Lathosterol side chain amides-A new class of human lathosterol oxidase inhibitors

Article abstract of DOI:10.1016/j.steroids.2007.10.015

Inhibition of cholesterol biosynthesis offers the opportunity for treatment of cardiovascular diseases. Numerous enzymes are involved in the post-squalene part of this biosynthesis, and selective inhibitors for almost all of the enzymes involved there hav

Full text of DOI:10.1016/j.steroids.2007.10.015

steroids 7 3 ( 2 0 0 8 ) 299–308
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Lathosterol side chain amides—A new class of human
lathosterol oxidase inhibitors
Martin Giera∗, Delphine Renard, Florian Plo¨ssl, Franz Bracher
¨
¨
¨
Department Pharmazie – Zentrum fur Pharmaforschung, Ludwig-Maximilians-Universitat Munchen,
Butenandtstrasse 5-13, 81377 Munich, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of cholesterol biosynthesis offers the opportunity for treatment of cardiovascular
diseases. Numerous enzymes are involved in the post-squalene part of this biosynthesis,
and selective inhibitors for almost all of the enzymes involved there have been described in
literature. The only exception is the enzyme lathosterol oxidase (EC 1.14.21.6), for which up
to now no selective inhibitor has been found. Up to date only triarimol has been reported
as a weak inhibitor. In this paper we report on lathosterol side chain amides as a new class
of selective lathosterol oxidase inhibitors. To study the influence of different sterol amides
on inhibition of this enzyme, numerous compounds were prepared and the sterol patterns
resulting from incubation of HL 60 cells with these enzyme inhibitors were monitored in a
whole cell screening assay by means of GC/MS analysis. Small alkyl residues at the amide
nitrogen (hydrogen and methyl) lead to an inhibition of the enzyme ꢀ24-reductase, the
N-ethyl and N-propyl derivatives show a dual action, inhibiting both ꢀ24-reductase and
lathosterol oxidase. Lathosterol-derived amides with larger substituents (butyl, isobutyl,
tert-butyl, pentyl) at the amide nitrogen were found to be selective inhibitors of lathosterol
oxidase. The corresponding 3␤-acetoxy derivatives showed comparable activities and can be
considered as prodrugs, since they are transformed into the 3␤-hydroxy derivatives under
the test conditions, as proven by HPLC analysis.
Received 11 June 2007
Received in revised form
23 October 2007
Accepted 31 October 2007
Published on line 17 November 2007
Keywords:
Lathosterol oxidase
EC 1.14.21.6
Cholesterol
Inhibitor
Biosynthesis
© 2007 Elsevier Inc. All rights reserved.
1.
Introduction
tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid disodium
salt) [4] and other hydrophilic chelators [1,2]. Further more
Lathosterol oxidase (5-DES, ꢀ7-sterol-C5(6)-desaturase,
EC 1.14.21.6) is a membrane bound microsomal enzyme
which catalyzes the conversion of lathosterol to 7-
dehydrocholesterol in the post-squalene pathway of
cholesterol biosynthesis [1]. This enzyme requires molec-
ular oxygen, a supernatant protein factor, and is strongly
promoted by NAD(P)H [2,3]. The enzyme system consists
of NADH-cytochrome b5 reductase, cytochrome b5 and the
terminal desaturase [3]. It is proposed to hold non-heme iron
in the catalytic center, because it can be inhibited by cyanide,
the enzyme is insensitive against carbon monoxide [2].
In 2002 Brunetti-Pierri et al. reported on lathosterolosis (5-
DES deficiency), a defect of cholesterol biosynthesis leading to
multiple malformations and mental retardation demonstrat-
ing the importance of this enzyme for embryogenesis [5,6].
The only, but quite weak inhibitor of lathosterol oxi-
dase reported up to date is triarimol ((RS)-2,4-dichloro-␣-
(pyrimidin-5-yl)benzhydryl alcohol), a pyrimidine fungicide
acting mainly on sterol C14-demethylase (EC 1.14.13.70),
but also initiating a weak accumulation of lathosterol, the
∗
Corresponding author. Tel.: +49 89 2180 77258; fax: +49 89 2180 77171.
E-mail address: martin.giera@cup.uni-muenchen.de (M. Giera).
0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2007.10.015

300
steroids 7 3 ( 2 0 0 8 ) 299–308
Scheme 1 – Synthesis of the lathosterol side chain amides, 4-DMAB: 4-dimethylaminobenzaldehyde.
[14,15]. Selective ozonolysis of the ꢀ22(23) double bond of 18
in dichloromethane/pyridine followed by reductive workup
using dimethyl sulfide gave the corresponding aldehyde 19
in yields ranging from 32 to 40% [16]. This aldehyde was
substrate of lathosterol oxidase [7]. Using our recently
described screening system for the identification of inhibitors
of enzymes in late cholesterol biosynthesis [8] we detected
a strong accumulation of lathosterol on treatment of HL 60
cells, a human promyelotic leukemia cell line (for a review
see Ref. [9]), with some side chain amides derived from lath-
osterol (see below). The observed lathosterol accumulation is
clearly associated to an inhibition of lathosterol oxidase, lead-
ing to the first class of selective inhibitors of this enzyme.
In order to study the qualitative and quantitative effects of
the lengths of the N-alkyl residues we prepared the com-
pounds listed below (Scheme 1, Table 1). All compounds
were subjected to a standard MTT assay [10] to determine
cytotoxic effects in a concentration range from 3.125 to
100 ␮M, to exclude unspecific cytotoxic effects (Table 2). The
compounds were submitted to qualitative testing for distal
cholesterol biosynthesis inhibition at concentrations of 1, 5
and 50 ␮M as previously described [8] (see also Fig. 1). The
percent inhibition of total cholesterol production was deter-
mined at a fixed concentration of 1 ␮M for all compounds
(Table 4), and the corresponding IC50 values were determined
for the selective inhibitors of lathosterol oxidase as recently
described (Table 5) [8]. Because the inhibitory behaviour
of the 3␤-alcohols (21b–30b) and the corresponding 3␤-
acetates (21a–30a) did not show significant differences, a HPLC
assay was performed to determine whether the 3␤-acetates
served as prodrugs for the 3␤-alcohols in our assay (see
below).
Table 1 – Lathosterol side chain amides
Compound
R1
R2
R3
21a
22a
23a
24a
25a
26a
27a
28a
29a
30a
21b
22b
23b
24b
25b
26b
27b
28b
29b
30b
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
Me
H
Me
Et
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OH
OH
OH
OH
OH
OH
OH
OH
OH
n-Pr
n-Bu
iso-Bu
t-Bu
Pent
Benzyl
n-Bu
H
Me
Et
n-Pr
n-Bu
iso-Bu
t-But
Pent
Benzyl
n-Bu
1.1.
Chemistry
Reaction of ergosterol with acetic anhydride and pyridine
gave ergosteryl acetate (17) [12]. Ester 17 was subjected
to selective hydrogenation using Raney nickel W2 [13] to
give 5␣,6-dihydroergosteryl acetate (18) in good yield (83%)
OH

steroids 7 3 ( 2 0 0 8 ) 299–308
301
Fig. 1 – Cholesterol biosynthesis and target enzymes for late cholesterol biosynthesis inhibitors [11]; (A) squalene
epoxidase, (B) lanosterol synthase, (C) ꢀ24-reductase, (D) sterol C14-demethylase, (E) ꢀ14-reductase, (F) C4-demethylase, (G)
ꢀ
8/7-isomerase, (H) lathosterol oxidase and (I) 7-dehydrocholesterol reductase. (1) squalene, (2) monoepoxysqualene, (3)
lanosterol, (4) dihydrolanosterol, (5) 4,4-dimethylcholesta-8,14-dien-3␤-ol, (6) 4,4-dimethylcholesta-8-en-3␤-ol, (7)
zymostenol, (8) lathosterol, (9) 7-dehydrocholesterol, (10) cholesterol, (11) desmosterol, (12) cholesta-5-7-24-trien-3␤-ol, (13)
cholesta-7,24-dien-3␤-ol, (14) zymosterol, (15) 4,4-dimethylcholesta-8,24-dien-3␤-ol and (16)
4,4-dimethylcholesta-8,14,24-trien-3␤-ol.
oxidized to the carboxylic acid 20 using potassium perman-
ganate in a tetrahydrofuran/water mixture in yields ranging
from 55 to 66%. Carboxylic acid 20 was transformed into
the corresponding acid chloride using oxalylchloride and
dimethylformamide. The crude acid chloride was reacted with
various primary amines to afford the corresponding amides
(21a–29a) in yields from 35 to 83%. The 3␤-acetate protec-
tive groups were conveniently removed by alkaline hydrolysis
using potassium carbonate in chloroform/methanol (1:1) to
give the free 3␤-alcohols (21b–29b) in very good yields
(Scheme 1). All new compounds are listed in Table 1.
Since in first biological screenings the N-butyl amide 25b
turned out to be a selective inhibitor of lathosterol oxidase,
we also prepared the tertiary amides 30a and 30b, containing
an additional N-methyl group in the same manner in order
to investigate biological effects of amides lacking a NH func-
tion. Both tertiary amides were found to be 1:1 mixtures of
rotamers, as determined by NMR.

302
steroids 7 3 ( 2 0 0 8 ) 299–308
was allowed to warm up to room temperature and stirred
2.
Experimental procedures
for another 2 h. 5 ml of a saturated NaHCO3 solution were
added and the reaction mixture was transferred into a sepa-
rating funnel, washed twice with a saturated NaHCO3 solution
(2 × 10 ml) and brine (2× 10 ml), dried over sodium sulfate and
evaporated to dryness under reduced pressure. The resulting
solid was subjected to silica column chromatography (SCC) as
specified below.
2.1.
Materials
RPMI 1640 medium and fetal bovine serum (FBS) were
from PAA Laboratories GmbH (Co¨lbe, Germany), Medium
fu¨ r HL 60 Zellen (lipid-free medium) and lipoprotein defi-
cient serum (LPDS) were purchased from PAN Biotech
(Aidenbach, Germany), the HL 60 cell line was from DSMZ
(Deutsche Sammlung fu¨ r Mikroorganismen und Zellkulturen,
Braunschweig, Germany). Culture flasks and 6-well plates
were from Peske (Aindling-Arnhofen, Germany). Cholestane
was obtained from Steraloids Inc. (Birmingham, UK). Silica
gel 60 (230–400 mesh) for SCC was from Merck (Darm-
stadt, Germany). All other chemicals were purchased from
Sigma–Aldrich (Schnelldorf, Germany). All solvents of a purity
degree lower than “pro analysi” were distilled before use.
2.3.2. General procedure B (hydrolysis of the 3ˇ-acetoxy
group)
50–100 mg of the ester was dissolved in 4 ml of chloro-
form/methanol (1:1) and three equivalents of potassium
carbonate were added under stirring. The solution was stirred
for 16 h at room temperature, evaporated to dryness under
reduced pressure and the residue was subjected to silica col-
umn chromatography (SCC) as specified below.
2.3.2.1. 5˛,6-Dihydroergosteryl acetate (18). The substance
was prepared according to Refs. [14,15] and recrystallized
twice from chloroform/MeOH (6.6 g, 83%).
2.2.
Equipment
mp 185 ◦C, Ref. [19] 181–182 ◦C. 1H NMR (CDCl3, 500 MHz)
ı 5.18 (m, 3H, H-7, H-22, H-23), 4.67 (m, 1H, H-3), 2.03 (s, 3H,
H-20), 2.02 (m, 2H, H-12 and H-20), 1.86–1.21 (m, 23H), 1.13
(ddd, 1H, H-1␣, 3J = 3.8 Hz, 2J = 3J = 13.5 Hz), 1.01 (d, 3H, H-21,
3J = 6.6 Hz), 0.91 (d, 3H, H-28, 3J = 6.8 Hz), 0.82 (2× d, 6H, H-26,
H-27, 3J = 6.8 Hz), 0.81 (s, 3H, H-19), 0.57 (s, 3H, H-18). HRMS
440.3689 (calcd 440.3654).
NMR spectra were recorded in CDCl3 or CDCl3/methanol-d4
using the chloroform peak as standard, on a Jeol GSX 400
or JNMR GX 500 instrument. High resolution mass spectra
were recorded on a GC Mate II (Jeol, Peabody, MA, USA).
Melting points were determined on a Bu¨ chi B 540 apparatus
(Bu¨ chi, Flavil, Switzerland). HPLC purity analysis was car-
ried out on a Shimadzu LC 10 AT equipped with a SIL 10
A autosampler, a SPD 10 AV UV–Vis detector (210 nm) and a
CTO 10 AC column oven (Shimadzu, Duisburg, Germany). A
LiChrospher 100 RP 18–250 mm × 4 mm (5 ␮m) column (Merck,
Darmstadt, Germany) was used at 35 ◦C. The binary pump sys-
tem supplied the specified mixture (see below) of MeOH and
phosphate buffer pH 5 (KH2PO4, 20 mM) isocratically at a flow
rate of 1 ml/min. All substances were dissolved in ethanol to
reach a final concentration of 1 mg/ml, 20 ␮l of these solu-
tions were injected. The purity was generally determined from
a 15–20 min run after a blank run (pure ethanol) subtrac-
tion.
Structure assignment for all prepared compounds was
done using 1H, 13C NMR, as well as DEPT, HMQC, and HMBC
techniques.
Cholesta-7,24-dien-3␤-ol (13) and 4,4-dimethylcholesta-
8,24-dien-3␤-ol (15) were identified on basis of their mass
spectral data [17,18] and their relative retention times. Quali-
fier ions are 13 441 (27), 343 (100) and 253 (18) [m/z]; 15 484 (52),
394 (71) and 379 (100) [m/z].
2.3.2.2. (3S,20S)-20-Formylpregn-7-en-3-yl acetate (19). To a
stirred solution of 18 (4.4 g, 10 mmol) and pyridine (5 ml) in
dichloromethane (1 l) was added at −70 ◦C ozone (flow rate
60 l O3/h, 16 mmol, 1.6 equiv.) over 10 min. After further 2 min
MeOH (50 ml) and dimethyl sulfide (1.5 ml) were added. The
reaction mixture was stirred for 30 min at −70 ◦C and then
was allowed to warm up to room temperature. The reac-
tion mixture was concentrated and the residue was purified
by SCC (pentane/diethyl ether, 90:10 to recover the starting
material, and then 85:15 to obtain 19) (1.2–1.5 g, 32–40%).
mp 139 ◦C Ref. [20] 144–145 ◦C. 1H NMR (CDCl3, 500 MHz) ı
9.58 (d, 1H, CHO, J = 3.2 Hz), 5.18 (m, 1H, H-7), 4.69 (m, 1H,
H-3), 2.36 (m, 1H, H-20), 2.02 (s, 3H, H3CCO), 1.96–1.20 (m,
20H), 1.01 (d, 3H, H-21, 3J = 6.8 Hz, with underneath 1H, H-1),
0.82 (s, 3H, H-19), 0.57 (s, 3H, H-18). HRMS 372.2685 (calcd
372.2665).
2.3.2.3. (3S,20S)-3ˇ-Acetoxypregn-7-en-20-yl-methanoic
acid (20). 19 (1.4 g, 3.77 mmol) was dissolved in 30 ml of a
mixture of tetrahydrofuran and water (4:1). A solution of
potassium permanganate (0.576 g, 3.60 mmol) dissolved in
8 ml water was added slowly under stirring until no further
decolourisation of the solution could be detected. Precipitated
manganese dioxide was filtered off and the filtrate was con-
centrated almost to dryness. The residue was washed with
acetic acid (6 ml) and then fully dried under reduced pressure.
The residue was subjected to SCC (pentane/diethyl ether 6:4)
(0.933 g, 63%). mp 197 ◦C, Ref. [21] 175–185 ◦C. 1H NMR (CDCl3,
500 MHz) ı 5.16 (m, 1H, H-7), 4.69 (m, 1H, H-3), 2.41 (m, 1H,
H-20), 2.03 (s, 3H, H3CCO), 1.96 (ddd, 1H, H-12␤, 3J = 3J = 3.3 Hz,
2J = 12.4 Hz), 1.89–1.31 (m, 18H), 1.25 (d, 3H, H-21, 3J = 6.9 Hz),
2.3.
Syntheses
2.3.1. General procedure A (amide synthesis)
To stirred solution of 200 mg (0.52 mmol) (3S,20S)-3␤-
a
acetoxypregn-7-en-20-yl-methanoic acid (20) in 3 ml of
dichloromethane 0.2 ml (1.03 mmol) of oxalyl chloride and
50 ␮l of dimethylformamide were added under nitrogen atmo-
sphere and the solution was stirred for 3 h at ambient
temperature. Then the solution was evaporated to dryness
under reduced pressure to give the acid chloride. This acid
chloride was dissolved in 5 ml of dichloromethane and four
equivalents of the amine were added at 0 ◦C. The solution

steroids 7 3 ( 2 0 0 8 ) 299–308
303
1.13 (ddd, 1H, H-1␣, 3J = 3.2 Hz, 2J = 3J = 13.0 Hz), 0.81 (s, 3H,
H-19), 0.56 (s, 3H, H-18). HRMS 388.2614 (calcd 388.2628).
ı 5.60 (s, 1H, NH), 5.16 (m, 1H, H-7), 4.68 (m, 1H, H-3), 3.22
(m, 2H, H-1ꢀ), 2.08 (m, 1H, H-20), 2.01 (s, 3H, H3CCO), 1.95
(ddd, 1H, H-12␤, 3J = 3.3 Hz, 3J = 2.6 Hz, 2J = 12.1 Hz), 1.88–1.23
(m, 22H), 1.18 (d, 3H, H-21, 3J = 6.6 Hz), 1.12 (ddd, 1H, H-1␣,
3J = 3.7 Hz, 2J = 3J = 13.5 Hz), 0.91 (t, 3H, H-4ꢀ, 3J = 7.3 Hz), 0.81 (s,
3H, H-19), 0.56 (s, 3H, H-18). HRMS 443.3416 (calcd 443.3399).
HPLC (85/15), 6.79 min (100%).
2.3.2.4. (3S,20S)-20-Carbamoylpregn-7-en-3ˇ-yl acetate (21a).
200 mg (0.52 mmol) 20 were treated as described in general
procedure A. An excess of gaseous ammonia was added at
−60 ◦C. The solution was allowed to warm up to 0 ◦C and
stirred for 30 min. The solution was warmed up to room
temperature and stirred until no further gas evolution was
observed. The crude product was subjected to SCC (diethyl
ether) (70 mg, 35%). mp 258 ◦C. 1H NMR (CDCl3, 500 MHz) ı 5.35
(m, 2H, NH2), 5.15 (m, 1H, H-7), 4.68 (m, 1H, H-3), 2.18 (m, 1H,
H-20), 2.02 (s, 3H, H3CCO), 1.96 (ddd, 1H, H-12␤, 3J = 3J = 3.6 Hz,
2J = 12.4 Hz), 1.9–1.3 (m, 19H), 1.22 (d, 3H, H-21, 3J = 6.6 Hz), 1.14
(ddd, 1H, H-1␣, 3J = 3.5 Hz, 2J = 3J = 13.1 Hz), 0.81 (s, 3H, H-19),
0.55 (s, 3H, H-18). HRMS 387.2773 (calcd 387.2744). HPLC (85/15),
4.70 min (97%).
2.3.2.9. (3S,20S)-20-N-(2-Methylpropyl)carbamoylpregn-7-en-
3ˇ-yl acetate (26a). 200 mg (0.52 mmol) 20 were treated as
described in procedure A. 0.21 ml of iso-butylamine (2.1 mmol)
were added and the crude product was subjected to SCC (pen-
tane/diethyl ether 3:7) (159 mg, 70%). mp 196 ◦C. 1H NMR
(CDCl3, 400 MHz) ı 5.42 (m, 1H, N–H), 5.14 (m, 1H, H-7), 4.68 (m,
1H, H-3), 3.05 (m, 2H, H-1ꢀ), 2.02 (s, 3H, H3CCO), 1.95 (ddd, 1H,
H-12␤, 3J = 3J = 3.7 Hz, 2J = 12.1 Hz), 1.9–1.2 (m, 20H), 1.19 (d, 3H,
H-21, 3J = 6.8 Hz, with underneath 1H, H-1␣), 0.91 (d, 6H, H-3ꢀ,
H-4ꢀ, 3J = 6.6 Hz), 0.80 (s, 3H, H-19), 0.54 (s, 3H, H-18). HRMS
443.3366 (calcd 443.3399). HPLC (85/15), 6.86 min (99%).
2.3.2.5. (3S,20S)-20-N-Methylcarbamoylpregn-7-en-3ˇ-yl
acetate (22a). 175 mg (0.45 mmol) 20 were treated as described
in general procedure A. 0.90 ml of methylamine in THF (2M,
1.8 mmol) were added and the crude product was subjected
to SCC (diethyl ether) (91 mg, 50%). mp 324 ◦C. 1H NMR (CDCl3,
400 MHz) ı 5.41 (m, 1H, N–H), 5.14 (m, 1H, H-7), 4.68 (m, 1H,
H-3), 2.79 (s, 0.5 × 3H, H-1ꢀ), 2.78 (s, 0.5 × 3H, H-1ꢀ), 2.02 (s,
3H, H3CCO), 1.95 (ddd, 1H, H-12␤, 3J = 3J = 3.7 Hz, 2J = 12.1 Hz),
1.9–1.2 (m, 20H), 1.18 (d, 3H, H-21, 3J = 6.8 Hz), 0.80 (s, 3H,
H-19), 0.53 (s, 3H, H-18). HRMS 401.2930 (calcd 401.2934). HPLC
(85/15), 4.57 min (95%).
2.3.2.10. (3S,20S)-20-N-tert-Butylcarbamoylpregn-7-en-3ˇ-yl
acetate (27a). 200 mg (0.52 mmol) 20 were treated as described
in procedure A. 0.21 ml of tert-butylamine (2.1 mmol) were
added and the crude product was subjected to SCC (pen-
tane/diethyl ether 1:1) (188 mg, 82%). mp 195 ◦C. 1H NMR
(CDCl3, 500 MHz) ı 5.16 (m, 2H, N–H, H-7), 4.69 (m, 1H, H-3),
2.02 (s, 3H, H3CCO), 1.95–1.35 (m, 20H), 1.33 (s, 9H, H-2ꢀ, H-3ꢀ,
H-4ꢀ), 1.15 (d, 3H, H-21, 3J = 6.6 Hz, with underneath 1H, H-1␣),
0.80 (s, 3H, H-19), 0.53 (s, 3H, H-18). HRMS 443.3399 (calcd
443.3364). HPLC (85/15), 7.42 min (95%).
2.3.2.6. (3S,20S)-20-N-Ethylcarbamoylpregn-7-en-3ˇ-yl
acetate (23a). 200 mg (0.52 mmol) 20 were treated as described
in general procedure A. 1.1 ml of ethylamine in THF (2M,
2.2 mmol) were added and the crude product was subjected
to SCC (diethyl ether) (78 mg, 37%). mp 255 ◦C. 1H NMR (CDCl3,
500 MHz) ı 5.35 (m, 1H, N–H), 5.15 (m, 1H, H-7), 4.68 (m, 1H,
H-3), 3.29 (m, 2H, H-1ꢀ), 2.02 (s, 3H, H3CCO), 1.95 (ddd, 1H,
H-12␤, 3J = 3J = 3.4 Hz, 2J = 12.1 Hz), 1.9–1.2 (m, 19H), 1.18 (d, 3H,
H-21, 3J = 6.6 Hz), 1.12 (t, 3H, H-2ꢀ, 3J = 7.4 Hz, with underneath
1H, H-1␣), 0.80 (s, 3H, H-19), 0.54 (s, 3H, H-18). HRMS 415.3086
(calcd 415.3055). HPLC (85/15), 4.86 min (80%).
2.3.2.11. (3S,20S)-20-N-Pentylcarbamoylpregn-7-en-3ˇ-yl
acetate (28a). 200 mg (0.52 mmol) 20 were treated as described
in procedure A. 0.24 ml of n-pentylamine (2.1 mmol) were
added and the crude product was subjected to SCC (pen-
tane/diethyl ether 3:7) (143 mg, 61%). mp 163 ◦C. 1H NMR
(CDCl3, 400 MHz) ı 5.35 (m, 1H, N–H), 5.14 (m, 1H, H-7), 4.69
(m, 1H, H-3), 3.22 (m, 2H, H-1ꢀ), 2.02 (s, 3H, H3CCO), 1.95
(ddd, 1H, H-12␤, 3J = 3J = 3.3 Hz, 2J = 12.1 Hz), 1.9–1.2 (m, 19H),
1.18 (d, 3H, H-21, 3J = 6.6 Hz), 1.14 (ddd, 1H, H-1␣, 3J = 3.9 Hz,
2J = 3J = 14.3 Hz), 0.90 (t, 3H, H-5ꢀ, 3J = 7.2 Hz), 0.80 (s, 3H, H-19),
0.54 (s, 3H, H-18). HRMS 457.3563 (calcd 457.3556). HPLC
(85/15), 8.06 min (98%).
2.3.2.7. (3S,20S)-20-N-Propylcarbamoylpregn-7-en-3ˇ-yl
acetate (24a). 200 mg (0.52 mmol) 20 were treated as described
in procedure A. 0.18 ml (2.1 mmol) of n-propylamine were
added and the crude product was subjected to SCC (pen-
tane/diethyl ether 7:3) (154 mg, 70%). mp 187 ◦C. 1H NMR
(CDCl3, 500 MHz) ı 5.37 (m, 1H, N–H), 5.14 (m, 1H, H-7), 4.69
(m, 1H, H-3), 3.19 (m, 2H, H-1ꢀ), 2.02 (s, 3H, H3CCO), 1.95
(ddd, 1H, H-12␤, 3J = 3J = 3.7 Hz, 2J = 12.2 Hz), 1.90–1.20 (m, 21H),
1.18 (d, 3H, H-21, 3J = 6.6 Hz), 1.13 (ddd, 1H, H-1␣, 3J = 3.3 Hz,
2J = 3J = 13.1 Hz), 0.92 (t, 3H, H-3ꢀ, 3J = 7.2 Hz), 0.80 (s, 3H, H-19),
0.50 (s, 3H, H-18). HRMS 429.3243 (calcd 429.3255). HPLC
(85/15), 5.27 min (99%).
2.3.2.12. (3S,20S)-20-N-Benzylcarbamoylpregn-7-en-3ˇ-yl
acetate (29a). 200 mg (0.52 mmol) 20 were treated as described
in procedure A. 0.23 ml of benzylamine (2.1 mmol) were added
and the crude product was subjected to SCC (pentane/diethyl
ether 3:7) (200 mg, 81%). mp 196 ◦C. 1H NMR (CDCl3, 400 MHz)
ı 7.36–7.26 (m, 5H, phenyl-H), 5.64 (m, 1H, N–H), 5.15 (m, 1H,
H-7), 4.69 (m, 1H, H-3), 4.51–4.36 (m, 2H, H-1ꢀ), 2.09 (m, 1H,
H-20), 2.02 (s, 3H, H3CCO), 1.96 (ddd, 1H, H-12␤, 3J = 3J = 3.7 Hz,
2J = 12.1 Hz), 1.9–1.2 (m, 18H), 1.23 (d, 3H, H-21, 3J = 6.8 Hz),
1.13 (ddd, 1H, H-1␣, 3J = 4.4 Hz, 2J = 3J = 14.3 Hz), 0.80 (s, 3H,
H-19), 0.53 (s, 3H, H-18). HRMS 477.3201 (calcd 477.3243). HPLC
(85/15), 6.35 min (96%).
2.3.2.8. (3S,20S)-20-N-Butylcarbamoylpregn-7-en-3ˇ-yl
acetate (25a). 200 mg (0.52 mmol) 20 were treated as described
in procedure A. 0.20 ml of n-butylamine (2.1 mmol) were added
and the crude product was subjected to SCC (pentane/diethyl
ether 3:7) (189 mg, 83%). mp 105 ◦C. 1H NMR (CDCl3, 500 MHz)
2.3.2.13. (3S,20S)-20-N-Methyl-N-butylcarbamoylpregn-7-en-
3ˇ-yl acetate (30a). 200 mg (0.52 mmol) 20 were treated as
described in procedure A. 0.25 ml of N-methyl-N-butylamine

304
steroids 7 3 ( 2 0 0 8 ) 299–308
2J = 12.1 Hz), 1.87–1.22 (m, 23 H), 1.18 (d, 3H, H-21, 3J = 6.6 Hz),
1.08 (ddd, 1H, H-1␣, 3J = 3.4 Hz, 2J = 3J = 13.3 Hz), 0.92 (t, 3H, H-4ꢀ,
3J = 7.3 Hz), 0.81 (s, 3H, H-19), 0.54 (s, 3H, H-18). HRMS 401.3331
(calcd 401.3294). HPLC (75/25) 6.12 min (99%).
(2.1 mmol) were added and the crude product was subjected
to SCC (pentane/diethyl ether 3:7) (158 mg, 67%). mp 173 ◦C.
1H NMR (CDCl3, 400 MHz) ı 5.14 (m, 1H, H-7), 4.69 (m, 1H,
H-3), 3.5–3.2 (m, 2H, H-1ꢀ), 3.02 (s, 0.5 × 3H, N–CH3), 2.91 (s,
0.5 × 3H, N–CH3), 2.67 (m, 1H, H-20), 2.02 (s, 3H, H3CCO),
1.95 (ddd, 1H, H-12␤, 3J = 3J = 3.3 Hz, 2J = 11.5 Hz), 1.9–1.2 (m,
23H), 1.15 (d, 0.5 × 3H, H-21, 3J = 6.8 Hz), 1.12 (d, 0.5 × 3H, H-21,
3J = 6.8 Hz), 0.95 (t, 0.5 × 3H, H-4ꢀ, 3J = 7.25 Hz), 0.92 (t, 0.5 × 3H,
H-4ꢀ, 3J = 7.25 Hz), 0.81 (s, 3H, H-19), 0.57 (s, 3H, H-18). HRMS
457.3536 (calcd 457.3556). HPLC (85/15), 12.92 min (99%).
2.3.2.19. (3S,20S)-20-N-(2-Methylpropyl)carbamoylpregn-7-
en-3ˇ-ol (26b). 100 mg (0.25 mmol) of 26a were treated as
described in procedure B. The crude product was subjected to
SCC (ethyl acetate) (91 mg, 100%). mp 130 ◦C. 1H NMR (CDCl3,
400 MHz) ı 5.40 (m, 1H, NH), 5.16 (m, 1H, H-7), 3.60 (m, 1H, H-3),
3.05 (m, 2H, H-1ꢀ), 2.05 (m, 1H, H-20), 1.96 (ddd, 1H, H-12␤,
3J = 3J = 3.5 Hz, 2J = 12.1 Hz), 1.75–1.20 (m, 20H), 1.19 (d, 3H, H-21,
3J = 6.6 Hz), 1.09 (ddd, 1H, H-1␣, 3J = 3.7 Hz, 2J = 3J = 13.0 Hz), 0.91
(d, 6H, H-3ꢀ, H-4ꢀ, 3J = 6.6 Hz), 0.79 (s, 3H, H-19), 0.55 (s, 3H, H-18).
HRMS 401.3293 (calcd 401.3294). HPLC (75/25) 6.11 min (99%).
2.3.2.14. (3S,20S)-20-Carbamoylpregn-7-en-3ˇ-ol
(21b).
50 mg (0.14 mmol) of 21a were treated as described in proce-
dure B. The crude product was subjected to SCC (ethyl acetate)
(43 mg, 97%). mp 233 ◦C. 1H NMR (CDCl3/MeOH-d4, 500 MHz)
ı 5.00 (m, 1H, H-7), 3.38 (m, 1H, H-3), 2.08 (m, 1H, H-20), 1.81
(ddd, 1H, H-12␤, 3J = 3J = 3.3 Hz, 2J = 12.1 Hz), 1.75–1.05 (m, 21H),
1.03 (d, 3H, H-21, 3J = 6.6 Hz), 0.92 (ddd, 1H, H-1␣, 3J = 3.3 Hz,
2J = 3J = 13.2 Hz), 0.63 (s, 3H, H-19), 0.40 (s, 3H, H-18). HRMS
345.2705 (calcd 345.2668). HPLC (65/35) 7.41 min (96%).
2.3.2.20. (3S,20S)-20-N-tert-Butylcarbamoylpregn-7-en-3ˇ-ol
(27b). 100 mg (0.25 mmol) of 27a were treated as described in
procedure B. The crude product was subjected to SCC (diethyl
ether) (93 mg, 98%). mp 216 ◦C. 1H NMR (CDCl3, 400 MHz) ı 5.17
(m, 2H, N-H, H-7), 3.60 (m, 1H, H-3), 1.95–1.34 (m, 19H), 1.33 (s,
9H, H-2ꢀ, H-3ꢀ, H-4ꢀ), 1.15 (d, 3H, H-21, 3J = 6.6 Hz), 1.08 (ddd, 1H,
H-1␣, 3J = 3J = 3.1 Hz, 2J = 12.9 Hz), 0.79 (s, 3H, H-19), 0.53 (s, 3H,
H-18). HRMS 401.3294 (calcd 401.3286). HPLC (75/25) 6.81 min
(99%).
2.3.2.15. (3S,20S)-20-N-Methylcarbamoylpregn-7-en-3ˇ-ol
(22b). 50 mg (0.14 mmol) of 22a were treated as described in
procedure B. The crude product was subjected to SCC (ethyl
acetate) (37 mg, 82%). mp 279 ◦C. 1H NMR (CDCl3/MeOH-d4,
500 MHz) ı 4.99 (m, 1H, H-7), 3.37 (m, 1H, H-3), 2.54 (s, 3H,
H-1ꢀ), 1.99 (m, 1H, H-20), 1.79 (ddd, 1H, H-12␤, 3J = 3J = 3.0 Hz,
2J = 13.6 Hz), 1.75–1.00 (m, 22H), 0.98 (d, 3H, H-21, 3J = 6.7 Hz),
0.91 (ddd, 1H, H-1␣, 3J = 3.0 Hz, 2J = 3J = 13.5 Hz), 0.62 (s, 3H,
H-19), 0.38 (s, 3H, H-18). HRMS 359.2835 (calcd 359.2824). HPLC
(65/35) 8.83 min (100%).
2.3.2.21. (3S,20S)-20-N-Pentylcarbamoylpregn-7-en-3ˇ-ol
(28b). 50 mg (0.12 mmol) of 28a were treated as described
in procedure B. The crude product was subjected to SCC
(diethyl ether) (45 mg, 100%). mp 114 ◦C. 1H NMR (CDCl3,
500 MHz) ı 5.35 (m, 1H, N-H), 5.16 (m, 1H, H-7), 3.59 (m, 1H,
H-3), 3.22 (m, 2H, H-1ꢀ), 2.03 (m, 1H, H-20), 1.96 (ddd, 1H, H-12␤,
3J = 3J = 3.3 Hz, 2J = 12.6 Hz), 1.9–1.2 (m, 19H), 1.18 (d, 3H, H-21,
3J = 6.6 Hz), 1.09 (ddd, 1H, H-1␣, 3J = 3.3 Hz, 2J = 3J = 13.2 Hz) 0.89
(t, 3H, H-5ꢀ, 3J = 7.1 Hz), 0.79 (s, 3H, H-19), 0.54 (s, 3H, H-18).
HRMS 415.3463 (calcd 415.3450). HPLC (75/25) 8.89 min (97%).
2.3.2.16. (3S,20S)-20-N-Ethylcarbamoylpregn-7-en-3ˇ-ol
(23b). 50 mg (0.13 mmol) of 23a were treated as described in
procedure B. The crude product was subjected to SCC (ethyl
acetate) (37 mg, 82%). mp 251 ◦C. 1H NMR (CDCl3/MeOH-d4,
500 MHz) ı 4.99 (m, 1H, H-7), 3.37 (m, 1H, H-3), 3.00 (m, 3H,
H-1ꢀ), 1.99 (m, 1H, H-20), 1.79 (ddd, 1H, H-12␤, 3J = 3J = 3.4 Hz,
2J = 12.2 Hz), 1.75–1.00 (m, 20H), 0.99 (d, 3H, H-21, 3J = 6.7 Hz),
0.93 (t, 3H, H-2ꢀ, 3J = 7.1 Hz, with underneath 1H, H-1␣), 0.61 (s,
3H, H-19), 0.38 (s, 3H, H-18). HRMS 373.2951 (calcd 373.2981).
HPLC (75/25) 4.69 min (95%).
2.3.2.22. (3S,20S)-20-N-Benzylcarbamoylpregn-7-en-3ˇ-ol
(29b). 80 mg (0.18 mmol) of 29a were treated as described
in procedure B. The crude product was subjected to silica
SCC (diethyl ether) (70 mg, 83%). mp 207 ◦C. 1H NMR (CDCl3,
400 MHz) ı 7.30 (m, 5H, phenyl-H), 5.69 (m, 1H, N–H), 5.15 (m,
1H, H-7), 4.42 (m, 2H, H-1ꢀ), 3.59 (m, 1H, H-3), 2.10 (m, 1H, H-
20), 1.96 (ddd, 1H, H-12␤, 3J = 3J = 3.3 Hz, 2J = 12.1 Hz), 1.90–1.25
(m, 19H), 1.22 (d, 3H, H-21, 3J = 6.6 Hz), 1.08 (ddd, 1H, H-1␣,
3J = 2.9 Hz, 2J = 3J = 12.9 Hz), 0.78 (s, 3H, H-19), 0.53 (s, 3H, H-18).
HRMS 435.3148 (calcd 435.3137). HPLC (75/25) 6.84 min (98%).
2.3.2.17. (3S,20S)-20-N-Propylcarbamoylpregn-7-en-3ˇ-ol
(24b). 100 mg (0.26 mmol) of 24a were treated as described in
procedure B. The crude product was subjected to SCC (diethyl
ether) (87 mg, 96%). mp 235 ◦C. 1H NMR (CDCl3, 400 MHz) ı 5.37
(m, 1H, N-H), 5.16 (m, 1H, H-7), 3.60 (m, 1H, H-3), 3.19 (m, 1H,
H-1ꢀ), 2.04 (m, 1H, H-20), 1.96 (ddd, 1H, H-12␤, 3J = 3J = 3.4 Hz,
2J = 12.0 Hz), 1.95–1.20 (m, 22H), 1.19 (d, 3H, H-21, 3J = 6.8 Hz),
1.08 (ddd, 1H, H-1␣, 3J = 3.1 Hz, 2J = 3J = 12.9 Hz), 0.92 (t, 3H, H-3ꢀ,
3J = 7.3 Hz), 0.80 (s, 3H, H-19), 0.54 (s, 3H, H-18). HRMS 387.3182
(calcd 387.3137). HPLC (75/25) 5.10 min (100%).
2.3.2.23. (3S,20S)-20-N-Methyl-N-butylcarbamoylpregn-7-
en-3ˇ-ol (30b). 75 mg (0.18 mmol) of 30a were treated as
described in procedure B. The crude product was subjected to
SCC (diethyl ether) (70 mg, 83%). mp 115 ◦C. 1H NMR (CDCl3,
400 MHz) ı 5.15 (m, 1H, H-7), 3.59 (m, 1H, H-3), 3.53–3.15 (m, 2H,
H-1ꢀ), 3.02 (s, 0.5 × 3H, N-CH3), 2.90 (s, 0.5 × 3H, N–CH3), 2.68
(m, 1H, H-20), 1.96 (ddd, 1H, H-12␤, 3J = 3J = 3.3 Hz, 2J = 11.7 Hz),
1.9–1.2 (m, 24H), 1.14 (d, 0.5 × 3H, H-21, 3J = 6.8 Hz), 1.12 (d, 0.5 ×
3H, H-21, 3J = 6.8 Hz), 0.95 (t, 0.5 × 3H, H-4ꢀ, 3J = 7.25 Hz), 0.91 (t,
0.5 × 3H, H-4ꢀ, 3J = 7.3 Hz), 0.79 (s, 3H, H-19), 0.57 (s, 3H, H-18).
HRMS 415.3494 (calcd 415.3450). HPLC (75/25) 12.24 min (99%).
2.3.2.18. (3S,20S)-20-N-Butylcarbamoylpregn-7-en-3ˇ-ol
(25b). 100 mg (0.25 mmol) of 25a were treated as described in
procedure B. The crude product was subjected to SCC (diethyl
ether) (90 mg, 98%). mp 120 ◦C. 1H NMR (CDCl3, 500 MHz) ı 5.38
(s, 1H, NH), 5.15 (m, 1H, H-7), 3.59 (m, 1H, H-3), 3.22 (m, 2H,
H-1ꢀ), 2.03 (m, 1H, H-20), 1.95 (ddd, 1H, H-12␤, 3J = 3J = 3.1 Hz,

steroids 7 3 ( 2 0 0 8 ) 299–308
305
2.4.
Cell culture
Table 2 – IC50 values of the prepared compounds in the
MTT cytotoxity assay
HL 60 cells were maintained in RPMI 1640 medium containing
10% FBS without antibiotics at 37 ◦C in a humidified atmo-
sphere containing 5% CO2.
Compound
IC50 (␮M)
21a
22a
23a
24a
25a
26a
27a
28a
29a
30a
21b
22b
23b
24b
25b
26b
27b
28b
29b
30b
>100
>50
>100
≈100
>100
>100
≈35
2.5.
Biological assays
The MTT test for cytotoxicity and cell proliferation was per-
formed on HL 60 cells as previously described [10]. The
determination of the inhibited enzyme(s) and IC50 values was
accomplished as described by us recently [8]. For IC50 deter-
mination no constraints were used.
≈50
≈50
>100
>100
≈50
2.6.
HPLC assay to determine the hydrolysis of the
>100
>100
>100
>100
≈100
>100
>100
>100
3ˇ-acetates during the test procedure
To determine whether the 3␤-acetates (21a–30a) were trans-
formed into the corresponding 3␤-alcohols 21b–30b the
following HPLC assay was employed using substance 26a as
model substance and substance 24b as internal standard (I.S.).
Five million HL 60 cells were seeded out into each well of a 6-
well plate, lipid-free medium was added to reach a volume of
4.975 ml. 25 ␮l of a solution of 26a in ethanol (1 mg/ml) corre-
sponding to 25 ␮g (about 10 ␮M) were added and incubated for
24 h, conditions as stated under cell culture. 25 ␮l of a solution
of 24b (I.S.) in ethanol (1 mg/ml, 25 ␮g) were added to each well
as internal standard. The content of each well was transferred
into a 15 ml plastic tube and each well was washed with 5 ml
water. To each sample 100 ␮l of acidic acid were added and the
samples were ultrasonicated for 10 min at 0 ◦C. 3 ml of tert-
butylmethylether (TBME) were added and the samples were
carefully shaken for 60 s. The samples were centrifuged for
3 min, at 5 ◦C and 4000 × g. The organic layer was transferred
into a glass tube. The extraction with TBME was repeated
twice in the same manner. The combined organic extracts
were evaporated to dryness at 50 ◦C under a mild stream of
nitrogen. The residue was dissolved in 500 ␮l of ethanol (30 s
ultrasonication) and centrifuged for 2 min at 15,000 × g. 20 ␮l
of this solution were injected into the HPLC system speci-
fied above. The binary pump supplied the following gradient
of methanol/KH2PO4 buffer (20 mM, pH 5) at a flow rate of
0.65 ml/min: 0 min 65% methanol, 12 min 95% methanol (4 min
equilibration time). The wavelength of the UV detector was
210 nm. The column used was a Phenomenex Gemini C-18,
150 mm × 2.00 mm (5 ␮m). The column oven was held at 35 ◦C.
A one-point calibration at a concentration of 50 ␮g/ml was
used for the determination of the response factors.
the concentration range used for quantitative screening for
cholesterol biosynthesis inhibition (0.06125–8 ␮M) were to be
expected.
3.2.
Determination of the inhibited enzyme(s)
Table 3 lists the mainly accumulating sterols (compare Fig. 1)
under treatment of HL 60 cells with the different compounds
Table 3 – Mainly accumulating sterols and resultant
enzyme inhibition
Compound
Mainly accumulating
sterol(s)
Inhibited
enzyme
21a
22a
23a
24a
25a
26a
27a
28a
29a
30a
21b
22b
23b
24b
25b
26b
27b
28b
29b
30b
11, 12a, 13a, 15
C
C
C + H
C + H
H
H
H
H
11, 12a, 13a, 15
11a, 13, 15
8, 13, 15a
8
8
8
8
15a
n.d.
n.d.
C
15a
11, 12a, 13a, 15a
11, 15a
8, 13, 15
8, 13
8
8
8
8
15a
15a
C
C + H
C + H
H
H
H
H
n.d.
n.d.
To determine the recovery of the substances 24b, 26a and
26b we spiked 5 million cells in 5 ml of RPMI 1640 medium with
25 ␮g each, mixed the samples and immediately performed
work up as described above.
3.
Results
n.d.: not determined; (8) lathosterol, (11) desmosterol, (12),
cholesta-5,7,24-trien-3␤-ol, (13) cholesta-7,24-dien-3␤-ol, (15) 4,4-
dimethylcholesta-8,24-dien-3␤-ol, (C) ꢀ24-reductase and (H) lath-
osterol oxidase.
3.1.
MTT assay for cytotoxic activity
All amides synthesized in the course of this work showed neg-
ligible cytotoxicity with IC50 values mainly greater than 100 ␮M
(see Table 2). Thus, no cytotoxic effects of the substances in
a
Sterol was only found in traces.

306
steroids 7 3 ( 2 0 0 8 ) 299–308
Fig. 2 – Extracted ion chromatograms—A1: control; A2: 21b, 5 ␮M (extracted ions m/z
217 + 357 + 253 + 129 + 349 + 323 + 394 + 379 + 343); B1: control; B2: 23b, 5 ␮M
(217 + 357 + 441 + 343 + 253 + 458 + 213 + 484 + 394 + 379); C1: control; C2: 26b, 5 ␮M (217 + 357 + 458 + 255 + 213), I.S.: internal
standard (cholestane), (8) lathosterol, (10) cholesterol, (11) desmosterol, (13) cholesta-7,24-dien-3␤-ol and (15)
4,4-dimethylcholesta-8,24-dien-3␤-ol.
(concentration 5 ␮M). From the resulting changes in the sterol
pattern we could deduce the enzymes inhibited by the single
compounds as shown previously [8]. As examples the obtained
extracted ion chromatograms for 21b, 23b and 26b are shown
in Fig. 2.
3.4.
Hydrolysis of the 3ˇ-acetoxy compounds
Preliminary the recovery for the compounds 24b, 26a and
26b was determined as described above for standard samples
which had not been incubated (see Table 6).
After a 24 h incubation period only 2.0% could be recovered
as 3␤-acetate 26a, 84.4% of the engaged ester were recovered
as the 3␤-alcohol 26b. The remaining 13.6% (3.4 ␮g) of the used
ester 26a could not be recovered, what suggests that the sub-
stance was transformed even further.
3.3.
IC50 values and % inhibition
For all compounds a % inhibition of total cholesterol pro-
duction at a fixed concentration of 1 ␮M was determined
in triplicate (see Table 4). For the compounds 25a–28a and
25b–28b, inhibiting lathosterol oxidase, IC50 values were
determined (see Table 5). As an example the dose–response
curve for compound 26a is shown in Fig. 3.
4.
Discussion
Most of the tested lathosterol-derived amides led to a specific
sterol accumulation which could be shown to be dependent on

steroids 7 3 ( 2 0 0 8 ) 299–308
307
Table 4 – Inhibition of total cholesterol production at a
fixed concentration of 1 ␮M
Compound
Inhibition (%)
21a
22a
23a
24a
25a
26a
27a
28a
29a
30a
21b
22b
23b
24b
25b
26b
27b
28b
29b
30b
94
75
84
2
2
1
58 11
68
99
29
29
22
64
97
61
71
45 12
34 13
99
35
9
1
3
7
6
2
1
7
9
1
8
49 12
19
55
6
6
Fig. 3 – Dose–response curve of compound 26a; error bars
The results are given with 1 S.D.
show 1 standard error of the mean (S.E.M.), the numerical
data shown for each data point indicates the “real”
concentration (␮g/ml).
Table 5 – IC50 values for the inhibition of total cholesterol
production
Compound
IC50 (␮M)
with 21a/b and 22a/b is 11, what clearly indicates inhibition
of the enzyme ꢀ24-reductase by these amides. The amides
bearing an N-ethyl or N-propyl residue showed an interme-
diate behaviour. The N-ethyl derivatives (23a and 23b) led to
an accumulation of 13 besides a weak accumulation of 15
(see above). The accumulation of 13 describes the intermedi-
ate position of the compounds inhibiting lathosterol oxidase
as well as ꢀ24-reductase. The N-propyl derivatives (24a and
24b) showed an accumulation of 8 and 13, indicating inhi-
bition of the same target enzymes as 23a and 23b, with a
stronger inhibition of lathosterol oxidase. The N-butyl, N-
isobutyl, N-tert-butyl and the N-pentyl derivatives (25a–28a
and 25b–28b) led to a significant accumulation of lathosterol
(8), regarding to the inhibition of lathosterol oxidase (H). Since
no significant accumulation of other sterols could be detected
under incubation with these amides, these substances can be
regarded as selective inhibitors of lathosterol oxidase. In con-
trast, the tertiary amides 30a and 30b, simply being N-methyl
derivatives of the selective lathosterol oxidase inhibitors 25a
and 25b (N-butyl amides) only caused a weak accumulation
of 4,4-dimethylcholesta-8,24-dien-3␤-ol (15), indicating that
only secondary amides act as selective inhibitors of lathos-
terol oxidase. Obviously selectivity for single enzymes only
depends on the residues at the side chain amide nitrogen.
Small alkyl residues lead to an inhibition of ꢀ24-reductase,
midsized residues to inhibition of both lathosterol oxidase and
25a
26a
27a
28a
25b
26b
27b
28b
0.91
0.45
0.95
1.10
0.70
0.33
1.05
1.16
The “best fit” values are listed.
the size of the substituent at the amide nitrogen. Compounds
having small residues like hydrogen or methyl (21a, 22a, 21b
and 22b) led to an accumulation of desmosterol (11), besides
a weak accumulation of cholesta-5,7,24-trien-3␤-ol (12) and
4,4-dimethylcholesta-8,24-dien-3␤-ol (15). The accumulation
of 11 is clearly associated to an inhibition of ꢀ24-reductase (C)
[8,22], the weak accumulation of 15 indicates a slight inhibi-
tion of lathosterol oxidase (H) and C4-demethylase complex (F)
(see Fig. 1). The N-methyl derivatives 22a and 22b additionally
showed a slight accumulation of cholesta-7,24-dien-3␤-ol (13)
regarding to the initial inhibition of lathosterol oxidase (H).
Nevertheless the mainly accumulating sterol upon treatment
Table 6 – Recovery for the compounds 24b, 26a and 26b,
RT: retention time
ꢀ
24-reductase, larger residues like butyl or pentyl lead to a
selective inhibition of lathosterol oxidase.
Compound
Recovery (%)
RT (min)
Interestingly, we found no significant differences in the
resulting sterol patterns upon treatment of the cells with
the 3␤-acetates (21a–30a) and the corresponding 3␤-alcohols
(21b–30b). Employing a HPLC assay for the model substance
24b
26b
26a
94
96
92
5.7
6.7
10.8

308
steroids 7 3 ( 2 0 0 8 ) 299–308
mental retardation syndrome due to deficiency of
3␤-hydroxysteroid-ꢀ5-desaturase. Am J Hum Genet
2002;71:952–8.
26a we could show that the ester was almost quanti-
tatively transformed into the 3␤-alcohol 26b under test
conditions. This proves for the thesis that the 3␤-acetates
simply served as prodrugs for the corresponding 3␤-hydroxy
compounds in the applied assay for late cholesterol biosyn-
thesis. Because not all of the engaged 3␤-acetate 26a could
be detected as 3␤-alcohol 26b or in its original form as ester
it seems possible that the substance was transformed even
beyond the 3␤-alcohol 26b. Nevertheless the ester 26a was
almost completely transformed into its corresponding alco-
hol 26b (84.4%) and hence served as prodrug in the described
assay.
Investigations on the quantitative effect of the new lathos-
terol oxidase inhibitors on total cholesterol production in HL
60 cells showed that the N-isobutyl derivatives 26a and 26b
exhibit the uppermost activity (IC50 values 0.45 and 0.33 ␮M).
Overall the IC50 values of the lathosterol oxidase inhibitors are
quite similar (see Table 5), although they have different % inhi-
bitions at 1 ␮M (see Table 4). This can be explained through the
intra-day precision of the assay which is about 25% (coefficient
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In conclusion, we present here the first selective and potent
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tion of triarimol more than 30 years ago [7]. Besides triarimol
up to date only very unspecific inhibitors of this enzyme
(i.e., cyanide and phenanthroline) have been reported. Our
new inhibitors could help to complete studies on this spe-
cial enzymatic step in late cholesterol biosynthesis for which
no efficient inhibitors have been available up to now [23,24].
Furthermore these new inhibitors might contribute to getting
better insight into the molecular backgrounds of lathosterolo-
sis.
Acknowledgments
We thank Ms. T. Ho¨ft for sample work-up, Dr. L. Allmendinger
and Dr. H. Lerche for NMR and HRMS measurements.
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