Synthesis and antimicrobial evaluation of water-soluble, dendritic derivatives of epimeric 5α-cholestan-3-amines and 5α-cholestan-3-yl aminoethanoates

Article abstract of DOI:10.1016/j.steroids.2007.04.001

To examine the effect of negatively charged steroidal amphiphiles on antimicrobial activity, two pairs of epimeric, dendritic tricarboxylato amphiphiles - 4-(2-carboxyethyl)-4-[3-(5α-cholestan-3-yl)ureido]heptanedioic acid (1) and 4-(2-carboxyethyl)-4-[3-(5α-cholestan-3-yloxycarbonylmethyl)ureido]heptanedioic acid (2) - were synthesized. A broad antimicrobial screen of 11 microbes revealed that these amphiphiles only showed good activity against a methicillin-resistant isolate of Staphylococcus aureus (MRSA) and modest activity against an unrelated strain of S. aureus. The best activity, a minimal inhibitory concentration (MIC) of 27 μM, was found for the 3β epimer of 1 against MRSA.

Full text of DOI:10.1016/j.steroids.2007.04.001

steroids 7 2 ( 2 0 0 7 ) 615–626
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Synthesis and antimicrobial evaluation of water-soluble,
dendritic derivatives of epimeric 5␣-cholestan-3-amines
and 5␣-cholestan-3-yl aminoethanoates
a
a
b
a,∗
Eko W. Sugandhi , Carla Slebodnick , Joseph O. Falkinham III , Richard D. Gandour
a
Department of Chemistry MC 0212, Virginia Tech, Blacksburg, VA 24061, United States
Department of Biological Sciences MC 0406, Virginia Tech, Blacksburg, VA 24061, United States
b
a r t i c l e i n f o
a b s t r a c t
Article history:
To examine the effect of negatively charged steroidal amphiphiles on antimicrobial activity,
two pairs of epimeric, dendritic tricarboxylato amphiphiles – 4-(2-carboxyethyl)-4-[3-(5␣-
cholestan-3-yl)ureido]heptanedioic acid (1) and 4-(2-carboxyethyl)-4-[3-(5␣-cholestan-3-
yloxycarbonylmethyl)ureido]heptanedioic acid (2) – were synthesized. A broad antimicrobial
screen of 11 microbes revealed that these amphiphiles only showed good activity against a
methicillin-resistant isolate of Staphylococcus aureus (MRSA) and modest activity against an
unrelated strain of S. aureus. The best activity, a minimal inhibitory concentration (MIC) of
27 ␮M, was found for the 3␤ epimer of 1 against MRSA.
Received 9 February 2007
Received in revised form
2
April 2007
Accepted 5 April 2007
Published on line 8 April 2007
Keywords:
Cholestan-3-amine
Dendritic amphiphile
Antimicrobial
© 2007 Elsevier Inc. All rights reserved.
1
.
Introduction
show selective activity against a broad spectrum of microbes.
Very long fatty chains, up to 22 carbon atoms comprise
Steroidal amphiphiles based on aminosterols have antibiotic
properties. Since the discovery of squalamine [1], a C-24-
sulfated cholestane conjugated to spermidine at C-3␤, several
researchers have made [2–9] or isolated [10] sterol-polyamine
conjugates that exhibit antimicrobial activity against Gram-
positive bacteria, Gram-negative bacteria, and yeast. Other
aminosterols linked to aminoglycosides [11] and aromatic
amides [12] show antimicrobial activity. The attachment of
polyamines to a steroid moiety enhances the aqueous sol-
ubility through the multiple positive charges created by
protonated amines. A dendritic polyamine-sterol [13] medi-
ates ion transport.
these one-tailed amphiphiles; two chains up to 12 car-
bons comprise the two-tailed amphiphiles. These trianionic
amphiphiles with the Newkome-type first-generation den-
dron [16] as a headgroup have >20 mM solubility in aqueous
triethanolamine for the longest chains. As a result of the rela-
tively high solubilities of these amphiphiles that contain very
hydrophobic chains, we can probe the inherent chain-length
specificities of antimicrobial activities without resorting to
organic solvents, suspensions, or emulsions to deliver the
agent.
To continue our probe of the structure of the hydrophobe
on antimicrobial selectivity and to examine if negatively
charged steroidal amphiphiles have antimicrobial activity,
we have synthesized two epimeric pairs of cholestanyl
amphiphiles, 1 and 2, and measured their antimicrobial
To overcome the challenge of low solubilities of saturated
fatty acids, we have synthesized one-tailed [14] and two-tailed
[
15], water-soluble, dendritic tricarboxylato amphiphiles that
∗
Corresponding author. Tel.: +1 540 231 3731; fax: +1 540 231 3255.
E-mail address: gandour@vt.edu (R.D. Gandour).
0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2007.04.001

6
16
steroids 7 2 ( 2 0 0 7 ) 615–626
activity against a broad spectrum of bacteria, yeast, and
fungi. Herein, we report the details of the syntheses and the
results of the antimicrobial assays. Further, we report the
X-ray crystal structure of the immediate precursor to 1a, a
cholestane with a bulky group attached at C-3.
2.2.
Chemical synthesis
2.2.1. 3ˇ-Methanesulfonyloxy-5˛-cholestane (4)
A 100-mL round bottom flask containing a stirred solu-
tion of 3b (5.12 g, 12.9 mmol) and Et N (2.55 mL, 18.2 mmol)
3
in dry CH2Cl2 was cooled in an ice–salt bath. When the
◦
temperature reached 0 C, MsCl (1.2 mL, 2.96 g, 15.4 mmol)
was added slowly through a syringe. Upon completion of
the addition, the solution was stirred for 15 min while
maintaining the low temperature. The resulting reaction
was quenched by washing successively with water (5 mL),
2-M HCl (5 mL), water (5 mL), saturated NaHCO3 (5 mL),
and water (5 mL). The organic solution was dried with
MgSO4, filtered, and concentrated to give an off-white solid
(
(
5.71 g, 12.2 mmol, 94%), which gave a single spot on TLC
CH2Cl2, R = 0.70). The 1H NMR spectrum of this crude
f
product suggested that the material was suitable for the
next step without further purification. 1H NMR (CDCl3):
ı 0.60–2.00 (m, 46H), 3.00 (s, 3H), 4.62 (m, 1H) (lit. [18]
2
2
3
8
00 MHz); 13C NMR (CDCl3): ı 12.1, 12.2, 18.7, 21.2, 22.6,
2.8, 23.8, 24.2, 28.0, 28.2, 28.5, 28.7, 31.9, 35.2, 35.3, 35.4,
5.8, 36.2, 36.8, 38.9, 39.5, 39.9, 42.6, 44.9, 54.1, 56.3, 56.4,
2.3.
2
.
Experimental
2.2.2. 3˛-Bromo-5˛-cholestane (5)
2
.1.
General
To a stirred solution of crude 4 (4.37 g, 9.36 mmol) in dry
THF (40 mL), LiBr (2.06 g, 23.5 mmol) was slowly added. The
◦
Chemicals were obtained from Sigma–Aldrich Corp. (St.
Louis, MO), Fischer Scientific/Acros Organics (Pittsburgh, PA)
and TCI America (Portland, OR); they were used without
further purification. Solvents were reagent grade or HPLC
grade; they were used as received unless otherwise spec-
ified. THF was distilled from sodium/benzophenone ketyl.
suspension was heated under reflux at 70 C for 14 h. The
resulting solution was concentrated, and the residue was
extracted into hexane (50 mL). The undissolved solid was fil-
tered off and the filtrate was concentrated to give a white
solid, which was recrystallized from EtOH to give a white
◦
◦
solid (3.15 g, 74%): mp 103.5–103.9 C (lit. mp 100–102 C [19],
WeisocyanateTM [O
prepared as described [17], except with a shorter reaction time
15 min). Analytical thin layer chromatography was performed
C
N–C(CH2CH2CO2 Bu)3, WeNCO] was
t
◦
1
102–104 C [20]); H NMR (CDCl3): ı 0.60–2.00 (m, 46H), 4.74
(m, 1H) (lit. 60 MHz [21], 400 MHz [20]); 13C NMR (CDCl3): ı
12.1, 13.3, 18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 27.9, 28.0, 28.2, 31.0,
31.8, 32.9, 35.4, 35.8, 36.17, 36.23, 37.3, 39.5, 39.95, 40.13, 42.6,
53.9, 56.16, 56.24, 56.4 (lit. [20] 100 MHz); IR: 2942, 2923, 2844,
694 (lit. [20] film); [␣]D25 +29.4 (c 1.62, CHCl3) (lit. [19] [␣]D
+26 (c 1.2, CHCl3)); HRMS: for C27H47Br calcd 450.2861, found
(
on polyester-coated silica gel 60 A˚ ; spots were visualized by
treating the plates with 10% ethanolic phosphomolybdic acid
reagent (20 wt.% solution in ethanol) followed by heating with
a heat gun for several minutes until the spots darkened. Flash
column chromatography was carried out on silica gel (60 A˚ );
◦
◦
450.2851.
samples were loaded as concentrated solutions in the elu-
ent; column diameter × height (1 3/4 in. × 6 in.), eluted samples
varied between ∼2.00 and 4.00 g, flow rate (∼1.5–2 in./min)
was controlled by air pressure. Solutions were concentrated
by rotary evaporation. Melting-point ranges were determined
in open capillary tubes and uncorrected. NMR spectra were
recorded at 400 and 100 MHz for 1H and 13C, respectively, and
reported in ppm relative to the known solvent residual peak.
Resonances were reported in the order of the chemical shifts
2.2.3. 3˛-Azido-5˛-cholestane (6a)
To a 250-mL round bottom flask containing a solution of crude
4 (4.97 g, 10.7 mmol) in DMF (75 mL), NaN3 (3.49 g, 53.2 mmol)
was slowly added. The resulting suspension was heated
◦
at 90 C for 4 h. Then, the reaction mixture was cooled to
room temperature before hexane (150 mL) and water (25 mL)
were added. The organic layer was separated and washed
successively with saturated NaHCO3 (25 mL) and saturated
NaCl (25 mL). The organic layer was dried with Na2SO4 and
concentrated to give a crude product, which was purified by
flash column chromatography (hexane) to give a white solid
(ı), followed by the splitting pattern, and the number of pro-
tons. Abbreviations used in the splitting pattern were as the
following: s, singlet; d, doublet; t, triplet; m, multiplet and b,
broad. IR spectra were recorded on neat samples with an FTIR
(3.73 g, 85%): mp 64.4–64.8 C (lit. [22] 62.5–63.5 C); 1H NMR
(CDCl3): ı 0.60–2.00 (m, 46H), 3.88 (bs, 1H) (lit. [23] 300 MHz);
13C NMR (CDCl3): ı 11.6, 12.1, 18.7, 20.8, 22.6, 22.9, 23.9, 24.2,
25.7, 28.0, 28.3, 28.4, 31.9, 32.6, 32.9, 35.5, 35.8, 35.9, 36.2, 39.5,
40.0, 40.1, 42.6, 54.2, 56.3, 56.5, 58.3; IR: 2931, 2865, 2852, 2084
◦
◦
equipped with a diamond ATR system, and reported in cm 1.
HRMS data were obtained on a dual-sector mass spectrometer
in FAB mode with 2-nitrobenzylalcohol as the proton donor.
Elemental analyses were performed by Atlantic Microlabs, Inc.
−
(lit. [23] film); [␣]D26 +18.6 (c 1.52, CHCl3) (lit. [22] [␣]D +18.6
◦
26
◦
(Norcross, GA). Optical rotations were measured at the sodium
line with a Perkin-Elmer 241 polarimeter in a 1-dm tube.
(c 1.01, CHCl3)).

steroids 7 2 ( 2 0 0 7 ) 615–626
617
◦
◦
◦
2
.2.4. 3ˇ-Azido-5˛-cholestane (6b)
To a 250-mL round bottom flask containing a solution of 5
2.36 g, 5.22 mmol) in DMF (36 mL), NaN3 (1.72 g, 26.1 mmol)
was slowly added. The resulting suspension was heated at
2.19 mmol; 65%): 85.8–86.4 C (lit. 104–106 C [23], 118–120 C
[20]).
(
Method B. Azide 6b (2.94 g, 7.10 mmol), LiAlH4 (851 mg,
21.3 mmol), dry THF (26 mL), and were combined and treated
as above to give a white solid (2.36 g, 6.09 mmol; 86%): mp
91.6–92.1 C (lit. 104–106 C [23], 118–120 C [20]); 1H NMR
(CDCl3): ı 0.60–2.00 (m, 47H), 2.64 (m, 1H) (lit. 300 MHz [23],
400 MHz [20]); 13C NMR (CDCl3): ı 12.1, 12.4, 18.7, 21.2, 22.6, 22.8,
23.8, 24.2, 28.0, 28.3, 28.8, 32.1, 32.6, 35.53, 35.55, 35.8, 36.2, 37.7,
39.4, 39.5, 40.1, 42.6, 45.6, 51.2, 54.5, 56.3, 56.5 (lit. 100 MHz [20]);
◦
9
0 C for 5 h. The reaction was cooled to room temperature
◦
◦
◦
before hexane (72 mL) and water (15 mL) were added. The
organic layer was separated and washed successively with
saturated NaHCO3 (15 mL) and saturated NaCl (15 mL). The
organic layer was dried with Na2SO4 and concentrated to give
a crude product, which was purified by flash column chro-
matography (hexane) to give a white solid (1.70 g, 79%): mp
IR: 3400, 2931, 2849, 1465, 1381(lit. film [20,23]); [␣]D25 +24.6 (c
1.80, CHCl3) (lit. [28] [␣]D +29 (c 0.9, CHCl3)).
◦
◦
◦
◦
◦
◦
6
6.3–66.8 C (lit. mp 71.5–72 C [24], 65–66 C [25], 65–67 C [20]);
1
H NMR (CDCl3): ı 0.60–2.00 (t, 47H), 3.25 (m, 1H) (lit. 300 MHz
23], 400 MHz [20]); 13C NMR (CDCl3): ı 12.1, 12.4, 18.7, 21.2, 22.6,
[
2.2.6. General procedure for the synthesis of 8a,b
2
3
1
2.8, 23.8, 24.2, 28.0, 28.3, 38.8, 32.1, 32.7, 35.55, 35.56, 35.8,
6.2, 37.7, 39.51, 39.53, 40.1, 42.6, 45.6, 51.2, 54.5, 56.3, 56.6 (lit.
00 MHz [20]); IR: 2965, 2929, 2848, 2090, 1463, 1256 (lit. film
To a 50-mL round bottom flask containing a solution of WeNCO
in CH2Cl2, an amine 7 was slowly added and then stirred at
room temperature for 24 h. The reaction was concentrated
to give a colorless oil that solidified under vacuum. Flash
column chromatography (hexane/THF/MeOH, 1:1:0.04, v/v/v)
gave a white solid, which gave a single spot on TLC (hex-
20,23]); [␣]D25 +25.6 (c 1.84, CHCl3) (lit. [25] [␣]D +26 (c 1.00,
◦ ◦
20
[
2
0
◦
CHCl3), (lit. [21] [␣]D +25.5 (c 1.00, CHCl3)).
2
5
.2.5. General procedure for the preparation of epimeric
˛-cholestan-3-amines (7a,b)
ane/THF/MeOH, 1:1:0.04, v/v/v, R = 0.56–0.63).
f
Catalytic hydrogenation. Method A. To a 250-mL hydrogenation
bottle was added an azide 6, 10% Pd/C (4 wt.% of azide), and
hexane so that the concentration of the azide was ∼0.13 M.
The resulting suspension was shaken at 60 psi of H2 at room
temperature for 6 h. After sitting overnight, the resulting sus-
pension was filtered; the filtrate was concentrated to give an
off-white solid.
2.2.6.1. Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(5˛-
cholestan-3˛-yl)ureido]heptanedioate (8a). Amine 7a (1.01 g,
2.61 mmol), WeNCO (1.15 g, 2.61 mmol), and CH2Cl2 (10 mL)
were combined and treated as above to give a white solid
(2.04 g, 2.46 mmol, 94%): mp 179.9–180.6 C; 1H NMR (CDCl3):
ı 0.60–2.0 (m, 46H), 1.44 (s, 27H), 1.95 (m, 6H), 2.23 (m, 6H),
3.67 (bs, 1H), 4.38–4.42 (m, 2H); 13C NMR (CDCl3): ı 11.4, 12.1,
18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.6, 28.0, 28.1, 28.3, 28.6, 30.0,
30.7, 32.0, 33.1, 33.5, 35.5, 35.8, 36.0, 36.2, 39.5, 40.1, 40.7, 42.6,
45.4, 54.5, 56.3, 56.5, 56.6, 80.5, 156.2, 173.1; IR: 2929, 2864, 1726,
◦
Reduction by lithium aluminum hydride. Method B. A 250-mL
round bottom flask containing a suspension of LiAlH4 in
dry THF was put in an ice–salt bath. When the temperature
◦
27
◦
reached 0 C, an azide 6 was added slowly to the flask, so
1628, 1551, 1456, 1366, 1247, 1146; [␣]D +15.6 (c 1.41, CHCl3);
+
that its concentration was ∼0.80 M. The resulting suspension
was then refluxed for 4 h. After refluxing and cooling to room
temperature, the mixture was diluted with THF to double the
volume. The solution was cooled in an ice bath; water (one-
eighth of THF volume), 10-M NaOH (one-fifth of THF volume),
and water (one-eighth of THF volume) were added succes-
sively. The solid that formed was filtered; the filtrate was
dried with Na2SO4, filtered, and concentrated to give a white
solid.
HRMS: for C50H89N2O7 (M + H) calcd 829.6670, found 829.6675.
Anal. Calcd for C50H88N2O7: C, 72.42; H, 10.70; N, 3.38. Found:
C, 72.18; H, 10.79; N, 3.38.
2.2.6.2. Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(5˛-
cholestan-3ˇ-yl)ureido]heptanedioate (8b). Amine 7b (1.22 g,
3.16 mmol), WeNCO (1.39 g, 3.15 mmol), and CH2Cl2 (14 mL)
were combined and treated as above to give a white solid
(2.35 g, 2.84 mmol, 90%): mp 187.4–187.8 C; 1H NMR (CDCl3):
ı 0.60–2.0 (m, 46H), 1.43 (s, 27H), 1.93 (m, 6H), 2.23 (m, 6H),
3.87 (sb, 1H), 3.88 (d, 1H), 4.46 (s, 1H); 13C NMR (CDCl3): ı 12.1,
12.3, 18.7, 21.2, 22.6, 22.8, 23.9, 24.2, 28.0, 28.1, 28.3, 28.6, 29.6,
30.0, 30.7, 32.0, 35.4, 35.5, 35.8, 36.19, 36.26, 37.6, 39.5, 40.0, 42.6,
45.1, 49.8, 54.4, 56.3, 56.5, 80.5, 156.2, 173.2; IR: 2929, 2864, 1724,
◦
2
(
.2.5.1. 5˛-Cholestan-3˛-amine (7a). Method A. Azide 6a
1.84 g, 4.46 mmol), 10% Pd/C (82.8 mg), and hexane (35 mL)
were combined and treated as above to give an off-white solid
◦
◦
(
1.18 g, 3.06 mmol; 68%): mp 84.6–85.4 C (lit. [26] 88–89 C).
2
7
◦
Method B. Azide 6a (1.00 g, 2.42 mmol), LiAlH4 (320 mg,
1625, 1558, 1457, 1365, 1308, 1148; [␣]D +9.6 (c 1.49, CHCl3);
+
8
.01 mmol), and dry THF (10 mL) were combined and treated
HRMS: for C50H89N2O7 (M + H) calcd 829.6670, found 829.6638.
as above to give a white solid (755 mg, 1.95 mmol; 80%):
Anal. Calcd for C50H88N2O7: C, 72.42; H, 10.70; N, 3.38. Found:
C, 72.67; H, 10.77; N, 3.35.
8.4–89.2 C (lit. [26] 88–89 C); 1H NMR (CDCl3): ı 0.60–2.00 (m,
7H), 3.18 (bs, 1 H) (lit. [23] 300 MHz); 13C NMR (CDCl3): ı 11.4,
2.1, 18.7, 20.8, 22.6, 22.8, 23.6, 24.2, 28.0, 28.3, 28.8, 29.3, 32.1,
2.2, 35.6, 35.8, 36.2, 36.37, 36.41, 39.2, 39.5, 40.1, 42.6, 45.8, 54.6,
6.3, 56.6; IR: 3400, 2930, 2849, 1465, 1380 (lit. [23] film); [␣]D25
◦
◦
8
4
1
3
5
+
2.2.7. General procedure for the synthesis of 1a,b
To a 50-mL round bottom flask containing 8 was added
HCOOH; the flask was warmed in a hot-water bath until a
transparent solution appeared. The resulting solution was
stirred overnight at room temperature. A completed reac-
tion was identified by the formation of a milky solution. The
solvent was evaporated to leave a white solid, which was
recrystallized by dissolving in hot AcOH and then cooled
◦
28
◦
26.4 (c 1.81, CHCl3) (lit. [27] [␣]D +28 (c 1.1, CHCl3)).
2
.2.5.2. ˛-Cholestan-3ˇ-amine (7b). Method A. Azide 6b (1.38 g,
3
.35 mmol), 10% Pd/C (54.3 mg), and hexane (26 mL) were com-
bined and treated as above to give an off-white solid (849 mg,

6
18
steroids 7 2 ( 2 0 0 7 ) 615–626
before adding hexane until the solution became cloudy. A
white solid eventually formed in a 1 or 2 days.
temperature overnight. The reaction was filtered; the filtrate
was concentrated to give the crude product, which was puri-
fied by flash column chromatography (CH2Cl2/hexane, 1:1, v/v)
to give a white solid, which gave a single spot on TLC in
2
.2.7.1. 4-(2-Carboxyethyl)-4-[3-(5˛-cholestan-3˛-yl)ureido]-
heptanedioic acid (1a). Compound 8a (1.53 g, 1.90 mmol) and
(CH2Cl2/hexane, 1:1, v/v, R = 0.33–0.41).
f
HCOOH (30 mL) were combined and treated as above to give
a white solid (922 mg, 1.39 mmol; 73%): dec. at 199.0 C; 1H
◦
2.2.9.1. 5˛-Cholestan-3˛-yl chloroethanoate (9a). Chloroacetyl
chloride (1.80 g, 15.6 mmol), 3a (3.80 g, 9.78 mmol), Et3N
(1.50 mL, 10.7 mmol), DMAP (60.0 mg, 0.49 mmol), and PhH
(58 mL) were combined and treated as above to give a white
NMR (DMSO-d6): ı 0.60–2.0 (m, 51H), 2.09 (m, 6H), 3.67 (bs, 1H),
5
1
2
4
1
.50 (s, 1H), 5.97 (d, 1H), 12.00 (s, 3H); 13C NMR (DMSO-d6): ı
1.6, 12.3, 18.9, 20.8, 22.8, 23.1, 22.8, 23.1, 23.7, 24.2, 26.7, 27.8,
8.2, 28.6, 30.6, 32.2, 33.0, 33.7, 35.4, 35.7, 36.0, 36.1, 39.4, 42.6,
4.0, 54.5, 55.5, 56.2, 56.7, 157.0, 175.0; IR: 3373, 2925, 2865,
◦
1
solid (2.59 g, 8.56 mmol, 57%): mp 134.7–135.2 C; H NMR
1
3
(CDCl3): ı 0.65–2.00 (m, 46H), 4.07 (s, 2 H), 5.12 (m, 1H); C NMR
(CDCl3): ı 11.3, 12.1, 18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.0, 28.0,
28.25, 28.32, 31.9, 32.7, 32.8, 35.5, 35.76, 35.82, 36.2, 39.5, 40.0,
41.4, 42.6, 54.2, 56.3, 56.5, 72.7, 166.8; IR: 2932, 2864, 2849, 1754,
705, 1635, 1559, 1288; [␣]D27 +19.9 (c 0.87, EtOH); HRMS: for
◦
C38H65N2O7 (M + H)+ calcd 661.4792, found 661.4780. Anal.
Calcd for C38H64N2O7: C, 69.06; H, 9.76; N, 4.24. Found: C, 69.10;
H, 9.80; N, 4.22.
1194, 1185; [␣]D27 +24.5 (c 1.18, CHCl3).
◦
2
.2.7.2. 4-(2-Carboxyethyl)-4-[3-(5˛-cholestan-3ˇ-yl)ureido]-
2.2.9.2. 5˛-Cholestan-3ˇ-yl chloroethanoate (9b). Chloroacetyl
chloride (1.804 g, 15.6 mmol), 3b (5.00 g, 12.6 mmol), Et3N
(1.90 mL, 13.5 mmol), DMAP (77.3 mg, 0.63 mmol), and PhH
(94 mL) were combined and treated as above to give a white
heptanedioic acid (1b). Compound 8b (907 mg, 1.09 mmol) and
HCOOH (26 mL) were combined and treated as above to give
a white solid (560 mg, 0.85 mmol; 78%): dec. at 194.3 C; 1H
◦
◦
NMR (DMSO-d6): ı 0.60–2.0 (m, 51H), 2.08 (m, 6H), 3.23 (bs, 1H),
solid (3.98 g, 8.56 mmol, 68%): mp 176.1–176.9 C (lit. mp
.38 (s, 1H), 5.50 (d, 1H), 12.02 (s, 3H); 13C NMR (DMSO-d6):
178–179 C [32], 180–181 C [33]); H NMR (CDCl3): ı 0.60–2.00
(t, 46H), 4.02 (s, 2H), 4.79 (m, 1H) (lit. 60 MHz [34]); 13C NMR
(CDCl3): ı 12.1, 12.2, 18.7, 21.2, 22.6, 22.8, 23.8, 24.0, 27.3, 28.0,
28.2, 28.6, 31.9, 33.8, 35.43, 35.45, 35.8, 36.2, 36.7, 39.5, 40.0,
41.3, 42.6, 44.6, 54.2, 56.3, 56.4, 76.1, 166.9; IR: 2935, 2917,
◦
◦
1
5
ı 12.3, 12.4, 19.0, 21.2, 22.8, 23.1, 23.7, 24.2, 27.8, 28.3, 28.6,
2
4
1
8.7, 29.5, 30.5, 32.1, 35.5, 35.7, 36.1, 36.2, 37.7, 42.6, 45.6,
8.9, 54.2, 55.5, 56.2, 56.4, 156.9, 175.0; IR: 3405, 2930, 2847,
704, 1613, 1559, 1295; [␣]D27 +14.8 (c 0.83, EtOH); HRMS: for
◦
C38H65N2O7 (M + H)+ calcd 661.4792, found 661.4794. Anal.
Calcd for C38H64N2O7: C, 69.06; H, 9.76; N, 4.24. Found: C, 68.85;
H, 9.71; N, 4.20.
2848, 1747, 1205, 1180 (lit. CCl4 [34]; [␣]D24 +11.3 (c 0.36,
◦
CHCl3)).
2.2.10. General procedure for the preparation of
2
.2.8. 5˛-Cholestan-3˛-ol (3a)
5˛-cholestan-3-yl azidoethanoate (10a,b)
To a 100-mL round bottom flask containing a colorless solu-
tion of 3b (4.90 g, 12.4 mmol) in dry THF (24 mL) was added
diisopropylazodicarboxylate (DIAD, 2.45 mL, 15.6 mmol) as
described previously [29]. The resulting solution turned from
colorless to orange. Addition was continued with CF3COOH
To a 0.20 M solution of 9 in DMF, NaN3 was slowly added. The
mixture was heated at 85–90 C for 10 h and turned yellow.
◦
After cooling to room temperature, hexane (as much as the
volume of DMF) and water (one-third volume of DMF) were
added to the mixture. Two layers formed; the organic layer was
separated and washed with saturated NaHCO3 (equal volume
to removed water layer) and saturated NaCl (equal volume to
removed water layer) successively. The light yellow organic
layer was dried and concentrated to give a light yellow solid.
Purification by flash column chromatography (hexane) gave
a white solid, which showed a single spot on TLC (hexane,
Rf = 0.15–0.21).
(
(
1.20 mL, 15.6 mmol), PPh3 (4.09 g, 15.5 mmol), and PhCOONa
2.25 g, 15.5 mmol), successively. The resulting colorless solu-
tion was stirred at room temperature overnight. The solution
was concentrated to give an off-white solid. After dilution
◦
with MeOH (24 mL), the resulting solution was heated at 50 C
overnight. The solvent was concentrated; the residue was
partitioned between CH2Cl2 (150 mL) and water (15 mL). The
organic layer was washed once with water (15 mL) and dried
with MgSO4. Concentration of the solution gave a white solid,
which was purified by flash column chromatography (CH2Cl2)
2.2.10.1. 5˛-Cholestan-3˛-yl azidoethanoate (10a). Combin-
ing NaN3 (1.24 g, 18.9 mmol), 9a (1.76 g, 3.79 mmol), and
DMF (19 mL) and treating as above gave a white solid
◦
to yield a white solid (3.84 g, 80%): mp 185.2–185.7 C (lit. [29]
mp 182–185 C); 1H NMR (CDCl3): ı 0.60–2.00 (m, 46H), 4.04 (bs,
◦
(846 mg, 1.80 mmol, 47%): mp 114.9–115.6 C; H NMR (CDCl3):
◦
1
H) (lit. [29] 300 MHz); 13C NMR (CDCl3): ı 11.2, 12.1, 18.7, 20.8,
2.6, 22.8, 23.8, 24.2, 28.0, 28.3, 28.6, 29.0, 32.0, 32.2, 35.5, 35.8,
5.9, 36.1, 36.2, 39.2, 39.5, 40.1, 42.6, 54.3, 56.2, 56.6, 66.7; IR:
ı 0.65–2.00 (m, 46H), 3.86 (s, 2H), 5.16 (m, 1H); C NMR (CDCl3):
13
1
2
3
ı 11.4, 12.1, 18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.1, 28.0, 28.2,
28.3, 31.9, 32.81, 32.83, 35.5, 35.76, 35.81, 36.2, 39.5, 39.99, 40.04,
42.6, 50.7, 54.2, 56.3, 56.5, 72.6, 167.8; IR: 2926, 2865, 2104,
2
7
◦
3
375, 2928, 2848, 1002 (lit. [30] bands); [␣]D +25.5 (c 1.78,
2
6.5
◦
+27 (c 1.92, CHCl3)).
1733, 1218, 1159, 975; [␣]D27 +29.8 (c 1.14, CHCl3); HRMS: for
C29H49N3O2 calcd 471.3825, found 471.3812. Anal. Calcd for
C29H49N3O2: C, 73.84; H, 10.47; N, 8.91. Found: C, 73.89; H, 10.53;
N, 8.91.
◦
CHCl3) (lit. [31] [␣]D
2
5
.2.9. General procedure for the synthesis of
˛-cholestan-3-yl chloroethanoate (9a,b)
Chloroacetyl chloride was added dropwise to a 100-mL round
bottom flask containing a solution of 3, Et3N, and DMAP in
CH2Cl2. The resulting solution was stirred in PhH at room
2.2.10.2. 5˛-Cholestan-3ˇ-yl azidoethanoate (10b). Combin-
ing NaN3 (798 mg, 12.2 mmol), 9b (1.85 g, 3.98 mmol), and

steroids 7 2 ( 2 0 0 7 ) 615–626
619
white solid (691 mg, 0.78 mmol, 64.0%): mp 82.9–83.3 C; 1H
◦
DMF (20 mL) and treating as above gave
(
a
white solid
915 mg, 1.94 mmol, 49%): mp 124.2–124.9 C; H NMR (CDCl3):
ı 0.60–2.00 (m, 46H), 3.83 (s, 2H), 4.81 (m, 1H); 13C NMR
CDCl3): ı 12.3, 12.5, 18.9, 21.4, 22.8, 23.1, 26.1, 24.4, 27.6,
◦
1
NMR (CDCl ): ı 0.60–2.00 (m, 79H), 2.24 (m, 6H), 3.94 (dd, 1H),
3.89 (dd, 1H), 4.65 (m, 1H; exchange with D O), 4.79 (s, 1H;
3
2
1
3
(
2 3
exchange with D O), 5.08 (m, 1H); C NMR (CDCl ): ı 11.3, 12.1,
2
4
2
8.2, 28.5, 28.8, 32.2, 34.1, 35.67, 35.68, 36.0, 36.4, 36.9, 39.7,
0.2, 42.8, 44.9, 50.8, 54.4, 56.5, 56.6, 76.0, 168.1; IR: 2934,
18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.1, 28.0, 28.1, 28.2, 28.3, 29.9,
30.6, 31.9, 32.8, 35.5, 35.7, 35.8, 36.2, 39.5, 39.9, 40.0, 42.3, 42.6,
54.1, 56.3, 56.5, 56.7, 71.6, 80.5, 156.1, 170.5, 173.0; IR: 2928,
849, 2108, 1738, 1218; [␣]D27 +14.5 (c 1.00, CHCl3); HRMS: for
◦
1727, 1365, 1203, 1149, 1100; [␣]D27 +6.3 (c 0.80, CHCl ); HRMS:
◦
C29H49N3O2 calcd 471.3825, found 471.3809. Anal. Calcd for
C29H49N3O2: C, 73.84; H, 10.47; N, 8.91. Found: C, 73.90; H, 10.60;
N, 8.92.
3
for C52H91N O (M + H)+ calcd 887.6725, found 887.6702. Anal.
2
9
Calcd for C52H90N O : C, 70.39; H, 10.22; N, 3.16. Found: C,
2
9
70.43; H, 10.30; N, 3.17.
2
5
.2.11. General procedure for the preparation of
˛-cholestan-3-yl aminoethanoate (11a,b)
2.2.12.2. Di-tert-butyl
4-(2-tert-butoxycarbonylethyl)-4-[3-
A cholestan-3-yl azidoethanoate (10) was added to a hydro-
genator vessel containing a suspension of 10% Pd/C (4 wt.%
of azide 10) in CH2Cl2 so that the concentration of azide was
(5˛-cholestan-3ˇ-yloxycarbonylmethyl)ureido]heptanedioate
(12b). Combining 11b (1.09 g, 2.44 mmol), WeNCO (989 mg,
2.24 mmol), and CH Cl (20 mL) and treating as above gave
2
2
◦
a white solid (1.21 g, 1.36 mmol, 61%): mp 195.8–196.2 C; 1H
◦
∼
0.11 M. The mixture was shaken at 60 psi of H2 at ∼ 60 C for
4
h. The reaction was cooled to room temperature and filtered
NMR (CDCl ): ı 0.60–2.00 (m, 79H), 2.20 (m, 6H), 3.84 (d, 2H),
3
through a pad of Celite® in vacuo; the filtrate was concentrated
to give an off-white solid.
4.62 (t, 1H; exchanges with D O), 4.70 (s, 1H; exchanges with
2
1
3
D O), 4.75 (m, 1H); C NMR (CDCl ): ı 11.7, 11.8, 18.3, 20.8,
2
3
22.2, 22.4, 23.5, 23.8, 27.0, 27.6, 27.7, 27.8, 28.2, 29.4, 30.1, 31.6,
2
.2.11.1. 5˛-Cholestan-3˛-yl aminoethanoate (11a). Combin-
33.5, 35.0, 35.1, 35.4, 35.8, 36.3, 39.1, 39.6, 41.8, 42.2, 44.2, 53.8,
55.9, 56.0, 56.1, 74.4, 80.0, 156.0, 170.4, 172.6; IR: 2927, 1726,
ing 10a (736 mg, 1.56 mmol), 10% Pd/C (29.7 mg), and CH2Cl2
1365, 1147, 1199, 1097; [␣]D27 +11.6 (c 1.50, CHCl ); HRMS:
◦
(
1
0
15 mL) and treating as above gave a white solid (615 mg,
3
.38 mmol, 88%): mp 109.2–110.1 C; 1H NMR (CDCl3):
◦
ı
for C52H91N O (M + H)+ calcd 887.6725, found 887.6744. Anal.
2
9
1
3
.65–2.00 (t, 49H), 3.43 (s, 2 H), 5.08 (m, 1H); C NMR (CDCl3):
2 9
Calcd for C52H90N O : C, 70.39; H, 10.22; N, 3.16. Found: C,
ı 11.3, 12.1, 18.7, 20.8, 22.6, 22.8, 23.9, 24.2, 26.2, 28.0, 28.3,
70.42; H, 10.31; N, 3.16.
2
4
1
8.4, 31.9, 32.87, 32.92, 35.5, 35.79, 35.82, 36.2, 39.5, 40.03, 40.05,
2.6, 44.3, 54.2, 56.3, 56.5, 70.9, 173.8; IR: 3400, 2931, 2865,
2.2.13. General procedure for the synthesis of 2a,b
728, 1206; [␣]D27 +28.2 (c 0.94, CHCl3); HRMS: for C29H52NO2
◦
To a 50-mL round bottom flask containing a tri-tert-butyl ester
12 was added HCOOH; the flask was warmed in a hot-water
bath until a transparent solution appeared. The resulting solu-
tion was removed from the hot-water bath and stirred at room
temperature for 5 h. The resulting reaction was concentrated
to dryness to leave a white solid. Recrystallization of the crude
material by dissolution in hot AcOH and then cooling before
adding hexane until the solution became cloudy gave a white
solid.
(
M + H)+ calcd 446.3998, found 446.4001. Anal. Calcd for
C29H51NO2: C, 78.15; H, 11.53; N, 3.14. Found: C, 78.17; H, 11.67;
N, 3.12.
2
.2.11.2. 5˛-Cholestan-3ˇ-yl aminoethanoate (11b). Combin-
ing 10b (2.70 g, 5.72 mmol), 10% Pd/C (110 mg), and CH2Cl2
(
4
50 mL) and treating as above gave an off-white solid (1.93 g,
.34 mmol, 76%): mp 157.5–158.3 C (lit. [35] 178–180 C); 1H
◦ ◦
NMR (CDCl3): ı 0.65–2.00 (m, 49H), 3.38 (s, 2H), 4.75 (m, 1H);
13C NMR (CDCl3): ı 12.0, 12.2, 18.6, 21.2, 22.5, 22.8, 23.8, 24.2,
2.2.13.1. 4-(2-Carboxyethyl)-4-[3-(5˛-cholestan-3˛-yloxycar-
bonylmethyl)ureido]heptanedioic acid (2a). Combining HCOOH
(6 mL) and 12a (418 mg, 0.472 mmol) and treating as above gave
a white solid (174 mg, 0.242 mmol, 51%): mp 192.5–192.9 C; H
NMR (DMSO-d6): ı 0.60–2.10 (m, 53H), 3.66 (d, 2H), 4.87 (s, 1H),
5.86 (s, 1H), 5.91 (t, 1H), 11.97 (bs, 1H); 13C NMR (DMSO-d6): ı
11.7, 12.5, 19.2, 21.0, 23.1, 23.3, 23.9, 24.4, 26.2, 28.0, 28.5, 28.6,
2
3
2
7.4, 27.97, 28.03, 28.2, 28.6, 31.9, 34.0, 35.4, 35.8, 36.1, 36.7,
9.5, 39.9, 42.5, 44.0, 44.6, 54.2, 56.2, 56.4, 74.4, 173.8; IR: 3400,
2
4
◦
◦
1
933, 2849, 1725, 1220; [␣]D +12.2 (c 0.43, CHCl3); HRMS: for
+
C29H52NO2 (M + H) calcd 446.3998, found 446.4003. Anal. Calcd
for C29H51NO2: C, 78.15; H, 11.53; N, 3.14. Found: C, 78.18; H,
1
1.57; N, 3.10.
28.8, 30.7, 32.1, 33.0, 33.1, 35.6, 35.9, 36.0, 36.3, 39.6, 42.1, 42.8,
2
.2.12. General procedure for the synthesis of 12a,b
54.2, 55.9, 56.4, 56.6, 70.5, 157.5, 171.2, 175.1; IR: 3409, 2933,
1751, 1702, 1559, 1204, 1158; [␣]D27 +15.7 (c 0.65, EtOH); HRMS:
for C40H67N2O9 (M + H)+ calcd 719.4847, found 719.4851. Anal.
Calcd for C40H66N2O9: C, 66.82; H, 9.25; N, 3.90. Found: C, 66.96;
H, 9.37; N, 3.89.
◦
An amine 11 was added to a solution containing WeNCO in
CH2Cl2. The solution was stirred at room temperature for 5 h.
The resulting transparent solution was concentrated to give
a white solid. Flash column chromatography (hexane/EtOAc,
2
.5:1, v/v) of the crude product gave a white solid, which
showed as a single spot on TLC (hexane/EtOAc, 2.5:1, v/v,
Rf = 0.33–0.35).
2.2.13.2. 4-(2-Carboxyethyl)-4-[3-(5˛-cholestan-3ˇ-yloxycar-
bonylmethyl)ureido]heptanedioic acid (2b). Combining HCOOH
(
10 mL) and 12b (891.4 mg, 1.00 mmol) and treating as above
◦
2
.2.12.1. Di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-(5˛-
gave a white solid (409 mg, 0.569 mmol, 57%): dec. at 194.2 C;
1H NMR (DMSO-d6): ı 0.50–2.10 (m, 59H), 3.62 (d, 2H), 4.54 (h,
1H), 5.84 (s, 1H), 5.91 (t, 1H), 11.96 (bs, 1H); 13C NMR (DMSO-d6):
ı 12.5, 12.6, 19.2, 21.4, 23.0, 23.3, 23.9, 24.5, 27.8, 28.0, 28.4,
cholestan-3˛-yloxycarbonylmethyl)ureido]heptanedioate (12a).
Combining 11a (542 mg, 1.22 mmol), WeNCO (534 mg,
1
.21 mmol), and CH2Cl2 (6 mL) and treating as above gave a

6
20
steroids 7 2 ( 2 0 0 7 ) 615–626
2
5
1
8.7, 30.6, 32.2, 34.3, 35.6, 35.9, 36.3, 36.8, 42.1, 42.8, 44.6, 54.2,
5.9, 56.4, 56.6, 74.1, 157.4, 171.2, 175.1; IR: 3410, 2933, 1728,
2.4.
Antimicrobial assays
704, 1564, 1221, 1187; [␣]D27 +9.8 (c 0.58, EtOH); HRMS: for
◦
2.4.1. Microbial strains, culture conditions, and
preparations of inocula for susceptibility testing
Strains of Escherichia coli strain C (ATCC # 13706), Klebsiella
pneumoniae (ATCC # 4352), Lactobacillus plantarum (ATCC #
C40H67N2O9 (M + H)+ calcd 719.4847, found 719.4851. Anal.
Calcd for C40H66N2O9: C, 66.82; H, 9.25; N, 3.90. Found: C, 66.72;
H, 9.41; N, 3.87.
14917), Staphylococcus aureus (ATCC # 6538), and Mycobacterium
2
.3.
X-ray analysis of 8a
smegmatis (ATCC # 607) were obtained from the American
Type Culture Collection (ATCC, Manassas, VA). A methicillin-
resistant isolate of S. aureus (MRSA) was obtained from
the Microbiology Laboratory, Danville Community Hospital
(Virginia) and Saccharomyces cerevisiae, Candida albicans, Cryp-
tococcus neoformans, Aspergillus niger, and Micrococcus luteus
strains were obtained from the Virginia Tech Microbiology
teaching culture collection. Colonies of E. coli, K. pneumoniae, S.
aureus, MRSA, M. luteus, S. cerevisiae, C. albicans, and C. neofor-
mans were grown on 1/10-strength Brain Heart Infusion Broth
(BHIB, BBL Microbiology Systems, Cockeysville, MD) contain-
ing 0.2% (wt./vol) sucrose (BHIB + S) and 1.5% (wt./vol) agar. L.
plantarum was grown on 1/4-strength Tryptic Soy Broth (TSB,
BBL Microbiology Systems, Cockeysville, MD) containing 0.2%
glucose (TSB + G) and 1.5% (wt./vol) agar. M. smegmatis was
grown on Middlebrook 7H10 agar (BBL Microbiology Systems,
Cockeysville, MD) and A. niger on Potato Dextrose Agar (PDA,
BBL Microbiology Systems, Cockeysville, MD). Streaked plates
Colorless plates (0.055 mm × 0.47 mm × 0.55 mm) were crys-
tallized from EtOH (Table 1). The chosen crystal was
mounted on a nylon CryoLoopTM (Hampton Research) with
Krytox® Oil (DuPont) and centered on the goniometer of
an Oxford Diffraction XcaliburTM diffractometer equipped
with
a
Sapphire 2TM CCD. The data collection routine,
unit cell refinement, and data processing were carried out
with the program CrysAlis [36]. The Laue symmetry and
systematic absences were consistent with the monoclinic
space groups P21 and P21/m. As the sample was known
to be enantiomerically pure from the synthetic procedure,
the chiral space group P21 was chosen. Because there are
no heavy atoms, the absolute configuration could not be
determined from the Friedel pairs; the Friedel pairs were
therefore merged for the final refinement. The structure was
solved by direct methods and refined by using the SHELXTL
NT program [37]. The asymmetric unit of the structure
comprises two crystallographically independent cholesterol
derivatives and two EtOH molecules. The final refinement
model involved anisotropic displacement parameters for non-
hydrogen atoms and a riding model for all hydrogen atoms.
The program SHELXTL NT [37] was used for molecular graphics
generation.
◦
were incubated at 37 C for 3–7 days, except for that of A. niger,
◦
which was incubated in the dark at 30 C. A single colony
for each microbe except A. niger was used to inoculate 5 mL
of 1/10-strength BHIB + S (E. coli, K. pneumoniae, M. luteus, S.
aureus, and MRSA), TSB (L. plantarum), Middlebrook 7H9 broth
(M. smegmatis), or yeast extract peptone maltose broth (S. cere-
◦
visiae, C. albicans and C. neoformans) and incubated at 37 C (S.
Table 1 – Crystal data and structure refinement for 8a
Category
Crystal data and structure refinement
Empirical formula
Formula weight
Temperature
C50H88N2O7·CH3CH2OH
875.29
100(2) K
Wavelength
0.71073 A˚
Crystal system
Space group
Unit cell dimension
Volume
Monoclinic
P21
◦
◦
◦
a = 12.1220(16) A˚ ; b = 12.8097(16) A˚ ; c = 35.589(3) A˚ ; ˛ = 90 ; ˇ = 99.166(9) ; ꢀ = 90
5455.7(11) A˚ 3
4
Z
3
Density (calculated)
Absorption coefficient
F(0 0 0)
1.066 mg/m
−1
0.070 mm
1936
Crystal size
Theta range for data collection
Index ranges
Reflections collected
Independent reflections
Completeness to theta = 25.07
0.550 mm × 0.465 mm × 0.055 mm
◦
2.81–25.07
−14 ≤ h ≤ 14, −14 ≤ k ≤ 15, −42 ≤ l ≤ 41
25,907
10,121 [R(int) = 0.0573]
99.6%
◦
Absorption correction
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2ꢁ(I)]
R indices (all data)
None
Full-matrix least-squares on F2
10121/1/1119
0.967
R1 = 0.0734, wR2 = 0.1820
R1 = 0.1180, wR2 = 0.2131
0.686 and −0.273 e A˚ −
3
Largest diff. peak and hole

Table 2 – Minimal inhibitory concentrations (MICs) of dendritic tricarboxylato amphiphiles against bacteria and yeast
Amphiphile
MIC (mg/L)
E. coli
K. pneumoniae
L. plantarum
S. aureus
MRSA
M. luteus
M. smegmatis
S. cerevisiae
C. albicans
C. neoformans
A. niger
a
140b
140b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
3(14)
3(16)
3(18)
3(20)
3(22)
140
71
71
36
71
1600b
390b
780b
1600b
1600b
1600b
780b
200b
1600b
1600
1600
280
3100
3100
780
780
560
3100
780
6300
6300
6300
3100
3100
3100
3100
6300
6300
3100
390
780
780
3100
3100
390
780
780
1600
1600
6300
>6300
>6300
>6300
>6300
>6300
3100
1600
6300
6300
280
71
18
36
71
280
280
71
71
18
390
200
390
1600
3100
3100
1600
1600
1600
780
390
780
24
780
1600
780
24
49
1600
3100
570
280
140
3100
1600
780
390
3100
390
200
3100
6300
a
a
a
a
b
b
b
b
b
140b
b
b
b
b
b
140b
280b
b
b
b
b
b
b
b
b
b
780
>6300
>6300
>6300
>6300
>6300
>6300
>6300
>6300
>6300
>6300
>6300
1600
c
c
c
b
b
b
b
780b
98b
>6300
>6300
>6300
1600b
390b
b
b
4(7,7)
4(8,8)
4(9,9)
>560
>560
>560
>560
>560
>6300
>6300
>6300
>6300
>6300
>6300
560
1600
780
780
390
390
1600
780
200
200
390
780
390
390
390
390
b
b
b
b
b
b
b
b
b
b
b
b
c
c
b
b
b
b
36b
b
4(10,10)
4(11,11)
6300
6300
b
b
b
b
b
6300
>560
560
140
6300
c
b
b
b
5(7,7)
>6300
>6300
>6300
>6300
>6300
>6300
200
18
200
780
>6300
>6300
>6300
>6300
>6300
>6300
3100
780
6300
6300
3100
3100
1600
6300
6300
6300
1600
1600
6300
6300
c
b
b
b
5(8,8)
c
b
b
36b
8.9
4.4
4.4
5(9,9)
>6300
>6300
>6300
>6300
c
c
c
b
b
b
5(10,10)
5(11,11)
5(12,12)
b
b
b
b
b
b
b
b
b
b
b
b
a
b
a
b
1600
1600
3100
3100
6300
390
6300
>6300
>6300
>6300
b
b
b
b
b
b
560
560
560
560
560
560
>6300
>6300
>6300
b
b
b
b
b
b
>6300
>6300
>6300
b
160b
b
b
b
b
>6300
>6300
>6300
a
b
MIC data from Ref. [14].
Incomplete inhibition. Inhibition was defined as the lowest concentration of drug resulting in a prominent visible decrease in turbidity. Complete inhibition (i.e., 100%) or incomplete inhibition (i.e.,
50%) reduction in visible turbidity compared to the drug-free control.
MIC data from Ref. [15].
≥
c

6
22
steroids 7 2 ( 2 0 0 7 ) 615–626
◦
cerevisiae and M. luteus at 30 C) for 4–7 days. After growth,
the resulting broth cultures were diluted with buffered saline
gelatin [BSG, gelatin (0.1 g/L), NaCl (8.5 g/L), KH2PO4 (0.3 g/L),
Na2HPO4 (0.6 g/L)] to equal the turbidity of a No. 1 McFarland
Standard. To check for viability and contamination, broth cul-
tures were streaked on Plate Count Agar (BBL Microbiology
◦
Systems, Cockeysville, MD); the plates were incubated at 37 C
for 3–4 days. Plates for M. luteus and S. cerevisiae were grown
◦
at 30 C. Spores of A. niger were scraped from the surface of
PDA and suspended in 5 mL of 1/10-strength BHIB + S and that
suspension transferred to a sterile test tube. The turbidity was
adjusted to a No. 1 McFarland Standard by dilution with BSG.
To check for viability and contamination, those spore suspen-
◦
sions were streaked on PDA and incubated at 37 C for 3–4
days.
2
.4.2. Quality assurance
For the work reported here, all cultures and suspensions that
were used as inocula were uncontaminated; colonies had the
expected morphologies. To check for viability and contami-
nation, broth cultures were streaked on plate count agar; the
◦
plates were incubated at 37 C for 4 days. All viable, uncontam-
◦
inated inocula were stored up to 14 days at 4 C until used.
2
.4.3. Measurement of minimal inhibitory concentrations
(
MICs)
Microtiter plates (96 wells: rows A–H, columns 1–12) were filled
by the following protocol to give twofold serial dilutions of
1
a (1b, 2a, and 2b). Aliquots (50 ␮L) of 1/10-strength BHIB + S
pH 7.4) were placed in all the wells except for those in col-
umn 1. Stock solutions of triethanolamine: 1a (1b, 2a, and
b) (100 ␮L) were placed in column 1. An aliquot (50 ␮L) was
(
2
removed from a well in column 1 and mixed with the BHIB + S
in the well of column 2. Then, an aliquot (50 ␮L) of this mix-
ture was removed and mixed with BHIB + S in column 3. This
process was repeated through column 11, at which point an
aliquot (50 ␮L) was removed and discarded. Column 12 was the
blank (positive growth) control, BHIB + S only, for each row. An
8
aliquot (50 ␮L) of the microbial inoculum (∼10 CFU/mL) was
added to all wells in a row. Aqueous triethanolamine without
1
a (1b, 2a, and 2b) was also tested for antimicrobial activ-
ity by using the same protocol; no antimicrobial activity was
found. The concentration of 1a (1b, 2a, and 2b) ranged from
Scheme 1 – Reagents and conditions. (i) MsCl, Et3N, CH2Cl2,
1
3
2.5 mg/mL to 1.1 ␮g/mL. After the plates were incubated at
7 C for 4 days, the MICs were read. The MIC of each com-
◦
◦
◦
◦
0 C, 15 min; (ii) LiBr, THF, 70 C, 14 h; (iii) NaN3, DMF, 90 C,
◦
4
h; (iv) LiAlH4, THF, 0 C then reflux, 4 h; (v) WeNCO,
pound was measured in triplicate and was defined as the
lowest concentration of drug resulting in a prominent visible
decrease in turbidity (incomplete; i.e., ≥50%) or a complete
absence of visible turbidity compared to the drug-free control.
CH2Cl2, room temperature, 24 h; (vi) HCOOH, room
temperature, 16 h.
b
In many cases (identified in Table 2 by superscript ), inhibition
(
5–7) for which we obtained narrow melting-temperature
was incomplete.
ranges. In order to synthesize the epimeric pair 1a and 1b,
the first step converted 3b into the corresponding known
mesylate 4. The reaction occurred quickly; the crude prod-
uct was suitable without purification for the next two steps.
Mesylate 4 was the key synthon to produce both epimers,
3
.
Results and discussion
3
.1.
Chemistry
1a and 1b. To synthesize epimer 1b, 4 reacted with lithium
3
.1.1. Syntheses of 1a and 1b
Our syntheses (Scheme 1) began with commercially available
␣-cholestan-3␤-ol (3b) and used several known compounds
bromide in THF to give 3␣-bromo-5␣-cholestane (5) in 74%
yield. Treating 4 and 5 with sodium azide in DMF gave 6a
and 6b in 85% and 79% isolated yield, respectively, after flash
5

steroids 7 2 ( 2 0 0 7 ) 615–626
623
column chromatography. Reduction of 6a and 6b was per-
formed by two methods – catalytic hydrogenation and lithium
aluminum hydride – to give amines 7a and 7b, respectively.
The work-up of catalytic hydrogenation was more conve-
nient than that of lithium aluminum hydride. However, the
product of the latter method was isolated in a higher yield,
with a higher melting temperature, and a whiter color. The
amines 7a and 7b added to WeNCO to give the tri-tert-butyl
esters 8a and 8b in 94% and 90% yields, respectively. Both 8a
and 8b were fully characterized. Recrystallization of 8a from
ethanol formed crystals suitable for X-ray analysis (see below).
Formolysis of the compounds 8a and 8b at room tempera-
ture produced the triacids 1a and 1b in 72% and 78% yields
after recrystallization, respectively. Both 1a and 1b were fully
characterized.
and 11b added to WeNCO to give the tri-tert-butyl esters 12a
and 12b in 64% and 61% yields, respectively. These com-
pounds were fully characterized. These epimers displayed
two noteworthy differences. The first was the large differ-
◦
ence in melting temperatures—12a, 82.9–83.3 C compared
◦
to 12b, 195.8–196.2 C. Second, the methylene protons of the
ethanoate moiety in 12a were magnetically nonequivalent
protons (ı 3.94 and 3.89 ppm); these protons in 12b were
magnetically equivalent. Formolysis of 12a and 12b at room
temperature produced the triacids 2a and 2b in 57% and 51%
yields, respectively. These triesters and triacids were fully
characterized.
3.1.3. X-ray structure of 8a
The crystal structure of 8a shows how the molecules with
a
bulky dendritic polar moiety and an extremely rigid
3
.1.2. Syntheses of 2a and 2b
hydrophobic moiety assemble in the solid state. Two differ-
ent crystallographic independent molecules are found in the
X-ray crystal structure arranged in parallel; the 21 screw axis
generates the antiparallel arrangement. In general, molecules
that are antiparallel are packed more closely than molecules
that are parallel. The closest interactions in the antiparallel
Our syntheses (Scheme 2) began with commercially available
5
3
␣-cholestan-3␤-ol (3b). Converting 3b into 5␣-cholestan-
␣-ol (3a) followed the literature procedure [29]; 3a was
obtained in 80% yield after column chromatography. Treating
the 5␣-cholestan-3-ols 3a and 3b with chloroacetyl chloride
gave 9a and 9b in 57% and 68% yields, respectively. Heat-
ing the chloroethanoates 9a and 9b with sodium azide in
DMF gave the azidoethanoates 10a and 10b in 47% and
orientation range from 3.8 to 4.1
A˚ and involve the side chain
of one cholestanyl moiety and rings B and C, including the
methyl groups at the A/B and C/D ring junctions on the other
cholestanyl moiety. In the parallel orientation, close interac-
4
9% yields, respectively. Catalytic hydrogenation reduced azi-
doethanoates 10a and 10b to aminoethanoates 11a and
1b in 88% and 76% yields, respectively. These azido- and
tions range from 4.0 to 4.1
A˚ and involve the side chain of one
1
cholestanyl moiety and rings B, C, and D, including the methyl
group at the A–B ring junction of the other cholestanyl moiety.
Ring A of this cholestane moiety displays a short intermolec-
ular contact to the headgroup of the other cholestanyl moiety,
ranging from 3.4 to 4.1 A˚ .
aminoethanoates were fully characterized. The amines 11a
Symmetrically equivalent molecules are separated by
1
2.12 A˚ (i.e., the a-axis) from each other. In the two inde-
pendent molecules, all six-membered rings are found in
full chair conformations, while all five-membered rings
are found in twisted envelope conformations. Two of the
three headgroups of each independent molecule adopt
anti conformations, while the third headgroup adopts a
gauche conformation. Torsion angles of two headgroups
◦
in the first molecule are −170.1(6) [C(1)–C(37)–C(38)–C(39)]
◦
and +177.2(6) [C(1)–C(44)–C(45)–C(46)], and two in the sec-
◦
ond molecule are −170.5(6) [C(51)–C(80)–C(81)–C(82)] and
◦
+
166.8(7) [C(51)–C(94)–C(95)–C(96)]. Torsion angles of the third
headgroup adopt gauche conformations, however, of oppo-
◦
site sign; those in the first and second molecules are −96.9(5)
◦
[
C(1)–C(30)–C(31)–C(32)] and +91.6(7) [C(51)–C(87)–C(88)–C89)],
respectively.
Molecules of ethanol co-crystallize with the steroid
molecules. Ethanols bridge the two steroid molecules, serving
as a hydrogen bond donor to one crystallographic molecule of
8a and a hydrogen bond acceptor to the other. Ethanols are
hydrogen-bond donors (O(16) and O(15)) to the carbonyl oxy-
gen on the ureido linker, namely O(1) and O(8), respectively
(Fig. 1). The ethanolic oxygens O(16) and O(15) are accep-
tors for the hydrogens on ureido nitrogens [N(4) and N(2),
respectively] connected to the cholestanyl moiety. In addi-
tion, the other ureido nitrogens [N(3) and N(1)] in the linker
form weak hydrogen bonds with the same ethanol molecules
(donor–acceptor distance >3.0 A˚ ). The hydrogen bond between
Scheme 2 – Reagents and conditions. (i) DIAD, CF3COOH,
Ph3P, PhCOONa, THF, room temperature, 16 h; (ii)
ClCH2COCl, Et3N, DMAP, PhH, room temperature, 16 h; (iii)
◦
◦
NaN3, DMF, 90 C, 10 h; (iv) H2, 10% Pd/C, CH2Cl2, 60 C, 4 h;
v) WeNCO, CH2Cl2, room temperature, 5 h; (vi) HCOOH,
room temperature, 5 h.
(

6
24
steroids 7 2 ( 2 0 0 7 ) 615–626
Fig. 1 – X-ray crystal structure of 8a, showing the packing and the intermolecular hydrogen bonding to ethanol.
◦
Hydrogen-bond donor (D)–acceptor (A) distances and approximate ∠DHA: O(16)· · ·O(1), 2.621(7) A˚ and 158.1 ; O(15)· · ·O(8),
◦
◦
◦
2
.625(6) A˚ and 161.0 ; O(4)· · ·O(16), 2.865(7) A˚ and 155.7 ; N(2)· · ·O(15), 2.849(8) and 159.6 . The other nitrogens on the ureido
◦
linker are just outside the range for significant hydrogen bonding. N(3)· · ·O(16), 3.035(7) A˚ and 141.6 ; N(1)· · ·O(15), 3.084(8) A˚
◦
and 146.0 .
ethanol and the carbonyl oxygen of the uriedo linker is the
shortest.
and E. coli showed that the 13(n) series was more active than
the other series.
3
.2.
Antimicrobial assays
The amphiphiles dissolved readily into aqueous tri-
ethanolamine solutions [14]. These aqueous solutions of
1
and 2 were 18.9 and 17.4 mM, respectively. We did not
pursue the maximum solubilities of these amphiphiles in
the aqueous triethanolamine solution because any com-
pound with a minimal inhibitory concentration (MIC) at
this concentration is not worth developing as a lead. The
molar ratios of 1:triethanolamine and 2:triethanolamine
were 1:17 and 1:19, respectively; these solutions had a pH of
8
–9.
The comparison of the antimicrobial activities of 1a, 1b,
a, and 2b with those of 13(n), 14(n,n), and 15(n,n) in a
2
broad screen of several microorganisms revealed several dif-
ferences (Table 2) [14,15]. In general, L. plantarum, M. luteus
and A. niger were simply not inhibited to any degree by any of
the amphiphiles; perhaps the weak activity of 14(8,8) against
A. niger was an exception. Compared to 13(n), 14(n,n), and
The only microorganisms that were relatively susceptible
to the steroidal amphiphiles were the unrelated S. aureus and
MRSA strains. For S. aureus, 2b showed the most activity, albeit
modest, among those amphiphiles tested. Both two-tailed
series were completely inactive; the one-tailed series was
weakly active. For the MRSA strain, all steroidal amphiphiles
except 2b were more active than any homologue in the first
three series. The MICs of 1a, 1b, 2a, and 2b against the MRSA
1
5(n,n), the steroidal amphiphiles 1a, 1b, 2a, and 2b, were
inactive against three yeast—S. cerevisiae, C. albicans, and C.
neoformans. In part, this may be due to the fact that ergosterol
is a component of the fungal cell membrane. For M. smegma-
tis, these steroidal amphiphiles showed a little activity, which
was less than the 13(n) series (Table 2). Similarly, the modest
activities of the steroidal amphiphiles against K. pneumoniae

steroids 7 2 ( 2 0 0 7 ) 615–626
625
strain were 300, 27, 270, and 1100 ␮M, respectively. The most
active amphiphile 1b was 10-fold more potent than 2a against
MRSA. These data suggested that stereochemistry and spacer
length of the amphiphiles affect their biological activities.
In summary, tricarboxylato amphiphiles with a cholestanyl
moiety appear to have selective antimicrobial properties,
namely, against the MRSA strain and, to a lesser extent,
the S. aureus strain. Other antimicrobial compounds with
cholestanyl moieties also show activity against S. aureus
in screens of antimicrobial activity. Charge and polar-
ity of cholestanyl amphiphiles might modulate activity as
squalamine [1], squalamine mimics [6,9], and cholestanyl
aminoglycoside [11] appear to be more active than 2b against S.
aureus. The steroidal ring might control antimicrobial selectiv-
ity as S. aureus and MRSA are quite susceptible to amphiphiles
with steroidal moieties. Now that derivatives with some
antimicrobial activity have been identified, large numbers of
MRSA and S. aureus strains can be tested to determine sus-
ceptibilities of many representatives of those pathogens. The
good activity of 1b against MRSA provides a potential lead to
the development of more potent analogs.
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