TRITERPENE SAPONIN ANALOGUES
20230219991 · 2023-07-13
Assignee
Inventors
- David Y. GIN (Lincoln, NE, US)
- Eric CHEA (Oakland, CA, US)
- Alberto FERNANDEZ-TEJADA (Logroño. La Rioja, ES)
- Jeffrey GARDNER (New York, NY, US)
- Jason LEWIS (New York, NY, US)
- Philip Livingston (New York, NY)
- J. Tyler MARTIN (Roca, NE, US)
- Lars NORDSTROEM (New York, NY, US)
- Naga Vara Kishore PILLARSETTY (Jackson Heights, NY, US)
- Govind RAGUPATHI (New York, NY, US)
- Derek TAN (New York, NY, US)
Cpc classification
C07H1/00
CHEMISTRY; METALLURGY
C07H13/04
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
C07J63/008
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K2039/10
HUMAN NECESSITIES
International classification
C07H1/00
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
C07H13/04
CHEMISTRY; METALLURGY
C07H13/08
CHEMISTRY; METALLURGY
Abstract
The present application relates to triterpene glycoside saponin-derived adjuvants, syntheses thereof, and intermediates thereto. The application also provides pharmaceutical compositions comprising compounds of the present invention and methods of using said compounds or compositions in the treatment of and immunization for infectious diseases.
Claims
1. A compound having the formula ##STR00092## wherein R.sup.1 is —CHO or —CH.sub.3, and R.sup.2 is —OH or —H, or a pharmaceutically acceptable salt thereof.
2. A pharmaceutical composition comprising a compound of the formula ##STR00093## wherein R.sup.1 is —CHO or —CH.sub.3, and R.sup.2 is —OH or —H, or a pharmaceutically acceptable salt thereof; and an immunologically effective amount of an antigen.
3. The pharmaceutical composition according to claim 2, wherein the antigen is associated with a bacteria or virus.
4. The pharmaceutical composition according to claim 3, wherein the antigen is associated with a bacterial or virus causing a disease selected from the group consisting of Hepatitis B, pneumococcus, diphtheria, tetanus, pertussis, or Lyme disease including the closely related spirochetes of the genus Borrelia such as, B. burgdorferi, B. garinii, B. afzelli, and B. japonica.
5. The pharmaceutical composition according to claim 4, wherein the antigen is an antigen associated with Hepatitis B virus.
6. The pharmaceutical composition according to claim 4, wherein the antigen is an antigen associated with pneumococcus bacterium.
7. The pharmaceutical composition according to claim 4, wherein the antigen is an antigen associated with Corynebacterium diphtheria bacterium.
8. The pharmaceutical composition according to claim 4, wherein the antigen is an antigen associated with Clostridium tetani bacterium.
9. The pharmaceutical composition according to claim 4, wherein the antigen is an antigen associated with Bordetella pertussis bacterium.
10. The pharmaceutical composition according to claim 4, wherein the antigen is an antigen associated with a bacterium causing Lyme disease or a spirochete of the genus Borrelia selected from the group consisting of B. burgdorferi, B. garinii, B. afzelli, and B. japonica.
11. A method of conferring resistance to an infection, the method comprising administering an antigen in combination with a pharmaceutical composition comprising a compound of the formula ##STR00094## wherein R.sup.1 is —CHO or —CH.sub.3, and R.sup.2 is —OH or —H, or a pharmaceutically acceptable salt thereof.
12. The method according to claim 11, wherein the antigen is an antigen associated with a bacteria or a virus.
13. The method according to claim 12, wherein the antigen is an antigen associated with a bacteria or a virus causing a disease selected from the group consisting of Hepatitis B, pneumococcus, diphtheria, tetanus, pertussis, or Lyme disease including the closely related spirochetes of the genus Borrelia such as, B. burgdorferi, B. garinii, B. afzelli, and B. japonica.
14. The method according to claim 13, wherein the antigen is associated with Hepatitis B virus.
15. The method according to claim 13, wherein the antigen is associated with pneumococcus bacterium.
16. The method according to claim 13, wherein the antigen is associated with Corynebacterium diphtheria bacterium.
17. The method according to claim 13, wherein the antigen is associated with Clostridium bacterium.
18. The method according to claim 13, wherein the antigen is associated with Bordetella pertussis bacterium.
19. The method according to claim 13, wherein the antigen is associated with a bacterium causing Lyme disease or a spirochete of the genus Borrelia selected from the group consisting of B. burgdorferi, B. garinii, B. afzelli, and B. japonica.
20. A method for obtaining the compound according to claim 1 comprising: providing the compound according to claim 1 and a second substance, and subsequently purifying the compound of claim 1 by removing at least a portion of the second substance.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0178]
[0179]
[0180]
[0181]
[0182]
[0183]
[0184]
[0185]
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0186] The clinical success of anticancer, antiviral and antimicrobial vaccines critically depends on the identification of, and access to, novel potent adjuvants with attenuated toxicity. In this context, specific fractions from extracts of the bark of Quillaja saponaria (QS) have proven to be exceedingly powerful adjuvants in immunotherapy. The QS-21 fraction (Kensil, C. R.; Patel, U.; Lennick, M.; Marciani, D. J. Immunol. 1991, 146, 431-437), comprising isomeric forms of a complex triterpene glycoside saponin (Soltysik, S.; Wu, J. Y.; Recchia, J.; Wheeler, D. A.; Newman, M. J.; Coughlin, R. T.; Kensil, C. R. Vaccine 1995, 13, 1403-1410; Kensil, C. R. Crit. Rev. Ther. Drug Carrier Syst. 1996, 13, 1-55), had previously been considered the most promising immuno-potentiator (Kim, S. K.; Ragupathi, G.; Musselli, C.; Choi, S. J.; Park, Y. S.; Livingston, P. O. Vaccine 2000, 18, 597-603) in several antitumor (melanoma, breast, small cell lung cancer, prostate) (Livingston, P. O.; Ragupathi, G. Hum. Vaccines 2006, 2, 137-143) and infectious-disease (HIV, malaria) vaccine therapies (Sasaki, S.; Sumino, K.; Hamajima, K.; Fukushima, J.; Ishii, N.; Kawamoto, S.; Mohri, H.; Kensil, C. R.; Okuda, K. J. Virol. 1998, 72, 4931-4939; Evans, T. G., et al. Vaccine 2001, 19, 2080-2091; Kashala, O., et al. Vaccine 2002, 20, 2263-2277; Carcaboso, A. M.; Hernandez, R. M.; Igartua, M.; Rosas, J. E.; Patarroyo, M. E.; Pedraz, J. L. Vaccine 2004, 22, 1423-1432).
[0187] However, the tolerated dose of QS-21 in cancer patients typically does not exceed 100-150 μg, above which significant local erythema and systemic flu-like symptoms arise. QS-21's inherent instability can lead to toxicities associated with its breakdown. It is also known that QS-21 is hemolytic, and this hemolytic activity had previously been hypothesized that at least some of QS-21's adjuvant activity was related to its hemolytic properties. Some of the various shortcomings of QS-21 have been partially addressed by formulation with emulsions (AS02 by GlaxoSmithKline (GSK) or liposomes (AS01, GSK)), however, these solutions are suboptimal and there remains a strong need for improved adjuvants that exhibit good adjuvant properties while maintaining a high degree of tolerability and/or reduced side-effects.
[0188] Now, surprisingly, the inventors of the present subject matter have found that compounds of the present application, which are in some embodiments synthetic analogues of QS-21 and other QS extraction fractions such as QS-7, possess significant stand-alone adjuvant activity as well as a high degree of tolerability and/or reduced side-effects. These new adjuvant compounds are more cost-effective to produce than natural QS-21, more stable, more efficacious, and less toxic for use in prophylactic and therapeutic vaccination programs. Some embodiments have no detectable toxicity in pharmacology/toxicology studies in mice at doses close to the likely 1000 mcg human dose. Some embodiments are surprisingly completely nonhemolytic while still retaining their adjuvant properties. This is surprising in part because it was initially thought that both QS-21 toxicity and potency were related to hemolysis and other cellular toxicity associated with QS-21. Some embodiments of the present application exhibit greater stability and less hemolytic activity by replacing the unstable ester linkage of the acyl chain in QS-21 with a very stable amide linkage, resulting in adjuvant active analogs of QS-21. Some embodiments also retain adjuvant activity despite having a simplified structure as compared to QS-21, resulting in higher synthetic yields and significantly reduced synthetic steps and cost of manufacture in comparison to synthetic QS-21.
[0189] The present application also provides efficient semi-synthetic methods of synthesizing the compounds of the present application, thereby significantly reducing the number of synthetic steps required to access this potent class of adjuvants.
[0190] The application also includes pharmaceutical compositions comprising the compounds of the present application together with an immunologically effective amount of an antigen associated with a bacterium or virus. Bacterium or viruses included in the subject matter of this application consist of those associated with Hepatitis B, pneumococcus, diphtheria, tetanus, pertussis, or Lyme disease including the closely related spirochetes of the genus Borrelia such as, B. burgdorferi, B. garinii, B. afzelli, and B. japonica.
[0191] The application also includes methods of vaccinating a human patient comprising administering an immunologically effective amount of a pharmaceutical compositions or of the compounds of the present application. The application also includes methods for increasing the immune response to a vaccine comprising administering an immunologically effective amount of a pharmaceutical compositions or of the compounds of the present application.
Compounds
[0192] Compounds of this invention include those described generally above, and are further illustrated by the classes, subclasses, and species disclosed herein. In some embodiments, provided compounds are analogs of naturally occurring triterpene glycoside saponins and intermediates thereto. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, and March's Advanced Organic Chemistry, 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.
Description of Exemplary Compounds
[0193] In some embodiments, provided compounds are analogs of Quillaja saponins. In some embodiments, provided compounds are prosapogenins. In certain embodiments, provided compounds are analogs of QS-7 and QS-21 and possess potent adjuvant activity.
In one aspect, the present application provides compounds of Formula I:
##STR00024##
or a pharmaceutically acceptable salt thereof, wherein [0194] is a single or double bond; [0195] W is —CHO; [0196] V is hydrogen or OR.sup.x; [0197] Y is CH.sub.2, —O—, —NR—, or —NH—; [0198] Z is hydrogen; a cyclic or acyclic, optionally substituted moiety selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, arylalkyl, heteroacyl, and heteroaryl; or a carbohydrate domain having the structure:
##STR00025## [0199] wherein each occurrence of R.sup.1 is R.sup.x or a carbohydrate domain having the structure:
##STR00026## [0200] wherein: [0201] each occurrence of a, b, and c is independently 0, 1, or 2; [0202] d is an integer from 1-5, wherein each d bracketed structure may be the same or different; with the proviso that the d bracketed structure represents a furanose or a pyranose moiety, and the sum of b and c is 1 or 2; [0203] R.sup.0 is hydrogen; an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates; or an optionally substituted moiety selected from the group consisting of acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10 membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 4-7 membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0204] each occurrence of R.sup.a, R.sup.b, R.sup.c, and R.sup.d is independently hydrogen, halogen, OH, OR, OR.sup.x, NR.sub.2, NHCOR, or an optionally substituted group selected from acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulfur; 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0205] R.sup.2 is hydrogen, halogen, OH, OR, OC(O)R.sup.4, OC(O)OR.sup.4, OC(O)NHR.sup.4, OC(O)NRR.sup.4, OC(O)SR.sup.4, NHC(O)R.sup.4, NRC(O)R.sup.4, NHC(O)OR.sup.4, NHC(O)NHR.sup.4, NHC(O)NRR.sup.4, NHR.sup.4, N(R.sup.4).sub.2, NHR.sup.4, NRR.sup.4, N.sub.3, or an optionally substituted group selected from C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10 membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0206] R.sup.3 is hydrogen, halogen, CH.sub.2OR.sup.1, or an optionally substituted group selected from the group consisting of acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, [0207] R.sup.4 is -T-R.sup.z, —C(O)-T-R.sup.z, —NH-T-R.sup.z, —O-T-R.sup.z, —S-T-R.sup.z, —C(O) NH-T-R.sup.z, C(O)O-T-R.sup.z, C(O)S-T-R.sup.z, C(O)NH-T-O-T-R.sup.z, —O-T-R.sup.z, -T-O-T-R.sup.z, -T-S-T-R.sup.z, or
##STR00027## [0208] wherein [0209] X is —O—, —NR—, or T-R.sup.z; [0210] T is a covalent bond or a bivalent C.sub.1-26 saturated or unsaturated, straight or branched, aliphatic or heteroaliphatic chain; and [0211] R.sup.z is hydrogen, halogen, —OR, —OR.sup.x, —OR.sup.1, —SR, NR.sub.2, —C(O)OR, —C(O)R, —NHC(O)R, —NHC(O)OR, NC(O)OR, or an optionally substituted group selected from acyl, arylalkyl, heteroarylalkyl, C.sub.1-6 aliphatic, 6-10-membered aryl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0212] each occurrence of R.sup.x is independently hydrogen or an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates; [0213] each occurrence of R is independently hydrogen, an optionally substituted group selected from acyl, arylalkyl, 6-10-membered aryl, C.sub.1-6 aliphatic, or C.sub.1-6 heteroaliphatic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, or: [0214] two R on the same nitrogen atom are taken with the nitrogen atom to form a 4-7-membered heterocyclic ring having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0215] In one aspect, the present application provides compounds of Formula II:
##STR00028##
or a pharmaceutically acceptable salt thereof, wherein [0216] is a single or double bond; [0217] W is ME, —CHO, or
##STR00029## [0218] V is hydrogen or OR.sup.x; [0219] Y is CH.sub.2, —O—, —NR—, or —NH—; [0220] Z is hydrogen; a cyclic or acyclic, optionally substituted moiety selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, arylalkyl, heteroacyl, and heteroaryl; or a carbohydrate domain having the structure:
##STR00030## [0221] wherein each occurrence of R.sup.1 is R.sup.x or a carbohydrate domain having the structure:
##STR00031## [0222] wherein: [0223] each occurrence of a, b, and c is independently 0, 1, or 2; [0224] d is an integer from 1-5, wherein each d bracketed structure may be the same or different; with the proviso that the d bracketed structure represents a furanose or a pyranose moiety, and the sum of b and c is 1 or 2; [0225] R.sup.0 is hydrogen; an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates; or an optionally substituted moiety selected from the group consisting of acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10 membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 4-7 membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0226] each occurrence of R.sup.a, R.sup.b, R.sup.c, and R.sup.d is independently hydrogen, halogen, OH, OR, OR.sup.x, NR.sub.2, NHCOR, or an optionally substituted group selected from acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulfur; 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0227] R.sup.2 is hydrogen, halogen, OH, OR, OC(O)R.sup.4, OC(O)OR.sup.4, OC(O)NHR.sup.4, OC(O)NRR.sup.4, OC(O)SR.sup.4, NHC(O)R.sup.4, NRC(O)R.sup.4, NHC(O)OR.sup.4, NHC(O)NHR.sup.4, NHC(O)NRR.sup.4, NHR.sup.4, N(R.sup.4).sub.2, NHR.sup.4, NRR.sup.4, N.sub.3, or an optionally substituted group selected from C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10 membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0228] R.sup.3 is hydrogen, halogen, CH.sub.2OR.sup.1, or an optionally substituted group selected from the group consisting of acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, [0229] R.sup.4 is -T-R.sup.z, —C(O)-T-R.sup.z, —NH-T-R.sup.z, —O-T-R.sup.z, —S-T-R.sup.z, —C(O)NH-T-R.sup.z, C(O)O-T-R.sup.z, C(O)S-T-R.sup.z, C(O)NH-T-O-T-R.sup.z, —O-T-R.sup.z, -T-O-T-R.sup.z, -T-S-T-R.sup.z, or
##STR00032## [0230] wherein [0231] X is —O—, —NR—, or T-R.sup.z; [0232] T is a covalent bond or a bivalent C.sub.1-26 saturated or unsaturated, straight or branched, aliphatic or heteroaliphatic chain; and [0233] R.sup.z is hydrogen, halogen, —OR, —OR.sup.x, —OR.sup.1, —SR, NR.sub.2, —C(O)OR, —C(O)R, —NHC(O)R, —NHC(O)OR, NC(O)OR, or an optionally substituted group selected from acyl, arylalkyl, heteroarylalkyl, C.sub.1-6 aliphatic, 6-10-membered aryl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0234] each occurrence of R.sup.x is independently hydrogen or an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates; [0235] R.sup.y is —OH, —OR, or a carboxyl protecting group selected from the group consisting of ester, amides, and hydrazides; [0236] R.sup.s is
##STR00033## [0237] each occurrence of R.sup.x′ is independently an optionally substituted group selected from 6-10-membered aryl, C.sub.1-6 aliphatic, or C.sub.1-6 heteroaliphatic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; or: [0238] two R.sup.x′ are taken together to form a 5-7-membered heterocyclic ring having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0239] each occurrence of R is independently hydrogen, an optionally substituted group selected from acyl, arylalkyl, 6-10-membered aryl, C.sub.1-6 aliphatic, or C.sub.1-6 heteroaliphatic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, or: [0240] two R on the same nitrogen atom are taken with the nitrogen atom to form a 4-7-membered heterocyclic ring having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0241] In one aspect, the present application provides compounds of Formula I:
##STR00034##
or a pharmaceutically acceptable salt thereof, wherein is a single or double bond;
W is —CHO;
V is —OH;
Y is —O—;
[0242] wherein Z is a carbohydrate domain having the structure:
##STR00035##
wherein: [0243] R.sup.1 is independently H or
##STR00036## [0244] R.sup.2 is NHR.sup.4; [0245] R.sup.3 is CH.sub.2OH; and [0246] R.sup.4 is -T-R.sup.z, —C(O)-T-R.sup.z, —NH-T-R.sup.z, —O-T-R.sup.z, —S-T-R.sup.z, —C(O)NH-T-R.sup.z, C(O)O-T-R.sup.z, C(O)S-T-R.sup.z, C(O)NH-T-O-T-R.sup.z, —O-T-R.sup.z, -T-O-T-R.sup.z, -T-S-T-R.sup.z, or
##STR00037## [0247] wherein: [0248] X is —O—, —NR—, or T-R.sup.z; [0249] T is a covalent bond or a bivalent C.sub.1-26 saturated or unsaturated, straight or branched, aliphatic or heteroaliphatic chain; and [0250] R.sup.z is hydrogen, halogen, —OR, —OR.sup.x, —OR.sup.1, —SR, NR.sub.2, —C(O)OR, —C(O)R, —NHC(O)R, —NHC(O)OR, NC(O)OR, or an optionally substituted group selected from acyl, arylalkyl, heteroarylalkyl, C.sub.1-6 aliphatic, 6-10-membered aryl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0251] It will be appreciated by one of ordinary skill in the art that the compounds of the present application include but are not necessarily limited to those compounds encompassed in the genus definitions set forth as part of the present section. The compounds encompassed by this application include at least all of the compounds disclosed in the entire specification as a whole, including all individual species within each genus.
[0252] In certain embodiments, V is OR.sup.x. In certain embodiments V is OH. In certain embodiments, V is H.
[0253] In certain embodiments, Y is —O—. In certain embodiments, Y is —NH—. In certain embodiments, Y is —NR—. In certain embodiments, Y is CH.sub.2.
[0254] In certain embodiments, Z is hydrogen. In certain embodiments, Z is a cyclic or acyclic, optionally substituted moiety. In certain embodiments, Z is an acyl. In certain embodiments, Z is an aliphatic. In certain embodiments, Z is a heteroaliphatic. In certain embodiments, Z is aryl. In certain embodiments Z is arylalkyl. In certain embodiments, Z is heteroacyl. In certain embodiments, Z is heteroaryl. In certain embodiments, Z is a carbohydrate domain having the structure:
##STR00038##
In some embodiments Z is a carbohydrate domain having the structure:
##STR00039##
wherein:
[0255] R.sup.1 is independently H or
##STR00040##
[0256] R.sup.2 is NHR.sup.4,
[0257] R.sup.3 is CH.sub.2OH, and
[0258] R.sup.4 is selected from:
##STR00041##
[0259] In some embodiments, R.sup.1 is R.sup.x. In other embodiments, R.sup.1 a carbohydrate domain having the structure:
##STR00042##
[0260] In some aspects, each occurrence of a, b, and c is independently 0, 1, or 2. In some embodiments, d is an integer from 1-5. In some embodiments, each d bracketed structure may be the same. In some embodiments, each d bracketed structure may be different. In some embodiments, the d bracketed structure represents a furanose or a pyranose moiety. In some embodiments, and the sum of b and c is 1 or 2.
[0261] In some embodiments, R.sup.0 is hydrogen. In some embodiments, R.sup.0 is an oxygen protecting group selected from the group. In some embodiments, R.sup.0 is an alkyl ether. In some embodiments, R.sup.0 is a benzyl ether. In some embodiments, R.sup.0 is a silyl ether. In some embodiments, R.sup.0 is an acetal. In some embodiments, R.sup.0 is ketal. In some embodiments, R.sup.0 is an ester. In some embodiments, R.sup.0 is a carbamate. In some embodiments, R.sup.0 is a carbonate. In some embodiments, R.sup.0 is an optionally substituted moiety. In some embodiments, R.sup.0 is an acyl. In some embodiments, R.sup.0 is a C.sub.1-10 aliphatic. In some embodiments, R.sup.0 is a C.sub.1-6 heteroaliphatic. In some embodiments, R.sup.0 is a 6-10-membered aryl. In some embodiments, R.sup.0 is a arylalkyl. In some embodiments, R.sup.0 is a 5-10 membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.0 is a 4-7 membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0262] In some embodiments, R.sup.a is hydrogen. In some embodiments, R.sup.a is a halogen. In some embodiments, R.sup.a is OH. In some embodiments, R.sup.a is OR. In some embodiments, R.sup.a is OR.sup.x. In some embodiments, R.sup.a is NR.sub.2. In some embodiments, R.sup.a is NHCOR. In some embodiments, R.sup.a an acyl. In some embodiments, R.sup.a is C.sub.1-10 aliphatic. In some embodiments, R.sup.a is C.sub.1-6 heteroaliphatic. In some embodiments, R.sup.a is 6-10-membered aryl. In some embodiments, R.sup.a is arylalkyl. In some embodiments, R.sup.a is 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulfur. In some embodiments, R.sup.a is 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0263] In some embodiments, R.sup.b is hydrogen. In some embodiments, R.sup.b is a halogen. In some embodiments, R.sup.b is OH. In some embodiments, R.sup.b is OR. In some embodiments, R.sup.b is OR.sup.x. In some embodiments, R.sup.b is NR.sub.2. In some embodiments, R.sup.b is NHCOR. In some embodiments, R.sup.b an acyl. In some embodiments, R.sup.b is C.sub.1-10 aliphatic. In some embodiments, R.sup.b is C.sub.1-6 heteroaliphatic. In some embodiments, R.sup.b is 6-10-membered aryl. In some embodiments, R.sup.b is arylalkyl. In some embodiments, R.sup.b is 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulfur. In some embodiments, R.sup.b is 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0264] In some embodiments, R.sup.b is hydrogen. In some embodiments, R.sup.b is a halogen. In some embodiments, R.sup.b is OH. In some embodiments, R.sup.b is OR. In some embodiments, R.sup.b is OR.sup.x. In some embodiments, R.sup.b is NR.sub.2. In some embodiments, R.sup.b is NHCOR. In some embodiments, R.sup.b an acyl. In some embodiments, R.sup.b is C.sub.1-10 aliphatic. In some embodiments, R.sup.b is C.sub.1-6 heteroaliphatic. In some embodiments, R.sup.b is 6-10-membered aryl. In some embodiments, R.sup.b is arylalkyl. In some embodiments, R.sup.b is 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulfur. In some embodiments, R.sup.b is 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0265] In some embodiments, R.sup.c is hydrogen. In some embodiments, R.sup.c is a halogen. In some embodiments, R.sup.c is OH. In some embodiments, R.sup.c is OR. In some embodiments, R.sup.c is OR.sup.x. In some embodiments, R.sup.c is NR.sub.2. In some embodiments, R.sup.c is NHCOR. In some embodiments, R.sup.c an acyl. In some embodiments, R.sup.c is C.sub.1-10 aliphatic. In some embodiments, R.sup.c is C.sub.1-6 heteroaliphatic. In some embodiments, R.sup.c is 6-10-membered aryl. In some embodiments, R.sup.c is arylalkyl. In some embodiments, R.sup.c is 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulfur. In some embodiments, R.sup.c is 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0266] In some embodiments, R.sup.d is hydrogen. In some embodiments, R.sup.d is a halogen. In some embodiments, R.sup.d is OH. In some embodiments, R.sup.d is OR. In some embodiments, R.sup.d is OR.sup.x. In some embodiments, R.sup.d is NR.sub.2. In some embodiments, R.sup.d is NHCOR. In some embodiments, R.sup.d an acyl. In some embodiments, R.sup.d is C.sub.1-10 aliphatic. In some embodiments, R.sup.d is C.sub.1-6 heteroaliphatic. In some embodiments, R.sup.d is 6-10-membered aryl. In some embodiments, R.sup.d is arylalkyl. In some embodiments, R.sup.d is 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulfur. In some embodiments, R.sup.d is 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0267] In some embodiments, R.sup.2 is hydrogen. In some embodiments, R.sup.2 is a halogen. In some embodiments, R.sup.2 is OH. In some embodiments, R.sup.2 is OR. In some embodiments, R.sup.2 is OC(O)R.sup.4. In some embodiments, R.sup.2 is OC(O)OR.sup.4. In some embodiments, R.sup.2 is OC(O)NHR.sup.4. In some embodiments, R.sup.2 is OC(O)NRR.sup.4. In some embodiments, R.sup.2 is OC(O)SR.sup.4. In some embodiments, R.sup.2 is NHC(O)R.sup.4. In some embodiments, R.sup.2 is NRC(O)R.sup.4. In some embodiments, R.sup.2 is NHC(O)OR.sup.4. In some embodiments, R.sup.2 is NHC(O)NHR.sup.4. In some embodiments, R.sup.2 is NHC(O)NRR.sup.4. In some embodiments, R.sup.2 is NHR.sup.4. In some embodiments, R.sup.2 is N(R.sup.4).sub.2. In some embodiments, R.sup.2 is NHR.sup.4. In some embodiments, R.sup.2 is NRR.sup.4. In some embodiments, R.sup.2 is N.sub.3. In some embodiments, R.sup.2 is C.sub.1-10 aliphatic. In some embodiments, R.sup.2 is C.sub.1-6 heteroaliphatic. In some embodiments, R.sup.2 is 6-10-membered aryl. In some embodiments, R.sup.2 is arylalkyl. In some embodiments, R.sup.2 is 5-10 membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, R.sup.2 is 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0268] In some embodiments, R.sup.3 is hydrogen. In some embodiments, R.sup.3 is a halogen. In some embodiments, R.sup.3 is CH.sub.2OR.sup.1. In some embodiments, R.sup.3 is an acyl. In some embodiments, R.sup.3 is C.sub.1-10 aliphatic. In some embodiments, R.sup.3 is C.sub.1-6 heteroaliphatic. In some embodiments, R.sup.3 is 6-10-membered aryl. In some embodiments, R.sup.3 is arylalkyl. In some embodiments, R.sup.3 is 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, R.sup.3 is 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0269] In some embodiments, R.sup.4 is -T-R.sup.z. In some embodiments, R.sup.4 is —C(O)-T-R.sup.z. In some embodiments, R.sup.4 is —NH-T-R.sup.z. In some embodiments, R.sup.4 is —O-T-R.sup.z. In some embodiments, R.sup.4 is —S-T-R.sup.z. In some embodiments, R.sup.4 is —C(O)NH-T-R.sup.z. In some embodiments, R.sup.4 is C(O)O-T-R.sup.z. In some embodiments, R.sup.4 is C(O)S-T-R.sup.z. In some embodiments, R.sup.4 is C(O)NH-T-O-T-R.sup.z. In some embodiments, R.sup.4 is —O-T-R.sup.z. In some embodiments, R.sup.4 is -T-O-T-R.sup.z. In some embodiments, R.sup.4 is -T-S-T-R.sup.z. In some embodiments, R.sup.4 is
##STR00043##
[0270] In some embodiments, X is —O—. In some embodiments, X is —NR—. In some embodiments, X is T-R.sup.z.
[0271] In some embodiments, T is a covalent bond or a bivalent C.sub.1-26 saturated or unsaturated, straight or branched, aliphatic or heteroaliphatic chain.
[0272] In some embodiments, R.sup.z is hydrogen. In some embodiments, R.sup.z is a halogen. In some embodiments, R.sup.z is —OR. In some embodiments, R.sup.z is —OR.sup.x. In some embodiments, R.sup.z is —OR.sup.1. In some embodiments, R.sup.z is —OR.sup.1′. In some embodiments, R.sup.z is —SR. In some embodiments, R.sup.z is NR.sub.2. In some embodiments, R.sup.z is —C(O)OR. In some embodiments, R.sup.z is —C(O)R. In some embodiments, R.sup.z is —NHC(O)R. In some embodiments, R.sup.z is —NHC(O)OR. In some embodiments, R.sup.z is NC(O)OR. In some embodiments, R.sup.z is an acyl. In some embodiments, R.sup.z is arylalkyl. In some embodiments, R.sup.z is heteroarylalkyl. In some embodiments, R.sup.z is C.sub.1-6 aliphatic. In some embodiments, R.sup.z is 6-10-membered aryl. In some embodiments, R.sup.z is 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.z is 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0273] In some embodiments, R.sup.x is hydrogen. In some embodiments, R.sup.x is an oxygen protecting group. In some embodiments, R.sup.x is an alkyl ethers. In some embodiments, R.sup.x is a benzyl ether. In some embodiments, R.sup.x is silyl ether. In some embodiments, R.sup.x is an acetal. In some embodiments, R.sup.x is ketal. In some embodiments, R.sup.x is ester. In some embodiments, R.sup.x is carbamate. In some embodiments, R.sup.x is carbonate.
[0274] In some embodiments, R.sup.y is —OH. In some embodiments, R.sup.y is —OR. In some embodiments, R.sup.y is a carboxyl protecting group. In some embodiments, R.sup.y is an ester. In some embodiments, R.sup.y is an amide. In some embodiments, R.sup.y is a hydrazide.
[0275] In some embodiments, R.sup.s is
##STR00044##
[0276] In some embodiments, R.sup.x′ is optionally substituted 6-10-membered aryl. In some embodiments, R.sup.x′ is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.x′ is optionally substituted or C.sub.1-6 heteroaliphatic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, two R.sup.x′ are taken together to form a 5-7-membered heterocyclic ring having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0277] In some embodiments, R is hydrogen. In some embodiments, R is an acyl. In some embodiments, R is arylalkyl. In some embodiments, R is 6-10-membered aryl. In some embodiments, R is C.sub.1-6 aliphatic. In some embodiments, R is C.sub.1-6 heteroaliphatic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, two R on the same nitrogen atom are taken with the nitrogen atom to form a 4-7-membered heterocyclic ring having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0278] In some embodiments, R.sup.1′ has the same embodiments as R.sup.1. Exemplary compounds of Formula I are set forth in Table 1 below:
TABLE-US-00001 TABLE 1 EXEMPLARY COMPOUNDS OF FORMULA I
[0279] It will be appreciated that it is not an object of the present subject matter to claim compounds disclosed in the prior art that are the result of isolation or degradation studies on naturally occurring prosapogenins or saponins.
Synthesis of Compounds
[0280] As described in U.S. Ser. No. 12/420,803, issued as U.S. Pat. No. 8,283,456 (and its parent/child U.S. applications and publications), the synthesis of QS-21 and at least some of its analogues can be carried out in part by obtaining semi-purified abstract from Quillaja saponaria (commercially available as Quil-A, Accurate Chemical and Scientific Corporation, Westbury, N.Y.) comprising a mixture of at least 50 distinct saponin species (van Setten, D. C.; Vandewerken, G.; Zomer, G.; Kersten, G. F. A. Rapid Commun. Mass Spectrom. 1995, 9, 660-666). Many of said saponin species include a triterpene-trisaccharide substructure as found in immunologically-active Quillaja saponins such as QS-21 and QS-7. Exposing these saponin species to base hydrolysis affords a mixture enriched with prosapogenins A, B, and C (shown below).
##STR00054##
[0281] U.S. Ser. No. 12/420,803, issued as U.S. Pat. No. 8,283,456 (and its parent/child U.S. applications and publications) presents a strategy that allows for the facile separation of derivatized prosapogenins A, B, and C via silica gel chromatography. It will be appreciated that some embodiments of the present application may be synthesized in part using the methods described in U.S. Ser. No. 12/420,803, issued as U.S. Pat. No. 8,283,456 (and its parent/child U.S. applications and publications), particularly the methods relating to facile separation of derivatized prosapogenins A, B, and C. In one aspect, separated derivatized prosapogenins A, B, and/or C may then be used to synthesize QS-21 or analogs thereof using the methods described herein.
[0282] In one embodiment, the present application provides semi-synthetic methods for synthesizing QS-7, QS-21, and related analogs, the method comprising coupling a triterpene compound with a compound comprising a saccharide to form a compound of Formula I or of Formula II. In some embodiments, the method comprises the steps of: [0283] (a) Providing a compound of Formula III:
##STR00055## [0284] wherein: [0285] is a single or double bond; [0286] Y′ is hydrogen, halogen, alkyl, aryl, OR, OR.sup.y, OH, NR.sub.2, NR.sub.3.sup.+, NHR, NH.sub.2, SR, or NROR; [0287] W is Me, —CHO, —CH.sub.2OR.sup.x, —C(O)R.sup.y, or
##STR00056## [0288] V is hydrogen or —OR.sup.x; [0289] R.sup.y is —OH, or a carboxyl protecting group selected from the group consisting of ester, amides, and hydrazides; [0290] each occurrence of R.sup.x′ is independently an optionally substituted group selected from 6-10-membered aryl, C.sub.1-6 aliphatic, or C.sub.1-6 heteroaliphatic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; or: [0291] two R.sup.x′ are taken together to form a 5-7-membered heterocyclic ring having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0292] each occurrence of R is independently hydrogen, an optionally substituted group selected from acyl, arylalkyl, 6-10-membered aryl, C.sub.1-12 aliphatic, or C.sub.1-12 heteroaliphatic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0293] each occurrence of R.sup.x is independently hydrogen or an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, and carbonates; [0294] (b) treating said compound of Formula III under suitable conditions with a compound of formula V:
LG-Z (V) [0295] wherein: [0296] Z is hydrogen; a cyclic or acyclic, optionally substituted moiety selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, arylalkyl, and heteroaryl; or a carbohydrate domain having the structure:
##STR00057## [0297] wherein: [0298] each occurrence of R1 is Rx or a carbohydrate domain having the structure:
##STR00058## [0299] wherein: [0300] each occurrence of a, b, and c is independently 0, 1, or 2; [0301] d is an integer from 1-5, wherein each d bracketed structure may be the same or different; with the proviso that the d bracketed structure represents a furanose or a pyranose moiety, and the sum of b and c is 1 or 2; [0302] R.sup.0 is hydrogen; an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates; or an optionally substituted moiety selected from the group consisting of acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10 membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 4-7 membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0303] each occurrence of R.sup.a, R.sup.b, R.sup.c, and R.sup.d is independently hydrogen, halogen, OH, OR, OR.sup.x, NR.sub.2, NHCOR, or an optionally substituted group selected from acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulfur; 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0304] R.sup.2 is hydrogen, halogen, OH, OR, OC(O)R.sup.4, OC(O)OR.sup.4, OC(O)NHR.sup.4, OC(O)NRR.sup.4, OC(O)SR.sup.4, NHC(O)R.sup.4, NRC(O)R.sup.4, NHC(O)OR.sup.4, NHC(O)NHR.sup.4, NHC(O)NRR.sup.4, NHR.sup.4, N(R.sup.4).sub.2, NHR.sup.4, NRR.sup.4, N.sub.3, or an optionally substituted group selected from C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10 membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0305] R.sup.3 is hydrogen, halogen, CH.sub.2OR.sup.1, or an optionally substituted group selected from the group consisting of acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, [0306] R.sup.4 is -T-R.sup.z, —C(O)-T-R.sup.z, —NH-T-R.sup.z, —O-T-R.sup.z, —S-T-R.sup.z, —C(O)NH-T-R.sup.z, C(O)O-T-R.sup.z, C(O)S-T-R.sup.z, C(O)NH-T-O-T-R.sup.z, —O-T-R.sup.z, -T-O-T-R.sup.z, -T-S-T-R.sup.z, or
##STR00059## [0307] wherein [0308] X is —O—, —NR—, or T-R.sup.z; [0309] T is a covalent bond or a bivalent C.sub.1-26 saturated or unsaturated, straight or branched, aliphatic or heteroaliphatic chain; and [0310] R.sup.z is hydrogen, halogen, —OR, —OR.sup.x, —OR.sup.1, —SR, NR.sub.2, —C(O)OR, —C(O)R, —NHC(O)R, —NHC(O)OR, NC(O)OR, or an optionally substituted group selected from acyl, arylalkyl, heteroarylalkyl, C.sub.1-6 aliphatic, 6-10-membered aryl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0311] each occurrence of R.sup.x is as defined for compounds of formula III; and [0312] LG is a suitable leaving group selected from the group consisting of halogen, imidate, alkoxy, sulphonyloxy, optionally substituted alkylsulphonyl, optionally substituted alkenylsulfonyl, optionally substituted arylsulfonyl, and diazonium moieties; [0313] (c) to give a compound of Formula I as described herein.
In some embodiments, the method comprises the steps of: [0314] (a) Providing a compound of Formula IV:
##STR00060## [0315] wherein: [0316] is a single or double bond; [0317] Y′ is hydrogen, halogen, alkyl, aryl, OR, OR.sup.y, OH, NR.sub.2, NR.sub.3.sup.+, NHR, NH.sub.2, SR, or NROR; [0318] W is Me, —CHO, —CH.sub.2OR.sup.x, —C(O)R.sup.y, or
##STR00061## [0319] V is hydrogen or —OR.sup.x; [0320] R.sup.y is —OH, or a carboxyl protecting group selected from the group consisting of ester, amides, and hydrazides; [0321] R.sup.s is
##STR00062## [0322] each occurrence of R.sup.x′ is independently an optionally substituted group selected from 6-10-membered aryl, C.sub.1-6 aliphatic, or C.sub.1-6 heteroaliphatic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; or: [0323] two R.sup.x′ are taken together to form a 5-7-membered heterocyclic ring having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0324] each occurrence of R is independently hydrogen, an optionally substituted group selected from acyl, arylalkyl, 6-10-membered aryl, C.sub.1-12 aliphatic, or C.sub.1-12 heteroaliphatic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0325] each occurrence of R.sup.x is independently hydrogen or an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, and carbonates; [0326] (b) treating said compound of Formula IV under suitable conditions with a compound of formula V:
LG-Z (V) [0327] wherein: [0328] Z is hydrogen; a cyclic or acyclic, optionally substituted moiety selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, arylalkyl, and heteroaryl; or a carbohydrate domain having the structure:
##STR00063## [0329] wherein: [0330] each occurrence of R1 is Rx or a carbohydrate domain having the structure:
##STR00064## [0331] wherein: [0332] each occurrence of a, b, and c is independently 0, 1, or 2; [0333] d is an integer from 1-5, wherein each d bracketed structure may be the same or different; with the proviso that the d bracketed structure represents a furanose or a pyranose moiety, and the sum of b and c is 1 or 2; [0334] R.sup.0 is hydrogen; an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates; or an optionally substituted moiety selected from the group consisting of acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10 membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 4-7 membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0335] each occurrence of R.sup.a, R.sup.b, R.sup.c, and R.sup.d is independently hydrogen, halogen, OH, OR, OR.sup.x, NR.sub.2, NHCOR, or an optionally substituted group selected from acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulfur; 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0336] R.sup.2 is hydrogen, halogen, OH, OR, OC(O)R.sup.4, OC(O)OR.sup.4, OC(O)NHR.sup.4, OC(O)NRR.sup.4, OC(O)SR.sup.4, NHC(O)R.sup.4, NRC(O)R.sup.4, NHC(O)OR.sup.4, NHC(O)NHR.sup.4, NHC(O)NRR.sup.4, NHR.sup.4, N(R.sup.4).sub.2, NHR.sup.4, NRR.sup.4, N.sub.3, or an optionally substituted group selected from C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10 membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0337] R.sup.3 is hydrogen, halogen, CH.sub.2OR.sup.1, or an optionally substituted group selected from the group consisting of acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, [0338] R.sup.4 is -T-R.sup.z, —C(O)-T-R.sup.z, —NH-T-R.sup.z, —O-T-R.sup.z, —S-T-R.sup.z, —C(O)NH-T-R.sup.z, C(O)O-T-R.sup.z, C(O)S-T-R.sup.z, C(O)NH-T-O-T-R.sup.z, —O-T-R.sup.z, -T-O-T-R.sup.z, -T-S-T-R.sup.z, or
##STR00065## [0339] wherein [0340] X is —O—, —NR—, or T-R.sup.z; [0341] T is a covalent bond or a bivalent C.sub.1-26 saturated or unsaturated, straight or branched, aliphatic or heteroaliphatic chain; and [0342] R.sup.z is hydrogen, halogen, —OR, —OR.sup.x, —OR.sup.1, —SR, NR.sub.2, —C(O)OR, —C(O)R, —NHC(O)R, —NHC(O)OR, NC(O)OR, or an optionally substituted group selected from acyl, arylalkyl, heteroarylalkyl, C.sub.1-6 aliphatic, 6-10-membered aryl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0343] each occurrence of R.sup.x is as defined for compounds of formula IV; and [0344] LG is a suitable leaving group selected from the group consisting of halogen, imidate, alkoxy, sulphonyloxy, optionally substituted alkylsulphonyl, optionally substituted alkenylsulfonyl, optionally substituted arylsulfonyl, and diazonium moieties; [0345] (c) to give a compound of formula II as described herein.
[0346] In another aspect, the present application provides a synthesis method comprising: [0347] (a) providing a compound of Formula III:
##STR00066## [0348] wherein: [0349] is a single or double bond; [0350] Y′ is hydrogen, halogen, alkyl, aryl, OR, OR.sup.y, OH, NR.sub.2, NR.sub.3.sup.+, NHR, NH.sub.2, SR, or NROR; [0351] W —CHO; [0352] V —OR.sup.x; [0353] R.sup.x is independently hydrogen or an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates; [0354] (b) treating said compound of Formula III under suitable conditions with a compound of formula V:
LG-Z (V) [0355] wherein: [0356] Z is a carbohydrate domain having the structure:
##STR00067## [0357] wherein: [0358] R.sup.1 is independently H or
##STR00068## [0359] R.sup.2 is NHR.sup.4; [0360] R.sup.3 is CH.sub.2OH; and [0361] R.sup.4 is -T-R.sup.z, —C(O)-T-R.sup.z, —NH-T-R.sup.z, —O-T-R.sup.z, —S-T-R.sup.z, —C(O)NH-T-R.sup.z, C(O)O-T-R.sup.z, C(O)S-T-R.sup.z, C(O)NH-T-O-T-R.sup.z, —O-T-R.sup.z, -T-O-T-R.sup.z, -T-S-T-R.sup.z, or
##STR00069## [0362] wherein: [0363] X is —O—, —NR—, or T-R.sup.z; [0364] T is a covalent bond or a bivalent C.sub.1-26 saturated or unsaturated, straight or branched, aliphatic or heteroaliphatic chain; and [0365] R.sup.z is hydrogen, halogen, —OR, —OR.sup.x, —OR.sup.1, —SR, NR.sub.2, —C(O)OR, —C(O)R, —NHC(O)R, —NHC(O)OR, NC(O)OR, or an optionally substituted group selected from acyl, arylalkyl, heteroarylalkyl, C.sub.1-6 aliphatic, 6-10-membered aryl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0366] (c) to give a compound of Formula I as described herein.
[0367] In another aspect, the present application provides a method of synthesizing a compound of Formula I, or an intermediate thereof, comprising the following steps: [0368] (a) providing a compound of Formula III:
##STR00070## [0369] wherein: [0370] is a single or double bond; [0371] Y′ is hydrogen, halogen, alkyl, aryl, OR, OR.sup.y, OH, NR.sub.2, NR.sub.3.sup.+, NHR, NH.sub.2, SR, or NROR; [0372] W —CHO; [0373] V —OH; [0374] wherein one or more substituents of the compound of Formula III are optionally protected; [0375] (b) reacting the compound of Formula III with a compound of Formula X:
##STR00071## [0376] wherein: [0377] R.sup.H is a halogen; [0378] R.sup.2 is hydrogen, N.sub.3, NH.sub.2, halogen, OH, OR, OC(O)R.sup.4, OC(O)OR.sup.4, OC(O)NHR.sup.4, OC(O)NRR.sup.4, OC(O)SR.sup.4, NHC(O)R.sup.4, NRC(O)R.sup.4, NHC(O)OR.sup.4, NHC(O)NHR.sup.4, NHC(O)NRR.sup.4, NHR.sup.4, N(R.sup.4).sub.2, NHR.sup.4, NRR.sup.4, N.sub.3, or an optionally substituted group selected from C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10 membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0379] R.sup.4 is -T-R.sup.z, —C(O)-T-R.sup.z, —NH-T-R.sup.z, —O-T-R.sup.z, —S-T-R.sup.z, —C(O)NH-T-R.sup.z, C(O)O-T-R.sup.z, C(O)S-T-R.sup.z, C(O)NH-T-O-T-R.sup.z, —O-T-R.sup.z, -T-O-T-R.sup.z, -T-S-T-R.sup.z, or
##STR00072## [0380] wherein: [0381] X is —O—, —NR—, or T-R.sup.z; [0382] T is a covalent bond or a bivalent C.sub.1-26 saturated or unsaturated, straight or branched, aliphatic or heteroaliphatic chain; [0383] R.sup.z is hydrogen, halogen, —OR, —OR.sup.x, —OR.sup.1′, —SR, NR.sub.2, —C(O)OR, —C(O)R, —NHC(O)R, —NHC(O)OR, NC(O)OR, or an optionally substituted group selected from acyl, arylalkyl, heteroarylalkyl, C.sub.1-6 aliphatic, 6-10-membered aryl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0384] R.sup.x is independently hydrogen or an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates; and [0385] R is independently hydrogen, an optionally substituted group selected from acyl, arylalkyl, 6-10-membered aryl, C.sub.1-6 aliphatic, or C.sub.1-6 heteroaliphatic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, or: [0386] two R on the same nitrogen atom are taken with the nitrogen atom to form a 4-7-membered heterocyclic ring having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0387] R.sup.1′ is R.sup.x or a carbohydrate domain having the structure:
##STR00073## [0388] wherein: [0389] each occurrence of a, b, and c is independently 0, 1, or 2; [0390] d is an integer from 1-5, wherein each d bracketed structure may be the same or different; with the proviso that the d bracketed structure represents a furanose or a pyranose moiety, and the sum of b and c is 1 or 2; [0391] R.sup.0 is hydrogen; an oxygen protecting group selected from the group consisting of alkyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates; or an optionally substituted moiety selected from the group consisting of acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10 membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 4-7 membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; [0392] each occurrence of R.sup.a, R.sup.b, R.sup.c, and R.sup.d is independently hydrogen, halogen, OH, OR, OR.sup.x, NR.sub.2, NHCOR, or an optionally substituted group selected from acyl, C.sub.1-10 aliphatic, C.sub.1-6 heteroaliphatic, 6-10-membered aryl, arylalkyl, 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, sulfur; 4-7-membered heterocyclyl having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
[0393] In one embodiment, the compound of Formula X is:
##STR00074##
[0394] In one embodiment, the method includes reacting the product of step (b) or a further downstream product with R.sup.4—OH. In one embodiment, the method includes reacting the product of step (b) or a compound obtained after modifying the product of step (b) with R.sup.4—OH. In one embodiment, the method includes reacting the product of step (b) or a compound obtained after modifying the product of step (b) with R.sup.4—OH. In one embodiment, the method includes reacting the product of step (b) or an intermediate with R.sup.4—OH. In one embodiment, R.sup.4—OH is HO—C(O)—(CH.sub.2).sub.10—C(O)—OR.sup.x. In one embodiment, Rx is H. In one embodiment, R.sup.x is Bn.
[0395] In another aspect, the present application provides a method of synthesizing a compound of Formula I, or an intermediate thereof, comprising at least one of the following steps:
##STR00075## ##STR00076##
[0396] In another aspect, the present application discloses a synthesis route for Compound I-4 (TQL-1055/TiterQuil-1-0-5-5), as shown, for example, in
[0397] In another aspect of the subject matter, synthesis of QS-21, QS-7, and/or analogs of these compounds may be undertaken by using one or more of the methods disclosed in the examples, including examples 1-10, described in this application. Although the synthesis of several compounds is disclosed in these examples, one of ordinary skill in the art will appreciate that these methods may be modified or adapted according to the knowledge of one of ordinary skill in the art to obtain other molecules.
[0398] In another aspect, the present application also includes methods for obtaining the compounds according the present application comprising providing a compound according to the application and a second substance, and subsequently purifying the compound of the application by removing at least a portion of the second substance.
Adjuvants
[0399] Most protein and glycoprotein antigens are poorly immunogenic or non-immunogenic when administered alone. Strong adaptive immune responses to such antigens often requires the use of adjuvants. Immune adjuvants are substances that, when administered to a subject, increase the immune response to an antigen or enhance certain activities of cells from the immune system. An adjuvant may also allow the use of a lower dose of antigen to achieve a useful immune response in a subject.
[0400] Common adjuvants include alum, Freund's adjuvant (an oil-in-water emulsion with dead mycobacteria), Freund's adjuvant with MDP (an oil-in-water emulsion with muramyl dipeptide, MDP, a constituent of mycobacteria), alum plus Bordetella pertussis (aluminum hydroxide gel with killed B. pertussis). Such adjuvants are thought to act by delaying the release of antigens and enhancing uptake by macrophages. Immune stimulatory complexes (ISCOMs) are open cage-like complexes typically with a diameter of about 40 nm that are built up by cholesterol, lipid, immunogen, and saponin such as Quil-A (a Quillaja saponin extract). ISCOMs deliver antigen to the cytosol, and have been demonstrated to promote antibody response and induction of T helper cell as well as cytotoxic T lymphocyte responses in a variety of experimental animal models.
[0401] Natural saponin adjuvant QS-21 is far more potent than currently used adjuvants, like alum. QS-21's superiority over more than 20 other adjuvants tested in preclinical models and over 7 other adjuvants used in the clinic has been demonstrated. Thus, QS-21 has been widely used despite its three major liabilities: dose limiting toxicity, poor stability, and the limited availability of quality product.
[0402] Use of QS-21 as an adjuvant has been associated with notable adverse biological effects. In humans, QS-21 has displayed both local and systemic toxicity. Maximum doses for cancer patients are 100-150 μg and for healthy patients are typically 50 μg (an immunology suboptimal dose). As a result, clinical success of non-cancer vaccines depends upon the identification of novel, potent adjuvants that are more tolerable.
[0403] The present application encompasses the recognition that synthetic access to and structural modification of QS-21 and related Quillaja saponins may afford compounds with high adjuvant potency and low toxicity, as well as having more stability and being more cost effective.
Vaccines
[0404] Compositions in this application are useful as vaccines to induce active immunity towards antigens in subjects. Any animal that may experience the beneficial effects of the compositions of the present application is within the scope of subjects that may be treated. In some embodiments, the subjects are mammals. In some embodiments, the subjects are humans.
[0405] The vaccines of the present application may be used to confer resistance to infection by either passive or active immunization. When the vaccines of the present application are used to confer resistance through active immunization, a vaccine of the present application is administered to an animal to elicit a protective immune response which either prevents or attenuates a proliferative or infectious disease. When the vaccines of the present application are used to confer resistance to infection through passive immunization, the vaccine is provided to a host animal (e.g., human, dog, or mouse), and the antisera elicited by this vaccine is recovered and directly provided to a recipient suspected of having an infection or disease or exposed to a causative organism.
[0406] The present application thus concerns and provides a means for preventing or attenuating a proliferative disease resulting from organisms which have antigens that are recognized and bound by antisera produced in response to the immunogenic antigens included in vaccines of the present application. As used herein, a vaccine is said to prevent or attenuate a disease if its administration to an animal results either in the total or partial attenuation (i.e., suppression) of a symptom or condition of the disease, or in the total or partial immunity of the animal to the disease.
[0407] The administration of the vaccine (or the antisera which it elicits) may be for either a “prophylactic” or “therapeutic” purpose. When provided prophylactically, the vaccine(s) are provided in advance of any symptoms of proliferative disease. The prophylactic administration of the vaccine(s) serves to prevent or attenuate any subsequent presentation of the disease. When provided therapeutically, the vaccine(s) is provided upon or after the detection of symptoms which indicate that an animal may be infected with a pathogen. The therapeutic administration of the vaccine(s) serves to attenuate any actual disease presentation. Thus, the vaccines may be provided either prior to the onset of disease proliferation (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual proliferation.
[0408] Thus, in one aspect the present application provides vaccines comprising an antigen associated with Hepatitis B, pneumococcus, diphtheria, tetanus, pertussis, or Lyme disease including the closely related spirochetes of the genus Borrelia such as, B. burgdorferi, B. garinii, B. afzelli, and B. japonica.
[0409] One of ordinary skill in the art will appreciate that vaccines may optionally include a pharmaceutically acceptable excipient or carrier. Thus, according to another aspect, provided vaccines may comprise one or more antigens that are optionally conjugated to a pharmaceutically acceptable excipient or carrier. In some embodiments, said one or more antigens are conjugated covalently to a pharmaceutically acceptable excipient. In other embodiments, said one or more antigens are non-covalently associated with a pharmaceutically acceptable excipient.
[0410] As described above, adjuvants may be used to increase the immune response to an antigen. According to the present application, provided vaccines may be used to invoke an immune response when administered to a subject. In certain embodiments, an immune response to an antigen may be potentiated by administering to a subject a provided vaccine in an effective amount to potentiate the immune response of said subject to said antigen.
Formulations
[0411] The compounds of the present application may be combined with a pharmaceutically acceptable excipient to form a pharmaceutical composition. In certain embodiments, formulations of the present application include injectable formulations. In certain embodiments, the pharmaceutical composition includes a pharmaceutically acceptable amount of a compound of the present application. In certain embodiments, the compounds of the application and an antigen form an active ingredient. In certain embodiments, the compound of the present application alone forms an active ingredient. The amount of active ingredient(s) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, and the particular mode of administration. The amount of active ingredient(s) that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%, or from about 1% to 99%, preferably from 10% to 90%, 20% to 80%, 30% to 70%, 40% to 60%, 45% to 55%, or about 50%.
[0412] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
[0413] Non-limiting examples of pharmaceutically-acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
[0414] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
[0415] Non-limiting examples of suitable aqueous and nonaqueous carriers, which may be employed in the pharmaceutical compositions of the present application include water, alcohols (including but not limited to methanol, ethanol, butanol, etc.), polyols (including but not limited to glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
[0416] These compositions may also contain additives such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
[0417] In some cases, in order to prolong the effect of a formulation, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which in turn, may depend upon crystal size and crystalline form.
[0418] Regardless of the route of administration selected, the compounds of the present application, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present application, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
[0419] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present application may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
[0420] The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present application employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
[0421] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the present application employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and then gradually increasing the dosage until the desired effect is achieved.
[0422] In some embodiments, a compound or pharmaceutical composition of the present application is provided to a subject chronically. Chronic treatments include any form of repeated administration for an extended period of time, such as repeated administrations for one or more months, between a month and a year, one or more years, or longer. In many embodiments, a chronic treatment involves administering a compound or pharmaceutical composition of the present application repeatedly over the life of the subject. Preferred chronic treatments involve regular administrations, for example one or more times a day, one or more times a week, or one or more times a month. In general, a suitable dose, such as a daily dose of a compound of the present application, will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
[0423] Generally, doses of the compounds of the present application for a patient, when used for the indicated effects, will range from about 0.0001 to about 100 mg per kg of body weight per day. Preferably the daily dosage will range from 0.001 to 50 mg of compound per kg of body weight, and even more preferably from 0.01 to 10 mg of compound per kg of body weight. However, lower or higher doses can be used. In some embodiments, the dose administered to a subject may be modified as the physiology of the subject changes due to age, disease progression, weight, or other factors.
[0424] In some embodiments, provided adjuvant compounds of the present application are administered as pharmaceutical compositions or vaccines. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 1-2000 μg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 1-1000 μg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 1-500 μg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 1-250 μg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 100-1000 μg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 100-500 μg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 100-200 μg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 250-500 μg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 10-1000 μg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 500-1000 μg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 50-250 μg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 50-500 μg.
[0425] In some embodiments, provided adjuvant compounds of the present application are administered as pharmaceutical compositions or vaccines. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 1-2000 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 1-1000 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 1-500 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 1-250 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 100-1000 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 100-500 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 100-200 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 250-500 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 10-1000 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 500-1000 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 50-250 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 50-500 mg. In certain embodiments, it is contemplated that the amount of adjuvant compound administered will be 0.01-215.4 mg.
[0426] In certain embodiments, it is contemplated that the amount of adjuvant administered will be 1000-5000 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 1000-4000 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 1000-3000 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 1000-2000 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 2000-5000 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 2000-4000 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 2000-3000 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 3000-5000 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 3000-4000 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 4000-5000 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 1-500 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 500-1000 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 1000-1500 μg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 1 mg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 2 mg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 3 mg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 4 mg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 5 mg/kg. In certain embodiments, it is contemplated that the amount of adjuvant administered will be 0.0029-5 mg/kg. In certain embodiments, the amount of adjuvant administered in females is less than the amount of adjuvant administered in males. In certain embodiments, the amount of adjuvant administered to infants is less than the amount of adjuvant administered to adults. In certain embodiments, the amount of adjuvant administered to pediatric recipients is less than the amount of adjuvant administered to adults. In certain embodiments, the amount of adjuvant administered to immunocompromised recipients is more than the amount of adjuvant administered to healthy recipients. In certain embodiments, the amount of adjuvant administered to elderly recipients is more than the amount of adjuvant administered to non-elderly recipients.
[0427] If desired, the effective dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
[0428] While it is possible for a compound of the present application to be administered alone, in certain embodiments the compound is administered as a pharmaceutical formulation or composition as described above.
[0429] The compounds according to the present application may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.
[0430] The present application provides kits comprising pharmaceutical formulations or compositions of a compound of the present application. In certain embodiments, such kits include the combination of a compound of formulae I and/or II and an antigen. The agents may be packaged separately or together. The kit optionally includes instructions for prescribing the medication. In certain embodiments, the kit includes multiple doses of each agent. The kit may include sufficient quantities of each component to treat one or more subject for a week, two weeks, three weeks, four weeks, or multiple months. The kit may include a full cycle of immunotherapy. In some embodiments, the kit includes a vaccine comprising one or more bacterial or viral-associated antigens, and one or more provided compounds.
EXAMPLES
[0431] The numbering associated with compounds in the Examples 1-9 is not meant to correspond with other formula or compound numbering appearing throughout the remainder of the application, including the Figures, the claims, or Example 10.
Example 1: Isolation and Selective Protection of Quillaic Acid Triterpene
[0432] ##STR00077##
Part A: Isolation of Quillaic Acid Triterpene 9 from Quil-A.
[0433] 1. In a 250-mL round-bottomed flask equipped with a reflux
condenser, Quil A (5 g) is suspended in distilled water (25 mL)
and concentrated HCl (17 mL) is added.
[0434] 2. The mixture is slowly heated to reflux for 7 h (Heating should be done slowly to avoid a foam-over when approaching reflux), then removed from heat, and filtered through filter paper. The dark brown solid is washed with hot (˜65° C.) distilled water (2×50 mL), collected and dried under high vacuum overnight.
[0435] 3. The dry solid is placed into a Soxhlet thimble and subjected to continuous extraction with diethyl ether (200 mL) for 24 h.
[0436] 4. The ether solution is concentrated, the residue is dissolved in MeOH (20 mL), and activated charcoal (˜5 g) is added. The mixture is filtered through celite, the solids are washed with MeOH (50 mL), and the solvent is removed by rotary evaporation.
[0437] 5. The resulting residue is purified by silica gel chromatography (CHCl 3/MeOH, 30:1 to 20:1 to 10:1) to afford the quillaic acid triterpene 9 (˜0.5 g, ˜10% mass yield) (Quillaic acid triterpene product is ˜80% pure. High purity is achieved after allylation reaction.).
Part B: Synthesis of Quillaic Acid Allyl Ester 10 by Allylation of C28 Carboxylic Acid of Quillaic Acid.
[0438] 1. In a 50-mL round-bottomed flask, the quillaic acid triterpene 9 (100 mg, 0.20 mmol, 1.0 equiv.) is dissolved in DMF (5 mL) and the solution is cooled to 0° C.
[0439] 2. Potassium bicarbonate (205 mg, 2.05 mmol, 10 equiv.) and allyl bromide (23 μL, 0.27 mmol, 1.3 equiv.) are added and the mixture is stirred and allowed to warm to room temperature (rt) overnight.
[0440] 3. The reaction is diluted with water (25 mL) and extracted with hexanes/EtOAc (1:1) (3×15 mL). The organic extracts are combined, washed with brine (15 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated.
[0441] 4. Purification by silica gel chromatography (hexanes/EtOAc, 8:1 to 2:1) affords quillaic acid allyl ester 10 (77 mg, 71%) as a white solid.
Part C: Synthesis of Protected Quillaic Acid Triterpene 11 by Silylation of C3 and C16 Hydroxyl Groups of Quillaic Acid Alkyl Ester 10
[0442] 1. In a 25-mL modified Schlenk flask, quillaic allyl ester 10 (77 mg, 0.15 mmol, 1.0 equiv.) is dissolved in DCM (5 mL) and the solution is cooled to 0° C. 2,6-Lutidine (0.17 mL, 1.46 mmol, 10 equiv.) is added, followed by TESOTf (0.17 mL, 0.73 mmol, 5.0 equiv.) via gas-tight syringe, and the mixture is stirred while the ice bath is allowed to melt.
[0443] 2. The reaction progress is monitored by TLC using CHCl.sub.3/MeOH (10:1) as eluent. If the reaction is not complete after 3 h, more TESOTf (33 μL, 0.15 mmol, 1.0 equiv.) is added and the mixture is stirred until the reaction is complete.
[0444] 3. The reaction mixture is diluted with water (10 mL) and the aqueous phase is extracted with EtOAc (10 mL×3). The combined organic phases are dried (anhydrous Na.sub.2SO.sub.4), filtered, and concentrated.
[0445] 4. Purification by silica gel chromatography (hexanes/acetone, 1:0 to 10:1) yields the TES-protected quillaic allyl ester 11 (93 mg, 84%) as a white solid.
Part D: Synthesis of TES-Protected Quillaic Acid Triterpene 12 by Deallylation of Protected Quillaic Acid
[0446] 1. In a 10-mL round-bottomed flask, fully protected quillaic acid 11 (93 mg, 0.12 mmol, 1.0 equiv.) is dissolved in DCM (2 mL) and pyrrolidine (51 μL, 0.61 mmol, 5.0 equiv.) is added, followed by Pd(PPh 3) 4 (7.0 mg, 0.006 mmol, 0.05 equiv.).
[0447] 2. The reaction mixture is stirred for 15 min, then directly subjected to purification by silica gel chromatography (hexanes/EtOAc, 2:1), to afford TES-protected quillaic acid 12 (88 mg, >99%) as a white solid.
Example 2: Synthesis of Truncated Linear Oligosaccharide Domain
[0448] Part A: Synthesis of Selectively Protected Monosaccharide Precursor 2,3,4-Tri-O-Benzyl-D-Xylose 15 from D-Xylose
##STR00078##
[0449] 1. Step A: Synthesis of 1-O-allyl-D-xylose 13 by selective allylation of D-xylose. In a 500-mL round-bottomed flask, a solution of allyl alcohol (50 mL, 0.74 mol, 9.0 equiv.) and AcCl (12.7 mL, 0.17 mol, 2.1 equiv.) is cooled to −10° C., then solid D-xylose (12.3 g, 0.08 mol, 1.0 equiv.) is added.
[0450] 2. Once all xylose has been added, the cooling bath is removed and the reaction mixture is stirred for 19 h at rt.
[0451] 3. Solid NaHCO.sub.3 (25 g) is added, the mixture is filtered through a pad of celite, and the volatile materials are removed by rotary evaporation.
[0452] 4. The residue is passed through a plug of silica gel eluted with DCM/MeOH (9:1) and the eluate is concentrated to afford the anomeric allyl xylose 13 (11.5 g), which is used in the next step without further purification.
[0453] 5. Step B: Synthesis of 1-O-allyl-2,3,4-tri-O-benzyl-D-xylose 14 by benzylation of 1-O-allyl-D-xylose 13. In a 500-mL round-bottomed flask, allyl xylose 13 (11.5 g, 60.5 mmol, 1.0 equiv.) is dissolved in DMF (200 mL), then the solution is cooled to 0° C. Sodium hydride (60% dispersion in oil, 15.7 g, 0.39 mol, 6.5 equiv.) (Caution: sodium hydride reacts violently with water) is added and the reaction mixture is stirred for 10 min.
[0454] 6. Benzyl bromide (47 mL, 0.39 mol, 6.5 equiv.) is added dropwise at 0° C., and the resulting suspension is stirred at rt for 16 h.
[0455] 7. The reaction mixture is cooled to 0° C. and quenched by slow addition of MeOH (150 mL) followed by water (600 mL). The mixture is extracted with hexanes/EtOAc (1:1) (3×250 mL) and the combined organic layers are washed with water (100 mL), brine (100 mL), dried with anhydrous MgSO.sub.4, filtered, and concentrated.
[0456] 8. Purification by silica gel chromatography (hexanes/EtOAc, 9:1) affords the fully protected xylose 14 (23 g, 83%).
[0457] 9. Step C: Synthesis of selectively protected 2,3,4-tri-O-benzyl-D-xylose 15 by deallylation of 1-O-allyl-2,3,4-tri-O-benzyl-D-xylose 14. In a 100-mL round-bottomed flask covered in aluminum foil, PPh.sub.3 (3.4 g, 13 mmol, 1.2 equiv.) and Pd(OAc) 2 (0.45 g, 2.2 mmol, 0.2 equiv.) are dissolved in DCM/MeOH (1:1) (20 mL), then Et.sub.2NH (15.8 mL, 0.15 mol, 14.0 equiv.) is added.
[0458] 10. A solution of the fully protected xylose 14 (5.0 g, 10.9 mmol, 1.0 equiv.) in DCM (100 mL) is added by cannula transfer, and the reaction mixture is stirred at 30° C. for 18 h.
[0459] 11. The solution is passed through a plug of silica gel eluted with hexanes/EtOAc (1:1) and the eluate is concentrated.
[0460] 12. Purification by silica gel chromatography (hexanes/EtOAc, 8:2 to 7:3) affords 2,3,4,-tri-O-benzyl xylose 15 (4.1 g, 90%) as a mixture of anomers (α:β, 2:1).
Part B: Synthesis of Selectively Protected
[0461] Monosaccharide Precursor 1-O-Allyl-2,3-O-Isopropylidene-L-Rhamnose 16 from L-Rhamnose
##STR00079##
[0462] 1. In a 250-mL round-bottomed flask, a solution of allyl alcohol (34 mL, 0.50 mol, 9.0 equiv.) and AcCl (8.1 mL, 0.12 mol,
2.1 equiv.) is cooled at −10° C., then L-rhamnose monohydrate (10 g, 0.055 mol, 1.0 equiv.) is added.
[0463] 2. The mixture is stirred for 20 h at rt, neutralized with Et.sub.3N, and concentrated.
[0464] 3. The residue is dissolved in toluene and the solution is concentrated to remove allyl alcohol; this process is repeated two more times.
[0465] 4. The residual syrup is dissolved in dry acetone (75 mL), and DMP (27 mL, 0.22 mol, 4.0 equiv.) and pTsOH monohydrate (95 mg, 0.5 mmol, 0.01 equiv.) are added.
[0466] 5. The reaction mixture is stirred for 16 h at rt and Et 3 N is then added.
[0467] 6. The reaction mixture is concentrated and purified by silica gel chromatography (hexanes/EtOAc, 8:2) to afford 1-O-allyl-2,3-O-isopropylidene-α-L-rhamnose (16) (8.9 g, 66%) as a colorless oil.
Part C: Synthesis of Selectively Protected Monosaccharide Precursor 4-Azido-4-Deoxy-3,6-Di-O-Benzyl-1-Otriisopropylsilyl-D-Galactose 21 from D-Glucal
##STR00080##
[0468] 1. Step A: Synthesis of 3,6-di-O-benzoyl-4-O-mesyl-D-glucal 17 by selective protection of D-glucal. In a 500-mL round-bottomed flask, D-glucal (10.0 g, 67.1 mmol, 1.0 equiv.) is dissolved in pyridine (165 mL) and the solution is cooled to 0° C., then BzCl (17 mL, 0.15 mol, 2.2 equiv.) is added dropwise.
[0469] 2. The reaction mixture is stirred at 0° C. for 1.5 h, then MsCl (10.3 mL, 0.13 mol, 2.0 equiv.) is added. The reaction mixture is stirred for 0.5 h while allowing the ice bath warm to rt, then quenched by slow addition of MeOH (20 mL) at 0° C. (Caution: exothermic reaction).
[0470] 3. The mixture is concentrated and the residue is partitioned between EtOAc (200 mL) and water (200 mL). The organic layer is washed with water (100 mL), brine (100 mL), dried with anhydrous MgSO.sub.4, filtered, and concentrated.
[0471] 4. Purification by silica gel chromatography (hexanes/EtOAc,
8:2) affords 3,6-di-O-benzoyl-4-O-mesyl-D-glucal (17) (19.4 g, 67%) as a syrup.
[0472] 5. Step B: Synthesis of 4-azido-4-deoxy-3,6-di-O-benzoyl-D-galactal 18 by azide substitution of mesylate 17. In a 250 mL roundbottomed flask, the mesyl-glucal 17 (5.1 g, 11.8 mmol, 1.0 equiv.) is dissolved in toluene (55 mL), then sodium azide (Caution: sodium azide is a toxic, hazardous substance that should not be acidified to avoid poisonous, explosive hydrazoic acid (HN.sub.3). The reaction should be carried out behind a blast shield due to risk of explosion of sodium azide when heated near its decomposition temperature (300° C.)) (2.8 g, 43.3 mmol, 3.7 equiv.) is added, followed by Bu.sub.4NCl (7.1 g, 25.6 mmol, 2.2 equiv.), and the flask is equipped with a reflux condenser.
[0473] 6. The reaction mixture is heated to reflux (110° C.) for 20 h. The resulting brown suspension is washed with water (2×100 mL), dried with anhydrous MgSO.sub.4, filtered, and concentrated to give an orange oil.
[0474] 7. Purification by silica gel chromatography (hexanes/EtOAc, 19:1 to 8:2) provides 4-azido-4-deoxy-3,6-di-0-benzoyl-D-galactal (18) (2.9 g, 66%) as a light yellow oil.
[0475] 8. Step C: Synthesis of 4-azido-4-deoxy-3,6-di-O-benzyl-D-galactal 19 by saponification and benzylation of dibenzoate 18. In 250-mL round-bottomed flask, the benzoyl-protected azidogalactal 18 (2.9 g, 8.1 mmol, 1.0 equiv.) is dissolved in MeOH (40 mL) and the solution is cooled to 0° C.
[0476] 9. Sodium hydroxide (0.12 g, 2.9 mmol, 0.36 equiv.) is added and the reaction mixture is stirred for 14 h at rt.
[0477] 10. The reaction mixture is concentrated to afford a sticky tan solid, then evaporated again from toluene (7 mL) to remove trace solvent.
[0478] 11. DMF (40 mL) is added to the residue and the resulting brown suspension is cooled to 0° C. Sodium hydride (60% dispersion in mineral oil, 0.98 g, 24.4 mmol, 3.0 equiv.) (Caution: sodium hydride reacts violently with water) is added, followed by benzyl bromide (4.8 mL, 40.3 mmol, 5.0 equiv.), and the mixture is stirred at 0° C. for 3 h.
[0479] 12. The resulting orange suspension is stirred for another 16 h at rt, and the reaction is quenched with MeOH (20 mL), diluted with DCM (100 mL), and washed with water (100 mL).
[0480] 13. The aqueous layer is extracted with DCM (80 mL), and the combined organic layers are washed with water (100 mL), dried with anhydrous MgSO.sub.4, filtered, and concentrated.
[0481] 14. Purification by silica gel chromatography (hexanes/EtOAc, 9:1 to 4:1) affords 4-azido-4-deoxy-3,6-di-O-benzyl-D-galactal (19) (2.2 g, 78%) as a yellow oil.
[0482] 15. Step D: Synthesis of 4-azido-4-deoxy-3,6-di-O-benzyl-D-galactose 20 by dihydroxylation of galactal 19. The benzyl-protected azidogalactal 19 (5.8 g, 16.5 mmol, 1.0 equiv.) is dissolved in a mixture of water/THF/tBuOH (1:3:7) (400 mL), then OsO.sub.4 (2.5 wt % in tBuOH) (5.1 mL, 0.4 mmol, 0.025 equiv.) is added. NMO (50% in water) (10.2 mL, 44.5 mmol, 3.0 equiv.) is added in three portions (1.0 equiv. each) over 8 h.
[0483] 16. The reaction mixture is stirred at rt overnight, then quenched with saturated aqueous Na.sub.2SO.sub.3 solution (30 mL) and EtOAc (200 mL).
[0484] 17. After 5 min, the phases are separated and the aqueous layer is extracted with EtOAc (2×75 mL) and DCM (2×50 mL). The combined organic phases are dried over anhydrous sodium sulfate, filtered, and concentrated.
[0485] 18. Purification by silica gel chromatography (hexanes/EtOAc,
4:1 to 1:1) affords 4-azido-4-deoxy-3,6-di-O-benzyl-D-galactose (20) (5.5 g, 88%) as a colorless oil.
[0486] 19. Step E: Synthesis of 4-azido-4-deoxy-3,6-di-O-benzyl-1-O-triisopropylsilyl-D-galactose 21 by selective silylation of diol 20. In a 10-mL modified Schlenk flask, the galactose diol 20 (0.96 g, 2.5 mmol, 1.0 equiv.) is dissolved in DMF (2.5 mL), then imidazole (0.41 g, 6.0 mmol, 2.4 equiv.) and DMAP (29 mg, 0.24 mmol, 0.1 equiv.) are added.
[0487] 20. TIPSCl (0.63 mL, 3.0 mmol, 1.2 equiv.) is added and the reaction mixture is stirred for 19 h at rt.
[0488] 21. The yellow solution is concentrated and purified by silica gel chromatography (hexanes/EtOAc, 19:1 to 9:1) to afford 4-azido-4-deoxy-3,6-di-O-benzyl-1-O-triisopropylsilyl-D-galactose (21) (0.8 g, 59%) as a colorless oil.
Part D: Synthesis of Protected Xylose-Rhamnose Disaccharide Hemiacetal 23 ([2,3,4-Tri-O-Benzyl-β-D-Xylopyranosyl-(1.fwdarw.4)]2,3-di-O-isopropylidene-L-rhamnopyranose) from protected D-xylose 15 and protected L-rhamnose 16
##STR00081##
[0489] 1. Step A: Dehydrative glycosylation of protected rhamnose 16 with protected xylose 15 (22): In a 25-mL modified Schlenk flask, azeotropically dried 2,3,4-tri-O-benzyl xylose (15)
(52 mg, 0.12 mmol, 1.7 equiv.), Ph.sub.2SO (69 mg, 0.34 mmol, 4.9 equiv.), and TBP (85 mg, 0.34 mmol, 4.9 equiv.) are dissolved in DCM (2 mL), injected via glass syringe.
[0490] 2. The solution is cooled to −78° C., Tf.sub.2O (29 μL, 0.17 mmol, 2.4 equiv.) is added via gas-tight syringe, and the reaction mixture is stirred for 2 h at −78° C.
[0491] 3. A precooled solution of protected rhamnose 16 (17 mg, 70 μmol, 1.0 equiv.) in toluene (1 mL) is then cannula transferred from a flame dried, 10-mL modified Schlenk flask, then additional toluene (1 mL) is added to rinse the source flask and transferred to the reaction flask.
[0492] 4. The reaction mixture is stirred at −60° C. for 12 h, at −42° C. for 30 min, and finally at 0° C. for 2 min.
[0493] 5. The reaction is quenched by addition of Et 3 N (0.1 mL) at −42° C., diluted with DCM (90 mL) and transferred to a separatory funnel. The organic layer is washed with saturated aqueous NaHCO.sub.3 solution (30 mL) and the aqueous layer is extracted with DCM (2×80 mL). The organic phases are combined, dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to afford the crude product as a tan oil (160 mg).
[0494] 6. Purification by silica gel chromatography (hexanes/EtOAc, 50:1 to 25:1) affords 0-allyl [2,3,4-tri-O-benzyl-β-D-xylopyranosyl-(1.fwdarw.4)]-2,3-O-isopropylidene-L-rhamnopyranoside (22) as a clear oil (32.1 mg, 71% yield).
[0495] 7. Step B: Anomeric deallylation of protected xylose-rhamnose disaccharide (23): In a 5-mL pear-shaped Schlenk flask equipped with a triangular stir bar, PPh.sub.3 (13 mg, 51 μmol, 1.2 equiv.) and Pd(OAc).sub.2 (2.4 mg, 11 μmol, 0.25 equiv.) are placed. A solution of DCM/MeOH (1:1) (0.2 mL) is added via syringe followed by Et.sub.2NH (62 μL, 0.6 mmol, 14.0 equiv.), which results in a change from a clear yellow-orange to a bright yellow solution.
[0496] 8. Allyl-protected disaccharide 22 (29 mg, 43 μmol, 1.0 equiv.) dissolved in DCM (0.4 mL) is cannula transferred to the reaction Schlenk flask and the source flask is rinsed with additional DCM (0.2 mL) that is transferred to the reaction flask.
[0497] 9. The solution is degassed by performing three freeze-thaw pump cycles (This degassing technique involves freezing the solvent under liquid nitrogen, evacuating the headspace for 4-5 min, and letting the solvent thaw under static vacuum, thereby allowing any gas bubbles trapped in the solvent to escape into the headspace of the flask. After the last cycle, the flask is refilled with Ar.) and then stirred at 30° C. for 18 h, at which point the turbid solution turns clear, dark yellow.
[0498] 10. The reaction mixture is passed through a plug of silica gel eluted with hexanes/EtOAc (2:1, 50 mL) and the eluate is concentrated to afford the crude product as a bright yellow oil (29 mg).
[0499] 11. Purification by silica gel chromatography (hexanes/EtOAc, 2:1) affords disaccharide hemiacetal (23) as an inseparable mixture of anomers (α:β, 9:1) as a clear oil (25.9 mg, >99%).
Step E: Synthesis of Protected Xylose-Rhamnose-Azidogalactose Trisaccharide Imidate 26 (O-Trichloroacetimidoyl {[2,3,4-Tri-O-Benzyl-β-D-Xylopyranosyl-(1.fwdarw.4)]-2,3-Oisopropylidene-L-Rhamnopyranosyl-(1.fwdarw.2)}-4-Azido-4-Deoxy-3,6-O-Benzyl-β-D-Galactopyranoside)
##STR00082##
[0500] 1. Step A: Synthesis of protected xylose-rhamnose-azidogalactose trisaccharide 24 by dehydrative glycosylation of protected 4-azido-4-deoxygalactose 21 with protected xylose-rhamnose disaccharide 23 (24): In a 25-mL modified Schlenk flask, Ph.sub.2SO (171 mg, 0.85 mmol, 3.2 equiv.) is dissolved in DCM (3.2 mL). To this clear, colorless solution, Tf.sub.2O (76 μL, 0.45 mmol, 1.7 equiv.) is injected via gas-tight syringe at −78° C. After 10 s, the solution turns pink, then purple, and quickly dissipates back to a clear, colorless solution.
[0501] 2. A precooled solution of azeotropically dried disaccharide
hemiacetal 23 (185 mg, 0.30 mmol, 1.1 equiv.) in DCM (1 mL) is added to the reaction mixture at −42° C. via cannula from a flame-dried, 5-mL pear-shaped Schlenk flask; then additional DCM (1 mL) is added to rinse the source flask and transferred to the reaction flask.
[0502] 3. The reaction mixture is stirred at −42° C. for 15 min, then TBP (190 mg, 0.77 mmol, 3.0 equiv.) is added, and the mixture is
further stirred at −42° C. for 1 h.
[0503] 4. A precooled solution of protected 4-azido-4-deoxygalactose 21 (141 mg, 0.26 mmol, 1.0 equiv.) in DCM (1 mL) is added to the reaction mixture via cannula from a flame-dried, 5-mL pear-shaped Schlenk flask, at which point white fumes develop. Additional DCM (1 mL) is added to rinse the source flask and transferred to the reaction flask.
[0504] 5. The reaction mixture is stirred at −42° C. for 16.5 h and at 0° C. for 1 h, then concentrated.
[0505] 6. Purification by silica gel chromatography (hexanes/EtOAc, 99:1 to 50:1 to 6:1) gives a mixture of monosaccharide starting material (21) and trisaccharide product (24) as a yellow oil (460 mg). Additional purification of this mixture by silica gel chromatography (hexanes/EtOAc, 10:1 to 6:1) provides the protected trisaccharide 24 (231 mg, 79%) as a clear oil.
[0506] 7. Step B: Synthesis of trisaccharide hemiacetal 25 by anomeric desilylation of protected xylose-rhamnose-azidogalactose trisaccharide 24. In a 250-mL modified Schlenk flask, the protected trisaccharide 24 (575 mg, 0.51 mmol, 1.0 equiv.) is dissolved in THF (50 mL) and the solution is cooled to 0° C.
[0507] 8. A precooled (0° C.) solution of commercially available TBAF (1 M in THF) (0.76 mL, 0.76 mmol, 1.5 equiv.) and AcOH (35 μL, 0.61 mmol, 1.2 equiv.) in THF (50 mL) is added dropwise via cannula to the reaction flask over 50 min at 0° C.
[0508] 9. The reaction mixture is stirred for an additional 5 min at 0° C., then quenched by addition of saturated aqueous NaHCO.sub.3 solution (20 mL).
[0509] 10. The contents are transferred to a separatory funnel, EtOAc (125 mL) and brine (50 mL) are added, and the organic phase is separated. The aqueous layer is extracted with EtOAc (2×200 mL) and the combined organic phases are dried over anhydrous magnesium sulfate, filtered, and concentrated.
[0510] 11. The resulting oil is passed through a plug of silica gel eluted with EtOAc, and the eluate is concentrated to afford the trisaccharide hemiacetal 25 (402 mg, 82%) as a white foam, which is taken directly to the next step without further purification.
[0511] 12. Step C: Synthesis of protected xylose-rhamnose-azidogalactose trisaccharide trichloroacetimidate 26 by activation of protected xylose-rhamnose-azidogalactose trisaccharide 25. In a 100-mL round-bottomed flask, the hemiacetal 25 (200 mg, 0.21 mmol, 1.0 equiv.) is dissolved in DCM (32 mL) and the solution is cooled to 0° C.
[0512] 13. Cl.sub.3CCN (0.32 mL, 3.2 mmol, 1.6 equiv.) is added followed by DBU (0.1 mL, 0.67 mmol, 3.3 equiv.) and the reaction is allowed to warm to rt.
[0513] 14. After stirring for 13.5 h, the mixture is concentrated to afford an oil.
[0514] 15. Purification by silica gel chromatography (hexanes/EtOAc, 6:1 with 0.5 vol % Et.sub.3N) (In absence of Et 3 N, prolonged chromatography on silica gel when purifying glycosyl trichloroacetimidates leads to progressive hydrolysis of the product.) affords the linear trisaccharide imidate 26 (230 mg, >99%) as a yellow foam.
Example 3: Modular, Convergent Assembly of Saponin Domain Fragments
Part A: Synthesis of Protected Aminogalactose Saponin
[0515] ##STR00083##
[0516] 1. Step A: Synthesis of protected azidogalactose saponin 29 by glycosylation of protected quillaic acid 12 with protected xylose-rhanose-azidogalactose linear trisaccharide 26. In a 25-mL modified Schienk flask, the selectively protected quillaic acid triterpene 12 (38 mg, 49 μmol, 1.05 equiv.) and the trisaccharide imidate 26 (52 mg, 47 μmol, 1.0 equiv.) are azeotroped from toluene (3×1 mL) under high vacuum, then dissolved in DCM (7 mL) and powdered 4 Å MS (80 mg) is added to the solution.
[0517] 2. The mixture is stirred for 30 m at rt, then cooled to −42° C. Freshly distilled BF.sub.3OEt.sub.2 (1.2 μL, 9.0 μmol, 0.2 equiv.) is injected via gas-tight syringe and the reaction mixture is stirred for another 30 min at −42° C.
[0518] 3. The reaction is quenched by addition of Et.sub.3N (0.2 mL) and
the mixture is concentrated by rotary evaporation.
[0519] 4. Purification by silica gel chromatography (benzene with 0.5 vol % Et.sub.3N to benzene/EtOAc, 97:3) affords the triterpene-linear trisaccharide conjugate 29 (56 mg, 72%) as a white solid.
[0520] 5. Step B: Synthesis of protected aminogalactose saponin 30 by reduction of protected azidogalactose saponin 29. In a 50-mL modified Schlenk flask, PhSeSePh (187 mg, 0.6 mmol, 1.0 equiv.) is dissolved in THF (6 mL) and H.sub.3PO.sub.2 (50% in water) (0.72 mL, 6.6 mmol, 11 equiv.) is added via syringe.
[0521] 6. The yellow solution is heated at 40° C. for 1 h until it turns colorless.
[0522] 7. The reaction mixture is removed from the heat, diluted with benzene (6 mL) and distilled water (6 mL), and stirred vigorously for 5 min under Ar. The lower aqueous phase of the resulting biphasic suspension is removed by glass pipette and the remaining organic layer is dried over anhydrous sodium sulfate while stirring.
[0523] 8. This freshly prepared solution of PhSeH (˜1.1 mmol, 30 equiv.) is then cannula transferred under Ar to a 100-mL reaction Schlenk flask containing a solution of the azeotropically dried saponin azide 29 (62 mg, 37 μmol, 1.0 equiv.) in Et.sub.3N (28 mL). Upon addition, a white precipitate is formed and the solution becomes bright yellow.
[0524] 9. The reaction mixture is stirred for 8 h at 38° C., then concentrated to afford a yellow-white solid.
[0525] 10. Purification by silica gel chromatography (benzene/EtOAc, 90:10 to 85:15) affords the truncated saponin amine 30 (49 mg, 80%) as a glassy solid.
Part B: Synthesis of Protected Aminoacyl Saponin 32
[0526] ##STR00084##
[0527] 1. In a 10-mL pear-shaped Schlenk flask, 6-(Boc-amino) hexanoic acid (45.0 mg, 0.20 mmol, 11.5 equiv.) is dissolved in THF (2.5 mL), then Et.sub.3N (213 μL, 1.53 mmol, 90 equiv.) is added. To this clear, colorless solution at 0° C., EtOCOCl (16 μL, 0.17 mmol, 10 equiv.) is injected via gas-tight syringe.
[0528] 2. The resulting turbid white mixture is stirred for 2.5 h at 0° C., and then cannula transferred at 0° C. into a 10-mL, Schlenk flask containing a neat film of azeotropically dried (3×1 mL toluene) saponin amine 30 (28 mg, 17.0 μmol, 1.0 equiv.).
[0529] 3. The turbid white reaction mixture is stirred for 1.5 h at 0° C., then quenched with water (0.2 mL) to give a clear, colorless solution.
[0530] 4. The mixture is diluted with saturated aqueous NaHCO.sub.3 solution (30 mL) and the aqueous phase is extracted with DCM (3×25 mL). The combined organic layers are dried over anhydrous sodium sulfate, filtered, and concentrated (After quenching the reaction with water, the mixture can also be directly concentrated by rotary evaporation without the need for performing the described aqueous work-up).
[0531] 5. Purification by silica gel chromatography (hexanes/EtOAc,
2:1 with 0.5 vol % Et 3 N) (Elution with 9:1 to 5:1 benzene/EtOAc (0.5 vol % Et 3 N) can also be used for the silica gel chromatography purification) affords the truncated, fully protected aminoacyl saponin 32 (28 mg, 88%) as a white glassy solid.
Example 4: Global Deprotection of Protected Aminoacylated Saponins
Part A: Synthesis of Aminoacyl Saponin 34 (Compound I-6) by Hydrogenolysis and Acid Hydrolysis of Protected Aminoacyl Saponin 32
[0532] ##STR00085##
[0533] 1. In a 50-mL round-bottomed flask, the fully protected truncated saponin 32 (68 mg, 36.6 μmol, 1.0 equiv.) is dissolved in THF/EtOH (1:1) (20 mL), then 10% (dry basis) Pd/C, wet Degussa type E101 NE/W (390 mg, 0.18 mmol, 5.0 equiv.) is added.
[0534] 2. The reaction mixture is stirred under an atmosphere of H.sub.2 (50 psi) for 24 h at rt using a high-pressure bomb reactor (In similar saponin triterpene variants lacking the branched trisaccharide domain, hydrogenolysis under hydrogen atmosphere at balloon pressure for 12 h is sufficient to provide the corresponding debenzylated products).
[0535] 3. The suspension is filtered through a 0.45 μm nylon syringe filter, washed with MeOH (3×30 mL) and concentrated. Successful debenzylation is assessed by the disappearance of aromatic resonances by .sup.1HNMR in methanol-d.sub.4.
[0536] 4. In a 25-mL round-bottomed flask, the resulting crude mixture is dissolved in a precooled (0° C.) solution of TFA/water (3:1) (8 mL).
[0537] 5. The reaction mixture is stirred for 2 h at 0° C. and then concentrated under high vacuum at 0° C. to give a white solid residue.
[0538] 6. This crude product is dissolved in water/MeCN (4:1) (20 mL) and purified by RP-HPLC using a linear gradient of 30.fwdarw.70% MeCN in water (0.05 vol % TFA) over 15 min. The fully deprotected, truncated saponin 34 elutes as a main, single peak and is obtained as a fluffy white solid (28 mg, 74%) after lyophilization.
Example 5: Late Stage Acylation of Acyl Chain Domain Amine to Form Fully Elaborated Saponin 4 (Compound I-8)
Step A: Synthesis of Fully Elaborated Saponin 4, (Compound I-8), Lacking the Branched Trisaccharide Domain, by Selective 4-Iodobenzoylation of Free Amine in Aminoacyl Saponin 34
[0539] ##STR00086##
[0540] 1. In a 5-mL pear-shaped flask equipped with a rubber septum fitted with an Ar inlet needle, amine-terminating truncated saponin 34 (2.1 mg, 2.0 μmol, 1.0 equiv.) is dissolved in DMF (0.4 mL). Et.sub.3N (11 μL, 0.08 mmol, 40 equiv.) is injected followed by dropwise addition of a solution of N-succinimidyl 4-iodobenzoate (4.0 mg, 10 μmol, 5.8 equiv.) in DMF (0.2 mL) under Ar via gas-tight syringe.
[0541] 2. The reaction mixture is stirred for 2 h at rt, then diluted with 30% MeCN/water (2.3 mL), and directly purified by RPHPLC using a linear gradient of 30.fwdarw.70% MeCN in water (0.05 vol % TFA) over 15 min.
[0542] 3. The fully elaborated, truncated saponin 4 (Compound I-8) (1.7 mg, 67%) is obtained as a white powder after lyophilization.
Example 6: Isolation and Selective Protection of Branched Trisaccharide-Triterpene Prosapogenin
[0543] ##STR00087##
Part A: Isolation of Branched Trisaccharide-Triterpene Prosapogenins from Quil A.
[0544] 1. In a 250-mL round-bottomed flask equipped with a reflux condenser, Quil A (1.15 g) and potassium hydroxide (0.97 g, 17 mmol) are suspended in EtOH/water (1:1) (50 mL), then the mixture is heated to 80 TC for 7 h.
[0545] 2. The reaction is cooled to 0° C., neutralized with 1.0 N HCl, and concentrated to approximately one-half volume (care must be taken to avoid excessive foaming and bumping; water bath should be kept at 35° C. and pressure decreased slowly).
[0546] 3. The mixture is frozen and lyophilized, and the resulting dry solid is purified by silica gel chromatography (CHCl.sub.3/MeOH/water/AcOH, 15:9:2:1). The major product corresponding to the main spot observed by TLC is isolated by concentrating the desired fractions.
[0547] 4. The resulting solid is dried by azeotropic removal of solvents with toluene (2×20 mL) and lyophilized in MeCN/water (1:1) (3×15 mL) to provide a mixture of prosapogenins (5:6, 2.5:1) as a light tan foam (˜0.55 g, 50% mass yield). These xylose- and rhamnose-containing prosapogenins 5 and 6, respectively, correspond to the two most abundant trisaccharide-triterpene fragments found in QS saponins, and are advanced to the next protection step without further purification.
Part B: Synthesis of Triethylsilyl (TES)-Protected Prosapogenin by Selective Protection of Prosapogenin Hydroxyl Groups
[0548] 1. In a 25-mL modified Schlenk flask, the solid mixture of prosapogenins 5 and 6 (˜0.55 g) is azeotroped from pyridine (5 mL), then additional pyridine (8 mL) is added, followed by TESOTf (2.0 mL, 8.8 mmol).
[0549] 2. The reaction mixture is stirred for 2.75 days, then TESOTf (0.3 mL, 1.3 mmol) is added, followed by two further additions (0.1 mL each, 0.44 mmol each) 24 h and 48 h later, respectively (the last extra addition of TESOTf is situation-dependent and only required if the reaction is still incomplete after the first 4 days).
[0550] 3. After a total of 5 days, the mixture is concentrated and passed through a short plug of silica gel eluted with hexanes/EtOAc (4:1 to 2:1). The eluate is concentrated, the resulting yellow oil is dissolved in MeOH/THF (1:1) (20 mL), and the solution is stirred for 3.5 days to remove the silyl esters by solvolysis.
[0551] 4. The reaction mixture is concentrated and the resulting mixture of xylose- and rhamnose-containing (TES).sub.9-protected prosapogenin diacids is separated by silica gel chromatography (hexanes/EtOAc, 4:1 to 2:1) to afford purified xylose-containing protected prosapogenin 7 (˜0.25 g, ˜22% yield) as a white solid.
Part C: Synthesis of Protected Quillaja Prosapogenin 8 by Selective Esterification of Glucuronic Acid Carboxylic Acid in Protected Prosapogenin 7
[0552] 1. In a 10-mL modified Schlenk flask, the prosapogenin diacid 7 (81 mg, 41 μmol, 1.0 equiv.) is dissolved in DCM (0.7 mL) and pyridine (30 μL, 0.37 mmol, 9.0 equiv.) and TBP (102 mg, 0.41 mmol, 10 equiv.) are added, followed by benzyl chloroformate (15 μL, 0.11 mmol, 2.6 equiv.).
[0553] 2. The reaction is stirred for 6 h, additional benzyl chloroformate (3.0 μL, 21 μmol, 0.51 equiv.) is added (the extra addition of CbzCl after the first 6 h depends on the progress of the reaction in each particular case; when purifying by silica gel chromatography, elution with benzene/EtOAc (100:0 to 24:1) can also be considered) and the reaction is stirred for another 20 h.
[0554] 3. The mixture is concentrated and purified by silica gel chromatography (hexanes/EtOAc, 20:1 to 7:1) to afford selectively glucuronate-protected prosapogenin 8 (58 mg, 68%) as a white solid.
Example 7: Modular, Convergent Assembly of Saponin Domain Fragments
Part A: Synthesis of Protected Aminogalactose Saponin 28
[0555] ##STR00088##
[0556] 1. Step A: Synthesis of protected azidogalactose saponin 27 by glycosylation of branched trisaccharide-triterpene prosapogenin 8 with protected xylose-rhamnose-azidogalactose linear trisaccharide 26. In a 50-mL modified Schlenk flask, the selectively protected prosapogenin 8 (653 mg, 0.32 mmol, 1.5 equiv.) and the trisaccharide imidate 26 (230 mg, 0.21 mmol, 1.0 equiv.) are azeotropically dried from toluene (3×3 mL) under high vacuum, then dissolved in DCM (10 mL).
[0557] 2. Powdered 4 Å MS (1 g) is added and the suspension is stirred for 2 h at rt. The opaque, white mixture is then cooled to −78° C. and freshly distilled BF.sub.3.Math.OEt.sub.2 (15 μL, 0.23 mmol, 1.1 equiv.) is injected via gas-tight syringe.
[0558] 3. The reaction mixture is stirred at −78° C. for 6 h, passed through a plug of silica gel, and the filtrate is concentrated.
[0559] 4. Purification by silica gel chromatography (hexanes/EtOAc, 9:1 to 4:1) affords the prosapogenin-linear trisaccharide conjugate 27 (322 mg, 73%) as a glassy solid.
[0560] 5. Step B: Synthesis of protected aminogalactose saponin 28 by reduction of protected azidogalactose saponin 27. In a 50-mL modified Schlenk flask, PhSeSePh (313 mg, 1.0 mmol, 1.0 equiv.) (Caution: selenium compounds are highly toxic and have an unpleasant odor. Phenylselenol itself is extremely noxious. The in-situ preparation of phenylselenol solution by reduction of diphenyldiselenide circumvents the need to handle phenylselenol directly, but manipulation of the selenide-containing solution that will be added to the reaction flask is necessary. Care should be taken when handling selenium reagents and all manipulations should be performed in a fumehood wearing protective gloves and safety glasses, including weighing of the diphenyldiselenide starting material. A bleach solution should be prepared in advance to treat all used glassware and possibly early column fractions as well, to oxidize any remaining trace selenides. Bleach solution should also be placed in the solvent trap of the rotary evaporator, which should be thoroughly cleaned after use and ideally contained within the fumehood.) is dissolved in THF (8 mL) and H.sub.3PO.sub.2 (50% in water) (1.2 mL, 11.0 mmol, 11 equiv.) is then added via syringe.
[0561] 6. The yellow solution is heated at 40° C. for 1 h until it turns colorless.
[0562] 7. The reaction mixture is removed from the heat, diluted with benzene (8 mL) and distilled water (8 mL), and stirred vigorously for 5 min under Ar. The lower aqueous phase of the resulting biphasic suspension is removed by syringe (or glass pipette) under positive pressure of Ar, and anhydrous sodium sulfate is added to the Schlenk flask to dry the remaining organic layer while stirring.
[0563] 8. This freshly prepared solution of PhSeH (˜1.9 mmol) is then added under Ar via cannula transfer to a 250-mL reaction Schlenk flask containing a solution of the azeotropically dried saponin azide 27 (322 mg, 0.11 mmol, 1.0 equiv.) in Et.sub.3N (50 mL). Upon addition, a white precipitate is formed and the solution turns bright yellow.
[0564] 9. The reaction mixture is stirred for 3 h at 40° C., then concentrated to give a yellow-white solid.
[0565] 10. Purification by silica gel chromatography (hexanes/EtOAc, 4:1 to EtOAc with 0.5 vol % Et.sub.3N) affords the saponin amine 28 (256 mg, 87%) as a glassy solid (another alternative experimental procedure to perform this azide reduction step to give the corresponding saponin amine is the treatment of the starting material in Et.sub.3N with hydrogen sulfide (gas) as follows: An excess of hydrogen sulfide from a steel cylinder is bubbled via cannula (long steel needle) through an ice-cooled solution of the saponin azide (˜45 mg, ˜0.015 mmol, 1.0 equiv.) in pyridine/Et.sub.3N (3.5:1) (4.5 mL) for 2 min. Vent needle and cannula are removed from septum, which is sealed with Teflon tape and parafilm, and the reaction mixture is stirred overnight at rt. The dark green solution is then purged of excess hydrogen sulfide with a stream of nitrogen, and the resulting light-orange solution is concentrated by rotary evaporation. Purification of the residue by silica gel chromatography (hexanes/EtOAc, 1.0 vol % Et.sub.3N) yields the desired saponin amine product (˜40 mg, 80-90% yield)).
Part B: Synthesis of Protect Aminoacyl Saponin 31
[0566] ##STR00089##
[0567] 1. In a 5-mL pear-shaped Schlenk flask, commercially available 6-(Boc-amino)hexanoic acid (HO.sub.2C(CH.sub.2).sub.5NHBoc) (19.9 mg, 86 μmol, 10 equiv.) is dissolved in THF (0.9 mL), then Et.sub.3N (0.11 mL, 0.77 mmol, 90 equiv.) is added. To this clear, colorless solution at 0° C., EtOCOCl (7.3 μL, 77 μmol, 9.0 equiv.) is injected via gas-tight syringe.
[0568] 2. The turbid white mixture is stirred for 3 h at 0° C. The prosapogenin-linear trisaccharide saponin amine 28 (26 mg, 8.6 μmol, 1.0 equiv.) is then added, and the reaction is stirred for 1.5 h at rt.
[0569] 3. Water (0.1 mL) is added to quench the reaction, at which point the solution turns from turbid white to clear yellow. After addition of more water (0.1 mL), the resulting immiscible mixture is concentrated.
[0570] 4. Purification by silica gel chromatography (toluene/EtOAc, 20:1 to 11:1) affords the aminoacyl, branched trisaccharide-containing saponin 31 (22 mg, 81%) as a white glassy solid.
Example 8: Global Deprotection of Protected Aminoacylated Saponins
Part A: Synthesis of Aminoacyl Saponin 33 by Hydrogenolysis and Acid Hydrolysis of Protected Aminoacyl Saponin 31
[0571] ##STR00090##
[0572] 1. In a 100-mL round-bottomed flask, fully protected, branched trisaccharide-containing saponin 31 (240 mg, 75 μmol, 1.0 equiv.) is dissolved in THF/EtOH (1:1) (20 mL), then 10% (dry basis) Pd/C, wet, Degussa type E101 NE/W (140 mg, 66 μmol, 0.9 equiv.) is added (Caution: hydrogenolysis reactions pose a significant fire hazard. Caution should be taken when handling flammable palladium on carbon as well as hydrogen gas, which increases the risk of explosion.).
[0573] 2. The reaction mixture is stirred under H.sub.2 atmosphere (50 psi) for 24 h at rt using a high-pressure bomb reactor, and the suspension is filtered through a 0.45 μm nylon syringe filter.
[0574] 3. The palladium is washed thoroughly with MeOH (3×100 mL) and the clear filtrate is concentrated. Successful debenzylation is assessed by disappearance of aromatic resonances by .sup.1HNMR in methanol-d.sub.4.
[0575] 4. In a 50-mL round-bottomed flask, the resulting crude mixture of partially desilylated products is dissolved in a precooled (0° C.) solution of TFA/water (4:1) (10 mL).
[0576] 5. The reaction mixture is stirred for 3 h at 0° C. and then concentrated under high vacuum at 0° C. to give a white solid residue (140 mg).
[0577] 6. This crude product is dissolved in a solution of water/MeCN (4:1) and purified by RP-HPLC using a linear gradient of 20.fwdarw.35% MeCN in water (0.05 vol % TFA) over 10 min. The fraction containing the major, single peak is collected and lyophilized to dryness to afford the fully deprotected, free amine-containing saponin 33 (88 mg, 78%) as a fluffy white solid.
Example 9: Late-Stage Acylation of Acyl Chain Domain Amine to Form Fully Elaborated Saponin 3
Part A: Synthesis of Fully Elaborated Saponin 3 by Selective 4-Iodobenzoylation of Free Amine in Aminoacyl Saponin 33
[0578] ##STR00091##
[0579] 1. In a 10-mL round-bottomed flask equipped with a rubber septum fitted with an Ar inlet needle, amine-terminating saponin 33 (9.0 mg, 6.0 μmol, 1.0 equiv.) is dissolved in DMF (2.0 mL) and Et.sub.3N (50 μL, 0.36 mmol, 60 equiv.) is injected via gastight syringe.
[0580] 2. The mixture is stirred for 50 min at rt and commercially available N-succinimidyl 4-iodobenzoate (20 mg, 60 μmol, 10 equiv.), dissolved in DMF (0.6 mL) under Ar, is then added dropwise via syringe from a 5-mL pear-shaped flask equipped with a rubber septum.
[0581] 3. The reaction mixture is stirred for 1 h at rt, diluted with water/MeCN (4:1) (10 mL), and directly purified by RP-HPLC using a linear gradient of 20.fwdarw.70% MeCN in water over 30 min.
[0582] 4. The fraction corresponding to the peak containing the desired product, as assessed by mass spectrometry, is collected and lyophilized to dryness to afford the fully elaborated saponin 3 (5.4 mg, 52%) as a white powder.
Example 10: Total Synthesis of Compound I-4 (TQL-1055)
[0583] The total synthesis of Compound I-4 (TiterQuil-1-0-5-5/TQL-1055) is depicted in
Example 11: Prevnar-13-CRM197 Conjugate Vaccine Adjuvanted with Synthetic Saponins
[0584] The impact of synthetic QS-21 and TQL-1055 (Compound I-4) on antibody titers induced by the FDA approved human pneumococcal-CRM197 conjugate vaccine, Prevnar-13, was tested. Mice were immunized with Prevnar-13 in the presence or absence of synthetic saponin adjuvants at two different Prevnar dose levels (0.04 mcg and 0.2 mcg). Mice were immunized once at Day 0 and bled on Day 21 for serum analysis.
Example 11: Impact of TQL-1055 (Compound I-4) and QS-21 on Tdap Vaccine Adacel Immunogenicity
[0585] Adacel doses containing 1, 0.3, and 0.1 mcg of pertussis toxin per mouse were administered subcutaneously (SC, with no immunological adjuvant), using 2 vaccinations 4 weeks apart, resulting in a mean of 1,618 mcg, 898 mcg, and 107 mcg respectively of anti-PT antibody per ml of serum drawn 2 weeks after the second vaccination. The 0.1 mcg dose was indistinguishable from unvaccinated controls (96 mcg/ml). A 0.5 mcg dose of Adacel was selected for a pharmacology/toxicology (pharm/tox) study. The serological results for this study are summarized in
Example 12: Impact of TiterQuil-1-0-5-5 and QS-21 on Hepatitis B Vaccine Engerix-B Immunogenicity
[0586] Experiments were conducted with Engerix-B (HBV adult vaccine) in groups of 10 mice. Initially 3 mcg, 1 mcg, 0.3 mcg, 0.1 mcg, and 0.03 mcg Engerix-B doses per mouse were tested. Mean resulting anti-HBsAg antibody levels were 92,512 mcg/ml, 64,255 mcg/ml, 24,847 mcg/ml, 3,682 mcg/ml, and 910 mcg/ml respectively, with the 0.03 dose being indistinguishable from controls (821 mcg/ml). The 0.3 mcg dose of Engerix-B was selected for further studies and this dose was used mixed with various doses of TiterQuil-1055 (TQL-1055/Compound I-4). The resulting geometric mean antibody concentrations are summarized in
Example 13: Results of a Pilot Pharmacology/Toxicology with Adacel QS-21 and TiterQuil-1055
[0587] A pharm/tox study was conducted in 7 groups of 5 mice: 1) PBS alone, 2) 50 mcg TiterQuil-1055, 3) 20 mcg QS-21, 4) Adacel 2.5 mcg pertussis toxin (⅕ the human dose), 5) Adacel+QS-21 (20 mcg QS-21), 6) Adacel+TiterQuil-1055 (50 mcg), 7) Adacel+TiterQuil-1055 (50 mcg). Mice were vaccinated SC on days 1 and 15, weighed daily, and bled and sacrificed on day 22, except for group 7 which was sacrificed on day 29. No changes in blood chemistry or hematology results were seen in any group. 7-9% weight loss was seen in all mice in groups 3 and 5 (in agreement with prior results of QS-21) and in no other mice. Histopathology of 33 different tissues was performed on all mice. Detected abnormalities were restricted to the liver. Moderate to severe hepatocellular cytoplasmic vacuolization was seen in all mice in groups 4-6 (completely attributable to the pertussis vaccine at this dose, groups 5 and 6 were no more severe than group 4) but no mice in groups 1 or 2. This abnormality was short lived and was no detected in group 7, which was sacrificed one week after groups 1-6. Mild vacuolar changes were seen in all mice in group 3 (QS-21 alone). No changes at all were seen in groups 1 and 2 (PBS and TiterQuil-1055).
Example 14: Stability and Hemolytic Activity of Compound I-4 (TQL-1055/TiterQuil-1-0-5-5)
[0588] Natural and synthetic QS-21 (SQS-21 or SAPONEX®) and a variety of analogs were tested for hemolytic activity. This data clearly demonstrates that QS-21 is highly hemolytically active whereas several of the structural analogs, particularly Compound I-4 (TiterQuil-1-0-5-5/TQL-1055), demonstrated much lower or undetectable hemolytic activity in addition to increased stability.