A new class of selective and potent 7-dehydrocholesterol reductase inhibitors

Article abstract of DOI:10.1021/jm3006096

We prepared a number of N-phenethyltetrahydroisoquinolines structurally related to protoberberines. They were tested for activity against bacteria, fungi, and human leukemia HL-60 cells and also for inhibition of biosynthesis: ergosterol in yeasts and cholesterol in human cells. In the latter assay panel, several of the compounds were distinguished by a strong and selective inhibition of 7-dehydrocholesterol reductase (7-DHCR, EC 1.3.1.21), an enzyme responsible for the conversion of 7-dehydrocholesterol to cholesterol in the last step of cholesterol biosynthesis. In a whole-cell assay, the most active compound 5f showed a much stronger inhibition of overall cholesterol biosynthesis (IC ₅₀ 2.3 nM) than BM 15.766 (IC₅₀ 500 nM), presently the most selective known inhibitor of 7-DHCR. Since a defect of 7-dehydrocholesterol reductase is associated with Smith-Lemli-Opitz syndrome (SLOS), the potent and selective inhibitors reported here will enable more detailed investigation of the pathogenesis of SLOS.

Full text of DOI:10.1021/jm3006096

Article
pubs.acs.org/jmc
A New Class of Selective and Potent 7‑Dehydrocholesterol Reductase
Inhibitors
Aline Horling,† Christoph Muller,‡ Richard Barthel,† Franz Bracher,‡ and Peter Imming*,†
̈
†Institut fur Pharmazie, Martin-Luther-Universitat Halle−Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle, Germany
̈
̈
‡Department fur Pharmazie, Zentrum fur Pharmaforschung, Ludwig-Maximilians-Universitat Munchen, Butenandtstrasse 5-13,
̈
̈
̈
̈
D-81377, Germany
S
* Supporting Information
ABSTRACT: We prepared a number of N-phenethyltetrahydroisoquinolines
structurally related to protoberberines. They were tested for activity against bacteria,
fungi, and human leukemia HL-60 cells and also for inhibition of biosynthesis:
ergosterol in yeasts and cholesterol in human cells. In the latter assay panel, several of
the compounds were distinguished by a strong and selective inhibition of 7-
dehydrocholesterol reductase (7-DHCR, EC 1.3.1.21), an enzyme responsible for the
conversion of 7-dehydrocholesterol to cholesterol in the last step of cholesterol
biosynthesis. In a whole-cell assay, the most active compound 5f showed a much
stronger inhibition of overall cholesterol biosynthesis (IC50 2.3 nM) than BM 15.766 (IC50 500 nM), presently the most selective
known inhibitor of 7-DHCR. Since a defect of 7-dehydrocholesterol reductase is associated with Smith−Lemli−Opitz syndrome
(SLOS), the potent and selective inhibitors reported here will enable more detailed investigation of the pathogenesis of SLOS.
lesterol and its oxidation products, have to be further
investigated.12 This needs selective 7-DHCR inhibitors that
inhibit cholesterol biosynthesis at the stage of 7-dehydrocho-
lesterol, simulating aspects of SLOS in vitro. Several 7-DHCR
inhibitors (AY 9944, BM 15.766, and YM 9429; Scheme 2)
have been published, but all of them have severe disadvantages.
AY 9944 inhibits several enzymes of cholesterol biosynthesis
including upstream enzymes, most notably Δ8,7-isomer-
ase.8,13,14 This inhibition of two enzymes leads to an
accumulation of other sterols, mainly zymostenol (cholesta-8-
en-3β-ol), blurring the similarity with the situation in SLOS.
The inhibitor BM 15.766 is a known selective inhibitor of 7-
dehydrocholesterol reductase but not a potent one.15 In rat
hepatocytes, cholesterol synthesis was reported to have been
almost totally inhibited by BM 15.766 at a concentration of as
much as 10 μM.15 BM 15.766 has lower toxicity than AY 9944,
the latter displaying higher lethality toward rats.16 YM 9429 was
investigated only in a few studies.17 Just as with AY 9944,
inhibition of related enzymes was not completely excluded, and
inhibition of 7-DHCR was even weaker than with BM 15.766.17
Furthermore, YM 9429 is considered to be potentially
teratogenic.17 Consequently, no suitable 7-DHCR inhibitor
for investigation of the SLOS pathobiochemistry mechanism is
available at present.
INTRODUCTION
■
Cholesterol has different functions in human physiology. It is
an important structural component of the mammalian cell
membrane and also a precursor molecule for glucocorticoids,
mineralocorticoids, neurosteroids, and steroid sex hormones. A
known autosomal recessive trait, the Smith−Lemli−Opitz
syndrome (SLOS), is associated with the pivotal role of
cholesterol, especially during embryonic development and
morphogenesis.1 The worldwide incidence of SLOS is 1 in
10 000−80 000 births, depending on ethnic group. It is
accompanied by severe abnormalities, observed in most of
the patients, such as microcephaly, short nose with anteverted
nares, retrognathia, cutaneous syndactyly of second to third toe,
feeding difficulties in the gastrointestinal tract, mental
retardation, and brain growth retardation.1 The syndrome
was first described by Smith, Lemli, and Opitz in 1964.2 As
recently as 1994, a defect of 7-dehydrocholesterol reductase (7-
DHCR, EC 1.3.1.21), the enzyme that catalyzes the reduction
of 7-dehydrocholesterol (cholesta-5,7-dien-3β-ol) to cholesterol
(cholesta-5-en-3β-ol) in the last step of cholesterol biosynthesis
(Scheme 1), was identified as the biochemical cause of this
clinically complex syndrome.3−5 The route of cholesterol
biosynthesis from lanosterol via 7-dehydrocholesterol is called
the Kandutsch−Russell pathway.6,7 A defect of 7-DHCR leads
to a deficit in cholesterol and an accumulation of 7-
dehydrocholesterol, which together with its oxidized derivatives
is embryotoxic, also having a negative influence on total
cholesterol transfer.5,8,9 An early diagnosis of SLOS with
external substitution of cholesterol will alleviate the symptoms
of SLOS.10,11
In various pharmacological studies, protoberberines have
shown antibacterial, antifungal, antileishmanial, and antispas-
modic effects.18−21 Protoberberines are characterized by many
reported activities and many possibilities of binding to targets.22
Berberine was recently found to inhibit the hepatic HMG-CoA
For a better understanding of SLOS, the molecular
Received: May 2, 2012
mechanisms, including details of the effects of 7-dehydrocho-
Published: August 13, 2012
© 2012 American Chemical Society
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a6
Scheme 1. Post-Squalene Pathway of Cholesterol Biosynthesis
a
(a) Catalyzed by Δ8,7-isomerase. (b) Catalyzed by 7-dehydrocholesterol reductase (7-DHCR).
RESULTS
Scheme 2. Structures of Known Inhibitors of 7-
Dehydrocholesterol Reductase and the Herein-Reported
Potent and Selective Inhibitor 5f
■
Chemistry. In the course of studies with protoberberines
and related alkaloid analogues,24 we prepared a series of
phenethyltetrahydroisoquinolines. Since the phenethylamine
moiety seems to be essential for the various pharmacological
effects of protoberberines, we decided to include this motif in
all synthetic analogues. Compared to the parent alkaloids
berberine and palmatine, ring B has formally been opened,
whereas ring C has been reduced to the tetrahydro form in the
synthetic analogues in order to avoid getting quaternary
ammonium compounds (Scheme 3). Drugs containing
Scheme 3. Target Phenethyltetrahydroisoquinoline Skeleton
in Comparison with Parent Alkaloids and Dopamine
reductase in hyperhomocysteinemic rats and to reduce the
cholesterol level in liver.23 HMG-CoA reductase catalyzes the
rate-limiting step in cholesterol biosynthesis. This prompted us
to take a closer look at the effect of some protoberberine
alkaloids on cholesterol biosynthesis in mammalian cells and on
ergosterol biosynthesis in fungal cells. Since the molecular
targets for inhibition (and thus the structural requirements for
inhibitors) were unknown, we decided to go for a broader
approach, including not only berberine but also the related
alkaloid tetrahydropalmatine (nonquarternary) and phenethyl-
tetrahydroisoquinolines (formal seco analogues having higher
conformational flexibility). The test compounds were selected
without taking the constitution of known 7-DHCR inhibitors
into account, as we did not know at the outset where the effect
on cholesterol catabolism and metabolism might reside. We
rather relied on the effectiveness of the assay method in
identifying a molecular target through a whole-cell approach. In
order to keep undesired effects in view, the compounds were
also screened for antibacterial and antifungal activities and
general cytotoxicity for human cells. The investigations led to
the discovery of the synthetic phenethyltetrahydroisoquinoline
5f as a highly potent and selective inhibitor of 7-DHCR in
cholesterol biosynthesis, displaying no effect on ergosterol
biosynthesis in fungi and very low cytotoxicity.
quaternary ammonium groups are characterized by high
hydrophilicity, causing low permeability through biological
membranes and restricted in vivo distribution. Several
investigations document the low absolute bioavailability of
berberine.25,26 Its log P value was determined to be −1.5.27 In
contrast, the nonquaternary tetrahydropalmatine was reported
to cross biological membranes rapidly. Its maximum concen-
tration in plasma after oral administration was reached within
30 min.28,29 The phenethyltetrahydroisoquinoline skeleton
retains the dopamine moiety included in (proto)berberine
alkaloids.
The phenethyltetrahydroisoquinoline 5a was synthesized in
four steps (Scheme 4). The synthetic strategy builds on
protocols previously described;30−32 however, the literature
contains many discrepancies and contradictions in experimental
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Scheme 4. Syntheses of Phenethyltetrahydroisoquinolines
detail. We have investigated each step extensively in order to
find the best strategy for different substitution patterns. In the
Experimental Section and Supporting Information, detailed
procedures are provided for the derivatives reported here. The
first step comprises a condensation reaction of the phenyl-
ethylamine 1a and the phenylacetic acid 2a without activation
of the carboxylic acid to give the amide 3a in excellent yield.
Normally, amines and carboxylic acids would form only a salt,
but in refluxing xylenes with azeotropic removal of the reaction
water, we were able to get the amides. This straightforward
procedure also relies on the thermostability of the educts and
products.
Due to its incompatibility with phosphorus oxychloride used
in a later step of the sequence, the phenolic moiety of 3a had to
be protected by acetylation. The protected amide 7 was treated
with phosphorus oxychloride to give an imidoyl chloride, which
in turn was smoothly converted to the amine 4a by a mild
reduction protocol.33 In the course of this reduction, the acetyl
protecting group was removed quantitatively. Mannich reaction
of 4a with formalin in methanol finally yielded 5a. For the ring
to be closed in the desired way, the solvent, concentration of
the starting material, and pH (1−2) had to be diligently chosen.
For the Mannich-type cyclization, forming ring C of
phenethyltetrahydroisoquinolines, an activated phenyl ring is
absolutely necessary. Derivatives that bear no +M or +I
substituents at the phenyl ring annulated to the future
piperidine ring cannot be closed by the Mannich reaction.
We have investigated this reaction under a variety of pH
conditions. The formation of phenethyltetrahydroisoquinolines
that were substituted only with a methylenedioxy group or
methoxy groups was achieved by Mannich reaction in methanol
independent of pH value. C-6-hydroxylated compounds, in
contrast, needed to be synthesized at pH 1−2. At higher pH,
two products were furnished by C-6 and C-8 hydroxylation.
In order to reduce the steps of the synthesis of further
phenethyltetrahydroisoquinolines and avoid the possible
formation of phosphoric acid esters, reduction of the amide
with phosphorus oxychloride followed by sodium borohydride
was replaced by a reduction with lithium aluminum hydride,
resulting in easier workup and better yields. With this method,
only three steps were needed to prepare the target compounds
5b−k since protection of the phenolic groups was no longer
required (Scheme 4). Again, the Mannich-type cyclizations
occurred regioselectively if reaction conditions were adjusted
properly (see Experimental Section). Demethylation of
dimethoxy compound 5b was achieved in refluxing 48%
aqueous hydrobromic acid, yielding the diphenol 6.
Pharmacology. Cytotoxic Activity. For the detection of
undesired cytotoxic activities, the substances were subjected to
a preliminary screening for unspecific cytotoxicity in an MTT
assay on HL-60 cells (human promyelocytic leukemia cells).34
The reference inhibitor cisplatin showed an IC50 value of 5 μM
in this assay, whereas none of the compounds under
investigation here showed significant cytotoxicity (IC50 values
>50 μM; data not shown).
Antibacterial and Antifungal Activity. The compounds
were subjected to a standard disk diffusion antimicrobial
sensivity test (agar diffusion assay) with different strains of
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a
Scheme 5. Chromatograms of Sterol Fractions Isolated after Treatment with or without Inhibitor
a
(A1−C1) Full scan mode (m/z 100−650); (A2−C2) selected ion mode for identification of 7-dehydrocholesterol (m/z 351, 325, 366). (A1) Blank
sample, full scan; (A2) blank sample, selected ion scan; (B1) BM 15.766, 1 μM, full scan; (B2) BM 15.766, 1 μM, selected ion scan; (C1) 5f, 1 μM,
full scan; (C2)5f, 1 μM, selected ion scan. (Peak 1) Cholestane (internal standard); (peak 2) cholesterol; (peak 3) 7-dehydrocholesterol.
Gram-positive or Gram-negative bacteria, yeasts, and fungi. The
activities, expressed as diameters of zone of inhibition, were
compared to those found for reference inhibitors, viz. the
antibacterial tetracycline and the antimycotic clotrimazole.35
None of the compounds showed significant antibacterial
activity, and in the screening for antifungal activity, only
compounds 5a, 5e, and 5i showed some moderate effects
against Hyphopichia burtonii (data not shown).
Screening for Ergosterol Biosynthesis Inhibition in Yeasts.
In the first step, MIC (minimal inhibitory concentration) values
against the yeasts Candida glabrata, Saccharomyces cerevisiae,
and Yarrowia lipolytica were determined according to DIN
58940-84.36 Clotrimazole as a positive control showed an MIC
value of <1 μg/mL; all other compounds gave values >20 μg/
mL. These values, in combination with the data from the agar
diffusion assay, showed that the antifungal potency of the
substances investigated here was orders of magnitude lower
than the 7-DHCR inhibition of some of them (v.i.).
post-squalene part of cholesterol biosynthesis are inhibited
(qualitative results). The inhibitory potency of a discrete
inhibitor (quantitative results) has to be investigated in an
additional test.
Qualitative Results. The sterol patterns resulting from
incubation with our compounds were compared to blank
samples obtained by incubation of HL-60 cells without
inhibitor. Compounds 5a and 5e caused an accumulation of
7-dehydrocholesterol (7-DHC; see Scheme 1) at low
concentration levels, and at higher levels an accumulation of
both 7-DHC and zymostenol. Consequently, these compounds
inhibit both Δ8,7-isomerase (marker sterol zymostenol) and 7-
dehydrocholesterol reductase (marker sterol 7-dehydrocholes-
terol).4,38 In contrast, compounds 5b, 5c, 5j, and 5k caused an
accumulation of zymostenol only (see Supporting Informa-
tion), indicative of a selective inhibition of the enzyme Δ8,7-
isomerase. Compound 5d had no effect on cholesterol
biosynthesis.
However, inhibition of single enzymes of ergosterol biosyn-
thesis need not necessarily result in cell death. Consequently,
the effect of our compounds on ergosterol biosynthesis was
studied in order to detect the inhibition of one or several
biosynthetic steps. The compounds were subjected to a whole-
cell assay with a liquid−liquid microextraction workup after cell
lysis for the isolation of sterols, and subsequent gas
chromatography−mass spectrometry (GC-MS) analysis of the
sterol pattern. Qualitative analysis of the effects of all
compounds, including the known inhibitors AY 9944 and
BM 15.766, on three different yeasts (C. glabrata, S. cerevisiae,
and Y. lipolytica) was performed at two different inhibitor
concentrations (20 and 10 μg/mL). The inhibition of any
enzyme of the post-squalene part of ergosterol biosynthesis
would lead to a characteristic change in the sterol
composition.37 With all compounds tested, GC-MS analysis
showed no significant change in the sterol pattern, and no
abnormal sterols were detected compared to blank samples.
Screening for Inhibition of Cholesterol Biosynthesis. The
effect on cholesterol biosynthesis was determined in an in vitro
assay developed by us, based on incubation with HL-60 cells,
followed by lysis of the cells, liquid−liquid microextraction, and
GC-MS analysis of the resulting sterol pattern.13,38 By simply
identifying accumulating sterols and comparing the sterol
patterns with those resulting from incubation with known
enzyme inhibitors, this assay indicates which enzymes in the
Most importantly, compounds 5f, 5g, 5h, 5i, and 6 displayed
a sterol pattern characteristic of selective 7-dehydrocholesterol
reductase inhibition. Compounds 5g and 6 caused an
accumulation of 7-dehydrocholesterol at higher concentration
levels only, whereas 5f, 5h, and 5i effected this at very low
levels.
The results obtained with known inhibitors of 7-DHCR as
reference compounds agreed with the literature, leading to the
expected accumulation effects and thus validating the data
found for the new compounds. AY 9944 inhibited both Δ8,7-
isomerase and 7-dehydrocholesterol reductase.7,8,13,14 Giera et
al.38 observed that AY 9944 inhibits 7-dehydrocholesterol
reductase at 0.1 μM and two upstream enzymes, Δ14-reductase
and Δ8,7-isomerase, at 10 μM. Similar results were published
by Fernan
́
dez et al.9 BM 15.766 was reported to be more
selective for 7-DHCR9,15 (see Scheme 5). The phenethyl-
amines 5f, 5h, and 5i showed the same effects on HL-60 cells
that BM 15.766 had (accumulation of 7-dehydrocholesterol)
but led to significantly stronger accumulation of the substrate
sterol at the inhibitor concentrations investigated here.
Quantitative Results. The next step was quantitative
determination of the inhibitory potency of the most promising
substances. They were selected by visual inspection of the peaks
of accumulating 7-dehydrocholesterol in the chromatograms
obtained in the qualitative assay described above. In order to
avoid having to use isolated enzymes of each step of cholesterol
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biosynthesis, we recently established a whole-cell assay for
determination of the effect of inhibitors on total cholesterol
biosynthesis.38 The IC50 values were determined for
phenethyltetrahydroisoquinolines 5f, 5h, and 5i and for BM
15.766. In order to distinguish newly synthesized cholesterol
from the large amounts of matrix cholesterol already present in
the cells, incubation of the cells was performed in the presence
of 2-13C-acetate in this assay. Due to the incorporation of
several labeled acetate units into cholesterol, newly synthesized
sterol can be determined selectively by GC-MS.38 The IC50
values determined in this way correspond to percent inhibition
of cholesterol biosynthesis in the whole-cell assay. The results
are shown in Table 1. Compound 5f displayed exceptionally
high inhibitory activity.
inhibit both Δ8,7-isomerase and 7-dehydrocholesterol reduc-
tase at higher concentration levels, while they exhibited
selective inhibition of 7-DHCR at lower concentrations.
Compounds 5b, 5c, 5j, and 5k selectively inhibited the enzyme
Δ8,7-isomerase. Compound 5d showed no effect on choles-
terol biosynthesis. Five compounds (5f−i and 6), were found
to inhibit 7-DHCR only, with 5f, 5h, and 5i showing the
strongest inhibitory activity (Table 1). The IC50 values
presented in Table 1 refer to inhibition of overall cholesterol
biosynthesis in a whole-cell assay, but since 7-DHCR is the very
last enzyme in this multistep biosynthesis, in all likelihood they
reflect the selective inhibition of this enzyme. This is
corroborated by two observations: (1) even at the much
higher inhibitor concentration of 50 μM, no other steroids were
found to accumulate; and (2) nonselective inhibitors (5a, 5e)
were detected by our method. Furthermore, 7-DHCR is the
very last enzyme in cholesterol biosynthesis, and for this reason
downstream effects concealing the inhibition of additional
enzymes in this biosynthesis can be excluded.
Table 1. IC50 Values for Inhibition of Total Cholesterol
Biosynthesis through Determination of 13C Incorporation
into Newly Synthesized Cholesterol
relative
molecular mass
IC50 value
(nM)
confidence
a
In view of the low number of compounds tested, structure−
activity relationships (SAR) have a very tentative nature at this
stage. However, all inhibitors of 7-DHCR (5f−i, 6), including
the nonselective inhibitors 5a and 5e, have a hydroxy group at
C-6 of the tetrahydroisoquinoline moiety in common. Addi-
tionally, all these compounds except 6 bear a methoxy group in
the 7-position.
b
compd
interval (nM)
R2
BM 15.766
sulfate
483.0
500
430−570
0.975
5f
5h
5i
317.8
313.4
343.4
2.3
120
2.0−2.6
95−150
6,100−8,700
b
0.979
0.908
0.958
7300
a
Confidence interval for the IC50 value was 95%. R2 = goodness of fit
None of the selective inhibitors of Δ8,7-isomerase (5b, 5c,
5j, and 5k) showed this substitution pattern. Derivatives that
lacked a hydroxy group at C-6 and methoxy group at C-7 did
not inhibit 7-DHCR. Hydroxylation at C-7 and introduction of
a methoxy group in position 6 of the phenethyltetrahydroiso-
quinoline scaffold (5d) resulted in a loss of effects on
cholesterol biosynthesis. So for the phenethyltetrahydroisoqui-
noline scaffold, the 6-hydroxy group appears to be a
requirement for inhibition of 7-DHCR. The known inhibitors
BM 15.766 and AY 9944 resemble 5f in that they all bear a
chlorine on one of the phenyl rings, but since 5h, which does
not bear an electron-attracting substituent on either phenyl
ring, also has higher activity than BM 15.766, the importance of
the substitution pattern of the phenyl ring annulated to the
piperidine ring appears to be much higher for (selective) 7-
DHCR inhibition than the pattern on the other aromatic
moiety.
Further studies will provide more inhibitory data for an SAR
analysis of the scaffold. This will address two important points,
closely connected to each other, that were out of the scope of
the present study: (1) determine the inhibition of isolated
enzymes of the 7-DHC pathway and (2) on the basis of this
with clogP and permeability data, detect the influence
lipophilicity and cell membrane permeability of the compounds
have on the effect seen in the present study. With some
compounds showing selective inhibition of one or two enzymes
of the human cholesterol biosynthesis, the phenethyltetrahy-
droisoquinoline scaffold holds promise for development of
selective inhibitors even for other enzymes of this pathway.
of the dose−response curves.
DISCUSSION
■
Since none of the new phenethyltetrahydroisoquinolines
showed considerable cytotoxicity toward HL-60 cells in a
MTT colorimetric assay (IC50 values >50 μM), the compounds
were considered to be nontoxic against human cells. In the agar
diffusion assay, all compounds of interest were found to be not
toxic against bacteria. Only compounds 5a, 5e, and 5i showed
poor inhibitory activity against one of the yeasts tested (H.
burtonii). Despite the poor antifungal activity found in the agar
diffusion assay, we investigated the effect of the phenethylte-
trahydroisoquinolines on ergosterol biosynthesis in three yeast
strains. None of the compounds gave rise to detectable changes
in the sterol pattern of the yeasts, indicating that the enzymes
of the post-squalene part of ergosterol biosynthesis are not
targets of the compounds. Particularly with regard to the results
obtained in the screening for inhibition of cholesterol
biosynthesis in human cells, this is not surprising because no
enzyme similar to the human 7-dehydrocholesterol reductase
(v.i.) exists in yeasts and fungi.39,40 Inhibition of other fungal
enzymes in ergosterol biosynthesis had nevertheless been
conceivable but was excluded with this assay.
In a whole-cell screening assay for inhibition of cholesterol
biosynthesis in human cells, two established inhibitors of
cholesterol biosynthesis from different chemical classes, AY
9944 and BM 15.766 (Scheme 2), were used as reference
substances. Biochemically, AY 9944 was known to inhibit both
Δ14-reductase and Δ8,7-isomerase at higher concentrations
and 7-dehydrocholesterol reductase at lower concentrations.9,41
BM 15.766 inhibits 7-dehydrocholesterol reductase selectively
but with low potency. By reproducing these results with our
screening system, we confirmed the validity of data obtained in
the first step, namely, identification of the target enzymes.
Phenethyltetrahydroisoquinolines 5a and 5e were found to
CONCLUSION
■
We have identified a new class of very active and selective
inhibitors of 7-dehydrocholesterol reductase, the last enzyme in
cholesterol biosynthesis. The compound 5f showed 200 times
stronger inhibition of cholesterol biosynthesis in a whole-cell
assay than the established (commercially available) inhibitor
BM 15.766 did under the same conditions. It was devoid of
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or dry tert-butyl methyl ether (TBME), stirred under argon, and
cooled to 0 °C. A 2.5−5.0-fold molar amount of lithium aluminum
hydride was added in one portion. The reaction was allowed to reach
room temperature and stirred for 14−16 h. The mixture was cooled to
0 °C, and water was added until no more hydrogen was released. The
solid was separated and extracted five times with trichloromethane/
methanol (9:1). The liquid layer was also extracted with trichloro-
methane/methanol (9:1). The organic layers were combined, washed
with water, and dried over anhydrous magnesium sulfate. Removal of
the solvent gave an oily residue, which was purified by silica column
chromatography (gradient, trichloromethane 100% to trichloro-
methane/methanol 93:7) to give the amine. Details and spectroscopic
data can be found in the Supporting Information; representative
examples are given below.
inhibitory activity in fungal ergosterol biosynthesis and showed
negligible cytotoxicity. The new chemotype of selective
inhibitors of 7-DHCR, phenethyltetrahydroisoquinolines, is
characterized by facile synthetic access and stability. Through
blocking 7-dehydrocholesterol reductase selectively, 5f will be a
useful tool for studying molecular details of the pathogenesis of
Smith−Lemli−Opitz syndrome, a disease with debilitating
effects on patients for which presently there is no cure.
EXPERIMENTAL SECTION
Chemistry. Melting points were determined on an electrically
■
heated Boetius microscope hot stage and are uncorrected. NMR
̈
spectra were recorded on a Varian Gemini 2000 spectrometer, with
1
5-{2-[2-(4-Chlorophenyl)ethylamino]ethyl}-2-methoxyphenol 4f.
Amide 3f (1.4 g, 4.3 mmol), dissolved in 50 mL of dry TBME, and
lithium aluminum hydride (1.0 g, 26.4 mmol) gave 0.4 g of 4f (33%)
as white crystals. The compound was used for the next step without
further purification.
400 MHz operating frequency for H NMR and 100 MHz operating
frequency for 13C NMR, or on a Varian Unity Inova 500, with 500
MHz operating frequency for 1H NMR and 125 MHz operating
frequency for 13C NMR. The spectra were recorded in trichloro-
methane-d or methanol-d4 as solvent. The chemical shifts are reported
in parts per million (ppm, δ) and rounded to two decimal places for
1H NMR and one decimal place for 13C NMR. J values are given in
5-{2-[2-(3,4-Dimethoxyphenyl)ethylamino]ethyl}-2-methoxyphe-
nol 4i. Amide 3i (1.4 g, 4.1 mmol), dissolved in 50 mL of dry TBME,
and lithium aluminum hydride (0.3 g, 6.8 mmol) gave 0.2 g of 4i
hertz.
1
(14%) as white crystals. H NMR (CDCl3, 400 MHz) δ = 6.80−6.61
Mass spectra (MS) were recorded on an AMD Intectra DP 10
instrument operating at an ionizing potential of 70 eV (EI-MS) or on a
Thermo Finnigan LCQ-Classic instrument (ESI-MS).
(m, 6H), 3.85 (s, 3H), 3.83 (s, 3H), 3 0.82 (s, 3H), 3.50 (s, 2H),
3.22−3.13 (m, 8H).
Synthesis of 5a−k by Mannich Reaction: General Procedure. For
preparation of the phenethyltetrahydroisoquinolines, the amines (4a−
k) and a few drops of concentrated aqueous hydrochloric acid were
stirred in dry methanol. An aqueous methanal solution (37%) was
added, and the mixture was heated under reflux for 3 h. Methanol was
distilled off, and the residue was basified with 10% aqueous ammonia
and extracted with trichloromethane three times. The extract was
washed with water, dried over anhydrous magnesium sulfate, and
evaporated to leave either a solid or an oily residue. Phenethylte-
trahydroisoquinolines 5a−k were finally purified by silica column
chromatography (gradient, trichloromethane 100% to trichloro-
methane/methanol 95:5). Details and spectroscopic data can be
found in the Supporting Information; representative examples are
given below.
Most chemicals were purchased from Sigma−Aldrich (Schnelldorf,
Germany) or Acros Organics (Geel, Belgium). All solvents were
commercial-grade and were distilled before use. Berberine chloride was
purchased from Alfa Aesar (Karlsruhe, Germany). Palmatine iodide
belonged to an old substance collection. Its identity and purity was
ascertained by NMR, MS, elementary analysis, and melting-point
determination. Reactions were monitored by thin-layer chromatog-
raphy (TLC) on precoated aluminum sheets of TLC silica gel 60 F254
from Merck (Darmstadt, Germany). Detection of the compounds on
TLC plates was achieved by using UV light at 254 nm or with iodine
vapor. Merck silica gel 60 (40−63 μm) was used as the stationary
phase for flash column chromatography. Elementary analysis was
performed with a LECO CHNS-932 instrument. Data are given as
percentages and rounded to two decimal places as the average of
duplicate determinations. Elementary analytical results were within
0.4% of the theoretical values, except when noted otherwise. The
purity of compounds 5h, 5i, and 5f was determined by HPLC (Agilent
1100 Series, Waldbronn, Germany) and found to be >97%.
Synthesis of Phenylacetamides 3a−k: General Procedure. The
phenylacetic acid derivative and 1.1 mol equiv of the respective
phenylethylamine were suspended in dry xylene and refluxed under
argon for 16 h. After evaporation of xylene, an oily residue was
chromatographed and a solid residue was recrystallized from ethyl
acetate/heptane 2:1, providing the amides (3a−k) as bright fluffy
crystals. Details and spectroscopic data can be found in the Supporting
Information; representative examples are given below.
2-[2-(4-Chlorophenyl)ethyl]-6-hydroxy-7-methoxy-1,2,3,4-tetra-
hydroisoquinoline 5f. Amine 4f (0.4 g, 1.4 mmol), dissolved in 15 mL
of dry methanol, and 15 mL of aqueous methanal solution (37%) gave
1
0.19 g (43%) of 5f as a yellow solid, mp 168−171 °C. H NMR
(CD3OD, 400 MHz) δ = 7.28 (dd, J = 2.35/8.61 Hz, 2H), 7.24 (dd, J
= 2.35/8.61 Hz, 2H), 6.62 (s, 1H), 6.56 (s, 1H), 3.81 (s, 3H), 3.64 (s,
2H), 2.92−2.88 (m, 2H), 2.80 (s, 4H), 2.76−2.72 (m, 2H). 13C NMR
(CD3OD, 125 MHz) δ = 145.0, 144.2, 138.8, 131.8, 130.1, 128.5,
126.8, 125.6, 114.3, 108.8, 59.8, 56.0, 55.8, 51.0, 33.3, 28.3. MS (ESI)
m/z 318.2 (100, M+ + 1). Anal. calcd for C18H20ClNO2: C, 68.03; H,
6.34; N, 4.41. Found: C, 68.22; H, 6.21; N, 4.35.
2-[2-(3,4-Dimethoxyphenyl)ethyl]-6-hydroxy-7-methoxy-1,2,3,4-
tetrahydroisoquinoline 5i. Amine 4i (210 mg, 0.6 mmol), dissolved
in 15 mL of dry methanol, and 15 mL of aqueous methanal solution
(37%) gave 79 mg (36%) of 5i as a pale yellow solid, mp 142−145 °C.
1H NMR (CD3OD, 400 MHz) δ = 6.87 (d, J = 7.83, 1H), 6.86 (s,
1H), 6.79 (dd, J = 1.96/7.83 Hz, 1H), 6.62 (s, 1H), 6.56 (s, 1H), 3.83
(s, 3H), 3.81 (s, 3H), 3.80 (s, 3H), 3.65 (s, 2H), 2.87−2.72 (m, 8H).
MS (ESI) m/z 344.2 (100, M+ + 1); Anal. calcd for C20H25NO4: C,
69.95; H, 7.34; N, 4.08. Found: C, 69.60; H, 7.30; N, 4.13.
The new selective inhibitors 5h, 5i, and 5f were subjected to HPLC
purity testing (see Supporting Information).
Synthesis of 2-(3-Acetoxy-4-methoxyphenyl)-N-[2-(4-
methoxyphenyl)ethyl]acetamide (7), 2-Methoxy-5-{2-[2-(4-
methoxyphenyl)ethylamino]ethyl}phenol (4a), and 2-[2-(4-
Hydroxyphenyl)ethyl]-1,2,3,4-tetrahydroisoquinolin-6-ol Hydrobro-
mide (6). Synthetic details and spectroscopic data can be found in the
Supporting Information.
N-[2-(4-Chlorophenyl)ethyl]-2-(3-hydroxy-4-methoxyphenyl)-
acetamide 3f. 3-Hydroxy-4-methoxyphenylacetic acid 2a (1.6 g, 8.6
mmol) and 4-chlorophenethylamine 1c (1.3 g, 8.5 mmol) in 25 mL of
xylene gave 1.8 g of 3f (65%) as pale beige crystals. 1H NMR (CDCl3,
500 MHz) δ = 7.17 (dd, J = 1.83/8.24 Hz, 2H), 6.94 (dd, J = 1.83/
8.55 Hz, 2H), 6.74 (d, J = 7.94 Hz, 1H), 6.71 (s, 1H), 6.59 (d, J = 7.94
Hz, 1H), 5.32 (br s, 1H), 3.89 (s, 3H), 3.43 3.38 (m, 4H), 2.67 (t, J =
6.71 Hz, 2H).
N-[2-(3,4-Dimethoxyphenyl)ethyl]-2-(3-hydroxy-4-
methoxyphenyl)acetamide 3i. 3-Hydroxy-4-methoxyphenylacetic
acid 2a (1.0 g, 5.5 mmol) and 2-(3,4-dimethoxyphenyl)ethylamine
1f (1.0 g, 5,5 mmol) in 17 mL of xylene gave 1.4 g of 3i (75%) as an
1
oily residue. H NMR (CDCl3, 400 MHz) δ = 6.75−6.71 (m, 3H),
6.62−6.54 (m, 3H), 5.57 (br s, 1H), 3.87 (s, 3H), 3.83 (s, 3H), 3.81
(s, 3H), 3.50 (s, 2H), 3.44 (dd, J = 5.87/6.26 Hz, 2H), 2.67 (t, J = 6.65
Hz, 2H).
Biological Assays, General. All cell cultures were obtained from
DSMZ (German Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany), and cultivated according to the recom-
Synthesis of 4b−k by Reduction with Lithium Aluminum
Hydride: General Procedure. For reduction of the amides (3b−k),
the respective substance was dissolved in dry tetrahydrofuran (THF)
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Article
mendations of DSMZ. The following strains were used: Aspergillus
brasiliensis (DSM 1988), Candida glabrata (DSM 11226), Escherichia
coli (DSM 426), Hyphopichia burtonii (DSM 70663) Pseudomonas
antimicrobia (DSM 8361), Saccharomyces cerevisiae (DSM 1333)
Staphylococcus equorum (DSM 20675), Streptococcus entericus (DSM
14446), and Yarrowia lipolytica (DSM 1345).
incubation procedure was identical to the one described above, but
only 980 μL of the cell suspension was used; 10 μL of inhibitor
solution and 10 μL of sterile sodium 13C-acetate solution (6.25 mg/
mL) were added to obtain a final volume of 1000 μL. A slight change
in the workup procedure was necessary. After cell lysis, 3 × 25 μL of
every cell lysate (total volume 1.0 mL) was not transferred back to the
microcentrifuge safe-lock tubes. These suspensions were separated for
a Bradford assay to determine the protein content.38,43 Further
treatment of the samples was the same as described above.
The overall inhibition of cholesterol biosynthesis was determined by
integration of the relevant MS signals of newly synthesized, labeled
cholesterol (quantifier ions m/z 372−379 and 462−469) and the
internal standard cholestane (quantifier ions m/z 203, 217, and 357),
with the results from the Bradford assay taken into account. The
percentage inhibition is calculated in eq 1:
HL 60 cells (DSM ACC3) were cultivated in RPMI 1640 medium
with 10% FBS (fetal bovine serum, both from PAA Laboratories,
Colbe, Germany) without the addition of antibiotics at 37 °C in a
̈
humidified atmosphere containing 5% CO2.
Stock solutions of all compounds and references were diluted with
absolute ethanol according to the SANCO guideline.42
The assays were performed under laminar flow cabinets: HeraSafe
12 from Heraeus Instruments (Hanau, Germany) for microorganisms,
and MSC Advantage from Thermo Fisher (Schwerte, Germany) for
HL 60 cells.
Assay for antibacterial and antifungal activities (agar diffusion
assay), screening assay for ergosterol biosynthesis inhibition, and MTT
assay are described in Supporting Information.
⎡
⎛
⎜
⎝
⎤
⎞
⎟
⎠
AS × AISC × PCC
% inhibition = ⎢1 −
⎥ × 100
A
× AISS × PCS
⎣
⎦
(1)
C
where AS is the area of the sample (13C-labeled cholesterol); AISC is the
area of the internal standard (cholestane); PCC is the protein content
control; AC is the area of the control (13C-labeled cholesterol); AISS is
the area of the internal standard sample (cholestane); and PCS is the
protein content sample.
Screening Assay for Cholesterol Biosynthesis Inhibition. This
assay is based on a method described by us previously,38 but workup
was slightly modified in analogy to the ergosterol biosynthesis assay.
Qualitative Screening (Identification of Target Enzyme). A
suspension of 1 × 106 HL 60 cells in a special HL 60 cell medium
The percentage inhibition (eq 1) relative to untreated control
samples (0% inhibition) was plotted against logarithmic inhibitor
concentration by use of Graph Pad Prism 4 (Graph Pad Inc., San
Diego, CA). A bottom level constant equal to zero was set as a
constraint, by use of a sigmoidal dose−response model with a variable
slope.38
(“Medium fur HL 60 Zellen, lipid free medium”; PAN Biotech,
̈
Aidenbach, Germany) was incubated with 10 μL of inhibitor solution
(concentrations 50 μM and 1 μM) in 24-well plates. Blank samples
were prepared with 10 μL of ethanol instead of 10 μL of inhibitor
solution. AY 9944 and BM 15.766 sulfate (both from Sigma−Aldrich,
Steinheim, Germany) were used as reference inhibitors for 7-
DHCR.9,15 After incubation for 24 h (conditions, 37 °C in a
humidified atmosphere containing 5% CO2), the suspensions were
transferred into 2.0-mL plastic microcentrifuge safe-lock tubes. The
tubes were centrifuged for 5 min at 9000g. The cell pellets were
suspended in 1 mL of 1 M aqueous sodium hydroxide solution,
vortexed for 30 s, and transferred into 4-mL glass vials with Teflon
septa. The vials were flooded with nitrogen and closed tightly. The
vials were stored for 1 h at 70 °C in a laboratory drying cabinet. While
cooling down to room temperature, the vials were ultrasonicated. The
whole solutions were transferred back to the microcentrifuge safe-lock
tubes, and 700 μL of TBME and 50 μL of cholestane as internal
standard (10 μg/mL in TBME) were added. After thorough shaking
for 1 min, the solutions were centrifuged (5 min, 9000g). The organic
supernatant was transferred to a second 2.0-mL plastic microcentrifuge
safe-lock tube containing 40 2 mg of a mixture of anhydrous sodium
sulfate/PSA (primary secondary amine sorbent, Bondesil PSA, Varian,
Darmstadt, Germany) 7:1. Two further extractions were performed
with 750 μL of TBME each. The combined organic extracts were
shaken for 1 min, followed by a last centrifugation step (5 min, 9000g).
Each 1.0 mL of the purified extracts obtained by this dispersive solid-
phase extraction was transferred into a brown glass vial. Under a gentle
stream of nitrogen, the extracts were brought to dryness. The residues
were taken up in 950 μL of TBME and 50 μL of a silylation reagent
mixture of N-methyl-N-(trimethylsilyl)trifluoroacetamide and N-
(trimethylsilyl)imidazole (MSTFA/TSIM 9:1). The samples were
stored for at least 30 min at room temperature and then subjected to
GC-MS analysis.
GC-MS Analysis of Sterols. A Varian Saturn 2200 ion trap was
coupled with a Varian 3800 gas chromatograph (Darmstadt,
Germany). The gas chromatograph was linked with a CombiPal
from CTC Analytics (Zwingen, Schweiz). A 1177 injector from Varian
(Darmstadt, Germany) was used in splitless injection mode during
injection. Data analysis and instrument control was made with Varian
Workstation 6.9 SP 1 software. A Varian VF-5 ms capillary column of
30 m length, 0.25 mm i.d., and 0.25 μm film thickness with a 10 m EZ-
Guard column was used for analysis. The GC-MS conditions were the
following: helium 5.0 (99.9990%), constant flow of 1.3 mL/min, inlet
temperature 250 °C, injection volume 1 μL, MS transfer line
temperature 270 °C. The initial GC oven temperature was 50 °C (1
min hold) ramped up to 260 °C (50 °C/min), followed by a gradient
of 4 °C per minute up to 310 °C (hold time 0.5 min). The total run
time was 18.2 min. The MS was operated in full scan mode from 9 to
12 min at a mass range from 50 to 450 m/z and from 12 to 18.2 min at
a mass range from 100 to 650 m/z. The sterols were identified by
comparison with authentic standard substances or by alignment with
data from NIST 2005 mass spectral database.37,38
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional text and one scheme with synthetic details and
spectroscopic data for intermediates and final substances. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Analysis was focused on identifying 7-DHC unequivocally. This was
accomplished by comparing the relative retention time and the
fragmentation of the sterols. Characteristic ions for zymostenol are m/
z 458, 353, and 213; for 7-dehydrocholesterol, m/z 351, 325, and 366;
and for cholestane, m/z 357, 217, and 203. The reference standard,
zymostenol, was synthesized from cholesta-8,14-dien-3β-ol;38 7-
dehydrocholesterol and cholestane were purchased from Sigma-
Aldrich (Steinheim, Germany). The investigations were performed
in duplicate.
AUTHOR INFORMATION
Corresponding Author
* Phone +49-(0)345-55-25175; e-mail peter.imming@
pharmazie.uni-halle.de.
■
Notes
The authors declare no competing financial interest.
Quantitative Screening. Six suitable concentrations of each potent
inhibitor (selected by semiquantitative analysis of the peak of
accumulating 7-dehydrocholesterol in the qualitative screening) plus
one blank sample, all in triplicate, were tested in a 24-well plate. The
ACKNOWLEDGMENTS
A.H. and R.B. thank Frau Ilona Fritsche, Halle, for valuable
synthetic support.
■
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Article
(19) Vollekova,
́
A.; Kost, D.; Kettmann, V.; Troth, J. Antifungal
ABBREVIATIONS
■
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7-DHC, 7-dehydrocholesterol; 7-DHCR, 7-dehydrocholesterol
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Antibacterial activity and structure-activity relationships of berberine
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