Differentially-protected steroidal triamines; scaffolds with potential for medicinal, supramolecular, and combinatorial chemistry

Article abstract of DOI:10.1039/b412298d

Cholic acid 2a has been converted into two new orthogonally-protected triamino scaffolds, 13 and 14. The synthesis proceeds via the bis-Boc-NH-substituted azide 10, for which an improved preparation is described. After removal of the Boc groups, the two a

Full text of DOI:10.1039/b412298d

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A R T I C L E
Differentially-protected steroidal triamines; scaffolds with potential
for medicinal, supramolecular, and combinatorial chemistry†
Vicente del Amo,a Laura Siracusa,a,b Theodoros Markidis,a Beatriz Baragaña,b
Khadga M. Bhattarai,a Marta Galobardes,a Gregorio Naredo,a M. Nieves Pérez-Payánb and
Anthony P. Davis*a
a
School of Chemistry, University of Bristol, Cantock’s Close, Bristol, UK BS8 1TS.
E-mail: anthony.davis@bristol.ac.uk; Fax: +44 (0) 117 929 8611; Tel: +44 (0) 117 954 6334
Trinity College, Dublin 2, Ireland
b
Received 10th August 2004, Accepted 14th September 2004
First published as an Advance Article on the web 15th October 2004
Cholic acid 2a has been converted into two new orthogonally-protected triamino scaffolds, 13 and 14. The
synthesis proceeds via the bis-Boc-NH-substituted azide 10, for which an improved preparation is described. After
removal of the Boc groups, the two axial amines are differentiated through a novel monoprotection employing
1-(2-nitrobenzenesulfonyloxy)-benzotriazole 29. Regioselectivity of 50:1 is achieved, presumably reflecting an
exceptional sensitivity to steric hindrance. Protection of the remaining amino group as Boc or Alloc gives the
scaffolds in 40% overall yield from cholic acid. Scaffold 13 has been sequentially deprotected and derivatised with
N-carbamoyl amino acids, to give a model for tripodal peptide libraries.
in well-spaced arrays. Thirdly, they are readily adapted for solid-
Introduction
Polyfunctionalised scaffolds
phase synthesis; the carboxyl-functionalised side-chain provides
a natural point of attachment to a resin. Fourthly, they provide
co-directed functionality, especially useful if the appended
groups are to act cooperatively (e.g. in synthetic receptors or
catalysts, TASPs, or most biological applications).
1 are key components for
many areas of chemistry. They are needed as core units for
combinatorial libraries,1 dendrimers,2 template-assembled
synthetic proteins (TASPs),3 glycoclusters,4 and podand-
type synthetic receptors.1e,f,5 For most of these applications,
scaffolds should possess well-defined architectures which confer
distinctive shapes on derived compounds or libraries. The
steroid nucleus is an attractive starting point for such systems,
being readily available, rigid, extended and chiral. Bile acids,
such as cholic acid 2a, have proved especially useful.1c–e,2b,3b,4c,5c
Firstly, they are inexpensive; cholic acid, at ca. €0.4 g−1,6 is the
second-cheapest of the commercially-available steroids (after
cholesterol). Secondly, they possess high degrees of functionality
In developing the bile acids as scaffolds, two issues must be
addressed. Firstly, the “natural” hydroxyl groups are relatively
unreactive and difficult to derivatise (especially the axial 7- and
12-OH). The problem can be solved in individual cases, but for
rapid, trouble-free elaboration (especially on solid phase), amino
groups are preferable. Secondly, differential and orthogonal
protection adds greatly to the versatility of any scaffold. The
“ideal” bile acid scaffold would therefore be of form 3. The
synthesis of such compounds, however, is not straightforward,
especially considering that gram quantities are needed for
most applications. The conversion of 2a to 3 requires efficient
stereocontrol at three sterically different centres, and also good
regiocontrol. A number of “less ambitious” amino-scaffolds
have been described (Fig. 1), including (a) the difunctional
4 and 5 due to Still;1e,7 (b) ketone 6 due to Kasal et al.;8
(c) undifferentiated triamine 7 due to Savage;9 (d) alcohol 8,10
tris-Boc-protected triamine 911 and bis-Boc-azide 10,12 all due to
our own group. However, only one fully-differentiated triamino
scaffold has been reported. Compound 11 was prepared by
Savage and coworkers in 15% overall yield from tetraol 12, in
a sequence, consisting of 12 steps.13 The process is workable on a
gram scale but, as all steps require chromatography, is probably
not ideal for routine use.
As mentioned above, we have previously described a partially-
differentiated triamino-scaffold, the azide 10. An intermediate
for tris-ammonium “facial amphiphiles”14 and “cholapod”anion
receptors,5c,15 this compound is available via a high-yielding,
large-scale process. In principle, deprotection at positions 7 and
12 followed by stepwise reprotection could give a synthesis of
3 in just three further steps. However, this requires a reagent
of exceptional selectivity, able to distinguish between two axial
† Electronic supplementary information (ESI) available: Regiochemical
assignment of monoprotected bile acid derivatives and estimation of
regioselectivities. See http://www.rsc.org/suppdata/ob/b4/b412298d/
3 3 2 0
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 3 2 0 – 3 3 2 8
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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Fig. 1 Amino-scaffolds derived from bile acids, previously reported in the literature.
amino groups. We now report a novel protection method which
demonstrates this capability, and enables practical syntheses of
two new orthogonally-protected triamino scaffolds, 13 and 14,
in 40% yield from cholic acid 2a. We also give details of an
updated preparation of 10, and show that 13 can be sequentially
deprotected and derivatised with N-carbamoyl amino acids to
give a model for tripodal peptide libraries.
Results and discussion
Our current, optimised preparation of 10 is summarised in
Scheme 1. Compared to the earlier process12 this version
incorporates a new oxidation method for 15 → 16 which avoids
toxic Cr(VI) reagents, a new method for 18 → 19, and modified
procedures for 16 → 17 and 20 → 10. Only the last two steps
require chromatography. The steps up to 19 are performed
routinely on a 20 g scale, and the final 2 steps on a 10 g scale.
The overall yield is 62% from cholic acid 2a. Treatment of 10
Scheme 1 Optimised synthesis of bis-differentiated scaffold 10. Reagents and conditions: (a) MeOAc, p-TsOH·H2O (cat.), reflux, 48 h, then MgSO4,
reflux, overnight; (b) Ca(ClO)2, KBr (cat.), AcOH/H2O, 0 °C to rt, overnight; then 2-propanol, rt, 1.5 h; (c) NH2OH·HCl, NaOAc, MeOH, reflux,
overnight; (d) H2, PtO2, AcOH, rt, 3 d then Zn, AcOH, rt, 24 h; (e) Boc2O, NaHCO3, THF/H2O, rt, 2 d; (f) NaOH, MeOH (dry), 0 °C., 4 h; (g) MsOH,
PPh3, DEAD, DMAP, THF, 0 °C then rt, 48 h; (h) NaN3, DMF, 47 °C, 3 d.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 3 2 0 – 3 3 2 8
3 3 2 1

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with TFA in DCM, followed by washing with sat. aq. Na2CO3,
gave diamine 21 for studies on regioselective protection.
The differentiation of positions 7 and 12 on the bile acid
skeleton is a long-standing problem in steroid chemistry. A
classical solution is the 3,7-bis-acetylation of methyl cholate
with acetic anhydride/pyridine, due to Fieser and Rajagopalan.16
Although this might imply that the 7-OH is less hindered,
detailed studies by Blickenstaff showed that, in the general
case, the 12-OH is more accessible; Fieser’s 3,7-bis-acetylation
relies on a specific acceleration of acetylation at 7 by a free
12-OH.17 Blickenstaff’s results accord with expectation, the
steroidal C4 is very close to a 7a substituent and seems likely to
lower its reactivity. Other acylation conditions can indeed lead
preferentially to 3,12-derivatisation.18 However, regioselectivities
are generally quite modest for both 3,7- and 3,12-bis-acylations.
These preparations are only useful because of fortuitious
crystallisations.
Initial studies on diamine 21 suggested similar tendencies
to the 7,12-diols. Treatment with 1 equivalent of the common
protecting agents (Boc)2O 25, BOC-ON 26 and AllocCl 27
gave mixtures in which the 12-protected monoamine 22 pre-
dominated (Scheme 2 and Table 1, entries 1–4). However, yields
were low, and substantial quantities of diprotected compound
24 were also present. Such product mixtures are expected if
the 12a-NH2 continues to react rapidly after monoprotection
at 7a-NH2, so that much of the 23 formed is converted to 24.‡
Separation of 22 and 23 could not be achieved, so none of these
methods could be considered useful.
monoprotected products 22 and 23 improved significantly.
However, once again, these regioisomers could not be
separated.
To increase sensitivity to steric hindrance, we considered
changing the leaving group. Methanesulfonyloxy benzotriazole
30 had been used as a selective N-mesylating agent, capable
of distinguishing between amino nitrogens in differing steric
environments.20 o-Nitrobenzenesulfonyl analogue 29 had been
employed as a peptide coupling agent,21 but not for N-protection.
29 was prepared from 28 and 1-hydroxybenzotriazole, and
applied to the monoprotection of 21. As shown in Table 1
(entry 7), the yield remained high and the regioselectivity did
indeed improve significantly. The mixture of 31 and 32 (50:1)
was treated with (Boc)2O and the crude product precipitated
from Et2O–hexane to give 13 (Scheme 3). The overall yield was
67% from 10 and 41% from cholic acid 2a. A 2.6 g batch of 13
has been prepared, and we estimate that up to 7 g should be
feasible with standard laboratory equipment.
Scheme 3
Steroid 13 conforms well to the ideal of 3, being furnished with
fully orthogonal protection (Boc removable with acid, oNs with
thiolate, N3 by treatment with R3P/H2O). None of the groups is
sensitive to the basic conditions required to hydrolyse the side-
chain methyl ester. To demonstrate its potential the groups were
unmasked and derivatised in turn as shown in Scheme 4. The
protecting groups were removed in the order 12-oNs, 7-Boc and
3-N3. The sequence proceeded without incident; the conditions
required to remove the oNs, though seemingly aggressive, did
not appear to affect any other functional group in 13.
In the hope that a sulfonyl-based electrophile might behave
differently to the carbonyl-based 25–27, we investigated
o-nitrobenzenesulfonyl chloride (oNsCl, 28). The o-nitro-
benzenesulfonyl group has been used to both activate and
protect primary amines.19 Removal is accomplished by nucleo-
philic attack with thiolate. Diamine 21 was treated with 28 in
the presence of triethylamine or 2,6-lutidine (Table 1, entries 5
and 6). Regioselectivities were still moderate, but the yields of
Finally, the scaffold 13 presents a potential difficulty if one
wishes to prepare template-assembled synthetic proteins, or
peptide libraries of general form 37, using Fmoc-based solid
‡ Kinetic modelling suggests that, for these entries, reaction at position
12 might be accelerated by 7-protection. This acceleration does not seem
to apply in the case of o-nitrobenzenesulfonyl protection (entries 5–7).
Scheme 2
3 3 2 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 3 2 0 – 3 3 2 8

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Table 1 Attempted regioselective protections of diamine 21a
Entry
Reagentb
Basec
Time/h
Ratio 22/23d
Yield 22 + 23 (%)e
Yield 24 (%)e
1.
2.
3.
4.
5.
6.
7.
Boc2O 25
Boc2O 25
BOC-ON 26
AllocCl 27
oNsCl 28
oNsCl 28
29
—
TEA
—
2,6-lutidine
TEA
2,6-lutidine
—
48
48
48
24
12
12
12
16:1
8:1
7:1
4:1
7:1
6:1
50:1
50
46
39
30
71
72
75
24
25
27
29
13
11
10
a All reactions were performed at room temperature with THF as solvent. b 1 equivalent. c 1.1 equivalents, where relevant. d Determined by 1H-NMR
integration (C18 methyl group). For further details, including regiochemical assignments, see electronic supplementary information.† e After isolation
by flash chromatography.
Scheme 4 Reagents and conditions: (a) PhSH, Cs2CO3, DMF, 55 °C, overnight; (b) N-Fmoc–L-Phe-OH, TBTU, HOBt, DIPEA, DMF/DCM;
(c) TFA, DCM, 0 °C, 2 h; (d) sat. Na2CO3 aq.; (e) N-Boc–L-Val-OH, TBTU, HOBt, DIPEA, DMF/DCM; (f) PMe3, THF, rt, 5 h then H2O, rt,
overnight; (g) N-Boc–Gly-OH, TBTU, HOBt, DIPEA, DMF/DCM.
phase synthesis. The standard N-Fmoc-protected amino acid
derivatives possess acid-labile protecting groups in their side-
chains, so acidic conditions cannot be used to reveal a steroid
nitrogen once a peptide chain is in place. The 7-Boc group in
13 must thus be the first to be removed. However, this means
that the 12-oNs must be removed later in the sequence, after
synthesis of at least one peptide. Although N-oNs protection has
been advanced as a general method for peptide synthesis,19b the
removal conditions are vigorous and might not be compatible
with all sequences and side-chain protections. On this basis, the
12-oNs should be removed before the others in 13. The conflict
may be resolved by replacing the 7-Boc protection with a group
removable by non-acidic conditions, and still orthogonal to
oNs and N3. Alloc fulfils these criteria, deprotection being
accomplished with a nucleophile under Pd catalysis. Derivative
14 was therefore prepared by treatment of 31 with reagent 27
(83% yield, 38% overall from cholic acid). Scaffold 14 should
complement 13, being suitable for tris-peptide libraries and
other cases where acidic conditions must be avoided.
benzenesulfonyl derivative 29. The results suggest that reagent
29 is exceptionally sensitive to steric hindrance, a property which
should find further use in synthesis. Scaffolds 13 and 14 may
be used to construct a wide range of tripodal molecules for use
as receptors, catalysts, pharmaceuticals etc. They are especially
well suited to the preparation of combinatorial libraries using
solid phase synthesis.
Experimental
All reagents and solvents were obtained from commercial
suppliers and used without further purification unless otherwise
stated. Methanol was distilled over calcium chloride, magnesium
and iodine. DMF was obtained dry from Aldrich. THF and
DCM were obtained dry from an Anhydrous Engineering
Solvent Purification System (AESPS). Analytical TLC was
carried out on DC-Alufolien Kieselgel 60F254 0.2 mm plates
(Merck) and compounds were visualised by UV fluorescence,
5% phosphomolybdic acid in ethanol, ninhydrine solution or by
charring over a Bunsen burner flame. Flash chromatography of
reaction products was carried out using Silica 60A, particle size
35–70 micron (Fischer Scientific). IR spectra were recorded on
a Perkin-Elmer Spectrum One spectrometer. The most intense
bands were only quoted. Melting points were obtained using
Gallekamp melting point blocks and are quoted as uncorrected
1
values. H-NMR and 13C-NMR spectra were recorded on a
Jeol Delta/GX270 or Jeol Delta/GX400 spectrometer, using
deuterated solvents and were referenced internally to the residual
solvent peak or TMS (dH = 0.00 ppm, dC = 0.00 ppm) signal.
Coupling constants (J-Values) are given in Hz. The DEPT 135°
technique was used to assign (CH2) signals. Chemical shifts are
reported as follows: value (description of absorption, coupling
constant(s) where applicable, number of protons, assignment).
NMR spectra assignation was aided by comparison with
literature values for similar compounds. In all this experimental
section only clear identifiable peaks are assigned.
Conclusion
In conclusion, we have developed practical syntheses for two
new orthogonally-protected triamino scaffolds based on the bile
acid framework. The key step in these preparations is a highly
regioselective monoprotection of 7a,12a-diamine 21 by o-nitro-
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 3 2 0 – 3 3 2 8
3 3 2 3

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Methyl 3a-acetoxy-7a,12a-dihydroxy-5b-cholan-24-oate 15.22
3 H, 18-H3), 1.15 (s, 3 H, 19-H3), 2.01 (s, 3 H, CH3CO2), 2.35–2.41
(m, 1 H), 2.44 (t, J = 11.2 Hz, 1 H), 3.10 (dd, J = 13.2, 1.4 Hz,
1 H), 3.25 (dd, J = 13.2, 4.80 Hz, 1 H), 3.66 (s, 3 H CO2CH3),
4.71 (m, 1 H, 3b-H); 13C NMR (100 MHz, CDCl3): d = 11.92
(18-CH3), 18.88 (21-CH3), 19.72 (CH2), 20.93 (CH3CO2), 22.23
(19-CH3), 25.06 (CH2), 25.84 (CH2), 27.17 (CH2), 27.63 (CH2),
30.45 (CH2), 31.40 (CH2), 32.36 (CH2), 34.07 (CH2), 35.60 (CH),
35.76 (C), 41.81 (CH), 43.71 (CH), 43.73 (CH), 45.93 (CH),
49.12 (C), 51.23 (CO2CH3), 52.52 (CH), 73.18 (CH), 158.78 (C),
163.77 (C), 170.94 (CH3CO2), 175.07 (CO2CH3); Anal. found:
C, 65.82; H, 8.54; N, 5.76%. C27H42N2O6 requires C, 66.10; H,
8.63; N, 5.71%.
Cholic acid 2a, (62.00 g, 151.70 mmol) and p-toluenesulfonic
acid monohydrate (3.00 g, 15.70 mmol) were suspended in
methyl acetate (750 mL), refluxed for 48 h, then allowed to cool
to room temperature. MgSO4 (20 g, 166.15 mmol) was added to
the resulting yellow solution and the mixture refluxed overnight.
NaHCO3 (1.32 g, 15.70 mmol) in H2O (15 mL) was added
dropwise. The mixture was filtered through a plug of silica (ca.
35 g) washing thoroughly with EtOAc to obtain a solution which
was evaporated under reduced pressure. The resulting white foam
was recrystallised from EtOAc–hexane in several crops to give
diol 15 (67.98 g, 96%) as a white powder. An analytical sample
was recrystallised from DCM–hexane. Mp 150–151 °C (lit.22
mp 153–155 °C); Rf = 0.46 (Et2O); IR (solid state): mmax = 3300,
2938, 1723 cm−1; 1H NMR (400 MHz, CDCl3): d = 0.70 (s, 3 H,
18-H3), 0.91 (s, 3 H, 19-H3), 0.98 (d, J = 6.3 Hz, 3 H, 21-H3), 2.01
(s, 3 H, CH3CO2), 2.18–2.41 (m, 4 H), 3.67 (s, 3 H, CO2CH3),
3.85 (broad s, 1 H, 7b-H), 3.99 (broad s, 1 H, 12b-H), 4.58 (m,
1 H, 3b-H); 13C NMR (100 MHz, CDCl3): d = 12.57 (18-CH3),
17.37 (21-CH3), 21.43 (CH3CO2), 22.54 (19-CH3), 23.15 (CH2),
26.66 (CH), 27.41 (CH2), 28.44 (CH2), 30.92 (CH2), 31.08 (CH2),
34.38 (CH2), 34.70 (C), 34.91 (CH2), 35.13 (CH), 35.25 (CH2),
39.65 (CH), 41.24 (CH), 42.09 (CH), 46.58 (C), 47.26 (CH),
51.47 (CO2CH3), 68.25 (CH), 72.88 (CH), 74.30 (CH), 170.71
(CH3CO2), 174.63 (CO2CH3); Anal. found: C, 69.64; H, 9.49%.
C27H44O6 requires: C, 69.79; H, 9.49%.
Methyl 3a-acetoxy-7a,12a-di[N-(t-butyloxycarbonyl)amino]-5b-
cholan-24-oate 18
A mixture of the dioximino compound 17 (10 g, 20.4 mmol)
and platinum (IV) oxide hydrate (Adams’ catalyst) (1 g, 10%
by weight) in glacial acetic acid (52 mL) was stirred under 1
atmosphere of H2 for 3 d, during which the initial slurry turned
into a greenish solution. The reaction mixture was filtered,§
washing with acetic acid, and zinc powder (20 g) was added to
the combined filtrates. The mixture was stirred for 24 h before
removing the zinc by filtration. The solvents were evaporated
under reduced pressure and the residue was dissolved in a
mixture of THF (162 mL) and saturated aqueous NaHCO3
(80 mL). Di-tert-butyl dicarbonate (11.3 g, 60.8 mmol) was
added to the mixture, which was then stirred for 2 d. The layers
were separated and the aqueous phase was extracted with ethyl
acetate. The organic volumes were combined, dried with MgSO4
and evaporated under reduced pressure to give bis-carbamate
18 (11.9 g, 90%) as a white foam. An analytical sample was
recrystallised from DCM–hexane. Mp 217–222 °C; Rf = 0.73
(hexane–EtOAc 1:1); IR (solid state): mmax = 3370, 2958, 1738,
1708 cm−1; 1H NMR (400 MHz, CDCl3): d = 0.79 (s, 3 H, 18-H3),
0.92 (d, J = 7.4 Hz, 3 H, 21-H3), 0.93 (s, 3 H, 19-H3), 1.43 (s,
18 H, (CH3)3C), 2.02 (s, 3 H, CH3CO2), 2.18–2.50 (m, 2 H), 3.68
(broad s, 1 H, 7b-H), 3.71 (s, 3 H, CO2CH3), 4.00 (broad s, 1 H,
12b-H), 4.57 (m, 1 H, 3b-H), 5.20 (broad s, 1 H, 7-CH–NHR),
5.40 (broad s, 1 H, 12-CH–NHR); 13C NMR (100 MHz, CDCl3):
d = 13.53 (18-CH3), 17.50 (21-CH3), 21.30 (CH3COO), 22.76
(19-CH3), 22.91 (CH2), 26.68 (CH2), 27.13 (CH2), 27.81 (CH2),
28.49 ((CH3)3C), 28.52 ((CH3)3C and CH), 30.48 (CH2), 31.72
(CH2), 32.23 (CH2), 34.76 (CH2 and C), 34.92 (CH2), 35.80
(CH), 37.06 (CH), 41.40 (CH), 44.31 (CH), 44.64 (C), 47.19
(CH), 49.49 (CH), 52.27 (CO2CH3), 53.23 (CH), 74.39 (CH),
78.30 ((CH3)3C), 78.48 ((CH3)3C), 155.44 (NHCO), 155.53
(NHCO), 170.08 (CH3CO2), 176.65 (CO2CH3); Anal. found:
C, 67.19; H, 9.47; N, 3.95%. C37N62N2O8 requires C, 67.04; H,
9.43; N, 4.23%.
Methyl 3a-acetoxy-7,12-dioxo-5b-cholan-24-oate 16
Calcium hypochlorite (65% by weight, 14.2 g, 64.6 mmol)
suspended in H2O (150 mL) was added dropwise to a solution
of diol 15 (20.00 g, 43.04 mmol), acetic acid (260 mL) and a
few crystals of potassium bromide at 0 °C. The reaction was
stirred overnight at room temperature before the addition of
isopropanol (6 mL). After 1.5 h the solution was poured into
an ice–water mixture under vigorous stirring. The resulting
white solid was filtered and washed thoroughly with water to
give diketone 16 (19.4 g, 98%) as a white powder. An analytical
sample was recrystallised from AcOH–H2O. Mp 159–160 °C
(lit.23 mp 163–164 °C); Rf = 0.31 (Toluene–EtOAc 4:1); IR
(solid state): mmax = 2970, 1738, 1714 cm−1; 1H NMR (400 MHz,
CDCl3): d = 0.84 (d, J = 6.2 Hz, 3 H, 21-H3), 1.04 (s, 3 H,
18-H3), 1.31 (s, 3 H, 19-H3), 2.00 (s, 3 H, CH3CO2), 2.21–2.31
(m, 3 H), 2.36–2.44 (m, 1 H), 2.71 (t, J = 12.8 Hz, 1 H), 2.80
(t, J = 11.7 Hz, 1H), 2.90 (q, J = 6.6 Hz, 1 H), 3.67 (s, 3 H,
CO2CH3), 4.67 (m, 1 H, 3b-H); 13C NMR (100 MHz, CDCl3):
d = 11.77 (18-CH3), 18.60 (21-CH3), 21.15 (CH3CO2), 22.40
(19-CH3), 25.13 (CH2), 25.80 (CH2), 27.61 (CH2), 30.49 (CH2),
31.28 (CH2), 33.14 (CH2), 33.75 (CH2), 35.51 (CH), 35.78 (C),
38.31 (CH2), 44.95 (CH), 45.18 (CH2), 45.46 (CH), 45.64 (CH),
48.92 (CH), 51.39 (CO2CH3), 51.82 (CH), 56.84 (C), 72.34 (CH),
170.41 (CH3CO2), 174.46 (CO2CH3), 209.07 (CO), 212.34
(CO); Anal. found: C, 70.18; H, 8.72%. C27H40O6 requires C,
70.41; H, 8.75%.
Methyl 3a-hydroxy-7a,12a-di[N-(t-butyloxycarbonyl)amino]-5b-
cholan-24-oate 19
The acetoxy cholanoate 18 (14 g, 21.6 mmol) was dissolved in dry
methanol (125 mL), and a solution of sodium hydroxide (1.26 g,
31.5 mmol) in dry methanol (10 mL) was added dropwise. The
mixture was stirred at 0 °C for 4 h before evaporating the solvent
under reduced pressure. The residue was redissolved in diethyl
ether, washed thoroughly with H2O, dried with MgSO4 and
evaporated under reduced pressure to give hydroxy cholanoate
19 (13.3 g, 99%) as a white foam. An analytical sample was
recrystallised from DCM–hexane. Mp 245–247 °C; Rf = 0.42
(hexane–EtOAc 1:1); IR (solid state): mmax = 3379, 2928,
1706 cm−1; 1H NMR (400 MHz, CDCl3): d = 0.80 (s, 3 H, 18-H3),
0.92 (d, J = 7.8 Hz, 3 H, 21-H3), 0.93 (s, 3 H, 19-H3), 1.43 (s,
18 H, (CH3)3C), 2.15–2.25 (m, 1 H), 2.45 (m, 1 H), 3.49 (m, 1 H,
3b-H), 3.71 (m, 4 H, 7b-H and CO2CH3), 3.98 (broad s, 1 H,
Methyl 3a-acetoxy-7,12-dioximino-5b-cholan-24-oate 17
The diketone 16 (14 g, 30.5 mmol), sodium acetate (12.5 g,
152 mmol) and hydroxylamine hydrochloride (6.32 g, 91 mmol)
were suspended in methanol (290 mL). The mixture was heated
under reflux overnight. The resulting pale suspension was
cooled, to give a precipitate which was collected by filtration. The
solid was suspended in water and again collected by filtration.
The filtrate from the initial filtration (methanol solvent) was
concentrated to a small volume (ca. 10 mL). Water (ca. 100 mL)
was added and the resulting precipitate was collected by
filtration. The solids were combined to give dioxime 17 (13.9 g,
93%) as a white powder. An analytical sample was recrystallised
from CHCl3–hexane. Mp >261 °C (decomp); Rf = 0.70 (Et2O);
1
IR (solid state): mmax = 3259, 2940, 1737, 1659 cm−1; H NMR
§ Provided the catalyst is washed thoroughly with acetic acid, it may be
(400 MHz, CDCl3): d = 0.92 (d, J = 6.0 Hz, 3 H, 21-H3), 0.93 (s,
dried, resuspended and used to hydrogenate further batches of 17.
3 3 2 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 3 2 0 – 3 3 2 8

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The bisammonium salt was dissolved in chloroform and
washed with three portions of saturated aqueous Na2CO3. The
organic phase was separated and dried over MgSO4. The solvent
was removed under reduced pressure to yield diamine 21 (1.38 g,
99%) as an off-white solid. Rf = 0.32 (ethyl acetate–methanol
12b-H), 5.21 (broad s, 1 H, 7-CH–NHR), 5.35 (broad s, 1 H, 12-
CH–NHR); 13C NMR (100 MHz, CDCl3): d = 13.72 (18-CH3),
17.37 (21-CH3), 22.89 (19-CH3), 23.05 (CH2), 26.95 (CH2), 27.41
(CH2), 28.50 ((CH3)3C), 28.54 ((CH3)3C), 29.04 (CH), 30.26
(CH2), 30.75 (CH2), 31.61 (CH2), 32.17 (CH2), 34.65 (C), 35.00
(CH2), 35.24 (CH), 35.26 (CH), 37.07 (CH), 39.14 (CH2), 41.48
(CH), 44.79 (C), 44.89 (CH), 48.89 (CH), 51.97 (CO2CH3), 53.29
(CH), 71.88 (CH), 79.00 ((CH3)3C × 2), 155.45 (NHCO), 155.47
(NHCO), 175.44 (CO2CH3); Anal. found: C, 67.51; H, 9.68; N,
4.49%. C35H60N2O7 requires C, 67.71; H, 9.74; N, 4.51%.
1
9:1); IR (solid state): mmax = 3370, 2917, 2090, 1736 cm−1; H
NMR (400 MHz, CDCl3): d = 0.73 (s, 3 H, 18-H3), 0.93 (s, 3 H,
19-H3), 0.97 (d, J = 6.3 Hz, 3 H, 21-H3), 2.13–2.49 (m, 2 H), 3.10
(s, 1 H, 7b-H), 3.15 (broad s, 1 H, 12b-H), 3.17 (m, 1 H, 3b-H),
3.66 (s, 3 H, CO2CH3); 13C NMR (100 MHz, CDCl3): d = 13.22
(18-CH3), 16.86 (21-CH3), 22.53 (19-CH3), 23.21 (CH2), 25.58
(CH), 26.59 (CH2), 27.27 (CH2), 28.10 (CH2), 30.60 (CH2), 30.73
(CH2), 34.42 (CH2), 34.76 (C), 34.82 (CH), 35.24 (CH2), 35.87
(CH2), 39.31 (CH), 41.50 (CH), 41.98 (CH), 45.84 (C), 47.16
(CH), 47.40 (CH), 51.03 (CH), 53.59 (CO2CH3), 61.16 (CH),
174.04 (CO2CH3); MS (FAB+): m/z (%): 446 (100) [M + H]+; 403
(40) [M − N3]+; 468 (10) [M + Na]+.
Methyl 3a-azido-7a,12a-di[N-(t-butyloxycarbonyl)amino]-5b-
cholan-24-oate 10, (via methyl 3b-methanesulfonyl-7a,12a-di[N-
(t-butyloxycarbonyl)amino]-5b-cholan-24-oate 20)
The 3a-hydroxy cholanoate 19 (5.00 g, 8.05 mmol), DMAP
(2.58 g, 21.4 mmol) and triphenylphosphine (6.35 g, 24.5 mmol)
were dissolved in dry THF (75 mL) under nitrogen and cooled
in an ice-bath. Methanesulfonic acid (1.40 mL, 17 mmol) was
added to the stirred solution to give a white suspension, and
DEAD (4.03 mL, 24 mmol) was added dropwise. With the
addition of DEAD the reaction mixture turned into a clear
yellow solution then after 3–5 min into a white slurry. After
15 min the mixture was warmed to room temperature and
was maintained with vigorous stirring for 48 h. The white
solid was removed by filtration, and the mother liquors were
evaporated under reduced pressure. The dry residue was
purified by flash chromatography (hexane–EtOAc, 3:1 to
1:1, loading the product mixed with silica) to give the required
3b-methanesulfonate derivative 20 (with small impurities of
DEAD and its reduced product; and triphenylphosphine and
its oxidised product). The 3b-methanesulfonate derivative 20
and sodium azide (6.9 g, 106 mmol) were stirred in dry DMF
(85 mL) under nitrogen at 47 °C for 3 d. The solvent was
evaporated under reduced pressure. The residue was redissolved
in ethyl acetate, washed with H2O, dried with MgSO4 and
evaporated under reduced pressure. The dry residue was purified
by flash chromatography (hexane–EtOAc, 4:1, dry loading by
evaporation from EtOAc onto silica) to give 3a-azide 10 (4.11 g,
79%) as a white solid. An analytical sample was recrystallised
from DCM–hexane. Mp 218–220 °C; Rf = 0.27 (hexane–EtOAc
1-(2-nitrobenzenesulfonyloxy)-benzotriazole 29.21
In a three-necked round bottomed flask equipped with two
dropping funnels was prepared a solution of 1-hydroxy
benzotriazole (4.05 g, 30.0 mmol), in chloroform (10 mL).
The temperature was brought to 0 °C with an ice-bath. To
this solution were added at the same time and under vigorous
stirring, 2-nitrobenzene sulfonyl chloride 28 (6.65 g, 30 mmol)
in chloroform (50 mL), and Na2CO3 (4.24 g, 40 mmol) in water
(50 mL). Once the additions were finished the reaction mixture
was allowed to reach room temperature and stirred overnight.
The two layers were separated, the organic phase dried (MgSO4)
and evaporated under reduced pressure. The residue was
crystallised from chloroform–hexane to obtain the desired
compound 29 (6.84 g, 71%) as a crystalline pale yellow solid.
IR (solid state) mmax = 3103, 2160, 1979, 1618, 1539, 1398, 1134,
1015 cm−1; 1H NMR (400 MHz, CDCl3): d = 7.48 (ddd, J = 8.4,
7.0, 1.1 Hz, 1 H, ArH), 7.64 (ddd, J = 7.0, 7.0, 0.9 Hz, 1 H,
ArH), 7.73 (dt, J = 8.4, 1.0 Hz, 1 H, ArH), 7.80 (ddd, J = 8.9,
7.4, 1.5 Hz, 1 H, ArH), 7.95–8.06 (m, 4 H, ArH); 13C NMR
(100 MHz, CDCl3): d = 109.63 (ArCH), 120.81 (ArCH), 125.97
(ArCH), 126.18 (ArCH), 128.38 (ArC), 130.13 (ArCH), 133.16
(ArCH), 133.18 (ArC), 133.44 (ArCH), 137.42 (ArCH), 143.28
(ArC), 150.75 (ArC).
1
4:1); IR (solid state): mmax = 3384, 2930, 2087, 1702 cm−1; H
NMR (400 MHz, CDCl3): d = 0.80 (s, 3 H, 18-H3), 0.92 (d,
J = 5.8 Hz, 3 H, 21-H3), 0.95 (s, 3 H, 19-H3), 1.42 (s, 9 H,
(CH3)3C), 1.43 (s, 9 H, (CH3)3C), 2.20–2.25 (m, 1 H), 2.45–2.55
(m, 1 H), 3.26 (broad s, 1 H, 3b-H), 3.65 (broad s, 1 H, 7b-H),
3.73 (s, 3 H, CO2CH3), 3.99 (broad s, 1 H, 12b-H), 5.25 (broad
s, 1 H, 7-CH–NHR), 5.47 (broad s, 1 H, 12-CH–NHR); 13C
NMR (100 MHz, CDCl3): d = 13.64 (18-CH3), 17.42 (21-CH3),
22.96 (CH2), 23.00 (19-CH3), 26.91 (CH2), 27.02 (CH2), 27.60
(CH2), 28.51 ((CH3)3C × 2), 28.78 (CH), 30.64 (CH2), 31.66
(CH2), 31.93 (CH2), 34.79 (C), 35.24 (CH2 × 2), 35.54 (CH),
37.03 (CH), 41.84 (CH), 44.52 (CH), 44.71 (C), 47.22 (CH),
49.21 (CH), 52.17 (CO2CH3), 53.21 (CH), 61.75 (CH), 78.87
((CH3)3C × 2), 155.45 ((CH3)3OCONH × 2), 176.92 (CO2CH3);
Anal. found: C, 64.99; H, 9.23; N, 11.10%. C35H59N5O6 requires
C, 65.09; H, 9.21; N, 10.84%.
Methyl 3a-azido-7a-amino, 12a-[N-(o-nitrobenzenesulfonyl)amino]-
5b-cholan-24-oate 31
Diamine 21 (2.07 g, 4.65 mmol) was dissolved in dry THF
(2.5 mL) under nitrogen and 1-(2-nitrobenzenesulfonyloxy)-
benzotriazole 29 (1.48 g, 4.62 mmol) was added at room
temperature under vigorous stirring, to give a yellow paste.
Stirring was continued overnight after which the solvent was
removed. The crude solid was redissolved in DCM, washed with
a saturated aqueous Na2CO3 solution (3 × 30 mL), the organic
phase dried (MgSO4) and the organic solvent evaporated under
reduced pressure. The crude product was purified by flash
chromatography (DCM–methanol 95:5) to yield 31 (2.21 g,
75%) as a yellowish solid. Rf = 0.14 (DCM–methanol 95:5);
IR (solid state): mmax = 3350, 2941, 2089, 1732, 1360, 1150 cm−1;
1H NMR (400 MHz, CDCl3): d = 0.14 (q, J = 12.4 Hz, 1 H),
0.67–0.79 (m, 1 H), 0.81 (s, 3 H, 18-H3), 0.83 (s, 3 H, 19-H3), 1.01
(d, J = 6.6 Hz, 3 H, 21-H3), 2.19–2.41 (m, 2 H), 2.92 (m, 1 H,
3b-H), 3.10 (broad s, 1 H, 7b-H), 3.66 (s, 3 H, CO2CH3), 4.12
(d, J = 6.8 Hz, 1 H, 12b-H), 5.79 (d, J = 9.8 Hz, 1 H, NHSO2),
7.68–7.74 (m, 2 H, ArH), 7.85–7.89 (m, 1 H, ArH), 8.09–8.13 (m,
1 H, ArH); 13C NMR (100 MHz, CDCl3): d = 13.23 (18-CH3),
17.41 (21-CH3), 22.52 (19-CH3), 23.56 (CH2), 25.66 (CH2), 25.90
(CH), 26.18 (CH2), 27.32 (CH2), 28.32 (CH), 30.64 (CH2), 30.93
(CH2), 34.56 (CH2), 35.12 (CH2), 35.17 (C), 35.34 (CH), 35.86
(CH2), 39.08 (CH), 41.79 (CH), 43.73 (CH), 45.52 (C), 47.02
(CH), 51.23 (CO2CH3), 58.09 (CH), 60.93 (CH), 125.26 (ArCH),
129.71 (ArCH), 132.65 (ArCH), 133.05 (ArCH), 136.51 (ArC),
148.27 (ArC), 174.35 (CO2CH3); MS (FAB+): m/z (%): 631 (100)
Methyl 3a-azido-7a, 12a-diamino-5b-cholan-24-oate 21
To a stirred solution of the biscarbamate 10 (2.02 g, 3.13 mmol)
in dry dichloromethane (50 mL) at 0 °C was added trifluoro-
acetic acid (30 mL). The solution was stirred for 1 h at 0 °C,
then allowed to reach room temperature and stirred for a further
2 h. The solvent was evaporated under reduced pressure and the
trifluoroacetic acid removed by addition and evaporation of
further dichloromethane, to give the bis(trifluoroacetate) salt
21·2TFA as a white foam. 1H NMR (400 MHz, CDCl3): d = 0.90
(s, 3 H, 18-H3), 0.99 (d, J = 6.3 Hz, 3 H, 21-H3), 1.01 (s, 3 H,
19-H3), 2.23–2.31 (m, 2 H), 2.34–2.41 (m, 2 H), 3.27 (m, 1 H,
3b-H), 3.60 (broad s, 1 H, 7b-H), 3.68 (s, 3 H, COOCH3), 3.82
+
(broad s, 1 H, 12b-H), 7.73 (broad s, 6 H, NH3 ).
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 3 2 0 – 3 3 2 8
3 3 2 5

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[M + H]+; 588 (25) [M − N3]+; 653 (12) [M + Na]+; HRMS (ES+):
m/z calcd for [C31H46N6O6S + H]+ 631.3272 found 631.3270.
1H-NMR analysis implied that the level of contamination by
regioisomer 32 was below 2%. See electronic supplementary
information (ESI).†
products and then EtOAc–TEA 9:1) gave 12a-amino cholanoate
33 (1.57 g, 80%) as a yellowish solid. Rf = 0.33 (hexane/EtOAc
1
1:1); IR (solid state): mmax = 3394, 2934, 2090, 1699 cm−1; H
NMR (400 MHz, CDCl3): d = 0.73 (s, 3 H, 18-H3), 0.94 (s, 3 H,
19-H3), 0.97 (d, J = 6.4 Hz, 3 H, 21-H3), 1.45 (s, 9 H, (CH3)3C),
2.15–2.41 (m, 2 H), 3.14–3.21 (m, 1 H, 3b-H), 3.23 (broad s,
1 H, 12b-H), 3.67 (m, 4 H, CO2CH3 + 7b-H), 4.95 (broad s, 1 H,
7-CH–NHBoc); 13C NMR (100 MHz, CDCl3): d = 13.59 (18-
CH3), 17.17 (21-CH3), 22.91 (19-CH3), 22.95 (CH2), 27.10 (CH2),
27.48 (CH2), 28.15 (CH), 28.49 ((CH3)3C), 28.84 (CH2), 30.91
(CH2), 31.15 (CH2), 31.42 (CH2), 34.66 (C), 35.11 (CH), 35.20
(CH2), 35.31 (CH2), 36.39 (CH), 37.38 (CH), 41.94 (CH), 42.29
(CH), 46.12 (C), 47.88 (CH), 51.45 (CO2CH3), 53.66 (CH), 61.27
(CH), 79.05 ((CH3)3C), 155.50 (NHCO), 174.43 (CO2CH3); MS
(FAB+) m/z (%): 547 (100) [M + 1]+; 569 (15), [M + Na]+.
Also isolated was methyl 3a-azido-7a,12a-di[N-(o-nitro-
benzenesulfonyl)amino]-5b-cholan-24-oate 24 (P = oNs)
(381 mg, 10%). Rf = 0.45 (DCM/methanol 95:5); IR (solid
state): mmax = 3265, 3097, 2951, 2872, 2090, 1732, 1699, 1540,
1
1442, 1423, 1353, 1162, 742, 730 cm−1; H NMR (400 MHz,
CDCl3): d = 0.16 (q, J = 12.7 Hz, 1 H), 0.80 (s, 3 H, 18-H3),
0.86 (s, 3 H, 19-H3), 0.96 (d, J = 6.8 Hz, 3 H, 21-H3), 2.17–2.25
(m, 1 H), 2.32–2.40 (m, 1 H), 2.87 (m, 1 H, 3b-H), 3.67 (s, 3 H,
CO2CH3), 3.78 (broad s, 1 H, 7b-H), 4.09 (d, J = 8.8 Hz, 1 H,
12b-H), 5.48 (d, J = 7.8 Hz, 1 H, 7-CH–NHSO2), 5.65 (d,
J = 8.8 Hz, 1 H, 12-CH–NHSO2), 7.68–7.75 (m, 4 H, ArH),
7.87–7.90 (m, 2 H, ArH), 8.08–8.11 (m, 2 H, ArH); 13C NMR
(100 MHz, CDCl3): d = 14.05 (18-CH3), 17.67 (21-CH3),
23.22 (19-CH3), 25.98 (CH2), 26.24 (CH2), 27.54 (CH2), 29.69
(CH), 30.99 (CH), 31.34 (C), 35.22 (CH), 35.60 (CH2), 40.42
(CH), 44.30 (CH), 46.01 (C), 48.05 (CH), 51.78 (CO2CH3), 57.74
(CH), 60.76 (CH), 61.09 (CH), 121.26 (ArCH), 121.42 (ArCH),
125.93 (ArCH), 126.09 (ArCH), 129.78 (ArCH), 133.03 (ArCH),
133.05 (ArCH), 174.79 (CO2CH3); MS (CI+): m/z (%) = 817 (7)
[M + H]+, 788 (19) [M − N2]+.
Compound 34
In a dry round bottomed flask N-Fmoc–L-phenylalanine
(355 mg, 0.92 mmol), TBTU (295 mg, 0.92 mmol), HOBt
(124 mg, 0.92 mmol) and DIPEA (160 lL, 0.92 mmol) were
dissolved in dry DMF (0.5 mL) and the solution was vigorously
sonicated for 5 min. To this activated solution was then
added amine 33 (201 mg, 0.37 mmol) dissolved in dry DCM
(0.2 mL). The reaction mixture was stirred for 24 h at room
temperature and under nitrogen atmosphere before the solvent
was removed under reduced pressure, the crude was washed
with brine, extracted with DCM, and dried over MgSO4. Flash
chromatography (diethyl ether–TEA 99:1) gave 34 (197 mg,
60%) as a white solid. Rf = 0.75 (diethyl ether–TEA 99:1) IR
(solid state): mmax = 3380, 2950, 2091, 1700, 1513, 1450, 1365,
Methyl 3a-azido-12a-N-(o-nitrobenzenesulfonyl)–amino-7a-[N-
(t-butyloxycarbonyl)–amino]-5b-cholan-24-oate 13
Amine 31 (2.50 g, 3.96 mmol) was dissolved in THF (34 mL)
and an aqueous saturated solution of NaHCO3 (17 mL). To
the solution, di-tert-butyl dicarbonate (1.73 g, 7.92 mmol) was
added and the reaction mixture stirred at room temperature for
4 d. The two layers were separated and the aqueous phase was
extracted with ethyl acetate. The organic extracts were dried
with MgSO4 and evaporated under reduced pressure. The crude
material was precipitated from diethyl ether–petroleum ether to
yield 13 (2.6 g, 90%) as a white solid. Mp 148–150 °C; Rf = 0.21
(hexane–EtOAc 3:1); IR (solid state): mmax = 3389, 2933, 2089,
1699, 1363, 1160 cm−1; 1H NMR (400 MHz, CDCl3): d = 0.22 (q,
J = 12.0 Hz, 1 H), 0.82 (s, 3 H, 18-H3), 0.88 (s, 3 H, 19-H3), 0.92
(d, J = 6.2 Hz, 3 H, 21-H3), 1.49 (s, 9 H, C(CH3)3), 2.14–2.22 (m,
1 H), 2.32–2.40 (m, 1 H), 2.96–3.04 (m, 1 H, 3b-H), 3.67 (broad s,
4 H, CO2CH3 + 7b-H), 4.05–4.08 (m, 1 H, 12b-H), 4.61 (broad s,
1 H, 7-CH–NHBoc), 5.59 (d, J = 9.1 Hz, 1 H, 12-CH–NHNos),
7.70–7.77 (m, 2 H, ArH), 7.86–7.88 (m, 1 H, ArH), 8.11–8.13
(m, 1 H, ArH); 13C NMR (100 MHz, CDCl3): d = 13.59 (18-
CH3), 17.33 (21-CH3), 22.81 (19-CH3), 23.34 (CH2), 25.92 (CH2),
26.33 (CH2), 27.25 (CH2), 28.22 ((CH3)3C), 28.42 (CH), 30.66
(CH2), 31.00 (CH2), 31.57 (C), 31.59 (CH), 34.89 (CH2), 35.02
(CH2), 35.21 (CH2), 35.33 (CH), 37.06 (CH), 41.44 (CH), 44.50
(CH), 45.64 (C), 46.82 (CH), 47.70 (CH), 51.48 (CO2CH3),
57.84 (CH), 60.55 (CH), 79.52 ((CH3)3C), 125.65 (ArCH),
129.82 (ArCH), 132.79 (ArCH), 133.34 (ArCH), 138.59 (ArC),
148.61 (ArC), 155.42 (NHCO), 174.46 (CO2CH3); MS (FAB+):
m/z (%) 753 (100) [M + Na]+; 631 (45) [M − Boc]+; Anal. found:
C, 59.56; H, 7.77; N, 11.18%. C36H54N6O8S requires: C, 59.16;
H, 7.45; N, 11.50%.
1
1247, 1165, 1053 cm−1; H NMR (400 MHz, CDCl3): d = 0.62
(d, J = 6.5 Hz, 3 H, 21-H3), 0.69 (s, 3 H, 18-H3), 0.90 (s, 3 H,
19-H3), 1.44 (s, 9 H, C(CH3)3), 2.01–2.16 (m, 1 H), 2.21–2.36
(m, 1 H), 3.06–3.21 (m, 3 H, PheCH2 + 3b-H), 3.65 (m, 4 H,
CO2CH3 + 7b-H), 3.90–4.58 (m, 5 H, FmocCH2 + 2 H + 12b-
H), 5.12 (broad s, 1 H, NH), 5.34 (broad s, 1 H, NH), 6.98–7.74
(m, 13 H, ArH); 13C NMR (100 MHz, CDCl3): d = 13.3 (18-
CH3), 17.1 (21-CH3), 23.1 (19-CH3), 23.2 (CH2), 24.5 (CH2),
28.3 (CH2), 28.5 (CH2), 28.6(C(CH3)3), 28.8 (CH), 30.8 (CH2),
31.1 (CH2), 34.8(C), 34.9 (CH), 33.6 (CH2), 35.7 (CH2), 35.8
(CH2), 37.2 (CH2), 39.2 (CH), 41.9 (CH), 44.2 (CH), 44.3 (CH),
44.8 (C), 45.6 (CH), 48.6 (CH), 51.5 (CO2CH3), 52.1 (CH), 55.0
(CH), 61.8 (CH), 66.6(CH2), 77.5 (C(CH3)3), 119.5 (ArCH),
126.8 (ArCH), 127.4 (ArCH), 128.0 (ArCH), 128.9 (ArCH),
137.3 (ArC), 141.2 (ArC), 143.8 (ArC), 155.4 (NHCO), 157.0
(NHCO), 173.4 (C), 174.5 (CO2CH3); MS (FAB+): m/z (%):
815 (100) [M − Boc]+, 937 (75) [M + Na]+, 915 (48) [M + 1]+;
HRMS (FAB+): m/z calcd. for [C54H70N6O7 + Na]+: 937.5204,
found 937.5201.
Compound 35
Compound 34 (200 mg, 0.22 mmol) was dissolved in DCM
(50 mL). TFA (5 mL) was added at 0 °C and the reaction was
stirred for 2 h. The solvent was evaporated under reduced
pressure and the resulting pale yellow foam was dissolved again
in DCM (15 mL), washed with saturated aqueous Na2CO3
(2 × 10 mL), and dried over MgSO4. Evaporation gave the
7a-amine (170 mg, 95%) as a pale yellow solid. Rf = 0.39 (ethyl
acetate–TEA 9.9:0.1); IR (solid state): mmax = 2917, 2090,
Methyl 3a-azido-7a-[N-(t-butyloxycarbonyl)–amino]-12a-
amino-5b-cholan-24-oate 33
1
1734, 1448, 1251, 1167 cm−1; H NMR (400 MHz, CDCl3,):
d = 0.66 (s, 3 H, 18-H3), 0.85–2.30 (m, 28 H), 3.00–3.21 (m, 4 H,
PheCH2 + 7b-H + 3b-H), 3.63 (s, 3 H, CO2CH3), 3.90–4.58
(m, 5 H, FmocCH2 + 2 H + 12b-H), 5.30 (broad s, 1 H, NH),
6.98–7.74 (m, 13 H, ArH).
To a solution of the above amine (150 mg, 0.18 mmol) in dry
DCM (0.2 mL) was added a pre-activated mixture (sonicated
for 5 min) of N-Boc–L-valine (80 mg, 0.37 mmol), TBTU
(115 mg, 0.36 mmol), HOBt (48 mg, 0.36 mmol) and DIPEA
(62 ll, 0.36 mmol) in dry DMF (0.4 mL). The reaction mixture
was stirred for 24 h at room temperature and under nitrogen
Thiophenol (3.65 mL, 35.5 mmol) was added to a suspension
of the scaffold 13 (2.6 g, 3.56 mmol) and caesium carbonate
(6.9 g, 21.2 mmol) in dry DMF (32 mL) under nitrogen at
room temperature. The reaction was heated at 55 °C overnight
before removing the DMF under reduced pressure. The mixture
obtained was dissolved in ethyl acetate, washed with saturated
aqueous NaHCO3, then brine, dried with MgSO4 and evaporated
under reduced pressure. Purification of the crude product by
flash chromatography (hexane–EtOAc 1:1 to remove all the by-
3 3 2 6
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 3 2 0 – 3 3 2 8

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atmosphere before the solvent was removed under reduced
pressure. The residue was dissolved in DCM, washed with brine
and dried over MgSO4. Flash chromatography (diethyl ether–
TEA 99:1) gave 35 (156 mg, 85%) as a white solid. Rf = 0.64
(ethyl acetate–TEA 99:1); IR (solid state): mmax = 3276, 2928,
2089, 1660, 1533, 1449, 1366, 1297, 1249, 1168, 1043 cm−1;
1H NMR (400 MHz, CDCl3): d = 0.60 (d, J = 6.5 Hz, 3 H,
21-H3), 0.74 (s, 3 H, 18-H3), 0.89(s, 3 H, 19-H3), 0.91–2.01 (m,
26 H), 1.46 (s, 9 H, C(CH3)3), 2.02–2.14 (m, 1 H), 2.20–2.58
(m, 2 H), 3.00–3.21 (m, 3 H, PheCH2 + 3b-H), 3.64 (m, 4 H,
CO2CH3 + 7b-H), 4.01–4.62 (m, 6 H, FmocCH2 + 2 H + 12b-
H + CH Val), 5.12 (broad s, 1 H, NH), 5.57 (broad s, 1 H, NH),
6.98–7.74 (m, 14 H, ArH + NH); 13C NMR (100 MHz, CDCl3):
d = 13.2 (18-CH3), 17.0 (21-CH3), 17.5 (CH3 Val), 19.7 (CH3
Val), 22.7 (19-CH3), 23.3 (CH2), 24.5 (CH2), 28.2 (CH2), 28.3
(C(CH3)3), 28.5 (CH2), 28.8 (CH), 30.4 (CH2), 31.3(CH), 31.9
(CH2), 34.7(C), 34.9 (CH), 35.7 (CH2), 35.8 (CH2), 37.2 (CH2),
39.0 (CH), 42.0 (CH), 44.2 (CH), 44.3 (CH), 44.8 (C), 46.2
(CH), 47.0(CH), 48.6 (CH), 51.5 (CO2CH3), 52.1 (CH), 55.0
(CH), 58.4(C), 61.1 (CH), 67.5(CH2), 80.1(CH), 119.5 (ArCH),
126.8 (ArCH), 127.4(ArCH), 128.0 (ArCH), 128.9 (ArCH),
136.6 (ArC), 141.3 (ArC), 143.7(ArC), 156.4 (NHCO), 157.0
(NHCO), 173.4 (C), 170.8 (C), 174.5 (CO2CH3). MS (FAB+):
m/z (%) 915, (100) [M − Boc]+, 1015 (80) [M + H]+, 1037 (64)
[M + Na]+; HRMS (FAB+): m/z calcd. for [C59H79N7O8 + Na]+:
1036.5889, found 1036.5883.
Methyl 3a-azido-12a-N-(o-nitrobenzenesulfonyl)–amino-7a-[N-
(allyloxycarbonyl)–amino]-5b-cholan-24-oate 14
Amine 31 (438 mg, 0.69 mmol) was dissolved in THF (7 mL) and
an aqueous saturated solution of NaHCO3 (3 mL) was added.
Allyl chloroformate (136 mg, 120 lL, 1.13 mmol) was added and
the reaction mixture was stirred for 4 d at room temperature.
The solvent was removed under reduced pressure, the residue
was redissolved in DCM and washed with water and the organic
phases were combined and dried with MgSO4. Flash chromato-
graphy (DCM to DCM–EtOAc 1:1) afforded 14 (409 mg, 83%)
as a white solid. Rf = 0.85. (EtOAc–MeOH–TEA 9:1:0.09); 1H
NMR (400 MHz, CDCl3): d = 0.23 (q, J = 11.9 Hz, 1 H), 0.82
(s, 3 H, 18-H3), 0.88 (s, 3 H, 19-H3), 0.91 (d, J = 6.6 Hz, 3 H, 21-
H3), 1.03–1.15 (m, 2 H), 2.16–2.27 (m, 1 H); 2.31–2.39 (m, 1H),
2.99–3.07 (m, 1 H, 3b-H), 3.66 (s, 3 H, CO2CH3), 3.77 (broad
s, 1 H, 7b-H), 4.06 (d, J = 9.0 Hz, 1 H, 12b-H), 4.59 (m, 2 H,
CO2CH2CHCH2), 4.86 (d, J = 8.7 Hz,1 H, NHAlloc), 5.24
(dd, J = 10.6, 0.9 Hz, 1 H, CH alkene), 5.34 (d, J = 17.6 Hz, 1 H,
CH alkene), 5.57 (d, J = 8.6 Hz, 1 H, NHSO2), 5.92–6.01 (m,
1 H, CO2CH2CHCH2), 7.69–7.76 (m, 2 H, ArH), 7.82–7.85
(m, 1 H, ArH), 8.09–8.12 (m, 1 H, ArH); 13C NMR (100 MHz,
CDCl3): d = 13.6 (18-CH3), 17.3 (19-CH3), 22.8 (21-CH3), 23.3
(CH2), 25.9 (CH2), 26.2 (CH2), 27.2 (CH2), 28.2 (CH), 30.6 (CH2),
30.9 (CH2), 31.8 (CH2), 34.8 (C), 35.0 (CH2), 35.3 (CH2), 35.4
(CH). 36.9 (CH), 41.4 (CH), 44.4 (CH), 45.6 (CH), 47.6 (7-CH),
51.5 (CO2CH3), 57.8 (CH), 60.7 (3-CH), 65.5 (OCH2CHCH2),
117.4 (OCH2CHCH2), 125.4 (OCH2CHCH2); 129.9 (ArCH);
132.8 (ArC), 132.9(ArCH); 133.4 (ArCH); 134.4 (ArC);
136.3 (ArC), 148.5 (NHCOO), 174.6 (CO2CH3); MS (ES−):
m/z (%) 713.5, (100) [M − H]−; HRMS (FAB+): m/z calcd. for
[C35H50N6O8S + NH4]+ 732.3749 found 732.3747.
Compound 36
Azide 35 (150 mg, 0.15 mmol) was dissolved in dry THF (3 mL).
Trimethylphosphine (1.0 M in THF, 180 mL, 0.18 mmol) was
added to the solution of the steroid and the mixture was stirred
at room temperature under nitrogen for 5 h before water (53 lL,
2.9 mmol) was added and the reaction was stirred overnight.
The solvents were removed by evaporation, addition of toluene
and re-evaporation to obtain the 3a-amine as a solid. The
formation of the product was monitored by TLC (Rf = 0.05,
ethyl acetate–TEA 99:1) and then confirmed by MS (FAB+):
m/z (%) 989 (62) [M + H]+, 1011 (30) [M + Na]+.
Acknowledgements
Financial support was provided by the European Commission,
EPSRC (GR/R42757) and BBSRC (7/B16122). Assistance
from the EPSRC National Mass Spectroscopy Service Centre,
Swansea, is gratefully acknowledged.
The above amine was dissolved in dry DCM (0.2 mL).
A mixture of N-Boc–glycine (53 mg, 0.30 mmol), TBTU
(96 mg, 0.30 mmol), HOBt (40 mg, 0.30 mmol) and DIPEA
(52 lL, 0.3 mmol), previously activated (sonicated for 5 min)
in 0.4 mL of dry DMF, was added to the solution of the steroid
and the resulting mixture was stirred for 24 h under nitrogen.
After removal of the solvents, the crude product was solved
in DCM, washed with brine, and dried over MgSO4. Flash
chromatography (diethyl ether–TEA 99:1) gave 36 (124 mg, 72%
over two steps) as a white solid. Rf = 0.72 (ethyl acetate–TEA
99:1); IR (solid state): mmax = 3789, 3696, 3661, 3298, 2958, 1659,
1534, 1450, 1250, 1367, 1250, 1169 cm−1; 1H NMR (400 MHz,
CDCl3): d = 0.60–2.58 (m, 38 H), 1.46 (s, 9 H, C(CH3)3),1.48
(s, 9 H, C(CH3)3), 3.01–3.29 (m, 2 H, PheCH2), 3.64 (m, 4 H,
CO2CH3 + 7b-H), 4.01–4.62 (m, 9 H, FmocCH2 + 2 H + 12b-
H + 3b-H + CH Val + CH2 Gly), 5.13 (broad s, 1 H, NH), 5.47
(broad s, 1 H, NH), 5.57 (broad s, 1 H, NH), 6.98–7.74 (m, 14 H,
ArH + NH), 7.90 (broad s, 1 H, NH); 13C NMR (100 MHz,
CDCl3): d = 13.1 (18-CH3), 17.2 (21-CH3), 17.4 (CH3), 19.7
(CH3), 22.5 (19-CH3), 23.3 (CH2), 24.5 (CH2), 28.1 (CH2), 28.3
(C(CH3)3 × 2), 28.6 (CH2), 28.8 (CH), 30.2 (CH2), 31.3 (CH),
32.0 (CH2), 34.7 (C), 34.9 (CH), 35.6 (CH2), 35.8 (CH2), 37.2
(CH2), 39.0 (CH), 42.0 (CH), 42.7 (CH2), 44.2 (CH), 44.3 (CH),
44.8 (C), 46.2 (CH), 47.0 (CH), 48.6 (CH), 51.5 (CO2CH3), 52.0
(CH), 55.2 (CH), 58.4 (C), 61.1 (CH), 67.5 (CH2), 80.1 (CH),
81.0 (C), 119.5 (ArCH), 126.8 (ArCH), 127.4(ArCH), 128.0
(ArCH), 128.9 (ArCH), 136.6 (ArC), 141.3 (ArC), 143.7 (ArC),
156.1 (NHCO), 156.4 (NHCO), 157.0 (NHCO), 173.4 (C), 170.8
(C), 174.5 (CO2CH3), 174.8 (C); MS (FAB+): m/z (%): 1145 (60)
[M + H]+, 1045 (50) [M − Boc], 1167 (32) [M + Na]+; HRMS
(FAB+): m/z calcd. for [C66H92N6O11 + H]+ 1145.6902 found
1145.6920.
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