3-AZASTEROID COMPOUNDS FOR THE TREATMENT OF DISEASES RELATED TO MITROCHONDRIAL FUNCTION

Abstract

Compounds of formula (I):

##STR00001##

wherein R.sup.1, R.sup.2 and R.sup.3 are as defined herein are able to rescue dysfunctional mitochondria and are therefore of use in the treatment and prevention of neurodegenerative disorders as well as acute radiation syndrome and myalgic encephalomyelitis (ME, chronic fatigue syndrome) or post viral syndrome, including chronic symptoms arising from infection with SARS-CoV2 (long COVID).

Claims

1-93. (canceled)

94. A compound of formula (I): ##STR00123## wherein: R.sup.1 is selected from H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C(O)R.sup.4 and C(O)OR.sup.5, wherein alkyl, alkenyl and alkynyl R.sup.1 groups are optionally substituted with one or more substituents independently selected from OR.sup.11a and N(R.sup.11a)(R.sup.11b); wherein each of R.sup.4 and R.sup.5 is independently C.sub.1-6 alkyl optionally substituted with one or more substituents selected from OR.sup.14a, N(R.sup.14a)(R.sup.14b), NH.sub.3.sup.+, C(O)N(R.sup.14a)(R.sup.14b), SR.sup.14 a 5- or 6-membered nitrogen-containing heterocyclic ring and a 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from OH, halo, NH.sub.2, NO.sub.2, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) and O(C.sub.1-6 haloalkyl); and R.sup.14a and R.sup.14b are each independently selected from H and C.sub.1-6 alkyl; and wherein each of R.sup.11a and R.sup.11b is independently selected from H and C.sub.1-4 alkyl; R.sup.2 is selected from O and OH, wherein when R.sup.2 is O, is a double bond and when R.sup.2 is OH, is a single bond; R.sup.3 is selected from C(O)OH, C(O)OR.sup.16, C(O)N(R.sup.6)X.sup.1R.sup.7, C(O)N(R.sup.8)(R.sup.9) and C(O)SR.sup.10; R.sup.16 is selected from C.sub.1-8 alkyl optionally substituted with one or more substituents selected from OH, halo and phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) O(C.sub.1-6 haloalkyl), O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; X.sup.1 is C.sub.1-6 alkylene optionally substituted with one or more substituents selected from halo, OR.sup.12a, SR.sup.12a, N(R.sup.12a)(R.sup.12b) C(O)OR.sup.12a, C(O)N(R.sup.12a)(R.sup.12b), N(R.sup.12a)C(NH)N(R.sup.12a)(R.sup.12b), N(R.sup.12a)C(N.sup.+H.sub.2)N(R.sup.12a)(R.sup.12b) and 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from halo, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl OR.sup.13a, N(R.sup.13a)(R.sup.13b), NO.sub.2, S(O).sub.2OH, and CN; R.sup.12a and R.sup.12b are each independently selected from H and C.sub.1-6 alkyl; R.sup.13a and R.sup.13b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; R.sup.6 is selected from H, methyl and ethyl; R.sup.7 is selected from C(O)OH, C(O)O(C.sub.1-6 alkyl), S(O).sub.2OH, and S(O).sub.2O(C.sub.1-6 alkyl); R.sup.8 is selected from H, C.sub.1-6 alkyl and a 3- to 6-membered carbocyclyl group optionally substituted with one or more substituents selected from C.sub.1-6 alkyl, OH, O(C.sub.1-6 alkyl), N(R.sup.19a)(R.sup.19b), C.sub.1-6 haloalkyl and halo; wherein R.sup.19a and R.sup.19b are each independently selected from H and C.sub.1-6 alkyl; R.sup.9 is selected from H, C.sub.1-6 alkyl, a 3- to 7-membered carbocyclyl group, a 3- to 7-membered heterocyclyl group, 6- to 14-membered aryl and 5- to 14-membered heteroaryl; wherein alkyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), halo, N(R.sup.19a)(R.sup.19b), phenyl, 3- to 7-membered carbocyclyl and 3- to 7-membered heterocyclyl; wherein R.sup.19a and R.sup.19b are each independently as defined above; wherein carbocyclyl and heterocyclyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), oxo, phenyl, benzyl and halo, provided that heteroatoms of a heterocyclyl group are not substituted with OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); and wherein aryl and heteroaryl are optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b), C(O)OH and C(O)O(C.sub.1-6 alkyl); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 4- to 10-membered heterocyclic group, optionally containing one or more further heteroatoms selected from O, N and S and optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), halo, C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), C(O)OH, C(O)O(C.sub.1-4 alkyl), phenyl, benzyl, CN, N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b) and oxo provided that heteroatoms of a heterocyclyl group are not substituted with CN, N(R.sup.15a)(R.sup.15b), OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); wherein alkyl groups are optionally substituted by one or more groups selected from O(C.sub.1-4 alkyl), O(C.sub.1-4 haloalkyl), N(R.sup.15a)(R.sup.15b), OH and C.sub.3-6 cycloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 5- to 10-membered heteroaryl group optionally containing one or more further heteroatoms selected from N, O and S and optionally substituted with one or more substituents selected halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)OH, C(O)N(R.sup.15a)(R.sup.15b) and C(O)O(C.sub.1-6 alkyl); wherein R.sup.15a and R.sup.15b are each independently as defined above; wherein alkyl groups are optionally substituted by one or more groups selected from OH and C.sub.3-6 cycloalkyl; wherein when the heteroaryl group contains a non-aromatic ring, the non-aromatic ring may also be substituted with oxo; R.sup.10 is C.sub.1-6 alkyl optionally substituted with OH, halo or phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently as defined above; and n is 1 or 2; or a salt or solvate thereof.

95. A compound, salt or solvate according to claim 94, wherein is a single bond and the compound is a compound of formula (IA) or (IB): ##STR00124## wherein R.sup.1 is selected from H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C(O)R.sup.4 and C(O)OR.sup.5, wherein alkyl, alkenyl and alkynyl R.sup.1 groups are optionally substituted with one or more substituents independently selected from OR.sup.11a and N(R.sup.11a)(R.sup.11b); wherein each of R.sup.4 and R.sup.5 is independently C.sub.1-6 alkyl optionally substituted with one or more substituents selected from OR.sup.14a, N(R.sup.14a)(R.sup.14b), NH.sub.3.sup.+, C(O)N(R.sup.14a)(R.sup.14b), SR.sup.14 a 5- or 6-membered nitrogen-containing heterocyclic ring and a 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from OH, halo, NH.sub.2, NO.sub.2, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) and O(C.sub.1-6 haloalkyl); and R.sup.14a and R.sup.14b are each independently selected from H and C.sub.1-6 alkyl; and wherein each of R.sup.11a and R.sup.11b is independently selected from H and C.sub.1-4 alkyl; R.sup.3 is selected from C(O)OH, C(O)OR.sup.16, C(O)N(R.sup.6)X.sup.1R.sup.7, C(O)N(R.sup.8)(R.sup.9) and C(O)SR.sup.10; R.sup.16 is selected from C.sub.1-8 alkyl optionally substituted with one or more substituents selected from OH, halo and phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) O(C.sub.1-6 haloalkyl), O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; X.sup.1 is C.sub.1-6 alkylene optionally substituted with one or more substituents selected from halo, OR.sup.12a, SR.sup.12a, N(R.sup.12a)(R.sup.12b) C(O)OR.sup.12a, C(O)N(R.sup.12a)(R.sup.12b), N(R.sup.12a)C(NH)N(R.sup.12a)(R.sup.12b), N(R.sup.12a)C(N.sup.+H.sub.2)N(R.sup.12a)(R.sup.12b) and 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from halo, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl OR.sup.13a, N(R.sup.13a)(R.sup.13b), NO.sub.2, S(O).sub.2OH, and CN; R.sup.12a and R.sup.12b are each independently selected from H and C.sub.1-6 alkyl; R.sup.13a and R.sup.13b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; R.sup.6 is selected from H, methyl and ethyl; R.sup.7 is selected from C(O)OH, C(O)O(C.sub.1-6 alkyl), S(O).sub.2OH, and S(O).sub.2O(C.sub.1-6 alkyl); R.sup.8 is selected from H, C.sub.1-6 alkyl and a 3- to 6-membered carbocyclyl group optionally substituted with one or more substituents selected from C.sub.1-6 alkyl, OH, O(C.sub.1-6 alkyl), N(R.sup.19a)(R.sup.19b), C.sub.1-6 haloalkyl and halo; wherein R.sup.19a and R.sup.19b are each independently selected from H and C.sub.1-6 alkyl; R.sup.9 is selected from H, C.sub.1-6 alkyl, a 3- to 7-membered carbocyclyl group, a 3- to 7-membered heterocyclyl group, 6- to 14-membered aryl and 5- to 14-membered heteroaryl; wherein alkyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), halo, N(R.sup.19a)(R.sup.19b), phenyl, 3- to 7-membered carbocyclyl and 3- to 7-membered heterocyclyl; wherein R.sup.19a and R.sup.19b are each independently as defined above; wherein carbocyclyl and heterocyclyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), oxo, phenyl, benzyl and halo, provided that heteroatoms of a heterocyclyl group are not substituted with OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); and wherein aryl and heteroaryl are optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b), C(O)OH and C(O)O(C.sub.1-6 alkyl); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 4- to 10-membered heterocyclic group, optionally containing one or more further heteroatoms selected from O, N and S and optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), halo, C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), C(O)OH, C(O)O(C.sub.1-4 alkyl), phenyl, benzyl, CN, N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b) and oxo provided that heteroatoms of a heterocyclyl group are not substituted with CN, N(R.sup.15a)(R.sup.15b), OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); wherein alkyl groups are optionally substituted by one or more groups selected from O(C.sub.1-4 alkyl), O(C.sub.1-4 haloalkyl), N(R.sup.15a)(R.sup.15b), OH and C.sub.3-6 cycloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 5- to 10-membered heteroaryl group optionally containing one or more further heteroatoms selected from N, O and S and optionally substituted with one or more substituents selected halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)OH, C(O)N(R.sup.15a)(R.sup.15b) and C(O)O(C.sub.1-6 alkyl); wherein R.sup.15a and R.sup.15b are each independently as defined above; wherein alkyl groups are optionally substituted by one or more groups selected from OH and C.sub.3-6 cycloalkyl; wherein when the heteroaryl group contains a non-aromatic ring, the non-aromatic ring may also be substituted with oxo; R.sup.10 is C.sub.1-6 alkyl optionally substituted with OH, halo or phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently as defined above; and n is 1 or 2.

96. A compound, salt or solvate according to claim 94, wherein is a double bond and the compound is a compound of formula (IC): ##STR00125## wherein R.sup.1 is selected from H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C(O)R.sup.4 and C(O)OR.sup.5, wherein alkyl, alkenyl and alkynyl R.sup.1 groups are optionally substituted with one or more substituents independently selected from OR.sup.11a and N(R.sup.11a)(R.sup.11b); wherein each of R.sup.4 and R.sup.5 is independently C.sub.1-6 alkyl optionally substituted with one or more substituents selected from OR.sup.14a, N(R.sup.14a)(R.sup.14b), NH.sub.3.sup.+, C(O)N(R.sup.14a)(R.sup.14b), SR.sup.14 a 5- or 6-membered nitrogen-containing heterocyclic ring and a 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from OH, halo, NH.sub.2, NO.sub.2, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) and O(C.sub.1-6 haloalkyl); and R.sup.14a and R.sup.14b are each independently selected from H and C.sub.1-6 alkyl; and wherein each of R.sup.11a and R.sup.11b is independently selected from H and C.sub.1-4 alkyl; R.sup.3 is selected from C(O)OH, C(O)OR.sup.16, C(O)N(R.sup.6)X.sup.1R.sup.7, C(O)N(R.sup.8)(R.sup.9) and C(O)SR.sup.10; R.sup.16 is selected from C.sub.1-8 alkyl optionally substituted with one or more substituents selected from OH, halo and phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) O(C.sub.1-6 haloalkyl), O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; X.sup.1 is C.sub.1-6 alkylene optionally substituted with one or more substituents selected from halo, OR.sup.12a, SR.sup.12a, N(R.sup.12a)(R.sup.12b) C(O)OR.sup.12a, C(O)N(R.sup.12a)(R.sup.12b) N(R.sup.12a)C(NH)N(R.sup.12a)(R.sup.12b), N(R.sup.12a)C(N.sup.+H.sub.2)N(R.sup.12a)(R.sup.12b) and 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from halo, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl OR.sup.13a, N(R.sup.13a)(R.sup.13b), NO.sub.2, S(O).sub.2OH, and CN; R.sup.12a and R.sup.12b are each independently selected from H and C.sub.1-6 alkyl; R.sup.13a and R.sup.13b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; R.sup.6 is selected from H, methyl and ethyl; R.sup.7 is selected from C(O)OH, C(O)O(C.sub.1-6 alkyl), S(O).sub.2OH, and S(O).sub.2O(C.sub.1-6 alkyl); R.sup.8 is selected from H, C.sub.1-6 alkyl and a 3- to 6-membered carbocyclyl group optionally substituted with one or more substituents selected from C.sub.1-5 alkyl, OH, O(C.sub.1-5 alkyl), N(R.sup.19a)(R.sup.19b), C.sub.1-6 haloalkyl and halo; wherein R.sup.19a and R.sup.19b are each independently selected from H and C.sub.1-6 alkyl; R.sup.9 is selected from H, C.sub.1-6 alkyl, a 3- to 7-membered carbocyclyl group, a 3- to 7-membered heterocyclyl group, 6- to 14-membered aryl and 5- to 14-membered heteroaryl; wherein alkyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), halo, N(R.sup.19a)(R.sup.19b), phenyl, 3- to 7-membered carbocyclyl and 3- to 7-membered heterocyclyl; wherein R.sup.19a and R.sup.19b are each independently as defined above; wherein carbocyclyl and heterocyclyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), oxo, phenyl, benzyl and halo, provided that heteroatoms of a heterocyclyl group are not substituted with OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); and wherein aryl and heteroaryl are optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b), C(O)OH and C(O)O(C.sub.1-6 alkyl); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 4- to 10-membered heterocyclic group, optionally containing one or more further heteroatoms selected from O, N and S and optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), halo, C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), C(O)OH, C(O)O(C.sub.1-4 alkyl), phenyl, benzyl, CN, N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b) and oxo provided that heteroatoms of a heterocyclyl group are not substituted with CN, N(R.sup.15a)(R.sup.15b), OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); wherein alkyl groups are optionally substituted by one or more groups selected from O(C.sub.1-4 alkyl), O(C.sub.1-4 haloalkyl), N(R.sup.15a)(R.sup.15b), OH and C.sub.3-6 cycloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 5- to 10-membered heteroaryl group optionally containing one or more further heteroatoms selected from N, O and S and optionally substituted with one or more substituents selected halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)OH, C(O)N(R.sup.15a)(R.sup.15b) and C(O)O(C.sub.1-5 alkyl); wherein R.sup.15a and R.sup.15b are each independently as defined above; wherein alkyl groups are optionally substituted by one or more groups selected from OH and C.sub.3-6 cycloalkyl; wherein when the heteroaryl group contains a non-aromatic ring, the non-aromatic ring may also be substituted with oxo; R.sup.10 is C.sub.1-6 alkyl optionally substituted with OH, halo or phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently as defined above; and n is 1 or 2.

97. A compound, salt or solvate according to claim 94, wherein R.sup.1 is H, methyl or ethyl.

98. A compound, salt or solvate according to claim 94, wherein R.sup.1 is C(O)R.sup.4, wherein R.sup.4 is selected from C.sub.1-6 alkyl optionally substituted with one or more substituents selected from OH, NH.sub.2, NH.sub.3, phenyl optionally substituted with one or more substituents selected from OH and halo, and a nitrogen-containing heteroaryl group selected from pyrrole, pyridine and indole, optionally substituted with one or more substituents selected from OH and halo.

99. A compound, salt or solvate according to claim 98, wherein R.sup.1 is an amino acid residue selected from residues of glycine, valine, isoleucine, leucine, tryptophan and tyrosine and salts of these amino acid residues.

100. A compound, salt or solvate according to claim 94, wherein R.sup.1 is C(O)OR.sup.5 wherein R.sup.5 is selected from C.sub.1-6 alkyl, benzyl or fluorenylmethyl.

101. A compound, salt or solvate according to claim 94, which is a salt of formula (ID), (IE), (IF) or (IG): ##STR00126## ##STR00127## wherein: R.sup.1 is selected from H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C(O)R.sup.4 and C(O)OR.sup.5, wherein alkyl, alkenyl and alkynyl R.sup.1 groups are optionally substituted with one or more substituents independently selected from OR.sup.11a and N(R.sup.11a)(R.sup.11b); wherein each of R.sup.4 and R.sup.5 is independently C.sub.1-6 alkyl optionally substituted with one or more substituents selected from OR.sup.14a, N(R.sup.14a)(R.sup.14b), NH.sub.3.sup.+, C(O)N(R.sup.14a)(R.sup.14b), SR.sup.14 a 5- or 6-membered nitrogen-containing heterocyclic ring and a 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from OH, halo, NH.sub.2, NO.sub.2, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) and O(C.sub.1-6 haloalkyl); and R.sup.14a and R.sup.14b are each independently selected from H and C.sub.1-6 alkyl; and wherein each of R.sup.11a and R.sup.11b is independently selected from H and C.sub.1-4 alkyl; R.sup.2 is selected from O and OH, wherein when R.sup.2 is O, is a double bond and when R.sup.2 is OH, is a single bond; R.sup.3 is selected from C(O)OH, C(O)OR.sup.16, C(O)N(R.sup.6)X.sup.1R.sup.7, C(O)N(R.sup.8)(R.sup.9) and C(O)SR.sup.10; R.sup.16 is selected from C.sub.1-8 alkyl optionally substituted with one or more substituents selected from OH, halo and phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) O(C.sub.1-6 haloalkyl), O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; X.sup.1 is C.sub.1-6 alkylene optionally substituted with one or more substituents selected from halo, OR.sup.12a, SR.sup.12a, N(R.sup.12a)(R.sup.12b) C(O)OR.sup.12a, C(O)N(R.sup.12a)(R.sup.12b), N(R.sup.12a)C(NH)N(R.sup.12a)(R.sup.12b), N(R.sup.12a)C(N.sup.+H.sub.2)N(R.sup.12a)(R.sup.12b) and 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from halo, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl OR.sup.13a, N(R.sup.13a)(R.sup.13b), NO.sub.2, S(O).sub.2OH, and CN; R.sup.12a and R.sup.12b are each independently selected from H and C.sub.1-6 alkyl; R.sup.13a and R.sup.13b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; R.sup.6 is selected from H, methyl and ethyl; R.sup.7 is selected from C(O)OH, C(O)O(C.sub.1-6 alkyl), S(O).sub.2OH, and S(O).sub.2O(C.sub.1-6 alkyl); R.sup.8 is selected from H, C.sub.1-6 alkyl and a 3- to 6-membered carbocyclyl group optionally substituted with one or more substituents selected from C.sub.1-6 alkyl, OH, O(C.sub.1-6 alkyl), N(R.sup.19a)(R.sup.19b), C.sub.1-6 haloalkyl and halo; wherein R.sup.19a and R.sup.19b are each independently selected from H and C.sub.1-6 alkyl; R.sup.9 is selected from H, C.sub.1-6 alkyl, a 3- to 7-membered carbocyclyl group, a 3- to 7-membered heterocyclyl group, 6- to 14-membered aryl and 5- to 14-membered heteroaryl; wherein alkyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), halo, N(R.sup.19a)(R.sup.19b), phenyl, 3- to 7-membered carbocyclyl and 3- to 7-membered heterocyclyl; wherein R.sup.19a and R.sup.19b are each independently as defined above; wherein carbocyclyl and heterocyclyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), oxo, phenyl, benzyl and halo, provided that heteroatoms of a heterocyclyl group are not substituted with OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); and wherein aryl and heteroaryl are optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b), C(O)OH and C(O)O(C.sub.1-6 alkyl); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 4- to 10-membered heterocyclic group, optionally containing one or more further heteroatoms selected from O, N and S and optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), halo, C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), C(O)OH, C(O)O(C.sub.1-4 alkyl), phenyl, benzyl, CN, N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b) and oxo provided that heteroatoms of a heterocyclyl group are not substituted with CN, N(R.sup.15a)(R.sup.15b), OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); wherein alkyl groups are optionally substituted by one or more groups selected from O(C.sub.1-4 alkyl), O(C.sub.1-4 haloalkyl), N(R.sup.15a)(R.sup.15b), OH and C.sub.3-6 cycloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 5- to 10-membered heteroaryl group optionally containing one or more further heteroatoms selected from N, O and S and optionally substituted with one or more substituents selected halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)OH, C(O)N(R.sup.15a)(R.sup.15b) and C(O)O(C.sub.1-6 alkyl); wherein R.sup.15a and R.sup.15b are each independently as defined above; wherein alkyl groups are optionally substituted by one or more groups selected from OH and C.sub.3-6 cycloalkyl; wherein when the heteroaryl group contains a non-aromatic ring, the non-aromatic ring may also be substituted with oxo; R.sup.10 is C.sub.1-6 alkyl optionally substituted with OH, halo or phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently as defined above; and n is 1 or 2; wherein the salt comprises a counterion Z.sup. selected from chloride, trifluoroacetate, mesylate, bromide, sulphate, and fumarate is present.

102. A compound, salt or solvate according to claim 94, wherein n is 1.

103. A compound, salt or solvate according to claim 94, wherein n is 2.

104. A compound, salt or solvate according to claim 94, wherein R.sup.3 is C(O)OH or C(O)O(C.sub.1-4 alkyl).

105. A compound, salt or solvate according to claim 94, wherein R.sup.3 is C(O)N(R.sup.6)X.sup.1R.sup.7, wherein R.sup.6 is H or methyl; and wherein X.sup.1 is unsubstituted or substituted with one or more substituents selected from fluoro, OH, methoxy, ethoxy, i-propyloxy, s-butyloxy, t-butyloxy, S-methyl, NH.sub.2, C(O)OH, phenyl and phenyl substituted with OH; and wherein R.sup.7 is selected from C(O)OH, C(O)O(C.sub.1-3 alkyl) and S(O).sub.2OH.

106. A compound, salt or solvate according to claim 94 wherein R.sup.3 is C(O)N(R.sup.8)(R.sup.9), where: R.sup.8 is selected from H, C.sub.1-6 alkyl and a 3- to 6-membered carbocyclyl group optionally substituted with one or more substituents selected from C.sub.1-6 alkyl, OH, O(C.sub.1-6 alkyl), N(R.sup.19a)(R.sup.19b), C.sub.1-6 haloalkyl and halo; wherein R.sup.19a and R.sup.19b are each independently selected from H and C.sub.1-6 alkyl; R.sup.9 is selected from H, C.sub.1-6 alkyl, a 3- to 7-membered carbocyclyl group, a 3- to 7-membered heterocyclyl group, 6- to 14-membered aryl and 5- to 14-membered heteroaryl; wherein alkyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), halo, N(R.sup.19a)(R.sup.19b), phenyl, 3- to 7-membered carbocyclyl and 3- to 7-membered heterocyclyl; wherein R.sup.19a and R.sup.19b are each independently as defined above; wherein carbocyclyl and heterocyclyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), oxo, phenyl, benzyl and halo, provided that heteroatoms of a heterocyclyl group are not substituted with OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); and wherein aryl and heteroaryl are optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b), C(O)OH and C(O)O(C.sub.1-6 alkyl); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 4- to 10-membered heterocyclic group, optionally containing one or more further heteroatoms selected from O, N and S and optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), halo, C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), C(O)OH, C(O)O(C.sub.1-4 alkyl), phenyl, benzyl, CN, N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b) and oxo provided that heteroatoms of a heterocyclyl group are not substituted with CN, N(R.sup.15a)(R.sup.15b), OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); wherein alkyl groups are optionally substituted by one or more groups selected from O(C.sub.1-4 alkyl), O(C.sub.1-4 haloalkyl), N(R.sup.15a)(R.sup.15b), OH and C.sub.3-6 cycloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 5- to 10-membered heteroaryl group optionally containing one or more further heteroatoms selected from N, O and S and optionally substituted with one or more substituents selected halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)OH, C(O)N(R.sup.15a)(R.sup.15b) and C(O)O(C.sub.1-6 alkyl); wherein R.sup.15a and R.sup.15b are each independently as defined above; wherein alkyl groups are optionally substituted by one or more groups selected from OH and C.sub.3-6 cycloalkyl; wherein when the heteroaryl group contains a non-aromatic ring, the non-aromatic ring may also be substituted with oxo.

107. A compound, salt or solvate according to claim 106, wherein R.sup.8 is selected from H, methyl, ethyl, unsubstituted cyclopentyl and unsubstituted cyclohexyl.

108. A compound, salt or solvate according to claim 106, wherein R.sup.8 is H and R.sup.9 is methyl, which is unsubstituted or substituted with a 5- or 6-membered heterocyclyl group selected from morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl and tetrahydrofuryl, wherein the heterocyclyl group is unsubstituted or substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), oxo, phenyl, benzyl and halo, provided that heteroatoms of a heterocyclyl group are not substituted with OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl).

109. A compound, salt or solvate according to claim 106, wherein R.sup.9 is selected from: a carbocyclyl group selected from cyclopentyl and cyclohexyl; a heterocyclyl group selected from pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl and morpholinyl wherein the heterocyclyl group is unsubstituted or substituted with oxo; phenyl or naphthyl wherein phenyl or naphthyl is unsubstituted or substituted with one or more substituents selected from halo, C(O)OH and C(O)O(C.sub.1-4 alkyl); and pyridyl which is unsubstituted or substituted with one or more substituents selected from halo, C(O)OH and C(O)O(C.sub.1-4 alkyl).

110. A compound, salt or solvate according to claim 109, wherein R.sup.8 is H.

111. A compound, salt or solvate according to claim 109, wherein R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 4- to 10-membered heterocyclic group wherein: the heterocyclic group is selected from piperidine, pyrrolidine, piperazine, morpholine and isothiazolidine, and is unsubstituted or is substituted as defined in claim 94; or wherein the heterocyclic group comprises two rings, which are fused or bridged or joined by a spiro linkage; or wherein the heterocyclic group is a piperidone or a pyrrolidone ring.

112. A compound, salt or solvate according to claim 109, wherein R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 5- to 10-membered heteroaryl group optionally containing one or more further heteroatoms selected from N, O and S, wherein the heteroaryl group is unsubstituted or is substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)OH and C(O)O(C.sub.1-6 alkyl).

113. A compound, salt or solvate according to claim 94, wherein R.sup.3 is C(O)SR.sup.10, where R.sup.10 is selected from C.sub.1-6 alkyl optionally substituted with OH, halo or phenyl.

114. A compound, salt or solvate according to claim 94, wherein R.sup.2 is OH, R.sup.3 is C(O)OH and the compound is of formula (IH) or a salt of formula (IJ): ##STR00128## where R.sup.1 is selected from H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C(O)R.sup.4 and C(O)OR.sup.5, wherein alkyl, alkenyl and alkynyl R.sup.1 groups are optionally substituted with one or more substituents independently selected from OR.sup.11a and N(R.sup.11a)(R.sup.11b); wherein each of R.sup.4 and R.sup.5 is independently C.sub.1-6 alkyl optionally substituted with one or more substituents selected from OR.sup.14a, N(R.sup.14a)(R.sup.14b), NH.sub.3.sup.+, C(O)N(R.sup.14a)(R.sup.14b), SR.sup.14 a 5- or 6-membered nitrogen-containing heterocyclic ring and a 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from OH, halo, NH.sub.2, NO.sub.2, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) and O(C.sub.1-6 haloalkyl); and R.sup.14a and R.sup.14b are each independently selected from H and C.sub.1-6 alkyl; and wherein each of R.sup.11a and R.sup.11b is independently selected from H and C.sub.1-4 alkyl; or wherein R.sup.1 is H, R.sup.2 is OH and n is 1 and the compound is of formula (IK) or a salt of formula (IL) ##STR00129## where R.sup.3 is selected from C(O)OH, C(O)OR.sup.16, C(O)N(R.sup.6)X.sup.1R.sup.7, C(O)N(R.sup.8)(R.sup.9) and C(O)SR.sup.10; R.sup.16 is selected from C.sub.1-8 alkyl optionally substituted with one or more substituents selected from OH, halo and phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) O(C.sub.1-6 haloalkyl), O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; X.sup.1 is C.sub.1-6 alkylene optionally substituted with one or more substituents selected from halo, OR.sup.12a, SR.sup.12a, N(R.sup.12a)(R.sup.12b) C(O)OR.sup.12a, C(O)N(R.sup.12a)(R.sup.12b), N(R.sup.12a)C(NH)N(R.sup.12a)(R.sup.12b), N(R.sup.12a)C(N.sup.+H.sub.2)N(R.sup.12a)(R.sup.12b) and 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from halo, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl OR.sup.13a, N(R.sup.13a)(R.sup.13b), NO.sub.2, S(O).sub.2OH, and CN; R.sup.12a and R.sup.12b are each independently selected from H and C.sub.1-6 alkyl; R.sup.13a and R.sup.13b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; R.sup.6 is selected from H, methyl and ethyl; R.sup.7 is selected from C(O)OH, C(O)O(C.sub.1-6 alkyl), S(O).sub.2OH, and S(O).sub.2O(C.sub.1-6 alkyl); R.sup.8 is selected from H, C.sub.1-6 alkyl and a 3- to 6-membered carbocyclyl group optionally substituted with one or more substituents selected from C.sub.1-6 alkyl, OH, O(C.sub.1-6 alkyl), N(R.sup.19a)(R.sup.19b), C.sub.1-6 haloalkyl and halo; wherein R.sup.19a and R.sup.19b are each independently selected from H and C.sub.1-6 alkyl; R.sup.9 is selected from H, C.sub.1-6 alkyl, a 3- to 7-membered carbocyclyl group, a 3- to 7-membered heterocyclyl group, 6- to 14-membered aryl and 5- to 14-membered heteroaryl; wherein alkyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), halo, N(R.sup.19a)(R.sup.19b), phenyl, 3- to 7-membered carbocyclyl and 3- to 7-membered heterocyclyl; wherein R.sup.19a and R.sup.19b are each independently as defined above; wherein carbocyclyl and heterocyclyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), oxo, phenyl, benzyl and halo, provided that heteroatoms of a heterocyclyl group are not substituted with OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); and wherein aryl and heteroaryl are optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b), C(O)OH and C(O)O(C.sub.1-6 alkyl); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 4- to 10-membered heterocyclic group, optionally containing one or more further heteroatoms selected from O, N and S and optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), halo, C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), C(O)OH, C(O)O(C.sub.1-4 alkyl), phenyl, benzyl, CN, N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b) and oxo provided that heteroatoms of a heterocyclyl group are not substituted with CN, N(R.sup.15a)(R.sup.15b), OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); wherein alkyl groups are optionally substituted by one or more groups selected from O(C.sub.1-4 alkyl), O(C.sub.1-4 haloalkyl), N(R.sup.15a)(R.sup.15b), OH and C.sub.3-6 cycloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 5- to 10-membered heteroaryl group optionally containing one or more further heteroatoms selected from N, O and S and optionally substituted with one or more substituents selected halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)OH, C(O)N(R.sup.15a)(R.sup.15b) and C(O)O(C.sub.1-6 alkyl); wherein R.sup.15a and R.sup.15b are each independently as defined above; wherein alkyl groups are optionally substituted by one or more groups selected from OH and C.sub.3-6 cycloalkyl; wherein when the heteroaryl group contains a non-aromatic ring, the non-aromatic ring may also be substituted with oxo; R.sup.10 is C.sub.1-6 alkyl optionally substituted with OH, halo or phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently as defined above.

115. A compound according to claim 94, selected from: tert-Butyl N-(benzyloxycarbony)-3-aza-7-hydroxy-5-cholan-24-oate (25a); tert-Butyl 3-aza-7-hydroxy-5-cholan-24-oate (27a); 3-Aza-7-hydroxy-5-cholan-24-oic acid (28a); tert-Butyl N-methyl-3-aza-7-hydroxy-5-cholan-24-oate (29a); N-Methyl-3-aza-7-hydroxy-5-cholan-24-oic acid (31a and 33); tert-Butyl N-ethyl-3-aza-7-hydroxy-5-cholan-24-oate (30); N-Ethyl-3-aza-7-hydroxy-5-cholan-24-oic acid (32); tert-Butyl N-glycolyl-3-aza-7-hydroxy-5-cholan-24-oate (34a); N-Glycolyl-3-aza-7-hydroxy-5-cholan-24-oic acid (40a); N-[(2S)-2-amino-3-methylbutanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid (41a); N-[(2S,3S)-2-Amino-3-methylpentanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid (42a); N-[(2S)-2-Amino-4-methylpentanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid (43a); N-[(2S)-2-Amino-3-(1H-indol-3-yl)propanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid (44a); N-[(2S)-2-Amino-3-(4-hydroxyphenyl)propanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid (45a); tert-Butyl N-(benzyloxycarbonyl)-3-aza-7-hydroxy-25-homo-5-cholan-25-oate (25b); tert-Butyl 3-aza-7-hydroxy-25-homo-5-cholan-25-oate (27b); 3-Aza-7-hydroxy-25-homo-5-cholan-25-oic acid (28b); tert-Butyl N-methyl-3-aza-7-hydroxy-25-homo-5-cholan-25-oate (29b); N-Methyl-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid (31b); tert-Butyl N-glycolyl-3-aza-7-hydroxy-25-homo-5-cholan-25-oate (34b); N-Glycolyl-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid (40b); N-[(2S)-2-Amino-3-methylbutanoyl]-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid (41b); N-[(2S,3S)-2-Amino-3-methylpentanoyl]-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid (42b); N-[(2S)-2-Amino-4-methylpentanoyl]-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid (43b, Example 25); N-[(2S)-2-Amino-3-(1H-indol-3-yl)propanoyl]-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid (44b); N-(benzyloxycarbony)-3-aza-7-hydroxy-5-cholan-24-oic acid (50a); N-(3-Aza-7-hydroxy-5-cholan-24-amide)-ethylsulfonic acid (51a); N-(3-Aza-7-hydroxy-5-cholan-24-amide)-acetic acid (52a); N-(3-Methyl-aza-7-hydroxy-5-cholan-24-oyl)-(2S)-2-amino-3-[(2-methylpropan-2-yl)oxy]propanoic acid (53a); N-(3-Methyl-aza-7-hydroxy-5-cholan-24-oyl)-(R)-3-amino-3-phenylpropanoic acid (54a); N-(3-Methyl-aza-7-hydroxy-5-cholan-24-oyl)-1-amino-4-fluorobenzene (55a); N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-morpholine (56a); N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-(S)-2-amino-3-hydroxypropanoic acid (57a); N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-3-amino-2-fluoropropanoic acid (58a); N-(Cyclohexyl)-N-(3-methyl-aza-7-hydroxy-5-cholan-24-oyl)-cyclohexanamine (59a); N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-4-aminobenzoic acid (60a); N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-(S)-2-amino-4-(methylthio)butanoic acid (61a); N-(3-Aza-7-hydroxy-5-cholan-24-amide)-propanoic acid (62a); N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-(isopropyl-4-aminobenzoate) (63a); S-(3-Aza-7-hydroxy-5-cholan-24-oyl)-phenylmethanethiol (64a); N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-(S)-2-amino-3-methylbutanoic acid (65a); N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-(2S,3S)-2-amino-3-methylpentanoic acid (66a); N-Methyl-N-(3-aza-7-hydroxy-5-cholan-24-oyl)-glycine (67a); N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-(S)-2-aminopropanoic acid (68a); N-(benzyloxycarbonyl)-3-aza-7-hydroxy-24-homo-5-cholan-25-oic acid (50b); N-(3-Aza-7-hydroxy-27-homo-5-cholan-27-oyl)-(S)-2-aminobutanedioic acid (70b); N-(3-Aza-7-hydroxy-27-homo-5-cholan-27-oyl)-(2S)-2-amino-3-[(2-methylpropan-2-yl)oxy]propanoic acid (71b); N-(3-Aza-7-hydroxy-27-homo-5-cholan-27-amide)-ethylsulfonic acid (72b); N-(3-Aza-7-hydroxy-27-homo-5-cholan-27-oyl)-(S)-2-amino-3-phenylpropanoic acid (73b); N-(3-Aza-7-hydroxy-27-homo-5-cholan-27-oyl)-3-aminobutanoic acid (74b); N-(3-Aza-7-hydroxy-27-homo-5-cholan-27-oyl)-(S)-2-amino-4-methylpentanoic acid (75b); N-(3-Aza-7-hydroxy-27-homo-5-cholan-27-oyl)-(2S)-2,6-diaminohexanoic acid (76b); N-(3-Aza-7-hydroxy-27-homo-5-cholan-27-oyl)-(2S)-2-amino-3-(4-hydroxyphenyl)propanoic acid (77b); N-{(benzyloxycarbonyl)-3-aza-7-hydroxy-5-cholan-24-oyl}-2-oxa-6-azospiro{3,3}heptane (78a); N-{(benzyloxycarbonyl)-3-aza-7-hydroxy-5-cholan-24-oyl}-4-piperidone (79a); N-{(benzyloxycarbonyl)-3-aza-7-hydroxy-25-homo-5-cholan-25-oyl}-3-aminotetrahydrofuran (80b); N-{(3-aza-7-hydroxy-5-cholan-25-oyl}-isoindoline (81a); N-{(3-aza-7-hydroxy-25-homo-5-cholan-25-oyl)}-3-aminotetrahydrofuran (82b); and salts and solvates thereof.

116. A method of preparing a compound of formula (I) or a salt or solvate thereof according to claim 94, the method comprising: A. for a compound of formula (I) in which R.sup.1 is C(O)OR.sup.5 and R.sup.3 is C(O)OR.sup.16: reacting a compound of formula (II): ##STR00130## wherein R.sup.2 is selected from O and OH, wherein when R.sup.2 is O, is a double bond and when R.sup.2 is OH, is a single bond; n is 1 or 2; R.sup.1a is C(O)OR.sup.5 wherein R.sup.5 is C.sub.1-6 alkyl optionally substituted with one or more substituents selected from OR.sup.14a, N(R.sup.14a)(R.sup.14b), NH.sub.3.sup.+, C(O)N(R.sup.14a)(R.sup.14b), SR.sup.14 a 5- or 6-membered nitrogen-containing heterocyclic ring and a 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from OH, halo, NH.sub.2, NO.sub.2, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) and O(C.sub.1-6 haloalkyl); and R.sup.14a and R.sup.14b are each independently selected from H and C.sub.1-6 alkyl; and R.sup.3a is C(O)OR.sup.16, wherein R.sup.16 is selected from C.sub.1-8 alkyl optionally substituted with one or more substituents selected from OH, halo and phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) O(C.sub.1-6 haloalkyl), O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; with methane sulfonyl chloride in an organic solvent at about 5 to 5 C.; B. for a compound of formula (I) in which R.sup.1 is H: hydrogenation of a compound of formula (I) in which R.sup.1 is C(O)OR.sup.5 over a palladium catalyst; C. for a compound of formula (I) in which R.sup.1 is C.sub.1-6 alkyl and R.sup.3 is C(O)OR.sup.16: reaction of a compound of formula (I) in which R.sup.1 is H and R.sup.3 is C(O)OR.sup.16 with a compound of formula (XX):
R.sup.1bC(O)H(XX) wherein R.sup.1b is C.sub.1-5 alkyl; followed by hydrogenation over a palladium/carbon catalyst; D. for a compound of formula (I) in which R.sup.1 is C.sub.2-6 alkenyl or C.sub.2-6 alkynyl and R.sup.3 is C(O)OR.sup.16: reaction compound of formula (I) in which R.sup.1 is H and R.sup.3 is C(O)OR.sup.16 with a compound of formula (XVIII): ##STR00131## wherein R.sup.1c is C.sub.2-6 alkenyl or C.sub.2-6 alkynyl and X is chloro or bromo; in the presence of a base; wherein when R.sup.2 is OH, the method further comprises the initial step of protecting R.sup.2 with a suitable protecting group and the final step of removing the protecting group; E. for a compound in which R.sup.3 is C(O)OH: acid or base hydrolysis of a compound of formula (I) in which R.sup.3 is C(O)OR.sup.16; F. for a salt of formula (I) in which the nitrogen atom to which R.sup.1 is attached is quaternised: treatment of a compound of formula (I) in which R.sub.1 is H with an acid. G. for a compound of formula (I) in which R.sup.1 is C(O)R.sup.4 and R.sup.3 is C(O)OR.sup.16: i. reaction of a compound of formula (I) in which R.sup.1 is H and R.sup.3 is C(O)OR.sup.16 with a compound of formula (XXI): ##STR00132## wherein R.sup.4 is C.sub.1-6 alkyl optionally substituted with one or more substituents selected from OR.sup.14a, N(R.sup.14a)(R.sup.14b), NH.sub.3.sup.+, C(O)N(R.sup.14a)(R.sup.14b), SR.sup.14 a protected OH group, a protected NH.sub.2 group, a protected C(O)NH.sub.2 group, a 5- or 6-membered nitrogen-containing heterocyclic ring and a 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from OH, halo, NH.sub.2, NO.sub.2, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) and O(C.sub.1-6 haloalkyl); under basic conditions, and in the presence of a coupling reagent; and ii. where necessary, removal of a protecting group to give a group R.sup.4 comprising an OH, NH.sub.2 or C(O)NH.sub.2; H. for a compound of formula (I) in which R.sup.1 is C(O)R.sup.4 and R.sup.3 is C(O)OH: reaction of the product of G(i) with trifluoroacetic acid; I. for a compound of formula (I) in which R.sup.3 is C(O)N(R.sup.6)X.sup.1R.sup.7: reaction of a compound of formula (I) in which R.sup.3 is C(O)OH with a compound of formula (XXV): ##STR00133## wherein X.sup.1 is C.sub.1-6 alkylene optionally substituted with one or more substituents selected from halo, OR.sup.12a, SR.sup.12a, N(R.sup.12a)(R.sup.12b) C(O)OR.sup.12a, C(O)N(R.sup.12a)(R.sup.12b), N(R.sup.12a)C(NH)N(R.sup.12a)(R.sup.12b), N(R.sup.12a)C(N.sup.+H.sub.2)N(R.sup.12a)(R.sup.12b) and 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein aryl and heteroaryl groups are optionally substituted with one or more substituents selected from halo, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl OR.sup.13a, N(R.sup.13a)(R.sup.13b), NO.sub.2, S(O).sub.2OH, and CN; R.sup.12a and R.sup.12b are each independently selected from H and C.sub.1-6 alkyl; R.sup.13a and R.sup.13b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; R.sup.6 is selected from H, methyl and ethyl; R.sup.7 is selected from C(O)OH, C(O)O(C.sub.1-6 alkyl), S(O).sub.2OH, and S(O).sub.2O(C.sub.1-6 alkyl); in the presence of a coupling reagent and under basic conditions, for example in the presence of an amine selected from diisopropylethylamine (DIPEA) or triethylamine (TEA) and in an organic solvent; J. for a compound of formula (I) in which R.sup.3 is C(O)N(R.sup.8)(R.sup.9): reaction of a compound formula (I) in which R.sup.3 is C(O)OH with a compound of formula (XXVI): ##STR00134## where: R.sup.8 is selected from H, C.sub.1-6 alkyl and a 3- to 6-membered carbocyclyl group optionally substituted with one or more substituents selected from C.sub.1-6 alkyl, OH, O(C.sub.1-6 alkyl), N(R.sup.19a)(R.sup.19b), C.sub.1-6 haloalkyl and halo; wherein R.sup.19a and R.sup.19b are each independently selected from H and C.sub.1-6 alkyl; R.sup.9 is selected from H, C.sub.1-6 alkyl, a 3- to 7-membered carbocyclyl group, a 3- to 7-membered heterocyclyl group, 6- to 14-membered aryl and 5- to 14-membered heteroaryl; wherein alkyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), halo, N(R.sup.19a)(R.sup.19b), phenyl, 3- to 7-membered carbocyclyl and 3- to 7-membered heterocyclyl; wherein R.sup.19a and R.sup.19b are each independently as defined above; wherein carbocyclyl and heterocyclyl groups are optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), oxo, phenyl, benzyl and halo, provided that heteroatoms of a heterocyclyl group are not substituted with OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); and wherein aryl and heteroaryl are optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b), C(O)OH and C(O)O(C.sub.1-6 alkyl); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 4- to 10-membered heterocyclic group, optionally containing one or more further heteroatoms selected from O, N and S and optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, OH, O(C.sub.1-4 alkyl), halo, C.sub.1-4 haloalkyl, O(C.sub.1-4 haloalkyl), C(O)OH, C(O)O(C.sub.1-4 alkyl), phenyl, benzyl, CN, N(R.sup.15a)(R.sup.15b), C(O)N(R.sup.15a)(R.sup.15b) and oxo provided that heteroatoms of a heterocyclyl group are not substituted with CN, N(R.sup.15a)(R.sup.15b), OH, O(C.sub.1-4 alkyl) or O(C.sub.1-4 haloalkyl); wherein alkyl groups are optionally substituted by one or more groups selected from O(C.sub.1-4 alkyl), O(C.sub.1-4 haloalkyl), N(R.sup.15a)(R.sup.15b), OH and C.sub.3-6 cycloalkyl; or R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 5- to 10-membered heteroaryl group optionally containing one or more further heteroatoms selected from N, O and S and optionally substituted with one or more substituents selected halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b), C(O)OH, C(O)N(R.sup.15a)(R.sup.15b) and C(O)O(C.sub.1-5 alkyl); wherein R.sup.15a and R.sup.15b are each independently as defined above; wherein alkyl groups are optionally substituted by one or more groups selected from OH and C.sub.3-6 cycloalkyl; wherein when the heteroaryl group contains a non-aromatic ring, the non-aromatic ring may also be substituted with oxo; under basic conditions and in the presence of a coupling agent; K. for a compound of formula (I) in which R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 5-membered heteroaryl ring: coupling reaction of a compound of formula (I) in which R.sup.3 is C(O)N(R.sup.8)(R.sup.9), where R.sup.8 and R.sup.9 together with the nitrogen atom to which they are attached combine to form a 5- or 6-membered heterocyclic ring substituted with oxo; L. for a compound of formula (I) in which R.sup.3 is C(O)SR.sup.10: reaction of a compound formula (I) in which R.sup.3 is C(O)OH with a compound of formula (XXVII): ##STR00135## where R.sup.10 is C.sub.1-6 alkyl optionally substituted with OH, halo or phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl.

117. A method for the treatment or prevention of a neurodegenerative disorder, acute radiation syndrome or myalgic encephalomyelitis (ME, chronic fatigue syndrome) or post viral syndrome, including chronic symptoms arising from infection with SARS-CoV2 (long COVID), the method comprising administering to a patient in need of such treatment an effective amount of a compound, salt or solvate according to claim 94.

118. A pharmaceutical composition comprising a compound, salt or solvate according to claim 94, and a pharmaceutically acceptable excipient or carrier.

119. A pharmaceutical composition according to claim 118, formulated for parenteral administration, oral administration topical administration to the skin (transdermal administration) or topical administration to the lung (by inhalation).

120. A compound of formula (II): ##STR00136## wherein R.sup.2 is selected from O and OH, wherein when R.sup.2 is O, is a double bond and when R.sup.2 is OH, is a single bond, n is 1 or 2, R.sup.1a is C(O)OR.sup.5 and R.sup.3a is C(O)OR.sup.16, wherein R.sup.5 is C.sub.1-6 alkyl optionally substituted with one or more substituents selected from OR.sup.14a, N(R.sup.14a)(R.sup.14b), NH.sub.3.sup.+, C(O)N(R.sup.14a)(R.sup.14b), SR.sup.14 a 5- or 6-membered nitrogen-containing heterocyclic ring and a 6- to 14-membered aryl or 5- to 14-membered heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from OH, halo, NH.sub.2, NO.sub.2, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) and O(C.sub.1-6 haloalkyl, and R.sup.16 is selected from C.sub.1-8 alkyl optionally substituted with one or more substituents selected from OH, halo and phenyl, wherein phenyl is optionally substituted with one or more substituents selected from halo, NO.sub.2, CN, S(O).sub.2OH, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, O(C.sub.1-6 alkyl) O(C.sub.1-6 haloalkyl), O(R.sup.15a), N(R.sup.15a)(R.sup.15b) and C(O)N(R.sup.15a)(R.sup.15b); wherein R.sup.15a and R.sup.15b are each independently selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; or a compound of formula (IP2): ##STR00137## wherein R.sup.2 is selected from O and OH, wherein when R.sup.2 is O, is a double bond and when R.sup.2 is OH, is a single bond, n is 1 or 2, R.sup.3a is as defined for formula (II) above and R.sup.4a is C.sub.1-6 alkyl substituted with a protected NH.sub.2 group, a protected OH group or a protected C(O)NH.sub.2 group.

Description

FIGURES AND EXAMPLES

[0378] The invention will now be further described with reference to the following examples and to the drawings in which:

[0379] FIG. 1 shows data obtained from measuring mitochondrial respiration using the Seahorse Mito Stress test. Data are presented from 3 control fibroblast lines and 3 sporadic Parkinson's disease patient fibroblast lines, each treated both with vehicle and with Compound 28b (Example 18). Each test was repeated at 3 independent passages. The bars represent mean values from the 3 passages and error bars represent the standard deviation. FIG. 1A shows spare respiratory capacity, FIG. 1B shows basal mitochondrial respiration and FIG. 1C shows ATP linked respiration.

[0380] FIG. 2 shows mitochondrial respiratory chain complex I activity in WT C57B6 mouse brain hemispheres (3 per group) in an untreated control group (Con un in the figure) and in a group treated with Compound 28b (Example 18) at 1, 4, 8, 12 and 24 hours after dosing. Complex I activity is elevated after dosing with Compound 28b 1 hour post dosing, remained elevated at the same level at 8 hours post dosing. The levels remained elevated, although to a lesser extent by 24 hours post dosing.

GENERAL EXPERIMENTAL PROCEDURES

[0381] Proton (.sup.1H) and carbon (.sup.13C) NMR-spectra were recorded on Bruker Avance (III)-500 spectrometer. Chemical shifts are reported in ppm relative to Me.sub.4Si (TMS, d 0), or residual solvent peaks as an internal standard set to d 7.26 and 77.00 (CDCl.sub.3), or d 3.34 and 49.05 (CDOD), or d 2.50 and 39.43 (d.sub.6-DMSO). NMR data is reported as follows: chemical shift in ppm, multiplicity (ap=apparent, s=singlet, d=doublet, t=triplet, q=quartet, sp=septet, br=broad, dd=doublet of doublets, td=triplet of doublets, dt=doublet of triplets, m=multiplet), coupling constant in Hz, integration.

[0382] Electrospray ionization (ESI) mass spectrometry (MS) experiments were performed on a QTOF Premier mass spectrometer (Micromass, UK) under normal conditions. Sodium formate solution was used as calibrant for high resolution mass spectra (HRMS) measurements. All reactions were monitored by thin layer chromatography (TLC) using 0.2 mm silica gel (Merck Kieselgel 60 F.sub.254) precoated aluminium plates, using UV light, ammonium molybdate, ninhydrin or potassium permanganate staining solution to visualize. Flash column chromatography was performed on Davisil silica gel (60, particle size 0.040-0.063 mm), or using Reveleris silica or C-18 reversed phase flash cartridges on a Grace Reveleris automated flash system with continuous gradient facility. Solvents for reactions and chromatography were analytical grade and were used as supplied unless otherwise stated. Chiral and achiral high performance liquid chromatography (HPLC) analyses were performed on an Agilent 1100 (Quaternary pump) HPLC system with a refractive index detector, employing columns as indicated. Data was processed with Agilent Cerity System software.

Example 1

tert-Butyl N-(benzyloxycarbony)-3-aza-7-hydroxy-5-cholan-24-oate (25a)

##STR00028##

A. tert-Butyl 3,7-dihydroxy-5-cholan-24-oate (12)

##STR00029##

[0383] To a solution of ursodeoxycholic acid (10.2 g, 26.0 mmol) in dry THF (225 mL) was added dropwise trifluoroacetic anhydride (30.0 mL, 216 mmol) at 0 C. After complete addition the ice bath was removed and the reaction was stirred for 1.5 h. Subsequently, tert-butanol (63.8 mL) was introduced portionwise at room temperature and the reaction was stirred at room temperature overnight. Then concentrated aqueous ammonia (53 mL) was added at room temperature and again the reaction was left stirring overnight. Saturated bicarbonate solution was added and the aqueous phase was extracted with ethyl acetate (3). The organic fractions were combined, washed with brine, dried over MgSO.sub.4 and concentrated. The crude product was purified by automated flash column chromatography (silica gel, ethyl acetate/petroleum ether 10-100%) to yield the product 12 quantitatively as a colourless foam.

[0384] .sup.1H NMR (500 MHz, CDCl.sub.3) 3.64-3.54 (m, 2H), 2.26 (ddd, 15.2, 9.9, 5.3 Hz, 1H), 2.13 (ddd, 15.3, 9.5, 6.8 Hz, 1H), 2.00 (dt, 12.5, 3.1 Hz, 1H), 1.96-1.86 (m, 1H), 1.84-1.72 (m, 4H), 1.71-1.64 (m, 2H), 1.64-1.54 (m, 2H), 1.53-1.36 (m, 6H; 1.44, s, 9H), 1.36-1.20 (m, 5H), 1.15 (td, 12.9, 3.8 Hz, 1H), 1.10-0.98 (m, 2H), 0.95 (s, 3H), 0.92 (d, 6.6 Hz, 3H), 0.68 (s, 3H); HRMS (ESI) m/z calcd for C.sub.28H.sub.48O.sub.4Na.sup.+ 471.3445, found 471.3440.

B. tert-Butyl 7-Hydroxy-3-oxo-5-cholan-24-oate (13)

##STR00030##

[0385] To a solution of 12, the product of step A (26.0 mmol) in dry dichloromethane (180 mL) was added (diacetoxyiodo)benzene (BAIB; 10.3 g, 32.0 mmol) and a catalytic amount of TEMPO (624 mg, 3.99 mmol). After being stirred overnight the reaction was quenched with saturated Na.sub.2S.sub.2O.sub.3 solution and diluted with water. The organic layer was separated and the aqueous layer was extracted another three times with dichloromethane. The organic phases were combined, washed with brine, dried over MgSO.sub.4 and concentrated. The residue was purified by automated flash column chromatography (silica gel, ethyl acetate/petroleum ether 2-100%) to give 11.4 g (98%) of the product 13 as a light yellowish foam.

[0386] .sup.1H NMR (500 MHz, CDCl.sub.3) 3.61 (ddd, 11.3, 8.7, 5.2 Hz, 1H), 2.52 (dd, 15.0, 13.9 Hz, 1H), 2.32-2.22 (m, 2H), 2.22-2.09 (m, 3H), 2.09-2.00 (overlapping signals: 2.06, dt, 13.1, 3.2 Hz, 1H; 2.02, ddd, 14.3, 5.3, 3.3 Hz, 1H), 1.98-1.88 (m, 2H), 1.87-1.73 (m, 3H), 1.62 (ddd, 13.5, 5.1, 2.4 Hz, 1H), 1.58-1.34 (m, 9H; 1.44, s, 9H), 1.34-1.24 (m, 2H), 1.21 (td, 12.6, 4.7 Hz, 1H), 1.09 (dt, 9.7, 9.6 Hz, 1H), 1.05 (s, 3H), 0.94 (d, 6.6 Hz, 3H), 0.72 (s, 3H); HRMS (ESI) m/z calcd for C.sub.28H.sub.46O.sub.4Na.sup.+ 469.3288, found 469.3292.

C. tert-Butyl 7-Hydroxy-4-oxa-3-oxo-4-homo-5-cholan-24-oate (Side Product 14a) and tert-butyl 7-Hydroxy-3-oxa-4-oxo-4-homo-5-cholan-24-oate (Required Product 15a)

##STR00031##

[0387] To a solution of 13, the product of step B (11.3 g, 25.3 mmol) in dichloromethane (300 mL)) was added meta-chloroperbenzoic acid (mCPBA, 57-86%; 13.8 g, 56.0 mmol) in one portion at room temperature and the resulting reaction mixture was stirred overnight. The reaction was quenched with saturated Na.sub.2S.sub.2O.sub.3 solution which was followed by addition of saturated bicarbonate solution. The organic layer was separated and the aqueous phase was extracted with ethyl acetate (3). The combined organic fractions were washed with brine, dried over MgSO.sub.4 and concentrated. The crude product was purified by automated flash column chromatography (silica gel, ethyl acetate/petroleum ether 2-80%) to yield 10.6 g (91%) of 14a and 15a as a 1:1 mixture of isomers (colourless foam).

[0388] .sup.1H NMR (500 MHz, CDCl.sub.3, mixture of isomers) 4.48 (dd, 13.1, 9.6 Hz, 1H), 4.17 (dd, 13.2, 10.2 Hz, 1H), 4.05 (ddd, 13.3, 6.5, 1.1 Hz, 1H), 3.98 (ap d, 12.9 Hz, 1H), 3.48-3.40 (m, 1H), 3.33-3.25 (m, 1H), 3.03 (ap t, 12.9 Hz, 1H), 2.63 (ap dd, 14.4, 13.1 Hz, 1H), 2.42-2.35 (m, 2H), 2.26 (ddd, 15.3, 9.9, 5.4 Hz, 2H), 2.13 (ddd, 15.4, 9.4, 6.8 Hz, 2H), 2.09-2.01 (m, 3H), 1.98-1.85 (m, 7H), 1.85-1.68 (m, 6H), 1.58-1.31 (m, 18H; 1.44, s, 18H), 1.31-1.15 (m, 6H), 1.12-1.03 (overlapping signals: m, 2H; 1.05, s, 3H; 1.04, s, 3H), 0.93 (d, 6.6 Hz, 3H), 0.92 (d, 6.6 Hz, 3H), 0.70 (s, 6H);

[0389] HRMS (ESI) m/z calcd for C.sub.28H.sub.46O.sub.5Na.sup.+ (mixture of isomers) 485.3237, found 485.3250.

D. tert-Butyl 4,7-dihydroxy-3,4-seco-5-cholan-24-oate-3-amide (Side Product 16a) and tert-butyl 2,7-dihydroxy-2,3-seco-5-cholan-24-oate-4-amide (Required Product 17a)

##STR00032##

[0390] A solution of a mixture of lactones 14a and 15a from step C (10.6 g, 22.9 mmol) in dry 7 N ammonia in methanol (ca. 165 mL) was heated in a 200 mL sealed tube with Teflon screw cap at 90 C. overnight (the sealed tube was filled to 4/5 of its volume and a blast shield was added). The reaction was concentrated and the residue purified by automated flash column chromatography (silica gel, methanol/ethyl acetate 0-25%) to give 8.95 g (81%) of a mixture of amides (16a and 17a) as a colourless oil. The product ratio (16a:17a) was determined to be 1:1.2 by HPLC analysis (Phenomenex Luna C18(2) 5 m 2504.6 mm; Phenomenex Security Guard C18 43 mm; mobile phase: 45:55:0.05 water/acetonitrile/trifluoroacetic acid; flow rate: 1 mL/min; sample solvent: methanol; column temperature: 35 C.; injection volume: 25 L; detection: refractive index). In addition 1.55 g (14%) of the corresponding esters 18a and 19a was isolated as colourless foam. The ratio of 18a:19a was determined to be 1:6.8 by HPLC analysis (Phenomenex Luna C18(2) 5 m 2504.6 mm; Phenomenex Security Guard C18 43 mm; mobile phase: 30:70:0.1 water/acetonitrile/trifluoroacetic acid; flow rate: 1 mL/min; sample solvent: methanol; column temperature: 35 C.; injection volume: 25 L; detection: refractive index). An analytical sample of each of the four compounds was recovered for spectroscopic characterization.

[0391] tert-Butyl 4,7-dihydroxy-3,4-seco-5-cholan-24-oate-3-amide (16a).

[0392] .sup.1H NMR (500 MHz, CDCl.sub.3) 6.44 (s.sub.br, 1H, NH.sub.2), 6.06 (s.sub.br, 1H, NH.sub.2), 3.76-3.68 (m, 1H), 3.56-3.49 (m, 1H), 3.49-3.41 (m, 1H), 2.36-2.20 (overlapping signals: m, 1H; 2.25, ddd, 15.3, 9.8, 5.5 Hz, 1H), 2.17-2.07 (m, 2H), 1.99-1.94 (m, 2H), 1.93-1.71 (m, 4H), 1.71-1.63 (m, 1H), 1.62-1.52 (m, 2H), 1.51-1.36 (m, 4H; 1.44, s, 9H), 1.36-1.21 (m, 3H), 1.21-1.02 (m, 3H), 1.02-0.94 (overlapping signals: m, 1H; 0.97, s, 3H), 0.91 (d, 6.5 Hz, 3H), 0.68 (s, 3H); H,HCOSY (500 MHz, CDCl.sub.3) 3.72 g 1.67, 3.53 g 1.67;

[0393] HRMS (ESI) m/z calcd for C.sub.28H.sub.49NO.sub.5Na.sup.+ 502.3503, found 502.3511.

[0394] tert-Butyl 2,7-dihydroxy-2,3-seco-5-cholan-24-oate-3-amide (17a). .sup.1H NMR (500 MHz, CDCl.sub.3) 6.75 (s.sub.br, 1H, NH.sub.2), 6.35 (s.sub.br, 1H, NH.sub.2), 3.82-3.72 (m, 1H), 3.72-3.63 (m, 1H), 3.63-3.54 (m, 1H), 3.49-2.39 (m, 1H), 2.25 (ddd, 15.3, 9.5, 5.2 Hz, 1H), 2.21-2.08 (m, 2H), 2.06-1.96 (m, 2H), 1.92-1.59 (m, 6H), 1.58-1.35 (m, 5H; 1.44, s, 9H), 1.35-1.11 (m, 5H), 1.11-1.01 (overlapping signals: m, 1H; 1.05, s, 3H), 1.00-0.87 (overlapping signals: m, 1H; 0.92, d, 6.4 Hz, 3H), 0.68 (s, 3H); H,HCOSY (500 MHz, CDCl.sub.3) 3.75 g 1.71/1.39, 3.67 g 1.71/1.39;

[0395] HRMS (ESI) m/z calcd for C.sub.28H.sub.49NO.sub.5Na.sup.+ 502.3503, found 502.3510. tert-Butyl 4,7-dihydroxy-3,4-seco-5-cholan-24-oate-3-methyl ester (18a). .sup.1H NMR (500 MHz, CDCl.sub.3) 3.70 (dd, 10.8, 4.6 Hz, 1H), 3.67 (s, 3H), 3.60-3.50 (m, 2H), 2.37 (ddd, 15.5, 12.1, 4.5 Hz, 1H), 2.31-2.20 (m, 2H), 2.16-2.01 (overlapping signals: 2.13, ddd, 15.4, 9.5, 6.8 Hz, 1H; m, 3H), 1.98 (dt, 12.7, 3.2 Hz, 1H), 1.94-1.85 (m, 1H), 1.84-1.71 (m, 3H), 1.68-1.63 (m, 1H), 1.61-1.53 (m, 2H), 1.52-1.35 (m, 4H; 1.44, s, 9H), 1.35-1.22 (m, 3H), 1.19 (ddd, 10.6, 12.3, 7.3 Hz, 1H),), 1.15-0.95 (overlapping signals: 1.11, td, 13.0, 3.9 Hz, 1H; m, 2H; 0.99, s, 3H), 0.91 (d, 6.6 Hz, 3H), 0.68 (s, 3H); H,HCOSY (500 MHz, CDCl.sub.3) 3.70 g 1.66, 3.56 g 1.66;

[0396] HRMS (ESI) m/z calcd for C.sub.29H.sub.50O.sub.6Na.sup.+ 517.3500, found 517.3512.

[0397] tert-Butyl 2,7-dihydroxy-2,3-seco-5-cholan-24-oate-3-methyl ester (19a). .sup.1H NMR (500 MHz, CDCl.sub.3) 3.77-3.68 (m, 2H), 3.68 (s, 3H), 3.58 (dt, 7.4, 9.4 Hz, 1H), 2.44 (dd, 14.8, 3.4 Hz, 1H), 2.33 (dd, 14.8, 11.4 Hz, 1H), 2.25 (ddd, 15.2, 9.9, 5.3 Hz, 1H), 2.12 (ddd, 15.3, 9.5, 6.8 Hz, 1H), 2.07-1.97 (m, 2H), 1.95-1.86 (m, 1H), 1.82-1.64 (m, 5H), 1.58-1.34 (m, 7H; 1.44, s, 9H), 1.34-1.24 (m, 3H), 1.21 (ddd, 10.6, 12.3, 7.3 Hz, 1H), 1.13 (td, 13.1, 4.0 Hz, 1H), 1.10-1.02 (overlapping signals: m, 1H; 1.08, s, 3H), 1.00-0.93 (m, 1H), 0.92 (d, 6.6 Hz, 3H), 0.68 (s, 3H); H,HCOSY (500 MHz, CDCl.sub.3) 3.72 (2H) g 1.74/1.39;

[0398] HRMS (ESI) m/z calcd for C.sub.29H.sub.50O.sub.6Na.sup.+ 517.3500, found 517.3510.

E. tert-Butyl 2-amino-4,7-dihydroxy-3-nor-3,4-seco-5-cholan-24-oate (Side Product, 20a) and tert-butyl 4-amino-2,7-dihydroxy-3-nor-3,4-seco-5-cholan-24-oate (Required Product, 21a)

##STR00033##

[0399] To a solution of the mixture of amide products from step D 16a and 17a (11.7 a. 24.4 mmol) in a 3:1 mixture of acetonitrile and water (590 mL/197 mL; ca. 30 mL of ethyl acetate was added to help solubilize the starting material) was added (diacetoxyiodo)benzene (BAIB; 9.09 g, 28.2 mmol) in one portion at room temperature. After being stirred overnight the reaction mixture was transferred into a 2 L flask and carefully concentrated to dryness. The residue was re-dissolved in methanol, passed along A 26-resin and concentrated. The crude product was then purified by automated flash column chromatography [silica gel, ethyl acetate/(methanol:28% aqueous ammonia solution 9:1) 0-80%] to yield 9.28 g (84%) of a mixture of the product amines 20a and 21a as a colourless semi solid.

[0400] .sup.1H NMR (500 MHz, CD.sub.3OD; mixture of isomers) 3.69 (dd, 10.9, 3.9 Hz, 1H), 3.68-3.56 (m, 2H), 3.52 (ap t, 10.4 Hz, 1H), 3.49-3.38 (m, 2H), 2.79-2.67 (m, 2H), 2.66-2.58 (m, 2H), 2.25 (ddd, 14.9, 9.2, 5.5 Hz, 2H), 2.14 (ddd, 15.1, 8.5, 7.7 Hz, 2H), 2.11-2.05 (m, 1H), 2.04-1.98 (m, 2H), 1.95-1.73 (overlapping signals: 1.92, ddd, 13.8, 4.9, 2.9 Hz, 1H; m, 6H), 1.68 (ddd, 13.5, 10.3, 5.3 Hz, 1H), 1.63-1.53 (m, 6H), 1.53-1.34 (m, 8H; 1.44, s, 18H), 1.35-1.11 (m, 11H), 1.10-0.96 (overlapping signals: m, 4H; 1.07, s, 3H; 1.05, s, 3H), 0.94 (d, 6.6 Hz, 6H), 0.72 (s, 6H); HRMS (ESI) m/z calcd for C.sub.27H.sub.49NO.sub.4H.sup.+452.3734, found 452.3730.

F. tert-Butyl N-(benzyloxycarbonyl)-2-amino-4,7-dihydroxy-3-nor-3,4-seco-5-cholan-24-oate (Side Product 22a) and tert-butyl N-(benzyloxycarbonyl)-4-amino-2,7-dihydroxy-3-nor-3,4-seco-5-cholan-24-oate (Required Product 23a)

##STR00034##

[0401] To a solution of a mixture of 20a and 21a, the products of Step E (10.9 g, 24.1 mmol) in a 1:1 mixture of dichloromethane (160 mL) and water (160 mL) was added sodium carbonate (13.1 g, 124 mmol) and the mixture was cooled to 0 C. Then, benzyl chloroformate (CbzCl 95%; 4.00 mL, 28.0 mmol) was introduced dropwise and the reaction was stirred for 40 min at 0 C. After complete reaction (TLC analysis) 28% aqueous ammonia solution (50 mL) was added and the resulting mixture was diluted with water. The mixture was extracted with ethyl acetate (3) and the combined organic phases were washed with brine, dried over MgSO.sub.4 and concentrated. The crude product mixture was purified by automated column chromatography (silica gel, ethyl acetate/petroleum ether 2-100%) to afford 5.95 g (42%) of the products 22a and 5.11 g (36%) of 23a, both as colourless foams.

tert-Butyl N-(benzyloxycarbonyl)-2-amino-4,7-dihydroxy-3-nor-3,4-seco-5-cholan-24-oate (22a)

[0402] .sup.1H NMR (500 MHz, CDCl.sub.3) 7.40-7.29 (m, 5H), 5.12-5.05 (m, 2H), 4.88-4.79 (m, 1H, NH), 3.92-3.84 (m, 1H), 3.61-3.52 (m, 1H), 3.51-3.44 (m, 1H), 3.44-3.34 (m, 1H), 3.14-3.04 (m, 1H), 2.37-2.20 (overlapping signals: s.sub.br, 1H, OH; 2.25, ddd, 15.3, 9.9, 5.3 Hz, 1H), 2.12 (ddd, 15.4, 9.3, 6.7 Hz, 1H), 2.06-1.94 (overlapping signals: m, 1H; 1.97, dt, 12.6, 3.1 Hz, 1H), 1.94-1.84 (m, 1H), 1.82-1.71 (m, 3H), 1.68-1.54 (m, 2H), 1.54-1.35 (m, 6H; 1.44, s, 9H), 1.35-1.21 (m, 3H), 1.17 (ddd, 12.1, 10.7, 7.4 Hz, 1H), 1.13-0.94 (overlapping signals: 1.10, td, 13.1, 3.8 Hz, 1H; m, 2H; 1.03, s, 3H), 0.91 (d, 6.6 Hz, 3H), 0.68 (s, 3H); H,HCOSY (500 MHz, CDCl.sub.3) 3.88 g 1.75, 3.57 g 1.75;

[0403] tert-Butyl N-(Benzyloxycarbonyl)-4-amino-2,7-dihydroxy-3-nor-3,4-seco-5-cholan-24-oate (23a). .sup.1H NMR (500 MHz, CDCl.sub.3) 7.41-7.29 (m, 5H), 5.09 (s, 2H), 4.96-4.90 (m, 1H, NH), 3.91-3.83 (m, 1H), 3.75-3.66 (m, 1H), 3.63-3.53 (m, 1H), 3.46-3.38 (m, 1H), 3.11 (ddd, 13.4, 11.5, 6.7 Hz, 1H), 2.25 (ddd, 15.3, 9.9, 5.3 Hz, 1H), 2.12 (ddd, 15.3, 9.5, 6.8 Hz, 1H), 2.02-1.95 (m, 1H), 1.96-1.86 (m, 1H), 1.83 (ddd, 13.7, 4.9, 2.7 Hz, 1H), 1.80-1.71 (m, 3H), 1.70-1.62 (m, 2H), 1.62-1.30 (m, 8H; 1.44, s, 9H), 1.30-1.17 (m, 3H), 1.17-1.08 (m, 1H), 1.08-0.97 (overlapping signals: m, 2H; 1.04, s, 3H), 0.91, d, 6.6 Hz, 3H), 0.68 (s, 3H); H,HCOSY (500 MHz, CDCl.sub.3) 3.87 g 1.76, 1.49, 3.71 g 1.76, 1.49;

[0404] HRMS (ESI) m/z calcd for C.sub.35H.sub.55NO.sub.6Na.sup.+ 608.3922, found 608.3931.

G. tert-Butyl N-(benzyloxycarbony)-3-aza-7-hydroxy-5-cholan-25-oate (25a)

##STR00035##

[0405] To a solution of 23a (2.83 g, 4.83 mmol) in dry pyridine (90 mL) was added dropwise methanesulfonyl chloride (0.45 mL, 5.81 mmol) at 0 C. The resulting reaction mixture was stirred for 2 h at this temperature and once only traces of staring material were detectable (TLC analysis) saturated bicarbonate solution (5.8 mL) was introduced. The reaction was allowed to warm to room temperature and subsequently stirred at 74 C. overnight. The solvent was evaporated and the residue was taken up in water. The mixture was extracted with ethyl acetate (3), the combined organic fractions were washed with brine, dried over MgSO.sub.4, filtered through a plug of celite and concentrated. The crude product was purified by automated column chromatography (silica gel, ethyl acetate/petroleum ether 0-60%) to yield 2.02 g (74%) of 25a as a colourless foam. In addition, a sample of the over-mesylated by-product 26 (24.2 mg) was recovered.

[0406] tert-Butyl N-(benzyloxycarbonyl)-3-aza-7-hydroxy-5-cholan-25-oate (25a). .sup.1H NMR (500 MHz, CDCl.sub.3) 7.39-7.28 (m, 5H), 5.18-5.05 (m, 2H), 3.98-3.75 (m, 2H), 3.57-3.45 (m, 1H), 3.08-2.91 (m, 1H), 2.89-2.73 (m, 1H), 2.25 (ddd, 15.3, 9.9, 5.3 Hz, 1H), 2.12 (ddd, 15.4, 9.5, 6.8 Hz, 1H), 2.01 (dt, 12.8, 3.1 Hz, 1H), 1.96-1.86 (m, 1H), 1.84-1.70 (m, 4H), 1.70-1.58 (m, 2H), 1.55-1.39 (m, 6H; 1.44, s, 9H), 1.39-1.11 (overlapping signals: m, 5H; 1.15, td, 12.9, 4.1 Hz, 1H), 1.07 (dt, 9.7, 9.7 Hz, 1H), 0.99 (s, 3H), 0.92 (d, 6.6 Hz, 3H), 0.69 (s, 3H);

[0407] HRMS (ESI) m/z calcd for C.sub.35H.sub.53NO.sub.5Na.sup.+ 590.3816, found 590.3815.

[0408] tert-Butyl N-(benzyloxycarbonyl)-3-aza-7-mesyloxy-5-cholan-25-oate (26). .sup.1H NMR (500 MHz, CDCl.sub.3) 7.39-7.28 (m, 5H), 5.12 (s.sub.br, 2H), 4.65-4.57 (m, 1H), 4.00-3.80 (m, 2H), 3.04-2.94 (overlapping signals: m, 1H; 2.98, s, 3H), 2.87-2.71 (m, 1H), 2.24 (ddd, 15.2, 9.7, 5.4 Hz, 1H), 2.16-2.05 (m, 3H), 2.01 (dt, 12.8, 3.2 Hz, 1H), 1.93-1.81 (m, 2H), 1.81-1.71 (m, 3H), 1.71-1.62 (m, 1H), 1.62-1.55 (m, 1H), 1.52-1.20 (m, 8H; 1.44, s, 9H), 1.16 (td, 12.9, 3.8 Hz, 1H), 1.07 (dt, 9.5, 9.0 Hz, 1H), 1.01 (s, 3H), 0.91 (d, 6.5 Hz, 3H), 0.68 (s, 3H);

[0409] HRMS (ESI) m/z calcd for C.sub.36H.sub.55NO.sub.7SNa.sup.+ 668.3591, found 668.3590.

Example 2

tert-Butyl 3-aza-7-hydroxy-5-cholan-24-oate (27a)

##STR00036##

[0410] To a solution of 25a from Example 1 (1.47 g, 2.58 mmol) in methanol (30 mL) was added 10% palladium on charcoal (92.7 mg) and the atmosphere was exchanged for hydrogen. The resulting reaction mixture was stirred at room temperature overnight. After complete deprotection (TLC analysis) the reaction was filtered through celite and concentrated. The residual colourless oil was purified by automated column chromatography [silica gel, (methanol/28% aqueous ammonia solution 9:1)/ethyl acetate 0-60%] to give 1.04 g (93%) of 27a as a colourless foam.

[0411] .sup.1H NMR (500 MHz, CDCl.sub.3) 3.48 (ddd, 10.9, 9.0, 5.2 Hz, 1H), 3.00-2.87 (m, 3H), 2.76 (td, 13.1, 2.0 Hz, 1H), 2.26 (ddd, 15.3, 9.9, 5.3 Hz, 1H), 2.13 (ddd, 15.4, 9.4, 6.8 Hz, 1H), 2.01 (dt, 12.7, 3.2 Hz, 1H), 1.97-1.87 (m, 1H), 1.87-1.72 (m, 5H), 1.64-1.56 (m, 1H), 1.55-1.19 (m, 10H; 1.44, s, 9H), 1.15 (td, 12.9, 4.3 Hz, 1H), 1.08 (dt, 9.7, 9.7 Hz, 1H), 1.03 (s, 3H), 0.92 (d, 6.6 Hz, 3H), 0.68 (s, 3H);

[0412] HRMS (ESI) m/z calcd for C.sub.27H.sub.47NO.sub.3H.sup.+ 434.3629, found 434.3627.

Example 3

3-Aza-7-hydroxy-5-cholan-24-oic acid hydrochloride salt (28a)

##STR00037##

[0413] To a solution of the compound of Example 2 (27a) (153 mg, 0.353 mmol) in dry dichloromethane (4 mL) was added trifluoroacetic acid (TFA; 3 mL) at 0 C. After being stirred at this temperature for 2.5 h the reaction was concentrated and the residue co-evaporated with a 3 M aqueous hydrochloric acid solution (3) followed by THF (1). The crude product was dried under high vacuum and re-dissolved in THF (60 mL). The resulting mixture was concentrated to a small volume in an open 100 mL round bottomed flask. The precipitate was filtered off, washed with THF and dried under high vacuum. Residual THF was subsequently removed by co-evaporation with methanol. The sample was then dried under high vacuum at 80 C. to afford 110 mg (75%) of the product 28a as an amorphous colourless solid.

[0414] .sup.1H NMR (500 MHz, CD.sub.3OD) 3.42-3.34 (m, 1H), 3.24 (t, 12.8 Hz, 1H), 3.15-3.05 (m, 2H), 3.01 (td, 13.5, 2.3 Hz, 1H), 2.33 (ddd, 15.3, 9.8, 5.4 Hz, 1H), 2.20 (ddd, 15.6, 9.2, 6.8 Hz, 1H), 2.10-1.99 (m, 2H), 1.96-1.76 (m, 5H), 1.66-1.38 (m, 8H), 1.38-1.22 (m, 4H), 1.16-1.05 (overlapping signals: m, 1H; 1.09, s, 3H), 0.96 (d, 6.6 Hz, 3H), 0.73 (s, 3H);

[0415] .sup.13C NMR (125 MHz, CD.sub.3OD) 178.17, 71.03, 56.92, 56.58, 45.02, 44.78, 44.35, 42.06, 41.16, 40.80, 39.98, 36.70, 35.17, 34.26, 33.55, 32.40, 32.09, 29.62, 27.91, 23.47, 22.54, 18.95, 12.62;

[0416] HRMS (ESI) m/z calcd for C.sub.23H.sub.39NO.sub.3H.sup.+ 378.3003, found 378.3004.

Example 4

tert-Butyl N-methyl-3-aza-7-hydroxy-5-cholan-24-oate (29a)

##STR00038##

[0417] To a solution of 25a, the product of Example 1 (203 mg, 0.358 mmol) in methanol (10 mL) was added formalin (37% in water; 0.15 mL, 2.02 mmol) and the mixture was stirred at room temperature for 1 h. Subsequently, 10% palladium on charcoal was added (37.8 mg) and the atmosphere was exchanged for hydrogen. After being stirred overnight the reaction was filtered through a plug of celite and the filtrate was concentrated. The crude product was purified by automated column chromatography [silica gel, (methanol:28% aqueous ammonia solution 9:1)/ethyl acetate 0-20%] to give 161 mg (quant.) of the product 29a as a colourless oil.

[0418] .sup.1H NMR (500 MHz, CDCl.sub.3) 3.53 (ddd, 11.0, 9.1, 5.2 Hz, 1H), 2.58-2.48 (m, 2H), 2.29-2.09 (overlapping signals: m, 1H; 2.26, s, 3H; 2.19, ap t, 11.7 Hz, 1H; 2.13, ddd, 15.4, 9.5, 6.7 Hz, 1H), 2.03-1.96 (m, 2H), 1.95-1.86 (m, 1H), 1.85-1.72 (m, 5H), 1.63-1.56 (m, 1H), 1.54-1.21 (m, 10H; 1.44, s, 9H), 1.16 (td, 12.9, 3.8 Hz, 1H), 1.08 (dt, 9.6, 9.6 Hz, 1H), 0.99 (s, 3H), 0.92 (d, 6.5 Hz, 3H), 0.68 (s, 3H);

[0419] HRMS (ESI) m/z calcd for C.sub.28H.sub.49NO.sub.3H.sup.+ 448.3785, found 448.3782.

Example 5

N-Methyl-3-aza-7-hydroxy-5-cholan-24-oic acid hydrotrifluoroacetate (31a)

##STR00039##

[0420] To a solution of 29a, the product of Example 4, (93.0 mg, 0.208 mmol) in dry dichloromethane (5 mL) was added trifluoroacetic acid (4 mL) at 0 C. and the resulting mixture was stirred for 2.5 h. Then the reaction was concentrated and the crude product was purified by automated column chromatography [C18 silica gel, acetonitrile/water (+0.5% trifluoroacetic acid) 2-40%]. The purified product was co-evaporated with dichloromethane (3-4) and methanol (1) at 60 C. to give 51.1 mg (51%) of 31a as a colourless foam.

[0421] .sup.1H NMR (500 MHz, CD.sub.3OD) 3.38 (ddd, 11.2, 9.8, 5.1, 1H), 3.30-3.18 (m, 3H), 3.05-2.97 (m, 1H), 2.86 (s, 3H), 2.33 (ddd, 15.4, 9.8, 5.4 Hz, 1H), 2.21 (ddd, 15.5, 9.3, 6.9 Hz, 1H), 2.10-2.02 (m, 2H), 1.96-1.77 (m, 5H), 1.66-1.55 (m, 2H), 1.53-1.37 (m, 6H), 1.37-1.23 (m, 4H), 1.16-1.06 (overlapping signals: m, 1H; 1.08, s, 3H); 0.96 (d, 6.6 Hz, 3H), 0.73 (s, 3H);

[0422] .sup.19F NMR (470 MHz, CD.sub.3OD) 77.00;

[0423] .sup.13C NMR (125 MHz, CD.sub.3OD) 178.13, 70.94, 56.79, 56.61, 55.41, 51.07, 44.78, 44.37, 43.75, 42.96, 41.10, 39.79, 36.69, 35.06, 34.42, 33.47, 32.39, 32.08, 29.61, 27.90, 23.05, 22.55, 18.95, 12.61;

[0424] HRMS (ESI) m/z calcd for C.sub.24H.sub.41NO.sub.3H.sup.+ 392.3159, found 392.3169.

Example 6

N-Methyl-3-aza-7-hydroxy-5-cholan-24-oic acid hydrochloride (33)

##STR00040##

[0425] To a solution of 29a, the product of Example 4, (161 mg, 0.360 mmol) in dry dichloromethane (5 mL) was added trifluoroacetic acid (4 mL) at 0 C. After being stirred for 2.5 h the reaction was concentrated and the residue purified by automated column chromatography [C18 silica gel, acetonitrile/water (+0.5% trifluoroacetic acid) 2-40%]. The purified product was co-evaporated with dichloromethane (3-4) and a solution of 3 M aqueous hydrochloric acid (3). The resulting product was dried under high vacuum and in a desiccator over potassium carbonate to yield 112 mg (73%) of 33 as a colourless foam.

[0426] .sup.1H NMR (500 MHz, CD.sub.3OD) 3.38 (ddd, 11.3, 9.8, 5.2, 1H), 3.33-3.17 (m, 3H), 3.06-2.97 (m, 1H), 2.86 (s, 3H), 2.33 (ddd, 15.3, 9.8, 5.4 Hz, 1H), 2.20 (ddd, 15.5, 9.3, 6.9 Hz, 1H), 2.12-2.03 (m, 2H), 1.96-1.77 (m, 5H), 1.67-1.55 (m, 2H), 1.54-1.38 (m, 6H), 1.38-1.23 (m, 4H), 1.16-1.07 (overlapping signals: m, 1H; 1.09, s, 3H), 0.96 (d, 6.6 Hz, 3H), 0.74 (s, 3H);

[0427] .sup.13C NMR (125 MHz, CD.sub.3OD) 178.10, 70.93, 56.76, 56.59, 55.43, 51.10, 44.78, 44.36, 43.81, 42.94, 41.10, 39.76, 36.69, 35.07, 34.43, 33.50, 32.39, 32.08, 29.63, 27.91, 23.04, 22.56, 18.96, 12.62;

[0428] HRMS (ESI) m/z calcd for C.sub.24H.sub.41NO.sub.3H.sup.+ 392.3159, found 392.3174.

Example 7

tert-Butyl N-ethyl-3-aza-7-hydroxy-5-cholan-24-oate (30)

##STR00041##

[0429] Using an analogous method to that of Example 4, the compound of Example 1 (25a) (111 mg, 0.196 mmol) was dissolved in methanol (6 mL), treated with acetaldehyde (0.06 mL, 1.07 mmol) and subsequently hydrogenated on 10% palladium on charcoal (22.4 mg) to afford 56.4 mg (63%) of the product 30 as a colourless foam.

[0430] .sup.1H NMR (500 MHz, CDCl.sub.3) 3.53 (ddd, 11.0, 9.3, 5.2, 1H), 2.69-2.58 (m, 2H), 2.40 (q, 7.2H, 2H), 2.25 (ddd, 15.3, 9.9, 5.3 Hz, 1H), 2.19-2.08 (m, 2H), 2.03-1.86 (m, 3H), 1.85-1.72 (m, 5H), 1.64-1.56 (m, 1H), 1.54-1.21 (m, 11H; 1.44, s, 9H), 1.15 (td, 12.9, 3.9 Hz, 1H), 1.12-1.04 (overlapping signals: m, 1H; 1.08, t, 7.2 Hz, 3H), 0.99 (s, 3H), 0.92 (d, 6.6 Hz, 3H), 0.68 (s, 3H);

[0431] HRMS (ESI) m/z calcd for C.sub.29H.sub.51NO.sub.3H.sup.+ 462.3942, found 462.3942.

Example 8

N-Ethyl-3-aza-7-hydroxy-5-cholan-24-oic acid hydrotrifluoroacetate (32)

##STR00042##

[0432] Using an analogous method to that of Example 5, the compound of Example 7 (30) (50.0 mg, 0.108 mmol) was dissolved in dry dichloromethane (5 mL) and reacted with trifluoroacetic acid (4 mL) at 0 C. for 2.5 h to give 26.4 mg (47%) of the product 32 as a colourless foam.

[0433] .sup.1H NMR (500 MHz, CD.sub.3OD) 3.39 (ddd, 11.3, 9.7, 5.2, 1H), 3.33-3.24 (m, 2H), 3.24-3.13 (overlapping signals: m, 1H; 3.17, q, 7.5 Hz, 2H), 2.97-2.88 (m, 1H), 2.33 (ddd, 15.3, 9.8, 5.4 Hz, 1H), 2.21 (ddd, 15.5, 9.2, 6.9 Hz, 1H), 2.12-2.00 (m, 2H), 1.97-1.77 (m, 5H), 1.69-1.62 (m, 1H), 1.61-1.23 (overlapping signals: m, 11H; 1.33, t, 7.4 Hz, 3H), 1.16-1.04 (overlapping signals: m, 1H; 1.09, s, 3H), 0.96 (d, 6.6 Hz, 3H), 0.73 (s, 3H);

[0434] .sup.19F NMR (470 MHz, CD.sub.3OD) 76.94;

[0435] .sup.13C NMR (125 MHz, CD.sub.3OD) 178.14, 70.94, 56.78, 56.60, 53.29, 53.25, 49.26 (from DEPT 135), 44.78, 44.36, 42.82, 41.10, 39.81, 36.69, 35.12, 34.23, 33.98, 32.39, 32.07, 29.62, 27.90, 23.12, 22.55, 18.95, 12.61, 9.78;

[0436] HRMS (ESI) m/z calcd for C.sub.25H.sub.43NO.sub.3H.sup.+ 406.3316, found 406.3325.

Example 9

tert-Butyl N-qlycolyl-3-aza-7-hydroxy-5-cholan-24-oate (34a)

##STR00043##

[0437] To a solution of the product of Example 2 (27a) (101 mg, 0.233 mmol) in dry dichloromethane (2 mL) was added sequentially glycolic acid (27.2 mg, 0.358 mmol; 1.5 equiv.), 1-hydroxybenzotriazole (HOBt; 34.9 mg, 0.258 mmol; 1.1 equiv.), N,N-diisopropylethylamine (0.089 mL, 0.511 mmol; 2.2 equiv.) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDCI; 59.9 mg, 0.313 mmol; 1.3 equiv.) at room temperature. After being stirred overnight the reaction was quenched with saturated NH.sub.4Cl solution and diluted with water and dichloromethane. The mixture was extracted with dichloromethane (3) and the combined organic phases were dried over MgSO.sub.4 and concentrated. The residue was purified by automated column chromatography (silica gel, petroleum ether/ethyl acetate 2-80%) to yield 85.8 mg (75%) of 34a as a colourless foam.

[0438] .sup.1H NMR (500 MHz, CDCl.sub.3) 4.33-4.25 (m, 1H), 4.19-4.06 (m, 2H), 3.68-3.63 (m, 1H, OH), 3.57-3.46 (m, 1H), 3.24-3.16 (m, 1H), 3.11 (dd, 13.6, 3.8 Hz, 0.5H), 3.01 (td, 13.4, 2.0 Hz, 0.5H), 2.90 (ap t, 13.2 Hz, 0.5H), 2.73 (td, 13.4, 1.6 Hz, 0.5H), 2.26 (ddd, 15.3, 9.8, 5.3 Hz, 1H), 2.13 (ddd, 15.5, 9.3, 6.7 Hz, 1H), 2.06-2.00 (m, 1H), 1.97-1.87 (m, 1H), 1.87-1.68 (m, 4.5H), 1.68-1.59 (m, 1.5H), 1.55-1.33 (m, 8H; 1.44, s, 9H), 1.33-1.12 (m, 4H), 1.08 (dt, 9.6, 9.6 Hz, 1H), 1.02 (s, 3H), 0.92 (d, 6.5 Hz, 3H), 0.70 (s, 3H);

[0439] HRMS (ESI) m/z calcd for C.sub.29H.sub.49NO.sub.5Na.sup.+ 514.3503, found 514.3503.

Example 10

N-Glycolyl-3-aza-7-hydroxy-5-cholan-24-oic acid (40a)

##STR00044##

[0440] To a solution of the compound of Example 9 (34a) (85.8 mg, 0.175 mmol) in dry dichloromethane (6 mL) was added trifluoroacetic acid (4 mL) at 0 C. After being stirred for 2.5 h the solvent was evaporated in vacuo and the residue purified by automated column chromatography [silica gel, acetone (+1% acetic acid)/dichloromethane (+1% acetic acid) 0-40%]. The purified product was then co-evaporated with dichloromethane (3-4) and methanol (1) at 60 C. to give 45.5 mg (60%) of the product 40a as a colourless foam.

[0441] .sup.1H NMR (500 MHz, CD.sub.3OD) 4.26-4.12 (m, 3H), 3.48-3.38 (m, 1.5H), 3.38-3.27 (m, 1H), 3.08 (td, 13.4, 1.5 Hz, 0.5H), 2.96 (ap t, 13.1 Hz, 0.5H), 2.79-2.71 (m, 0.5H), 2.33 (ddd, 15.3, 9.9, 5.4 Hz, 1H), 2.20 (ddd, 15.5, 9.3, 6.8 Hz, 1H), 2.08-2.01 (m, 1H), 1.95-1.77 (m, 5H), 1.68-1.54 (m, 3H), 1.54-1.36 (m, 5H), 1.36-1.17 (m, 4.5H), 1.16-1.06 (m, 1.5H), 1.03 (s, 3H), 0.96 (d, 6.6 Hz, 3H), 0.73 (s, 3H);

[0442] .sup.13C NMR (125 MHz, CD.sub.3OD) 178.16, 171.77, 71.57, 71.54, 61.23, 61.16, 57.23, 56.59, 45.82, 44.97, 44.80, 44.35, 43.86, 41.36, 40.81, 40.19, 38.80, 37.06, 36.71, 36.46, 36.06, 35.91, 35.05, 32.42, 32.08, 29.64, 27.93, 23.78, 23.74, 22.63, 22.60, 18.98, 12.69;

[0443] HRMS (ESI) m/z calcd for C.sub.25H.sub.41NO.sub.5Na.sup.+ 458.2877, found 458.2877.

Example 11

N-[(2S)-2-amino-3-methylbutanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid hydrotrifluoroacetate (41a)

##STR00045##

A. tert-Butyl N-{(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}-3-aza-7-hydroxy-5-cholan-24-oate (35a)

##STR00046##

[0444] Deploying the same procedure as for Example 9, the compound of Example 2 (27a) (100 mg, 0.231 mmol) was dissolved in dry dichloromethane (2.5 mL) and sequentially treated with Boc-L-valine (Boc-Val-OH; 75.7 mg, 0.348 mmol), 1-hydroxybenzotriazole (HOBt; 34.6 mg, 0.256 mmol), N,N-diisopropylethylamine (0.089 mL, 0.511 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDCI; 58.3 mg, 0.304 mmol) to afford 122 mg (84%) of the product 35a as a colourless foam.

[0445] .sup.1H NMR (500 MHz, CDCl.sub.3) 5.38-5.30 (m, 1H, NH), 4.49-4.41 (m, 1H), 4.39-4.33 (m, 0.5H), 4.27 (dd, 13.6, 3.7 Hz, 0.5H), 3.69-3.63 (m, 0.5H), 3.63-3.57 (m, 0.5H), 3.56-3.47 (m, 1H), 3.25 (ap t, 13.1 Hz, 0.5H), 3.12 (td, 13.3, 1.5 Hz, 0.5H), 2.81 (ap t, 13.3 Hz, 0.5H), 2.61 (td, 13.3, 1.9 Hz, 0.5H), 2.26 (dddd, 15.2, 9.8, 5.3, 1.6 Hz, 1H), 2.13 (ddd, 15.4, 9.3, 6.8 Hz, 1H), 2.06-1.99 (m, 1H), 1.97-1.74 (m, 6H), 1.74-1.53 (m, 2H), 1.53-1.33 (m, 8H; 1.44, s, 9H; 1.432, s, 4.5H; 1.427, s, 4.5H), 1.33-1.12 (m, 4H), 1.11-1.05 (m, 1H), 1.02 (s, 1.5H), 0.99 (s, 1.5H), 0.96 (d, 6.7 Hz, 1.5H), 0.95 (d, 6.6 Hz, 1.5H), 0.93 (d, 6.5 Hz, 3H), 0.89 (d, 6.8 Hz, 1.5H), 0.85 (d, 6.7 Hz, 1.5H), 0.696 (s, 1.5H), 0.694 (s, 1.5H);

[0446] HRMS (ESI) m/z calcd for C.sub.37H.sub.64N.sub.2O.sub.6H.sup.+633.4837, found 633.4839.

B. N-[(2S)-2-amino-3-methylbutanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid hydrotrifluoroacetate (41a)

##STR00047##

[0447] Applying the same method of deprotection as for Example 10, the product of step A (35a) (122 mg, 0.193 mmol) dissolved in dry dichloromethane (5 mL) was treated with trifluoroacetic acid (4 mL) at 0 C. for 2.5 h and the crude product was purified by automated column chromatography [C18 silica gel, acetonitrile/water (+0.5% trifluoroacetic acid) 0-50%]. The purified product was co-evaporated with dichloromethane (3-4) and methanol (1) at 60 C. to give 67.9 mg (60%) of 41a as a colourless foam.

[0448] .sup.1H NMR (500 MHz, CD.sub.3OD; mixture of rotamers) 4.35 (d, 4.9 Hz, 0.5H), 4.32-4.26 (m, 0.5H), 4.24 (d, 5.0 Hz, 0.5H), 4.22-4.16 (m, 0.5H), 3.65-3.59 (m, 0.5H), 3.59-3.53 (m, 0.5H), 3.49-3.38 (m, 1.5H), 3.21 (td, 13.3, 1.5 Hz, 0.5H), 2.99 (ap t, 13.3 Hz, 0.5H), 2.79 (td, 13.3, 1.7 Hz, 0.5H), 2.33 (ddd, 15.3, 9.9, 5.4 Hz, 1H), 2.25-2.12 (m, 2H), 2.09-2.02 (m, 1H), 1.98-1.77 (m, 5H), 1.74-1.59 (m, 2.5H), 1.59-1.37 (m, 5.5H), 1.37-1.16 (m, 5H), 1.16-1.07 (overlapping signals: m, 1H; 1.10, d, 7.3 Hz, 1.5H; 1.09, d, 7.3 Hz, 1.5H), 1.05 (s, 1.5H), 1.03 (s, 1.5H), 1.01 (d, 7.0 Hz, 1.5H), 0.99 (d, 7.0 Hz, 1.5H), 0.96 (d, 6.5 Hz, 3H), 0.74 (s, 3H);

[0449] .sup.19F NMR (470 MHz, CD.sub.3OD) 77.00;

[0450] .sup.13C NMR (125 MHz, CD.sub.3OD) 178.17, 168.04, 167.89, 71.57, 71.52, 57.28, 57.10, 56.59, 56.34, 48.01, 45.75, 44.81, 44.45, 44.37, 44.18, 42.36, 41.38, 41.32, 40.46, 40.15, 39.21, 37.22, 36.71, 36.47, 36.04, 35.78, 35.19, 34.95, 32.41, 32.06, 30.94, 29.64, 27.94, 23.78, 23.67, 22.64, 22.58, 19.31, 19.10, 18.95, 17.04, 16.82, 12.66; HRMS (ESI) m/z calcd for C.sub.28H.sub.48N.sub.2O.sub.4H.sup.+ 477.3687, found 477.3696.

Example 12

N-[(2S,3S)-2-Amino-3-methylpentanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid hydrotrifluoroacetate (42a)

##STR00048##

A. tert-Butyl N-{(2S,3S)-2-[(tert-butoxycarbonyl)amino]-3-methylpentanoyl}-3-aza-7p-hydroxy-5-cholan-24-oate (36a)

##STR00049##

[0451] Using the same protocol as for Example 9, the compound of Example 2 (27a) (101 mg, 0.233 mmol) was dissolved in dry dichloromethane (2.5 mL) and sequentially treated with Boc-L-isoleucine (Boc-Ile-OH; 82.4 mg, 0.356 mmol), 1-hydroxybenzotriazole (35.0 mg, 0.259 mmol), N,N-diisopropylethylamine (0.09 mL, 0.517 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (59.0 mg, 0.308 mmol) to afford 129 mg (86%) of 36a as a colourless foam.

[0452] .sup.1H NMR (500 MHz, CDCl.sub.3) 5.37-5.30 (m, 1H, NH), 4.51-4.43 (m, 1H), 4.39-4.32 (m, 0.5H), 4.28 (dd, 13.6, 3.7 Hz, 0.5H), 3.75-3.62 (m, 1H), 3.56-3.47 (m, 1H), 3.25 (ap t, 13.0 Hz, 0.5H), 3.18-3.09 (m, 0.5H), 2.82 (apt, 13.3 Hz, 0.5H), 2.62 (td, 13.4, 1.9 Hz, 0.5H), 2.26 (dddd, 15.2, 9.8, 5.3, 1.8 Hz, 1H), 2.13 (ddd, 15.4, 9.3, 6.7 Hz, 1H), 2.06-1.99 (m, 1H), 1.97-1.61 (m, 7.5H), 1.60-1.14 (m, 13.5H; 1.44, s, 9H; 1.424, s, 4.5H; 1.422, s, 4.5H), 1.14-1.04 (m, 2H), 1.02 (s, 1.5H), 0.99 (s, 1.5H), 0.95-0.85 (overlapping signals: m, 3H, 1.25, d, 6.6 Hz, 3H; 0.88, t, 7.4 Hz, 1.5H; 0.87, t, 7.4 Hz, 1.5H), 0.697 (s, 1.5H), 0.694 (s, 1.5H);

[0453] HRMS (ESI) m/z calcd for C.sub.38H.sub.66N.sub.2O.sub.6Na.sup.+669.4813, found 669.4822.

B. N-[(2S,3S)-2-Amino-3-methylpentanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid hydrotrifluoroacetate (42a)

##STR00050##

[0454] Applying the same method as for Example 11, step B, the compound of step A (36a) (116 mg, 0.179 mmol) dissolved in dry dichloromethane (5 mL) was reacted with trifluoroacetic acid (4 mL) at 0 C. for 2.5 h to give 74.2 mg (68%) of the product 42a as a colourless foam.

[0455] .sup.1H NMR (500 MHz, CD.sub.3OD; mixture of rotamers) 4.36 (d, 4.9 Hz, 0.5H), 4.32-4.23 (overlapping signals: m, 0.5H; 4.25, d, 4.9 Hz, 0.5H), 4.19 (dd, 13.7, 3.4 Hz, 0.5H), 3.64-3.52 m, 1H), 3.50-3.38 (m, 1.5H), 3.21 (td, 13.3, 1.6 Hz, 0.5H), 2.99 (ap t, 13.3 Hz, 0.5H), 2.79 (td, 13.3, 1.9 Hz, 0.5H), 2.33 (ddd, 15.3, 9.8, 5.4 Hz, 1H), 2.20 (ddd, 15.5, 9.3, 6.8 Hz, 1H), 2.09-2.02 (m, 1H), 1.99-1.77 (m, 6H), 1.73-1.58 (m, 2.5H), 1.58-1.37 (m, 6.5H), 1.37-1.09 (m, 7H), 1.08 (d, 7.1 Hz, 1.5H), 1.06 (d, 7.2 Hz, 1.5H), 1.05 (s, 1.5H), 1.03 (s, 1.5H), 1.00-0.94 (m, 6H), 0.74 (s, 3H);

[0456] .sup.19F NMR (470 MHz, CD.sub.3OD) 76.95;

[0457] .sup.13C NMR (125 MHz, CD.sub.3OD; mixture of rotamers) 178.16, 168.02, 167.87, 71.57, 71.51, 57.28, 57.08, 56.64, 56.58, 56.35, 56.01, 48.12, 45.77, 44.81, 44.52, 44.44, 44.38, 44.24, 42.40, 41.38, 41.31, 40.49, 40.15, 39.24, 37.71, 37.67, 37.33, 36.71, 36.46, 36.04, 35.81, 35.20, 34.97, 32.41, 32.06, 29.64, 27.95, 27.93, 24.68, 24.51, 23.78, 23.73, 22.64, 22.58, 18.96, 15.81, 15.46, 12.68, 12.65, 12.07;

[0458] HRMS (ESI) m/z calcd for C.sub.29H.sub.50N.sub.2O.sub.4H.sup.+491.3843, found. 491.3857.

Example 13

N-[(2S)-2-Amino-4-methylpentanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid hydrotrifluoroacetate (43a)

##STR00051##

A. tert-Butyl N-{(2S)-2-[(tert-butoxycarbonyl)amino]-4-methylpentanoyl}-3-aza-7-hydroxy-5-cholan-24-oate (37a)

##STR00052##

[0459] Deploying the same procedure as for Example 9, the compound of Example 2 (27a) (102 mg, 0.235 mmol) was dissolved in dry dichloromethane (2.5 mL) and reacted with Boc-L-leucine (Boc-Leu-OH; 81.7 mg, 0.353 mmol), 1-hydroxybenzotriazole (35.3 mg, 0.261 mmol), N,N-diisopropylethylamine (0.09 mL, 0.517 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (59.4 mg, 0.310 mmol) to give 134 mg (88%) of the product 37a as a colourless foam.

[0460] .sup.1H NMR (500 MHz, CDCl.sub.3) 5.31 (s, 0.5H, NH), 5.29 (s, 0.5H, NH), 4.68-4.59 (m, 1H), 4.36-4.29 (m, 0.5H), 4.25 (dd, 13.7, 3.8 Hz, 0.5H), 3.66-3.56 (m, 1H), 3.56-3.46 (m, 1H), 3.23 (ap t, 13.1 Hz, 0.5H), 3.17-3.08 (m, 0.5H), 2.81 (ap t, 13.2 Hz, 0.5H), 2.59 (td, 13.4, 1.9 Hz, 0.5H), 2.26 (dddd, 15.2, 9.8, 5.3, 1.5 Hz, 1H), 2.13 (ddd, 15.4, 9.3, 6.7 Hz, 1H), 2.06-1.99 (m, 1H), 1.97-1.55 (m, 8H), 1.55-1.12 (m, 14H; 1.44, s, 9H; 1.428, s, 4.5H; 1.426, s, 4.5H), 1.12-1.04 (m, 1H), 1.02 (s, 1.5H), 1.01-0.96 (overlapping signals: m, 3H; 1.00, s, 1.5H); 0.94-0.90 (m, 6H), 0.70 (s, 3H);

[0461] HRMS (ESI) m/z calcd for C.sub.38H.sub.66N.sub.2O.sub.6Na.sup.+ 669.4813, found 669.4803.

B. N-[(2S)-2-Amino-4-methylpentanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid hydrotrifluoroacetate (43a)

##STR00053##

[0462] Following the same method as for Example 11, step B, the compound of step A (37a) (120 mg, 0.186 mmol) was dissolved in dry dichloromethane (5 mL) and stirred in the presence of trifluoroacetic acid (4 mL) to give rise to 73.3 mg (65%) of the product 43a as a colourless foam.

[0463] .sup.1H NMR (500 MHz, CD.sub.3OD; mixture of rotamers) 4.43 (dd, 9.2, 3.7 Hz, 0.5H), 4.35 (dd, 9.1, 3.5 Hz, 0.5H), 4.29-4.22 (m, 0.5H), 4.17 (dd, 13.6, 3.6 Hz, 0.5H), 3.52-3.38 (m, 2.5H), 3.28-3.19 (m, 0.5H), 3.00 (ap t, 13.3 Hz, 0.5H), 2.77 (td, 13.3, 1.9 Hz, 0.5H), 2.33 (ddd, 15.3, 9.8, 5.4 Hz, 1H), 2.20 (ddd, 15.5, 9.2, 6.9 Hz, 1H), 2.10-2.02 (m, 1H), 1.99-1.78 (m, 5H), 1.78-1.54 (m, 6H), 1.54-1.15 (m, 10H), 1.15-1.07 (m, 1H), 1.06 (s, 1.5H), 1.05-0.99 (overlapping signals: 1.04, s, 1.5H; m, 6H), 0.96 (d, 6.5 Hz, 3H), 0.74 (s, 3H);

[0464] .sup.19F NMR (470 MHz, CD.sub.3OD) 76.95;

[0465] .sup.13C NMR (125 MHz, CD.sub.3OD; mixture of rotamers) 178.16, 168.82, 168.73, 71.54, 57.27, 57.10, 56.64, 56.61, 50.46, 47.65, 45.44, 44.81, 44.41, 44.38, 44.25, 44.11, 42.21, 41.55, 41.38, 41.32, 41.02, 40.45, 40.17, 39.18, 37.06, 36.71, 36.42, 35.99, 35.98, 35.12, 35.02, 32.41, 32.07, 29.63, 27.95, 25.51, 25.46, 23.77, 23.64, 23.61, 23.59, 22.64, 22.58, 21.82, 21.72, 18.96, 12.67;

[0466] HRMS (ESI) m/z calcd for C.sub.29H.sub.50N.sub.2O.sub.4H.sup.+ 491.3843, found 491.3857

Example 14

N-[(2S)-2-Amino-3-(1H-indol-3-yl)propanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid hydrotrifluoroacetate (44a)

##STR00054##

A. tert-Butyl N-{(2S)-2-[(tert-butoxycarbonyl)amino]-3-[1H-indol-3-yl]propanoyl}-3-aza-7-hydroxy-5-cholan-24-oate (38a)

##STR00055##

[0467] Applying the same protocol as for Example 9, the compound of Example 2 (27a) (90.2 mg, 0.208 mmol) was dissolved in dry dichloromethane (2.5 mL) and treated with Na-Boc-L-tryptophan (Boc-Trp-OH; 95.3 mg, 0.313 mmol), 1-hydroxybenzotriazole (31.1 mg, 0.230 mmol), N,N-diisopropylethylamine (0.08 mL, 0.459 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (51.9 mg, 0.271 mmol) to afford 122 mg (82%) of the product 38a as a colourless oil.

[0468] .sup.1H NMR (500 MHz, CDCl.sub.3; mixture of rotamers) 8.20 (s, 0.5H, NH), 8.10 (s, 0.5H, NH), 7.69-7.64 (m, 0.5H), 7.58 (d, 7.8 Hz, 0.5H), 7.36-7.30 (m, 1H), 7.21-7.08 (m, 2H), 7.08-7.03 (m, 1H), 5.66 (d, 8.4 Hz, 0.5H, NH), 5.48 (d, 8.7 Hz, 0.5H, NH), 4.99-4.92 (m, 0.5H), 4.92-4.85 (m, 0.5H), 4.16-4.07 (m, 1H), 3.44-3.35 (m, 0.5H), 3.29-3.19 (m, 1H), 3.18-3.12 (m, 1H), 3.12-3.01 (overlapping signals: 3.08, dd, 14.0, 9.5 Hz, 0.5H; m, 0.5H), 2.85-2.75 (m, 1H), 2.61 (ap t, 13.2 Hz, 0.5H), 2.34-2.06 (overlapping signals: 2.29, ddd, 15.3, 10.0, 5.4 Hz, 0.5H; m, 2H), 2.01-1.84 (m, 2H), 1.84-1.49 (m, 4H), 1.49-0.97 (m, 14H; 1.46, s, 4.5H; 1.45, s, 4.5H; 1.44, s, 4.5H; 1.43, s, 4.5H), 0.94 (d, 6.5 Hz, 1.5H), 0.91-0.82 (overlapping signals: 0.88, d, 6.5 Hz, 1.5H; m, 0.5H; 0.84, s, 1.5H), 0.82-0.73 (m, 0.5H); 0.65-0.59 (overlapping singlets: 0.63, 1.5H; 0.62, 3H), 0.36 (td, 13.4, 2.8 Hz, 0.5H);

[0469] HRMS (ESI) m/z calcd for C.sub.43H.sub.65N.sub.3O.sub.6Na.sup.+ 742.4766, found 742.4764.

B. N-[(2S)-2-Amino-3-(1H-indol-3-yl)propanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid hydrotrifluoroacetate (44a)

##STR00056##

[0470] Employing the same reaction conditions as Example 11, step B, the compound of step A (38a) (122 mg, 0.169 mmol) dissolved in dry dichloromethane (5 mL) was treated with trifluoroacetic acid (4 mL) at 0 C. for 2.5 h to give 83.7 mg (73%) of the product 44a as a colourless foam.

[0471] .sup.1H NMR (500 MHz, CD.sub.3OD; mixture of rotamers) 7.55-7.51 (m, 0.5H), 7.46-7.35 (m, 1.5H), 7.22 (s, 0.5H), 7.20 (s, 0.5H), 7.19-7.07 (m, 1.5H), 7.06-7.02 (m, 0.5H), 4.65-4.57 (m, 1H), 4.04-3.96 (m, 1H), 3.36-3.20 (overlapping signals: m, 2H; 3.23, dd, 13.7, 10.4 Hz, 0.5H), 3.15-3.08 (m, 0.5H), 2.95 (dd, 13.4, 3.8 Hz, 0.5H), 2.84 (td, 13.2, 1.9 Hz, 0.5H), 2.77-2.65 (m, 1H), 2.38 (ddd, 15.3, 9.8, 5.4 Hz, 0.5H), 2.33-2.13 (m, 2H), 2.00-1.91 (m, 1.5H), 1.91-1.74 (m, 2.5H), 1.64-1.00 (m, 14H), 1.00-0.94 (overlapping signals: m, 1H; 0.99, d, 6.5 Hz, 1.5H), 0.91 (d, 6.5 Hz, 1.5H) 0.86 (s, 1.5H), 0.69-0.63 (overlapping signals: m, 1H; 0.66, d, 6.5 Hz, 3H), 0.60 (s, 1.5H), 0.52 (td, 13.8, 3.1 Hz, 0.5H);

[0472] .sup.19F NMR (470 MHz, CD.sub.3OD) 76.94;

[0473] .sup.13C NMR (125 MHz, CD.sub.3OD; mixture of rotamers) 178.20, 178.14, 169.37, 168.65, 138.15, 138.05, 128.81, 128.72, 125.76, 125.50, 123.33, 123.05, 120.53, 120.45, 119.31, 119.11, 112.99, 112.90, 108.63, 108.30, 71.41, 71.07, 57.21, 56.86, 56.77, 56.58, 52.13, 51.27, 46.43, 44.77, 44.73, 44.22, 44.09, 43.98, 43.86, 43.13, 42.59, 41.30, 41.19, 40.16, 40.11, 38.69, 36.75, 36.67, 35.94, 35.85, 35.57, 35.21, 34.11, 32.45, 32.37, 32.11, 32.04, 29.66, 29.57, 29.26, 29.17, 27.87, 27.77, 23.22, 23.03, 22.44, 22.34, 19.00, 18.91, 12.61;

[0474] HRMS (ESI) m/z calcd for C.sub.34H.sub.49N.sub.3O.sub.4H.sup.+ 564.3796, found 564.3799.

Example 15

N[R(2S)-2-Amino-3-(4-hydroxyphenyl)propanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid hydrotrifluoroacetate (45a)

##STR00057##

A. tert-Butyl N-{(2S)-2-[(tert-butoxycarbonyl)amino]-3-[4-hydroxyphenyl]propanoyl}-3-aza-7-hydroxy-5-cholan-24-oate (39a)

##STR00058##

[0475] Using the same method as for Example 9, the compound of Example 2 (27a) (91.9 mg, 0.212 mmol) was dissolved in dry dichloromethane (2.5 mL) and reacted with Boc-L-tyrosine (Boc-Tyr-OH; 91.0 mg, 0.324 mmol), 1-hydroxybenzotriazole (31.8 mg, 0.235 mmol), N,N-diisopropylethylamine (0.08 mL, 0.459 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (53.3 mg, 0.278 mmol) to give 110 mg (75%) of 39a as a colourless foam.

[0476] .sup.1H NMR (500 MHz, CDCl.sub.3; mixture of rotamers) 7.04 (AAXX, J.sub.AX=8.3 Hz, 1H), 7.01 (AAXX, J.sub.AX=8.4 Hz, 1H), 6.78-6.72 (m, 2H), 5.56 (d, 8.6 Hz, 0.5H, NH), 5.41 (d, 8.7 Hz, 1H, NH), 4.85-4.73 (m, 1H), 4.25-4.15 (m, 1H), 3.49-3.39 (m, 1H), 3.30-3.18 (m, 1H), 3.01-2.79 (overlapping signals: m, 2H; 2.82, dd, 13.3, 8.7 Hz, 0.5H), 2.71 (ap t, 13.2 Hz, 0.5H), 2.52 (ap t, 12.9 Hz, 0.5H), 2.32-2.20 (m, 1.5H), 2.18-2.08 (m, 1H), 2.05-1.94 (m, 1H), 1.94-1.84 (m, 1H), 1.84-1.59 (m, 4H), 1.56-1.00 (m, 15.5H; 1.45, s, 4.5H; 1.44, s, 4.5H; 1.43, s, 4.5H; 1.42, s, 4.5H), 0.95-0.89 (overlapping signals: 0.93, d, 6.5 Hz, 1.5H; 0.93, s, 1.5H; 0.91, d, 6.6 Hz, 1.5H), 0.84 (s, 1.5H), 0.67 (s, 1.5H), 0.66 (s, 1.5H), 0.21 (td, 13.4, 2.8 Hz, 0.5H);

[0477] HRMS (ESI) m/z calcd for C.sub.41H.sub.64N.sub.2O.sub.7H.sup.+697.4786, found 697.4785.

B. N-[(2S)-2-Amino-3-(4-hydroxyphenyl)propanoyl]-3-aza-7-hydroxy-5-cholan-24-oic acid hydrotrifluoroacetate (45a)

##STR00059##

[0478] Using the same method as for Example 11, step B, the compound of step A (39a) (110 mg, 0.158 mmol) was dissolved in dry dichloromethane (5 mL) and reacted with trifluoroacetic acid (4 mL) at 0 C. for 2.5 h to yield 56.0 mg (54%) of the product 45a as a colourless foam.

[0479] .sup.1H NMR (500 MHz, CD.sub.3OD; mixture of rotamers) 7.07 (AAXX, J.sub.AX=8.5 Hz, 1H), 7.05 (AAXX, J.sub.AX=8.5 Hz, 1H), 6.79 (AAXX, J.sub.AX=8.6 Hz, 1H), 6.76 (AAXX, J.sub.AX=8.6 Hz, 1H), 4.60 (dd, 9.9, 5.7 Hz, 0.5H), 4.54 (dd, 9.7, 5.8 Hz, 0.5H), 4.17-4.09 (m, 1H), 3.40-3.26 (m, 1H), 3.13-2.82 (m, 4H), 2.58 (td, 13.2, 2.2 Hz, 0.5H), 2.39-2.27 (m, 1H), 2.26-2.15 (m, 1H), 2.08-1.98 (m, 1.5H), 1.95-1.68 (m, 4.5H), 1.59 (ddd, 13.6, 5.0, 2.1 Hz, 0.5H), 1.53-1.04 (m, 13.5H), 0.99-0.92 (overlapping signals: 0.97, d, 6.5 Hz, 1.5H; 0.95, s, 1.5H; 0.94, d, 6.6 Hz, 1.5H), 0.85 (s, 1.5H), 0.704 (s, 1.5H), 0.698 (s, 1.5H), 0.05 (td, 13.8, 4.2 Hz, 0.5H); .sup.19F NMR (470 MHz, CD.sub.3OD) 76.98; .sup.13C NMR (125 MHz, CD.sub.3OD; mixture of rotamers) 178.22, 178.16, 168.60, 168.00, 158.73, 158.60, 132.12, 131.98, 125.88, 125.85, 117.03, 116.68, 71.46, 71.33, 57.24, 56.89, 56.60, 56.35, 52.31, 51.74, 46.72, 44.77, 44.40, 44.32, 44.27, 44.05, 43.79, 42.63, 41.34, 40.54, 40.24, 38.53, 38.49, 38.27, 36.72, 36.69, 36.12, 35.99, 35.85, 35.68, 34.56, 34.51, 32.44, 32.39, 32.06, 29.65, 29.60, 27.91, 27.87, 23.46, 23.27, 22.54, 22.47, 18.97, 18.94, 12.67, 12.65;

[0480] HRMS (ESI) m/z calcd for C.sub.32H.sub.48N.sub.2O.sub.5H.sup.+ 541.3636, found 541.3632

Example 16

tert-Butyl N-(benzyloxycarbonyl)-3-aza-7-hydroxy-25-homo-5-cholan-25-oate (25b)

##STR00060##

A. Methyl 3,7-3,7-dihydroxy-24-nor-5-cholan-23-oate

[0481] The title compound was prepared from ursodeoxycholic acid by the method of D'Amore et al 2014.

B. Methyl 3,7-bis(tert-butyldimethylsilyloxy)-24-nor-5-cholan-23-oate (3)

##STR00061##

[0482] To a solution of the product of step A (diol 2; 37.8 g, 96.3 mmol) in dry DMF (1 L) was added imidazole (33.4 g, 491 mmol) and tert-butyl(chloro)dimethylsilane (60.3 g, 400 mmol) at room temperature. After being stirred at 60 C. overnight the reaction was diluted with water and extracted with ethyl acetate (3). The combined organic layers were washed with water (2) and brine, dried over MgSO.sub.4 and concentrated. The crude product was purified by flash column chromatography (silica gel, ethyl acetate/petroleum ether 1:19, 1:9 and 1:6) to yield 53.6 g (90%) of the product 3 as a colourless amorphous solid.

[0483] .sup.1H NMR (500 MHz, CDCl.sub.3) 3.71-3.62 (overlapping signals: m, 1H; 3.66, s, 3H), 3.52 (tt, 10.7, 4.7 Hz, 1H), 2.43 (dd, 14.5, 3.3 Hz, 1H), 2.03-1.86 (m, 3H), 1.86-1.71 (m, 4H), 1.64 (dt, 11.1, 12.9 Hz, 1H), 1.58-1.33 (m, 8H), 1.32-1.20 (m, 3H), 1.18-1.00 (m, 3H), 1.00-0.91 (overlapping signals: m, 1H; 0.97, d, 6.5 Hz, 3H, 0.92, s, 3H), 0.893 (s, 9H), 0.885 (s, 9H), 0.68 (s, 3H), 0.060 (s, 3H), 0.057 (s, 3H), 0.049 (s, 3H), 0.046 (s, 3H);

[0484] HRMS (ESI) m/z calcd for C.sub.36H.sub.68O.sub.4Si.sub.2Na.sup.+ 643.4548, found 643.4553.

[0485] Procedure in: Yan, S.; Ding, N.; Zhang, W.; Wang, P.; Li, Y.; Li, M. Carbohydr. Research 2012, 354, 6-20.

C. 3,7-bis(tert-Butyldimethylsilyloxy)-24-nor-5-cholan-23-ol (4)

##STR00062##

[0486] To a solution of the product of step B, the methyl ester 3 (53.5 g, 86.1 mmol) in dry THF (2 L) and methanol (25 mL, 617 mmol) was added lithium borohydride (13.3 g, 611 mmol) at 0 C. After 30 min the ice bath was removed and the reaction was stirred at room temperature overnight. Subsequently, the reaction the reaction was carefully quenched with 1 M sodium hydroxide solution (1 L). The organic phase was separated and the aqueous phase was extracted with ethyl acetate (2). The organic phases were combined, washed with water and brine, dried over MgSO.sub.4 and concentrated. The crude product was purified by flash column chromatography (silica gel, ethyl acetate 1:19, 1:9 and 1:4) to 50.0 g (98%) of the product, alcohol 4, as a dry colourless gel.

[0487] .sup.1H NMR (500 MHz, CDCl.sub.3) 3.74-3.60 (m, 3H), 3.52 (tt, 10.6, 4.7 Hz, 1H), 1.96 (dt, 12.5, 3.2 Hz, 1H), 1.86-1.69 (m, 5H), 1.64 (dt, 11.2, 12.9 Hz, 1H), 1.59-1.19 (m, 13H), 1.17-1.00 (m, 4H), 1.00-0.91 (overlapping signals: m, 1H; 0.95, d, 6.6 Hz, 3H; 0.92, s, 3H), 0.894 (s, 9H), 0.889 (s, 9H), 0.66 (s, 3H), 0.061 (s, 3H), 0.059 (s, 3H), 0.054 (s, 3H), 0.049 (s, 3H);

[0488] HRMS (ESI) m/z calcd for C.sub.35H.sub.68O.sub.3Si.sub.2Na.sup.+ 615.4599, found 615.4609.

D. 3,7-bis(tert-Butyldimethylsilyloxy)-24-nor-5-cholan-23-al (5)

##STR00063##

[0489] To a solution of oxalyl chloride (5.32 mL, 62.9 mmol) in dry THF (400 mL) was added dropwise dry DMSO (4.75 mL, 66.8 mmol) at 78 C. After 20 min a solution of 4, the product of step C (23.3 g, 39.3 mmol) in dry THF (100 mL) was introduced dropwise via cannula. Then, the reaction mixture was allowed to warm to 60 C. before dry triethylamine (16.4 mL, 118 mmol) was added and the reaction was allowed to slowly warm to room temperature. Water was added and the aqueous phase was extracted with ethyl acetate (3). The combined organic phases were washed with brine, dried over MgSO.sub.4 and concentrated. The crude product was purified by automated column chromatography (silica gel, ethyl acetate/petroleum ether 2-10%) to afford 19.2 g (83%) of the required product, 5, as an amorphous, colourless solid.

[0490] .sup.1H NMR (500 MHz, CDCl.sub.3) 9.75 (dd, 3.4, 1.4 Hz, 1H), 3.70-3.63 (m, 1H), 3.52 (tt, 10.7, 4.7 Hz, 1H), 2.46 (ddd, 15.9, 3.1, 1.0 Hz, 1H), 2.16 (ddd, 15.9, 9.2, 3.4 Hz, 1H), 2.07-1.98 (m, 1H), 1.96 (dt, 12.5, 3.3 Hz, 1H), 1.88-1.72 (m, 4H), 1.64 (dt, 10.9, 13.0 Hz, 1H), 1.58-1.35 (m, 8H), 1.35-1.20 (m, 3H), 1.20-1.06 (m, 3H), 1.03-0.91 (overlapping signals: 1.01, d, 6.6 Hz, 3H; m, 1H; 0.93, s, 3H), 0.894 (s, 9H), 0.886 (s, 9H), 0.70 (s, 3H), 0.062 (s, 3H), 0.059 (s, 3H), 0.050 (s, 3H), 0.047 (s, 3H);

[0491] HRMS (ESI) m/z calcd for C.sub.35H.sub.66O.sub.3Si.sub.2Na.sup.+ 613.4443, found 613.4448.

E. Methyl 3,7-bis(tert-butyldimethylsilyloxy)-25-homo-5-cholan-23-en-25-oate (7a) and tert-butyl 3,7-bis(tert-butyldimethylsilyloxy)-25-homo-5-cholan-23-en-25-oate (7b)

##STR00064##

Methyl Ester

[0492] To a solution of aldehyde 5, the product of step D (8.48 g, 14.3 mmol) in dry dichloromethane (188 mL) was added methyl (triphenylphosphoranylidene) acetate (14.9 g, 44.6 mmol) at room temperature. After being stirred overnight the reaction was concentrated to small volume and directly loaded onto a high performance cartridge packed with silica gel. Gradient elution with ethyl acetate and petroleum ether (0-15%) using an automated chromatography system yielded 8.74 g (94%) of the desired methyl ester product 7a as an amorphous, colourless solid.

[0493] .sup.1H NMR (500 MHz, CDCl.sub.3) 6.95 (ddd, 15.5, 8.8, 6.6 Hz, 1H), 5.84-5.78 (m, 1H), 3.73 (s, 3H), 3.70-3.63 (m, 1H), 3.52 (tt, 10.7, 4.7 Hz, 1H), 2.33-2.26 (m, 1H), 2.01-1.91 (m, 2H), 1.87-1.71 (m, 4H), 1.64 (dt, 11.0, 13.1 Hz, 1H), 1.61-1.34 (m, 9H), 1.34-1.20 (m, 3H), 1.18-1.00 (m, 3H), 1.00-0.91 (overlapping signals: m, 1H; 0.94, d, 6.7 Hz, 3H; 0.92, s, 3H), 0.893 (s, 9H), 0.890 (s, 9H), 0.66 (s, 3H), 0.059 (s, 3H), 0.056 (s, 6H), 0.050 (s, 3H);

[0494] HRMS (ESI) m/z calcd for C.sub.38H.sub.70O.sub.4Si.sub.2Na.sup.+ 669.4705, found 669.4708.

Tert-Butyl Ester

[0495] Employing the same procedure as for the preparation of methyl ester 7a aldehyde 5 (19.2 g, 32.5 mmol) was dissolved in dry dichloromethane (520 mL) and reacted with (tert-butoxycarbonylmethylene)triphenylphosphorane (43.0 g, 114 mmol) at room temperature for 72 h to give 21.8 g (97%) of the tert-butyl ester 7b as a colourless foam.

[0496] .sup.1H NMR (500 MHz, CDCl.sub.3) 6.83 (ddd, 15.4, 8.7, 6.5 Hz, 1H), 5.75-5.69 (m, 1H), 3.70-3.63 (m, 1H), 3.52 (tt, 10.6, 4.7 Hz, 1H), 2.31-2.24 (m, 1H), 1.97-1.87 (m, 2H), 1.86-1.71 (m, 4H), 1.64 (dt, 11.0, 13.0 Hz, 1H), 1.59-1.32 (m, 9H), 1.49 (s, 9H), 1.32-1.19 (m, 3H), 1.18-1.00 (m, 3H), 1.00-0.91 (overlapping signals: m, 4H; 0.92, s, 3H), 0.893 (s, 9H), 0.890 (s, 9H), 0.65 (s, 3H), 0.059 (s, 3H), 0.056 (s, 6H), 0.050 (s, 3H);

[0497] HRMS (ESI) m/z calcd for C.sub.41H.sub.76O.sub.4Si.sub.2Na.sup.+ 711.5174, found 711.5175.

F. Methyl 3,7-dihydroxy-25-homo-5-cholan-25-oate (9a) and tert-butyl 3,7-dihydroxy-25-homo-5-cholan-25-oate (9b)

##STR00065##

Methyl Ester

[0498] To a solution of alkene 7a, the product of step E (8.07 g, 12.5 mmol) in ethyl acetate (220 mL) was added 10% palladium on charcoal (673 mg) and the atmosphere was exchanged for hydrogen. After being stirred at room temperature overnight the reaction was filtered through celite and concentrated. The crude bis(tert-Butyldimethylsilyloxy reaction product 8a was re-dissolved in a mixture of THF (165 mL) and methanol (250 mL) followed by the addition of a 37% aqueous hydrochloric acid solution (57 mL). After being stirred at room temperature for 2 h the reaction was carefully quenched with saturated bicarbonate solution and extracted with ethyl acetate (3). The combined organic layers were washed with brine, dried over MgSO.sub.4 and concentrated. The crude product was purified by automated column chromatography (silica gel, ethyl acetate/petroleum ether 2-100%) to yield 4.69 g (89%) of the required product 9a as a colourless foam.

[0499] Methyl 3,7-bis(tert-Butyldimethylsilyloxy)-25-homo-5-cholan-25-oate (8a). .sup.1H NMR (500 MHz, CDCl.sub.3) 3.69-3.62 (overlapping signals: m, 1H; 3.67, s, 3H), 3.52 (tt, 10.7, 4.7 Hz, 1H), 2.33-2.20 (m, 2H), 1.95 (dt, 12.5, 3.2 Hz, 1H), 1.84-1.60 (m, 6H); 1.58-1.15 (m, 14H), 1.15-1.03 (m, 3H), 1.03-0.91 (overlapping signals: m, 2H; 0.92, d, 3H; 0.92, s, 3H), 0.894 (s, 9H), 0.887 (s, 9H), 0.64 (s, 3H), 0.061 (s, 3H), 0.058 (s, 3H), 0.051 (s, 3H), 0.046 (s, 3H);

[0500] HRMS (ESI) m/z calcd for C.sub.38H.sub.72O.sub.4Si.sub.2Na.sup.+ 671.4861, found 671.4858.

[0501] Methyl 3,7-Dihydroxy-25-homo-5-cholan-25-oate (9a). .sup.1H NMR (500 MHz, CDCl.sub.3) 3.67 (s, 3H), 3.62-3.55 (m, 2H), 2.34-2.21 (m, 2H), 2.00 (dt, 12.6, 3.2 Hz, 1H), 1.91-1.83 (m, 1H), 1.83-1.75 (m, 3H), 1.75-1.64 (m, 3H), 1.63-1.54 (m, 2H), 1.54-1.35 (m, 10H), 1.33-1.19 (m, 4H), 1.14 (td, 12.9, 3.9 Hz, 1H), 1.11-0.98 (m, 3H), 0.95 (s, 3H), 0.94 (d, 6.7 Hz, 3H), 0.67 (s, 3H);

[0502] HRMS (ESI) m/z calcd for C.sub.26H.sub.44O.sub.4Na.sup.+ 443.3132, found 443.3138.

Tert-Butyl Ester

[0503] Using the same procedure as outlined for the preparation of methyl ester 8a, alkene 7b from Step E (21.7 g, 31.5 mmol) was dissolved in ethyl acetate (400 mL) and hydrogenated on 10% palladium on charcoal (867 mg) at room temperature overnight to afford 21.9 g (quant.) of 8b as a colourless resin.

[0504] .sup.1H NMR (500 MHz, CDCl.sub.3) 3.66 (ddd, 11.2, 8.6, 4.9 Hz, 1H), 3.52 (tt, 10.7, 4.7 Hz, 1H), 2.26-2.08 (m, 2H), 1.95 (dt, 12.5, 3.2 Hz, 1H), 1.86-1.71 (m, 4H), 1.70-1.59 (2H), 1.58-1.16 (m, 14H; 1.45, s, 9H), 1.15-0.99 (m, 4H), 0.99-0.91 (overlapping signals: m, 1H; 0.92, d, 3H; 0.92, s, 3H), 0.894 (s, 9H), 0.887 (s, 9H), 0.64 (s, 3H), 0.061 (s, 3H), 0.058 (s, 3H), 0.051 (s, 3H), 0.046 (s, 3H);

[0505] HRMS (ESI) m/z calcd for C.sub.41H.sub.78O.sub.4Si.sub.2Na.sup.+ 713.5331, found 713.5339.

[0506] A solution of silyl protected tert-butyl ester 8b (21.6 g, 31.3 mmol) in THF (100 mL) was treated with a 1 M solution of tetra-butyl ammonium fluoride in THF (144 mL, 144 mmol) at 50 C. for 72 h. After deprotection was complete (TLC analysis) water was added and the aqueous phase was extracted with ethyl acetate (3). The organic phases were combined, washed with brine, dried over MgSO.sub.4 and concentrated. The crude product was purified by automated column chromatography (silica gel, ethyl acetate/petroleum ether 5-80%) to give 13.9 g (96%) of the required product 9b as a colourless oil.

[0507] .sup.1H NMR (500 MHz, CDCl.sub.3) 3.63-3.55 (m, 2H), 2.24-2.11 (m, 2H), 2.00 (dt, 12.6, 3.2 Hz, 1H), 1.92-1.83 (m, 1H), 1.83-1.75 (m, 3H), 1.71-1.63 (m, 3H), 1.63-1.54 (m, 2H), 1.53-1.34 (m, 10H; 1.45, s, 9H), 1.33-1.19 (m, 4H), 1.14 (td, 12.9, 3.8 Hz, 1H), 1.11-0.98 (m, 3H), 0.95 (s, 3H), 0.94 (d, 6.6 Hz, 3H), 0.67 (s, 3H);

[0508] HRMS (ESI) m/z calcd for C.sub.29H.sub.50O.sub.4Na.sup.+ 485.3601, found 485.3611.

G. tert-Butyl 7p-hydroxy-3-oxo-25-homo-5-cholan-25-oate (11)

##STR00066##

[0509] To a solution of 9b, the product of step F (13.9 g, 30.0 mmol) in dry dichloromethane (210 mL) was added (diacetoxyiodo)benzene (BAIB; 10.9 g, 33.8 mmol) followed by a catalytic amount of TEMPO (713 mg, 4.56 mmol) and the resulting reaction mixture was stirred at room temperature overnight. Subsequently, the reaction was quenched with saturated Na.sub.2S.sub.2O.sub.3 solution and diluted with water. The organic layer was separated and the aqueous layer was extracted another three times with dichloromethane. The organic layers were combined, washed with brine, dried over MgSO.sub.4 and concentrated. The residue was purified by automated column chromatography (silica gel, ethyl acetate/petroleum ether 2-80%) to afford 13.3 g (96%) of the required product 11 as a colourless foam.

[0510] .sup.1H NMR (500 MHz, CDCl.sub.3) 3.61 (ddd, 11.2, 8.8, 5.2 Hz, 1H), 2.52 (dd, 14.9, 14.1 Hz, 1H), 2.28 (td, 14.6, 5.4 Hz, 1H), 2.24-2.11 (m, 4H), 2.10-2.00 (m, 2H), 1.95-1.78 (m, 4H), 1.72-1.60 (m, 2H), 1.59-1.34 (m, 10H; 1.45, s, 9H), 1.34-1.16 (m, 3H), 1.14-1.03 (overlapping signals: m, 2H; 1.05, s, 3H), 0.95 (d, 6.6 Hz, 3H), 0.72 (s, 3H);

[0511] HRMS (ESI) m/z calcd for C.sub.29H.sub.4804Na.sup.+ 483.3445, found 483.3447.

[0512] The product may also be prepared from 9a by the same method.

H. tert-Butyl 7p-hydroxy-3-oxa-4-oxo-4a,25-bishomo-5-cholan-25-oate (side Product 14b) and methyl 7p-hydroxy-4-oxa-3-oxo-4a,25-bishomo-5-cholan-25-oate (Required Product 15b)

##STR00067##

[0513] Following the same procedure as outlined for Example 1C, the product of Step G (11) (11.9 g, 25.8 mmol) in dry dichloromethane (200 mL) was treated with meta-chloroperbenzoic acid (mCPBA, 57-86%; 13.5 g, 54.8 mmol) at room temperature overnight to yield 11.1 g (90%) of the products 14b and 15b as a 1:1 mixture of isomers (colourless foam).

[0514] .sup.1H NMR (500 MHz, CDCl.sub.3, mixture of isomers) 4.48 (dd, 13.1, 9.6 Hz, 1H), 4.18 (dd, 13.3, 10.2 Hz, 1H), 4.05 (ddd, 13.2, 6.5, 1.0 Hz, 1H), 3.98 (ap d, 12.9 Hz, 1H), 3.48-3.40 (m, 1H), 3.33-3.25 (m, 1H), 3.03 (ap t, 12.9 Hz, 1H), 2.63 (ap dd, 14.5, 13.1, 1H), 2.42-2.35 (m, 2H), 2.24-2.11 (m, 4H), 2.08-2.01 (m, 3H), 1.98-1.84 (m, 7H), 1.84-1.75 (m, 2H), 1.74-1.61 (m, 4H), 1.58-1.31 (m, 20H; 1.44, s, 18H), 1.31-1.14 (m, 6H), 1.13-1.02 (overlapping signals: m, 4H; 1.05, s, 3H; 1.04, s, 3H), 0.939 (d, 6.6 Hz, 3H), 0.936 (d, 6.6 Hz, 3H), 0.70 (6H);

[0515] HRMS (ESI) m/z calcd for C.sub.29H.sub.48O.sub.5Na.sup.+ (mixture of isomers) 499.3394, found 499.3398.

I. tert-Butyl 4,7-dihydroxy-25-homo-3,4-seco-5-cholan-25-oate-3-amide (side Product 16b) and tert-butyl 2,7-dihydroxy-25-homo-2,3-seco-5-cholan-25-oate-3-amide (Required Product 17b)

##STR00068##

[0516] Deploying the same procedure as for Example 1D, the mixture of lactones 14b and 15b from Step H (10.9 g, 22.9 mmol) was dissolved in dry 7 N ammonia in methanol (155 mL) and heated at 92 C. in a 200 mL sealed tube with Teflon screw cap (the sealed tube was filled to 4/5 of its volume and a blast shield was added) overnight to give 8.52 g (76%) of a mixture of isomeric amides 16b and 17b as a colourless foam. The product ratio (16b:17b) was determined to be 1:1.3 by HPLC analysis (Phenomenex Luna C18(2) 5 m 2504.6 mm; Phenomenex Security Guard C18 43 mm; mobile phase: 45:55:0.05 water/acetonitrile/trifluoroacetic acid; flow rate: 1 mL/min; sample solvent: methanol; column temperature: 35 C.; injection volume: 25 L; detection: refractive index). In addition, the isomeric methyl esters 18b (230 mg, 2%; colourless oil) and 19b (1.55 g, 13%, colourless foam) were isolated as by-products. An analytical sample of each of the four compounds was recovered for spectroscopic characterization.

[0517] tert-Butyl 4,7-dihydroxy-25-homo-3,4-seco-5-cholan-25-oate-3-amide (16b). .sup.1H NMR (500 MHz, CDCl.sub.3) 6.00 (s.sub.br, 1H, NH.sub.2), 5.66 (s.sub.br, 1H, NH.sub.2), 3.76 (dd, 11.0, 5.1 Hz, 1H), 3.56 (dd, 11.0, 7.4 Hz, 1H), 3.49-3.41 (m, 1H), 2.41-2.29 (m, 1H), 2.22-2.08 (m, 3H), 2.03-1.95 (m, 2H), 1.91-1.74 (m, 3H), 1.74-1.68 (m, 1H), 1.68-1.54 (m, 3H), 1.53-1.33 (m, 6H; 1.44, s, 9H), 1.32-1.22 (m, 2H), 1.22-1.14 (m, 1H), 1.14-1.01 (m, 3H), 1.01-0.95 (overlapping signals: m, 1H; 0.98, s, 3H), 0.92 (d, 6.5 Hz, 3H), 0.68 (s, 3H); H,HCOSY (500 MHz, CDCl.sub.3) 3.76 g 1.71, 3.56 g 1.71;

[0518] HRMS (ESI) m/z calcd for C.sub.29H.sub.51NO.sub.5Na.sup.+ 516.3659, found 516.3669.

[0519] tert-Butyl 2,7-dihydroxy-25-homo-2,3-seco-5-cholan-24-oate-3-amide (17b). .sup.1H NMR (500 MHz, CDCl.sub.3) 6.32 (s.sub.br, 1H, NH.sub.2), 6.00 (s.sub.br, 1H, NH.sub.2), 3.82-3.74 (m, 1H), 3.74-3.66 (m, 1H), 3.64-3.55 (m, 1H), 2.50-2.42 (m, 1H), 2.25-2.10 (m, 3H), 2.08-1.97 (m, 2H), 1.90-1.59 (m, 6H), 1.57-1.34 (m, 8H; 1.44, s, 9H), 1.33-1.23 (m, 3H), 1.23-1.10 (m, 2H), 1.10-1.00 (overlapping signals: m, 2H; 1.06, s, 3H), 1.00-0.90 (overlapping signals: m, 1H; 0.93, d, 6.4 Hz, 3H), 0.68 (s, 3H); H,HCOSY (500 MHz, CDCl.sub.3) 3.78 g 1.72/1.41, 3.70 g 1.72/141;

[0520] HRMS (ESI) m/z calcd for C.sub.29H.sub.51NO.sub.5H.sup.+494.3840, found 494.3846.

[0521] tert-Butyl 4,7-dihydroxy-25-homo-3,4-seco-5-cholan-25-oate-3-methyl ester (18b). .sup.1H NMR (500 MHz, CDCl.sub.3) 3.72 (dd, 10.7, 4.6 Hz, 1H), 3.67 (s, 3H), 3.61-3.50 (overlapping signals: 3.58, dd, 10.8, 9.3 Hz, 1H; 3.54, ddd, 9.6, 11.5, 4.9 Hz, 1H), 2.37 (ddd, 15.5, 12.1, 4.5 Hz, 1H), 2.29 (dd, 12.0, 5.2 Hz, 1H), 2.22-2.12 (m, 2H), 2.07 (ddd, 13.4, 4.9, 2.9 Hz, 1H), 1.99 (dt, 12.8, 3.3 Hz, 1H), 1.95-1.71 (m, 3H), 1.71-1.61 (m, 2H), 1.61-1.53 (m, 2H), 1.53-1.33 (m, 8H; 1.44, s, 9H), 1.33-1.22 (m, 2H), 1.18 (ddd, 10.7, 12.4, 7.3 Hz, 1H), 1.15-1.04 (m, 3H), 1.04-0.96 (overlapping signals: m, 1H; 0.99, s, 3H), 0.92 (d, 6.6 Hz, 3H), 0.68 (s, 3H); H,HCOSY (500 MHz, CDCl.sub.3) 3.72 g 1.67, 3.58 g 1.67;

[0522] HRMS (ESI) m/z calcd for C.sub.30H.sub.52O.sub.6Na.sup.+ 531.3656, found 531.3670.

[0523] tert-Butyl 2,7-dihydroxy-25-homo-2,3-seco-5-cholan-25-oate-3-methyl ester (19b). .sup.1H NMR (500 MHz, CDCl.sub.3) 3.77-3.68 (m, 2H), 3.68 (s, 3H), 3.58 (dt, 7.5, 9.3 Hz, 1H), 2.44 (dd, 14.9, 3.4 Hz, 1H), 2.33 (dd, 14.9, 11.4 Hz, 1H), 2.24-2.11 (m, 2H), 2.06-1.97 (m, 2H), 1.91-1.82 (m, 1H), 1.81-1.70 (m, 2H), 1.70-1.58 (m, 3H), 1.57-1.51 (m, 1H), 1.51-1.32 (m, 8H; 1.44, s, 9H), 1.32-1.24 (m, 2H), 1.20 (ddd, 10.7, 12.4, 7.3 Hz, 1H); 1.17-1.02 (overlapping signals: 1.13, td, 13.1, 4.0 Hz, 1H; m, 2H; 1.08, s, 3H), 1.00-0.91 (overlapping signals: m, 1H; 0.93, d, 6.6 Hz, 3H), 0.68 (s, 3H); H,HCOSY (500 MHz, CDCl.sub.3) 3.72 (2H) g 1.74, 1.39;

[0524] HRMS (ESI) m/z calcd for C.sub.30H.sub.52O.sub.6Na.sup.+ 531.3656, found 531.3662.

J. tert-butyl 2-Amino-4,7-dihydroxy-25-homo-3-nor-3,4-seco-5-cholan-25-oate (Side Product 20b) and tert-butyl 4-Amino-2,7-dihydroxy-25-homo-3-nor-3,4-seco-5-cholan-25-oate (Required Product 21b)

##STR00069##

[0525] Employing the same procedure as for Example 1E, a mixture of the products of step I (16b and 17b) (7.85 g, 15.9 mmol) dissolved in a 3:1 mixture of acetonitrile and water (400 mL/132 mL; additionally 20 mL of ethyl acetate was added to help solubilize the starting material) was treated with (diacetoxyiodo)benzene (BAIB; 5.92 g, 18.4 mmol) at room temperature overnight to afford 5.55 g (75%) of a mixture of 20b and 21b as a colourless foam.

[0526] .sup.1H NMR (500 MHz, CD.sub.3OD; mixture of isomers) 3.71-3.57 (overlapping signals: 3.69, dd, 10.9, 4.6 Hz, 1H; m, 2H), 3.53 (dd, 10.9, 9.5 Hz, 1H), 3.46-3.37 (m, 2H), 2.84-2.75 (overlapping signals: 2.82, dd, 12.1, 4.8 Hz, 1H; m, 1H), 2.73-2.62 (overlapping signals: 2.70, td, 12.0, 4.7 Hz, 1H; 2.65, dd, 12.7, 10.9 Hz, 1H), 2.23-2.12 (m, 4H), 2.09-1.99 (m, 3H), 1.94-1.79 (m, 5H), 1.72-1.52 (m, 9H), 1.52-1.28 (m, 15H; 1.44, s, 18H), 1.28-1.11 (m, 6H), 1.11-0.98 (overlapping signals: m, 6H; 1.07, s, 3H; 1.06, s, 3H), 0.95 (d, 6.5 Hz, 6H), 0.72 (s, 6H);

[0527] HRMS (ESI) m/z calcd for C.sub.28H.sub.51NO.sub.4H.sup.+ 466.3891, found 466.3894.

K. tert-Butyl N-(benzyloxycarbonyl)-2-amino-4,7-dihydroxy-25-homo-3-nor-3,4-seco-5-cholan-25-oate (Side Product 22b) and tert-butyl N-(benzyloxycarbonyl)-4-amino-2,7-dihydroxy-25-homo-3-nor-3,4-seco-5-cholan-25-oate (Required Product 23b)

##STR00070##

[0528] Following the same procedure as for Example 1F, the mixture of amines from Step J (20b and 21b) (5.48 g, 11.8 mmol) was dissolved in a 1:1 mixture of dichloromethane (80 mL) and water (80 mL) and treated with benzyl chloroformate (CbzCl 95%, 2.00 mL, 14.0 mmol) in the presence of sodium carbonate (6.44 g, 60.8 mmol) at 0 C. to yield 3.53 g (50%) of product 22b and 2.60 g (37%) of product 23b, both as colourless foams.

[0529] N-(Benzyloxycarbonyl)-2-amino-4,7-dihydroxy-25-homo-3-nor-3,4-seco-5-cholan-25-oate (22b) .sup.1H NMR (500 MHz, CDCl.sub.3) 7.39-7.29 (m, 5H), 5.12-5.05 (m, 2H), 4.89-4.80 (m, 1H, NH), 3.92-3.83 (m, 1H), 3.60-3.52 (m, 1H), 3.51-3.44 (m, 1H), 3.44-3.34 (m, 1H), 3.14-3.04 (m, 1H), 2.34 (s.sub.br, 1H, OH), 2.23-2.10 (m, 2H), 2.06-1.94 (m, 2H), 1.90-1.81 (m, 1H), 1.81-1.71 (m, 2H), 1.70-1.55 (m, 3H), 1.53-1.33 (m, 8H; 1.44, s, 9H), 1.32-1.21 (m, 2H), 1.20-1.13 (m, 1H), 1.13-0.94 (overlapping signals: m, 4H; 1.03, s, 3H), 0.92 (d, 6.6 Hz, 3H), 0.67 (s, 3H); H,HCOSY (500 MHz, CDCl.sub.3) 3.88 g 1.74, 3.56 g 1.74;

[0530] HRMS (ESI) m/z calcd for C.sub.36H.sub.57NO.sub.6H.sup.+ 600.4259, found 600.4269.

[0531] tert-Butyl N-(Benzyloxycarbonyl)-4-amino-2,7-dihydroxy-25-homo-3-nor-3,4-seco-5-cholan-25-oate (23b). .sup.1H NMR (500 MHz, CDCl.sub.3) 7.41-7.29 (m, 5H), 5.09 (s, 2H), 4.98-4.90 (m, 1H, NH), 3.92-3.82 (m, 1H), 3.75-3.66 (m, 1H), 3.63-3.53 (m, 1H), 3.46-3.38 (m, 1H), 3.11 (ddd, 13.3, 11.5, 6.7 Hz, 1H), 2.23-2.11 (m, 2H), 2.02-1.95 (m, 1H), 1.90-1.80 (m, 3H), 1.80-1.70 (m, 2H), 1.70-1.61 (m, 2H), 1.61-1.33 (m, 9H; 1.44, s, 9H), 1.33-1.16 (m, 3H), 1.16-0.97 (overlapping signals: m, 4H, 1.04, s, 3H), 0.93 (d, 6.5 Hz, 3H), 0.67 (s, 3H); H,HCOSY (500 MHz, CDCl.sub.3) 3.87 g 1.75, 1.49, 3.71 g 1.75, 1.49;

[0532] HRMS (ESI) m/z calcd for C.sub.36H.sub.57NO.sub.6Na.sup.+ 622.4078, found 622.4082.

L. tert-Butyl N-(benzyloxycarbonyl)-3-aza-7-hydroxy-25-homo-5-cholan-25-oate

##STR00071##

[0533] According to the protocol outlined for Example 1G, compound 23b of Step K (2.51 g, 4.18 mmol) dissolved in dry pyridine (80 mL) was treated with methanesulfonyl chloride (0.39 mL, 5.01 mmol) at 0 C. for 5 h, until only traces of starting material were detectable by TLC analysis. Then, saturated bicarbonate solution (4.5 mL) was added and the reaction mixture was stirred at 74 C. overnight. The crude product was purified by automated column chromatography (silica gel, ethyl acetate/petroleum ether 0-50%) to give 1.75 g (72%) of the product 25b as a colourless foam.

[0534] .sup.1H NMR (500 MHz, CDCl.sub.3) 7.39-7.28 (m, 5H), 5.18-5.06 (m, 2H), 3.98-3.75 (m, 2H), 3.57-3.45 (m, 1H), 3.08-2.90 (m, 1H), 2.90-2.73 (m, 1H), 2.23-2.11 (m, 2H), 2.02 (dt, 12.7, 3.01 Hz, 1H), 1.92-1.83 (m, 1H), 1.83-1.71 (m, 3H), 1.70-1.59 (m, 3H), 1.53-1.26 (m, 10H; 1.44, s, 9H), 1.26-1.02 (m, 5H), 0.99 (s, 3H), 0.93 (d, 6.6 Hz, 3H), 0.68 (s, 3H);

[0535] HRMS (ESI) m/z calcd for C.sub.36H.sub.55NO.sub.5H.sup.+582.4153, found 582.4154.

Example 17

tert-Butyl 3-aza-7-hydroxy-25-homo-5-cholan-25-oate (27b)

##STR00072##

[0536] Using the same procedure as described for Example 2, the compound of Example 16 (25b) (865 mg, 1.48 mmol) dissolved in methanol (35 mL) was hydrogenated on 10% palladium on charcoal (93.8 mg) to yield 675 mg (quant.) of the product 27b as a colourless foam.

[0537] .sup.1H NMR (500 MHz, CDCl.sub.3) 3.51 (ddd, 11.6, 9.5, 5.1 Hz, 1H), 2.88 (ap t, 12.5 Hz, 1H), 2.77-2.63 (m, 3H), 2.24-2.11 (m, 2H), 2.04-1.98 (m, 1H), 1.92-1.73 (m, 6H), 1.71-1.62 (m, 1H), 1.62-1.54 (m, 3H), 1.52-1.33 (m, 6H; 1.44, s, 9H), 1.33-1.22 (m, 3H), 1.22-1.02 (m, 4H), 0.99 (s, 3H), 0.94 (d, 6.6 Hz, 3H), 0.68 (s, 3H);

[0538] HRMS (ESI) m/z calcd for C.sub.28H.sub.49NO.sub.3H.sup.+ 448.3785, found 448.3787.

Example 18

3-Aza-7-hydroxy-25-homo-5-cholan-25-oic acid hydrochloride (28b)

##STR00073##

[0539] Applying the same protocol as for Example 3, tert-butyl ester 27b from Example 17 (151 mg, 0.337 mmol) dissolved in dry dichloromethane (4 mL) was deprotected with trifluoroacetic acid (TFA; 3 mL) at 0 C. to yield 113 mg (78%) of 28b as a colourless foam.

[0540] .sup.1H NMR (500 MHz, CD.sub.3OD) 3.41-3.34 (m, 1H), 3.24 (apt, 13.0 Hz, 1H), 3.15-3.05 (m, 2H), 3.01 (td, 13.5, 2.3 Hz, 1H), 2.31-2.18 (m, 2H), 2.09-1.99 (m, 2H), 1.95-1.81 (m, 4H), 1.74-1.65 (m, 1H), 1.65-1.60 (m, 1H), 1.60-1.36 (m, 9H), 1.35-1.21 (m, 3H), 1.16-1.05 (overlapping signals: m, 2H; 1.09, s, 3H), 0.97 (d, 6.6 Hz, 3H), 0.73 (s, 3H);

[0541] .sup.13C NMR (125 MHz, CD.sub.3OD) 177.78, 71.06, 56.95, 56.61, 45.03, 44.75, 44.37, 42.08, 41.19, 40.80, 40.00, 36.87, 36.64, 35.41, 35.17, 34.26, 33.56, 29.70, 27.91, 23.48, 22.75, 22.55, 19.27, 12.61;

[0542] HRMS (ESI) m/z calcd for C.sub.24H.sub.41NO.sub.3H.sup.+ 392.3159, found 392.3162.

Example 19

tert-Butyl N-methyl-3-aza-7-hydroxy-25-homo-5-cholan-25-oate (29b)

##STR00074##

[0543] Using the same procedure as for Example 4, compound 25b of Example 16 (70.1 mg, 0.121 mmol) dissolved in methanol (5 mL) was treated with formalin (37% in water, 0.1 mL, 1.34 mmol) and stirred under an atmosphere of hydrogen in the presence of 10% palladium on charcoal (20.3 mg) to give 52.2 mg (94%) of 29b as a colourless oil.

[0544] .sup.1H NMR (500 MHz, CDCl.sub.3) 3.53 (ddd, 10.8, 9.3, 5.0, 1H), 2.56-2.46 (m, 2H), 2.25 (s, 3H), 2.23-2.11 (m, 3H), 2.03-1.94 (m, 2H), 1.91-1.83 (m, 1H), 1.83-1.73 (m, 4H), 1.71-1.56 (m, 2H), 1.52-1.33 (m, 8H; 1.44, s, 9H), 1.33-1.20 (m, 3H), 1.15 (td, 13.0, 3.9 Hz, 1H), 1.12-1.02 (m, 2H), 0.99 (s, 3H), 0.94 (d, 6.6 Hz, 3H), 0.68 (s, 3H);

[0545] HRMS (ESI) m/z calcd for C.sub.29H.sub.51NO.sub.3H.sup.+ 462.3942, found 462.3953.

Example 20

N-Methyl-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid hydrotrifluoroacetate (31b)

##STR00075##

[0546] Employing the same reaction protocol as for Example 5, the tert-butyl ester 29b of Example 19 (52.2 mg, 0.113 mmol) dissolved in dry dichloromethane (5 mL) was treated with trifluoroacetic acid (4 mL) at 0 C. to afford 48.8 mg (83%) of the product 31 b as a colourless foam.

[0547] .sup.1H NMR (500 MHz, CD.sub.3OD) 3.38 (ddd, 11.4, 9.7, 5.1, 1H), 3.30-3.17 (m, 3H), 3.05-2.97 (m, 1H), 2.85 (s, 3H), 2.31-2.18 (m, 2H), 2.10-2.02 (m, 2H), 1.95-1.80 (m, 4H), 1.75-1.66 (m, 1H), 1.66-1.54 (m, 2H), 1.54-1.37 (m, 8H), 1.327-1.22 (m, 3H), 1.16-1.04 (overlapping signals: m, 2H; 1.08, s, 3H), 0.97 (d, 6.6 Hz, 3H), 0.73 (s, 3H);

[0548] .sup.19F NMR (470 MHz, CD.sub.3OD) 76.92;

[0549] .sup.13C NMR (125 MHz, CD.sub.3OD) 177.71, 70.95, 56.81, 56.63, 55.40, 51.06, 44.74, 44.36, 43.74, 42.94, 41.12, 39.79, 36.86, 36.63, 35.39, 35.06, 34.40, 33.47, 29.70, 27.91, 23.06, 22.75, 22.55, 19.27, 12.60;

[0550] HRMS (ESI) m/z calcd for C.sub.25H.sub.43NO.sub.3H.sup.+ 406.3316, found 406.3315.

Example 21

tert-Butyl N-glycolyl-3-aza-7-hydroxy-25-homo-5-cholan-25-oate (34b)

##STR00076##

[0551] Using the same procedure as for Example 9, compound 27b of Example 17 (102 mg, 0.228 mmol) was dissolved in dry dichloromethane (2.5 mL) and sequentially treated with glycolic acid (27.7 mg, 0.364 mmol), 1-hydroxybenzotriazole (HOBt; 33.8 mg, 0.250 mmol), N,N-diisopropylethylamine (0.087 mL, 0.499 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDCI; 60.8 mg, 0.317 mmol) to yield 107 mg (93%) of 34b as a colourless foam.

[0552] .sup.1H NMR (500 MHz, CDCl.sub.3; mixture of rotamers) 4.34-4.24 (m, 1H), 4.19-4.06 (m, 2H), 3.66 (s.sub.br, 1H, OH), 3.57-3.47 (m, 1H), 3.25-3.15 (m, 1H), 3.11 (dd, 13.5, 4.2 Hz, 0.5H), 3.01 (td, 13.4, 1.9 Hz, 0.5H), 2.90 (ap t, 13.2 Hz, 0.5H), 2.77-2.68 (m, 0.5H), 2.24-2.11 (m, 2H), 2.07-2.00 (m, 1H), 1.93-1.75 (m, 4H), 1.76-1.60 (m, 3H), 1.59-1.34 (m, 9H; 1.44, s, 9H), 1.34-1.05 (6H), 1.02 (s, 3H), 0.94 (d, 6.3 Hz, 3H), 0.70 (s, 3H);

[0553] HRMS (ESI) m/z calcd for C.sub.30H.sub.51NO.sub.5Na.sup.+ 528.3659, found 528.3658.

Example 22

N-Glycolyl-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid (40b)

##STR00077##

[0554] Employing the same protocol as for Example 10, compound 34b of Example 21 (93.8 mg, 0.186 mmol) dissolved in dry dichloromethane (5 mL) was treated with trifluoroacetic acid (4 mL) at 0 C. for 2.5 h to afford 45.1 mg (54%) of the product 40b as a colourless foam.

[0555] .sup.1H NMR (500 MHz, CD.sub.3OD; mixture of rotamers) 4.25-4.12 (m, 3H), 3.48-3.38 (m, 1.5H), 3.38-3.27 (m, 1H), 3.09 (td, 13.4, 1.6 Hz, 0.5H), 2.96 (apt, 13.1 Hz, 0.5H), 2.80-2.71 (m, 0.5H), 2.31-2.17 (m, 2H), 2.08-2.02 (m, 1H), 1.94-1.80 (m, 4H), 1.74-1.54 (m, 4H), 1.54-1.35 (m, 7H), 1.33-1.05 (m, 6H), 1.03 (s, 3H), 0.97 (d, 6.5 Hz, 3H), 0.73 (s, 3H);

[0556] .sup.13C NMR (125 MHz, CD.sub.3OD; mixture of rotamers) 177.75, 171.78, 71.61, 71.57, 61.24, 61.17, 57.27, 57.25, 56.66, 45.84, 44.99, 44.77, 44.38, 43.87, 41.40, 40.82, 40.22, 38.81, 37.07, 36.90, 36.66, 36.47, 36.07, 35.93, 35.42, 35.06, 29.72, 27.95, 23.78, 23.73, 22.79, 22.64, 22.60, 19.29, 12.67;

[0557] HRMS (ESI) m/z calcd for C.sub.26H.sub.43NO.sub.5Na.sup.+ 472.3033, found 472.3028.

Example 23

N-[(2S)-2-Amino-3-methylbutanoyl]-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid hydrotrifluoroacetate (41b)

##STR00078##

A. tert-Butyl N-{(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}-3-aza-7-hydroxy-25-homo-5-cholan-25-oate (35b)

##STR00079##

[0558] Following the same procedure as for Example 5, compound 27b of Example 17 (100 mg, 0.223 mmol) was dissolved in dry dichloromethane (2.5 mL) and reacted with Boc-L-valine (74.6 mg, 0.343 mmol), 1-hydroxybenzotriazole (33.4 mg, 0.247 mmol), N,N-diisopropylethylamine (0.086 mL, 0.494 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (56.3 mg, 0.294 mmol) to give 154 mg (quant.) of the product 35b as a colourless oil.

[0559] .sup.1H NMR (500 MHz, CDCl.sub.3; mixture of rotamers) 5.68-5.57 (m, 1H, NH), 4.52-4.41 (m, 1H), 4.38-4.31 (m, 0.5H), 4.27 (dd, 13.6, 3.6 Hz, 0.5H), 3.78-3.67 (m, 1H), 3.60-3.49 (m, 1H), 3.28 (ap t, 13.0 Hz, 0.5H), 3.23-3.14 (m, 0.5H), 2.87 (ap t, 13.3 Hz, 0.5H), 2.67 (td, 13.3, 1.9 Hz, 0.5H), 2.25-2.12 (m, 2H), 2.07-2.01 (m, 1H), 1.97-1.75 (m, 5H), 1.75-1.54 (m, 3H), 1.53-1.34 (m, 8H; 1.45, s, 9H; 1.429, s, 4.5H; 1.425, s, 4.5H), 1.34-1.12 (m, 4H), 1.12-1.04 (m, 2H), 1.03 (s, 1.5H), 1.00 (s, 1.5H), 0.97-0.91 (overlapping signals: m, 1.5H; 0.96, d, 6.8 Hz, 1.5H; 0.94, d, 6.7 Hz, 3H; 0.93, d, 6.8 Hz, 1.5H); 0.89 (d, 6.8 Hz, 1.5H), 0.695 (s, 1.5H), 0.692 (s, 1.5H);

[0560] HRMS (ESI) m/z calcd for C.sub.38H.sub.66N.sub.2O.sub.6Na.sup.+669.4813, found 669.4817.

B. N-[(2S)-2-Amino-3-methylbutanoyl]-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid hydrotrifluoroacetate (41b)

##STR00080##

[0561] Following the same method as or Example 12, step B, compound 35b of step A (137 mg, 0.212 mmol) was dissolved in dry dichloromethane (5 mL) and treated with trifluoroacetic acid (4 mL) to yield 80.1 mg (63%) of the product 41b as a colourless foam.

[0562] .sup.1H NMR (500 MHz, CD.sub.3OD; mixture of rotamers) 4.35 (d, 3.9 Hz, 0.5H), 4.32-4.26 (m, 0.5H), 4.24 (d, 5.0 Hz, 0.5H), 4.21-4.16 (m, 0.5H), 3.64-3.59 (m, 0.5H), 3.59-3.53 (m, 0.5H), 3.49-3.38 (m, 1.5H), 3.20 (td, 13.5, 1.6 Hz, 0.5H), 2.99 (ap t, 13.3 Hz, 0.5H), 2.79 (td, 13.4, 1.9 Hz, 0.5H), 2.31-2.12 (m, 3H), 2.09-2.03 (m, 1H), 1.97-1.80 (m, 4H), 1.74-1.58 (m, 3.5H), 1.58-1.35 (m, 7.5H), 1.34-1.19 (m, 3.5H), 1.19-1.07 (overlapping signals: m, 2.5H; 1.10, d, 7.3 Hz, 1.5H; 1.09, d, 7.25 Hz, 1.5H), 1.05 (s, 1.5H), 1.03 (s, 1.5H), 1.01 (d, 7.0 Hz, 1.5H), 0.99 (d, 7.0 Hz, 1.5H), 0.97 (d, 6.5 Hz, 3H), 0.74 (s, 3H);

[0563] .sup.19F NMR (470 MHz, CD.sub.3OD) 76.97;

[0564] .sup.13C NMR (125 MHz, CD.sub.3OD; mixture of rotamers) 177.74, 168.04, 167.88, 71.59, 71.54, 57.31, 57.12, 56.62, 56.34, 48.02, 45.75, 44.77, 44.45, 44.38, 44.18, 42.36, 41.41, 41.35, 40.47, 40.17, 39.21, 37.22, 36.89, 36.65, 36.47, 36.04, 35.79, 35.39, 35.19, 34.96, 30.95, 29.72, 27.96, 23.79, 23.68, 22.75, 22.64, 22.58, 19.29, 19.10, 17.04, 16.83, 12.65;

[0565] HRMS (ESI) m/z calcd for C.sub.29H.sub.50N.sub.2O.sub.4H.sup.+ 491.3843, found 491.3856.

Example 24

N-[(2S,3S)-2-Amino-3-methylpentanoyl]-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid hydrotrifluoroacetate (42b)

##STR00081##

A. tert-Butyl N-{(2S,3S)-2-[(tert-butoxycarbonyl)amino]-3-methylpentanoyl}-3-aza-7p-hydroxy-25-homo-5-cholan-25-oate (36b)

##STR00082##

[0566] Employing the same protocol as for Example 9, compound 27b of Example 17 (61.5 mg, 0.137 mmol) was dissolved in dry dichloromethane (2 mL) and reacted with Boc-L-isoleucine (48.3 mg, 0.209 mmol), 1-hydroxybenzotriazole (20.9 mg, 0.155 mmol), N,N-diisopropylethylamine (0.053 mL, 0.304 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (34.8 mg, 0.182 mmol) to afford 88.1 mg (97%) of the product 36b as a colourless oil.

[0567] .sup.1H NMR (500 MHz, CDCl.sub.3; mixture of rotamers) 5.33-5.25 (m, 1H, NH), 4.50-4.42 (m, 1H), 4.39-4.33 (m, 0.5H), 4.28 (dd, 13.5, 3.5 Hz, 0.5H), 3.74-3.60 (m, 1H), 3.56-3.46 (m, 1H), 3.25 (ap t, 13.0 Hz, 0.5H), 3.16-3.08 (m, 0.5H), 2.81 (ap t, 13.3 Hz, 0.5H), 2.61 (td, 13.3, 1.9 Hz, 0.5H), 2.24-2.11 (m, 2H), 2.04-2.00 (m, 1H), 1.93-1.75 (m, 4H), 1.73-1.59 (m, 3.5H), 1.59-1.32 (m, 10.5H; 1.44; s, 9H; 1.425, s, 4.5H; 1.423, s, 4.5H), 1.32-1.13 (m, 4H), 1.13-1.04 (m, 3H), 1.02 (s, 1.5H), 0.99 (s, 1.5H), 0.96-0.85 (overlapping signals: 0.94, d, 6.5 Hz, 3H; 0.93, d, 7.2 Hz, 1.5H; 0.92, d, 7.2 Hz, 1.5H; 0.88, t, 7.3 Hz, 1.5H; 0.87, t, 7.3 Hz, 1.5H), 0.694 (s, 1.5H), 0.692 (s, 1.5H);

[0568] HRMS (ESI) m/z calcd for C.sub.39H.sub.68N.sub.2O.sub.6H.sup.+661.5150, found 661.5152.

B. N-[(2S,3S)-2-Amino-3-methylpentanoyl]-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid hydrotrifluoroacetate (42b)

##STR00083##

[0569] According to the method of Example 11, step B, compound 36b of step A (88.1 mg, 0.133 mmol) was dissolved in dry dichloromethane (4 mL) and treated with trifluoroacetic acid (3 mL) to give 38.8 mg (47%) of the product 42b as a colourless foam.

[0570] .sup.1H NMR (500 MHz, CD.sub.3OD; mixture of rotamers) 4.36 (d, 4.8 Hz, 0.5H), 4.32-4.24 (overlapping signals: m, 0.5H; 4.25, d, 4.9 Hz, 0.5H), 4.18 (dd, 13.7, 3.6 Hz, 0.5H), 3.63-3.52 (overlapping signals: m, 0.5H; 3.55, dd, 13.5, 3.0 Hz, 0.5H), 3.50-3.38 (m, 1.5H), 3.25-3.17 (m, 0.5H), 2.99 (ap t, 13.3 Hz, 0.5H), 2.79 (td, 13.2, 1.6 Hz, 0.5H), 2.31-2.18 (m, 2H), 2.09-2.03 (m, 1H), 1.99-1.81 (m, 5H), 1.74-1.57 (m, 3.5H), 1.57-1.35 (m, 8.5H), 1.34-1.15 (m, 5H), 1.15-1.04 (overlapping signals: m, 2H; 1.08, d, 7.0 Hz, 1.5H; 1.06, d, 7.0 Hz, 1.5H; 1.05, s, 1.5H), 1.03 (s, 1.5H), 1.00-0.94 (m, 6H), 0.74 (s, 3H);

[0571] .sup.19F NMR (470 MHz, CD.sub.3OD) 76.96;

[0572] .sup.13C NMR (125 MHz, CD.sub.3OD; mixture of rotamers) 177.73, 168.03, 167.87, 71.59, 71.53, 57.30, 57.11, 56.67, 56.62, 56.33, 56.00, 48.13, 45.76, 44.77, 44.52, 44.44, 44.38, 44.24, 42.40, 41.41, 41.34, 40.50, 40.16, 39.25, 37.71, 37.67, 37.33, 36.88, 36.65, 36.45, 36.04, 35.80, 35.39, 35.20, 34.97, 29.73, 27.96, 24.69, 24.51, 23.78, 23.74, 22.75, 22.64, 22.58, 19.28, 15.80, 15.46, 12.64, 12.06, 11.86;

[0573] HRMS (ESI) m/z calcd for C.sub.30H.sub.52N.sub.2O.sub.4H.sup.+505.4000, found 505.4010.

Example 25

N-[(2S)-2-Amino-4-methylpentanoyl]-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid hydrotrifluoroacetate (43b)

##STR00084##

A. tert-Butyl N-{(2S)-2-[(tert-butoxycarbonyl)amino]-4-methylpentanoyl}-3-aza-7-hydroxy-25-homo-5-cholan-25-oate (37b)

##STR00085##

[0574] Using the same procedure as for Example 9, compound 27b of Example 17 (60.4 mg, 0.135 mmol) was dissolved in dry dichloromethane (2 mL) and reacted with Boc-L-isoleucine (46.9 mg, 0.203 mmol), 1-hydroxybenzotriazole (20.5 mg, 0.152 mmol), N,N-diisopropylethylamine (0.052 mL, 0.299 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (33.9 mg, 0.177 mmol) to give 71.1 mg (80%) of the product 37b as a colourless oil.

[0575] .sup.1H NMR (500 MHz, CDCl.sub.3; mixture of rotamers) 5.33 (s, 0.5H, NH), 5.32 (s, 0.5H, NH), 4.68-4.60 (m, 1H), 4.36-4.29 (m, 0.5H), 4.25 (dd, 13.6, 3.8 Hz, 0.5H), 3.65-3.56 (m, 1H), 3.56-3.46 (m, 1H), 3.23 (ap t, 13.1 Hz, 0.5H), 3.17-3.08 (m, 0.5H), 2.81 (ap t, 13.2 Hz, 0.5H), 2.59 (td, 13.3, 1.9 Hz, 0.5H), 2.24-2.11 (m, 2H), 2.06-2.00 (m, 1H), 1.93-1.75 (m, 4H), 1.75-1.54 (m, 4H), 1.54-1.13 (m, 15H; 1.44, s, 9H; 1.429, s, 4.5H; 1.425, s, 4.5H); 1.13-1.04 (m, 2H); 1.02 (s, 1.5H), 1.01-0.97 (overlapping signals: 1.00, s, 1.5H; m, 3H), 0.96-0.90 (overlapping signals: 0.94, d, 6.6 Hz, 3H; m, 3H), 0.69 (s, 3H);

[0576] HRMS (ESI) m/z calcd for C.sub.39H.sub.68N.sub.2O.sub.6Na.sup.+ 683.4970, found 683.4970.

B. N-[(2S)-2-Amino-4-methylpentanoyl]-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid hydrotrifluoroacetate (43b)

##STR00086##

[0577] Employing the same reaction conditions as for Example 11, step B, compound 37b of step A (71.1 mg, 0.108 mmol) was dissolved in dry dichloromethane (3 mL) was treated with trifluoroacetic acid (2 mL) at 0 C. for 2.5 h to yield 50.8 mg (76%) of the product 43b as a colourless foam.

[0578] .sup.1H NMR (500 MHz, CD.sub.3OD; mixture of rotamers) 4.43 (dd, 9.3, 4.0 Hz, 0.5H), 4.35 (dd, 9.1, 3.7 Hz, 0.5H), 4.28-4.22 (m, 0.5H), 4.17 (dd, 13.6, 3.8 Hz, 0.5H), 3.52-3.38 (m, 2.5H), 3.27-3.19 (m, 0.5H), 2.99 (ap t, 13.2 Hz, 0.5H), 2.77 (td, 13.3, 1.5 Hz, 0.5H), 2.31-2.18 (m, 2H), 2.10-2.02 (m, 1H), 1.99-1.93 (m, 0.5H), 1.93-1.81 (m, 3.5H), 1.80-1.55 (m, 7H), 1.55-1.36 (m, 7H), 1.35-1.16 (m, 4H), 1.16-1.07 (m, 2H), 1.06 (s, 1.5H), 1.05-0.99 (overlapping signals: 1.04, s, 1.5H; m, 6H), 0.97 (d, 6.5 Hz, 3H), 0.74 (s, 3H);

[0579] .sup.19F NMR (470 MHz, CD.sub.3OD) 76.90;

[0580] .sup.13C NMR (125 MHz, CD.sub.3OD; mixture of rotamers) 177.73, 168.82, 168.74, 71.56, 71.53, 57.29, 57.12, 56.68, 56.64, 50.45, 47.65, 45.43, 44.77, 44.41, 44.39, 44.25, 44.11, 42.21, 41.55, 41.40, 41.34, 41.02, 40.46, 40.18, 39.18, 37.06, 36.88, 36.65, 36.42, 36.00, 35.97, 35.39, 35.12, 35.02, 29.72, 27.96, 25.51, 25.45, 23.77, 23.64, 23.60, 23.58, 22.75, 22.63, 22.58, 21.83, 21.72, 19.28, 12.66;

[0581] HRMS (ESI) m/z calcd for C.sub.30H.sub.52N.sub.2O.sub.4H.sup.+ 505.4000, found 505.4011.

Example 26

N-[(2S)-2-Amino-3-(1H-indol-3-yl)propanoyl]-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid hydrotrifluoroacetate (44b)

##STR00087##

A. tert-Butyl N-{(2S)-2-[(tert-butoxycarbonyl)amino]-3-[1H-indol-3-yl]propanoyl}-3-aza-7-hydroxy-25-homo-5-cholan-25-oate (38b)

##STR00088##

[0582] Applying the same method as for Example 9, compound 27b of Example 17 (49.8 mg, 0.111 mmol) was dissolved in dry dichloromethane (2 mL) and treated with Na-Boc-L-tryptophan (51.3 mg, 0.169 mmol), 1-hydroxybenzotriazole (16.6 mg, 0.123 mmol), N,N-diisopropylethylamine (0.043 mL, 0.247 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (30.4 mg, 0.159 mmol) to give 75.5 mg (93%) of the product 38b as a colourless oil.

[0583] .sup.1H NMR (500 MHz, CDCl.sub.3; mixture of rotamers) 8.32 (s, 0.5H, NH), 8.22 (s, 0.5H, NH), 7.68-7.62 (m, 0.5H), 7.59-7.55 (m, 0.5H), 7.36-7.29 (m, 1H), 7.20-7.07 (m, 2H), 7.06-7.01 (m, 1H), 5.69 (d, 8.4 Hz, 0.5H, NH), 5.52 (d, 8.7 Hz, 0.5H, NH), 4.99-4.92 (m, 0.5H), 4.92-4.84 (m, 0.5H), 4.15-4.05 (m, 1H), 3.43-3.33 (m, 0.5H), 3.29-3.19 (m, 1H), 3.18-3.12 (m, 1H), 3.12-2.99 (overlapping signals: 3.08, dd, 14.0, 9.5 Hz, 0.5H; 3.03, dd, 13.4, 3.5 Hz, 0.5H), 2.84-2.74 (m, 1H), 2.60 (ap t, 13.2 Hz, 0.5H), 2.27-2.09 (m, 2.5H), 1.99-0.98 (m, 31H; 1.46, s, 4.5H, 1.43, s, 4.5H), 0.95 (d, 6.6 Hz, 1.5H), 0.92-0.80 (overlapping signals: 0.90, d, 6.6 Hz, 1.5H; m, 0.5H; 0.83, s, 1.5H), 0.80-0.72 (m, 0.5H), 0.64-0.59 (overlapping singlets: 0.62, 1.5H; 0.61, 3H), 0.34 (td, 13.2, 2.8 Hz, 0.5H);

B. N-[(2S)-2-Amino-3-(1H-indol-3-yl)propanoyl]-3-aza-7-hydroxy-25-homo-5-cholan-25-oic acid hydrotrifluoroacetate (44b)

##STR00089##

[0584] Deploying the same reaction conditions as for Example 11, step B compound 38b of step A (75.5 mg, 0.103 mmol) dissolved in dry dichloromethane (5 mL) was treated with trifluoroacetic acid (4 mL) to afford 47.6 mg (67%) of the product 44b as a colourless foam.

[0585] .sup.1H NMR (500 MHz, CD.sub.3OD; mixture of rotamers) 7.55-7.51 (m, 0.5H), 7.46-7.36 (m, 1.5H), 7.22 (s, 0.5H), 7.20 (s, 0.5H), 7.19-7.07 (m, 1.5H), 7.06-7.02 (m, 0.5H), 4.65-4.58 (m, 1H), 4.03-3.96 (m, 1H), 3.36-3.20 (overlapping signals: m, 2H; 3.23, dd, 13.8, 10.5 Hz, 0.5H), 3.14-3.08 (m, 0.5H), 2.94 (dd, 13.5, 3.7 Hz, 0.5H), 2.84 (td, 13.2, 1.9 Hz, 0.5H), 2.77-1.65 (m, 1H), 2.34-2.15 (m, 2.5H), 2.00-1.63 (m, 4H), 1.63-1.01 (m, 16.5H), 1.01-0.93 (overlapping signals: m, 0.5H; 1.00, d, 6.5 Hz, 1.5H), 0.92 (d, 6.6 Hz, 1.5H), 0.85 (s, 1.5H), 0.70-0.63 (overlapping signals: m, 1H; 0.66, s, 1.5H; 0.65, s, 1.5H), 0.60 (s, 1.5H), 0.52 (td, 13.7, 4.1 Hz, 0.5H);

[0586] .sup.19F NMR (470 MHz, CD.sub.3OD) 76.92;

[0587] .sup.13C NMR (125 MHz, CD.sub.3OD; mixture of rotamers) 177.78, 177.73, 169.38, 168.66,138.14, 138.04, 128.82, 128.73, 125.76, 125.50, 123.32, 123.05, 120.52, 120.44, 119.31, 119.12, 112.98, 112.90, 108.64, 108.31, 71.44, 71.08, 57.23, 56.88, 56.78, 56.61, 52.13, 51.27, 46.43, 44.73, 44.69, 44.22, 44.09, 43.97, 43.86, 43.12, 42.59, 41.32, 41.21, 40.17, 40.13, 38.68, 36.91, 36.83, 36.70, 36.61, 35.94, 35.85, 35.56, 35.42, 35.36, 35.21, 34.10, 29.74, 29.65, 29.26, 29.16, 27.87, 27.77, 23.22, 23.04, 22.79, 22.72, 22.44, 22.34, 19.33, 19.24, 12.60;

[0588] HRMS (ESI) m/z calcd for C.sub.35H.sub.51N.sub.3O.sub.4H.sup.+ 578.3952, found 578.3958.

Example 27

Conjugates of of 3-aza-7-hydroxy-5-cholan-24-oic acid (Compound 28a of Example 3) and 3-aza-7-hydroxy-25-homo-5-cholan-24-oic acid (Compound 28b of Example 18)

I. Preparation of N-(benzyloxycarbony)-3-aza-7-hydroxy-5-cholan-24-oic acid (50a)

##STR00090##

[0589] To a solution of tert-butyl N-(benzyloxycarbonyl)-3-aza-7-hydroxy-5-cholan-25-oate (Compound 25a of Example 1) (2.6 g, 4.5 mmol) in anhydrous DCM (67 mL, 26 vol) was added trifluoroacetic acid (TFA; 52 mL, 20 vol) at 0 C. After being stirred for 3.5 h at 0 C. the reaction was concentrated and the residue co-evaporated with water (10 mL) followed by THF (10 mL) and DCM (10 mL). The crude product was purified by column chromatography (heptane:ethyl acetate) to afford 1.5 g (2.9 mmol, 64%) of 19 as a colourless foam.

[0590] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.39-7.28 (m, 5H), 5.12 (s, 2H), 3.87 (s, 2H), 3.51 (s, 1H), 3.00 (s, 1H), 2.81 (s, 1H), 2.40 (ddd, J=15.4, 10.0, 5.1 Hz, 1H), 2.27 (ddd, J=15.8, 9.5, 6.5 Hz, 1H), 2.05-1.98 (m, 1H), 1.94-1.57 (m, 6H), 1.54-1.02 (m, 13H), 0.99 (s, 3H), 0.94 (d, J=6.4 Hz, 3H), 0.69 (s, 3H).

[0591] HRMS (ESI) m/z calcd for C.sub.31H.sub.45NNaO.sub.5.sup.+ 534.3195, found 534.3193.

II. Preparation of N-(benzyloxycarbonyl)-3-aza-7-hydroxy-24-homo-5-cholan-25-oic acid (50b)

##STR00091##

[0592] To a solution of tert-butyl N-(benzyloxycarbonyl)-3-aza-7-hydroxy-25-homo-5-cholan-25-oate (Compound 25b of Example 21) (930 mg, 1.6 mmol) in anhydrous DCM (25 mL, 27 vol) was added trifluoroacetic acid (TFA; 19 mL, 20 vol) at 0 C. After being stirred for 3.5 h at 0 C. the reaction was concentrated and the residue co-evaporated with water (10 mL) followed by THF (10 mL) and DCM (10 mL). The crude product was purified by column chromatography (heptane:ethyl acetate) to afford 560 mg (1.1 mmol, 69%) of 28 as a colourless foam.

[0593] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.39-7.27 (m, 5H), 5.12 (s, 2H), 3.88 (s, 2H), 3.51 (s, 1H), 3.00 (s, 1H), 2.82 (s, 1H), 2.40-2.22 (m, 2H), 2.04-1.96 (m, 1H), 1.90-1.57 (m, 6H), 1.56-1.03 (m, 15H), 0.99 (s, 3H), 0.94 (d, J=6.5 Hz, 3H), 0.68 (s, 3H).

[0594] HRMS (ESI) m/z calcd for C.sub.32H.sub.47NNaO.sub.5.sup.+ 548.3352, found 548.3346.

IIIa. General Procedure A for the Formation of Conjugates

[0595] Cbz-protected bile acid from step I (50a) or step II (50b) (1 equiv.) was dissolved in anhydrous DMF (25 vol). HATU (2.0 equiv.), DIPEA (3.0 equiv.) and the amine (1.2 equiv.) was added, and the reaction stirred at RT until complete by TLC. Upon completion, the reaction mixture was diluted with H.sub.2O, extracted with ethyl acetate (3), the organic layers were combined and washed with saturated LiCl (aq.) and concentrated. The crude residue was purified via column chromatography [C18 silica gel, gradient elution of H.sub.2O/MeOH] to yield the conjugate as either a DIPEA salt or the free acid. DIPEA salts were treated with a sodium ion exchange column to yield the desired sodium salts of the residues.

IIIb. General Procedure B for the Formation of Conjugates

[0596] Cbz-protected bile acid from step I (50a) or step II (50b) (1 equiv.) was dissolved in THF (25 vol) and cooled to 0 C. Ethyl chloroformate (1.2 eq) was added followed by triethylamine (1.2 eq) and the reaction stirred for 1 h at 0 C. After complete conversion of the starting material by TLC a solution of amine/thiol (1.5 eq) and NaHCO.sub.3 (1.5 eq) in H.sub.2O (25 vol) is added in one portion then stirred 0 C. for 2 h or until complete by TLC. Upon completion the mixture is concentrated under reduced pressure, diluted with H.sub.2O and acidified to pH<2 with 2M HCl (aq.). The suspension was extracted (ethyl acetate or THF; 3); the organic layers were combined and washed with brine, dried over magnesium sulphate and concentrated to afford a crude residue. The crude material was purified via column chromatography.

IV. General Procedure C for Deprotection of Cbz Protecting Group

[0597] To a solution of Cbz-protected conjugate of Step IIIa or Step IIIb (1 equiv.) in methanol (8 mL) was added 10% palladium on charcoal (10 mol %) and the atmosphere was exchanged for hydrogen. The resulting reaction mixture was stirred at room temperature overnight. After complete conversion of the starting material by TLC the reaction was filtered and concentrated in vacuo. The crude residue was purified by column chromatography [silica gel, gradient elution of ethyl acetate/methanol:triethylamine (9:1)] to give the conjugate as a colourless foam.

N-(3-Aza-7-hydroxy-5-cholan-24-amide)-ethylsulfonic acid (Compound 51a)

##STR00092##

[0598] Using general procedure A compound (50a) (50 mg, 0.098 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (51a) as a colourless glass (8.5 mg, 18%).

[0599] .sup.1H NMR (400 MHz, CD.sub.3OD) 3.60 (ddt, 17.2, 13.9, 7.0 Hz, 2H), 3.45-3.33 (m, 1H), 3.30-3.01 (m, 4H), 2.96 (t, 6.8 Hz, 2H), 2.27 (ddd, 14.6, 9.6, 5.4 Hz, 1H), 2.17-2.08 (m, 1H), 2.08-1.99 (m, 2H), 1.95-1.10 (m, 18H), 1.11 (s, 3H), 0.98 (d, 6.4 Hz, 3H), 0.73 (s, 3H).

[0600] HRMS (ESI) m/z calcd for C.sub.25H.sub.45N.sub.2O.sub.5S 485.3044, found 485.3053; TLC Rf 0.27.

N-(3-Aza-7-hydroxy-5-cholan-24-amide)-acetic acid (Compound 52a)

##STR00093##

[0601] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (52a) as a colourless powder (10.4 mg, 31%).

[0602] .sup.1H NMR (400 MHz, CD.sub.3OD) 3.74 (d, 2.8 Hz, 2H), 3.44-3.34 (m, 1H), 3.20-2.93 (m, 4H), 2.30 (td, 10.6, 10.0, 5.3 Hz, 1H), 2.15 (ddd, 12.5, 9.3, 5.3 Hz, 1H), 2.11-1.96 (m, 2H), 1.88-1.21 (m, 18H), 1.09 (d, 5.9 Hz, 3H), 0.98 (d, 6.5 Hz, 3H), 0.74 (s, 3H).

[0603] HRMS (ESI) m/z calcd for C.sub.25H.sub.43N.sub.2O.sub.4.sup.+ 435.3217, found 435.3223.

[0604] TLC: Rf 0.16

N-(3-Methyl-aza-7-hydroxy-5-cholan-24-oyl)-(2S)-2-amino-3-[(2-methylpropan-2-yl)oxy]propanoic acid (Compound 53a)

##STR00094##

[0605] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, during which N-methylation occurred, to yield compound (53a) as a colourless glass (15.1 mg, 37%).

[0606] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.34 (t, 3.8 Hz, 1H), 3.75 (dd, 9.1, 4.2 Hz, 1H), 3.65 (dd, 9.0, 3.5 Hz, 1H), 3.39 (dq, 8.8, 4.1, 3.4 Hz, 1H), 3.07-2.99 (m, 3H), 2.78 (t, 13.0 Hz, 1H), 2.70 (s, 3H), 2.32 (ddd, 15.0, 10.5, 5.1 Hz, 1H), 2.23-2.07 (m, 1H), 2.07-1.97 (m, 2H), 1.92-1.78 (m, 3H), 1.65-1.22 (m, 15H), 1.16 (s, 9H), 1.07 (s, 3H), 0.99 (d, 6.5 Hz, 3H), 0.73 (s, 3H).

[0607] HRMS (ESI) m/z calcd for C.sub.31H.sub.55N.sub.2O.sub.5.sup.+ 535.4105, found 535.4115; TLC: Rf 0.62.

N-(3-Methyl-aza-7-hydroxy-5-cholan-24-oyl)-(R)-3-amino-3-phenylpropanoic acid (Compound 54a)

##STR00095##

[0608] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, during which N-methylation occurred, to yield compound (54a) as a colourless glass (13.1 mg, 32%).

[0609] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.39-7.31 (m, 2H), 7.26 (dd, 8.4, 6.8 Hz, 2H), 7.21-7.14 (m, 1H), 5.27 (t, 6.8 Hz, 1H), 3.44-3.33 (m, 1H), 3.02-2.86 (m, 3H), 2.74-2.60 (m, 5H), 2.27 (ddd, 14.2, 9.4, 5.2 Hz, 1H), 2.20-2.10 (m, 1H), 2.00 (dd, 25.4, 13.7 Hz, 2H), 1.90-1.74 (m, 5H), 1.66-1.11 (m, 14H), 1.05 (s, 3H), 0.97 (dd, 6.5, 4.2 Hz, 3H), 0.68 (s, 3H).

[0610] HRMS (ESI) m/z calcd for C.sub.33H.sub.51N.sub.2O.sub.4.sup.+ 539.3843, found 539.3844; TLC: Rf 0.51.

N-(3-Methyl-aza-7-hydroxy-5-cholan-24-oyl)-1-amino-4-fluorobenzene (Compound 55a)

##STR00096##

[0611] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, during which N-methylation occurred, to yield compound (55a) as a colourless glass (4.6 mg, 13%).

[0612] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.68-7.44 (m, 2H), 7.09-6.96 (m, 2H), 3.46-3.35 (m, 1H), 2.65-2.49 (m, 2H), 2.51-2.34 (m, 2H), 2.33-2.21 (m, 4H), 2.20-2.09 (m, 1H), 2.09-1.99 (m, 1H), 1.97-1.69 (m, 6H), 1.65-1.09 (m, 13H), 1.04-0.98 (m, 6H), 0.73 (s, 3H). .sup.19F NMR (470 MHz, CD.sub.3OD) 120.83.

[0613] HRMS (ESI) m/z calcd for C.sub.30H.sub.46FN.sub.2O.sub.2.sup.+ 485.3538, found 485.3543; TLC Rf 0.62.

N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-morpholine (Compound 56a)

##STR00097##

[0614] Using general procedure A compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (56a) as an off white glass (13.8 mg, 40%).

[0615] .sup.1H NMR (400 MHz, CD.sub.3OD) 3.65 (ddd, 12.5, 5.4, 3.7 Hz, 4H), 3.60-3.50 (m, 4H), 3.43-3.33 (m, 1H), 3.25-3.07 (m, 1H), 2.92 (dt, 25.2, 14.6 Hz, 3H), 2.44 (ddd, 14.6, 10.6, 5.2 Hz, 1H), 2.31 (ddd, 14.7, 10.3, 6.0 Hz, 1H), 2.06 (dt, 12.8, 2.9 Hz, 1H), 2.01-1.69 (m, 6H), 1.65-1.54 (m, 2H), 1.54-1.18 (m, 9H), 1.18-1.09 (m, 2H), 1.07 (s, 3H), 0.99 (d, 6.5 Hz, 3H), 0.74 (s, 3H).

[0616] HRMS (ESI) m/z calcd for C.sub.27H.sub.47N.sub.2O.sub.3.sup.+ 447.3581, found 447.3589; TLC Rf 0.20.

N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-(S)-2-amino-3-hydroxypropanoic acid (Compound 57a)

##STR00098##

[0617] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (57a) as an off white powder (8.8 mg, 19%).

[0618] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.29 (t, 4.8 Hz, 1H), 3.80 (dd, 4.8, 2.0 Hz, 2H), 3.45-3.32 (m, 1H), 3.29-2.92 (m, 4H), 2.39-2.26 (m, 1H), 2.23-2.10 (m, 1H), 2.04 (dd, 18.9, 13.2 Hz, 2H), 1.83 (d, 13.0 Hz, 3H), 1.67-1.21 (m, 14H), 1.21-1.07 (m, 4H), 0.99 (d, 6.6 Hz, 3H), 0.74 (s, 3H).

[0619] HRMS (ESI) m/z calcd for C.sub.26H.sub.45N.sub.2O.sub.5.sup.+ 465.3323, found 465.3322; TLC Rf 0.17.

N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-3-amino-2-fluoropropanoic acid (Compound 58a)

##STR00099##

[0620] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (58a) as a colourless glass (11.2 mg, 24%).

[0621] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.68 (dd, 7.6, 3.3 Hz, 1H), 3.75 (dt, 25.8, 12.4 Hz, 2H), 3.59-3.35 (m, 1H), 3.30-2.89 (m, 4H), 2.27 (ddt, 14.5, 9.8, 4.9 Hz, 1H), 2.14 (ddt, 13.6, 7.1, 3.6 Hz, 1H), 2.04 (t, 14.5 Hz, 2H), 1.86 (dd, 19.5, 9.0 Hz, 4H), 1.68-1.22 (m, 13H), 1.19-1.01 (m, 4H), 0.98 (d, 6.4 Hz, 3H), 0.73 (s, 3H). .sup.19F NMR (470 MHz, CD.sub.3OD) 187.60 (d, 17.7 Hz).

[0622] HRMS (ESI) m/z calcd for C.sub.26H.sub.44FN.sub.2O.sub.4.sup.+ 467.3280, found 467.3287; TLC Rf 0.22.

N-(Cyclohexyl)-N-(3-methyl-aza-7-hydroxy-5-cholan-24-oyl)-cyclohexanamine (Compound 59a)

##STR00100##

[0623] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, during which methylation occurred, to yield compound (59a) as an off-white glass (12.7 mg, 24%).

[0624] HRMS (ESI) m/z calcd for C.sub.36H.sub.63N.sub.2O.sub.2.sup.+ 555.4884, found 555.4892; TLC Rf 0.48.

N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-4-aminobenzoic acid (Compound 60a)

##STR00101##

[0625] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (60a) as a yellow glass (14.2 mg, 37%).

[0626] .sup.1H NMR (400 MHz, CD.sub.3OD) 8.09-7.82 (m, 2H), 7.64-7.47 (m, 2H), 3.27-3.09 (m, 1H), 3.09-2.90 (m, 3H), 2.45 (ddd, 14.1, 9.6, 5.1 Hz, 1H), 2.30 (dt, 14.6, 7.7 Hz, 1H), 2.10-1.96 (m, 2H), 1.85 (d, 9.5 Hz, 1H), 1.61 (dd, 12.2, 4.9 Hz, 1H), 1.56-1.16 (m, 16H), 1.11 (t, 7.3 Hz, 1H), 1.07 (s, 3H), 1.03 (t, 6.4 Hz, 3H), 0.72 (s, 3H).

[0627] HRMS (ESI) m/z calcd for C.sub.30H.sub.45N.sub.2O.sub.4.sup.+ 497.3374, found 497.3380; TLC RF 0.12.

N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-(S)-2-amino-4-(methylthio)butanoic acid (Compound 61a)

##STR00102##

[0628] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (61a) as a colourless glass (5.2 mg, 13%); TLC Rf: 0.62.

N-(3-Aza-7-hydroxy-5-cholan-24-amide)-propanoic acid (Compound 62a)

##STR00103##

[0629] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (62a) as an off-white glass (13.8 mg, 39%).

[0630] .sup.1H NMR (400 MHz, CD.sub.3OD) 3.43-3.32 (m, 3H), 3.28-2.93 (m, 5H), 2.35 (t, 6.8 Hz, 2H), 2.23 (ddd, 13.7, 10.0, 5.3 Hz, 1H), 2.13-1.98 (m, 3H), 1.96-1.71 (m, 2H), 1.67-1.21 (m, 14H), 1.14-1.01 (m, 4H), 0.97 (d, 6.5 Hz, 3H), 0.73 (s, 3H).

[0631] HRMS (ESI) m/z calcd for C.sub.26H.sub.45N.sub.2O.sub.4.sup.+ 449.3374, found 449.3379; TLC Rf 0.16.

N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-(isopropyl-4-aminobenzoate) (Compound 63a)

##STR00104##

[0632] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (62a) as a colourless glass (3.2 mg, 8%).

[0633] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.94 (d, 8.8 Hz, 2H), 7.67 (d, 8.8 Hz, 2H), 5.19 (p, 6.3 Hz, 1H), 3.45-3.36 (m, 2H), 3.24-2.76 (m, 6H), 2.46 (ddd, 14.7, 9.9, 5.3 Hz, 1H), 2.38-2.20 (m, 2H), 2.12-1.77 (m, 6H), 1.65-1.41 (m, 4H), 1.4-1.32 (m, 6H), 1.09 (s, 3H), 1.05 (s, 3H), 1.02 (d, 6.5 Hz, 3H), 0.96 (d, 6.5 Hz, 3H), 0.73 (s, 8.0 Hz, 3H).

[0634] HRMS (ESI) m/z calcd for C.sub.33H.sub.51N.sub.2O.sub.4.sup.+ 539.3843, found 539.3853; TLC Rf 0.16.

S-(3-Aza-7-hydroxy-5-cholan-24-oyl)-phenylmethanethiol (Compound 64a)

##STR00105##

[0635] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (64a) as a colourless glass (7.0 mg, 19%).

[0636] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.48-7.18 (m, 5H), 5.10 (s, 2H), 3.89-3.75 (m, 2H), 3.41 (br, 1H), 3.10 (br, 1H), 2.91 (br, 1H), 2.36 (ddd, 15.1, 9.7, 5.3 Hz, 1H), 2.30-2.16 (m, 1H), 2.08-1.99 (m, 2H), 1.95-1.72 (m, 5H), 1.63-1.05 (m, 13H), 1.01 (s, 3H), 0.95 (d, 6.5 Hz, 3H), 0.72 (s, 3H); TLC Rf 0.78.

N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-(S)-2-amino-3-methylbutanoic acid (Compound 65a)

##STR00106##

[0637] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (65a) as an off-white powder (14.9 mg, 40%).

[0638] .sup.1H NMR (400 MHz, D.sub.2O) 4.12 (d, 5.7 Hz, 1H), 3.54 (d, 8.8 Hz, 1H), 3.41-3.12 (m, 4H), 3.07 (t, 13.4 Hz, 1H), 2.52-2.38 (m, 1H), 2.33-2.19 (m, 1H), 2.19-2.04 (m, 3H), 1.93-1.74 (m, 4H), 1.68 (dd, 11.6, 5.1 Hz, 1H), 1.63-1.16 (m, 12H), 1.12 (s, 3H), 1.01 (d, 6.5 Hz, 3H), 0.96 (dd, 13.5, 6.8 Hz, 6H), 0.74 (s, 3H).

[0639] HRMS (ESI) m/z calcd for C.sub.28H.sub.49N.sub.2O.sub.4.sup.+ 477.3687, found 477.3691; TLC Rf 0.22.

N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-(2S,3S)-2-amino-3-methylpentanoic acid (Compound 66a)

##STR00107##

[0640] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (66a) as a colourless glass (8.0 mg, 21%).

[0641] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.26 (d, 5.2 Hz, 1H), 3.42-3.35 (m, 1H), 3.25-2.93 (m, 6H), 2.38-2.25 (m, 1H), 2.23-2.21 (m, 1H), 2.11-1.97 (m, 2H), 1.91-1.78 (m, 2H), 1.71-1.22 (m, 14H), 1.21-1.06 (m, 6H), 0.99 (d, 6.5 Hz, 3H), 0.97-0.85 (m, 6H), 0.73 (s, 3H).

[0642] HRMS (ESI) m/z calcd for C.sub.29H.sub.51N.sub.2O.sub.4.sup.+ 491.3843, found 491.3853; TLC Rf 0.24.

N-Methyl-N-(3-aza-7-hydroxy-5-cholan-24-oyl)-glycine (Compound 67a)

##STR00108##

[0643] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (67a) as a colourless glass (9.9 mg, 28%).

[0644] .sup.1H NMR (400 MHz, CD.sub.3OD) 3.87 (s, 2H), 3.28-3.01 (m, 5H), 2.95 (s, 3H), 2.40-2.26 (m, 1H), 2.26-2.12 (m, 1H), 2.05 (dd, 18.1, 9.3 Hz, 4H), 1.89-1.69 (m, 1H), 1.64-1.23 (m, 15H), 1.19-1.05 (m, 4H), 0.97 (d, 6.5 Hz, 3H), 0.73 (s, 3H).

[0645] HRMS (ESI) m/z calcd for C.sub.26H.sub.45N.sub.2O.sub.4.sup.+ 449.3374, found 449.3381; TLC Rf 0.16. N-(3-Aza-7-hydroxy-5-cholan-24-oyl)-(S)-2-aminopropanoic acid (Compound 68a)

##STR00109##

[0646] Using general procedure B compound (50a) (40 mg, 0.078 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (68a) as a colourless glass (10.3 mg, 29%).

[0647] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.20 (q, 7.1 Hz, 1H), 3.45-3.33 (m, 1H), 3.27-3.17 (m, 2H), 3.17-2.84 (m, 3H), 2.28 (ddd, 13.9, 10.4, 5.3 Hz, 1H), 2.19-2.10 (m, 1H), 2.10-1.96 (m, 2H), 1.96-1.75 (m, 4H), 1.66-1.24 (m, 16H), 1.09 (s, 3H), 0.98 (d, 6.5 Hz, 3H), 0.73 (s, 3H).

[0648] HRMS (ESI) m/z calcd for C.sub.26H.sub.45N.sub.2O.sub.4.sup.+ 449.3374, found 449.3380; TLC Rf 0.18.

N-(3-Aza-7-hydroxy-25-homo-5-cholan-25-oyl)-(S)-2-aminobutanedioic acid (Compound 70b)

##STR00110##

[0649] Using general procedure B compound (50b) (40 mg, 0.076 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (70b) as an off-white powder (7.5 mg, 19%).

[0650] HRMS (ESI) m/z calcd for C.sub.28H.sub.47N.sub.2O.sub.6.sup.+ 507.3429, found 507.3436; TLC Rf 0.04.

N-(3-Aza-7-hydroxy-25-homo-5-cholan-25-oyl)-(2S)-2-amino-3-[(2-methylpropan-2-yl)oxy]propanoic acid (Compound 71b)

##STR00111##

[0651] Using general procedure B compound (50b) (40 mg, 0.076 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (71b) as an off white powder (4.6 mg, 11%).

[0652] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.35 (t, 3.9 Hz, 2H), 3.75 (dd, 9.0, 4.3 Hz, 1H), 3.65 (dd, 8.8, 3.5 Hz, 1H), 3.39-2.92 (m, 5H), 3.09-2.95 (m, 1H), 2.29-2.16 (m, 2H), 2.10-1.96 (m, 2H), 1.61-1.20 (m, 18H), 1.16 (d, 1.4 Hz, 9H), 1.08 (s, 3H), 0.97 (d, 6.1 Hz, 3H), 0.73 (s, 3H).

[0653] HRMS (ESI) m/z calcd for C.sub.31H.sub.55N.sub.2O.sub.5.sup.+ 535.4105, found 535.4096; TLC Rf 0.24.

N-(3-Aza-7-hydroxy-25-homo-5-cholan-25-amide)-ethylsulfonic acid (Compound 72b)

##STR00112##

[0654] Using general procedure B compound (50b) (40 mg, 0.076 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (72b) as a colourless glass (5.2 mg, 14%).

[0655] .sup.1H NMR (400 MHz, CD.sub.3OD) 3.59 (t, 6.8 Hz, 2H), 3.28-3.00 (m, 5H), 2.96 (t, 6.8 Hz, 2H), 2.22-1.97 (m, 4H), 1.96-1.77 (m, 2H), 1.61-1.10 (m, 18H), 1.08 (s, 3H), 0.96 (d, 6.6 Hz, 3H), 0.73 (s, 3H).

[0656] HRMS (ESI) m/z calcd for C.sub.26H.sub.47N.sub.2O.sub.5S 499.3200, found 499.3207; TLC Rf 0.25.

N-(3-Aza-7-hydroxy-25-homo-5-cholan-25-oyl)-(S)-2-amino-3-phenylpropanoic acid (Compound 73b)

##STR00113##

[0657] Using general procedure B compound (50b) (40 mg, 0.076 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (73b) as an off-white powder (18.7 mg, 46%).

[0658] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.36-7.04 (m, 5H), 4.52 (dd, 7.6, 4.9 Hz, 1H), 3.39 (dd, 10.6, 5.1 Hz, 1H), 3.27-3.14 (m, 4H), 3.11-2.93 (m, 3H), 2.19-1.95 (m, 4H), 1.89-1.74 (m, 4H), 1.68-1.13 (m, 15H), 1.08 (s, 3H), 0.92 (d, 6.5 Hz, 3H), 0.72 (s, 3H).

[0659] HRMS (ESI) m/z calcd for C.sub.33H.sub.51N.sub.2O.sub.4.sup.+ 539.3843, found 539.3849; TLC Rf 0.21.

N-(3-Aza-7-hydroxy-25-homo-5-cholan-25-oyl)-3-aminobutanoic acid (Compound 74b)

##STR00114##

[0660] Using general procedure B compound (50b) (40 mg, 0.076 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (74b) as an off-white glass (12.2 mg, 34%).

[0661] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.21 (h, 6.6 Hz, 1H), 3.44-3.34 (m, 1H), 3.29-2.89 (m, 6H), 2.38 (dd, 14.3, 5.7 Hz, 1H), 2.26 (dd, 14.3, 7.2 Hz, 1H), 2.19-1.96 (m, 3H), 1.90-1.77 (m, 1H), 1.77-1.22 (m, 18H), 1.18 (d, 6.5 Hz, 3H), 1.09 (s, 3H), 0.96 (d, 6.5 Hz, 3H), 0.73 (s, 3H).

[0662] HRMS (ESI) m/z calcd for C.sub.28H.sub.49N.sub.2O.sub.4.sup.+ 477.3687, found 477.3678; TLC Rf 0.15.

N-(3-Aza-7-hydroxy-25-homo-5-cholan-25-oyl)-(S)-2-amino-4-methylpentanoic acid (Compound 75b)

##STR00115##

[0663] Using general procedure B compound (50b) (40 mg, 0.076 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (75b) as a white powder (4.5 mg, 12%).

[0664] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.36 (dd, 9.7, 4.0 Hz, 1H), 3.27-2.91 (m, 5H), 2.28-1.94 (m, 3H), 1.89-1.76 (m, 2H), 1.74-1.20 (m, 22H), 1.08 (s, 3H), 0.99-0.90 (m, 9H), 0.73 (s, 3H).

[0665] HRMS (ESI) m/z calcd for C.sub.30H.sub.53N.sub.2O.sub.4.sup.+ 505.4000, found 505.4007; TLC Rf 0.20.

N-(3-Aza-7-hydroxy-25-homo-5-cholan-25-oyl)-(2S)-2,6-diaminohexanoic acid (Compound 76b)

##STR00116##

[0666] Using general procedure B compound (50b) (40 mg, 0.076 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (76b) as a yellow glass (12.9 mg, 33%).

[0667] .sup.1H NMR (400 MHz, CD.sub.3OD) 3.54-3.45 (m, 1H), 3.44-3.35 (m, 1H), 3.28-2.74 (m, 6H), 2.25-1.96 (m, 5H), 1.92-1.70 (m, 3H), 1.70-1.18 (m, 24H), 1.09 (s, 3H), 0.97 (d, 6.4 Hz, 3H), 0.73 (s, 3H).

[0668] HRMS (ESI) m/z calcd for C.sub.30H.sub.54N.sub.3O.sub.4.sup.+ 520.4109, found 520.4114; TLC Rf 0.07.

N-(3-Aza-7-hydroxy-25-homo-5-cholan-25-oyl)-(2S)-2-amino-3-(4-hydroxyphenyl)propanoic acid (Compound 77b)

##STR00117##

[0669] Using general procedure B compound (50b) (40 mg, 0.076 mmol) was conjugated, followed by deprotection using general procedure C, to yield compound (77b) as a yellow glass (11.0 mg, 26%).

[0670] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.03 (d, 8.5 Hz, 2H), 6.66 (d, 8.5 Hz, 2H), 4.47 (dd, 7.3, 4.9 Hz, 1H), 3.28-2.80 (m, 7H), 2.19-1.97 (m, 2H), 1.90-1.78 (m, 3H), 1.67-1.19 (m, 19H), 1.09 (s, 3H), 0.93 (d, 6.5 Hz, 3H), 0.72 (s, 3H).

[0671] HRMS (ESI) m/z calcd for C.sub.33H.sub.51N.sub.2O.sub.5.sup.+ 555.3792, found 555.3803; TLC Rf 0.14.

N-{(benzyloxycarbonyl)-3-aza-7-hydroxy-5-cholan-24-oyl}-2-oxa-6-azospiro{3,3}heptane (Compound 78a)

##STR00118##

[0672] Using general procedure B compound (50a) (33 mg, 0.06 mmol) was conjugated, to yield compound (78a) as a colourless glass (17 mg, 45%).

[0673] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.38-7.28 (m, 5H), 5.11 (br. s, 2H), 4.80 (d, J=5.8 Hz, 2H), 4.77 (d, J=5.4 Hz, 2H), 4.26 (s, 2H), 4.13 (s, 2H), 3.97-3.77 (br. m, 2H), 3.57-3.45 (br. m, 1H), 3.07-2.91 (br. m, 1H), 2.89-2.73 (br. m, 1H), 2.15-2.05 (m, 1H), 2.05-1.98 (m, 1H), 1.98-1.86 (m, 2H), 1.83-1.73 (m, 4H), 1.67-1.62 (m, 2H), 1.52-1.38 (m, 5H), 1.35-1.27 (m, 3H), 1.24-1.03 (m, 4H), 0.99 (s, 3H), 0.92 (d, J=6.5, 3H), 0.69 (s, 3H). .sup.13C NMR (100 MHz, CDCl.sub.3) 173.6, 155.4, 137.0, 128.5 (2C), 127.9, 127.8 (2C), 80.8 (2C), 71.1, 67.0, 59.8, 57.2, 56.4, 55.7, 55.0, 44.4, 43.8, 43.5, 42.7, 40.0, 39.3, 38.7, 37.6, 35.5, 34.3, 33.6, 30.7, 28.6, 28.4, 26.8, 23.4, 21.3, 18.5, 12.2.

N-{(benzyloxycarbonyl)-3-aza-7-hydroxy-5-cholan-24-oyl}-4-piperidone (Compound 79a)

##STR00119##

[0674] Using general procedure B compound (50a) (40 mg, 0.08 mmol) was conjugated, to yield compound (79a) as a colourless glass (20 mg, 43%).

[0675] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.38-7.28 (m, 5H), 5.11 (br. s, 2H), 3.96-3.79 (br. m, 2H), 3.88 (br. t, J=6.0 Hz, 2H), 3.75 (br. t, J=6.0 Hz, 2H), 3.57-3.46 (br. m, 1H), 3.08-2.92 (br. m, 1H), 2.89-2.73 (br. m, 1H), 2.49-2.42 (m, 5H), 2.31 (ddd, J=15.0, 10.5, 5.9 Hz, 1H), 2.05-1.99 (m, 1H), 1.97-1.89 (m, 1H), 1.89-1.71 (m, 3H), 1.69-1.55 (m, 2H.sup.+H.sub.2O), 1.53-1.30 (m, 8H), 1.28-1.12 (m, 4H), 1.18-1.14 (m 1H), 0.99 (s, 3H), 0.96 (d, J=6.4, 3H), 0.70 (s, 3H).

[0676] .sup.13C NMR (100 MHz, CDCl.sub.3) 206.8, 172.3, 155.4, 136.9, 128.5 (2C), 127.9, 127.8 (2C), 71.1, 67.0, 55.7, 55.1, 44.4, 44.2, 43.8, 43.5, 42.7, 41.3 (2C), 40.9, 40.0, 39.3, 38.7, 35.5, 34.3, 33.6, 31.4, 30.3, 29.7, 28.7, 26.9, 23.4, 21.3, 18.6, 12.2.

N-{(benzyloxycarbonyl)-3-aza-7-hydroxy-25-homo-5-cholan-25-oyl}-3-aminotetrahydrofuran (Compound 80b)

##STR00120##

[0677] Using general procedure B compound (50b) (35 mg, 0.07 mmol) was conjugated, to yield compound (80b) as a colourless glass (18 mg, 44%).

[0678] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.38-7.28 (m, 5H), 5.69 (d, J=7.4 Hz, 1H, NH), 5.12 (br. s, 2H), 4.53 (m, 1H), 3.96-3.75 (br. m, 5H), 3.65 (dd, J=9.4, 2.5, 1H), 3.57-3.46 (br. m, 1H), 3.08-2.92 (br. m, 1H), 2.89-2.73 (br. m, 1H), 2.32-2.21 (m, 1H), 2.19-1.98 (m, 3H), 1.92-1.58 (m, 8H), 1.56-1.33 (m, 5H.sup.+H.sub.2O), 1.33-1.17 (m, 6H), 1.16-1.02 (m, 3H), 0.99 (s, 3H), 0.94 (d, J=6.5, 3H), 0.68 (s, 3H).

N-{(3-aza-7-hydroxy-5-cholan-25-oyl}-isoindoline (Compound 81a)

##STR00121##

[0679] Using general procedure B compound (50a) (40 mg, 0.08 mmol) was conjugated followed by deprotection using general procedure C, to yield compound (81a) as a pale yellow glass (5.6 mg, 15%).

[0680] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.37-7.27 (m, 4H), 4.90 (br. s, 2H), 4.74 (br. s, 2H), 3.43-3.34 (m, 1H), 3.28-2.95 (m, 4H), 2.51 (ddd, J=15.6, 10.6, 5.4 Hz, 1H), 2.37 (ddd, J=15.6, 10.1, 6.0 Hz, 1H), 2.13-1.99 (m, 2H), 1.97-1.81 (m, 5H), 1.66-1.22 (m, 1.10, 12H), 1.22-1.14 (m, 1H), 1.10 (s, 3H), 1.04 (d, J=6.6 Hz, 3H), 0.76 (s, 3H).

[0681] .sup.13C NMR (100 MHz, CD.sub.3OD) 175.3, 137.7, 137.3, 128.9, 128.7, 124.0, 123.8, 71.1, 57.0, 56.7, 53.7, 53.2, 45.0, 44.8, 44.3, 42.2, 41.2, 40.8, 40.0, 37.0, 35.2, 34.3, 33.7, 32.4, 32.2, 29.7, 27.9, 23.5, 22.5, 19.2, 12.6.

N-{(3-aza-7-hydroxy-25-homo-5-cholan-25-oyl)}-3-aminotetrahydrofuran (Compound 82b)

##STR00122##

[0682] Using general procedure C compound (80b) (17.6 mg, 0.03 mmol) was deprotected to yield compound (82b) as a pale yellow glass (4.0 mg, 29%).

[0683] .sup.1H NMR (400 MHz, CD.sub.3OD) 4.38-4.32 (m, 1H), 3.94-3.75 (m, 3H), 3.58 (dd, J=9.1, 3.6 Hz, 1H), 3.42-3.34 (m, 1H), 3.12-2.99 (m, 1H), 2.95-2.76 (m, 3H), 2.25-2.09 (m, 3H), 2.09-2.00 (m, 2H), 1.95-1.77 (m, 4H), 1.76-1.65 (m, 2H), 1.64-1.54 (m, 2H), 1.54-1.38 (m, 7H), 1.38-1.17 (m, 5H), 1.16-1.04 (m, 1H), 1.05 (s, 3H), 0.96 (d J=6.6 Hz, 3H), 0.72 (s, 3H).

[0684] .sup.13C NMR (100 MHz, CD.sub.3OD) 176.3, 74.1, 71.5, 68.0, 57.2, 56.6, 51.5, 46.3, 44.8, 44.4, 43.7, 41.4, 41.3, 40.2, 37.2, 36.8, 35.8, 34.6, 33.3, 30.8, 29.7, 27.9, 24.0, 23.8, 23.6, 22.5, 19.3, 12.6.

Biological Example

A. Culture of Primary Fibroblasts, Generation and Culture of iNPC's

[0685] Primary fibroblast cells were cultured continuously in Glucose based medium (high glucose (4500 mg/L) Dulbecco's Modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 IU/mL penicillin, 100 g/mL streptomycin (Lonza), 1 mM sodium pyruvate (Sigma-Aldrich) and 50 g/mL uridine (Sigma-Aldrich)). Unless otherwise stated, 24 hours prior to analysis, the Glucose based medium was exchanged for Galactose based medium (glucose-free DMEM (Gibco) with the same supplementation and in addition, 5 mM galactose (Sigma-Aldrich)). All cells were assessed between passage 6-10.

[0686] Induced neural progenitor cells (iNPC's) were generated as previously described (Meyer et al, Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS Proc Natl Acad Sci USA 2014). iNPC's were maintained in DMEM/Ham F12 (Invitrogen); N.sub.2, B27 supplements (Invitrogen) and FGFb (Peprotech) in fibronectin (Millipore) coated tissue culture dishes and routinely subcultured every 2-3 days using accutase to detach them. The fibroblasts lines and iNPC lines have previously been characterised and published in Carling et al, 2020.

B. Dopaminergic Neuron Differentiation of iNPC's

[0687] Briefly, iNPCs are plated in a 6-well plate and cultured for 2 days in DMEM/F-12 medium with Glutamax supplemented with 1% NEAA, 2% B27 (Gibco) and 2.5 M of DAPT. On day 3, DAPT is removed and the medium is supplemented with 1 M smoothened agonist (SAG) and FGF8 (75 ng/ml) for additional 10 days. Neurons are replated at this stage. Subsequently SAG and FGF8 are withdrawn and replaced with BDNF (30 ng/ml), GDNF (30 ng/ml), TGF-b3 (2 mM) and dcAMP (2 mM, Sigma) for 15 days, as previously described Schwartzentruber et al, 2020 and Carling et al, 2020. Dopaminergic neurons were treated with compounds at concentrations of 0.1 nM, 1 nM, 3 nM, 10 nM, 30 nM and 100 nM dosing every 3 days for the last 12 days of differentiation.

C. Immunofluorescence Staining

[0688] Cells are plated into 96 well plates and fixed using 4% paraformaldehyde for 30 minutes. After PBS washes cells are permeabilised using 0.1% Triton X-100 for 10 minutes and blocked using 5% goat serum for 1 hour. Cells are incubated with primary antibodies III tubulin (Millipore); activated caspase 3 (Cell Signaling); at 4 C. for 16 hours. Cells are washed using PBS-Tween and incubated with Alexa Fluor-conjugated secondary antibodies 488 and 568 (Invitrogen) and Hoescht (Sigma) 1 M prior to imaging. Imaging was performed using the Opera Phenix high content imaging system (Perkin Elmer).

MMP Protocol

[0689] Fibroblasts were cultured and plated into a greiner black 384 Clear plate at a concentration of 1000 cells per well in 50 l of media volume. The plates are left overnight in an incubator to allow the fibroblasts to adhere to the plate surface. The following morning the Glucose based medium is replaced with 25 l of Galactose based medium. The plates were then dosed with the compounds using an ECHO 550 liquid handling system. The wells were dosed to provide an 8-point concentration range of 0.06 nM-300 nM of compound. After dosing the wells are topped up with a further 25 l of Galactose based medium and then left in an incubator for 24 hours. After 24 hours, the medium is removed from the wells and replaced with 25 l phenol free Minimal essential medium supplemented with 10% FBS, 1% Penicillin-streptomycin, 1% sodium pyruvate, 0.1% uridine, 1% non-essential amino acids and 1% MEM vitamins with 80 nM TMRM (Sigma) and 10 M Hoechst Stain (Sigma). The plate is returned to the incubator for another hour after which the stain medium is removed and replaced with 25 l Phenol free MEM. The plate is then imaged using an IN Cell high content microscope (GE Healthcare) with fields of view per well in 2 channels, Cy3 excitation 542 nm, emission 604-64 nm; and the DAPI excitation 350 nm, emission 450-55 nm at 37 C. with CO.sub.2. After imaging the plate is disposed of and the images are Data mined using the INCell developer Toolkit (GE Healthcare).

Full ATP Protocol

[0690] The ATP protocol is generally as described in Mortiboys et al 2008. Briefly, fibroblasts were cultured as and plated into white 384 well plates at a concentration of 5000 cells per well in 50 l of media volume. The plates are left overnight in an incubator to allow the fibroblasts to adhere to the plate surface. The following morning the Glucose based medium is replaced with 25 l of Galactose based medium. The plates were then spiked with the compounds using a ECHO 550 liquid handling system. The wells were dosed to provide an 8-point concentration range of 0.06 nM-300 nM of compound. After dosing the wells are topped up with a further 25 l of Galactose based medium and then left in an incubator for 24 hours. Following this incubation, the medium is removed from the plate and the wells are washed twice with sterile PBS. The wells are filled with 25 l of Sterile PBS followed by 12.5 l of Lysis solution from the ATPlite Luminescence ATP detection assay system (Perkin Elmer), including 16 cell free wells to use as blank controls. The plate is then placed on a rotary shaker for 5 mins at 700 rpm. Following the shaking 12.5 l of ATP substrate solution (Perkin Elmer) is added to each well and a further 5 min of shaking. The plate is then placed in darkness for 10 minutes prior to reading. Using a PHERAStar plate reader, luminescence intensity is recorded. Following the ATP assay the plates are immediately assayed for DNA content in a CyQUANT assay.

[0691] Immediately following The ATP assay DNA content is assessed with the CyQUANT NF Cell Proliferation Assay Kit (ThermoFisher). CyQUANT buffer is prepared immediately before the assay and is comprised of 1 l CyQUANT dye per ml1 HBSS solution. 12.5 l of CyQUANT buffer is added to each well. Plate left in incubator for 1 hour then read on a PHERAStar Plate reader with excitation at 497 nm and emission at 520 nm.

[0692] ATP Quantification for each well is determined using the following formula:

[00001]$\mathrm{ATP}\mathrm{score}=\frac{\mathrm{ATPlite}\mathrm{blank}\mathrm{corrected}\mathrm{value}}{\mathrm{CyQUANT}\mathrm{blank}\mathrm{corrected}\mathrm{value}}$

Data Analysis for Primary Screen Assays.

[0693] After the assays had been repeated in triplicate per line and compound the data was then inputted into Graph pad Prism 7 software suite where a dose response curve is generated using the default [Agonist] vs response(three parameters) equation.

[00002]$y=\mathrm{Bottom}+x\frac{\mathrm{Top}-\mathrm{Bottom}}{\mathrm{EC}50+x}$

[0694] From this EC.sub.50 values, lowest response and maximal response were taken and used to calculate the Geometric mean between the 5 different lines assessed.

[0695] Results derived from compounds that showed an Ambiguous result from the [Agonist] vs response (three parameters) equation were excluded from the Geometric mean calculations due to the high skew that was introduced by their inclusion.

Preliminary ATP Protocol

[0696] In some cases, a preliminary ATP protocol was used in which EC.sub.50 was not determined. The preliminary ATP protocol is generally as described in Mortiboys et al 2008. Briefly, fibroblasts were cultured and plated into white, clear-bottom 384 well plates at a concentration of 4000 cells per well in 50 l of media volume. The plates are left overnight in an incubator to allow the fibroblasts to adhere to the plate surface. The following morning the Glucose based medium is replaced with 25 l of Galactose based medium. The cells were then dosed with 100 nM and 1 M concentrations of the compounds using a ECHO 550 liquid handling system. After dosing the wells are topped up with a further 25 l of Galactose based medium and then left in an incubator for 24 hours. Following this incubation, the medium is removed from the plate and the wells are washed twice with sterile PBS. The wells are filled with 20 l of Sterile PBS followed by 10 l of Lysis solution from the ATPlite Luminescence ATP detection assay system (Perkin Elmer), including 14 cell free wells to use as blank controls. The plate is then placed on a rotary shaker for 10 mins at 700 rpm. Following the shaking 10 l of ATP substrate solution (Perkin Elmer) is added to each well and a further 5 min of shaking. The plate is then incubated in the dark for 10 minutes prior to reading. Using a FLUOstar Omega plate reader, luminescence intensity is recorded. Following the ATP assay the plates are immediately assayed for DNA content in a CyQUANT assay.

[0697] Immediately following The ATP assay DNA content is assessed with the CyQUANT NF Cell Proliferation Assay Kit (ThermoFisher). CyQUANT buffer is prepared immediately before the assay and is comprised of 2 l CyQUANT dye per ml1 HBSS solution. 10 l of CyQUANT buffer is added to each well. Cell plates were then incubated for 1 hour before being read on a FLUOStar Plate reader with excitation at 497 nm and emission at 520 nm.

[0698] The ATP Quantification for each well is determined as set out above.

Respiration Measurements

[0699] Oxygen consumption rate (OCR) was measured by the Agilent Seahorse Mito Stress test using a 24-well Agilent Seahorse XF analyzer machine (Agilent). Human fibroblasts where plated at a density of 60,000 cells per well. Cells were treated with compound 50 nM for 24 hours prior to measurement. 3

[0700] measurements of OCR were taken in each state: basal state, after addition of oligomycin (0.5 M), FCCP (0.5 M) and rotenone (1 M). A cell count was then done on a fixed assay plate using a Hoechst dye (1 M). Data presented is normalized to cell number.

Complex I Assay

[0701] Ex vivo mice brain was homogenated in a buffer of 250 mM sucrose, 20 mM HEPES, 3 mM EDTA, pH 7.5 at 4 C. Homogenisation was carried out using a Dounce homogenizer, for cortex samples, and by repetitive passage through a 0.5 mm syringe for isolated striatum. Samples were then incubated with 30 l of detergent from the AbCam colorimetric Complex I assay kit on ice for 20 minutes. Samples are then centrifuged at 13,000 rpm for 30 mins. Triplicate samples per condition were blocked using the kit blocking buffer on the AbCam colorimetric Complex I assay kit plate for 3 hours. Samples are then washed using the kit wash buffer 3 times before the addition of the kit assay buffer containing NADH and colorimetric dye. The assay plate is read on a plate reader in a kinetic assay programme reading 450 nm in a 30 second interval for 50 minutes.

D. Mitochondrial Function and Morphology Measurements in iNeurons

[0702] iNeurons were treated every 3 days for the last 12 days of the differentiation protocol. Cells are plated in 96 well plates; for live imaging cells are incubated for one hour at 37 C. with 80 nM tetramethlyrhodamine (TMRM), 1 M LysoTracker Green (Invitrogen) and Hoechst Stain solution (Sigma) at 1 M before imaging using Opera Phenix. Cellular ATP measurements are undertaken using ATPlite kit (Perkin Elmer) as per manufacturer's instructions. Mitochondrial reactive oxygen species generation was assessed using mitochondrial NpFR2 (probe; a kind gift from Dr Liz New, University of Sydney, Australia) at 20 M and Hoechst stain solution at 1 M for 30 mins at 37 C., then the dyes are removed and cells images using Opera Phenix. Images generated from the live imaging experiments were analysed using Harmony (Perkin Elmer software). We developed protocols in order to segment nucleus, cell boundary and processes, mitochondria, lysosomes, autophagosomes. We only analysed the z projection images collected from the z stacks.

Results

Fibroblasts

[0703] The mitochondrial membrane potential was measured in fibroblasts from 3 patients with sporadic Parkinson's disease (sPD) when treated with Compounds of the invention. The results are shown in Table 1, where Bottom=max response with lowest dose of compound (0.06 nM) and top=max response with highest dose of compound (300 nM).

TABLE-US-00001 TABLE 1 Mitochondrial Membrane Potential data from 3 sporadic PD patient fibroblasts MMP % of vehicle Example bottom % top % EC.sub.50 (nM) 28a 96.8 119.8 4.58 28b 92.8 100.6 4.03 40a 93.5 105.6 65.3 41b 84.6 91.13 17.6 40b 95.3 130.83 26.08 41b 94.3 123.23 2.8 42a 71.3 69.2 129.6 42b 92.2 96.6 5.1 43a 84.1 100.1 0.2 43b 86.9 102.6 1.1 44a 73.5 101.5 0.09 44b 77.8 98.8 0.11 45a 82.3 95.1 0.18 33 82.3 92.2 18.3 31b 86.5 95.1 6.1 32 92.6 121.8 5.4 NB: The sPD patients have a mean reduction in MMP compared to controls of 18%; therefore increase of MMP from the vehicle treated sPD patient level of 118% would restore MMP to control levels.

[0704] Cellular ATP levels were measured in fibroblasts from 3 patients with sporadic Parkinson's disease when treated with Compounds of the invention. The results are shown in Table 2, where Bottom=max response with lowest dose of compound (0.06 nM) and top=max response with highest dose of compound (300 nM).

TABLE-US-00002 TABLE 2 Cellular ATP levels data from 3 sporadic PD patient fibroblasts in full ATP assay ATP % of vehicle Example bottom % top % EC.sub.50 (nM) 28a 98.3 137.03 10.9 28b 94.6 138.12 32.9 40a 89.9 103.1 22.8 41b 78.9 99.3 33.7 40b 101.3 129.7 19.53 41b 76.4 120.8 3.13 42a 89.6 64.5 133.7 42b 91.3 98.5 25.4 43a 90.2 85.98 8.9 43b ~ 80.6 ~ 44a 65.1 122.4 0.23 44b ~ 98.4 ~ 45a 83.8 97.2 0.25 33 88.8 103.1 0.75 31b 91.7 160.8 0.266 32 93.2 140.2 1.6 NB: sPD fibroblasts have an average reduction of 24% of cellular ATP levels as compared to controls. Therefore a % of vehicle treated sPD fibroblasts of 124% is an increase to control ATP levels.

[0705] The MMP and ATP assays described above along with a toxicity measure comprise the primary screen of the Compounds in primary patient fibroblasts. When considering which compound is most active in the primary screens all information is taken into account including EC50 values indicating potency and % maximal responses for both assays; based upon the combined activity expert biologists take decisions for each compound.

[0706] Oxygen Consumption data from 3 sporadic PD patient fibroblast lines and 3 controls are shown in FIGS. 1A, 1B and 1C, from which it can be seen that when treated with vehicle, sPD fibroblasts show a reduction of basal mitochondrial respiration of 30%, spare respiratory capacity of 42% and ATP linked respiration of 23% compared with the control cell lines. Treatment with Compound 28b increases basal respiration in the sPD fibroblasts to control levels (* p<0.05). Increases are also seen in ATP linked respiration and spare capacity in the sPD fibroblasts although to a lesser extent. Note Compound 28b was dosed at 50 nM and therefore it is clear Compound 28b provides an increase in mitochondrial function as measured by oxygen consumption.

[0707] The above data shows the mitochondrial protective effects of the compounds in primary fibroblasts from sPD patients however the cell type which is primarily affected in PD is the dopaminergic neuron. The data below shows the results obtained from dopaminergic neurons derived from sPD patients; these cultures are approximately 96% dopaminergic neurons and currently this methodology is the only protocol to generate such a pure dopaminergic culture from patient cells (method developed by Mortiboys, University of Sheffield published previously Schwarztentruber et al, 2020, Carling et al, 2020); therefore this is the patient derived model which represents most closely the neurons affected in PD.

[0708] Table 3 below shows the results for mitochondrial function and neuronal morphology measurements in iNeurons from sPD patients vs controls when untreated or when treated with either UDCA, Compound 28a or Compound 28b.

TABLE-US-00003 TABLE 3 iNeurons % of level in iNeurons from Controls sPD sPD sPD patients patients Untreated patients treated with treated with sPD treated with Compound Compound Parameter patients UDCA 28a 28b ATP normalisation 50 82 89.6 94.3 (% of controls) ATP EC.sub.50 (nM) 5 6.5 5 MMP normalisation 52 73 71 76 (% of controls) MMP EC.sub.50 (nM) 7.3 10.3 8.2 ROS normalisation 115 110 111 109 (% of controls) Neuronal morphology 48 64 Not tested 68 (elongation; % controls) Activated caspase 3 levels 162 143 Not tested 138

[0709] The data in Table 3 show very clearly that Compound 28b provides a protective effect on both mitochondrial parameters in sPD derived dopaminergic neurons in addition to improving neuronal morphology and reducing apoptosis levels (as measured by activated caspase 3 levels). Apoptosis is a major mechanism of cell death of the dopaminergic neurons in culture and of dopaminergic neurons in patients with PD.

In Vivo Mouse Data with Compound 28b.

[0710] FIG. 2 shows that mitochondrial respiratory chain complex I activity is increased in wild type mouse whole brain homogenate after dosing with Compound 28b 2 mg/kg IV and collecting brain hemisphere samples (from 3 animals per group) at 1, 4, 8, 12 and 24 hours post injection. Complex I activity was increased 125% 1 hour after dosing, the levels remained elevated to a similar extent at 4 and 8 hours post injection. By 12 hours post injection complex I activity began to reduce again and was elevated by 98% above control. By 24 hours post injection, complex I activity was still elevated by 35% above control levels.

Preliminary Assay of Compounds of Example 27

[0711] A preliminary assay with additional compounds of the invention was carried out to determine cellular ATP levels in fibroblasts from 3 patients with sporadic Parkinson's disease. The results are shown in Table 4.

TABLE-US-00004 TABLE 4 Cellular ATP levels data from 3 sporadic PD patient fibroblasts in Preliminary ATP assay Average ATP Average ATP Response Response Compound 100 nM 1 M UDCA 104.8 98.91 51a 101.3 107.3 52a 106.4 110.3 53a 108.7 102.2 UDCA 104.8 98.91 54a 109.2 107.4 55a 109.7 109.0 56a 109.4 100.1 57a 108.6 106.7 58a 102.2 102.57 59a 106.1 99.71 60a 101.7 102.5 61a 104.7 107.9 62a 108.3 108.3 63a 106.3 103.8 64a 118.2 111.4 65a 110.7 108.4 66a 109.1 111.0 67a 108.1 107.2 68a 108.6 99.01 69a 106.8 102.48 70b 105.68 106.9 71b 105.8 103.7 72b 101.5 112.4 73b 99.61 100.4 74b 102.8 98.5 75b 112.4 105.6 76b 104.8 106.5 77b 109.2 113.2 50b 101.2 99.8 78a 120.6 120.1 79a 116.0 121.6 80b 121.9 120.4 81a 94.15 102.5 82b 96.15 97.45 NB: sPD fibroblasts have an average reduction of 18.33% of cellular ATP levels as compared to controls. Therefore a % of vehicle treated sPD fibroblasts of 118% or more is an increase to control ATP levels or higher than control ATP levels. Most compounds of the invention are more active at lower concentration ranges which may explain the drop off in activity seen at 1 M versus 100 nM.

[0712] The data in Table 4 show that some compounds, for example Compounds 52a, 54a, 55a, 57a, 62a, 64a, 65a, 66a, 67a, 75b, 77b, 78a, 79a and 80b show particularly good recovery of ATP production across both concentrations, with Compound 64a at 100 nM returning ATP production to control levels. ATP is the cellular energy component and important for many processes required for the health of these cells, therefore increasing ATP production is key for prolonging the lifespan of neurons in neurodegenerative diseases. Since, as discussed above, mitochondrial dysfunction is also thought to play a role in acute radiation syndrome, myalgic encephalomyelitis and long COVID, increasing ATP function is also likely to be beneficial for treating these conditions.

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