R′ is a C1-C4 alkyl group, or when Z is a moiety CR6, R2 may form, together with R6 and the carbon and nitrogen atoms which connect R2 and R6 in the formula (I), a 6-membered heterocyclic ring, said cycloalkyl group typically being unsubstituted, said heterocyclic ring typically being unsubstituted or substituted with one C1-C4 alkyl group, said phenyl group typically being substituted with one halogen substituent, L′ is a C1-C4 alkylene group, and A2 is a phenyl group, which is unsubstituted or substituted with one or two substituents selected from halogen, C1-C2 alkyl, C1-C2 alkoxy, C1-C2 haloalkyl, C1-C2 haloalkoxy —SO2R, and —NH(C═O)R″ substituents wherein R and R″ are the same or different and each represents a C1-C2 alkyl group.
More preferably, when W is CR8, R2 is a hydrogen atom, or a C1-C3 alkyl, -L′-A2, cyclopropyl, —COOtBu group, or a phenyl group which is unsubstituted or substituted with a fluorine atom, or when Z is a moiety CR6, R2 may form, together with R6 and the carbon and nitrogen atoms which connect R2 and R6 in the formula (I), a morpholine or piperidine group, said morpholine and piperidine groups being unsubstituted or substituted with a C1-C2 alkyl group, L′ is a —CH2— group, and A2 is a phenyl group which is unsubstituted or substituted by one or two substituents selected from chlorine, fluorine, C1-C2 alkyl, C1-C2 alkoxy, CF3O—, —SO2Me, and —NHAc substituents.
Typically, R3 is a hydrogen atom, or —NR′R″ group, wherein R′ and R″ are the same or different and each represent a hydrogen atom or C1-C4 alkyl group.
Preferably, R3 is hydrogen or —NH2.
R4 and R5 may form, together with the carbon atoms bonded to R4 and R5, a 5- to 6-membered heterocyclic ring. Said heterocyclic ring typically contains one, two or three, preferably two, heteroatoms selected from N and O, preferably O. Said heterocyclic ring is typically a 5-membered ring. Said heterocyclic ring is preferably a dioxolane ring, more preferably a 1,3-dioxolane ring. Said heterocyclic ring is preferably unsubstituted. In one particularly preferred embodiment, said heterocyclic ring is an unsubstituted 1,3-dioxolane ring. In this embodiment, said compound of formula (I) is preferably a compound of formula (Ie):
wherein W, Z, R1, R2, R3, R7 and R9 are as defined herein.
Typically, when R4 and R5 form, together with the carbon atoms bonded to R4 and R5, a 5- to 6-membered heterocyclic ring, then R2 does not form, together with R6 and the carbon and nitrogen atoms which connect R2 and R6 in the formula (I), a 5- to 6-membered heterocyclic ring.
Typically, R4 is a hydrogen atom, a halogen atom, or a hydroxy, C1-C8 alkyl, C1-C8 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, nitro, —NR′R″, —CO2R′″, 5- to 10-membered heteroaryl, C6-C10 aryl or —CO—(C1-C6 alkyl) group, wherein R′, R″ and R′″ are the same or different and each represent a hydrogen atom or C1-C6 alkyl group, or when Y is a moiety CR5, R4 may form, together with R5 and the carbon atoms bonded to R4 and R5, a 5- to 6-membered heterocyclic ring.
Preferably, R4 is a hydrogen atom, a halogen atom, or a C1-C8 alkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, nitro, —NR′R″, —CO2R′″, 5- to 6-membered heteroaryl, C6-C10 aryl or —CO—(C1-C4 alkyl) group, wherein R′, R″ and R′″ are the same or different and each represent a hydrogen atom or C1-C4 alkyl group, or when Y is a moiety CR5, R4 may form, together with R5 and the carbon atoms bonded to R4 and R5, a 5- to 6-membered heterocyclic ring, said heterocyclic and heteroaryl groups being unsubstituted or substituted with one or two substituents selected from halogen, C1-C4 alkyl and C1-C4 alkoxy substituents, said aryl groups being unsubstituted or substituted with one or two substitutents selected from halogen, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, —NR′(C═O)R″, or —COOR substituents, wherein R, R′ and R″ are the same or different and each represents a hydrogen atom or C1-C4 alkyl group.
More preferably, R4 is a hydrogen atom, a halogen atom, or a C1-C6 alkyl, C1-C2 alkoxy, C1-C2 haloalkoxy, nitro, —NR′R″, —CO2R′″, 5- or 6-membered heteroaryl, phenyl or —CO—(C1-C2 alkyl) group, wherein R′ and R″ are the same or different and each represent a C1-C4 alkyl group, and R′″ is a hydrogen atom, or when Y is a moiety CR5, R4 may form, together with R5 and the carbon atoms bonded to R4 and R5, a 5-membered heterocyclic ring, wherein the 5-membered heterocyclic and 5- or 6-membered heteroaryl groups are unsubstituted and the phenyl group is unsubstituted or substituted with a C1-C2 hydroxyalkyl, —NH(C═O)R″, or —COOR substituent, wherein R and R″ are the same or different and each represents a C1-C2 alkyl group.
Most preferably, R4 is a hydrogen, bromine or fluorine atom, or a C1-C6 alkyl, methoxy, trifluoromethoxy, nitro, —NMe2, —CO2H, unsubstituted thiophene, unsubstituted pyridine, phenyl or —COMe group, or when Y is a moiety CR5, R4 may form, together with R5 and the carbon atoms bonded to R4 and R5, an unsubstituted 1,3-dioxolane group, said phenyl group being substituted with one —COOH, —CH2OH or —NH(C═O)Me substituent.
Typically, R4 is a bromine atom, or a C6-C10 aryl or 5- to 10-membered heteroaryl group, said aryl and heteroaryl groups being unsubstituted or substituted with one or more substituents selected from halogen, hydroxy, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, —SOR, —SO2R, —NR′(C═O)R″, —COOR, nitro and cyano substituents, wherein R, R′ and R″ are the same or different and each represents a hydrogen atom or C1-C4 alkyl group.
Preferably, R4 is a bromine atom, or a 5- to 10-membered heteroaryl or C6-C10 aryl group, said heteroaryl groups being unsubstituted or substituted with one or two substituents selected from halogen, C1-C4 alkyl and C1-C4 alkoxy substituents, and said aryl groups being unsubstituted or substituted with one or two substitutents selected from halogen, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, —NR′(C═O)R″, or —COOR substituents, wherein R, R′ and R″ are the same or different and each represents a hydrogen atomor C1-C4 alkyl group.
More preferably, R4 is a bromine atom, or a 5- or 6-membered heteroaryl or phenyl group, wherein the 5- or 6-membered heteroaryl groups are unsubstituted and the phenyl group is unsubstituted or substituted with a C1-C2 hydroxyalkyl, —NH(C═O)R″, or —COOR substituent, wherein R and R″ are the same or different and each represents a hydrogen atom or C1-C2 alkyl group.
Most preferably, R4 is a bromine atom, or an unsubstituted thiophene group, an unsubstituted pyridine group or a phenyl group which is substituted with one —COOH, —CH2OH or —NH(C═O)Me substituent.
R4 and/or R5 may be a 5 to 10-membered heterocyclyl group. Said group is typically a monocyclic or bicyclic, saturated C5-C10 carbocyclic ring in which one or more, for example 1 or 2 of the carbon atoms are replaced with a moiety selected from N, O, and S, preferably N and O. Examples of such groups include piperidine, piperazine and octahydro-1H-pyrrolo[3,4-b]pyridine. Said heterocyclyl group is typically 6 to 9-membered. Said heterocyclyl group is typically unsubstituted or substituted with one, two or three substituents selected from halogen atoms, C1-C4 alkyl and hydroxy groups, preferably one or two substituents selected from C1-C2 alkyl and hydroxy groups. Particularly preferred examples are unsubstituted octahydro-1H-pyrrolo[3,4-b]pyridine, unsubstituted piperazine, N-methyl-piperazine, dimethylpiperazine, and hydroxypiperidine.
Typically, when R5 is a 5 to 10-membered heterocyclyl group, R4 is other than a 5 to 10-membered heterocyclyl group.
Typically, R5 is a hydrogen atom, a halogen atom, or a C1-C6 alkyl, C1-C6 haloalkyl, 5 to 10-membered heterocyclyl, or —CO2R′ group, wherein R′ is a hydrogen atom or C1-C6 alkyl group, or when X is a moiety CR4, R5 may form, together with R4 and the carbon atoms bonded to R4 and R5, a 5- to 6-membered heterocyclic ring.
Preferably, R5 is a hydrogen atom, or a C1-C4 alkyl, C1-C4 haloalkyl, 5 to 10-membered heterocyclyl, or —CO2R′ group, wherein R′ is a hydrogen atom or a C1-C4 alkyl group, or when X is a moiety CR4, R5 may form, together with R4 and the carbon atoms bonded to R4 and R5, a 5- to 6-membered heterocyclic ring, said heterocyclicgroups typically being unsubstituted or substituted with one or two substituents selected from halogen, hydroxyl, C1-C4 alkyl and C1-C4 alkoxy substituents.
More preferably, R5 is a hydrogen atom, or a C1-C2 alkyl, C1-C2 haloalkyl, 6 to 9-membered heterocyclyl, or —CO2H group, said 6 to 9-membered heterocyclyl group typically being unsubstituted or substituted with one or two substituents selected from hydroxyl and C1-C2 alkyl groups, or when X is a moiety CR4, R5 may form, together with R5 and the carbon atoms bonded to R4 and R5, a 5-membered heterocyclic ring which is typically unsubstituted.
Most preferably, R5 is a hydrogen atom, or a methyl, trifluoromethyl, unsubstituted octahydro-1H-pyrrolo[3,4-b]pyridine, unsubstituted piperazine, N-methyl-piperazine, dimethylpiperazine, hydroxypiperidine, or —CO2H group, or when X is a moiety CR4, R5 may form, together with R5 and the carbon atoms bonded to R4 and R5, an unsubstituted 1,3-dioxolane group.
Typically, R6 is a hydrogen atom, a halogen atom, or a C1-C6 alkyl, C1-C6 alkoxy, or —CO2R′ group, wherein R′ is a hydrogen atom or C1-C4 alkyl group, or when W is a moiety CR8, R6 may form, together with R2 and the carbon and nitrogen atoms which connect R6 and R2 in the formula (I), a 5- to 6-membered heterocyclic ring, said heterocyclic ring being typically unsubstituted or substituted with one or two substituents selected from halogen, C1-C4 alkyl and C1-C4 alkoxy substituents.
Preferably, R6 is a hydrogen atom, a halogen atom, or a C1-C4 alkyl, C1-C2 alkoxy, or —CO2H group, or when W is a moiety CR8, R6 may form, together with R2 and the carbon and nitrogen atoms which connect R6 and R2 in the formula (I), a 6-membered heterocyclic ring, said heterocyclic ring being unsubstituted or substituted with one C1-C4 alkyl substituent.
More preferably, R6 is a hydrogen atom, or a C1-C3 alkyl, methoxy, or —CO2H group, or when W is a moiety CR8, R6 may form, together with R2 and the carbon and nitrogen atoms which connect R6 and R2 in the formula (I), a morpholine or piperidine group, said morpholine and piperidine groups being unsubstituted or substituted with a methyl group.
Typically, R7 is hydrogen.
Typically, not more than three of V, W, X, Y and Z are N.
Preferably, not more than two of V, W, X, Y and Z are N.
In a preferred embodiment, the compounds of formula (I) are of formula (If):
wherein R1-R7 are as defined herein.
In a further preferred embodiment, the compounds of formula (I) are of formula (Ig):
wherein R1-R5 and R7 are as defined herein.
In a further preferred embodiment, the compounds of formula (I) are of formula (Ih):
wherein R1-R3, R5 and R7 are as defined herein.
In a further preferred embodiment, the compounds of formula (I) are of formula (Ij):
wherein Y, R1, R3, R4, R6 and R7 are as defined herein.
Preferred compounds of the invention are compounds in which:
Further preferred compounds of the invention are compounds in which:
Most preferably, compounds of the invention are compounds in which:
Typically, in compounds of formula (If),
Preferably, in compounds of formula (If):
Preferably, in compounds of formula (If):
More preferably, in compounds of formula (If):
Preferred compounds of the invention are
tautomers thereof, pharmaceutically acceptable salts thereof, and/or N-oxides thereof.
Particularly preferred compounds are compounds 28, 57, 59, 60, 61, 62, 63, 85, 87, 88, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 116, 117, 118, 119, 134, 139, 142, 178, 187, 202, 200, 189, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311 and 312, tautomers thereof, pharmaceutically acceptable salts thereof, and/or N-oxides thereof.
Compounds 28, 61, 63, 88, 111, 112, 114, 116, 117, 118, 119, 134 and 187, tautomers thereof, pharmaceutically acceptable salts thereof, and/or N-oxides thereof are more preferred.
Compounds 28, 118 and 187, tautomers thereof, pharmaceutically acceptable salts thereof, and/or N-oxides thereof are even more preferred.
Compounds 28, 118, tautomers thereof, pharmaceutically acceptable salts thereof, and/or N-oxides thereof are particularly preferred.
Compounds of the invention can be made in accordance with known methods.
When W is CR8, and R8 and R9 together form a bond, compounds of formula I″ can be prepared following the reaction scheme below.
wherein B, V, X, Y, Z, R2 and R7 are as defined herein, provided that R2 together with R6 and the carbon and nitrogen atoms which connect R2 and R6 so not form a 5- to 6-membered heterocyclic ring. The first step of the reaction is typically carried out at 120-130 degrees C. for between 2 and 3 hours. The second step of the reaction is typically carried out by refluxing in diphenyl ether for 8 hours. Compounds of formula II and III are typically commercially available or can be prepared by analogy with known methods.
When W is N, and R2 and R9 are linked to form a bond, compounds of formula Ia″ can be prepared following the reaction scheme below.
wherein B, V, X, Y, Z, and R7 are as defined herein. The first step of the reaction is typically carried out at 120-130 degrees C. for between 2 and 3 hours. The second step of the reaction is typically carried out by refluxing in diphenyl ether for 8 hours. Compounds of formula IIa and III are typically commercially available or can be prepared by analogy with known methods.
When R2 is a C1-C6 alkyl, C1-C6 haloalkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, 5- to 10-membered heterocyclyl, C3-C10 cycloalkyl, or —COOR′ group, wherein R′ is a hydrogen atom or C1-C6 alkyl group, compounds of formula I″ can be prepared following the reaction scheme below.
wherein LG is a good leaving group, typically a halogen atom, triflate or mesylate group, preferably a halogen atom, more preferably a bromine atom. Compounds of formula I′″ by analogy with the method described above.
When B is —(C═O)R1, and R1 is a proteinogenic α amino acid, which is optionally esterified at the α carboxylic acid group with a C1-C6 alkyl group or a C6-C10 aryl group, compounds of formula I″ can be prepared by reacting a compound of IIV with N,N-dimethylacetamide, and N-ethyldiisopropylamine followed by O-(7-Azabenzotriazol-1-yl)-N,N,N′N′-tetramethylutonium hexafluorophosphate and the hydrochloride salt of the proteinogenic α amino acid, which is optionally esterified at the α carboxylic acid group with a C1-C6 alkyl group or a C6-C10 aryl group.
wherein W, V, X, Y, Z, R2, R7 and R9 are as defined herein.
Compounds of formula IIV can be prepared by saponifying a compound of formula IV
wherein W, V, X, Y, Z, R2, R7 and R9 are as defined herein, and R′ is a C1-C6 alkyl group or a C1-C6 haloalkyl group. Compounds of formula IV can be prepared by analogy with the method for producing compounds of formula I from compounds of formula II and III above.
The present invention also provides a pharmaceutical composition comprising a compound of the invention and one or more pharmaceutically acceptable diluents or carriers.
The compounds of the invention may be administered in a variety of dosage forms.
Thus, they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. The compounds of the invention may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The compounds may also be administered as suppositories.
A compound of the invention is typically formulated for administration with a pharmaceutically acceptable carrier or diluent. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.
Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
Solutions for intravenous administration or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
A therapeutically effective amount of a compound of the invention is administered to a patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific compound, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.
The present invention also provides a product containing (i) a compound of the invention and (ii) a further therapeutic agent. The further therapeutic agent is typically a therapeutic agent that is used in the treatment of cardiovascular disease. Examples of such therapeutic agents include statins, anti-platelet agents and anti-hypertensives. Thus, the compounds of the present invention may be administered as a monotherapy or in combination with one or more further therapeutic agents as defined herein. The potent therapeutic effect of the compounds of the present invention is such that they are particularly suitable for administration as a monotherapy.
Statins are a well known class of drug that lowers blood cholesterol levels. A person skilled in the art would have no difficulty in choosing a statin for combination with compounds of the present invention. Examples of statins suitable for use in combination with compounds of the present invention include atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.
Anti-platelet agents are a well known class of drug that decreases blood platelet aggregation and inhibits thrombus formation. A person skilled in the art would have no difficulty in choosing an anti-platelet agent for combination with compounds of the present invention. Examples of anti-platelet agents suitable for use in combination with compounds of the present invention include aspirin, clopidogrel, ticlopidine, cilostazol, abciximab, eptifibatide, tirofiban, defibrotide and dipyridamole. Preferred anti-platelet agents are aspirin and clopidogrel.
Anti-hypertensives are a well known class of drug that decrease blood pressure. A person skilled in the art would have no difficulty in choosing an anti-hypertensive agent for combination with compounds of the present invention. Examples of anti-hypertensive agents include diuretics, adrenergic receptor antagonists, calcium channel blockers, renin inhibitors, ACE inhibitors, angiotensin II receptor antagonists, aldosterone antagonists, vasodilators, alpha-2 agonists, and endothelin receptor blockers. Specific examples of anti-hypertensive agents suitable for use in combination with compounds of the present invention include bumetanide, ethacrynic acid, furosemide, torsemide, epitizide, hydrochlorothiazide, chlorothiazide, bendroflumethiazide, indapamide, chlorthalidone, metolazone, amiloride, triamterene, spironolactone, atenolol, metoprolol, nadolol, nebivolol, oxprenolol, pindolol, propranolol, timolol, doxazosin, phentolamine, indoramin, phenoxybenzamine, prazosin, terazosin, tolazoline, bucindolol, carvedilol, labetalol, amlodipine, Cilnidipine, felodipine, isradipine, lercanidipine, nicardipine, nifedipine, nimodipine, nitrendipine, diltiazem, verapamil, aliskiren, captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, trandolapril, benazepril, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan, eplerenone, spironolactone, sodium nitroprusside, hydralazine, Clonidine, Guanabenz, Methyldopa, Moxonidine, Guanethidine, Reserpine and bosentan, and combinations thereof.
The compounds of the present invention are capable of agonising C-type natriuretic peptide receptors. They can therefore be used to treat or prevent any disease or condition that responds to agonism of the C-type natriuretic peptide receptors. The present invention therefore also provides a compound, composition or product of the invention for use in the treatment of a disease or condition alleviated by agonism of the natriuretic peptide receptors, in particular the natriuretic peptide receptor C.
Diseases and conditions alleviated by agonism of the natriuretic peptide receptors include cardiovascular and/or heart disease, peripheral vascular disease, and sepsis.
Examples of cardiovascular diseases which can be treated or prevented with the compounds of the invention include arteriosclerosis, atherosclerosis, myocardial ischemia, angina, congestive heart failure, ischemic vascular disease, stroke, myocardial infarction, aneurism, hypertension, restenosis, hypercholesterolaemia, hypertensive heart disease, ischemia/reperfusion injury and pulmonary hypertension.
In certain embodiments, the cardiovascular disease is heart disease.
In certain embodiments, the disease which can be treated or prevented is hypertension.
In other embodiments, the disease which can be treated or prevented is myocardial infarction.
The compounds of the present invention have been found to be vasodilators. Thus, the present invention also provides a compound of formula I, a tautomer thereof, or a pharmaceutically acceptable salt or N-oxide thereof as defined herein for use in the treatment or prevention of cardiovascular disease as defined herein by vasodilation. Also provided is a compound of formula I, a tautomer thereof, or a pharmaceutically acceptable salt or N-oxide thereof as defined herein for use in a method of vasodilation in a patient suffering from cardiovascular disease as defined herein.
The present invention also provides use of a compound of the invention in the manufacture of a medicament, for use in the treatment or prevention of a disease or condition that responds to agonism of the natriuretic peptide receptors, as defined herein.
The present invention also provides a method of treating or preventing a condition that responds to agonism of the natriuretic peptide receptors, as defined herein, in a patient, which method comprises administering to said patient an effective amount of a compound of the present invention.
The present invention also provides a compound of formula I, a tautomer thereof, or a pharmaceutically acceptable salt or N-oxide thereof as defined herein for use in the treatment or prevention of an inflammatory disease. Also provided is a compound of formula I, a tautomer thereof, or a pharmaceutically acceptable salt or N-oxide thereof as defined herein for use as an anti-inflammatory in a patient suffering from cardiovascular disease as defined herein.
The present invention also provides use of a compound of the invention in the manufacture of a medicament, for use in the treatment or prevention of an inflammatory disease or condition.
The present invention also provides a method of treating or preventing of an inflammatory disease or condition in a patient, which method comprises administering to said patient an effective amount of a compound of the present invention.
Examples of inflammation and inflammatory diseases or conditions which can be treated or prevented with the compounds of the invention include acne vulgaris, Alzheimer's, ankylosing spondylitis, arthritis (including e.g. osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis), asthma, atherosclerosis, autoimmune diseases, celiac disease, chronic prostatitis, colitis, crohn's disease, dermatitis, diverticulitis, fibromyalgia, glomerulonephritis, hepatitis, hypersensitivities, inflammatory bowel diseases, interstitial cystitis, irritable bowel syndrome (IBS), lupus erythematous, nephritis, Parkinson's disease, pelvic inflammatory disease, peritonitis, reperfusion injury, rheumatoid arthritis, sarcoidosis, systemic lupus erythematous (SLE), systemic sclerosis, transplant rejection, ulcerative colitis and vasculitis.
Typically, the patient treated in accordance with the present invention is a human.
Generally, the patient treated in accordance with the present invention does not have type 2 diabetes; and/or has not been diagnosed as having type 2 diabetes; and/or is not being treated with antidiabetic treatment; and/or has not been treated with antidiabetic treatment.
In compounds of formula (1), typically one of criteria (a) to (d) applies. Alternatively, more than one of (a) to (d) applies.
Thus, in certain embodiments (a) and (b) both apply, in which case the present invention provides compounds of formula (1) wherein:
Typically, preferred substituent definitions for compounds of formula (1) are the same as those for compounds of formula (I).
Typically, in (a), B is —(C═O)R1, and R1 is a 5- to 10-membered heterocyclyl group, or R1 is a group of formula —NH—CHRIV—CO2RV, wherein RV is a hydrogen atom or C1-C6 alkyl group, and RIV is a hydrogen atom, or a methyl, —(CH2)3—NH—(C═NH)—NH2, —CH2CONH2, —CH2CO2H, —CH2SH, —(CH2)2CO2H, —(CH2)2CONH2, —CH(CH3)CH2CH3, CH2CH(CH3)2, —(CH2)4NH2, —(CH2)2SCH3, —CH2Ph, —CH2OH, —CH(CH3)OH, —CH2-p-hydroxy-Ph, —CH(CH3)2, —CH2SeH group, or a
group; or R1 is a group of formula —NRIV-L′″-COOR wherein RIV is a hydrogen atom, L′″ is a C2-C4 alkylene group, and R is a hydrogen atom or C1-C4 alkyl group, provided that the compound is other than
More typically in (a), R1 is a 5-membered heterocyclyl group, or R1 is a group of formula —NH—CHRIV—CO2RV, wherein RV is a hydrogen atom or C1-C6 alkyl group, and RIV is a hydrogen atom, or a methyl, —CH2CH(CH3)2, —CH2-Ph or —CH(CH3)2 group; or R1 is a group of —NH—(CH2)2—COOR, where R is hydrogen or C1-C4 alkyl provided that the compound is other than
Generally in (a), R1 is
or R1 is a group of formula —NH—CHRIV—CO2RV, wherein RV is a hydrogen atom or C1-C4 alkyl group, and RIV is a hydrogen atom, or a methyl, —CH2CH(CH3)2, —CH2-Ph or —CH(CH3)2 group; or R1 is —NH—(CH2)2—COOH or —NH—(CH2)2—COOCH3, provided that the compound is other than
Typically in (b), R4 is a bromine atom, nitro group, or a C6-C10 aryl or 5- to 6-membered heteroaryl group, said heteroaryl groups being unsubstituted or substituted with one or two substituents selected from halogen, C1-C4 alkyl and C1-C4 alkoxy substituents, and said aryl groups being unsubstituted or substituted with one or two substitutents selected from halogen, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, —NR′(C═O)R″, or —COOR substituents, wherein R, R′ and R″ are the same or different and each represents a hydrogen atom or C1-C4 alkyl group, providing that the compound is other than
More typically in (b), R4 is a bromine atom, nitro group, or a phenyl or 5- or 6-membered heteroaryl group wherein the heteroaryl groups are unsubstituted and the phenyl group is unsubstituted or substituted with a C1-C2 hydroxyalkyl, —NH(C═O)R″, or —COOR substituent, wherein R and R″ are the same or different and each represents a C1-C2 alkyl group, providing that the compound is other than
Generally in (b), R4 is a bromine atom, nitro group, or a phenyl or unsusbstituted thiophene or pyridine group, said phenyl group being substituted with one —COOH, —CH2OH or —NH(C═O)Me substituent, providing that the compound is other than
Typically in (c), the compound is of formula (Ij) as defined herein, providing that the compound is other than
Typically in (d), R2 is -L′-A2 or —COOR′, wherein R′ is a hydrogen atom or C1-C4 alkyl group, and wherein L′ is a C1-C6 alkylene group, and A2 is a phenyl group, which is substituted with two substituents selected from C1-C4 alkyl substituents.
More typically in (d), R2 is -L′-A2 or —COOR′, wherein R′ is a C1-C4 alkyl group, and wherein L′ is a C1-C4 alkylene group, and A2 is a phenyl group, which is substituted with two substituents selected from C1-C4 alkyl substituents.
Generally in (d), R2 is -L′-A2 or —COOtBu, wherein L′ is a —CH2— group, and A2 is a phenyl group which is substituted by two C1-C2 alkyl substituents.
The present invention also provides a compound, a tautomer thereof, or a pharmaceutically acceptable salt and//or N-oxide thereof, the compound being selected from compounds 6, 27, 28, 29, 59, 60, 61, 62, 63, 64, 82, 83, 87, 88, 116, 117, 118, 119, 120, 127, 128, 152, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205 301, 302, 303, 304, 305, 306, 307, 308 and 309, whose structures are given above.
Compounds 28, 59, 60, 61, 62, 63, 87, 88, 116, 117, 118, 119, 178, 187, 189, 200, 202, 301, 302, 303, 304, 305, 306, 307, 308 and 309, tautomers thereof, pharmaceutically acceptable salts and//or N-oxides thereof are preferred.
Compounds 28, 61, 63, 88, 116, 117, 118, 119 and 187, tautomers thereof, pharmaceutically acceptable salts and//or N-oxides thereof are more preferred.
Compounds 28, 118, and 187, tautomers thereof, pharmaceutically acceptable salts and//or N-oxides thereof are even more preferred.
Compounds 28, and 118, tautomers thereof, pharmaceutically acceptable salts and//or N-oxides thereof are still more preferred. The structures of these compounds are shown below.
The present invention also provides a compound as defined herein or product as defined herein, for use in a method of treatment of the human or animal body.
The present invention also provides use of a compound as defined herein or product as defined herein, in the manufacture of a medicament for the treatment of the human or animal body.
The following Examples illustrate the invention.
1. General Experimental
Solvents
N,N-Dimethylformamide, tetrahydrofuran, 1,4-dioxane were purchased dry from the Aldrich Chemical Company in sure-Seal™ bottles. Water was distilled. All other solvents were used as supplied without further purification (Analytical or HPLC grade).
Reagents
Reactions performed under an atmosphere of nitrogen or hydrogen gas were maintained by an inflated balloon.
Chromatography
Thin layer chromatography (TLC) was performed on Whatman glass plates coated with MK6F Silica Gel 60 Å, visualized using UV light and/or iodine gas. Purification via flash column chromatography was performed on Biotage Isolute® pre-packed columns (Flash Si II type) unless otherwise stated.
Melting Points
Melting points were recorded on a Gallenkamp Melting Point Apparatus.
Nuclear Magnetic Resonance Spectroscopy
Nuclear Magnetic Resonance (NMR) Spectra were recorded on Bruker DRX 500, AMX 300 spectrometers in the deuterated solvent stated. Chemical shifts (δ) are quoted in ppm and coupling constants (J) in Hz. Coupling constants are quoted twice. Residual signals from the solvents were used as an internal reference.
Infra-Red Spectroscopy
Infra-red (IR) spectra were recorded on a Perkin-Elmer Spectrum One IR Fourier Transform spectrophotometer and using thin films on KBr or Ge plates as stated. Only the characteristic peaks are quoted and in units of cm−1.
Mass Spectrometry
High resolution mass spectra (HRMS) were measured on a VG70 SE mass spec or a Thermo Finnigan MAT900xp mass spec Electrospray-Thermo Finnigan MAT900xp.
Elemental Microanalysis
Elemental analyses were performed by the microanalysis service of the Department of Chemistry, University College London, London.
Polarimetry
Optical rotations, [α]D, were measured on a Perkin-Elmer Model 343 polarimeter with a path length of 1.0 dm. Concentrations are quoted in g 100 ml−1. The Na lamp was set at 589 nm.
Reference paper: H. Koga, Itoh, S. Murayama, S. Suzue, T. Irikura, J. Med. Chem., 1980, 23, 1358. 4-Bromoaniline (3.440 g, 20.00 mMol) was stirred in diethyl ethoxymethylenemalonate (4.04 mL, 20.00 mMol) at 120-130° C. for 2 hours and 20 minutes. T.l.c. analysis (ethyl acetate:cyclohexane, 1:2) showed the presence of one UV-active product (Rf 0.56) and complete consumption of both starting materials (Rf 0.42 and 0.45). Upon cooling down of the reaction solution to room temperature, intermediate 1 solidified (a white crystalline solid). The solid was then re-heated (86° C.) and concentrated in vacuo in order to remove any ethanol formed during the reaction. M.p. 94.5-97° C. (dichloromethane); HRMS (EI): found 341.02539 [M]+ C14H16NO479Br requires 341.02572; found 343.02298 [M]+ C14H16NO481Br requires 343.02338; peak ratio: 49.7%:50.3%; νmax (thin film): 3258, 3199, 3164 (w, NH, hydrogen-bonding), 3105, 3069 (w, ArC—H), 2981, 2938, 2902 (m, alkyl C—H), 1686 (s, 2×intramolecularly hydrogen-bonded C═O conjugated with C═C), 1641, 1614 (s, C═C conjugated with C═O) cm−1; δH (CDCl3, 300 MHz): 1.32 (3H, t, JCH3,CH2 7.2 Hz, CH3), 1.37 (3H, t, JCH3,CH2 7.2 Hz, CH3), 4.24 (2H, q, JCH2,CH3 7.2 Hz, CH2), 4.30 (2H, q, JCH2,CH3 7.1 Hz, CH2), 7.01 (2H, d, J 9.1 Hz, 2×ArCH—C—Br), 7.48 (2H, d, J 8.8 Hz, 2×ArCH—C—N), 8.45 (1H, d, JCH,NH 13.3 Hz, CH—NH), 11.00 (1H, d, JNH,CH 13.4 Hz, CH—NH); δC (CDCl3, 75 MHz): 14.3, 14.4 (2×CH3), 60.2, 60.5 (2×CH2), 94.4 (O═C—C—C═O), 117.6 (ArCquat-Br), 118.7 (2×ArCH—Cquat-NH), 132.8 (2×ArCH—Cquat-Br), 138.4 (ArCquat-NH), 151.4 (NH—CH), 165.6 (C═O), 169.0 (hydrogen bonded C═O). Diphenyl ether (100 g) was then added to the crude solid and the mixture was refluxed at 260° C. for 8 hours. After 1.5 hours a white solid started crushing out of solution in the reaction flask. T.l.c. analysis (ethyl acetate:cyclohexane, 1:4) showed the presence of one UV-active product (Rf 0.75) and complete consumption of the intermediate starting material 1 (Rf 0.49). Upon cooling of the reaction solution to room temperature, more ethyl 6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylate 2 crushed out of solution as a white powder (at 230° C.). The white solid was filtered, washed with toluene and dried in vacuo to afford ethyl 6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylate 2 (2.587 g, 43.4%) as a pale yellow/white powder. M.p. 285-290° C. (decomposition: gas evolved); HRMS (EI): found 294.98314 [M]+ C12H10NO379Br requires 294.98386; found 296.98073 [M]+ C12H10NO381Br requires 296.98152; peak ratio: 49.5%:50.5%; νmax (Ge): 3421 (br s, NH), 3149, 3088 (w, ArC—H), 2981 (m, alkyl C—H), 1694 (s, 2×intramolecularly hydrogen-bonded C═O conjugated with C═C), 1615 (s, C═C conjugated with C═O) cm−1; Elemental Analysis: found C, 48.36%, H, 3.34%, N, 4.64%; required C, 48.67%, H, 3.40%, N, 4.73%.
Crude 6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylate 2 (1.000 g, 3.377 mMol) was stirred in DMF (6.7 mL) with potassium carbonate (1.159 g, 8.386 mMol) at 40° C. After 5 minutes benzyl bromide (1.99 mL, 16.771 mMol) was added dropwise and the temperature raised to 80° C. After 24.5 hours, LCMS analysis showed the presence of one product only (m/z 385-387) and complete consumption of the starting material (m/z 297-299). The reaction mixture was concentrated in vacuo and co-evaporated twice (toluene). The residue was partitioned between water and dichloromethane and the aqueous layer was washed with dichloromethane 3 times. The organic layers were collected, dried (magnesium sulphate), filtered and concentrated in vacuo to give crude ethyl 1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylate 3 as a sticky bright pale yellow solid. The solid residue was then dissolved in dichloromethane, pre-absorbed onto silica gel and purified by flash-chromatography (ethyl acetate to ethyl acetate:methanol 5% to ethyl acetate:methanol 10%) to afford ethyl 1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylate 3 (1.280 g, 98.1%) as a white crystalline solid. M.p. 166-168° C. (ethyl acetate/methanol); HRMS (EI): found 385.03131 [M]+ C19H16NO379Br requires 385.03081; found 387.02894 [M]+ C19H16NO381Br requires 387.02847; peak ratio: 51.9%:48.1%; νmax (thin film): 3035 (w, ArC—H), 2964, 2928, (m, alkyl C—H), 1725 (s, C═O ketone), 1686 (s, C═O ester, conjugated with C═C), 1623, 1606 (s, C═C conjugated with C═Os) cm−1; δH (CDCl3, 300 MHz): 1.32 (3H, t, JCH3,CH2 7.2 Hz, CH3), 4.30 (2H, q, JCH2,CH3 7.2 Hz, CH2), 5.30 (2H, s, CH2, Bn), 7.04-7.09 & 7.22-7.32 (5H, 2×m, 5×ArHs, Bn), 7.10 (1H, d, JH-C,H-B 9.0 Hz, ArHC), 7.50 (1H, dd, JH-B,H-C 9.0 Hz, JH-B,H-A 2.3 Hz, ArHB), 8.47 (1H, s, CH—N-Bn), 8.50 (1H, d, JH-A,H-B 2.3 Hz, ArHA); δC (CDCl3, 75 MHz): 14.4 (CH3), 57.6 (CH2, Bn), 61.0 (OCH2CH3), 111.5 (O═C—C—C═O), 118.6 (ArCquatC), 119.2 (Br-Cq), 126.0, 128.7, 129.4 (5×ArCs, Bn), 130.4 (CA), 130.5 (CA-Cq-C═O), 133.9 (Cq (Bn)), 135.5 (CB), 138.0 (CC-Cq-N-Bn), 149.7 (CD), 165.2 (CO2tBu), 173.1 (O═C—C═C). Soluble in DCM and a bit soluble in acetone.
Ethyl 1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylate 3 (1.000 g, 2.589 mMol) was refluxed at 150° C. in a 2 M sodium hydroxide aqueous solution (20 mL). After 3 hours the reaction mixture turned into a thick white slurry. LCMS analysis revealed the presence of one product (m/z 359-361) and complete consumption of the starting material (m/z 387-389). The reaction mixture was allowed to cool down to room temperature, distilled water was added (20 mL) to it and was subsequently neutralised with acetic acid (2.3 mL). The resulting precipitate was filtered, washed with water, ground and dried in vacuo to give 1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4 (786 mg, 84.7%) as a white solid. M.p.>250° C. (water); HRMS (TOF MS ES+): found 358.0096 [M+H+]+ C17H13NO379Br requires 358.0079; found 360.0077 [M+H+]+ νmax (Ge): 3034 (w, ArC—H), 1717 (s, COOH), 1675 (w, intramolecularly hydrogen-bonded C═O conjugated with C═C), 1615 (s, C═C conjugated with C═O) cm−1; δH (DMSO, 300 MHz): 14.76 (1H, s, COOH), 9.29 (1H, s, HD), 8.43 (1H, d, JHA,HB 2.3 Hz, HA), 8.01 (1H, dd, JHB,HC 9.2 Hz, JHB,HA 2.4 Hz, HB), 7.80 (1H, d, JHC,HB 9.2 Hz, HC), 7.42-7.14 (5H, m, 5×ArHs (Bn)), 5.86 (2H, s, CH2 (Bn)); δC (DMSO, 75 MHz): 56.5 (CH2(Bn)), 108.7 (O═C—C—C═O), 119.3 (Cq-Br), 121.1 (CC), 126.5, 127.3, 128.9 (5×ArHs (Bn)), 127.9 (CA), 128.0 (CA-Cq-C═O), 135.1 (Cq (Bn)), 136.6 (CB), 138.5 (CC-Cq-N-Bn), 150.5 (CD), 165.5 (COOH), 177.7 (O═C—C═C).
1-Benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4 (218 mg, 0.605 mMol) was stirred in N,N-dimethylacetamide (6 mL) and N-ethyldiisopropylamine (0.207 mL, 1.210 mMol). After 5 minutes O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (460 mg, 1.210 mMol) and L-alanine-tert-butyl ester hydrochloride (220 mg, 1210 mMol) were added (a yellow reaction solution) and the temperature was raised to 50° C. After 27 hours L.C.M.S. analysis revealed the presence of various compounds and the starting material (m/z 359-361) in the reaction solution, hence the temperature was raised to 70° C. and let to run for an extra 23 hours. L.C.M.S. and t.l.c. (ethyl acetate:methanol, 5%) analyses showed solely the presence of the product (m/z 486-488, Rf 0.80) and complete consumption of the starting material (Rf 0.00). The reaction solution turned into a deep purple red/red solution and a N,N-dimethylacetamide-soluble white crystalline residue collected on the internal wall of the flask. The reaction solution was co-evaporated in vacuo three times (methanol). The residue was pre-absorbed on silica gel and purified by flash-chromatography (ethyl acetate:cyclohexane, 1:2) to give (S)-tert-butyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)propanoate 5 (134 mg, 45.6%) as a white crystalline solid). M.p. 144.5-147° C. (ethyl acetate/cyclohexane); HRMS (ESI): found 507.09005 [M+Na]+ C24H25N2O479BrNa requires 507.08954; found 509.08684 [M+Na]+ C24H25N2O481BrNa requires 509.08749; peak ratio: 49.6%:50.4%; [α]D25+16.176 (c, 0.20 in dichloromethane); νmax (thin film): 3222, 3192 (w, NH, h-bonded), 3037 (m, ArC—H), 2980, 2933, 2874 (m, alkyl C—H), 1738 (m, C═OOtBu ester), 1729 (m, C═O ketone conjugated to C═C), 1660 (s, C═ONH, I and C═C conjugated with C═O), 1543 (m, CONH, II), 1601 (m, aromatic ring and C═C conjugated with C═O), 1482 (m, aromatic ring) cm−1; δH (Acetone-d6, 500 MHz): 1.44 (3H, d, JCH3,CH 7.2 Hz, Alanine CH3), 1.48 (9H, s, C(CH3)3), 4.55 (1H, a-quint, J 7.2 Hz, Alanine-CαH), 5.84 (2H, s, CH2 (Bn)), 7.25-7.40 (5H, m, 5×ArHs), 7.72 (1H, d, 3JH-C,H-B 9.0 Hz, HC), 7.81 (1H, dd, 3JH-B,H-C 9.1 Hz, 4JH-B,H-A 2.5 Hz, HB), 8.54 (1H, d, 4JH-A,H-B 2.5 Hz, HA), 8.98 (1H, s, HD), 10.23 (1H, d, JNH,CH 6.9 Hz, CONH); δC (Acetone-d6, 125 MHz): 18.8 (Alanine-CH3), 28.1 (C(CH3)3), 49.5 (Alanine-Cα), 57.6 (CH2 (Bn)), 81.5 (C(CH3)3), 112.9 (O═C—C—C═O), 119.1 (Br-Cq), 121.0 (CC), 127.4, 129.8, 129.9 (5×ArCs (Bn)), 129.0 (CA), 130.4 (CA-Cq-C═O), 136.2 (CB), 136.4 (Cq (Bn)), 139.5 (CC-Cq-N-Bn), 150.0 (CD), 164.2 (C═ONH), 172.7 (C═OOtBu), 175.7 (O═C—C═C); Elemental Analysis: found C, 59.54%, H, 5.56%, N, 5.60%, required C, 59.39%, H, 5.19%, N, 5.77%.
1-Benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4 (250 mg, 0.694 mMol) was stirred in N,N-dimethylacetamide (6.9 mL) and N-ethyldiisopropylamine (0.24 mL, 1.388 mMol). After 5 minutes O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (527 mg, 1.388 mMol) and L-leucine-tert-butyl ester hydrochloride (310 mg, 1.388 mMol) were added (a pale yellow reaction mixture) and the temperature was raised to 50° C. After 23 hours the reaction mixture turned pale brown and L.C.M.S. analysis revealed the presence of the starting material (m/z 359-361) and of the product (m/z 528-530), hence the temperature was raised to 70° C. and let to run for an extra 22 hours. L.C.M.S. and t.l.c. (ethyl acetate:methanol, 5%) analysis showed solely the presence of the product (Rf 0.86) and complete consumption of the starting material (Rf 0.00). The reaction mixture was filtered through Celite (acetone), the filtrate was co-evaporated three times (acetone) and the residue was pre-absorbed on silica gel and purified by flash-chromatography (ethyl acetate:cyclohexane, 1:2 to ethyl acetate) to give (S)-tert-butyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)-4-methylpentanoate 6 (92 mg, 25.1%) as a white crystalline solid. M.p. 185-186° C. (ethyl acetate/cyclohexane); HRMS (FAB+): found 549.13552 [M+Na]+ C27H31N2O479BrNa requires 549.13648; found 551.13342 [M+Na]+ C27H31N2O481BrNa requires 551.13414; peak ratio: 50.0%:50.0%; [α]D25 −7.540 (c, 0.25 in dichloromethane); νmax (thin film): 3214, 3180 (w, NH, h-bonded), 3034 (m, ArC—H), 2957, 2871 (m, alkyl C—H), 1724 (m with shoulder, C═OOtBu ester & C═O ketone conjugated to C═C), 1654 (s, C═ONH, I and C═C conjugated with C═O), 1535 (m, CONH, II), 1596 (m, aromatic ring and C═C conjugated with C═O), 1478 (m, aromatic ring) cm−1; δH (CDCl3, 300 MHz): 0.91 (6H, d, JCH3,CH 5.8 Hz, (CH3)2CH-leucine), 1.49 (9H, s, C(CH3)3), 1.55-1.70 (2H, m, CH2 (Leucine)), 1.77-1.87 (1H, m, CH (Leucine)), 4.68 (1H, m, J 7.2 Hz, CαH (Leucine)), 5.42 (2H, s, CH2 (Bn)), 7.10-7.15 (2H, m, 2×ArHs (Bn)), 7.22-7.38 (4H, m, 3×ArHs (Bn) & HC), 7.64 (1H, d, J 8.8 Hz, HB), 8.65 (1H, s, HA), 8.90 (1H, s, HD), 10.26 (1H, d, JNH,CαH 7.0 Hz, NH); δC (CDCl3, 125 MHz): 22.0, 23.1 (Leucine (CH3)2), 25.2 (Leucine CH), 28.1 (C(CH3)3), 41.6 (Leucine CH2), 51.9 (Leucine Cα), 57.9 (CH2 (Bn)), 81.5 (C(CH3)3), 112.4 (O═C—C—C═O), 118.7 (CC), 119.3 (Br-Cq), 126.1, 128.8, 129.5 (5×ArCs (Bn)), 129.6 (CA-Cq-C═O), 130.2 (CA), 133.9 (Cq (Bn)), 135.9 (CB), 138.2 (CC-Cq-N-Bn), 148.8 (CD), 164.4 (C═ONH), 172.6 (C═OOtBu), 175.6 (O═C—C═C).
1-Benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4 (200 mg, 0.558 mMol) was stirred in N,N-dimethylacetamide (5.5 mL). Subsequently N,N-diisopropylethylamine (191 μL, 1.117 mMol), O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (425 mg, 1.117 mMol) and L-phenylalanine tert-butyl ester hydrochloride (288 mg, 1.117 mMol) were added and the yellow reaction solution was allowed to run at 50° C. After 16.5 hours L.C.M.S. analysis of the light brown/red reaction solution showed the presence of one product (m/z 560-562) and the presence of some starting material (m/z 357-359). T.l.c. analysis (ethyl acetate:methanol, 5%) showed the presence of one product (Rf 0.71). The reaction solution was allowed to cool down, the solvent removed by co-evaporation (2 times with toluene, once with acetone). The residue was pre-absorbed on silica gel and purified by flash column chromatography (ethyl acetate:cyclohexane, 1:1 to ethyl acetate) to give (S)-tert-butyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-phenylpropanoate 7 (313 mg, quantitative) as a white crystalline solid. M.p. 162° C. (DCM) (starts decomposing (turning brown)), 178-182° C. (decomposes and partially melts (turns brown throughout)); HRMS (TOF MS ES+): found 583.1173 [M+Na]+ C30H29N2O479BrNa requires 583.1208; found 585.1201 [M+Na]+ C30H29N2O481BrNa requires 585.1188; peak ratio: 50.7%:49.3%; [α]D25 −52.451 (c, 0.20 in dichloromethane); νmax (thin film): 3221, 3177 (w, NH, h-bonded), 3087, 3033 (m, ArC—H), 2976, 2931 (m, alkyl C—H), 1744 (m, C═OOtBu ester), 1729 (m, C═O ketone conjugated to C═C), 1661 (s, C═ONH, I and C═C conjugated with C═O), 1542 (m, CONH, II), 1599 (m, aromatic ring and C═C conjugated with C═O), 1481 (m, aromatic ring) cm−1; δH (CDCl3, 300 MHz): 1.41 (9H, s, C(CH3)3), 3.18 (1H, d, JH-E,H-F 14.0 Hz, JH-E,CH 6.9 Hz, HE), 3.23 (1H, d, JH-F,H-E 14.0 Hz, JH-F,CH 6.2 Hz, HE), 4.87-4.97 (1H, a-q, JCH,H-E 7.0 Hz, JCH,H-F 6.3 Hz NHCH), 5.93 (2H, s, CH2, Phenylalanine), 7.08-7.38 (11H, m, 10×ArHs & ArHC), 7.61 (1H, dd, JH-B,H-C 9.1 Hz, J 2.6 Hz, ArHB), 8.62 (1H, d, J 2.2 Hz, ArHA), 8.83 (1H, s, HD), 10.41 (1H, d, J 8.0 Hz, NH); δC (CDCl3, 125 MHz): 28.1 (C(CH3)3), 38.7 (CH2, Phenylalanine), 54.6 (Phenylalanine-Cα), 57.9 (CH2, Bn), 81.9 (C(CH3)3), 112.3 (O═C—C—C═O), 118.7 (CC), 119.3 (Br-Cq), 126.2 126.9 127.1, 128.4, 128.8, 128.9, 129.4, 129.6, 129.7, (10×ArCs), 129.5 (CA-Cq-C═O), 130.2 (CA), 134.0 (Cq (Bn)), 135.8 (CB), 137.0 (Cq, Phenylalanine), 138.1 (CC-Cq-N-Bn), 148.7 (CD), 164.2 (C═ONH), 170.7 (CO2tBu), 175.5 (O═C—C═C).
1-Benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4 (200 mg, 0.558 mMol) was stirred in N,N-dimethylacetamide (5.5 mL). Subsequently N,N-diisopropylethylamine (191 μL, 1.117 mMol), O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (425 mg, 1.117 mMol) and L-glycine tert-butyl ester hydrochloride (187 mg, 1.117 mMol) were added and the yellow reaction solution was allowed to run at 50° C. After 16.5 hours L.C.M.S. analysis of the yellow/white reaction suspension showed the presence of one product (m/z 470-472) and complete consumption of the starting material (m/z 357-359). T.l.c. analysis (ethyl acetate:methanol, 5%) showed the presence of one product (Rf 0.74). The reaction mixture was allowed to cool down, the solvent removed by co-evaporation (3 times with toluene). The residue was pre-absorbed on silica gel and purified by flash column chromatography (ethyl acetate:cyclohexane, 1:2 to 1:1) to give tert-butyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetate 8 (196 mg, 74.5%) as a white crystalline solid. M.p. 150° C. (decomposition only: turned brown); HRMS (TOF MS ES+): found 493.0728 [M+Na]+ C23H23N2O479BrNa requires 493.0739; found 495.0677 [M+Na]+ C23H23N2O481BrNa requires 495.0718; peak ratio: 52.7%:47.3%; νmax (thin film): 3186 (w, broad, NH, h-bonded), 3035 (m, ArC—H), 2981, (m, alkyl C—H), 1732 (m with shoulder, C═OOtBu ester & C═O ketone conjugated to C═C), 1663 (s, C═ONH, I and C═C conjugated with C═O), 1547 (m, CONH, II), 1600 (m, aromatic ring and C═C conjugated with C═O), 1466 (m, Aromatic ring) cm−1; δH (CD2Cl2, 300 MHz): 1.49 (9H, s, C(CH3)3), 4.11 (2H, d, JCH2,NH 5.9 Hz, NHCH2C═O), 5.48 (2H, s, CH2 (Bn)), 7.12-7.20 & 7.30-7.40 (6H, 2×m, 5×ArHs (Bn) & ArHC), 7.68 (1H, dd, JH-B,H-C 9.1 Hz, JH-B,H-A 2.4 Hz, ArHB), 8.61 (1H, d, JH-A,H-B 2.4 Hz, ArHA), 8.90 (1H, s, 8.83 (1H, s, HD), 10.21 (1H, a-t, J 5.00 Hz, NH); δC (CD2Cl2, 125 MHz): 28.2 (C(CH3)3), 42.5 (Glycine-Cα), 58.2 (CH2 (Bn)), 82.0 (C(CH3)3), 112.3 (O═C—C—C═O), 119.3 (CC), 119.5 (Br-Cq), 126.6 129.0, 129.6 (5×ArCs), 129.9 (CA-Cq-C═O), 130.1 (CA), 134.5 (Cq (Bn)), 136.1 (CB), 138.7 (CC-Cq-N-Bn), 149.2 (CD), 165.0 (C═ONH), 169.4 (CO2tBu), 175.8 (O═C—C═C).
1-Benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4 (150 mg, 0.419 mMol) was stirred in N,N-dimethylacetamide (4.1 mL). Subsequently N,N-diisopropylethylamine (143 μL, 0.838 mMol), 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (318 mg, 0.838 mMol) and L-valine tert-butyl ester hydrochloride (176 mg, 0.838 mMol) were added and the light brown reaction solution was allowed to run at 50° C. After 19.5 hours t.l.c. analysis (ethyl acetate:methanol, 5%) of the light brown/white reaction suspension showed the presence of one product of one product (Rf 0.60). The reaction mixture was allowed to cool down, the solvent removed by co-evaporation (3 times with toluene). The residue was pre-absorbed on silica gel (methanol) and purified by flash column chromatography (ethyl acetate:cyclohexane, 2:3) to give (S)-tert-butyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-methylbutanoate 9 (144 mg, 66.9%) as a white crystalline solid. M.p. 218-219° C. (melting and decomposition); HRMS (TOF MS ES+): found 535.1195 [M+Na]+ C26H29N2O479BrNa requires 535.1208; found 537.1179 [M+Na]+ C26H29N2O481BrNa requires 537.1188; peak ratio: 50.6%:49.4%; [α]D25 +19.000 (c, 0.20 in dichloromethane); νmax (thin film): 3232, 3179 (w, NH, h-bonded), 3035 (m, ArC—H), 2963, 2930, 2871 (m, alkyl C—H), 1726 (m with shoulder, C═O ketone conjugated to C═C & C═OOtBu ester), 1663 (s, C═ONH, I and C═C conjugated with C═O), 1543 (m, CONH, II), 1599 (m, aromatic ring and C═C conjugated with C═O), 1482 (m, aromatic ring) cm−1; δH (CD2Cl2, 300 MHz): 0.93 (3H, d, JCH3,CH 6.9 Hz, Valine (CH3)2), 0.94 (3H, d, JCH3,CH 6.9 Hz, Valine (CH3)2), 1.40 (9H, s, C(CH3)3), 2.21 (1H, d septet, JCH,CH3 6.8 Hz, JCH,CαH 4.7 Hz, CHCH(CH3)2), 4.46 (1H, dd, JCH,CH 4.7 Hz, JCH,NH 8.5 Hz, Valine CαH), 5.38 (2H, s, CH2, Bn), 7.05-7.12 (2H, m, 2×ArHs (Bn)), 7.20-7.30 (4H, m, 3×ArHs (Bn) & ArHC), 7.59 (1H, dd, JH-B,H-C 9.1 Hz, JH-B,H-A 2.3 Hz, ArHB), 8.56 (1H, d, JH-A,H-B 2.4 Hz, ArHA), 8.79 (1H, s, HD), 10.18 (1H, d, JNH,CH 8.6 Hz, NH); δC (CD2Cl2, 125 MHz): 18.0, 19.5 (Valine CH(CH3)2), 28.2 (C(CH3)3), 31.6 (Valine CH(CH3)2), 58.1 (CH2 (Bn)), 58.4 (Valine-Cα), 81.6 (C(CH3)3), 112.8 (O═C—C—C═O), 119.2 (CC), 119.3 (Br-Cq), 126.6 128.9 129.6 (5×ArCs (Bn)), 130.0 (CA-Cq-C═O), 130.2 (CA), 134.6 (Cq (Bn)), 136.0 (CB), 138.7 (CC-Cq-N-Bn), 149.1 (CD), 164.5 (C═ONH), 171.3 (CO2tBu), 175.9 (O═C—C═C). Elemental Analysis: found C, 60.79%, H, 5.86%, N, 5.19%, required C, 60.82%, H, 5.69%, N, 5.46%.
1-Benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4 (200 mg, 0.5584 mMol) was stirred in dichloromethane (5 mL) followed by the addition of triethylamine (257 μL, 1.8426 mMol) and DPPA (156 μL, 0.7259 mMol). After 5 minutes L-valine methyl ester hydrochloride (112 mg, 0.6681 mMol) were added to the stirring solution at room temperature and under an atmosphere of nitrogen. After 18 hours t.l.c. analysis (ethyl acetate:cyclohexane, 1:1) showed the presence of one product of one product (Rf 0.31). The reaction mixture was filtered (ethyl acetate), the solvents were removed in vacuo and the residue was pre-absorbed on silica gel and purified by flash column chromatography (ethyl acetate:cyclohexane, 1:1 to 3:1 to ethyl acetate) followed by a recrystallisation of an impurity from dichloromethane/cyclohexane to give (S)-methyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-methylbutanoate 27 (242 mg, 91.9%) as a white fluffy solid. M.p. 230-232° C.; HRMS (ES+): found 493.0715 [M+Na]+ C23H23N2O479BrNa requires 493.0739; found 495.0708 [M+Na]+ C23H23N2O481BrNa requires 495.0718; peak ratio: 50.0%:50.0%; [α]D19.8 +83.8 (c, 0.14 in chloroform); νmax (thin film): 3194 (w, sharp, 2 bands, sec. NH), 3038 (w, ArC—H), 2962, 2872 (w, alkyl C—H), 1752 (w, C═O ester), 1729 (m, C═O ketone), 1662 (s, C═ONH, I), 1570 (m, C═ONH, II), 1600 (s, C═C conjugated with C═Os) cm−1; δH (CDCl3, 500 MHz): 10.44 (1H, d, JNH,CαH 7.5 Hz, NH), 8.93 (1H, s, HD), 8.68 (1H, s, ArHA), 7.68 (1H, d, JH-B,H-C 9.0 Hz, ArHB), 7.53-7.22 (4H, m, 3×ArHs (Bn) & ArHC), 7.16 (2H, m, 2×ArHs (Bn)), 5.48 (2H, s, CH2(Bn)), 4.72 (1H, dd, JCαH,NH 7.9, JCαH,CH 5.1 Hz, CαH), 3.76 (3H, s, OMe), 2.38 (1H, septet, JCH,CH3 6.6 Hz, CH (Valine)), 1.10 (6H, t, JCH3,CH 6.8 Hz, 2×CH3 (Valine)); δC (CDCl3, 125 MHz): 18.1, 19.5 (2×CH3 (Valine)), 31.0 (CH (Valine)), 52.1 (OCH3), 57.9 (Cα (Valine)), 58.0 (CH2 (Bn)), 112.2 (O═C—C—C═O), 118.8 (CC), 119.4 (Br-Cq), 126.2, 128.9, 129.5 (5×ArCs (Bn)), 129.4 (CC-Cq-N-Bn), 130.1 (CA), 133.9 (Cq(Bn)), 136.0 (CB), 138.2 (CA-Cq-C═O), 148.8 (CD), 164.8 (C═ONH), 172.4 (C═OOMe), 175.7 (O═C—C═C).
1-Benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4 (500 mg, 1.3959 mMol) was stirred in acetonitrile (20 mL) followed by triethylamine (973 μL, 6.9796 mMol) and 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (763 mg, 1.9543 mMol). After 5 minutes L-alanine methyl ester hydrochloride (273 mg, 1.9543 mMol) was added to the stirring solution. After 21 hours t.l.c. analysis (ethyl acetate:methanol, 5%) of the reaction mixture showed the presence of one product (Rf 0.88). The reaction mixture was filtered (ethyl acetate) and the filtrate was concentrated in vacuo. The residue was pre-absorbed on silica gel and purified by flash column chromatography (ethyl acetate:cyclohexane, 1:1, to 3:1, to 1:0) to give (S)-methyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)propanoate 14 (522 mg, 84%) as a white crystalline solid. M.p. 182-184° C.; HRMS (ES+): found 465.0434 [M+Na]+ C21H19N2O479BrNa requires 465.0426; found 467.0415 [M+Na]+ C21H19N2O481BrNa requires 467.0405; peak ratio: 51.7%: 49.3%; [α]D19.8 +83.8 (c, 0.14 in chloroform); νmax (thin film): 3220, 3189 (w, sharp, sec. NH), 3039 (w, ArC—H), 2997 (w, alkyl C—H), 1755 (m, C═O ester), 1743 (s, C═O ketone), 1662 (s, C═ONH, I), 1574 (m, C═ONH, II), 1602 (s, C═C conjugated with C═Os) cm−1; δH (CDCl3, 500 MHz): 1.52 (3H, d, JCH3,CαH 7.2 Hz, CH3 (Ala)), 3.74 (3H, s, OCH3), 4.74 (1H, a-quint, JCαH,CH3=JCαH,NH 7.2 Hz, CαH), 5.43 (2H, s, CH2(Bn)), 7.08-7.12 (2H, m, 2×ArHs (Bn)), 7.24-7.32 (4H, m, 3×ArHs (Bn) & ArHC), 7.61 (1H, dd, JH-B,H-C 9.0 Hz, JH-B,H-A 2.3 Hz, ArHB), 8.51 (1H, d, JH-A,H-B 2.2 Hz, ArHA), 8.86 (1H, s, HD), 10.30 (1H, d, JNH,CαH 7.1 Hz, NH); δC (CDCl3, 125 MHz): 18.3 (Alanine-CH3), 48.3 (Alanine-Cα), 52.4 (OCH3), 57.9 (CH2 (Bn)), 112.0 (O═C—C—C═O), 118.9 (CC), 119.3 (Br-Cq), 126.2, 128.8, 129.4 (5×ArCs (Bn)), 129.3 (CC-Cq-N-Bn), 129.9 (CA), 134.0 (Cq(Bn)), 135.9 (CB), 138.1 (CA-Cq-C═O), 148.8 (CD), 164.2 (C═ONH), 173.4 (C═OOMe), 175.4 (O═C—C═C).
1-Benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4 (500 mg, 1.3959 mMol) was stirred in acetonitrile (20 mL) followed by triethylamine (973 μL, 6.9796 mMol) and 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (763 mg, 1.9543 mMol). After 5 minutes glycine methyl ester hydrochloride (245 mg, 1.9543 mMol) was added to the stirring solution. After 21 hours t.l.c. analysis (ethyl acetate:methanol, 5%) of the reaction mixture showed the presence of one product (Rf 0.71). The reaction mixture was filtered (ethyl acetate) and the filtrate was concentrated in vacuo. The residue was pre-absorbed on silica gel and purified by flash column chromatography (ethyl acetate:cyclohexane, 1:1, to 3:1, to 1:0) to give methyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetate 15 (170 mg, 28%) as a white crystalline solid. M.p. 227-228.5° C. melting and decomposition (turned brown); HRMS (ES+): found 451.0268 [M+Na]+ C20H17N2O479BrNa requires 451.0269; found 453.0245 [M+Na]+ C20H17N2O481BrNa requires 453.0249; peak ratio: 49.7%:50.3%; νmax (thin film): 3473, 3411 (w, sharp, sec. NH), 3050 (w, ArC—H), 2980, 2945 (w, alkyl C—H), 1754 (w, C═O ester), 1742 (s, C═O ketone), 1658 (s, C═ONH, I), 1578 (m, C═ONH, II), 1612 (s, C═C conjugated with C═Os) cm−1; δH (CDCl3, 500 MHz): 3.77 (3H, s, OCH3), 4.25 (2H, d, JCαH,NH 5.7 Hz, CαH2), 5.43 (2H, s, CH2(Bn)), 7.10-7.12 (2H, m, 2×ArHs (Bn)), 7.26-7.36 (4H, m, 3×ArHs (Bn) & ArHC), 7.65 (1H, dd, JH-B,H-C 9.0 Hz, JH-B,H-A 2.4 Hz, ArHB), 8.64 (1H, d, JH-A,H-B 2.2 Hz, ArHA), 8.88 (1H, s, HD), 10.31 (1H, t, JH-B,CαH 5.5 Hz, NH); δC (CDCl3, 125 MHz): 41.4 (Cα), 52.3 (OCH3), 57.9 (CH2 (Bn)), 112.1 (O═C—C—C═O), 118.8 (CC), 119.4 (Br-Cq), 126.1, 128.9, 129.5 (5×ArCs (Bn)), 129.5 (CC-Cq-N-Bn), 130.1 (CA), 133.8 (Cq(Bn)), 136.0 (CB), 138.2 (CA-Cq-C═O), 148.8 (CD), 165.0 (C═ONH), 170.3 (C═OOMe), 175.6 (O═C—C═C).
1-Benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4 (500 mg, 1.3959 mMol) was stirred in acetonitrile (20 mL). Subsequently triethylamine (973 μL, 6.9796 mMol), 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (763 mg, 1.9543 mMol) and leucine methyl ester hydrochloride (355 mg, 1.9543 mMol) were added. After 27 hours t.l.c. analysis (ethyl acetate:cyclohexane, 1:1) of the reaction mixture showed the presence of one product (Rf 0.40). The reaction mixture was filtered (ethyl acetate) and the filtrate was concentrated in vacuo. The residue was pre-absorbed on silica gel and purified by flash column chromatography (ethyl acetate:cyclohexane, 1:4, to 1:1) to give (S)-methyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)-4-methylpentanoate 16 (310 mg, 46%) as a pale yellow crystalline solid. M.p. at 120° C. a gas started being released, at 210° C. a pale brown colour started developing, at 250° C. a dark brown colour was produced. Decomposition before melting; HRMS (ES+): found 507.0872 [M+Na]+ C24H25N2O479BrNa requires 507.0895; found 509.0872 [M+Na]+ C24H25N2O481BrNa requires 509.0875; peak ratio: 48.1%:51.9%; νmax (thin film): 3178 (w, sharp with shoulder, sec. NH), 3038 (w, ArC—H), 2956, 2870 (w, alkyl C—H), 1736 (m, C═O ester), 1662 (s, C═ONH, I), 1545 (m, C═ONH, II), 1600 (s, C═C conjugated with C═Os) cm−1; δH (CDCl3, 500 MHz): 10.27 (1H, d, JNH,CαH 7.3 Hz, NH), 8.90 (1H, s, HD), 8.61 (1H, d, JH-A,H-B 2.3, HA), 7.65 (1H, dd, JH-B,H-C 9.0 Hz, JH-B,H-A 2.4, HB), 7.36-7.27 (4H, m, 4×ArHs (Bn)), 7.29 (1H, d, JH-C,H-B 9.0 Hz, HC), 7.14-7.10 (1H, m, ArH), 5.44 (2H, s, CH2 (Bn)), 4.77 (1H, ddd, J 13.0 Hz, J 5.4 Hz, J 3.3 Hz, CαH), 3.74 (3H, s, OMe), 1.92-1.69 (3H, m, CH & CH2 (Leucine)), 0.99 (3H, d, J 6.2 Hz, CH3 (Leucine)), 0.97 (1H, d, J 6.3 Hz, CH3 (Leucine)); δC (CDCl3, 125 MHz): 21.4, 22.7 (2×CH3 (Leucine)), 24.7 (CH (Leucine)), 40.7 (CH2 (Leucine)), 50.8 (Cα), 51.9 (OMe), 57.6 (CH2 (Bn)), 112.0 (O═C—C—C═O), 118.5 (CC), 119.5 (Br-Cq), 125.8, 128.4, 129.1 (5×ArCs (Bn)), 129.4 (CA-Cq-C═O), 129.6 (CA), 133.9 (Cq (Bn)), 135.6 (CB), 138.2 (CC-Cq-N-Bn), 148.5 (CD), 164.7 (C═ONH), 173.4 (C═OOMe), 175.6 (O═C—C═C).
1-Benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 4 (200 mg, 0.5584 mMol) was stirred in dichloromethane (5 mL) followed by triethylamine (257 μL, 1.8426 mMol) and DPPA (156 μL, 0.7259 mMol). After 5 minutes L-phenyl methyl ester hydrochloride (145 mg, 0.6700 mMol) was added. After 17.5 hours t.l.c. analysis (ethyl acetate:cyclohexane, 1:1) of the reaction mixture showed the presence of one product (Rf 0.35). The reaction mixture was pre-absorbed on silica gel and purified by flash column chromatography (ethyl acetate:cyclohexane, 17:83, to 1:3, to 1:1) and recrystallised from dichloromethane/cyclohexane to give (S)-methyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-phenylpropanoate 17 (201 mg, 69%) as a pale yellow crystalline solid. M.p. 223-224° C. melted and decomposed (turned brown); HRMS (ES+): found 541.0732 [M+Na]+ C27H23N2O479BrNa requires 541.0739; found 543.0728 [M+Na]+ C27H23N2O481BrNa requires 543.0718; peak ratio: 49.3%:50.7%; [α]D20 −5.067 (c, 0.25 in chloroform); νmax (thin film): 3216, 3174 (w, sharp, sec. NH), 3033 (w, ArC—H), 2951 (w, alkyl C—H), 1755 (m, C═O ester), 1744 (m, C═O ketone), 1661 (s, C═ONH, I), 1600 (s, C═C conjugated with C═Os), 1572 (m, C═ONH, II), 1542 (m, C═C benzene) cm−1; δH (CDCl3, 500 MHz): 3.18 (1H, a-dd, JCHxHy,CHxHy 14.0 Hz, JCHxHy,CαH 8.0 Hz, CHXHY (Phenylalanine)), 3.27 (1H, a-dd, JCHxHy,CHxHy 13.9 Hz, JCHxHy,CαH 5.6 Hz, CHXHY (Phenylalanine)), 3.72 (3H, s, OMe), 5.01 (1H, ddd, JCαH,NH 7.6 Hz, JCαH,CHxCHy 5.7 Hz, J 13.2 Hz, CαH), 5.39 (2H, s, CH2(Bn)), 7.07-7.13 (2H, m, 2×ArHs (Bn)), 7.20-7.36 (9H, m, 8×ArHs (Bn) & ArHC), 7.62 (1H, dd, 9.1 Hz, JH-B,H-A 2.5 Hz, ArHB), 8.61 (1H, d, JH-A,H-B 2.6 Hz, ArHA), 8.81 (1H, s, HD), 10.38 (1H, d, JNH,CαH 7.6 Hz, NH); δC (CDCl3, 125 MHz): 38.4 (CH2 (Phenylalanine)), 52.3 (OMe), 54.2 (Cα), 57.9 (CH2 (Bn)), 112.1 (O═C—C—C═O), 118.7 (CC), 119.3 (Br-Cq), 126.1, 127.0, 128.6, 128.8, 129.4 (10×ArCs (Bn & Phenylalanine), 129.5 (CA-Cq-C═O), 130.1 (CA), 133.8 (Cq (Bn)), 135.9 (CB), 136.6 (Cq, Phenylalanine), 138.2 (CC-Cq-N-Bn), 148.7 (CD), 164.5 (C═ONH), 172.1 (C═OOMe), 175.5 (O═C—C═C); Elemental Analysis: found C, 62.96%, H, 4.75%, N, 5.36%; required C, 62.44%, H, 4.46%, N, 5.39%.
A cloudy mixture of (S)-methyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-methylbutanoate 27 (75 mg, 0.159 mmol), acetone (2.5 mL) and an aqueous solution of sodium hydroxide (2M, 5 mL) was stirred at room temperature for 24 hours. T.l.c. analysis (ethyl acetate:methanol, 5%) of the solution showed the presence of one UV-active product (Rf 0.00-0.10). The reaction solution was acidified with an aqueous solution of hydrochloric acid (1M), a white solid crushed out of solution and was filtered (water). The solid was dried under reduced pressure to give (S)-2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-methylbutanoic acid 28 (65 mg, 89%) as a white crystalline solid. M.p. 247-248° C. melting and decomposition (turned brown); HRMS (ES+): found 493.0715 [M+Na]+ C23H23N2O479BrNa requires 493.0739; found 495.0708 [M+Na]+ C23H23N2O481BrNa requires 495.0718; peak ratio: 50.0%:50.0%; νmax (Ge): 3223 (w, sec. CONH), 3067 (w, ArC—H), 2964 (w, alkyl C—H), 3200-2700 (m, h-bonded COOH), 1733 (w, C═O ketone), 1714 (w, C═OOH), 1657 (s, C═ONH, I), 1601 (m, C═C conjugated with C═Os), 1543 (s with shoulder, C═ONH, II & C═C benzene) cm−1; δH (Pyridine d5, 500 MHz): 11.15 (1H, d, JNH,CαH 8.4 Hz, NH), 9.52 (1H, s, HD), 8.87 (1H, s, HA), 7.64 (1H, d, JHB,HC 9.0 Hz, HB), 7.60 (1H, d, JHC,HB 8.9 Hz, HC), 7.41-7.20 (5H, m, 5×ArH (Bn)), 5.75 (2H, s, CH2 (Bn)), 5.38 (1H, dd, JCαH,NH 8.3 Hz, JCαH,CH 4.4 Hz, CαH), 2.82-2.64 (1H, m, J 6.5 Hz, CH (Valine)), 1.36 (3H, d, JCH3,CH 6.7 Hz, CH3 (Valine)), 1.30 (3H, d, JCH3,CH 6.7 Hz, CH3 (Valine)); δC (Pyridine d5, 125 MHz): 19.6, 21.2 (6Hs, 2×CH3 (Valine)), 32.9 (CH (Valine)), 58.5 (CH2 (Bn)), 59.8 (CαH), 114.4 (O═C—C—C═O), 120.4 (Br-Cq), 121.4 (CC), 128.1, 129.8, 130.8 (5×ArCs (Bn)), 131.1 (CA-Cq-C═O), 131.2 (CA), 137.0 (Cq (Bn)), 138.8 (CB), 140.0 (Cq-N-Bn), 151.0 (CD), 166.3 (C═ONH), 176.1 (COOH), 177.1 (O═C—C═C).
A cloudy mixture of (S)-methyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-phenylpropanoate 17 (60 mg, 0.116 mmol) in acetone (2 mL) and an aqueous solution of sodium hydroxide (2 M, 4 mL) was stirred at room temperature for 24 hours. T.l.c. analysis (ethyl acetate/methanol, 5%) showed the presence of one UV-active product (Rf 0.00-0.10) and complete consumption of the starting material (Rf 0.88). The reaction solution was acidified with an aqueous solution of hydrochloric acid (1 M) until a white solid crushed out. The reaction mixture was filtered, the solid washed with water and dried under reduced pressure to yield (S)-2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-phenylpropanoic acid 29 (56 mg, 96%) as a pale yellow crystalline solid. M.p. phase transition occurs at 140-145° C. At higher emperature, decomposition occurs (turns brown); HRMS (ES+): found 527.0582 [M+Na]+ C26H21N2O479BrNa requires 527.0582; found 529.0552 [M+Na]+ C26H21N2O481BrNa requires 529.0562; peak ratio: 51.7%:49.3%; νmax (Ge): 3225 (w, sec. CONH), 3060, 3031 (w, ArC—H), 2922 (w, alkyl C—H), 3200-2700 (w, h-bonded COOH), 1734 (m with shoulder, C═O ketone & C═OOH), 1658 (s, C═ONH, I), 1601 (m, C═C conjugated with C═Os), 1543 (s with shoulder, C═ONH, II & C═C benzene) cm−1; δH (pyridine d5, 500 MHz): 11.10 (1H, d, JNH,CαH 7.7 Hz, NH), 9.44 (1H, s, HD), 8.79 (1H, d, JHA,HB 2.1 Hz, HA), 8.69-7.77 (1H, br s, COOH), 7.66 (2H, m, 2×ArHs), 7.60 (1H, dd, JHB,HC 9.0 Hz, JHB,HA 2.3 Hz, HB), 7.55 (1H, d, JHC,HB 9.0 Hz, HC), 7.43-7.10 (8H, m, 8×ArHs), 5.79-5.59 (3H, m, CH2 (Bn) & CαH), 3.73 (1H, dd, JCHxHy,CHxHy 13.8 Hz, JCHxHy,NH 5.1 Hz, CHXHY (Bn)), 3.56 (1H, dd, JCHyHx,CHyHx 13.8 Hz, JCHyHx,NH 7.8 Hz, CHXHY (Bn)); δC (Pyridine d5, 125 MHz): 39.32 (CH2 (Phenylalanine)), 55.57 (Cα), 55.67 (CH2 (Bn)), 113.38 (O═C—C—C═O), 119.47 (CC), 120.47 (Br-Cq), 127.2, 129.4, 130.0, 130.6 (10×ArCs), 130.3 (CA), 165.2 (C═ONH), 175.1 (C═OOH), 176.1 (C═O).
(S)-methyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)-4-methylpentanoate 16 (20 mg, 4.1206×10−2 mMol) was stirred in acetone (2 mL) and an aqueous solution of NaOH (2M, 2 mL) for 21.5 hours at room temperature. T.l.c. (ethyl acetate:cyclohexane, 1:1) revealed the presence of a product (Rf 0.00) and complete consumption of the starting material (Rf 0.40). Amberlite IR 120[H+] was added to the stirring reaction solution and after 15 minutes the reaction mixture was filtered (acetone/water) and the solvents were removed in vacuo to give a residue which was purified by a silica plug (acetone to acetone:methanol, 40%) to give (S)-2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)-4-methylpentanoic acid 30 (10 mg, 52%) as a white solid. M.p. decomposition occurred above 210° C. (turned brown); HRMS (TOF MS ES−): found 469.0776 [M]+ C23H22N2O479Br requires 469.0763; found 471.0751 [M]+ C23H22N2O481Br requires 471.0742; peak ratio: 49.6%:51.4%; νmax (Ge): 3235 (w, sec. CONH), 3067 (w, ArC—H), 2954, 2930 (w, alkyl C—H), 3400-3000 (w, h-bonded COOH), 1734 (w, C═O ketone), 1650 (s, C═ONH, I), 1597 (m, C═C conjugated with C═Os), 1579 (s, COO−), 1542 (s, C═ONH, II) cm−1; δH (methanol d4, 600 MHz): 9.00 (1H, s, HD), 8.38 (1H, br s, HA), 7.76 (1H, d, J 8.5 Hz, HB), 7.58 (1H, d, J 8.1 Hz, HC), 7.36-7.15 (5Hs, m, 5×ArHs (Bn)), 5.67 (1H, s CH2 (Bn)), 4.63 (1H, m, CαH (Leucine)), 1.87-1.73 (3H, m, CH & CH2 (Leucine)), 1.02 (3H, d, J 5.4 Hz, CH3 (Leucine)), 0.99 (3H, d, J 4.8 Hz, CH3 (Leucine)); δC (methanol d4, 150 MHz): 21.0, 22.2 (Leucine 2×CH3), 25.0 (Leucine CH), 41.3 (Leucine CH2), 53.1 (Leucine Cα), 57.1 (CH2 (Bn)), 111.1 (O═C—C—C═O), 119.8 (CC), 119.8 (Br-Cq), 126.2, 128.0, 128.8 (5×ArCs (Bn)), 128.7 (CA-Cq-C═O), 128.7 (CA), 134.9 (Cq (Bn)), 135.6 (CB), 138.2 (CC-Cq-N-Bn), 149.2 (CD), 164.9 (C═ONH), 171.6 (C═OOH), 175.2 (O═C—C═C).
A cloudy mixture of (S)-methyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)propanoate 14 (100 mg, 0.2256 mMol) was stirred in acetone (3 mL) and an aqueous solution of NaOH (2M, 7 mL) for 2 hours at room temperature. T.l.c. analysis (ethyl acetate:methanol, 5%) of the reaction solution revealed the presence of a product (Rf 0.00-0.20) and complete consumption of the starting material (Rf 0.88). The reaction solution was acidified with an aqueous solution of hydrochloric acid (1M) and a white solid crushed out. The mixture was then filtered (water, then briefly with an acetone:toluene, 1:1 mixture) and the solvents were removed in vacuo to give (S)-2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)propanoic acid 31 (70 mg, 72%) as a pale yellow crystalline solid. M.p. 234-236° C. melting and decomposition (turned brown); HRMS (ES+): found 451.0264 [M+Na]+ C20H17N2O479BrNa requires 451.0269; found 453.0219 [M+Na]+ C20H17N2O481BrNa requires 453.0249; peak ratio: 52.8%:47.2%; νmax (Ge): 3228 (m, sec. CONH), 3063 (m, ArC—H), 2935 (w, alkyl C—H), 3200-2400 (w, h-bonded COOH), 1729 (m, C═O ketone), 1658 (s, C═ONH, I), 1600 (m, C═C conjugated with C═Os), 1580 (s, COO−), 1543 (s, C═ONH, II) cm−1; δH (pyridine d5, 500 MHz): 11.09 (1H, d, J 7.2), 11.00-9.70 (1H, br s, COOH), 9.51 (1H, s, HD), 8.85 (1H, d, JHA,HB 2.1 Hz, HA), 7.64 (1H, dt, JHB,HC 9.1 Hz, JHB,HA 2.3 Hz, HB), 7.60 (1H, d, JHC,HB 9.1 Hz, HC), 7.38-7.23 (5H, m, 5×ArHs (Bn)), 5.76 (2H, s, CH2 (Bn)), 5.33 (1H, quintet, JCαH,NH=JCαH,CH3 7.2 Hz, CαH (Alanine)), 1.80 (3H, d, JCH3,CαH 7.2 Hz, CH3 (Alanine)); δC (pyridine d5, 125 MHz): 19.6 (Alanine-CH3), 49.8 (Alanine-Cα), 57.7 (CH2 (Bn)), 113.5 (O═C—C—C═O), 119.6 (Br-Cq), 120.6 (CB), 127.3, 129.0, 130.0 (5×ArCs (Bn)), 130.3 (CA), 130.5 (CC-Cq-N-Bn), 136.2 (CC & Cq(Bn)), 139.2 (CA-Cq-C═O), 150.1 (CD), 165.0 (C═ONH), 176.1 (O═C—C═C), 176.4 (C═OOH).
Methyl 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetate 15 (15 mg, 3.4944×10−2 mMol) was stirred in acetone (2 mL) and an aqueous solution of NaOH (2M, 2 mL) for 50 hours at room temperature. T.l.c. analysis (ethyl acetate:cyclohexane, 1:1) of the reaction solution revealed the presence of a product (Rf 0.00) and complete consumption of the starting material (Rf 0.71). The reaction mixture was acidified by stirring Amberlite IR 120[H+] in it for 15 minutes. The mixture was then filtered (water) and the solvents were removed in vacuo to give a residue which was purified by silica plug (acetone, then acetone/methanol 20%) to give 2-(1-benzyl-6-bromo-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetic acid 32 (10 mg, 69%) as a pale yellow solid. M.p. 190° C. decomposition (turned brown); HRMS (ES+): found 437.0106 [M+Na]+ C19H15N2O479BrNa requires 437.0113; found 439.0076 [M+Na]+ C19H15N2O481BrNa requires 439.0092; peak ratio: 48.5%:51.5%; νmax (Ge): 3401, 3307 (s broad, sec. CONH), 3600-2800 (m, COOH), 3067 (m, ArC—H), 2917, 2850 (w, alkyl C—H), 1652 (s, C═ONH, I), 1599 (m, C═C conjugated with C═Os), 1540 (s, C═ONH, II) cm−1; δH (methanol d4, 500 MHz): 8.98 (1H, s, HD), 8.52 (1H, d, JHA,HB 2.3 Hz, HA), 7.77 (1H, dd, JHB,HC 9.1 Hz, JHB,HA 2.4 Hz, HB), 7.60 (1H, d, JHC,HB 9.1 Hz, HC), 7.38-7.22 (5H, m, 5×ArHs (Bn)), 5.66 (2H, s, CH2 (Bn)), 4.04 (2H, s, CαH2); δC (methanol d4, 150 MHz): 44.6 (Glycine-Cα), 58.4 (CH2 (Bn)), 113.1 (O═C—C—C═O), 120.1 (Br-Cq), 121.2 (CB), 127.6, 129.5, 130.3 (5Cs, 5×ArCs (Bn)), 130.2 (CA), 130.3 (CC-Cq-N-Bn), 136.4 (Cq(Bn)), 137.0 (CC), 139.8 (CA-Cq-C═O), 150.5 (CD), 166.2 (C═ONH), 176.2 (O═C—C═C), 176.7 (C═OOH).
Aniline (2.733 mL, 29.99 mMol) was stirred in diethyl ethoxymethylenemalonate (6.063 mL, 30.00 mMol) at 120-130° C. for 16.5 hours. T.l.c. analysis (ethyl acetate:cyclohexane, 1:1) showed the presence of one UV-active product (Rf 0.84) and complete consumption of both starting materials. Upon cooling down of the reaction solution to room temperature, intermediate diethyl 2-((phenylamino)methylene)malonate 35 solidified (as dark yellow crystalline solid, 7.899 g, quant.). M.p. 36-37° C.; HRMS (EI+): found 263.11531 [M]+ C14H17NO4 requires 263.11521; νmax (thin film): 3265, 3184 (w, NH), 3050 (w, ArC—H), 2981, 2936, 2904, 2871 (m, alkyl C—H), 1717 (s, 2×intramolecularly hydrogen-bonded C═O conjugated with C═C), 1691 (s, C═C—NH), 1655 (s, C═N—), 1623 (s, aryl conjugated C═C), 1255 (s, C—N stretch) cm−1; δH (CD3CN, 500 MHz): 1.31 (3H, t, JCH3,CH2 7.1 Hz, CH3), 1.32 (3H, t, JCH3,CH2 7.2 Hz, CH3), 4.19 (2H, q, JCH2,CH3 7.2 Hz, CH2), 4.25 (2H, q, JCH2,CH3 7.1 Hz, CH2), 7.16 (1H, tt, JparaArH,metaArHs 7.4 Hz, JparaArH,orthoArHs 1.1 Hz, para-ArH), 7.20 (2H, dt, JorthoArHs,metaArHs 7.6 Hz, JorthoArHs,paraArH 1.0 Hz, 2×orthoArHs), 7.38 (2H, m, J 7.4 Hz, 2×metaArHs), 8.48 (1H, d, JCH,NH 13.8 Hz, CH—NH), 10.81 (1H, d, JNH,CH 13.6 Hz, CH—NH); δC (CD3CN, 125 MHz): 14.1, 14.2 (2×CH3), 60.3, 60.6 (2×CH2), 93.9 (O═C—C—C═O), 117.6 (2×orthoArCs), 125.1 (paraArC), 130.1 (2×metaArCs), 139.8 (ArCquat-NH), 151.9 (NH—CH), 165.6 (C═O), 168.8 (hydrogen bonded C═O). Diphenyl ether (50 g) was then added to diethyl 2-((phenylamino)methylene)malonate 5 and the mixture (7.4145 g, 28.161 mMol) was refluxed at 250° C. After 6 hours the reaction mixture was allowed to cool down to room temperature and diluted in cyclohexane. A brown solid crushed out of solution. Filtration (eluant cyclohexane) yielded a brown solid which was dried by concentration in vacuo. The solid was then washed with acetone, filtered (eluant acetone) and concentrated in vacuo to yield ethyl 4-oxo-1,4-dihydroquinoline-3-carboxylate 36 (2.431 g, 37.3%) as a white/grey solid. M.p. 235° C. (gas evolved, decomposition); HRMS (EI+): found 217.07455 [M]+ C12H11NO3 requires 217.07334; νmax (thin film): 3409 (s broad, NH), 2974, 2899 (m, alkyl C—H), 1682 (s with shoulder, 2×C═O and C═C—NH), 1619, 1610 (s, aryl conjugated C═C) m−1; δH (DMSO-d6, 500 MHz): 1.27 (3H, t, JCH3,CH2 7.0 Hz, CH3), 4.20 (2H, q, JCH2,CH3 6.9 Hz, CH2), 7.40 (1H, m, J 7.6 Hz, J 7.0 Hz, HB), 7.60 (1H, d, JHD,HC 7.8 Hz, HD), 7.69 (1H, m, J 7.8 Hz, J 6.9 Hz, HC), 8.14 (1H, d, JHA,HB 7.9 Hz, HA), 8.54 (1H, s, CH—NH), 12.10-12.40 (1H, br s, NH); δC (DMSO-d6, 125 MHz): 14.3 (CH3), 59.6 (CH2), 109.8 (O═C—C—C═O), 118.7 (ArCD), 124.7 (ArCB), 125.6 (ArCA), 127.3 (ArCq-C═O), 132.4 (ArCC), 139.0 (ArCq-NH), 144.9 (NH—CH), 164.8 (C═OOEt), 173.4 (C═O).
Ethyl 4-oxo-1,4-dihydroquinoline-3-carboxylate 6 (3.00 g, 13.811 mMol) was stirred in DMF (120 mL) with potassium carbonate (4.77 g, 34.527 mMol) at 40° C. under an atmosphere of nitrogen. After 5 minutes benzyl bromide (8.2 mL, 69.054 mMol) was added dropwise and the temperature raised to 80° C. After 4.5 hours, t.l.c. analysis (dichloromethane:acetone, 4:1) showed the presence of one UV-active product (Rf 0.87) and complete consumption of the starting material (Rf 0.10). The reaction mixture was filtered and the filtrate was concentrated in vacuo, pre-absorbed on silica and purified by flash-column chromatography (ethyl acetate:cyclohexane, 3:7, to 1:1, to 7:1, to ethyl acetate) to afford ethyl 1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylate 7 (2.70 g, 64%) as a white/pale yellow crystalline solid. M.p. 150-158° C. (ethyl acetate/cyclohexane; melted over the range); HRMS (EI): found 307.12088 [M]+ C19H17NO3 requires 307.12029; νmax (thin film): 3109, 3048 (w, ArC—H), 2979, 2935, 2904, 2871 (m, alkyl C—H), 1722 (s, C═O ketone), 1692 (s, C═O ester, conjugated with C═C), 1622, 1610 (s, C═C conjugated with C═Os), 1234 (s, C—N stretch) cm−1; δH (CD3CN, 500 MHz): 1.36 (3H, t, JCH3,CH2 7.1 Hz, CH3), 4.31 (2H, q, JCH2,CH3 7.1 Hz, CH2), 5.52 (2H, s, CH2-Bn), 7.25-7.29 (2H, m, 2×ArHs (Bn)), 7.32-7.40 (3H, m, 3×ArHs (Bn)), 7.43 (1H, ddd, JHB,HA 8.0 Hz, JHB,HC 7.1 Hz, JHB,HD 1.0 Hz, HB), 7.51 (1H, d, JHD,HC 8.6 Hz, HD), 7.62 (1H, ddd, JHC,HD 8.6 Hz, JHC,HB 7.0 Hz, JHC,HA 1.6 Hz, HC), 8.38 (1H, dd, JHA,HB 8.5 Hz, JHA,HC 1.6 Hz, HA), 8.74 (1H, s, HE); δC (CD3CN, 125 MHz): 14.1 (CH3), 57.0 (CH2 (Bn)), 60.5 (CH2), 111.1 (O═C—C—C═O), 117.8 (ArCD), 125.3 (ArCB), 127.1 (ArCA), 126.9, 128.5, 129.4 (5×ArCs (Bn) & ArCq-C═O), 132.8 (ArCC), 136.0 (Cq (Bn)), 139.8 (ArCq-NH), 150.5 (ArCE), 165.2 (C═OOEt), 174.2 (C═O).
Ethyl 1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylate 7 (2.65 g, 8.622 mmol) was srirred in acetone (135 mL) and an aqueous solution of sodium hydroxide (2 M, 63 mL) for 90 minutes at room temperature and for four hours at 70° C. T.l.c. analysis (dichloromethane:acetone, 4:1) showed the presence of one product (Rf 0.00-0.23) and complete consumption of the starting material (Rf 0.57). the reaction solution was allowed to cool down to room temperature and acidified with an aqueous solution of hydrochloric acid (1 M). A white solid crushed out of solution, was filtered (water) and the solid was dried under reduced pressure to give 1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 8 (2.40 g, quant.) as a white fluffy solid. M.p. 109-110° C.; HRMS (EI+): found 280.09755 [M+H]+ C17H14NO3 requires 280.09737; νmax (thin film): 3200-2000 (m, hydrogen-bonded of OH of carboxylic acid), 1708 (m, C═O, carboxylic acid), 1614 (s, C═C conjugated with C═Os) cm−1; δH (pyridine d5, 500 MHz): 15.50-15.90 (1H, br s, COOH), 9.55 (1H, s, HE), 8.63 (1H, dd, JHA,HB 8.0 Hz, JHA,HC 1.5 Hz, HA), 7.76 (1H, d, JHD,HC 8.6 Hz, HD), 7.58 (1H, ddd, JHC,HD 11.5 Hz, JHC,HB 6.5 Hz, JHC,HA 3.0 Hz, HC), 7.41 (1H, ddd, JHB,HA 8.0 Hz, JHB,HC 7.1 Hz, JHB,HD 0.9 Hz, HB), 7.36-7.22 (5H, m, 5×ArHs (Bn)), 5.86 (2H, s, CH2 (Bn)); δC (pyridine d5, 125 MHz): 58.9 (CH2 (Bn)), 109.7 (O═C—C—C═O), 118.7 (ArCD), 126.7 (ArCA), 127.3 (ArCq-C═O), 127.4 (ArCB), 127.2, 129.1, 129.9 (5×ArCs (Bn), 134.5 (ArCC), 135.8 (Cq (Bn) mostly masked by the pyridine d6 signal), 140.5 (ArCq-NH), 150.7 (ArCE), 167.2 (COOH), 179.3 (C═O).
To a stirred mixture of 1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 8 (100 mg, 0.358 mmol), triethylamine (165 μL, 1.182 mmol) and dichloromethane (5 mL), diphenylphosphoryl azide (100 μL, 0.465 mmol) was added dropwise at room temperature under an atmosphere of nitrogen. After 5 minutes L-glycine tert-butyl ester hydrochloride (72 mg, 0.430 mmol) was added and left to stir for 36 hours. T.l.c. analysis (acetone:cyclohexane, 1:1) showed the presence of one UV-active product (Rf 0.62). The reaction mixture was washed with an aqueous solution of hydrochloric acid (1 M, 10 mL), the organic layer was then dried (magnesium sulphate), filtered and purified by flash-column chromatography (acetone:cyclohexane, 1:3, to 1:1) to give tert-butyl 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetate 10 (110 mg, 78%) as a pale yellow crystalline solid. M.p. 189-190° C.; HRMS (CI+): found 393.18078 [M+H]+ C23H25N2O4 requires 393.18143; νmax (thin film): 3169 (m, 2 peaks, CONH) 3042 (m, ArC—H), 2984, 2929 (m, alkyl C—H), 1732 (s with shoulder, C═O, ester & C═O, ketone), 1660 (s, CONH, I & C═C conjugated with C═O), 1552 (m, CONH, II), 1605 (s, aromatic ring and C═C conjugated with C═O), 1492 (s, aromatic ring) cm−1; δH (CDCl3, 500 MHz): 10.44 (1H, t, JNH,CH 5.2 Hz, NH), 8.87 (1H, s, HE), 8.52 (1H, dd, JH-A,H-B 8.1 Hz, JH-A,H-C 1.4 Hz, ArHA), 7.56 (1H, ddd, JHC,HD 8.6 Hz, JHC,HB 7.1 Hz, JHC,HA 1.6 Hz, HC), 7.40 (1H, ddd, JHB,HA 7.9 Hz, JHB,HC 6.9 Hz, JHB,HD 1.0 Hz, HB), 7.37 (1H, d, JHD,HC 8.8 Hz, HD), 7.34-7.25 (3H, m, 3×ArHs (Bn)), 7.16-7.09 (2H, m, 2×ArHs (Bn)), 5.43 (2H, s, CH2 (Bn)), 4.15 (2H, d, JCαH,NH 5.5 Hz, CαH2),), 1.48 (9H, s, C(CH3)3); δC (CDCl3, 125 MHz): 28.1 (C(CH3)3), 42.2 (Glycine-Cα), 57.7 (CH2 (Bn)), 81.8 (C(CH3)3), 111.8 (O═C—C—C═O), 116.8 (CD), 125.2 (CB), 126.2 128.6 129.4 (5×ArCs (Bn)), 127.6 (CA), 128.2 (CA-Cq-C═O), 132.9 (CC), 134.3 (Cq (Bn)), 139.4 (CC-Cq-N-Bn), 148.6 (CE), 165.2 (C═ONH), 169.1 (CO2tBu), 176.8 (O═C—C═C).
To a stirred mixture of 1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 8 (100 mg, 0.358 mmol), triethylamine (164 μL, 1.182 mmol) and dichloromethane (5 mL) diphenylphosphoryl azide (100 μL, 0.465 mmol) was added dropwise at room temperature under an atmosphere of nitrogen. After 5 minutes L-valine tort-butyl ester hydrochloride (90 mg, 0.430 mmol) was added and left to stir for 72 hours. T.l.c. analysis (acetone:cyclohexane, 1:1) showed the presence of one UV-active product (Rf 0.63). The reaction mixture was washed with an aqueous solution of hydrochloric acid (1 M), dried (magnesium sulphate), filtered and purified by flash-column chromatography (ethyl acetate:cyclohexane, 1:9, to 1:5) to give (S)-tert-butyl 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-methylbutanoate 10 (155 mg, quant.) as a white crystalline solid. M.p. 197-198° C.; HRMS (TOF MS ES+): found 457.2099 [M+Na]+ C26H30N2O4Na requires 457.2103; [α]D25 +21.032 (c, 0.25 in dichloromethane); νmax (thin film): 3179 (m, 2 peaks, CONH) 3042, 3008 (m, ArC—H), 2932, 2873 (m, alkyl C—H), 1733 (m, C═O, ester), 1724 (m, C═O, ketone), 1662 (s, CONH, I & C═C conjugated with C═O), 1551 (m, CONH, II), 1605 (m, aromatic ring and C═C conjugated with C═O), 1491 (m, aromatic ring) cm−1; δH (CD3CN, 500 MHz): 10.44 (1H, d, JNH,CH 8.4 Hz, NH), 8.91 (1H, s, HE), 8.47 (1H, dd, JH-A,H-B 8.1 Hz, JH-A,H-C 1.6 Hz, ArHA), 7.67 (1H, ddd, JHC,HD 8.6 Hz, JHC,HB 7.0 Hz, JHC,HA 1.6 Hz, HC), 7.59 (1H, d, JHD,HC 8.6 Hz, HD), 7.47 (1H, ddd, JHB,HC 8.0 Hz, JHB,HC 7.0 Hz, JHB,HD 1.0 Hz, HB), 7.39-7.26 (3H, m, 3×ArHs (Bn)), 7.26-7.18 (2H, m, 2×ArHs (Bn)), 5.58 (2H, s, CH2 (Bn)), 4.46 (1H, dd, JCαH,NH 8.4 Hz, JCαH,CH 4.8 Hz, CαH), 2.25 (1H, d septet, JCH,CαH 3.5 Hz, JCH,CH3 6.9 Hz, CH (Valine)), 1.47 (9H, s, C(CH3)3), 1.03 (3H, d, JCH3,CH 6.9 Hz, CH3 (Valine)), 1.02 (3H, d, JCH3,CH 6.9 Hz, CH3 (Valine)); δC (CD3CN, 150 MHz): 16.9, 18.4 (Valine CH(CH3)2), 26.9 (C(CH3)3), 30.5 (Valine CH(CH3)2), 56.6 (CH2 (Bn)), 57.6 (Valine-Cα), 80.7 (C(CH3)3), 111.0 (O═C—C—C═O), 117.0 (CD), 124.7 (CB), 126.2, 127.8, 128.7 (5×ArCs (Bn)), 126.3 (CA), 132.5 (CC), 127.7 (CA-Cq-C═O), 135.2 (Cq (Bn)), 139.2 (CC-Cq-N-Bn), 148.5 (CE), 164.2 (C═ONH), 170.8 (CO2tBu), 176.3 (O═C—C═C).
To a stirring solution of 1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 8 (200 mg, 0.716 mmol) and triethylamine (329 μL, 2.363 mmol) in dichloromethane (5 mL), diphenylphosphoryl azide (200 μL, 0.931 mmol) was added dropwise at room temperature under an atmosphere of nitrogen. After 5 minutes L-phenylalanine methyl ester hydrochloride (185 mg, 0.859 mmol) was added to the stirring milky-coloured reaction mixture. After 90 hours, t.l.c. analysis (ethyl acetate/dichloromethane, 1:1) of the pale yellow semi-transparent suspension showed the presence of one new UV-active spot (Rf 0.91). The reaction mixture was pre-absorbed on silica and purified by flash-column chromatography (ethyl acetate:cyclohexane, 1:3, to 1:1, to ethyl acetate) to yield (S)-methyl 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-phenylpropanoate 20 (248 mg, 79%) as a pale yellow crystalline solid. M.p. at 165° C. started turning brown. At 174-177° C. melted and decomposed (continued turning brown); [α]D19.8 −10.873 (c, 0.42 in chloroform); νmax (thin film): 3207, 3171 (w, sharp, sec. NH), 3046 (w, ArC—H), 2950, 2848 (w, alkyl C—H), 1755 (m, C═O ester), 1743 (m, C═O ketone), 1660 (s, C═ONH, I), 1571 (m, C═ONH, II), 1605 (s, C═C conjugated with C═Os) cm−1; δH (CDCl3, 500 MHz): 3.16 (1H, dd, JCHxHy,CHxHy 13.7 Hz, JCHxHy,CαH 7.7 Hz, CHXHY (Phenylalanine)), 3.26 (1H, dd, JCHxHy,CHxHy 13.9 Hz, JCHxHy,CαH 5.7 Hz, CHXHY (Phenylalanine)), 3.69 (3H, s, OMe), 5.01 (1H, (1H, td, JCαH,NH=JCαH,CHxHy 7.6 Hz Hz, JCαH,CHxCHy 5.7 Hz, CαH), 5.36 (2H, s, CH2(Bn)), 7.08-7.11 (2H, m, 2×ArHs), 7.18-7.31 (10H, m, 8×ArHs), 7.33 (1H, d, JHD,HC 8.8 Hz, HD), 7.35 (1H, m, J 7.3 Hz, HB), 7.49 (1H, ddd, JHC,HD 8.7 Hz, JHC,HB 7.3 Hz, JHC,HA 1.6 Hz, HC), 8.45 (1H, dd, JHA,HB 7.9 Hz, JHA,HC 1.6 Hz, HA), 8.78 (1H, s, HE), 10.56 (1H, d, JNH,CαH 7.5 Hz, NH); δC (CDCl3, 125 MHz): 38.8 (CH2 (Phenylalanine)), 52.6 (OMe), 54.5 (CH), 57.9 (CH2 (Bn)), 111.4 (O═C—C—C═O), 117.3 (CB), 125.6, 126.6, 127.3, 128.9, 129.6, 129.7 (10×ArCs (Bn) & (Phenylalanine)), 128.4 (CA-Cq-C═O), 128.9 (CD), 133.3 (CC), 134.8 (Cq (Bn)), 137.1 (Cq (Phenylalanine)), 139.6 (Cq-N-Bn), 148.9 (CE), 164.9 (C═ONH), 172.2 (C═OOMe), 176.7 (O═C—C═C).
To a stirring solution of 1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 8 (200 mg, 0.716 mmol) and triethylamine (329 μL, 2.363 mmol) in dichloromethane (5 mL), diphenylphosphoryl azide (200 μL, 0.931 mmol) was added dropwise at room temperature under an atmosphere of nitrogen. After 5 minutes L-alanine methyl ester hydrochloride (119 mg, 0.859 mmol) was added to the stirring milky-coloured reaction mixture. After 90 hours, t.l.c. analysis (ethyl acetate) of the pale yellow semi-transparent suspension showed the presence of one new UV-active spot (Rf 0.90). The reaction mixture was pre-absorbed on silica and purified by flash-column chromatography (ethyl acetate:cyclohexane, 1:3, to 1:1, to ethyl acetate) to yield (S)-methyl 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)propanoate 21 (188 mg, 72%) as a white solid. M.p. 168-170° C.; HRMS (CI+): found 365.15043 [M+H]+ C21H21N2O4 requires 365.15012; [α]D20 +49.770 (c, 0.29 in chloroform); νmax (thin film): 3200 (w, sharp, 2 bands, sec. NH), 3049 (w, ArC—H), 2983, 2952 (w, alkyl C—H), 1738 (s, C═O ester & C═O ketone), 1659 (s, C═ONH, I), 1583 (m, C═ONH, II), 1606 (s, C═C conjugated with C═Os) cm−1; δH (CDCl3, 500 MHz): 1.53 (3Hs, d, JCH3,CαH 7.3 Hz, CH3 (Alanine)), 3.74 (3Hs, s, OMe), 4.76 (1H, a-quint, JCαH,CH3=JCαH,NH 7.3 Hz, CH), 7.10-7.14 (2Hs, m, 2×ArHs (Bn)), 7.24-7.32 (3Hs, m, 3×ArHs (Bn)), 7.36-7.41 (2Hs, m, HB & HD), 7.55 (1H, ddd, J 8.9 Hz, J 6.9 Hz, JHC,HA 1.6 Hz, HC), 8.47 (1H, dd, JHA,HB 8.2 Hz, JHA,HC 1.5 Hz, HA), 8.86 (1H, s, HE), 10.45 (1H, d, JNH,CαH 7.4 Hz, NH); δC (CDCl3, 125 MHz): 18.3 (CH3 (Alanine)), 48.2 (CαH), 52.4 (OMe), 57.7 (CH2 (Bn)), 111.6 (O═C—C—C═O), 116.9, 125.3 (CB & CD), 126.2, 128.6, 129.3 (5×ArCs (Bn)), 127.3 (CA), 128.1 (CA-Cq-C═O), 133.0 (CC), 134.3 (Cq (Bn)), 139.3 (Cq-N-Bn), 148.6 (CE), 164.7 (C═ONH), 173.4 (C═OOMe), 176.8 (O═C—C═C).
To a stirring solution of 1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 8 (200 mg, 0.716 mmol) and triethylamine (329 μL, 2.363 mmol) in dichloromethane (5 mL), diphenylphosphoryl azide (200 μL, 0.931 mmol) was added dropwise at room temperature under an atmosphere of nitrogen. After 5 minutes L-valine methyl ester hydrochloride (144 mg, 0.859 mmol) was added to the stirring milky-coloured reaction mixture. After 90 hours, t.l.c. analysis (ethyl acetate) of the pale yellow semi-transparent suspension showed the presence of one new UV-active spot (Rf 0.90). The reaction mixture was pre-absorbed on silica and purified by flash-column chromatography (ethyl acetate:cyclohexane, 1:3, to 1:1, to ethyl acetate) to yield (S)-methyl 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-methylbutanoate 22 (231 mg, 82%) as a white solid. M.p. 194-196° C.; HRMS (CI+): found 393.18079 [M+H]+ C23H25N2O4 requires 393.18142; [α]D20 +45.591 (c, 0.31 in chloroform); νmax (thin film): 3173 (w, sharp, 2 bands, sec. NH), 3039 (w, ArC—H), 2961, 2876 (w, alkyl C—H), 1733 (s, C═O ester & C═O ketone), 1661 (s, C═ONH, I), 1574 (m, C═ONH, II), 1604 (s, C═C conjugated with C═Os) cm−1; δH (CDCl3, 500 MHz): 1.01 (3Hs, d, JCH3,CH 6.9 Hz, CH3 (Valine)), 1.03 (3Hs, d, JCH3,CH 6.8 Hz, CH3 (Valine)), 2.29 (1H, d septet, JCH,CH3 6.8 Hz, JCH,CαH 5.2 Hz, CH (Valine)), 3.69 (3Hs, s, OMe), 4.64 (1H, dd, JCαH,NH 8.2 Hz, JCαH,CH 5.1 Hz, CαH), 5.63 (2Hs, s, CH2 (Bn)), 7.07-7.11 (2Hs, m, 2×ArHs (Bn)), 7.19-7.28 (3Hs, m, 3×ArHs (Bn)), 7.34 (1H, ddd, JHB,HA 8.0 Hz, JHB,HC 7.1 Hz, JHB,HD 1.0 Hz, HB), 7.37 (1H, d, JHD,HC 8.5 Hz, HD), 7.51 (1H, ddd, JHC,HD 8.7 Hz, JHC,HB 7.1 Hz, JHC,HA 1.6 Hz, HC), 8.43 (1H, dd, JHA,HB 8.1 Hz, JHA,HC 1.6 Hz, HA), 8.86 (1H, s, HE), 10.52 (1H, d, JNH,CαH 8.2 Hz, NH); δC (CDCl3, 125 MHz): 18.2, 19.5 (6Hs, 2×CH3 (Valine)), 31.0 (CH (Valine)), 52.0 (OMe), 57.6 (CH2 (Bn)), 57.8 (CαH), 111.7 (O═C—C—C═O), 117.0 (CD), 125.2 (CB), 126.2, 128.7, 129.3 (5×ArCs (Bn)), 127.2 (CA), 128.5 (CA-Cq-C═O), 132.9 (CC), 134.4 (Cq (Bn)), 139.3 (Cq-N-Bn), 148.6 (CE), 165.2 (C═ONH), 172.5 (C═OOMe), 176.9 (O═C—C═C).
To a stirring solution of 1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 8 (200 mg, 0.716 mmol) and triethylamine (329 μL, 2.363 mmol) in dichloromethane (10 mL), diphenylphosphoryl azide (200 μL, 0.931 mmol) was added dropwise at room temperature under an atmosphere of nitrogen. After 5 minutes L-glycine methyl ester hydrochloride (108 mg, 0.859 mmol) was added to the stirring milky-coloured reaction mixture. After 90 hours, t.l.c. analysis (ethyl acetate) of the pale yellow semi-transparent suspension showed the presence of one new UV-active spot (Rf 0.90). The reaction mixture was pre-absorbed on silica and purified by flash-column chromatography (ethyl acetate:cyclohexane, 1:3, to 1:1, to ethyl acetate) to yield methyl 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetate 23 (110 mg, 44%) as a white crystalline solid. M.p. 244-245° C. melting and decomposition (turned brown); HRMS (CI+): found 351.13406 [M+H]+ C20H19N2O4 requires 351.13448; νmax (thin film): 3169 (w, sharp, 2 bands, sec. NH), 3044, 3008 (w, ArC—H), 2959, 2909 (w, alkyl C—H), 1743 (s, C═O ester & C═O ketone), 1657 (s, C═ONH, I), 1574 (m, C═ONH, II), 1607 (s, C═C conjugated with C═Os) cm−1; δH (CDCl3, 500 MHz): 3.78 (3Hs, s, OMe), 4.27 (1H, d, JCαH2,NH 5.7 Hz, CαH2), 5.46 (2Hs, s, CH2 (Bn)), 7.13-7.18 (2Hs, m, 2×ArHs (Bn)), 7.28-7.37 (3Hs, m, 3×ArHs (Bn)), 7.40 (1H, d, JHD,HC 8.9 Hz, HD), 7.44 (1H, ddd, JHB,HA 8.2 Hz, JHB,HC 6.9 Hz, JHB,HD 1.1 Hz, HB), 7.60 (1H, ddd, JHC,HD 8.7 Hz, JHC,HB 7.1 Hz, JHC,HA 1.6 Hz, HC), 8.54 (1H, dd, JHA,HB 8.0 Hz, JHA,HC 1.4 Hz, HA), 8.90 (1H, s, HE), 10.48 (1H, t, JNH,CαH2 5.7 Hz, NH); δC (CDCl3, 125 MHz): 41.4 (CαH2), 52.3 (OMe), 57.8 (CH2 (Bn)), 111.7 (O═C—C—C═O), 116.8 (CD), 125.3 (CB), 126.2, 128.7, 129.4 (5×ArCs (Bn)), 127.5 (CA), 128.2 (CA-Cq-C═O), 133.0 (CC), 134.2 (Cq (Bn)), 139.4 (Cq-N-Bn), 148.7 (CE), 165.5 (C═ONH), 170.4 (C═OOMe), 176.9 (O═C—C═C).
To a stirring solution of 1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 8 (200 mg, 0.716 mmol) and triethylamine (329 μL, 2.363 mmol) in dichloromethane (5 mL), diphenylphosphoryl azide (200 μL, 0.931 mmol) was added dropwise at room temperature under an atmosphere of nitrogen. After 5 minutes L-leucine methyl ester hydrochloride (156 mg, 0.859 mmol) was added to the stirring milky-coloured reaction mixture. After 90 hours, t.l.c. analysis (ethyl acetate) of the pale yellow semi-transparent suspension showed the presence of one new UV-active spot (Rf 0.90). The reaction mixture was pre-absorbed on silica and purified by flash-column chromatography (ethyl acetate:cyclohexane, 1:3, to 1:1, to ethyl acetate) to yield (S)-methyl 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)-4-methylpentanoate 24 (229 mg, 79%) as a pale yellow crystalline solid. M.p. 116-117° C. melting and decomposition (turned brown); HRMS (CI+): found 407.19636 [M+H]+ C24H27N2O4 requires 407.19707; [α]D20 +33.866 (c, 0.25 in chloroform); νmax (thin film): 3170 (w, sharp, 2 bands, sec. NH), 3040 (w, ArC—H), 2956, 2929 (w, alkyl C—H), 1748 (m, C═O ester), 1738 (s, C═O ketone), 1661 (s, C═ONH, I), 1571 (m, C═ONH, II), 1605 (s, C═C conjugated with C═Os) cm−1; δH (CDCl3, 500 MHz): 0.95 (3Hs, d, JCH3,CH 6.3 Hz, CH3 (Leucine)), 0.97 (3Hs, d, JCH3,CH 6.6 Hz, CH3 (Leucine)), 1.70-1.90 (1H, m, CH (Leucine)), 1.76 (2Hs, dd, JCH2,CH 14.1 Hz, JCH2,CαH 8.6 Hz, CH2 (Leucine)), 3.71 (3Hs, s, OMe), 4.77 (1H, ddd, JCαH,CHH 9.1 Hz, JCαH,NH 7.5 Hz, JCαH,CHH 5.6 Hz, CαH), 5.43 (2Hs, s, CH2 (Bn)), 7.10-7.14 (2Hs, m, 2×ArHs (Bn)), 7.23-7.32 (3Hs, m, 3×ArHs (Bn)), 7.36-7.41 (2Hs, m, HB & HD), 7.55 (1H, ddd, JHC,HD 8.7 Hz, JHC,HB 6.9 Hz, JHC,HA 1.6 Hz, HC), 8.46 (1H, dd, JHA,HB 8.3 Hz, JHA,HC 1.4 Hz, HA), 8.87 (1H, s, HE), 10.38 (1H, d, JNH,CαH 7.3 Hz, NH); δC (CDCl3, 125 MHz): 21.8, 23.1 (6Hs, 2×CH3 (Leucine)), 25.1 (CH (Leucine)), 41.2 (CH2 (Leucine)), 51.1 (CαH), 52.2 (OMe), 57.7 (CH2 (Bn)), 111.6 (O═C—C—C═O), 116.9 (CD), 125.3 (CB), 126.2, 128.6, 129.3 (5×ArCs (Bn)), 127.3 (CA), 128.1 (CA-Cq-C═O), 133.0 (CC), 134.3 (Cq (Bn)), 139.3 (Cq-N-Bn), 148.7 (CE), 165.0 (C═ONH), 173.5 (C═OOMe), 176.9 (O═C—C═C).
(S)-Methyl 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)-4-methylpentanoate 24 (60 mg, 0.172 mmol) was stirred in acetone (2 mL) and an aqueous solution of sodium hydroxide (2 M, 5 mL) at room temperature for 24 hours. T.l.c. analysis (ethyl acetate) showed the presence of one UV-active product and complete consumption of the starting material. The reaction solution was acidified with an aqueous solution of hydrochloric acid (2 M) until a white solid crushed out. The reaction mixture was filtered, the solid washed with water and dried under reduced pressure to yield (S)-2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)-4-methylpentanoic acid 25 as a white solid. HRMS (TOF MS ES+): found 415.1635 [M+Na]+ C23H24N2O4Na requires 415.1634; νmax (Ge): 3500-2700 (m, COOH), 3243 (m, broad, sec. NH), 3066 (w, ArC—H), 2955, 2930, 2870 (m, alkyl C—H), 1652 (s broad, C═ONH, I), 1602 (s, C═C conjugated with C═Os), 1548 (m, C═ONH, II) cm−1; δH (methanol d4, 500 MHz): 0.95-1.00 (6Hs, m, 2×CH3 (Leucine)), 1.70-1.87 (3H, m, CH & CH2 (Leucine)), 4.55-4.63 (1H, m, CαH (Leucine)), 5.69 (2Hs, s, CH2 (Bn)), 7.18-7.34 (5Hs, m, 5×ArHs (Bn)), 7.39-7.46 (1Hs, m, HB), 7.62-7.67 (2Hs, m, HC & HD), 8.35 (1H, d, J 7.8 Hz, HA), 8.97 (1H, s, HE); δC (methanol d4, 150 MHz): 21.6, 23.0 (2Cs, 2×CH3 (Leucine)), 25.6 (CH (Leucine)), 42.4 (CH2 (Leucine)), 54.1 (CαH), 57.6 (CH2 (Bn)), 111.5 (O═C—C—C═O), 117.4 (CD), 125.7 (CB), 126.7, 128.5, 129.4 (5×ArCs (Bn)), 126.9 (CA), 128.1 (CA-Cq-C═O), 133.5 (CC), 135.8 (Cq (Bn)), 139.9 (Cq-N-Bn), 149.5 (CE), 165.7 (C═ONH), 177.3 (COOH), 179.2 (O═C—C═C).
(S)-Methyl 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-phenylpropanoate 20 (60 mg, 0.136 mmol) was stirred in acetone (2 mL) and an aqueous solution of sodium hydroxide (2 M, 5 mL) at room temperature for 24 hours. T.l.c. analysis (ethyl acetate) showed the presence of one UV-active product (Rf 0.00-0.10) and complete consumption of the starting material (Rf 0.94). The reaction solution was acidified with an aqueous solution of hydrochloric acid (2 M) until a white solid crushed out. The reaction mixture was filtered, the solid washed with water and dried under reduced pressure to yield (S)-2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-phenylpropanoic acid 26 as a white solid. M.p. 227-228° C. melted and decomposed (turned brown); HRMS (TOF MS ES+): found 427.1644 [M+H]+ C26H23N2O4 requires 427.1658, found 449.1496 [M+Na]+ C26H22N2O4Na requires 449.1477; νmax (Ge): 3132 (m, broad, sec. NH), (masked ArC—H & alkyl C—H), 1713 (m, C═OOH), 1661 (s broad, C═ONH, I), 1598, 1586 (s, C═C conjugated with C═Os), 1552 (s, C═ONH, II) cm−1; δH (pyridine d5, 500 MHz): 11.34 (1H, d, JNH,CαH 7.7 Hz, NH), 9.47 (1H, s, HE), 8.78 (1H, d, JHA,HB 8.0 Hz, HA), 7.68 (2H, d, J 7.3 Hz, 2×ArHs), 7.64 (1H, d, JHD,HC 8.5 Hz, HD), 7.51 (1H, ddd, JHC,HD 8.5 Hz, JHC,HB 7.2 Hz, JHC,HA 1.6 Hz, HC), 7.42-7.19 (8H, m, 7×ArHs & HB), 5.73 (1H, m, J 5.2 Hz, CαH), 5.71 (2H, s, CH2 (Bn)), 3.78 (1H, dd, JCHyHx,CHxHy 13.8 Hz, JCHyHx,CαH 5.1 Hz, CHYHX (Phenylalanine)), 3.59 (1H, dd, JCHxHy,CHyHx 13.8 Hz, JCHyHx,CαH 7.8 Hz, CHYHX (Phenylalanine)); δC (pyridine d5, 125 MHz): 39.3 (CH2 (Phenylalanine)), 55.4 (Phenylalanine Cα), 57.4 (CH2 (Bn)), 112.7 (O═C—C—C═O), 118.1 (CD), 125.5 (CB), 127.0 127.3, 128.7, 129.1 129.7, 130.4 (10×ArCs (Bn & Phenylalanine)), 127.6 (CA), 128.9 (CA-Cq-C═O), 133.2 (CC), 136.2 (Cq (Bn)), 138.7 (Cq Phenylalanine)), 140.1 (CC-Cq-N-Bn), 149.7 (CE), 165.4 (C═ONH), 175.0 (CO2H), 177.3 (O═C—C═C).
(S)-Methyl 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)propanoate 21 (60 mg, 0.165 mmol) was stirred in acetone (2 mL) and an aqueous solution of sodium hydroxide (2 M, 5 mL) at room temperature for 24 hours. T.l.c. analysis (ethyl acetate) showed the presence of one UV-active product and complete consumption of the starting material. The reaction solution was acidified with an aqueous solution of hydrochloric acid (2 M) until a white solid crushed out. The reaction mixture was filtered, the solid washed with water and dried under reduced pressure to yield (S)-2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)-2-methylethanoic acid 27 as a white solid. M.p. Decomposition before melting. At 205° C. the compound started turning brown. Kept turning brown until at 230° C. it was dark brown; HRMS (CI+): found 351.13380 [M+H]+ C20H19N2O4 requires 351.13448; νmax (Ge): 3500-2700 (m, COOH), (masked sec. NH & ArC—H), 2945 (w, alkyl C—H), (masked C═OOH), 1654 (s broad, C═ONH, I), 1603 (s, C═C conjugated with C═Os), 1546 (m, C═ONH, II) cm−1; δH (methanol d4, 500 MHz) 8.98 (1H, s, HE), 8.30 (1H, d, JHA,HB 7.0 Hz, HA), 7.62 (2H, m, HC & HD), 7.43-7.37 (1H, m, HB), 7.35-7.11 (5H, m, 5×ArHs (Bn)), 5.64 (2H, s, CH2 (Bn)), 4.54 (1H, dd, J 12.9 Hz, 6.0 Hz, CαH), 1.50 (3H, d, J 7.0 Hz, CH3 (Alanine)). δC (methanol d4, 150 MHz): 19.1 (CH3 (Alanine)), 51.3 (CαH), 58.4 (CH2 (Bn)), 112.1 (O═C—C—C═O), 119.0 (CD), 126.6 (CB), 127.5, 129.4, 130.2 (5×ArCs (Bn)), 127.6 (CA), 128.9 (CA-Cq-C═O), 134.3 (CC), 136.6 (Cq (Bn)), 140.7 (Cq-N-Bn), 150.4 (CE), 166.4 (C═ONH), 177.9 (COOH), 179.2 (O═C—C═C).
(S)-Methyl 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-methylbutanoate 22 (55 mg, 0.140 mmol) was stirred in acetone (2 mL) and an aqueous solution of sodium hydroxide (2 M, 5 mL) at room temperature for 24 hours. T.l.c. analysis (ethyl acetate) showed the presence of one UV-active product and complete consumption of the starting material. The reaction solution was acidified with an aqueous solution of hydrochloric acid (2 M) until a white solid crushed out. The reaction mixture was filtered, the solid washed with water and dried under reduced pressure to yield (S)-2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)-3-methylbutanoic acid 28 (31 mg) as a white solid. HRMS (TOF MS ES+): found 401.1477 [M+Na]+ C22H22N2O4Na requires 401.1477; νmax (Ge): 3400 (m broad, COOH), 3251 (m, broad, sec. NH), 3065 (w, ArC—H), 2964, 2931, 2873 (m, alkyl C—H), 1655 (s broad with shoulder, C═ONH, I & C═OOH), 1603 (s, C═C conjugated with C═Os), 1546 (m, C═ONH, II) cm−1; δH (methanol d4, 500 MHz) 8.97 (1H, s, HE), 8.45 (1H, d, JHA,HB 8.1 Hz, HA), 7.69 (2H, m, HC & HD), 7.48 (1H, dt, JHB,HA 8.1 Hz, J 3.9 Hz HB), 7.37-7.21 (5H, m, 5×ArHs (Bn)), 5.68 (2H, s, CH2 (Bn)), 4.50 (1H, d, J 4.5 Hz, CαH (Valine)), 2.40 (1H, d septet, JCH,CH3 6.8 Hz, JCH,CαH 4.6 Hz, CH (Valine)), 1.07 (3Hs, d, JCH3,CH 6.9 Hz, CH3 (Valine)), 1.05 (3Hs, d, JCH3,CH 6.9 Hz, CH3 (Valine)). δC (methanol d4, 150 MHz): 18.3, 20.6 (2×CH3 (Valine)), 32.5 (1H, CH (Valine)), 58.4 (CH2 (Bn)), 61.5 (CαH), 112.7 (O═C—C—C═O), 119.0 (CD), 126.5 (CB), 127.6, 129.4, 130.2 (5×ArCs (Bn)), 127.7 (CA), 129.1 (CA-Cq-C═O), 134.2 (CC), 136.7 (Cq (Bn)), 140.8 (Cq-N-Bn), 150.3 (CE), 166.5 (C═ONH), 178.2 (COOH), 178.8 (O═C—C═C).
Methyl 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetate 23 (60 mg, 0.171 mmol) was stirred in acetone (2 mL) and an aqueous solution of sodium hydroxide (2 M, 5 mL) at room temperature for 24 hours. T.l.c. analysis (ethyl acetate) showed the presence of one UV-active product and complete consumption of the starting material. The reaction solution was acidified with an aqueous solution of hydrochloric acid (2 M) until a white solid crushed out. The reaction mixture was filtered, the solid washed with water and dried under reduced pressure to yield 2-(1-benzyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetic acid 29 as a pale yellow solid. M.p. At 182° C. decomposition occurred (turned brown); HRMS (TOF MS ES+): found 359.1018 [M+Na]+ C19H16N2O4Na requires 359.1008; νmax (Ge): 3487 (m broad, COOH), 3281 (m, broad, sec. NH), 3066 (w, ArC—H), 2920, 2851 (w, alkyl C—H), 1654 (s broad with shoulder, C═ONH, I & C═OOH), 1602 (s, C═C conjugated with C═Os), 1545 (m, C═ONH, II) cm−1; δH (methanol d4, 500 MHz) 8.99 (1H, s, HE), 8.44 (1H, d, JHA,HB 7.9 Hz, HA), 7.70-7.66 (2H, m, HC & HD), 7.48 (1H, dt, JHB,HA 8.0 Hz, J 4.1 Hz HB), 7.37-7.21 (5H, m, 5×ArHs (Bn)), 5.67 (2H, s, CH2 (Bn)), 4.04 (1H, s, CαH2 (Glycine)); δC (methanol d4, 150 MHz): 44.7 (CαH2), 58.3 (CH2 (Bn)), 112.6 (O═C—C—C═O), 119.0 (CD), 126.5 (CB), 127.6, 129.4, 130.2 (5×ArCs (Bn)), 127.8 (CA), 129.1 (CA-Cq-C═O), 134.2 (CC), 136.7 (Cq (Bn)), 140.9 (Cq-N-Bn), 150.3 (CE), 166.6 (C═ONH), 176.3 (COOH), 178.1 (O═C—C═C).
A mixture of aniline (9.12 mL, 0.1 mol) and diethylethoxymethylmalonate (20 mL, 0.1 mol) were stirred at 120° C. for 2 h. The ethanol formed was removed under reduced pressure to give a yellow solid which was used directly in the next step.
To diethyl 2-((phenylamino)methylene)malonate from the previous step (max 0.1 mol) was added Ph2O (500 mL). In three batches the reaction mixture was heated to 260° C. for 4 h. Upon cooling to room temperature a white precipitate formed which was collected by filtration, washed with toluene and dried under vacuum to give the title product (12.4 g, 57% over two steps) as a white powder.
HRMS m/z (EI) 217.07352, calculated for C12H11NO3+217.07334; 1H NMR (500 MHz, DMSO) δ 12.32 (s, 1H, NH), 8.55 (s, 1H, aromatic), 8.19-8.13 (m, 1H, aromatic), 7.74-7.67 (m, 1H, aromatic), 7.62 (d, J=8.1, 1H, aromatic), 7.42 (t, J=7.1, 1H, aromatic), 4.22 (q, J=7.1, 2H, CH2CH3), 1.28 (t, J=7.1, 3H, CH2CH3); 13C NMR (126 MHz, DMSO) δ 173.40 (CO), 164.80 (CO), 144.87 (aromatic), 138.95 (aromatic), 132.37 (aromatic), 127.25 (aromatic), 125.60 (aromatic), 124.66 (aromatic), 118.77 (aromatic), 109.76 (aromatic), 59.54 (CH2CH3), 14.32 (CH2CH3).
To a suspension of ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (1 g, 4.60 mmol) and K2CO3 (1.20 g, 8.60 mmol) in DMF (7 mL) at 40° C. was added 2-fluorobenzyl bromide (2.02 mL, 16.75 mmol) dropwise. The reaction mixture was stirred at 80° C. for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 0-20% MeOH in EtOAc to give the title product (1.106 g, 74%) as a white powder.
HRMS m/z (EI) 325.11090, calculated for C19H16NO3F+ 325.11087.
To a suspension of ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (1 g, 4.60 mmol) and K2CO3 (1.20 g, 8.60 mmol) in DMF (7 mL) at 40° C. was added 4-trifluoromethoxybenzyl bromide (2.68 mL, 16.75 mmol) dropwise. The reaction mixture was stirred at 80° C. for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 0-20% MeOH in EtOAc to give the title product (1.56 g, 87%) as a white powder.
HRMS m/z (EI) 391.10268, calculated for C20H16NO4F3+ 391.10259.
To a suspension of ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (1 g, 4.60 mmol) and K2CO3 (1.20 g, 8.60 mmol) in DMF (7 mL) at 40° C. was added bromoethane (1.25 mL, 16.75 mmol) dropwise. The reaction mixture was stirred at 80° C. for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 0-20% MeOH in EtOAc to give the title product (921 mg, 82%) as a white powder.
HRMS m/z (EI) 245.10481, calculated for C14H15NO3+ 245.10464; 1H NMR (500 MHz, CDCl3) δ 8.53 (s, 1H, aromatic), 8.51 (dd, J=8.1, 1.6, 1H, aromatic), 7.66 (ddd, J=8.6, 7.1, 1.6, 1H, aromatic), 7.45 (d, J=8.5, 1H, aromatic), 7.43-7.38 (m, 1H, aromatic), 4.37 (q, J=7.1, 2H, CH2CH3), 4.26 (q, J=7.3, 2H, CH2CH3), 1.52 (t, J=7.3, 3H, CH2CH3), 1.38 (t, J=7.1, 3H, CH2CH3); 13C NMR (126 MHz, CDCl3) δ 174.40 (CO), 166.22 (CO), 148.85 (aromatic), 138.82 (aromatic), 132.88 (aromatic), 129.29 (aromatic), 128.28 (aromatic), 125.31 (aromatic), 115.76 (aromatic), 111.08 (aromatic), 61.15 (CH2CH3), 49.11 (CH2CH3), 14.71 (CH2CH3), 14.62 (CH2CH3).
To a suspension of ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (1 g, 4.60 mmol) and K2CO3 (1.20 g, 8.60 mmol) in DMF (7 mL) at 40° C. was added 3-chlorobenzyl bromide (2.2 mL, 16.75 mmol) dropwise. The reaction mixture was stirred at 80° C. for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 0-20% MeOH in EtOAc to give the title product (805 mg, 51%) as a white powder.
HRMS m/z (EI) 341.08162, calculated for C19H16NO335Cl+ 341.08132; 1H NMR (500 MHz, CDCl3) δ 8.62 (s, 1H, aromatic), 8.52 (dd, J=8.1, 1.5, 1H, aromatic), 7.55 (ddd, J=8.6, 7.1, 1.6, 1H, aromatic), 7.43-7.37 (m, 1H, aromatic), 7.30-7.25 (m, 3H, aromatic), 7.15 (s, 1H, aromatic), 7.03-6.98 (m, 1H, aromatic), 5.38 (s, 2H, Bn-CH2), 4.39 (q, J=7.1, 2H, CH2CH3), 1.40 (t, J=7.1, 3H, CH2CH3); 13C NMR (126 MHz, CDCl3) δ 174.50 (CO), 165.99 (CO), 149.96 (aromatic), 139.18 (aromatic), 136.59 (aromatic), 135.63 (aromatic), 133.08 (aromatic), 130.91 (aromatic), 129.24 (aromatic), 129.08 (aromatic), 128.23 (aromatic), 126.40 (aromatic), 125.65 (aromatic), 124.29 (aromatic), 116.56 (aromatic), 111.57 (aromatic), 61.37 (Bn-CH2), 57.02 (CH2CH3), 14.63 (CH2CH3).
To a suspension of ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (1 g, 4.60 mmol) and K2CO3 (1.20 g, 8.60 mmol) in DMF (7 mL) at 40° C. was added 4-methoxybenzyl chloride (2.28 mL, 16.75 mmol) dropwise. The reaction mixture was stirred at 80° C. for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 0-20% MeOH in EtOAc to give the title product (960 mg, 62%) as a white powder.
HRMS m/z (EI) 337.13145, calculated for C20H19NO4+ 337.13086; 1H NMR (500 MHz, CDCl3) δ 8.68 (s, 1H, aromatic), 8.51 (dd, J=8.2, 1.6, 1H, aromatic), 7.56 (ddd, J=8.7, 7.2, 1.6, 1H, aromatic), 7.43-7.37 (m, 2H, aromatic), 7.28 (d, J=8.5, 1H, aromatic), 7.10 (d, J=8.7, 1H, aromatic), 6.85 (dd, J=8.7, 3.5, 2H, aromatic), 5.37 (s, 2H, Bn-CH2), 4.39 (q, J=7.1, 2H, CH2CH3), 3.75 (s, 3H, O—CH3), 1.40 (t, J=7.1, 3H, CH2CH3); 13C NMR (126 MHz, CDCl3) δ 174.54 (CO), 166.20 (CO), 159.97 (aromatic), 149.87 (aromatic), 139.41 (aromatic), 132.97 (aromatic), 130.46 (aromatic), 129.11 (aromatic), 128.83 (aromatic), 128.04 (aromatic), 127.90 (aromatic), 126.13 (aromatic), 125.55 (aromatic), 116.84 (aromatic), 114.93 (aromatic), 114.12 (aromatic), 61.34 (Bn-CH2), 57.31 (CH2CH3), 55.52 (O—CH3), 14.62 (CH2CH3).
To a suspension of ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (1 g, 4.60 mmol) and K2CO3 (1.20 g, 8.60 mmol) in DMF (7 mL) at 40° C. was added 3,5-dimethylbenzyl bromide (3.33 g, 16.75 mmol) dropwise. The reaction mixture was stirred at 80° C. for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 0-20% MeOH in EtOAc to give the title product (850 mg, 55%) as a white powder.
HRMS m/z (EI) 335.15192, calculated for C21H21NO3+ 335.15160; 1H NMR (500 MHz, CDCl3) δ 8.61 (s, 1H, aromatic), 8.54 (d, J=8.0, 1H, aromatic), 7.55 (t, J=7.7, 1H, aromatic), 7.39 (t, J=7.5, 1H, aromatic), 7.34 (d, J=8.5, 1H, aromatic), 6.94 (s, 1H, aromatic), 6.75 (s, 2H, aromatic), 5.32 (s, 2H, Bn-CH2), 4.41 (q, J=7.1, 2H, CH2CH3), 2.26 (s, 6H 2×aromatic-CH3), 1.42 (t, J=7.1, 3H, CH2CH3); 13C NMR (126 MHz, CDCl3) δ 174.56 (CO), 166.08 (CO), 149.96 (aromatic), 139.34 (aromatic), 139.20 (aromatic), 134.24 (aromatic), 132.73 (aromatic), 130.32 (aromatic), 129.26 (aromatic), 127.93 (aromatic), 125.27 (aromatic), 123.83 (aromatic), 116.70 (aromatic), 111.11 (aromatic), 61.10 (Bn-CH2), 57.59 (CH2CH3), 21.36 (aromatic-CH3), 14.52 (CH2CH3).
To a suspension of ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (1 g, 4.60 mmol) and K2CO3 (1.20 g, 8.60 mmol) in DMF (7 mL) at 40° C. was added 2-chlorobenzyl bromide (2.16 mL, 16.75 mmol) dropwise. The reaction mixture was stirred at 80° C. for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 0-20% MeOH in EtOAc to give the title product (452 mg, 29%) as a white powder.
HRMS m/z (EI) 341.08157, calculated for C19H16NO335Cl+ 341.08132; 1H NMR (500 MHz, CDCl3) δ 8.61 (s, 1H, aromatic), 8.51 (dd, J=8.1, 1.5, 1H, aromatic), 7.57 (ddd, J=8.6, 7.1, 1.6, 1H, aromatic), 7.46 (dd, J=8.0, 0.9, 1H, aromatic), 7.41-7.36 (m, 1H, aromatic), 7.32-7.25 (m, 1H, aromatic), 7.22 (d, J=8.6, 1H, aromatic), 7.14 (td, J=7.7, 1.1, 1H, aromatic), 6.78 (d, J=7.1, 1H, aromatic), 5.49 (s, 2H, Bn-CH2), 4.37 (q, J=7.1, 2H, CH2CH3), 1.38 (t, J=7.1, 3H, CH2CH3); 13C NMR (126 MHz, CDCl3) δ 174.96 (CO), 165.88 (CO), 150.05 (aromatic), 139.29 (aromatic), 136.94 (aromatic), 133.23 (aromatic), 132.57 (aromatic), 131.89 (aromatic), 130.92 (aromatic), 130.32 (aromatic), 130.04 (aromatic), 128.13 (aromatic), 127.88 (aromatic), 127.49 (aromatic), 125.67 (aromatic), 116.59 (aromatic), 61.33 (Bn-CH2), 55.11 (CH2CH3), 14.61 (CH2CH3).
A suspension of ethyl 1-ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (500 mg, 2.04 mmol) in 2 M NaOH (aq) (10 mL, 20 mmol) was stirred at 100° C. for 2 h. After cooling to room temperature the reaction mixture was diluted with water (10 mL) and acidified by dropwise addition of conc. HCl. The resulting white precipitate was collected by filtration and dried under vacuum to give the title product (345 mg, 78%) as a white powder.
HRMS m/z (ES+) 218.0806, calculated for C12H12NO3+ 218.0817; 1H NMR (500 MHz, DMSO) δ 15.25 (s, OH), 9.07 (s, 1H, aromatic), 8.40 (d, J=7.8, 1H, aromatic), 8.06 (d, J=8.6, 1H, aromatic), 7.98 (t, J=7.5, 1H, aromatic), 7.67 (t, J=7.4, 1H, aromatic), 4.61 (q, J=7.0, 2H, CH2CH3), 1.42 (t, J=7.1, 3H, CH2CH3); 13C NMR (126 MHz, DMSO) δ 177.70 (CO), 166.06 (CO), 149.19 (aromatic), 139.04 (aromatic), 134.30 (aromatic), 126.29 (aromatic), 125.96 (aromatic), 125.53 (aromatic), 118.08 (aromatic), 107.66 (aromatic), 48.98 (CH2CH3), 14.54 (CH2CH3).
A suspension of ethyl 1-(3-chlorobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (500 mg, 1.46 mmol) in 2 M NaOH (aq) (10 mL, 20 mmol) was stirred at 100° C. for 2 h. After cooling to room temperature the reaction mixture was diluted with water (10 mL) and acidified by dropwise addition of conc. HCl. The resulting white precipitate was collected by filtration and dried under vacuum to give the title product (410 mg, 90%) as a white powder.
HRMS m/z (ES+) 314.0600, calculated for C17H13NO335Cl+ 314.0584; 1H NMR (500 MHz, DMSO) δ 15.13 (s, OH), 9.17 (s, 2H, aromatic), 8.37 (d, J=8.0, 1H, aromatic), 7.93-7.71 (m, 2H, aromatic), 7.56 (t, J=7.4, 1H, aromatic), 7.41 (s, 1H, aromatic), 7.37 (d, J=5.0, 2H, aromatic), 7.17 (t, J=3.6, 1H, aromatic), 5.82 (s, 2H Bn-CH2); 13C NMR (126 MHz, DMSO) δ 177.49 (CO), 166.15 (CO), 149.96 (aromatic), 139.31 (aromatic), 138.33 (aromatic), 133.51 (aromatic), 130.82 (aromatic), 127.95 (aromatic), 126.54 (aromatic), 126.13 (aromatic), 125.67 (aromatic), 125.12 (aromatic), 118.09 (aromatic), 55.37 (Bn-CH2).
To a suspension of ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (500 mg, 2.30 mmol) in 1:1 H2O/1,4-dioxane (4 mL) was added Et3N, (700 μL, 5.02 mmol) followed by di-tert-butyl dicarbonate (750 mg, 3.44 mmol). The reaction mixture was stirred at room temperature for 18 h. Water (25 mL0 and EtOAc were added to the reaction mixture and the layers separated. The aqueous layer was extracted with EtOAc (2×50 mL) and the combined organic fractions were dried over MgSO4, filtered and evaporated to give the title product (691 mg, 95%) as a white powder. HRMS m/z (EI) 317.12627, calculated for C17H19NO5+ 317.12577.
1H NMR (500 MHz, CDCl3) δ 9.12 (s, 1H, aromatic), 8.47 (d, J=8.8, 1H, aromatic), 8.43 (dd, J=8.0, 1.7, 1H, aromatic), 7.65 (ddd, J=8.8, 7.1, 1.8, 1H, aromatic), 7.44 (ddd, J=8.0, 7.1, 0.9, 1H, aromatic), 4.39 (q, J=7.1, 2H, CH2CH3), 1.68 (s, 9H, C(CH3)3), 1.39 (t, J=7.1, 3H, CH2CH3); 13C NMR (126 MHz, CDCl3) δ 175.11 (CO), 165.04 (CO), 149.45 (CO), 145.22 (aromatic), 137.72 (aromatic), 133.07 (aromatic), 128.22 (aromatic), 127.59 (aromatic), 126.27 (aromatic), 119.96 (aromatic), 113.58 (aromatic), 88.12 (C(CH3)3), 61.54 (CH2CH3), 28.09 (C(CH3)3), 14.53 (CH2CH3).
A suspension of ethyl 1-(2-fluorobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (500 mg, 1.54 mmol) in 2 M NaOH (aq) (10 mL, 20 mmol) was stirred at 100° C. for 2 h. After cooling to room temperature the reaction mixture was diluted with water (10 mL) and acidified by dropwise addition of conc. HCl. The resulting white precipitate was collected by filtration and dried under vacuum to give the title product (379 mg, 83%) as a pale pink powder.
HRMS m/z (ES+) 298.0871, calculated for C17H13NO3F+ 298.0879; 1H NMR (500 MHz, DMSO) δ 15.10 (s, 1H, OH), 9.27 (s, 1H, aromatic), 8.41 (d, J=7.8, 1H, aromatic), 7.87 (dd, J=17.7, 7.6, 2H, aromatic), 7.64 (t, J=7.3, 1H, aromatic), 7.39 (d, J=5.9, 1H, aromatic), 7.32-7.25 (m, 1H, aromatic), 7.18 (dd, J=19.1, 7.2, 2H, aromatic), 5.93 (s, 2H, Bn-CH2); 13C NMR (126 MHz, DMSO) δ 178.02 (CO), 165.89 (CO), 150.53 (aromatic), 139.43 (aromatic), 134.33 (aromatic), 130.39 (aromatic), 128.99 (aromatic), 126.43 (aromatic), 126.03 (aromatic), 125.58 (aromatic), 124.98 (aromatic), 122.24 (aromatic), 118.16 (aromatic), 115.93 (aromatic), 115.76 (aromatic), 107.95 (aromatic), 51.38 (Bn-CH2).
A suspension of ethyl 4-oxo-1-(4-(trifluoromethoxy)benzyl)-1,4-dihydroquinoline-3-carboxylate (500 mg, 1.28 mmol) in 2 M NaOH (aq) (10 mL, 20 mmol) was stirred at 100° C. for 2 h. After cooling to room temperature the reaction mixture was diluted with water (10 mL) and acidified by dropwise addition of conc. HCl. The resulting white precipitate was collected by filtration and dried under vacuum to give the title product (282 mg, 61%) as a white powder.
HRMS m/z (ES+) 364.0804, calculated for C18H13NO4F3+ 364.0797.
A suspension of ethyl 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (500 mg, 1.48 mmol) in 2 M NaOH (aq) (10 mL, 20 mmol) was stirred at 100° C. for 2 h. After cooling to room temperature the reaction mixture was diluted with water (10 mL) and acidified by dropwise addition of conc. HCl. The resulting white precipitate was collected by filtration and dried under vacuum to give the title product (420 mg, 92%) as a pale pink powder.
HRMS m/z (ES+) 332.0883, calculated for C18H15NO4Na+ 332.0899; 1H NMR (500 MHz, DMSO) δ 15.18 (s, 1H, OH), 9.26 (s, 1H, aromatic), 8.39 (d, J=7.9, 1H, aromatic), 7.95 (d, J=8.6, 1H, aromatic), 7.88 (dd, J=11.4, 4.2, 1H, aromatic), 7.63 (t, J=7.5, 1H, aromatic), 7.27 (d, J=8.5, 2H, aromatic), 6.91 (d, J=8.6, 2H, aromatic), 5.78 (s, 2H, Bn-CH2), 3.71 (s, 3H, O—CH3); 13C NMR (126 MHz, DMSO) δ 177.90 (CO), 166.02 (CO), 159.02 (aromatic), 149.84 (aromatic), 139.44 (aromatic), 134.12 (aromatic), 128.40 (aromatic), 127.88 (aromatic), 127.00 (aromatic), 126.36 (aromatic), 125.89 (aromatic), 125.70 (aromatic), 118.72 (aromatic), 114.31 (aromatic), 113.42 (aromatic), 107.77 (aromatic), 55.96 (Bn-CH2), 55.08 (O—CH3).
A suspension of ethyl 1-(3.5-dimethylbenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (500 mg, 1.49 mmol) in 2 M NaOH (aq) (10 mL, 20 mmol) was stirred at 100° C. for 2 h. After cooling to room temperature the reaction mixture was diluted with water (10 mL) and acidified by dropwise addition of conc. HCl. The resulting white precipitate was collected by filtration and dried under vacuum to give the title product (411 mg, 90%) as an off white powder.
HRMS m/z (EI) 307.12077, calculated for C19H17NO3+ 307.12029.
A suspension of ethyl 1-(2-chlorobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (350 mg, 1.02 mmol) in 2 M NaOH (aq) (10 mL, 20 mmol) was stirred at 100° C. for 2 h. After cooling to room temperature the reaction mixture was diluted with water (10 mL) and acidified by dropwise addition of conc. HCl. The resulting white precipitate was collected by filtration and dried under vacuum to give the title product (198 mg, 62%) as a white powder.
HRMS m/z (ES+) 314.0574, calculated for C17H13NO3Cl+ 314.0584; 1H NMR (500 MHz, DMSO) δ 15.06 (s, 1H, OH), 9.19 (s, 1H, aromatic), 8.42 (dd, J=8.0, 1.4, 1H, aromatic), 7.91-7.82 (m, 1H, aromatic), 7.61 (ddd, J=11.6, 9.0, 5.4, 3H, aromatic), 7.44-7.32 (m, 1H, aromatic), 7.24 (dd, J=10.9, 4.3, 1H, aromatic), 6.87 (d, J=7.7, 1H, aromatic), 5.91 (s, 2H, Bn-CH2); 13C NMR (126 MHz, DMSO) δ 177.99 (CO), 165.89 (CO), 150.36 (aromatic), 139.52 (aromatic), 134.31 (aromatic), 132.62 (aromatic), 131.57 (aromatic), 129.85 (aromatic), 129.78 (aromatic), 127.86 (aromatic), 127.82 (aromatic), 126.31 (aromatic), 126.08 (aromatic), 125.77 (aromatic), 124.09 (aromatic), 118.06 (aromatic), 54.33 (Bn-CH2).
A suspension of 1-tert-butyl 3-ethyl 4-oxoquinoline-1,3(4H)-dicarboxylate (500 mg, 1.58 mmol) in 2 M NaOH (aq) (10 mL, 20 mmol) was stirred at 100° C. for 2 h. After cooling to room temperature the reaction mixture was diluted with water (10 mL) and acidified by dropwise addition of conc. HCl. The resulting white precipitate was collected by filtration and dried under vacuum. LCMS showed that the product had lost the Boc group during the reaction work up. The amine (128 mg, 0.677 mmol) was taken up in H2O/dioxane (1:1) (2 mL). Et3N (180 μL, 1.29 mmol) and di-tert-butyl dicarbonate (155 mg, 0.70 mmol) were added. The reaction mixture was stirred at room temperature for 2 h. Water (5 mL) and EtOAc (10 mL) were added to the reaction mixture and the layers separated. The aqueous layer was extracted with EtOAc (2×10 mL) and the combined organic fractions were dried over MgSO4, filtered and evaporated to give the title product (180 mg, 40% over two steps) as a white powder.
HRMS m/z (ES+) 190.0495, calculated for C10H8NO3+ (M-Boc+) 190.0504; 1H NMR (500 MHz, CDCl3) δ 14.25 (s, 1H, OH), 9.36 (s, 1H, aromatic), 8.56 (d, J=8.9, 1H, aromatic), 8.50 (dd, J=8.1, 1.6, 1H, aromatic), 7.83 (ddd, J=8.8, 7.1, 1.7, 1H, aromatic), 7.59 (dd, J=11.2, 4.0, 1H, aromatic), 1.73 (s, 9H, C(CH3)3); 13C NMR (126 MHz, CDCl3) δ 180.16 (CO), 165.79 (CO), 148.81 (CO), 146.95 (aromatic), 146.15 (aromatic), 138.42 (aromatic), 134.73 (aromatic), 127.13 (aromatic), 127.05 (aromatic), 125.89 (aromatic), 120.31 (aromatic), 110.01 (aromatic), 89.81 (C(CH3)3), 28.02 (C(CH3)3).
To a suspension of 1-ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (173 mg, 0.797 mmol) in anhydrous DMF (5 mL) was added DIEA (555 μL, 3.188 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (288 mg, 0.757 mmol). After stirring for 2 min, Glycine methylester hydrochloride (200 mg, 1.594 mmol) was added and the reaction mixture was stirred at room temperature for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 0-100% EtOAc in cyclohexane, some product precipitated on the top of the column and was also collected to afford the title product (165 mg, 72%) as a white powder.
HRMS m/z (EI) 288.11017, calculated for C15H16O4N2+ 288.11045; 1H NMR (500 MHz, CDCl3) δ 10.39 (s, 1H, NH), 8.79 (s, 1H, aromatic), 8.53 (dd, J=8.1, 1.5, 1H, aromatic), 7.82-7.68 (m, 1H, aromatic), 7.56 (d, J=8.6, 1H, aromatic), 7.49 (t, J=7.5, 1H, aromatic), 4.35 (q, J=7.2, 2H, CH2CH3), 4.23 (d, J=5.6, 2H, Gly-CH2), 3.75 (s, 3H, O—CH3), 1.54 (t, J=7.3, 3H, CH2CH3); 13C NMR (126 MHz, CDCl3) δ 176.65 (CO), 170.39 (CO), 165.93 (CO), 162.91 (aromatic), 147.50 (aromatic), 139.01 (aromatic), 133.37 (aromatic), 127.77 (aromatic), 125.56 (aromatic), 116.11 (aromatic), 55.83 (O—CH3), 49.50 (CH2CH3), 41.54 (Gly-CH2), 14.79 (CH2CH3).
To a suspension of 1-(3-chlorobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (250 mg, 0.797 mmol) in anhydrous DMF (5 mL) was added DIEA (555 μL, 3.188 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (288 mg, 0.757 mmol). After stirring for 2 min, Glycine methylester hydrochloride (200 mg, 1.594 mmol) was added and the reaction mixture was stirred at room temperature for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 50-100% EtOAc in cyclohexane, some product precipitated on the top of the column and was also collected to afford the title product (211 mg, 69%) as a white powder.
HRMS m/z (EI) 384.08751, calculated for C20H17O4N2Cl+ 384.08713; 1H NMR (500 MHz, CDCl3) δ 10.45 (s, 1H, NH), 8.96 (s, 1H, aromatic), 8.52 (dd, J=8.1, 1.5, 1H, aromatic), 7.65-7.56 (m, 1H, aromatic), 7.46 (t, J=7.5, 1H, aromatic), 7.36 (d, J=8.6, 1H, aromatic), 7.32-7.19 (m, 2H, aromatic), 7.15 (s, 1H, aromatic), 7.00 (d, J=6.9, 1H, aromatic), 5.46 (s, 2H, Bn-CH2), 4.25 (d, J=5.4, 2H, Gly-CH2), 3.76 (s, 3H, O—CH3); 13C NMR (126 MHz, CDCl3) δ 176.86 (CO), 170.32 (CO), 165.74 (CO), 148.79 (aromatic), 139.33 (aromatic), 136.40 (aromatic), 135.61 (aromatic), 133.59 (aromatic), 130.89 (aromatic), 129.15 (aromatic), 127.71 (aromatic), 126.48 (aromatic), 125.87 (aromatic), 124.40 (aromatic), 116.90 (aromatic), 57.38 (Bn-CH2), 52.50 (O—CH3), 41.56 (Gly-CH2).
To a suspension of 1-(2-fluorobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (237 mg, 0.797 mmol) in anhydrous DMF (5 mL) was added DIEA (555 μL, 3.188 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (288 mg, 0.757 mmol). After stirring for 2 min, Glycine methylester hydrochloride (200 mg, 1.594 mmol) was added and the reaction mixture was stirred at room temperature for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 50-100% EtOAc in cyclohexane, some product precipitated on the top of the column and was also collected to afford the title product (250 mg, 85%) as a white powder.
HRMS m/z (EI) 368.11711, calculated for C20H17O4N2F+ 368.11668; 1H NMR (500 MHz, CDCl3) δ 10.48 (s, 1H, NH), 8.95 (s, 1H, aromatic), 8.55 (d, J=7.9, 1H, aromatic), 7.66 (t, J=7.4, 1H, aromatic), 7.46 (dd, J=16.3, 8.3, 2H, aromatic), 7.32 (d, J=6.8, 1H, aromatic), 7.15 (t, J=9.2, 1H, aromatic), 7.06 (t, J=7.5, 1H, aromatic), 6.95 (t, J=7.2, 1H, aromatic), 5.51 (s, 2H, Bn-CH2), 4.27 (d, J=5.4, 2H, Gly-CH2), 3.78 (s, 3H, O—CH3); 13C NMR (126 MHz, CDCl3) δ 176.84 (CO), 170.30 (CO), 165.46 (CO), 160.97 (aromatic), 159.00 (aromatic), 148.65 (aromatic), 139.25 (aromatic), 133.26 (aromatic), 130.66 (aromatic), 128.00 (aromatic), 127.63 (aromatic), 125.51 (aromatic), 125.05 (aromatic), 121.41 (aromatic), 116.33 (aromatic), 116.17 (aromatic), 116.01 (aromatic), 54.22 (O—CH3), 51.43 (Bn-CH2), 41.41 (Gly-CH2).
To a suspension of 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (246 mg, 0.797 mmol) in anhydrous DMF (5 mL) was added DIEA (555 μL, 3.188 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (288 mg, 0.757 mmol). After stirring for 2 min, Glycine methylester hydrochloride (200 mg, 1.594 mmol) was added and the reaction mixture was stirred at room temperature for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 50-100% EtOAc in cyclohexane, some product precipitated on the top of the column and was also collected to afford the title product (231 mg, 76%) as a white powder.
HRMS m/z (EI) 380.13589, calculated for C21H20O5N2+ 380.13667; 1H NMR (500 MHz, CDCl3) δ 10.46 (s, 1H, NH), 8.93 (s, 1H, aromatic), 8.51 (d, J=8.1, 1H, aromatic), 7.61 (t, J=7.8, 1H, aromatic), 7.52-7.39 (m, 2H, aromatic), 7.09 (d, J=8.6, 2H, aromatic), 6.83 (d, J=8.7, 2H, aromatic), 5.40 (s, 2H, Bn-CH2), 4.24 (d, J=5.2, 2H, Gly-CH2), 3.75 (s, 3H, O—CH3), 3.74 (s, 3H, O—CH3); 13C NMR (126 MHz, CDCl3) δ 176.85 (CO), 170.40 (CO), 165.83 (CO), 159.98 (aromatic), 148.55 (aromatic), 139.51 (aromatic), 133.26 (aromatic), 127.98 (aromatic), 127.54 (aromatic), 126.03 (aromatic), 125.59 (aromatic), 117.08 (aromatic), 114.90 (aromatic), 111.33 (aromatic), 57.59 (Bn-CH2), 55.51 (O—CH3), 52.45 (O—CH3), 41.52 (Gly-CH2).
To a suspension of 1-(3.5-dimethylbenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (245 mg, 0.797 mmol) in anhydrous DMF (5 mL) was added DIEA (555 μL, 3.188 mmol) and O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (288 mg, 0.757 mmol). After stirring for 2 min, Glycine methylester hydrochloride (200 mg, 1.594 mmol) was added and the reaction mixture was stirred at room temperature for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 50-100% EtOAc in cyclohexane, some product precipitated on the top of the column and was also collected to afford the title product (246 mg, 81%) as a white powder.
HRMS m/z (EI) 378.15705, calculated for C22H22O4N2+ 378.15740; 1H NMR (500 MHz, CDCl3) δ 10.46 (s, 1H, NH), 9.03 (s, 1H, aromatic), 8.53 (dd, J=8.1, 1.4, 1H, aromatic), 7.68-7.60 (m, 1H, aromatic), 7.52-7.42 (m, 2H, aromatic), 6.91 (s, 1H, aromatic), 6.74 (s, 2H, aromatic), 5.43 (s, 2H, Bn-CH2), 4.26 (d, J=4.1, 2H, Gly-CH2), 3.77 (s, 3H, O—CH3), 2.23 (s, 6H, 2×aromatic-CH3); 13C NMR (126 MHz, CDCl3) δ 176.77 (CO), 170.24 (CO), 166.18 (CO), 148.88 (aromatic), 139.63 (aromatic), 139.36 (aromatic), 134.04 (aromatic), 133.68 (aromatic), 130.89 (aromatic), 130.61 (aromatic), 127.44 (aromatic), 125.94 (aromatic), 124.12 (aromatic), 117.38 (aromatic), 58.41 (Bn-CH2), 52.51 (O—CH3), 41.59 (Gly-CH2), 21.46 (aromatic-CH3s).
To a suspension of 4-oxo-1-(4-(trifluoromethoxy)benzyl)-1,4-dihydroquinoline-3-carboxylic acid (127 mg, 0.325 mmol) in anhydrous DMF (2.5 mL) was added DIEA (230 μL, 1.30 mmol) and O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (117 mg, 0.308 mmol). After stirring for 2 min, Glycine methylester hydrochloride (82 mg, 0.65 mmol) was added and the reaction mixture was stirred at room temperature for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 50-100% EtOAc in cyclohexane, some product precipitated on the top of the column and was also collected to afford the title product (101 mg, 72%) as a white powder.
HRMS m/z (EI) 434.10909, calculated for C21H17O5N2F3+ 434.10840; 1H NMR (500 MHz, CDCl3) δ 10.40 (s, 1H, NH), 8.91 (s, 1H, aromatic), 8.59-8.41 (m, 1H, aromatic), 7.69-7.56 (m, 1H, aromatic), 7.46 (td, J=7.8, 4.3, 1H, aromatic), 7.36 (dd, J=8.5, 4.0, 1H, aromatic), 7.18 (d, J=3.8, 4H, aromatic), 5.47 (s, 2H, Bn-CH2), 4.25 (s, 2H, Gly-CH2), 3.76 (s, 3H, O—CH3); 13C NMR (126 MHz, CDCl3) δ 176.89 (CO), 170.41 (CO), 165.56 (CO), 149.48 (aromatic), 148.67 (aromatic), 139.35 (aromatic), 133.46 (aromatic), 133.01 (aromatic), 128.14 (aromatic), 127.87 (aromatic), 127.78 (aromatic), 125.75 (aromatic), 121.99 (aromatic), 116.79 (aromatic), 111.82 (aromatic), 57.12 (Bn-CH2), 52.47 (O—CH3), 41.53 (Gly-CH2).
To a suspension of 1-(2-chlorobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (150 mg, 0.478 mmol) in anhydrous DMF (3.5 mL) was added DIEA (335 μL, 1.912 mmol) and O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (173 mg, 0.454 mmol). After stirring for 2 min, Glycine methylester hydrochloride (120 mg, 0.956 mmol) was added and the reaction mixture was stirred at room temperature for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 50-100% EtOAc in cyclohexane, some product precipitated on the top of the column and was also collected to afford the title product (142 mg, 77%) as a white powder.
HRMS m/z (EI) 384.08671, calculated for C20H17O4N2Cl+ 384.08713; 1H NMR (500 MHz, CDCl3) δ 10.44 (s, 1H, NH), 8.86 (s, 1H, aromatic), 8.54 (dd, J=8.1, 1.5, 1H, aromatic), 7.61 (ddd, J=8.6, 7.2, 1.5, 1H, aromatic), 7.45 (t, J=7.3, 2H, aromatic), 7.27 (d, J=9.2, 2H, aromatic), 7.12 (td, J=7.7, 1.0, 1H, aromatic), 6.75-6.67 (m, 1H, aromatic), 5.52 (s, 2H, Bn-CH2), 4.24 (d, J=5.5, 2H, Gly-CH2), 3.75 (s, 3H, O—CH3); 13C NMR (126 MHz, CDCl3) δ 176.97 (CO), 170.43 (CO), 165.45 (CO), 148.77 (aromatic), 139.43 (aromatic), 133.43 (aromatic), 132.42 (aromatic), 131.83 (aromatic), 130.29 (aromatic), 130.04 (aromatic), 128.18 (aromatic), 127.85 (aromatic), 127.73 (aromatic), 127.35 (aromatic), 125.65 (aromatic), 116.67 (aromatic), 112.03 (aromatic), 55.14 (Bn-CH2), 52.44 (O—CH3), 41.50 (Gly-CH2).
Compound 170. tert-Butyl 3-(2-tert-butoxy-2-oxoethylcarbamoyl)-4-oxoquinoline-
To a suspension of 1-tert-butoxycarbonyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (138 mg, 0.478 mmol) in anhydrous DMF (3.5 mL) was added DIEA (335 μL, 1.912 mmol) and O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (173 mg, 0.454 mmol). After stirring for 2 min, Glycine tert-butylester hydrochloride (160 mg, 0.956 mmol) was added and the reaction mixture was stirred at room temperature for 18 h. Solvent was removed under reduced pressure and the product purified by flash chromatography using a gradient of 50-100% EtOAc in cyclohexane, some product precipitated on the top of the column and was also collected to afford the title product (63 mg, 33%) as a pale yellow oil.
HRMS m/z (ES−) 301.1192, calculated for C16H17N2O4 (M-Boc−) 301.1188; 1H NMR (500 MHz, CDCl3) δ 10.56 (t, J=5.3, 1H, NH), 8.60 (s, 1H, aromatic), 8.13 (dd, J=8.2, 1.1, 1H, aromatic), 7.54 (ddd, J=8.4, 7.1, 1.4, 1H, aromatic), 7.43 (d, J=8.2, 1H, aromatic), 7.29-7.26 (m, 1H, aromatic), 4.10 (d, J=5.4, 2H, Gly-CH2), 1.45 (s, 9H C(CH3)3), 1.42 (s, 9H, C(CH3)3); 13C NMR (126 MHz, CDCl3) δ 177.28 (CO), 170.28 (CO), 166.60 (CO), 166.31 (CO), 144.08 (aromatic), 138.86 (aromatic), 133.11 (aromatic), 126.30 (aromatic), 125.98 (aromatic), 125.60 (aromatic), 118.92 (aromatic), 110.46 (aromatic), 84.98 (C(CH3)3), 82.96 (C(CH3)3), 41.73 (Gly-CH2), 28.19 (C(CH3)3), 27.99 (C(CH3)3).
To a solution of methyl 2-(1-ethyl-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetate (50 mg, 0.173 mmol) in THF (2 mL) was added 1 M NaOH (aq) (1 mL, 1 mmol), the reaction mixture was stirred at room temperature for 18 h. The pH of the mixture was then adjusted to 2 by addition of 1 M HCl(aq). Water (10 mL) was added and the product was extracted with EtOAc (3×25 mL). The combined organic fractions were dried over MgSO4, filtered and evaporated. NMR showed some impurities so the product was purified by flash chromatography using a gradient of 0-50% MeOH in EtOAc to give the title product (21.5 mg, 45%) as a white powder.
HRMS m/z (ES+) 297.0864, calculated for C14H14N2O4Na+ 297.0851;
To a solution of methyl 2-(1-(3-chlorobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetate (40 mg, 0.104 mmol) in THF (2 mL) was added 1 M NaOH (aq) (1 mL, 1 mmol), the reaction mixture was stirred at room temperature for 18 h. LCMS showed starting material remaining so a further 1 mL 1M NaOH (aq) was added and the reaction mixture stirred at room temperature for 72 h. The pH of the mixture was then adjusted to 2 by addition of 1 M HCl (aq). Water (10 mL) was added and the product was extracted with EtOAc (3×25 mL). The combined organic fractions were dried over MgSO4, filtered and evaporated to give the title product (15 mg, 39%) as a white powder.
HRMS m/z (ES+) 371.0781, calculated for C19H16N2O4Cl+ 371.0799;
To a solution of methyl-2-(1-(2-fluorobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetate (50 mg, 0.136 mmol) in THF (2 mL) was added 1 M NaOH (aq) (1 mL, 1 mmol), the reaction mixture was stirred at room temperature for 18 h. The pH of the mixture was then adjusted to 2 by addition of 1 M HCl (aq). Water (10 mL) was added and the product was extracted with EtOAc (3×25 mL). The combined organic fractions were dried over MgSO4, filtered and evaporated. NMR showed some impurities so the product was purified by flash chromatography using a gradient of 0-50% MeOH in EtOAc to give the title product (27 mg, 56%) as a white powder. HRMS m/z (ES+) 377.0908, calculated for C19H15N2O4FNa+ 377.0914; 1H NMR (500 MHz, MeOH) δ 9.00 (s, 1H, aromatic), 8.48-8.42 (m, 1H, aromatic), 7.70 (t, J=9.1, 2H, aromatic), 7.49 (t, J=6.9, 1H, aromatic), 7.39-7.31 (m, 1H, aromatic), 7.22-7.02 (m, 3H, aromatic), 5.72 (s, 2H, Bn-CH2), 4.03 (s, 2H Gly-CH2);
To a solution of methyl 2-(1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetate (40 mg, 0.105 mmol) in THF (2 mL) was added 1 M NaOH (aq) (1 mL, 1 mmol), the reaction mixture was stirred at room temperature for 18 h. The pH of the mixture was then adjusted to 2 by addition of 1 M HCl (aq). Water (10 mL) was added and the product was extracted with EtOAc (3×25 mL). The combined organic fractions were dried over MgSO4, filtered and evaporated to give the title product (22 mg, 57%) as a white powder.
HRMS m/z (ES+) 389.1115, calculated for C20H18N2O5Na+ 389.1113;
To a solution of methyl 2-(1-(3.5-dimethylbenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxamido)acetate (30 mg, 0.0793 mmol) in THF (2 mL) was added 1 M NaOH (aq) (1 mL, 1 mmol), the reaction mixture was stirred at room temperature for 18 h. The pH of the mixture was then adjusted to 2 by addition of 1 M HCl (aq). Water (10 mL) was added and the product was extracted with EtOAc (3×25 mL). The combined organic fractions were dried over MgSO4, filtered and evaporated. NMR showed some impurities so the product was purified by flash chromatography using a gradient of 0-50% MeOH in EtOAc to give the title product (16 mg, 55%) as a white powder. HRMS m/z (ES+) 387.1310, calculated for C21H20N2O4Na+ 387.1321;
To a solution of methyl 2-(4-oxo-1-(4-(trifluoromethoxy)benzyl)-1,4-dihydroquinoline-3-carboxamido)acetate (50 mg, 0.115 mmol) in THF (2 mL) was added 1 M NaOH (aq) (1 mL, 1 mmol), the reaction mixture was stirred at room temperature for 18 h. The pH of the mixture was then adjusted to 2 by addition of 1 M HCl(aq). Water (10 mL) was added and the product was extracted with EtOAc (3×25 mL). The combined organic fractions were dried over MgSO4, filtered and evaporated to give the title product (38 mg, 79%) as a white powder.
HRMS m/z (ES+) 443.0817, calculated for C20H15N2O5F3Na+ 443.0831; 1H NMR (500 MHz, MeOH) δ 9.02 (s, 1H), 8.45 (dd, J=8.1, 1.2, 1H), 7.71 (ddt, J=18.1, 14.8, 4.9, 2H), 7.51 (ddd, J=8.0, 6.9, 1.1, 1H), 7.35 (d, J=8.8, 2H), 7.26 (d, J=8.1, 2H), 5.73 (s, 2H), 4.22 (s, 2H); 13C NMR (126 MHz, MeOH) δ 178.33, 173.10, 167.42, 150.59, 140.95, 136.07, 134.65, 133.76, 132.55, 130.02, 129.60, 129.24, 127.98, 126.87, 122.88, 119.00, 112.36, 111.16, 57.63, 42.14.
To a solution of methyl 2-(1-(2-chlorobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxyamido)acetate (30 mg, 0.0780 mmol) in THF (2 mL) was added 1 M NaOH (aq) (1 mL, 1 mmol), the reaction mixture was stirred at room temperature for 18 h. LCMS showed starting material remaining so a further 1 mL 1M NaOH (aq) was added and the reaction mixture stirred at room temperature for 72 h. The pH of the mixture was then adjusted to 2 by addition of 1 M HCl (aq). Water (10 mL) was added and the product was extracted with EtOAc (3×25 mL). The combined organic fractions were dried over MgSO4, filtered and evaporated to give the title product (21 mg, 73%) as a white powder.
HRMS m/z (ES+) 393.0628, calculated for C19H15N2O4NaCl+ 393.0618;
To a solution of tert-butyl 3-(2-tert-butoxy-2-oxoethylcarbamoyl)-4-oxoquinoline-1(4H)-carboxylate (30 mg, 0.0745 mmol) in DCM (2 mL) was added TFA/m-cresol (95:5), (1 mL). The reaction mixture was stirred at room temperature for 3 h. Solvent was removed under educed pressure and the product was purified by precipitation from Et2O then dried under vacuum to give the title product (12 mg, 65%) as a white powder.
HRMS m/z (ES+) 269.0526, calculated for C12H10N2O4Na+ 269.0538; 1H NMR (500 MHz, MeOH) δ 8.76 (s, 1H, aromatic), 8.35 (dd, J=8.2, 1.0, 1H, aromatic), 7.77 (ddd, J=8.4, 7.1, 1.4, 1H, aromatic), 7.63 (d, J=8.3, 1H, aromatic), 7.55-7.44 (m, 1H, aromatic), 4.20 (s, 2H, Gly-CH2).
To 118 (165.6 mg, 0.4 mmol) and thiophene boronic acid or ester (0.4 mmol) in isopropanol (2.6 mL) was added K2CO3 (300 mg, 2 mmol) in water (1.2 mL) followed by Pd.CH2Cl2.dppf (8 mg, 0.04 mmol). The reaction mixture was stirred and purged with nitrogen by bubbling with a needle inserted below the level of the solvents for 5 min. The reaction vessel was sealed and transferred to a microwave reactor. The reaction mixture was heated at 120° for 10 min. On cooling the reaction mixture was poured onto 20% aq. NaHSO4 (20 mL) and stirred for 30 mins. Clumps of solid were broken up by placing in an ultrasonic bath for 5 min. The resulting suspension was filtered and washed extensively with water. The resultant solid was dried in vacuo to give the title product.
1H NMR (500 MHz, DMSO) δ 12.68 (s, 1H), 10.26 (t, J=5.5 Hz, 1H), 9.06 (s, 1H), 8.58 (d, J=2.2 Hz, 1H), 8.09 (dd, J=8.9, 2.2 Hz, 1H), 8.01 (dd, J=2.8, 1.3 Hz, 1H), 7.77 (d, J=9.0 Hz, 2H), 7.67 (dd, J=5.0, 2.9 Hz, 1H), 7.62 (dd, J=5.0, 1.2 Hz, 1H), 7.41-7.17 (m, 5H), 5.80 (s, 2H), 4.11 (d, J=5.5 Hz, 2H).
13C NMR (126 MHz, DMSO) δ 175.49, 171.32, 164.26, 148.66, 139.85, 138.11, 135.96, 132.02, 131.03, 128.96, 127.90, 127.77, 127.66, 126.50, 126.08, 122.51, 122.23, 118.77, 110.85, 55.86, 40.98.
HRMS (TOF MS ES+) Calc. for C23H19N2O4 419.1064 found 419.1066.
Example 67 was repeated, except that a boronic acid or ester of benzoic acid was used in place of thiophene boronic acid or ester.
1H NMR (500 MHz, DMSO) δ 10.21 (t, J=5.4 Hz, 1H), 9.09 (s, 1H), 8.58 (d, J=1.9 Hz, 1H), 8.24 (s, 1H), 8.10 (dd, J=8.9, 1.9 Hz, 1H), 7.98 (dd, J=14.4, 7.7 Hz, 2H), 7.86 (d, J=9.0 Hz, 1H), 7.62 (t, J=7.7 Hz, 1H), 7.40-7.21 (m, 5H), 5.82 (s, 2H), 4.11 (d, J=5.5 Hz, 2H).
13C NMR (151 MHz, DMSO) δ 175.54, 171.37, 167.13, 164.24, 149.05, 138.89, 138.79, 135.95, 135.92, 131.70, 131.52, 131.28, 129.64, 129.03, 128.79, 127.96, 127.78, 127.42, 126.52, 123.69, 119.07, 111.07, 55.90, 41.01.
HRMS (TOF MS ES+) Calc. for C26H21N2O6 457.1400 found 457.1359.
Example 67 was repeated, except that a boronic acid or ester of hydroxymethyl benzene was used in place of thiophene boronic acid or ester.
1H NMR (500 MHz, DMSO) δ 12.68 (s, 1H), 10.24 (t, J=5.4 Hz, 1H), 9.08 (s, 1H), 8.58 (d, J=2.0 Hz, 1H), 8.04 (dd, J=8.8, 2.2 Hz, 1H), 7.83 (d, J=9.0 Hz, 1H), 7.69 (s, 1H), 7.59 (d, J=7.7 Hz, 1H), 7.44 (t, J=7.6 Hz, 1H), 7.37-7.23 (m, 7H), 5.81 (s, 2H), 5.28 (broad s, 1H), 4.58 (d, J=3.6 Hz, 2H), 4.11 (d, J=5.5 Hz, 6H).
13C NMR (126 MHz, DMSO) δ 175.57, 171.32, 164.24, 148.84, 143.56, 138.47, 138.28, 136.98, 135.94, 131.40, 128.98, 127.91, 127.73, 126.49, 126.09, 125.13, 124.81, 123.38, 118.82, 110.94, 62.79, 55.86, 40.98.
HRMS (TOF MS ES+) Calc. for C26H23N2O5 443.1607 found 443.1617.
Example 67 was repeated, except that a boronic acid or ester of acetamidomethyl benzene was used in place of thiophene boronic acid or ester.
1H NMR (500 MHz, DMSO) δ 12.68 (s, 1H), 10.24 (t, J=5.1 Hz, 1H), 10.07 (s, 1H), 9.08 (s, 1H), 8.55 (s, 1H), 8.00 (d, J=5.0 Hz, 2H), 7.84 (d, J=8.8 Hz, 1H), 7.61 (d, J=3.4 Hz, 1H), 7.32 (ddd, J=25.6, 19.6, 10.0 Hz, 7H), 5.79 (d, J=17.9 Hz, 2H), 4.11 (d, J=5.2 Hz, 2H), 3.16 (s, 2H), 2.06 (s, 3H).
13C NMR (126 MHz, DMSO) δ 175.54, 171.32, 168.51, 164.23, 148.86, 140.11, 138.90, 138.57, 136.71, 135.91, 131.24, 129.58, 128.97, 127.91, 127.71, 126.51, 123.33, 121.39, 118.88, 118.48, 117.16, 110.98, 55.85, 40.99, 24.08.
HRMS (TOF MS ES+) Calc. for C27H23N3O5 470.1716 found 470.1741.
Example 67 was repeated, except that a boronic acid or ester of pyridine was used in place of thiophene boronic acid or ester. The title compound was obtained as a beige solid.
1H NMR (500 MHz, DMSO) δ 10.19 (t, J=5.3 Hz, 1H), 9.15 (s, 1H), 9.11 (s, 1H), 8.76 (d, J=4.9 Hz, 1H), 8.68 (d, J=2.1 Hz, 1H), 8.56 (d, J=7.6 Hz, 1H), 8.21-8.10 (m, 1H), 7.90 (d, J=8.9 Hz, 1H), 7.87-7.77 (m, 1H), 7.42-7.16 (m, 5H), 5.84 (s, 2H), 4.11 (d, J=5.5 Hz, 2H).
13C NMR (151 MHz, DMSO) δ 175.44, 171.36, 164.10, 149.31, 144.96, 144.21, 139.37, 139.09, 135.91, 135.83, 132.16, 131.67, 129.03, 127.98, 127.80, 126.51, 125.70, 124.66, 119.26, 111.32, 55.92, 48.64.
HRMS (TOF MS ES+) Calc. for C24H20N3O4 414,1454 found 414.1447.
To the quinoline compound shown above (2.94 g, 10 mmol) in DMF (60 mL) was added K2CO3 (5.5 g, 40 mmol) followed by 4-chloromethylmethlsulfone (Fluorochem) (2.04 g, 10 mmol) and the reaction mixture heated to 90° for 4 h. The DMF was removed on a rotary evaporator (1 mm Hg) and water (200 mL) and ethyl acetate (100 mL) added. The mixture was stirred for 30 mins and the light grey solid product filtered off and washed with ethyl acetate (100 mL). 3.58 g, 82%.
To the ester obtained in the previous step and shown above (2.32 g, 5 mmol) was added sodium hydroxide (1.6 g, 40 mmol) in water (25 mL) and the reaction mixture heated under reflux for 8 h. The grey solid product was filtered off and the product used directly for the next step.
To the quinoline carboxylic acid sodium salt obtained in the previous step and shown above (91 mg, 0.2 mmol) in DMF was added PyBOP (104 mg, 0.2 mmol) followed by DIPEA (90 μL) and the reaction stirred for 16 h. The volatiles were removed under a stream of nitrogen and the residue stirred in DCM (20 mL) and 1 M HCl (20 mL) for 2 h. The mixture was filtered through Whatman filter paper (no. 3)10025-33. The DCM was separated off and Silica gel (1 g) added. The DCM was removed on a rotary evaporator and the dry silica gel loaded onto a silica column (Pur1flash, SiO2, 50 μm) and eluted with methanol/DCM to obtain the title product (58 mg, 0.114 mM, 57%).
1H NMR (600 MHz, DMSO) δ 10.10 (t, J=5.8 Hz, 1H), 9.14 (s, 1H), 8.43 (d, J=2.4 Hz, 1H), 7.92 (dd, J=9.1, 2.4 Hz, 1H), 7.89 (d, J=8.5 Hz, 2H), 7.65 (d, J=9.2 Hz, 1H), 7.46 (d, J=8.5 Hz, 2H), 5.94 (s, 2H), 4.19 (d, J=5.7 Hz, 2H), 3.67 (s, 3H), 3.19 (s, 3H).
13C NMR (151 MHz, DMSO) δ 174.45, 170.44, 164.10, 149.68, 141.72, 140.29, 138.19, 135.83, 128.95, 128.43, 127.70, 127.28, 120.52, 118.46, 111.50, 55.46, 51.86, 45.90, 45.87, 43.44.
HMRS (TOF MS ES+), Calculated for C21H20BrN2O6S 507.0225, found 507.0231.
To the methyl ester 311 (102 mg, 200 μmol) in methanol (2 mL) was added sodium hydroxide (400 μL, of a 1 M solution in water) and the reaction stirred for 48 h. The volatiles were removed on a rotary evaporator and the residue partitioned between water (10 mL) and DCM. The aqueous layer was acidified to pH 2-3 with 1M HCl. The DCM was separated and the aqueous phase extracted with DCM (2×20 mL). The combined DCM extracts were dried (MgSO4), 38 mg crude as a white solid. The crude product was purified using a SiO2 Puriflash column (6 g) with DCM and a gradient elution of methanol containing 1% acetic acid. The title product was obtained as a white solid, yield 31 mg, 63 mol, 32%.
1H NMR (500 MHz, DMSO) δ 10.04 (t, J=5.6 Hz, 1H), 9.12 (s, 1H), 8.42 (d, J=2.4 Hz, 1H), 7.91-7.86 (m, 3H), 7.64 (d, J=9.2 Hz, 1H), 7.44 (d, J=8.5 Hz, 2H), 5.92 (s, 2H), 4.09 (d, J=5.7 Hz, 3H), 3.16 (d, J=6.5 Hz, 3H).
13C NMR (126 MHz, DMSO) δ 174.40, 171.20, 163.81, 149.54, 141.67, 140.31, 138.18, 135.74, 128.94, 128.43, 127.64, 127.26, 120.44, 118.35, 111.67, 55.41, 43.43, 41.00.
HMRS (TOF MS ES+), Calculated for C20H18N2O6SBr, 493.0069 found 493.0080.
To the quinoline carboxylic acid shown above (374 mg, 1.0 mmol) in DMF (10 mL) was added PyBOP (520 mg, 1.0 mmol) followed by DIPEA (260 mg, 351 μL, 2 mmol) and beta-alanine, methyl ester (139.6 mg, 1.0 mmol). The reaction mixture was stirred at 30° for 3 hours. The volatiles were removed under a stream of nitrogen at room temperature. The resulting solid was partitioned between DCM (10 mL) and water (20 mL). The DCM was separated and the water extracted with DCM (2×10 mL). The combined DCM extracts were dried over MgSO4. The crude product was purified using a silica column eluting with 0-15% acetone in DCM, to give the title compound as a cream coloured solid. Yield (290 mg, 0.52 mmol, 52%).
To the methyl ester 312 (55 mg, 125 μmol) in THF (1 mL) was added sodium hydroxide (1 mL, of a 1 mM solution in water) and the reaction stirred for 48 h. Aqueous 1 M HCl (2 mL) was added and the reaction mixture stirred for 1 h. The precipitated solid was filtered off and dried in vacuo to give the title compound. Yield 33.5 mg, 78.3 μmol, 63%.
Mesenteric artery smooth muscle cells were isolated from male Sprague-Dawley rats (200-250 g). Mesenteric arteries were dissected, cleared of extraneous tissue and incubated in an enzyme solution containing collagenase type I (1 mg/ml), soybean trypsin inhibitor (0.25 mg/ml), bovine albumin (1 mg/ml), CaCl2 (0.2 mM), 100 U/ml penicillin (100 U/ml) and streptomycin (100 μg/ml) in a humidified incubator with 5% CO2 in air. The enzyme solution was replenished at 30 min intervals and cells were pelleted by centrifugation (200 g, 5 min) and differentially plated (×2) for 15 min in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), L-Glutamine (2 mM) and 10% heat inactivated New Zealand foetal calf serum. Cells were passaged by trypsinisation and viability determined by trypan blue exclusion.
Measurements of Ca2+ flux in response to Angiotensin II were conducted using a Fluorescent Imaging Plate Reader (FLIPR; Molecular Devices). Cells were plated at a density of 10,000 cells/well into black, clear-bottomed 96-well plates in DMEM supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), L-Glutamine (2 mM) and 10% heat inactivated New Zealand foetal calf serum. 24 hrs later the cells were incubated with Fluo-3-AM (4 μM) for 60 min at 37° C. Once dye-loaded, the cells were washed thoroughly with the proprietary assay buffer to remove any unincorporated dye. The cells were then incubated with varying concentrations of a number of representative NPR-C agonist molecules of the invention, C-type natriuretic peptide or vehicle (DMSO) and placed into the FLIPR. After 15 mins, Angiotensin II (100 nM) was automatically dispensed into each well and the fluorescence signal followed for 5 mins. Potency was determined by comparison of the peak fluorescence in each test well (run in triplicate) with that of the control (Angiotensin II alone). Background fluorescence (assay buffer only) was subtracted from all values.
The results for a number of compounds of the invention are shown in the Table below.
It can be seen from the above results that the NPR-C agonist compounds of the invention inhibit the Ca2+ flux in rat smooth muscle cells, indicating that they will be effective in reducing the associated contractile response.
The Table above shows the structure-activity relationship (SAR) for a selection of compounds of the present invention using an identical cell based assay using AngII-mediated increases in Ca2+ as readout.
Male rats (Sprague-Dawley; 200-250 g) were stunned and killed by cervical dislocation. The mesentery was removed and third-order arteries mounted in an automated tension myograph (Danish Myotechnology). After an equilibration period of 45 min, vessels were normalized and diameter determined. Following normalization, each vessel was contracted repeatedly with the thromboxane A2-mimetic 9,11-dideoxy-11α,9α-epoxymethano-prostaglandin F2α (U46619; 1 μM) until the response was reproducible. The vessels were then washed to restore basal tone before contracting to approximately 50% of the maximum U46619-induced response. Once a stable response to U46619 was achieved, cumulative concentration-response curves were constructed with a variety of NPR-C agonists of the invention at varying concentrations (0.001-30 μM), or vehicle (DMSO). In some studies, concentration-response curves to NPR-C agonists were conducted in the presence of the NPR-C antagonist M372049 (10 μM). Only one curve to any one agonist was constructed in any single tissue.
The results for a number of compounds of the invention are shown in the Table below.
EC50 values were also calculated for a number of compounds, as detailed in the Table below.
The results above demonstrate the potent vasorelaxant properties of the compounds of the invention.
Blood samples (35 ml) were taken from healthy volunteers who had abstained from aspirin, non-steroidal anti-inflammatory drugs or paracetamol for 14 days. Blood was collected by venepuncture into tri-sodium citrate (3.2% w/v) and centrifuged at 175 g for 15 min to obtain platelet rich plasma (PRP). Platelet poor plasma (PPP) was obtained by centrifugation of PRP at 15000 g for 5 min. The PRP (100 μl) was then added to the wells of 96-well plates and incubated for 15 mins with representative NPR-C agonists of the invention or vehicle (DMSO). Subsequently, platelet agonists (10 μl each) were added to wells in triplicate: adenosine diphosphate (ADP, 0.1-30 μM), arachidonic acid (AA, 0.03-1.3 mM), collagen (type I equine tendon 0.1-30 μg/ml), adrenaline (0.001-100 μM), ristocetin (0.2-3 mg/ml), TRAP6 (thrombin receptor activating peptide) amide (SFLLRN; 0.1-30 μM), U46619 (0.1-30 μM) or vehicle. Plates were then immediately placed in a 96-well plate reader and absorbance determined at 595 nm every 15 s for 16 min between vigorous shaking at 37° C. Changes in absorbance were converted to % aggregation by reference to the absorbances of PRP and PPP.
The results for a number of compounds of the invention are shown in the Table below.
These results show that the compounds of the present invention have strong antiaggregatory properties in human platelets.
Male mice (C57BL6, 8 to 12 weeks old) were killed by neck dislocation and the mesentery was removed and placed in physiological salt solution (PSS) composed of (in mmol/L) NaCl 119, KCl 4.7, CaCl2.2H2O 2.5, MgSO4.7H2O 1.2, NaHCO3 25, KH2PO4 1.2, and glucose 5.5. Second or third order arteries were mounted in an automated tension myograph (Danish Myotechnology) and bathed in PSS (37° C. gassed with 5% CO2 in O2). After equilibration of 45 min, vessels were normalized according to published protocols and vessel diameter determined (Mulvany M. J., Halpern W (1977). Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res 41:19-26.).
Following normalization, each vessel was contracted repeatedly with the thromboxane A2-mimetic 9,11-dideoxy-11α,9α-epoxymethano-prostaglandin F2α (U46619; 1 μM) until the response was reproducible. The vessels were then washed to restore basal tone before contracting to approximately 50% of the maximum U46619-induced response. Once a stable response to U46619 was achieved, cumulative concentration-response curves were constructed to the following representative compounds of of the present invention: 118, 304(Szk1r), 305(Szk2r), 306(Szk4r), 307(Szk5r) and 310(Szk6r).
The results obtained are shown as
Mice were maintained under anesthesia (isoflurane, 1-1.5%, in 100% O2 at 0.4 L·min) throughout the experiment and body temperature was kept constant at 37.5° C. A Millar pressure transducer catheter (Millar Instruments, Houston, Tex., USA) was placed in the carotid artery for arterial blood pressure (BP) measurements and a tube catheter (polyvinyl tubing, 0.61 mm outside diameter) in the jugular vein for intravenous bolus doses of 118 (0.001 g-3 mg/kg) and vehicle (10% DMSO in PBS) using a 0.5 mL insulin syringe. A pump (flow 0.5 ml/h) was used for infusion of 118 (1 mg/kg/min) or vehicle (10% dimethylsulfoxide; DMSO in PBS) during 10 min. BP was evaluated throughout.
It can be seen from the results depicted in
All SPR analysis was performed on a BIAcore T200 system using series S CM5 sensor chips. Data processing and analysis were performed using BIAevaluation software and Scrubber2. All sensorgrams were double referenced by subtracting the response on a reference flowcell and a blank sample.
Human NPR-C (OriGene, Rockville, USA), was covalently attached to a CM5 chip via amine coupling. A surface density of 2700 RU was used for measurements with natriuretic peptides and the NPR-C antagonist M372049, and a density of 5200 RU for measurements with compound 118. Sequential injections of CNP (0.25, 0.50, 1, 2 & 4 nM) were performed at a flow rate of 30 μL/min for (240 s for each), followed by a dissociation time of 3600 s. Binding site saturation was observed and the surface was regenerated by two injections of 1M NaCl (200 s each). Binding of M372049 (0.94, 1.88, 3.75, 7.5, 15, 30 & 60 nM) and Compound 118 (1.17, 2.34, 4.70, 9.38, 18.75, 37.50, 75 & 150 μM) was analysed by sequential injections (120 s for M372049, 30 s for compound 118) followed by undisturbed dissociation (600 s for M372049, 30 s for compound 118), during which curves returned to baseline. Kinetic parameters were calculated assuming a simple 1:1 (Langmuir) binding.
In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
This application is a continuation of International Application No. PCT/GB2015/051500, filed on May 21, 2015, which in turn claims the benefit of Great Britain Application No. 1409044.3, filed on May 21, 2014. These prior applications are incorporated herein in their entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4014877 | Gerster | Mar 1977 | A |
| 4621088 | Laruelle et al. | Nov 1986 | A |
| 4959363 | Wentland | Sep 1990 | A |
| Number | Date | Country |
|---|---|---|
| 0811613 | Dec 1997 | EP |
| 1568962 | Jun 1980 | GB |
| WO 199704779 | Feb 1997 | WO |
| WO 200134570 | May 2001 | WO |
| WO 2006117660 | Nov 2006 | WO |
| WO 2007059108 | May 2007 | WO |
| WO 2015131259 | Sep 2015 | WO |
| Entry |
|---|
| Lager, et al, 4-Quinoline derivatives: High-affinity Ligands at the Benzodiazepine Site of Brain GABAA Receptors. Synthesis, Pharmacology, and Pharmacophore Modeling, J. of Med. Chem., Am. Chem. Soc., vol. 49, No. 8, 2526-2533 (2006). |
| Wentland, et al, 3-Quinolinecarboxamides. A Series of Novel Orally-active Antiherpetic Agents, J. of Med. Chem., Am. Chem. Soc., vol. 36, No. 11, 1580-1596 (1993). |
| RN: 1153437-25-2 with a publication date of Mar. 10, 2010. |
| Ahluwalia et al., “Endothelium-derived C-type natriuretic peptide: more than just a hyperpolarizing factor,” Trends in Pharmacological Sciences 26(3): 162-167, Mar. 31, 2005. |
| Belicova et al., “Biological activity of new aza analogues of quinolones.” Folia Microbiologica 42(3): 193-198, Jun. 1, 1997. |
| Chauhan et al., “Release of C-type natriuretic peptide accounts for the biological activity of endothelium-derived hyperpolarizing factor.” Proceedings of the National Academy of Sciences 100(3): 1426-1431 Feb. 4, 2003. |
| CHEMCATS Accession No. 0017369117, Mar. 10, 2010, Compound Registry No. 1153437-25-2. |
| CHEMCATS Accession No. 0018410795, Mar. 10, 2010, Compound Registry No. 1098367-70-4. |
| Clemenzia, et al., “Behavior of certain forms of enzymuria in subjects treated with pipemidic acid,” Minerva Medica 77(16): 621-626, Apr. 1986 (English abstract provided). |
| Erkens et al., “Use of fluorquinolones is associated with a reduced risk of coronary heart disease in Diabetes mellitus type 2 patients,” European Heart Journal 23(20): 1575-1579, Oct. 1, 2002. |
| Golub et al., “Evaluation of 3-Carboxy-4 (1 H)-quinolones as Inhibitors of Human Protein Kinase CK2,” Journal of Medicinal Chemistry 49(22): 6443-6450, Nov. 2, 2006. |
| He et al., “Design, synthesis, and biological evaluation of novel 4-hydro-quinoline-3-carboxamide derivatives as an immunomodulatory,” Bioorganic & Medicinal Chemistry Letters 15(12): 2980-2985, Jun. 15, 2005. |
| Hobbs et al., “Natriuretic peptide receptor-C regulates coronary blood flow and prevents myocardial ischemia/reperfusion injury,” Circulation 110(10): 1231-1235, Aug. 30, 2004. |
| International Search Report and Written Opinion from International Application No. PCT/GB2015/051500 dated Oct. 7, 2015. |
| Koga et al., “Structure-activity relationships of antibacterial 6, 7-and 7, 8-disubstituted 1-alkyl-1, 4-dihydro-4-oxoquinoline-3-carboxylic acids,” Journal of Medicinal Chemistry 23(12): 1358-1363, Dec. 1, 1980. |
| Lager et al., “4-Quinolone derivatives: high-affinity ligands at the benzodiazepine site of brain GABAA receptors. Synthesis, pharmacology, and pharmacophore modeling,” Journal of Medicinal Chemistry 49(8): 2526-2533, Apr. 20, 2006. |
| Loew et al., “Piromidic acid and beta-hydroxypiromidic acid: therapeutic effectiveness on experimental E. coli pyelonephritis in the rat,” International Journal of Clinical Pharmacology, Therapy and Toxicology 7(2): 234-237, Apr. 7, 1973. |
| Macinga et al., “Unique biological properties and molecular mechanism of 5, 6-bridged quinolones.,” Antimicrobial Agents and Chemotherapy 47(8): 2526-2537, Aug. 1, 2003. |
| Moyes et al., “Endothelial C-type natriuretic peptide maintains vascular homeostasis,” The Journal of Clinical Investigation 124(9): 4039-4051, Sep. 2, 2014. |
| Ogino et al., “In vivo and in vitro effects of fluoroquinolones on lipopolysaccharide-induced pro-inflammatory cytokine production,” Journal of Infection and Chemotherapy 15(3): 168-173, Jan. 1, 2009. |
| Quinteiro et al., “Design and development of novel non-peptide agonists at NPR-C,” BMC Pharmacology 11(S1): P54, Jun. 24, 2011. |
| Rimola et al., “Partially-absorbable quinolones in the prophylaxis of spontaneous bacterial peritonitis recurrence in cirrhosis,” J Hepatol 7(1): S72, 1988 (abstract only). |
| Scotland et al., “C-type natriuretic peptide inhibits leukocyte recruitment and platelet-leukocyte interactions via suppression of P-selectin expression,” Proceedings of the National Academy of Sciences of the United States of America 102(40): 14452-14457, Oct. 4, 2005. |
| Search Report issued by UK IPO on related British patent application (GB 1012899.9) dated Nov. 17, 2000. |
| Search Report issued by UK IPO on British priority application (GB 1409044.3) dated Jan. 13, 2015. |
| Taniguchi et al., “Clinical studies with antimicrobic agent ‘Dolcol’ for chronic prostatitis,” Hinyokika Kiyo. Acta Urologica Japonica 31(9): 1661-1665, Sep. 31, 1985 (English abstract provided). |
| Tao et al., “The inhibitory Effects of Quinolones on Hepatitis B Virus in Vitro [J],”Virologica Sinica 9(2): 113-118, 1994 (English abstract provided). |
| Villar et al., “Definitive role for natriuretic peptide receptor-C in mediating the vasorelaxant activity of C-type natriuretic peptide and endothelium-derived hyperpolarising factor,” Cardiovascular Research 74(3): 515-525, Jun. 1, 2007. |
| Wang et al., “CoMFA and DISCOtech studies of 4-quinolone derivative agonists for GABAA/BZ receptor,” Jisuanji Yu Yingyong Huaxue Bianjibu (Computers and Applied Chemistry), 25(10): 1197-1201, CAS Abstract Accession No. 2009:173425, 2008 (English abstract only). |
| Wentland et al., “3-Quinolinecarboxamides. A series of novel orally-active antiherpetic agents,” Journal of Medicinal Chemistry 36(11): 1580-1596, May 1, 1993. |
| Yang et al., “Parallel synthesis of N-biaryl quinolone carboxylic acids as selective M1 positive allosteric modulators,” Bioorganic & Medicinal Chemistry Letters 20(2): 531-536, Nov. 24, 2009. |
| Zhang et al., “Besitioxacin, a novel fluoroquinolone antimicrobial agent, exhibits potent inhibition of pro-inflammatory cytokines in human THP-1 monocytes,” Journal of Antimicrobial Chemotherapy 61(1): 111-116, Oct. 25, 2007. |
| Number | Date | Country | |
|---|---|---|---|
| 20170066722 A1 | Mar 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/GB2015/051500 | May 2015 | US |
| Child | 15356311 | US |
