(12) United States Patent (10) Patent No.: US 7,727,976 B2 Bhat Et Al

US007727976B2

(12) United States Patent (10) Patent No.: US 7,727,976 B2 Bhat et al. (45) Date of Patent: Jun. 1, 2010

(54) BILE-ACID DERIVED COMPOUNDS FOR WO WOOOf 666.11 11, 2000 ENHANCING ORAL ABSORPTION AND OTHER PUBLICATIONS SYSTEMC BOAVAILABILITY OF DRUGS Chaplinet al., “Steroid series. IV. Some basic derivatives.” Journal of (75) Inventors: Laxminarayan Bhat, Santa Clara, CA the Chemical Society, pp. 3194-3202, 1959.* (US); Mark A. Gallop, Los Altos, CA Baringhaus, et al., “Substrate Specificity of the Ileal and the Hepatic (US); Bernd Jandeleit, Menlo Park, CA Na+/bile acid Cotransporters of the Rabbit. II. A Relaible 3D QSAR Pharmacophore Model for the Ileal Na+/bile acid Cotransporter'. J. (US) of Lipid Res., vol. 40, pp. 2158-2168 (1999). (73) Assignee: XenoPort, Inc., Santa Clara, CA (US) Begum, et al., “Synthesis of 2B-Hydroxyursolic Acid and Other Ursane Analogues from UrSonic Acid', Aust. J. Chem, vol. 46, pp. (*) Notice: Subject to any disclaimer, the term of this 1067-1071 (1993). patent is extended or adjusted under 35 Bellini, et al., “Studi Su 5beta-chetosteroidi—Nota VII. Derivati U.S.C. 154(b) by 1130 days. pirimidinici e pteridinici dell'acido 3.7.12-trichetocolanico', Il Farmaco—Ed. Sc., vol. 25, fax. 3, pp. 226-233, (1970). (21) Appl. No.: 11/234,310 Bellini, et al., “Studi Su 5f-cheto-steroidi. Nota IV. Derivati (22) Filed: Sep. 26, 2005 eterociclici Sugi anelli A e B dell'acido 3.7.12-tricheto-colanico”. Gazetta Chimica Italiana, vol. 99, fasc. XII, pp. 1243-1251 (1969). (65) Prior Publication Data Brouwer, et al., “An Efficient Synthesis of N-Protected B-Aminoethansulfonyl Chlorides'. Synthesis, No. 11, pp. 1579-1584 US 2006/OO30551A1 Feb. 9, 2006 (2000). Related U.S. Application Data Clinton, et al., “Steroidal 3.2-cpyrazoles. II. Androstanes, 19-Norandrostanes and their Unsaturated Analogs”. J. of American (62) Division of application No. 10/229.565, filed on Aug. Chem. Soc., vol. 83, pp. 1478-1491 (1961). 28, 2002, now Pat. No. 7,053,076. Ho, et al., “Utilizing Bile Acid Carrier Mechanisms to Enahnce Liver and Small Intestine Absorption'. Ann. New York Acad. Sci., pp. (60) Provisional application No. 60/316,182, filed on Aug. 315-329 (1987). 29, 2001. Junjappa, H., et al., “O-Oxoketene-SS-, N.S- And N,N-Acetals: Ver satile Intermediates in Organic Synthesis”, Tetrahedron Report, No. (51) Int. Cl. 278, pp. 5423-5506 (1990). A6 IK3I/58 (2006.01) Kim, et al., “Evaluation of Bile Acid Transporter in Enhancing Intes CO7I 71/00 (2006.01) tinal Permeaility of Renin-Inhibitory Peptides'. J. Drug TargetingI. vol. 1, pp. 347-359 (1993). (52) U.S. Cl...... 514/176; 514/172:540/49; Kramer, et al., “Substrate Specificity of the Ileal and the Hepatic 540/50:540/51; 540/52 Na + Bile Acid Cotransporters of the Rabbit. I. Transport Studies (58) Field of Classification Search ...... 540/49, with Membrane Vesicles and Cell Lines Expressing the Cloned 540/50, 51, 52: 514/172, 176 Transporters”. J. of Lipid Res., vol. 40, pp. 1604-1617 (1999). See application file for complete search history. Kramer, et al., “Liver-Specific Drug Targeting by Coupling to Bile Acids”. J. Biol. chem., vol. 267, pp. 18598-18604 (1992). (56) References Cited Kramer, et al., “Intestinal Absporption of Peptides by Coupling to Bile Acids”. J. of Biol. Chem., vol. 269, No. 14, pp. 10621-10627 U.S. PATENT DOCUMENTS (1994). Kramer, et al., “Bile Acid Derived HMG-CoA Reductase Inhibitors', 4,848,349 A 7, 1989 Sherman et al. Biochim. Biophys. Acta, vol. 1227, pp. 137-154 (1994). 4.866,044 A 9, 1989 Sato et al. Kullak-Ublick, et al., “Hepatobiliary Transport”. J. Hepatology, vol. 5,462.933 A 10, 1995 Kramer et al. 32, pp. 3-18 (2000). 5,646,272 A 7, 1997 Kramer et al. Navia, et al., “Design Principles for Orally Bioavailable Drugs'. 5,668,126 A 9, 1997 Kramer et al. Drug Discovery Today, vol. 1, pp. 179-189 (1996). Paquette, L.A., et al., “2-Chloro-1-Formyl-1-Cyclohexene'. Organic FOREIGN PATENT DOCUMENTS Syntheses, vol. 5, pp. 215-232. EP O 285 285 10, 1988 * cited by examiner EP O 624593 11, 1994 EP O 676 410 10, 1995 Primary Examiner Barbara P Badio EP O 702 026 6, 1997 (74) Attorney, Agent, or Firm D. Byron Miller; Lucy S. JP 10-286453 10, 1998 Chang JP 11-605.94 3, 1999 WO WO90, 13298 11, 1990 (57) ABSTRACT WO WO94/OO 126 1, 1994 WO WO95/03056 2, 1995 Disclosed are compounds that exhibit high transport across WO WO97, 18816 5, 1997 the intestinal wall of an animal. The compounds may option WO WO97/.44043 11, 1997 ally be linked to drugs that are poorly absorbed or poorly WO WO98/O1159 1, 1998 transported across the intestinal wall after oral administration WO WO 99/15179 4f1999 to provide for enhanced therapeutic, and optionally pro WO WO99/52932 10, 1999 longed therapeutic, systemic blood concentrations of the WO WOOO/20437 4/2000 drugs upon oral administration of the drug-compound conju WO WOOO34461 6, 2000 gate. Also disclosed are pharmaceutical compositions con WO WOOO,38738 T 2000 taining and methods of using Such compounds. WO WOOOf 40965 T 2000 WO WOOOf 57915 10, 2000 30 Claims, 39 Drawing Sheets

U.S. Patent Jun. 1, 2010 Sheet 2 of 39 US 7.727,976 B2

Scheme 4. Preparation of 2-hydroxymethylene-3-oxosteroidal building blocks 4

toluene, rt, 12-24 h

Scheme 5. Preparation of 2-dimethylaminomethylene-3-oxo steroidal building blocks 5

1. O R2 - OR CC) Bredereck's reagent G Human-asTHFor DME, reflux

Free OH group(s) protected with appropriate protecting group (e.g. Ac, TMS, TBDMS) in 1 Scheme 6. Preparation of oxoketene S,S-acetal derivatives of bile acids 6

1. Base/CS/toluene, rt Mes1N

2. Mei?toluene, rt H 1 Base: EtONa or BuONa/Toluene or LDAVTHF 6 1, 6 a; R = R2 = H; b; R = R2 = a-OH;c; R = a-OH, R2 = H, d; R = H, R2 = o-OH; e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Me, Et or other alkyl groups, Bu, CHPh Free OH group(s) protected with appropriate protecting group (e.g. TES, TBDMS) in 1

FIG. B.

U.S. Patent Jun. 1, 2010 Sheet 4 of 39 US 7.727,976 B2

Scheme 9: Preparation of 3-oxo-2-(B-oxomethyl)substituted bile acid building blocks 9 (1,4-dicarbonyl steroidal building blocks)

1. O R5

or20 5 xiida -la-la-ars THF H 9 1, 9 a; R = R2 = H;b; R = R2 = a-OH; c; R = a-OH, R2 = H; d; R = H, R2 = a-OH; e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Me, Et or other alkyl groups, Bu, CHPh 9, 20 R5 = H, alkyl, ary and alkary substituents with or without functional groups RS = H, alkyl, aryland alkaryi substituents with or without functional groups; Alkoxide, aryloxide and alkaryoxide substituents with or without functional groups; Thioalkoxide, thioaryioxide and thioalkaryloxide substituents with or without functional groups; 10 and 2° amines 20, X = Cl, Br, l, OTs, OMs, OTf Free OH group(s) protected with appropriate protecting group (e.g. TES, TBDMS) in 1

Schene 10: Preparation of 2-propargy derivatives of bile acids 10

1, 10a; R = R2 = H;b; R = R2 = a-OH;c; R = a-OH, R2 = H; d; R = H, R2 = o-OH; e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Me, Etor other alkyl groups, Bu, CHPh 10, 21, R = H, alkyl, ary or alkary substituents with or without functional groups R = ether, amine, ester, amide functional groups; H, alkyi, ary or alkary substituents with or without functional groups 21, X = Cl, Br, I, OTs, OMs, OTf Free OH group(s) protected with appropriate protecting group (e.g. TES, TBDMS) in 1

FIG. 1D U.S. Patent Jun. 1, 2010 Sheet 5 of 39 US 7,727,976 B2

Schene 11: Preparation of vinyl ether or ester derivatives of bile acids 11

1. O OR Base/RX, 22 a-male Solvent O R1 RO H 1 11 1, 11a; R = R2 = H;b; R = R2 = a-OH; c.; R1 = a-OH, R2 = H; d; R = H, R2 = a-OH: e; R1 = B-OH, R2 = H; f; R1 = B-OH, R2 = c-OH R = H, Me, Et or other alkyl groups, Bu, CHPh 11, 22, R = TMS, TES, TBDMS, alkyl, aryl, acetyl, tosyl, mesyi, trify groups 22, X = Cl, Br, I, OTs, OMs, OTf Free OH group(s) protected with appropriate protecting group (e.g. Ac, TES, TBDMS) in 1 For R9 = Ac: Reaction conditions-isopropeny acetatelcat. ptSA/toluene, reflux Scheme 12: Preparation of 2-methylene-3-oxo derivatives (enones) of bile acids 12

1, 12 a; R = R2 = H; b; R = R2 = a-OH; c; R = a-OH, R2 = H; d; R = H, R2 = c-OH; e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Me, Et or other alkyl groups, Bu, CHPh 12, 23, R, R = H, alkyi, aryi, alkary substituents with or without functional groups Free OH group(s) protected with appropriate protecting group (e.g. TMS, TES, TBDMS) in 1

F.G. 1E U.S. Patent Jun. 1, 2010 Sheet 6 of 39 US 7,727,976 B2

Preparation of Amino Acid Building Blocks

a-Amino acid derivatives 13 and taurine 14 are commercially available. The 2 aminoethylthioesters 15 are not commercially available and they can be easily prepared from the corresponding amino acids by the reported protocol in overall good yields. R13 ? R13 OR' - R14 OR" HirO HN1N1iO OH 2 n-s-MeO N1) O 13 14 15 30 R = H, Me, Et or other alkyl groups, 'Bu, CHPh R = H, alkyl, aryl, alkary substituents with or without functional groups R = alkyl, ary, alkary substituents with or without functional groups Scheme 13. Preparation of 2-aminoethylthioacetate 15 R13 * SE R13 MSC/TEA R13 a-ul-Ha-e- ---OH Hrams 1N-OMs PGHN OH 2. NaBH Ho PGHN DCM, OpC PGHN 24 O 25 26 CSCOJHSC(O)Ne DMF, it

R13 Piperidine/DCMperidin R13 His- S Me t PGHN S Me 1s (For PG = Fmoc) 27 PG = Fmoc, Cbz, Boc R = H, alkyl, aryl, alkary Substituents with or without functional groups

Scheme 14. Preparation of N-substituted a-amino acid esters 30

OR TEAWDCM y R14-NH, + x1 --- R14N N OR 28 29 30 R = Me, Et or other alkyl groups, 'Bu, CHPh R = alky, aryl, alkary substituents with or without functional groups X = Cl, Br, I, OTs, OMs, OTf

FIG. 2 U.S. Patent Jun. 1, 2010 Sheet 7 of 39 US 7.727,976 B2

Synthetic strategy for 5-membered 2,3-fused heterocycles

General structure:

Template IVA

a, R = R2 = H; b, R = R2 = a-OH; c, R = c-OH, R2 = H; d, R = H, R2 = a-OH; e, R = B-OH, R2 = H; f, R = B-OH, R2 = a-OH R = H, Me, Et or other alkyl groups, Bu, CHPh, CPh F = Fused aromatic and non-aromatic carbocycles and heterocycles with one or more hetero atoms S, S = H, alkyl, ary and alkary substituents with or without functional groups FG= Functional Groups (e.g. OH, SH, NH, NHR, COH, COR, CONH CONHR,

5-Membered core structures:

Furans Pyrroles Indoles Dihydropyrroles Pyrazoles Dihydropyrazoles Isoxazoles Triazoles

FIG. 3A U.S. Patent Jun. 1, 2010 Sheet 8 of 39 US 7.727,976 B2

Scheme 15: Synthetic strategy for 2,3-fused furan derivatives of bile acids 31

OR fert-BuOK or NaH amal DMF

10 10, 31 a; R = R2 = H;b; R = R2 = a-OH; c; R = a-OH, R2 = H; d; R = H, R2 = a-OH; e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Me, Et or other alkyl groups, Bu, CHPh 10, R = H, alkyl, ary or alkary substituents with or without functional groups R = ether, amine, ester, amide functional groups; H, alkyl, ary or alkary substituents with or without functional groups Free OH group(s) protected with appropriate protecting group (e.g. TES, TBDMS) in 10 Scheme 16: Synthetic strategy for 2,3-fused pyrrole derivatives of bile acids 33

1. O

OR HNR15lcat. ptSA 32 R8 u-mas O R toluene, heat 10 10, 33a; R = R2 = H, b; R = R2 = a-OH; c.; R = a-OH, R2 = H, d; R = H, R2 = a-OH; e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Bu, CPh 10, 32, 33 R = H, alkyl, ary or alkary substituents with or without functional groups R = ether, amine, ester, amide functional groups; H, alkyl, ary or alkary substituents with or without functional groups R5 = H, alkyl or ary substituents with or without functional groups Free OH group(s) protected with appropriate protecting group (e.g. TES, TBDMS) in 10

FIG. 3B U.S. Patent Jun. 1, 2010 Sheet 9 Of 39 US 7.727,976 B2

Scheme 17: Synthetic strategy for 2,3-fused pyrrole derivatives of bile acids 34

HNR15lcat.pTSA 32 -aa-areases O H R1 toluene, heat

9,34 a; R = R2 = H;b; R = R2 = a-OH; c; R = a-OH, R2 = H, d; R = H, R2 = a-OH; e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Bu, CPh 32, 34 R5 = H, alkyl, ary and alkary substituents with or without functional groups R15 = H, alkyl, ary, alkary substituents with or without functional groups 9 R = H, alkyl, aryland alkary substituents with or without functional groups; Alkoxide, aryloxide and alkaryioxide substituents with or without functional groups; Thioalkoxide, thioaryioxide and thioalkaryloxide substituents with or without functional groups; 10 and 20 amines 34 RB = OH; H, alkyl, aryl, alkary substituents with or without functional groups Free OH group(s) may be protected with appropriate protecting group (e.g. TES, TBDMS) in 9

Scheme 18: Synthetic strategy for 2,3-fused pyrrole derivatives of bile acids 35.

% O v 1RHNnoR/DMF OR 30 O 2) NaH or LDAVTHF ROC

3,35a; R = R2 = H;b; R = R2 = a-OH; c; R = a-OH, R2 = H; d; R = H, R2 = a-OH; e; R1 = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R, R = H, Me, Et or other alkyl groups, Bu, CHPh 30, 35 R = H, alkyl, aryl, alkary substitutions with or without functional groups

FIG. 3C

U.S. Patent Jun. 1, 2010 Sheet 11 of 39 US 7,727,976 B2

Scheme 21: Synthetic strategy for 2,3-fused pyrrole derivatives of bile acids 40.

12, 39, 40 a; R = R2 = H; b; R = R2 = a-OH; c; R1 = a-OH, R2 = H; d; R = H, R2 = o-OH; e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R, R = H, Me, Et or other alkyl groups, Bu, CHPh 12, 30, 39, 40 R, R, R = H, alkyl, ary, alkary substitutions with or without functional groups

FIG. 3E U.S. Patent Jun. 1, 2010 Sheet 12 of 39 US 7.727,976 B2

Scheme 22: Synthetic strategy for 2,3-fused indole derivatives of bile acids 44.

e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Bu, CPh. 42, 44, R, R = H, alkyl, aryl, alkary substituents with or without functional groups, OH, alkoxy, amino, acy, COR where R= H, alkyi, ary, alkary substituents with or without functional groups 43, 44 R = H, alkyl, ary, alkary substituents with or without functional groups, 43 X = Cl, Br, l, OTs, OMs, OTf

FIG. 3F U.S. Patent Jun. 1, 2010 Sheet 13 of 39 US 7.727,976 B2

Scheme 23: Synthetic strategy for 2,3-fused 2(1)-substituted pyrazole derivatives of bile acids 46 and 47

HNHNR18

HNHNR19 45 TEA or toluene R19-N,

3, 46,47a; R = R2 = H;b; R = R2 = a-OH; c; R = a-OH, R2 = H; d; R1 = H, R2 = c-OH; e; R = B-OH, R2 = H; f; R1 = B-OH, R2 = a-OH R = H, Bu, CPh 46 R = ary or electron withdrawing substituents with or without functional groups 47 R = H, alky substituents with or without functional groups

FIG. 3G U.S. Patent Jun. 1, 2010 Sheet 14 of 39 US 7.727,976 B2

Scheme 24: Synthetic strategy for 2,3-fused 2(1)-substituted pyrazole derivatives of bile acids 48 and 49. HNHNR18 45 ROH or toluene

HNHNR19 45 ROH or toluene R19 H 49 4, 5, 48, 49 a; R = R2 = H; b; R = R2 = a-OH;c; R = a-OH, R2 = H; d; R1 = H, R2 = c-OH: e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Me, Et or other alkyl groups, Bu, CHPh, CPh. 48 R = H, alkyl substituents with or without functional groups 49 R = ary or electron withdrawing substituents with or without functional groups

FIG. 3H U.S. Patent Jun. 1, 2010 Sheet 15 of 39 US 7.727,976 B2

Scheme 25: Synthetic strategy for 2,3-fused 3'-aminosubstituted pyrazole derivatives of bile acids 5.

OR HNHNR19 MeS N i 45 O R1 ROH or DMF H 6, X = SMe 6-8 1. In-CPBAVHF

HNHNR19 2. HNR3R/DMF 2 8, X FNR'R' ROH or DMF

6-8, 50, 51a; R = R2 = H; b; R = R2 = a-OH; c; R = c-OH, R2 = H; d; R = H, R2 = a-OH; e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Me, Et or other alkyl groups, 'Bu, CHPh, CPh R, R = H, alkyi, ary, alkary substituents with or without functional groups R = H, alkyl substituents with or without functional groups

FIG. 3

U.S. Patent Jun. 1, 2010 Sheet 19 of 39 US 7.727,976 B2

Synthetic strategy for 6-membered 2,3-fused heterocycles

General Structure:

Template IVB

a, R e R1 R = H, Me, Et or other alkyl groups, Bu, CHPh, CPh. F = Fused aromatic and non-aromatic 6-membered carbocycles and heterocycles with one of more hetero atoms S, S = H, alkyl, ary and alkary substituents with or without functional groups FG = Functional Groups (e.g. OH, SH, NH, NHR, COH, COR, CONH CONHR,

6-Membered core structures: Six membered aromatic and non-aromatic rings Pyran and dihydropyran Pyridine

FIG. 4A U.S. Patent Jun. 1, 2010 Sheet 20 of 39 US 7.727,976 B2

Scheme 31: Synthetic strategy for 2,3-fused carbocyclic derivatives of bile acids 61

R2O R212

OR R22 n 59 R23 toluene, heat

M O

1. A.

11, 60, 61 a R = R2 = H; b; R = R2 = a-OH; c; R = a-OH, R2 = H; d; R = H, R2 = a-OH; e; R = B-OH, R2 = H; f; R1 = B-OH, R2 = a-OH R = H, Me, Et or other alkyl groups, Bu, CHPh 11, 60; R = TMS, TES, TBDMS, alkyl, ary, acetyl, tosy, mesyi, trify groups 59-61: R2-R = H, alkyl, aryl and alkary substituents with or without functional groups (functional groups e.g. OH, SH, OR, NH, NR, COH, COR, CONH CONHR, etc)

FIG. 4B

U.S. Patent Jun. 1, 2010 Sheet 25 of 39 US 7.727,976 B2

Scheme 37: Synthetic strategy for 2,3-fused 3'-substituted pyrimidine derivatives of bile acids 90-93

86-89 R11 = | DDO/toluene

12, 86-93 a; R = R2 = H; b; R = R2 = a-OH; c; R = a-OH, R2 = H; d; R1 = H, R2 = c-OH; e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Me, Et or other alkyl groups, 'Bu, CHPh R, R = H, alkyl, ary and alkary substituents with or without functional groups 70, 86, 90: X = OH; 71, 87,91: X = SH; 72, 88,92: X = NH 73, 89,93: X = R32 = H, alky, ary and alkary substituents with or without functional groups

FIG. 4G U.S. Patent Jun. 1, 2010 Sheet 26 of 39 US 7.727,976 B2

Synthetic strategy for 7-and 8-membered 2,3-fused heterocycles

General Structures:

Template IVC Template IVD

a, R = R2 = H; b, R = R2 = a-OH; c, R = a-OH, R2 = H; d, R = H, R2 = a-OH; R = H, Me, Et or other alkyl groups, 'Bu, CHPh, CPh F = Fused aromatic and non-aromatic 7- and 8-membered carbocycles and heterocycles with one or more hetero atoms S, S = H, alkyl, ary and alkaryi substituents with or without functional groups FG = Functional Groups (e.g. OH, SH, NH, NHR, COH, COR, CONH CONHR,

FIG. SA U.S. Patent Jun. 1, 2010 Sheet 27 Of 39 US 7.727,976 B2

Scheme 38: Synthetic strategy for 2,3-fused diazepine derivatives of bile acids 95

%. O Rss N H R1D. NH, s. DMF Rs

94, 95: R33, R3 = H, alkyl, aryl, alkary substituents with functional groups (functional groups e.g. OH, OR, COR etc); Also R3 and R' may be linked in a ring

Scheme 39: Synthetic strategy for 2,3-fused diazepine derivatives of bile acids 96

12 O e Ras NH, OR CE 94 R. NH, EtOH, reflux.

4, L = OH; 5, L = NMe

94, 96: R38, R* = H, alkyl, aryl, alkary substituents with functional groups (functional groups e.g. OH, OR, COR etc); Also R33 and R' may be linked in a ring Diazepines prepared from 4a NH 96a-1, R = Et and Me (-9:1) N R = R2 = H R4 1 R3 = OH; R4 = H N 96a-2, R = Et; R = R2 = H R3 R = H; R = CoMe

FIG.SB U.S. Patent Jun. 1, 2010 Sheet 28 of 39 US 7.727,976 B2

Scheme 40: Synthetic strategy for 2,3-fused diazepine derivatives of bile acids 97

12, 97a; R = R2 = H;b; R = R2 = a-OH; c; R = a-OH, R2 = H; d; R = H, R2 = a-OH; e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Bu, CPh. R, R = H, alkyl, ary, alkary substituents with or without functional groups 94, 97: R, R = H, alkyl, aryl, alkary substituents with functional groups (functional groups e.g. OH, OR, COR etc); Also R and R' may be linked in a ring

Scheme 41: Synthetic strategy for 2,3-fused diazepine derivatives of bile acids 99

DMF, heat 6, L = SMe

1. Oxone or n-CPBA 2. HNR3R/DMF

7, L = HNR3 8, L = NR3R4 941 DMF, heat

6-8, 98, 99 a; R = R2 = H; b; R = R2 = a-OH; c; R = a-OH, R2 = H; d; R = H, R2 = a-OH; e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Bu, CPh R, R = H, alkyl, aryl, alkary substituents with or without functional groups 98, 99: R, R = H, alkyl, ary, alkary substituents with functional groups (functional groups e.g. OH, OR, COR etc); Also R33 and R3 may be linked in a ring

FIG. 5C

U.S. Patent Jun. 1, 2010 Sheet 30 of 39 US 7.727,976 B2

Scheme 44: Synthetic strategy for 2,3-fused diazepine derivatives of bile acids 103

R, R = H, alkyl, ary, alkary substituents with or without functional groups 100, 103: R = H, O, OH, alkyi, aryl, alkary alkoxy, aryloxy, alkaryloxy substituents with or without functional groups

Scheme 45: Synthetic strategy for 2,3-fused diazepine derivatives of bile acids 105

35 100 NH DMF, heat 6, L = SMe R 104. 1. Oxone of n-CPBA 2. HNR3R4/DMF

4. S. 100/ DMF, heat

6-8, 104,105 a; R = R2 = H; b; R = R2 = a-OH; c; R = a-OH, R2 = H; d; R1 = H, R2 = c-OH: e; R = B-OH, R2 = H; f; R = B-OH, R2 = a-OH R = H, Bu, CPh, R, R = H, alkyl, ary, alkary substituents with or without functional groups 100,104, 105; R = H, O,OH, alkyi, aryl, alkaryl alkoxy, aryloxy, alkaryloxy substituents with or without functional groups

FIG. 5E U.S. Patent Jun. 1, 2010 Sheet 31 of 39 US 7.727,976 B2

Synthesis of 2,3-fused carbocyclic and heterocyclic derivatives of glyco- and taurocholanoic acids

General Structure: Template II ? O

a-Amino acid or Taurine derivatives

109-111, 113-114 a, R = R2 = H; b, R = R2 = a-OH; c, R = a-OH, R2 = H; d, R = H, R2 = a-OH; e, R1 = B-OH, R2 = H; f, R1 = B-OH, R2 = a-OH R = H, Me, Et or other alkyl groups, 'Bu, CHPh, CPh F = Fused aromatic and non-aromatic carbocycles and heterocycles with one or more hetero atoms S, SF H, alkyl, ary and alkary substituents with or without functional groups FG' = Functional Groups (e.g. OH, SH, NH, NHR, COH, COR, CONH

FIG. 6A U.S. Patent Jun. 1, 2010 Sheet 32 of 39 US 7.727,976 B2

Scheme 46: Synthesis of 2,3-fused carbocyclic and heterocyclic derivatives of glycocholanoic acids having general formula 109

1. O 24 OR

NaOH | HO FG'-s; R1 -la-Ho-- FG-S'- H 106 coupling agent 1 base OR" THF or DCM HN

Hydrolysis

a, R = R2 = H; b, R = R2 = c-OH; c, R1 = c-OH, R2 = H; d, R = H, R2 = a-OH; e, R = B-OH, R2 = H; f, R1 = B-OH, R2 = a-OH R, R = H, Me, Etor other alkyl groups, Bu, CHPh, CPh R2 = H, alkyl, ary, alkary substituents with or with out functional groups F = Fused aromatic and non-aromatic carbocycles and heterocycles with one or more hetero atoms S, S2 = H, alkyl, ary and alkary substituents with or without functional groups FG' = Functional Groups (e.g. OH, SH, NH, NHR, COH, COR, CONH CONHR,

Deprotection conditions; R = Me, Et, other simple alkyl- NaOH, H.O, THF; R = CHPh:- Pd/C, H, EtOAC; R = Bu, CPhi-TFA or HCl, dioxane Coupling conditions: DCC, DIC, EDAC, EDC, ethyl chloroformate and base like TEA, DIEA, Pyridine Hydrolysis conditions: TFA or HCl, dioxane (R = 'Bu); NaOH, HO, THF (R = alkyl)

FIG. 6B U.S. Patent Jun. 1, 2010 Sheet 33 of 39 US 7.727,976 B2

Scheme 47: Synthesis of 2,3-fused carbocyclic and heterocyclic derivatives of taurocholanoic acid having the general structure 110.

1. CIC(O)CEt 1 TBA 1 Dioxane O 2. aq.NaOH HN1\1i 2 O 14

110 (X = H) and 111 (X = Na) a, R = R2 = H; b, R = R2 = a-OH; c, R1 = a-OH, R2 = H; d, R = H, R2 = a-OH; F = Fused aromatic and non-aromatic carbocycles and heterocycles with one or ?nore heterO atoms S, S = H, alkyl, ary and alkary substituents with or without functional groups FG' = Functional Groups (e.g. OH, SH, NH, NHR, COH, COR, CONH CONHR,

FIG. 6C U.S. Patent Jun. 1, 2010 Sheet 34 of 39 US 7.727,976 B2

Scheme 48: Synthesis of 2,3-fused carbocyclic and heterocyclic derivatives of taurocholanoic acids having the general structure 113.

O R13 24 N H S Me 30%O HO/HOAC FG-S; O H R1 112 1.30% HO/HOAC 2. NaOAC

R13 O S-ONaf O

114

a, R = R2 = H; b, R = R2 = a-OH; c, R = ot-OH, R2 = H; d, R = H, R2 = c-OH: e, R = B-OH, R2 = H; f, R1 = B-OH, R2 = a-OH R, R = H, Me, Etor other alkyl groups, Bu, CHPh, CPh R = H, alkyl, aryl, alkaryl substituents with or with out functional groups F = Fused aromatic and non-aromatic cartbocycles and heterocycles with one or more hetero atoms S’, S2= H, alkyl, aryland alkary substituents with or without functional groups FG' = Functional Groups (e.g. OH, SH, NH, NHR, COH, COR, CONH CONHR, C(=S)OR, C(=O)SR) Coupling conditions: DCC, DIC, EDAC, EDC, ethyl chloroformate and base like TEA, DEA, Pyridine

FIG. 6D U.S. Patent Jun. 1, 2010 Sheet 35 of 39 US 7.727,976 B2

Conjugation of drug molecules or surrogates to carbocyclic and heterocyclic derivatives of glyco- and taurocholanoic acids General structure: Template Ill 2. O 24N o-Amino acid of Taurine

H derivatives

Drug candidates -S Or surrogates 2 R1

a, R = R2 = H; b, R = R2 = a-OH; c, R = a-OH, R2 = H; d, R = H, R2 = c-OH: e, R = 3-OH, R2 = H; f, R = B-OH, R2 = a-OH F = Fused aromatic and non-aromatic carbocycles and heterocycles with one or more hetero atoms S’, S2= H, alkyl, ary and alkary substituents with or without functional groups X = Bond that connects drug candidates or surrogates to fused ring

R12 or = ro COR' O 106-1: FG' = OH 106-2: FG's NHR 106-3: FG' = COH

us- S FG SC(O)Ne O 112-1: FG' = OH 112-2: FG' = NHR 112-3: FG' = COH

BSE-FG = FG"-DIs) 116-2:15: ESCOFG = NHR 117.3; FG= OH a, R1 = R2 = H; b, R1 = R2 = o-OH; C, R1 = a-OH, R2s H; d, R1 = H, R2 = o-OH: e, R = B-OH, R2 = H; f, R = B-OH, R2 = a-OH R = H, Me, Etor other alkyl groups, Bu, CHPh, CPh, R, R, R = H, alkyi, aryi, alkary substituents with or with out functional groups F = Fused aromatic and non-aromatic carbocycles and heterocycles with one or Thore hetero atons S, S = H, alkyl, ary and alkary substituents with or without functional groups FG' and FG' = OH, COH, NHR

FIG. 7A U.S. Patent Jun. 1, 2010 Sheet 36 of 39 US 7,727,976 B2

Scheme 49: Conjugation of drug molecules to XenoPorter"106-1 (RF Bu) with a hydroxy functional group on the fused ring 1) Dis-COH 115 oxalyl chloride/DMF/DMAPIDCM O or CBC/TEAWDMAP/DC M O-xenoporter-co.H 2) TFA or HC/Dioxane 118

1) Dis-NHR 116 O I CD THF (Osiyo-error-co 2) TFA or HCJ Dioxane R 119 106-1 1) Dis-OH 117 O I CD (NaHITHF (Oslo'o-gapor-co. 2) TFA or HC / Dioxane 120

Ester confuciates synthesized

118-2 (R1 = H, R = fBu) : S:5) 118-3 (R1 = OH, R = tBu) 118-5 (R1 = R = H) 118-6 (R = OH, R = H) Carbamate conjugates Synthesized O O or 119-3119-1 (R = H)fBu) R1 = MeO or's H O

ro-/N H 119-4(R192 RBu) = H) R- MeO Ou 119, 120 Carbonate confugates synthesized

120-1 (R = fBu) 120-2(R = H) R1 =

FIG. 7B U.S. Patent Jun. 1, 2010 Sheet 37 of 39 US 7.727,976 B2

Scheme 50: Conjugation of drug molecules to XenoPorter" 106-2 (R = Bu) with an amine functional group on the fused ring 1) COH 115 1 DCC or EDAC/ O TEAI DCM or THF -xenoPorter-coh 2) TFA for HCII Dioxane 121 R

1) Dis-NHR 116 /CD or phosgene?TEAV DIS MOf RHN-xenoPorter-Colbu - 2)POMOTHE TFA for HCI Dioxane RN Nit COH 106-2 122

or phosgen TEA / DCM or THF (Disso- COH 2) TFA for HC 1 Dioxane 123 R

Scheme 51: Conjugation of drug molecules to XenoPorter" 106-3 (R = Bu) with a carboxyl functional group on the fused ring

1) Dis-NHR 116 | DCC or EDAC O EAI DCM or THF N COH 2) TFA or HC/Dioxane R 124

1) OH 117 O 106-3 Oxalyl chloride/DMF/DMAP/DCM or TCBC/TEAVOMAP/DCM 2) TFA or HC / Dioxane 125

FIG. 7C U.S. Patent Jun. 1, 2010 Sheet 38 of 39 US 7.727,976 B2

Schene 52: Conjugation of drug molecules to XenoPorter" 112-1 with a hydroxy functional group on the fused ring

1) Dis-COH 115 / Oxalyl chloride/DMF/DMAP/DCM O or TCBC/EAWDMAP/DCM O SOX 2) HoAcOH 3) NaOAC 126 (X = H) & 134 (X = Na) 1) Dis-NHR 116 O l CD or phosgene 1 l TEAI DCM or THF

2) HOAcOH Dish-soR SOX 3) NaOAC 112-1 127(X = H) & 135 (X = Na) 1) Dis-OH 117 O fCD/NaH TEA/ DCM or HF 2) HOJACOH 3) NaOAC 128 (X = H) & 136 (X = Na)

Schene 53: Conjugation of drug molecules to XenoPorter" 112-2 with an amine functional group on the fused ring

1) CoH 115 DCC of EDACTEAV O 2) HOACOH Dis R X 3) NaOAC 129 (X = H) & 137 (X = Na) ) NHR 116 O /CD orphosgene 1 l TEAI DCM or THF DIS -xenoporter- or a hoaco R X 112-2 3) NaOAC 130 (X = H) & 138 (X = Na) 1) OH 117 O 1 CDI DCM or THF 2) HOJACOH (poord-sex 3) NaOAC 131 (X = H) & 139 (X = Na)

FIG. 7D U.S. Patent Jun. 1, 2010 Sheet 39 of 39 US 7.727,976 B2

Scheme 54: Conjugation of drug molecules to XenoPorter" 112-3 with a carboxyl functional group on the fused ring

1) NHR 116 I DCC or EDAC O TEA / DCM or THF

2) HO/AcOH Ds) SOX 3) NaOAC R 132 (X = H) & 140 (X = Na) O 1) OH 117 112-3 DIS | Oxalyl chloride/DMFIDMAP/DCM or TCBCTEAWDMAPIDCM 133 (X = H) & 141 (X = Na) 2) HOJACOH 3) NaOAC

FIG. 7E US 7,727,976 B2 1. 2 BILE-ACD DERVED COMPOUNDS FOR good intestinal permeability for a drug by designing in rec ENHANCING ORAL ABSORPTION AND ognition and uptake by a nutrient transport system. SYSTEMC BOAVAILABILITY OF DRUGS Incomplete bioavailability of drugs that, nevertheless, are orally delivered necessitates the administration of a larger This application is a divisional of U.S. patent application dose of such drug to compensate for that amount of drug not Ser. No. 10/229,565, filed Aug. 28, 2002, now U.S. Pat. No. delivered to the systemic blood circulation. Such larger doses 7,053,076, which claims priority under 35 U.S.C. S 119 to of the drug, however, may result in greater variability in drug U.S. Provisional Application Ser. No. 60/316,182, entitled exposure, more frequent occurrence of side effects, decrease Bile-Acid Derived Compounds for Enhancing Oral Absorp in patient compliance, or alternatively, require use of tion and Systemic Bioavailability of Drugs, filed on Aug. 29, 10 parenteral delivery routes. 2001, both of which are hereby incorporated by reference in One attractive pathway that might be exploitable for oral their entirety. delivery of such drugs is the intestinal bile acid transport system (Swaan et al., 1996). Bile acids are hydroxylated ste BACKGROUND OF THE INVENTION roids that play a key role indigestion and absorption offat and 15 lipophilic vitamins. After synthesis in the liver, they are 1. Field of the Invention secreted into bile and excreted by the gall bladder into the This invention is directed to compounds that provide for intestinal lumen where they emulsify and help solubilize the enhanced and prolonged systemic blood concentrations of lipophilic substances. Bile acids are conserved in the body by drugs that are incompletely translocated across the intestinal active uptake from the terminal ileum via the Sodium-depen wall after oral delivery to animals. This invention is also dent transporter IBAT (or ASBT) and subsequent hepatic directed to pharmaceutical compositions containing and extraction by the transporter NTCP located in the sinusoidal methods using Such compounds. membrane of hepatocytes. This efficient mechanism to pre 2. State of the Art serve the bile acid pool is termed the enterohepatic circulation Incomplete or poor oral bioavailability of both existing and (see FIG. 1). In man, the total bile acid pool (3-5 g) recircu developmental stage therapeutic and/or prophylactic com 25 lates 6-10 times per day giving rise to a daily uptake of pounds represents a major impediment to effective pharma approximately 20-30 g of bile acids. ceutical drug development. Though multiple factors influ The high transport capacity of the bile acid pathway has ence the bioavailability of drugs (including solubility, been a key reason for interest in this system for drug delivery dissolution rate, first-pass metabolism, p-glycoprotein and purposes. Several papers have postulated that chemical con related efflux mechanisms, etc), low intestinal cell permeabil 30 jugates of bile acids with drugs could be used to provideliver ity is a particularly significant reason for the poor systemic site-directed delivery of a drug to bring about high therapeutic absorption of many compounds. concentrations in the diseased liver with minimization of Compound uptake from the gut is significantly curtailed by general toxic reactions elsewhere in the body; and gallblad the network of tight junctions formed by the intestinal epi der-site delivery systems of cholecystographic agents and thelial cell layer, and the majority of drugs that are orally 35 cholesterol gallstone dissolution accelerators' (Ho, 1987). absorbed traverse this epithelial barrier by passive diffusion Several groups have explored these concepts in some detail, across the apical and basolateral membranes of these cells. using the C-24 carboxylic acid, C-3, C-7, and C-12 hydroxyl The physicochemical features of a molecule that favor its groups of cholic acid (and other bile acids) as handles for passive uptake from the intestinal lumen into the systemic chemically conjugating drugs or drug Surrogates. (Kramer, et circulation include low molecular weight (e.g. <500 Da). 40 al., 1992, Kim, et al., 1993). adequate solubility, and a balance of hydrophobic and hydro The most rigorous drug targeting studies using the bile acid philic character (logP generally 1.5-4.0) (Navia and transport pathway to date relate to work with bile acid con Chaturvedi, 1996). Polar or hydrophilic compounds are typi jugates of HMG-CoA reductase inhibitors (Kramer et al. cally poorly absorbed through an animals intestine as there is 1994b; Petzinger et al., 1995; Kramer and Wess, 1995; a Substantial energetic penalty for passage of Such com 45 Kramer et al., 1997b). Coupling of the HMG-CoA reductase pounds across the lipid bilayers that constitute cellular mem inhibitor HR 780 via an amide linkage to the C-3 position of branes. Many nutrients that result from the digestion of cholate, taurocholate and glycocholate afforded Substrates ingested foodstuffs in animals, such as amino acids, di- and for both the ileal and liver bile acid transporter proteins (FIG. tripeptides, monosaccharides, nucleosides and water-soluble 2). Upon oral dosing of rats, the cholate conjugate S3554 led Vitamins, are polar compounds whose uptake is essential to 50 to specific inhibition of HMG-CoA reductase in the liver, and the viability of the animal. For these substances there exist in contrast to the parent compound HR 780, gave significantly specific mechanisms for active transport of the solute mol reduced inhibition of the enzyme in extra-hepatic organs. ecules across the apical membrane of the intestinal epithelia. Companion studies that looked at the tissue distribution of This transport is frequently energized by co-transport of ions radiolabeled drugs two hours after i.V., administration down a concentration gradient. Solute transporter proteins are 55 through the mesenteric vein of rats also showed dramatically generally single Sub-unit, multi-transmembrane spanning lower systemic levels for the bile acid conjugate relative to the polypeptides, and upon binding of their substrates are parent. Because inhibition of HMG-CoA reductase requires believed to undergo conformational changes which result in the presence of the free carboxylic acid moiety in HR 780 this movement of the Substrate(s) across the membrane. data was taken to indicate that S3554 served as a prodrug of Over the past 10-15 years, it has been found that a number 60 HR 780, undergoing hydrolysis (and other uncharacterized of orally administered drugs are recognized as Substrates by metabolism) in the ratliver. Interestingly, uptake of S3554 by Some of these transporter proteins, and that this active trans liver did not appear to depend on the liverbile acid transporter port may largely account for the oral absorption of these NTCP (which prefers taurocholate conjugates), but may molecules (Tsuji and Tamai, 1996). While in most instances instead have involved another multispecific organic anion the transporter Substrate properties of these drugs were unan 65 transport system on the sinusoidal hepatocyte membrane. ticipated discoveries made through retrospective analysis, it Syntheses of substituted steroids are well known in the art. has been appreciated that, in principle, one might achieve By way of example, hetercyclic derivatives of 3,7,12-triketo US 7,727,976 B2 3 4 cholanic acid, including diaminopyridine, diamino-, and R" is H, OH, alkylene-R, substituted alkylene-R, diketopteridine derivatives, in which the heterorings arefused cycloalkylene-R, substituted cycloalkylene-R, alk to both the A and Brings of steroidic compounds in positions enylene-R, substituted alkenylene-R, cycloalk 2, 3 and 6, 7 or 3, 4 and 6, 7, are known. (Bellini et al., 1969; enylene-R, substituted cycloalkenylene-R, alky Bellini et al; Rocchi et al). In addition, heterosteroids con 5 nylene-R, substituted alkynylene-R, arylene-R, taining a dihydroethisterone skeleton have been prepared and substituted arylene-R, heteroarylene-R, substituted have been shown to displace substance P in receptor binding heteroarylene-R, heterocyclene-R, or substituted het assays. (Venepalli et al., 1992). erocyclene-R or R and R together with the carbon In Summary, while the concept of harnessing the intestinal atoms to which they are attached form a cycloalkyl-R. bile acid uptake pathway to enhance the absorption of poorly 10 substituted cycloalkyl-R, cycloalkenyl-R, substituted absorbed drugs is well appreciated, the existing art has merely cycloalkenyl-R, heterocycloalkyl-R, substituted het demonstrated that bile acid-drug conjugates may be effec erocycloalkyl-R, heterocycloalkenyl-R, substituted tively trafficked to the liver and generally excreted into the heterocycloalkenyl-R, aryl-R, substituted aryl-R, bile, either unchanged or as some type of metabolite. The art heteroaryl-R or substituted heteroaryl-R ring: gives no guidance as to how one prepares a composition that 15 R is H, OH, alkylene-R', substituted alkylene-R', exploits the bile acid transport pathway and simultaneously cycloalkylene-R', substituted cycloalkylene-R', alk provides therapeutically meaningful levels of a drug Sub enylene-R', substituted alkenylene-R', cycloalk stance outside of the enterohepatic circulation. The art further enylene-R', substituted cycloalkenylene-R', alky gives no guidance as to bile acid derivatives that can be used nylene-R', substituted alkynylene-R', arylene-R', in Such a composition. substituted arylene-R', heteroarylene-R', substituted heteroarylene-R', heterocyclene-R', or substituted SUMMARY OF THE INVENTION heterocyclene-R' or RandR together with the carbon atoms to which they are attached form a cycloalkyl-R', This invention is directed to the surprising discovery that substituted cycloalkyl-R', cycloalkenyl-R', substi the bile acid transport system can be utilized to enhance the 25 tuted cycloalkenyl-R', heterocycloalkyl-R', substi systemic bioavailability of orally delivered drugs which are tuted heterocycloalkyl-R', heterocycloalkenyl-R', incompletely translocated across the intestinal wall of an substituted heterocycloalkenyl-R', aryl-R', substi animal. This invention, therefore, permits enhanced oral bio tuted aryl-R'', heteroaryl-R' or substituted heteroaryl availability in animals of Such incompletely translocated R" ring: drugs and, in addition, permits therapeutic or prophylactic 30 R is H, OH, alkylene-R'', substituted alkylene-R'', systemic blood concentrations of orally delivered drugs cycloalkylene-R', substituted cycloalkylene-R', alk which heretofore could not be achieved by oral administra enylene-R', substituted alkenylene-R', cycloalk tion. enylene-R'', substituted cycloalkenylene-R'', alky In one aspect, the invention is directed to compounds of nylene-R'', substituted alkynylene-R'', arylene-R'', formula (I): 35 substituted arylene-R'', heteroarylene-R'', substituted heteroarylene-R'', heterocyclene-R' or substituted heterocyclene-R'': provided that one and only one of (I) R/R, R/R or R/R form a ring: R’ is OH, an alkylamino group substituted with a moiety 40 that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, a di-substituted alkylamino group substituted with L-D and a moiety that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid 45 nucleus, L-D or pharmaceutically acceptable salts thereof; R is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero 50 cycloalkyl, heteroaryl, substituted heteroaryl, OH, wherein OR', SH, SR', NH, NHR', N(R'), COH, R is CH, CHOH or CR-OH: COR, CONH CONHR, CONCR), or L-D; R is CH, CHOH or CROH: R is H, alkyl, substituted alkyl, cycloalkyl, substituted R is H, OH, alkylene-R, substituted alkylene-R, cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi cycloalkylene-R, substituted cycloalkylene-R, alk 55 tuted heteroalkyl, heterocycloalkyl, substituted hetero enylene-R, substituted alkenylene-R, cycloalk cycloalkyl, heteroaryl, substituted heteroaryl, OH, enylene-R, substituted cycloalkenylene-R, alky OR', SH, SR', NH, NHR', N(R'), COH, nylene-R, substituted alkynylene-R, arylene-R, COR, CONH CONHR, CONCR'), or L-D; substituted arylene-R, heteroarylene-R, substituted R" is H, alkyl, substituted alkyl, cycloalkyl, substituted heteroarylene-R, heterocyclene-R, or substituted het 60 cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi erocyclene-R or R and R together with the carbon tuted heteroalkyl, heterocycloalkyl, substituted hetero atoms to which they are attached form a cycloalkyl-R, cycloalkyl, heteroaryl, substituted heteroaryl, OH, substituted cycloalkyl-R, cycloalkenyl-R, substituted OR', SH, SR', NH, NHR', N(R'), COH, cycloalkenyl-R, heterocycloalkyl-R, substituted het COR, CONH CONHR, CONCR'), or L-D; erocycloalkyl-R, heterocycloalkenyl-R, substituted 65 R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted heterocycloalkenyl-R, aryl-R, substituted aryl-R, cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi heteroaryl-R or substituted heteroaryl-R ring: tuted heteroalkyl, heterocycloalkyl, substituted hetero US 7,727,976 B2 5 6 cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, -continued COR, CONH CONHR, CONCR'), or L-D; R" is alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl or substi- 5 tuted heteroaryl; R" is alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl or substi tuted heteroaryl; dashed lines represent possible sites of unsaturation; 10 L is a covalent bond or a linking group; D is a drug; or a pharmaceutically acceptable salt thereof. provided that 15 not more than one of the substituents R. R. R. R. and R’ includes moiety L-D; when R' and Rare CHOH, Rand Rare H, and R7 is OH, then RandR together with the carbon atoms to which they are attached do not form 2O

and when RandR are CHOH, RandR are H, and R7 is OH, then RandR together with the carbonatoms to which they are attached do not form

40 Preferably, the compound of formula (I) is one of the following compounds:

wherein the substituents are as defined above. 60 In one class of preferred compounds, Rand R together with the carbon atoms to which they are attached form a 5-membered ring, wherein the ring is heterocycloalkyl-R, substituted heterocycloalkyl-R, heteroaryl-R or substituted 65 heteroaryl-R.9 In another class of preferred compounds, R and R' together with the carbon atoms to which they are attached US 7,727,976 B2 7 form a 5-membered ring, wherein the ring is heterocy cloalkyl-R, substituted heterocycloalkyl-R, heteroaryl-R or substituted heteroaryl-R. R9 R R 14 In another class of preferred compounds, R'' and R' N together with the carbon atoms to which they are attached N / NM 7 form a 6-membered ring, wherein the ring is cycloalkyl-R, N V R9 substituted cycloalkyl-R, cycloalkenyl-R, substituted O N O cycloalkenyl-R, aryl-R, substituted aryl-R, heterocy cloalkyl-R, substituted heterocycloalkyl-R, heteroaryl-R 10 R9 R 15 or substituted heteroaryl-R. In another class of preferred compounds, R and R' together with the carbon atoms to which they are attached R 14 R 14 O N form a 6-membered ring, wherein the ring is cycloalkyl-R, A substituted cycloalkyl-R, cycloakenyl-R, substituted 15 R9 cycloalkenyl-R, aryl-R, substituted aryl-R, heterocy cloalkyl-R, substituted heterocycloalkyl-R, heteroaryl-R or substituted heteroaryl-R. In another class of preferred compounds, R and R' together with the carbon atoms to which they are attached form a 7-membered ring, wherein the ring is heterocy cloalkyl-R, substituted heterocycloalkyl-R, heterocy cloalkenyl-R, substituted heterocycloalkenyl-R, het eroaryl-R or substituted heteroaryl-R. 25 In another class of preferred compounds, R and R' together with the carbon atoms to which they are attached form a 7-membered ring, wherein the ring is heterocy cloalkyl-R, substituted heterocycloalkyl-R, heterocy 30 cloalkenyl-R, substituted heterocycloalkenyl-R, het R 15 R9 eroaryl-R or substituted heteroaryl-R. s s In another class of preferred compounds, R and R' R91 N R 151 N together with the carbon atoms to which they are attached 35 e e form an 8-membered ring, wherein the ring is heterocy R 14 R 14 cloalkyl-R, substituted heterocycloalkyl-R, heterocy cloalkenyl-R or substituted heterocycloalkenyl-R. In another class of preferred compounds, R and R' 40 M. together with the carbon atoms to which they are attached N form an 8-membered ring, wherein the ring is heterocy N cloalkyl-R, substituted heterocycloalkyl-R, heterocy A cloalkenyl-R or substituted heterocycloalkenyl-R. R9 In certain preferred compounds, R is an alkylamino group 45 R9 Substituted with a moiety that is negatively charged at physi s ological pH and located 5 to 15 atoms from C-22 of the bile N acid nucleus, wherein the moiety is selected from the group R 14 Y N e consisting of —COOH, -SOH, -SOH, - POH, N –OPOH, - OSOH, -C(O)NHOH, -tetrazole, -catechol 50 and pharmaceutically acceptable salts thereof. In other preferred compounds, R7 is a di-substituted alky lamino group Substituted with L-D and a moiety that is nega tively charged at physiological pH and located 5 to 15 atoms 55 from C-22 of the bile acid nucleus, and wherein the moiety is selected from the group consisting of —COOH, -SOH, - SOH, - POH, OPOH, OSOH, C(O)NHOH, -tetrazole, -catechol and pharmaceutically acceptable salts thereof. 60 In other preferred compounds, R is OH. wherein In other preferred compounds, R is L-D. R is L-D; Where in the compound RandR together with the carbon R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted atoms to which they are attached form a 5-membered ring, the 65 cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi 5-membered ring is preferably one of the following 5-mem tuted heteroalkyl, heterocycloalkyl, substituted hetero bered rings: cycloalkyl, heteroaryl, substituted heteroaryl, OH, US 7,727,976 B2 9 10 OR', SH, SR', NH, NHR', N(R'), COH,

COR, CONH CONHR' or CONCR'); -continued R" is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR or CONCR); and A R9 R 14 R 14 R9 4 R" and R are independently H, halo, alkyl, substituted 10 alkyl, cycloalkyl, Substituted cycloalkyl, aryl, Substi tuted aryl, heteroalkyl, substituted heteroalkyl, hetero cycloalkyl, substituted heterocycloalkyl, heteroaryl, R13 9 substituted heteroaryl, OH, OR, SH, SR', NH, NN N RS 15 \ NHR', N(R'), COH, COR, CONH CONHR'. NF or CONCR'). R9 Where in the compound RandR together with the carbon si-KV A. R50 atoms to which they are attached form a 5-membered ring, the 5-membered ring is preferably one of the following 5-mem bered rings: wherein R is L-D; R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted 25 cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, 30 COR, CONH CONHR or CONCR'); R" is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero 35 cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR' or CONCR); and R" and R are independently H, halo, alkyl, substituted alkyl, cycloalkyl, Substituted cycloalkyl, aryl, Substi 40 tuted aryl, heteroalkyl, substituted heteroalkyl, hetero cycloalkyl, substituted heterocycloalkyl, heteroaryl, substituted heteroaryl, OH, OR, SH, SR', NH, NHR', N(R'), COH, COR, CONH, CONHR'. 45 or CONCR'). R13 R13 Where in the compound RandR together with the carbon NN N NN N atoms to which they are attached form a 6-membered ring, the 6-membered ring is preferably one of the following 6-mem bered rings: R9 R 15 R15 R9 50

R 16 R 16

15 % R 2 55

b-( )-( R 14 R17 R9 R17 R9 R 14 R9 R9 R 14 60 R 16 R9 R R18 R13 a R a \- \- R 15 R17 R15 R17 A A 65 R9 R 14 R15 R 14 US 7,727,976 B2 11 12

-continued -continued

6 R 16 R1 N RIS N RI 5 n n RIS RIS R 14 2 R9 2

R 14 R9 R9 R 14 10 R 15 R9 R 14 RI 6 R9 n N R RIS 15 R 15 21 R9 4. R 14 R15 R15 R15 R9 4 RI R 14 R 9 R 15 R 15 R15 2O N N R13 R9 2 2 R 14 N R 14 N

R9 O R 14 O 25 R 14 R9

R9 R15

O O R17 N R9 N R 15 R9 35 DC 2 2 R9 RIS R9 N R 14 N

R 14 O R 14 O 40 R17 R17 wherein R is L-D; R 15 R 15 R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi N1N N N 45 tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, 2 OR', SH, SR', NH, NHR', N(R'), COH, R 14 21 R9 COR, CONH CONHR or CONCR'); R9 C 50 R" is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi R9 R15 tuted heteroalkyl, heterocycloalkyl, substituted hetero R13 cycloalkyl, heteroaryl, substituted heteroaryl, OH, N1N N OR', SH, SR', NH, NHR', N(R'), COH, 55 COR, CONH CONHR' or CONCR'); 2 R" is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi cR 14 N R9 tuted heteroalkyl, heterocycloalkyl, substituted hetero 5 R cycloalkyl, heteroaryl, substituted heteroaryl, OH, 60 OR', SH, SR', NH, NHR', N(R'), COH, R RIS COR, CONH CONHR' or CON(R'); and, N N R7 is OH, OP, NHP or NRP, wherein R is alkyl oraryl N and P is a protecting group. ul' 21 Where in the compound RandR together with the carbon R1 65 atoms to which they are attached form a 6-membered ring, the 4 4 6-membered ring is preferably one of the following 6-mem bered rings:

US 7,727,976 B2 15 16

-continued -continued

N l N21 N21 RI 41S N 1S N S 10 14 14 R9 R 14 RI 6 wherein S.5 R is L-D; 15 R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted R9 cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi RI 4 tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR or CONCR'); N R" is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi R 15 tuted heteroalkyl, heterocycloalkyl, substituted hetero 25 cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, 6 COR, CONH CONHR or CONCR'); RI R" is H. alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi 30 tuted heteroalkyl, heterocycloalkyl, substituted hetero R9 cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, 3.4RI 4 COR, CONH CONHR' or CONCR'); and, R7 is 454 35 RIRIR1 6 OH, OP, NHP or NRP, wherein R is alkyl or aryland RI P is a protecting group. Where in the compound RandR together with the carbon N atoms to which they are attached form a 7-membered ring, the 7-membered ring is preferably one of the following 7-mem- 40 RI 5 R 15 beredered rings:ring RRRRRRRR\915\91515 3.RI 4 3.3. 6 8 R1 RI R 16 R 16 RI 5 45 Nse- Nie

R15 R 15 R 14 N N ^ 14 9 RI 4 R k R ka 50 R 16 R9

Na- Na

55 R9 R 16 N N R 15 ka R 15 ka R 14 R 16 R 16 60 Na- N2

R9 2 N 65 3. US 7,727,976 B2

-continued -continued R9 R 16 R18 R 16 R9 R18 N N N N 5

Rs.1\15 2 Ris?\15 2 N N R 14 R 14 10 wherein R’ is L-D; R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR's, NH, NHR's, N(R'), CO.H, COR, CONH CONHR or CONCR'); R" is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, 25 OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR' or CONCR'); R' is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroalkyl, substi- 30 tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR or CONCR); and, R" is H. alkyl, substituted alkyl, cycloalkyl, substituted 35 cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, 40 COR, CONH CONHR or CONCR). Where in the compound RandR together with the carbon atoms to which they are attached form a 7-membered ring, the 7-membered ring is preferably one of the following 7-mem- as bered rings:

50 R6N S R6N N

R15 A R9 R 15 A R 14 55 N N R 14 R9

60 R6 R9 N N N N N21 N21 R18 R18 R 9 7-R414 R I6 7 NR 14 R9 R16 R 14 R 16 N N N N 65 \ V R15 R 15 R 14 R 15 R9 R 15 US 7,727,976 B2 19 20

-continued

5 R 16 N21 N21 R18 R18 R15 R 14 I6 R 14 9 N R N R 10 R15 Ye R 15 Y. R 16

15 R9 N21 N21 R18 R18 R9 y^eI6 R "Y14 ?eI6 R 14 Ys R9 Y is R18

R 16 25 N21 N21 R18 R18 R 14 N />RicI6 RRN 14 /> 9 R1 R15 Y. R 15 Y. 30 R14 wherein R’ is L-D; 35 R" is H. alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, 40 OR', SH, SR, NH, NHR', N(R'), COH, . COR, CONH CONHR' or CONCR'); 9 6 R" is H, alkyl, substituted alkyl, cycloalkyl, substituted R RI cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero- 45 R' N cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR' or CONCR'); R' is H, alkyl, substituted alkyl, cycloalkyl, substituted so cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH, CONHR or CONCR); and, 55 R" is H. alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, 60 OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR' or CONCR'). Where in the compound RandR together with the carbon atoms to which they are attached form a 8-membered ring, the 65 8-membered ring is preferably one of the following 8-mem bered rings: US 7,727,976 B2 21 22

-continued -continued RI 6 R9 R1 5 RI 6 V \ R1 N Y R13 N Y 5

O NZ C. NZ R 14 R9 10 wherein R is L-D; R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted 15 cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR, NH, NHR', N(R'), COH, COR, CONH CONHR or CONCR'); R" is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, 25 OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR' or CONCR'); R' is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero 30 cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR or CONCR); and, R" is H. alkyl, substituted alkyl, cycloalkyl, substituted 35 R 15 cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR' or CONCR). 40 N2 Where in the compound RandR together with the carbon \ R18 atoms to which they are attached form a 8-membered ring, the 8-membered ring is preferably one of the following 8-mem N bered rings: 45

R9 R 14 50 N N N N N N R13 R / R18 R18 N N RI 5 R15 55 R 16 R 16

R 14 R 14 60 N N N N RIS N RIS N R18 R18 N N R9 R 16 65 US 7,727,976 B2 23 24 In certain preferred compounds, L is a cleavable linking -continued group. In particularly preferred compounds, L is a linking group selected from —OC(O)(CH.)NHC(O)— —OC(O)(alky lene-O)-alkylene-, and —C(O)O(alkylene-O)-alkylene, wherein q is an integer of from 1 to 20. In another aspect, the invention is directed to pharmaceu tical compositions comprising a pharmaceutically acceptable excipient and a compound of formula (I): 10

(I) wherein 15 R’ is L-D; R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, Substituted heteroaryl, OH. 20 OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR or CONCR'); R" is H. alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, Substituted hetero- 25 cycloalkyl, heteroaryl, substituted heteroaryl, OH, wherein OR', SH, SR', NH, NHR', N(R'), COH, R is CH, CHOH or CROH: COR, CONH CONHR' or CONCR'); R is CH, CHOH or CROH: R' is H, alkyl, substituted alkyl, cycloalkyl, substituted R is H, OH, alkylene-R, substituted alkylene-R, cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi- 30 cycloalkylene-R, substituted cycloalkylene-R, alk tuted heteroalkyl, heterocycloalkyl, substituted hetero enylene-R, substituted alkenylene-R, cycloalk cycloalkyl, heteroaryl, Substituted heteroaryl, OH, enylene-R, substituted cycloalkenylene-R, alky OR', SH, SR', NH, NHR', N(R'), COH, nylene-R, substituted alkynylene-R, arylene-R, COR, CONH CONHR or CONCR); and, substituted arylene-R, heteroarylene-R, substituted R" is H, alkyl, substituted alkyl, cycloalkyl, substituted 35 heteroarylene-R, heterocyclene-R, or substituted het cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi- erocyclene-R or R and R together with the carbon tuted heteroalkyl, heterocycloalkyl, substituted heteroatoms to which they are attached form a cycloalkyl-R, cycloalkyl, heteroaryl, substituted heteroaryl, OH, substituted cycloalkyl-R, cycloalkenyl-R, substituted OR', SH, SR', NH, NHR', N(R'), COH, cycloalkenyl-R, heterocycloalkyl-R, substituted het COR, CONH CONHR' or CONCR'). 40 erocycloalkyl-R, heterocycloalkenyl-R, substituted Preferably, L in the compounds is a linking group, wherein heterocycloalkenyl-R, aryl-R, substituted aryl-R, the linking group is of the formula —X—Y—Z: heteroaryl-R or substituted heteroaryl-R ring: wherein R is H, OH, alkylene-R, Substituted alkylene-R, X is selected from the group consisting of ester, amide, cycloalkylene-R , substituted cycloalkylene-R , alk acyloxyalkyl ester, alkoxycarbonyloxyalkyl ester, gly- enylene-R, substituted alkenylene-R’. cycloalk colamide ester, amidomethyl ester, carbonate, carbam- enylene.R. , substituted cycloalkenylene-R, ally ate, acyloxyalkyl ether, alkoxycarbonyloxyalkyl ether, nylene-R s substituted alkynylene-R 9 arylene-R s acyloxyalkyl carbonate, acyloxyalkyl carbamate substituted arylene-R, heteroarylene-R, substituted alkoxycarbonyloxyalkyl carbamate, Mannich base. heteroarylene-R, heterocyclene-R O substituted het imide, N-acyloxyalkylphosphoramidate, and N-alkoxy- 50 erocyclene-R or R" and R together with the carbon carbonyloxyalkyl phosphoramidate; atoms to which they are attached form a cycloalkyl-R s Y is of the formula –(R')m(R')n(R')p-, wherein Substituted cycloalkyl-R s cycloalkenyl-R s substituted each of R', R'' and R'' are independently selected cycloalkenyl-R 3 heterocycloalkyl-R, substituted het from the group consisting of alkylene, Substituted alky- E. s crysley s bill lene, alkenylene, Substituted alkenylene, alkynylene, 55 E. eny SE , Su y ary -R, Substituted alkynylene, cycloalkylene, Substituted 5 eteroary -R OSU stitute eteroary -K ring: O cycloalkylene, cycloalkenylene, Substituted cycloalk- R is H, OH, alkylene-R , substituted alkylene-R', enylene, arylene, Substituted arylene, heteroarylene, cycloalkylene-R s substituted cycloalkylene-R , alk substituted heteroarylene, heterocyclene and substituted enylene-R, substituted alkenylene-R', ycloalk heterocyclene; 60 enylene-R''. substituted cycloalkenylene-R', alky s nylene-R', substituted alkynylene-R', arylene-R', each of m, n and pare independently an integer from 0 to 3: substituted arylene-R', heteroarylene-R', substituted and, heteroarylene-R', heterocyclene-R', or substituted Z is selected from the group consisting of ether, thioether, heterocyclene-R' or RandR together with the carbon ester, carbonate, carbamate, phosphate, phosphonate, 65 atoms to which they are attached form a cycloalkyl-R". phosphoramidate, amide, amine, urea, thiourea, Sul substituted cycloalkyl-R', cycloalkenyl-R', substi fonamide, sulfoxide, sulfone, thioester, and disulfide. tuted cycloalkenyl-R', heterocycloalkyl-R', substi US 7,727,976 B2 25 26 tuted heterocycloalkyl-R'', heterocycloalkenyl-R', substituted heterocycloalkenyl-R', aryl-R', substi tuted aryl-R'', heteroaryl-R' or substituted heteroaryl R" ring: R is H, OH, alkylene-R'', substituted alkylene-R'', cycloalkylene-R'', substituted cycloalkylene-R'', alk enylene-R', substituted alkenylene-R'', cycloalk enylene-R', substituted cycloalkenylene-R'', alky and when R' and Rare CHOH, Rand Rare H, and R' nylene-R', substituted alkynylene-R'', arylene-R'', is OH, then RandR together with the carbonatoms substituted arylene-R'', heteroarylene-R', substituted 10 to which they are attached do not form heteroarylene-R'', heterocyclene-R' or substituted het erocyclene-R'; with the proviso that only one of R/R, R/R or R/R form a ring: R’ is OH, an alkylamino group substituted with a moiety 15 that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, a di-substituted alkylamino group substituted with L-D In another aspect, the invention is directed to methods for and a moiety that is negatively charged at physiological achieving prolonged therapeutic or prophylactic concentra pH and located 5 to 15 atoms from C-22 of the bile acid tions of a drug or active metabolite thereof in the systemic nucleus, L-D or pharmaceutically acceptable salts circulation of an animal comprising orally administering a thereof; compound of formula (I) to the animal: R is H. alkyl, substituted alkyl, cycloalkyl, substituted (I) cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi 25 tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR, NH, NHR', N(R'), COH, COR, CONH CONHR, CONCR'), or L-D; R is H, alkyl, substituted alkyl, cycloalkyl, substituted 30 cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR, CONCR), or L-D; 35 R" is H, alkyl, substituted alkyl, cycloalkyl, substituted wherein cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi R" is CH, CHOH or CROH: tuted heteroalkyl, heterocycloalkyl, substituted hetero R° is CH, CHOH or CROH: cycloalkyl, heteroaryl, substituted heteroaryl, OH, 40 R is H, OH, alkylene-R, substituted alkylene-R, OR', SH, SR', NH, NHR', N(R'), COH, cycloalkylene-R, substituted cycloalkylene-R, alk COR, CONH CONHR, CONCR), or L-D; enylene-R, substituted alkenylene-R, cycloalk R'' is H. alkyl, substituted alkyl, cycloalkyl, substituted enylene-R, substituted cycloalkenylene-R, alky cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi nylene-R, substituted alkynylene-R, arylene-R, 45 substituted arylene-R, heteroarylene-R, substituted tuted heteroalkyl, heterocycloalkyl, substituted hetero heteroarylene-R, heterocyclene-R, or substituted het cycloalkyl, heteroaryl, substituted heteroaryl, OH, erocyclene-R or R and R together with the carbon OR', SH, SR', NH, NHR', N(R'), COH, atoms to which they are attached form a cycloalkyl-R, COR, CONH CONHR, CONCR), or L-D; substituted cycloalkyl-R, cycloalkenyl-R, substituted R’ is alkyl, substituted alkyl, cycloalkyl, substituted 50 cycloalkenyl-R, heterocycloalkyl-R, substituted het cycloalkyl, aryl, substituted aryl, heteroaryl or substi erocycloalkyl-R, heterocycloalkenyl-R, substituted tuted heteroaryl; heterocycloalkenyl-R, aryl-R, substituted aryl-R, R" is alkyl, substituted alkyl, cycloalkyl, substituted heteroaryl-R or substituted heteroaryl-R ring: cycloalkyl, aryl, substituted aryl, heteroaryl or substi R is H, OH, alkylene-R, substituted alkylene-R, tuted heteroaryl; 55 cycloalkylene-R, substituted cycloalkylene-R, alk enylene-R, substituted alkenylene-R, cycloalk dashed lines represent possible sites of unsaturation; enylene-R, substituted cycloalkenylene-R, alky L is a covalent bond or a linking group; nylene-R, substituted alkynylene-R, arylene-R, D is a drug; substituted arylene-R, heteroarylene-R, substituted or a pharmaceutically acceptable salt thereof. 60 heteroarylene-R, heterocyclene-R, or substituted het erocyclene-R or R and R together with the carbon provided that atoms to which they are attached form a cycloalkyl-R, not more than one of the substituents R. R. R. R. and substituted cycloalkyl-R, cycloalkenyl-R, substituted R7 includes moiety L-D; cycloalkenyl-R, heterocycloalkyl-R, substituted het when R' and Rare CHOH, Rand Rare H, and R7 is 65 erocycloalkyl-R, heterocycloalkenyl-R, substituted OH, then RandR together with the carbon atoms to heterocycloalkenyl-R, aryl-R, substituted aryl-R, which they are attached do not form heteroaryl-R or substituted heteroaryl-R ring: US 7,727,976 B2 27 28 R is H, OH, alkylene-R', substituted alkylene-R', metabolite thereof thereby providing a therapeutic or cycloalkylene-R', substituted cycloalkylene-R', alk prophylactic systemic blood concentration in said ani enylene-R', substituted alkenylene-R', cycloalk mal; enylene-R', substituted cycloalkenylene-R', alky or a pharmaceutically acceptable salt thereof; nylene-R', substituted alkynylene-R', arylene-R', 5 substituted arylene-R', heteroarylene-R', substituted provided that heteroarylene-R'', heterocyclene-R', or substituted not more than one of the substituents R. R. R. R. and heterocyclene-R' or RandR together with the carbon R7 includes moiety L-D; atoms to which they are attached form a cycloalkyl-R', when R' and Rare CHOH, Rand Rare H, and R7 is substituted cycloalkyl-R', cycloalkenyl-R', substi 10 OH, then RandR together with the carbonatoms to tuted cycloalkenyl-R", heterocycloalkyl-R', substi which they are attached do not form tuted heterocycloalkyl-R', heterocycloalkenyl-R', substituted heterocycloalkenyl-R', aryl-R', substi tuted aryl-R'', heteroaryl-R' or substituted heteroaryl R" ring: 15 R is H, OH, alkylene-R', substituted alkylene-R'', cycloalkylene-R', substituted cycloalkylene-R'', alk enylene-R', substituted alkenylene-R'', cycloalk enylene-R'', substituted cycloalkenylene-R'', alky nylene-R', substituted alkynylene-R'', arylene-R'', substituted arylene-R'', heteroarylene-R'', substituted and when R' and Rare CHOH, Rand Rare H, and R' heteroarylene-R'', heterocyclene-R' or substituted is OH, then RandR together with the carbon atoms heterocyclene-R'; with the proviso that only one of to which they are attached do not form R/R, R/R or R/R form a ring: R’ is OH, an alkylamino group substituted with a moiety 25 that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, a di-substituted alkylamino group substituted with L-D and a moiety that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid 30 nucleus, L-D or pharmaceutically acceptable salts thereof; In a preferred method, at least a portion of the cleavable R is H, alkyl, substituted alkyl, cycloalkyl, substituted linker cleaves in the contents of the intestinal lumen of said cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi animal. tuted heteroalkyl, heterocycloalkyl, substituted hetero 35 In another preferred method, at least a portion of the cleav cycloalkyl, heteroaryl, substituted heteroaryl, OH, able linker cleaves in the intestinal cells of said animal. OR', SH, SR', NH, NHR', N(R'), COH, In another preferred method, at least a portion of the cleav COR, CONH CONHR, CONCR), or L-D; able linker cleaves in the blood of said animal. R is H. alkyl, substituted alkyl, cycloalkyl, substituted In another preferred method, at least a portion of the cleav cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi 40 able linker cleaves in the liver cells of said animal. tuted heteroalkyl, heterocycloalkyl, substituted hetero In another preferred method, at least a portion of the cleav cycloalkyl, heteroaryl, substituted heteroaryl, OH, able linker cleaves in the biliary tract of said animal. OR', SH, SR', NH, NHR', N(R'), COH, In another aspect, the invention is directed to methods for COR, CONH CONHR, CONCR'), or L-D; enhancing the systemic bioavailability of a drug or an active R" is H, alkyl, substituted alkyl, cycloalkyl, substituted 45 metabolite thereof in an animal by increasing the amount of cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi drug translocated across the intestinal wall of said animal tuted heteroalkyl, heterocycloalkyl, substituted hetero comprising orally administering a compound of formula (I) to cycloalkyl, heteroaryl, substituted heteroaryl, OH, said animal: OR', SH, SR', NH, NHR', N(R'), COH,CO.R., CONH CONHR, CON(R'), or L-D; 50

R'' is H. alkyl, substituted alkyl, cycloalkyl, substituted (I) cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, 55 COR, CONH CONHR, CONCR), or L-D; R" is alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl or substi tuted heteroaryl; R" is alkyl, substituted alkyl, cycloalkyl, substituted 60 cycloalkyl, aryl, substituted aryl, heteroaryl or substi tuted heteroaryl; dashed lines represent possible sites of unsaturation; D is a drug; 65 wherein L is a cleavable linking group, wherein a Sufficient amount R is CH, CHOH or CROH: of the linking group is cleaved to release D or an active R is CH, CHOH or CROH: US 7,727,976 B2 29 30 R is H, OH, alkylene-R, substituted alkylene-R, R is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkylene-R, substituted cycloalkylene-R, alk cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi enylene-R, substituted alkenylene-R, cycloalk tuted heteroalkyl, heterocycloalkyl, substituted hetero enylene-R, substituted cycloalkenylene-R, alky cycloalkyl, heteroaryl, substituted heteroaryl, OH, nylene-R, substituted alkynylene-R, arylene-R, OR', SH, SR, NH, NHR', N(R'), COH, substituted arylene-R, heteroarylene-R, substituted COR, CONH CONHR, CONCR), or L-D; heteroarylene-R, heterocyclene-R, or substituted het R" is H, alkyl, substituted alkyl, cycloalkyl, substituted erocyclene-R or R and R together with the carbon cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi atoms to which they are attached form a cycloalkyl-R, tuted heteroalkyl, heterocycloalkyl, substituted hetero substituted cycloalkyl-R, cycloalkenyl-R, substituted 10 cycloalkyl, heteroaryl, substituted heteroaryl, OH, cycloalkenyl-R, heterocycloalkyl-R, substituted het OR', SH, SR', NH, NHR', N(R'), COH, erocycloalkyl-R, heterocycloalkenyl-R, substituted COR, CONH CONHR, CONCR), or L-D; heterocycloalkenyl-R, aryl-R, substituted aryl-R, R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted heteroaryl-R or substituted heteroaryl-R ring: cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi R is H, OH, alkylene-R, substituted alkylene-R, 15 tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkylene-R, substituted cycloalkylene-R, alk cycloalkyl, heteroaryl, substituted heteroaryl, OH, enylene-R, substituted alkenylene-R, cycloalk OR', SH, SR', NH, NHR', N(R'), COH, enylene-R, substituted cycloalkenylene-R, alky COR, CONH CONHR, CONCR), or L-D; nylene-R, substituted alkynylene-R, arylene-R, R'' is alkyl, substituted alkyl, cycloalkyl, substituted substituted arylene-R, heteroarylene-R, substituted cycloalkyl, aryl, substituted aryl, heteroaryl or substi heteroarylene-R, heterocyclene-R, or substituted het tuted heteroaryl; erocyclene-R or R and R together with the carbon R" is alkyl, substituted alkyl, cycloalkyl, substituted atoms to which they are attached form a cycloalkyl-R, cycloalkyl, aryl, substituted aryl, heteroaryl or substi substituted cycloalkyl-R, cycloalkenyl-R, substituted tuted heteroaryl; cycloalkenyl-R, heterocycloalkyl-R, substituted het 25 dashed lines represent possible sites of unsaturation; erocycloalkyl-R, heterocycloalkenyl-R, substituted D is a drug which is incompletely translocated across the heterocycloalkenyl-R, aryl-R, substituted aryl-R, intestinal wall of the animal; heteroaryl-R or substituted heteroaryl-R ring: L is a cleavable linking group, wherein a sufficient amount R is H, OH, alkylene-R', substituted alkylene-R', of the linking group is cleaved to release D or an active cycloalkylene-R', substituted cycloalkylene-R', alk 30 metabolite thereof thereby providing a therapeutic or enylene-R', substituted alkenylene-R', cycloalk prophylactic systemic blood concentration in the ani enylene-R', substituted cycloalkenylene-R', alky mal; nylene-R', substituted alkynylene-R', arylene-R', or a pharmaceutically acceptable salt thereof; substituted arylene-R', heteroarylene-R', substituted provided that heteroarylene-R'', heterocyclene-R', or substituted 35 not more than one of the substituents R. R. R. R. and heterocyclene-R' or RandR together with the carbon R7 includes moiety L-D; atoms to which they are attached form a cycloalkyl-R', when R' and Rare CHOH, Rand Rare H, and R7 is substituted cycloalkyl-R', cycloalkenyl-R', substi OH, then RandR together with the carbonatoms to tuted cycloalkenyl-R', heterocycloalkyl-R', substi which they are attached do not form tuted heterocycloalkyl-R'', heterocycloalkenyl-R', 40 substituted heterocycloalkenyl-R', aryl-R', substi tuted aryl-R', heteroaryl-R' or substituted heteroaryl R" ring: R is H, OH, alkylene-R'', substituted alkylene-R'', cycloalkylene-R', substituted cycloalkylene-R'', alk 45 enylene-R', substituted alkenylene-R'', cycloalk enylene-R'', substituted cycloalkenylene-R'', alky nylene-R', substituted alkynylene-R'', arylene-R'', substituted arylene-R'', heteroarylene-R'', substituted and when R' and Rare CHOH, Rand Rare H, and R' heteroarylene-R'', heterocyclene-R' or substituted 50 is OH, then RandR together with the carbonatoms heterocyclene-R'; with the proviso that only one of to which they are attached do not form R/R, R/R or R/R form a ring: R’ is OH, an alkylamino group substituted with a moiety that is negatively charged at physiological pH and 55 located 5 to 15 atoms from C-22 of the bile acid nucleus, a di-substituted alkylamino group substituted with L-D and a moiety that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, L-D or pharmaceutically acceptable salts 60 thereof; In a preferred method, at least a portion of the cleavable R is H, alkyl, substituted alkyl, cycloalkyl, substituted linker cleaves in the contents of the intestinal lumen of said cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi animal. tuted heteroalkyl, heterocycloalkyl, substituted hetero In another preferred method, at least a portion of the cleav cycloalkyl, heteroaryl, substituted heteroaryl, OH, 65 able linker cleaves in the intestinal cells of said animal. OR', SH, SR, NH, NHR', N(R'), COH, In another preferred method, at least a portion of the cleav COR, CONH CONHR, CONCR'), or L-D; able linker cleaves in the blood of said animal. US 7,727,976 B2 31 32 In another preferred method, at least a portion of the cleav R’ is alkyl, substituted alkyl, cycloalkyl, substituted able linker cleaves in the liver cells of said animal. cycloalkyl, aryl, substituted aryl, heteroaryl or substi In another preferred method, at least a portion of the cleav tuted heteroaryl; able linker cleaves in the biliary tract of said animal. L is a covalent bond or a linking group; and In another aspect, the invention is directed to methods for 5 modulating cholesterol metabolism in an animal comprising D is a drug. orally administering to the animala compound of formula (I). Preferably, R is alkyl, R' is alkyl and R’ is alkyl. In a further aspect, the invention is directed to a method of In a particularly preferred compound, R' is methyl, R is treating a condition in a mammal selected from the group methyl and R’ is methyl. consisting of gallstone, a gastrointestinal inflammatory dis order, colorectal cancer, a viral infection, and a fungal infec In another aspect, the invention is directed to compounds of tion, comprising orally administering to the animal a com formula (III), which are useful for the production of com pound of formula (I). pounds of formula (I): In an additional aspect, the invention is directed to a method for increasing oral or nasal mucosal permeability, 15 comprising applying a compound of formula (I) to the oral or

nasal mucosa of an animal. (III) In a further aspect, the invention is directed to a method of inhibiting apoptosis in an animal, comprising orally admin istering to the animal a compound of formula (I). In an additional aspect, the invention is directed to a method of delivering a therapeutic drug to the liver of an animal comprising orally administering to the animal a com pound of formula (I), wherein the compound contains L-D. 25 A further aspect of the invention is directed to a method of delivering a diagnostic agent to the liver of an animal com prising orally administering to the animal a compound of formula (I). In another aspect, the invention is directed to compounds of 30 formula (II), which are useful for the production of com pounds of formula (I): wherein R" is CH, CHOH or CROH: R is CH, CHOH or CROH: (II) 35 R’ is OH, an alkylamino group substituted with a moiety that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, a di-substituted alkylamino group substituted with L-D 40 and a moiety that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, L-D or pharmaceutically acceptable salts thereof; R" is alkyl, aryl or substituted aryl; 45 R" is O, CH(OH), CHN(R)(R'), C(SR)(SR), wherein C(SRO)NH(R), C(SRO)N(R)(R) or C(R)(R): R is CH, CHOH or CROH: R’ is alkyl, substituted alkyl, cycloalkyl, substituted R is CH, CHOH or CROH: cycloalkyl, aryl, substituted aryl, heteroaryl or substi R’ is OH, an alkylamino group substituted with a moiety 50 tuted heteroaryl; that is negatively charged at physiological pH and R’ is alkyl, substituted alkyl, cycloalkyl, substituted located 5 to 15 atoms from C-22 of the bile acid nucleus, cycloalkyl, aryl, substituted aryl, heteroaryl or substi a di-substituted alkylamino group substituted with L-D tuted heteroaryl; and a moiety that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid 55 R’ is alkyl, substituted alkyl, cycloalkyl, substituted nucleus, L-D or pharmaceutically acceptable salts cycloalkyl, aryl, substituted aryl, heteroaryl or substi thereof; tuted heteroaryl; R" is alkyl, aryl or substituted aryl; L is a covalent bond or a linking group; and R is O, CH(OH), CHN(R)(R), C(SR2)(SRI), 60 D is a drug. C(SRO)NH(R), C(SRO)N(R)(R) or C(R)(R): Preferably, R is alkyl, R' is alkyl and R’ is alkyl. R" is alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl or substi In a particularly preferred compound, R” is methyl, R is tuted heteroaryl; methyl and R’ is methyl. R’ is alkyl, substituted alkyl, cycloalkyl, substituted 65 In another aspect, the invention is directed to compounds of cycloalkyl, aryl, substituted aryl, heteroaryl or substi formula (IV), which are useful for the production of com tuted heteroaryl; pounds of formula (I): US 7,727,976 B2 33 34 L is a covalent bond or a linking group; and D is a drug. (IV) Preferably, R is TMS, TBDMS, acetyl, tosyl, mesyl or triflyl. 5 In another aspect, the invention is directed to compounds of formula (VI), which are useful for the production of com pounds of formula (I):

10 (VI)

wherein 15 R" is CH, CHOH or CROH: R2 is CH2, CHOH or CR12OH: R7 is OH, an alkylamino group substituted with a moiety that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, a di-substituted alkylamino group substituted with L-D and a moiety that is negatively charged at physiological wherein pH and located 5 to 15 atoms from C-22 of the bile acid R" is CH, CHOH or CR'? OH: nucleus, L-D or pharmaceutically acceptable salts R is CH, CHOH or CROH: thereof; 25 R’ is OH, an alkylamino group substituted with a moiety R" is alkyl, aryl or substituted aryl; that is negatively charged at physiological pH and R’ is halo or OR; located 5 to 15 atoms from C-22 of the bile acid nucleus, R" is H, alkyl or substituted alkyl; a di-substituted alkylamino group substituted with L-D R is a protecting group; and a moiety that is negatively charged at physiological L is a covalent bond or a linking group; and 30 pH and located 5 to 15 atoms from C-22 of the bile acid D is a drug. nucleus, L-D or pharmaceutically acceptable salts Preferably, R is TMS, TBDMS, acetyl, tosyl, mesyl or thereof; triflyl. R" is alkyl, aryl or substituted aryl; In another aspect, the invention is directed to compounds of R’ is halo or OR; formula (V), which are useful for the production of com R is H, alkyl or substituted alkyl: pounds of formula (I): R is a protecting group; L is a covalent bond or a linking group; and D is a drug. (V) Preferably, R is TMS, TBDMS, acetyl, tosyl, mesyl or 40 triflyl. In another aspect, the invention is directed to compounds of formula (VII), which are useful for the production of com pounds of formula (I): 45

(VII)

50 wherein R" is CH, CHOH or CROH: R is CH, CHOH or CROH: 55 R’ is OH, an alkylamino group substituted with a moiety that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, a di-substituted alkylamino group substituted with L-D and a moiety that is negatively charged at physiological 60 wherein pH and located 5 to 15 atoms from C-22 of the bile acid R is CH, CHOH or CROH: nucleus, L-D or pharmaceutically acceptable salts R is CH, CHOH or CROH: thereof; R’ is OH, an alkylamino group substituted with a moiety R" is alkyl, aryl or substituted aryl; that is negatively charged at physiological pH and R’ is halo or OR; 65 located 5 to 15 atoms from C-22 of the bile acid nucleus, R is H. alkyl or substituted alkyl: a di-substituted alkylamino group substituted with L-D R is a protecting group; and a moiety that is negatively charged at physiological US 7,727,976 B2 35 36 pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, L-D or pharmaceutically acceptable salts

thereof; (IX) R’ is alkyl, aryl or substituted aryl; R’ is halo or OR; R is H. alkyl or substituted alkyl: R is a protecting group;

L is a covalent bond or a linking group; and 10 D is a drug. Preferably, R is TMS, TBDMS, acetyl, tosyl, mesyl or triflyl.

In another aspect, the invention is directed to compounds of 15 formula (VIII), which are useful for the production of com wherein pounds of formula (I): R" is CH, CHOH or CROH: R is CH, CHOH or CROH: R’ is OH, an alkylamino group substituted with a moiety that is negatively charged at physiological pH and (VIII) located 5 to 15 atoms from C-22 of the bile acid nucleus, a di-substituted alkylamino group substituted with L-D and a moiety that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid 25 nucleus, L-D or pharmaceutically acceptable salts thereof; R" is alkyl, aryl or substituted aryl; R’ is alkyl or substituted alkyl: L is a covalent bond or a linking group; and 30 D is a drug. Preferably, substituents R-R- are as follows: R2 is CH(R27)C(O)R’ or CH(R)CCR; R7 is H, alkyl, substituted alkyl, aryl or substituted aryl; wherein R’ is H, alkyl, substituted alkyl, aryl, substituted aryl, 35 OR', SR or NRR2; R is CH, CHOH or CROH: R’ is H, alkyl, substituted alkyl, aryl or substituted aryl; R is CH, CHOH or CROH: R" is H, alkyl, substituted alkyl, aryl or substituted aryl; R’ is OH, an alkylamino group substituted with a moiety R" is H, alkyl, substituted alkyl, aryl or substituted aryl; that is negatively charged at physiological pH and R is H, alkyl, substituted alkyl, aryl or substituted aryl. 40 located 5 to 15 atoms from C-22 of the bile acid nucleus, BRIEF DESCRIPTION OF THE DRAWINGS a di-substituted alkylamino group substituted with L-D FIGS. 1A-1E illustrate the preparation of steroidal building and a moiety that is negatively charged at physiological blocks that are used in the preparation of compounds of pH and located 5 to 15 atoms from C-22 of the bile acid formula (I). nucleus, L-D or pharmaceutically acceptable salts 45 FIG. 2 illustrates the preparation of amino acid building thereof; blocks that are used in the preparation of compounds of formula (I). R" is alkyl, aryl or substituted aryl; FIGS. 3A-3L illustrate synthetic strategies for obtaining R’ is alkyl or substituted alkyl; 50 compounds of formula (I), wherein the compounds have a L is a covalent bond or a linking group; and 5-membered fused heterocycle. FIGS. 4A-4G illustrate synthetic strategies for obtaining D is a drug. compounds of formula (I), wherein the compounds have a Preferably, substituents R-R'' are as follows: 6-membered fused heterocycle. FIGS. 5A-5E illustrate synthetic strategies for obtaining 55 compounds of formula (I), wherein the compounds have R7 is H. alkyl, substituted alkyl, aryl or substituted aryl; either a 7-membered or 8-membered fused heterocycle. R’ is H, alkyl, substituted alkyl, aryl, substituted aryl, FIGS. 6A-6D illustrate synthetic strategies for obtaining OR', SR or NR'R''. compounds of formula (I), wherein the compounds are R’ is H. alkyl, substituted alkyl, aryl or substituted aryl; derivatives of either glyco- or taurocholanic acids. 60 FIGS. 7A-7E illustrate synthetic strategies for conjugating R" is H. alkyl, substituted alkyl, aryl or substituted aryl; drug molecules or Surrogates to derivatives of either glyco- or R" is H. alkyl, substituted alkyl, aryl or substituted aryl; taurocholanic acids. R’ is H. alkyl, substituted alkyl, aryl or substituted aryl. DETAILED DESCRIPTION OF THE INVENTION In another aspect, the invention is directed to compounds of 65 formula (IX), which are useful for the production of com This invention provides compositions and methods for pro pounds of formula (I): viding enhanced systemic blood concentrations of orally US 7,727,976 B2 37 38 delivered drugs that are incompletely translocated across the unsaturation. This term is exemplified by groups such as intestinal wall of an animal. This invention also provides ethenylene (-CH=CH-), propenylene methods and compositions for the Sustained release of drugs, (—CH-CH=CH-), and the like. whether poorly or readily bioavailable via oral delivery to “Alkoxy' refers to the group “alkyl-O-” which includes, by animals. This invention further provides methods and com way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, positions for modulating cholesterol metabolism. However, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1.2- prior to describing this invention in further detail, the follow dimethylbutoxy, and the like. ing terms will first be defined: Alkyl refers to alkyl groups preferably having from 1 to 20 carbon atoms and more preferably 1 to 6 carbon atoms. Definitions 10 This term is exemplified by groups such as methyl, t-butyl, n-heptyl, octyl, dodecyl and the like. As used herein, the term “animal' refers to various species Alkylene' refers to a divalent alkylene group preferably Such as mammalian and avian species including, by way of having from 1 to 20 carbon atoms and more preferably 1 to 6 example, humans, cattle, sheep, horses, dogs, cats, turkeys, carbon atoms. This term is exemplified by groups such as chicken, and the like. Preferably, the animal is a mammal and 15 methylene (—CH2—), ethylene (-CH2CH2—), the propy even more preferably is a human. lene isomers (e.g., —CH2CHCH - and —CH(CH) Active metabolite of a drug” refers to products of in vivo CH ) and the like. modification of a compound of formula (I) which have thera Alkynyl refers to alkynyl group preferably having from 2 peutic or prophylactic effect. to 20 carbon atoms and more preferably 3 to 6 carbon atoms Active transport or active transport mechanism” refers to and having at least 1 and preferably from 1-2 sites of alkynyl the movement of molecules across cellular membranes that: unsaturation. a) is directly or indirectly dependent on an energy mediated Alkynylene' refers to a divalent alkynylene group prefer process (i.e. driven by ATP hydrolysis, ion gradient, etc); ably having from 2 to 20 carbon atoms and more preferably 1 b) occurs by facilitated diffusion mediated by interaction to 6 carbon atoms and having from 1 to 2 sites of alkynyl with specific transporter proteins; or 25 unsaturation. This term is exemplified by groups such as c) occurs through a modulated solute channel. ethynylene, propynylene and the like. Acyl refers to the groups H C(O)—, alkyl-C(O)—, Amidino” refers to the group HNC(=NH)— and the substituted alkyl-C(O)—, alkenyl-C(O)—, substituted alk term “alkylamidino” refers to compounds having 1 to 3 alkyl enyl-C(O)—, alkynyl-C(O)—, substituted alkynyl-C(O)— groups (e.g., alkylHNC(=NH)—). cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—, aryl-C 30 “Aminoacyl refers to the groups —NRC(O)alkyl, - NRC (O) , substituted aryl-C(O)—, heteroaryl-C(O)—, substi (O)Substituted alkyl, - NRC(O)cycloalkyl, —NRC(O)Sub tuted heteroaryl-C(O), heterocyclic-C(O)—, and substituted stituted cycloalkyl, - NRC(O)alkenyl, - NRC(O)Substi heterocyclic-C(O)— wherein alkyl, substituted alkyl, alk tuted alkenyl, - NRC(O)alkynyl, - NRC(O)Substituted enyl, Substituted alkenyl, alkynyl, Substituted alkynyl, alkynyl, - NRC(O)aryl, - NRC(O)substituted aryl, - NRC cycloalkyl, Substituted cycloalkyl, aryl, Substituted aryl, het 35 (O)heteroaryl, - NRC(O)substituted heteroaryl, - NRC(O) eroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, and —NRC(O)Substituted heterocyclic where heterocyclic are as defined herein. R is hydrogen or alkyl and wherein alkyl, substituted alkyl, Acylamino” refers to the group —C(O)NRR where each alkenyl, Substituted alkenyl, alkynyl. Substituted alkynyl, R is independently selected from the group consisting of cycloalkyl, Substituted cycloalkyl, aryl, Substituted aryl, het hydrogen, alkyl, Substituted alkyl, alkenyl, Substituted alk 40 eroaryl, substituted heteroaryl, heterocyclic and substituted enyl, alkynyl. Substituted alkynyl, aryl, Substituted aryl, heterocyclic are as defined herein. cycloalkyl, substituted cycloalkyl, heteroaryl, substituted “Aminocarbonyloxy' refers to the groups —NRC(O)O- heteroaryl, heterocyclic, substituted heterocyclic and where alkyl, —NRC(O)O-substituted alkyl, - NRC(O)O-alkenyl, each R is joined to form together with the nitrogen atom a —NRC(O)O-substituted alkenyl, - NRC(O)O-alkynyl, heterocyclic or substituted heterocyclic ring wherein alkyl, 45 —NRC(O)O-substituted alkynyl, - NRC(O)O-cycloalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, sub - NRC(O)O-substituted cycloalkyl, - NRC(O)O-aryl, stituted alkynyl, cycloalkyl, Substituted cycloalkyl, aryl, Sub - NRC(O)O-substituted aryl, - NRC(O)O-heteroaryl, stituted aryl, heteroaryl, substituted heteroaryl, heterocyclic - NRC(O)O-substituted heteroaryl, - NRC(O)O-heterocy and substituted heterocyclic are as defined herein. clic, and —NRC(O)O-substituted heterocyclic where R is Acyloxy' refers to the groups alkyl-C(O)O— substituted 50 hydrogen or alkyl and wherein alkyl, Substituted alkyl, alk alkyl-C(O)O , alkenyl-C(O)O—, substituted alkenyl-C(O) enyl, Substituted alkenyl, alkynyl, Substituted alkynyl, O— alkynyl-C(O)O— substituted alkynyl-C(O)O—, aryl cycloalkyl, Substituted cycloalkyl, aryl, Substituted aryl, het C(O)O substituted aryl-C(O)O—, cycloalkyl-C(O)O , eroaryl, substituted heteroaryl, heterocyclic and substituted substituted cycloalkyl-C(O)O—, heteroaryl-C(O)O—, sub heterocyclic are as defined herein. stituted heteroaryl-C(O)O heterocyclic-C(O)O—, and 55 “Aminocarbonylamino” refers to the groups —NRC(O) substituted heterocyclic-C(O)C)— wherein alkyl, substituted NRR, NRC(O)NR-alkyl, - NRC(O)NR-substituted alkyl, alkyl, alkenyl, Substituted alkenyl, alkynyl, Substituted alky - NRC(O)NR-alkenyl, - NRC(O)NR-substituted alkenyl, nyl, cycloalkyl, Substituted cycloalkyl, aryl, Substituted aryl, - NRC(O)NR-alkynyl, - NRC(O)NR-substituted alkynyl, heteroaryl, substituted heteroaryl, heterocyclic and substi - NRC(O)NR-aryl, - NRC(O)NR-substituted aryl, - NRC tuted heterocyclic are as defined herein. 60 (O)NR-cycloalkyl, - NRC(O)NR-substituted cycloalkyl, Alkenyl refers to alkenyl group preferably having from 2 - NRC(O)NR-heteroaryl, and NRC(O)NR-substituted to 20 carbon atoms and more preferably 2 to 6 carbon atoms heteroaryl, - NRC(O)NR-heterocyclic, and NRC(O)NR and having at least 1 and preferably from 1-2 sites of alkenyl substituted heterocyclic where each R is independently unsaturation. hydrogen, alkyl or where each R is joined to form together Alkenylene' refers to a divalent alkenylene group prefer 65 with the nitrogen atom a heterocyclic or substituted hetero ably having from 2 to 20 carbon atoms and more preferably 1 cyclic ring as well as where one of the amino groups is to 6 carbon atoms and having from 1 to 2 sites of alkenyl blocked by conventional blocking groups such as Boc, Cbz, US 7,727,976 B2 39 40 formyl, and the like and wherein alkyl, substituted alkyl, “Cycloalkylene' refers to divalent cyclic alkylene groups alkenyl, Substituted alkenyl, alkynyl. Substituted alkynyl, of from 3 to 8 carbon atoms having a single cyclic ring cycloalkyl, Substituted cycloalkyl, aryl, Substituted aryl, het including, by way of example, cyclopropylene, cyclobuty eroaryl, substituted heteroaryl, heterocyclic and substituted lene, cyclopentylene, cyclooctylene and the like. heterocyclic are as defined herein. “N,N-Dimethylcarbamyloxy” refers to the group —OC Aminothiocarbonylamino” refers to the groups —NRC (O)N(CH). (S)NRR, NRC(S)NR-alkyl, - NRC(S)NR-substituted “Drug/cleavable linker/transporter compound” (which alkyl, - NRC(S)NR-alkenyl, - NRC(S)NR-substituted alk Sometimes is referred to as the "drug-transporter compound'. enyl, - NRC(S)NR-alkynyl, - NRC(S)NR-substituted alky “drug/linker/transporter compound” and “drug/cleavable nyl, - NRC(S)NR-aryl, - NRC(S)NR-substituted aryl, 10 linker/transporter conjugate' refers to a compound of for - NRC(S)NR-cycloalkyl, - NRC(S)NR-substituted mula (I). cycloalkyl, - NRC(S)NR-heteroaryl, and NRC(S)NR “Drugs that are either completely or incompletely translo substituted heteroaryl, - NRC(S)NR-heterocyclic, and cated across the intestinal wall into the systemic blood circu —NRC(S)NR-substituted heterocyclic where each R is inde lation of an animal' refer to any of the well known orally pendently hydrogen, alkyl or where each R is joined to form 15 delivered drugs currently delivered by oral administration as together with the nitrogen atom a heterocyclic or Substituted well as drugs which cannot be orally administered because heterocyclic ring as well as where one of the amino groups is Such drugs are insufficiently translocated across the intestinal blocked by conventional blocking groups such as Boc, Cbz, wall of an animal to provide therapeutic or prophylactic blood formyl, and the like and wherein alkyl, substituted alkyl, concentrations in said animal. alkenyl, Substituted alkenyl, alkynyl. Substituted alkynyl, Preferably, drugs that fall into the following categories: cycloalkyl, Substituted cycloalkyl, aryl, Substituted aryl, het i) drugs which are insufficiently translocated across the eroaryl, substituted heteroaryl, heterocyclic and substituted intestinal wall to provide therapeutic or prophylactic heterocyclic are as defined herein. blood concentrations; Aryl or 'Ar' refers to a monovalent unsaturated aromatic ii) incompletely translocated drugs; or carbocyclic group of from 6 to 14 carbon atoms having a 25 iii) drugs that are either completely or incompletely trans single ring (e.g., phenyl) or multiple condensed rings (e.g., located across the intestinal wall into the systemic blood naphthyl or anthryl) which condensed rings may or may not circulation of an animal be aromatic (e.g., 2-benzoxazolinone, 2H-1,4-benzoxazin-3 contain Suitable functionality to provide points of linkage (4H)-one-7yl, and the like). Preferred aryls include phenyl informing a compound of formula (I) above. Such func and naphthyl. 30 tionality includes, by way of example, carboxyl groups, Arylene' refers to a divalent unsaturated aromatic car amine groups and hydroxyl groups. bocyclic group of from 6 to 14 carbon atoms having a single Examples of drugs containing carboxyl groups include, for ring (e.g., phenylene) or multiple condensed rings (e.g., naph instance, angiotensin-converting enzyme inhibitors such as thylene oranthrylene) which condensed rings may or may not alecapril, captopril, 1-4-carboxy-2-methyl-2R,4R-pen be aromatic. Preferred arylenes include phenylene and naph 35 tanoyl-2,3-dihydro-2S-indole-2-carboxylic acid, enalaprilic thylene. acid, lisinopril, N-cyclopentyl-N-3-(2,2-dimethyl-1-oxo Aryloxy' refers to the group aryl-O which includes, by propyl)thio-2-methyl-1-oxopropylglycine, pivopril. (2R, way of example, phenoxy, naphthoxy, and the like. 4R)-2-hydroxyphenyl)-3-(3-mercaptopropionyl)-4-thiazo Aryloxyaryl' refers to the group -aryl-O-aryl. lidinecarboxylic acid, (S) benzamido-4-oxo-6- 40 phenylhexenoyl-2-carboxypyrrolidine. 2S-1R*(R*))2C. “Bile acid transport system” refers to any membrane trans 3CfB.7O?3-12-1-carboxy-3-phenylpropyl-amino-1- porter protein capable of causing a bile acid or a derivative oxopropyloctahydro-1H-indole-2-carboxylic acid, 3S-1 thereof to be translocated across a membrane of a cell of the R*(R)).3R)-2-[2-(1-carboxy-3-phenylpropyl-amino gastrointestinal tract or liver. 1-oxopropyl-1,2,3,4-tetrahydro-3-isoquinolone carboxylic “Cleavable linker or cleavable linking group' refers to linkers that contain one or more functional groups which 45 acid and tiopronin; cephalosporin antibiotics Such as cefaclor, permit cleavage of Such groups in vivo by, for example, cefadroxil, cefamandole, cefatrizine, cefazedone, cefaZuflur, endogenous enzymes. Preferably, the functional group Sub cefazolin, cefbuperaZone, cefinenoxime, cefinetazole, cefo ject to cleavage in the cleavable linker is attached adjacent the dizime, cefonicid, cefoperaZone, ceforamide, cefotaxime, drug moiety, D. Such that upon cleavage, the free drug is cefotefan, cefotiam, cefoxitin, cefpimizole, cefpirome, released. The cleavable linker preferably comprises one or 50 cefroxadine, cefsulodin, ce?piramide, ceftazidime, ceftezole, more functional groups such as ester groups, amide groups, ceftizoxime, ceftriaxone, cefuroxime, cephacetrile, cephal glycolamide ester groups, amidomethyl esters, acyloxyalkyl exin, cephaloglycin, cephaloridine, cephalosporin, cepha esters, alkoxycarbonyloxyalkyl esters, and the like. FIGS. 7A none, cephradine and latamoxef penicillins such as amoxy through 7D illustrate suitable cleavable linker functionality cillin, amplicillin, apalcillin, azidocillin, azlocillin, 55 benzylpencillin, carbenicillin, carfecillin, carindacillin, clox which can be used. acillin, cyclacillin, dicloxacillin, epicillin, flucloxacillin, het “Conjugating refers to the formation of a covalent bond. acillin, methicillin, mezlocillin, nafcillin, oxacillin, phenethi “Cycloalkenyl refers to cyclic alkenyl groups of form 3 to cillin, piperazillin, Sulbeniclin, temocillin and ticarcillin; 8 carbon atoms having a single cyclic ring. non-steroidal antiinflammatory agents such as acametacin, “Cycloalkenylene' refers to a divalent cyclic alkenylene 60 alclofenac, alminoprofen, aspirin (acetylsalicylic acid), 4-bi groups of form 3 to 8 carbonatoms having a single cyclic ring. phenylacetic acid, bucloxic acid, carprofen, cinchofen, cim “Cycloalkoxy' refers to —O-cycloalkyl groups. netacin, clometacin, clonixin, diclenofac, diflunisal, etod “Cycloalkyl refers to cyclic alkyl groups of from 3 to 8 olac, fenbufen, fenclofenac, fenclosic acid, fenoprofen, carbonatoms having a single cyclic ring including, by way of ferobufen, flufenamic acid, flufenisal, flurbiprofin, fluprofen, example, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl 65 flutiazin, ibufenac, ibuprofen, indomethacin, indoprofen, and the like. Excluded from this definition are multi-ring ketoprofen, ketorolac, lonazolac, loxoprofen, meclofenamic alkyl groups such as adamantanyl, etc. acid, mefenamic acid, 2-(8-methyl-10,11-dihydro-11-oxod US 7,727,976 B2 41 42 ibenzb.foxepin-2-yl)propionic acid, naproxen, nifluminic Sone, budenosid, chlorprednison, clobetasol, clobetaSone, acid, O-(carbamoylphenoxy)acetic acid, oxoprozin, pirpro corticosteron, cortisone, cortodexon, dexamethason, flucor fen, prodolic acid, Salicylic acid, salicylsalicylic acid, Sulin tolon, fludrocortisone, flumethasone, flunisolid, flupredniso dac, Suprofen, tiaprofenic acid, tolfenamic acid, tolmetin and lon, flurandrenolide, flurandrenolon acetonide, hydrocorti Zopemirac, prostaglandins such as ciprostene, 16-deoxy-16 5 Sone, meprednisone, methylpresnisolon, paramethasone, hydroxy-16-vinyl prostaglandin E, 6, 16-dimethylprostag prednisolon, prednisol, triamcinolon and triamcinolon landin E, epoprostostenol, meteneprost, nileprost, prostacy acetonide; narcotic agonists and antagonists such as apomor clin, prostaglandins E, E, or F, and thromboxane A2; phine, buprenorphine, butorphanol, codein, cyclazocin, quinolone antibiotics Such as acrosoxacin, cinoxacin, cipro hydromorphon, ketobemidon, levallorphan, levorphanol, floxacin, enoxacin, flumequine, naladixic acid, norfloxacin, 10 metazocin, morphine, nalbuphin, nalmefen, naloxon, nalor ofloxacin, oxolinic acid, pefloxacin, pipemidic acid and piro phine, naltrexon, oxycodon, oxymorphon and pentazocin, midic acid. stimulants such asmazindol and pseudoephidrine; anaesthet Representative drugs containing amine groups include: ics such as hydroxydion and propofol; B-receptor blockers acebutalol, albuterol, alprenolol, atenolol, bunolol, butopam Such as acebutolol, albuterol, alprenolol, atenolol, betazolol. ine, butoxamine, carbuterol, cartelolol, colterol, deterenol, 15 bucindolol, cartelolol, celiprolol, cetamolol, labetalol, dexpropanolol, diacetolol, dobutamine, exaprolol, expre levobunelol, metoprolol, metipranolol, nadolol, oxyprenolol. nolol, fenoterol, fenyripol, labotolol, levobunolol, metolol. pindolol, propanolol and timolol, C-sympathomimetics Such metaproterenol, metoprolol, nadolol, pamatolol, penbutalol, as adrenalin, metaraminol, midodrin, norfenefrin, octapam pindolol, pirbuterol, practolol, prenalterol, primidolol, prizi ine, Oxedrin, oxillofrin, Oximetazolin and phenylefrin; B-sym dilol, procaterol, propanolol, quinterenol, rimiterol, ritodrine, pathomimetics such as bamethan, clenbuterol, fenoterol, solotol, soterenol, sulfiniolol, sulfinterol, Sulictidil, tazaolol, hexoprenalin, isoprenalin, isoxSuprin, orciprenalin, reprot terbutaline, timolol, tiprenolol, tipridil, tolamolol, thiabenda erol, salbutamol and terbutalin; bronchodilators such as car Zole, albendazole, albutoin, alinidine, alizapride, amiloride, buterol, dyphillin, etophyllin, fenoterol, pirbuterol, rimiterol aminorex, aprinocid, cambendazole, cimetidine, clonidine, and terbutalin; cardiotonics Such as digitoxin, dobutamin, cyclobenzadole, etintidine, fenbendazole, fenmetazole, 25 etillefrin and prenalterol; antimycotics such as amphotericin flubendazole, fludorex, lobendazole, mebendazole, metazo B, chlorphenesin, nystatin and perimycin; anticoagulants line, nocodazole, Oxfendazole, oxibendazole, OXmetidine, Such as acenocoumarol, dicoumarol, phenprocoumon and parbendazole, ranitidine, tetrahydrazoline, tiamenidine, tina warfarin; vasodilators such as bamethan, dipyrimadol, dipro Zoline, tiotidine, tolaZoline, tramaZoline, Xylometazoline, phyllin, isoxSuprin, Vincaminand Xantinol nicotinate; antihy dimethoxyphenethylamine, N-3(R)-2-piperidin-4-yl) 30 pocholesteremics Such as compactin, eptastatin, mevinolin ethyl-2-piperidone-1-yl)acetyl-3(R)-methyl-B-alanine, and simvastatin; miscellaneous drugs such as bromperidol adrenolone, aletamine, amidephrine, amphetamine, aspar (antipsychotic), dithranol (psoriasis) ergotamine (migraine) tame, bamethan, betahistine, clorprenaline, chlortermine, ivermectin (antihelminthic), metronidazole and secnizadole dopamine, ephrinephrine etryptamine, fenfluramine, methyl (antiprotozoals), nandrolon (anabolic), propafenon and qui dopamine, norepinephrine, tocainide, enviroXime, nife 35 nadine (antiarythmics), serotonin (neurotransmitter) and sily dipine, nimodipine, triamterene, norfloxacin and similar bin (hepatic disturbance). compounds Such as pipedemic acid, 1-ethyl-6-fluoro-1,4-di “Drugs which are insufficiently translocated across the hydro-4-oxo-7-(1-piperazinyl)-1.8-napthyridine-3-carboxy intestinal wall to provide therapeutic or prophylactic blood lic acid, 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(piper concentrations' refers to drugs which, when administered azinyl)-3-quinolinecarboxylic acid. 40 orally at tolerable doses or using a practical dosage regimen, Representative drugs containing hydroxy groups include: cannot provide blood concentrations of the drug or active steroidal hormones such as allylestrenol, cingestol, dehy metabolite thereof sufficient to effect either disease therapy or droepiandrosteron, dienostrol, diethylstilbestrol, dimethis prophylaxis. Examples of Such drugs include, for instance: teron, ethyneron, ethynodiol, estradiol, estron, ethinyl estra (i) the antibiotics, cefepime, ceftazidime, ceftriaxone, diol, ethisteron, lynestrenol, mestranol, methyl testosterone, 45 aztreonam, meropenem, imipenem; norethindron, norgestrel, norvinsteron, oxogeston, (ii) the anticancer agents, paclitaxel, docetaxel, doxorubi quinestrol, testosteron and tigestol; tranquilizers such as cin, fludarabine, gemcitabine, pentostatin, camptoth dofexazepam, hydroxy Zin, lorazepam and oxazepam; neuro eC1n, leptics such as acetophenazine, carphenazine, fluphenazine, (iii) the thrombin inhibitors, argatroban, melagatran, nap perpheny Zine and piperaetazine; cytostatics such as aclarubi 50 Sagatran; cin, daunorubicin, dihydro-5-azacytidine, doxorubicin, epi (iv) the renin inhibitors, enalkiren, ciprokiren, terlakiren: rubicin, estramustin, etoposide, 7-hydroxychlorpromazin, (v) the HIV protease inhibitors, kynostatin, A-77003, neplanocin A, pentostatin, podophyllotoxin, vinblastin, Vin SB-206343, XM-323; cristin, Vindesin; hormones and hormone antagonists such as (vi) the gpIb/IIIa inhibitors, lamifiban; orbofiban; buSerilin, gonadoliberin, icatibrant and leuprorelin acetate; 55 fradafiban, FK-633; and antihistamines such as terphenadine; analgesics such as (vii) the influenza neuraminidase inhibitors, Zanamivir, diflunisal, naproxol, paracetamol, salicylamide and Salicyclic BCX-1812. acid; antibiotics Such as azidamphenicol, cefamandol, “Guanidino” refers to the groups - NRC(=NR)NRR, chloramphenicol, clavulanic acid, clindamycin, comptoth NRC(=NR)NR-alkyl, - NRC(—NR)NR-substituted ecin, demeclocyclin, doxycyclin, imipenem, latamoxef. 60 alkyl, - NRC(=NR)NR-alkenyl, - NRC(=NR)NR-sub novobiocin, oleandomycin, oxytetracyclin, tetracyclin and stituted alkenyl, - NRC(—NR)NR-alkynyl, - NRC(=NR) thiamenicol; prostaglandins such as arbaprostil, carboprost NR-substituted alkynyl, - NRC(—NR)NR-aryl, - NRC and prostacydin, antidepressives such as 8-hydroxychlorimi (—NR)NR-substituted aryl, - NRC(—NR)NR-cycloalkyl, pramine and 2-hydroxyimipramine; antihypertonics such as - NRC(—NR)NR-heteroaryl, - NRC(=NR)NR-substi Sotarol and fenoldopam; anticholinerogenics Such as piperi 65 tuted heteroaryl, - NRC(=NR)NR-heterocyclic, and dine, carbidopa, procyclidin and trihexyphenidal; antialler - NRC(—NR)NR-substituted heterocyclic where each R is genics such as cromolyn; glucocorticoids such as betametha independently hydrogen and alkyl as well as where one of the US 7,727,976 B2 43 44 amino groups is blocked by conventional blocking groups gen within the ring wherein, in fused ring systems, one or such as Boc, Cbz, formyl, and the like and wherein alkyl, more the rings can be aryl or heteroaryl. substituted alkyl, alkenyl, substituted alkenyl, alkynyl, sub “Heterocyclyloxy' refers to the group —O-heterocyclic stituted alkynyl, cycloalkyl, Substituted cycloalkyl, aryl, Sub and “substituted heterocyclyloxy' refers to the group —O- stituted aryl, heteroaryl, substituted heteroaryl, heterocyclic 5 substituted heterocyclic. and substituted heterocyclic are as defined herein. “Incompletely translocated drugs’ refer to those drugs “Guanidinosulfone' refers to the groups —NRC(=NR) wherein less than 90%, typically less than 75%, and more NRSO-alkyl, - NRC(—NR)NRSO-substituted alkyl, typically less than 50% of the drug delivered orally to an - NRC(—NR)NRSO-alkenyl, NRC(=NR)NRSO animal is absorbed into the systemic blood circulation of the substituted alkenyl, - NRC(—NR)NRSO-alkynyl, - NRC 10 animal as the drug itself or as an active metabolite thereof, (—NR)NRSO-substituted alkynyl, - NRC(—NR)NRSO wherein incomplete absorption is due, at least in part, to aryl, - NRC(=NR)NRSO-substituted aryl, - NRC(=NR) incomplete translocation of the drug or active metabolite NRSO-cycloalkyl, - NRC(=NR)NRSO-substituted thereof across the intestinal wall of the animal. Examples of cycloalkyl, - NRC(=NR)NRSO-heteroaryl, and - NRC incompletely translocated drugs include, for instance, bis (—NR)NRSO-substituted heteroaryl, - NRC(=NR) 15 phosphonates such as alendronate, clondronate, ibandronate, NRSO-heterocyclic, and - NRC(—NR)NRSO-substi incadronate, pamidronate, risedronate, tiludronate, Zoledr tuted heterocyclic where each R is independently hydrogen Onate. and alkyl and wherein alkyl, substituted alkyl, alkenyl, sub "Leaving group' refers to a chemical moiety that can either stituted alkenyl, alkynyl. Substituted alkynyl, cycloalkyl, Sub be displaced by a nucleophile (SN2 reaction) or eliminated in stituted cycloalkyl, aryl, substituted aryl, heteroaryl, substi the presence of a base (E2 reaction). Examples of leaving tuted heteroaryl, heterocyclic and substituted heterocyclic are groups include, without limitation, the following: tosyl, as defined herein. mesyl and triflyl. "Halo’ or “halogen refers to fluoro, chloro, bromo and “A moiety selected to permit a compound of formula (I) to iodo and preferably is either chloro or bromo. be translocated across the intestinal wall of an animal via the “Heteroaryl” refers to an aromatic carbocyclic group of 25 bile acid transport system” refers to compounds which, when from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected conjugated to the drug/cleavable linker moiety, are translo from the group consisting of oxygen, nitrogen and Sulfur cated across the intestinal wall via the bile acid transport within the ring. Such heteroaryl groups can have a single ring system. Evaluation of which candidate compounds can be so (e.g., pyridyl or furyl) or multiple condensed rings (e.g., translocated across the intestinal wall can be conducted by the indolizinyl or benzothienyl). Preferred heteroaryls include 30 in vitro assay set forth in Examples 50 and 51 below. pyridyl, pyrrolyl, indolyl and furyl. “Orally delivered drugs’ refer to drugs which are admin “Heterocycle' or "heterocyclic” refers to a saturated or istered to an animal in an oral form, preferably, in a pharma unsaturated group having a single ring or multiple condensed ceutically acceptable diluent. Oral delivery includes inges rings, from 1 to 10 carbonatoms and from 1 to 4 hetero atoms tion of the drug as well as oral gavage of the drug. selected from the group consisting of nitrogen, Sulfur or oxy 35 "Oxycarbonylamino” refers to the groups —OC(O)NH, gen within the ring wherein, in fused ring systems, one or –OC(O)NRR, OC(O)NR-alkyl, OC(O)NR-substituted more the rings can be aryl or heteroaryl. alkyl, —OC(O)NR-alkenyl, —OC(O)NR-substituted alk Examples of heterocycles and heteroaryls include, but are enyl, —OC(O)NR-alkynyl, —OC(O)NR-substituted alky not limited to, aZetidine, pyrrole, imidazole, pyrazole, pyri nyl, - OC(O)NR-cycloalkyl, - OC(O)NR-substituted dine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, 40 cycloalkyl, —OC(O)NR-aryl, —OC(O)NR-substituted aryl, indole, dihydroindole, indazole, purine, quinolizine, iso –OC(O)NR-heteroaryl, - OC(O)NR-substituted het quinoline, quinoline, phthalazine, naphthylpyridine, qui eroaryl, —OC(O)NR-heterocyclic, and —OC(O)NR-substi noxaline, quinazoline, cinnoline, pteridine, carbazole, carbo tuted heterocyclic where R is hydrogen, alkyl or where each line, phenanthridine, acridine, phenanthroline, isothiazole, R is joined to form, together with the nitrogen atom a hetero 45 cyclic or Substituted heterocyclic ring and wherein alkyl, phenazine, isoxazole, phenoxazine, phenothiazine, imidazo substituted alkyl, alkenyl, substituted alkenyl, alkynyl, sub lidine, imidazoline, piperidine, piperazine, indoline, phthal stituted alkynyl, cycloalkyl, Substituted cycloalkyl, aryl, Sub imide, 1.2.3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahy stituted aryl, heteroaryl, substituted heteroaryl, heterocyclic drobenzobthiophene, thiazole, thiazolidine, thiophene, and substituted heterocyclic are as defined herein. benzobthiophene, morpholinyl, thiomorpholinyl (also “Oxythiocarbonylamino” refers to the groups —OC(S) referred to as thiamorpholinyl), piperidinyl, pyrrolidine, tet NH, OC(S)NRR, OC(S)NR-alkyl, - OC(S)NR-sub rahydrofuranyl, and the like. stituted alkyl, —OC(S)NR-alkenyl, —OC(S)NR-substituted “Heteroarylene' refers to a divalent aromatic carbocyclic alkenyl, —OC(S)NR-alkynyl, —OC(S)NR-substituted alky group of from 2 to 10 carbon atoms and 1 to 4 heteroatoms nyl, - OC(S)NR-cycloalkyl, - OC(S)NR-substituted selected from the group consisting of oxygen, nitrogen and 55 cycloalkyl, —OC(S)NR-aryl, —OC(S)NR-substituted aryl, Sulfur within the ring. Such heteroarylene groups can have a –OC(S)NR-heteroaryl, OC(S)NR-substituted heteroaryl, single ring (e.g., pyridylene or furylene) or multiple con —OC(S)NR-heterocyclic, and —OC(S)NR-substituted het densed rings (e.g., indolizinylene or benzothienylene). Pre erocyclic where R is hydrogen, alkyl or where each R is ferred heteroarylenes include pyridylene, pyrrolylene, joined to form together with the nitrogenatom a heterocyclic indolylene and furylene. 60 or substituted heterocyclic ring and wherein alkyl, substituted “Heteroaryloxy” refers to the group —O-heteroaryl and alkyl, alkenyl, Substituted alkenyl, alkynyl, Substituted alky “substituted heteroaryloxy' refers to the group —O-substi nyl, cycloalkyl, Substituted cycloalkyl, aryl, Substituted aryl, tuted heteroaryl. heteroaryl, substituted heteroaryl, heterocyclic and substi “Heterocyclene' refers to a divalent saturated or unsatur tuted heterocyclic are as defined herein. ated group having a single ring or multiple condensed rings, 65 "Pharmaceutically acceptable excipient” refers to an from 1 to 10 carbon atoms and from 1 to 4 hetero atoms excipient that is useful in preparing a pharmaceutical compo selected from the group consisting of nitrogen, Sulfur or oxy sition that is generally safe, non-toxic and neither biologically US 7,727,976 B2 45 46 nor otherwise undesirable, and includes an excipient that is groups such as Boc, Cbz, formyl, and the like or alkenyl/ acceptable for veterniary use as well as human pharmaceuti substituted alkenyl groups substituted with —SO-alkyl, cal use. "A pharmaceutically acceptable excipient as used in —SO-substituted alkyl, —SO-alkenyl, -SO-substituted the specification and claims includes both one and more than alkenyl, -SO-cycloalkyl, -SO-substituted cycloalkyl, one such excipient. 5 —SO-aryl, -SO-substituted aryl, -SO-heteroaryl, “Pharmaceutically acceptable salt” refers to pharmaceuti —SO-substituted heteroaryl, -SO-heterocyclic. —SO cally acceptable salts of a compound of the present invention substituted heterocyclic and—SONRR where Rishydrogen which salts are derived from a variety of organic and inor or alkyl. ganic counter ions well known in the art and include, by way “Substituted alkenylene' refers to alkenylene groups hav of example only, sodium, potassium, calcium, magnesium, 10 ing from 1 to 5 substituents selected from the group consisting ammonium, tetraalkylammonium, and the like; and when the of alkoxy, Substituted alkoxy, acyl, acylamino, thiocarbony molecule contains a basic functionality, salts of organic or lamino, acyloxy, amino, amidino, alkylamidino, thioamidino, inorganic acids, such as hydrochloride, hydrobromide, tar aminoacyl, aminocarbonylamino, aminothiocarbonylamino, trate, mesylate, acetate, maleate, oxalate and the like. aminocarbonyloxy, aryl, Substituted aryl, aryloxy, Substituted “Practical dosage regimen” refers to a schedule of drug 15 aryloxy, aryloxyaryl, Substituted aryloxyaryl, halogen, administration that is practical for a patient to comply with. hydroxyl, cyano, nitro, carboxyl, carboxylalkyl, carboxyl For human patients, a practical dosage regimen for an orally substituted alkyl, carboxyl-cycloalkyl, carboxyl-substituted administered drug is likely to be an aggregate dose of less than cycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxy 10 g/day. lheteroaryl, carboxyl-substituted heteroaryl, carboxylhetero “Protecting group' refers to a chemical moiety used to cyclic, carboxyl-substituted heterocyclic, cycloalkyl, Substi prevent certain functional groups from undergoing undesired tuted cycloalkyl, guanidino, guanidinosulfone, thiol, reactions. Examples of protecting groups include, without thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl, limitation, the following: TMS, TBDMS and acetyl. thiocycloalkyl, substituted thiocycloalkyl, thioheteroaryl, “Substituted alkenyl refers to alkenyl groups having from substituted thioheteroaryl, thioheterocyclic, substituted thio 1 to 5 substituents selected from the group consisting of 25 heterocyclic, heteroaryl, substituted heteroaryl, heterocyclic, alkoxy, Substituted alkoxy, acyl, acylamino, thiocarbony Substituted heterocyclic, cycloalkoxy, substituted lamino, acyloxy, amino, amidino, alkylamidino, thioamidino, cycloalkoxy, heteroaryloxy, Substituted heteroaryloxy, het aminoacyl, aminocarbonylamino, aminothiocarbonylamino, erocyclyloxy, Substituted heterocyclyloxy, oxycarbony aminocarbonyloxy, aryl, Substituted aryl, aryloxy, Substituted lamino, oxythiocarbonylamino, —OS(O)-alkyl, —OS(O)- aryloxy, aryloxyaryl, Substituted aryloxyaryl, halogen, 30 substituted alkyl, —OS(O)-aryl, —OS(O)-substituted aryl, hydroxyl, cyano, nitro, carboxyl, carboxylalkyl, carboxyl —OS(O)-heteroaryl, —OS(O)-substituted heteroaryl, substituted alkyl, carboxyl-cycloalkyl, carboxyl-substituted —OS(O)-heterocyclic. —OS(O)-substituted heterocyclic, cycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxy —OSO NRR where R is hydrogen or alkyl, - NRS(O)- lheteroaryl, carboxyl-substituted heteroaryl, carboxylhetero alkyl, - NRS(O)-substituted alkyl, —NRS(O)-aryl, cyclic, carboxyl-substituted heterocyclic, cycloalkyl, Substi 35 —NRS(O)-substituted aryl, - NRS(O)-heteroaryl, - NRS tuted cycloalkyl, guanidino, guanidinosulfone, thiol, (O)-substituted heteroaryl, —NRS(O)-heterocyclic, thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl, —NRS(O)-substituted heterocyclic, NRS(O) NR thiocycloalkyl, substituted thiocycloalkyl, thioheteroaryl, alkyl, - NRS(O), NR-substituted alkyl, - NRS(O)— substituted thioheteroaryl, thioheterocyclic, substituted thio NR-aryl, - NRS(O) NR-substituted aryl, - NRS(O)— heterocyclic, heteroaryl, substituted heteroaryl, heterocyclic, 40 NR-heteroaryl, - NRS(O), NR-substituted heteroaryl, Substituted heterocyclic, cycloalkoxy, substituted —NRS(O), NR-heterocyclic, NRS(O), NR-substi cycloalkoxy, heteroaryloxy, Substituted heteroaryloxy, het tuted heterocyclic where R is hydrogen or alkyl, mono- and erocyclyloxy, Substituted heterocyclyloxy, oxycarbony di-alkylamino, mono- and di-(Substituted alkyl)amino, lamino, oxythiocarbonylamino, —OS(O)-alkyl, —OS(O)- mono- and di-arylamino, mono- and di-substituted ary substituted alkyl, —OS(O)-aryl, —OS(O)-substituted aryl, 45 lamino, mono- and di-heteroarylamino, mono- and di-substi —OS(O)-heteroaryl, —OS(O)-substituted heteroaryl, tuted heteroarylamino, mono- and di-heterocyclic amino, —OS(O)-heterocyclic, —OS(O)-substituted heterocyclic, mono- and di-substituted heterocyclic amino, unsymmetric —OSO NRR where R is hydrogen or alkyl, - NRS(O)- di-substituted amines having different substituents selected alkyl, - NRS(O)-substituted alkyl, - NRS(O)-aryl, from the group consisting of alkyl, Substituted alkyl, aryl, —NRS(O)-substituted aryl, - NRS(O)-heteroaryl, - NRS 50 substituted aryl, heteroaryl, substituted heteroaryl, heterocy (O)-substituted heteroaryl, - NRS(O)-heterocyclic, clic, Substituted heterocyclic and Substituted alkenyl groups —NRS(O)-substituted heterocyclic, NRS(O) NR having amino groups blocked by conventional blocking alkyl, —NRS(O), NR-substituted alkyl, - NRS(O)— groups such as Boc, Cbz, formyl, and the like or alkenyl/ NR-aryl, - NRS(O), NR-substituted aryl, - NRS(O) substituted alkenyl groups substituted with —SO-alkyl, NR-heteroaryl, —NRS(O) NR-substituted heteroaryl, 55 —SO-substituted alkyl, —SO-alkenyl, -SO-substituted —NRS(O), NR-heterocyclic, NRS(O), NR-substi alkenyl, -SO-cycloalkyl, -SO-substituted cycloalkyl, tuted heterocyclic where R is hydrogen or alkyl, mono- and —SO-aryl, -SO-substituted aryl, -SO-heteroaryl, di-alkylamino, mono- and di-(substituted alkyl)amino, —SO-substituted heteroaryl, -SO-heterocyclic. —SO mono- and di-arylamino, mono- and di-substituted ary substituted heterocyclic and—SONRR where R is hydrogen lamino, mono- and di-heteroarylamino, mono- and di-Substi 60 or alkyl. tuted heteroarylamino, mono- and di-heterocyclic amino, “Substituted alkoxy' refers to the group “substituted alkyl mono- and di-substituted heterocyclic amino, unsymmetric O . di-substituted amines having different substituents selected “Substituted alkyl refers to an alkyl group, preferably of from the group consisting of alkyl, Substituted alkyl, aryl, from 1 to 20 carbon atoms, having from 1 to 5 substituents substituted aryl, heteroaryl, substituted heteroaryl, heterocy 65 selected from the group consisting of alkoxy, Substituted clic, Substituted heterocyclic and Substituted alkenyl groups alkoxy, acyl, acylamino, thiocarbonylamino, acyloxy, amino, having amino groups blocked by conventional blocking amidino, alkyl amidino, thioamidino, aminoacyl, aminocar US 7,727,976 B2 47 48 bonylamino, aminothiocarbonylamino, aminocarbonyloxy, lamino, oxythiocarbonylamino, —OS(O)-alkyl, —OS(O)- aryl, Substituted aryl, aryloxy, Substituted aryloxy, aryloxy substituted alkyl, —OS(O)-aryl, —OS(O)-substituted aryl, laryl, Substituted aryloxyaryl, cyano, halogen, hydroxyl, —OS(O)-heteroaryl, —OS(O)-substituted heteroaryl, nitro, carboxyl, carboxylalkyl, carboxyl-substituted alkyl, —OS(O)-heterocyclic. —OS(O)-substituted heterocyclic, carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, car —OSO NRR where R is hydrogen or alkyl, - NRS(O)- boxylaryl, carboxyl-substituted aryl, carboxylheteroaryl, car alkyl, - NRS(O)-substituted alkyl, —NRS(O)-aryl, boxyl-substituted heteroaryl, carboxylheterocyclic, car —NRS(O)-substituted aryl, - NRS(O)-heteroaryl, - NRS boxyl-substituted heterocyclic, cycloalkyl, substituted (O)-substituted heteroaryl, —NRS(O)-heterocyclic, cycloalkyl, guanidino, guanidinosulfone, thiol, thioalkyl, —NRS(O)-substituted heterocyclic, NRS(O), NR substituted thioalkyl, thioaryl, substituted thioaryl, thiocy 10 alkyl, - NRS(O), NR-substituted alkyl, - NRS(O)— cloalkyl, substituted thiocycloalkyl, thioheteroaryl, substi NR-aryl, - NRS(O) NR-substituted aryl, - NRS(O)— tuted thioheteroaryl, thioheterocyclic, substituted thiohetero NR-heteroaryl, - NRS(O) NR-substituted heteroaryl, cyclic, heteroaryl, substituted aryl, substituted heteroaryl, —NRS(O), NR-heterocyclic, NRS(O), NR-substi heterocyclic, Substituted heterocyclic, cycloalkoxy, Substi tuted heterocyclic where R is hydrogen or alkyl, mono- and tuted cycloalkoxy, heteroaryloxy, Substituted heteroaryloxy, 15 di-alkylamino, mono- and di-(Substituted alkyl) amino, heterocyclyloxy, Substituted heterocyclyloxy, oxycarbony mono- and di-arylamino, mono- and di-substituted ary lamino, oxythiocarbonylamino, —OS(O)-alkyl, —OS(O)- lamino, mono- and di-heteroarylamino, mono- and di-substi substituted alkyl, —OS(O)-aryl, —OS(O)-substituted aryl, tuted heteroarylamino, mono- and di-heterocyclic amino, —OS(O)-heteroaryl, —OS(O)-substituted heteroaryl, mono- and di-substituted heterocyclic amino, unsymmetric —OS(O)-heterocyclic. —OS(O)-substituted heterocyclic, di-substituted amines having different substituents selected —OSO NRR where R is hydrogen or alkyl, - NRS(O)- from the group consisting of alkyl, Substituted alkyl, aryl, alkyl, - NRS(O)-substituted alkyl, - NRS(O)-aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocy —NRS(O)-substituted aryl, - NRS(O)-heteroaryl, - NRS clic, Substituted heterocyclic and Substituted alkenyl groups (O)-substituted heteroaryl, - NRS(O)-heterocyclic, having amino groups blocked by conventional blocking —NRS(O)-substituted heterocyclic, NRS(O) NR 25 groups such as Boc, Cbz, formyl, and the like or alkenyl/ alkyl, —NRS(O), NR-substituted alkyl, - NRS(O)— substituted alkenyl groups substituted with —SO-alkyl, NR-aryl, - NRS(O) NR-substituted aryl, - NRS(O)— —SO-substituted alkyl, —SO-alkenyl, -SO-substituted NR-heteroaryl, - NRS(O), NR-substituted heteroaryl, alkenyl, -SO-cycloalkyl, -SO-substituted cycloalkyl, —NRS(O), NR-heterocyclic, NRS(O), NR-substi —SO-aryl, -SO-substituted aryl, -SO-heteroaryl, tuted heterocyclic where R is hydrogen or alkyl, mono- and 30 —SO-substituted heteroaryl, -SO-heterocyclic. —SO di-alkylamino, mono- and di-(substituted alkyl)amino, substituted heterocyclic and—SONRR where R is hydrogen mono- and di-arylamino, mono- and di-substituted ary or alkyl. lamino, mono- and di-heteroarylamino, mono- and di-Substi “Substituted alkynyl refers to alkynyl groups having from tuted heteroarylamino, mono- and di-heterocyclic amino, 1 to 5 substituents selected from the group consisting of mono- and di-substituted heterocyclic amino, unsymmetric 35 alkoxy, Substituted alkoxy, acyl, acylamino, thiocarbony di-substituted amines having different substituents selected lamino, acyloxy, amino, amidino, alkylamidino, thioamidino, from the group consisting of alkyl, Substituted alkyl, aryl, aminoacyl, aminocarbonylamino, aminothiocarbonylamino, substituted aryl, heteroaryl, substituted heteroaryl, heterocy aminocarbonyloxy, aryl, Substituted aryl, aryloxy, Substituted clic and Substituted heterocyclic and Substituted alkyl groups aryloxy, aryloxyaryl, Substituted aryloxyaryl, halogen, having amino groups blocked by conventional blocking 40 hydroxyl, cyano, nitro, carboxyl, carboxylalkyl, carboxyl groups such as Boc, Cbz, formyl, and the like or alkyl/sub substituted alkyl, carboxyl-cycloalkyl, carboxyl-substituted stituted alkyl groups substituted with —SO-alkyl, -SO cycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxy substituted alkyl, -SO-alkenyl, -SO-substituted alkenyl, lheteroaryl, carboxyl-substituted heteroaryl, carboxylhetero —SO-cycloalkyl, -SO-substituted cycloalkyl, -SO cyclic, carboxyl-substituted heterocyclic, cycloalkyl, Substi aryl, —SO-substituted aryl, -SO-heteroaryl, -SO-sub 45 tuted cycloalkyl, guanidino, guanidinosulfone, thiol, stituted heteroaryl, -SO-heterocyclic. —SO-substituted thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl, heterocyclic and —SONRR where R is hydrogen or alkyl. thiocycloalkyl, substituted thiocycloalkyl, thioheteroaryl, “Substituted alkylene' refers to alkylene groups having substituted thioheteroaryl, thioheterocyclic, substituted thio from 1 to 5 substituents selected from the group consisting of heterocyclic, heteroaryl, substituted heteroaryl, heterocyclic, alkoxy, Substituted alkoxy, acyl, acylamino, thiocarbony 50 Substituted heterocyclic, cycloalkoxy, substituted lamino, acyloxy, amino, amidino, alkylamidino, thioamidino, cycloalkoxy, heteroaryloxy, Substituted heteroaryloxy, het aminoacyl, aminocarbonylamino, aminothiocarbonylamino, erocyclyloxy, Substituted heterocyclyloxy, oxycarbony aminocarbonyloxy, aryl, Substituted aryl, aryloxy, Substituted lamino, oxythiocarbonylamino, —OS(O)-alkyl, —OS(O)- aryloxy, aryloxyaryl, Substituted aryloxyaryl, halogen, substituted alkyl, —OS(O)-aryl, —OS(O)-substituted aryl, hydroxyl, cyano, nitro, carboxyl, carboxylalkyl, carboxyl 55 —OS(O)-heteroaryl, —OS(O)-substituted heteroaryl, substituted alkyl, carboxyl-cycloalkyl, carboxyl-substituted —OS(O)-heterocyclic. —OS(O)-substituted heterocyclic, cycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxy —OSO NRR where R is hydrogen or alkyl, - NRS(O)- lheteroaryl, carboxyl-substituted heteroaryl, carboxylhetero alkyl, - NRS(O)-substituted alkyl, —NRS(O)-aryl, cyclic, carboxyl-substituted heterocyclic, cycloalkyl, Substi —NRS(O)-substituted aryl, - NRS(O)-heteroaryl, - NRS tuted cycloalkyl, guanidino, guanidinosulfone, thiol, 60 (O)-substituted heteroaryl, —NRS(O)-heterocyclic, thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl, —NRS(O)-substituted heterocyclic, NRS(O), NR thiocycloalkyl, substituted thiocycloalkyl, thioheteroaryl, alkyl, - NRS(O), NR-substituted alkyl, - NRS(O)— substituted thioheteroaryl, thioheterocyclic, substituted thio NR-aryl, - NRS(O), NR-substituted aryl, - NRS(O) heterocyclic, heteroaryl, substituted heteroaryl, heterocyclic, NR-heteroaryl, - NRS(O) NR-substituted heteroaryl, Substituted heterocyclic, cycloalkoxy, substituted 65 —NRS(O), NR-heterocyclic, NRS(O), NR-substi cycloalkoxy, heteroaryloxy, Substituted heteroaryloxy, het tuted heterocyclic where R is hydrogen or alkyl, mono- and erocyclyloxy, Substituted heterocyclyloxy, oxycarbony di-alkylamino, mono- and di-(Substituted alkyl)amino, US 7,727,976 B2 49 50 mono- and di-arylamino, mono- and di-substituted ary —SO-substituted heteroaryl, -SO-heterocyclic. —SO lamino, mono- and di-heteroarylamino, mono- and di-Substi substituted heterocyclic and—SONRR where Rishydrogen tuted heteroarylamino, mono- and di-heterocyclic amino, or alkyl. mono- and di-substituted heterocyclic amino, unsymmetric “Substituted aryl refers to aryl groups which are substi di-substituted amines having different substituents selected tuted with from 1 to 3 substituents selected from the group from the group consisting of alkyl, Substituted alkyl, aryl, consisting of hydroxy, acyl, acylamino, thiocarbonylamino, substituted aryl, heteroaryl, substituted heteroaryl, heterocy acyloxy, alkyl, Substituted alkyl, alkoxy, Substituted alkoxy, clic, Substituted heterocyclic and Substituted alkynyl groups alkenyl, Substituted alkenyl, alkynyl. Substituted alkynyl, having amino groups blocked by conventional blocking amidino, alkylamidino, thioamidino, amino, aminoacyl, ami 10 nocarbonyloxy, aminocarbonylamino, aminothiocarbony groups such as Boc, Cbz, formyl, and the like or alkynyl/ lamino, aryl, Substituted aryl, aryloxy, Substituted aryloxy, substituted alkynyl groups substituted with —SO-alkyl, cycloalkoxy, Substituted cycloalkoxy, heteroaryloxy, Substi —SO-substituted alkyl, —SO-alkenyl, -SO-substituted tuted heteroaryloxy, heterocyclyloxy, substituted heterocy alkenyl, -SO-cycloalkyl, -SO-substituted cycloalkyl, clyloxy, carboxyl, carboxylalkyl, carboxyl-substituted alkyl, —SO-aryl, -SO-substituted aryl, -SO-heteroaryl. 15 carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, car —SO-substituted heteroaryl, - SO-heterocyclic. —SO boxylaryl, carboxyl-substituted aryl, carboxylheteroaryl, car substituted heterocyclic and—SONRR where R is hydrogen boxyl-substituted heteroaryl, carboxylheterocyclic, car or alkyl. boxyl-Substituted heterocyclic, carboxylamido, cyano, thiol, “Substituted alkynylene' refers to alkynylene groups hav thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl, ing from 1 to 5 substituents selected from the group consisting thioheteroaryl, substituted thioheteroaryl, thiocycloalkyl, of alkoxy, Substituted alkoxy, acyl, acylamino, thiocarbony substituted thiocycloalkyl, thioheterocyclic, substituted thio lamino, acyloxy, amino, amidino, alkylamidino, thioamidino, heterocyclic, cycloalkyl, Substituted cycloalkyl, guanidino, aminoacyl, aminocarbonylamino, aminothiocarbonylamino, guanidinosulfone, halo, nitro, heteroaryl, Substituted het aminocarbonyloxy, aryl, Substituted aryl, aryloxy, Substituted eroaryl, heterocyclic, Substituted heterocyclic, cycloalkoxy, aryloxy, aryloxyaryl, Substituted aryloxyaryl, halogen, 25 substituted cycloalkoxy, heteroaryloxy, substituted het hydroxyl, cyano, nitro, carboxyl, carboxylalkyl, carboxyl eroaryloxy, heterocyclyloxy, Substituted heterocyclyloxy, substituted alkyl, carboxyl-cycloalkyl, carboxyl-substituted oxycarbonylamino, oxythiocarbonylamino. —S(O)-alkyl, cycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxy —S(O)-substituted alkyl, -S(O)-cycloalkyl, - SCO)- lheteroaryl, carboxyl-substituted heteroaryl, carboxylhetero substituted cycloalkyl, —S(O)-alkenyl, - S(O)-substituted cyclic, carboxyl-substituted heterocyclic, cycloalkyl, Substi 30 alkenyl, -S(O)-aryl. —S(O)-substituted aryl, - S(O)- tuted cycloalkyl, guanidino, guanidinosulfone, thiol, heteroaryl, —S(O)-substituted heteroaryl, —S(O)-hetero thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl, cyclic. —S(O)-substituted heterocyclic. —OS(O)-alkyl, thiocycloalkyl, substituted thiocycloalkyl, thioheteroaryl, —OS(O)-substituted alkyl, —OS(O)-aryl, —OS(O)-sub substituted thioheteroaryl, thioheterocyclic, substituted thio stituted aryl, —OS(O)-heteroaryl, —OS(O)-substituted heterocyclic, heteroaryl, substituted heteroaryl, heterocyclic, 35 heteroaryl, —OS(O)-heterocyclic, —OS(O)-substituted Substituted heterocyclic, cycloalkoxy, substituted heterocyclic. —OSO NRR where R is hydrogen or alkyl, cycloalkoxy, heteroaryloxy, Substituted heteroaryloxy, het —NRS(O)-alkyl, —NRS(O)-substituted alkyl, erocyclyloxy, Substituted heterocyclyloxy, oxycarbony —NRS(O)-aryl, - NRS(O)-substituted aryl, - NRS(O)- lamino, oxythiocarbonylamino, —OS(O)-alkyl, —OS(O)- heteroaryl, - NRS(O)-substituted heteroaryl, - NRS(O)- substituted alkyl, —OS(O)-aryl, —OS(O)-substituted aryl, 40 heterocyclic, NRS(O)-substituted heterocyclic, NRS —OS(O)-heteroaryl, —OS(O)-substituted heteroaryl, (O) NR-alkyl, - NRS(O), NR-substituted alkyl, —OS(O)-heterocyclic, —OS(O)-substituted heterocyclic, —NRS(O), NR-aryl, - NRS(O), NR-substituted aryl, —OSO NRR where R is hydrogen or alkyl, - NRS(O)- - NRS(O), NR-heteroaryl, - NRS(O) NR-substituted alkyl, - NRS(O)-substituted alkyl, - NRS(O)-aryl, heteroaryl, - NRS(O) NR-heterocyclic, NRS(O)— —NRS(O)-substituted aryl, - NRS(O)-heteroaryl, - NRS 45 NR-substituted heterocyclic where R is hydrogen or alkyl, (O)-substituted heteroaryl, - NRS(O)-heterocyclic, mono- and di-alkylamino, mono- and di-(substituted alkyl) —NRS(O)-substituted heterocyclic, NRS(O), NR amino, mono- and di-arylamino, mono- and di-substituted alkyl, —NRS(O), NR-substituted alkyl, - NRS(O)— arylamino, mono- and di-heteroarylamino, mono- and di NR-aryl, - NRS(O), NR-substituted aryl, - NRS(O) Substituted heteroarylamino, mono- and di-heterocyclic NR-heteroaryl, —NRS(O) NR-substituted heteroaryl, 50 amino, mono- and di-substituted heterocyclic amino, unsym —NRS(O), NR-heterocyclic, NRS(O), NR-substi metric di-substituted amines having different substituents tuted heterocyclic where R is hydrogen or alkyl, mono- and selected from the group consisting of alkyl, Substituted alkyl, di-alkylamino, mono- and di-(substituted alkyl)amino, aryl, substituted aryl, heteroaryl, substituted heteroaryl, het mono- and di-arylamino, mono- and di-substituted ary erocyclic and Substituted heterocyclic and amino groups on lamino, mono- and di-heteroarylamino, mono- and di-Substi 55 the Substituted aryl blocked by conventional blocking groups tuted heteroarylamino, mono- and di-heterocyclic amino, such as Boc, Cbz, formyl, and the like or substituted with mono- and di-substituted heterocyclic amino, unsymmetric —SONRR where R is hydrogen or alkyl. di-substituted amines having different substituents selected “Substituted arylene' refers to arylene groups which are from the group consisting of alkyl, Substituted alkyl, aryl, substituted with from 1 to 3 substituents selected from the substituted aryl, heteroaryl, substituted heteroaryl, heterocy 60 group consisting of hydroxy, acyl, acylamino, thiocarbony clic, Substituted heterocyclic and Substituted alkenyl groups lamino, acyloxy, alkyl, Substituted alkyl, alkoxy, Substituted having amino groups blocked by conventional blocking alkoxy, alkenyl, Substituted alkenyl, alkynyl. Substituted groups such as Boc, Cbz, formyl, and the like or alkenyl/ alkynyl, amidino, alkylamidino, thioamidino, amino, ami substituted alkenyl groups substituted with —SO-alkyl, noacyl, aminocarbonyloxy, aminocarbonylamino, ami —SO-substituted alkyl, —SO-alkenyl, -SO-substituted 65 nothiocarbonylamino, aryl, Substituted aryl, aryloxy, Substi alkenyl, -SO-cycloalkyl, -SO-substituted cycloalkyl, tuted aryloxy, cycloalkoxy, Substituted cycloalkoxy, —SO-aryl, -SO-substituted aryl, -SO-heteroaryl, heteroaryloxy, Substituted heteroaryloxy, heterocyclyloxy, US 7,727,976 B2 51 52 Substituted heterocyclyloxy, carboxyl, carboxylalkyl, car fone, halo, nitro, heteroaryl, substituted heteroaryl, heterocy boxyl-substituted alkyl, carboxyl-cycloalkyl, carboxyl-sub clic, Substituted heterocyclic, cycloalkoxy, Substituted stituted cycloalkyl, carboxylaryl, carboxyl-substituted aryl, cycloalkoxy, heteroaryloxy, Substituted heteroaryloxy, het carboxylheteroaryl, carboxyl-substituted heteroaryl, car erocyclyloxy, Substituted heterocyclyloxy, oxycarbony boxylheterocyclic, carboxyl-substituted heterocyclic, car lamino, oxythiocarbonylamino, —S(O)-alkyl, -S(O)- boxylamido, cyano, thiol, thioalkyl, Substituted thioalkyl, substituted alkyl, —S(O)-cycloalkyl, - S(O)-substituted thioaryl, substituted thioaryl, thioheteroaryl, substituted thio cycloalkyl. —S(O)-alkenyl. —S(O)-substituted alkenyl, heteroaryl, thiocycloalkyl, substituted thiocycloalkyl, thio —S(O)-aryl, -S(O)-substituted aryl. —S(O)-heteroaryl, heterocyclic, substituted thioheterocyclic, cycloalkyl, substi —S(O)-substituted heteroaryl, —S(O)-heterocyclic, tuted cycloalkyl, guanidino, guanidinosulfone, halo, nitro, 10 —S(O)-substituted heterocyclic, —OS(O)-alkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted —OS(O)-substituted alkyl, —OS(O)-aryl, —OS(O)-sub heterocyclic, cycloalkoxy, Substituted cycloalkoxy, het stituted aryl, —OS(O)-heteroaryl, —OS(O)-substituted eroaryloxy, Substituted heteroaryloxy, heterocyclyloxy, Sub heteroaryl, —OS(O)-heterocyclic. —OS(O)-substituted stituted heterocyclyloxy, oxycarbonylamino, oxythiocarbo heterocyclic, —OSO NRR where R is hydrogen or alkyl, nylamino, —S(O)-alkyl, —S(O)-substituted alkyl, 15 —NRS(O)-alkyl, —NRS(O)-substituted alkyl, —S(O)-cycloalkyl, —S(O)-substituted cycloalkyl, —NRS(O)-aryl, - NRS(O)-substituted aryl, - NRS(O)- —S(O)-alkenyl, —S(O)-substituted alkenyl, —S(O)-aryl, heteroaryl, - NRS(O)-substituted heteroaryl, - NRS(O)- —S(O)-substituted aryl, - S(O)-heteroaryl, - S(O)-sub heterocyclic. —NRS(O)-substituted heterocyclic, NRS stituted heteroaryl, —S(O)-heterocyclic. —S(O)-substi (O) NR-alkyl, - NRS(O), NR-substituted alkyl, tuted heterocyclic. —OS(O)-alkyl, —OS(O)-substituted —NRS(O), NR-aryl, - NRS(O), NR-substituted aryl, alkyl, —OS(O)-aryl, —OS(O)-substituted aryl, —OS(O)- - NRS(O), NR-heteroaryl, - NRS(O), NR-substituted heteroaryl, —OS(O)-substituted heteroaryl, —OS(O)-het heteroaryl, - NRS(O) NR-heterocyclic, NRS(O)— erocyclic. —OS(O)-substituted heterocyclic. —OSO NR-substituted heterocyclic where R is hydrogen or alkyl, NRR where R is hydrogen or alkyl, —NRS(O)-alkyl, mono- and di-alkylamino, mono- and di-(substituted alkyl) —NRS(O)-substituted alkyl, - NRS(O)-aryl, NRS(O)- 25 amino, mono- and di-arylamino, mono- and di-substituted substituted aryl, —NRS(O)-heteroaryl, —NRS(O)-substi arylamino, mono- and di-heteroarylamino, mono- and di tuted heteroaryl, - NRS(O)-heterocyclic, NRS(O)-sub Substituted heteroarylamino, mono- and di-heterocyclic stituted heterocyclic, NRS(O), NR-alkyl, - NRS(O) amino, mono- and di-substituted heterocyclic amino, unsym NR-substituted alkyl, - NRS(O), NR-aryl, - NRS(O)— metric di-substituted amines having different substituents NR-substituted aryl, -NRS(O), NR-heteroaryl, - NRS 30 selected from the group consisting of alkyl, Substituted alkyl, (O) NR-substituted heteroaryl, - NRS(O) NR aryl, substituted aryl, heteroaryl, substituted heteroaryl, het heterocyclic, NRS(O), NR-substituted heterocyclic erocyclic and substituted heterocyclic and amino groups on where R is hydrogen or alkyl, mono- and di-alkylamino, the Substituted aryl blocked by conventional blocking groups mono- and di-(Substituted alkyl)amino, mono- and di-ary such as Boc, Cbz, formyl, and the like or substituted with lamino, mono- and di-substituted arylamino, mono- and di 35 —SONRR where R is hydrogen or alkyl. heteroarylamino, mono- and di-substituted heteroarylamino, “Substituted cycloalkoxy' refers to —O-substituted mono- and di-heterocyclic amino, mono- and di-substituted cycloalkyl groups. heterocyclic amino, unsymmetric di-substituted amines hav “Substituted cycloalkyl and “substituted cycloalkenyl ing different Substituents selected from the group consisting refers to an cycloalkyl or cycloalkenyl group, preferably of of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, 40 from 3 to 8 carbon atoms, having from 1 to 5 substituents substituted heteroaryl, heterocyclic and substituted heterocy selected from the group consisting of oxo (=O), thioxo clic and amino groups on the Substituted aryl blocked by (=S), alkoxy, Substituted alkoxy, acyl, acylamino, thiocar conventional blocking groups such as Boc, Cbz, formyl, and bonylamino, acyloxy, amino, amidino, alkylamidino, thioa the like or substituted with —SONRR where R is hydrogen midino, aminoacyl, aminocarbonylamino, aminothiocarbo or alkyl. 45 nylamino, aminocarbonyloxy, aryl, Substituted aryl, aryloxy, “Substituted aryloxy' refers to substituted aryl-O- Substituted aryloxy, aryloxyaryl, Substituted aryloxyaryl, groups. halogen, hydroxyl, cyano, nitro, carboxyl, carboxylalkyl, car “Substituted aryloxyaryl refers to aryloxyaryl groups sub boxyl-substituted alkyl, carboxyl-cycloalkyl, carboxyl-sub stituted with from 1 to 3 substituents on either or both aryl stituted cycloalkyl, carboxylaryl, carboxyl-substituted aryl, rings selected from the group consisting of hydroxy, acyl, 50 carboxylheteroaryl, carboxyl-substituted heteroaryl, car acylamino, thiocarbonylamino, acyloxy, alkyl, Substituted boxylheterocyclic, carboxyl-substituted heterocyclic, alkyl, alkoxy, Substituted alkoxy, alkenyl, Substituted alkenyl, cycloalkyl, Substituted cycloalkyl, guanidino, guanidinosul alkynyl. Substituted alkynyl, amidino, alkylamidino, thioami fone, thiol, thioalkyl, substituted thioalkyl, thioaryl, substi dino, amino, aminoacyl, aminocarbonyloxy, aminocarbony tuted thioaryl, thiocycloalkyl, substituted thiocycloalkyl, lamino, aminothiocarbonylamino, aryl, Substituted aryl, ary 55 thioheteroaryl, substituted thioheteroaryl, thioheterocyclic, loxy, Substituted aryloxy, cycloalkoxy, Substituted substituted thioheterocyclic, heteroaryl, substituted het cycloalkoxy, heteroaryloxy, Substituted heteroaryloxy, het eroaryl, heterocyclic, Substituted heterocyclic, cycloalkoxy, erocyclyloxy, Substituted heterocyclyloxy, carboxyl, car substituted cycloalkoxy, heteroaryloxy, substituted het boxylalkyl, carboxyl-substituted alkyl, carboxyl-cycloalkyl, eroaryloxy, heterocyclyloxy, Substituted heterocyclyloxy, carboxyl-substituted cycloalkyl, carboxylaryl, carboxyl-sub 60 oxycarbonylamino, oxythiocarbonylamino, —OS(O)-alkyl, stituted aryl, carboxylheteroaryl, carboxyl-substituted het —OS(O)-substituted alkyl, —OS(O)-aryl, —OS(O)-sub eroaryl, carboxylheterocyclic, carboxyl-substituted hetero stituted aryl, —OS(O)-heteroaryl, —OS(O)-substituted cyclic, carboxylamido, cyano, thiol, thioalkyl, Substituted heteroaryl, —OS(O)-heterocyclic, —OS(O)-substituted thioalkyl, thioaryl, substituted thioaryl, thioheteroaryl, sub heterocyclic. —OSO NRR where R is hydrogen or alkyl, stituted thioheteroaryl, thiocycloalkyl, substituted thiocy 65 —NRS(O)-alkyl, —NRS(O)-substituted alkyl, cloalkyl, thioheterocyclic, substituted thioheterocyclic, —NRS(O)-aryl, - NRS(O)-substituted aryl, - NRS(O)- cycloalkyl, Substituted cycloalkyl, guanidino, guanidinosul heteroaryl, - NRS(O)-substituted heteroaryl, - NRS(O)- US 7,727,976 B2 53 54 heterocyclic, NRS(O)-substituted heterocyclic. —NRS stituted alkyl, aryl, substituted aryl, heteroaryl, substituted (O), NR-alkyl, - NRS(O), NR-substituted alkyl, heteroaryl, heterocyclic and substituted heterocyclic and sub —NRS(O), NR-aryl, —NRS(O), NR-substituted aryl, stituted alkynyl groups having amino groups blocked by con - NRS(O), NR-heteroaryl, - NRS(O) NR-substituted ventional blocking groups such as Boc, Cbz, formyl, and the heteroaryl, - NRS(O) NR-heterocyclic. —NRS(O)— like or alkynyl/substituted alkynyl groups substituted with NR-substituted heterocyclic where R is hydrogen or alkyl, —SO-alkyl, -SO-substituted alkyl, -SO-alkenyl, mono- and di-alkylamino, mono- and di-(substituted alkyl) —SO-substituted alkenyl, -SO-cycloalkyl, -SO-Sub amino, mono- and di-arylamino, mono- and di-substituted stituted cycloalkyl, -SO-aryl, -SO-substituted aryl, arylamino, mono- and di-heteroarylamino, mono- and di —SO-heteroaryl, - SO-substituted heteroaryl, - SO-het Substituted heteroarylamino, mono- and di-heterocyclic 10 amino, mono- and di-substituted heterocyclic amino, unsym erocyclic. —SO-substituted heterocyclic and —SONRR metric di-substituted amines having different substituents where R is hydrogen or alkyl. selected from the group consisting of alkyl, Substituted alkyl, “Substituted heteroaryl refers to heteroaryl groups which aryl, substituted aryl, heteroaryl, substituted heteroaryl, het are substituted with from 1 to 3 substituents selected from the erocyclic and substituted heterocyclic and substituted alkynyl 15 group consisting of hydroxy, acyl, acylamino, thiocarbony groups having amino groups blocked by conventional block lamino, acyloxy, alkyl, Substituted alkyl, alkoxy, Substituted ing groups such as Boc, Cbz, formyl, and the like or alkynyl/ alkoxy, alkenyl, Substituted alkenyl, alkynyl. Substituted substituted alkynyl groups substituted with —SO-alkyl, alkynyl, amidino, alkylamidino, thioamidino, amino, ami —SO-substituted alkyl, —SO-alkenyl, -SO-substituted noacyl, aminocarbonyloxy, aminocarbonylamino, ami alkenyl, -SO-cycloalkyl, -SO-substituted cycloalkyl, nothiocarbonylamino, aryl, Substituted aryl, aryloxy, Substi —SO-aryl, -SO-substituted aryl, -SO-heteroaryl. tuted aryloxy, cycloalkoxy, Substituted cycloalkoxy, —SO-substituted heteroaryl, - SO-heterocyclic. —SO heteroaryloxy, Substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclic and—SONRR where Rishydrogen Substituted heterocyclyloxy, carboxyl, carboxylalkyl, car or alkyl. boxyl-substituted alkyl, carboxyl-cycloalkyl, carboxyl-sub “Substituted cycloalkylene' and “substituted cycloalk 25 stituted cycloalkyl, carboxylaryl, carboxyl-substituted aryl, enylene' refers to a cycloalkylene or cycloalkenylene group, carboxylheteroaryl, carboxyl-substituted heteroaryl, car preferably of from 3 to 8 carbon atoms, having from 1 to 5 boxylheterocyclic, carboxyl-substituted heterocyclic, car Substituents selected from the group consisting of oxo (=O), boxylamido, cyano, thiol, thioalkyl, Substituted thioalkyl, thioxo (=S), alkoxy, Substituted alkoxy, acyl, acylamino, thioaryl, substituted thioaryl, thioheteroaryl, substituted thio thiocarbonylamino, acyloxy, amino, amidino, alkylamidino, 30 heteroaryl, thiocycloalkyl, substituted thiocycloalkyl, thio thioamidino, aminoacyl, aminocarbonylamino, aminothio heterocyclic, substituted thioheterocyclic, cycloalkyl, substi carbonylamino, aminocarbonyloxy, aryl, substituted aryl, tuted cycloalkyl, guanidino, guanidinosulfone, halo, nitro, aryloxy, Substituted aryloxy, aryloxyaryl, Substituted ary heteroaryl, substituted heteroaryl, heterocyclic, substituted loxyaryl, halogen, hydroxyl, cyano, nitro, carboxyl, carboxy heterocyclic, cycloalkoxy, Substituted cycloalkoxy, het lalkyl, carboxyl-substituted alkyl, carboxyl-cycloalkyl, car 35 eroaryloxy, Substituted heteroaryloxy, heterocyclyloxy, Sub boxyl-substituted cycloalkyl, carboxylaryl, carboxyl stituted heterocyclyloxy, oxycarbonylamino, oxythiocarbo substituted aryl, carboxylheteroaryl, carboxyl-substituted nylamino, —S(O)-alkyl, —S(O)-substituted alkyl, heteroaryl, carboxylheterocyclic, carboxyl-substituted het —S(O)-cycloalkyl, —S(O)-substituted cycloalkyl, erocyclic, cycloalkyl, Substituted cycloalkyl, guanidino, —S(O)-alkenyl, —S(O)-substituted alkenyl, —S(O)-aryl, guanidinosulfone, thiol, thioalkyl, Substituted thioalkyl, thio 40 —S(O)-substituted aryl, - S(O)-heteroaryl, - S(O)-sub aryl, substituted thioaryl, thiocycloalkyl, substituted thiocy stituted heteroaryl, S(O)-heterocyclic. —S(O)-substi cloalkyl, thioheteroaryl, substituted thioheteroaryl, thiohet tuted heterocyclic, —OS(O)-alkyl, —OS(O)-substituted erocyclic, substituted thioheterocyclic, heteroaryl, alkyl, —OS(O)-aryl, —OS(O)-substituted aryl, —OS(O)- substituted heteroaryl, heterocyclic, substituted heterocyclic, heteroaryl, —OS(O)-substituted heteroaryl, —OS(O)-het cycloalkoxy, Substituted cycloalkoxy, heteroaryloxy, Substi 45 erocyclic. —OS(O)-substituted heterocyclic. —OSO tuted heteroaryloxy, heterocyclyloxy, substituted heterocy NRR where R is hydrogen or alkyl, —NRS(O)-alkyl, clyloxy, oxycarbonylamino, oxythiocarbonylamino, —OS —NRS(O)-substituted alkyl, NRS(O)-aryl, NRS(O)- (O)-alkyl, —OS(O)-substituted alkyl, —OS(O)-aryl, substituted aryl, —NRS(O)-heteroaryl, —NRS(O)-substi —OS(O)-substituted aryl, —OS(O)-heteroaryl, tuted heteroaryl, - NRS(O)-heterocyclic, NRS(O)-sub —OS(O)-substituted heteroaryl, —OS(O)-heterocyclic, 50 stituted heterocyclic. —NRS(O), NR-alkyl, —NRS(O)— —OS(O)-substituted heterocyclic, —OSO NRR where R NR-substituted alkyl, - NRS(O) NR-aryl, - NRS(O)— is hydrogen or alkyl, - NRS(O)-alkyl, —NRS(O)-substi NR-substituted aryl, - NRS(O), NR-heteroaryl, - NRS tuted alkyl, - NRS(O)-aryl, - NRS(O)-substituted aryl, (O) NR-substituted heteroaryl, - NRS(O) NR —NRS(O)-heteroaryl, - NRS(O)-substituted heteroaryl, heterocyclic, NRS(O), NR-substituted heterocyclic —NRS(O)-heterocyclic, —NRS(O)-substituted heterocy 55 where R is hydrogen or alkyl, mono- and di-alkylamino, clic, NRS(O), NR-alkyl, - NRS(O), NR-substituted mono- and di-(Substituted alkyl)amino, mono- and di-ary alkyl, - NRS(O) NR-aryl, - NRS(O) NR-substituted lamino, mono- and di-substituted arylamino, mono- and di aryl, - NRS(O), NR-heteroaryl, - NRS(O), NR-substi heteroarylamino, mono- and di-substituted heteroarylamino, tuted heteroaryl, —NRS(O), NR-heterocyclic, mono- and di-heterocyclic amino, mono- and di-substituted —NRS(O), NR-substituted heterocyclic where R is hydro 60 heterocyclic amino, unsymmetric di-substituted amines hav gen or alkyl, mono- and di-alkylamino, mono- and di-(Sub ing different Substituents selected from the group consisting stituted alkyl)amino, mono- and di-arylamino, mono- and of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, di-Substituted arylamino, mono- and di-heteroarylamino, substituted heteroaryl, heterocyclic and substituted heterocy mono- and di-substituted heteroarylamino, mono- and di clic and amino groups on the Substituted aryl blocked by heterocyclic amino, mono- and di-substituted heterocyclic 65 conventional blocking groups such as Boc, Cbz, formyl, and amino, unsymmetric di-substituted amines having different the like or substituted with —SONRR where R is hydrogen Substituents selected from the group consisting of alkyl, Sub or alkyl. US 7,727,976 B2 55 56 “Substituted heteroarylene' refers to heteroarylene groups boxyl-substituted heterocyclic, cycloalkyl, substituted which are substituted with from 1 to 3 substituents selected cycloalkyl, guanidino, guanidinosulfone, thiol, thioalkyl, from the group consisting of hydroxy, acyl, acylamino, thio substituted thioalkyl, thioaryl, substituted thioaryl, thiocy carbonylamino, acyloxy, alkyl, Substituted alkyl, alkoxy, Sub cloalkyl, substituted thiocycloalkyl, thioheteroaryl, substi stituted alkoxy, alkenyl, Substituted alkenyl, alkynyl, Substi tuted thioheteroaryl, thioheterocyclic, substituted thiohetero tuted alkynyl, amidino, alkylamidino, thioamidino, amino, cyclic, heteroaryl, Substituted heteroaryl, heterocyclic, aminoacyl, aminocarbonyloxy, aminocarbonylamino, ami Substituted heterocyclic, cycloalkoxy, substituted nothiocarbonylamino, aryl, Substituted aryl, aryloxy, Substi cycloalkoxy, heteroaryloxy, Substituted heteroaryloxy, tuted aryloxy, cycloalkoxy, Substituted cycloalkoxy, het —C(O)C-aryl, —C(O)O-substituted aryl, heterocyclyloxy, eroaryloxy, Substituted heteroaryloxy, heterocyclyloxy, 10 Substituted heterocyclyloxy, oxycarbonylamino, oxythiocar Substituted heterocyclyloxy, carboxyl, carboxylalkyl, car bonylamino, —OS(O)-alkyl, —OS(O)-substituted alkyl, boxyl-substituted alkyl, carboxyl-cycloalkyl, carboxyl-sub —OS(O)-aryl, —OS(O)-substituted aryl, —OS(O)-het stituted cycloalkyl, carboxylaryl, carboxyl-substituted aryl, eroaryl, —OS(O)-substituted heteroaryl, —OS(O)-hetero carboxylheteroaryl, carboxyl-substituted heteroaryl, car cyclic. —OS(O)-substituted heterocyclic, —OSO, NRR boxylheterocyclic, carboxyl-substituted heterocyclic, car 15 where R is hydrogen or alkyl, —NRS(O)-alkyl, boxylamido, cyano, thiol, thioalkyl, Substituted thioalkyl, —NRS(O)-substituted alkyl, NRS(O)-aryl, NRS(O)- thioaryl, substituted thioaryl, thioheteroaryl, substituted thio substituted aryl, —NRS(O)-heteroaryl, —NRS(O)-substi heteroaryl, thiocycloalkyl, substituted thiocycloalkyl, thio tuted heteroaryl, - NRS(O)-heterocyclic, NRS(O)-sub heterocyclic, substituted thioheterocyclic, cycloalkyl, substi stituted heterocyclic. —NRS(O), NR-alkyl, —NRS(O)— tuted cycloalkyl, guanidino, guanidinosulfone, halo, nitro, NR-substituted alkyl, - NRS(O) NR-aryl, - NRS(O)— heteroaryl, substituted heteroaryl, heterocyclic, substituted NR-substituted aryl, -NRS(O), NR-heteroaryl, - NRS heterocyclic, cycloalkoxy, Substituted cycloalkoxy, het (O) NR-substituted heteroaryl, - NRS(O) NR eroaryloxy, Substituted heteroaryloxy, heterocyclyloxy, Sub heterocyclic, NRS(O), NR-substituted heterocyclic stituted heterocyclyloxy, oxycarbonylamino, oxythiocarbo where R is hydrogen or alkyl, mono- and di-alkylamino, nylamino, —S(O)-alkyl, —S(O)-substituted alkyl, 25 mono- and di-(Substituted alkyl)amino, mono- and di-ary —S(O)-cycloalkyl, —S(O)-substituted cycloalkyl, lamino, mono- and di-substituted arylamino, mono- and di —S(O)-alkenyl, —S(O)-substituted alkenyl, —S(O)-aryl, heteroarylamino, mono- and di-substituted heteroarylamino, —S(O)-substituted aryl, - S(O)-heteroaryl, - S(O)-sub mono- and di-heterocyclic amino, mono- and di-substituted stituted heteroaryl, —S(O)-heterocyclic. —S(O)-substi heterocyclic amino, unsymmetric di-substituted amines hav tuted heterocyclic, —OS(O)-alkyl, —OS(O)-substituted 30 ing different Substituents selected from the group consisting alkyl, —OS(O)-aryl, —OS(O)-substituted aryl, —OS(O)- of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, heteroaryl, - OS(O)-substituted heteroaryl, - OS(O)-het substituted heteroaryl, heterocyclic and substituted heterocy erocyclic. —OS(O)-substituted heterocyclic. —OSO clic and Substituted alkynyl groups having amino groups NRR where R is hydrogen or alkyl, —NRS(O)-alkyl, blocked by conventional blocking groups such as Boc, Cbz, —NRS(O)-substituted alkyl, - NRS(O)-aryl, NRS(O)- 35 formyl, and the like or alkynyl/substituted alkynyl groups substituted aryl, —NRS(O)-heteroaryl, —NRS(O)-substi substituted with —SO-alkyl, -SO-substituted alkyl, tuted heteroaryl, - NRS(O)-heterocyclic, NRS(O)-sub —SO-alkenyl, -SO-substituted alkenyl, -SO-cy stituted heterocyclic. —NRS(O), NR-alkyl, —NRS(O)— cloalkyl, -SO-substituted cycloalkyl, -SO-aryl, -SO NR-substituted alkyl, - NRS(O), NR-aryl, - NRS(O)— substituted aryl, -SO-heteroaryl, -SO-substituted het NR-substituted aryl, -NRS(O), NR-heteroaryl, - NRS 40 eroaryl, -SO-heterocyclic, -SO-substituted heterocyclic (O) NR-substituted heteroaryl, - NRS(O) NR and —SONRR where R is hydrogen or alkyl. heterocyclic, NRS(O), NR-substituted heterocyclic “Substituted heterocyclic” refers to heterocycle groups where R is hydrogen or alkyl, mono- and di-alkylamino, which are substituted with from 1 to 3 substituents selected mono- and di-(Substituted alkyl)amino, mono- and di-ary from the group consisting of oxo (=O), thioxo (=S), alkoxy, lamino, mono- and di-substituted arylamino, mono- and di 45 Substituted alkoxy, acyl, acylamino, thiocarbonylamino, acy heteroarylamino, mono- and di-substituted heteroarylamino, loxy, amino, amidino, alkylamidino, thioamidino, aminoacyl, mono- and di-heterocyclic amino, mono- and di-substituted aminocarbonylamino, aminothiocarbonylamino, aminocar heterocyclic amino, unsymmetric di-substituted amines hav bonyloxy, aryl, Substituted aryl, aryloxy, Substituted aryloxy, ing different Substituents selected from the group consisting aryloxyaryl, Substituted aryloxyaryl, halogen, hydroxyl, of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, 50 cyano, nitro, carboxyl, carboxylalkyl, carboxyl-substituted substituted heteroaryl, heterocyclic and substituted heterocy alkyl, carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, clic and amino groups on the Substituted aryl blocked by carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl, conventional blocking groups such as Boc, Cbz, formyl, and carboxyl-substituted heteroaryl, carboxylheterocyclic, car the like or substituted with —SONRR where R is hydrogen boxyl-substituted heterocyclic, cycloalkyl, substituted or alkyl. 55 cycloalkyl, guanidino, guanidinosulfone, thiol, thioalkyl, “Substituted heterocyclene' refers to heterocyclene groups substituted thioalkyl, thioaryl, substituted thioaryl, thiocy which are substituted with from 1 to 3 substituents selected cloalkyl, substituted thiocycloalkyl, thioheteroaryl, substi from the group consisting of oxo (=O), thioxo (=S), alkoxy, tuted thioheteroaryl, thioheterocyclic, substituted thiohetero Substituted alkoxy, acyl, acylamino, thiocarbonylamino, acy cyclic, heteroaryl, Substituted heteroaryl, heterocyclic, loxy, amino, amidino, alkylamidino, thioamidino, aminoacyl, 60 Substituted heterocyclic, cycloalkoxy, substituted aminocarbonylamino, aminothiocarbonylamino, aminocar cycloalkoxy, heteroaryloxy, Substituted heteroaryloxy, bonyloxy, aryl, Substituted aryl, aryloxy, Substituted aryloxy, —C(O)C-aryl, —C(O)O-substituted aryl, heterocyclyloxy, aryloxyaryl, Substituted aryloxyaryl, halogen, hydroxyl, Substituted heterocyclyloxy, oxycarbonylamino, oxythiocar cyano, nitro, carboxyl, carboxylalkyl, carboxyl-substituted bonylamino, —OS(O)-alkyl, —OS(O)-substituted alkyl, alkyl, carboxyl-cycloalkyl, carboxyl-Substituted cycloalkyl, 65 —OS(O)-aryl, —OS(O)-substituted aryl, —OS(O)-het carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl, eroaryl, —OS(O)-substituted heteroaryl, —OS(O)-hetero carboxyl-substituted heteroaryl, carboxylheterocyclic, car cyclic. —OS(O)-substituted heterocyclic. —OSO NRR US 7,727,976 B2 57 58 where R is hydrogen or alkyl, —NRS(O)-alkyl, “Thioaryl refers to the group —S-aryl and “substituted —NRS(O)-substituted alkyl, - NRS(O)-aryl, NRS(O)- thioaryl refers to the group —S-substituted aryl. substituted aryl, —NRS(O)-heteroaryl, —NRS(O)-substi “Thiocarbonylamino” refers to the group —C(S)NRR tuted heteroaryl, - NRS(O)-heterocyclic, NRS(O)-sub where each R is independently selected from the group con stituted heterocyclic. —NRS(O), NR-alkyl, —NRS(O)— 5 sisting of hydrogen, alkyl, Substituted alkyl, alkenyl, Substi NR-substituted alkyl, - NRS(O), NR-aryl, - NRS(O)— tuted alkenyl, alkynyl, Substituted alkynyl, aryl, Substituted NR-substituted aryl, -NRS(O), NR-heteroaryl, - NRS aryl, cycloalkyl, Substituted cycloalkyl, heteroaryl, Substi (O) NR-substituted heteroaryl, - NRS(O) NR tuted heteroaryl, heterocyclic, substituted heterocyclic and heterocyclic, NRS(O), NR-substituted heterocyclic where each R is joined to form, together with the nitrogen where R is hydrogen or alkyl, mono- and di-alkylamino, 10 atom a heterocyclic or Substituted heterocyclic ring wherein mono- and di-(Substituted alkyl)amino, mono- and di-ary alkyl, Substituted alkyl, alkenyl, Substituted alkenyl, alkynyl, lamino, mono- and di-substituted arylamino, mono- and di Substituted alkynyl, cycloalkyl, Substituted cycloalkyl, aryl, heteroarylamino, mono- and di-substituted heteroarylamino, substituted aryl, heteroaryl, substituted heteroaryl, heterocy mono- and di-heterocyclic amino, mono- and di-substituted clic and substituted heterocyclic are as defined herein. heterocyclic amino, unsymmetric di-substituted amines hav 15 “Thiocycloalkyl refers to the groups—S-cycloalkyl. ing different Substituents selected from the group consisting “Thioheteroaryl refers to the group —S-heteroaryl and of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, “substituted thioheteroaryl” refers to the group —S-substi substituted heteroaryl, heterocyclic and substituted heterocy tuted heteroaryl. clic and Substituted alkynyl groups having amino groups “Thioheterocyclic” refers to the group —S-heterocyclic blocked by conventional blocking groups such as Boc, Cbz, formyl, and the like or alkynyl/substituted alkynyl groups and “substituted thioheterocyclic” refers to the group —S- substituted with —SO-alkyl, -SO-substituted alkyl, substituted heterocyclic. —SO-alkenyl, -SO-substituted alkenyl, -SO-cy “Thiol” refers to the group - SH. cloalkyl, -SO-substituted cycloalkyl, -SO-aryl, -SO “Tissue of the enterohepatic circulation” refers to the substituted aryl, —SO-heteroaryl, -SO-substituted het 25 blood, plasma, intestinal contents, intestinal cells, liver cells, eroaryl, -SO-heterocyclic. —SO-substituted heterocyclic biliary tract or any fraction, Suspension, homogenate, extract and —SONRR where R is hydrogen or alkyl. or preparation thereof. “Substituted thioalkyl refers to the group —S-substituted “Translocation across the intestinal wall” refers to move alkyl. ment of a drug or drug conjugate by a passive or active “Substituted thiocycloalkyl refers to the group —S-sub 30 mechanism, or both, across an epithelial cell membrane of stituted cycloalkyl. any region of the gastrointestinal tract. “Sustained release” refers to release of a compound of the invention (i.e., a compound whether or not linked to a drug D) Compound Preparation into the gutlumen over a prolonged period of time relative to The compounds of this invention can be prepared from that achieved by administration of a conventional orally 35 readily available starting materials using the following gen administered formulation of the compound. A number of eral methods and procedures. (See Examples 1-49 and FIGS. known Sustained release oral dosage forms can be used to 1-7.) It will be appreciated that where typical or preferred achieve Sustained release of the compounds of the present process conditions (i.e., reaction temperatures, times, mole invention including compound-releasing beads, tablets, erod ratios of reactants, solvents, pressures, etc.) are given, other ible and non-erodible polymers, enteric-coated materials, 40 process conditions can also be used unless otherwise stated. lipid matrices, wax matrices, osmotic delivery systems such Optimum reaction conditions may vary with the particular as the OROSR) systems made by ALZA Corporation of reactants or solvent used, but such conditions can be deter Mountain View, Calif., and tiny timed-release pills. Regard mined by one skilled in the art by routine optimization pro less of the specific form of Sustained release oral dosage form cedures. used, the compound is preferably released from the oral Sus 45 Additionally, as will be apparent to those skilled in the art, tained release dosage form over a period of at least 6 hours, conventional protecting groups may be necessary to prevent more preferably over a period of at least about 8 hours, and certain functional groups from undergoing undesired reac most preferably over a period of at least about 12 hours. tions. Suitable protecting groups for various functional Further the dosage form preferably releases from 0 to 20% of groups as well as Suitable conditions for protecting and the compound in 0 to 2 hours, from 20 to 50% of the com 50 deprotecting particular functional groups are well known in pound in 2 to 12 hours, from 50 to 85% of the compound in 3 the art. For example, numerous protecting groups are to 20 hours and greater than 75% of the compound in 5 to 18 described in T. W. Greene and G. M. Wuts, Protecting Groups hours. Preferably, the dosage forms are administered no more in Organic Synthesis, Second Edition, Wiley, New York, frequently than twice per day, more preferably no more fre 1991, and references cited therein. quently than once per day. 55 Furthermore, the compounds of this invention will typi “Systemic bioavailability” refers to the rate and extent of cally contain one or more chiral centers. Accordingly, if systemic exposure to a drug or a metabolite thereof as desired. Such compounds can be prepared or isolated as pure reflected by the area under the systemic blood concentration Stereoisomers, i.e., as individual enantiomers or diastere Versus time curve. omers, or as Stereoisomer-enriched mixtures. All Such stere “Therapeutic or prophylactic blood concentrations' refers 60 oisomers (and enriched mixtures) are included within the to systemic exposure to a Sufficient concentration of a drug or scope of this invention, unless otherwise indicated. Pure ste an active metabolite thereof over a sufficient period of time to reoisomers (or enriched mixtures) may be prepared using, for effect disease therapy or to prevent the onset or reduce the example, optically active starting materials or stereoselective severity of a disease in the treated animal. reagents well-known in the art. Alternatively, racemic mix “Thioalkyl refers to the groups—S-alkyl. 65 tures of such compounds can be separated using, for example, “Thioamidino” refers to the group RSC(=NH) where R chiral column chromatography, chiral resolving agents and is hydrogen or alkyl. the like. US 7,727,976 B2 59 60 Utility 94/00126; Atkinson et al., European Patent Application No. In one embodiment of the present invention, the com EP 285285A2). In a preferred embodiment of this method, pounds and methods described herein permit significant the compounds described herein do not contain L-D. increases in Systemic blood levels of drugs or active metabo In another embodiment of the present invention, the com lites thereof upon oral dosing of animals with the drug/linker/ 5 pounds described herein are useful as antifungal agents (for transporter compounds (relative to blood levels achieved with example, see Stretton, International Publication No. WO the parent compounds). In addition, the selection of cleavable 90/13298). In a preferred embodiment of this method, the linker also permits the drug/linker/transporter compounds compounds described herein do not contain L-D. described herein to provide sustained release of the drug or In another embodiment of the present invention, the com active metabolite thereof relative to oral dosing with the par 10 pounds described herein are inhibitors of apoptosis (for ent drug itself. In this regard, the enterohepatic recycling of example, see Steer et al. International Publication No. WO the bile acid conjugates creates a reservoir for the active 99/15179). In a preferred embodiment of this method, the agent. The above are molar comparisons, i.e., they are based compounds described herein do not contain L-D. on molar amounts of compounds of the present invention and In another embodiment of the present invention, the com parent drugs. The above comparisons are also based on 15 pounds described herein are useful for the liver-specific deliv approximately the same oral dosage forms, which release at ery of therapeutic (e.g. antineoplastics) or diagnostic agents approximately the same location in the gastrointestinal tract (e.g. MRI contrast agents, etc) (for example, see Anelli et al. of the animal being dosed. International Publication No. WO 00/38738: Sherman et al, In another embodiment of the present invention, the com U.S. Pat. No. 4,848.349). In an embodiment of this method, pounds described herein are inhibitors of the intestinal bile the compounds described herein contain L-D. In an additional acid transporter and have utility as anti-hypercholesterolemic embodiment of this method, the compounds described herein and anti-atherogenic agents (for example, see Stenglelin et al. do not contain L-D. International Publication No. WO 00/20437; Wess et al, European Patent Application No. EP 702026 A2; Wess et al. Pharmaceutical Formulations European Patent Application No. EP 624593 A2). 25 When employed as pharmaceuticals, the compounds of the In another embodiment of the present invention, the com present invention are usually administered in the form of pounds described herein are anticholestatic agents and are pharmaceutical compositions that are administered by oral useful in the treatment of biliary calculosis (i.e. gallstone routes. Such compositions are prepared in a manner well dissolution) (for example, see Gilat et al. International Pub known in the pharmaceutical art and comprise at least one lication No. WO 99/52932; Marchi et al., European Patent 30 active compound. Application No. EP 676410 A2: Krameretal, Ger. Offen. DE This invention also includes pharmaceutical compositions 98-19824123). In a preferred embodiment of this method, the that contain, as the active ingredient, one or more of the compounds described herein do not contain L-D. compounds of the present invention associated with a phar In another embodiment of the present invention, the com maceutically acceptable excipient. In making the composi pounds described herein modulate cholesterol metabolism 35 tions of this invention, the active ingredient is usually mixed via interaction with nuclear hormone receptors selected from with an excipient, diluted by an excipient, and optionally the group consisting of FXR and LXR (for example, see enclosed within a pharmaceutically acceptable carrier, which Forman et al, International Publication No. WO 00/57915; carrier can be in the form of a capsule, Sachet, paper or other Shanetal, International Publication No.WO 00/40965; Man container. When the excipient serves as a diluent, it can be a gelsdorf et al. International Publication No. WO 00/34461; 40 Solid, semi-solid, or liquid material, which acts as a vehicle or Liao et al. International Publication No. WO 00/66611). In a medium for the active ingredient. Thus, the compositions can preferred embodiment of this method, the compounds be in the form of tablets, pills, powders, lozenges, Sachets, described herein do not contain L-D. cachets, elixirs, Suspensions, emulsions, Solutions, syrups, In another embodiment of the present invention, the com etc. containing, for example, up to 10% by weight of the pounds described herein are mucosal permeability enhancers 45 active compound using, for example, Soft and hard gelatin useful in oral or nasal drug delivery systems (for example, see capsules. Illum et al, International Publication No. WO 98/01159; In preparing a formulation, it may be necessary to mill the Takahashi et al., Jpn. Kokai Tokkyo Koho JP 10286453 A2; active compound to provide the appropriate particle size prior Okada, Jpn. Kokai Tokkyo Koho JP 11060594). In a preferred to combining with other ingredients. If the active compound embodiment of this method, the compounds described herein 50 is substantially insoluble, it ordinarily is milled to a particle do not contain L-D. size of less than 200 mesh. If the active compound is substan In another embodiment of the present invention, the com tially water soluble, the particle size is normally adjusted by pounds described herein are inhibitors of gastrointestinal milling to provide a substantially uniform distribution in the inflammatory disorders (for example, see Setchelletal, Inter formulation, e.g. ~40 mesh. national Publication No. WO 97/18816). In a preferred 55 Some examples of Suitable excipients include lactose, dex embodiment of this method, the compounds described herein trose, Sucrose, Sorbitol, mannitol, starches, gum acacia, cal do not contain L-D. cium phosphate, alginates, tragacanth, gelatin, calcium sili In another embodiment of the present invention, the com cate, microcrystalline cellulose, polyvinylpyrrolidone, pounds described herein are useful in the prevention and cellulose, water, syrup, and methyl cellulose. The formula treatment of colorectal cancer (for example, see Gibson et al. 60 tions can additionally include: lubricating agents such as talc, International Publication No. WO 97/44043). In a preferred magnesium Stearate, and mineral oil; wetting agents; emulsi embodiment of this method, the compounds described herein fying and Suspending agents; preserving agents such as do not contain L-D. methyl- and propylhydroxy-benzoates; Sweetening agents; In another embodiment of the present invention, the com and flavoring agents. The compositions of the invention can pounds described herein are useful as antiviral agents (for 65 be formulated so as to provide quick, Sustained or delayed example, see Ozeki, International Publication No. WO release of the active ingredient after administration to the 95/03056; Berlati et al, International Publication No. WO patient by employing procedures known in the art. US 7,727,976 B2 61 62 The compositions are preferably formulated in a unit dos sions with edible oils such as cottonseed oil, Sesame oil, age form, each dosage containing from about 0.1 to about 500 coconut oil, or peanut oil, as well as elixirs and similar phar mg, more usually about 10 to about 100 mg. of the active maceutical vehicles. ingredient. The term “unit dosage forms’ refers to physically The following formulation examples illustrate representa discrete units suitable as unitary dosages for human Subjects tive pharmaceutical compositions of the present invention. and other animals, each unit containing a predetermined quantity of active material calculated to produce the desired Formulation Example 1 therapeutic effect, in association with a Suitable pharmaceu tical excipient. Hard gelatin capsules containing the following ingredients In therapeutic use in warm-blooded animals, the com 10 are prepared: pounds or pharmaceutical compositions thereof will be administered orally at a dosage and at a frequency per day to obtain and maintain a concentration, that is, an amount or Quantity blood-level of active component in the animal undergoing Ingredient (mg capsule) treatment, that will be therapeutically effective. Generally, 15 Such therapeutically effective amount of dosage of active Active Ingredient 3O.O component (i.e., an effective dosage) will be in the range of Starch 3OS.O about 0.1 to about 100, more preferably about 0.1 to about 10 Magnesium Stearate S.O mg/kg of body weight/day. The frequency of administration may be varied or adjusted The above ingredients are mixed and filled into hardgelatin widely depending upon the dosage of active component, that capsules in 340 mg quantities. is, the compound according to the Subject invention, in the pharmaceutical composition, the particular application, the Formulation Example 2 potency of the particular compound, and the desired concen tration. The frequency of administration may be in the range 25 A tablet formula is prepared using the ingredients below: of about once to three times per 24 hours, preferably once per 24 hours. The active compound is effective over a wide dosage range Quantity and is generally administered in a pharmaceutically effective Ingredient (mg tablet) amount. It, will be understood, however, that the amount of 30 the compound actually administered will be determined by a Active Ingredient 2S.O Cellulose, microcrystalline 2OO.O physician, in the light of the relevant circumstances, includ Colloidal silicon dioxide 1O.O ing the condition to be treated, the chosen route of adminis Stearic acid S.O tration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the 35 patient’s symptoms, and the like. The components are blended and compressed to form tab For preparing Solid compositions such as tablets, the prin lets, each weighing 240 mg. cipal active ingredient is mixed with a pharmaceutical excipi ent to form a solid preformulation composition containing a Formulation Example 3 homogeneous mixture of a compound of the present inven 40 tion. When referring to these preformulation compositions as Tablets, each containing 30 mg of active ingredient, are homogeneous, it is meant that the active ingredient is dis prepared as follows persed evenly throughout the composition so that the compo sition may be readily subdivided into equally effective unit 45 dosage forms such as tablets, pills and capsules. This solid Quantity preformulation is then subdivided into unit dosage forms of Ingredient (mg tablet) the type described above containing from, for example, 0.1 mg to about 2 g of the active ingredient of the present inven Active Ingredient 30.0 mg tion. Starch 45.0 mg 50 Microcrystalline cellulose 35.0 mg The tablets or pills of the present invention may be coated Polyvinylpyrrollidone 4.0 mg (as 10% solution in sterile water) or otherwise compounded to provide a dosage form affording Sodium carboxymethyl starch 4.5 mg the advantage of prolonged action. For example, the tablet or Magnesium stearate 0.5 mg pill can comprise an inner dosage and an outer dosage com Talc 1.0 mg ponent, the latter being in the form of an envelope over the 55 former. The two components can be separated by an enteric Total 120 mg layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum The active ingredient, starch and cellulose are passed or to be delayed in release. A variety of materials can be used through a No. 20 mesh U.S. sieve and mixed thoroughly. The for Such enteric layers or coatings, such materials including a 60 solution of polyvinylpyrrolidone is mixed with the resultant number of polymeric acids and mixtures of polymeric acids powders, which are then passed through a 16 mesh U.S. sieve. with Such materials as shellac, cetyl alcohol, and cellulose The granules so produced are dried at 50° C. to 60° C. and acetate. passed through a 16 mesh U.S. sieve. The sodium carboxym The liquid forms in which the novel compositions of the ethyl starch, magnesium Stearate, and talc, previously passed present invention may be incorporated for administration 65 through a No. 30 mesh U.S. sieve, are then added to the orally or by injection include aqueous solutions Suitably fla granules which, after mixing, are compressed on a tablet Vored syrups, aqueous or oil suspensions, and flavored emul machine to yield tablets each weighing 120 mg. US 7,727,976 B2 63 64 Formulation Example 4 lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the Capsules, each containing 40 mg of medicament are made compound preparations typically will be between 3 and 11, as follows: more preferably from 5 to 9 and most preferably from 7 and 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts. Quantity Toxicity and therapeutic efficacy of Such compounds can Ingredient (mg capsule) be determined by Standard pharmaceutical procedures in cell Active Ingredient 40.0 mg 10 cultures or experimental animals, e.g., for determining the Starch 109.0 mg L-Ds (the dose lethal to 50% of the population) and the EDs Magnesium Stearate 1.0 mg (the dose therapeutically effective in 50% of the population). Total 150.0 mg The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio L-Dso/ 15 EDso. Compounds that exhibit large therapeutic indices are The active ingredient, starch and magnesium Stearate are preferred. blended, passed through a No. 20 mesh U.S. sieve, and filled The data obtained from the cell culture assays and animal into hard gelatin capsules in 150 mg quantities. studies can be used informulating a range of dosage for use in humans. The dosage of Such compounds lies preferably Formulation Example 5 within a range of circulating concentrations that include the EDs with little or no toxicity. The dosage may vary within Suspensions, each containing 50 mg of medicament per 5.0 this range depending upon the dosage form employed and the mL dose are made as follows: route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose 25 can be estimated initially from cell culture assays. A dose may Ingredient Amount be formulated in animal models to achieve a circulating plasma concentration range which includes the ICso (the con Active Ingredient 50.0 mg Xanthan gum 4.0 mg centration of the test compound which achieves a half-maxi Sodium carboxymethyl cellulose (11%) 50.0 mg mal inhibition of symptoms) as determined in cell culture. Microcrystalline cellulose (89%) 30 Such information can be used to more accurately determine Sucrose 1.75 g useful doses in humans. Levels in plasma may be measured, Sodium benzoate 10.0 mg for example, by high performance liquid chromatography. Flavor and Color G.V. Purified water to 5.0 mL. The following synthetic and biological examples are offered to illustrate this invention and are not to be construed 35 in any way as limiting the scope of this invention. Unless The active ingredient, Sucrose and Xanthan gum are otherwise stated, all temperatures are in degrees Celsius. blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made Solution of the microcrystal EXAMPLES line cellulose and sodium carboxymethyl cellulose in water. The sodium benzoate, flavor, and color are diluted with some 40 In the examples below, the following abbreviations have of the water and added with stirring. Sufficient water is then the following meanings. If an abbreviation is not defined, it added to produce the required Volume. has its generally accepted meaning. Formulation Example 6 45 Atm = atmosphere Cbz = carbobenzyloxy CPM = counts per minute Quantity DCM = dichloromethane Ingredient (mg capsule) DMAP = 4-N,N-dimethylaminopyridine 50 DMEM = Dulbecco's minimun eagle medium Active Ingredient 15.0 mg DMF = N,N-dimethylformamide Starch 407.0 mg DMSO = dimethylsulfoxide Magnesium Stearate 3.0 mg FMOC = 9-fluorenylmethyloxycarbonyl g = gram Total 425.0 mg h = hour 55 HBSS = Hank's buffered saline solution IBAT = intestinal bile acid transporter kg = kilograms The active ingredient, starch, and magnesium Stearate are LBAT = liver bile acid transporter blended, passed through a No. 20 mesh U.S. sieve, and filled : liter into hard gelatin capsules in 425.0 mg quantities. LCMS = liquid chromatography/mass spectroscopy 60 Other suitable formulations for use in the present invention : molar can be found in Remington's Pharmaceutical Sciences, Mace min = minute Publishing Company, Philadelphia, Pa., 17th ed. (1985). mL = milliliter As noted above, the compounds administered to a patient mmol = millimols NTCP = Na+ taurocholate cotransporting are in the form of pharmaceutical compositions described polypeptide above. These compositions may be sterilized by conventional 65 PBS = phosphate buffered saline sterilization techniques, or may be sterile filtered. The result TBDMS = tert-butyldimethylsilyl ing aqueous solutions may be packaged for use as is, or US 7,727,976 B2 65 66 ride (12 mmol). The reaction mixture is slowly warmed to -continued room temperature. Then, a Solution of the appropriate 3-oxo TEOC-C = trimethylsilylethyl chloroformate cholanoic acid ester 1 (10 mmol) in 50 mL of trichloroethyl TEA = triethylamine ene is added to the reaction mixture at Such a rate that the TES = triethylsilyl temperature does not rise above 60° C. Prior to chloroformy THF = tetrahydrofuran lation, any free C-7 and/or C-12 hydroxy groups on the chol TFA = trifluoroacetic acid TMS = trimethylsilyl anoic acid derivative 1 are protected with an appropriate LIL = microliter protecting group, preferably with acetyl or trialkylsilyl group. M = micromolar When the addition is completed, the reaction mixture is wfv = volume to volume 10 heated at 55-60° C. for 3-12 h (monitored by TLC or LC/MS). The reaction mixture is cooled to room temperature and then a solution of sodium acetate (400 g) in water (500 mL) is Experimental Methods cautiously added dropwise through a dropping funnel. The organic layer is separated, washed with brine (15 mLX2) and I. General Procedures for the Preparation of Steroidal 15 dried over anhydrous Sodium sulfate. After evaporating the Building Blocks 1-12 solvent under reduced pressure, the residue is purified by passing through a short silica gel column using a gradient of Example 1 ethyl acetate and hexane as eluents to give the corresponding pure 3-chloro-2-formyl cholenoic acid ester 3. Preparation of 3-oxocholanoic acid alkyl esters 1 (Scheme 1) Example 4 The 3-oxocholanoic acid methyl esters were prepared Preparation of 2-hydroxymethylene-3-oxocholanoic according to the protocol reported by Tserang, K-Y (J. Lipid acid esters 4 (Scheme 4) Res. 1978, 32, 977-983). To a solution of the appropriate 25 cholanoic acid methyl ester 16 (25 mmol) in 250 mL of To a stirred suspension of sodium hydride (11 mmol) in anhydrous toluene was added silver carbonate, -50 wt.% on anhydrous toluene (25 mL) under nitrogen atmosphere at Celite (28.5g, 50 mmol). The reaction flask was then attached room temperature was added dropwise a solution of the to a Dean-Stark apparatus and refluxed for 2-10 h. The reac appropriate 3-oxocholanoic acid ester (R-Me) 1 (10 mmol) tion mixture was filtered, the precipitate washed with hot 30 and ethyl formate (30 mmol) in anhydrous toluene (25 mL). toluene (25 mLX3) and then the combined filtrate was evapo After having stirred for 30 min, 2-3 drops of anhydrous etha rated. The residue was passed through a short silica gel col nol was introduced into the reaction mixture. The mixture was umn using 10-25% ethyl acetate and hexane as eluents to give further stirred at room temperature for 18-24 h (monitored by the corresponding pure 3-oxocholanoic acid methyl esters TLC). Then, the reaction mixture was cautiously poured onto 1a-d in 70-92% overall yields (Table 1). Similarly, other 35 crushed ice and acidified with 5% HC1. The organic layer was 3-oxocholanoic acid alkyl esters are prepared from the cor separated and the aqueous layer was extracted with ethyl responding alkyl esters following this protocol. acetate (25 mLx2). The combined extract was washed with brine (50 mL), dried over anhydrous sodium sulfate and Example 2 evaporated to give a mixture of 2-hydroxymethylene-3-oxo 40 cholanoic acid ethyl and methyl esters, 4a-d in ~3:1 to 5:1 Preparation of 2,3-dioxocholanoic acid acids and ratios. The crude 4a-dgave satisfactory "H NMR and LS/MS their alkyl esters 2 (Scheme 2) spectra (Table 2). Similarly, the compounds 4e-f (R=Et/Me) are prepared A stirred suspension of selenium dioxide (15 mmol) in from the corresponding 3-oxocholanoic acids 1e-f. The crude dioxane (50 mL) and few drops of water is stirred at 50–55°C. 45 2-hydroxymethylene derivatives 4a-d were used for the syn for 10-20 minto give a clear solution. Then, a solution of the thesis of corresponding fused pyrazoles and pyrimidine appropriate 3-oxocholanoic acid or its ester 1 (10 mmol) in 50 derivatives without any further purification. mL of dioxane is introduced into the flask and the mixture is heated at 50-85°C. for 4-10 h (monitored by TLC or LC/MS). Example 5 Prior to oxidation, any free C-7 and/or C-12 hydroxy groups 50 on the cholanoic acid derivative 1 are protected with an appro Preparation of priate protecting group, preferably with acetyl or trialkylsilyl 2-dimethylaminomethylene-3-oxocholanoic acid group. The reaction mixture is filtered through a sintered methyl esters 5a-c (Scheme 5) funnel, the precipitate washed with dioxane (15 mLx2) and the combined filtrate is evaporated on a rotavapor under 55 To a stirred solution of the appropriate 3-oxocholanoic acid reduced pressure. The residue is purified by silica gel column ester 1 (10 mmol) in anhydrous THF or dioxane (30 mL) chromatography using 10-25% gradient of ethyl acetate and under nitrogen atmosphere was added Bredereck’s reagent, hexane as eluents to give the pure 2. tert-butoxybis(dimethylamino)methane (22 mmol) and the mixture was refluxed for 3-24 h (monitored by TLC or Example 3 60 LC/MS). After concentrating the reaction mixture under reduced pressure the residue was diluted with ethyl acetate Preparation of 3-chloro-2-formyl derivatives of (50 mL). Then, it was washed with water (25 mL) and brine cholenoic acid alkyl esters 3 (Scheme 3) (25 mL). Solvent was evaporated after drying over anhydrous sodium sulfate. The crude 5a-c gave satisfactory "H NMR and To a stirred solution of anhydrous N,N-dimethylforma 65 LS/MS spectra. (Table 3). mide (15 mmol) in 50 mL of trichloroethane under nitrogen Similarly, the compounds 5d-f(R=Me) are prepared from atmosphere at 0°C. is added dropwise phosphorous oxychlo the corresponding 3-oxocholanoic acids 1d-f. The crude US 7,727,976 B2 67 68 2-hydroxymethylene derivatives 5a-c were used for the syn sodium ethoxide as a base (Scheme 7). The 7a-1 gave satis thesis of fused pyrazoles derivatives 48 without any further factory "H NMR spectral data. The Electrospray mass spec purification. trometry showed the expected molecular ion at m/z 552 Example 6 (M+H). Preparation of Example 8 2-bis(thiomethyl)methylene-3-oxocholanoic acid Preparation of oxoketene N.S-acetal derivatives of esters 6 (Scheme 6) cholanoic acid esters 8 (Scheme 8) To a stirred solution of sodium ethoxide or tert-butoxide 10 (22 mmol) in anhydrous toluene (100 mL) under nitrogen A solution of the appropriate oxoketene S.S-acetal 6 (10 atmosphere at 0-5°C. is dropwise added a solution of the mmol) and aliphatic amine 19 (10 mmol) in methanol or appropriate 3-oxocholanoic acid ester 1 (10 mmol) and car ethanol (50 mL) is refluxed for 12-18 h (monitored by TLC). bon disulfide (10 mmol) in anhydrous toluene (50 mL). Any Acetic acid is used as solvent if 19 is an aromatic amine. After free C-7 and/or C-12 hydroxy groups on the cholanoic acid 15 completion of the reaction, the solvent is evaporated on a derivative 1 are protected with an appropriate protecting rotavapor under reduced pressure and the residue is diluted group, preferably with a trialkylsilyl group. The reaction mix with ethyl acetate (100 mL). The organic layer is washed with ture is slowly warmed to room temperature and stirring is water (25 mLX2), dried over anhydrous sodium sulfate, and continued further at room temperature for 12-15 h. Then, a evaporated. The crude product is purified by silica gel chro solution of iodomethane (25 mmol) in anhydrous toluene (25 matography using 10-25% gradient of ethyl acetate and hex mL) is added dropwise at 0-5°C. and stirred further for 12-15 ane as eluents to give the pure N.S-acetal 8. hat room temperature. The reaction mixture is poured onto crushed ice and the organic layer is separated. The aqueous Example 9 layer is extracted with ethyl acetate (25 mL) and the com 25 Preparation of 3-oxo-2-(B-oxomethyl) substituted bined extract is washed with water (25 mL) then dried over anhydrous sodium sulfate. The solvent is evaporated under bile acid building blocks 9 (Scheme 9) reduced pressure and the residue is purified by silica gel column chromatography using 0-25% gradient of ethyl To a stirred solution of the appropriate 3-oxocholanoic acid acetate and hexane as eluents. ester derivative 1 (10 mmol) in anhydrous THF (50 mL) under 30 nitrogen atmosphere at -78°C. is added dropwise a solution Example 7 of freshly prepared L-DA (10.5 mmol) in THF. The reaction mixture is slowly warmed to 0°C. After having stirred at 0°C. Preparation of oxoketene N.S-acetal derivatives of for 1 h, the reaction mixture is cooled to -78° C. and a cholanoic acid esters 7 (Scheme 7) solution of the appropriate 20 (10 mmol) in anhydrous THF 35 (25 mL) added dropwise. The reaction mixture is slowly To a stirred solution of sodium ethoxide or tert-butoxide warmed to room temperature and further stirred at room (12 mmol) in anhydrous toluene (50 mL) under nitrogen temperature for 12-18h. The mixture is poured into saturated atmosphere at 0-5°C. is added dropwise a solution of the ammonium chloride solution (250 mL) and extracted with appropriate 3-oxocholanoic acid ester 1 (10 mmol) and ethyl acetate (50 mLX3). The combined extract is washed isothiocyanate 17 (10 mmol) in anhydrous toluene (50 mL). 40 with water (25 mL), dried over anhydrous sodium sulfate, and Any free C-7 and/or C-12 hydroxy groups on the cholanoic finally the solvent evaporated under reduced pressure. The acid derivative 1 are protected with an appropriate protecting residue is purified by silica gel column chromatography using group, preferably with a trialkylsilyl group. After having 5-25% gradient of ethyl acetate and hexane as eluents to give stirred at room temperature for 12-18 h, the reaction mixture the pure 9. is poured onto crushed ice. The organic phase is separated and 45 the aqueous layer is extracted with ethyl acetate (25 mLx2). Example 10 The combined extract is washed with water (50 mL), dried over anhydrous Sodium Sulfate and evaporated under reduced Preparation of 3-oxo-2-propargyl substituted bile pressure. The crude B-ketothioamide 18 is purified by silica acid building blocks 10 (Scheme 10) gel column chromatography using 0-25% gradient of ethyl 50 acetate and hexane as eluents. The pure thioamide 18 is added To a stirred solution of the appropriate 3-oxocholanoic acid to a stirred Suspension of potassium carbonate (10 mmol) in ester derivative 1 (10 mmol) in anhydrous THF (50 mL) under anhydrous acetone (100 mL) and refluxed for 3–4 h. After nitrogen atmosphere at -78°C. is added dropwise a solution cooling the reaction mixture to 0° C., a solution of of freshly prepared L-DA (10.5 mmol) in THF. The reaction iodomethane (15 mmol) in anhydrous acetone (25 mL) is 55 mixture is slowly warmed to 0°C. After having stirred at 0°C. added dropwise and stirred at room temperature for 12-18 h. for 1 h, the reaction mixture is cooled to -78° C. and a Then, the mixture is filtered through a sintered funnel and the Solution of appropriate propargyl halide or tosylate 21 (10 precipitate is washed with acetone (25 mLx2). The combined mmol) in anhydrous THF (25 mL) added dropwise. The reac filtrate is concentrated on a rotavoporunder reduced pressure. tion mixture is slowly warmed to room temperature and fur The residue is diluted with ethyl acetate (200 mL) and washed 60 ther stirred at room temperature for 12-18 h. The mixture is with water (25 mLx2), dried over anhydrous sodium sulfate, poured into saturated ammonium chloride solution (250 mL) and the solvent evaporated. The crude product is passed and extracted with ethyl acetate (50 mLX3). The combined through a short silica gel column using 0-25% gradient of extract is washed with water, dried over anhydrous sodium ethyl acetate and hexane as eluents to give the corresponding sulfate, and finally evaporated the solvent under reduced pres pure oxoketene N.S-acetal 7. 65 Sure. The residue is purified by silica gel column chromatog Following this protocol, the NS-acetal 7a-1 (R=Et, raphy using 5-25% gradient of ethyl acetate and hexane as R-R-H and R=CHs) was prepared in 25% yield using eluents to give the pure 10. US 7,727,976 B2 69 70 Example 11 drous Sodium Sulfate the solvent is evaporated under vacuum. The residue is crystallized from DCM/hexane to give the pure Preparation of Vinyl Ether or Ester Derivatives of mesylates 26. Bile Acid Building Blocks 11 (Scheme 11) To a stirred Suspension of cesium carbonate (4 mmol) in anhydrous DMF (25 mL) is added thioacetic acid (6 mmol) To a stirred solution of the appropriate 3-oxocholanoic acid followed by the appropriate mesylate 26 (5 mmol) in one ester derivative 1 (10 mmol) in anhydrous THF (50 mL) under portion and the reaction mixture stirred at room temperature nitrogen atmosphere at -78°C. is added dropwise a solution for 18 h. The mixture is poured into cold water and extracted of freshly prepared L-DA (10.5 mmol) in THF. The reaction with ethyl acetate (50 mLX3). The combined extract is mixture is slowly warmed to 0°C. After having stirred at 0°C. 10 washed with water (200 mL), sodium bicarbonate solution for 1 h, the reaction mixture is cooled to -78° C. and a (55 w/w, 200 mL) and finally with brine (100 mL). The Solution of the appropriate alkylating or silylating agent 22 organic layer is dried over anhydrous sodium sulfate and (10 mmol) in anhydrous THF (25 mL) added dropwise. The concentrated under reduced pressure to give the crude prod reaction mixture is slowly warmed to room temperature and uct 15, which is purified by Silica gel column chromatography further stirred at room temperature for 12-18 h. The mixture 15 using DCM as eluent. is poured into saturated ammonium chloride solution (250 mL) and extracted with ethyl acetate (50 mLX3). The com Example 14 bined extract is washed with brine (25 mL), dried over anhy drous sodium Sulfate, and finally evaporated the Solvent under Preparation of N-substituted C.-amino acid esters 30 reduced pressure. The crude product 11 is carried further for (Scheme 14) the synthesis of various 2.3-fused carbocyclic and heterocy clic derivatives without purification. The corresponding vinyl To a stirred solution of the appropriate primary amine 28 acetate derivatives (R-Ac) are prepared by refluxing the (10 mmol) in anhydrous DCM (50 mL) at room temperature appropriate 1 with excess ofisopropenyl acetate in anhydrous is added TEA (11 mmol). The reaction mixture is cooled to 0° toluene in the presence of catalytic amounts of p-toluene 25 C. and then a solution of an appropriate O-activated acetate Sulfonic acid. ester 29 (10 mmol) in anhydrous DCM (25 mL) is added dropwise. The reaction mixture is stirred at 0°C. for 3 hand Example 12 at room temperature for 12-18 h. After completion of the reaction, the mixture is filtered and the precipitate washed Preparation of 2-methylene-3-oxo derivatives of bile 30 with DCM (15 mL). The combined filtrate is concentrated on acid building blocks 12 (Scheme 12) a rotavapor under reduced pressure and the residue is purified by column chromatography on silica gel using a gradient of To a stirred solution of the appropriate 3-oxocholanoic acid ethyl acetate and hexane as eluents to give the pure product ester derivative 1 (10 mmol) in anhydrous THF (50 mL) under 3O. nitrogen atmosphere at -78°C. is added dropwise a solution 35 III. General Procedures for the Synthesis of 5-Membered of freshly prepared L-DA (10.5 mmol) in THF. The reaction Heterocycles mixture is slowly warmed to 0°C. After having stirred at 0°C. for 1 h, the reaction mixture is cooled to -78° C. and a Example 15 solution of the appropriate aldehyde or ketone 23 (10 mmol) in anhydrous THF (25 mL) added dropwise. The reaction 40 Synthesis of Fused Furan Derivatives of Cholanoic mixture is slowly warmed to room temperature and further Acid Esters 31 (Scheme 15) stirred at room temperature for 12-18h. The mixture is poured into saturated ammonium chloride solution (250 mL) and To a stirred solution of potassium tert-butoxide or sodium extracted with ethyl acetate (50 mLX3). The combined extract hydride (6 mmol) in anhydrous DMF (10 mL) under nitrogen is washed with water (25 mL), dried over anhydrous sodium 45 atmosphere at room temperature is added a solution of the sulfate, and finally the solvent evaporated under reduced pres appropriate 3-oxo-2-propargylcholanoic acid ester 10 (5 Sure. The residue is purified by silica gel column chromatog mmol) in anhydrous DMF (15 mL). The reaction mixture is raphy using 5-25% gradient of ethyl acetate and hexane as stirred at room temperature to reflux temperature for 5-24h eluents to give pure 12. depending upon the Substituents on the propargyl side chain. II. General Procedures for the Preparation of Amino Acid 50 After completion of the reaction as judged by TLC or LC/MS, Building Blocks 15 and 30 the reaction mixture is poured onto crushed ice and extracted with ethyl acetate (25 mLX3), dried over anhydrous sodium Example 13 sulfate and finally the solvent evaporated under reduced pres Sure using a rotavapor. The crude product 31 is purified by Preparation of 2-aminoethylthioacetate 15 55 silica gel chromatography using a gradient of ethyl acetate (Scheme 13) and hexane as eluents. The N-protected amino acid alcohols 25 are either pur Example 16 chased from commercial sources or prepared in high yield according to the protocol reported by Rodriguez et al. To a 60 Synthesis of Fused Pyrrole Derivatives of Cholanoic stirred solution of the appropriate alcohol 25 (10 mmol) in Acid Esters 33 (Scheme 16) and 34 (Scheme 17) anhydrous DCM (50 mL) is added triethylamine (12 mmol) at room temperature. After cooling to 0° C. methanesulfonyl To a solution of the appropriate 3-oxo-2-propargylchol chloride (12 mmol) is added dropwise. Stirring is continued anoic acid ester 10 (e.g. R-Bu) (5 mmol) and an amine 32 (6 for 1-5 hat room temperature followed by addition of DCM 65 mmol) in anhydrous toluene (25 mL) is added catalytic (50 mL). The mixture is washed with a KHSO (1M, 50 mL), amounts of p-toluenesulfonic acid and the mixture refluxed water (50 mL), and brine (50 mL). After drying over anhy for 5-24 h. The progress of the reaction is monitored by TLC. US 7,727,976 B2 71 72 The mixture is poured into cold water (100 mL) and extracted sphere at -78°C. The reaction mixture is slowly warmed to with ethyl acetate (25 mLx2). The combined extract is room temperature and further stirred at room temperature for washed with brine (25 mL), dried over anhydrous sodium 12-18 h. Then, the mixture is poured onto crushed ice, acidi sulfate and finally the solvent evaporated under reduced pres fied with 5% HCl, and extracted with ethyl acetate (25 Sure using a rotavapor. The residue is passed through a short mLX3). The combined extract is washed with water (25 mL), silica gel column using a gradient of ethyl acetate and hexane brine (25 mL), and dried over anhydrous sodium sulfate. to give the pure pyrrole 33 (Scheme 16). After evaporation of the solvent, the residue is purified by Treatment of the appropriate cholanoic acid esters 9 (e.g. silica gel column chromatography using a gradient of ethyl R=TBu) with amines 32 under identical conditions affords the acetate and hexane as eluents to give the pure pyrrole 37. corresponding 2.3-fused pyrrole derivatives 34 (Scheme 17). 10 The pyrrole 37 (3 mmol) is treated with m-chloroperben Zoic acid (7 mmol) in THF (25 mL) at room temperature for Example 17 12-24 h (monitored by TLC or LC/MS). The reaction mixture is filtered, the filtrate is diluted with water (25 mL), and Synthesis of Fused Pyrrole Derivatives of Cholanoic extracted with ethyl acetate (25 mLX3). The combined extract Acid Esters 35 (Scheme 18) and 36 (Scheme 19) 15 is washed with saturated sodium bicarbonate (15 mlx2), water (25 mL), dried over anhydrous sodium sulfate, and To a stirred solution of the appropriate 3-chloro-2-formyl cautiously evaporated under reduced pressure. The crude Sul 2-cholenoic acid ester 3 (5 mmol) in anhydrous THF (15 mL) fone is then heated at ~70° C. with the appropriate amine (3 at 0°C. under nitrogen atmosphere is added dropwise a solu mmol) in DMF (25 mL) for 12-24 h (monitored by TLC). The tion of an N-substituted amino acid ester 30 (5 mmol) and Solvent is evaporated and the residue chromatographed on triethylamine (6 mmol) in THF (15 mL). The reaction mixture silica gel column using a gradient of ethyl acetate and hexane is warmed to room temperature and further stirred for 5-18h as eluents to give the pure aminopyrrole 38 (Scheme 20). (monitored by TLC or LC/MS). Once the reaction is com The aminopyrroles 38 can also be prepared from the cor plete, the reaction mixture is filtered to remove the triethy responding N.S-acetals 8 (RandR'zH) in two steps under lamine hydrochloride salt and the filtrate is concentrated on a 25 identical reaction conditions as described for the synthesis of rotavapor under reduced pressure. The residue is diluted with pyrrole 37. Initial treatment of N.S-acetals 8 (5 mmol) with ethyl acetate (50 mL), washed with water (25 mLx2), dried amines 30 gives the corresponding N,N-acetals, which after over anhydrous sodium sulfate, and the solvent evaporated aqueous workup and purification as described above, are under reduced pressure to afford the corresponding enaminal cyclized by treating with a suitable base (e.g. NaH or L-DA) cholanoic acid ester derivative. The crude enaminal is then 30 to give the corresponding aminopyrroles 38 in good yields dissolved in anhydrous THF (15 mL) and added dropwise to (Scheme 20). a stirred suspension of sodium hydride (5 mmol) in THF (15 mL) at 0°C. under nitrogen atmosphere. The reaction mixture Example 19 is warmed to room temperature. If L-DA is used as the base, a freshly prepared solution of L-DA (5 mmol) in THF (15 mL) 35 Synthesis of Fused Pyrrole Derivatives of Cholanoic is added to stirred solution of crude enaminal at -78° C. and Acid Esters 40 (Scheme 21) slowly warmed to room temperature. The mixture is stirred further at room temperature for 2-15 h. The mixture is poured A solution of enone 12 (5 mmol) and amino acid ester 30 (5 into crushed ice, acidified with 5% HCl solution, and then mmol) in anhydrous DMF or ethanol (25 mL) is heated at extracted with ethyl acetate (25 mLX3). The combined extract 40 70-90° C. (monitored by TLC). The mixture is cooled to room is washed with water (25 mL), brine (25 mL), dried over temperature, poured into cold water (50 mL), extracted with anhydrous sodium Sulfate and evaporated. The crude pyrrole ethyl acetate (25 mLX3), and dried over anhydrous sodium 35 is purified by silica gel column chromatography using a sulfate. After evaporating the solvent, the residue is dissolved gradient of ethyl acetate and hexane as eluents. in anhydrous THF (15 mL) and added dropwise to a suspen The pyrrole derivatives 36 are also prepared under identical 45 sion of sodium hydride (5 mmol) in THF (15 mL) at 0°C. If reaction conditions by treating 2-hydroxymethylene-3-oxo L-DA is used instead as the base, a freshly prepared solution cholanoic acid esters 4 or 2-dimethylamino-methylene-3- of L-DA (5 mmol) in THF is added to the corresponding oxocholanoic acid esters 5 with amine 30 (Scheme 19). amino acid ester adduct solution in THF (15 mL) at -78°C. The reaction mixture is slowly warmed to room temperature Example 18 50 and further stirred for 12-15 h. The mixture is poured onto crushed ice and extracted with ethyl acetate (25 mLX3). The Synthesis of Fused Pyrrole Derivatives of Cholanoic combined extract is washed with water (25 mL), brine (25 Acid Esters 38 (Scheme 20) mL) and dried over anhydrous sodium sulfate. After evapo ration of the solvent, the crude product37 is treated with DDQ A solution of the appropriate C-oxoketene S.S-acetal 6 (5 55 (6 mmol) in toluene at reflux temperature for 12-24 h (moni mmol) and amino acid ester 30 (5 mmol) in anhydrous DMF tored by TLC). The reaction mixture is evaporated and the (25 mL) is heated at 80-90° C. for 5-12 h. The progress of the residue is chromatographed on silica gel column using a reaction is monitored by TLC. The reaction mixture is poured gradient of ethylacetate and hexane as eluents to give the pure into cold water (25 mL), extracted with ethyl acetate (25 pyrrole 40 (Scheme 21). mLX3), and dried over anhydrous sodium sulfate. The solvent 60 is evaporated under reduced pressure to give the correspond Example 20 ing NS-acetal. The crude N.S-acetal is dissolved in anhy drous THF (15 mL) and added dropwise to a stirred suspen Synthesis of Fused Indole Derivatives of Cholanoic sion of sodium hydride (5 mmol) in THF (15 mL) under Acid Esters 42 and 44 (Scheme 22) nitrogen atmosphere at 0°C. If L-DA is used as the base, a 65 solution of L-DA (5 mmol) in THF is added to a solution of To a solution of the appropriate 3-oxocholanoic acid ester N.S-acetal (5 mmol) in THF (15 mL) under nitrogen atmo 1 (e.g. R="Bu) (5 mmol) in glacial acetic acid (50 mL) is US 7,727,976 B2 73 74 added phenylhydrazine 41 (6 mmol) followed by anhydrous Similarly, pyrazole derivatives 48 were also synthesized Zinc chloride (10 mmol). The mixture is refluxed for 12-24h from the corresponding. 2-dimethylaminomethylene-3-oxo (monitored by TLC). The reaction mixture is cooled to room cholanoic acid esters 5 and hydrazines 45 under identical temperature and poured into cold water (50 mL), extracted reaction conditions (Scheme 24). with ethyl acetate (25 mLX3). The combined extract is washed with saturated sodium bicarbonate (25 mL), water Example 23 (25 mL), dried over anhydrous Sodium Sulfate, and evapo rated to dryness. The residue is purified by silica gel column Synthesis of Fused Pyrazole Derivatives of chromatography using a gradient of ethyl acetate and hexane Cholanoic Acid Esters 50 and 51 (Scheme 25) as eluents to give the pure indole derivative 42 (Scheme 22). 10 The indole 42 may be alkylated with various alkylating A solution of the appropriate C-oxoketene S.S-acetal 6 (5 agents under mild reaction conditions (Scheme 22). To a mmol) and hydrazine 45 (5 mmol) in absolute alcohol or stirred Suspension of potassium carbonate (3 mmol) in anhy anhydrous DMF (25 mL) is heated at 80-90° C. for 5-12 h. drous THF (25 mL) is added the appropriate indole 42 (3 The progress of the reaction is monitored by TLC. The reac mmol) and then the mixture refluxed for 3 h. The reaction 15 tion mixture is poured into cold water (50 mL), extracted with mixture is cooled to 0°C. and added dropwise a solution of ethyl acetate (25 mLX3) and dried over anhydrous sodium the appropriate alkyl or acyl halide 43 (4 mmol) in THF (10 sulfate. After evaporating the solvent, the residue is purified mL). The cooling bath is removed and the mixture is stirred at by column chromatography on silica gel using a gradient of room temperature for 12-18h. The reaction mixture is filtered ethyl acetate and hexane as eluents to give the pure thiometh and the filtrate is evaporated. The residue is dissolved in ethyl ylpyrazole derivative 50. Under identical reaction conditions acetate (100 mL) and washed with water (25 mLx2). The N.S-acetals 7 and 8 afford the corresponding aminopyrazole organic phase is dried over anhydrous Sodium Sulfate and the derivatives 51. Solvent evaporated. The residue is passed through a short The amino pyrazoles 51 can also be prepared from the silica gel column using a gradient of ethyl acetate and hexane corresponding pyrazoles 50 (Scheme 25). The pyrazole 50 (3 as eluents to give the corresponding pure N-alkylated oracy 25 mmol) is treated with m-chloroperbenzoic acid (7 mmol) in lated indole 44 (Scheme 22). THF (25 mL) at room temperature for 12-24 h (monitored by TLC or LC/MS). The reaction mixture is filtered, the filtrate Example 21 is diluted with water (25 mL) and extracted with ethyl acetate (25 mLX3). The combined extract is washed with saturated Synthesis of Fused Pyrazole Derivatives of 30 sodium bicarbonate (15 mLx2), water (25 mL), dried over Cholanoic Acid Esters 46 and 47 (Scheme 23) anhydrous sodium Sulfate, and cautiously evaporated under reduced pressure. The crude sulfone thus obtained is heated at To a solution of the appropriate 3-chloro-2-formyl chole ~70° C. with the appropriate amine 14 (3 mmol) in DMF (25 noic acid ester 3 (e.g. R-Bu) (5 mmol) and hydrazine 45 (5 mL) for 12-24 h (monitored by TLC). The solvent is evapo mmol) in anhydrous toluene (25 mL) is added triethylamine 35 rated and the residue chromatographed on silica gel column (6 mmol). The mixture is refluxed for 5-24 h (monitored by using a gradient of ethyl acetate and hexane as eluents to give TLC). After cooling to room temperature, the reaction mix the corresponding pure aminopyrazole 51 (Scheme 25). ture is poured into cold water (50 mL) and extracted with ethyl acetate (25 mLX3). The combined extract is washed Example 24 with water (25 mL), dried over anhydrous sodium sulfate, and evaporated. The residue is purified by Silica gel column chro 40 Synthesis of Fused Dihydropyrazole and Pyrazole matography using a gradient of ethyl acetate and hexane as Derivatives of Cholanoic Acid Esters 52 and 53 eluents to give the pure pyrazole derivative 46 or 47. The (Scheme 26) N-alkyl substituted hydrazines 45 afford the 2'-substituted pyrazole 47 whereas N-aryl or electron withdrawing substi A solution of enone 12 (5 mmol) and hydrazine 45 in tuted hydrazines 45 afford the corresponding 1'-substituted 45 absolute ethanol or anhydrous DMF (25 mL) is heated at pyrazoles 46 (Scheme 23). 70-90° C. (monitored by TLC). The mixture is cooled to room temperature, poured into cold water (50 mL), extracted with Example 22 ethyl acetate (25 mLX3) and dried over anhydrous sodium sulfate. After evaporating the solvent, the residue is purified Synthesis of Fused Pyrazole Derivatives of 50 by silica gel column chromatography on silica gel using a Cholanoic Acid Esters 48 and 49 (Scheme 24) gradient of ethylacetate and hexane as eluents to give the pure dihydropyrazole derivative 52. A solution of appropriate 2-hydroxymethylene-3-oxochol The purified or crude 52(R''=H) (2 mmol) is refluxed with anoic acid ester 4 (5 mmol) and hydrazine 45 (5 mmol) in DDQ (3 mmol) in anhydrous toluene (25 mL) for 12-24 h absolute ethanol (25 mL) was refluxed for 5-24 h (monitored 55 (monitored by TLC). After evaporation of the solvent, the by TLC). The reaction mixture was evaporated on a rotavapor residue is purified by silica gel column chromatography using under reduced pressure, and the residue was diluted with a gradient of ethyl acetate and hexane as eluents to give the ethyl acetate (50 mL). It was washed with water (15 mLx2), pure pyrazole derivative 53. dried over anhydrous sodium sulfate and evaporated. The residue was purified by silica gel column chromatography 60 Example 25 using a 10-50% gradient of ethyl acetate and hexane as elu ents to give the pure 2'-substituted pyrazole 48 in good yield. Synthesis of Fused Isoxazole Derivatives of Similarly, the N-aryl or electron withdrawing substituted Cholanoic Acid Esters 54 (Scheme 27) and 55 hydrazines afforded the corresponding 1'-substituted pyra (Scheme 28) Zoles 49 in good yields (Scheme 24). The pyrazole derivatives 65 48 and 49 prepared by this method and their corresponding To a stirred suspension of hydroxylamine hydrochloride (6 analytical data are given in Table 4. mmol) in anhydrous THF (15 mL) is added freshly prepared US 7,727,976 B2 75 76 sodium ethoxide (15 mmol) followed by the appropriate IV. General Procedures for the Synthesis of Fused 6-Mem 3-chloro-2-formyl derivative of cholenoic acid ester 3 (5 bered Carbocyclic and Heterocyclic Derivatives of Bile Acids mmol) in THF (25 mL). The reaction mixture is refluxed for 12-24 h (monitored by TLC). The mixture is cooled to room Example 28 temperature, diluted with water (50 mL) and extracted with ethyl acetate (25 mLX3). The combined extract is washed Synthesis of 2,3-benzofused cholanoic acid esters 61 with water (25 mL), dried over anhydrous sodium sulfate and (Scheme 31) evaporated. The residue is purified by Silica gel column chro matography using a gradient of ethyl acetate and hexane as A solution of the appropriate vinyl ester or ether 11 (5 eluents to give the corresponding pure isoxazole derivative 54 10 mmol) and diene 59 (5 mmol) in anhydrous toluene (25 mL) (Scheme 27). is heated in a sealed tube at 110-115°C. for 8-14 h (monitored The isoxazole derivatives 55 are synthesized from the by TLC). After evaporation of the solvent under reduced 2-hydroxymethylene 4, or 2-dimethylaminomethylene-3- pressure the residue is purified by silica gel chromatography oxocholanoic acid ester 5 with hydroxylamine hydrochloride using a gradient of ethyl acetate and hexane as eluents to give under identical reaction conditions (Scheme 28). 15 the pure adduct 60 (Scheme 31). The cycloadduct 60 is treated with DDQ (6 mmol) in Example 26 anhydrous toluene (25 mL) at reflux temperature for 12-24h (monitored by TLC). The solventis evaporated under reduced Synthesis of Fused Isoxazole Derivatives of pressure and the residue is purified by silica gel column Cholanoic Acid Esters 57 (Scheme 29) chromatography using a gradient of ethyl acetate and hexane as eluents to give the pure benzofused cholanoic acid ester 61 To a stirred suspension of hydroxylamine hydrochloride (6 (Scheme 31). mmol) in anhydrous THF (25 mL) is added sodium ethoxide or methoxide (15 mmol) followed by a solution of the appro Example 29 priate C-oxoketene S.S-acetal 6 (5 mmol). Then, the mixture 25 is refluxed for 8-24 h (monitored by TLC). The reaction Synthesis of Fused Dihydropyran Derivatives of mixture is poured into cold water (50 mL), extracted with Cholanoic Acid Esters 64 (Scheme 32) ethyl acetate (25 mLX3) and dried over anhydrous sodium sulfate. After evaporating the solvent, the residue is purified Following the protocol described above for the synthesis of by column chromatography on silica gel using a gradient of 30 benzofused derivatives 61 (Scheme 31), the fused dihydro ethyl acetate and hexane as eluents to give the pure thiometh pyran derivatives of cholanoic acid esters 64 are prepared by ylisoxazole derivative 56. Under identical reaction conditions treating the appropriate vinyl ester or ether 11 with C.f3 N.S-acetals 7 and 8 afford the corresponding aminoisoxazole unsaturated carbonyl compounds 62 (Scheme 32). derivatives 57. Example 30 The aminoisoxazoles 57 can also be prepared from the 35 corresponding isoxazole 56 (Scheme 29). The appropriate isoxazole 56 (3 mmol) is treated with m-chloroperbenzoic Synthesis of Fused Pyridine Derivatives of acid (7 mmol) in THF (25 mL) at room temperature for 12-24 Cholanoic Acid Esters 67 (Scheme 33) h (monitored by TLC or LC/MS). The reaction mixture is Following the protocol described above for the synthesis of filtered, the filtrate is diluted with water (25 mL) and extracted 40 benzofused derivatives 61 (Scheme 31), the fused pyridine with ethyl acetate (25 mLX3). The combined extract is derivatives of cholanoic acid esters 64 are prepared by treat washed with saturated sodium bicarbonate (15 mLx2), water (25 mL), dried over anhydrous Sodium sulfate, and cautiously ing the appropriate vinyl ester or ether 11 with C.f3-unsatur evaporated under reduced pressure. Then, a solution the crude ated imines 65 (Scheme 33). Sulfone and the appropriate amine 14 (3 mmol) is heated at 45 Example 31 ~70° C. in DMF (25 mL) for 12-24 h (monitored by TLC). The solvent is evaporated under reduced pressure and the Synthesis of Fused Pyrazine Derivatives of residue is chromatographed on silica gel column using a Cholanoic Acid Esters 69 (Scheme 34) gradient of ethyl acetate and hexane as eluents to give the corresponding pure aminoisoxazole 57 (Scheme 29). 50 A solution of the appropriate 2,3-dioxocholanoic acid ester 2 (e.g. R=tEu) (5 mmol) and 1,2-diamine 68 (5 mmol) in Example 27 DMF is heated at 100-120° C. for 5-24 h (monitored by TLC). After cooling to room temperature, the mixture is diluted with Synthesis of Fused Triazole Derivatives of Cholanoic water (50 mL) and extracted with ethyl acetate (25 mLX3). Acid Esters 58 (Scheme 30) 55 The combined extract is washed with water (25 mL), dried over anhydrous sodium Sulfate, and evaporated under To a stirred solution of the appropriate vinyl acetate or reduced pressure. The residue is purified by silica gel chro ether 11 (5 mmol) in anhydrous DMF (15 mL) in a wide matography using a gradient of ethyl acetate and hexane as mouth reaction vessel is added a solution of alkyl orarylazide eluents to give the corresponding pure fused pyrazine 69. (5 mmol) in DMF (15 mL). The reaction mixture is heated at 60 100-120° C. for 8-24 h (monitored by TLC). After cooling, Example 32 the reaction mixture is poured into ice-cold water and extracted with ethyl acetate (25 mLX3). The combined extract Synthesis of Fused Pyrimidine Derivatives of is washed with water (25 mL), dried over anhydrous sodium Cholanoic Acid Esters 74-77 (Scheme 35) sulfate, and evaporated. The residue is purified by silica gel 65 column chromatography using a gradient of ethyl acetate and A solution of the appropriate cholanoic acid ester building hexane as eluents to give the pure triazole 58. block 4 (5 mmol) and urea 70 (6 mmol) in ethanol (25 mL) US 7,727,976 B2 77 78 was refluxed for 5-24 h (monitored by TLC). The reaction Example 34 mixture was concentrated under reduced pressure. The resi due was diluted with ethyl acetate (50 mL), washed with Synthesis of Fused Pyrimidine Derivatives of water (25 mLx2) and dried over dried over anhydrous sodium Cholanoic Acid Esters 90-93 (Scheme 37) sulfate. After evaporation of the solvent the residue was puri fied by silica gel chromatography using a gradient of ethyl A solution of enone 12 (5 mmol) and urea (70) in anhy drous DMF (25 mL) is heated at 100-120° C. for 8-24 h acetate and hexane as eluents to give the corresponding pure (monitored by TLC). The mixture is cooled to room tempera fused pyrimidone derivative 74 (Scheme 35). ture, diluted with cold water (50 mL), extracted with ethyl Under identical reaction conditions, treatment of steroidal 10 acetate (25 mLX3) and dried over anhydrous sodium sulfate. building blocks 4 with thiourea 71 afforded the corresponding After evaporating the solvent, the residue is purified by silica fused 2-thiopyrimidones 75 and the amidine 73 gel column chromatography using a gradient of ethyl acetate (X=CHCONH2) afforded the corresponding fused pyrimi and hexane as eluents to give the pure dihydropyrimidone 86. dine 77 (Scheme 35). Representative pyrimidine derivatives Similarly, the reaction of 12 with thiourea (71) affords the 15 corresponding dihydrothiopyrimidone 87; reaction with synthesized and their analytical data are given Table 5. guanidine (72) affords dihydroamino-pyrimidine 88; and Similarly, other pyrimidine derivatives having the general reaction with amidine 73 affords the dihydropyrimidine 89 structure 74-77 are prepared by reacting the steroidal building (Scheme 37). blocks 3, 4, and 5 with various 70-73 either in ethanol or DMF The purified or crude dihydropyrimidines 86-89 (R''=H) (Scheme 35). (2 mmol) are refluxed with DDQ (3 mmol) in anhydrous toluene (25 mL) for 12-24 h (monitored by TLC). After Example 33 evaporation of the solvent, the residues are purified by silica gel column chromatography using a gradient of ethyl acetate Synthesis of Fused Pyrimidine Derivatives of and hexane as eluents to give the pure pyrimidine derivatives 25 90-93 (Scheme 37). Cholanoic Acid Esters 82-85 (Scheme 36) V. General Procedures for the Synthesis of Fused Diaz epine Derivative of Bile Acids A solution of the appropriate oxoketene S.S-acetal deriva Example 35 tive 6 (5 mmol) and urea (70) (5 mmol) in DMF (25 mL) is heated at 100-120° C. for 5-24 h (monitored by TLC). After 30 Synthesis of Fused Diazepine Derivatives of cooling to room temperature, the mixture is diluted with Cholanoic Acid Esters 95 and 97 water (50 mL) and extracted with ethyl acetate (25 mLX3). (Schemes 38 and 40) The combined extract is washed with water (25 mL), dried over anhydrous Sodium Sulfate and evaporated under reduced A solution of the appropriate 3-chloro-2-formyl cholenoic pressure. The residue is purified by silica gel chromatography 35 acid ester 3 (e.g. R=TBu) (5 mmol) and 1,2-diamine 94 (5 using a gradient of ethyl acetate and hexane as eluents to give mmol) in DMF is heated at 100-120° C. for 5-24 h (monitored the corresponding pure fused thiomethylpyrimidone 78. by TLC). After cooling to room temperature, the mixture is Under identical reaction conditions, treatment of 6 with thio diluted with water (50 mL) and extracted with ethyl acetate urea (71) affords the corresponding thiomethyl 2-thiopyrimi (25 mLX3). The combined extract is washed with water (25 done 79; guanidine (72) affords 2-aminopyrimidine 80; and 40 mL), dried over anhydrous Sodium sulfate, and evaporated the amidine 73 affords pyrimidine 81 (Scheme36). Following under reduced pressure. The residue is purified by silica gel the same protocol, the treatment of N.S-acetals 7 and 8, with chromatography using a gradient of ethyl acetate and hexane urea 70 affords the corresponding fused aminopyrimidones as eluents to give the corresponding pure fused diazepine 95 82; thiourea 71 affords the fused amino 2-thiopyrimidones 45 (Scheme 38). 83; guanidine 72 affords the fused diaminopyrimidines 84; Under identical reaction conditions, the reaction of 2-dim and the amidines 73 afford the corresponding aminopyrim ethylaminomethylene-3-oxocholanoic acid esters 5 with appropriate 1,2-diamines 94 affords the corresponding diaz idines 85 (Scheme 36). epines 96 (Scheme 39). Similarly, treatment of enones 12 The amino substituted pyrimidine derivatives 82-85 are with 1,2-diamines 94 affords the corresponding diazepines 97 also prepared by the nucleophilic displacement of the thiom 50 (Scheme 40). ethyl moiety on the pyrimidines 78-81 with various amines 14 (Scheme 36). The appropriate pyrimidine 78-81 (3 mmol) is Example 36 treated with m-chloroperbenzoic acid (7 mmol) in THF (25 mL) at room temperature for 12-24 h (monitored by TLC or Synthesis of Fused Diazepine Derivatives of LC/MS). The reaction mixture is filtered and the the filtrate is 55 Cholanoic Acid Esters 96 (Scheme 39) diluted with water (25 mL) followed by extraction with ethyl A solution of the appropriate 2-hydroxymethylenechole acetate (25 mLX3). The combined extract is washed with noic acid ester 4 (5 mmol) and 1,2-diamine 94 (5 mmol) in saturated sodium bicarbonate (15 mlx2), water (25 mL), absolute ethanol was refluxed for 24 h (monitored by TLC). dried over anhydrous Sodium sulfate, and cautiously evapo 60 The reaction mixture was concentrated under reduced pres rated under reduced pressure. The crude sulfone is heated at sure. The residue was diluted with ethyl acetate (50 mL), 70° C. with the appropriate amine 14 (3 mmol) in DMF (25 washed with water (25 mLx2) and dried over dried over mL) for 12-24 h (monitored by TLC). The solvent is evapo anhydrous sodium sulfate. After evaporation of the solvent rated and the residue is chromatographed on silica gel column the residue was purified by silica gel chromatography using a using a gradient of ethyl acetate and hexane as eluents to give 65 gradient of ethyl acetate and hexane as eluents to give the the corresponding pure aminopyrimidines 82-85 (Scheme corresponding pure fused diazepine derivative 96 (Scheme 36). 39). US 7,727,976 B2 79 80 Two representative diazepine derivative 96a-1 and 96a-2 appropriate oxoketene S.S-acetals 6 with 1,3-diamines 100 were synthesized by treating 4a with 2,3-diaminophenol and affords the corresponding thiomethyloctadiazepine deriva methyl 3,4-diaminobenzoate respectively (Scheme 39). tives 104. Similarly, the NS-acetals 7 and 8 afford the corre Diazepine 96a-1: Yield, 24%. LC/MS (m/z): 519 (M+H, sponding amino-Substituted octadiazepines 105 (Scheme R=Et) and 505 (M+H, R-Me). Calcd. 518 (CHNO, 5 45). R=Et) and 504 (CHNO, R=Me). The amino-substituted octadiazepines 105 (Scheme 45) Diazepine 96a-2: Yield, 35%. LC/MS (m/z): 561 (M+H). are also prepared from the corresponding thiomethyl-substi Calcd. 560 (CHNO). tuted octadiazepines 104 by the nucleophilic displacement of the Sulfone group with appropriate amines 14 following the Example 37 10 procedure described for the synthesis of aminodiazepines 99 from the diazepines 98 (Scheme 41). Synthesis of Fused Diazepine Derivatives of VII. General Procedure for the Synthesis of Fused Car Cholanoic Acid Esters 98 and 99 (Scheme 41) bocyclic and Heterocyclic Derivatives of Glycocholanic Acid Esters Having the General Structure 10 (Scheme 46) A solution of the appropriate oXoketene S.S.-acetal 6 (e.g. 15 R-Bu) (5 mmol) and 1,2-diamine 94 (5 mmol) in anhydrous Example 40 DMF is heated at 100-120° C. for 8-24 h (monitored by TLC). After cooling to room temperature, the reaction mixture is Synthesis of Fused Cholanoic Acids Having the poured into cold water (50 mL), extracted with ethyl acetate General Structure 107 (Scheme 46) (25 mLX3) and dried over anhydrous sodium sulfate. The solvent is evaporated and the residue is purified by column Method 1. For R=Me, Et and other simple alkyl: chromatography on silica gel using a gradient of ethyl acetate To a solution of a fused carbocyclic or heterocyclic deriva and hexane as eluents to give the pure thiomethyldiazepine tive of cholanoic acid ester having the general structure 106 (5 derivative 98. Under identical reaction conditions NS-acetals mmol) in THF (25 mL) was added 2 equivalents of 2MNaOH 7 and 8 afford the corresponding aminodiazepine derivatives 25 and the reaction stirred 12-24 h (monitored by TLC). The 99. reaction mixture was diluted with ice-cold water (25 mL) and The aminodiazepines 99 can also be prepared from the acidified with 5% HC1. Then, the reaction mixture was corresponding diazepines 98 (Scheme 41). The appropriate extracted with ethyl acetate (20 mLX3) and the combined diazepine 98 (3 mmol) is treated with m-chloroperbenzoic extract was washed with brine (20 mL), dried over anhydrous acid (7 mmol) in THF (25 mL) at room temperature for 12-24 30 sodium sulfate and the solvent evaporated under reduced h (monitored by TLC or LC/MS). The reaction mixture is pressure. The crude product was purified by silica gel column filtered and the filtrate is diluted with water (25 mL) and chromatography using a 0-50% gradient of methanol and extracted with ethyl acetate (25 mLX3). The combined extract ethyl acetate as eluents to give the corresponding free fused is washed with saturated sodium bicarbonate (15 mLx2), cholanoic acid 107 (Scheme 46). The yields and analytical water (25 mL), dried over anhydrous sodium sulfate, and 35 data for representative fused heterocyclic derivatives of bile cautiously evaporated under reduced pressure. The crude Sul acids having the general structure 107 are given in Table 6. fone is heated with the appropriate amine 14 (3 mmol) in Method 2. For R=TBu DMF (25 mL) at ~70° C. for 12-24 h (monitored by TLC). A fused carbocyclic or heterocyclic derivative of cholanoic The Solventis evaporated and the residue is chromatographed acid ester having the general structure 106 (5 mmol) is treated on silica gel column using a gradient of ethyl acetate and 40 with 15 mL of TFA or 4M HCl in dioxane at room tempera hexane as eluents to give the corresponding pure aminodiaz ture for 3-12 h (monitored by TLC). The reaction mixture is epine 99 (Scheme 41). concentrated under reduced pressure and diluted with ethyl VI. General Procedures for the Synthesis of Fused Octadi acetate (50 mL). Then, it is washed with water, dried over azepine Derivatives of Bile Acids anhydrous sodium sulfate. The solvent is evaporated under 45 reduced pressure. The crude product is purified by silica gel Example 38 column chromatography using a 0-50% gradient of methanol Synthesis of Fused Octadiazepine Derivatives of and ethyl acetate as eluents to give the corresponding free fused cholanoic acid 107 (Scheme 46). Cholanoic Acid Esters 101-103 (Schemes 42-44) Method 3. For R=CHPh Following the protocol described for the synthesis of fused 50 A fused carbocyclic or heterocyclic derivative of cholanoic diazepine derivatives 95-97 (Schemes 38-40) the reaction of acid ester having the general structure 106 (5 mmol) is dis the appropriate 3-chloro-2-formyl cholenoic acid esters 3 solved in 25 mL of ethyl acetate and stirred with 10% Pd C with 1,3-diamines 100 affords the corresponding octadiaz (50 mg) under 1 atm hydrogen gas for 4 h (monitored by epines 101 (Scheme 42), the 2-hydroxymethylene 4, and TLC). The reaction is worked-up and purified as described 2-dimethylaminomethylene-3-oxocholanoic acid esters 5 55 above in Method 2. with 100 affords the corresponding octadiazepines 102 (Scheme 43), and the enones 12 afford the corresponding Example 41 octadiazepines 103 (Scheme 44). Synthesis of Fused Glycocholanic Acid Esters Example 39 60 Having the General Structure 108 (R'=tert-butyl) (Scheme 46) Synthesis of Fused Amino-Substituted Octadiazepine Derivatives of Cholanoic Acid Esters 105 To a stirred Suspension containing fused cholanoic acid (Scheme 45) derivative 107 (5 mmol), EDAC (15 mmol), and glycine 65 tert-butyl ester (10 mmol) in anhydrous THF (25 mL) was Following the protocol described for the synthesis of fused added DIEA (50 mmol) at room temperature under a nitrogen diazepine derivatives 98 (Scheme 41) the reaction of the atmosphere. The reaction mixture was stirred for 12-24 h US 7,727,976 B2 81 82 (monitored by TLC). Then, the mixture was diluted with cold 25-100% gradient ethyl acetate and hexane as eluents to give water (50 mL) and extracted with ethyl acetate (25 mLX3). the corresponding thioacetate 112. Then, a mixture of H.O. The combined extract was washed with water (25 mL), brine 30% w/w in HO (5 mL) and acetic acid (10 mL) is added to (25 mL) and dried over anhydrous sodium sulfate. After a solution of thioacetate 112 dissolved in acetic acid (5 mL). evaporation of the solvent, the residue was purified by silica After stirring 12-18hat room temperature 10% Pd/C (25 mg) gel column chromatography using 25-100% gradient ethyl is added to destroy the excess peroxide. For synthesis of the acetate and hexane as eluents to give the corresponding gly corresponding sodium Sulfonate 114, Sodium acetate (1.1 cine tert-butyl ester 108. Representative glycocholanoic acid equiv) is added before adding the Pd/C, followed by stirring at esters 108 synthesized and their analytical data are given in room temperature for 1 h. Then the reaction mixture is filtered Table 7. 10 and the filtrate is concentrated. The crude taurocholanoic acid derivative 113 is purified by silica gel column chromatogra Example 42 phy using 25-100% methanol and chloroform as eluents. However, for the sodium salt of 114 ion-exchange resins are Synthesis of Fused Glycocholanoic Acids Having the used instead of silica gel for the purification. General Structure 109 (Scheme 46) 15 X. General Procedures for the Conjugation of Drug Mol ecules to Fused Carbocylic and Heterocyclic Derivative of The ester 108 was treated with 25 mL of 4M HCl in diox Glyco- and Taurocholanoic Acids ane at room temperature for 3-12 h to cleave off the tert-butyl group. After completion, the reaction mixture was diluted Example 45 with water (50 mL) and extracted with ethyl acetate (25 mLX3). The combined extract was washed with brine (25 Conjugation of Drug Molecules or Surrogates to the mL), dried over anhydrous sodium sulfate and evaporated. Fused Ring of the Glycocholanoic Acids Via an Ester The residue was purified by silica gel column chromatogra Bond. Synthesis of 118 (Scheme 49), 125 (Scheme phy using a gradient of 0-20% methanol and ethyl acetate as 51) 126 (Scheme 52), and 133 (Scheme 54) eluents to give the corresponding pure fused glycocholanoic 25 acid derivative 109 in good yield. Trifluroacetic acid was also Method 1. Synthesis of conjugate ester 118-1 using oxalyl used in some reactions instead of 4M HCl to cleave the chloride and DMF as coupling agents (Scheme 49). tert-butyl group. Representative glycocholanoic acids 109 To a stirred solution of naproxen (1.0 mmol. 230.3 mg) in synthesized and the analytical data are given in Table 8. anhydrous benzene under nitrogen atmosphere at room tem VIII. General Procedure for the Synthesis of Fused Car 30 perature was added a catalytic amount of DMF (10-20 uL) bocyclic and Heterocyclic Derivatives of Taurocholanic Acid followed by oxalyl chloride (3.0 mmol, 380.8 mg). The reac Having the General Structure 110 (Scheme 47) tion mixture was stirred at room temperature until all solids were dissolved, the gas evolution stopped and the reaction Example 43 mixture turned yellow. The benzene was evaporated under 35 reduced pressure using a rotary evaporator and the residue To a stirred solution of a fused carbocyclic or heterocyclic was diluted in anhydrous dichloromethane (DCM) to a total derivative of cholanoic acid 107 (5 mmol) and tributylamine volume of 5 mL (stock solution). The acid chloride stock (7 mmol) in anhydrous dioxane (15 mL) at 0°C. is added solution (0.60 mL) was added dropwise to a stirred solution of ethyl chloroformate (6 mmol). After stirring for 30 min, a pyrazole 108-48a-2 (0.01 to 0.005 mmol) and DMAP (5-10 solution of taurine (5 mmol) in 1MNaOH (5 mL) and dioxane 40 mol %) in dichloromethane (2 mL) under nitrogen atmo (10 mL) is added in one portion. The mixture is stirred 3-5 h sphere at room temperature. The reaction mixture was stirred at room temperature (monitored by TLC). The reaction mix at room temperature for 18 h (monitored by TLC). The reac ture is diluted with water (25 mL), acidified with 5% HCl and tion was quenched with a saturated Solution of sodium bicar extracted with ethyl acetate (25 mLx4). The combined extract bonate (5 mL) and diluted with ethyl acetate (40 mL). After is washed with brine (20 mL), dried over anhydrous sodium 45 separating the organic layer the aqueous layer was extracted sulfate and evaporated. The crude product is purified by silica with ethyl acetate (40 mLx2). The combined extracts were gel column chromatography using 25-100% gradient of Successively washed with an aqueous solution of potassium methanol and chloroform as eluents to give the pure tauro hydrogen sulfate, water, brine and finally dried over anhy cholanoic acid derivative 110. The treatment of 110 with one drous MgSO. After evaporation of the solvent the crude equivalent of NaOH followed by evaporation affords the cor 50 product was purified by flash chromatography on silica gel responding Sodium salt 111. using a gradient of hexane and ethyl acetate to give the pure IX. General Procedure for the Synthesis of Fused Carbocy products as white solids (Scheme 49). clic and Heterocyclic Derivatives of Taurocholanic Acid Hav Method 2. Synthesis of ester conjugates 118-2 and 118-3 ing the General Structure 113 (Scheme 48) using trichlorobenzoyl chloride (TCBC) as coupling agent 55 (Scheme 49). Example 44 To a stirred solution of naproxen (0.12 mmol) in anhydrous dichloromethane (2 mL) under nitrogen atmosphere at room To a stirred Suspension containing fused cholanoic acid temperature was added trichlorobenzoyl chloride (0.12 derivative 107 (5 mmol), EDAC (15 mmol), and thioacetate mmol. 29.2 mg) followed by triethylamine (0.12 mmol. 12.2 15 (10 mmol) in anhydrous THF (25 mL) is added DIEA (50 60 mg). The reaction mixture was stirred at room temperature for mmol) at room temperature under nitrogen atmosphere. The 1 h. The resulting mixed anhydride intermediate was added to reaction mixture is stirred for 12-24 h (monitored by TLC). a stirred solution of the appropriate pyrazole (i.e. 108-48a-3 Then, the mixture is diluted with cold water (50 mL) and or 108-48c-3) (0.01 to 0.005 mmol) and DMAP (5-10 mol%) extracted with ethyl acetate (25 mLX3). The combined extract in anhydrous dichloromethane (2 mL) under nitrogen atmo is washed with water (25 mL), brine (25 mL) and dried over 65 sphere at room temperature. The reaction mixture was stirred anhydrous sodium sulfate. After evaporation of the solvent, at room temperature for 18 h (monitored by TLC). The reac the residue is chromatographed on silica gel column using tion was quenched with Saturated solution of sodium bicar US 7,727,976 B2 83 84 bonate (5 mL) and diluted with ethyl acetate (40 mL). After J=7.2 Hz, CHCHC(=O); 3.892H, d, J=5.2 Hz, NHCHC separating the organic layer the aqueous layer was extracted (=O): 3.90 (3H, s, OCH); 4.041H, q, J–7.2 HZ, CHCHC with ethyl acetate (40 mLx2). The combined extracts were (=O): 5.10 (1H, d, J=16.0 Hz, NCHHAr); 5.17 (1H, d, Successively washed with an aqueous solution of potassium J=16.4 Hz, NCHHAr); 5.88 (1H, br. t, J=5.2 Hz, NHCHC hydrogen sulfate, water, brine and finally dried over anhy (=O): 6.72-7.02 (3H, m, Ar H); 7.10-7.30 (4H, m, Ar H, drous MgSO4. After evaporation of the solvent the crude Pyrazole-H): 743-748 (1H, dd, J–8.4, 1.2 Hz, Ar H); 7.69 product was purified by flash chromatography on silica gel 7.76 (3H, m, Ar H) ppm; "C NMR (CDC1, 100 MHz) & using a gradient of methanol and ethyl acetate to give the pure 8.57; 12.31; 18.55; 18.78; 21.37:22.55; 22.98; 24.03; 25.50; ester conjugate tert-butyl esters 118-2 and 118-3 (Scheme 25.68; 27.99; 29.66; 31.54; 31.87; 33.26:35.10; 35.40; 35.73; 49). 10 The tert-butyl group was cleaved by treating the appropri 39.48; 39.80:40.35; 41.94:42.65:45.49; 45.70:52.23:55.29; ate drug and/or drug Surrogate conjugated pyrazole derivative 55.89:56.32: 82.23; 105.54; 114.81; 119.08:119.95; 120.54; of glycocholanoic acid tert-butyl ester (0.1 mmol) with 5 mL 124.07: 126.04; 126.09: 127.35; 128.92; 129.29; 129.59; of neat TFA (for conjugates derived from glycolithocholic 133.76; 135.01; 136.80; 137.59; 138.93; 151.01: 157.71; acid derivatives) or with a 4N solution of HCl in 1,4-dioxane 15 169.29; 172.98; 173.45. LC/MS (m/z): found, 830 (M+H). (for conjugates derived from hydroxyl group-bearing bile Calcd.: 829 (C5H7NO). acids such as glycocholic-, glycochenodeoxy-, or glycode Ester Conjugate 118-3 (R-OH, R=tBu) (Method 2) oxy acid derivatives). The reaction mixture was stirred for Yield: 73%. "H NMR (CDC1, 400 MHz, characteristic 5-24 h at room temperature and course of the reaction was signals) 8 0.64 (3H, s, CH-19); 0.87 (3H, d, J=6.4 HZ, CH monitored by TLC and/or LC/MS. Upon complete consump 21); 1.01 (3H, s, CH-18): 1.449H, s, C(CH); 1.65 [3H, d, tion of the starting materials, the TFA or the HC1/1,4-dioxane Solution was concentrated under reduced pressure using a J=7.2 HZ, CHCHC(=O): 3.55 (1H, m, CHOH); 3.872H, rotary evaporator. The residue was subjected to purification d, J=5.2 Hz, NHCHC(=O); 3.89 (3H, s, OCH); 3.671H, by preparative HPLC. q, J–7.2 HZ, CHCHC(=O): 4.04 1H, q, J–7.2 Hz, Similarly other drug and drug Surrogates conjugated via an 25 CHCHC(=O); 5.07 (1H, d, J=14.8 Hz, NCHHAr); 5.17 ester bond linkage: i.e. 118 (Scheme 49), 125 (Scheme 51), (1H, d, J=14.8 Hz, NCHHAr); 5.92 (1H, br. t, J=5.2 Hz, 126 (Scheme 52), and 133 (Scheme 54) are synthesized by NHCHC(=O); 6.83-6.92 (2H, m, Ar H); 6.99 (1H, br. d, following above described methods or by using standard pro J=8.0 Hz, Ar H); 7.09-7.16 (2H, m, Ar H); 7.20 (1H, s, tocols reported in the literature (vide infra). In the case of Pyrazole-H): 7.25 (1H, t, J–8.0 Hz, Ar H); 7.31 (1H, dd, taurocholanoic acid derivatives, after conjugation of drug 30 J=8.8, 2.0 Hz, Ar-H); 7.63-7.76 (3H, m, Ar H) ppm. "C molecules or drug Surrogates to 112, the oxidation of the NMR (CDC1, 100 MHz) & 8.57; 12.04; 18.28; 18.51; 21.37: thioacetate group to the corresponding sulfonic acid deriva 22.55; 22.98; 24.03; 25.50; 25.68; 27.99; 28.17; 29.66; 31.54; tives 126 and 133, and conversion to their sodium salts 134 31.81:33.26:35.10; 35.40; 35.73:39.48; 39.80:40.35; 41.94: and 141 are performed according to the procedures described 42.65; 45.49; 52.23; 55.29; 55.89; 56.32; 82.23; 105.54; for the synthesis of 113 and 114 (Scheme 48). 35 114.81; 119.08: 119.95; 120.54; 124.07: 126.04; 126.09: Ester Conjugate 118-1 (R'=H, R=tBu) (Method 1) 127.35; 128.93; 129.29; 129.59; 133.76; 135.01: 136.80; Yield: 64-69%. "H NMR (CDC1,400 MHz, characteristic 137.59; 138.93; 15101: 157.71; 169.29; 127.98; 173.45. signals, dr ca. 3:1, de ca. 50%) 8 0.58 (3H, s, CH-19, minor LC/MS (m/z): found, 846 (M+H). Calcd.: 845 diastereoisomer): 0.60 (3H, s, CH-19, major diastereoiso (Cs2H67NO.7). mer); 0.80 (3H, d. J=6.4 Hz, CH-21, minor diastereoisomer); 40 Ester Conjugate 118-4 (R'=H, R=H) 0.84 (3H, d. J=6.4 HZ, CH-21, major diastereoisomer); 0.97 Yield: 60-80%. "H NMR (CDC1,400 MHz, drca. 3:1, de (3H, s, CH-18, major diastereoisomer); 0.98 (3H, s, CH-18, ca. 50%): 8 0.56 (3H, s, CH-19, minor diastereoisomer); minor diastereoisomer); 1.44 (9H, s, C(CH), both diastere 0.60 (3H, s, CH-19, major diastereoisomer); 0.78 (3H, d, oisomers); 1.51 (3H, d, J=7.6 Hz, CHCHC(=O), minor J=6.4 HZ, CH-21, minor diastereoisomer): 0.84 (3H, d, diastereoisomer: 1.52 (3H, d, J=7.6 HZ, CHCHC(=O), 45 J=6.8 Hz, CH-21, major diastereoisomer): 0.92 (3H, s, CH major diastereoisomer: 3.78 1H, q, J–7.2 HZ, CHCHC 18, major diastereoisomer); 0.94 (3H, s, CH-18, minor dias (=O), minor diastereoisomer; 3.82 1H, q, J–7.2 Hz, tereoisomer); 1.493H, d, J=6.8 Hz, CHCHC(=O), minor CHCHC(=O).), major diastereoisomer; 3.86-3.92 (5H, m, diastereoisomer: 1.53 3H, d, J=6.8 Hz, CHCHC(=O), OCH NHCHC(=O), both diastereoisomers; 4.04-4.20 major diastereoisomer: 3.75 (1H, q, J=6.8 Hz, CHCHC (2H, m, OCH2CHN, both diastereoisomers); 4.26-4.50 (2H, 50 (=O), minor diastereoisomer; 3.83 1H, q, J=6.8 Hz, m, OCHCHN, both diastereoisomers);5.83 (1H, br. t, J=5.2 CHCHC(=O).), major diastereoisomer; 3.88 (3H, m, HZ, NHCHC(=O), minor diastereoisomer; 5.88 (1H, br. t. OCH minor diastereoisomer) 3.89 (3H, m, OCH major J–4.8 Hz, NHCHC(=O), major diastereoisomer: 7.06-7.36 diastereoisomer); 3.97 (2H, br. m., HCHC(=O), both dias (4H, m, Ar H. Pyrazole-H); 7.52-7.70 (3H, m, Ar H, both tereoisomers); 4.20-4.40 (2H, br. m., OCHCHN, both dias diastereoisomers) ppm. "C NMR (CDC1, 100 MHz, major 55 tereoisomers); 4.40-4.60 (2H, br. m., OCHCHN, both dias diastereoisomer) & 12.04; 18.12: 18.30; 21.32; 23.02; 23.81; tereoisomers); 6.57-6.72 (1H, br. m, NHCHC(=O), both 24.01; 25.41; 25.89; 27.99; 29.67; 31.21: 31.54; 33.24; 35.18: diastereoisomers; 7.06-7.21 (2H, m, Ar—H, both diastere 35.28; 35.44; 39.76; 40.14; 41.94:42.64; 45.38: 50.39; 55.25: oisomers); 7.26 (1H, dd, J–8.4, 1.6 Hz, Ar—H, minor dias 55.80; 56.14; 63.60; 82.23; 105.52; 114.52; 119.10; 126.00: tereoisomer); 7.31 (1H, dd, J–8.4, 1.6 Hz, Ar—H, major 126.17; 126.52; 127.20; 128.86; 129.22; 129.26; 133.69; 60 diastereoisomer); 7.53 (1H, s, Pyrrazole-H, minor diastere 135.29; 137.08; 137.83; 148.64; 157.68: 169.30; 173.46; oisomer); 7.60 (1H, s, Pyrazole-H, major diastereoisomer); 174.09. LC/MS (m/z): found, 768 (M+H). Calcd.: 767 7.61-7.73 (3H, m, Ar H, both diastereoisomers) ppm. "C (C7H5NO). NMR (CDC1, 100 MHz, major diastereoisomer) & 11.95: Ester Conjugate 118-2 (R'=H, R=tBu) (Method 2) 17.65; 18.06:18.14; 21.25: 21.50; 22.52; 23.85:25.06; 27.97; Yield: 68-79%. "H NMR (CDC1,400 MHz, characteristic 65 29.56; 29.67; 31.56:32.73:35.03:35.28:38.42:39.48:40.23; signals) 8 0.60 (3H, s, CH-19); 0.85 (3H, d. J=6.4 Hz, CH 41.54; 42.45:42.54:45.24; 49.91:55.26:55.32:55.47:55.95: 21); 1.03 (3H, s, CH-18): 1.449H, s, C(CH); 1.653H, d, 62.17; 105.55; 116.58; 119.25; 119.53; 125.97: 126.09: US 7,727,976 B2 85 86 127.40; 128.82; 129.28: 130.72; 146.44; 157.97; 173.63: plete consumption of the starting materials, the TFA or the 176.O.O. HC1/1,4-dioxane solution was concentrated under reduced Ester Conjugate 118-5 (R'=H, R=H) pressure on rotary evaporator. The residue was Subjected to Yield: 60-80%. "H NMR (CDC1,400 MHz) & 0.61 (3H, s, purification by preparative HPLC. CH-19); 0.86 (3H, d, J=6.4 HZ, CH-21); 1.09 (3H, s, CH Similarly other drug and drug Surrogates conjugated via a 18); 1.65 [3H, d, J=7.6 Hz, CHCHC(=O); 3.91 (3H, s, OCH); 4.042H, d, J=5.2 Hz, NHCHC(=O); 4.07 1H, q, carbamate bond linkage: 119 (Scheme 49), 123 (Scheme 50), J=7.2 Hz, CHCHC(=O): 5.35 (1H, d, J=16.4 Hz, NCH 127 (Scheme 52), and 131 (Scheme 53) are synthesized by HAr); 5.49 (1H, d, J=16.4 Hz, NCHHAr); 6.37 (1H, br. t, following above described method or by using standard pro J=5.2 Hz, NHCHC(=O); 6.86-6.90 (1H, m, Ar-H); 6.92 10 tocols reported in the literature (vide infra). In the case of 7.00 (2H, m, Ar H); 7.10-7.18 (2H, m, Ar H); 7.31 (1H, t, taurocholanoic acid derivatives, after conjugation of drug J=8.0 Hz, Ar H); 7.46 (1H, dd, J–8.4, 2.0 Hz, Ar H); 7.62 molecules or drug Surrogates to 112, the oxidation of the (1H, s, Pyrazole-H); 7.69-7.76 (3H, br. m, Ar H). LC/MS thioacetate group to the corresponding Sulfonic acid deriva (m/z): found, 774 (M+H): 772 (M-H). Calcd.: 773 tives 127 and 131 and conversion to their sodium salts 135 and (Cashson-Os). 15 139 are performed according to the procedures described for Ester Conjugate 118-6 (R-OH, RH) the synthesis of 113 and 114 (Scheme 48). Yield: 81%. "H NMR (CDC1, 400 MHz): 8 0.60 (3H, s, Carbamate Conjugate 119-1 (R=tBu) CH-19); 0.85 (3H, d, J=6.4 HZ, CH-21); 1.02 (3H, s, CH Yield: 81% quant. "H NMR (CDC1, 400 MHz, charac 18); 1.65 [3H, d, J=7.6 Hz, CHCHC(=O): 3.77 (1H, br. m. teristic signals, line broadening): 8 0.59 (3H, s, CH-19); 0.81 CHOH); 3.89 (3H, s, OCH); 3.95 2H, d, J=5.6 Hz, NHCHC(=O): 406 1H, q, J=7.6 Hz, CHCHC(=O); (3H, br. d, CH-21); 1.03 (3H, br. s. CH-18): 1.43 9H, br. s. 5.28 (2H, br. m, NCHAr); 6.67 (1H, br. t, J=5.2 Hz, C(CH): 3.50-3.68 (2H, br. m. ArCHCH-NH); 3.81 (6H, NHCHC(=O); 6.89-7.01 (2H, m, Ar H): 707-7.17 (3H, br. s, OCH); 3.882H, d. J=5.2 Hz, NHCHC(=O): 4.20 m, Ar H); 7.31 (1H, t, J=8.0 Hz, Ar-H); 7.36 (1H, s, 4.32 (2H, br. m., OCHCHN); 4.33-4.45 (4H, br. m. Pyrazole-H); 7.43 (1H, dd, J–8.4, 2.0 Hz, Ar-H); 7.67-7.73 25 OCHCHN, ArCHN); 5.92 (1H, br. t, NHCHC(=O); (3H, br. m, Ar H). LC/MS (m/z): found, 790 (M+H): 788 7.00 (2H, br.s, Ar-H): 7.20(1H, br.s, Pyrazole-H) ppm. "C (M-H). Calcd.: 789 (CH5NO2). NMR (CDC1,100MHz) & 12.01; 18.26; 21.38; 23.05:23.09: 23.89; 23.97; 25.42; 25.90; 27.97; 28.11:31.50; 31.60:33.19; Example 46 35.18:35.30; 35.62:39.82:40.20; 41.91; 42.63:45.30:50.89; 30 55.81; 55.87; 56.18; 66.11; 77.20; 82.18; 108.78: 109.02; Conjugation of Drug Molecules or Surrogates to the 109.23: 111.33; 111.43; 113.92; 114.60; 125.01; 124.50; Fused Ring of Glycocholanoic Acid Derivatives Via 125.97: 126.41: 147.60; 148.88: 155.03; 169.26; 173.44. a Carbamate Bond. Synthesis of 119 (Scheme 49), LC/MS (m/z): found, 776 (M+H). Calcd.: 775 123 (Scheme 50), 127 and 135 (Scheme 52), and 131 (Casso N4O7). and 139 (Scheme 53) 35 Carbamate Conjugate 119-2 (R=tBu) Synthesis of Carbamate Conjugates 119 (Scheme 49) Yield: 67%. "H NMR (CDC1, 400 MHz, characteristic To a stirred solution of pyrazole 108-48a-3 (1.0 mmol) in signals, line broadening): 8 0.59 (3H, s, CH-19); 0.81 (3H, d, anhydrous THF (1 mL) under nitrogen atmosphere at room J=6.0 Hz, CH-21); 1.02 (3H, s, CH-18): 1.42 9H, s, temperature was added a solution of N,N'-carbonyldiimida 40 C(CH): 3.36 (2H, br. quint., J=6.4 Hz, ArCHCH-NH); Zole (162.2 mg, 1.0 mmol) in THF (4.8 mL). The reaction 3.81 (3H, s, OCH); 3.82 (3H, s, OCH); 3.872H, d, J=4.8 mixture was heated at 65° C. till the starting pyrazole was Hz, NHCHC(=O): 4.12-4.27 (2H, m, OCHCHN); 4.28 completely converted to the corresponding N-imidazolyl car 4.45 (2H, m, OCHCHN); 4.86 (1H, br. t, J=4.0 Hz, bamate intermediate (~12 h, monitored by TLC). Then, a ArCHCH-NH), 5.96 (1H, br. t, NHCHC(=O); 6.67 (1H, Solution of appropriate amine drug or drug Surrogate (0.2 45 br. d, J–7.2 Hz, Ar H); 6.75 (1H, br. d, J=8.0 Hz, Ar H): mmol) in THF (1 mL) was added at room temperature. The 6.95 (1H, br.s, Ar-H); 7.17 (1H, br.s, Pyrazole-H) ppm. "C reaction mixture was stirred at room temperature for 24 h. NMR (CDC1,100MHz) & 11.99; 18.23; 23.05; 23.11; 23.30; (monitored by TLC). The reaction mixture was diluted with 23.89; 25.39; 25.89; 27.94:28.10:31:49: 31.53:33.16:35.18: ethyl acetate (40 mL). After separating the organic layer the 35.31:35.59:35.73; 39.76; 39.80; 40.16; 41.88:42.20:42.62: aqueous layer was extracted with ethyl acetate (40 mLX2). 50 50.93; 55.76; 55.82; 55.90; 56.15; 63.46; 82.13; 111.23; The combined extracts were successively washed with an 111.77; 114.57; 120.59; 126.39; 130.99; 131.04; 136.90; aqueous Solution of potassium hydrogen Sulfate, water, brine 147.60; 148.85; 148.91; 155.87; 169.25; 173.45. LC/MS and finally dried over anhydrous MgSO4. After evaporation (m/z): found: 764 (M--H). Calcd.: 763 (CHNO2). of the solvent the crude product was purified by flash chro Carbamate Conjugate 119-3 (R=H) matography on silica gel using a gradient of hexane and ethyl 55 acetate to give the pure carbamate tert-butyl esters conjugates Yield: 45% unoptimized. LC/MS (m/z): found, 719 119 (Scheme 49). (M+H); 717 (M-H). Calcd.: 718 (CHNO). The tert-butyl group was cleaved by treating the appropri Carbamate Conjugate 119-4 (R=H) ate drug and/or drug Surrogate conjugated pyrazole glyco Yield: 60%. "H NMR (CDC1, 400 MHz, characteristic cholanoic acid tert-butyl ester derivative (0.1 mmol) with 5 60 signals, line broadening): 8 0.60 (3H, s, CH-19); 0.82 (3H, mL of neat TFA (for conjugates derived from glycolitho br. d, CH-21); 1.09 (3H, s, CH-18): 3.20-3.40 (2H, br. m. cholic acid derivatives) or with a 4N solution of HCl in ArCHCH-NH); 3.76-3.88 (8H, m, 2xOCH NHCHC 1,4-dioxane (for conjugates derived from hydroxyl group (=O): 3.92-4.04 (2H, br. m. ArCHCH-NH); 4.30-4.60 (4H, bearing bile acids such as glycocholic-, glycochenodeoxy-, or br. m., OCHCHN, OCHCHN); 5.52-5.75 (1H, br. m. glycodeoxy acid derivatives). The reaction mixture was 65 ArCHCH-NH); 6.55-6.80 (4H, br. m, Ar H, NHCHC stirred for 5-24 h at room temperature and course of the (=O): 7.27 (1H, br. s. Pyrazole-H). LC/MS (m/z): found, reaction was monitored by TLC and/or LC/MS. Upon com 707 (M+H): 705 (M-H). Calcd.: 706 (CHssNO). US 7,727,976 B2 87 88 Example 47 (2H, m, OCHCHN, both diastereoisomers); 5.91 (1H, br. t. J–4.4 Hz, NHCHC(=O): 7.10-7.31 (6H, m, Pyrazole-H, Conjugation of Drug Molecules or Surrogates to the Ar H, both diastereoisomers) ppm. C NMR (CDC1, 100 Fused Ring of Glycocholanoic Acid Derivatives Via MHz, major diastereoisomer) & 11.66: 12.04; 18.28; 21.39; a Carbonate Bond. Synthesis of 120 (Scheme 49) 5 23.12; 23.88; 24.02:25.10; 25.43; 25.91; 27.98; 28.15; 29.64: and 128 and 136 (Scheme 52) 31.55:33.23:35.23:35.35; 35.62:39.78; 39.82;40. 19:41.93; 42.67; 46.63; 50.35; 55.82; 56.18; 66.37; 71.79; 82.20; Synthesis of Carbonate Conjugate 120-1 (Scheme 49) 114.73; 126.64; 126.79; 127: 86; 128.45; 137.13: 141.02: To a stirred solution of B-ethylphenylethylalcohol (150.2 149.08: 154.73; 169.28; 173.47. LC/MS (m/z): found: 733 mg, 1.0 mmol) in anhydrous THF (3 mL) under nitrogen 10 (M+H). Calcd.: 732 (CHNO). atmosphere at 0°C. was added a solution of N,N'-carbonyl Carbonate Conjugate 120-2 diimidazole (162.2 mg, 1.0 mmol) in THF (1.6 mL). After Yield: ca. quant. "H NMR (CDC1, 400 MHz, both diaste having stirred at 0°C. for 10 min the reaction mixture was reoisomers, characteristic signals, line broadening, drca. 2:1, further stirred at room temperature for 2 h to give the N-imi de ca. 33%): 8 0.62 (3H, s, CH-19, both diastereoisomers); dazolylcarbamate intermediate. The carbamate intermediate 15 0.77 (3H, t, J=7.6HZ, CH(CHCH), minordiastereoisomer), was added dropwise to a stirred solution of pyrazole 108 0.79 (3H, t, J=7.6 Hz, CH(CHCH), major diastereoisomer); 48a-3 (0.1 mmol) and catalytic amounts of sodium hydride 0.84 (3H, br. d, J=6.4 HZ, CH-21, both diastereoisomers); (2-3 mg) in THF (1 mL) at 0°C. The reaction mixture was 1.15 (3H, s, CH-18, both diastereoisomers); 4.03 2H, d, stirred at 0°C. for 30 min and then at room temperature for 18 J–4.4 Hz, NHCHC(=O), both diastereoisomers; 4.16-4.32 h (monitored by TLC). The reaction was diluted with ethyl (4H, m, CH(CHO), OCH2CHN, both diastereoisomers); acetate (40 mL). After separating the organic layer the aque 4.32-4.70 (2H, m, OCHCHN, both diastereoisomers); 6.86 ous layer was extracted with ethyl acetate (40 mLX2). The (1H, br. t, NHCHC(=O), both diastereoisomers: 7.10-7.40 combined extracts were successively washed with an aqueous (6H, m, Pyrazole-H, Ar—H, both diastereoisomers). LC/MS Solution of potassium hydrogen Sulfate, water, brine and (m/z): found, 676 (M--H). Calcd.: 675 (CoH.N.O.). finally dried over anhydrous MgSO4. After evaporation of the 25 Solvent the crude product was purified by flash chromatogra Example 48 phy on silica gel using a gradient of hexane and ethyl acetate to give the pure carbonate tert-butyl ester conjugate 120-1 Conjugation of Drug Molecules or Surrogates to the (Scheme 49). Fused Ring of Glycocholanoic Acid Derivatives Via The tert-butyl group was cleaved by treating the appropri 30 an Amide Bond. Synthesis of 121 (Scheme 50), 124 ate drug and/or drug Surrogate conjugated pyrazole glyco (Scheme 51), 129 and 137 (Scheme 53), and 132 and cholanoic acid tert-butyl ester derivative (0.1 mmol) with 5 140 (Scheme 54) mL of neat TFA (for conjugates derived from glycolitho cholic acid derivatives) or with a 4N solution of HCl in To a stirred Suspension containing 106-2 (5 mmol), bearing 1,4-dioxane (for conjugates derived from hydroxyl group 35 a free amino group on the fused ring, EDAC or an equivalent bearing bile acids such as glycocholic-, glycochenodeoxy-, or coupling agent such as DCC, DIC (15 mmol), and a drug glycodeoxy acid derivatives). The reaction mixture was molecule or Surrogate bearing a carboxyl group 115 (5 mmol) stirred for 5-24 h at room temperature and course of the in anhydrous THF (25 mL) is added DIEA (50 mmol) at room reaction was monitored by TLC and/or LC/MS. Upon com temperature under a nitrogen atmosphere. The reaction mix plete consumption of the starting materials, the TFA or the 40 ture is stirred for 12-24 h (monitored by TLC). Then, the HC1/1,4-dioxane solution was concentrated under reduced mixture is diluted with cold water (50 mL) and extracted with pressure on rotary evaporator. The residue was Subjected to ethyl acetate (25 mLX3). The combined extract is washed purification by preparative HPLC. with water (25 mL), brine (25 mL) and dried over anhydrous Similarly other drug and drug Surrogates conjugated via a sodium sulfate. After evaporation of the solvent, the residue is carbonate bond linkage: 121 (Scheme 49), 128 (Scheme 52) 45 purified by silica gel column chromatography using 25-100% are synthesized by following above described method or by gradient ethyl acetate and hexane as eluents to give the cor using standard protocols reported in the literature (vide infra). responding amide conjugate tert-butyl esters (Scheme 50). In case of taurocholanoic acid derivatives, after conjugation The tert-butyl group was cleaved by treating the appropri of drug molecules or drug Surrogates to 112, the oxidation of ate drug and/or drug Surrogate conjugated pyrazole derivative the thioacetate group to the corresponding Sulfonic acid 50 of glycocholanoic acid tert-butyl ester (0.1 mmol) with 5 mL derivatives 128 and conversion to their sodium salts 136 are of neat TFA (for conjugates derived from glycolithocholic performed according to the procedures described for the Syn acid derivatives) or with a 4N solution of HCl in 1,4-dioxane thesis of 113 and 114 (Scheme 48). (for conjugates derived from hydroxyl group-bearing bile Carbonate Conjugate 120-1 (R=tBu) acids such as glycocholic-, glycochenodeoxy-, or glycode Yield: 57%. "H NMR (CDC1, 400 MHz, characteristic 55 oxy acid derivatives). The reaction mixture was stirred for signals, both diastereoisomers, dr ca. 2:1, de ca. 33%): 8 0.61 5-24 h at room temperature and course of the reaction was (3H, s, CH-19, both diastereoisomers): 0.78 (3H, t, J=7.6 Hz, monitored by TLC and/or LC/MS. Upon complete consump CH(CHCH), minor diastereoisomer), 0.79 (3H, t, J–7.6 Hz, tion of the starting materials, the TFA or the HC1/1,4-dioxane CH(CH2CH), major diastereoisomer); 0.84 (3H, d. J=6.4 Solution was concentrated under reduced pressure on rotary HZ, CH-21, major diastereoisomer); 0.86 (3H, d. J=6.4 Hz, 60 evaporator. The crude residue was subjected to purification by CH-21, minor diastereoisomer); 1.04 (3H, s, CH-18, major preparative HPLC. diastereoisomer); 1.05 (3H, s, CH-18, minor diastereoiso Similarly other drug and drug Surrogate conjugated via an mer); 1.43 9H, s, C(CH), major diastereoisomer: 1.44 amide bond linkage: 121 (Scheme 50), 124 (Scheme 51), 129 9H, s, C(CH), minor diastereoisomer; 2.74-2.88 (1H, m, (Scheme 53) and 132 (Scheme 54) are synthesized by follow CH(CHCH), both diastereoisomers); 3.892H, d, J=5.2 Hz, 65 ing above described method or by using standard protocols NHCHC(=O), both diastereoisomers; 4.14-4.26 (4H. m. reported in the literature (vide infra). In case of taurochol CH(CHO), OCHCHN, both diastereoisomers); 4.32-4.44 anoic acid derivatives, after conjugation of drug molecules or US 7,727,976 B2 89 90 drug Surrogates to 112, the oxidation of the thioacetate group to the corresponding sulfonic acid derivatives 129 and 132 TABLE 1-continued and conversion to their sodium salts 137 and 140 are per formed according to the procedures described for the synthe 3-Oxocholanoic acid methyl esters la-d prepared (Scheme 1 sis of 113 and 114 (Scheme 48). Yield Mol. Formula Compound (%) (Calcd. exact M. Wit.) LC-MS Data Example 49 1b 79 C25HoOs (420) 841 (2M + H) 1c 85 C2s HoO (404) 387 (M-OH); Conjugation of Drug Molecules or Surrogates to the 809 (2M + H) Fused Ring of Glycocholanoic Acid Derivatives Via 10 1d 70 C25HoO (404) 387 (M-OH); a Urea Bond. Synthesis of 122 (Scheme 50) and 130, 809 (2M + H) 138 (Scheme 53) To a stirred solution of 106-2 (1.0 mmol) in anhydrous THF TABLE 2 (1 mL) under nitrogen atmosphere at room temperature was 15 2-Hydroxymethylene-3-oxocholanoic acid ethyl methyl added a solution of NN'-carbonyldiimidazole (162.2 mg, 1.0 esters 4a-c prepared (Scheme 4 mmol) in THF (4.8 mL). The reaction mixture was heated at 65° C. till complete conversion of the starting 106-2 to the Compound %Yield, NMRI and LC-MS Data corresponding N-imidazolyl carbamate intermediate (~12 h, 4a Yield, 94%. "H NMR(CDC1,400 MHz): 8 0.65 (3H, s, monitored by TLC). Then, a solution of appropriate amine R = H CH-19); 0.93 (3H, d, J = 6.4 Hz, CH-21); 1.01 (3H, s, R2 = H CH-18), 1.10-2.80 (CH & CH-steroidal and OCH2CH), drug or drug surrogate 116 (0.2 mmol) in THF (1 mL) was 4.10 (2H, q, J = 6.8 Hz, OCH2); 8.24 (1H, s, =CH). added at room temperature. The reaction mixture was stirred LC/MS (m/z): found, 431 (M+H, R = Et):417 (M+H, at room temperature for 24 h (monitored by tic). The reaction R = Me) and calcd., 430 (C27H2O, mixture was diluted with ethyl acetate (40 mL). After sepa R = Et), 416 (C26HoO, R = Me). 25 4b Yield, 81%. H NMR(CDC1,400 MHz): 8 0.68 (3H, s, rating the organic layer the aqueous layer was extracted with R = OH CH-19); 0.91 (3H, d, J = 6.0 HZ, CH-21); 1.01 (3H, s, ethyl acetate (40 mLx2). The combined extracts were succes R2 = OH CH-18), 1.10-2.80 (CH & CH-steroidal and OCH2CH), sively washed with an aqueous Solution of potassium hydro 3.80 (1H, bs, CH-12); 3.88 (1H, bs, CH-7); 4.12 (2H, q, gen Sulfate, water, brine and finally dried over anhydrous J = 6.8 Hz, OCH); 9.12 (1H, s, —CH). LC/MS (m/z): found, 925 (2M + H, R = Et);461 (M-H, R = Et); MgSO4. After evaporation of the solvent the crude product 447 (M-H, R = Me). Calcd., 462 (C27H2O, was purified by flash chromatography on silica gel using a 30 R = Et) and 448 (C26HoO, R = Me). gradient of hexane and ethyl acetate to give the pure urea 4c Yield, 93%. "H NMR(CDC1,400 MHz): 8 0.66 (3H, s, tert-butyl ester conjugates (Scheme 50). R = OH, CH-19); 0.91 (3H, d, J = 6.4 Hz, CH-21); 1.02 (3H, s, R = H CH-18), 1.10-2.80 (CH & CH-steroidal and OCH2CH), The tert-butyl group was cleaved by treating the appropri 3.82 (1H, m, CH-7); 4.12 (2H, q, J = 6.8 Hz, OCH2); ate drug and/or drug Surrogate conjugated pyrazole glyco 9.13 (1H, s, =CH). LC/MS (m/z): found, 447 (M+H, cholanoic acid tert-butyl ester derivative (0.1 mmol) with 5 35 R = Et); 445 (M-H, R = Et); 431 (M-H, R = Me). Calcd., mL of neat TFA (for conjugates derived from glycolitho 446 (C27H12O5, R = Et) and 432 (C26HoOs, R = Me). cholic acid derivatives) or with a 4N solution of HCl in 4d Yield, 9%. LC/MS (m/z): found, 447 (M+H, R = Et); 1,4-dioxane (for conjugates derived from hydroxyl group R = H, 445 (M-H, R = Et): 431 (M-H, R = Me). Calcd., bearing bile acids such as glycocholic-, glycochenodeoxy-, or R2 = OH 446 (C27H12O5, R = Et) and 432 (C26HoOs, R = Me). 40 glycodeoxy acid derivatives). The reaction mixture was The products 4a-d were obtained as a mixture of ethyl and methyl esters stirred for 5-24 h at room temperature and course of the (R = Et and Me) in a ~3:1 to 5:1 ratio and their ratios were determined by 'H reaction was monitored by TLC and/or LC/MS. Upon com NMR spectral data. plete consumption of the starting materials, the TFA or the "H NMR data of the major isomers (R = Et) are reported herein. HC1/1,4-dioxane solution was concentrated under reduced pressure on rotary evaporator. The crude residue was Sub 45 TABLE 3 jected to purification by preparative HPLC. 2-Dimethylaminomethylene-3-oxocholanoic acid methyl Similarly other drug and drug Surrogate conjugates via esters 5a-c prepared (Scheme 5 urea bond linkage are synthesized by following above described method or by using standard protocols reported in Mol. Formula 50 Yield (Calcd. the literature (vide infra). In case of taurocholanoic acid Compound (%) exact M. Wit.) LCMS Data derivatives, after conjugation of drug molecules or drug Sur 5a 79 CHNO (443) m/z = 444 (M+H) rogates to 112, the oxidation of the thioacetate group to the Sb 55 C2HNOs (475) m/z = 476 (M+H), 457 corresponding Sulfonic acid derivatives 130 and conversion (M - H2O) to their sodium salts 138 are performed according to the Sc 66 C2HNO (459) m/z = 460 (M+H) procedures described for the synthesis of 113 and 114 55 (Scheme 48). XI. Analytical Data TABLE 4 TABLE 1. 2,3-Fused pyrazole derivative cholanoic acid ethyl methyl 60 esters having general structures 48 and 49 Synthesized (Scheme 24 3-OXocholanoic acid methyl esters la-d prepared (Scheme 1 Com Yield Mol. Formula pound % Yield, NMRI and LC-MS Data Compound (%) (Calcd. exact M. Wit.) LC-MS Data 48a-1 Yield, 84%. "H NMR(CDC1,400 MHz): 8 0.65 (3H, s, CH,- 1a. 92 C25HoO (388) 389 (M+H): 65 R = H, 19); 0.86 (3H, bd, J = 6.0 Hz, CH-21); 1.10 (3H, s, CH-18), 777 (2M+H) R2 = H 1.12-2.89 (CH & CH-steroidal and OCH2CH), 4.12 (2H, q,

US 7,727,976 B2 97 98 XII. In Vitro Compound Transport Assays with hIBAT and from electrogenic transport and was directly proportional to hLBAT-Expressing Cell Lines tranport rate. Recordings were made from a single oocyte for up to 60 min, enabling 30-40 separate compounds to be tested Example 50 per oocyte. To compare results between oocytes expressing different levels of transport activity, a saturating concentra Inhibition of Radiolabeled Taurocholate Uptake tion of glycodeoxycholate (300 LM) was used as a common reference to normalize results from test compounds. Using CHO cells transfected with either the hEBAT or hEBAT this normalization procedure V (i.e. maximal induced cur transporter were seeded into 96-well microtiter plates at 100, rent) for different compounds tested on different oocytes 000 cells/well in 100 uL DMEM containing 10% serum, 10 glutamine and Penstrep. After overnight incubation the media could be compared. was removed and test compound (25uL) added at 2x the final desired concentration. Tritiated taurocholate (50,000 CPM/ TABLE 9 well) was diluted with cold substrate to a final concentration In vitro transport(inhibition of tritiated taurocholate uptake) data for of 5uM and 25uL/well of this mixture was added to the plate. 15 2,3-fused pyrazole and pyrimidine derivatives of cholanoic acids After incubating for 1 hat room temperature the solution was 07 and glycocholanoic acids 109 on hIBAT and hLBAT removed and the plate washed 4x with PBS at 4° C. 200 expressing cells. uL/well of scintillant is added and the plate then read in a hIBAT, hLBAT, Wallac microbeta counter. The inhibition data is processed by Compoun ICso (IM) ICso (IM) standard methods to calculate an inhibition constant ICso for the test compound. O7-48a-2 >300 110 Example 51 R = R2 = Hi R' = HOCHCH O7-48a-3 240 0.7 R = R = H; R'= m-HOCHCH Analysis of Electrogenic Transport in Xenopus 25 O7-48c-1 2.5 O.6 Oocytes R = OH: R2 = R = H

RNA preparation: Human IBAT and LBAT Transporter O7-48c-3 4.5 0.7 cDNAs were subcloned into a modified pCEM plasmid that R = OH; R = H; R = m-HOCHCH contains 5' and 3' untranslated sequences from the Xenopus 30 B-actin gene. These sequences increase RNA stability and protein expression. Plasmid c)NA was linearized and used as template for in vitro transcription (Epicentre Technologies transcription kit, 4:1 methylated:non-methylated GTP). Xenopus oocyte isolation. Xenopus laevis frogs were anes 35 thetized by immersion in Tricaine (1.5 g/mL in deionized water) for 15 min. Oocytes were removed and digested in frog ringer solution (90 mM NaCl, 2 mM KCl, 1 mM MgCl, 10 mM NaHEPES, pH 7.45, no CaCl) with 1 mg/mL collage O9-48c-1 1.6 O.3 nase (Worthington Type 3) for 80-100 min with shaking. The 40 R = OH, R = R = H oocytes were washed 6 times, and the buffer changed to frog O9-48c-2 22 O.2 ringer solution containing CaCl (1.8 mM). Remaining fol R = OH, R2 = H, R = HOCHCH licle cells were removed if necessary. Cells were incubated at 16°C., and each oocyte injected with 10-20 ug RNA in 45 uL O9-77a-1 140 2 Solution. 45 R = R2 = H; X = p-CHCONH Electrophysiology measurements. Transport currents were measured 2-14 days after injection, using a standard two ICso values from radiolabelled competition assay in transporter-expressing electrode electrophysiology set-up (Geneclamp 500 ampli CHO cells fier, Digidata 1320/PCLAMP software and ADInstruments hardware and Software were used for signal acquisition). 50 TABLE 10 Electrodes (2-4 mS2) were microfabricated using a Sutter In vitro transport(Oocyte assay) data for 2,3-fused pyrazole and Instrument puller and filled with 3M KC1. The bath was pyrimidine derivatives of cholanoic acids 107 and glycocholanoic directly grounded (transporter currents were less than 0.3 acids 109 on hiBAT expressing cells? LA). Bath flow was controlled by an automated perfusion system (ALA Scientific Instruments, Solenoid valves). 55 %. Max For transporter pharmacology, oocytes were clamped at Compound GDC -60 to -90 mV, and continuous current measurements 107-48a-2 40 acquired using PowerLab Software and an ADInstruments R = R2 = H; R = HOCHCH digitizer. Current signals were lowpass filtered at 20 Hz and 107-48c-1 18 R = OH: R2 = R = H acquired at 4-8 HZ. All bath and compound-containing solu 60 107-48c-2 7 tions were frog ringers solution containing CaCl. Com R = OH; R = Hi R'= HOCHCH pounds were applied for 10-30 seconds until the induced 107-74a-1 40 R = R2 = H; X = O current reached a new steady-state level, followed by a con 109-48a-1 26 trol solution until baseline currents returned to levels that R = R2 = R = H preceded compound application. The difference current 65 109-48a-3 27 (baseline Subtracted from peak current during compound application) reflected the net movement of charge resulting US 7,727,976 B2 99 100 Bellini, A.M.; Rocchi, R.; Fomasini, G.; Benassi, C. A. Studi TABLE 10-continued su 53-chetosteroidi. Nota VII. Derivati pirimidinici e pteridinici dell'acido 3.7.12-trichetocolanico. Il Far In vitro transport(Oocyte assay) data for 2,3-fused pyrazole and maco—Ed Sc., Vol. 25, fasc. 3, 226-233. pyrimidine derivatives of cholanoic acids 107 and glycocholanoic Brouwer, A.; Monnee, M. C. F.; Liskamp, R. M.J. A efficient acids 109 on hIBAT expressing cells. synthesis of N-protected B-aminoethanesolfonyl chlo %. Max rides: Versatile building blocks for the synthesis of oli Compound GDC gopeptidosulfonamides. Synthesis 2000, 1579-1584. 109-48b-1 8 Clinton, R. O. Manson, A. J.; Stonner, F. W.; Neumann, H. R = R2 = OH: R' = m-HOCHCH 10 C.; Christiansen, R. G.; Clarke, R. L.; Ackerman, J. H.; 109-48c-1 21 R = O; R2 = R = H Page, D. F.; Dean, J. W.; Dickison, W. B.: Carabateas, C. 109-48c-2 13 Steroidal 3.2-cpyrazoles II. Androstanes, 19-norandrast R = OH: R2 = Hi R' = HOCHCH anes and their Unsaturated Analogs. J. Am. Chem. Soc. 109-48c-1 8 1961, 83, 1478-1491. R = OH: R = H; R'= m-HOCHCH 15 Ho, N. F. H. Utilizing bile acid carrier mechanisms to enhance 109-77a-1 8 liver and small intestine absorption. Ann. N.Y. Acad. Sci. 1987, 507, 315-329. % Max values are relative to glycodeoxycholic acid (GDC) in transporter Kim, D.-C.; Harrison, A.W.; Ruwart, M. J.; Wilkinson, K. F.; expressing oocytes at a test sample concentration of 100 M Fisher, J. F.; Hidalgo, I.J.; Borchardt, R. T. Evaluation of bile acid transporter in enhancing intestinal permeability of TABLE 11 renin-inhibitory peptides. J. Drug Targeting 1993, 1,347 359. In vitro transport(inhibition of tritiated taurocholate uptake) data for Kramer, W.; Wess, G.; Schubert, G.; Bickel, M.: Girbig. F.; the drug and drug Surrogates conjugated to 2,3-fused pyrazole Gutahr, U.: Kowalewski, S.; Baringhaus, K.-H.; Enhsen, derivatives of glycocholanoic acids 118-120 on hIBAT and 25 A., Glombik, H.; Mullner, S.; Neckermann, G.; Schulz, S.; hLBAT expressing cells? Petzinger, E. Liver-specific drug targeting by coupling to Compound hIBAT, ICso (M) hLBAT, ICso (IM) bile acids. J. Biol. Chem. 1992, 267, 18598-18604. 118-4 143 0.4 Kramer, W.; Wess, G., Neckermann, G.; Schubert, G.; Fink, 118-5 >300 63 J. Girbig. F.; Gutahr, U.: Kowalewski, S.; Baringhaus, 118-6 >300 3.4 30 K.-H.; Boger, G.; Enhsen, A.; Falk, E.; Friedrich, M.: 119-3 32 O.22 Glombik, H.; Hoffmann, A.; Pittius, C.; Urmann, M. Intes 119-4 57 O.28 tinal absorption of peptides by coupling to bile acids. J. 120-2 >300 3.2 Biol. Chem. 1994a, 269, 10621-10627. ICso values from radiolabelled competition assay in transporter-expressing Kramer, W.; Wess, G.; Enhsen, A.; Bock, K., Falk, E.; Hoff CHO cells 35 mann, A.; Neckerman, G., Gantz, D.; Schulz, S.; Nickau, L.; Petzinger, E.; Turley, S.; Dietschy, J. M. Bile acid derived HMG-CoA reductase inhibitors. Biochim. Bio TABLE 12 phys. Acta 1994b, 1227, 137-154. In vitro transport(Oocyte assay) data for the drug and drug Kramer, W.; Wess, G. Modified bile acid conjugates, and their Surrogates conjugated to 2,3-fused pyrazole and derivatives of 40 use as pharmaceuticals. U.S. Pat. No. 5,462.933, 1995. glycocholanoic acids 118-120 on hiBAT expressing cells? Kramer, W.; Wess, G. Bile acid conjugates of proline Compound %. Max GDC hydroxylase inhibitors. U.S. Pat. No. 5,646.272, 1997a. Kramer, W.; Wess, G. Bile acid derivatives, processes for their 118-4 12 118-5 O preparation, and use as pharmaceuticals. U.S. Pat. No. 118-6 12 45 5,668,126, 1997b. 119-3 59 Kramer, W.; Stengelin, S.; Baringhaus, K.-H.; Enhsen, A.; 119-4 18 Heuer, H.; Becker, W.; Corsiero, D.: Girbig, F.; Noll, R.; 120-2 O Weyland, C. Substrate specificity of the ileal and hepatic % Max values are relative to glycodeoxycholic acid (GDC) in transporter Na"/bile acid cotransporters of the rabbit. I. Transport stud expressing oocytes at a test sample concentration of 100 M 50 ies with membrane vesicles and cell lines expressing the cloned transporters. J. Lipid Res. 1999, 40, 1604-1617. Kullak-Ublick, G. A.; Beuers, U.; Paumgartner, G. Hepato REFERENCES biliary transport.J. Hepatology 2000, 32 (Suppl. 1), 3-18. Navia, M. A.; Chaturvedi, P. R. Design principles for orally Baringhaus, K.-H.; Matter, H.; Stengelin, S.; Kramer, W. 55 bioavailable drugs. Drug Discovery Today 1996, 1, 179 Substrate specificity of the ileal and hepatic Na"/bile acid 189. cotransporters of the rabbit. II. A reliable 3D QSAR phar Paquette, L. A.; Johnson, B. A.; Hinga, F. M. 2-Chloro-1- macophore model for the ileal Na/bile acid cotransporter. formyl-1-cyclohexene. Organic synth. Col. Vol. XX, 215 J. Lipid Res. 1999, 40, 2158-2168. 232, XXX. 60 Petzinger, E.; Nickau, L.; Horz, J. A.; Schulz, S.; Wess, G.; Begum, S.; Adil, Q.: Siddiqui, B. S.; Siddiqui, S. Synthesis of Enhsen, A.; Falk, E.; Baringhaus, K.-H.; Glombik. H.; 2B-hydroxyursolic acid and other ursane analogues from Hoffmann, A.; Mullner, S.; Neckermann, G. Kramer, W. urSonic acid. Aust. J. Chem. 1993, 46, 1067-1071. Hepatobiliary transport of hepatic 3-hydroxy-3-methyl Bellini, A. M.; Rocchi, R.; Benassi, C. A. Studi su 5B cheto glutaryl coenzyme A reductase inhibitors conjugated with steroidi. Nota IV. Derivati eterociclici suglianelli Ae B 65 bile acids. Hepatology 1995, 22, 1801-1811. dell'acido 3,7,12-tricheto-colanico. Gazzetta Chimica Rocchi, R.; Bellini, A.M.; Formasini, G.; Benassi, C. A. Studi Italiana, 1969, vol. 99, fasc. 12, 1243-1251. su 53-cheto-steroidi. Nota VI. Derivati pirimidinici e US 7,727,976 B2 101 102 pteridinici degli acidi3,12-dicheto-e 7,12-dichetocolanici. substituted arylene-R'', heteroarylene-R', substituted Il Farmaco—Ed. Sc., Vol. 25, fasc. 2, 125-132. heteroarylene-R'', heterocyclene-R' or substituted Spivey, S.C.; Diaper, C. M.: Adams, H. A new germanium heterocyclene-R'': based linker for solid phase synthesis of aromatics: Syn R’ is an alkylamino group substituted with a moiety that is thesis of a pyrazole library. J. Org. Chem. 2000, 65,5253 negatively charged at physiological pH and located 5 to 5263 and the references cited therein. 15 atoms from C-22 of the bile acid nucleus, a di-sub Swaan, P. W.; Szoka, F. C. Oie. S. Use of the intestinal and stituted alkylamino group Substituted with L-D and a hepatic bile acid transporters for drug delivery. Adv. Drug moiety that is negatively charged at physiological pH Delivery Rev. 1996, 20, 59-82. and located 5 to 15 atoms from C-22 of the bile acid Tsering, K-Y. A convenient synthesis of 3-keto bile acids by 10 nucleus, or pharmaceutically acceptable salts thereof; selective oxidation of bile acids with silver carbonate R is H, alkyl, substituted alkyl, cycloalkyl, substituted Celite. J. Lipid. Res. 1978, 19, 501-504. cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi Tsuji, A.; Tamai, I. Carrier-mediated intestinal transport of tuted heteroalkyl, heterocycloalkyl, substituted hetero drugs. Pharm. Res. 1996, 13,963-977. cycloalkyl, heteroaryl, substituted heteroaryl, OH, Venepalli, B. P.; Aimone, L. D.; Appell, K. C.: Bell, M. R.: 15 OR', SH, SR', NH, NHR', N(R'), COH, Dority, J. A.; Goswami, R.; Hall, P. L.; Kumar, V.; COR, CONH CONHR, CONCR), or L-D; Lawrence, K. B.: Logan, M. E.; Scensny, P. M.: Seelye, J. R" is H, alkyl, substituted alkyl, cycloalkyl, substituted A.: Tomczuk, B. E.; Yanni, J. M. Synthesis and Substance cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi P Receptor Binding Activiy of Androstano.3.2-bipyrimio tuted heteroalkyl, heterocycloalkyl, substituted hetero 1,2-abenzimidazoles. J. Med. Chem. 1992. 35.374-378. cycloalkyl, heteroaryl, substituted heteroaryl, OH, For a review of chemistry of oxoketene S.S.-, N.S- and OR', SH, SR', NH, NHR', N(R'), COH, N.N-acetals preparation and their chemistry see: Junjappa, COR, CONH CONHR, CONCR'), or L-D; H.; Ila, H.; and Asokan, C. V. C.-Oxoketene S.S.-, N.S- and R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted N,N-acetals: Versatile intermediates in organic synthesis. Tet cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi 25 tuted heteroalkyl, heterocycloalkyl, substituted hetero rahedron 1990, 46, 5423-5506. cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, What is claimed is: COR, CONH CONHR, CONCR), or L-D; 1. A compound of formula (I): R" is alkyl, substituted alkyl, cycloalkyl, substituted 30 cycloalkyl, aryl, substituted aryl, heteroaryl or substi tuted heteroaryl; (I) R" is alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl or substi tuted heteroaryl; 35 dashed lines represent possible sites of unsaturation; L is a covalent bond or a linking group; and D is a drug; provided that not more than one of the substituents R. R. R. R. and R' 40 includes moiety L-D. 2. The compound according to claim 1, wherein the com pound is selected from a group consisting of the following compounds:

or a pharmaceutically acceptable salt thereof, wherein 45 R" is CH, CHOH or CROH: R is CH, CHOH or CROH: RandR together with the carbonatoms to which they are attached form a cycloalkyl-R, substituted cycloalkyl R. cycloalkenyl-R, substituted cycloalkenyl-R, het erocycloalkyl-R, substituted heterocycloalkyl-R, het 50 erocycloalkenyl-R, substituted heterocycloalkenyl-R, aryl-R, substituted aryl-R, heteroaryl-R or substi tuted heteroaryl-R ring: R is H, OH, alkylene-R', substituted alkylene-R', 55 cycloalkylene-R', substituted cycloalkylene-R', alk enylene-R', substituted alkenylene-R', cycloalk enylene-R', substituted cycloalkenylene-R', alky nylene-R', substituted alkynylene-R', arylene-R', substituted arylene-R', heteroarylene-R', substituted 60 heteroarylene-R'', heterocyclene-R', or substituted heterocyclene-R' R is H, OH, alkylene-R'', substituted alkylene-R'', cycloalkylene-R', substituted cycloalkylene-R'', alk enylene-R', substituted alkenylene-R'', cycloalk 65 enylene-R'', substituted cycloalkenylene-R'', alky nylene-R', substituted alkynylene-R'', arylene-R'', US 7,727,976 B2 103 104 SOH, - POH, OPOH, OSOH, C(O)NHOH, -continued —tetrazole, -catechol and pharmaceutically acceptable salts

122. thereof. 5. The compound according to claim 4, wherein R and R' together with the carbon atoms to which they are attached form a 5-membered ring selected from the group consisting of the following 5-membered rings:

45 RN N N %

\-N \,O f

50 R 14 R9

RIS RIS 55 21 / 21 / , and N N 3. The compound according to claim 1, wherein Rand R' R 14 R9 R9 R 14 together with the carbon atoms to which they are attached form a 5-membered ring, and wherein the ring is heterocy cloalkyl-R, substituted heterocycloalkyl-R, heteroaryl-R 60 or substituted heteroaryl-R. R13 4. The compound according to claim 1, wherein R is an n1N \ alkylamino group Substituted with a moiety that is negatively NF charged at physiological pH and located 5 to 15 atoms from 65 C-22 of the bile acid nucleus, and wherein the moiety is R9 selected from the group consisting of —COOH, -SOH, US 7,727,976 B2 105 106 wherein R is H, alkyl, substituted alkyl, cycloalkyl, substituted R’ is L-D; cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi cycloalkyl, heteroaryl, substituted heteroaryl, OH, tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH CONHR, CONCR'), or L-D; COR, CONH CONHR' or CONCR'); R" is H, alkyl, substituted alkyl, cycloalkyl, substituted R" is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero tuted heteroalkyl, heterocycloalkyl, substituted hetero 10 cycloalkyl, heteroaryl, substituted heteroaryl, OH, cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, OR', SH, SR'', NH, NHR', N(R'), COH, COR, CONH CONHR, CONCR), or L-D; COR, CONH CONHR' or CONCR'). R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted 6. A pharmaceutical composition comprising a pharma 15 cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi ceutically acceptable excipient and a compound of claim 1. tuted heteroalkyl, heterocycloalkyl, substituted hetero 7. The composition of claim 6, wherein the compound cycloalkyl, heteroaryl, substituted heteroaryl, OH, contains L-D. OR', SH, SR', NH, NHR', N(R'), COH, 8. The composition of claim 6 further comprising a phar COR, CONH CONHR, CONCR), or L-D; maceutically acceptable carrier. R" is alkyl, substituted alkyl, cycloalkyl, substituted 9. A compound of formula (I): cycloalkyl, aryl, substituted aryl, heteroaryl or substi tuted heteroaryl; (I) dashed lines represent possible sites of unsaturation; 25 L is a covalent bond or a linking group; and D is a drug; provided that not more than one of the substituents R. R. R. R. and R7 includes moiety L-D; and 30 when R' and Rare CHOH, Rand Rare H, and R7 is OH, then RandR together with the carbonatoms to which they are attached do not form

s or a pharmaceutically acceptable salt thereof, wherein: 35 R is CHOH: HN R is CHOH: RandR together with the carbonatoms to which they are attached form a cycloalkyl-R, substituted cycloalkyl R. cycloalkenyl-R, substituted cycloalkenyl-R, het 40 erocycloalkyl-R, substituted heterocycloalkyl-R, het 10. The compound according to claim 9, wherein the com erocycloalkenyl-R, substituted heterocycloalkenyl-R. pound is selected from a group consisting of the following aryl-R, substituted aryl-R, heteroaryl-R or substi compounds: tuted heteroaryl-R ring: R is H, OH, alkylene-R', substituted alkylene-R', 45 cycloalkylene-R', substituted cycloalkylene-R', alk enylene-R', substituted alkenylene-R', cycloalk enylene-R', substituted cycloalkenylene-R', alky nylene-R', substituted alkynylene-R', arylene-R', substituted arylene-R', heteroarylene-R', substituted 50 heteroarylene-R'', heterocyclene-R', or substituted heterocyclene-R' R is H, OH, alkylene-R'', substituted alkylene-R'', cycloalkylene-R'', substituted cycloalkylene-R'', alk enylene-R', substituted alkenylene-R'', cycloalk 55 enylene-R'', substituted cycloalkenylene-R'', alky nylene-R', substituted alkynylene-R'', arylene-R'', substituted arylene-R'', heteroarylene-R'', substituted heteroarylene-R'', heterocyclene-R' or substituted heterocyclene-R'': 60 R’ is OH, an alkylamino group substituted with a moiety that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, a di-substituted alkylamino group substituted with L-D and a moiety that is negatively charged at physiological 65 pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, or pharmaceutically acceptable salts thereof. US 7,727,976 B2 107 108

-continued

122.

N-N NFN

10 Y.

O N O N RN N N 15

R9 R 14 R 14 R9 R 14 R15

R13N N R13N N

25 R9

R9 a 30 \ { A R 15 R 14

35 R R, and n/N N4 \-N \,O f 40 R 14 R9

R1 RIS 45 21 / 21 / , and A N R 14 R9 R9 R 14

50 R13NN N \-N 55 R9 11. The compound according to claim 9, wherein RandR together with the carbon atoms to which they are attached wherein form a 5-membered ring, and wherein the ring is heterocy R is L-D; 60 R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted cloalkyl-R, substituted heterocycloalkyl-R, heteroaryl-R cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi or substituted heteroaryl-R. tuted heteroalkyl, heterocycloalkyl, substituted hetero 12. The compound according to claim 9, wherein R is OH. cycloalkyl, heteroaryl, substituted heteroaryl, OH, 13. The compound according to claim 11, wherein R and OR', SH, SR', NH, NHR', N(R'), COH, R" together with the carbonatoms to which they are attached 65 COR, CONH CONHR' or CONCR'); form a 5-membered ring selected from the group consisting R" is H, alkyl, substituted alkyl, cycloalkyl, substituted of the following 5-membered rings: cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi US 7,727,976 B2 109 110 tuted heteroalkyl, heterocycloalkyl, substituted hetero OR', SH, SR', NH, NHR', N(R'), COH, cycloalkyl, heteroaryl, substituted heteroaryl, OH, COR, CONH CONHR, CONCR'), or L-D; OR', SH, SR', NH, NHR', N(R'), COH, R" is H, alkyl, substituted alkyl, cycloalkyl, substituted COR, CONH CONHR' or CONCR'). cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi 14. A pharmaceutical composition comprising a pharma tuted heteroalkyl, heterocycloalkyl, substituted hetero ceutically acceptable excipient and a compound of claim 9. cycloalkyl, heteroaryl, substituted heteroaryl, OH, 15. The composition of claim 14, wherein the compound contains L-D. OR', SH, SR', NH, NHR', N(R'), COH, 16. The composition of claim 14 further comprising a COR, CONH CONHR, CONCR), or L-D; pharmaceutically acceptable carrier. 10 R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted 17. A compound of formula (I): cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, 15 OR', SH, SR', NH, NHR', N(R'), COH, (I) COR, CONH CONHR, CONCR'), or L-D; R" is alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl or substi tuted heteroaryl; R" is alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl or substi tuted heteroaryl; dashed lines represent possible sites of unsaturation; 25 L is a covalent bond or a linking group; and D is a drug; provided that not more than one of the substituents R. R. R. R. and R' includes moiety L-D. or a pharmaceutically acceptable salt thereof, wherein: 30 18. The compound according to claim 17, wherein the R is CROH: compound is selected from a group consisting of the follow R’ is CROH: ing compounds: RandR together with the carbonatoms to which they are attached form a cycloalkyl-R, substituted cycloalkyl R. cycloalkenyl-R, substituted cycloalkenyl-R, het 35 erocycloalkyl-R, substituted heterocycloalkyl-R, het erocycloalkenyl-R, substituted heterocycloalkenyl-R. aryl-R, substituted aryl-R, heteroaryl-R or substi tuted heteroaryl-R ring: R is H, OH, alkylene-R', substituted alkylene-R', 40 cycloalkylene-R', substituted cycloalkylene-R', alk enylene-R', substituted alkenylene-R', cycloalk enylene-R', substituted cycloalkenylene-R', alky nylene-R', substituted alkynylene-R', arylene-R', substituted arylene-R', heteroarylene-R', substituted 45 heteroarylene-R'', heterocyclene-R', or substituted heterocyclene-R' R is H, OH, alkylene-R'', substituted alkylene-R'', cycloalkylene-R', substituted cycloalkylene-R'', alk enylene-R', substituted alkenylene-R'', cycloalk 50 enylene-R', substituted cycloalkenylene-R'', alky nylene-R', substituted alkynylene-R'', arylene-R'', substituted arylene-R'', heteroarylene-R', substituted heteroarylene-R'', heterocyclene-R' or substituted heterocyclene-R'': 55 R’ is OH, an alkylamino group substituted with a moiety that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, a di-substituted alkylamino group substituted with L-D 60 and a moiety that is negatively charged at physiological pH and located 5 to 15 atoms from C-22 of the bile acid nucleus, or pharmaceutically acceptable salts thereof. R is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi 65 tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, US 7,727,976 B2 111 112

-continued -continued 12.1.

O N O N RN N N

R9 R 14 R 14 R9 R 14 R 15

R13NN N R13N N 15

R9 21 ( / 25 R 15 R 14 R, and RN 1n N4 30 \-N \,O f R 14 R9

35 R13 RIS 2 / 2 / , and A. N 40 R 14 R9 R9 R 14

R13NN N 45 \- 19. The compound according to claim 17, wherein Rand N R" together with the carbonatoms to which they are attached R9 form a 5-membered ring, and wherein the ring is heterocy cloalkyl-R, substituted heterocycloalkyl-R, heteroaryl-R or substituted heteroaryl-R. 50 wherein 20. The compound according to claim 17, wherein R is R’ is L-D; OH. R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted 21. The compound according to claim 19, wherein Rand cycloalkyl, aryl, substituted aryl, heteroalkyl, substi R" together with the carbonatoms to which they are attached tuted heteroalkyl, heterocycloalkyl, substituted hetero 55 cycloalkyl, heteroaryl, substituted heteroaryl, OH, form a 5-membered ring selected from the group consisting OR', SH, SR', NH, NHR', N(R'), COH, of the following 5-membered rings: COR, CONH CONHR' or CONCR'); R" is H. alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroalkyl, substi tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, N N RN N COR, CONH CONHR' or CONCR). 22. A pharmaceutical composition comprising a pharma WN-N \-NFN V 65 ceutically acceptable excipient and a compound of claim 17. R9 23. The composition of claim 22, wherein the compound contains L-D. US 7,727,976 B2 113 114 24. The composition of claim 22 further comprising a pharmaceutically acceptable carrier. -continued 25. A compound of formula (I):

R N N %

\-N yO f R 14 R9 10

RIS RIS 2 / a / , and 15 N N A A R 14 R9 R9 R 14

R13n/N or a pharmaceutically acceptable salt thereof, wherein \- R is CH, CHOH or CROH: N R is CH, CHOH or CROH: 25 R9 RandR together with the carbonatoms to which they are attached form a 5-membered ring selected from the wherein group consisting of the following 5-membered rings: R is L-D; R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted 30 cycloalkyl, aryl. Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted het erocycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, COR, CONH, CONHR' or CON(R'); and 35 R" is H. alkyl, substituted alkyl, cycloalkyl, substituted W x RS V N N-N N cycloalkyl, aryl. Substituted aryl, heteroalkyl, Substi V tuted heteroalkyl, heterocycloalkyl, substituted het erocycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NHR', N(R'), COH, COR', 40 CONH CONHR' or CON(R'); R is H, OH, alkylene-R', substituted alkylene-R', cycloalkylene-R', substituted cycloalkylene-R', alk enylene-R', substituted alkenylene-R', cycloalk enylene-R', substituted cycloalkenylene-R', alky 45 nylene-R', substituted alkynylene-R', arylene-R', substituted arylene-R', heteroarylene-R', substituted heteroarylene-R', heterocyclene-R', or substituted heterocyclene-R': R is H, OH, alkylene-R'', substituted alkylene-R'', 50 cycloalkylene-R', substituted cycloalkylene-R'', alk enylene-R', substituted alkenylene-R'', cycloalk enylene-R'', substituted cycloalkenylene-R'', alky nylene-R'', substituted alkynylene-R'', arylene-R'', substituted arylene-R'', heteroarylene-R', substituted 55 heteroarylene-R'', heterocyclene-R' or substituted R9 R 15 R15 R9 heterocyclene-R'': R’ is OH, an alkylamino group substituted with a moiety that is negatively charged at physiological pH and 60 located 5 to 15 atoms from C-22 of the bile acid nucleus, a di-substituted alkylamino group substituted with L-D and a moiety that is negatively charged at physiological R9 a pH and located 5 to 15 atoms from C-22 of the bile acid \ . nucleus, or pharmaceutically acceptable salts thereof; A 65 R" is H, alkyl, substituted alkyl, cycloalkyl, substituted R15 R 14 cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi tuted heteroalkyl, heterocycloalkyl, substituted hetero US 7,727,976 B2 115 116 cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, -continued COR, CONH CONHR, CONCR'), or L-D; 12 R'' is H, alkyl, substituted alkyl, cycloalkyl, substituted a. cycloalkyl, aryl, Substituted aryl, heteroalkyl, Substi 5 tuted heteroalkyl, heterocycloalkyl, substituted hetero cycloalkyl, heteroaryl, substituted heteroaryl, OH, OR', SH, SR', NH, NHR', N(R'), COH, R COR, CONH CONHR, CONCR), or L-D; R" is alkyl, substituted alkyl, cycloalkyl, substituted 10 cycloalkyl, aryl, substituted aryl, heteroaryl or substi tuted heteroaryl; R3

R" is alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl or substi 1% O tuted heteroaryl; 15 dashed lines represent possible sites of unsaturation; L is a covalent bond or a linking group; and D is a drug; provided that not more than one of the substituents R. R. R. R. and R7 includes moiety L-D. 2O 26. The compound according to claim 25, wherein the compound is selected from a group consisting of the follow ing compounds:

27. The compound according to claim 25, wherein R is OH. 28. A pharmaceutical composition comprising a pharma 50 ceutically acceptable excipient and a compound of claim 25. 29. The composition of claim 28, wherein the compound contains L-D. 30. The composition of claim 28 further comprising a 55 pharmaceutically acceptable carrier.