Preparation of a protected triamino analogue of cholic acid and sequential incorporation of amino acids in solution and on a solid support

Article abstract of DOI:10.1021/ol006336v

We have prepared a triamine derivative of cholic acid with protecting groups on the amines that allow sequential amide formation. The triamine was formed from 3α,7α,12α-trihydroxycholan-24-ol with good stereoselectivity. Sequential removal of the amine pr

Full text of DOI:10.1021/ol006336v

ORGANIC
LETTERS
2000
Vol. 2, No. 19
3015-3018
Preparation of a Protected Triamino
Analogue of Cholic Acid and Sequential
Incorporation of Amino Acids in
Solution and on a Solid Support
Xiao-Ti Zhou, Atiq-ur Rehman, Chunhong Li, and Paul B. Savage*
Department of Chemistry and Biochemistry, Brigham Young UniVersity,
ProVo, Utah 84602
saVage@byu.edu
Received July 15, 2000
ABSTRACT
We have prepared a triamine derivative of cholic acid with protecting groups on the amines that allow sequential amide formation. The
triamine was formed from 3r,7r,12r-trihydroxycholan-24-ol with good stereoselectivity. Sequential removal of the amine protecting groups
and amide formation was achieved in high-yielding steps and was performed in solution and on a solid support.
Steroid nuclei have been used extensively in peptide
mimicry,1 in the preparation of receptors for various mol-
ecules,2 and in the development of antimicrobial agents.3
Cholic acid has attracted significant attention primarily due
to the orientation of its three hydroxyl groups on one face
of the steroid, and many derivatives of cholic acid have been
prepared. To increase the facial amphiphilicity of cholic acid,
Davis and co-workers4 have prepared a triamino analogue
of cholic acid in which each of the hydroxyl groups in the
molecule was replaced with an amine group (with retention
of stereochemistry). We have used a similar triamine
derivative of cholic acid to prepare antimicrobial agents.5
Recently, Davis and co-workers reported the preparation of
a diamine derivative of cholic acid suited for combinatorial
chemistry.6 The compound offers three potentially reactive
sites directly on the steroid nucleus: two amine groups and
a hydroxyl group. In our work with amino acid functionalized
cholic acid derivatives, we have found that attachment of
amino acid groups to cholic acid via ester linkages can yield
compounds that are not stable under mildly basic conditions.7
To avoid stability problems with ester groups, we have
(1) For example see: (a) Wess, G.; Bock, K.; Kleine, H.; Kurz, M.; Guba,
W.; Hemmeric, H.; Lopez-Calle, E.; Baringhaus, K.-H.; Glombic, H.;
Enhsen, A.; Kramer, W. Angew. Chem., Int. Ed. Engl. 1996, 35, 2222. (b)
Hirschmann, R.; Sprengeler, P. A.; Kawasake, T.; Leahy, J. W.; Shakes-
peare, W. C.; Smith, A. B., III. Tetrahedron 1993, 49, 3665. (c) Hirschmann,
R.; Sprengeler, P. A.; Kawasake, T.; Leahy, J. W.; Shakespeare, W. C.;
Smith, A. B., III. J. Am. Chem. Soc. 1992, 114, 9699.
(2) For recent reviews see: (a) Wallimann, P.; Marti, T.; Fu¨rer, A.;
Diederich, F. Chem. ReV. 1997, 97, 1567. (b) Li, Y. X.; Dias, J. R. Chem.
ReV. 1997, 97, 283. (c) Davis, A. P. Chem. Soc. ReV. 1993, 22, 243.
(3) (a) Bellini, A. M.; Quaglio, M. P.; Guarneri, M.; Cavazzini, G. Eur.
J. Med. Chem. 1983, 18, 185. (b) Kikuchi, K.; Bernard, E. M.; Sadownik,
A.; Regen, S. L.; Armstrong, D. Antimicrob. Agents Chemother. 1997, 41,
1433. (c) Li, C.; Lewis, M. R.; Gilbert, A. B.; Noel, M. D.; Scoville, D.
H.; Allman, G. W.; Savage, P. B. Antimicrob. Agents. Chemother. 1999,
43, 1347. (d) Li, C.; Budge, L. P.; Driscoll, C. D.; Willardson, B. M.;
Allman, G. W.; Savage, P. B. J. Am. Chem. Soc. 1999, 121, 931.
(4) (a) Broderick, S.; Davis, A. P.; Williams, R. P. Tetrahedron Lett.
1998, 39, 6083. (b) Davis, A. P.; Pe´rez-Paya´n, M. N. Synth. Lett. 1999,
991.
(5) Rehman, A.; Li, C.; Budge, L. P.; Street, S. E.; Savage, P.
B.Tetrahedron Lett. 1999, 40, 1865.
(6) Barry, J. F.; Davis, A. P.; Pe´rez-Paya´n, M. N.; Elsegood, M. R.;
Jackson, R. F. W.; Gennari, C.; Piarulli, U.; Gude, M. Tetrahedron Lett.
1999, 40, 2849.
(7) Guan, Q.; Li, C.; Schmidt, E. J.; Boswell, J. S.; Walsh, J. P.; Allman,
G. W.; Savage, P. B. Org. Lett., in press.
10.1021/ol006336v CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/31/2000

prepared an appropriately protected triamine derivative of
cholic acid (1; Figure 1) that allows sequential amide
the two former positions and differentiation of groups at C-7
(or C-12) and C-3 would be straightforward.
To differentiate C-7 and C-12 and to initiate the prepara-
tion of 1, triacetate 4 was formed in good yield (Scheme 1).
Oxidation of the C-12 alcohol, oxime formation, and
reduction using sodium in n-propanol9 gave amine 5. The
1
stereochemistry at C-12 was confirmed via H NMR; i.e.,
the proton R to the amine was clearly equatorial. The C-12
amine was protected as the (tert-butyloxy)carbamate, and a
trityl ether was formed from the primary alcohol. Oxidation
of the remaining alcohol groups on 6, oxime formation, and
reduction gave a mixture of 7a and 7b, which were readily
separable. Stereochemical assignments of 7a and 7b were
1
made on the basis of H NMR comparisons to previously
prepared compounds.8 This step was the lowest yielding of
the series. Nevertheless, because of the ease by which 6 was
prepared and because of the high yields of subsequent steps,
it was possible to bring gram quantities of material through
this step. After isolation of 7a, a variety of protecting groups
were used in attempts to differentiate the amine groups at
C-3 and C-7. Some protecting groups, including Fmoc
chloride and trimethylsilylethoxychloroformate, offered little
selectivity between these amines. Allyloxychloroformate
(Alloc chloride) proved to selectively react with the amine
at C-3. Protection of the remaining amine group with Fmoc
chloride and deprotection of the primary alcohol yielded 1.
Figure 1. Structures of tricarbamate 1 and triamide 2.
formation at C-3, C-7, and C-12 on the steroid, yielding
triamides such as 2. In addition, we have shown that
sequential deprotection of the three amine groups and amide
formation can be accomplished on a solid support.
To verify that the three protecting groups could be
removed independently of one another and in the presence
of an ester group at C-24, we prepared triamide 2 in solution
(Scheme 2). The benzoyl ester of 1 (9) was formed in
anticipation of immobilization of 1 on a solid support via
an ester linkage. Because we planned on using alcohol 1 in
solid-phase synthesis, it was important that the yield of each
step was optimized, and all yields of the reactions in Scheme
2 were near or above 90%. We elected to use Boc-protected
amino acids; consequently, it was necessary to first deprotect
the amine at C-12, followed by amide formation with Boc-
In our earlier work on the synthesis of a triamine derivative
of cholic acid,8 the amine groups were incorporated in a
single step. However, to provide for sequential amide
formation with the amine groups, orthogonal protection of
the amine groups was required. Therefore, it was necessary
to introduce the amine groups in separate steps. On the
steroid nucleus, functionality at C-7 and C-12 is more
hindered than that at C-3. Consequently, we reasoned that
if we could differentiate functional groups at C-7 and C-12,
reactions at C-3 would be much more rapid than at either of
Scheme 1. Preparation of Tricarbamate 1
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Org. Lett., Vol. 2, No. 19, 2000

Scheme 2. Preparation of Triamide 2
phenylalanine, giving 10. Deprotection of the amine at C-7
on the steroid scaffolding, the orthogonality of the nitrogen
protecting groups on 1 is expected to allow incorporation of
a Boc-protected amino acid at C-12, an Fmoc-protected
amino acid at C-7, and an Alloc-protected amino acid at C-3.
Subsequently, these protecting groups could be removed in
turn and additional amino acids could be incorporated. Thus,
three peptide chains of designed or random sequence could
be incrementally built upon a cholic acid scaffolding.
To verify that preparation of triamides could be ac-
complished on a solid support, compound 1 was coupled to
an acid chloride containing resin (Scheme 3). The loading
was ∼33% of the theoretical amount, and methyl esters were
formed from the remaining acid groups. Subsequently, the
sequence described in Scheme 2 was followed. At the
in the presence of a benzoyl ester proved challenging. Many
of the nitrogen bases typically used to remove Fmoc groups
cleaved the ester. We found, however, that 50% dicyclo-
hexylamine in DMF effectively removed the Fmoc group
without cleaving the benzoyl ester. After the Fmoc group
was removed, Boc-glycine was incorporated. Deprotection
of the amine at C-3 and amide formation with Boc-alanine
yielded 12. The amino acids were deprotected followed by
cleavage of the ester, giving 2 in 63% overall yield from 9.
In contrast to cholic acid derivatives in which amino acids
were attached to the steroid via ester linkages, triamide 2
was stable under basic conditions.
A potentially important consideration is that while only
Boc-protected amino acids were used to form the amides
Scheme 3. Preparation of Triamide 2 via Solid-Phase
Synthesis
Figure 2. FAB-mass spectra (thioglycerol/Na+ matrix) of 2: (A)
spectrum of the purified compound prepared in solution (Scheme
2); (B) spectrum of 2 prepared on a solid support without
purification (Scheme 3).
Org. Lett., Vol. 2, No. 19, 2000
3017

phase syntheses were identical on TLC, and 2 was clearly
the major product of the solid-phase synthesis. FAB-MS
confirmed that 2 was formed (Figure 2), and capillary
electrophoresis demonstrated that 2 was the major product
of the solid-phase sequence (Figure 3).
Compound 1 offers a means of sequentially forming robust
amide bonds to C-3, C-7, and C-12 on a cholic acid nucleus.
We have demonstrated that three different groups can be
added to the cholic acid scaffolding via solution synthesis
or on a solid support. In addition, C-24 can be functionalized
or used to link the compound to a solid support. We
anticipate that this compound will facilitate designed syn-
theses of novel small molecule receptors formed from three
peptide strands. It may also be possible to use 1 as the
nucleus of peptide triple-helix bundles. We are currently
using the compounds in combinatorial synthesis of receptors
for selected small-molecule targets, and our results will be
published in due course.
Acknowledgment. This work was generously supported
by the National Institutes of Health (GM 54619). We thank
Roopa S. Shah and Qunying Guan for help with electro-
phoresis and chromatography experiments.
Figure 3. (A) Electropherogram of the purified compound prepared
in solution (Scheme 2). (B) Electropherogram of 2 prepared on a
solid support without purification (Scheme 3).
Supporting Information Available: Experimental details
for the preparation of 1-12 and capillary electrophoresis.
This material is available free of charge via the Internet at
http://pubs.acs.org.
conclusion of each step, a small amount of the resin was
removed and the steroid was cleaved from the resin. TLC
was used to monitor the progress of the synthesis. After
incorporation of the three amino acids and cleavage of the
product from the resin, the resulting compound was com-
pared to purified 2. The products from solution and solid-
OL006336V
(8) Li, C.; Rehman, A.; Dalley, N. K.; Savage, P. B. Tetrahedron Lett.
1999, 40, 1861.
(9) Hsieh, H.-P.; Muller, J. G.; Burrows, C. J. Bioorg. Med. Chem. 1995,
3, 823.
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