Steroidomimetic aminomethyl spiroacetals as novel inhibitors of the enzyme Δ8,7-sterol isomerase in cholesterol biosynthesis

Article abstract of DOI:10.1002/ardp.201300296

Grundmann's ketone is converted to a spiroacetal containing a 5-hydroxymethyl-5-nitro-1,3-dioxane moiety whose hydroxymethyl group can be esterified or directly substituted with primary and secondary amines. Among the resulting aminomethyl spiroacetals, several ones bearing diamino residues were found to be inhibitors of the enzyme Δ8,7-isomerase in cholesterol biosynthesis. The complex bicyclic building block derived from Grundmann's ketone could be replaced by a properly substituted tetraline scaffold, without noteworthy loss in activity. This opens the opportunity to perform further structural modifications for the design of new steroidomimetic inhibitors of human Δ8,7-isomerase. Aminomethyl spiroacetals derived from Grundmann's ketone were identified as a new chemotype of inhibitors of human Δ8,7-sterol isomerase. In a second step, an equipotent but not cytotoxic tetraline analog was developed as potent inhibitor of cholesterol biosynthesis.

Full text of DOI:10.1002/ardp.201300296

108
Arch. Pharm. Chem. Life Sci. 2014, 347, 108–122
Full Paper
Steroidomimetic Aminomethyl Spiroacetals as Novel Inhibitors
of the Enzyme D8,7-Sterol Isomerase in Cholesterol
Biosynthesis
Melanie Krojer, Christoph Müller, and Franz Bracher
Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians-University of Munich, Munich,
Germany
Grundmann’s ketone is converted to a spiroacetal containing a 5-hydroxymethyl-5-nitro-1,3-dioxane
moiety whose hydroxymethyl group can be esterified or directly substituted with primary and
secondary amines. Among the resulting aminomethyl spiroacetals, several ones bearing diamino
residues were found to be inhibitors of the enzyme D8,7-isomerase in cholesterol biosynthesis. The
complex bicyclic building block derived from Grundmann’s ketone could be replaced by a properly
substituted tetraline scaffold, without noteworthy loss in activity. This opens the opportunity to
perform further structural modifications for the design of new steroidomimetic inhibitors of human
D8,7-isomerase.
Keywords: Acetal / Cholesterol biosynthesis / Enzyme inhibitors / Isomerase / Steroidomimetic
Received: August 9, 2013; Revised: September 16, 2013; Accepted: September 17, 2013
DOI 10.1002/ardp.201300296
Introduction
side, the inborn lack of late enzymes of cholesterol
biosynthesis is known to cause some rare but very serious
disorders. For example, CHILD (congenital hemidysplasia
with ichthyosiform erythroderma and limb defects) syndrome
[6] and Conradi-Hünermann-Happle syndrome [7] are dysmor-
phogenetic syndromes of variable severity due to mutations of
the gene encoding human 3b-hydroxysteroid D8,7-sterol
isomerase [hSI; EC 5.3.3.5; also known as emopamil-binding
protein (EBP)] [8]. Specific inhibitors of such enzymes are
expected to be useful tools for getting deeper insight into
these disorders [9].
Ergosterol is the main sterol in the cytoplasmatic
membranes of fungi and trypanosomes, and disruption of
ergosterol biosynthesis is the main strategy for treatment of
fungal [10] and trypanosomal infections [11]. Further,
inhibitors of ergosterol biosynthesis in plant-pathogenic
fungi play a central role in crop protection [12]. The azoles
are the most widely used antifungal drugs, but due to
emerging resistance against these drugs (e.g., fluconazole),
there is urgent need for developing novel antifungals [13]. The
post-squalene part of ergosterol biosynthesis still offers new
targets to be exploited [14].
The enzymes of the biosynthetic pathways leading to
cholesterol in man and ergosterol in fungi and trypanosomes
are important targets for therapy with low-molecular drugs.
Cholesterol is essential for the various functions of vertebral
plasma membranes [1], and relevant for the embryonic
progress and morphogenesis [2]. Further, cholesterol is the
biosynthetic precursor for steroid hormones and bile acids.
Disruptions in cholesterol metabolism cause various diseases,
among them cardiovascular indispositions induced by
increased blood cholesterol levels (hypercholesterinaemia)
[3]. Presently, these disorders are treated mainly with statins,
inhibitors of 3-hydroxy-3-methylglutaryl (HMG)-CoA reduc-
tase, an enzyme acting early in the pre-squalene part of
cholesterol biosynthesis. A number of severe side-effects of the
statins [4] led to efforts aimed at developing new cholesterol-
lowering drugs with other molecular mechanisms of action.
For this purpose the post-squalene part of the cholesterol
biosynthesis pathway offers attractive targets [5]. On the other
Correspondence: Prof. Franz Bracher, Department of Pharmacy – Center
for Drug Research, Ludwig-Maximilians-University of Munich, Bute-
nandtstr. 5–13, 81377 Munich, Germany.
E-mail: franz.bracher@cup.uni-muenchen.de
Fax: þ49-89-218077802
Due to the tremendous importance of steroids as hormones
and membrane constituents, numerous approaches have
been developed for the inhibition of relevant enzymes in
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Arch. Pharm. Chem. Life Sci. 2014, 347, 108–122
Aminomethyl Spiroacetals as Inhibitors of D8,7-Isomerase
109
sterol biosynthesis, and a wide variety of chemical structures
can be found in compounds targeting the biosynthesis of
cholesterol and ergosterol. Since the single steps of these
biosynthetic pathways are understood in detail for centuries,
considerable effort has been undertaken to design new
inhibitors on a rational basis. In order to mimic the substrates
or high-energy intermediates (HEI) of these enzymatic
reactions, numerous steroids bearing modifications in the
tetracyclic core [15–19] or the side chain [20–26] have been
developed as inhibitors of sexual hormone, cholesterol, and
ergosterol biosynthesis. Alternatively, steroidomimetic com-
pounds have been described to efficiently inhibit particular
enzymes in steroid biosynthesis. In estrogen biosynthesis the
enzymes 17a-hydroxylase and 17,20-lyase can be inhibited by
compounds that mimic some of the four rings of the steroid
scaffold, e.g., biphenyls as ring AC mimics [27], (partially
hydrogenated) arylindenes and hydroxyphenylnaphthols as
ring A-CD mimics [28–30], and vinylbenzo[b]thiophenes as
ring AB mimics [31]. N-substituted azadecalines mimic rings A
and B in plant [32] and fungal sterol biosynthesis [33].
Recently, our group demonstrated that Grundmann’s ketone
(1) [34], easily accessible by ozonolysis of cholecalciferol
(vitamin D3), is a suitable, correctly configurated building
block providing rings C and D, as well as the side chain of
steroids for the synthesis of sterol biosynthesis inhibitors.
Through suitable chemical modifications we obtained amino-
protonated at physiological pH, and thus are able to mimic
carbocationic HEIs of enzymatic transformations. Since
mimics of the HEI have a higher affinity for the active site
of the enzyme than the regular substrates, steroidomimetics
containing protonable nitrogen atoms in pertinent positions
have the potential to strongly inhibit enzymes in ergosterol
and cholesterol biosynthesis [37]. On the other hand, it is
difficult to predict the minute target enzyme, since numerous
enzymatic transformations in the post-squalene part of sterol
biosynthesis go through carbocationic HEIs, e.g., those
catalyzed by the enzymes sterol D8,7-isomerase, D7-sterol
reductase, D24-sterol reductase, and D14-sterol reductase [37].
For this reason most of the established protonable sterol
biosynthesis inhibitors show low selectivity. Besides inhibi-
tion of D8,7-sterol isomerase, ifenprodil and MDL28815 also
inhibit D14-sterol reductase [38]; trifluperidol, AY9944, and
fenpropimorph show additional inhibition of D7-sterol
reductase [39], trifluoperazine, tamoxifen, and U18666A
additionally inhibit D24-sterol reductase [40].
In this project we aimed at the introduction of amino
groups via conversion of Grundmann’s ketone (1) to
spiroacetals (A) using 1,2- or 1,3-diols bearing the required
protonable residues or precursors thereof. We selected the
spiroacetal moiety due to its expected easy introduction and
sufficient stability. Further, the antifungal spiroxamine (4), a
sterol D8,7-isomerase inhibitor used in crop protection [41],
shows a comparable spiroacetal moiety, and cyclic acetal (1,3-
dioxolane) groups are also present in the azole antifungals
(inhibitors of lanosterol demethylase) ketoconazole (5) and
itraconazole (Fig. 2). Since we have previously developed
screening systems that allow the identification of inhibitors of
any enzymes in the post-squalene part of cholesterol
biosynthesis [42] and the post-lanosterol part of ergosterol
biosynthesis [43], we had a tool in hand to detect any
emerging effect in this field, no matter which of the
considered enzyme(s) were inhibited.
propylindenes of type
2 as inhibitors of the enzyme
oxidosqualene cyclase (lanosterol synthase) from various
organisms [35], and aminoalcohols of type 3 as potent,
selective inhibitors of mammalian D8,7-sterol isomerase [36]
(Fig. 1).
In continuation of this work, we intended to introduce new
aminoalkyl residues to the scaffold 1. Aliphatic amino groups
are of special interest in this context, since they are
Results and discussion
Chemistry
H
O
Grundmann’s ketone (1) was obtained from cholecalciferol by
ozonolysis using our recently improved protocol [36] on a
multigram scale. For the attempted introduction of the amino
residues at C-4 of ketone 1, we first worked out a suitable
protocol for the acetalization with diols. In a model reaction, 1
was reacted with 1,2-ethanediol in the presence of various
catalysts that had been described for related reactions in
the literature before. Standard protocols using Brønsted acids
(e.g., 4-toluenesulfonic acid [44]) or iodine [45] as catalysts gave
no conversion to the 1,3-dioxolane 6 at all. Finally, we found
that the Lewis acid scandium(III) triflate [46] catalyzes this
reaction well, and after some optimization (additives,
R2N
1
2
N
OH
3
Figure 1. Grundmann’s ketone (1) and recently developed
inhibitors of enzymes in sterol biosynthesis (2 and 3) derived
thereof.
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a
1
O
O
6
O
O
O
O
N
NR2
A
4
O
O
O
O
Cl
Cl
N
O
NR2
R
NHR
N
O
7
8
O
Scheme 1. Synthesis of the dioxolane 6. (a) 1,2-Ethanediol,
Sc(OTf)3 (3 mol%), HC(OMe)3, acetonitrile, microwave irradiation
(57%). Target structures 7 and 8.
N
N
5
O
dioxane (9) in 37% yield. Ozonolysis of methylene compound 9
gave the 1,3-dioxan-5-one 10, a formal dihydroxyacetone
acetal of Grundmann’s ketone, in poor yield (25%). The poor
yields in both of these steps are most likely due to steric
hindrance, since analogous conversions starting from cyclo-
hexanone proceed in high yields [49]. Unfortunately, and
despite broad variations of reagents and reaction conditions,
we did not succeed in converting ketone 10 into correspond-
ing 4-amino-1,3-dioxanes 11 (Scheme 2).
Figure 2. General structure of the target compounds of this work
(A) and two inhibitors of ergosterol biosynthesis containing acetal
moieties: spiroxamine (4) and ketoconazole (5).
solvents, reaction conditions) we found that acetalization can
be performed in 45% yield using 3 mol% of the catalyst,
trimethyl orthoformate as dehydrating agent [47] in acetoni-
trile under reflux, and the yield can even be enhanced to 57%
by microwave irradiation (Scheme 1).
For the preparation of analogous spiroacetals of type 7,
containing aminoalkyl residues in the 1,3-dioxolane ring, we
investigated the acetalization of 1 with various 3-amino-1,2-
propandiols (3-amino, 3-N-methylamino, 3-N,N-dimethyla-
mino, 3-pyrrolidin-1-yl, 3-morpholin-4-yl), but did not observe
any conversions. This is most likely due to the fact that the
Lewis acid catalyst is deactivated by the amino residues.
Tris(hydroxymethyl)aminomethane (TRIS) and 2-amino-1,3-
diol could not be converted at all, and even the N-Cbz
derivative of 2-amino-1,3-diol [48], in which the basicity of the
amino group is eliminated, did not give the desired 5-Cbz-
amino-1,3-dioxane of type 8 (Scheme 1). Therefore, we
intended to prepare a spiroacetal bearing a non-basic residue
first, and then convert this to the desired amino(alkyl)
derivative. Acetalization of Grundmann’s ketone (1) with
glycerol under the conditions described above resulted in
almost quantitative conversion of the ketone, but gave an
inseparable mixture of isomeric 1,3-dioxolanes and 1,3-
dioxanes. For this reason we focussed on symmetric acetal
rings exclusively in our further investigations. Acetalization
with 2-methylene-1,3-propanediol gave the 5-methylene-1,3-
Eventually, we found that tris(hydroxymethyl)nitrometh-
ane (12) readily undergoes acetalization with ketone 1 under
scandium(III) triflate catalysis to give the 5-hydroxymethyl-5-
nitro-1,3-dioxane 13.
Reduction of the nitro group should give a primary amine
14, which upon N-alkylation should be a central building
block for the preparation of a library of aminoacetals. To our
surprise, first attempts to reduce the nitro group by transfer
hydrogenation with ammonium formate, as previously
described for related substrates with Raney nickel [50] or
palladium catalysts [51], failed completely. Finally, hydro-
genation with Raney nickel and hydrazine as the hydrogen
transfer agent gave the desired primary amine 14 in 25%
yield (Scheme 3). The poor yield in this reduction step as well
as the expectable difficulties in functionalizations of this
sterically hindered amine prevented us from further inves-
tigations on 14.
But the 5-hydroxymethyl-5-nitro-1,3-dioxane 13 opened the
opportunity to prepare a large collection of amino derivatives
16a–m by direct displacement of the primary hydroxyl group
with primary and secondary amines. As postulated by
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Aminomethyl Spiroacetals as Inhibitors of D8,7-Isomerase
111
a
b
1
O
O
O
O
O
O
O
NR2
11
9
10
Scheme 2. Approach through dihydroxyacetone acetal 10: (a) 2-Methylene-1,3-diol, Sc(OTf)3 (3 mol%), HC(OMe)3, acetonitrile, microwave
irradiation (37%); (b) O3, CH2Cl2, pyridine, ꢀ78°C (25%).
OH OH
a
b
1
+
O
O
O
O
NO2
OH
12
HO
HO
NO2
NH2
13
14
R
_
N
H
15a m
_
c
d
RR'N
=
R'
H3C
N
H
a
b
c
d
(H3C)2N
(H3C)2N
(H3C)2N
H
N
O
O
NO2
O
O
N
CH3
R
O
RR'N
_
NO2 16a m
H3C
N
N
e
O
H
H
N
17a R =
17b R =
17c R =
N
h
g
N
N
f
i
O
N
N
C
N
N
H
C
N
N
j
CH3
H2
H2
F3C
O
N
H
N
N
k
l
C
N
m
N
N
H
H2
Scheme 3. Aminomethyl spiroacetals and related esters: (a) Sc(OTf)3 (3 mol%), HC(OMe)3, acetonitrile, microwave irradiation (43%);
(b) Raney nickel, hydrazine hydrate, EtOH, 20°C (25%); (c) acid chloride, pyridine, 20°C (35–41%); (d) amines 15a–m, EtOH, 70°C
(21–52%).
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Malinowski [52], the hydroxyl group is activated by a
hydrogen bond with the nitro group and therefore can be
displaced by simply heating with appropriate amines. A large
number of primary and secondary amines 15 (for structures
see Scheme 3) were converted to the respective 5-amino-
methyl-5-nitro-1,3-dioxanes 16 by heating in ethanol for 16 h
in yields ranging from 21 to 52%. Amines containing more
than one reactive group (1,2-ethanediamine, N-methyl-1,2-
ethanediamine, 2-(aminomethyl)piperidine) did not give the
desired products.
reflux) gave the desired spiroacetal 19a in only 13% yield, so we
examined other catalysts. Finally, we found that acetalization
proceeds very well under solvent-free conditions by employing
1-tetralone (18a), four equivalents of tris(hydroxymethyl)-
nitromethane, one equivalent of triethyl orthoformate, and
the catalyst tetrabutylammonium tribromide (1 mol%) [53] at
room temperature for 48 h [54]. Aminolysis of the resulting 5-
hydroxymethyl-5-nitro-1,3-dioxane 19a with 4-amino-1-benzyl-
piperidine (15i) gave the tetraline-derived aminomethyl
spiroacetal 20a in poor yield. This product did not show the
expected bioactivity, and we attributed this to the absence of
an equivalent of the sterol side chain in this molecule. Hence,
we prepared two analogs bearing readily introducible alkoxy
residues at a geometrically proper ring position (C-6 of the 1-
tetralone building block). The methoxy (20b) and the
isohexyloxy derivatives were prepared in the same manner
as 20a from appropriate alkoxytetralones 18b and 18c
(Scheme 4).
The primary hydroxyl group in 5-hydroxymethyl-5-nitro-
1,3-dioxanes is accessible for esterification [52]; therefore,
the benzoate (17a) and nicotinate esters (17b), as well as the
morpholino carbamate (17c), were prepared using the
respective acid chlorides and pyridine as a base (Scheme 3).
The results of the screenings on inhibition of cholesterol
and ergosterol biosynthesis (see below) clearly demonstrated
that the N-benzylpiperidin-4-ylamino derivative 16i was the by
far most active compound obtained so long. This prompted us
to investigate the role of the perhydroindene scaffold derived
from Grundmann’s ketone (1), which had been introduced in
order to get maximum mimicry of the rings C and D and the
lipophilic side chain of the steroidal substrates of the
enzymes. Obviously, our concept worked well, yielding 16i
as a promising lead structure, but the high costs for
preparation of precursor 1 and the poor yields obtained in
several conversions were disadvantageous. So we investigated
whether Grundmann’s ketone (1) can be replaced by other,
cheaper and more reactive bicyclic ketones. Inspired by
reports in the literature that tetralines can function as ring
CD equivalents in steroidomimetics [27, 28], we prepared
tetraline analogs of compound 16i starting from appropriate
1-tetralones (18a–c) (Scheme 4). The acetalization of 1-tetralone
(18a) with tris(hydroxymethyl)nitromethane (12) under the
conditions described above (3 mol% scandium(III) triflate,
acetonitrile, trimethyl orthoformate, microwave irradiation,
Screenings
The compounds were subjected to our in-house cellular
screening systems for the identification of target enzymes in
the post-squalene part of cholesterol biosynthesis in the
human leukemia cell line HL 60. Whereas untreated control
cells contain cholesterol as the only detectable sterol (see
upper row of chromatograms in Fig. 3), incubation with
enzyme inhibitors leads to the accumulation of other sterols,
which are identified by GC-MS. In this assay the compounds 6,
9, 10, 13, 14, esters 17a–c, and 19a–c were found to be
completely inactive. In the series of the aminomethyl
spiroacetals 16 derived from Grundmann’s ketone, most of
the compounds showed no effect, the (pyrrolidin-1-yl)ethyl-
amino compound 16g caused accumulation of several new
sterols, indicating multienzyme inhibition, whereas with the
homologous (piperidin-1-yl)ethylamino compound 16f only
cholesta-8-en-3b-ol (zymostenol), a substrate of human sterol
R
R
R
a
b
O
O
O
O
H
N
NO2
NO2
O
OH
N
_
_
_
18a c
19a c
20a c
a R = H
b R = OCH3
c R = OCH2-CH2-CH2-CH(CH3)2
Scheme 4. Synthesis of the tetraline analogs of lead compound 16i: (a) Bu4NBr3 (1 mol%), HC(OEt)3, 20°C (50–89%); (b) 15i, EtOH, 70°C
(6–48%).
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Arch. Pharm. Chem. Life Sci. 2014, 347, 108–122
Aminomethyl Spiroacetals as Inhibitors of D8,7-Isomerase
113
Figure 3. Extracted ion chromatograms of the accumulating sterols after treatment of HL 60 cells with inhibitors; A1–A3: blank sample
analysis; B1–B3: samples treated with inhibitors; A1: control (m/z 217, 368, 349, 351); B1: compound 20b (50 mM) (m/z 217, 368, 349, 351);
A2: control (m/z 217, 368, 458); B2: compound 16i (50 mM) (m/z 217, 368, 458); A3: control (m/z 217, 368, 458); B3: compound 20c (50 mM)
(m/z 217, 368, 458); peaks: I.S. ¼ cholestane (internal standard), 1 ¼ cholesterol, 2 ¼ cholesta-5,7,24-trien-3b-ol (7-dehydrocholesterol),
3 ¼ cholesta-5,7-dien-3b-ol, 4 ¼ cholesta-8-en-3b-ol (zymostenol).
D8,7-isomerase [36], accumulated. The same effect was found
upon incubation with the diamines 16c, 16i, and 16j. In the
tetraline series ring-unsubstitited tetraline 20a was complete-
ly inactive; the methoxy analog 20b led to an accumulation of
several sterols, among them cholesta-5,7,24-trien-3b-ol and
cholesta-5,7-dien-3b-ol (7-dehydrocholesterol), substrates of
the enzymes 7-dehydrocholesterol reductase and D24-reduc-
tase. Incubation with compound 20c, however, led to a
significant accumulation of zymostenol, a clear indication
for an inhibition of human sterol D8,7-isomerase (Table 1 and
Fig. 3).
For the most active D8,7-isomerase inhibitors (identified by
semiquantitative evaluation of the chromatograms) of this
series we determined their effect on overall cholesterol
biosynthesis in a cellular assay [42]. In this whole-cell assay HL
60 cells are incubated with various concentrations of test
substances in the presence of 2–13C-acetate. Due to the
incorporation of 4–7 13C atoms into cholesterol molecules
biosynthesized during the incubation period, newly synthe-
sized cholesterol can be distinguished from unlabeled matrix
cholesterol that was present in the cells before incubation, by
GC-MS. From the dose-response curves obtained by determi-
nation of 13C-labeled cholesterol and after normalization to
the protein content, the IC50 values describing the overall
effects of the inhibitors on cholesterol biosynthesis in the
cellular system are calculated (Table 1).
The N-benzylpiperidin-4-ylamino derivative 16i was the
most active (IC50 ¼ 1.7 mM) compound; its N-methyl analog
16j (IC50 ¼ 4.7 mM) and the isohexyloxytetraline analog 20c
(IC50 ¼ 4.5 mM) were slightly less active.
Since in a routine agar diffusion assay for antimicrobial
activities on dermatophytes, molds, and yeasts (data not
shown here), none of the compounds described here showed
notable antifungal activity, inhibition of ergosterol biosyn-
thesis by these compounds was not probable. Nevertheless,
the cholesterol biosynthesis inhibitors identified above were
subjected to our in-house qualitative assay [43] for the
identification of target enzymes in the post-squalene part of
ergosterol biosynthesis in three different strains. All com-
pounds were found to be completely inactive in this assay.
All compounds were further subjected to an MTT assay [55]
for general cytotoxicity (Table 1). Cisplatin (IC50 ¼ 5 mM) was
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Arch. Pharm. Chem. Life Sci. 2014, 347, 108–122
Table 1. Results of the screening on inhibition of cholesterol
biosynthesis and general cytotoxicity (MTT assay). All experi-
ments were performed in triplicate.
A very promising structural element in these diamines are a
secondary amino group next to the 1,3-dioxane ring; the
corresponding N-methyl analogs are inactive (see 16c/16d) or
at least less active (see 16i/16j). A distal N-benzyl residue leads
to the by far most active inhibitors (16i/16j). Due to the very
costly synthesis of the most active compound 16i and its
undesired slight cytotoxicity, we prepared steroidomimetic
analogs 20a–c of this compound in which the building block
derived from expensive Grundmann’s ketone (1) is replaced by
a tetraline ring. The screening results of these compounds
clearly indicate that it is not sufficient to introduce the
tetraline ring as a steroid ring CD mimic, but the lipophilic
side chain has to be covered as well. By this way we invented
an easy-to-prepare isohexyloxytetraline-derived analog of
compound 16i, which is a potent inhibitor of the enzyme
D8,7-isomerase as well (IC50 ¼ 4.5 vs. 1.7 mM) and fortunately
free of cytotoxicity.
In contrast to the inhibitors derived from Grundmann’s
ketone (1), the tetraline-type inhibitor 20c can be prepared
from an inexpensive precursor in a few steps, and being a
solid, it has better physicochemical properties and stability.
Most importantly, the tetralone building block offers the
opportunity to perform a broad range of structural mod-
ifications for further optimization, whereas the hydrocarbon
parts of Grundmann’s ketone (representing rings C and D and
the side chain of the sterols) are not suitable substrates for
chemical modifications.
Effect on
cholesterol
IC50 (mM) for Cytotoxicity
inhibition of
in MTT
biosynthesis/target total cholesterol
assay,
IC50 (mM)
Compound
enzyme
biosynthesis
6
9
–
23
22
–
10
–
19
13
–
23
14
–
24
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
17a
17b
17c
19a
19b
19c
20a
20b
20c
–
18
23
16
26
11
22
17
8
13
30
>50
14
50
28
46
20
–
D8,7-Isomerase
–
–
D8,7-Isomerase
Multienzyme inhibitor
–
D8,7-Isomerase
D8,7-Isomerase
1.7
4.7
–
–
–
–
–
–
–
>50
>50
>50
>50
>50
>50
–
–
–
Multienzyme inhibitor
D8,7-Isomerase
Work is in progress to further optimize the enzyme
inhibitors described here.
4.5
Experimental
used as a reference. None of the aminomethyl spiroacetals 16
derived from Grundmann’s ketone showed strong cytotoxici-
ty, most of them are very slightly cytotoxic (IC50 ¼ 10–20 mM);
in contrast, the aminomethyl spiroacetals 20a–c derived from
tetralones are devoid of cytotoxicity.
Materials and methods
IR-spectra: FT-IR Paragon 1000, Perkin-Elmer (Wellesley, USA); MS:
5989 A MS-Engine, Hewlett Packard (Agilent Technologies, Palo
Alto, USA); electron ionization (EI) 70 eV, chemical ionization (CI)
with CH4 (300 eV); HR-MS: GC/MS system JMS-GCmate II, Jeol
(Peabody, USA); NMR: Jeol GSX 400 (1H: 400 MHz, 13C: 100 MHz)
(Peabody, USA); melting points: Büchi Melting Point B-540 (Flawil,
Switzerland); flash column chromatography (FCC): silica gel 60
(230–400 mesh, E. Merck, Darmstadt, Germany); optical rotation:
Polarimeter 241 MC (Perkin Elmer; Waltham, USA); microwave
reactor: Discovery (CEM, Matthews, USA); ozone generator:
Ozonova Typ OG700-10WC (Jeske Ozontechnik, Rudersberg,
Germany).
Discussion
We have worked out convenient protocols for the acetaliza-
tion of Grundmann’s ketone (1), and the spiroacetal 13
containing
a 5-hydroxymethyl-5-nitro-1,3-dioxane moiety
provides an easy access to esters (17a–c) and variably
substituted aminomethyl spiroacetals 16. Whereas the esters
and compounds bearing monoamino residues (16a,b,h,k,l,m)
are inactive in a cholesterol biosynthesis assay, diamino side-
chains lead to significant effects. Depending on structural
features that are not yet fully understood, the compounds are
either multienzyme inhibitors in the post-squalene part of
cholesterol biosynthesis (16g) or selective inhibitors of the
enzyme D8,7-isomerase.
Chemistry
General Procedure 1: Scandium(III) triflate-catalyzed
acetalization of ketones 1 and 18a–c
To a solution of the ketone (1.0 equiv.) and the required diol
(2.0 equiv.) dissolved in acetonitrile (2 mL per mmol ketone),
scandium(III) triflate (3 mol%) and trimethyl orthoformate
(2.0 equiv.) are added, and the mixture is refluxed for 2 h under
microwave irradiation (P ¼ 80 W). Then sodium carbonate
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Arch. Pharm. Chem. Life Sci. 2014, 347, 108–122
Aminomethyl Spiroacetals as Inhibitors of D8,7-Isomerase
115
solution (10%; 4 mL per mmol ketone) is added and the mixture is
extracted with dichloromethane (3 ꢁ 2 mL per mmol ketone). The
combined organic layers are dried over magnesium sulfate and
evaporated. The residue is purified by FCC (isohexane/ethyl
acetate mixtures).
(100 MHz, CD2Cl2): d (ppm) ¼ 141.73 (spiro-C), 107.35 (5-CH2),
100.76 (C-5), 63.16 (C-4), 63.12 (C-6), 55.13 (C-10), 50.88 (C-3a0),
44.52 (C-7a0), 39.51 (C-50), 36.94 (C-60), 33.99 (C-100), 33.84
(C-70), 28.46 (C-20), 28.14 (C-500), 25.79 (C-30), 25.27 (C-300), 25.03
(C-200), 22.57 (C-600), 22.39 (7a0-CH3), 21.54 (500-CH3), 19.66 (100-CH3),
18.92 (C-400); MS (CI): m/z (%) ¼ 335 (100, Mþþ1), 247 (20); IR
(CH2Cl2, film) n (cmꢀ1): 2954, 1674, 1467, 1367, 1367, 1092, 901;
General Procedure 2: Tetrabutylammonium tribromide-
catalyzed acetalization of ketones 1 and 18a–c
20
½aꢂD ¼ 74.5 (c ¼ 0.010, CH2Cl2); HRMS: calcd. for C22H38O2:
334.2872. Found: 334.2879.
A mixture of the ketone (1.0 equiv.), tris(hydroxymethyl)nitro-
methane (12, 4.0 equiv.), triethyl orthoformate (1.0 equiv.), and
tetrabutylammonium tribromide (1 mol%) is stirred at room
temp. for 48 h. Then sodium carbonate solution (10%; 10 mL per
mmol ketone) is added and the mixture is extracted with
dichloromethane (3 ꢁ 10 mL per mmol ketone). The combined
organic layers are dried over magnesium sulfate and evaporated.
The residue is purified using the protocol described for the
particular product below.
(10R,3a0R,7a0R,100R)-7a0-Methyl-10-(1,5-dimethylhexyl)-
spiro[1,3-dioxane-2,40-octahydroinden]-5-one (10)
A solution of 1.66 g (4.93 mmol) of the methylene acetal 9 in 3 mL
dichloromethane and 1 mL pyridine is cooled to ꢀ78°C and ozone
(flow: 25 L hꢀ1) is bubbled through the stirred solution until a
blue color persists (about 10 min). Then sodium carbonate
solution (10%; 20 mL) is added and the mixture is extracted
with dichloromethane (3 ꢁ 30 mL). The combined organic layers
are dried over magnesium sulfate and evaporated. The residue is
purified by FCC (isohexane/ethyl acetate 98:2).
General Procedure 3: Conversion of hydroxymethylacetals
13 and 19a–c to aminomethylacetals 16a–m and 20a–c
A solution of the hydroxymethylacetal (1.0 equiv.) and the
required primary or secondary amine (3 equiv. unless stated
otherwise) in anhydrous ethanol (1 mL per mmol hydroxyme-
thylacetal) is heated in a closed vessel at 70°C for 16 h. After
cooling to room temperature the mixture is poured into sodium
carbonate solution (10%; 20 mL per mmol hydroxymethylacetal)
and extracted with dichloromethane (3 ꢁ 20 mL per mmol
hydroxymethylacetal). The combined organic layers are dried
over magnesium sulfate and evaporated. The residue is purified
by FCC using an appropriate eluent.
Yield: 617 mg (37%); colorless oil; 1H NMR (400 MHz, CD2Cl2): d
(ppm) ¼ 4.29–4.05 (m, 4H, 4-H, 6-H), 2.11–2.07 (m, 1H, 3a0-H), 1.94–
1.25 (m, 19H, 10-H, 20-H, 30-H, 50-H, 60-H, 70-H, 100-H, 200-H, 300-H, 400-H,
500-H), 1.08 (s, 3H, 7a0-CH3), 0.88–0.86 (m, 9H, 100-CH3, 600-H, 500-CH3).
13C NMR (100 MHz, CD2Cl2): d (ppm) ¼ 208.96 (C-5), 103.03 (spiro-
C), 67.05 (C-4), 67.03 (C-6), 56.91 (C-10), 51.48 (C-3a0), 45.02 (C-7a0),
39.99 (C-50), 37.43 (C-60), 34.25 (C-100), 33.99 (C-70), 28.63 (C-20), 28.30
(C-500), 27.30 (C-30), 25.85 (C-300), 25.05 (C-200), 23.05 (C-600), 22.87 (7a0-
CH3), 21.75 (500-CH3), 20.19 (100-CH3), 19.63 (C-400); MS (CI): m/z
(%) ¼ 337 (82, Mþþ1), 247 (100); IR (CH2Cl2, film) n (cmꢀ1): 2954,
20
1801, 1749, 1709, 1467, 1375, 1235, 1095, 929; ½aꢂD ¼ 73.8
(c ¼ 0.016, CH2Cl2); HRMS: calcd. for C22H36O3: 336.2665. Found:
(10R,3a0R,7a0R,100R)-7a0-Methyl-10-(1,5-dimethylhexyl)-
336.2664.
spiro[1,3–dioxolane–2,40–octahydroindene] (6)
Prepared following General Procedure 1 from 104 mg (0.40 mmol)
Grundmann’s ketone (1) and 1,2-ethanediol. Eluent: isohexane/
ethyl acetate 98:2.
(10R,3a0R,7a0R,100R)-5-Hydroxymethyl-7a0-methyl-10-(1,5-
dimethylhexyl)-5-nitrospiro[1,3-dioxane-2,40-
octahydroindene] (13)
a. Prepared following General Procedure 1 from 6.83g (25.8 mmol)
Grundmann’s ketone (1) and tris(hydroxymethyl)nitromethane.
Eluent: isohexane/ethyl acetate 8:2. Yield: 4.36 g (43%); light
brown oil.
b. Prepared following General Procedure 2 from 1.65g (6.23 mmol)
Grundmann’s ketone (1) and tris(hydroxymethyl)nitromethane.
Yield: 854 mg (34%).
Yield: 72 mg (57%); colorless oil; 1H NMR (400 MHz, MeOH-d4): d
(ppm) ¼ 3.94–3.87 (m, 4H, 4-H, 5-H), 1.90–1.78 (m, 1H, 3a0-H), 1.71–
1.10 (m, 19H, 10-H, 20-H, 30-H, 50-H, 60-H, 70-H, 100-H, 200-H, 300-H, 400-H,
500-H), 0.98 (s, 3H, 7a0-CH3), 0.86 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C
NMR (100 MHz, MeOH-d4): d (ppm) ¼ 111.38 (spiro-C), 64.58 (C-4),
63.91 (C-5), 54.99 (C-10), 51.42 (C-3a0), 45.10 (C-7a0), 39.49 (C-50),
35.88 (C-60), 34.96 (C-70), 34.49 (C-100), 32.17 (C-500), 28.01 (C-20), 27.13
(C-30), 24.91 (C-300), 24.36 (C-200), 22.99 (C-600), 22.79 (C-7a0-CH3), 22.56
(500-CH3), 19.66 (100-CH3), 19.38 (C-400); MS (CI): m/z (%) ¼ 309 (100,
Mþþ1), 265 (16); IR (CH2Cl2, film) n (cmꢀ1): 2952, 2871, 1467, 1381,
1H NMR (400 MHz, CD2Cl2): d (ppm) ¼ 4.41 (d, 1H, J ¼ 12.9 Hz)
and 4.38 (d, 1H, J ¼ 12.8 Hz) (4-Heq, 6-Heq), 4.03 (d, 1H, J ¼ 12.8 Hz)
and 3.98 (d, 1H, J ¼ 13.0 Hz) (4-Hax, 6-Hax), 3.95 (s, 2 H, 1000-H), 2.19
(s, 1H, 1000-OH), 1.99–1.96 (m, 1H, 3a0-H), 1.88–1.78 (m, 2H, 50-H),
1.59–1.49 (m, 9H, 10-H, 20-H, 30-H, 60-H, 70-H), 1.34–1.29 (m, 3H, 100-
H, 200-H), 1.24–1.12 (m, 5H, 300-H, 400-H, 500-H), 0.97 (s, 3H, 7a0-CH3),
0.86–0.84 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C NMR (100 MHz,
CD2Cl2): d (ppm) ¼ 101.89 (spiro-C), 86.79 (C-5), 63.73 (C-1000), 60.85
(C-6), 60.55 (C-4), 53.63 (C-10), 51.59 (C-3a0), 44.54 (C-7a0), 39.40 (C-
50), 36.43 (C-60), 34.38 (C-70), 34.21 (C-100), 28.07 (C-500), 27.69 (C-20)
26.06 (C-30), 25.05 (C-300), 24.89 (C-200), 22.53 (C-600), 22.38 (7a0-CH3),
22.10 (500-CH3), 19.57 (100-CH3), 18.65 (C-400); MS (CI): m/z (%) ¼ 398
(14, Mþþ1), 247 (100); IR (CH2Cl2, film) n (cmꢀ1): 2956, 1710, 1550,
20
1336, 1146, 1057, 928; ½aꢂD ¼ 36.5 (c ¼ 0.014, CH2Cl2); HRMS:
calcd. for C20H36O2: 308.2715. Found: 308.2719.
(10R,3a0R,7a0R,100R)-7a0-Methyl-5-methylene-10-(1,5-
dimethylhexyl)-spiro[1,3-dioxane-2,40-octahydroindene] (9)
Prepared following General Procedure 1 from 250 mg (0.950 mmol)
Grundmann’s ketone (1) and 2-methylene-1,3-propandiol. Eluent:
isohexane/ethyl acetate 96:4.
Yield: 117 mg (37%); colorless oil; 1H NMR (400 MHz, CD2Cl2):
d (ppm): 4.82 (2d, 2H, J ¼ 2.4, 2.4 Hz, 5-CH2), 4.32–4.24 (m, 4H, 4-H,
6-H), 2.13 (t, 1H, J ¼ 8.9 Hz, 3a0-H), 1.83–1.33 (m, 15H, 10-H, 20-H, 30-
H, 50-H, 70-H, 100-H, 300-H, 400-H, 500-H), 1.18 (m, 4H, 60-H, 200-H), 1.04 (s,
3H, 7a0-CH3), 0.88–0.84 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C NMR
20
1469, 1348, 1262, 1216, 1115, 1062, 758; ½aꢂD ¼ 38.7 (c ¼ 0.012,
CH2Cl2); HRMS: calcd. for C22H39NO5: 397.2828. Found: 397.2825.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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116
M. Krojer et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 108–122
(10R,3a0R,7a0R,100R)-5-Amino-5-hydroxymethyl-7a0-
methyl-10-(1,5-dimethylhexyl)-spiro[1,3-dioxane-2,40-
octahydroindene] (14)
To freshly prepared Raney nickel (prepared from 2.5 g Ni–Al alloy,
3.1 g sodium hydroxide and 12 mL water at 70°C for 2 h, then
washed with 3 ꢁ 40 mL water and with 4 ꢁ 50 mL ethanol) a
solution of 750 mg (1.89 mmol) nitro compound 13 in 15 mL EtOH
is added with stirring, followed by 5.2 mL (103 mmol) hydrazine
hydrate. The suspension is stirred at room temperature for 16 h
and then filtered through celite. The filtrate is evaporated and the
residue purified by FCC (isohexane/ethyl acetate/triethylamine
6:4:0.1).
300-H, 400-H, 500-H), 0.94 (s, 3H, 7a0-CH3), 0.86–0.84 (m, 9H, 100-CH3, 600-
H, 500-CH3). 13C NMR (100 MHz, CD2Cl2): d (ppm) ¼ 101.93 (spiro-C),
87.16 (C-5), 62.79 (C-6), 62.63 (C-4), 62.38 (C-1000), 53.01 (C-10), 52.99
(C-3a0), 47.89 (N(CH3)2), 45.07 (C-7a0), 39.89 (C-50), 36.76 (C-60), 35.23
(C-70), 34.99 (C-100), 34.90 (C-500), 28.57 (C-20), 27.25 (C-30), 25.44 (C-
200), 24.75 (C-300), 23.12 (7a0-CH3), 23.05 (100-CH3), 22.86 (C-600), 20.02
(500-CH3), 19.07 (C-400); MS (CI): m/z (%) ¼ 495 (12, Mþþ1), 128 (100);
IR (CH2Cl2, film) n (cmꢀ1): 2954, 1549, 1457, 1373, 1332, 1143,
20
1080, 758; ½aꢂD ¼ 40.1 (c ¼ 0.011, CH2Cl2); HRMS: calcd. for
C24H44N2O4: 424.3301. Found: 424.3299.
(10R,3a0R,7a0R,100R)-7a0-Methyl-5-[N-(N,N-
dimethylaminoeth-2-yl-aminomethyl)-10-(1,5-
dimethylhexyl)-5-nitrospiro[1,3-dioxane-2,40-
Yield: 170 mg (25%); brown oil; 1H NMR (400 MHz, CD2Cl2):
d (ppm): 4.42 (d, 1H, J ¼ 12.9 Hz) and 4.40 (d, 1H, J ¼ 12.8 Hz)
(4-Heq, 6-Heq), 4.04 (d, 1H, J ¼ 13.0 Hz) and 3.99 (d, 1H, J ¼ 12.9 Hz)
(4-Hax, 6-Hax), 3.95 (s, 2H, 1000-H), 1.98 (t, 1H J ¼ 8.1 Hz, 3a0-H), 1.83
(m, 1H, 30-H), 1.63–1.08 (m, 18H, 10-H, 20-H, 30-H, 60-H, 70-H, 100-H, 200-
H, 300-H, 400-H, 500-H), 0.99 (s, 3H, 7a0-CH3), 0.88–0.84 (m, 9H, 100-CH3,
600-H, 500-CH3). 13C NMR (100 MHz, CD2Cl2): d (ppm) ¼ 102.40 (spiro-
C), 87.31 (C-5), 64.20 (C-1000), 61.35 (C-6), 61.05 (C-4), 54.09 (C-10),
52.16 (C-3a0), 45.04 (C-7a0), 39.90 (C-50), 36.93 (C-60), 34.90 (C-70),
34.72 (C-100), 28.56 (C-500), 28.16 (C-20), 26.60 (C-30), 25.54 (C-300),
25.37 (C-200), 23.04 (C-600), 22.89 (7a0-CH3), 22.63 (500-CH3), 20.07
(100-CH3), 19.14 (C-400); MS (CI): m/z (%) ¼ 368 (60, Mþþ1), 247
(100); IR (CH2Cl2, film) n (cmꢀ1): 2954, 1546, 1466, 1366, 1339,
octahydroindene] (16c)
Prepared following General Procedure 3 from 700 mg (1.76 mmol)
13 and N,N-dimethylethylenediamine (15c). Eluent: dichloro-
methane/methanol 95:5.
Yield: 175 mg (21%); yellow oil; 1H NMR (400 MHz, CD2Cl2): d
(ppm) ¼ 4.41 (d, 1H, J ¼ 12.3 Hz) and 4.33 (d, 1H, J ¼ 12.6 Hz) (4-Heq
,
,
6-Heq), 4.02 (d, 1H, J ¼ 12.9 Hz) and 3.98 (d, 1H, J ¼ 12.8 Hz) (4-Hax
6-Hax), 3.06 (s, 2H, 1000-H), 2.61 (t, 2H, J ¼ 5.8 Hz, N(CH3)2-CH2-CH2),
2.31 (t, 2H, J ¼ 5.8 Hz, N(CH3)2-CH2), 2.21 (d, 1H, J ¼ 9.0 Hz, 3a0-H),
2.18 (s, 6H, N(CH3)2), 1.86–1.69 (m, 2H, 50-H), 1.67–1.06 (m, 17H, 10-
H, 20-H, 30-H, 60-H, 70-H, 100-H, 200-H, 300-H, 400-H, 500-H), 1.03 (s, 3H, 7a0-
CH3), 0.89–0.84 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C NMR (100 MHz,
CD2Cl2): d (ppm) ¼ 101.49 (spiro-C), 86.63 (C-5), 61.86 (C-6), 61.80
(C-4), 58.75 (N(CH3)2-CH2), 55.10 (C-10), 52.65 (C-1000), 48.46 (C-3a0),
47.89 (N(CH3)2-CH2-CH2), 45.23 (N(CH3)2), 44.45 (C-7a0), 39.54 (C-50),
36.80 (C-60), 34.00 (C-100), 33.98 (C-70), 29.54 (C-20), 28.08 (C-500), 25.87
(C-30), 25.18 (C-200, C-300), 22.60 (C-600), 22.52 (100-CH3), 21.43 (7a0-CH3),
19.69 (500-CH3), 18.68 (C-400); MS (CI): m/z (%) ¼ 468 (Mþþ1, 56), 101
(100); IR (CH2Cl2, film) n (cmꢀ1): 2954, 1547, 1467, 1366, 1338,
20
1118, 1026, 757; ½aꢂD ¼ 45.7 (c ¼ 0.012, CH2Cl2); HRMS: calcd. for
C22H41NO3: 367.3086. Found: 367.3099.
(10R,3a0R,7a0R,100R)-7a0-Methyl-5-(N-methylamino)-
methyl-10-(1,5-dimethylhexyl)-5-nitrospiro[1,3-dioxane-
2,40-octahydroindene] (16a)
Prepared following General Procedure 3 from 571 mg (1.44 mmol)
13 and 2.00 mL of an ethanolic methylamine solution (33%).
Eluent: isohexane/ethyl acetate 7:3.
20
1216, 1115, 1034, 757; ½aꢂD ¼ 40.2 (c ¼ 0.013, CH2Cl2); HRMS:
Yield: 183 mg (32%); brown oil; 1H NMR (400 MHz, CD2Cl2):
d (ppm) ¼ 4.46–4.42 (m, 2 H, 4-Heq, 6-Heq), 4.01 (d, 1H, J ¼ 13.1 Hz)
and 3.96 (d, 1H, J ¼ 13.0 Hz) (4-Hax, 6-Hax), 2.90 (s, 2H, 1000-H), 2.36 (s,
3H, N-CH3), 2.00–1.94 (m, 1H, 3a0-H), 1.80–1.75 (m, 2H, 30-H), 1.58–
1.48 (m, 8 H, 20-H, 50-H, 60-H, 70-H), 1.34–1.13 (m, 9 H, 10-H, 100-H, 200-
H, 300-H, 400-H, 500-H), 0.95 (s, 3H, 7a0-CH3), 0.87–0.85 (m, 9H, 100-CH3,
600-H, 500-CH3). 13C NMR (100 MHz, CD2Cl2): d (ppm) ¼ 102.12 (spiro-
C), 87.49 (C-5), 62.60 (C-6), 62.35 (C-4), 55.04 (C-1000), 54.27 (C-10),
53.04 (C-3a0), 45.08 (C-7a0), 39.91 (C-50), 37.50 (N-CH3), 36.76 (C-60),
35.22 (C-70), 34.92 (C-100), 28.58 (C-500), 27.22 (C-20), 27.09 (C-30), 25.45
(C-300), 24.80 (C-200), 23.14 (C-600), 23.03 (7a0-CH3), 22.87 (500-CH3),
20.02 (100-CH3), 19.07 (C-400); MS (CI): m/z (%) ¼ 411 (100, Mþþ1), 147
(40); IR (CH2Cl2, film) n (cmꢀ1): 2954, 1548, 1468, 1468, 1366, 1336,
calcd. for C26H49N3O4: 467.3723. Found: 467.3730.
(10R,3a0R,7a0R,100R)-7a0-Methyl-5-[N-(N,N-
dimethylaminoeth-2-yl)-N-methylaminomethyl-10-(1,5-
dimethylhexyl)-5-nitrospiro[1,3-dioxane-2,40-
octahydroindene] (16d)
Prepared following General Procedure 3 from 1.00 g (2.52 mmol)
13 and N,N,N0-trimethylethylenediamine (15d). Eluent: isohexane/
ethyl acetate/triethylamine 6:4:0.1.
Yield: 331 mg (27%); colorless oil; 1H NMR (400 MHz, CD2Cl2): d
(ppm): 4.32 (d, 1H, J ¼ 12.7 Hz) and 4.26 (d, 1H, J ¼ 12.7 Hz) (4-Heq
,
6-Heq), 4.03 (2d, 2H, J ¼ 12.6 Hz, 12.4 Hz, 4-Hax, 6-Hax), 2.97 (s, 2H,
1000-H), 2.54–2.43 (m, 2H, N(CH3)2-CH2-CH2), 2.28 (t, 2H, J ¼ 6.6 Hz,
N(CH3)2-CH2), 2.23 (s, 3H, N-CH3), 2.15 (s, 6H, N(CH3)2), 2.13–2.09
(m, 1H, 3a0-H), 1.82–1.76 (m, 2H, 50-H), 1.65–1.08 (m, 17H, 10-H, 20-
H, 30-H, 60-H, 70-H, 100-H, 200-H, 300-H, 400-H, 500-H), 1.01 (s, 3H, 7a0-CH3),
0.88–0.84 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C NMR (100 MHz,
CD2Cl2): d (ppm) ¼ 101.82 (spiro-C), 86.26 (C-5), 62.23 (C-1000), 61.97
(C-6), 61.72 (C-4), 58.26 (N(CH3)2-CH2), 58.01 (N(CH3)2-CH2-CH2),
54.93 (C-10), 53.94 (C-3a0), 50.02 (C-7a0), 46.16 (N-CH3), 45.05 (N-
CH3)2, 45.00 (N-CH3)2, 40.15 (C-50), 37.16 (C-60), 34.77 (C-100), 34.63
(C-70), 29.58 (C-20), 28.63 (C-500), 26.06 (C-200), 26.03 (C-300), 25.67 (C-
30), 23.14 (C-600), 23.02 (100-CH3), 22.23 (7a0-CH3), 20.17 (500-CH3),
19.12 (C-400); MS (CI): m/z (%) ¼ 482 (16, Mþþ1), 115 (100); IR
(CH2Cl2, film) n (cmꢀ1): 2953, 1546, 1464, 1366, 1337, 1117, 1030,
20
1118, 1092, 758; ½aꢂD ¼ 43.5 (c ¼ 0.011, CH2Cl2); HRMS: calcd. for
C23H42N2O4: 410.3144. Found: 410.3205.
(10R,3a0R,7a0R,100R)-7a0-Methyl-5-(N,N-
dimethylaminomethyl)-10-(1,5-dimethylhexyl)-5-
nitrospiro[1,3-dioxane-2,40-octahydroindene] (16b)
Prepared following General Procedure 3 from 1.00 g (2.52 mmol)
13, 0.300 g (3.73 mmol) dimethylamine hydrochloride, and 0.5 mL
triethylamine. Eluent: isohexane/ethyl acetate 8:2.
Yield: 245 mg (23%); yellow oil; 1H NMR (400 MHz, CD2Cl2): d
(ppm): 4.40 (2d, 2H, J ¼ 13.0 Hz, 12.9 Hz, 4-Heq, 6-Heq), 4.00 (d, 1H,
J ¼ 13.0 Hz) and 3.96 (d, 1H, J ¼ 12.9 Hz) (4-Hax, 6-Hax), 2.71 (s, 2H,
1000-H), 2.20 (s, 6H, N(CH3)2), 1.99–1.93 (m, 1H, 3a0-H), 1.77–1.75 (m,
2H, 50-H), 1.56–1.12 (m, 17H, 10-H, 20-H, 30-H, 60-H, 70-H, 100-H, 200-H,
20
756; ½aꢂD ¼ 27.8 (c ¼ 0.016, CH2Cl2); HRMS: calcd. for C27H51N3O4:
481.3880. Found: 481.3863.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 108–122
Aminomethyl Spiroacetals as Inhibitors of D8,7-Isomerase
117
(10R,3a0R,7a0R,100R)-7a0-Methyl-10-(1,5-dimethylhexyl)-5-
(4-methylpiperazin-1-yl)-methyl-5-nitrospiro[1,3-dioxane-
6-Heq), 4.02 (d, 1H, J ¼ 13.1 Hz) and 3.98 (d, 1H, J ¼ 12.8 Hz) (4-Hax
,
6-Hax), 3.05 (s, 2H, 1000-H), 2.64 (t, 2H, J ¼ 6.0 Hz, pyrrolidin-CH2),
2.48 (t, 2H, J ¼ 6.0 Hz, pyrrolidine-CH2-CH2), 2.46–2.39 (m, 4H,
pyrrolidine-2-H, pyrrolidine-5-H), 2.19 (t, 1H, J ¼ 8.8 Hz, 3a0-H),
1.87–1.76 (m, 1H, 10-H), 1.69–1.76 (m, 4H, pyrrolidine-3-H,
pyrrolidine-4-H), 1.61–1.10 (m, 18-H, 20-H, 30-H, 50-H, 60-H, 70-H,
100-H, 200-H, 300-H, 400-H, 500-H), 1.02 (s, 3H, 7a0-CH3), 0.87–0.84 (m, 9H,
100-CH3, 600-H, 500-CH3). 13C NMR (100 MHz, CD2Cl2): d (ppm)
¼ 101.52 (spiro-C), 86.69 (C-5), 61.89 (C-6), 61.79 (C-4), 55.52
(pyrrolidine-CH2), 55.09 (C-10), 54.05 (pyrrolidine-C-2), 54.04
(pyrrolidine-C-5), 52.67 (C-1000), 49.23 (pyrrolidine-CH2-CH2),
48.35 (C-3a0), 44.46 (C-7a0), 39.53 (C-50), 36.77 (C-60), 33.97 (C-100,
C-70), 29.62 (C-20), 28.09 (C-500), 28.09 (C-200), 25.86 (C-30), 25.19
(pyrrolidine-C-3), 25.15 (pyrrolidine-C-5), 23.52 (C-300), 22.56 (C-600),
22.48 (100-CH3), 21.37 (7a0-CH3), 19.65 (500-CH3), 18.67 (C-400); MS (CI):
m/z (%) ¼ 494 (32, Mþþ1), 127 (100); IR (CH2Cl2, film) n (cmꢀ1):
2,40-octahydroindene] (16e)
Prepared following General Procedure 3 from 460 mg (1.16 mmol)
13 and 1-methylpiperazine (15e). Eluent: isohexane/ethyl acetate/
triethylamine 6:4:0.1.
Yield: 215 mg (39%); yellow oil; 1H NMR (400 MHz, CD2Cl2):
d (ppm) ¼ 4.35 (d, 1H, J ¼ 10.0 Hz) and 4.28 (d, 1H, J ¼ 10.7 Hz) (4-
H
eq, 6-Heq), 4.02 (d, 1H, J ¼ 9.7 Hz) and 3.99 (d, 1H, J ¼ 9.4 Hz) (4-Hax
,
6-Hax), 2.93–2.86 (m, 2H, 1000-H), 2.49 (t, 4H, J ¼ 4.8 Hz, piperazine-2-
H, piperazine-6-H), 2.32 (s, 4H, piperazine-3-H, piperazine-5-H),
2.19 (s, 3H, N-CH3), 2.12 (t, 1H, J ¼ 8.5 Hz, 3a0-H), 1.85–1.79 (m, 2H,
50-H), 1.57–1.11 (m, 17H, 10-H, 20-H, 30-H, 60-H, 70-H, 100-H, 200-H, 300-H,
400-H, 500-H), 1.02 (s, 3H, 7a0-CH3), 0.90–0.87 (m, 9H, 100-CH3, 600-H, 500-
CH3). 13C NMR (100 MHz, CD2Cl2): d (ppm) ¼ 101.94 (spiro-C), 86.24
(C-5), 62.29 (C-6), 62.13 (C-4), 60.93 (C-1000), 55.77 (piperazine-C-3,
piperazine-C-5), 55.39 (piperazine-C-2, piperazine-C-6), 54.75 (C-10),
50.03 (C-3a0), 46.30 (N-CH3), 45.03 (C-7a0), 40.09 (C-50), 37.13 (C-60),
34.80 (C-70), 34.61 (C-100), 29.73 (C-500), 28.62 (C-20), 26.24 (C-30), 25.52
(C-200), 23.09 (C-300), 23.01 (7a0-CH3), 22.26 (100-CH3), 20.14 (C-600),
20.14 (500-CH3), 19.10 (C-400); MS (CI): m/z (%) ¼ 480 (12, Mþþ1), 113
(100); IR (CH2Cl2, film) n (cmꢀ1): 2962, 1540, 1456, 1261, 1166,
20
2956, 1547, 1466, 1366, 1348, 1215, 1140, 1117, 757; ½aꢂD ¼ 38.3
(c ¼ 0.016, CH2Cl2); HRMS: calcd. for C28H51N3O4: 493.3880.
Found: 493.3903.
(10R,3a0R,7a0R,100R)-7a0-Methyl-10-(1,5-dimethylhexyl)-5-
(2,6-dimethylmorpholin-4-yl)-methyl-5-nitrospiro[1,3-
20
dioxane-2,40-octahydroindene] (16h)
1097, 1016, 800; ½aꢂD ¼ 32.7 (c ¼ 0.015, CH2Cl2); HRMS: calcd. for
C27H49N3O4: 479.3723. Found: 479.3725.
Prepared following General Procedure 3 from 1.00 g (2.52 mmol)
13 and 2,6-dimethylmorpholine (15h). Eluent: isohexane/ethyl
acetate 95:5.
(10R,3a0R,7a0R,100R)-7a0-Methyl-10-(1,5-dimethylhexyl)-5-
nitro-5-{[2-(piperidin-1-yl)ethyl]aminomethyl}spiro[1,3-
Yield: 215 mg (28%); colorless oil; 1H NMR (400 MHz, CD2Cl2): d
(ppm) ¼ 4.39 (d, 2H, J ¼ 12.8 Hz), (4-Heq, 6-Heq), 4.00 (d, 1H,
J ¼ 13.1 Hz) and 3.96 (d, 1H, J ¼ 12.9 Hz) (4-Hax, 6-Hax), 3.58–3.50
(m, 2H, morpholine-2-H, morpholine-6-H), 2.76 (s, 2H, 1000-H), 2.46
(m, 2H, morpholine-3-H, morpholine-5-H), 1.98–1.94 (m, 3H, 3a0-H,
morpholine-3-H, morpholine-5-H), 1.86–1.75 (m, 2H, 50-H), 1.55–
1.13 (m, 17H, 10-H, 20-H, 30-H, 60-H, 70-H, 100-H, 200-H, 300-H, 400-H, 500-
H), 1.04 (d, 6H, J ¼ 6.3 Hz, morpholine-2-CH3, morpholine-6-CH3),
0.96 (s, 3H, 7a0-CH3), 0.87–0.85 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C
NMR (100 MHz, CD2Cl2): d (ppm) ¼ 101.44 (spiro-C), 86.17 (C-5),
71.65 (morpholine-C-2), 71.64 (morpholine-C-6), 62.10 (C-6), 61.73
(C-4), 60.92 (morpholine-C-3), 60.90 (morpholine-C-5), 60.54 (C-1000),
52.99 (C-10), 52.74 (C-3a0), 44.56 (C-7a0), 39.40 (C-50), 36.32 (C-60),
34.69 (C-70), 34.39 (C-100), 30.12 (C-500), 28.06 (C-20), 26.63 (C-30), 24.99
(C-200), 24.38 (C-300), 22.58 (7a0-CH3), 22.53 (100-CH3), 22.41 (C-600),
19.57 (500-CH3), 18.71 (morpholine-2-CH3, morpholine-6-CH3),
18.61 (C-400); MS (CI): m/z (%) ¼ 425 (100, Mþþ1), 114 (6); IR
(CH2Cl2, film) n (cmꢀ1): 3417, 2953, 1722, 1550, 1466, 1366, 1336,
dioxane-2,40-octahydroindene] (16f)
Prepared following General Procedure 3 from 500 mg (1.26 mmol)
13 and 1-(2-aminoethyl)piperidine (15f). Eluent: isohexane/ethyl
acetate 7:3.
Yield: 136 mg (21%); yellow oil; 1H NMR (400 MHz, CD2Cl2): d
(ppm) ¼ 4.41 (d, 1H, J ¼ 12.6 Hz) and 4.34 (d, 1H, J ¼ 12.7 Hz) (4-Heq
,
,
6-Heq), 4.02 (d, 1H, J ¼ 12.6 Hz) and 3.99 (d, 1H, J ¼ 12.4 Hz) (4-Hax
6-Hax), 3.28 (s, 2H, piperidine-2-H, piperidine-6-H), 3.13 (d, 2H,
J ¼ 4.3 Hz, 1000-H), 2.85 (t, 2H, J ¼ 6.5 Hz, piperidine-CH2), 2.63 (t, 2H,
J ¼ 6.9 Hz, piperidine-CH2-CH2), 2.27 (s, 2H, piperidine-2-H, piperi-
dine-6-H), 2.20 (dd, 1H, J ¼ 4.8 Hz, 8.3 Hz, 3a0-H), 1.84–1.80 (m, 2 H,
50), 1.61–1.15 (m, 26H, 10-H, 20-H, 30-H, 60-H, 70-H, 100-H, 200-H, 300-H,
400-H, 500-H, piperidine-3-H, piperidine-4-H, piperidine-5-H), 1.02 (s,
3H, 7a0-CH3), 0.88–0.86 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C NMR
(100 MHz, CD2Cl2): d (ppm) ¼ 102.06 (spiro-C), 87.24 (C-5), 62.43 (C-
6), 62.32 (C-4), 58.64 (C-1000), 55.63 (piperidine-CH2), 55.14 (C-10),
53.17 (piperidine-CH2CH2), 48.85 (C-3a0), 47.73 (C-7a0), 44.96 (C-50),
40.03 (C-60), 37.28 (C-100), 34.50 (C-70), 34.47 (C-500), 30.14 (C-20), 28.60
(piperidine-C-2), 28.58 (piperidine-C-6), 26.62 (C-30), 26.38 (piperi-
dine-C-3), 26.21 (piperidine-C-5), 25.69 (piperidin-C-4), 25.68 (C-200),
25.01 (C-300), 24.28 (7a0-CH3), 23.05 (100-CH3), 22.96 (C-600), 21.88 (500-
CH3), 19.18 (C-400); MS (CI): m/z (%) ¼ 508 (22, Mþþ1), 141 (100); IR
(CH2Cl2, film) n (cmꢀ1): 2933, 1547, 1466, 1366, 1348, 1117, 1041,
20
1261, 1112, 1047, 802; ½aꢂD ¼ 37.9 (c ¼ 0.011, CH2Cl2); HRMS:
calcd. for C28H50N2O5: 494.3720. Found: 494.3726.
(10R,3a0R,7a0R,100R)-5-(N-Benzylpiperidin-4-yl)-
aminomethyl-7a0-methyl-10-(1,5-dimethylhexyl)-5-
nitrospiro[1,3-dioxane-2,40-octahydroindene] (16i)
Prepared following General Procedure 3 from 500 mg (1.26 mmol)
13 and 4-amino-1-benzylpiperidine (15i). Eluent: isohexane/ethyl
acetate 7:3.
20
756; ½aꢂD ¼ 30.4 (c ¼ 0.007, CH2Cl2); HRMS: calcd. for C29H53N3O4:
508.4114. Found: 508.4112.
(10R,3a0R,7a0R,100R)-7a0-Methyl-10-(1,5-dimethylhexyl)-5-
nitro-5-{[2-(pyrrolidin-1-yl)ethyl]aminomethyl}spiro[1,3-
Yield: 190 mg (26%); yellow oil; 1H NMR (400 MHz, CD2Cl2): d
(ppm) ¼ 7.31–7.29 (m, 4H, phenyl-2-H, phenyl-3-H, phenyl-5-H,
phenyl-6-H), 7.26–7.21 (m, 1H, phenyl-4-H), 4.40 (d, 1H, J ¼ 10.1 Hz)
and 4.32 (d, 1H, J ¼ 10.0 Hz) (4-Heq, 6-Heq), 4.03 (d, 1H, J ¼ 10.2 Hz)
and 3.99 (d, 1H, J ¼ 10.0 Hz) (4-Hax, 6-Hax), 3.46 (s, 2H, 1000-H), 3.10
(s, 2 H, phenyl-CH2), 2.79 (d, 2H, J ¼ 9.3 Hz, piperidine-2-H,
piperidine-6-H), 2.40–2.35 (m, 1H, piperidine-4-H), 2.17 (t, 1H,
J ¼ 7.0Hz, 3a0-H), 1.99 (t, 2H, J ¼ 8.7, piperidine-2-H, piperidine-6-H),
dioxane-2,40-octahydroindene] (16g)
Prepared following General Procedure 3 from 854 mg (2.15 mmol)
13 and 1-(2-aminoethyl)pyrrolidine (15g). Eluent: isohexane/ethyl
acetate/triethylamine 6:4:0.1.
Yield: 253 mg (26%); yellow oil; 1H NMR (400 MHz, CD2Cl2): d
(ppm) ¼ 4.41 (d, 1H, J ¼ 12.7 Hz) and 4.33 (d, 1H, J ¼ 12.6 Hz) (4-Heq
,
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www.archpharm.com

118
M. Krojer et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 108–122
1.67–1.52 (m, 4H, piperidine-3-H, piperidine-5H), 1.64–1.12
(m, 17H, 10-H, 20-H, 30-H, 60-H, 70-H, 100-H, 200-H, 300-H, 400-H, 500-H),
1.04 (s, 3H, 7a0-CH3), 0.91–0.87 (m, 9H, 100-CH3, 600-H, 500-CH3).
13C NMR (100MHz, CD2Cl2): d (ppm) ¼ 139.36 (phenyl-C-1), 129.59
(phenyl-C-2, phenyl-C-6), 128.64 (phenyl-C-3, phenyl-C-5), 127.41
(phenyl-C-4), 102.06 (spiro-C), 87.11 (C-5), 63.43 (C-1000), 62.31 (C-6),
62.23 (C-4), 55.95 (piperidine-C-4), 55.35 (C-10), 52.77 (piperidine-C-2,
piperidine-C-6), 49.99 (phenyl-CH2), 49.25 (C-3a0), 45.01 (C-7a0), 40.08
(C-50), 37.17 (C-60), 34.49 (C-100), 32.51 (piperidine-C-5), 30.28 (C-70),
29.94 (piperidine-C-3), 29.56 (C-500), 28.56 (C-20), 26.24 (C-30), 23.08
(C-200), 22.89 (C-300), 20.12 (7a0-CH3), 19.16 (100-CH3), 17.88 (C-600), 17.86
(500-CH3), 15.06 (C-400); MS (CI): m/z (%) ¼ 570 (16, Mþþ1), 203 (100); IR
(CH2Cl2, film) n (cmꢀ1): 2952, 1546, 1467, 1366, 1340, 1113, 1026,
(s, 3H, 7a0-CH3), 0.88–0.85 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C NMR
(100 MHz, CD2Cl2): d (ppm) ¼ 131.10 (q, J ¼ 31.6 Hz, phenyl-C-3),
125.86 (phenyl-C-6), 124.51 (q, J ¼ 267 Hz, CF3), 123.15 (phenyl-C-
5), 118.85 (q, J ¼ 1.2 Hz, phenyl-C-2), 115.61 (phenyl-C-1), 112.03 (q,
J ¼ 3.6 Hz, phenyl-C-4), 101.53 (spiro-C), 86.15 (C-5), 62.08 (C-6),
61.74 (C-4), 60.27 (C-1000), 54.62 (piperazine-C-3, piperazine-
C-5), 53.33 (C-10), 53.23 (C-3a0), 48.83 (piperazine-C-2, piperazine-
C-6), 44.57 (C-7a0), 39.39 0(C-50), 36.33 (C-60), 34.64 (C-70), 34.36 (C-100),
28.07 (C-500), 26.53 (C-2 ), 24.99 (C-30), 24.93 (C-200), 24.45 (C-300),
22.55 (7a0-CH3), 22.44 (100-CH3), 22.38 (C-600), 19.54 (500-CH3), 18.62
(C-400); MS (CI): m/z (%) ¼ 610 (52, Mþþ1), 247 (100); IR (CH2Cl2, film)
n (cmꢀ1): 2954, 1708, 1610, 1548, 1450, 1319, 1237, 1164, 1124,
20
949, 757; ½aꢂD ¼ 34.5 (c ¼ 0.012, CH2Cl2); HRMS: calcd. for
20
757, 698; ½aꢂD ¼ 36.0 (c ¼ 0.012, CH2Cl2); HRMS: calcd. for
C33H50F3N3O4: 609.3753. Found: 609.3765.
C34H55N3O4: 569.4193. Found: 569.4200.
(10R,3a0R,7a0R,100R)-7a0-Methyl-10-(1,5-dimethylhexyl)-5-
nitro-5-(3-pyridinyl)methylaminomethyl-spiro[1,3-dioxane-
(10R,3a0R,7a0R,100R)-5-(N-Benzylpiperidin-4-yl)-N-
2,40-octahydroindene] (16l)
methylaminomethyl-7a0-methyl-10-(1,5-dimethylhexyl)-5-
nitrospiro[1,3-dioxane-2,40-octahydroindene] (16j)
Prepared following General Procedure 3 from 1.00 g (2.52 mmol)
13 and 1-benzyl-4-(N-methylamino)piperidine (15j). Eluent: iso-
hexane/ethyl acetate/triethylamine 8:2:0.1.
Prepared following General Procedure 3 from 460 mg (1.16 mmol)
13 and 1.06 g (9.82 mmol, 8.5 equiv.) 3-(aminomethyl)pyridine
(15l). Eluent: isohexane/ethyl acetate/triethylamine 6:4:0.1.
Yield: 292 mg (52%); brown oil; 1H NMR (400 MHz, CD2Cl2):
d (ppm) ¼ 8.47–8.46 (m, 2H, pyridine-2-H, pyridine-6-H), 7.62
(d, 1H, J ¼ 7.8 Hz, pyridine-4-H), 7.24 (dd, 1H, J ¼ 4.8 Hz, 7.8 Hz,
pyridine-5-H), 4.41 (d, 1H, J ¼ 12.7 Hz) and 4.33 (d, 1H, J ¼ 12.8 Hz)
(4-Heq, 6-Heq), 4.00 (d, 1H, J ¼ 12.7 Hz) and 3.9 (d, 1H, J ¼ 12.8 Hz)
(4-Hax, 6-Hax), 3.77 (s, 2H, pyridine-CH2), 3.07 (s, 2 H, 1000-H), 2.08
(t, 1H, J ¼ 8.7 Hz, 3a0-H), 1.77–1.72 (m, 2H, 50-H), 1.53–1.12 (m, 17
H, 10-H, 20-H, 30-H, 60-H, 70-H, 100-H, 200-H, 300-H, 400-H, 500-H), 1.00
(s, 3H, 7a0-CH3), 0.87–0.84 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C NMR
(100 MHz, CD2Cl2): d (ppm) ¼ 150.17 (pyridine-C-2), 149.29 (pyri-
dine-C-6), 136.06 (pyridine-C-5), 135.43 (pyridin-C-3), 123.89
(pyridine-C-4), 102.20 (spiro-C), 86.77 (C-5), 62.31 (C-6), 62.22
(C-4), 55.30 (C-10), 51.82 (C-1000), 51.81 (pyridine-CH2), 49.65 (C-3a0),
44.88 (C-7a0), 39.99 (C-50), 37.20 (C-60), 34.62 (C-70), 34.46 (C-100),
29.56 (C-20), 28.56 (C-500), 26.20 (C-30), 25.98 (C-200), 25.65 (C-300),
23.05 (C-600), 22.94 (7a0-CH3), 22.08 (500-CH3), 20.11 (100-CH3), 19.08
(C-400); MS (CI): m/z (%) ¼ 488 (66, Mþþ1), 121 (100); IR (CH2Cl2,
film) n (cmꢀ1): 2954, 1546, 1466, 1366, 1339, 1118, 1026, 757;
Yield: 248 mg (26%); yellow oil; 1H NMR (400 MHz, CD2Cl2):
d (ppm) ¼ 7.31–7.28 (m, 4H, phenyl-2-H, phenyl-3-H, phenyl-5-H,
phenyl-6-H), 7.25–7.21 (m, 1H, phenyl-4-H), 4.41 (d, 2H, J ¼ 12.8 Hz,
4-Heq, 6-Heq), 4.01 (d, 1H, J ¼ 13.1 Hz) and 3.97 (d, 1H, J ¼ 12.9 Hz)
(4-Hax, 6-Hax), 3.44 (s, 2H, 1000-H), 2.89 (d, 2H, J ¼ 11.5 Hz, piperidine-
2-H, piperidine-6-H), 2.85 (s, 2H, 2 H, phenyl-CH2), 2.21 (s, 3H, N-
CH3), 2.19 (t, 1H, J ¼ 7.0 Hz, 3a0-H), 2.03–1.96 (m, 1H, piperidine-4-
H), 1.89 (t, 2H, J ¼ 10.8 Hz, piperidine-2-H, piperidine-6-H), 1.79–
1.78 (m, 2H, 30-H), 1.62–1.12 (m, 23H, piperidine-3-H, piperidine-
5H, 10-H, 20-H, 60-H, 70-H, 100-H, 200-H, 300-H, 400-H, 500-H), 0.96 (s, 3H,
7a0-CH3), 0.88–0.86 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C NMR
(100 MHz, CD2Cl2): d (ppm) ¼ 139.43 (phenyl-C-1), 129.55 (phenyl-
C-2, phenyl-C-6), 128.66 (phenyl-C-3, phenyl-C-5), 127.43 (phenyl-C-
4), 101.80 (spiro-C), 87.41 (C-5), 63.81 (C-1000), 63.37 (N-CH3), 62.62
(C-6), 62.30 (C-4), 58.25 (piperidine-C-4), 53.79 (C-10), 52.90
(piperidine-C-2, piperidine-C-6), 45.08 (phenyl-CH2), 40.30 (C-3a0),
39.91 (C-7a0), 36.79 (C-50), 35.30 (C-60), 34.96 (C-100), 28.58
(piperidine-C-5), 28.44 (C-70), 28.40 (piperidine-C-3), 28.39 (C-500),
27.35 (C-20), 27.22 (C-30), 25.46 (C-200), 24.71 (C-300), 23.22 (7a0-CH3),
23.10 (100-CH3), 22.91 (C-600), 20.06 (500-CH3), 19.10 (C-400); MS (CI): m/z
(%) ¼ 584 (42, Mþþ1), 217 (100); IR (CH2Cl2, film) n (cmꢀ1): 2952,
20
½aꢂD ¼ 50.6 (c ¼ 0.009, CH2Cl2); HRMS: calcd. for C28H45N3O4:
487.3410. Found: 487.3401.
(10R,3a0R,7a0R,100R)-7a0-Methyl-10-(1,5-dimethylhexyl)-5-
nitro-5-[N-methyl-N-((E)-phenylprop-1-en-3-yl)-
20
1548, 1455, 1366, 1338, 1117, 1050, 757; ½aꢂD ¼ 32.0 (c ¼ 0.015,
CH2Cl2); HRMS: calcd. for C35H57N3O4: 583.4349. Found: 583.4344.
aminomethyl]spiro[1,3-dioxane-2,40-octahydroindene]
(16m)
(10R,3a0R,7a0R,100R)-7a0-Methyl-10-(1,5-dimethylhexyl)-5-
Prepared following General Procedure 3 from 770 mg (1.94 mmol)
13 and (E)-N-methyl-(phenylprop-1-en-3-yl)amine (15m). Eluent:
isohexane/ethyl acetate 9:1.
nitro-5-[4-(3-trifluoromethylphenyl)piperazin-1-ylmethyl]-
spiro[1,3-dioxane-2,40-octahydroindene] (16k)
Prepared following General Procedure 3 from 260 mg (0.87 mmol)
13 and 1-(3-trifluoromethylphenyl)piperazine (15k). Eluent: iso-
hexane/ethyl acetate 9:1.
Yield: 295 mg (29%); colorless oil; 1H NMR (400 MHz, CD2Cl2):
d (ppm) ¼ 7.38 (d, 2H, J ¼ 7.2 Hz, phenyl-2-H, phenyl-6-H), 7.31
(t, 2H, J ¼ 7.5 Hz, phenyl-3-H, phenyl-5-H), 7.23 (t, 1H, J ¼ 7.2 Hz,
phenyl-4-H), 6.48 (d, 1H, J ¼ 15.9 Hz, phenyl-CH), 6.16 (dt, 1H,
Yield: 112 mg (21%); yellow oil; 1H NMR (400 MHz, CD2Cl2):
d (ppm) ¼ 7.34 (t, 1H, J ¼ 7.8 Hz, phenyl-5-H), 7.07–7.03 (m, 3 H,
–
J ¼ 15.8 Hz, 6.7 Hz, phenyl-CH CH), 4.38 (d, 1H, J ¼ 12.7 Hz) and
–
phenyl-2-H, phenyl-4-H, phenyl-6-H), 4.43 (d, 2H, J ¼ 12.9 Hz, 4-Heq
6-Heq), 4.04 (d, 1H, J ¼ 13.0 Hz) and 4.01 (d, 1H, J ¼ 12.8 Hz) (4-Hax
,
,
4.32 (d, 1H, J ¼ 13.6 Hz) (4-Heq, 6-Heq), 4.04 (d, 1H, J ¼ 12.5 Hz) and
–
4.01 (d, 1H, J ¼ 12.4 Hz) (4-Hax, 6-Hax), 3.18 (d, 2H, J ¼ 6.7 Hz, CH-
–
6-Hax), 3.17–3.14 (m, 4 H, piperazine-2-H, piperazine-6-H),3.05
(s, 2H, 1000-H), 2.34–2.32 (m, 4 H, piperazine-2-H, piperazine-3-H,
piperazine-6-H), 1.97 (t, 1H, J ¼ 7.2 Hz, 3a0-H), 1.56–1.13 (m, 19 H,
10-H, 20-H, 30-H, 50-H, 60-H, 70-H, 100-H, 200-H, 300-H, 400-H, 500-H), 0.97
CH2), 2.98 (d, 2H, J ¼ 4.2 Hz, 1000-H), 2.30 (s, 3H, N-CH3), 2.12 (t, 1H,
J ¼ 8.5 Hz, 3a0-H), 1.84–1.75 (m, 2H, 50-H), 1.60–1.15 (m, 17H, 10-H,
20-H, 30-H, 60-H, 70-H, 100-H, 200-H, 300-H, 400-H, 500-H), 1.00 (s, 3H, 7a0-
CH3), 0.87–0.85 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C NMR (100 MHz,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2014, 347, 108–122
Aminomethyl Spiroacetals as Inhibitors of D8,7-Isomerase
119
CD2Cl2): d (ppm) ¼ 137.37 (phenyl-C-1), 133.57 (phenyl-CH), 129.10
20-H, 30-H, 60-H, 70-H, 100-H, 200-H, 300-H, 400-H, 500-H), 1.25 (s, 3H, 7a0-
CH3), 0.90–0.86 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C NMR (100 MHz,
–
–
(phenyl-C-3, phenyl-C-5), 128.12 (phenyl-C-4), 127.04 (phenyl-CH
–
CH), 126.88 (phenyl-C-2, phenyl-C-6), 101.86 (spiro-C), 86.37 (C-5),
CD Cl ): d (ppm) ¼ 164.25 (C O), 154.22 (pyridine-C-6), 150.92
–
2
2
62.33 (C-6), 62.25 (C-4), 62.10 ( CH–CH ), 60.18 (C-1000), 54.84 (C-10),
(pyridine-C-2), 137.05 (pyridine-C-5), 124.78 (pyridine-C-3), 123.52
(pyridine-C-4), 102.20 (spiro-C), 84.41 (C-5), 64.33 (C-1000), 60.97 (C-6),
60.70 (C-4), 53.56 (C-10), 51.58 (C-3a0), 44.57 (C-7a0), 39.39 (C-50),
36.38 (C-60), 34.42 (C-70), 34.22 (C-100), 30.07 (C-500), 28.07 (C-20), 27.68
(C-30), 26.12 (C-200), 25.04 (C-300), 24.87 (7a0-CH3), 22.53 (100-CH3),
22.37 (C-600), 22.12 (500-CH3), 19.55 (C-400); MS (CI): m/z (%) ¼ 503 (100,
Mþþ1), 307 (26), 247 (26); IR (CH2Cl2, film) n (cmꢀ1) ¼ 3319, 2954,
1737, 1591, 1555, 1468, 1367, 1347, 1276, 1110, 1025, 738;
–
–
2
49.63 (C-3a0), 44.95 (N-CH3), 44.79 (C-7a0), 40.09 (C-50), 37.09 (C-60),
34.76 (C-70), 34.62 (C-100), 29.84 (C-20), 28.56 (C-500), 26.19 (C-30), 26.00
(C-200), 25.55 (C-300), 23.04 (100-CH3), 22.98 (C-600), 22.16 (7a0-CH3),
20.09 (500-CH3), 19.07(C-400); MS (CI): m/z (%) ¼ 527 (10, Mþþ1), 160
(100); IR (CH2Cl2, film) n (cmꢀ1): 2954, 1681, 1548, 1538, 1455,
20
1337, 1117, 1026, 968, 757; ½aꢂD ¼ 44.3 (c ¼ 0.007, CH2Cl2); HRMS:
calcd. for C32H50N2O4: 526.3771. Found: 526.3780.
20
½aꢂD ¼ 35.3 (c ¼ 0.012, CH2Cl2); HRMS: calcd. for C28H42N2O6:
502.3043. Found: 502.3045.
(10R,3a0R,7a0R,100R)-5-Benzoyloxymethyl-7a0-methyl-10-
(1,5-dimethylhexyl)-5-nitrospiro[1,3-dioxane-2,40-
octahydroindene] (17a)
A solution of 453 mg (1.14 mmol) 13 in 8 mL pyridine is cooled to
0°C and 183 mg (1.30 mmol) benzoyl chloride is added in small
portions with stirring. The mixture is stirred at 0°C for 30 min
and at room temperature for 1 h, then sodium carbonate solution
(10%; 50 mL) is added, followed by extraction with dichloro-
methane (3 ꢁ 50 mL). The combined organic layers are dried over
magnesium sulfate and evaporated. The residue is purified by FCC
(isohexane/ethyl acetate 9:1).
(10R,3a0R,7a0R,100R)-7a0-Methyl-10-(1,5-dimethylhexyl)-5-
(morpholin-4-yl-carbonyloxymethyl)-5-nitrospiro[1,3-
dioxane-2,40-octahydroindene] (17c)
A solution of 470 mg (1.18 mmol) 13 and 205 mg (1.37 mmol) 4-
morpholinocarbonyl chloride in 8 mL pyridine is stirred at room
temp. for 16 h; then sodium carbonate solution (10%; 25 mL) is
added, followed by extraction with dichloromethane (3 ꢁ 30 mL).
The combined organic layers are dried over magnesium sulfate
and evaporated. The residue is purified by FCC (isohexane/ethyl
acetate/triethylamine 6:4:0.1.
Yield: 200 mg (35%); colorless oil; 1H NMR (400 MHz, CD2Cl2): d
(ppm) ¼ 7.97 (d, 2H, J ¼ 8.0 Hz, phenyl-2-H, phenyl-6-H), 7.61 (t, 1H,
J ¼ 7.6 Hz, phenyl-4-H), 7.47 (t, 2H, J ¼ 7.7 Hz, phenyl-3-H, phenyl-5-
H), 4.65 (s, 2H, 1000-H), 4.55 (d, 1H, J ¼ 13.1 Hz) and 4.54 (d, 1H,
J ¼ 12.7 Hz) (4-Heq, 6-Heq), 4.16 (d, 1H, J ¼ 13.0 Hz) and 4.11 (d, 1H,
J ¼ 13.0 Hz) (4-Hax, 6-Hax), 1.97 (t, 2H, J ¼ 8.0 Hz, 50-H), 1.87–1.78 (m,
1H, 3a0-H), 1.63–1.15 (m, 17H, 10-H, 20-H, 30-H, 60-H, 70-H, 100-H, 200-H,
Yield: 238 mg (40%); yellow oil; 1H NMR (400 MHz, CD2Cl2): d
(ppm) ¼ 4.44 (m, 2H, 4-Heq, 6-Heq), 4.40 (s, 2H, 1000-H), 4.09–3.93 (m,
2H, 4-Hax
,
6-Hax), 3.61 (d, 4H, J ¼ 16.2 Hz, morpholine-2-H,
morpholine-6-H), 3.38 (d, 4H, J ¼ 23.9 Hz, morpholine-3-H, mor-
pholine-5-H), 1.99–1.85 (m, 2H, 50-H), 1.80 (m, 1H, 3a0-H), 1.37–1.09
(m, 17 H, 10-H, 20-H, 30-H, 60-H, 70-H, 100-H, 200-H, 300-H, 400-H, 500-H),
0.97 (s, 3H, 7a0-CH3), 0.88–0.86 (m, 9H, 100-CH3, 600-H, 500-CH3). 13C
–
300-H, 400-H, 500-H), 1.00 (s, 3H, 7a0-CH3), 0.89–0.87 (m, 9H, 100-CH3, 600-
13
H, 500-CH3). C NMR (100 MHz, CD Cl ): d (ppm) ¼ 165.84 (C O),
–
–
2
2
NMR (100 MHz, CD Cl ): d (ppm): 152.87 (C O), 101.19 (spiro-C),
–
2
2
134.25 (phenyl-C-1), 131.01 (phenyl-C-6), 130.20 (phenyl-C-2),
129.47 (phenyl-C-4), 129.31 (phenyl-C-3), 129.16 (phenyl-C-5),
102.61 (spiro-C), 85.17 (C-5), 64.61 (C-1000), 61.51 (C-6), 61.25 (C-
4), 53.79 (C-10), 52.70 (C-3a0), 45.09 (C-7a0), 39.90 (C-50), 36.85 (C-60),
35.03 (C-70), 34.79 (C-100), 28.58 (C-500), 27.88 (C-20), 26.84 (C-30), 25.53
(C-200), 25.23 (C-300), 23.07 (7a0-CH3), 22.91 (100-CH3), 22.80 (C-600),
20.08 (500-CH3), 19.12 (C-400); MS (CI): m/z (%) ¼ 502 (44, Mþþ1), 105
(100); IR (CH2Cl2, film) n (cmꢀ1): 2954, 1731, 1602, 1556, 1452,
84.23 (C-5), 65.66 (morpholine-C2), 65.46 (morpholine-C-6), 63.85
(C-1000), 60.10 (C-6), 59.83 (C-4), 52.18 (C-10), 51.99 (C-3a0), 43.76 (C-
7a0), 43.50 (morpholine-C-5), 43.27 (morpholine-C-3), 38.57 (C-60),
35.45 (C-50), 33.77 (C-70), 33.50 (C-100), 27.26 (C-500), 26.16 (C-30), 25.63
(C-20), 24.17 (C-200), 23.72 (C-300), 21.72 (100-CH3), 21.58 (C-600), 21.55
(500-CH3), 18.71 (7a0-CH3), 17.75 (C-400); MS (CI): m/z (%) ¼ 510 (42,
Mþþ1), 354 (100); IR (CH2Cl2, film) n (cmꢀ1): 3450, 2955, 1717,
20
1553, 1437, 1365, 1347, 1240, 1118, 1025, 763; ½aꢂD ¼ 44.4
20
1347, 1266, 1109, 758, 709; ½aꢂD ¼ 42.0 (c ¼ 0.016, CH2Cl2); HRMS:
(c ¼ 0.011, CH2Cl2); HRMS: calcd. for C27H46N7O6: 510.3305.
calcd. for C29H43NO6: 501.3090. Found: 501.3088.
Found: 510.3307.
(10R,3a0R,7a0R,100R)-7a0-Methyl-10-(1,5-dimethylhexyl)-5-
nitro-5-(pyridin-3-yl-carbonyloxymethyl)spiro[1,3-dioxane-
10,20,30,40-Tetrahydro-5-hydroxymethyl-5-nitrospiro[1,3-
dioxane-2,10-naphthaline] (19a)
2,40-octahydroindene] (17b)
Prepared following General Procedure 2 from 1.10 g (7.52 mmol)
1-tetralone (18a). The crude product is recrystallized from
ethanol.
A solution of 400 mg (1.01 mmol) 13 and 540 mg (3.03 mmol)
nicotinoyl chloride hydrochloride in 2 mL pyridine and 1 mL
ethanol is stirred at room temp. for 48 h; then sodium carbonate
solution (10%; 50 mL) is added, followed by extraction with
dichloromethane (3 ꢁ 50 mL). The combined organic layers are
dried over magnesium sulfate and evaporated. The residue is
purified by FCC (isohexane/ethyl acetate/triethylamine 6:4:0.1.
Yield: 205 mg (41%); pale yellow oil; 1H NMR (400 MHz, CD2Cl2):
d (ppm) ¼ 9.12 (s, 1H, pyridine-2-H), 8.79 (d, 1H, J ¼ 6.8 Hz,
pyridine-6-H), 8.21 (d, 1H, J ¼ 8.0 Hz, pyridine-4-H), 7.41 (dd, 1H,
J ¼ 8.0 Hz, 7.0 Hz, pyridine-5-H), 4.70 (s, 2H, 1000-H), 4.54 (d, 1H,
J ¼ 13.0 Hz) and 4.51 (d, 1H, J ¼ 12.8 Hz) (4-Heq, 6-Heq), 4.15 (d, 1H,
J ¼ 13.1 Hz) and 4.10 (d, 1H, J ¼ 12.9 Hz) (4-Hax, 6-Hax), 2.00 (t, 1H,
J ¼ 7.9 Hz, 3a0-H), 1.95–1.76 (m, 2H, 50-H), 1.65–1.11 (m, 17H, 10-H,
Yield: 1.61 g (77%); light brown powder; m.p.: 133.1°C; 1H NMR
(400 MHz, CDCl3): d (ppm) ¼ 7.56 (d, 1H, J ¼ 7.0 Hz, 80-H), 7.25–7.18
(m, 2H, 50-H, 60-H), 7.08 (d, 1H, J ¼ 7.0 Hz, 50-H), 4.71 (d, 2H,
J ¼ 13.4 Hz, 4-Heq, 6-Heq), 4.19 (d, 2H, J ¼ 13.3 Hz, 4-Hax, 6-Hax), 3.92
(s, 2H, 100-H), 2.81 (t, 2H, J ¼ 6.4 Hz, 40-H), 2.27–2.21 (m, 2H, 20-H),
1.99–1.83 (m, 2H, 30-H). 13C NMR (100 MHz, CDCl3): d (ppm): 136.49
(C-60), 135.65 (C-4a0), 128.15 (C-8a0), 127.72 (C-70), 125.80 (C-80),
125.70 (C-50), 97.47 (spiro-C), 86.93 (C-5), 63.26 (C-4, C-6), 61.37 (C-
100), 28.36 (C-40), 26.26 (C-20), 18.81 (C-30); MS (CI): m/z (%) ¼ 280 (100,
Mþþ1), 103 (26); IR (CH2Cl2, film) n (cmꢀ1): 2936, 1549, 1458, 1327,
1130, 1058, 906, 763; HRMS: calcd. for C14H17NO5: 279.1107.
Found: 279.1112.
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120
M. Krojer et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 108–122
10,20,30,40-Tetrahydro-5-hydroxymethyl-60-methoxy-5-
4.72–4.68 (m, 2H, 4-Heq, 6-Heq), 4.45–4.32 (m, 2H, 4-Hax, 6-Hax), 3.54
(s, 2H, phenyl-CH2), 3.46 (s, 2H, 100-H), 2.84–2.73 (m, 6H, 20-H, 40-H,
piperidine-6-H), 2.50 (s, 1H, piperidine-4-H), 2.28 (m, 4H,
piperidine-5-H, piperidine-2-H), 1.89–1.84 (m, 4H, piperidine-3-
nitrospiro[1,3-dioxane-2,10-naphthaline] (19b)
Prepared following General Procedure 2 from 1.23 g (7.00 mmol)
6-methoxy-1-tetralone (18b). The crude product is recrystallized
from ethanol.
13
H, 30-H). C NMR (100 MHz, CDCl3): d (ppm) ¼ 138.37 (C-8a0),
137.75 (phenyl-C-1), 137.74 (C-4a0), 137.16 (C-60), 129.49 (phenyl-C-
2), 129.12 (phenyl-C-6), 128.70 (phenyl-C-4), 128.62 (phenyl-C-3),
127.46 (phenyl-C-5), 127.34 (C-70), 126.63 (C-50), 126.51 (C-80), 98.67
(spiro-C), 85.42 (C-5), 63.40 (C-100), 62.55 (C-4), 60.54 (C-6), 52.82
(piperidine-C-4), 52.81 (piperidine-C-2, piperidine-C-6), 49.72
(phenyl-CH2), 29.71 (piperidine-C-3), 29.52 (piperidine-C-6), 27.34
(C-40), 26.75 (C-20), 20.18 (C-30); MS (CI): m/z (%) ¼ 482 (70, Mþþ1),
103 (100); IR (CH2Cl2, film) n (cmꢀ1): 2937, 1540, 1454, 1351, 1337,
1030, 1110, 924, 760; HRMS: calcd. for C26H33N3O4: 452.2549.
Found: 452.2544.
Yield: 1.92 g (89%); pale red powder; m.p.: 139.0°C; 1H NMR
(400 MHz, CD2Cl2): d (ppm): 7.38 (d, 1H, J ¼ 8.7 Hz, 70-H), 6.72 (d,
1H, J ¼ 8.7 Hz, 80-H), 6.58 (s, 1H, 50-H), 4.65 (d, 2H, J ¼ 13.4 Hz, 4-Heq
,
6-Heq), 4.17 (d, 2H, J ¼ 13.4 Hz, 4-Hax, 6-Hax), 3.88 (s, 2H, 100-H), 3.75
(s, 3H, OCH3), 2.76 (t, 2H J ¼ 6.4 Hz, 40-H), 2.23–2.18 (m, 2H, 20-H),
1.89–1.83 (m, 2H, 30-H). 13C NMR (100 MHz, CD2Cl2): d (ppm)
¼ 160.18 (C-60), 139.61 (C-8a0), 129.92 (C-70), 128.71 (C-4a0), 112.91
(C-50), 112.83 (C-80), 98.64 (spiro-C), 88.06 (C-5), 64.49 (C-100), 62.64
(C-4, C-6), 55.68 (OCH3), 29.95 (C-40), 27.32 (C-20), 20.27 (C-30); MS
(CI): m/z (%) ¼ 310 (100, Mþþ1), 280 (50); IR (CH2Cl2, film) n (cmꢀ1):
2939, 1656, 1596, 1549, 1466, 1348, 1250, 1125, 1032; HRMS:
calcd. for C15H19NO6: 309.1212. Found: 309.1215.
10,20,30,40-Tetrahydro-5-(N-benzylpiperidin-4-yl)-
aminomethyl-60-methoxy-5-nitrospiro[1,3-dioxane-2,10-
10,20,30,40-Tetrahydro-5-hydroxymethyl-5-nitro-60-(4-
methylpentyloxy)-spiro[1,3-dioxane-2,10-naphthaline]
naphthaline] (20b)
Prepared following General Procedure 3 from 1.00 g (3.23 mmol)
tetralone acetal 19b and 4-amino-1-benzylpiperidine (15i). The
crude product is recrystallized from dichloromethane/isohexane.
Yield: 768 mg (48%); white powder; m.p.: 126.3°C; 1H NMR
(400 MHz, CD2Cl2): d (ppm) ¼ 7.35 (d, 1H, J ¼ 8.7 Hz, 70-H), 7.31–
7.28 (m, 4H, phenyl-2-H, phenyl-3-H, phenyl-5-H, phenyl-6-H),
7.25–7.23 (m, 1H, phenyl-4-H), 6.71 (dd, 1H, J ¼ 8.7 Hz, 2.7 Hz, 80-H),
6.57 (s, 1H, 50-H), 4.64 (d, 2H, J ¼ 13.3 Hz, 4-Heq, 6-Heq), 4.15 (d, 2H,
J ¼ 13.3 Hz, 4-Hax, 6-Hax), 3.75 (s, 3H, OCH3), 3.45 (s, 2H, 100-H), 2.96
(s, 2H, phenyl-CH2), 2.80–2.73 (m, 4H, 40-H, piperidine-6-H), 2.35–
2.32 (m, 1H, piperidine-4-H), 2.27–2.19 (m, 2H, 20-H), 1.97 (t, 2H,
J ¼ 10.8 Hz, piperidine-2-H), 1.87–1.81 (m, 2H, 30-H), 1.78–1.75 (m,
2H, piperidine-3-H), 1.34–1.17 (m, 2H, piperidine-5-H). 13C NMR
(100 MHz, CD2Cl2): d (ppm) ¼ 160.08 (C-60), 139.56 (C-8a0), 139.47
(phenyl-C-1), 130.20 (C-4a0), 129.46 (phenyl-C-2, phenyl-C-6), 128.80
(phenyl-C-4), 128.61 phenyl-C-3, phenyl-C-5), 127.34 (C-70), 112.92
(C-50), 112.69 (C-80), 98.41 (spiro-C), 87.95 (C-5), 63.78 (C-4, C-6),
63.38 (C-100), 56.04 (piperidine-C-4), 55.66 (OCH3), 52.75 (piperi-
dine-C-2, piperidine-C-6), 50.06 (phenyl-CH2), 33.32 (piperidine-C-3,
piperidine-C-6), 30.01 (C-40), 27.12 (C-20), 20.35 (C-30); MS (CI): m/z
(%) ¼ 482 (30, Mþþ1), 203 (100); IR (CH2Cl2, film) n (cmꢀ1): 2937,
1610, 1546, 1504, 1466, 1346, 1248, 1129, 1035, 755; HRMS: calcd.
for C27H35N3O5: 483.2733. Found: 483.2960.
(19c)
690 mg (4.25 mmol) 6-hydroxy-1-tetralone and 1.17 g (8.50 mmol)
potassium carbonate are dispersed in 4.3 mL of dimethylform-
amide; then 0.93 mL (6.38 mmol) of 1-bromo-4-methylpentane is
added, and the mixture is stirred at room temperature for 16 h.
Then 50 mL water is added and the mixture is extracted with
ethyl acetate (3 ꢁ 50 mL). The combined organic layers are washed
with 1 M hydrochloric acid (2 ꢁ 50 mL) and with water, then dried
over magnesium sulfate, and evaporated.
The crude product 6-(4-methylpentyloxy)-1-tetralone (18c;
936 mg) is converted to the acetal following General Procedure
2 using 2.29 g (15.2 mmol) tris(hydroxymethyl)nitromethane,
20 mg (0.04 mmol) tetrabutylammonium tribromide, and
0.5 mL triethyl orthoformate. The crude product is recrystallized
from dichloromethane/isohexane.
Overall yield: 810 g (50%); pale yellow solid; m.p.: 112.6°C; 1H
NMR (400MHz, CD2Cl2): d (ppm) ¼ 7.35 (d, 1H, J ¼ 8.7Hz, 70-H), 6.70
(d, 1H, J ¼ 8.7Hz, 80-H), 6.56 (s, 1H, 50-H), 4.69–4.59 (m, 2H, 4-Heq, 6-
Heq), 4.17 (2d, 2H, J ¼ 13.4Hz, 13.5 Hz, 4-Hax, 6-Hax), 3.90 (t, 2H,
J ¼ 6.7Hz, 100-H), 3.85 (s, 2H, 1000-H), 2.74 (t, 2H, J ¼ 6.3 Hz, 40-H), 2.25–
2.16 (m, 2H, 20-H), 1.90–1.80 (m, 2H, 30-H), 1.79–1.69 (m, 2H, 200-H),
1.64–1.53 (m, 1H, 400-H), 1.34–1.25 (m, 2H, 300-H), 0.91 (d, 3H,
J ¼ 7.3Hz, 400-CH3), 0.90 (d, 3H, J ¼ 6.6, 500-H). 13C NMR (100MHz,
CD2Cl2): d (ppm) ¼ 159.72 (C-60), 139.56 (C-8a0), 129.75 (C-70), 128.66
(C-4a0), 113.46 (C-50), 113.39 (C-80), 98.64 (spiro-C), 88.09 (C-5), 68.76
(C-100), 64.37 (C-1000), 62.66 (C-4, C-6), 35.57 (C-300), 29.96, (C-40) 28.38 (C-
400), 27.65 (C-200), 27.28 (C-20), 22.83 (400-CH3, C-500), 20.30 (C-30); MS (CI):
m/z (%) ¼ 380 (100, Mþþ1), 350 (74); IR (CH2Cl2, film) n (cmꢀ1): 3416,
2955, 1609, 1551, 1537, 1504, 1470, 1437, 1347, 1247, 1170, 1133,
1053, 845; HRMS: calcd. for C20H29NO6: 379.1995. Found: 379.1995.
10,20,30,40-Tetrahydro-5-(N-benzylpiperidin-4-yl)-
aminomethyl-5-nitro-60-(4-methylpentyloxy)-spiro[1,3-
dioxane-2,10-naphthaline] (20c)
Prepared following General Procedure 3 from 810 mg (2.13 mmol)
tetralone acetal 19c and 4-amino-1-benzylpiperidine (15i). The
crude product is recrystallized from dichloromethane/isohexane.
Yield: 410 mg (34%); light brown solid; m.p.: 88.7°C; 1H NMR
(400 MHz, CD2Cl2): d (ppm) ¼ 7.31–7.23 (m, 6H, 70-H, phenyl-2-H,
phenyl-3-H, phenyl-4-H, phenyl-5-H, phenyl-6-H), 6.69 (d, 1H,
J ¼ 7.0 Hz, 80-H), 6.56 (s, 1H, 50-H), 4.64 (d, 2H, J ¼ 10.7 Hz, 4-Heq, 6-
Heq), 4.17 (d, 2H, J ¼ 10.6 Hz, 4-Hax, 6-Hax), 3.89 (t, 2H, J ¼ 5.3 Hz, 100-
H), 3.45 (s, 4H, 1000-H, phenyl-CH2), 2.77 (m, 4H, piperidine-4-H,
piperidine-6-H), 2.74 (t, 2H, J ¼ 5.2 Hz, 40-H), 2.35–2.34 (m, 1H,
piperidine-4H), 2.22–2.20 (m, 2H, 20-H), 1.97–1.95 (m, 4H,
piperidine-3-H, piperidine-5-H), 1.85–1.82 (m, 2H, 30-H), 1.76–
1.72 (m, 2H, 200-H), 1.60–1.57 (m, 1H, 400-H), 1.33–1.26 (m, 2H, 300-H),
0.91 (d, 6H, J ¼ 5.3 Hz, 400-CH3, 500-H). 13C NMR (100 MHz, MeOH-d4):
10,20,30,40-Tetrahydro-5-(N-benzylpiperidin-4-yl)-
aminomethyl-5-nitrospiro[1,3-dioxane-2,10-naphthaline]
(20a)
Prepared following General Procedure 3 from 960 mg (3.44 mmol)
tetralone acetal 19a and 4-amino-1-benzylpiperidine (15i). Eluent:
isohexane/ethyl acetate 7:3.
Yield: 99 mg (6%); white solid; m.p.: 100.6°C; 1H NMR (400 MHz,
CDCl3): d (ppm) ¼ 7.29 (m, 3H, 50-H, 60-H, phenyl-6-H), 7.25–7.07 (m,
6H, 70-H, 80-H, phenyl-2-H, phenyl-3-H, phenyl-4-H, phenyl-5-H),
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Arch. Pharm. Chem. Life Sci. 2014, 347, 108–122
Aminomethyl Spiroacetals as Inhibitors of D8,7-Isomerase
121
d (ppm) ¼ 157.22 (C-60), 139.69 (C-8a0), 138.62 (phenyl-C-1), 130.86
(C-4a0), 129.45 (phenyl-C-2, phenyl-C-6), 128.80 (phenyl-C-4), 128.62
(phenyl-C-3, phenyl-C-5), 128.16 (C-70), 113.44 (C-50), 110.16 (C-80),
101.47 (spiro-C), 85.15 (C-5), 63.92 (C-1000), 53.11 (C-4, C-6), 48.46 (C-
100), 45.67 (piperazine-C-4), 40.95 (piperazine-C-2, piperazine-C-6),
38.79 (phenyl-CH2), 33.80 (C-300), 31.45 (piperazine-C-3, piperazine-
C-6), 28.48 (C-40), 25.30 (C-400), 23.11 (C-200), 20.99 (400-CH3), 20.86 (C-
500), 17.52 (C-20), 14.10 (C-30); MS (CI): m/z (%) ¼ 582 (80, Mþþ1), 280
(100); IR (CH2Cl2, film) n (cmꢀ1): 2939, 1608, 1545, 1453, 1345,
1246, 1123, 741, 699; HRMS: calcd. for C32H45N3O5: 552.3437.
Found: 552.3435.
MTT assay for general cytotoxicity [55]
HL 60 cells were maintained in RPMI 1640 medium (PAA
Laboratories, Cölbe, Germany) containing 10% fetal bovine
serum (FBS; PAA Laboratories, Cölbe, Germany) without
antibiotics at 37°C in a humidified atmosphere containing 5%
CO2. Solutions of the compounds in DMSO (1 mL, concen-
trations ranging from 10ꢀ9 to 10ꢀ4 mol Lꢀ1) were incubated
with 99 mL of a suspension of HL 60 cells (9 ꢁ 105 cells mLꢀ1) in
RPMI 1640 medium with 10% FBS in 96-well plates for 24 h at
37°C. Then, 10 mL of MTT solution in PBS (5 mg mLꢀ1) was
added and the plate was incubated for another 2 h. The cells
were quenched with 190 mL of DMSO, and after 1 h of
continuous shaking of the plates, a photometric evaluation
on an ELISA plate reader MRX II (Dynex Technologies,
Denkendorf, Germany; Software: Revelation 4.06) using the
wavelength of 550 nm followed. The IC50 values were
calculated by using Prism 4 (GraphPad Software, La Jolla,
USA). Cisplatin was used as the reference drug.
Screenings
Cholesterol biosynthesis assay
This assay for the identification of the target enzyme in the
post-squalene part of cholesterol biosynthesis is based on the
GC-MS analysis of changes in the sterol pattern after
incubation with a test substance (qualitative assay).
For qualitative analysis a Varian 3800 gas chromatograph
was coupled with a Varian Saturn 2200 ion trap. The
autosampler was a CombiPal from CTC Analytics (Zwingen,
Switzerland). Instrument control and data analysis were
conducted with a Varian Workstation 6.9 SP 1. The injector
(Varian 1177, Darmstadt, Germany) was held at 250°C and
operated in splitless mode (hold time 1 min). For separation
an Agilent VF-5ms capillary column (30 m ꢁ 0.25 mm,
0.25 mm film thickness, 10 m EZ-Guard column; Böblingen,
Germany) was used with helium (purity 99.999%) as carrier
gas at a constant flow rate of 1.4 mL minꢀ1. The transfer line
was held at 270°C. The oven program started at 50°C held for
1 min, and ramped to 260°C with 50°C minꢀ1, followed by a
gradient of 4°C minꢀ1 to 310°C held for 0.5 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 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 the NISTTM 2005 mass spectral database.
The authors thank Martina Stadler for technical assistance and
Dr. Lars Allmendinger for support in NMR analysis.
The authors have declared no conflict of interest.
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