1,2,3,4,8,9,10,11-Octahydrobenzo[j]phenanthridine-7,12-diones as new leads against Mycobacterium tuberculosis

Article abstract of DOI:10.1021/jm401735w

Tuberculosis (TB) continues to be a worldwide health problem with over 1.4 million deaths each year. Despite efforts to develop more effective vaccines, more reliable diagnostics, and chemotherapeutics, tuberculosis remains a threat to global health, fueled by the HIV pandemic and the rapid generation of drug resistance the exploration of novel drugs to serve as a companion drug for existing drugs is of paramount importance. As part of our program to design new 2-aza-anthraquinones with antimycobacterial activity, various tetrahydro- and octahydrobenzo[j]phenanthridinediones were synthesized these compounds showed high in vitro potency against Mycobacterium tuberculosis, the etiological agent of TB and against other clinically relevant mycobacterial species at submicromolar concentrations the susceptibility of a multidrug resistant strain toward these compounds and their ability to target intracellular replicating Mycobacterium tuberculosis was demonstrated. Next to the acute toxicity, the genotoxicity of these compounds was investigated. Often overlooked in studies, genotoxicity could be dismissed for the investigated compounds, making them a promising scaffold in TB drug research.

Full text of DOI:10.1021/jm401735w

Article
pubs.acs.org/jmc
1,2,3,4,8,9,10,11-Octahydrobenzo[j]phenanthridine-7,12-diones as
New Leads against Mycobacterium tuberculosis
Davie Cappoen,†,¶ Pieter Claes,‡,¶ Jan Jacobs,‡ Roel Anthonissen,⊥ Vanessa Mathys,§ Luc Verschaeve,⊥,∥
Kris Huygen,*,† and Norbert De Kimpe‡
†Service Immunology, O.D. Communicable & Infectious Diseases, Scientific Institute of Public Health (Site Ukkel), Engelandstraat
642, B-1180 Ukkel, Belgium
‡Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links
653, B-9000 Ghent, Belgium
§Program Tuberculosis and Mycobacteria, Service Bacterial Diseases, O.D. Communicable & Infectious Diseases, Scientific Institute
of Public Health (Site Ukkel), Engelandstraat 642, B-1180 Ukkel, Belgium
⊥Service Toxicology, O.D. Public Health and Surveillance, Scientific Institute of Public Health (Site Elsene), J. Wytsmanstraat 14,
B-1050 Brussels, Belgium
∥Department of Biomedical Sciences, Antwerp University, Universiteitsplein 1, B-2610 Wilrijk, Belgium
S
* Supporting Information
ABSTRACT: Tuberculosis (TB) continues to be a worldwide
health problem with over 1.4 million deaths each year. Despite
efforts to develop more effective vaccines, more reliable
diagnostics, and chemotherapeutics, tuberculosis remains a
threat to global health, fueled by the HIV pandemic and the
rapid generation of drug resistance. The exploration of novel
drugs to serve as a companion drug for existing drugs is of
paramount importance. As part of our program to design new 2-
aza-anthraquinones with antimycobacterial activity, various
tetrahydro- and octahydrobenzo[j]phenanthridinediones were
synthesized. These compounds showed high in vitro potency
against Mycobacterium tuberculosis, the etiological agent of TB
and against other clinically relevant mycobacterial species at
submicromolar concentrations. The susceptibility of a multidrug
resistant strain toward these compounds and their ability to target intracellular replicating Mycobacterium tuberculosis was
demonstrated. Next to the acute toxicity, the genotoxicity of these compounds was investigated. Often overlooked in studies,
genotoxicity could be dismissed for the investigated compounds, making them a promising scaffold in TB drug research.
any known antibiotic that usually targets an active metabolism
and replication.7−9 This not only complicates the development
of new drugs but also current treatment, which takes
considerable time and relies on access to high quality
antibiotics.
Lack of compliance results in the generation of drug resistant
strains: multidrug resistant (MDR) strains with resistance to
both isoniazid and rifampin and extensively drug resistant
(XDR) strains with additional resistance to fluoroquinolones
and one of the injectable aminoglycoside or polypeptide
antibiotics.10−12 Fueled by the HIV/AIDS pandemic, tuber-
culosis is not a mere disease from the past, and this fact can be
found in the report and figures presented by the World Health
Organization (WHO) in 2013. This report stated that 8.6
million people were infected with TB in 2012, of which 450 000
INTRODUCTION
■
The history of tuberculosis drug development is a story of great
success and failure. Despite the undeniable impact of
tuberculosis on global mortality, the discovery and develop-
ment of antibiotics against Mycobacterium tuberculosis (M.tb)
has been insufficient to eradicate the disease completely. Due to
close host−pathogen coevolution, M.tb has grown to a highly
resilient organism.1−3 In an arms race with its host,
mycobacteria have developed a thick lipid-rich cell wall as a
defense against xenobiotic and harmful effector molecules
unleashed by their host. It is the same mycobacterial cell wall
that poses an impermeable barrier and is responsible for the
high tolerance of M.tb to many commonly used antibiotics.4−6
After promoting its own phagocytosis within the host, M.tb can
proliferate within macrophages or enter into a dormant state
depending on the conditions. Adapting metabolic quiescence
not only helps the bacilli to survive harmful microenvironments
within the host but also makes them virtually invulnerable for
Received: November 12, 2013
Published: March 10, 2014
© 2014 American Chemical Society
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Scheme 1. Benzophenanthridinediones 1, Tetrahydrobenzophenanthridinediones 2, 3 & 4 and
Octahydrobenzophenanthridinediones 5
Scheme 2. Synthesis of Tetrahydrobenzophenanthridinediones 3 and 4
had multidrug-resistant pulmonary TB. Of those infected, 1.3
million patients died, including 320 000 people that were TB/
HIV coinfected.13,14
RESULTS AND DISCUSSION
■
Chemistry. Pyranonaphthoquinones can be synthesized by
means of the addition of a Stork enamine to a 2-(1-
hydroxyalkyl)-1,4-naphthoquinone followed by aerobic oxida-
tion.19 Thus, the synthesis of tetrahydrobenzophenanthridine-
diones 3 and 4 was envisaged starting from the addition of
Stork enamines 7 to 2-(1,3-dioxolan-2-yl)-1,4-naphthoquinone
6a20 and 2-acetyl-1,4-naphthoquinone 6b followed by ammo-
nia-mediated conversion of the enamine adduct and aerobic
oxidation toward the corresponding 2-aza-anthraquinones 3 or
4.21 Even though a precedent of this methodology exists in the
literature,20 we could not reproduce this protocol, even upon
several trials. Therefore, several enamines and conditions for
the ammonia-mediated ring opening−ring closure were
investigated, but no satisfying conditions were found.22 The
best results were obtained using 10 equivalents of 7 M NH3 in
MeOH as the nitrogen source. Using these conditions, a set of
D-ring-substituted derivatives 3 and 4, bearing either a methyl
substituent or no substituent at C-6 were synthesized in low
yields (Scheme 2). When these compounds were tested against
Mycobacterium tuberculosis, no significant activity was observed.
However, it was found that the compounds 3 with no
substitution at C-6 were more active than the corresponding
During a previous study, various benzo[j]phenantridine-7−
12-diones 1 were tested against Mycobacterium tuberculosis. It
was found that 3-methylbenzo[j]phenanthridine-7,12-dione 1a
showed promising antimycobacterial activity (MIC = 0.64 μM).
However, this activity was accompanied with a relatively high
cytotoxicity (IC50 = 3.21 μM), resulting in a selectivity index of
only 15. As it is thought that this cytotoxicity is due to potential
intercalation resulting from the flat nature of compounds 1, the
synthesis of more “out of plane” derivatives such as 1,2,3,4-
tetrahydrobenzo[j]phenanthridinediones 3 and 4 with an
aliphatic D-ring was envisaged. Structurally similar compounds
2, bearing a carbonyl function on the 1-position, have been
evaluated as antitumor compounds.16 These compounds were
synthesized by means of an oxidative addition17 or a hetero
Diels−Alder methodology.18 In a next step, the synthesis of
octahydrobenzo[j]phenathridinediones 5 with an aliphatic A-
and D-ring was envisaged (Scheme 1).
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Scheme 3. Synthesis of the Starting 2-(1,3-Dioxolan-2-yl)-1,4-naphthoquinone 10
Scheme 4. Synthesis of Octahydrobenzo[j]phenanthridinediones 5a−5g
methyl-substituted derivatives 4. Therefore, the synthesis of 2-
aza-anthraquinones with an aliphatic A- and D-ring and no
substitution at C-6 was envisaged.
The synthesis of methano-, ethano-, and epoxy-bridged
octahydrobenzo[j]phenanthridinediones 5 was envisaged using
the above-mentioned methodology. The starting 2-(1,3-
dioxolan-2-yl)-1,4-naphthoquinone 10 were prepared starting
from 1,4-dimethoxy-5,6,7,8-tetrahydronaphthalene-2-carboxal-
dehydes 8.23 These aldehydes 8 were converted to acetal by
reaction with ethylene glycol followed by oxidative demethy-
lation with cerium(IV) ammonium nitrate (CAN) to afford 6-
(1,3-dioxolan-2-yl)-1,2,3,4-tetrahydronaphthalene-5,8-diones
10a and 10b. As these compounds only had a limited stability,
they had to be used immediately in the next step. 2-(5,8-
Dimethoxy-1,4-dihydro-1,4-epoxynaphthalen-6-yl)-1,3-dioxo-
lane 10c could not be prepared using this methodology and was
prepared by means of a Diels−Alder reaction of 2-(3-bromo-
2,5-dimethoxyphenyl)-1,3-dioxolane with furan 11. Hydro-
genation of the isolated double bound followed by oxidative
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demethylation with CAN yielded the desired quinone 10c in
81% yield (Scheme 3).
Table 1. Growth Inhibition of M.tb by
Octahydrobenzo[j]phenanthridinediones and Their Acute
No reaction was observed when enamine 7c was added to 6-
(1,3-dioxolan-2-yl-1,2,3,4-tetrahydro-1,4-methanonaphthalene-
5,8-dione 10a under the above-mentioned conditions. After
investigation of the reaction conditions,22 it was found that
octahydrobenzophenanthridinediones 4 could be synthesized
upon addition of 10 mol % of BF3·OEt2 to the reaction mixture.
In the case of 6-(1,3-dioxan-2-yl)-1,2,3,4-tetrahydro-1,4-meth-
anonaphthalene-5,8-dione 10b, ammonium acetate in HOAc
had to be used to form the desired octahydrobenzo-
phenanthridine 5b. For O-bridged tert-butyl-substituted de-
rivative 4c, the ring-opening step was performed with 7 M NH3
in MeOH as the reaction in acetic acid led to ring-opening of
the epoxy-bridge (Scheme 4). Starting from ethyl 4-(pyrrolidin-
1-yl)cyclohex-3-enecarboxylate 13, two ester-substituted de-
rivatives 5d and 5e were synthesized. The reaction with 6-(1,3-
dioxolan-2-yl)-1,2,3,4-tetrahydro-1,4-epoxynaphthalene-5,8-
dione 10c resulted in a complex reaction mixture. Next,
substitution of a carbon with nitrogen was envisaged. When the
enamine addition was performed with 1-methyl-4-(pyrrolidin-
1-yl)-1,2,3,6-tetrahydropyridine 14a, a complex reaction
mixture was obtained for both quinones 10a and 10b. Upon
treatment of quinone 10a with 1-tert-butoxycarbonyl-4-
(pyrrolidin-1-yl)-1,2,3,6-tetrahydropyridine 14b, 2-tert-butoxy-
carbonyl-1,2,3,4,8,9,10,11-octahydro-2-aza-8,11-methanobenzo-
[j]phenanthridine-7,12-dione 5g was isolated in 17% yield. Due
to the low yields of the addition reaction, no further
derivatization of the obtained compounds was attempted.
Biology. Following the synthesis described here, the series
of pentacyclic quinones 3a−3e, 4a, 4d, 4e, and 5a−5g were
tested for their antimycobacterial activity against Mycobacterium
tuberculosis. A model based on a luminescent M.tb H37Rv strain
(H37Rvlux) was used. As reported previously in the literature,
this technique offers a sensitive and reproducible tool, able to
replace enumeration of bacteria by fastidious plating on
agar.24−27 Antitubercular properties of the synthesized
compounds are reported by a reduction of luminescence
emitted by a culture exposed to the compound, compared with
a negative control culture. After 6 days of exposure of M.tb
H37Rvlux to serial dilutions of the compounds, the potency was
calculated as the minimal inhibitory concentration (MIC) at
which the mycobacterial growth is reduced by 99%. In parallel,
the acute toxicity of the analogues against eukaryotic J774 A.1
cells, a murine macrophage like monocyte cell line was studied
in a neutral uptake assay. The macrophage model was chosen as
macrophages are the primary host cell for M.tb in a tuberculosis
infection. The neutral red uptake assay relies on the ability of
viable cells to bind and incorporate the neutral red dye.28 The
acute toxic concentration (IC50) of a compound is defined as
the concentration at which the uptake of the neutral red dye by
the cells is reduced by 50%. By dividing the IC50 with the MIC,
the selectivity index (SI) can be calculated.
Toxicity against Macrophages
a
b
c
compd
MIC
IC50
SI
(μM)
3a
3b
3c
3d
3e
4a
4d
4e
5a
5b
5c
5d
5e
5g
9.88
9.61
9.64
5.3
7.34
1.31
0.74
0.14
1.19
1.53
1.03
0.61
2.61
1.74
11.49
8.13
14.43
17.79
9.85
17.68
0.59
8.56
7.21
0.22
0.26
12.88
0.13
0.19
14.91
10.9
25.68
30.79
51.35
22.95
53.23
8.49
87.03
2.68
7.38
38.59
191.38
0.57
49.76
7.33
g
INH
>100
>100
>769.23
>526.31
e
RIF
a
Minimal inhibitory concentration (MIC) at which 99% growth
inhibition of the M.tb H37Rvlux lab strain was observed as calculated
b
from triplicate cultures (SD values <10%). Inhibitory concentration
(IC50) at which viability of the J774 A.1 macrophages was reduced by
50% as calculated from triplicate cultures (SD values <10%).
c
g
e
Selectivity index (SI), calculated as IC50/MIC. INH, RIF the first
line M.tb antibiotics, isoniazid and rifampin, used as a positive control.
μM, SI = 1.53). This reinforced the notion of importance for
substitution of the annellated ring of the 2-aza-anthraquinones.
Further attempts to optimize the structure by methyl
substitution at the C-6 backbone position were unsuccessful.
Compounds 4a−4e showed further decreased toxicity, but this
went along with a significantly lower activity against M.tb.
Compound 4d was the most active one, again bearing a methyl
substitution at the meta position of the annellated aliphatic ring
(MIC = 9.85 μM, IC50 = 25.68 μM, SI = 2.61).
In the next step, methano-, ethano-, and epoxy-bridged
octahydrobenzo[j]phenanthridinediones 5 were submitted to
biotesting. Bridging the ring in compounds 5a−5c did have an
impact on the acute toxicity of compound 3c with a 2-t-Bu
substitution at the para position. The methano-bridged
derivative 5a showed not only increased activity but also
lower acute toxicity, resulting in a more favorable SI (MIC =
0.59 μM, IC50 = 51.35 μM, SI = 87.03). In the case of the
ethano-bridged compound 5b, both toxicity and activity were
lower (MIC = 8.56 μM, IC50 = 22.95 μM, SI = 2.68). This was
also the case for epoxy-bridged derivative 5c (MIC = 7.21 μM,
IC50 = 53.23 μM, SI = 7.38). Two ester analogues 5d and 5e of
the bridged compounds were tested as well. Compound 5d
with the methano-bridge (MIC = 0.22 μM, IC50 = 8.49 μM, SI
= 38.59) showed an in vitro potency against M.tb close to the
potency of isoniazid (INH), tested as a positive control in the
present model and a well-known first line drug in tuberculosis
chemotherapy. Although acute toxicity could be observed at
relatively low concentrations, the potency at low concentrations
resulted in an acceptable SI. Ethano-bridged derivative 5e
displayed a lower activity than 5d but with a mild toxicity for
the J774 A.1 macrophages (MIC = 0.22 μM, IC50 = 49.76 μM,
SI = 191.38). In summary, compound 5e was the most
promising compound showing the most favorable properties.
Tetrahydrobenzophenanthridinedione 3a (MIC = 9.88 μM,
IC50 = 7.34 μM, SI = 0.74) with an aliphatic D-ring did not
show an increase in SI over the previous reported benzo[j]-
phenantridine-7−12-diones (Table 1).15 Although a decreased
toxicity was observed, the activity against M.tb was lower as
well. By alkyl substitution of the cyclohexane ring, two
compounds with improved activity were found: derivative 3c
with a 2-t-Bu substitution at the para position (MIC = 9.64 μM,
IC50 = 11.49 μM, SI = 1.19) and derivative 3d with a methyl
substitution at the meta position (MIC = 5.30 μM, IC50 = 8.13
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Compound 5g, a methano-bridged analogue bearing a tert-
butoxycarbonyl protecting group at the para position, only
showed low activity against M.tb, and IC50 was also very low
(MIC = 12.88 μM, IC50 = 7.33 μM, SI = 0.57).
In order to obtain information on the metabolic stability, an
in vitro assay was set up in which the compounds 5a, 5d, and
5e were exposed to S9 liver extract mixture. After 15 and 30
min of incubation, serial dilutions of the compounds exposed to
the S9 mixture were incubated with M.tb. After 6 days, the
antitubercular activity was investigated. As shown in Figure 1,
be linked to Crohn’s disease in humans.32 M. ulcerans causes
Buruli ulcer, a necrotizing skin disease affecting mostly children
in certain rural areas of West-Africa and may lead to irreversible
disabilities when left untreated.33 M.tb Beijing isolate 17919
showed the same susceptibility as M.tb H37Rv for the three
tested compounds (Table 2). The same was true for the
selected M. bovis strain. The two M. avium subspecies showed a
somewhat lower susceptibility. Finally, the MIC of the three
compounds against M. ulcerans 1615lux was found to be
significant higher than the MIC for M. tuberculosis, with IC90
values, respectively, 6-fold higher for compound 5a, 10-fold
higher for compound 5e, and even 50-fold higher for
compound 5d (data not shown).
To study the potential of the compound class for the
treatment of drug resistant tuberculosis, the susceptibility
toward the compounds of a multidrug resistant M.tb LAM-1
strain was tested, as shown in Figure 2. The LAM-1 strain was
spoligotyped, and resistance against isoniazid, rifampin,
rifabutine, and prothionamide was confirmed by the National
Reference Laboratory of Belgium. The critical concentration at
†
Minimal inhibitory concentration of compounds 5a, 5d, and 5e
against M.tb measured after 6 days following 15 and 30 min exposure
to S9 mixture. Presented values in the graph are the mean of eight
replications with SD.
1
MIC against M.tb of compounds 5a, 5d, 5e after exposure to
†
S9 mixture
no significant reduction in potency of compounds 5a, 5d, and
5e was observed after an incubation of 15 min with the S9
mixture. However, after 30 min, the MIC of compounds 5a, 5d,
and 5e increased significantly from 0.60 μM to 1.05 μM, from
0.21 μM to 0.66 μM, and from 0.26 to 0.65 μM, respectively.
The results indicate that the compounds were subjected to
enzymatic degradation by the S9 liver extract, resulting in a
decreased potency. As a negative control, we confirmed that the
growth of M.tb H37Rv was not inhibited by the presence of the
S9 mixture itself.
The susceptibility of other clinically relevant mycobacterial
species and an additional M.tb stain, M.tb Beijing 17919, to
compounds 5a, 5d and 5e is shown in Table 2. The M.tb
Table 2. In Vitro MIC of Compounds 5a, 5d, and 5e against
Other Clinically Relevant Mycobacteria
MIC (μM)
5a
5d
5e
INH
M. tuberculosis H37Rvlux
M. tuberculosis Bejing 17919lux
M.bovis AN5lux
0.56
0.33
0.78
0.69
0.11
0.14
0.09
0.44
0.22
0.16
0.57
0.21
0.13
0.09
0.08
0.99
M. avium subsp. paratuberculosis
ATCC19698lux
M. avium subsp. avium ATCC15769lux
0.8
1.22
0.37
0.76
In vitro growth inhibition was measured by luminometry after 6 days
exposure to the compounds. Inhibition calculated from triplicates
cultures (SD values <10%).
Beijing 17919 is a wild type strain originating from a clinical
isolate. M.tb Beijing strains are generally considered to be more
virulent than the M.tb H37Rv labstrain with possible
implications for drug susceptibility.29 M. bovis, together with
M. africanum and M. microti, constitute the M. tuberculosis
complex.30 M. avium subsp. avium is pathogenic for birds but
can cause opportunistic infections in AIDS patients,31 whereas
M. avium subsp. paratuberculosis is the etiological agent of
Johne’s disease or paratuberculosis (in ruminants) which may
Growth of MDR M.tb was monitored in BACTEC MGIT 960.
Growth was compared to an untreated bacterial suspension and an
untreated bacterial suspension diluted 100 times (C/100) to study the
minimal inhibitory concentration MIC. Resistance was confirmed with
isoniazid (INH). Mfx moxifloxacin was used as a positive control at
1.14 μM.
2
Susceptibility of an MDR M.tb LAM-1 strain to the
compounds 5a, 5d, and 5e.
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which the mycobacterial growth is reduced by 99% was
determined with fluorometric BACTEC MGIT 960 Mycobac-
teria Growth Indicator Tubes.34 The growth of a control
culture, 100 times diluted upon inoculation (C/100), was
measured simultaneously. By comparing the growth index (GI)
of this culture with the cultures exposed to the compound, the
critical concentration could be determined. At day 6, the C/100
had reached a GI of 890. For compound 5a, the GI at 0.50 μM
was 1982, whereas at 2.50 μM, the GI was 537, placing the
critical concentration of 5a between these two concentrations
and confirming the susceptibility of the MDR strain for the
derivative. Compound 5d was more active with a GI of 1970 at
0.25 μM and a GI of 367 at 0.5 μM. The critical concentration
of 5e was slightly less active than 5d with a GI of 2382 at 0.25
μM and a GI of 638 at 0.5 μM.
For most of its infectious cycle, M.tb resides within the early
endosome of the macrophages where it replicates.35 Therefore,
it was important to investigate whether the selected
compounds, 5a, 5d, and 5e were able to reach the bacteria
through the macrophage membrane. For this reason, J774 A.1
murine macrophage-like monocytes were infected with the M.tb
H37Rvlux strain. At 24 h post infection, the infected
macrophage monolayers were treated with serial dilutions of
the compounds. After 1 to 4 days, the monolayers were washed,
and the number of luminescent bacteria was measured in the
cell lysate.
As shown in Figure 3, at low concentrations of 0.03 and 0.05
μM, the selected compounds 5a, 5d, and 5e had no effect. At
0.25 and 0.50 μM, the bacterial replication was reduced by,
respectively, 43.83 6.41% and 52.65 7.37% for 5a and by
63.88 6.79 and 86.66 0.79% for compound 5d, whereas at
the same concentrations, compound 5e reduced the intra-
cellular growth by, respectively, 55.8 8.79 and 94.33 8.03%.
The MIC in this assay was reached at 2.48 μM for compound
5a, and when dividing the IC50 by this value, a SI of 20.73 was
calculated. Compound 5d showed a similar MIC at 2.38 μM,
but as a consequence of its lower IC50, the calculated SI was
10.75. Finally, the potency of 5e to inhibit intracellular M.tb
replication was similar, with a MIC at 2.41 μM, resulting in a
calculated SI of 79.31.
Growth inhibition of M.tb H37Rv inside J774 A.1 macrophages.
Results are presented as mean
SEM RLU of triplicate cultures.
The possibility of genotoxicity is often overlooked in the
initial phase of screening research and drug development.
Nevertheless, for treatment of TB, which can take 6 months to
2 years, it is crucial that genotoxicity is ruled out. For economic
reasons, genotoxic compounds are better discarded before
more costly assays are envisaged in an early stage. The
possibility of genotoxicity caused by the octahydrobenzo-
phenanthridinediones 5a, 5d, and 5e or their metabolites was
studied in a VITOTOX assay from Gentaur, as shown in Figure
4.36,37 Briefly, this model makes use of two recombinant
Salmonella typhimurium reporter strains, the TA104 recN2-4
(Genox strain) and TA104 pr1 (Cytox strain). The Cytox
strain expresses episomally encoded bacterial luciferase. A
reduction in the signal/noise of the luminescent signal indicates
cytotoxicity, whereas an increase indicates interference of the
compound with the luminescent signal itself. The Genox strain
carries an integrated lux operon under transcriptional control of
the recN promoter. When a compound is genotoxic, tran-
scription of the DNA repair mechanism will lead to an increase
of the luminescent signal. Addition of rat liver S9 fraction is
used to mimic the mammalian metabolic conditions so that the
mutagenic potential of metabolites derived from a parent
compound in the hepatic system can be assessed. The
Treatment of the infected cells started 24 h post infection and lasted
for 3 days. The first line drug INH was used as a positive control at 0.1
μM.
Inhibition of intracellular replication of M.tb inside macro-
phages by compounds 5a, 5d, and 5e.
3
luminescence is measured for 4 h with a 5 min interval, and
generally, when the signal-to-noise ratio in the Cytox strain
model is reduced below 0.8, the compound is regarded as
cytotoxic for Salmonella typhimurium, and the genotoxicity
cannot be studied. On the other hand, when the signal-to-noise
ratio in the Genox strain model is above 1.5, the DNA repair
mechanism is activated by the cell as an early response to
genotoxicity by the compound.
Serial dilutions of the compounds 5a, 5e from 0.25 μM to
50.00 μM were tested but for compound 5d the serial dilution
was limited form 0.25 μM to 5.00 μM to avoid exceeding the
IC50 of the compound. The tested concentrations of the
compounds did not reduce the signal-to-noise ratio of the
treated Cytox cultures below 0.8. This was also the case after
addition of the S9 liver extract in the TA104 pr1 strain,
indicating lack of toxicity toward the S. typhimurium model and
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Genomic DNA fragmentation after 24 h exposure to different
concentrations of compounds 5a, 5d, and 5e. The results show the
mean % of genomic DNA detected in the comet tail of 200 stained
nuclei. The Mann−Whitney U test was used to calculate statistical
significance.
5
Detection of DNA fragmentation by the Comet assay.
significant increase of genomic DNA in the TAIL was detected.
It was concluded that there was no DNA fragmentation caused
by compounds 5a, 5d, or 5e observable with the comet assay.
CONCLUSION
■
The presented library of annellated 2-azaanthraquinone
derivatives in this study represents a promising group of
antitubercular compounds. Not only did the compounds show
an increased potency compared to previous reported benzo-
[j]phenantridinediones, they also showed reduced acute
toxicity. The best compounds reported in the study were 5a
(MIC = 0.59 μM, IC50 = 51.35 μM, SI = 87.03), 5d ((MIC =
0.22 μM, IC50 = 8.49 μM, SI = 38.59), and 5e (MIC = 0.22 μM,
IC50 = 49.76 μM, SI = 191.38). The observed potency of these
compounds against MDR M.tb and intracellular replicating M.tb
encourage further study of this compound class. Combined
with the lack of observable in vitro genotoxicity, this compound
class would be suited for PK studies and a detailed in vivo
dose−response study. To counter metabolic instability, the
addition of protective groups to the scaffold is envisaged. The
SI and absence of observable toxicity against the Salmonella
typhimurium reporter strain in the VITOTOX suggest a certain
degree of selectivity of the investigated compounds. The
antimicrobial activity against other bacterial strains will be
tested in a continuation of the project. Furthermore we will
investigate the precise mechanism of action of the most potent
compounds 5a, 5d, and 5e.
Maximum recorded S/N in a time span of 4 h by the Genox (recN 2−
4) and Cytox (pr1) reporter strains. 4NQO; 4-nitroquinolone 1-oxide
genotoxic positive control in samples without S9 liver fraction. Bap;
benzo[a]pyrene, the positive control, only turns genotoxic after S9
metabolization.
VITOTOX assay for the detection of early signs of
genotoxicity caused by the investigated new compounds.
4
enabling to test for genotoxicity. The signal-to-noise ratio of the
luminescence emitted by the Genox strain did not exceed 1.5
before and after addition of the S9 liver extract. At the tested
concentrations, no activation of the SOS DNA repair
mechanism or early signs of genotoxicity elicited by the
compounds or their possible metabolites could be observed.
Intercalation or interaction of compounds with the genomic
DNA can have serious mutagenic consequences for the cell,
causing DNA breakage and fragmentation. The possibility of
DNA breakage caused by compounds 5a, 5d, and 5e was
investigated in a Comet assay.38 Briefly, JJ74 A.1 cells were
exposed to serial dilutions of the octahydrobenzophenan-
thridinediones 5. Possible DNA fragmentation caused by the
compound was measured by submitting the cell nuclei to
electrophoresis. Through pore exclusion of agarose, small
fragments will migrate further (COMET TAIL) than larger
fragments and unfragmented genomic DNA (COMET
HEAD). The ratio DNA in TAIL to DNA in HEAD is a
measure of DNA fragmentation. As shown in Figure 5, a serial
dilution of compounds 5a and 5e was tested, ranging from 0.25
μM to 50.00 μM. Due to acute toxicity of compound 5d, the
serial dilutions of this compound for the Comet assay was
limited from 0.25 μM to 5.00 μM. The test results were
submitted to a Mann−Whitney U probability test, and no
EXPERIMENTAL SECTION
■
General Biology. Materials. 7H9 growth medium was purchased
as a powder from BD Science (Franklin Lakes, NJ). DMEM, glutamax,
nonessential amino acids, sodium pyruvate, gentamycin, 2-mercaptoe-
thanol, and PBS were from GIBCO Invitrogen (Carlsbad, CA).
OADC, mycobactin J, penicillin, fungizone, and hygromycin were from
Roche (Basel, CH), and Triton X-100, glycerol, Tween 80, and n-
decanal were purchased from Sigma Aldrich (St. Louis, MO)
Strains and Growth Conditions. Mycobacterium tuberculosis H37Rv
(American Type Culture Collection 27294) is known to be sensitive to
the five first line antituberculosis drugs (streptomycin, isoniazid,
rifampin, ethambutol, and pyrazinamide). The LAM-1 strain is a
clinical isolate from a patient diagnosed with TB. This strain has been
spoligotyped for identification and characterized for antibiotic
resistance by the national TB reference lab of the Institute of Public
Health of Belgium. With the resistance to isoniazid, rifampin, rifabutin,
and prothionamid, this strain was classified as a multidrug resistant
(MDR) strain. Four other strains used for screening were M. bovis
strain AN5, M. avium ssp. avium ATCC 15769, M. avium ssp.
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paratuberculosis ATCC19698, and M. ulcerans 1615. All strains were
cultivated in 7H9 medium supplemented with 10% oleic acid−
albumin−dextrose−catalase (OADC) and 0.2% glycerol. For M. bovis,
the glycerol was replaced with 0.05% Tween 80. The growth medium
of M. avium ssp. paratuberculosis was further supplemented with
mycobactin J (2 μg/mL) required for optimal in vitro growth. Both M.
avium strains and the M. bovis strain were grown at 39 °C,whereas M.
ulcerans and M.tb were grown at 32 and 37 °C, respectively.
Transforming Mycobacteria. The general procedure for trans-
formation of the different mycobacterial species is to cultivate until an
O.D. between 0.8 and 1.0 was reached. The cultures were centrifuged
at 6000 rpm for 15 min, and the pellet was dissolved in 20 mL of 10%
glycerol. This step was repeated, but the pellet was dissolved in
decreasing volumes of 10% glycerol until a volume of 5 mL. This 5 mL
suspension was incubated together with the 10 ng/mL pSMT1
plasmid harboring the lux AB genes of Vibrio harveyi under control of a
mycobacterial specific promoter for 1 h at 37 °C. Afterwards, the
bacterial suspension was transferred to a cuvette and electroporated at
1000−1500 Ω and a time constant of 1.0 to 10.0 s. The cultures were
left to recover for 2−3 h, after which they were plated on selective
7H11 agar plates. After 2−3 weeks of incubation, isolates were selected
from the plate, grown, and tested for luminescene. After electro-
poration and selection, the M.tb Beijing strain was spoligotyped by the
Belgian reference lab to confirm the strain.
Monitoring Mycobacterial Growth by Luminometry. The minimal
inhibitory concentration (MIC) against mycobacteria of all synthe-
sized compounds was evaluated by testing serial dilutions. The in vitro
assay was based on a method in which luminescent mycobacteria
transformed with pSMT1 luciferase reporter plasmid is used. The
tested compounds were solubilized in DMSO (Sigma-Aldrich) at stock
concentrations of 10 mM. Serial dilutions of each compound were
made in liquid 7H9 medium [Middlebrook 7H9 broth based (Difco)]
+ 10% FCS (Gibco). Volumes of 20 μL of the serial dilutions were
added in triplicate to 96-well, flat-bottomed micro-well plates. The
bacterial suspension was made by thawing and dissolving a frozen
mycobacteria pellet in 7H9−10% FCS. The dissolved pellet was
passed through a 5.0 μM filter (Millipore) to eliminate clumps and left
for 1 h to recover at 37 °C, 5% CO2. Next, the bacterial suspension
was diluted in 7H9−10% FCS to obtain 50 000 relative light units
(RLU)/mL and a volume of 180 μL of bacteria was added to each
well. A bacterial replication was analyzed by luminometry after 6 days
of incubation. The bacterial suspension from each well was collected,
and transferred to a black 96-well plate to evade cross luminescence
between wells. The luminescent signal was evoked by addition of the
substrate for the bacterial luciferase, 1% n-decanal in ethanol to each
well by the multi plus reader from Promega and the light emission in
each well was measured.
Activity Post Exposure to S9 Extract. S9 mixture from Sigma-
Adrich is conditioned by addition of reagents A (NADPH) and B (G-
6-P), as proposed by the manufacturer. The compounds (100 μM)
were incubated with S9 mixture at 37 °C, 5% CO2 for 15 and 30 min.
In the next step, serial dilutions of both the S9 conditioned and S9
unconditioned compound solutions were made, and the MIC against
M.tb H37Rv was investigated as outlined above.
Activity against Multidrug Resistant M.tb. Antimicrobial activity of
the compounds was tested using the LAM-1 strain using the BACTEC
MGIT 960 TB detection system. The compounds were solubilized in
DMSO at stock concentrations of 1 mg/mL. Serial dilutions of each
compound were made in 7H9 containing 10% OADC, at 83-fold the
final concentrations. M.tb LAM-1 was precultured in a 4 mL BACTEC
vial to a growth index (GI) of 300. Then, 100 μL of this preculture was
inoculated into a new 4 mL BACTEC vial together with 100 μL of the
serial dilutions of the compounds. As a positive control for the
resistance, the MDR LAM-1 culture was inoculated with 0.1 μM
isoniazid. The GI was measured each day. To determine the IC99, the
cultures treated with the compounds were compared with an untreated
culture diluted 100 times upon inoculation.
complete DMEM medium until a semi-confluent layer was formed.
The macrophages were washed in fresh complete DMEM medium and
seeded in a flat-bottomed 96-well microwell plate at a cell density of
40 000 cells per well. The cells were left to recover overnight and were
washed three times in complete DMEM medium. M.tb H37Rvlux was
grown at 37 °C in 7H9 containing 10% FCS and 0.2% hygromycin to
an OD580 of 0.6−1.0. The fully grown bacterial suspension was
measured and brought into complete DMEM-Pen/Fung [DMEM
medium containing 0.1% penicillin and 0.8% fungizone but without
gentamicin]. The synthesized compounds were solubilized in DMSO
at stock concentrations of 10 mM. Serial dilutions of the peptides were
made in DMEM-Pen/Fung at 2 times the concentration of each
compound to be tested. A volume of 100 μL of the bacterial
suspension in DMEM-Pen/Fung containing 4000 RLU of bacteria
(multiplicity of infection of 0.1), and 100 μL of the serial compound
dilutions were added to the macrophage cultures. To measure the
effects of the compounds on intracellular growth of M.tb, the infected
macrophages were washed three times on day 5 to remove all
extracellular bacteria, incubated 1 h with 1% gentamicin to kill the
residual extracellular bacteria, lysed with 200 μL of 1% Triton X-100
(Sigma), and the wells were washed four times with 200 μL of PBS.
The lysate was transferred in a 2.5 mL tube together with the four PBS
washings. One hundred microliters of 1% n-decanal in ethanol was
added to the tube, and the luminescence was measured. RLU values
shown were obtained from six replicate cultures. Cell viability of the
macrophage culture was observed by Trypan blue with a microscope.
Assessment of Cytotoxicity. Inhibitory effects on the J774 A.1
murine macrophage cell line were determined for the derivatives by a
neutral red uptake assay as described before. The J774 A.1 cells were
grown in DMEM + 10% FCS until a semi-confluent layer of cells was
obtained. The cells were trypsinized, washed, and 40 000 cells were
seeded per well of a 96-well plate and left for recovery at 37 °C, 5%
CO2. The following days, the compounds were solubilized in DMSO
to stock concentrations of 10 μM. A serial dilution of each compound
was made in DMEM + 10% FCS. The J774 A.1 cells were washed and
exposed to the derivatives by adding the serial dilutions of the
compounds to the wells. The plates were left for incubation at 37 °C,
5% CO2 for 24 h. After exposure, the cells were washed with 200 μL of
PBS, and 200 μL of neutral red working solution (Sigma) was added
per well. Subsequently, the plates were incubated for 3 h at 37 °C, 5%
CO2. The wells were washed with 200 μL of PBS and 200 μL of an
ethanol/acetic acid (50%) mixture. The plates were left on the shaker
until the color became homogeneous purple and the optical density
was measured at 530 nm (NR max) and 620 nM (reference
wavelength) with the Paradigm detection platform.
Vitotox Assay. Observations on genotoxicity were done with
Vitotox (Gentaur, Kampenhout, BE), and the included protocol was
followed. In brief, TA104 RecN2-4 (Genox) and TA104 (Cytox)
Salmonella typhimurium bacteria were cultivated at 36 °C for 16 h in
poor 869 medium. The bacterial culture was diluted 250 times,
incubated for 1 h at 36 °C, and kept on ice. The bacterial culture was
divided by 10 once more. S9 was added to the designated +S9 cultures
to test the genotoxic effects of the metabolites of the compounds. The
bacterial suspensions were then incubated, with shaking at 36 °C, and
the luminescent signal was measured for 4 h with a 5 min interval.
Comet Assay. Possible DNA breakage effects of the derivatives on
J774 A.1 cells were investigated by the alkaline comet assay. The J774
A.1 cells were grown in DMEM + 10% FCS until a semi-confluent
layer of cells was obtained. The cells were trypsinized, washed, and
seeded at 100 000 cells per well of a 24-well plate and left for recovery
at 37 °C, 5% CO2. The following day, the testing compounds were
dissolved in DMSO as a stock concentration of 1 mM. Serial dilutions
of each compound were made in DMEM + 10% FCS to obtain the
final concentrations. The J774 A.1 cells were washed and exposed to
the derivatives by adding 1 mL of the serial dilutions to each well. The
plates were incubated at 37 °C, 5% CO2 for 24 h. After incubation, the
cells were trypsinized, washed with PBS, and 10 μL of cell suspension
was dissolved in 300 μL of low-melting-point agarose. The dissolved
cell suspension was then placed onto a frosted microscope slide and
left on ice for 5 min. The slide was subsequently placed in a jar
Inhibition of Intracellular M.tb Growth. The compounds were
tested on the murine J774 A.1 macrophage cell line infected with M.tb
H37Rvlux. The J774 macrophages were grown at 37 °C, 5% CO2 in
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containing lysing solution for 1 h. After lysis, the agarose cell
suspension was subjected to electrophoresis for 20 min at 300 mA.
The slides were washed with neutralization buffer for 5 min and dried
in ice cold ethanol for 10 min. Staining of the DNA was done with gel
red (Sigma-Aldrich). For the quantification of the DNA migration, a
fluorescence microscope was used, and the percentage of DNA in the
comet tail of the cells nuclei core was calculated in proportion to the
total DNA present in the nuclei (comet head + tail) by appropriate
imager software from Metasystems (Altlussheim, Germany).
(CH), 2873 (CH), 1672(CO), 1557 (CHAr), 1288, 716 cm−1. MS:
m/z (%) 278 ([M + H]+, 100).
2-tert-Butyl-1,2,3,4-tetrahydrobenzo[j]phenanthridine-7,12-
dione 3c. Yield 38%, amber crystals, mp 194.1 °C. 1H NMR (CDCl3):
δ 1.06 (9H, s, C(CH3)3), 1.45−1.54 (2H, m, CH2), 2.11−2.19 (1H, m,
CH), 2.92−3.13 (2H, m, CH2), 3.25 (1H, dd, J = 3.2, 19.1 Hz, CH),
3.64 (1H, dd, J = 3.2, 19.1 Hz, CH), 7.79−7.84 (2H, m, CH-9 and 10),
8.21−8.29 (2H, m, CH-8 and 11), 9.34 (1H, CH-6). 13C NMR
(CDCl3): δ 23.35 (CH2), 27.40 (C(CH3)3), 29.98 (CH2), 32.71
(C(CH3)3), 35.54 (CH2), 44.44 (CH-2), 125.18 (Cquat), 126.63 and
127.35 (CH-8 and 11), 132.46 (Cquat), 133.61 (Cquat), 134.28 and
134.49 (CH-9 and 10), 134.55 (Cquat), 146.87 (CH-6), 158.22 (Cquat),
166.43 (Cquat), 183.27 (CO), 185.58 (CO). IR (ATR): ν 2948
(CH), 2868 (CH), 1675 (CO), 1660 (CO), 1595 (CHAr), 1298,
709 cm−1. MS: m/z (%) 320 ([M + H]+, 100).
General Chemistry. Column chromatography was carried out
using a glass column with silica gel (Aldrich, particle size 0.035−0.070
mm, pore diameter ca. 6 nm). Solvent systems were determined via
initial TLC analysis on silica gel (Merck, Kieselgel 60F254, precoated
0.25 mm). Compounds were revealed by UV light or KMnO4
1
oxidation. H NMR (300 MHz) and 13C NMR (75 MHz) spectra
were recorded with a Jeol JNM-EX 300 NMR spectrometer. Peak
assignments were performed with the aid of HSQC and HMBC
techniques. The NMR samples were prepared with commercially
available deuterated solvents with Me4Si as an internal standard. Low-
resolution mass spectra were recorded using an Agilent 1100 series VS
(ESI, 4000 V) mass spectrometer via a direct inlet or via LC-MS
coupling [Phenomenex luna column; 250 × 3 mm length, 5 μm
particle size, 100 Å pore size with 5 mM NH4OAc in H2O and
acetonitrile as eluents]. High-resolution mass spectra were recorded on
a Finnigan MAT 95 XPAPI-GC-Trap tandem mass spectrometer or a
tandem spectrometer Agilent 6220 TOF-LC/MS. Infrared spectra
were recorded with a Perkin-Elmer BX FT-IR spectrometer. Melting
points were recorded on a Buchi Melting point B-540 apparatus and
are uncorrected. Final compounds were isolated with a purity higher
than 95% (HPLC). Purity was assessed at 221, 255, and 281 nm. THF
and CH2Cl2 were dried over CaH2/benzophenone. All reagents were
used without further purification, and all glassware was oven-dried
prior to use. Stork enamines 7, 13, and 14 were prepared according to
Stork et al.39 and used immediately after high vacuum distillation.
1,2,3,4-Tetrahydrobenzo[j]phenanthridine-7,12-diones 3 and 4.
A stirred solution of 2-(1,3-dioxolan-2-yl)naphthoquinone 6a6 (1.5
mmol) or 2-acetylnaphthoquinone 6b (1.5 mmol) in dry THF (10
mL) under a nitrogen atmosphere was cooled to 0 °C. Freshly distilled
enamine 7 (1.65 mmol) was added dropwise in 2 mL of dry THF.
Then, 7 M NH3 in MeOH (1.4 mL, 10 equiv) was added dropwise,
and the reaction was warmed to room temperature and stirred open to
the air overnight. After careful evaporation of the solvents, the reaction
mixture was redissolved in EtOAc (15 mL) and washed with brine (2
× 10 mL). Drying over MgSO4 and evaporation of the solvent in
vacuo gave a crude mixture, which was purified by means of
preparative TLC (hexane/ethyl acetate) and subsequent recrystalliza-
tion from EtOH to yield the desired 1,2,3,4-tetrahydrobenzo[j]-
phenanthridine-7,12-diones 3 and 4.
3-Methyl-1,2,3,4-tetrahydrobenzo[j]phenanthridine-7,12-dione
1
3d. Yield 24%, yellow solid, mp 168 °C. H NMR (CDCl3): δ 1.15
(3H, d, J = 6.1 Hz, CHCH3), 1.36−1.50 (1H, m, CH), 1.94−2.01 (2H,
m, CH2), 2.71 (1H, dd, J = 10.3, 18.4 Hz), 3.17−3.32 (2H, m, CH2),
3.58−3.66 (1H, m, CH), 7.79−7.84 (2H, m, CH-9 and 10), 8.20−8.28
(2H, m, CH-8 and 11), 9.36 (1H, s, CH-6). 13C NMR (CDCl3): δ
21.57 (CH3), 27.86 (CH2), 28.18 (CH2), 30.82 (CH2), 43.00 (CH),
125.24 (Cquat), 126.66 and 127.39 (CH-8 and 11), 132.51 (Cquat),
132.95 (Cquat), 132.75 (Cquat), 134.34 and 134.51 (CH-9 and 10),
135.41 (Cquat), 147.09 (CH-6), 166.26 (Cquat), 183.35 (CO), 185.62
(CO). IR (ATR): ν 2952 (CH), 2925 (CH), 1672 (CO), 1559
(CHAr), 1299, 1277, 710 cm−1. MS: m/z (%) 278 ([M + H]+, 100).
HRMS (ESI): calcd for [C18H16NO2]+, 278.1181; found, 278.1181.
4-Methyl-1,2,3,4-tetrahydrobenzo[j]phenanthridine-7,12-dione
1
3e. Yield 16%, yellow solid, mp 123 °C. H NMR (CDCl3): δ 1.44
(3H, d, J = 7.2 Hz, CHCH3), 1.67−2.13 (4H, m, 2xCH2), 3.19 (1H,
sextet, J = 6.6 Hz, CH-4), 3.35 (1H, td, J = 6.6, 19.3 Hz, CH), 3.46
(1H, td, J = 6.6, 19.3 Hz, CH), 7.79−7.84 (2H, m, CH-9 and 10),
8.21−8.29 (2H, m, CH-8 and 11), 9.41 (1H, s, CH-6). 13C NMR
(CDCl3): δ 19.84 (CH2), 21.57 (CH3), 28.70 (CH2), 29.94 (CH2),
37.57 (CH-4), 124.92 (Cquat), 126.52 and 127.32 (CH-8 and 11),
132.41 (Cquat), 132.95 (Cquat), 134.29 and 134.41 (CH-9 and 10),
134.55 (Cquat), 135.45 (Cquat), 146.86 (CH-6), 170.17 (Cquat), 183.26
(CO), 185.52 (CO). IR (ATR): ν 2944 (CH), 2874 (CH), 1672
(CO), 1588 (CHAr), 1559 (CHAr), 1298, 1281, 720 cm−1. MS: m/z
(%) 278 ([M + H]+, 100).
6-Methyl-1,2,3,4-tetrahydrobenzo[j]phenanthridine-7,12-dione
1
4a. Yield 15%, orange needles, mp 180.7 °C. H NMR (CDCl3): δ
1.83−1.98 (4H, m, 2xCH2), 3.02 (3H, s, CH3), 3.06 (2H, t, J = 6.3 Hz,
CH2), 3.34 (2H, t, J = 6.3 Hz, CH2), 7.74−7.83 (2H, m, CH-9 and
10), 8.14−8.22 (2H, m, CH-8 and 11). 13C NMR (CDCl3): δ 22.13
(CH2), 22.99 (CH2), 26.81 (CH2), 28.41 (CH2), 34.73 (CH3), 123.97
(Cquat), 126.66 (CH-8 and 11), 131.51 (Cquat), 133.73 (Cquat), 133.79
(CH-9 or 10), 133.94 (Cquat), 134.23 (CH-9 or 10), 135.42 (Cquat),
158.17 (Cquat), 164.45 (Cquat), 185.03 (CO), 186.46 (CO). IR
(ATR): ν 2942 (CH), 2868 (CH), 1670 (CO), 1589 (CHAr), 1537
(CHAr), 1288, 722, 713 cm−1. MS: m/z (%) 278 ([M + H]+, 100).
HRMS (ESI): calcd for [C18H16NO2]+, 278.1181; found, 278.0819.
2,6-Dimethyl-1,2,3,4-tetrahydrobenzo[j]phenanthridine-7,12-
dione 4b. Yield 23%, orange crystals, mp 164.2 °C. 1H NMR
(CDCl3): δ 1.18 (3H, d, J = 6.6 Hz, CHCH3), 1.48−1.61 (1H, m,
CH), 1.79−1.91 (1H, m, CH), 1.97−2.06 (1H, m, CH), 2.84 (1H, dd,
J = 11.0, 18.7 Hz, CH), 3.00 (1H, s, CH3), 3.04−3.12 (2H, m, CH2),
3.50 (1H, ddd, J = 1.7, 4.5, 18.7 Hz, CH), 7.72−7.81 (2H, m, CH-9
and 10), 8.12−8.20 (2H, m, CH-8 and 11). 13C NMR (CDCl3): δ
21.97 (CH3), 26.81 (CH3), 29.20 (CH), 30.16 (CH2), 34.39 (CH2),
36.76 (CH2), 123.88 (Cquat), 126.64 (CH-8 and 11), 131.01 (Cquat),
133.71 (Cquat), 133.80 (CH-9 or 10), 133.91 (Cquat), 134.23 (CH-9 or
10), 137.71 (Cquat), 158.22 (Cquat), 164.23 (Cquat), 184.97 (CO),
188.48 (CO). IR (ATR): ν 2933 (CH), 2869 (CH), 1667 (CO),
1590 (CHAr), 1289, 724 cm−1. MS: m/z (%) 292 ([M + H]+, 100).
3,6-Dimethyl-1,2,3,4-tetrahydrobenzo[j]phenanthridine-7,12-
dione 4d. Yield 36%, yellow needles, mp 168 °C. 1H NMR (CDCl3):
δ 1.14 (3H, d, J = 6.6 Hz, CHCH3), 1.33−1.47 (1H, m, CH), 1.95−
2.07 (2H, m, CH2), 2.65 (1H, dd, J = 10.5, 18.2 Hz), 3.02 (3H, s,
1,2,3,4-Tetrahydrobenzo[j]phenanthridine-7,12-dione 3a. Yield
1
43%, yellow needles, mp 149.2 °C. H NMR (CDCl3): δ 1.86−2.00
(4H, m, 2xCH2), 3.14 (2H, t, J = 6.1 Hz, CH2), 3.42 (2H, t, J = 6.1 Hz,
CH2), 7.79−7.85 (2H, m, CH-9 and 10), 8.21−8.29 (2H, m, CH-8
and 11), 9.37 (1H, s, CH-6). 13C NMR (CDCl3): δ 21.98 (CH2),
22.68 (CH2), 28.21 (CH2), 34.73 (CH2), 125.13 (Cquat), 126.58 and
127.33 (CH-8 and 11), 132.41 (Cquat), 133.19 (Cquat), 134.28 and
134.45 (CH-9 and 10), 133.91 (Cquat), 135.42 (Cquat), 146.86 (CH-6),
166.33 (Cquat), 183.20 (CO), 185.47 (CO). IR (ATR): ν 2941
(CH), 2867 (CH), 1674 (CO), 1660 (CO), 1557 (CHAr), 1297,
1288, 712 cm−1. MS: m/z (%) 264 ([M + H]+, 100).
2-Methyl-1,2,3,4-tetrahydrobenzo[j]phenanthridine-7,12-dione
1
3b. Yield 28%, yellow crystals, mp 125.9 °C. H NMR (CDCl3): δ
1.20 (3H, d, J = 6.6 Hz, CHCH3), 1.50−1.64 (1H, m, CH), 1.84−1.96
(1H, m, CH), 2.00−2.09 (1H, m, CH), 2.88 (1H, dd, J = 10.7, 19.0
Hz, CH), 3.06−3.27 (2H, m, CH2), 3.66 (1H, ddd, J = 1.7, 4.8, 19.1
Hz, CH), 7.79−7.85 (2H, m, CH-9 and 10), 8.21−8.30 (2H, m, CH-8
and 11), 9.37 (1H, s, CH-6). 13C NMR (CDCl3): δ 21.94 (CH3),
28.96 (CH2), 30.06 (CH2), 34.39 (CH2), 36.53 (CH), 125.13 (Cquat),
126.63 and 127.33 (CH-8 and 11), 132.48 (Cquat), 132.75 (Cquat),
134.31 and 134.49 (CH-9 and 10), 135.42 (Cquat), 146.93 (CH-3),
166.16 (Cquat), 183.24 (CO), 185.59 (CO). IR (ATR): ν 2948
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CH3), 3.10−3.30 (2H, m, CH2), 3.46−3.55 (1H, m, CH), 7.73−7.82
(2H, m, CH-9 and 10), 8.12−8.24 (2H, m, CH-8 and 11). 13C NMR
(CDCl3): δ 21.58 (CH3), 26.79 (CH3), 27.95 (CH2), 28.23 (CH2),
31.05 (CH2), 43.02 (CH), 123.88 (Cquat), 126.61 and 126.64 (CH-8
and 11), 130.92 (Cquat), 133.64 (Cquat), 133.74 (CH-9 or 10), 133.83
(Cquat), 134.19 (CH-9 or 10), 137.48 (Cquat), 158.31 (Cquat), 164.28
(Cquat), 184.86 (CO), 186.30 (CO). IR (ATR): ν 2948 (CH),
2928 (CH), 1667 (CO), 1591 (CHAr), 1294, 1264, 724 cm−1. MS:
m/z (%) 292 ([M+H]+, 100). HRMS (ESI): calcd for [C19H18NO2]+,
292.1338; found, 292.0978.
4,6-Dimethyl-1,2,3,4-tetrahydrobenzo[j]phenanthridine-7,12-
dione 4e. Yield 18%, orange needles, mp 161.5 °C. 1H NMR
(CDCl3): δ 1.42 (3H, d, J = 7.2 Hz, CHCH3), 1.64−1.81 (2H, m,
CH2), 1.87−1.99 (1H, m, CH), 2.02−2.11 (1H, m, CH), 3.01 (3H, s,
CH3), 3.11 (1H, sextet, J = 6.6 Hz, CH-4), 3.25 (1H, td, J = 6.6, 19.3
Hz, CH), 3.35 (1H, td, J = 6.6, 19.3 Hz, CH), 7.71−7.80 (2H, m, CH-
9 and 10), 8.11−8.19 (2H, m, CH-8 and 11), 9.41 (1H, s, CH-6). 13C
NMR (CDCl3): δ 20.19 (CH3), 21.66 (CH2), 26.90 (CH3), 28.91
(CH2), 30.10 (CH2), 37.49 (CH-4), 123.71 (Cquat), 126.58 and 126.64
(CH-8 and 11), 131.12 (Cquat), 132.95 (Cquat), 133.73 (CH-9 or 10),
134.00 (Cquat), 134.14 (CH-9 or 10), 137.76 (Cquat), 158.11 (Cquat),
168.25 (Cquat), 185.09 (CO), 185.52 (CO). IR (ATR): ν 2981
(CH), 2935 (CH), 1666 (CO), 1590 (CHAr), 1280, 725 cm−1. MS:
m/z (%) 292 ([M+H]+, 100). HRMS (ESI): calcd for [C19H16NO2]−,
290.1181; found, 290.1336.
Synthesis of 6-(1,3-dioxolan-2-yl)-1,2,3,4-tetrahydronaphtha-
lene-5,8-diones 10a and 10b. A solution of 5,8-dimethoxy-1,2,3,4-
tetrahydronaphthalene-6-carbaldehydes 8a or 8b or 3-bromo-2,5-
dimethoxybenzaldehyde40 (20 mmol), ethylene glycol (80 mmol), and
p-TsOH.H2O (0.2 mmol, 38 mg) in PhMe (40 mL) was equipped
with a Dean−Stark apparatus and boiled under reflux for 4 h. Next, the
reaction mixture was cooled to room temperature, diluted with EtOAc
(40 mL), washed once with NaHCO3,aq, sat (40 mL) and 3 times with
brine (40 mL). Drying over MgSO4 and evaporation of the solvent
yielded pure 2-(5,8-dimethoxy-1,2,3,4-tetrahydronaphthalen-6-yl)-1,3-
dioxolanes 9a or 9b or 2-(3-bromo-2,5-dimethoxyphenyl)-1,3-
dioxolane 11. Due to the limited stability of dioxolanes 9a, 9b, and
11, no MS or HRMS could be recorded.
IR (ATR): ν 2888 (CH), 1475 (CHAr), 1219, 1131 (C−O), 995 cm−1.
MS: m/z (%) 291/289 ([M + H]+, 100/97).
A solution of freshly prepared LDA (2 equiv, 17.5 mmol) in dry
THF (10 mL) was cooled to −78 °C, and furan (10 mL) was added
dropwise. Next, 2-(3-bromo-2,5-dimethoxyphenyl)-1,3-dioxolane 11
(2.5 g, 8.65 mmol) dissolved in 2.5 mL THF was slowly added
dropwise. The reaction mixture was stirred for 1 h at −78 °C and
quenched with 10 mL of H2O. The reaction mixture was diluted with
20 mL of EtOAc and washed twice with brine (10 mL). Drying over
MgSO4, evaporation of the solvent in vacuo, followed by
recrystallization from Et2O gave pure 2-(5,8-dimethoxy-1,4-dihydro-
1,4-epoxynaphthalen-6-yl)-1,3-dioxolane 12.
2-(5,8-Dimethoxy-1,4-dihydro-1,4-epoxynaphthalen-6-yl)-1,3-di-
oxolane 12. Yield 89%, pale white solid, mp 131 °C. 1H NMR
(CDCl3): δ 3.81 (3H, s, OCH3), 3.88 (3H, s, OCH3), 3.98−4.07 (2H,
m, CH2O), 4.08−4.18 (2H, m, CH2O), 5.90 (1H, d, J = 1.7 Hz, CH-1
or 8), 6.05 (CH(OCH2)2), 6.02 (1H, d, J = 1.7 Hz, CH-1 or 8), 6.83
(1H, s, CH-5), 7.03 (1H, dd, J = 1.7, 5.5 Hz, CH-9 or CH-10), 7.03
(1H, dd, J = 1.7, 5.5 Hz, CH-9 or CH-10). 13C NMR (CDCl3): δ
56.14 (OCH3), 61.46 (OCH3), 65.17 (OCH2CH2O), 79.76 and 81.30
(CH-1 and CH-8), 99.39 (CH(OCH2)2), 109.54 (CH-5), 128.20
(Cquat), 138.03 (Cquat), 139.09 (Cquat), 142.29 and 142.97 (CH-9 and
CH-10), 146.66 (Cquat), 148.36 (Cquat). IR (ATR): ν 2897 (CH),
1478, 1392, 1222, 1055 cm−1. MS: m/z (%) 277 ([M + H]+, 100).
HRMS (ESI): calcd for [C15H17O5]+, 277.1076; found, 277.1072.
2-(5,8-Dimethoxy-1,2,3,4-tetrahydro-1,4-epoxynaphthalen-6-yl)-1,3-
dioxolane 9c was prepared using the same procedure as 5,8-
dimethoxy-1,2,3,4-tetrahydronaphthalene-6-carbaldehydes 8.
2-(5,8-Dimethoxy-1,2,3,4-tetrahydro-1,4-epoxynaphthalen-6-yl)-
1,3-dioxolane 9c. Yield 99%, white crystals, mp 81−82.5 °C. 1H NMR
(CDCl3): δ 1.35−1.51 (2H, m, CH2), 1.99−2.13 (2H, m, CH2), 3.82
(3H, s, OCH3), 3.90 (3H, s, OCH3), 4.01−4.07 (2H, m, CH2O),
4.08−4.18 (2H, m, CH2O), 5.56 (1H, d, J = 4.4 Hz, CH-1 or 8), 5.66
(1H, d, J = 4.4 Hz, CH-1 or 4), 6.04 (CH(OCH2)2), 6.93 (1H, s, CH-
7). 13C NMR (CDCl3): δ 26.09 (CH2), 26.71 (CH2), 55.97 (OCH3),
61.40 (OCH3), 65.34 (OCH2CH2O), 76.47 and 77.72 (CH-1 and
CH-8), 99.45 (CH(OCH2)2), 108.89 (CH-5), 128.60 (Cquat), 136.02
(Cquat), 136.92 (Cquat), 145.16 (Cquat), 147.47 (Cquat). IR (ATR): ν
2954 (CH), 1482, 1394, 1069 cm−1. MS: m/z (%) 279 ([M + H]+,
100). HRMS (ESI): calcd for [C15H19O5]+, 279.1233; found,
279.1231.
To a stirred solution of 1,3-dioxolanes 9a, 9b, or 9c (1.81 mmol) in
CH3CN (10 mL) was added a solution of CAN (5.43 mmol, 2.98 g) in
H2O (10 mL) in one portion at room temperature. After 3 min, the
reaction mixture was diluted with EtOAc (20 mL) and washed twice
with brine (10 mL). Drying over MgSO4 and evaporation of the
solvent in vacuo yielded the crude quinones 10, which were used as
such. All dioxolanylnaphthoquinones 10 were unstable and had to be
used directly in the next step.
2-(5,8-Dimethoxy-1,2,3,4-tetrahydro-1,4-methanonaphthalen-6-
1
yl)-1,3-dioxolane 9a. Yield 82%, pale white solid, mp 106.4 °C. H
NMR (CDCl3): δ 1.14−1.27 (2H, m, CH2), 1.48 (1H, d, J = 8.8 Hz,
CHAHB), 1.70 (1H, d, J = 8.8 Hz, CHAHB), 1.84−1.98 (2H, m, CH2),
3.57 (1H, br s, CH-1 or CH-4), 3.61 (1H, br s, CH-1 or CH-4), 3.82
(3H, s, OCH3), 3.84 (3H, s, OCH3), 4.01−4.09 (2H, m, CH2O),
4.10−4.21 (2H, m, CH2O), 6.05 (CH(OCH2)2), 6.84 (1H, s, CHAr).
13C NMR (CDCl3): δ 26.42 (CH2), 26.90 (CH2), 39.84 (CH), 41.19
(CH), 49.14 (CH2), 55.92 (OCH3), 62.15 (OCH3), 65.37 (OCH2),
65.40 (OCH2), 99.88 (CH(OCH2)2), 106.93 (CHAr), 127.45 (Cquat),
138.45 (Cquat), 140.77 (Cquat), 146.71 (Cquat), 149.22 (Cquat). IR
(ATR): ν 2951 (CH), 2868 (CH), 1489, 1458, 1387, 1218, 1110,
1054, 1021 cm−1.
6-(1,3-Dioxolan-2-yl)-1,2,3,4-tetrahydro-1,4-methanonaphtha-
1
lene-5,8-dione 10a. Yield 60%, orange solid, mp 68.4 °C. H NMR
(CDCl3): δ 1.05−1.15 (2H, m, CH2), 1.32−1.36 (1H, m, CH), 1.54−
1.59 (1H, m, CH), 1.81−1.90 (2H, m, CH2), 3.39 (1H, d, J = 1.4 Hz,
CH-1 or CH-4), 3.42 (1H, d, J = 1.4 Hz, CH-1 or CH-4), 3.93−3.98
(4H, m, OCH2CH2O), 5.79 (CH(OCH2)2), 6.60 (1H, s, CH-7). 13C
NMR (CDCl3): δ 24.88 (CH2), 24.93 (CH2), 40.47 (CH), 40.53
(CH), 47.83 (CH2), 65.31 (OCH2), 65.45 (OCH2), 98.00 (CH-
(OCH2)2), 131.64 (CHAr), 142.26 (Cquat), 151.67 (Cquat), 151.93
(Cquat), 183.46 (CO), 184.65 (CO). IR (ATR): ν 2953 (CH),
2878 (CH), 1645 (CO), 1337, 1088, 943 cm−1.
2-(5,8-Dimethoxy-1,2,3,4-tetrahydro-1,4-ethanonaphthalen-6-
1
yl)-1,3-dioxolane 9b. Yield 89%, pale white crystals, mp 113 °C. H
NMR (CDCl3): δ 1.26−1.38 (4H, m, 2xCH2), 1.69−1.81 (4H, m,
2xCH2), 3.36−3.37 (1H, m, CH), 3.45−3.46 (1H, m, CH), 3.78 (3H,
s, OCH3), 3.82 (3H, s, OCH3), 4.03−4.11 (2H, m, CH2O), 4.13−4.21
(2H, m, CH2O), 6.11 (1H, s, CH(OCH2)2), 6.92 (1H, s, CHAr). 13C
NMR (CDCl3): δ 25.61 (2xCH2), 25.74 (2xCH2), 26.10 (CH), 27.46
(CH), 55.88 (OCH3), 63.28 (OCH3), 65.42 (OCH2CH2O), 99.92
(CH(OCH2)2), 105.62 (CHAr), 127.09 (Cquat), 134.86 (Cquat), 137.97
(Cquat), 147.88 (Cquat), 154.54 (Cquat). IR (ATR): ν 2939 (CH), 1487,
1383, 1217, 1114 cm−1.
6-(1,3-Dioxolan-2-yl)-1,2,3,4-tetrahydro-1,4-ethanonaphtha-
1
lene-5,8-dione 10b. Yield 83%, red oil. H NMR (CDCl3): δ 1.29
2-(3-Bromo-2,5-dimethoxyphenyl)-1,3-dioxolane 11. Yield 95%,
colorless oil. H NMR (CDCl3): δ 3.77 (3H, s, OCH3), 3.85 (3H, s,
(4H, br d, J = 8.0 Hz, 2xCH2), 1.73 (4H, br d, J = 8.0 Hz, 2xCH2),
3.33 (1H, s, CH), 3.37 (1H, m, CH), 4.05 (2H, s, CH2O), 4.05 (2H, s,
CH2O), 5.93 (1H, s, CH(OCH2)2), 6.80 (1H, s, CHAr). 13C NMR
(CDCl3): δ 24.79 (4xCH2), 26.00 (2xCH), 65.06 (OCH2CH2O),
97.75 (CH(OCH2)2), 131.08 (CHAr), 141.82 (Cquat), 147.70 (Cquat),
147.90 (Cquat), 182.91 (CO), 184.10 (CO). IR (ATR): ν 2949
(CH), 2868 (CH), 1648 (CO), 751 cm−1.
1
OCH3), 4.01−4.09 (2H, m, CH2O), 4.09−4.17 (2H, m, CH2O, 6.06
(CH(OCH2)2), 7.03 (1H, d, J = 3.0 Hz, CHAr), 7.10 (1H, d, J = 3.0
Hz, CHAr). 13C NMR (CDCl3): δ 55.91 (OCH3), 62.38 (OCH3),
65.48 (OCH2CH2O), 99.44 (CH(OCH2)2), 111.63 (CHAr), 117.62
(Cquat), 119.65 (CHAr), 133.36 (Cquat), 149.59 (Cquat), 156.26 (Cquat).
2904
dx.doi.org/10.1021/jm401735w | J. Med. Chem. 2014, 57, 2895−2907

Journal of Medicinal Chemistry
Article
6-(1,3-Dioxolan-2-yl)-1,2,3,4-tetrahydro-1,4-epoxynaphthalene-
(4H, m, 2xCH2), 2.06−2.17 (3H, m, CH2 and CH), 2.73 (1H, dd, J =
19.1, 10.9, CH), 3.02−3.08 (1H, m, CH), 3.16−3.19 (1H, m, CH),
3.72 (1H, dd, J = 3.9, 17.6 Hz, CH), 5.57 (2H, dd, J = 4.4, 11.0, CH-8
and CH-10), 9.08 (1H, CH-6). Minor isomer δ 1.02 (9H, s,
C(CH3)3), 1.37−1.51 (4H, m, 2xCH2), 2.06−2.17 (3H, m, CH2 and
CH), 2.87 (1H, dd, J = 19.1, 10.9, CH), 2.96−3.00 (1H, m, CH),
3.23−3.25 (1H, m, CH), 3.40 (1H, dd, J = 3.9, 17.6 Hz, CH), 5.57
(2H, dd, J = 4.4, 11.0, CH-8 and CH-10), 9.07 (1H, CH-6). Major/
minor 3/1. 13C NMR (CDCl3): major isomer δ 23.19 (CH2), 24.47
(CH2), 24.61 (CH2), 27.31 (C(CH3)3), 29.94 (CH2), 32.73
(C(CH3)3), 35.54 (CH2), 35.54 (2xCH), 44.27 (CH-2), 124.76
(Cquat), 133.61 (Cquat), 145.48 (CH-6), 150.66 (Cquat), 153.51 (Cquat),
167.19 (Cquat), 182.92 (CO), 183.87 (CO). Minor isomer δ
23.46 (CH2), 24.55 (CH2), 24.76 (CH2), 27.31 (C(CH3)3), 29.17
(CH2), 32.65 (C(CH3)3), 35.37 (2xCH), 42.76 (CH-2), 125.06
(Cquat), 134.54 (Cquat), 145.48 (CH-6), 158.00 (Cquat), 159.44 (Cquat),
167.07 (Cquat), 180.92 (CO), 183.87 (CO). IR (ATR): ν 2958
(CH), 2869 (CH), 1661 (CO), 1561 (CHAr), 1320 cm−1. MS: m/z
(%) 338 ([M + H]+, 100). HRMS (ESI): calcd for [C21H24NO3]+,
338.1756; found, 338.1755.
1
5,8-dione 10c. Yield 81%, red solid, mp 79 °C (decomp.). H NMR
(CDCl3): δ 1.30−1.45 (2H, m, CH2), 2.02−2.12 (2H, m, CH2), 4.04
(4H, s, OCH2CH2O), 5.46 (2H, t, J = 5.0 Hz, CH-1 and CH-4), 5.87
(CH(OCH2)2), 6.73 (1H, s, CH-5). 13C NMR (CDCl3): δ 23.77
(CH2), 23.83 (CH2), 64.93 (OCH2), 65.11 (OCH2), 76.27 and 76.34
(CH-1 and CH-4), 97.38 (CH(OCH2)2), 131.30 (CHAr), 142.26
(Cquat), 149.78 (Cquat), 150.00 (Cquat), 181.90 (CO), 182.92 (C
O). IR (ATR): ν 2980 (CH), 2885 (CH), 1655 (CO), 1305, 1286,
875 cm−1.
Synthesis of Octahydrobenzophenanthridinediones 5a−5g.
Method A. A stirred solution of 2-(1,3-dioxolan-2-yl)naphthoquinones
10 (1.5 mmol) in dry THF (10 mL) under a nitrogen atmosphere was
cooled to 0 °C, and BF3·OEt2 was added dropwise (19 μL, 0.1 equiv).
Next, freshly distilled enamine 7c, 13, or 14b (1.65 mmol) was added
dropwise in 2 mL of dry THF. The reaction mixture was allowed to
warm to room temperature and stirred for 3 h. Then, 7 M NH3 in
MeOH (1.4 mL, 10 equiv) was added dropwise, and the reaction was
stirred open to the air for 15 h. After careful evaporation of the
solvents, the reaction mixture was redissolved in EtOAc (15 mL) and
washed with brine (2 × 10 mL). Drying over MgSO4 and evaporation
of the solvent in vacuo gave a crude mixture which was purified by
means of preparative TLC (hexane/ethyl acetate). Recrystallization
from EtOH yielded the desired 1,2,3,4-octahydrobenzo[j]-
phenanthridine-7,12-diones 5.
Method B. After the enamine addition, the reaction mixture was
evaporated in vacuo and subsequently redissolved in 10 mL of HOAc.
After the addition of NH4OAc (15 mmol, 1.16 g), the reaction mixture
was stirred at 60 °C for 1 h open to the air. Next, the reaction mixture
was evaporated in vacuo, redissolved in 15 mL of EtOAc, and washed
with both NaHCO3,aq,sat (2 × 10 mL) and brine (10 mL). Further
purification proceeded as in method A.
2-tert-Butyl-1,2,3,4,8,9,10,11-octahydro-8,11-methanobenzo[j]-
phenanthridine-7,12-dione 5a. Yield 48%, yellow solid, mp 140.0 °C.
1H NMR (CDCl3): δ 1.02 (9H, s, C(CH3)3), 1.22−1.31 (2H, m,
CH2), 1.38−1.49 (3H, m, CH2 and CH), 1.66−1.72 (1H, m, CH),
1.95−2.05 (2H, m, CH2), 2.08−2.13 (1H, m, CH), 2.75 (1H, dd, J =
11.0, 18.7 Hz, CH), 2.91−3.06 (1H, m, CH), 3.15−3.22 (1H, m, CH),
3.61 (2H, s, 2xCH), 3.73 (1H, d, J = 18.7 Hz), 9.06 (1H, CH-6). 13C
NMR (CDCl3): major isomer δ 23.28 (CH2), 25.20 (CH2), 25.26
(CH2), 27.31 (C(CH3)3), 29.04 (CH2), 32.70 (C(CH3)3), 35.51
(CH2), 40.81 (CH), 41.19 (CH), 44.33 (CH2), 47.72 (CH-2), 124.80
(Cquat), 133.09 (Cquat), 134.75 (Cquat), 145.33 (CH-6), 152.43 (Cquat),
155.33 (Cquat), 182.05 (CO), 184.89 (CO). Minor isomer δ
23.51 (CH2), 25.26 (CH2), 25.46 (CH2), 27.31 (C(CH3)3), 29.71
(CH2), 32.64 (C(CH3)3), 35.34 (CH2), 40.91 (CH), 41.45 (CH),
44.33 (CH2), 46.93 (CH-2), 124.86 (Cquat), 133.02 (Cquat), 135.32
(Cquat), 145.33 (CH-6), 152.51 (Cquat), 155.41 (Cquat), 182.16 (C
O), 185.07 (CO). Major/minor 3.7/1. IR (ATR): ν 2954 (CH),
2872 (CH), 1656 (CO), 1321 cm−1. MS: m/z (%) 336 ([M + H]+,
100). HRMS (ESI): calcd for [C22H26NO2]+, 336.1964; found,
336.1957.
2-Ethoxycarbonyl-1,2,3,4,8,9,10,11-octahydro-8,11-
methanobenzo[j]phenanthridine-7,12-dione 5d. Yield 18%, light
1
brown solid, mp 61.5 °C. H NMR (CDCl3): Isomer I δ 1.10−1.14
(2H, m, CH2), 1.18 (3H, t, J = 7.2 Hz, CH3CH2), 1.37 (1H, d, J = 1.5
Hz, CHAHB), 1.58−1.60 (1H, m, CHAHB), 1.85−1.90 (2H, m, CH2),
1.95−1.97 (1H, m, CHCHD), 2.07−2.13 (1H, m, CHCHD), 2.61−2.71
(1H, m, CH-2), 2.91−3.00 (2H, m, CH2), 3.29 (1H, dd, J = 8.8, 19.1
Hz, CHEHF), 3.47 (1H, dd, J = 5.0, 19.1 Hz, CHEHF), 3.52 (2H, br. s,
2xCH), 4.09 (2H, q, J = 7.2 Hz, CH2CH3), 8.93 (1H, CH-6). Isomer
II δ 1.13−1.72 (2H, m, CH2), 1.22 (3H, t, J = 7.2 Hz, CH3CH2), 1.40
(1H, d, J = 1.5 Hz, CHAHB), 1.60−1.63 (1H, m, CHAHB), 1.90−1.92
(2H, m, CH2), 1.98−2.07(1H, m, CHCHD), 2.13−2.22 (1H, m,
CHCHD), 2.72−2.81 (1H, m, CH-2), 3.00−3.12 (2H, m, CH2), 3.33
(1H, dd, J = 10.7, 19.1 Hz, CHEHF), 3.52 (2H, br. s, 2xCH), 3.62 (1H,
dd, J = 5.8, 19.1 Hz, CHEHF), 4.12 (2H, q, J = 7.2 Hz, CH2CH3), 8.93
(1H, CH-6). Isomer I/II 1/1. 13C NMR (CDCl3): isomer I δ 14.26
(CH3), 24.27 (CH2), 25.16 (CH2), 25.25 (CH2), 29.27 (CH2), 32.46
(CH2), 38.82 (CH-2), 40.79 (CH), 41.25 (CH), 47.10 (CH2), 124.93
(Cquat), 130.31 (Cquat), 134.98 (Cquat), 145.53 (CH-6), 152.49 (Cquat),
155.33 (Cquat), 164.42 (Cquat), 174.54 (CO), 181.71 (CO),
184.37 (CO). Isomer II δ 14.26 (CH3), 24.55 (CH2), 25.16 (CH2),
25.30 (CH2), 29.92 (CH2), 32.91 (CH2), 39.34 (CH-2), 40.84 (CH),
41.33 (CH), 47.37 (CH2), 124.93 (Cquat), 130.37 (Cquat), 135.20
(Cquat), 145.61 (CH-6), 152.55 (Cquat), 155.36 (Cquat), 164.42 (Cquat),
174.59 (CO), 181.72 (CO), 184.43 (CO). IR (ATR): ν 2956
(CH), 2875 (CH), 1728 (CO), 1655 (CO), 1566 (CHAr), 1179
cm−1. MS: m/z (%) 352 ([M + H]+, 100). HRMS (ESI): calcd for
[C21H22NO4]+, 352.1549; found, 352.1527.
2-Ethoxycarbonyl-1,2,3,4,8,9,10,11-octahydro-8,11-
ethanobenzo[j]phenanthridine-7,12-dione 5e. Yield 10%, light
1
brown solid, mp 118−120 °C. H NMR (CDCl3): δ 1.30 (3H, t, J
= 7.2 Hz, CH3CH2), 1.35 (4H, br d, J = 8.3 Hz, 2xCH2), 1.79 (4H, m,
br d, J = 8.3 Hz, 2xCH2), 2.02−2.15 (1H, m, CH), 2.22−2.32 (1H, m,
CH), 2.77−2.87 (1H, m, CH), 3.04−3.21 (2H, m, CH2), 3.45 (1H,
dd, J = 18.6, 8.1 Hz, CH), 3.51 (2H, br. S, CH2), 3.70 (1H, dd, J =
18.6, 5.5 Hz), 4.20 (2H, q, J = 7.2 Hz, OCH2CH3), 9.17 (1H, CH-6).
13C NMR (CDCl3): δ 14.35 (CH3CH2), 24.56 (CH2), 25.22 (CH2),
2-tert-Butyl-1,2,3,4,8,9,10,11-octahydro-8,11-ethanobenzo[j]-
phenanthridine-7,12-dione 5b. Yield 33%, yellow solid, mp 154.0 °C.
1H NMR (CDCl3): δ 1.03 (9H, s, C(CH3)3), 1.23−1.34 (4H, m,
2xCH2), 1.40−1.49 (2H, m, CH2), 1.73−1.85 (4H, m, 2xCH2), 2.09−
2.16 (1H, m, CH), 2.86 (1H, dd, J = 11.0, 18.6 Hz, CH), 2.96−3.08
(1H, m, CH), 3.16−3.24 (1H, m, CH), 3.51 (2H, s, 2xCH), 3.60 (1H,
dd, J = 18.6, 3.6 Hz), 9.14 (1H, CH-6). 13C NMR (CDCl3): δ 23.46
(CH2), 25.20 (CH2), 25.25 (CH2), 25.40 (CH2), 25.48 (CH2), 26.52
(CH-8 or CH-11), 26.87 (CH-8 or CH-11), 27.39 (C(CH3)3), 29.65
(CH2), 32.72 (C(CH3)3), 35.52 (CH2), 44.47 (CH-2), 124.34 (Cquat),
133.10 (Cquat), 134.61 (Cquat), 145.71 (CH-6), 149.18 (Cquat), 151.84
(Cquat), 181.96 (CO), 184.45 (CO). IR (ATR): ν 2944 (CH),
2867 (CH), 1660 (CO), 1618 (CHAr), 1566 (CHAr), 1297 cm−1.
MS: m/z (%) 350 ([M + H]+, 100). HRMS (ESI): calcd for
[C23H28NO2]+, 350.2120; found, 350.2119.
25.25 (CH2), 25.29 (2xCH2), 25.34 (CH2), 26.53 and 27.06 (CH-8
and CH-11), 29.89 (CH2), 32.91 (CH2), 39.29 (CH-2), 124.48
(Cquat), 130.37 (Cquat), 134.64 (Cquat), 146.11 (CH-6), 149.29 (Cquat),
151.87 (Cquat), 164.34 (Cquat), 174.69 (CO), 181.68 (CO),
184.02 (CO). IR (ATR): ν 2980 (CH), 1725 (CO), 1654 (C
O), 1301, 1240, 1180 cm−1. MS: m/z (%) 366 ([M + H]+, 100).
HRMS (ESI): calcd for [C22H24NO4]+, 366.1705; found, 366.1692.
2-tert-Butoxycarbonyl-1,2,3,4,8,9,10,11-octahydro-2-aza-8,11-
methanobenzo[j]phenanthridine-7,12-dione 5g. Yield 17%, orange
solid, mp 98 °C. 1H NMR (CDCl3): 1H NMR (CDCl3): δ 1.23−1.26
(2H, m, CH2), 1.02 (10H, s+m, C(CH3)3 and CHAHB), 1.69−1.72
(1H, m, CHAHB), 2.00 (2H, dd, J = 2.5, 6.9, CH2), 3.15 (2H, t, J = 6.1
Hz,CH2-4), 3.63 (2H, s, CH-8 and CH-11), 3.73 (1H, dt, J = 12.9, 6.1
2-tert-Butyl-1,2,3,4,8,9,10,11-octahydro-8,11-epoxybenzo[j]-
phenanthridine-7,12-dione 5c. Yield 14%, yellow solid, mp 128.5 °C.
1H NMR (CDCl3): major isomer δ 1.02 (9H, s, C(CH3)3), 1.37−1.51
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Hz, CHAHB-3), 3.87 (1H, dt, J = 12.9, 6.1 Hz, CHAHB-3), 9.15 (1H,
CH-6). 13C NMR (CDCl3): δ 25.23 (CH2), 25.28 (CH2), 28.40
(C(CH3)3), 33.77 (CH2-4), 40.89 (CH2-3), 41.31 (CH-8 and CH-9),
45.11 (CH2-1), 47.45 (CH2-13), 80.52 (C(CH3)3), 124.86 (Cquat),
124.86 (Cquat), 129.19 (Cquat), 134.11 (Cquat), 146.25 (CH-6), 152.98
(Cquat), 154.66 (Cquat), 155.21 (Cquat), 162.84 (CO), 181.58 (2xC
O). IR (ATR): ν 2980 (CH), 1697 (CO), 1659 (CO), 1160,
1150, cm−1. MS: m/z (%) 381 ([M + H]+, 70). HRMS (ESI): calcd
for [C22H25N2O4]+, 381.1814; found, 381.1806. Synthesis of enamine
adduct 15c.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Additional experimental info and H and 13C NMR spectra of
1
all new compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: khuygen@wiv-isp.be. Fax: 32 2 373 33 67. Tel.: 32 2
373 33 70.
■
Author Contributions
¶Both authors contributed equally to the paper.
Notes
The authors declare no competing financial interest.
(15) Cappoen, D.; Jacobs, J.; Nguyen Van, T.; Claessens, S.; Diels,
G.; Anthonissen, R.; Einarsdottir, T.; Fauville, M.; Verschaeve, L.;
Huygen, K.; De Kimpe, N. Straightforward palladium-mediated
synthesis and biological evaluation of benzo[j]phenanthridine-7,12-
diones as anti-tuberculosis agents. Eur. J. Med. Chem. 2012, 48, 57−68.
ACKNOWLEDGMENTS
■
This work was partially supported by the Research Foundation
Flanders (FWO-Vlaanderen) (Grant G.0020.10N) and by
funding from the European Community’s Seventh Framework
Programme (FP7/2007-2013) under grant agreement FP7-
223681.
(16) (a) Valderrama, J. A.; Araya-Maturana, R.; Gonzal
́
es, F.; Tapia,
R.; Farina, F.; Paredes, M. C. Studies on quinones. Part 21.
̃
Regioselective synthesis of tetracyclic quinones related to rabelomycin.
J. Chem. Soc., Perkin Trans. 1 1991, 555−559. (b) Valderrama, J. A.;
ABBREVIATIONS USED
■
́ ́
Colonelli, P.; Vasquez, D.; Florencia Gonzalez, M.; Rodríguez, J. A.;
AIDS,acquired immune deficiency syndrome; HIV,human
immunodeficiency virus; IC50,neutral red uptake inhibition of
50%; MIC,concentration at which the growth is reduced by
99%; M. tb,Mycobacterium tuberculosis; M. avium,Mycobacterium
avium; M. bovis,Mycobacterium bovis; M. ulcerans,Mycobacterium
ulcerans; Spp,subspecies; TB,tuberculosis; WHO,World Health
Organization
Theoduloz, C. Studies on quinones. Part 44: Novel angucyclinone N-
heterocyclic analogues endowed with antitumoral activity. Bioorg. Med.
Chem. 2008, 16, 10172−10181. (c) Iribarra, J.; Vasquez, D.;
́
Theoduloz, C.; Benites, J.; Ríos, D.; Valderrama, J. Synthesis and
antitumor evaluation of 6-Aryl-substituted benzo[j]phenanthridine-
and benzo[g]pyrimido[4,5-c]isoquinolinequinones. Molecules 2012,
17, 11616−11629.
́ ́
(17) Valderrama, J. A.; Gonzalez, M. F.; Colonelli, P.; Vasquez, D.
Design and synthesis of angucyclinone 5-Aza analogues. Synlett 2006,
17, 2777−2780.
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