2,4-Dialkyl-8,9,10,11-tetrahydrobenzo[g]pyrimido[4,5-c]isoquinoline-1,3,7, 12(2H,4H)-tetraones as new leads against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2014.03.024

Given the re-emergence of tuberculosis in Europe and beyond, the search for novel bio-active compound classes against this disease is of utmost importance. As a result of a high intrinsic tolerance of the etiological agent, Mycobacterium tuberculosis, towards most antibiotics and xenobiotics, the search for such new compounds is far from trivial. Further exacerbated by the rapid generation and spread of drug resistant M. tuberculosis and fuelled by the HIV/AIDS pandemic, halting the tuberculosis epidemic is of paramount importance. As part of our program to design new 2-aza-anthraquinones with anti-mycobacterial activity, various dialkyltetrahydrobenzo[g]pyrimido[4,5-c] isoquinolinetetraones were designed and synthesised. The compounds were submitted to a biological evaluation in which the activity against M.tb H37Rv^lux was observed, as well as the acute toxicity towards J774 A.1 macrophages. From these results, the selectivity index was calculated. Furthermore, the activity of the most promising compounds was further studied against a multi-drug resistant LAM-1 strain and against intracellular replicating M.tb. The study was further extended with a comet assay and a VITOTOX assay to investigate the possibility of observable genotoxic effects caused by these compounds.

Full text of DOI:10.1016/j.ejmech.2014.03.024

European Journal of Medicinal Chemistry 77 (2014) 409e421
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
2,4-Dialkyl-8,9,10,11-tetrahydrobenzo[g]pyrimido[4,5-c]isoquinoline-
1,3,7,12(2H,4H)-tetraones as new leads against Mycobacterium
tuberculosis
,
,
Pieter Claes a 1, Davie Cappoen b 1, Cynthia Uythethofken a, Jan Jacobs a, Birgit Mertens d,
Vanessa Mathys c, Luc Verschaeve d e, Kris Huygen b , Norbert De Kimpe
a Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent,
,
,
a,
*
*
Belgium
b Scientific Service Immunology, O.D. Communicable & Infectious Diseases, Scientific Institute of Public Health (Site Ukkel), Engelandstraat 642, B-1180
Ukkel, Belgium
c Program Tuberculosis and Mycobacteria, O.D. Communicable & Infectious Diseases, Scientific Institute of Public Health (Site Ukkel), Engelandstraat 642,
B-1180 Ukkel, Belgium
d Program Toxicology, O.D. Public Health and Surveillance, Scientific Institute of Public Health (Site Elsene), J. Wytsmanstraat 14, B-1050 Brussels, Belgium
e Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Given the re-emergence of tuberculosis in Europe and beyond, the search for novel bio-active compound
classes against this disease is of utmost importance. As a result of a high intrinsic tolerance of the
etiological agent, Mycobacterium tuberculosis, towards most antibiotics and xenobiotics, the search for
such new compounds is far from trivial. Further exacerbated by the rapid generation and spread of drug
resistant M. tuberculosis and fuelled by the HIV/AIDS pandemic, halting the tuberculosis epidemic is of
paramount importance. As part of our program to design new 2-aza-anthraquinones with anti-
mycobacterial activity, various dialkyltetrahydrobenzo[g]pyrimido[4,5-c]isoquinolinetetraones were
designed and synthesised. The compounds were submitted to a biological evaluation in which the ac-
tivity against M.tb H37Rvlux was observed, as well as the acute toxicity towards J774 A.1 macrophages.
From these results, the selectivity index was calculated. Furthermore, the activity of the most promising
compounds was further studied against a multi-drug resistant LAM-1 strain and against intracellular
replicating M.tb. The study was further extended with a comet assay and a VITOTOXÔ assay to inves-
tigate the possibility of observable genotoxic effects caused by these compounds.
Received 5 October 2013
Received in revised form
10 March 2014
Accepted 10 March 2014
Available online 12 March 2014
Keywords:
Benzo[g]pyrimido[4,5-c]
isoquinolinetetraones
Tuberculosis
Mycobacterium tuberculosis
MDR M.tb
Acute toxicity
Genotoxicity
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Resistant (MDR) TB. To tackle this problem, the development of
new compounds able to get around the intrinsic tolerance of
Tuberculosis (TB) still poses a tremendous challenge for public
health. Especially in less industrialised countries where healthcare
is less obvious, the figures presented by the World Health Organi-
sation (WHO) are still alarming. In 2011 an estimated 8.7 million
people fell ill with TB and a total of 1.4 million people died including
430 000 co-infected with HIV [1]. Of the 8.7 million people that fell
ill with TB in 2011, 630 000 were diagnosed with Multi Drug
Mycobacterium tuberculosis (M.tb) towards most broad spectrum
antibiotics is needed [2].
During our program to design new 2-aza-anthraquinones with
anti-mycobacterial activity, it was found that benzo[j]phenan-
tridine-7-12-diones 1 were active against M.tb having GI50’s in the
micromolar range accompanied with a cytotoxicity of the same
order of magnitude [3]. However, as these compounds are
completely planar, they are potential DNA intercalators. In order to
synthesize more ‘out of plane’ compounds to counter potential
intercalation, the synthesis of dialkyltetrahydromethanobenzo[g]
* Corresponding authors.
pyrimido[4,5-c]isoquinolinetetraones
2
and dialkyltetrahy-
E-mail addresses: kris.huygen@wiv-isp.be (K. Huygen), norbert.dekimpe@
UGent.be (N. De Kimpe).
droethanobenzo[g]pyrimido[4,5-c]isoquinolinetetraones
3
was
1
envisaged (Scheme 1).
Both authors contributed equally to the paper.
http://dx.doi.org/10.1016/j.ejmech.2014.03.024
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

410
P. Claes et al. / European Journal of Medicinal Chemistry 77 (2014) 409e421
Next, the synthesis of a hexahydro-8,11-methanobenzo[j]phe-
nanthridine 11 was envisaged. Enaminones 10 were prepared from
4-isopropylcyclohexane-1,3-dione 9 [12] and ammonium acetate in
toluene at 110 ꢀC. These enamines were obtained as an inseparable
mixture of 3-amino-6-isopropyl-cyclohex-2-enone 10a and 3-
amino-4-isopropyl-cyclohex-2-enone 10b. Oxidative addition of
enaminones 10 to dihydroxynaphthalene 7a under the conditions
mentioned above yielded a mixture of regioisomeric 2-aza-an-
thraquinones 11, which were separated my means of preparative
HPLC (Scheme 3).
Scheme 1. Benzophenanthridinediones
dromethanobenzo[g]pyrimido[4,5-c]isoquinolinetetraones
droethanobenzo[g]pyrimido[4,5-c]isoquinolinetetraones 3.
1 and the proposed target dialkyltetrahy-
2.2. Biological activity assays
2
and dialkyltetrahy-
In previous publications, we and others have shown that
luminometry is a rapid and reproducible tool to test the anti-
mycobacterial activity of novel antituberculous compounds [13e
15]. Screening the library of compounds for in vitro activity
against M.tb, was done with the luminescent M.tb H37Rv laboratory
strain (H37Rvlux). The M.tb H37Rvlux strain has multiple copies of
pSMT1 plasmid carrying the necessary genes to express the ATP
independent bacterial luciferase of Vibrio harveyi driven by a
constitutive hsp60 promoter. Addition of the luciferase substrate n-
decanal, elicits a luminescent signal of green-blue light (490 nm)
which can be measured by luminometry. The influence of com-
pounds or other xenobiotics on the bacillary growth can be
measured as a reduction in the intensity of the luminescent signal.
Serial dilutions of the investigated compounds were used to
calculate the concentrations at which 50%, 90% and 99% of the M.tb
growth was inhibited after 6 days (Table 1). Acute toxicity of the
compounds was examined in a Neutral Red dye Uptake assay (NRU)
[16], also shown in Table 1. This test method makes use of the ability
of living cells to bind and incorporate neutral red in their lysosome
structures. The J774 A.1 murine macrophage cell line was selected
as a cellular model since the role of macrophages as host cell for
M.tb. Toxicity is measured as reduced uptake of the neutral red dye
by the macrophage culture and the NI50 is determined as the
concentration at which this reduction reaches 50%. The Selectivity
Index (SI) was calculated as a function of the NI50 divided with the
GI50. The bacillary growth was reduced by half at concentrations
2. Results and discussion
2.1. Synthesis of the target compounds
5,8-Dimethoxy-1,4-dihydronaphthalenes 4 [4] were hydroge-
nated over palladium on carbon and subsequently formylated
[5] using dichloromethyl methyl ether and titanium(IV)
chloride. The resulting 5,8-dimethoxy-1,4-dihydronaphthalene-
6-carboxaldehydes 6 were then subjected to a boron(III) bromide
mediated
demethylation
towards
5,8-dihydroxy-1,2,3,4-
tetrahydronaphthalene-6-carboxaldehydes 7a and 7b in 81% and
61% yield, respectively. The synthesis of 2-aza-anthraquinones 2
and 3 was effectuated by means of an oxidative addition of 6-
amino-1,3-dialkyluracils
8 [6,7] onto 5,8-dihydroxy-1,2,3,4-
tetrahydronaphthalene-6-carboxaldehydes 7. This type of reac-
tion allows for formation of the isoquinoline skeleton under mild
reaction conditions [8e11] (Scheme 2).
In general the yields of the oxidative addition were lower for the
short chained compounds and higher for the longer chained com-
pounds. No significant differences in yields were observed between
the methano- and the ethano-bridged series. In case of
6-aminouracil 8a (R ¼ H), no reaction was observed even after
24
h for both 5,8-dihydroxy-1,2,3,4-tetrahydronaphthalene-6-
carboxaldehydes 7.
Scheme 2. Synthesis of the target dialkyltetrahydromethanobenzo[g]pyrimido[4,5-c]isoquinolinetetraones 2 and 3.

P. Claes et al. / European Journal of Medicinal Chemistry 77 (2014) 409e421
411
Scheme 3. Synthesis of the hexahydro-8,11-methanobenzo[j]phenanthridine 11.
below
GI50 ¼ 0.52
compound 3e; GI50 ¼ 0.73
which 99% of the bacillary growth was reduced was observed to be
significantly higher for these components: 2e; GI99 ¼ 3.00 g/mL,
g/mL. In general, the
methano bridged quinones exerted a higher potency than the
corresponding ethano bridged analogues carrying the same sub-
stitutions. However, the nature of the dialkyl substitution showed a
1
m
g/mL for the methano bridged compounds 2e;
g/mL, 2k; GI50 ¼ 0.63 g/mL and ethano bridged
g/mL. However the concentration at
bigger influence on the activity shown by the compounds than the
ethano/methano bridge. Compounds carrying the same sub-
stitutions from both the ethano and the methano bridged group, for
example 2e and 3e with an i-Pr substitution were both identified
for their activity. This was also true for the n-heptyl substituted
compounds 2k and 3k which only differ from each other in the
ethano or methano bridge. The increase in potency of the
substituted compounds could be possibly attributed to the increase
in LogP in the methano bridged series 2fe2k and the ethano
bridged series 3fe3k as the potency within these series increases
and decreases as function of the LogP confirming the observations
reported in previous publications [13]. This rule did not apply for
compounds 2e and 3e which show a higher potency relative to the
other compounds despite the lower calculated lipophilicity. To
evaluate if the branched i-propyl group was responsible for the
increased potency, compounds with a n-butyl (2f, 3f) were
compared with i-butyl (2g, 3h) analogues and n-pentyl (2h, 3h)
with i-pentyl (2i, 3i) substituted derivatives. Within the group of
these derivatives, branched carbon chain substitutions did not lead
to increased activity of the compounds over their linear carbon
chain substituted analogues. As both regioisomers 11 showed a
very poor activity against M.tb, no further derivatives were
synthesized.
Macrophages are the most important target cell for M.tb infec-
tion [17]. For most of its infectious cycle, M.tb resides within the
early endosome of the host macrophages where it replicates.
Therefore it is important to investigate if the selected derivatives
2e, 2k, 3e and 3k are able to reach their target through the mem-
brane structure of the macrophages. In the macrophage infection
assay set up, the murine J774 A.1 macrophage like monocyte cell
line was first infected with M.tb H37Rvlux. After 24 h of infection,
the infected macrophages were washed and treated with a serial
dilution of the compounds for 4 days. Treatment was followed by
washing and subsequent lysis of the J774 monolayer. The lumi-
nescence emitted from the cell lysate was compared with infected
macrophages which received no treatment. If the bacterial load
inside the cells was diminished it was concluded that the com-
pound was able to reach its target and kill it. The observations
visualised in Fig. 1 confirm that the compounds reduced the
intracellular M.tb replication. The potency to do so for compound 2e
m
m
m
m
2k; GI99 ¼ 5.57
mg/mL and 3e; GI99 ¼ 4.89
m
Table 1
Growth
quinolinetetraones
inhibition
of
and
dialkyletrahydromethanobenzo[g]pyrimido[4,5-c]iso-
and hexahydro-2H-8,11-methanobenzo[j]phenan-
2
3
thridine-1,7,12-triones 11 on M.tb and acute toxicity against macrophages.
a
b
c
d
Cmpd.
R
GI50
GI90
GI99
NI50
SIe
miLogPf
m
g/mL
2b
2c
2d
2e
2f
2g
2h
2i
Me
Et
n-Pr
i-Pr
1.58
1.69
1.73
0.52
1.46
2.08
1.26
2.41
1.22
0.63
2.83
2.55
2.59
0.73
1.84
4.36
1.49
4.9
5.87
5.32
4.28
1.5
4.59
5.3
10.43
9.21
6.23
3.00
7.23
11.01
9.54
16.32
11.6
15.36
18.23
18.7
9.71
9.5
9.72
0.047
0.799
1.804
1.525
2.923
2.291
3.933
3.353
4.943
5.954
0.317
1.069
2.075
1.795
3.193
2.562
4.203
3.623
5.214
6.224
-
10.79
18.67
10.81
6.51
8.06
10.61
9.32
9.90
42.00
5.94
7.79
8.50
16.14
5.36
4.69
10.99
4.77
10.07
24.84
N.D.
n-Bu
i-Bu
n-Pe
i-Pe
n-Hex
n-Hept
Me
Et
n-Pr
i-Pr
n-Bu
i-Bu
n-Pe
i-Pe
n-Hex
n-Hept
H
16.76
13.37
22.47
12.08
26.46
16.8
19.87
22.01
11.78
9.87
20.43
16.37
23.36
13.7
32.79
N.D.g
N.D.
4.09
8.11
5.43
2.45
6.98
4.33
5.98
2.32
5.2
9.31
4.78
10.33
6.13
3.78
39.2
24.66
0.04
2j
2k
3b
3c
3d
3e
3f
3g
3h
3i
5.57
15.43
12.06
7.34
4.89
9.6
12.28
13.37
22.88
14.89
6.89
>100
48.06
0.06
3j
1.36
1.32
20.38
5.55
0.01
3k
11a
11b
INHh
H
N.D.
N.D.
-
-
N.D.
a,b,c
The concentration of the analogues at which a growth inhibition (GI) of 50%,
90% and 99%, respectively, was observed of the M.tb H37Rv lab strain compared to
the negative control.
d
The concentration of the analogues at which viability of the macrophages was
reduced by 50%.
was tested at a serial dilution ranging from (0.78
mL). Within this range significant reduction of 57.72% at 3.13
and 98.03% at 6.25 g/mL compared with the negative control was
m
g/mLe6.25
mg/
e
The selectivity index (S.I) was calculated by dividing the NIb50 by the GI5c0
miLogP; the Octanolewater partition coefficient calculated on a web based
.
m
g/mL
f
m
algorithm on www.molinspiration.com.
g
observed after 4 days of treatment. A significant reduction of
intracellular replicating M.tb could not be shown at the lower
N.D. Not Determined.
INH, the first line M.tb antibiotic isoniazid used as a positive control.
h

412
P. Claes et al. / European Journal of Medicinal Chemistry 77 (2014) 409e421
Fig. 1. Inhibition of intracellular growth of M.tb inside macrophages by compounds 2e, 2k, 3e and 3k. Growth inhibition of M.tb H37Rv lab strain inside J774 A.1 macrophages
mean ꢁ SD of triplicate cultures. Treatment of the infected cells started 24 h post infection and lasted for 4 days.
concentrations. Concentrations of 12.5
m
g/mL and higher exceed
fluorescence within the MGIT tube when visualised under UV light.
The intensity of fluorescence is directly proportional to the extent
of oxygen depletion and the growth of the mycobacterial. Drug
susceptibility can be measured in 4e6 days.
The growth inhibition induced by a compound was deduced by
comparing the growth index of the M.tb culture exposed to com-
pound concentration with the growth index of an untreated control
culture 100 times (C/100), respectively. As a resistant M.tb strain, a
LAM-1 clinical isolate was selected. This MDR M.tb strain shows
resistance against isoniazid, rifampicin, rifabutine and
prothionamide.
the critical NI50 value observed in the Neutral red uptake assay, the
data could not be considered reliable due to the acute toxicity to-
wards the monocytes. The qualitative viability of the cells before
lysation was guaranteed by trypan blue staining. Compound 2k,
characterised by a lower acute toxicity towards the monocytes was
tested at a serial dilution ranging from (0.78
For the concentrations of 2k tested a reduction of bacillary repli-
cation of 65.5% at 3.13 g/mL, 80.4% at 6.25 g/mL and 91.3% at
12.5 g/mL was observed. Compound 3e was tested at concentra-
tions ranging from 0.78 g/mL to 6.25 g/mL and induced a sig-
nificant reduction in intracellular M.tb replication in this assay of
58.7% at 3.13 g/mL and 82.3% at 6.25 g/mL. By exposing the
infected monolayer to a range of 0.78 g/mLe12.50 g/mL of
compound 3k a significant reduction by 68.8% and 92.0% could be
observed at 6.25 g/mL and 12.5 g/mL, respectively. Although
mg/mLe12.50 mg/mL).
m
m
m
m
m
The growth index of the C/100 tube after 6 days was situated
m
m
between 5
that the critical concentration to induce a 99% growth reduction
was situated between 5 g/mL and 10 g/mL. The critical concen-
tration towards this MDR LAM-1 M.tb strain for compound 2k, 3e
and 3k was situated between 10 g/mL and 20 g/mL, confirming
the sensitivity of the strain towards the derivatives. 0.1 g/mL INH
mg/mL and 10 mg/mL for compound 1e. This indicated
m
m
m
m
m
m
slowed down by exposure to the serial dilutions of the compounds,
the bacillary load inside the monolayer increased progressively
with time, indicating a recovery of the intracellular bacterial
replication. However this recovery was not observed when the
m
m
m
did not seem to inhibit the growth which at this critical concen-
tration confirms at least the resistance against INH.
monolayer was treated with 6.25
m
g/mL of derivative 2e.
Next, the potential genotoxicity of the target molecules was
assessed. Early signs of genotoxicity can be observed by studying
the activation of DNA repair mechanisms. The regulatory SOS
operon is key to the repair of early cellular DNA damage and its
activation is mediated by a multiple component system. As a result
of DNA interruption, binding of ssDNA fragments to the recA pro-
tein increases the enzymatic affinity for the lexA repressor protein.
The decrease in lexA frees the SOS box consensus sequence and
thereby facilitates the binding of the general transcription ma-
chinery to the recN promoter sequence and transcription of the SOS
To extend the study, antimicrobial activity against a multidrug
resistant M.tb isolate was determined with the fluorometric BAC-
TEC MGIT 960Ô Mycobacteria Growth Indicator [18] (Fig. 2). This
standardised automated fluorometric assay relies on an oxygen-
quenched fluorochrome, tris(4,7-diphenyl-1,10-phenanthroline)
ruthenium(II) chloride pentahydrate, embedded in silicone at the
bottom of the tube. During mycobacterial growth within the tube,
free oxygen is utilised and is replaced with CO2. With depletion of
free oxygen, the fluorochrome is no longer inhibited, resulting in

P. Claes et al. / European Journal of Medicinal Chemistry 77 (2014) 409e421
413
Fig. 2. Susceptibility of an MDR LAM-1 M.tb strain towards compounds 2e, 2k, 3e and 3k. Growth of MDR M.tb was monitored in BACTEC MGIT 960Ô. a,bGrowth was compared to
untreated bacterial suspension and untreated bacterial suspension diluted 100 times (C/100) to study the Minimal Inhibitory concentration MIC. cResistance of the MDR LAM-1
strain was confirmed for isoniazid (INH).
operon. Based on this molecular mechanism, the VITOTOXÔ model
from Gentaur can detect activation of the SOS operon by an inte-
grated lux operon. The model employs two recombinant Salmonella
typhimurium reporter strains. The integrated lux operon of the
Genox strain T104 (recN2-4) is expressed under transcriptional
control of the recN promoter and in the Cytox pr1 strain it is driven
by the strong constitutive pr1 promoter, the latter strain mainly
functioning as an internal control. Both recombinant reporter
strains lack the necessary oxidative machinery to metabolize xe-
nobiotics. By adding S9 liver fraction to genotoxicity of the inves-
tigated substance can be distinguished from that of its metabolites
[19]. Closely correlated with the AMES test (91%), with a high
specificity of 94%, this model allows an early elimination of novel
compound classes due to genotoxicity [20].
regarded a false positive. S/N values lower than 0.8 for the Cytox
strain are indicative of direct cytotoxicity and exclude false nega-
tives. As shown in Fig. 3, compounds 2e, 2k, 3e, 3k resulted in
values of the Cytox control strain between 0.8 and 1.5 with and
without the S9 liver extract. The luminescence emitted from the
Genox strain did not exceed S/N values significant higher as 1.5
after addition of the compounds in absence and presence of S9
extract. No activation the SOS operon could be observed as a sign of
early genotoxicity caused by the compounds 2e, 2k, 3e, 3k or their
metabolites.
Further study of the genotoxic properties was performed for
compounds 2e, 2k, 3e and 3k using a Comet assay [21]. With this
assay, fragmentation of the genomic DNA can be detected. Briefly,
JJ74 A.1 cells were exposed to a compound concentration and
immobilised onto agarose. Immobilised cells were lysed and the
remaining nuclei were submitted to electrophoresis. Through pore
exclusion of the agarose, fragmented DNA migrated further with
the applied current. After staining with gel red it can be observed
that the genomic DNA of an undamaged nucleus maintains its
spherical organisation (HEAD) whereas fragmented DNA migrates
further across the gel (TAIL). The ratio (DNA in the TAIL)/(DNA in the
HEAD þ TAIL) is a measure of DNA fragmentation due to the gen-
otoxicity of the compounds for the J774 A.1 murine macrophage
Emission of luminescence by the strains was recorded in real
time after the addition of the compounds every five minutes during
a four hour period. The compounds were tested at 10
mg/mL, 1 mg/
mL and 0.1 g/mL in the presence and absence of the metabolizing
m
S9 fraction. Genotoxicity was deduced from the signal to noise ratio
(S/N), being the light produced by the bacterial suspension exposed
to the compounds divided by the light produced by a non-exposed
bacterial suspension. S/N values for the Genox (recN2-4) higher
than 1.5 were interpreted as positive and implied genotoxic prop-
erties by the compounds. For the Cytox (pr1) strain, S/N values
above 1.5 are considered as direct influence of the compound on
the bacterial luciferase or the luminescent signal itself and
like monocytes. For the concentrations 0.5
and 10 g/mL of derivatives 2e, 2k, 3e and 3k, there was no dif-
ference in structural organisation of the genomic DNA due to DNA
mg/mL, 1 mg/mL, 5 mg/mL
m

414
P. Claes et al. / European Journal of Medicinal Chemistry 77 (2014) 409e421
Fig. 3. The VITOTOXÔ assay for detection of early signs of genotoxicity caused by compounds 2e, 2k, 3e and 3k. Maximum recorded S/N in a time span of 4 h by the Genox (recN2-
4) and Cytox (pr1) reporter strains. 4NQO; 4-nitroquinoline-1-oxide genotoxic positive control in samples without S9 liver fraction. Bap; benzo[a]pyrene, the positive control, only
turns genotoxic after S9 metabolisation.
breakage effects detectable (Fig. 4). A significant increase in the
DNA ratio in the comet tail to DNA in the Comet head compared
with the negative control was not observable (ManneWhitney
test). To avoid unreliability of the observations by using concen-
trations of the compounds exceeding the NI50 values obtained with
the NRU assay, higher concentrations were not tested.
mL (2k) and at 1.32
and 3e (GI50 ¼ 0.73
m
m
g/mL (3k). Compounds 2e (GI50 ¼ 0.52
mg/mL)
g/mL), substituted with two i-propyl groups,
showed a higher potency despite the low LogP of these compounds.
The other branched compounds 2f, 3f and 2h, 3h did not show
increased activity over their linear carbon chain substituted ana-
logues (2g, 3g) and (2i, 3i).
Although the present compounds showed a lower potency to
reduce the growth of intracellular replicating M.tb as compared
with previous reported 2-aza-anthraquinones, they also showed a
lower acute toxicity towards the host cells and therefore higher
concentrations of the compounds could be used [3]. For the most
potent compound of this study, compound 2e, a reduction by 98.0%
3. Conclusion
A library of ten dialkyltetrahydromethanobenzo[g]pyrimido
[4,5-c]isoquinolinetetraones
droethanobenzo[g]pyrimido[4,5-c]isoquinolinetetraones
2
and
ten
dialkyltetrahy-
was
3
synthesised by means of an oxidative addition methodology. Both
the methano and ethano bridged derivatives showed the potency to
reduce in vitro bacillary growth of the M. tuberculosis lab strain
H37Rvlux. Study on the structure-activity relation showed that the
methano bridged derivatives were more potent than the ethano
bridged derivatives but that the nature of the dialkyl substitution is
decisive for the potency of the molecule against the M.tb strain. For
the majority of the compounds, the observed activity correlated
with the calculated LogP of the compounds as a result of the carbon
chain substitution pattern, which confirms previous observations
[13]. With increasing LogP, an increased potency could be observed.
The same rule was observed both for the methano and the ethano
bridged derivatives in which compounds with a n-hexyl substitu-
was shown at 6.25 mg/mL. The potency of the compounds against
the intracellularly replicating M.tb H37Rv strain was lower than the
activity against extracellularly replicating M.tb H37Rv in which we
observed a 99.0% inhibition at 3 mg/mL for compound 2e. This could
be due to the partial inability of the compound to cross the plasma
and phagosome membrane of the monocytes resulting in less
efficient bio-distribution. The reduced activity could also be a
consequence of the differences in metabolic state between intra-
and extracellular replicating M.tb. A third possible explanation is
the possible metabolisation of the compound by the host cell.
Bacterial replication, initially slowed down by the compound
recovered from time point 2 in the macrophage infection assay for
all concentrations of the compounds tested except for 6.25
mg/mL of
tion showed a 50% growth inhibition (GI50) against M.tb at 0.63 mg/
compound 2e. The recovery of the bacillary replication could also

P. Claes et al. / European Journal of Medicinal Chemistry 77 (2014) 409e421
415
4.1.3. Monitoring mycobacterial growth by luminometry
The growth inhibition (GI) against mycobacteria of all syn-
thesised 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 synthetic compounds were solubilised in DMSO
(SigmaeAldrich) at stock concentrations of 10 mM. Serial dilutions
of each compound were made in liquid 7H9 medium [Middlebrook
7H9 broth based (Difco)] þ 10% FCS (Gibco). Volumes of 20
mL of the
serial dilutions were added in triplicate to 96 well, flat-bottomed
microwell 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 mM 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 mL of
bacteria was added to each well. Bacterial replication was analysed
by luminometry after 6 days of culture. 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.
Fig. 4. Detection of DNA fragmentation for compounds 2e, 2k, 3e and 3k by the Comet
assay. Genomic DNA fragmentation after 24 h exposure to different concentrations of
compounds 2e, 2k, 3e and 3k. ManneWhitney was used to test the significance.
be the result of the inactivation of the compound through enzy-
matic degradation by the host cell. Sensitivity of the MDR LAM-1
strain was observed with the BACTEC MGIT 960Ô Mycobacteria
Growth Indicator. The MIC found by luminometry was lower than
the MIC found by the fluorometric based MGIT 960Ô for the
compounds. For example for compound 2e, in luminometry, the
observed critical concentration at which 99% of M.tb H37Rv growth
4.1.4. Assessment of cytotoxicity
Inhibitory effects on the J774 A.1 murine macrophage cell line
were determined for the compounds by a neutral red uptake assay
as described before. The J774 A.1 cells were grown in DMEM þ 10%
FCS until a semi confluent layer of cells was obtained. The cells were
trypsinised, 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 solubilised in DMSO to stock concentrations
was reduced was 3
method was shown between 5
m
g/mL while the MIC with the MGIT 960Ô
g/mL and 10 g/mL by comparing
m
m
with the C/100. An explanation for this could be the differences in
the metabolic states of the slower growing MDR LAM-1 strain and
the H37Rv M.tb strain or a difference in the test set up but lies not
within the scope of this study. Activation of the SOS operon in the
VITOTOXÔ model could not be observed as an early sign of geno-
toxic properties of the compounds and their metabolites for the
concentrations tested. DNA breakage could also not be shown in the
comet assay. Although further tests to investigate toxicity and
genotoxicity are needed, the present compounds show promising
biological features despite their lower potency than the previously
reported aza-anthraquinone compounds [3,15]. Given the low
cytotoxicity, the reported scaffold could be used as a base for the
synthesis of further compounds.
of 10
mg/mL. A serial dilution of each compound was made in
DMEM þ 10% FCS. The J774 A.1 cells were washed and exposed to
the compounds 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
200 L 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 L PBS and 200 L of an ethanol/acetic
mL PBS and
m
m
m
acid (50%) mixture. The plates were left on the shaker until the
colour became homogeneous purple and the optical density was
measured at 530 nm (NR max) and 620 nM (reference wavelength)
with the Paradigm detection platform.
4. Experimental section
4.1.5. Activity against multi-drug resistant M. tuberculosis
4.1. Biological data
Antimicrobial activity of the compounds was tested using the
LAM-1 strain using the BACTECÔ MGITÔ 960-TB detection system.
The compounds were solubilised 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 pre-cultured in a 4 mL BACTEC vial to a growth index
4.1.1. Materials
7H9 growth medium was purchased as a powder from BD Sci-
ence (Franklin Lakes, NJ, USA). DMEM, glutamax, non-essential
amino acids, sodium pyruvate, gentamycin, 2-mercaptoethanol,
PBS were from GIBCO Invitrogen (Carlsbad, CA, USA). 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, USA).
(GI) of 300. Then 100
new 4 mL BACTEC vial together with 100
the compounds. As a positive control for resistance, the MDR LAM-
1 culture was inoculated with 0.1 g/mL isoniazid. The GI was
mL of this pre-culture was inoculated into a
mL of the serial dilutions of
m
measured each day. To determine the MIC99, the cultures, treated
with the compounds were compared with an untreated culture
diluted 100 times upon inoculation.
4.1.2. Strains and growth conditions
M. tuberculosis H37Rv (American Type Culture Collection 27294)
is known to be sensitive to the five first line anti-tuberculosis drugs
(streptomycin, isoniazid, rifampicin, ethambutol and pyr-
azinamide). The strain was cultivated in 7H9 medium supple-
mented with 10% oleic acidealbuminedextroseecatalase (OADC)
and 0.2% glycerol. M.tb was grown at 37 ꢀC.
4.1.6. Inhibition of intracellular M.tb growth
The compounds were tested on the murine J774.A1 macrophage
cell line infected with M.tb. H37Rvlux. The J774 macrophages were
grown at 37 ꢀC, 5% CO2 in complete DMEM medium until a semi

416
P. Claes et al. / European Journal of Medicinal Chemistry 77 (2014) 409e421
confluent layer was formed. The macrophages were washed in
fresh complete DMEM medium and seeded in a flat-bottomed 96-
well microtiter 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 0.6e1.0.
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 syn-
thesised compounds were solubilised in DMSO at stock concen-
trations of 10 mM. Serial dilutions of the peptides were made in
DMEM-Pen/Fung at two times the concentration of each compound
4.2. Chemical data
4.2.1. General experimental methods
Column chromatography was carried out using a glass column
with silica gel (Aldrich, particle size 0.035e0.070 mm, pore diam-
eter 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 oxidation. 1H
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 LCeMS coupling [Phe-
to be tested. A volume of 100
DMEM-Pen/Fung containing 4000 RLU of bacteria (multiplicity of
infection of 0.1) and 100 L of the serial compound dilutions were
mL of the bacterial suspension in
m
added to the macrophage cultures. To measure the effects of the
compounds on intracellular growth of M.tb, the infected macro-
phages were washed three times on day 5 to remove all extracel-
lular bacteria, incubated 1 h with 1% gentamicin to kill residual
nomenex luna column; 250 ꢂ 3 mm length, 5
mm particle size, 100
A 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 spec-
trometer 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 un-
corrected. THF was dried over Na/benzophenone and CH2Cl2 was
distilled from CaH2. With the exception of Ag2O, all reagents were
used without further purification and all glassware was oven-dried
prior to use.
extracellular bacteria, lysed with 200
mL 1% Triton X-100 (Sigma)
and the wells washed four times with 200
mL PBS. The lysate was
transferred in a 2.5 mL tube together with the 4 PBS washings. One
hundred microlitres 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 macro-
phage culture is observed by Trypan blue with a microscope.
4.1.7. VITOTOXÔ assay
4.2.2. Synthesis of 6-amino-1,3-dialkyluracils 8
Observations on genotoxicity were done with VITOTOXÔ
(Gentaur, Kampenhout, BE) and the included protocol was fol-
lowed. In brief, TA104 recN2-4 (Genox) and TA104 (Cytox)
S. 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 þS9 cultures to test the
genotoxic effects of the metabolites of the compounds. The bacte-
rial suspensions were then incubated, shaking at 36 ꢀC, and the
luminescent signal was measured for 4 h with a 5 min interval.
6-Aminouracil 8a and 6-amino-1,3-dimethyluracil 8b are
commercially available. All other 6-amino-1,3-dialkyluracils were
synthesised from triphosgene, a primary amine and cyanoacetic
acid following literature protocols [6,7]. The following 6-amino-1,3-
dialkyluracils 8 were not reported previously in the literature:
4.2.2.1. 6-Amino-1,3-di-n-pentyluracil 8h. 71%, white solid, m.p.
<50 ꢀC. 1H NMR (CDCl3):
d
0.87 (3H, t, J ¼ 6.6 Hz, CH3), 0.89 (3H, t,
J ¼ 6.6 Hz, CH3), 1.24e1.36 (8H, m, 4 ꢂ CH2), 1.53e1.57 (2H, m,
NCH2CH2), 1.66e1.71 (2H, m, NCH2CH2), 3.81e3.90 (4H, m,
2 ꢂ NCH2), 4.97 (1H, s, CH-5), 5.89 (2H, s, NH2). 13C NMR (CDCl3):
4.1.8. Comet assay
d 13.92 (CH3), 13.98 (CH3), 22.35 (CH2), 22.44 (CH2), 27.71 (CH2),
Possible DNA breakage effects of the compounds on C3A cells
were investigated by the alkaline comet assay. The C3A cells were
grown in DMEM þ 10% FCS until a semi confluent layer of cells was
obtained. The cells were trypsinised, 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 di-
lutions of each compound were made in DMEM þ 10% FCS to obtain
the final concentrations. The C3A cells were washed and exposed to
the compounds by adding 1 mL of the serial dilutions to each well.
The plates were incubated at 37 ꢀC, 5% CO2 for 24 h. After incuba-
27.77 (CH2), 28.82 (CH2), 29.13 (CH2), 41.14 (NCH2), 42.76 (NCH2),
77.47 (CH-5), 151.58 (Cquat), 154.32 (Cquat), 163.26 (Cquat). IR (ATR):
3347 (NH), 3204 (NH), 2956 (CH), 2931 (CH), 1607 (C]O),
1492 cmꢃ1. MS m/z (%): 268 ([MþH]þ, 100). HRMS (ESþ) calcd. for
[C14H26N3O2]þ: 268.2025, found 268.2027.
n
4.2.2.2. 6-Amino-1,3-di-iso-pentyluracil 8i. 64%, white solid, m.p.
142e143 ꢀC. 1H NMR (CDCl3):
d
0.94 (6H, d, J ¼ 6.6 Hz, 2 ꢂ CH3), 0.98
(6H, d, J ¼ 6.6 Hz, 2 ꢂ CH3), 1.44e1.73 (6H, m, 2 ꢂ CH2, 2 ꢂ CH),
3.84e3.92 (4H, m, 2 ꢂ NCH2), 4.69e4.81 (2H, m, NH2), 4.97 (1H, s,
CH-5). 13C NMR (CDCl3):
d 22.52 (CH3), 22.64 (CH3), 26.41 (CH),
tion, the cells were trypsinised, washed with PBS, and 10
mL of cell
26.49 (CH), 36.76 (CH2), 36.93 (CH2), 39.95 (NCH2), 41.51 (NCH2),
suspension was dissolved in 300 L low melting point agarose. The
m
77.57 (CH-5), 151.55 (Cquat), 154.03 (Cquat), 163.29 (Cquat). IR (ATR): n
dissolved cell suspension was then placed onto a frosted micro-
scope slide and left on ice for 5 min. The slide was subsequently
placed in a jar 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 neutralisation buffer for
5 min and dried in ice cold ethanol for 10 min. Staining of the DNA
was done with gel red (SigmaeAldrich). For the quantification of
the DNA migration, a fluorescence microscope was used and the
percentage DNA in the comet tail of the cells nuclei core was
calculated in proportion to the total DNA present in the nuclei
(comet head þ tail) by appropriate imager software from Meta-
systems (Altlussheim, Germany).
3351 (NH), 3216 (NH), 2957 (CH), 1608 (C]O), 1583, 1495 cmꢃ1. MS
m/z (%): 268 ([MþH]þ, 100). HRMS (ESþ) calcd. for [C14H26N3O2]þ:
268.2025, found 268.2021.
4.2.2.3. 6-Amino-1,3-di-n-heptyluracil 8k. Quant., white solid, m.p.
<50 ꢀC. 1H NMR (CDCl3):
d
0.82e0.91 (6H,m, 2 ꢂ CH3), 1.17e1.38
(16H, m, 8 ꢂ CH2), 1.53e1.71 (4H, m, 2 ꢂ NCH2CH2), 3.81e3.90 (4H,
m, 2 ꢂ NCH2), 4.99 (1H, s, CH-5), 5.28 (2H, br s, NH2). 13C NMR
(CDCl3):
d 14.13 (CH3), 14.16 (CH3), 22.64 (CH2), 22.70 (CH2), 26.97
(CH2), 27.08 (CH2), 28.10 (CH2), 28.26 (CH2), 29.03 (CH2), 29.17
(CH2), 41.34 (NCH2), 42.87 (NCH2), 78.03 (CH-5), 151.61 (Cquat),
153.79 (Cquat), 163.38 (Cquat). IR (ATR): n 3347 (NH), 2923 (CH), 1698

P. Claes et al. / European Journal of Medicinal Chemistry 77 (2014) 409e421
417
(C]O),1511,1411 cmꢃ1. MS m/z (%): 324 ([MþH]þ,100). HRMS (ESþ)
(%): 247 ([MþH]þ, 100). HRMS (ESþ) calcd. for [C19H19O3]þ:
calcd. for [C18H34N3O2]þ: 324.2651, found 324.2648.
247.1334, found 247.1338.
4.2.5. Synthesis of 5,8-dihydroxy-1,2,3,4-tetrahydronaphthalene-6-
carboxaldehydes 7
4.2.3. Synthesis of aminoisopropylcyclohexenones 10
A pressure vial was loaded with 4-isopropylcyclohexane-1,3-
dione 9 [12] (1.17 g, 7.59 mmol) and ammonium acetate (585 mg,
7.59 mmol) and toluene (6 mL). The vial was sealed and the reaction
was heated at 110 ꢀC for 1 h. After cooling, the solvent was evap-
orated in vacuo to yield crude aminoisopropylcyclohexenones 9,
which were recrystallised from EtOH as colourless prisms. At-
tempts to separate both regioisomers by means of column chro-
matography were unsuccessful.
A solution of 5,8-dimethoxy-1,2,3,4-tetrahydronaphthalene-6-
carboxaldehyde 6a or 6b (20 mmol) in anhydrous CH2Cl2
(100 mL) under a nitrogen atmosphere was cooled to ꢃ60 ꢀC. Next,
BBr3 (120 mmol, 11.6 mL) was added dropwise and the reaction was
allowed to room temperature. After 2.5 h, the reaction was
cautiously quenched with water (80 mL) and extracted with CH2Cl2
(2 ꢂ 50 mL). The combined organic extracts were dried over MgSO4,
evaporated in vacuo and purified by means of column chromatog-
raphy (PE/EA 4/1) to yield the desired 5,8-dihydroxy-1,2,3,4-
tetrahydronaphthalene-6-carboxaldehyde 7a or 7b.
Quant., colourless prisms, m.p. <50 ꢀC. 1H NMR (CDCl3):
d 0.83
(3H, d, J ¼ 6.9 Hz, CH3), 0.96 (3H, d, J ¼ 6.9 Hz, CH3), 1.74e1.87 (1H,
m, CH),1.91e2.50 (2H, m, CH2), 2.31e2.50 (3H, m, CH and CH2), 4.92
(2H, br s, NH2), 5.23 (1H, s, CH-2). 13C NMR (CDCl3):
d
18.53 (CH3),
4.2.5.1. 5,8-Dihydroxy-1,2,3,4-tetrahydro-1,4-methanonaphthalene-
20.94 (CH3), 21.80 (CH2), 26.48 (CH2), 27.87 (CH), 50.07 (CH), 100.02
6-carboxaldehyde 7a. 81%, yellow solid, m.p. 140 ꢀC. 1H NMR
(CH-2), 166.36 (Cquat-NH2), 199.70 (C]O). IR (ATR):
n 3354 (NH),
3157 (NH), 2954 (CH), 1538, 1531, 1207, 1189 cmꢃ1. MS m/z (%): 154
([MþH]þ, 100). HRMS (ESþ) calcd. for [C9H16NO]þ: 154.1232, found
154.1232.
(CDCl3):
d
1.20e1.24 (2H, m, 2 ꢂ CHxHn), 1.55e1.58 (1H, m, CHsHa),
1.73e1.79 (1H, m, CHsHa), 1.93e1.98 (2H, d, J ¼ 9.4 Hz, 2 ꢂ CHxHn),
3.56 (1H, d, J ¼ 1.4 Hz, CH-1 or CH-4), 3.71 (1H, d, J ¼ 1.4 Hz, CH-1 or
CH-4), 4.60e4.62 (1H, m, OH), 6.79 (1H, s, CH-7), 9.73 (1H, s, OH),
10.60 (1H, s, CHO). 13C NMR (CDCl3):
d 25.92 and 26.01 (CH2-2 and
4.2.4. Synthesis of 5,8-dimethoxy-1,2,3,4-tetrahydro-6-
carboxaldehydes 6
CH2-3), 39.54 and 40.58 (CH-1 and CH-4), 49.13 (CH2-9), 117.68
(CH-7), 119.56 (Cquat), 136.52 (Cquat), 142.69 (Cquat), 146.03 (Cquat),
To
a
Parr
bottle
were
added
1,4-dihydro-5,8-
149.45 (Cquat),196.11 (C]O). IR (ATR): n 3331 (OH), 2960 (CH), 1629,
dimethoxynaphthalene 4a or 4b (35 mmol), Pd/C (1 m/m%), EtOH
(25 mL) and EtOAc (125 mL). The reaction was stirred for 3 h at
room temperature under a hydrogen atmosphere of 5 bar. Next,
the reaction mixture was filtered over a pad of CeliteÒ and
evaporated in vacuo to afford pure 1,2,3,4-tetrahydro-5,8-
dimethoxynaphthalene 5a or 5b. These compounds 5 (30 mmol)
were dissolved in anhydrous CH2Cl2 (100 mL), cooled to 0 ꢀC and
under a nitrogen atmosphere, TiCl4 (33 mmol, 3.62 mL) was slowly
added dropwise. Next, Cl2CHOMe (33 mmol, 2.99 mL) was slowly
added dropwise. When the addition was complete, the reaction
was stirred for 3 h at 0 ꢀC and cautiously quenched with ice-cold
water (50 mL). The reaction mixture was washed with brine
(2 ꢂ 50 mL), dried over MgSO4 and evaporated in vacuo. Purification
by means of column chromatography yielded 5,8-dimethoxy-
1,2,3,4-tetrahydronaphthalene-6-carboxaldehydes 6a or 6b as pale
white solids.
1477 (CHAr), 1309, 1243 cmꢃ1. MS m/z (%): 205 ([MþH]þ, 100).
HRMS (ESþ) calcd. for [C12H13O3]þ: 205.0865, found 205.0861.
4.2.5.2. 5,8-Dihydroxy-1,2,3,4-tetrahydro-1,4-ethanonaphthalene-6-
carboxaldehyde 7b. 61%, green solid, m.p. 181 ꢀC. 1H NMR (CDCl3):
d
1.29e1.41 (4H, m, 2 ꢂ CH2),1.75e1.87 (4H, m, 2 ꢂ CH2), 3.39 (1H, s,
CH-1 or CH-4), 3.58 (1H, s, CH-1 or CH-4), 4.79 (1H, s, OH), 6.85 (1H,
s, CH-7), 9.76 (1H, s, OH), 10.80 (1H, s, CHO). 13C NMR (CDCl3):
d
25.17 (2 ꢂ CH2), 25.39 (2 ꢂ CH2), 25.86 and 27.69 (CH-1 and CH-4),
115.28 (CH-7), 117.99 (Cquat), 133.25 (Cquat), 142.31 (Cquat), 143.41
(Cquat), 150.81 (Cquat), 196.20 (C]O). IR (ATR):
n 3309 (OH), 2980
(CH), 1626, 1328, 1188, 670 cmꢃ1. MS m/z (%): 219 ([MþH]þ, 100).
HRMS (ESþ) calcd. for [C13H15O3]þ: 219.1021, found 219.1015.
4.2.6. Synthesis of 2,4-dialkyl-8,9,10,11-tetrahydrobenzo[g]
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraones 2 and 3
To
a
stirred solution of 5,8-dihydroxy-1,2,3,4-tetra
4.2.4.1. 5,8-Dimethoxy-1,2,3,4-tetrahydro-1,4-methanonaphthalene-
hydronaphthalene-6-carboxaldehyde 7a or 7b (0.5 mmol), a 6-
amino-1,3-dialkyluracil 8 (0.5 mmol) and MgSO4 (2.5 mmol,
302 mg) in anhydrous dichloromethane was added freshly pre-
pared Ag2O (2 mmol, 464 mg). The reaction was allowed to stir at
room temperature for 2 h and subsequently filtered over a pad of
celiteÒ and evaporated in vacuo. The residue was purified by means
of preparative TLC (CH2Cl2) to yield pure 2,4-dialkyl-8,9,10,11-
tetrahydrobenzo[g]pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-
tetraones 2 or 3.
6-carboxaldehyde 6a. 96%, pale white solid, m.p. 89 ꢀC. 1H NMR
(CDCl3):
d 1.18e1.30 (2H, m, CH2), 1.52e1.55 (1H, m, CHAHB), 1.73e
1.78 (1H, m, CHAHB), 2.91e2.05 (2H, m, CH2), 3.63 (1H, br d,
J ¼ 1.1 Hz, CH-1 or CH-4), 3.69 (1H, br d, J ¼ 1.1 Hz, CH-1 or CH-4),
3.84 (3H, s, OCH3), 3.94 (3H, s, OCH3), 7.13 (1H, s, CH-5),10.32 (1H, s,
CHO). 13C NMR (CDCl3):
d 25.97 (CH2), 26.73 (CH2), 40.23 (CH), 41.19
(CH), 49.02 (CH2), 55.78 (OCH3), 62.81 (OCH3),106.96 (CHAr),126.87
(Cquat), 140.93 (Cquat), 145.80 (Cquat), 149.58 (Cquat), 151.93 (Cquat),
189.81 (C]O). IR (ATR):
n 2949 (CH), 2868 (CH), 1677 (C]O), 1589,
1475, 1387, 1309, 1212, 1021 cmꢃ1. MS (ESþ) m/z (%): 233 ([MþH]þ,
4.2.6.1. 2,4-Dimethyl-8,9,10,11-tetrahydro-8,11-methanobenzo[g]
100). HRMS (ESþ) calcd. for [C14H17O3]þ: 233.1178, found 233.1179.
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 2b. 54%, yel-
low needles, m.p. 227 ꢀC. 1H NMR (CDCl3):
d
1.25 (2H, d, J ¼ 9.4 Hz,
4.2.4.2. 5,8-Dimethoxy-1,2,3,4-tetrahydro-1,4-ethanonaphthalene-6-
2 ꢂ CHxHn), 1.48 (1H, dd, J ¼ 1.1, 9.4 Hz, CHsHa), 1.71 (1H, d, 9.4 Hz,
CHsHa), 2.02 (2H, d, J ¼ 9.4 Hz, 2 ꢂ CHxHn), 3.48 (3H, s, NCH3), 3.62
(1H, br s, CH-8 or CH-11), 3.68 (1H, br s, CH-8 or CH-11), 3.75 (3H, s,
carboxaldehyde 6b. 97%, pale white solid, m.p. 85 ꢀC. 1H NMR
(CDCl3):
d
1.28e1.41 (4H, m, 2 ꢂ CH2), 1.75e1.86 (4H, m, 2 ꢂ CH2),
3.44 (1H, br s, CH), 3.54 (1H, br s, CH), 3.85 (3H, s, OCH3), 3.86 (3H, s,
NCH3), 9.22 (1H, s, CH-6). 13C NMR (CDCl3):
d 25.61 and 25.89 (CH2-
OCH3), 7.19 (1H, s, CH-5), 10.37 (1H, s, CHO). 13C NMR (CDCl3):
9 and CH2-10), 29.17 (NCH3), 30.59 (NCH3), 41.26 and 42.87 (CH-8
and CH-11), 45.98 (CH2-13), 106.93 (Cquat), 123.93 (Cquat), 144.71
(Cquat), 150.87 (Cquat), 152.23 (CH-6), 152.63 (Cquat), 154.52 (Cquat),
157.33 (NC]O), 158.54 (NC]O), 179.21 (C]O), 181.44 (C]O). IR
d
25.20 (2 ꢂ CH2), 25.46 (2 ꢂ CH2), 26.59 (CH), 27.17 (CH), 55.66
(OCH3), 64.55 (OCH3), 105.13 (CHAr), 126.41 (Cquat), 138.69 (Cquat),
142.03 (Cquat), 150.93 (Cquat), 153.13 (Cquat), 189.70 (C]O). IR (ATR):
n
2943 (CHO), 2863 (CH),1675 (CHAr),1391,1114 cmꢃ1. MS (ESþ) m/z
(ATR): n 2978 (CH), 1720 (C]O), 1670 (C]O), 1658 (C]O), 1574

418
P. Claes et al. / European Journal of Medicinal Chemistry 77 (2014) 409e421
(CHAr), 1546, 1316, 1287 (CeN), 1273 (CeN), 742 cmꢃ1. MS m/z (%):
338 ([MþH]þ, 100). HRMS (ESþ) calcd. for [C18H16N3O4]þ: 338.1141,
found 338.1148.
29.78 (CH2), 30.04 (CH2), 41.26 (CH-8 or CH-11), 42.56 (NCH2),
42.85 (CH-8 or CH-11), 43.46 (NCH2), 45.97 (CH2-13), 107.15 (Cquat),
123.91 (Cquat), 145.00 (Cquat), 150.37 (Cquat), 152.35 (CH-6),
152.64 (Cquat), 154.28 (Cquat), 157.30 (NC]O), 158.51 (NC]O),
4.2.6.2. 2,4-Diethyl-8,9,10,11-tetrahydro-8,11-methanobenzo[g]pyr-
179.35 (C]O), 181.82 (C]O). IR (ATR): n 2959 (CH), 2874 (CH), 1720
imido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 2c. 86%, yellow
(C]O), 1668 (C]O), 1657 (C]O), 1575 (CHAr), 1547 (CHAr), 1285
(CeN), 1273 (CeN), 755, 740 cmꢃ1. MS m/z (%): 422 ([MþH]þ, 100).
HRMS (ESþ) calcd. for [C24H28N3O4]þ: 422.2080, found 422.2085.
needles, m.p. 97e98 ꢀC. 1H NMR (CDCl3):
d
1.18 (2H, d, J ¼ 9.1 Hz,
2 ꢂ CHxHn), 1.23e1.28 (6H, m, 2 ꢂ CH3), 1.41 (1H, d, J ¼ 9.1 Hz,
CHsHa), 1.64 (1H, d, J ¼ 9.1 Hz, CHsHa), 1.95 (2H, d, J ¼ 9.1 Hz,
2 ꢂ CHxHn), 3.56 (1H, br s, CH-8 or CH-11), 3.62 (1H, br s, CH-8 or
CH-11), 3.99e4.17 (2H, m, NCH2), 4.30e4.50 (2H, m, NCH2), 9.19
4.2.6.6. 2,4-Di-iso-butyl-8,9,10,11-tetrahydro-8,11-methanobenzo[g]
(1H, s, CH-6). 13C NMR (CDCl3):
d12.90 (CH3), 13.20 (CH3), 25.58 and
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 2g. 67%, yel-
low viscous oil, m.p. 135 ꢀC. 1H NMR (CDCl3):
d 0.88e0.95 (12H, m,
25.86 (CH2-9 and CH2-10), 37.85 (NCH2), 38.88 (NCH2), 41.20 and
42.81 (CH-8 and CH-11), 45.89 (CH2-13), 107.21 (Cquat), 123.85
(Cquat), 144.92 (Cquat), 149.90 (Cquat), 152.31 (CH-6), 152.55 (Cquat),
154.04 (Cquat), 157.24 (NC]O), 158.25 (NC]O), 179.24 (C]O),
4 ꢂ CH3), 1.19 (2H, d, J ¼ 9.6 Hz, 2 ꢂ CHxHn), 1.42 (1H, d, J ¼ 9.4 Hz,
CHsHa), 1.65 (1H, d, J ¼ 9.4 Hz, CHsHa), 1.96 (2H, d, J ¼ 9.6 Hz,
2 ꢂ CHxHn), 2.10e2.25 (2H, m, 2 ꢂ CH(CH3)2), 3.57 (1H, br s, CH-8 or
CH-11), 3.64 (1H, br s, CH-8 or CH-11), 3.83e3.99 (2H, m, NCH2),
4.15e4.27 (2H, m, NCH2), 9.18 (1H, s, CH-6). 13C NMR (CDCl3):
181.66 (C]O). IR (ATR): n 2982 (CH), 1715 (C]O), 1663 (C]O), 1655
(C]O), 1574 (CHAr), 1545 (CHAr), 1316, 1289 (CeN), 1270 (CeN),
740 cmꢃ1. MS m/z (%): 366 ([MþH]þ, 100). HRMS (ESþ) calcd. for
[C20H20N3O4]þ: 366.1454, found 366.1459.
d
19.98 (2 ꢂ CH3), 20.13 (CH3), 20.24 (CH3), 25.63 and 25.89 (CH2-9
and CH2-10), 27.28 (CH(CH3)2), 27.34 (CH(CH3)2), 41.25 and 42.82
(CH-8 and CH-11),45.97 (CH2-13), 49.28 (NCH2), 50.00 (NCH2),
107.01 (Cquat), 123.93 (Cquat), 144.96 (Cquat), 151.01 (Cquat),
152.17 (CH-6), 152.64 (Cquat), 154.58 (Cquat), 157.24 (NC]O), 158.78
4.2.6.3. 2,4-Di-n-propyl-8,9,10,11-tetrahydro-8,11-methanobenzo[g]
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 2d. 85%, yel-
low crystals, m.p. 125 ꢀC. 1H NMR (CDCl3):
d
0.90 (6H, t, J ¼ 7.4 Hz,
(NC]O), 179.32 (C]O), 181.79 (C]O). IR (ATR):
n 2959 (CH), 2874
(CH), 1721 (C]O), 1657 (C]O), 1575 (CHAr), 1548 (CHAr), 1286 (Ce
N), 1272 (CeN), 753, 740 cmꢃ1. MS m/z (%): 422 ([MþH]þ, 100).
HRMS (ESþ) calcd. for [C24H28N3O4]þ: 422.2080, found 422.2083.
2 ꢂ CH3), 1.16 (2H, d, J ¼ 8.8 Hz, 2 ꢂ CHxHn), 1.39 (1H, d, J ¼ 9.9 Hz,
CHsHa), 1.61e1.70 (5H, m, CHsHa and 2 ꢂ CH2CH3), 1.93 (2H, d,
J ¼ 8.8 Hz, 2 ꢂ CHxHn), 3.53 (1H, br s, CH-8 or CH-11), 3.60 (1H, br s,
CH-8 or CH-11), 3.87e4.04 (2H, m, NCH2), 4.17e4.35 (2H, m, NCH2),
9.13 (1H, s, CH-6). 13C NMR (CDCl3):
d 11.13 (CH3), 11.29 (CH3), 20.96
4.2.6.7. 2,4-Di-n-pentyl-8,9,10,11-tetrahydro-8,11-methanobenzo[g]
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 2h. 71%, yel-
(CH2CH3), 21.14 (CH2CH3), 25.52 and 25.80 (CH2-9 and CH2-10),
41.16 and 42.73 (CH-8 and CH-11), 44.04 (NCH2), 44.93 (NCH2),
45.86 (CH2-13), 107.01 (Cquat), 123.79 (Cquat), 144.83 (Cquat), 150.26
(Cquat), 152.16 (CH-6), 152.49 (Cquat), 154.17 (Cquat), 157.13 (NC]O),
low viscous oil. 1H NMR (CDCl3):
d
0.81e0.89 (6H, m, 2 ꢂ CH3), 1.18
(2H, d, J ¼ 9.4 Hz, 2 ꢂ CHxHn), 1.27e1.36 (8H, m, 4 ꢂ CH2), 1.41 (1H,
J ¼ 9.4 Hz, CHsHa), 1.63e1.69 (5H, m, 2 ꢂ CH2 and CHsHa), 1.95 (2H,
d, J ¼ 9.4 Hz, 2 ꢂ CHxHn), 3.56 (1H, br s, CH-8 or CH-11), 3.62 (1H, br
s, CH-8 or CH-11), 3.92e4.09 (2H, m, NCH2), 4.22e4.40 (2H, m,
158.36 (NC]O), 179.15 (C]O), 181.59 (C]O). IR (ATR): n 2964 (CH),
2877 (CH), 1720 (C]O), 1660 (C]O), 1575 (CHAr), 1547 (CHAr), 1315,
1285 (CeN), 1273 (CeN), 752, 740 cmꢃ1. MS m/z (%): 394 ([MþH]þ,
100). HRMS (ESþ) calcd. for [C22H24N3O4]þ: 394.1767, found
394.1779.
NCH2), 9.17 (1H, s, CH-6). 13C NMR (CDCl3):
d
14.00 (2 ꢂ CH3), 22.36
(4 ꢂ CH2), 25.58 and 25.84 (CH2-9 and CH2-10), 28.87 (CH2), 29.06
(CH2), 41.20 (CH-8 or CH-11), 42.68 (NCH2), 42.79 (CH-8 or CH-11),
43.54 (NCH2), 45.91 (CH2-13), 107.08 (Cquat), 123.82 (Cquat), 144.90
(Cquat), 150.26 (Cquat), 152.25 (CH-6), 152.54 (Cquat), 154.20 (Cquat),
157.21 (NC]O), 158.40 (NC]O), 179.24 (C]O), 181.70 (C]O). IR
4.2.6.4. 2,4-Di-iso-propyl-8,9,10,11-tetrahydro-8,11-methanobenzo
[g]pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone
2e.
1.19 (2H, d, J ¼ 9.6 Hz,
33%, yellow viscous oil. 1H NMR (CDCl3):
d
(ATR):
n 2956 (CH), 2631 (CH), 1721 (C]O), 1668 (C]O), 1578
(CHAr), 1547 (CHAr), 1285 (CeN), 1272 (CeN), 754, 740 cmꢃ1. MS m/z
(%): 450 ([MþH]þ, 100). HRMS (ESþ) calcd. for [C26H32N3O4]þ:
450.2393, found 450.2398.
2 ꢂ CHxHn), 1.43 (1H, d, J ¼ 9.1 Hz, CHsHa), 1.51e1.56 (12H, m,
4 ꢂ CH3), 1.66 (1H, d, J ¼ 9.1 Hz, CHsHa), 1.97 (2H, d, J ¼ 9.6 Hz,
2 ꢂ CHxHn), 3.59 (1H, br s, CH-8 or CH-11), 3.66 (1H, br s, CH-8 or
CH-11), 5.14 (1H, septet, J ¼ 6.8 Hz, NCH), 5.70 (1H, septet,
J ¼ 6.8 Hz, NCH), 9.16 (1H, s, CH-6). 13C NMR (CDCl3):
d 18.81 (CH3),
19.66 (CH3), 19.82 (CH3), 20.03 (CH3), 25.54 and 25.83 (CH2-9 and
CH2-10), 41.18 and 42.70 (CH-8 and CH-11), 45.93 (CH2-13), 47.76
(NCH), 48.48 (NCH), 108.24 (Cquat), 123.53 (Cquat), 144.34 (Cquat),
149.89 (Cquat), 151.34 (CH-6), 152.68 (Cquat), 154.30 (Cquat), 157.00
4.2.6.8. 2,4-Di-iso-pentyl-8,9,10,11-tetrahydro-8,11-methanobenzo
[g]pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone
2i. 93%,
0.91e0.94
yellow crystals, m.p. 146e147 ꢀC. 1H NMR (CDCl3):
d
(12H, m, 4 ꢂ CH3), 1.17 (2H, d, J ¼ 9.6 Hz, 2 ꢂ CHxHn), 1.40 (1H, d,
J ¼ 9.4 Hz, CHsHa), 1.46e1.57 (4H, m, 2 ꢂ CH2), 1.58e1.67 (3H,
2 ꢂ CH(CH3)2 and CHsHa), 1.94 (2H, d, J ¼ 9.6 Hz, 2 ꢂ CHxHn), 3.54
(1H, br s, CH-8 or CH-11), 3.62 (1H, br s, CH-8 or CH-11), 3.94e34.09
(2H, m, NCH2), 4.23e4.41 (2H, m, NCH2), 9.16 (1H, s, CH-6). 13C NMR
(NC]O), 159.31 (NC]O), 179.38 (C]O), 181.74 (C]O). IR (ATR):
n
2971 (CH), 1721 (C]O), 1657, 1575 (CHAr), 1548 (CHAr), 1285 (CeN),
1273 (CeN), 756, 740 cmꢃ1. MS m/z (%): 394 ([MþH]þ, 100). HRMS
(ESþ) calcd. for [C22H24N3O4]þ: 394.1767, found 394.1774.
(CDCl3):
d 22.41 (CH3), 22.50 (3xCH3), 25.54 and 25.81 (CH2-9 and
4.2.6.5. 2,4-Di-n-butyl-8,9,10,11-tetrahydro-8,11-methanobenzo[g]
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 2f. 57%, amber
CH2-10), 26.24 (CH(CH3)2), 26.38 (CH(CH3)2), 36.21 (CH2), 36.52
(CH2), 41.16(CH-8 or CH-11), 41.31 (NCH2), 42.23 (CH-8 or CH-11),
42.76 (NCH2), 45.85 (CH2-13), 107.06 (Cquat), 123.76 (Cquat), 144.81
(Cquat), 150.11 (Cquat), 152.22 (CH-6), 152.51 (Cquat), 154.10 (Cquat),
157.15 (NC]O), 158.28 (NC]O), 179.17 (C]O), 181.64 (C]O). IR
viscous oil. 1H NMR (CDCl3):
d
0.93 (6H, t, J ¼ 7.2 Hz, 2 ꢂ CH3), 1.21
(2H, d, J ¼ 8.8 Hz, 2 ꢂ CHxHn), 1.35e1.45 (5H, m, 2 ꢂ CH2CH3 and
CHsHa), 1.61e1.71 (5H, m, 2 ꢂ CH2CH3 and CHsHa), 1.97 (2H, d,
J ¼ 8.8 Hz, 2 ꢂ CHxHn), 3.59 (1H, br s, CH-8 or CH-11), 3.66 (1H, br s,
CH-8 or CH-11), 3.96e4.13 (2H, m, NCH2), 4.26e4.44 (2H, m, NCH2),
(ATR): n 2956 (CH), 2931 (CH), 2872 (CH), 1717 (C]O), 1656 (C]O),
1670 (C]O), 1576 (CHAr), 1548 (CHAr), 1317 (CeN), 1286 (CeN)
cmꢃ1. MS m/z (%): 450 ([MþH]þ, 100). HRMS (ESþ) calcd. for
[C26H32N3O4]þ: 450.2393, found 450.2397.
9.21 (1H, s, CH-6). 13C NMR (CDCl3):
d
13.86 (2 ꢂ CH3), 20.12
(CH2CH3), 20.32 (CH2CH3), 25.64 and 25.92 (CH2-9 and CH2-10),

P. Claes et al. / European Journal of Medicinal Chemistry 77 (2014) 409e421
419
4.2.6.9. 2,4-Di-n-hexyl-8,9,10,11-tetrahydro-8,11-methanobenzo[g]
4.2.6.13. 2,4-Di-n-propyl-8,9,10,11-tetrahydro-8,11-ethanobenzo[g]
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 2j. 93%, amber
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 3d. 83%, yel-
viscous oil. 1H NMR (CDCl3):
d
0.82e0.91 (6H, m, 2 ꢂ CH3), 1.21 (2H,
low crystals, m.p. 153 ꢀC. 1H NMR (CDCl3):
d
0.98 (6H, t, J ¼ 7.2 Hz,
d, J ¼ 9.6 Hz, 2 ꢂ CHxHn), 1.26e1.39 (12H, m, 6xCH2), 1.43 (1H, d,
J ¼ 9.4 Hz, CHsHa),1.61e1.72 (5H, m, 2 ꢂ CH2 and CHsHa),1.97 (2H, d,
J ¼ 9.6 Hz, 2 ꢂ CHxHn), 3.59 (1H, br s, CH-8 or CH-11), 3.65 (1H, br s,
CH-8 or CH-11), 3.95e4.11 (2H, m, NCH2), 4.25e4.43 (2H, m, NCH2),
2 ꢂ CH3), 1.31e1.45 (4H, m, 2 ꢂ CH2), 1.64e1.81 (8H, m, 2 ꢂ CH2,
2 ꢂ CH2CH3), 3.47 (1H, s, CH-8 or CH-11), 3.50 (1H, s, CH-8 or CH-
11), 4.03 (2H, t, J ¼ 7.7 Hz, NCH2), 4.34 (2H, t, J ¼ 7.7 Hz, NCH2), 9.30
(1H, s, CH-6). 13C NMR (CDCl3):
d 11.18 (CH3), 11.34 (CH3), 20.99
9.21 (1H, s, CH-6). 13C NMR (CDCl3):
d
14.12 (2 ꢂ CH3), 22.64
(CH2CH3), 21.21 (CH2CH3), 25.28 (2 ꢂ CH2), 25.33 (2 ꢂ CH2), 26.72
and 28.41 (CH-8 and CH-11), 44.12 (NCH2), 44.95 (NCH2), 106.77
(Cquat), 123.17 (Cquat), 144.24 (Cquat), 148.85 (Cquat), 150.33 (Cquat),
152.71 (CH-6), 153.81 (Cquat), 154.12 (NC]O), 158.47 (NC]O),
(2 ꢂ CH2), 25.66 and 25.94 (CH2-9 and CH2-10), 26.52 (CH2), 29.71
(CH2), 27.68 (CH2), 27.91 (CH2), 31.54 (2 ꢂ CH2), 41.28 (CH-8 or CH-
11), 42.84 (NCH2), 42.87 (CH-8 or CH-11), 43.68 (NCH2), 46.00 (CH2-
13), 107.18 (Cquat), 123.91 (Cquat), 145.01 (Cquat), 150.36 (Cquat),
152.37 (CH-6), 152.65 (Cquat), 154.29 (Cquat), 157.32 (NC]O), 158.51
178.91 (C]O),180.06 (C]O). IR (ATR): n 2960 (CH), 2871 (CH), 1720
(C]O), 1668 (C]O), 1656 (C]O), 1578 (CHAr), 1288 (CeN), 751,
741 cmꢃ1. MS m/z (%): 408 ([MþH]þ, 100). HRMS (ESþ) calcd. for
[C23H26N3O4]þ: 408.1923, found 408.1928.
(NC]O), 179.38 (C]O), 181.82 (C]O). IR (ATR):
n 2956 (CH), 2928
(CH), 1721 (C]O), 1669 (C]O), 1657 (C]O), 1576 (CHAr), 1285 (Ce
N), 1273 (CeN) cmꢃ1. MS m/z (%): 478 ([MþH]þ, 100). HRMS (ESþ)
calcd. for [C28H36N3O4]þ: 478.2706, found 478.2714.
4.2.6.14. 2,4-Di-iso-propyl-8,9,10,11-tetrahydro-8,11-ethanobenzo[g]
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 3e. 48%, yel-
low crystals, m.p. 89 ꢀC. 1H NMR (CDCl3):
d 1.31e1.43 (4H, m,
4.2.6.10. 2,4-Di-n-heptyl-8,9,10,11-tetrahydro-8,11-methanobenzo[g]
2 ꢂ CH2), 1.55 (6H, d, J ¼ 6.8 Hz, 2 ꢂ CH3), 1.57 (6H, d, J ¼ 6.8 Hz,
2 ꢂ CH3), 1.74e1.85 (4H, m, 2 ꢂ CH2), 3.48 (1H, s, CH-8 or CH-11),
3.50 (1H, s, CH-8 or CH-11), 5.16 (1H, septet, J ¼ 6.8 Hz, NCH),
5.72 (1H, septet, J ¼ 6.8 Hz, NCH), 9.25 (1H, s, CH-6). 13C NMR
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 2k. 88%, yel-
low oil. 1H NMR (CDCl3):
d
0.76e0.85 (6H, m, 2 ꢂ CH3), 1.11e1.35
(22H, m, 2 ꢂ CHxHn, 10xCH2), 1.38 (1H, d, J ¼ 9.4 Hz, CHsHa), 1.55e
1.69 (5H, m, 2 ꢂ CH2 and CHsHa), 1.94 (2H, d, J ¼ 8.0 Hz, 2 ꢂ CHxHn),
3.54 (1H, br s, CH-8 or CH-11), 3.61 (1H, br s, CH-8 or CH-11), 3.91e
(CDCl3):
d
19.42 (2 ꢂ CH3), 19.76 (2 ꢂ CH3), 25.28 (2 ꢂ CH2), 25.33
4.07 (2H, m, NCH2), 4.21e4.38 (2H, m, NCH2), 9.15 (1H, s, CH-6). 13
C
(2 ꢂ CH2), 26.72 and 28.32 (CH-8 and CH-11), 47.73 (NCH), 48.43
(NCH), 107.91 (Cquat), 122.85 (Cquat), 143.61 (Cquat), 148.97 (Cquat),
149.89 (Cquat), 151.78 (CH-6), 153.62 (Cquat), 154.18 (NC]O), 159.35
NMR (CDCl3):
d
14.06 (2 ꢂ CH3), 22.56 (2 ꢂ CH2), 25.55 and 25.81
(CH2-9 and CH2-10), 26.71 (CH2), 26.91 (CH2), 27.62 (CH2), 27.84
(CH2), 28.93 (2 ꢂ CH2), 31.71 (2 ꢂ CH2), 41.17 (CH-8 or CH-11), 42.70
(NCH2), 42.76 (CH-8 or CH-11), 43.54 (NCH2), 45.86 (CH2-13),107.05
(Cquat), 123.77 (Cquat), 144.86 (Cquat), 150.22 (Cquat), 152.19 (CH-6),
152.49 (Cquat), 154.17 (Cquat), 157.16 (NC]O), 158.32 (NC]O), 179.17
(NC]O), 179.06 (C]O), 181.05 (C]O). IR (ATR):
n 2947 (CH), 1722
(C]O), 1667 (C]O), 1659 (C]O), 1578 (CHAr), 1287 (CeN), 753,
740 cmꢃ1. MS m/z (%): 408 ([MþH]þ, 100). HRMS (ESþ) calcd. for
[C23H26N3O4]þ: 408.1923, found 408.1927.
(C]O), 181.62 (C]O). IR (ATR):
n 2955 (CH), 2926 (CH), 2856 (CH),
4.2.6.15. 2,4-Di-n-butyl-8,9,10,11-tetrahydro-8,11-ethanobenzo[g]
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 3f. 90%, yel-
1721 (C]O), 1670 (C]O), 1658 (C]O), 1576 (CHAr), 1285 (CeN),
1273 (CeN) cmꢃ1. MS m/z (%): 506 ([MþH]þ, 100). HRMS (ESþ)
calcd. for [C30H40N3O4]þ: 506.3019, found 506.3015.
low viscous oil. 1H NMR (CDCl3):
d
0.93 (3H, t, J ¼ 7.2 Hz, CH3), 0.94
(3H, t, J ¼ 7.2 Hz, CH3), 1.30e1.45 (8H, m, 2 ꢂ CH2, 2 ꢂ CH2CH3), 1.66
(4H, pentet, J ¼ 7.4 Hz, 2 ꢂ NCH2CH2), 1.74e1.82 (4H, m, 2 ꢂ CH2),
3.44 (1H, s, CH-8 or CH-11), 3.48 (1H, s, CH-8 or CH-11), 4.04 (2H, t,
J ¼ 7.4 Hz, NCH2), 4.35 (2H, t, J ¼ 7.4 Hz, NCH2), 9.28 (1H, s, CH-6).
4.2.6.11. 2,4-Dimethyl-8,9,10,11-tetrahydro-8,11-ethanobenzo[g]pyr-
imido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 3b. 48%, yellow
needles, m.p. 266 ꢀC. 1H NMR (CDCl3):
d
1.34e1.47 (4H, m, 2 ꢂ CH2),
1.75e1.87 (4H, m, 2 ꢂ CH2), 3.50 (5H, s, NCH3 and CH-8 and CH-11),
3.77 (3H, s, NCH3), 9.33 (1H, s, CH-6). 13C NMR (CDCl3):
25.28
13C NMR (CDCl3):
d
13.86 (2 ꢂ CH3), 20.12 (CH2CH3), 21.32
(CH2CH3), 25.35 (2 ꢂ CH2), 25.42 (2 ꢂ CH2), 26.80 and 28.50 (CH-8
and CH-11), 29.78 (NCH2CH2), 30.04 (NCH2CH2), 42.59 (NCH2),
43.43 (NCH2), 106.87 (Cquat), 123.23 (Cquat), 144.34 (Cquat), 148.92
(Cquat), 150.37 (Cquat), 152.83 (CH-6), 153.93 (Cquat), 154.19 (NC]O),
d
(2 ꢂ CH2), 25.35 (2 ꢂ CH2), 26.75 and 28.44 (CH-8 and CH-11), 29.15
(NCH3), 30.54 (NCH3), 106.63 (Cquat), 123.26 (Cquat), 144.07 (Cquat),
148.94 (Cquat), 150.88 (Cquat), 152.77 (CH-6), 153.95 (Cquat), 154.28
158.52 (NC]O), 179.03 (C]O), 181.17 (C]O). IR (ATR): n 2958 (CH),
(NC]O), 158.59 (NC]O), 178.91 (C]O), 180.85 (C]O). IR (ATR):
n
2870 (CH), 1720 (C]O), 1668 (C]O), 1657 (C]O), 1578 (CHAr), 1289
(CeN), 753, 734 cmꢃ1. MS m/z (%): 436 ([MþH]þ, 100). HRMS (ESþ)
calcd. for [C25H30N3O4]þ: 436.2236, found 436.2243.
2952 (CH), 1717 (C]O),1688 (C]O),1668 (C]O),1652 (C]O), 1573
(CHAr), 1486, 1292 (CeN), 1274 (CeN), 838 cmꢃ1. MS m/z (%): 352
([MþH]þ, 100). HRMS (ESþ) calcd. for [C19H18N3O4]þ: 352.1297,
found 352.1303.
4.2.6.16. 2,4-Di-iso-butyl-8,9,10,11-tetrahydro-8,11-ethanobenzo[g]
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 3g. 89%, yel-
4.2.6.12. 2,4-Diethyl-8,9,10,11-tetrahydro-8,11-ethanobenzo[g]pyr-
imido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 3c. 89%, yellow
low crystals, m.p. 71 ꢀC. 1H NMR (CDCl3):
d
0.91 (6H, d, J ¼ 7.2 Hz,
2 ꢂ CH3), 0.94 (6H, d, J ¼ 7.2 Hz, 2 ꢂ CH3), 1.29e1.41 (4H, m,
2 ꢂ CH2), 1.76e1.78 (4H, m, 2 ꢂ CH2), 2.19 (2H, nonuplet, J ¼ 7.1 Hz,
2 ꢂ CH(CH3)2), 3.45 (1H, s, CH-8 or CH-11), 3.49 (1H, s, CH-8 or CH-
11), 3.92 (2H, t, J ¼ 7.2 Hz, NCH2), 4.23 (2H, t, J ¼ 7.2 Hz, NCH2), 9.26
needles, m.p. 192 ꢀC. 1H NMR (CDCl3):
d
1.28e1.33 (6H, m, 2 ꢂ CH3),
1.34e1.41 (4H, m, 2 ꢂ CH2), 1.75e1.83 (4H, m, 2 ꢂ CH2), 3.47 (1H, s,
CH-8 or CH-11), 3.49 (1H, s, CH-8 or CH-11), 4.13 (2H, q, J ¼ 7.0 Hz,
NCH2), 4.45 (2H, q, J ¼ 7.0 Hz, NCH2), 9.30 (1H, s, CH-6). 13C NMR
(1H, s, CH-6). 13C NMR (CDCl3):
d
20.00 (2 ꢂ CH3), 20.19 (2 ꢂ CH3),
(CDCl3):
d
12.89 (CH3),13.21 (CH3), 25.28 (2 ꢂ CH2), 25.34 (2 ꢂ CH2),
25.35 (2 ꢂ CH2), 25.40 (2 ꢂ CH2), 26.79 (CH-8 or CH-11), 27.28
(CH(CH3)2), 27.37 (CH(CH3)2), 28.45 (CH-8 or CH-11), 49.31 (NCH2),
49.94 (NCH2), 106.75 (Cquat), 123.27 (Cquat), 144.31 (Cquat), 148.95
(Cquat), 151.04 (Cquat), 152.66 (CH-6), 153.85 (Cquat), 154.49 (NC]O),
26.72 and 28.43 (CH-8 and CH-11), 37.84 (NCH2), 38.84 (NCH2),
106.89 (Cquat), 123.17 (Cquat), 144.27 (Cquat), 148.82 (Cquat),
149.89 (Cquat), 152.76 (CH-6), 153.84 (Cquat), 153.93 (NC]O), 158.27
(NC]O), 178.91 (C]O), 180.02 (C]O). IR (ATR):
n
2956 (CH), 2938
158.83 (NC]O), 179.01 (C]O), 181.15 (C]O). IR (ATR): n 2958 (CH),
(CH), 1719 (C]O), 1682 (C]O), 1667 (C]O), 1574 (CHAr), 1552, 1291
(CeN), 753 cmꢃ1. MS m/z (%): 380 ([MþH]þ,100). HRMS (ESþ) calcd.
for [C21H22N3O4]þ: 380.1610, found 380.1614.
2871 (CH), 1721 (C]O), 1676 (C]O), 1657 (C]O), 1579 (CHAr), 1290
(CeN), 752, 734 cmꢃ1. MS m/z (%): 436 ([MþH]þ, 100). HRMS (ESþ)
calcd. for [C25H30N3O4]þ: 436.2236, found 436.2240.

420
P. Claes et al. / European Journal of Medicinal Chemistry 77 (2014) 409e421
4.2.6.17. 2,4-Di-n-pentyl-8,9,10,11-tetrahydro-8,11-ethanobenzo[g]
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 3h. 64%,
amber viscous oil. 1H NMR (CDCl3):
0.84e0.92 (6H, m, 2 ꢂ CH3),
1579 (CHAr), 1289 (CeN), 754 cmꢃ1. MS m/z (%): 520 ([MþH]þ, 100).
HRMS (ESþ) calcd. for [C31H42N3O4]þ: 520.3175, found 520.3180.
d
1.30e1.45 (12H, m, 6xCH2), 1.62e1.73 (4H, m, 2 ꢂ NCH2CH2), 1.77e
1.80 (4H, m, 2 ꢂ CH2), 3.46 (1H, s, CH-8 or CH-11), 3.49 (1H, s, CH-8
or CH-11), 4.04 (2H, t, J ¼ 7.7 Hz, NCH2), 4.35 (2H, t, J ¼ 7.7 Hz,
4.2.7. Synthesis of 3,4,8,9,10,11-hexahydro-2H-8,11-methanobenzo
[j]phenanthridine-1,7,12-triones 11
To a stirred solution of 5,8-dihydroxy-1,2,3,4-tetrahydro-1,4-
methanonaphthalene-6-carboxaldehyde 7a (204 mg, 1 mmol),
aminoisopropylcyclohexenones 10 (153 mg, 1 mmol) and MgSO4
(5 mmol, 604 mg) in anhydrous dichloromethane (10 mL) was
added freshly prepared Ag2O (4 mmol, 927 mg). The reaction was
allowed to stir at room temperature for 4 h and subsequently
filtered over a pad of celiteÒ and evaporated in vacuo. The residue
was purified by means of column chromatography (petroleum
ether/ethyl acetate) followed by preparative HPLC (CH3CN/H2O) to
yield 3,4,8,9,10,11-hexahydro-2H-8,11-methanobenzo[j]phenan-
thridine-1,7,12-triones 11a and 11b. It was not possible to unam-
biguously assign the regioisomers based on 2D-NMR techniques.
NCH2), 9.28 (1H, s, CH-6). 13C NMR (CDCl3):
d
14.07 (2 ꢂ CH3), 22.44
(2 ꢂ CH2CH3), 25.37 (22 ꢂ CH2), 25.44 (2 ꢂ CH2), 26.80 (CH-8 or CH-
11), 27.38 (CH2), 27.64 (CH2), 28.50 (CH-8 or CH-11), 28.94
(NCH2CH2), 29.16 (NCH2CH2), 42.81 (NCH2), 43.60 (NCH2), 106.89
(Cquat), 123.24 (Cquat), 144.35 (Cquat), 148.93 (Cquat), 150.37 (Cquat),
152.84 (CH-6), 153.93 (Cquat), 154.19 (NC]O), 158.54 (NC]O),
179.04 (C]O), 181.18 (C]O). IR (ATR): n 2956 (CH), 2870 (CH), 1721
(C]O), 1669 (C]O), 1657 (C]O), 1578 (CHAr), 1289 (CeN),
754 cmꢃ1. MS m/z (%): 464 ([MþH]þ, 100). HRMS (ESþ) calcd. for
[C27H34N3O4]þ: 464.2549, found 464.2552.
4.2.6.18. 2,4-Di-iso-pentyl-8,9,10,11-tetrahydro-8,11-ethanobenzo[g]
4.2.7.1. Regioisomer 11a. 40%, yellow solid, m.p. 63 ꢀC. 1H NMR
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 3i. 90%, yel-
low crystals, m.p. 132e133 ꢀC. 1H NMR (CDCl3):
d 0.94 (6H, d,
(CDCl3):
d
1.10 (3H, d, J ¼ 6.6 Hz, CH3), 1.18 (3H, d, J ¼ 6.6 Hz, CH3),
J ¼ 5.5 Hz, 2 ꢂ CH3), 0.96 (6H, d, J ¼ 5.5 Hz, 2 ꢂ CH3), 1.29e1.42 (4H,
m, 2 ꢂ CH2), 1.51e1.58 (4H, m, 2 ꢂ NCH2CH2), 1.61e1.70 (2H, m,
2 ꢂ CH(CH3)2), 1.76e1.78 (4H, m, 2 ꢂ CH2), 3.44 (1H, s, CH-8 or CH-
11), 3.47 (1H, s, CH-8 or CH-11), 4.04 (2H, t, J ¼ 7.7 Hz, NCH2), 4.36
1.22 (1H, m, CH), 1.50 (1H, d, J ¼ 9.4 Hz, CH), 1.70 (1H, d, J ¼ 9.4 Hz,
CH), 1.93e2.13 (3H, m, CH and CH2), 2.20e2.32 (2H, m, CH2), 2.50e
2.57 (1H, m, CH), 3.01 (1H, ddd, J ¼ 17.6, 9.1, 5.0 Hz, CH), 3.23 (1H,
ddd, J ¼ 17.6, 6.6, 5.0 Hz, CH), 3.64 (2H, br s, CH-8 and CH-11), 9.22
(2H, t, J ¼ 7.7 Hz, NCH2), 9.27 (1H, s, CH-6). 13C NMR (CDCl3):
d 22.53
(1H, s, CH-6). 13C NMR (CDCl3):
d19.35 (CH3), 20.99 (CH3), 23.58
(4 ꢂ CH3), 25.34 (2 ꢂ CH3), 25.39 (2 ꢂ CH2), 26.33 (CH(CH3)2), 26.50
(CH(CH3)2), 26.78 and 28.47 (CH-8 and CH-11), 36.27 (CH2), 36.61
(CH2), 41.46 (NCH2), 42.30 (NCH2), 106.87 (Cquat), 123.19 (Cquat),
144.25 (Cquat), 148.89 (Cquat), 150.22 (Cquat), 152.81 (CH-6),
153.88 (Cquat),154.10 (NC]O),158.42 (NC]O),178.98 (C]O),181.11
(CH2), 25.37 (CH2), 25.68 (CH2), 29.17 (CH2), 31.37 (CH), 41.14 and
42.18 (CH-8 and CH-11), 46.32 (CH2-13), 55.63 (CH), 126.51 (Cquat),
128.91 (Cquat), 140.16 (Cquat), 149.81 (CH-6), 152.98 (Cquat), 156.14
(Cquat), 168.37 (Cquat), 180.56 (C]O), 181.69 (C]O), 201.66 (C]O).
IR (ATR): n
2958, 2874,1697,1657,1567,1318,1273, 739 cmꢃ1. MS m/
z (%): 336 ([MþH]þ, 100). HRMS (ESþ) calcd. for [C21H22NO3]þ:
(C]O). IR (ATR):
n 2956 (CH), 2870 (CH), 1720 (C]O), 1668 (C]O),
1579 (CHAr), 1290 (CeN), 753 cmꢃ1. MS m/z (%): 464 ([MþH]þ, 100).
336.1600, found 336.1596.
HRMS (ESþ) calcd. for [C27H34N3O4]þ: 464.2549, found 464.2551.
4.2.7.2. Regioisomer 11b. 13%, yellow solid, m.p. 95 ꢀC. 1H NMR
4.2.6.19. 2,4-Di-n-hexyl-8,9,10,11-tetrahydro-8,11-ethanobenzo[g]
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 3j. 90%, yel-
(CDCl3):
d
1.03 (3H, d, J ¼ 6.6 Hz, CH3), 1.15 (3H, d, J ¼ 6.6 Hz, CH3),
1.22e1.26 (1H, m, CH),1.45e1.49 (1H, m, CH),1.69e1.73 (1H, m, CH),
1.68e2.01 (3H, m, CH and CH2), 2.00e2.31 (1H, m, CH), 2.34e2.44
(1H, m, CH), 2.66e2.44 (1H, m, CH), 3.15 (1H, ddd, J ¼ 18.3, 10.6,
6.1 Hz, CH), 3.28 (1H, ddd, J ¼ 17.6, 3.5, 5.0 Hz, CH), 3.63 (2H, br s,
low viscous oil. 1H NMR (CDCl3):
d
0.79e0.88 (6H, m, 2 ꢂ CH3),
1.19e1.42 (16H, m, 8 ꢂ CH2), 1.60e1.71 (4H, m, 2 ꢂ NCH2CH2), 1.75e
1.82 (4H, m, 2 ꢂ CH2), 3.43 (1H, s, CH-8 or CH-11), 3.46 (1H, s, CH-8
or CH-11), 4.01 (2H, t, J ¼ 7.7 Hz, NCH2), 4.33 (2H, t, J ¼ 7.7 Hz,
CH-8 and CH-11), 9.22 (1H, s, CH-6). 13C NMR (CDCl3):
d 19.13 (CH3),
NCH2), 9.25 (1H, s, CH-6). 13C NMR (CDCl3):
d
14.04 (2 ꢂ CH3), 22.56
21.28 (CH3), 25.03 (CH2), 25.29 (CH2), 25.49 (CH2), 27.60 (CH2),
32.52 (CH), 41.10 and 41.92 (CH-8 and CH-11), 47.30 (CH2-13), 55.34
(CH), 126.02 (Cquat), 127.25 (Cquat), 139.44 (Cquat), 149.87 (CH-6),
153.26 (Cquat), 155.94 (Cquat), 167.96 (Cquat), 180.66 (C]O), 181.30
(2 ꢂ CH2CH3), 25.29 (2 ꢂ CH2), 25.36 (2 ꢂ CH2), 26.44 (CH2), 26.64
(CH2), 26.73 (CH-8 or CH-11), 27.60 (CH2), 27.84 (CH2), 28.42 (CH-8
or CH-11), 31.46 (2 ꢂ CH2), 42.76 (NCH2), 43.56 (NCH2), 106.81
(Cquat), 123.16 (Cquat), 144.25 (Cquat), 148.83 (Cquat), 150.28 (Cquat),
152.72 (CH-6), 153.84 (Cquat), 154.11 (NC]O), 158.42 (NC]O),
(C]O), 201.69 (C]O). IR (ATR): n 2961, 2874,1703,1657, 1569,1323,
1291 cmꢃ1. MS m/z (%): 336 ([MþH]þ, 100). HRMS (ESþ) calcd. for
178.92 (C]O), 181.06 (C]O). IR (ATR):
n 2955 (CH), 2928 (CH),
[C21H22NO3]þ: 336.1600, found 336.1596.
2868 (CH), 1721 (C]O), 1670 (C]O), 1657 (C]O), 1578 (CHAr), 1289
(CeN), 754 cmꢃ1. MS m/z (%): 492 ([MþH]þ,100). HRMS (ESþ) calcd.
for [C29H38N3O4]þ: 492.2862, found 492.2869.
Acknowledgements
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.
4.2.6.20. 2,4-Di-n-heptyl-8,9,10,11-tetrahydro-8,11-ethanobenzo[g]
pyrimido[4,5-c]isoquinoline-1,3,7,12(2H,4H)-tetraone 3k. 78%, yel-
low viscous oil. 1H NMR (CDCl3):
d
0.78e0.88 (6H, m, 2 ꢂ CH3),
1.20e1.27 (10H, m, 5 ꢂ CH2), 1.28e1.41 (10H, m, 5 ꢂ CH2), 1.60e1.70
(4H, m, 2 ꢂ NCH2CH2), 1.73e1.81 (4H, m, 2 ꢂ CH2), 3.43 (1H, s, CH-8
or CH-11), 3.47 (1H, s, CH-8 or CH-11), 4.01 (2H, t, J ¼ 7.7 Hz, NCH2),
4.33 (2H, t, J ¼ 7.7 Hz, NCH2), 9.25 (1H, s, CH-6). 13C NMR (CDCl3):
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.024.
d
14.10 (2 ꢂ CH3), 22.61 (2 ꢂ CH2CH3), 25.32 (2 ꢂ CH2), 25.37
(2 ꢂ CH2), 26.76 (2 ꢂ CH2), 26.97 (CH-8 or CH-11), 27.69 (CH2), 27.92
(CH2), 28.45 (CH-8 or CH-11), 28.99 (2 ꢂ CH2), 31.77 (2 ꢂ CH2),
42.81 (NCH2), 43.57 (NCH2), 106.83 (Cquat), 123.18 (Cquat), 144.28
(Cquat), 148.84 (Cquat), 150.29 (Cquat), 152.75 (CH-6), 153.85 (Cquat),
154.14 (NC]O), 158.45 (NC]O), 178.95 (C]O), 181.09 (C]O). IR
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