S. N ꢀe grel and J.M. Brunel
Tetrahedron 82 (2021) 131954
þ
þ
5
8
14
. General procedure for the synthesis of derivatives 12 and
m/z ([MþH] ) for C34
H
50
N
2
O
: calcd 569.3949, found 569.3953.
8
C1)
.4. Synthesis of 3-spermino methyl chenodeoxycholate (Claramine
8.1. Synthesis of methyl 3-oxo-chola-L-phenylalalinate 12
In a 10e20 mL microwave reaction vial were introduced in
0 mL of toluene and 6 mL of acetone, 299 mg of methyl chola-L-
In a 100 mL two necked round flask was introduced methyl 3-
oxo-chenodeoxycholate 2 (3.5 g, 8.6 mmol) dissolved into 50 mL
of methanol. 4 equivalents of titanium (IV) isopropoxide (10 mL,
4 mmol), then 3 equivalents of spermine (5.3 g, 26 mmol) were
1
phenylalaninate 11 (0.52 mmol) and 2 equivalents of aluminum
sec-butoxide (274 mg, 1.1 mmol). Microwave reaction vial was
sealed with a cap and a septum. Reaction was performed under
3
subsequently introduced. The mixture was stirred at room tem-
ꢀ
microwave irradiations (400 Watt) at 150 C for 10 min using a
ꢀ
perature for 24 h. After cooling at 0 C, 4 equivalents of sodium
normal mode and a pre-stirring of 20 s 5 mL of NH
solution was added and the mixture was stirred for 15 min. Reac-
4
ꢁOH (30%)
borohydride (1.3 g, 34 mmol) were then added, and the resulting
mixture was stirred for additional 2 h. The reaction was then
quenched by adding water (4 mL). Stirring was continued at room
temperature for 1 h then the reaction mixture was filtrated over a
tion mixture was filtered over a pad of Celite which was rinsed with
CH
pected methyl 3-oxo-chola-
yellow oil in 81% yield after filtration.
(ppm) ¼ 7.23e6.88 (m, 3H), 6.80e6.57 (m, 2H), 4.95 (s, 1H), 4.79
s, 1H), 4.57 (q, 1H), 4.24e3.74 (m, 4H), 3.65 (s, 2H), 3.31 (s, 4H),
2
Cl
2
. The mixture was concentrated in vacuo to afford the ex-
L
-phenylalalinate 12 obtained as a pale
pad of Celite which was subsequently rinsed with NH
and methanol. The mixture was concentrated in vacuo to afford the
expected crude compound which was purified by flash chroma-
4
ꢁOH (32%)
1
H
3
NMR (CD OD):
d
(
3
3
1
4
tography on silica gel using CH
2
Cl
2
/MeOH/NH
4
ꢁOH (32%) 7:3:1 as
.24e2.98 (m, 2H), 2.85e2.35 (m, 6H), 2.24e1.45 (m, 20H), 0.68 (t,
eluent. The expected Claramine C1 was obtained as a viscous yellow
1
3
H). C NMR (CD
3
OD):
d
(ppm) ¼ 216.26, 176.49, 173.69, 157.32,
1
oil in 35% yield. H NMR (CD
3
OD):
d
(ppm) ¼ 3.84 (m, 1H), 3.69 (s,
31.10, 128.66, 116.23, 73.73, 68.70, 55.32, 52.60, 47.49, 46.55, 44.71,
2
0
d
H), 3.02e3.44 (m, 3H), 2.92e2.35 (m, 14H), 2.03e2.31 (m, 3H),
.98e1.98 (m, 40H), 0.74 (s, 3H).
2.89, 40.87, 37.74, 37.58, 36.94, 36.71, 35.96, 35.80, 35.01, 33.73,
3.07, 29.82, 29.57, 28.33, 26.25, 24.12, 22.15, 17.72, 13.07, 9.75.
13
C
3
NMR (CD OD):
3
(ppm) ¼ 176.53, 69.02, 59.43, 577.66, 52.06, 51.08, 48.28, 45.83,
5.72, 43.90, 43.64, 41.28, 41.07, 40.70, 40.64, 36.82, 36.72, 36.02,
4.35, 32.46, 32.15, 31.70, 29.27, 27.92, 24.73, 23.56, 21.96, 18.98,
þ
þ
6
:
HRMS (ESI-TOF): m/z ([M
85.3898, found 585.3898.
þ
NH
4
] ) for
C
34
50
H N
2
O
calcd
4
3
1
5
þ
þ
2.37. HRMS (ESI-TOF): m/z ([MþH] ) for C35
67 4
H N O : calcd
3
8
.2. Synthesis of methyl 3-oxo-chola-L-glycinate 14
5
91.5168, found 591.5169.
.5. Determination of minimal inhibitory concentrations
Antimicrobial activity of the compounds was studied by deter-
Performed according the same procedure than involved for the
synthesis for 12. The expected product methyl 3-oxo-chola- -gly-
cinate 14 was obtained as yellow oil in a quantitative yield. H NMR
CD OD):
.63e1.49 (m, 24H), 0.73 (t, 3H).
8
L
1
(
2
d
3
d
(ppm) ¼ 5.11e4.60 (m, 11H), 4.06e3.68 (m, 5H),
13
mination of minimal inhibitory concentrations (MIC) according to
the NCCLS guidelines M7-A2 using the microbroth dilution
C
3
NMR (CD OD):
(ppm) ¼ 216.57, 177.31, 171.16, 73.85, 68.79, 48.06, 47.60, 46.62,
4.92, 43.02, 42.25, 40.98, 36.89, 36.08, 35.05, 33.79, 33.14, 29.81,
8.71, 28.38, 24.16, 22.13, 22.00, 19.70,17.70, 13.03, 9.98. HRMS (ESI-
methods. The bacteria strains were grown on trypticase soy agar
4
2
ꢀ
(
Becton Dickinson) at 37 C for 24 h. Inocula were prepared in TCE
þ
þ
27 6
C H45NO : calcd 478.3124, found
(tryptone 0.1%, NaCl 8%, wt/vol) by adjusting the turbidity at
TOF): m/z ([MþH] ) for
5
6
23 nm to obtain 1-3 10 CFU/mL. Antimicrobial activities of the
4
78.3162.
Claramine C1 were determined by using a broth microdilution
method performed in sterile 96-well microplates. The compound
was solubilized in water at a concentration of 10 mM and were
transferred to each microplate well in order to obtain a two-fold
8
16
.3. Synthesis of methyl 3-oxo-chenodeoxychola-L-phenylalalinate
serial dilution in 100 mL of broth and 100 mL of inoculum contain-
In a 10e20 mL microwave reaction vial were introduced in
0 mL of toluene and 6 mL of acetone, 250 mg of methyl cheno-
deoxychola- -phenylalalinate 15 (0.45 mmol) and 4 equivalents of
5
ing 2e6 10 CFU of each bacterium were added to each well. Some
wells were reserved for positive controls and inoculum viability.
After 24 h incubation, MIC was defined for each agent from
duplicate observations as the lowest concentration of compound
allowing no visible growth.
1
L
aluminum sec-butoxide (291 mg, 1.8 mmol). Microwave reaction
vial was sealed with a cap and a septum. Reaction was performed
ꢀ
under microwave irradiations (400 Watt) at 150 C for 1 h using a
normal mode and a pre-stirring of 20 s 5 mL of H
was added and the mixture was stirred for 15 min. The organic
phase was washed with 5 mL of NaHCO (10%). The aqueous phases
were extracted twice with 5 mL of dichloromethane and the
combined organic phases where dried over Na SO and filtered.
The mixture was concentrated in vacuo to afford the expected crude
compound which was purified by chromatography on silica gel
using petroleum ether-ethylacetate (1:1) as eluent. The expected
2 4
SO (2N) solution
Declaration of competing interest
3
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
2
4
Acknowledgments
product methyl 3-oxo-chenodeoxychola-
obtained as yellow oil in 58% yield.
(ppm) ¼ 7.49e7.37 (m, 5H), 5.61 (s,1H), 3.95e3.93 (m,1H), 3.77 (s,
L
-phenylalalinate 16 was
1
a
3
H NMR (CD OD):
S. N. thanks Biosqual company for a CIFRE PhD grant.
d
1
3
3
H), 3.60 (q, 1H), 2.73e0.94 (m, 35H), 0.82e0.75 (m, 3H). C NMR
OD):
(ppm) ¼ 215.95, 176.04, 172.52, 137.45, 129.81, 129.43,
28.69, 68.65, 57.97, 57.22, 52.95, 51.34, 46.52, 44.66, 43.59, 42.12,
(
CD
3
d
Appendix A. Supplementary data
1
4
2
0.82, 40.60, 37.87, 37.66, 36.68, 36.31, 35.05, 34.42, 33.43, 29.53,
9.15, 24.52, 22.42, 22.09, 18.98, 18.15, 15.11, 12.34. HRMS (ESI-TOF):
7