Cytotoxic ring A-modified steroid analogues derived from Grundmann's ketone

Article abstract of DOI:10.1016/j.ejmech.2011.04.036

A series of steroid and azasteroid analogues containing a six-membered ring A with various functionalities were synthesized. Furthermore, the syntheses of tetracyclic analogues bearing a five-membered A-ring and the syntheses of a number of bicyclic secosteroid analogues were carried out. All compounds were tested for their antibacterial, antifungal and cytotoxic activities. Among all tested compounds 7 and 9 showed outstanding cytotoxic activities but were devoid of antimicrobial activities. The cytotoxic activities of compounds 7, 9 and 10 were initially verified by the National Cancer Institute (NCI) in a one-dose 60 cell assay. In accordance with our results 7 and 9 satisfied pre-determined threshold inhibition criteria for progression to the 5-dose NCI screening, which revealed a selective activity profile for both candidates.

Full text of DOI:10.1016/j.ejmech.2011.04.036

European Journal of Medicinal Chemistry 46 (2011) 3227e3236
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cytotoxic ring A-modified steroid analogues derived from Grundmann’s ketone
*
Christoph D. Mayer , Franz Bracher
Department Pharmazie e Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, D-81377 Munich, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of steroid and azasteroid analogues containing a six-membered ring A with various function-
Received 10 December 2010
Received in revised form
8 April 2011
Accepted 12 April 2011
Available online 16 April 2011
alities were synthesized. Furthermore, the syntheses of tetracyclic analogues bearing a five-membered
A-ring and the syntheses of
a number of bicyclic secosteroid analogues were carried out. All
compounds were tested for their antibacterial, antifungal and cytotoxic activities. Among all tested
compounds 7 and 9 showed outstanding cytotoxic activities but were devoid of antimicrobial activities.
The cytotoxic activities of compounds 7, 9 and 10 were initially verified by the National Cancer Institute
(NCI) in a one-dose 60 cell assay. In accordance with our results 7 and 9 satisfied pre-determined
threshold inhibition criteria for progression to the 5-dose NCI screening, which revealed a selective
activity profile for both candidates.
Dedicated to Prof. Dr. Gunther Seitz,
Marburg, at the occasion of his 75th
birthday.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Steroid analogues
Cycloaddition
Cytotoxic activity
1. Introduction
A large number of bioactive sterols oxidized in various rings
have been isolated from marine organisms [5], and related semi-
Steroids play pivotal roles in human metabolism by acting as
hormones (androgens, estrogens, gestagens, glucocorticoids,
mineralocorticoids) or essential parts of cell membranes (choles-
terol). Typically, ring A of these physiological sterols consists of
a 3-hydroxycyclohexane moiety (cholesterol), a cyclohexenone
(androgens, gestagens, glucocorticoids, mineralocorticoids) or
a phenol ring (estrogens). Introduction of other structural motifs
into ring A of sterols frequently results in enhanced or completely
different biological activities. In glucocorticoids an additional
synthetic epoxysterols show potent cytotoxicity [6]. Recently, the
withanolides [7], a class of more than 300 oxidized sterols isolated
from plants largely belonging to the Solanaceae family, have
attracted considerable attention due to their cytotoxic [8,9], anti-
fungal [10], leishmanicidal, trypanocidal [11], insecticidal, anti-
inflammatory, anti-Alzheimer [12] and other activities. The vast
majority of bioactive withanolides, e.g. withanolide D (Fig. 1) [10],
contains an enone structure in ring A of the steroidal ring system,
frequently accompanied by an epoxide group at ring B.
double bond (
D
1,2) results in enhanced potency and receptor
Among the metabolites of estradiol, both 2-hydroxy- and
4-hydroxyestradiol can be oxidized to the corresponding ortho-
quinones (Fig. 1) in vivo and then undergo Michael-type additions
with physiological nucleophiles like thiols and amino groups
[13,14] and DNA bases like adenine [15].
The evident bioreactivity of Michael acceptor systems in the ring
A of steroids prompted us to investigate novel steroid and azaste-
roid analogues with miscellaneous functionalities in ring A.
selectivity [1], whereas the potent antiandrogen and gestagen
cyproterone acetate contains a cyclopropane ring annulated at the
1,2-bond [1]. Additional amino groups at ring A are found in the
plakinamines, a class of marine steroid alkaloids with cytotoxic and
antifungal activities [2], the 2-hydroxy-3-aminosteroid amafolone
shows antiarrhythmic activity [3], the muscle relaxant pan-
curonium bromide contains a quaternary ammonium group at C-2
[1]. Among the large group of biologically active heterosteroids the
5a-reductase inhibitors finasteride and dutasteride (4-azasteroids)
gained particular importance in the treatment of benign prostatic
hyperplasia and alopecia [4].
2. Results and discussion
2.1. Chemistry
Steroid analogues with a high structural diversity in ring A were
prepared following a protocol worked out by De Riccardis et al.
[16,17] based on fundamental investigations of Alberola et al. [18].
* Corresponding author. Tel.: þ49 89 2180 77297; fax: þ49 89 2180 77171.
E-mail address: christoph.mayer@cup.uni-muenchen.de (C.D. Mayer).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.036

3228
C.D. Mayer, F. Bracher / European Journal of Medicinal Chemistry 46 (2011) 3227e3236
Thus, Grundmann’s ketone (1), readily available by ozonolysis of
vitamine D3, was converted to the vinyl triflate 2 under kinetic
control, the latter then convertedtothe 1,3-diene 3 byStille coupling
with vinyltributyltin, catalyzed by tetrakis(triphenylphosphine)-
palladium(0). This diene had already been converted to the
1,4-dioxocholestane 7 by Diels-Alder cycloaddition with p-benzo-
quinone (4) [16] and to several moderately cytotoxic
steroid-anthraquinone hybrids by cycloaddition/oxidation with
naphthoquinones by De Riccardis (Scheme 1) [17].
In order to supply the compound for screenings, we prepared
product 7 following the reported protocol [17]. The stereochemistry
of the single isomer formed in this cycloaddition was confirmed by
extensive NOE experiments (Supplementary data, Fig. 1). Further,
we prepared a series of heterosteroidal congeners of 7 by cyclo-
addition of 3 with various cyclic dienophiles and diazadienophiles.
Phthalazine-1,4-dione (5), prepared in situ by oxidation of phthal-
hydrazide with lead tetraacetate [19], gave the pentacyclic
H
H
HO
H
O
O
O
O
H
H
H
H
H
O
O
OH
O
withanolide D
3,4-estrone-o-quinone
Fig. 1. Structures of withanolide D [10] and 3,4-estrone-o-quinone [15].
O
R
O
H
4
O
H
H
(c)
H
H
O
O
7
1
O
(a)
R
N
N
O
O
R
R
5
O
N
N
H
H
(b)
(d)
H
H
8
OTf
2
3
O
R
N
N
R =
O
6
O
N
H
H
N
(e)
O
9
(f)
R
O
O
H
N
N
H
H
10
Scheme 1. Reagents and conditions: (a) Sodium bis(trimethylsilyl)amide, THF, ꢀ78 ꢁC, 1 h, then N-phenyl-bis(trifluoromethanesulfonimide), ꢀ78 ꢁ
C to r.t., 2.2 h (67%);
(b) (PPh3)4Pd, LiCl, vinyltributyltin, THF, 75 ꢁC, 2 h (98%); (c) p-benzoquinone, toluene, 120 ꢁC, 12 h (36%); (d) phthalhydrazide, Pb(OAc)4, toluene, 120 ꢁC, 12 h (76%); (e) 3,6-pyr-
idazinedione (prepared in situ [20]), acetone, ꢀ78 ꢁC to r.t., 1 h (20%); (f) H2, PtO2, MeOH, r.t., 20 bar, 48 h (76%).

C.D. Mayer, F. Bracher / European Journal of Medicinal Chemistry 46 (2011) 3227e3236
3229
Diels-Alder product 8. For preparing the tetracyclic diazacholestane
analogue 9, the diazadienophile 3,6-pyridazinedione (6) had to be
prepared freshly by tert-butylhypochlorite oxidation of the
monopotassium salt of 3,6-dihydroxypyridazine [20]. Subsequent
cycloaddition with diene 3 at low temperature gave product 9 in
poor yield (20%). Comparably low yields in cycloadditions with
3,6-pyridazinedione had been reported by others [21]. Since 9 was
identified as being highly cytotoxic in early investigations, and we
supposed that the Michael acceptor system in ring A of this
compound might be crucial for this activity, we converted 9 to its
tetrahydro analogue 10 by catalytic hydrogenation of both olefinic
moieties (Scheme 1). Once again the exact stereochemistry of the
product was elucidated by extensive NMR experiments
(Supplementary data, Figure 2).
A second series of compounds consisted in Diels-Alder products
of diene 3 with various five-membered dienophiles (11, 12aee).
Maleic anhydride [22], maleimide, as well as its N-methyl, N-ethyl
and N-propyl derivatives underwent smooth cycloaddition with 3
to give the tetracycles 13 and 14aed. In an analogous manner
4-methyl-1,2,4-triazoline-3,5-dione [23] was converted to the tri-
aza tetracycle 14e (Scheme 2).
(PPh3)2PdCl2 under microwave conditions. Catalytic hydrogenation
of 16 gave an epimeric mixture of the fully saturated alcohol 17,
a/b
which was not separable by column chromatography. Thus, the
crude mixture was used in the next step. The maleimides 18, 19 and
20 were prepared from the corresponding alcohols 15, 16 and 17
and maleimide in poor to moderate yields by Mitsunobu reaction
under microwave conditions [27]. Under conventional Mitsunobu
conditions (maleimide, DIAD, triphenylphosphine and the appro-
priate alcohol) no conversions were recorded at all.
Finally, we intended to elucidate whether or not the complete
steroid-like tetracyclic ring system is required for the extraordi-
nary biological activity of diazasteroid 9. For this purpose the
natural terpene b-myrcene (21) was selected as a suitable diene
component, and reacted with 3,6-pyridazinedione (6) to give the
bicyclic pyridazino[1,2-a]pyridazine-1,4-dione 22. This product
contains the ring A þ B motif of 9, but rings C and D of the steroid
and the side chain are only adumbrated by a flexible side chain
(Scheme 4).
2.2. Pharmacology
Since the cycloadducts 7 and 9, standing out due to the Michael
acceptor systems in ring A, showed by far the highest cytotoxic
activities of all compounds prepared until then (Table 1), we
prepared a third series of compounds. These products are charac-
terized by having a maleimide group attached by a rigid or flexible
C3- or C4-spacer to a bicyclic ring system derived from vinyl triflate
2, the latter resembling sterol rings C and D and the lipophilic sterol
side chain. The target compounds still are Michael acceptors, but do
no longer contain the complete, rigid sterol backbone (Scheme 3).
Heck reaction [24,25] of 2 and allyl alcohol catalyzed by tri-o-
tolylphosphine and palladium(II) acetate under microwave irradi-
ation gave the hydroxymethyl-1,3-diene 15 in moderate yield. In
contrast acetylenic alcohol 16 was obtained in excellent yield by
Sonogashira coupling [26] of 2 and 3-butyn-1-ol catalyzed by
2.2.1. Cytotoxic activity
A first screening for cytotoxic activities of the compounds was
performed in a MTT assay [28] on human leukemia HL-60 cells.
Cisplatin was used as reference. The results are shown in Table 1.
Significant cytotoxicities (IC50 < 10 mM) were recorded only for
three tetracyclic compounds: The N-propylmaleimide adduct 14d
showed cytotoxic potency comparable to cisplatin, whereas the
diones 7 and 9 stand out with IC50-values of 0.7 mM and 0.03 mM.
2.2.2. Antimicrobial activity
The compounds were subjected to a standard disc diffusion
antimicrobial sensitivity test (Agar diffusion assay). Table 2 summa-
rizes the results obtained for the zones of inhibition of all tested
compounds against different strains of Gram-positive bacteria,
O
O
O
H
11
O
O
H
H
(a)
13
H
O
O
H
Y
Y
3
R
N
O
12a-e
O
Y
R
N
H
H
Y
(b)
14a-e
O
14 a: R = H; Y = CH
14 b: R = CH3; Y = CH
14 c: R = C2H5; Y = CH
14 d: R = C3H7; Y = CH
14 e: R = CH3; Y = N
Scheme 2. Reagents and conditions: (a) maleic anhydride, toluene, 120 ꢁC, 12 h (77%); (b) dienophile (a ¼ maleimide, b ¼ N-methylmaleimide, c ¼ N-ethylmaleimide, d ¼ N-pro-
pylmaleimide, e ¼ 4-methyl-1,2,4-triazoline-3,5-dione), toluene, 120 ꢁC, 12 h (41e81%).

3230
C.D. Mayer, F. Bracher / European Journal of Medicinal Chemistry 46 (2011) 3227e3236
Table 1
activity. Among the maleimides only 14a showed good activity. The
open-chain compounds 15e20 showed moderate antimicrobial
activities. Bicyclic compound 22 was completely inactive.
Cytotoxic activities of investigated compounds against HL-60 cells determined by
MTT test.
Compound
Molecular formula
IC50
5
[mM]
2.2.3. Primary single high dose (10ꢀ5 M) full NCI 60 cell panel
in vitro assay
Cisplatin
3
7
8
9
Cl2H6N2Pt
C20H34
C26H38O2
C28H38N2O2
C24H36N2O2
C24H40N2O2
C24H36O3
C24H37NO2
C25H39NO2
C26H41NO2
C27H43NO2
C23H37N3O2
>100
0.7
17
0.03
30
30
16
15
13
7
13
32
38
13
32
46
14
41
A selection of compounds described above (7, 9,10) was accepted
by the National Cancer Institute (NCI, USA) for testing initially at
a single high dose cell assay. This assay was performed in a 60 cell
panel representing leukemia, melanoma and cancers of lung, colon,
brain, breast, ovary, kidney and prostate in accordance with the
protocol of the NCI [29,30]. The compounds were incubated at
a single concentration (10ꢀ5 M) for 48 h, end point determinations
were made with a protein binding dye, sulforhodamine B (SRB).
Results for each compound were reported as a Mean Graph of the
growth percent of the treated cells when compared to the untreated
control cells (Supplementary data, Table 1). After analysis of
historical Developmental Therapeutics Program (DTP) compounds 7
and 9, which satisfied pre-determined threshold inhibition criteria
were selected for the NCI full panel 5-dose assay.
10
13
14a
14b
14c
14d
14e
15
16
17
18
19
C
21H36
O
O
O
C22H36
C22H42
C25H37NO2
C26H37NO2
C26H43NO2
C14H18N2O2
20
22
2.2.4. In vitro 5-dose full NCI 60 cell panel assay
different strains of Gram-negative bacteria, yeasts, a mould and
a dermatophyte. The results are expressed as diameter of zone (mm).
Tetracycline (antibiotic) and clotrimazole (antimycotic) were used as
references.
The 60 cell lines, representing nine tumor subpanels, were
incubated at five different concentrations (10ꢀ8,10ꢀ7,10ꢀ6,10ꢀ5 and
10ꢀ4 M) with the compounds and log concentration versus % growth
inhibition curves and three response parameters (GI50, TGI and LC50
)
The DielseAlder products 7, 8 and 9 did not show noteworthy
antimicrobial activity, whereas the saturated product 10 has broad
were calculated from the outcomes for each cell line. The GI50 value
(growth inhibitory activity) corresponds to the concentration of the
R
R
R
(a)
(b)
H
H
H
OTf
2
15
18
O
(c)
HO
N
O
R
R
(b)
H
H
O
16
19
OH
N
O
(d)
R
R
R =
O
(b)
H
H
HO
N
O
17
20
Scheme 3. Reagents and conditions: (a) allyl alcohol, tributylamine, tri-o-tolylphosphine, Pd(OAc)2, MW: 80 W, 150 ꢁC, 10 min (36%); (b) PPh3, maleimide, THF, DIAD, MW: 50 W,
76 ꢁC, 30 min (8e44%); (c) 3-butyn-1-ol, diethylamine, DMF, CuI, (PPh3)2PdCl2, MW: 300 W, 120 ꢁC, 5 min (98%); (d) H2, PtO2, ethyl acetate, r.t., 48 h (95%).

C.D. Mayer, F. Bracher / European Journal of Medicinal Chemistry 46 (2011) 3227e3236
3231
O
of sensitivity. The cytotoxic activity of compound 9 is remarkable
and exhibits a selective activity profile. Very low LC50-values
between 9.0 mM and 0.6 mM could be observed against all cell lines
except the complete leukemia sub panel and some very few other
cell lines (HCT-15, OVCAR-8 and HS 578T).
N
N
O
O
6
O
N
N
(a)
3. Conclusion
In conclusion, we have synthesized a number of steroid and
secosteroid analogues starting from Grundmann’s ketone (1),
which itself is readily available from commercial vitamin D3.
DielseAlder cycloadditions of the 1,3-diene 3 with (aza)dien-
ophiles gave steroid analogues 7e9, 13 and 14aee, all products
21
22
Scheme 4. Reagents and conditions: (a) 3,6-pyridazinedione (prepared in situ),
acetone, ꢀ78 ꢁC to r.t., 1 h (11%).
containing two carbonyl groups in the ring
A equivalent.
compound causing 50% decrease in net cell growth, the TGI value
(cytostatic activity) is the concentration of the compound resulting
in total growth inhibition and LC50 value (cytotoxic activity) is the
concentration of the compound causing net 50% loss of initial cells at
the end of the incubation period of 48 h.
Within this subclass compounds 7 and 9 stand out due to
their Michael acceptor system in ring A. Seco analogues con-
taining N-alkylmaleimide moieties, which are Michel accep-
tors, too, were prepared from vinyl triflate 2 via Pd-catalyzed
coupling reactions with C3 and C4-spacers, followed by
attaching the maleimide moiety under Mitsunobu conditions.
Finally, a bicyclic analogon 22 of compound 9 was obtained
The results of the assays are summarized as GI50-, TGI- and LC50
-
values of each cell line in Table 3. A cell line, which found to be
insensitive at the highest tested concentration (100 mM), is signed
with “>” as prefix to the concentration.
by DielseAlder cycloaddition of
b-myrcene (21) with
3,6-pyridazinedione.
The observed GI50 values of compound 7 are, with only two
exceptions, below 1 M for all tested cell lines. Very high activities
The compounds were tested for their antibacterial, antifungal
and cytotoxic activities. The tetra- and pentacyclic DielseAlder
products 7, 8 and 9 were almost inactive in the screenings for anti-
microbial activities, the tetrahydro analogue 10 showed moderate
activity. Maleimides 14aee and the open-chain analogues contain-
ing terminal maleimido groups also showed moderate antimicrobial
activities.
m
were recorded against the non-small cell lung cancer cells A549/
ATCC and HOP-92, the colon cancers COLO 205 and HCT-116, the
ovarian cancer OVCAR-8, the renal cancer UO-31 and the prostate
cancer PC-3 with GI50 values in a low nM range (<100 nM). The
obtained TGI-data for compound 7 represent a characteristic
sensitivity profile that showed 13 cell lines with total growth
In a preliminary in-house screening for cytotoxic activity on
HL-60 cells the tetracyclic compounds 7 and 9 showed extremely
promising effects. These results were reinforced by screening
results at the NCI. The single high dose 60 cell panel assays revealed
that the (diaza)quinone derivatives 7 and 9 exhibit remarkable
cytotoxic activities, whereas the hydrogenated analogue 10 showed
only moderate activity. Compounds 7 and 9 were further investi-
gated in a 5-dose 60 cell panel assay, and the diazaquinone 9 was
identified as being highly active against a broad panel of cancer cell
lines, except leukemia cells.
inhibitory values less than 1
cell lines HCT-116, LOX IMVI, OVCAR-4, OVCAR-8 and UO-31
ranging from 8.10 M to 1.75 M. The highest cytotoxic activity of
compound 7 was observed against the colon cancer cell line COLO
205 with a LC50 value of 0.54 M.
The diaza analogue 9 showed even higher activity against all cell
lines with GI50 values between 1.8 M and 110 nM. The obtained
TGI values of 9 are in the range of 4.79 M to 0.29 M. Neither the
mM. Notable are the LC50-values of the
m
m
m
m
m
m
GI50- nor the TGI Mean Graph shows any characteristic fingerprint
Obviously a Michael acceptor system in ring A, which is exclu-
sively found in compounds 7 and 9, is essential for high cytotoxic
activity in the classes of compounds investigated here. Benzo-
anullation (compound 8) as well as hydrogenation of the double
bond (10) lead to a drastic decrease of activity, and also the maleic
anhydride (13) and maleimido adducts (14aee) are by far less
active.
Table 2
Antimicrobial activities of investigated compounds determined by disk diffusion
sensitivity test expressed as zone of inhibition (diameter of zone in mm).
Ec
Pa
Seq
Sen
Yl
Cg
An
Hb
Tetracycline
Clotrimazole
3
7
8
9
10
13
14a
14b
14c
14d
14e
15
16
18
19
20
22
30
n.i.
e
25
n.i.
e
30
n.i.
10
e
22
n.i.
9
n.i.
18
e
n.i.
18
e
n.i.
13
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
n.i.
20
e
Furthermore, an intact tetracyclic steroid-like ring system seems
to be indispensable for cytotoxic activity. The secosteroids 18e20,
only consisting of the rings C þ D fragment of the steroid backbone,
which is attached to a Michael acceptor system via a flexible alkyl
side chain to mimic the rings A þ B fragment, showed only very
e
e
e
e
e
e
e
7
8
12
10
11
7
e
e
e
10
8
7
e
e
7
7
15
e
10
15
10
e
11
7
24
10
7
9
e
10
12
15
10
11
7
e
8
poor cytotoxic activities. The
b-myrcene cycloaddition product 22,
14
e
e
15
10
8
6
e
e
14
7
14
e
which compared to the most active compound 9 lacks the rings C
and D of the steroid backbone, is completely inactive.
7
7
8
e
e
7
e
10
e
Further it is worth mentioning that the compounds 7 and 9
showing outstanding activities against tumor cell lines are
almost inactive against bacteria and fungi, thus suggesting that
these compounds act by a specific mechanism of action, and not
by unselective cytotoxicity. First investigations aimed at the
identification of the target revealed that the steroid analogues 7
and 9 have no effect on cholesterol biosynthesis in the human
cell line HL 60 [31]. Further investigations are under way in
order to elucidate the molecular mode of action of these
compounds.
e
10
12
6
11
11
11
e
e
10
10
9
15
10
e
18
11
10
13
9
14
14
15
15
12
e
e
18
10
7
10
8
22
11
8
12
9
e
e
e
e
e
e
e
e
Ec, Escherichia coli; Pa, Pseudomonas antimicrobia; Seq, Staphylococcus equorum;
Sen, Streptococcus entericus; Yl, Yarrowia lipolytica; Cg, Candida glabrata; An,
Aspergillus niger; Hb, Hyphopichia burtonii; n.i., not investigated; e, inactive;
concentration, 1% (w/v) DMSO solution of the testing compound.

3232
C.D. Mayer, F. Bracher / European Journal of Medicinal Chemistry 46 (2011) 3227e3236
Table 3
NCI DTP in vitro testing results of compounds 7 and 9 (values expressed in mM).
Compound panel
Leukemia
Cell line
7 (NSC 750440/1)
9 (NSC 750441/1)
GI50
TGI
LC50
GI50
TGI
LC50
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
0.189
0.153
0.249
0.312
0.107
0.207
0.058
0.545
0.630
0.028
0.480
0.147
0.305
0.333
0.803
0.080
0.274
0.055
0.212
0.839
0.338
0.141
0.566
0.141
0.478
0.654
0.094
0.227
0.399
0.299
0.229
3.37
0.302
0.309
0.230
0.752
0.502
0.159
0.212
0.058
0.275
0.688
0.311
0.384
0.162
0.279
0.201
0.269
2.65
>100
n.d.
>100
>100
2.23
>100
0.780
>100
>100
0.086
n.d.
n.d.
0.849
5.59
>100
0.220
0.855
1.03
>100
>100
>100
>100
>100
>100
>100
>100
>100
n.d.
>100
>100
>100
>100
>100
0.536
>100
3.91
>100
>100
>100
>100
>100
>100
>100
>100
n.d.
0.360
0.113
0.309
0.545
0.181
0.137
0.325
1.03
1.33
0.175
1.27
1.42
1.45
0.739
0.175
0.255
0.431
0.142
0.418
0.616
0.443
0.375
1.46
0.110
1.47
1.52
0.438
0.288
1.44
0.285
0.354
0.286
0.512
0.563
0.433
0.544
0.861
0.686
1.05
0.317
0.417
1.63
0.713
1.06
>100
0.490
2.02
4.13
0.601
0.758
1.24
2.75
2.67
1.03
3.26
3.58
2.91
2.25
0.459
0.623
1.64
0.291
2.22
2.07
1.51
1.36
2.88
0.627
2.89
3.02
1.59
1.05
3.03
1.01
1.78
0.774
1.65
1.83
1.55
1.95
2.11
2.11
2.87
1.58
1.90
2.99
1.95
2.27
2.32
0.577
2.30
1.69
1.26
1.15
1.11
2.26
1.24
2.54
4.79
0.876
1.62
2.56
1.73
>100
>100
>100
>100
>100
>100
4.04
7.33
5.33
3.76
8.33
9.00
5.84
5.73
1.54
2.36
4.99
0.594
>100
5.51
4.44
5.33
5.70
3.76
5.64
6.01
4.18
3.52
6.38
3.32
7.68
3.39
4.14
4.52
4.32
5.45
4.78
5.63
7.84
>100
2.28
5.47
4.43
4.87
5.19
3.47
5.43
5.28
3.60
4.19
4.83
4.89
4.26
6.25
>100
2.97
6.29
9.42
7.76
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
MDA-MB231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
Colon cancer
n.d.
>100
>100
n.d.
>100
0.539
n.d.
>100
1.23
1.17
2.00
n.d.
0.997
>100
n.d.
CNS cancer
Melanoma
4.10
>100
>100
>100
>100
>100
n.d.
>100
>100
>100
2.65
>100
8.12
>100
>100
n.d.
1.00
0.610
>100
>100
0.498
>100
1.18
n.d.
n.d.
1.50
1.12
Ovarian cancer
Renal cancer
n.d.
n.d.
n.d.
n.d.
1.35
1.04
0.633
0.995
>100
>100
0.359
1.57
>100
0.757
1.76
>100
2.55
>100
0.462
6.45
0.179
0.952
0.388
0.356
0.237
0.243
1.04
0.285
1.04
1.80
>100
>100
1.75
>100
>100
n.d.
>100
>100
>100
>100
n.d.
0.072
0.059
0.632
0.112
0.343
0.817
0.427
0.337
0.215
0.269
Prostate cancer
Breast cancer
0.312
0.255
0.628
0.489
MID
63.1
n.d., not determined; MID, average sensitivity of all cell lines in
mM.
4. Experimental
tetramethylsilane as an internal standard. All spectra were recor-
ded in CDCl3 as solvent and chemical shifts are reported in parts per
4.1. Chemistry
million (ppm, d). J values are given in Hz. Mass spectra (MS) were
run by chemical impact (CI): Hewlett Packard 5989 A Mass Spec-
trometer with 59980 B Particle Beam LC/MS Inerterface and by
electron impact (EI): at 70 eV on a Jeol JMS GCmate II. Anhydrous
reactions were carried out in a nitrogen atmosphere unless other-
wise described. Solvents were of HPLC grade or p.a. grade, if not
they were distilled before use. All chemicals were purchased from
Melting points were determined by open tube capillary method
on a Büchi melting point B-450 apparatus and are uncorrected. IR
spectra were obtained on a Perkin Elmer FT-IR: Paragon 1000
spectrometer. NMR spectra were recorded on Jeol JNMR-GX 400
(400 MHz) and Jeol JNMR-GX 500 (500 MHz) spectrometers with

C.D. Mayer, F. Bracher / European Journal of Medicinal Chemistry 46 (2011) 3227e3236
3233
SigmaeAldrich (Schnelldorf, Germany) and Acros Organics (Geel,
Belgium). Microwave-promoted syntheses were performed on
a single-mode microwave reactor (Discover) equipped with an IR
temperature sensor from CEM (Kamp-Lintfort, Germany). Reactions
were monitored by thin-layer chromatography (TLC) using pre-
coated plastic sheets POLYGRAMÒ SIL G/UV254 from Macherey-
Nagel (Düren, Germany). Compounds on TLC plates were detected
under UV light at 254 nm and visualized by immersion in a solution
of 5% (NH4)6Mo7O24$4 H2O, 0.2% Ce(SO4)2$4 H2O and 5% conc.
H2SO4. Merck silica gel 60 was used as stationary phase for flash
column chromatography. Purity of the synthesized compounds was
>95% (HPLC).
General procedure for synthesis of Diels-Alder adducts 7, 13,
14a-e. Diene 3 [17] (0.20 g, 0.73 mmol) and dienophile (0.73 mmol)
were dissolved in toluene (2.2 mL) and heated under reflux at
120 ꢁC for 12 h. The reaction mixture was concentrated under
reduced pressure. Purification was performed as described below
for the single compounds.
Synthesis of 9. To a stirred suspension of 3,6-dihydroxypyr-
idazine potassium salt (0.31 g, 2.10 mmol) and acetone (15 mL) was
added tert-butylhypochlorite solution (0.24 mL, 2.10 mmol)
at ꢀ78 ꢁC. After stirring for 3 h a solution of diene 3 (0.55 g,
2.00 mmol) and acetone (5 mL) was added. The reaction mixture
was slowly warmed at r.t., stirred for further 1 h and then quenched
with water (50 mL). The volatile organic components were evap-
orated and the resulting aqueous residue was extracted with
diethyl ether (3 ꢃ 20 mL), the combined organic layers were dried
over Na2SO4 and concentrated under reduced pressure. The
product was purified by silica column chromatography (ethyl
acetate) to yield 0.15 g (20%) of 9 as a pale yellow solid. m.p.
~
148e150 ꢁC; ½a 2D0
ꢂ
¼ þ 339.2; IR (KBr):
n
¼ 3423, 2954, 1627, 1467,
¼ 6.89 (d,
1416, 1342, 1129, 857 cmꢀ1; 1H NMR (CDCl3, 400 MHz):
d
J ¼ 9.9 Hz, 1 H), 6.85 (d, J ¼ 9.9 Hz, 1 H), 5.70 (m, 1 H), 5.14 (dd,
J ¼ 16.8/6.8 Hz, 1 H), 5.03 (d, J ¼ 12.2 Hz, 1 H), 3.78 (d, J ¼ 16.8 Hz, 1
H), 2.64 (m, 1 H), 2.55 (m, 1 H), 2.04e1.91 (m, 2 H, 6-H), 1.89e185
(m, 2 H), 1.57e0.97 (m, 11 H), 0.90 (d, J ¼ 6.1 Hz, 3 H), 0.87 (d,
J ¼ 6.5 Hz, 3 H), 0.86 (d, J ¼ 6.5 Hz, 3 H), 0.84 (s, 3 H); 13C NMR
(5S,9S,10R,13R,14R,17R)-17-((R)-1,5-Dimethylhexyl)-13-methyl-6,
9,10,11,12,13,14,15,16,17-decahydro-5H-cyclopenta[a]phenanthrene-
1,4-dione (7) [16]. Dienophile: p-benzoquinone (4). The product was
purified by silica column chromatography (hexane/ethyl acetate 4:1)
to give 0.10 g (36%) 7 as pale yellow solid. m.p. 134e135 ꢁC (for
a complete analytical characterization see Supplementary data).
(1R,3aR,11bR,13aR)-1-((R)-1,5-Dimethylhexyl)-13a-methyl-2,3,
3a,5,11b,12,13,13a-octahydro-1H-5a,11a-diazaindeno[5,4-a]anthra-
cene-6,11-dione (8). A suspension of diene 3 (0.27 g, 1.00 mmol),
phthalhydrazide (0.11 g, 1.00 mmol), lead(IV) acetate (0.44 g,
1.00 mmol) and toluene (3.0 mL) was heated at 120 ꢁC for 12 h. The
reaction mixture was concentrated under reduced pressure. The
product was purified by silica column chromatography (hexane/
ethyl acetate 4:1) to give 0.33 g (76%) 8 as pale yellow solid. m.p.
~
(CDCl3, 100 MHz):
d
¼ 157.9, 157.4, 138.6, 135.5, 133.8, 113.6, 57.5,
56.1, 49.7, 43.8, 40.7, 39.4, 35.9, 35.8, 35.7, 28.7, 28.0, 24.8, 23.9, 22.8,
22.5, 22.7, 18.9, 18.2; MS (CI): m/z (%) ¼ 385 [M þ H]þ (29), 282
(100); HRMS (EI): m/z ¼ 384.2784 [M]þ, calcd for C24H36N2O2:
384.2776.
(5aR,7aR,8R,10aS,10bS)-8-((R)-1,5-Dimethylhexyl)-7a-methyl-do-
decahydro-cyclopenta[f]pyridazino[1,2-a]cinnoline-1,4-dione (10).
Platinum(IV) oxide (30 mg, 0.13 mmol) was added to a solution of 9
(0.13 g, 0.34 mmol) and methanol (10 mL). The reaction mixture was
stirred for 48 h under hydrogen atmosphere (20 bar) using a hydro-
genation set-up. The mixture was filtered (celite) and concentrated
under reduced pressure. The product was purified by silica column
chromatography (ethyl acetate)togive 0.10 g (76%)10 as colourlessoil.
~
123e124 ꢁC; ½a 2D0
ꢂ
¼ ꢀ222.6; IR (KBr):
n
¼ 3422, 2953, 1632, 1467,
¼ 8.28 (dd,
½
a 2D0
ꢂ
¼ ꢀ152.5; IR (NaCl):
n
¼ 3568, 2949, 1667, 1467, 1384, 1269, 1181,
¼ 4.57 (dt, J ¼ 6.6/5.5 Hz,1 H),
1357, 1241, 1141, 790 cmꢀ1; 1H NMR (CDCl3, 500 MHz):
d
717 cmꢀ1; 1H NMR (CDCl3, 400 MHz):
d
J ¼ 9.4/6.7 Hz, 2 H), 7.79 (dd, J ¼ 9.4/6.7 Hz, 2 H), 5.78 (dd, J ¼ 7.3/
2.7 Hz, 1 H), 5.32 (dd, J ¼ 16.4/7.0 Hz, 1 H), 5.26 (d, J ¼ 12.5 Hz, 1 H),
3.86 (dt, J ¼ 16.3/5.2 Hz, 1 H), 2.61e2.54 (m, 2 H), 2.05e1.84 (m, 4
H),1.56e0.98 (m,12 H), 0.90 (d, J ¼ 6.3 Hz, 3 H), 0.89 (s, 3 H), 0.87 (d,
J ¼ 6.6 Hz, 3 H), 0.86 (d, J ¼ 6.6 Hz, 3 H); 13C NMR (CDCl3, 100 MHz):
3.97 (dt, J ¼ 12.5/8.8 Hz, 1 H), 3.48 (dt, J ¼ 12.5/4.6 Hz, 1 H), 2.62e2.51
(m, 4 H), 2.03e1.98 (m, 2 H), 1.93e1.75 (m, 4 H), 1.72e1.47 (m, 4 H),
1.41e0.98 (m, 11 H), 0.90 (d, J ¼ 6.0 Hz, 3 H), 0.87 (d, J ¼ 6.6 Hz, 3 H),
0.86 (d, J ¼ 6.6 Hz, 3 H), 0.75 (s, 3 H); 13C NMR (CDCl3, 100 MHz):
d
¼ 169.0, 168.6, 56.3, 52.8, 47.1, 41.5, 41.3, 39.4, 35.9, 35.8, 35.6, 31.4,
d
¼ 158.9, 158.1, 139.1, 133.4, 133.1, 129.8, 128.9, 127.8, 127.2, 114.3,
29.6, 29.1, 28.1, 28.0, 25.1, 25.0, 23.7, 23.0, 22.8, 22.6,18.2,12.9; MS (CI):
m/z (%) ¼ 389 [M þ H]þ (100); HRMS (EI): m/z ¼ 388.3095 [M]þ, calcd
for C24H40N2O2: 388.3089.
57.5, 55.8, 49.9, 44.2, 40.9, 39.5, 36.0, 35.9, 35.7, 28.7, 28.0, 25.1, 23.9,
22.8, 22.6 (2 C), 19.0, 18.3; MS (CI): m/z (%) ¼ 435 [M þ H]þ (100);
HRMS (EI): m/z ¼ 434.2936 [M]þ, calcd for C28H38N2O2: 434.2933.
(5aR,7aR,8R,10aR)-8-((R)-1,5-Dimethylhexyl)-7a-methyl-
(3aR,3bS,5aR,6R,8aR,10aS)-6-((R)-1,5-Dimethylhexyl)-5a-methyl-
3b,4,5,5a,6,7,8,8a,10,10a-decahydro-3aH-indeno[5,4-e]isobenzofur-
an-1,3-dione (13). Dienophile: maleic anhydride. The product was
purified by silica column chromatography (hexane/ethyl acetate 4:1)
6,7,7a,8,9,10,10a,12-octahydro-5aH-cyclopenta[f]-pyridazino[1,2-a]
cinnoline-1,4-dione (9). The dienophile 3,6-pyridazinedione (6)
was prepared freshly by oxidization of 3,6-dihydroxypyridazine
potassium salt with tert-butylhypochlorite.
Preparation of tert-butylhypochlorite solution. Protected from
light a cooled (10 ꢁC) aqueous solution of sodium hypochlorite (13%
active chlorine, 250 mL, 46.0 mmol) was added to a vigorously
stirred mixture of tert-butanol (4.1 mL, 43.0 mmol) and glacial
acetic acid (2.6 mL, 46.0 mmol). The reaction mixture was stirred
for further 3 min, then the organic layer was separated and washed
with sodium carbonate solution (10 mL, 10%) and water (10 mL).
The dried (calcium chloride) organic layer was used immediately
for oxidization of 3,6-dihydropyridazine potassium salt.
to give 0.21 g (77%) 13 as
a
white solid. m.p. 139e141 ꢁC;
a 2D0
ꢂ
¼ þ53.6; IR (KBr):
n
¼ 2957, 1710, 1466, 1376, 1193, 926,
~
½
813 cmꢀ1
;
1H NMR (CDCl3, 500 MHz):
d
¼ 5.50 (m, 1 H), 3.40 (dt,
J ¼ 9.6/1.7 Hz, 1 H), 3.31 (dd, J ¼ 9.6/6.5 Hz, 1 H), 2.75 (dd, J ¼ 15.1/
7.0 Hz,1H), 2.42e2.31(m, 2H), 2.29e2.16(m, 2H), 2.03e1.92(m, 2H),
1.76 (m, 1 H), 1.62e0.98 (m, 13 H), 0.90 (d, J ¼ 6.5 Hz, 3 H), 0.87 (d,
J ¼ 6.6 Hz, 3 H), 0.86 (d, J ¼ 6.6 Hz, 3 H), 0.52 (s, 3 H); 13C NMR (CDCl3,
125 MHz):
d
¼ 174.6, 171.8, 145.6, 116.8, 56.3, 48.9, 44.2, 42.6, 40.9,
39.5, 36.2, 36.2, 35.9, 34.2, 28.9, 28.0, 24.0, 23.9, 23.0, 22.8, 22.5, 21.6,
18.4, 18.3; MS (CI): m/z (%) ¼ 373 [M þ H]þ (100); HRMS (EI):
m/z ¼ 372.2658 [M]þ, calcd for C24H36O3: 372.2665.
Synthesis of 3,6-dihydroxypyridazine potassium salt.
A
(3aR,3bS,5aR,6R,8aR,10aS)-6-((R)-1,5-Dimethylhexyl)-5a-methyl-
3b,4,5,5a,6,7,8,8a,10,10a-decahydro-3aH-indeno[5,4-e]isoindol-1,3-
dione (14a). Dienophile: maleimide. The product was purified by
silica column chromatography (hexane/ethyl acetate 4:1) to give
suspension of 3,6-dihydroxypyridazine (1.00 g, 8.92 mmol) in
water (4.0 mL) was treated with potassium hydroxide (0.50 g,
8.91 mmol). The reaction mixture was stirred until the solution
became clear. The solvent was removed under reduced pressure
and the achieved 3, 6-dihydroxypyridazine potassium salt was
finely pestled and dried in an exsiccator (drying agent: phosphorus
pentoxide).
0.22 g (81%) 14a as a white solid. m.p.148e150 ꢁC; ½a D20
ꢂ
¼ ꢀ125.2; IR
¼ 3190, 2957,1711,1466,1354,1196,1003, 814 cmꢀ1; 1H NMR
~
(KBr):
n
(CDCl3, 400 MHz):
d
¼ 7.87 (s, 1 H), 5.44 (m, 1 H), 3.16e3.07 (m, 2 H),
2.70 (dd, J ¼ 15.1/7.0 Hz, 1 H), 2.40e2.25 (m, 3 H), 2.12 (m, 1 H),

3234
C.D. Mayer, F. Bracher / European Journal of Medicinal Chemistry 46 (2011) 3227e3236
2.00e1.90 (m, 2 H), 1.74 (m, 1 H), 1.58e0.99 (m, 13 H), 0.90 (d,
J ¼ 6.2 Hz, 3 H), 0.88(d, J ¼ 6.6 Hz, 3 H), 0.86(d, J ¼ 6.6 Hz, 3 H), 0.51 (s,
(E)-3-[(1R,3aR,7aR)-1-((R)-1,5-Dimethylhexyl)-7a-methyl-2,3,3a,
6,7,7a-hexahydro-1H-inden-4-yl]-prop-2-en-1-ol (15). To a solution
of triflate 2 [17] (0.20 g, 0.50 mmol), acetonitrile (1.0 mL) and tri-n-
butylamine (1.7 mL) was added tri-o-tolylphosphine (4.3 mg,
3 H); 13C NMR (CDCl3, 100 MHz):
d
¼ 180.1, 178.2, 144.9, 116.3, 56.2,
48.9, 44.6, 42.7, 41.6, 39.5, 36.4, 36.2, 35.9, 34.7, 28.9, 28.0, 23.9, 23.8,
23.1, 22.8, 22.5, 21.6,18.4,18.3; MS (CI): m/z (%) ¼ 372 [M þ H]þ (100);
HRMS (EI): m/z ¼ 371.2864 [M]þ, calcd for C24H37NO3: 371.2824.
(3aR,3bS,5aR,6R,8aR,10aS)-6-((R)-1,5-Dimethylhexyl)-2,5a-dime-
thyl-3b,4,5,5a,6,7,8,8a,10,10a-decahydro-3aH-indeno[5,4-e]isoindol-
1,3-dione (14b). Dienophile: N-methylmaleimide. The product was
purified by silica column chromatography (hexane/ethyl acetate 4:1)
14
mmol), palladium(II) acetate (1.6 mg, 7.0
mmol) and allyl alcohol
(34
mL, 0.50 mmol). The reaction was performed in a sealed vessel in
a single-mode microwave reactor at a maximum output of 80 W,
a maximum temperature of 150 ꢁC and a reaction time of 10 min. The
resulting mixture was diluted with diethyl ether (20 mL) and washed
with water (2 ꢃ 20 mL) and brine (2 ꢃ 20 mL). The organic layer was
dried with MgSO4 and concentrated under reduced pressure. The
product was purified by silica column chromatography (hexane/ethyl
acetate 10:0 / 8:2) to give 54 mg (36%) 15 as yellow oil.
~
to give 0.22 g (78%) 14b as colourless oil. ½a D20
ꢂ
¼ ꢀ118.9; IR (KBr):
ꢀ1
;
1H NMR
~
n
¼ 3952, 1699, 1436, 1381, 1286, 1128, 1033, 990 cm
(CDCl3, 500 MHz):
d
¼ 5.37 (m, 1 H), 3.09e3.02 (m, 2 H), 2.90 (s, 3 H),
2.72 (dd, J ¼ 15.0/7.2 Hz, 1 H), 2.40e2.32 (m, 3 H), 2.13 (m, 1 H),
1.99e1.89 (m, 2 H), 1.78 (m, 1 H), 1.56e0.97 (m, 13 H), 0.90 (d,
J ¼ 6.2 Hz, 3 H), 0.88(d, J ¼ 6.6 Hz, 3 H), 0.86(d, J ¼ 6.6 Hz, 3 H), 0.50(s,
½
a 2D0
803 cmꢀ1
ꢂ
¼ þ28.9; IR (NaCl):
n
¼ 3328, 2956, 1467, 1377, 1181, 1082, 965,
;
1H NMR (CDCl3, 500 MHz):
d
¼ 6.01 (d, J ¼ 15.7 Hz, 1 H),
5.85 (dt, J ¼ 15.7/6.1 Hz, 1 H), 5.68 (dd, J ¼ 6.6/3.3 Hz, 1 H), 4.14 (d,
J ¼ 6.2 Hz, 2 H), 2.41 (sbr, 1 H), 2.33 (m, 1 H), 2.20e2.16 (m, 2 H),
2.01e1.93 (m, 2 H),1.74 (m,1 H),1.52 (m,1 H),1.45e1.08 (m, 10 H), 1.01
(m, 1 H), 0.94 (d, J ¼ 6.6 Hz, 3 H), 0.87 (d, J ¼ 6.6 Hz, 3 H), 0.86 (d,
3 H); 13C NMR (CDCl3, 125 MHz):
d
¼ 180.1, 178.1, 144.6, 115.9, 56.0,
48.6, 43.2, 42.5, 39.9, 39.2, 36.2, 35.9, 35.6, 34.5, 28.7, 27.8, 24.5, 23.7,
23.6, 23.1, 22.5, 22.3, 21.4, 18.2, 18.0; MS (CI): m/z (%) ¼ 386 [M þ H]þ
(100); HRMS (EI): m/z ¼ 385.2937 [M]þ, calcd for C25H39NO2:
385.2981.
J ¼ 6.6 Hz, 3 H), 0.70 (s, 3 H); 13C NMR (CDCl3, 125 MHz):
¼ 137.0,
d
133.8, 126.4, 125.9, 64.4, 54.0, 49.9, 42.7, 39.5, 36.2, 36.1, 35.9, 28.0,
27.9, 24.9, 24.1, 23.9, 22.8, 22.6, 18.8, 11.2; MS (CI): m/z (%) ¼ 305
[M þ H]þ (34), 287 (100); HRMS (EI): m/z ¼ 304.2791 [M]þ, calcd for
C21H36O: 304.2766.
(3aR,3bS,5aR,6R,8aR,10aS)-6-((R)-1,5-Dimethylhexyl)-2-ethyl-5a-
methyl-3b,4,5,5a,6,7,8,8a,10,10a-decahydro-3aH-indeno[5,4-e]isoin-
dol-1,3-dione (14c). Dienophile: N-ethylmaleimide. The product was
purified by silica column chromatography (hexane/ethyl acetate 4:1)
4-[(1R,3aR,7aR)-1-((R)-1,5-Dimethylhexyl)-7a-methyl-2,3,3a,6,7,
7a-hexahydro-1H-inden-4-yl]-but-3-yn-1-ol (16). To a solution of
triflate 2 [17] (0.35 g, 0.90 mmol) and diethylamine (1.5 mL) in
DMF (0.5 mL) copper(I) iodide (6.9 mg, 0.04 mmol), bis-
to give 0.12 g (41%) 14c as colourless oil. ½a D20
ꢂ
¼ þ 97.5; IR (KBr):
ꢀ1
;
1H NMR (CDCl3,
~
n
¼ 3446, 2952, 1697, 1457, 1403, 1228, 1138 cm
400 MHz):
d
¼ 5.44 (m, 1 H), 3.46 (q, J ¼ 7.3 Hz, 2 H), 3.07e2.94 (m, 2
H), 2.70 (dd, J ¼ 15.1/7.0 Hz, 1 H), 2.41e2.27 (m, 3 H), 2.11 (m, 1 H),
2.00e1.88 (m, 2 H), 1.76 (m, 1 H), 1.60e0.96 (m, 16 H), 0.90 (d,
J ¼ 6.1 Hz, 3 H), 0.88(d, J ¼ 6.6 Hz, 3 H), 0.86(d, J ¼ 6.6 Hz, 3 H), 0.50 (s,
(triphenylphosphine)palladium(II) dichloride (12.8 mg, 18.2 mmol)
and3-butyn-1-ol (0.07 mL,1.0 mmol)was added. The reaction mixture
was irradiated in a single-mode microwave reactor in a sealed vessel at
a maximum output of 300 W and a maximum temperature of 120 ꢁC
for 5 min. The resulting mixture was diluted with diethyl ether (20 mL)
and washed with water (2 ꢃ 20 mL) andbrine(2 ꢃ 20 mL). Theorganic
layer was dried with MgSO4 and concentrated under reduced pressure.
The product was purified by silica column chromatography (hexane/
3 H); 13C NMR (CDCl3, 100 MHz):
d
¼ 180.0, 178.0, 144.7, 116.1, 56.2,
48.9, 43.3, 42.7, 40.2, 39.5, 36.5, 36.2, 35.9, 34.8, 33.5, 28.9, 28.0, 24.0,
23.9, 23.2, 22.8, 22.5, 21.6, 18.4, 18.3, 13.1; MS (CI): m/z (%) ¼ 400
[M þ H]þ (100); HRMS (EI): m/z ¼ 399.3138 [M]þ, calcd for
C26H41NO2: 399.3137.
(3aR,3bS,5aR,6R,8aR,10aS)-6-((R)-1,5-Dimethylhexyl)-5a-methyl-
2-propyl-3b,4,5,5a,6,7,8,8a,10,10a-decahydro-3aH-indeno[5,4-e]iso-
indol-1,3-dione (14d). Dienophile: N-propylmaleimide. The product
was purified by silica column chromatography (hexane/ethyl acetate
ethyl acetate 10:0 / 9:1) to give 0.24 g (98%) 16 as yellow oil. ½a D20
ꢂ
¼ þ
¼ 3342, 2953, 1428, 1378, 1366 1044, 944 cmꢀ1; 1H
~
n
29.7; IR (NaCl):
NMR (CDCl3, 500 MHz):
d
¼ 5.90 (dd, J ¼ 6.8/3.3 Hz, 1 H), 3.71 (q,
J ¼ 5.7 Hz, 2 H), 2.57 (t, J ¼ 6.1 Hz, 2 H), 2.20e2.12 (m, 3 H), 2.01e1.88
(m, 2 H),1.80e1.73 (m, 2 H),1.52 (m,1 H),1.44e1.08 (m,10 H),1.00 (m,1
H), 0.93 (d, J ¼ 6.6 Hz, 3 H), 0.87 (d, J ¼ 6.6 Hz, 3 H), 0.86 (d, J ¼ 6.6 Hz, 3
4:1) to give 0.16 g (53%) 14d as colourless oil. ½a D20
ꢂ
¼ þ55.0; IR (KBr):
ꢀ1
;
1H NMR (CDCl3,
~
n
¼ 3373, 2956, 1699, 1404, 1205, 1045, 830 cm
400 MHz):
d
¼ 5.40 (m, 1 H), 3.40 (t, J ¼ 7.2 Hz, 2 H), 3.07e2.99 (m, 2
H), 0.68 (s, 3 H); 13C NMR (CDCl3,100 MHz):
d
¼ 133.2,122.1, 84.2, 82.8,
H), 2.73 (dd, J ¼ 15.1/7.0 Hz, 1 H), 2.38e2.27 (m, 3 H), 2.12 (m, 1 H),
61.3, 54.8, 50.0, 41.8, 39.5, 36.2, 36.1, 35.9, 28.0, 27.9, 25.0, 24.1, 23.9,
23.8, 22.8, 22.5,18.7,11.0; MS (CI): m/z (%) ¼ 317 [M þ H]þ (100); HRMS
(EI): m/z ¼ 316.2749 [M]þ, calcd for C22H36O: 316.2766.
2.01e1.89 (m, 2 H),1.77 (m,1 H),1.65e0.96(m,15 H), 0.93e0.85(m,12
H), 0.51 (s, 3 H); 13C NMR (CDCl3, 100 MHz):
d
¼ 180.3, 178.3, 144.8,
116.3, 56.2, 48.9, 43.3, 42.7, 40.2, 40.1, 39.5, 36.4, 36.2, 35.9, 34.8, 28.9,
28.0, 24.1, 23.9, 23.2, 22.8, 22.5, 21.9, 20.1, 18.4,18.3,11.2; MS (CI): m/z
(%) ¼ 386 [M þ H]þ (100); HRMS (EI): m/z ¼ 413.3342 [M]þ, calcd for
C27H43NO2: 413.3293.
4-[(1R,3aS,4RS,7aR)-1-((R)-1,5-Dimethylhexyl)-7a-methyl-octa-
hydroinden-4-yl]-butan-1-ol (17). Platinum(IV) oxide (15 mg,
0.17 mmol) was added to a solution of alkynol 16 (1.00 g, 3.10 mmol)
and ethyl acetate (30 mL). The reaction mixture was stirred at r.t. for
48 h under hydrogen atmosphere at ambient pressure. The reaction
mixture was filtered (celite) and concentrated under reduced pres-
sure. The raw product (0.95 g, 95%, colourless oil) was used directly
for the next reaction step without further purification.
General procedure for synthesis of N-substituted maleimides
18e20. Triphenylphosphine (0.83 g, 3.16 mmol) and maleimide
(0.31 g, 3.16 mmol) were dissolved in THF (12.6 mL). The corre-
sponding alcohol (15/16/17) (1.56 mmol) and diisopropyl azodi-
carboxylate (DIAD) (0.62 mL, 3.16 mmol) was added. The reaction
was performed in open flask fitted with a reflux condenser in
a single-mode microwave reactor. The reaction mixture was irra-
diated with a maximum output of 50 W and a maximum temper-
ature of 76 ꢁC for 30 min. After removal of the solvent, the product
was purified by silica column chromatography (hexane/ethyl
acetate 4:1) to yield the N-substituted maleimides 18e20.
(3bR,5aR,6R,8aR)-6-((R)-1,5-Dimethylhexyl)-2,5a-dimethyl-4,5,
5a,6,7,8,8a,10-octahydro-3bH-2,3a,10a-triazadicyclopenta[a,f]naph-
thalen-1,3-dione (14e). Dienophile: 4-methyl-1,2,4-triazoline-3,5-
dione. The product was purified by silica column chromatography
(hexane/ethylacetate4:1)togive0.22 g(78%)14e aswhitesolid. m.p.
ꢁ
~
n
150e151 C; IR (KBr):
¼ 3459, 2954, 1772, 1702, 1466, 1261, 1013,
759 cmꢀ1 1H NMR (CDCl3, 400 MHz):
;
d
¼ 5.43 (m, 1 H), 4.36e4.27
(m, 2 H), 3.91 (m, 1 H), 3.07 (s, 3 H), 3.01 (m, 1 H), 2.54 (m, 1 H),
2.00e1.70 (m, 5 H), 1.52 (sept, J ¼ 6.6 Hz, 1 H), 1.40e0.97 (m, 10 H),
0.90 (d, J ¼ 5.5 Hz, 3 H), 0.87 (d, J ¼ 6.6 Hz, 3 H), 0.86 (d, J ¼ 6.6 Hz, 3
H), 0.74 (s, 3 H); 13C NMR (CDCl3, 100 MHz):
d
¼ 155.3, 152.8, 138.6,
113.4, 57.5, 56.5, 48.4, 42.9, 40.5, 39.5, 35.9, 35.7, 35.6, 28.5, 28.0, 24.9,
24.2, 23.9, 23.4, 22.8, 22.5 (2 C), 18.7, 18.3; MS (CI): m/z (%) ¼ 388
[M þ H]þ (100); HRMS (EI): m/z ¼ 387.2918 [M]þ, calcd for
C23H37N3O2: 387.2885.

C.D. Mayer, F. Bracher / European Journal of Medicinal Chemistry 46 (2011) 3227e3236
3235
1-(E)-{3-[(1R,3aR,7aR)-1-((R)-1,5-Dimethylhexyl)-7a-methyl-
4.2.1. MTT assay
2,3,3a,6,7,7a-hexahydro-1H-inden-4-yl]-prop-2-enyl}-pyrrole-2,5-
Cytotoxicity of synthesized compounds was determined by MTT
test according to the method of Mosmann [28]. Solutions of the
dione (18). Yield: 20 mg (8%), pale yellow solid; m.p. 81e82 ꢁC;
~
½
a 2D0
972 cmꢀ1
ꢂ
¼ þ 22.7; IR (KBr):
n
¼ 2936, 1708, 1462, 1385, 1375, 1244,
compounds in DMSO (1 m
L, concentrations ranging from 10ꢀ9 to
;
1H NMR (CDCl3, 500 MHz):
d
¼ 6.70 (s, 2 H), 6.21 (d,
10ꢀ4 mol/L) were incubated with 99
mL of a suspension of HL 60
J ¼ 6.3 Hz, 1 H), 5.86e5.78 (m, 2 H), 4.16 (dd, J ¼ 5.5/1.4 Hz, 2 H),
2.23e2.08 (m, 3 H), 2.01e1.92 (m, 2 H), 1.68 (m, 1 H), 1.55e0.98
(m, 12 H), 0.91 (d, J ¼ 6.4 Hz, 3 H), 0.88 (d, J ¼ 6.5 Hz, 3 H), 0.87
(d, J ¼ 6.6 Hz, 3 H), 0.66 (s, 3 H); 13C NMR (CDCl3, 125 MHz):
cells (9 ꢃ 105 cells/mL) 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/mL) were added and the plate was incubated for another 2 h.
The cells were quenched with 190 L DMSO and after 1 h of
m
d
¼ 171.2 (2 C), 136.0 (2 C), 132.9, 131.2, 131.1, 124.7, 55.1, 49.3,
continuously 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).
40.9, 40.5, 39.4, 36.0, 35.9, 35.8, 28.0, 27.9, 25.2, 23.9, 23.8, 22.7,
22.6, 18.6, 11.4; MS (CI): m/z (%) ¼ 384 [M þ H]þ (100); HRMS
(EI): m/z ¼ 383.2836 [M]þ, calcd for C25H37NO2: 383.2824.
1-{4-[(1R,3aR,7aR)-1-((R)-1,5-Dimethylhexyl)-7a-methyl-2,3,3a,
6,7,7a-hexahydro-1H-inden-4-yl]-but-3-ynyl}-pyrrole-2,5-dione
(19). Yield: 0.27 g (44%), pale yellow solid; m.p. 81e83 ꢁC;
~
4.2.2. Assay for antibacterial and antifungal activities
½
a 2D0
692 cmꢀ1
ꢂ
¼ þ18.3; IR (KBr):
n
¼ 3453, 2954, 1703, 1444, 1384, 1147, 842,
All the work was performed under sterile conditions in
a laminar flow cabinet (HeraSafe 12, Heraeus Instruments, Hanau,
Germany). The bacteria and fungi (see Table 2) were cultivated on
an AC agar (SigmaeAldrich) except Aspergillus niger, which was
cultivated on a potato dextrose broth agar (SigmaeAldrich). Each
strain was spread evenly on the agar medium. To prepare the drug
formulations 1% (w/v) DMSO solutions of the testing compound
;
1H NMR (CDCl3, 400 MHz):
d
¼ 6.71 (s, 2 H), 5.86 (dd,
J ¼ 6.3/3.3 Hz, 1 H), 3.70 (t, J ¼ 7.0 Hz, 2 H), 2.57 (t, J ¼ 7.0 Hz, 2 H),
2.17e2.05 (m, 3 H), 1.99e1.87 (m, 2 H), 1.69 (m, 1 H), 1.50 (m, 1 H),
1.44e1.08 (m, 10 H), 1.00 (m, 1 H), 0.92 (d, J ¼ 6.5 Hz, 3 H), 0.87 (d,
J ¼ 6.6 Hz, 3 H), 0.86 (d, J ¼ 6.6 Hz, 3 H), 0.66 (s, 3 H); 13C NMR (CDCl3,
100 MHz):
d
¼ 170.5 (2 C), 134.1 (2 C), 133.3, 122.1, 83.5, 82.4, 54.7,
49.9, 41.8, 39.5, 36.8, 36.2, 36.1, 35.9, 28.0, 27.9, 25.0, 24.0, 23.9, 22.8,
22.6, 19.2, 18.7, 11.0; MS (CI): m/z (%) ¼ 396 [M þ H]þ (20), 163 (100);
HRMS (EI): m/z ¼ 395.2820 [M]þ, calcd for C26H37NO2: 395.2824.
1-{4-[(1R,3aS,4RS,7aR)-1-((R)-1,5-Dimethylhexyl)-7a-methyl-
octahydro-inden-4-yl]-butyl}-pyrrole-2,5-dione (20). Yield: 0.12 g
were made. Three paper disks were prepared with 5
mL of each test
solution. As a control a further paper disk was prepared with 5
mL of
DMSO. All paper discs were placed evenly separated on a culture
plate. The plates were covered and incubated for 36 h at 32 ꢁC
(bacteria) and 28 ꢁC (fungi), respectively. After incubation the
diameters of the zones of inhibition (defined as diameter of
inhibited bacterial growth) were measured in millimeters.
(20%), pale yellow solid; m.p. 78e80 ꢁC; ½a D20
ꢂ
¼ þ39.8; IR (KBr):
n
¼ 2936, 1708, 1467, 1408, 1375, 1244, 1098 cmꢀ1; 1H NMR (CDCl3,
~
500 MHz):
d
¼ 6.68 (s, 2 H), 3.51 (dt, J ¼ 7.3/3.0 Hz, 2 H), 1.90 (m, 1
H), 1.80e0.96 (m, 26 H), 0.91 (d, J ¼ 6.7 Hz, 3 H), 0.87 (d, J ¼ 6.6 Hz, 3
Acknowledgments
H), 0.86 (d, J ¼ 6.6 Hz, 3 H), 0.69 (s, 3 H); 13C NMR (CDCl3, 125 MHz):
d
¼ 170.9 (2 C), 134.1 (2 C), 57.1, 52.6, 42.2, 41.0, 39.5, 38.0, 37.0, 36.2,
The authors thank CEM GmbH for equipment support. For the
in vitro anti cancer screening of compounds the authors thank all
staff members of the National Cancer Institute (NCI), USA. We wish
to thank Maximilian Koppenwallner for technical support.
36.0, 35.6, 29.9, 28.8, 28.0, 27.6, 27.4, 23.8, 23.7, 22.8, 22.5, 20.7, 18.7,
14.0; MS (CI): m/z (%) ¼ 402 [M þ H]þ (100); HRMS (EI): m/
z ¼ 401.3250 [M]þ, calcd for C26H43NO2: 401.3294.
7-(4-Methylpent-3-enyl)pyridazino[1,2a]pyridazin-1,4(6H,9H)-
dione (22). The dienophile 3,6-pyridazinedione was prepared
freshly as follows: To a stirred suspension of 3,6-dihydroxypyr-
idazine potassium salt (1.00 g, 6.66 mmol) and acetone (25 mL)
at ꢀ78 ꢁC was added tert-butylhypochlorite solution (0.75 mL,
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.04.036.
6.66 mmol). After stirring for 3 h a solution of 21 (b-myrcene)
(1.00 mL, 6.00 mmol) was added. The reaction mixture was slowly
warmed at r.t., stirred for further 1 h and then quenched with water
(30 mL). The mixture was extracted with diethyl ether (3 ꢃ 20 mL).
The combined organic layers were washed with saturated sodium
chloride solution, dried over Na2SO4 and concentrated under
reduced pressure. The product was purified by silica column
chromatography (silica gel 60, ethyl acetae) to give 0.17 g (11%) 22
~
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as yellow solid. IR (KBr):
1334, 1136 cmꢀ1 1H NMR (CDCl3):
5.08 (s, 1 H), 4.51 (s, 2 H), 4.43 (s, 2 H), 2.19 (s, 4 H), 1.70 (s, 3 H), 1.61
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