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Synthesis and In-Vitro Cytotoxicity Evaluation of Novel Naphtindolizinedione Derivatives Part IIImproved Activity for Aza-Analogues.

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80
Arch. Pharm. Chem. Life Sci. 2009, 342, 80 – 86
Full Paper
Synthesis and In-Vitro Cytotoxicity Evaluation of Novel
Naphtindolizinedione Derivatives, Part II: Improved Activity
for Aza-Analogues
Andrea Defant, Graziano Guella, and Ines Mancini
Laboratorio di Chimica Bioorganica, Dipartimento di Fisica, Universit degli studi di Trento, Povo Trento, Italy
Our previous investigation on potential antitumor agents now got enriched by the evaluation of
in-vitro activity against a full panel of NCI cancer cell lines for five new compounds. The concurrent presence in the molecular structure of a nitrogen atom in the aromatic system and a N,Ndimethylaminoethyl amide chain play a decisive role to enhance cytotoxicity. The N,N-anti compound 14 shows a higher activity than its N,N-syn isomer, exhibiting the best selective inhibition
against the melanoma MALME-3M cell line, with a GI50-value (= 30 nM) corresponding to a 330fold increase in activity compared to the corresponding deaza-analogue. Compound 14 is efficiently synthesized by aminolysis of the ester obtained as a single regio-isomer by an one-pot
three-component procedure involving metal-assisted cyclization under microwave irradiation
conditions.
Keywords: Antitumor activity / Cytotoxic activity / DNA-topoisomerase / Indolizinoquinoline-5,12-dione derivatives / Microwave-assisted synthesis /
Received: September 22, 2008; accepted: November 3, 2008
DOI 10.1002/ardp.200800177
Introduction
Molecules with naphtindolizinedione skeleton are
known as good candidates for antitumor agents, due to
their approved cytotoxic activity against human cancer
cell lines and their inhibitory activity of DNA topoisomerases I or II [1]. Ethyl ester 1 and the structurally modified
analogues reported in Fig. 1 belong to this class of compounds. Recently, we have studied the multi-component
cyclization to aza-derivatives 2 and 3 starting from 6,7dichloroquinoline-5,8-dione showing that the regio-selective formation of the N,N-syn 2 and N,N-anti 3 isomers
depends on solvent and metal-ion chelation effects [2].
Moreover, we have also successfully exploited microwave
irradiation to gain the same heterocyclic products,
Correspondence: Ines Mancini, Laboratorio di Chimica Bioorganica, Dipartimento di Fisica, Universit degli studi di Trento, via Sommarive 14, I38100 Povo, Trento, Italy.
E-mail: mancini@science.unitn.it
Fax : +39 0461 881696
Abbreviations: flash-chromatography (FC); mean graph medium
(MGM); microwave (MW)
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Chemical structures of already reported cytotoxic
molecules.
which can be obtained more advantagously this way
(higher yields, shorter reaction times, and lower formation of by-products) than using conventional heating [3].
Thus, we have previously demonstrated that the microwave (MW) irradiation is an effective and versatile
method to synthesize almost pure regio-isomers which,
Arch. Pharm. Chem. Life Sci. 2009, 342, 80 – 86
in turn, can be used as precursors in the synthesis of
potential antitumour agents [3, 4].
Very recently, Cheng et al., [4] have synthesized a series
of indolizinoquinoline-5,12-dione derivatives, including
the known ethyl esters 1, N,N-syn compound 2, and its
N,N-anti isomer 3 together with new related analogues
bearing substitutions on the C-1 position of the D ring.
These compounds have been studied for their cytotoxic
activities toward a limited number of human cancer cell
lines and as topoisomerase-I inhibitors [4]. Evaluation of
the structure – activity relationships indicates that the
existence of a nitrogen atom in the A ring is relevant to
increase the cytotoxicity, whereas its ring position causes
a variable and moderate effect. In addition, the presence
of a functional group in C-1 position of the D ring (i.e., Cl,
Br, Me, OH, or NH2) does not induce an increased bioactivity.
It is known that the occurrence of basic nitrogen
centres, as one or two chains bearing amine terminal
functions or aza-units on a planar polycyclic aromatic
system, are able to increase the antitumor activity by
non-covalent interactions with DNA, interfering with its
conformation and affecting the functions of enzymes as
topoisomerases which bind DNA, [5, 6]. Based on the
structural similarity with known antitumor agents such
as ellipticine, daunorubicin, mitoxantrone, and derivatives of DACA (= N-[2-(dimethylamino)ethyl]acridine-4carboxamide), we have recently [7] designed and synthesized a series of analogues of ester 1. These compounds
were evaluated in the NCI panel of human tumor cell
lines, from which the N,N-dimethylethylamino chain (as
in compound 4) turned out to be the most relevant structural modification to induce the highest activity and
selectivity towards leukaemia, colon, and renal cancer
cell lines with GI50 values from lower than 10 nM to
0.2 lM [7].
We now report on the microwave-assisted synthesis
(Scheme 1) and cytotoxic evaluation of the new compounds 10 – 14 against a large panel of human tumor cell
lines. These results are compared with those of previous
investigations of the known molecules 1 – 4.
Results and discussion
Compounds tested for their cytotoxic evaluation are
obtained according to the synthetic sequence reported in
Scheme 1. Methyl esters 5a / 5b, 6, 7 and 8, 9 are prepared
starting from 2,3-dichloro-1,4-naphtoquinone or 6,7dichloroquinoline-5,8-dione [2], respectively and suitable
nucleophilic reagents through an one-pot cyclization
assisted by microwave irradiation. The involvement of
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Synthesis / Evaluation of Naphtindolizinedione Derivatives
81
Reagents and conditions: (i) Methyl acetoacetate, 3-methylpyridine, neat, MW irradiation, 4 min, 81%; (ii) methyl acetoacetate, methyl nicotinate or methylisonicotinate,
neat, MW irradiation, 4 min, 76 and 78%, resp.; (iii) methylacetoacetate, pyridine, tBuOH, MW irradiation, 4 min, syn / anti = 82 : 18, 90%, FC purification; (iv) a) MgCl2
(1.3 molar equiv.), CH3CN, r.t., 1 h, b) methylacetoacetate, pyridine, neat, MW irradiation, 4 min, anti / syn 100 : 0, 64%; (v) N,N-dimethylaminoethylamine in excess, 808C,
3 h, 98%.
Scheme 1. Synthesis of naphthindolizinedione derivatives 10 –
14.
the preformed complex of 6,7-dichloroquinoline-5,8dione with the eco-friendly salt MgCl2 is able to induce a
highly regio-selective production of the N,N-anti ester 9
[3], whereas the N,N-syn isomer 8 (Scheme 1) is obtained
by the most convenient procedure (see Experimental, Section 4) selected among the series of experimental conditions previously investigated [2, 3]. It is noteworthy that
in the reaction using 2,3-dichloro-1,4-naphtoquinone,
methylacetoacetate, and 3-methylpyridine, products 5a /
5b were formed in the ratio 72 : 28. This can be explained
from the proposed cyclization mechanism, implying
both C- and N-nucleophile attacks with displacement of
chlorine atoms, followed by the ring closure [2, 8]. The
latter step can involve the 6- or 2-positions in 3-methylpyridine, leading to product 5a or 5b, respectively. Otherwise, when methyl nicotinate (= methyl pyridine-3-carboxylate) is used, 6 is obtained as a single isomer, as
deduced from NMR analysis of the crude reaction mixture. Its formation stems from involvement of the 6-position, whereas the steric effect of COOCH3 group in
methyl-nicotinate hinders the closure at the 2-position.
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82
A. Defant et al.
The corresponding amides are prepared by heating
esters 5a / 5b and 6 – 9 with an excess of N,N-dimethylaminoethylamine [3]. Their molecular composition is established by mass-spectrometric measurements including
high resolution (HR-EIMS) experiments, whereas their
structures are fully characterized by extensive NMR analysis, including 1D- and 2D-one bond and long range 1H13
C hetero-correlated experiments (see Experimental, Section 4).
Compounds 10 – 14 are evaluated for their in-vitro activity against cancer cell lines by the National Cancer Institute (NCI). The data summarized in Table 1 may serve to
illustrate structure – activity relationships.
From the values of mean graph medium (MGM),
defined as the average GI50 (lM) for each compound over
all the cell lines investigated, it can be noticed that the
presence of: (i) a methyl group in the D ring, as in compound 10, does not affect the cytotoxicity, although
inducing selectivity on some special cell lines, (ii) two
side chains, as in compounds 11 and 12, recalling the
structure of the antineoplastic agent mitoxantrone used
in the treatment of certain types of cancer (such as metastatic breast cancer and acute myeloid leukaemia) cause
an activity decrease, with the second amide chain at the
3-position of D ring inducing the highest effect, (iii) a
nitrogen atom on the A ring, as in the two regio-isomers
13 and 14, has an increasing effect on the bioactivity
level, with the N,N-anti position exhibiting the highest
effect. This correlation is properly indicated by the values
obtained on the MCF7 breast cell line for esters 2 and 3
with respect to 1, and amides 13 and 14 with respect to 4
(Table 1). Whereas N,N-anti amide 14 exhibits the highest
effect on the whole cell line panel, Cheng et al. [4] find
that N,N-syn ester 2 has a better effect than its
regioisomer 3, although tested on only a limited number
of cell lines. This discrepancy is difficult to explain, but a
different DNA-binding mode for esters and amides may
be called on. In addition, the relevant role of the amide
chain in improving the effect is also proved by the data
reported by Cheng et al. [4] against this cell line (GI50 in
the range 1 – 12 lM) for compound 1 and for the N,N-syn
and N,N-anti analogues with an acetyl or a nitrile group
in the place of the ethoxycarbonyl unit of the regioisomers 2 and 3. Finally, the important role of the amide chain
was expected by comparison of the values for amide 4 [7]
with ethyl ester 1, both on the non small cell lung A549/
ATCC [1] and on leukaemia HL60 (TB) lines [4] (Table 1).
From the whole human tumor cell lines panel, the best
values were achieved for compound 14 against the melanoma MALME-3M, with GI50 = 30 nM. In comparison to
compound 4, a 6-, 155-, and 330-fold increase can be
observed for the presence of a methyl group on the D
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2009, 342, 80 – 86
Figure 2. Growth of MALME-3M melanoma cell line as a function of the concentration for amides bearing a single chain (4,
10, 13, and 14) and two side chains (11 and 12).
ring (compound 10) or a nitrogen atom on the A ring in
the N,N-syn compound 13 and in the N,N-anti isomer 14,
respectively. Although the molecules with two amide
chains tested here, exhibit a lower activity towards all
the cell lines, it must be noticed that compound 12 shows
an increased effect against the same MALME-3M cell line,
with respect to regio-isomer 11. The results are illustrated
in Fig.2, displaying the growth of this melanoma cell line
as a function of the concentration of all the compounds
under investigation. It is noteworthy that the in-vitro
cytotoxicity of compound 14 against the same NCI cell
line is 1300-fold higher than the one obtained for dacarbazine (DTIC), [9] one of the clinically most used anti-melanoma drugs.
Conclusions
In our study, focused on the design and synthesis of
naphthindolizinedione derivatives with potential antitumor activity, we have considered the new compounds
10 – 14. They are available through an efficient microwave-assisted synthesis and their in-vitro activity against
cancer cell lines has been evaluated by the National Cancer Institute (NCI). Compound 14 was found to be the
most potent cytotoxic agent within the series. Our results
against MCF7 cell line compared with the ones recently
reported by Cheng et al. for esters 2 and 3 [4], indicate
that the presence of a N,N-dimethylaminoethyl amide
chain plays a decisive role to improve bioactivity. Compound 14 comes out as the most promising novel molecule in the series of naphthindolizinedione derivatives.
Its structural variations include the presence of a nitrogen atom on A ring (with the possibility of generating
regio-isomers), substituents on the D ring, and alkoxycarbonyl and carboxamide groups on the C ring (with the
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Arch. Pharm. Chem. Life Sci. 2009, 342, 80 – 86
Synthesis / Evaluation of Naphtindolizinedione Derivatives
83
Table 1. Inhibition of in vitro human cancer cell lines by new compounds 10 – 14 in comparison with the known cytotoxic agents 1 – 4.
Cytotoxicity GI50 (lM)a)
Type of cancer Cell line
1b, c)
2c)
3c)
4d)
> 25.0
0.88
14.45
10
11
12
13
14
CCRF-CEM
HL60(TB)
K562
MOLT-4
RPMI-8226
SR
1.24
2.48
0.415
0.448
n. d.
0.0913
3.64
13.8
5.39
3.14
15.0
1.92
0.646
3.70
1.68
2.46
1.97
1.24
1.05
0.989
0.269
0.350
n. d.
0.238
0.226
0.312
0.324
0.165
0.246
0.104
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
0.666
1.82
0.979
0.812
1.69
1.02
0.810
0.447
1.26
5.25
24.2
6.25
2.22
6.66
10.5
14.6
8.17
6.12
1.18
2.13
3.14
0.766
4.33
3.09
2.18
1.72
1.72
0.273
0.554
0.795
2.49
1.13
0.239
0.365
0.153
1.11
0.175
0.494
0.357
0.930
0.449
0.299
0.164
0.128
0.449
Colon
COLO 205
HCC2998
HCT-116
HCT-15
HT29
KM12
SW-620
0.607
0.581
0.371
0.518
0.419
0.803
0.379
6.08
14.9
4.18
36.8
4.96
14.2
5.59
2.06
3.88
0.977
3.92
1.56
2.81
1.31
0.169
0.232
0.219
0.232
0.324
0.376
0.298
0.155
0.319
0.214
0.263
0.255
0.336
0.284
0.31 l 0.21
0.600 l 0.529
0.194 l 0.184
0.277 l 0.23
0.146 l 0.094
6.55 l 6.05
1.68 l 1.27
CNS
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
0.494
0.812
2.61
0.718
0.793
0.461
7.34
20.4
11.5
7.35
6.37
4.93
3.10
0.623
2.46
2.79
3.61
2.88
0.554
0.350
1.22
0.804
1.77
0.343
0.711
0.262
0.390
0.409
1.14
0.279
17.8 l 12.91
5.61 l 4.79
1.39 l 0.08
0.574 l 0.22
7.39 l 5.41
0.829 l 0.781
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
0.927
1.69
2.89
3.12
4.40
2.93
4.49
4.27
6.33
23.3
21.3
16.7
20.2
17.5
34.4
14.8
1.80
0.779
7.26
5.44
4.95
5.35
5.06
7.45
0.305
0.0651
0.489
1.66
1.57
0.174
1.33
0.629
0.195
0.0303
0.275
0.826
1.38
0.146
0.170
0.185
0.275 l 0.25
10.1 l 8.32
1.31 l 0.772
9.76 l 7.05
6.56 l 3.55
1.39 l 0.77
4.31 l 0.83
0.607 l 0.533
Ovarian
IGROV-1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
0.575
1.47
1.41
3.04
0.821
2.94
6.20
10.3
5.34
17.5
5.00
20.1
0.719
3.10
1.53
5.00
1.17
4.76
0.326
0.219
0.215
1.03
0.527
1.34
0.203
0.242
0.151
1.09
0.305
1.02
2.65 l 2.0
9.14 l 7.45
1.69 l 0.62
0.703 l 0.607
0.444 l 0.084
2.06 l 0.125
Renal
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
3.35
1.68
0.472
1.26
1.28
0.341
2.06
0.496
11.9
22.3
24.3
39.7
0.671
1.92
23.2
30.0
3.55
6.58
5.17
1.41
0.486
2.01
2.01
2.80
1.39
1.61
0.502
1.25
0.570
0.291
1.57
1.01
1.37
1.36
0.371
0.324
0.452
0.225
0.323
0.321
0.530 l 0.469
2.14 l 0.38
0.169 l 0.159
5.66 l 5.14
2.61
0.278 l 0.25
6.63 l 5.47
3.51 l 3.10
Prostate
PC-3
DU-145
1.15
0.851
8.34
5.57
1.02
1.77
0.585
0.452
0.259
0.229
1.86 l 0.015
1.47 l 0.17
Leukemia
Non Small
Cell Lung
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
16.3
n. d.
a 0.25
1.90
1.64 l 0.94
a 0.0100
0.992 l 0.01
0.268 l 0.025
0.853 l 0.48
0.318 l 0.11
0.395 l 0.097
1.44 l 0.265
0.988 l 0.952
1.28 l 0.70
0.412 l 0.37
0.719 l 0.631
6.92 l 6.08
3.91 l 0.5
4.24 l 3.03
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Arch. Pharm. Chem. Life Sci. 2009, 342, 80 – 86
Table 1. Continued.
Cytotoxicity GI50 (lM)a)
Type of cancer Cell line
Breast
10
11
12
13
14
1b, c)
2c)
3c)
4d)
MCF7
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
BT-540
T-47D
0.865
0.960
3.66
0.548
2.70
2.23
2.23
3.16
A 100
15.9
4.89
17.7
36.3
14.2
1.29
27.0
2.65
2.68
3.25
n. d.
6.24
0.349
1.08
1.33
0.516
0.194
1.97
1.19
0.163
0.828
0.282
0.637
0.174
n. d.
0.248
> 25.0
0.96
6.38
5.89 l 0.94
0.638 l 0.59
0.573 l 0.49
2.40 l 0.17
6.82 l 5.48
10.9
2.22 l 0.935
MGMe)
1.07
10.0
2.40
0.550
0.316
0.944
a)
GI50values, defined as the concentration that inhibits growth by 50% from NCI screening.
values from Cheng et al. [4].
c)
value for non small cell lung line A549/ATCC evaluated by Park et al. [1].
d)
Average values from re-testing of the compound [7].
n. d. = not determined.
e)
Mean graph medium (MGM) as average GI50 (lM) over all cell lines investigated.
b)
N,N-dimethylaminoethyl chain previously selected as the
best one in our investigation). In particular, N,N-anti compound 14 is more active than the N,N-syn regio-isomer 13
in the complete evaluation against the large panel of
human tumor cell lines; more specifically, its best selective inhibition is observed against the melanoma
MALME-3M cell line. Also the efficient synthesis of 14
reported here could open potential applications of this
molecule for treatment of melanoma, for which chemotherapy is usually not so effective as some other types of
cancer.
GmbH, Rheinstetten, Germany); 1H at 400 MHz; d values in ppm
rel. to SiMe4 (= 0 ppm), J values in Hz; structural assignments are
from 1H,1H-COSY, heteronuclear single quantum correlation
(HSQC) and Heteronuclear Multiple Bond Correlation (HMBC)
experiments; for compounds 10 – 14 the reported 13C-NMR data
were deduced by HMBC correlations. Electron-impact (EI) mass
spectra (m/z; rel.%) and HR-EI data were taken with a KratosMS80 mass spectrometer (Kratos. Ltd., Manchester, UK) with
home-built computerized acquisition software. ESI-MS data, and
tandem fragmentation spectra (MSn), were taken with a Bruker
Esquire-LCTM spectrometer (Bruker Daltonik GmbH, Bremen,
Germany), equipped with an electrospray ionization ion source
used in positive ion mode by injection of the sample into the
source from a methanol solution.
We would like to thank the National Cancer Institute (NCI) at
Bethesda, USA for the antitumor screening tests of our compounds. We are grateful to Mr. A. Sterni for mass spectra
recording.
Preparation of esters 5 – 9
The authors have declared no conflict of interest.
Experimental
Chemistry
All evaporations were carried out at room temperature at
reduced pressure. MW irradiation was done according to the
procedure reported in Defant et al. [3]. Yields are given for
reacted compounds. All reagents and solvents were purchased
from Sigma Aldrich Europe (Milan, Italy) and were used without
purification. TLC was carried on Kieselgel 60 PF254 and RP-18 F254
(Merck, Darmstadt, Germany) and flash-chromatography (FC)
was carried out on Merck silica gel 60, 20 – 50 lm. Melting points
were determined on Reichert Thermovapor microscope (Reichert, Austria) and the data are uncorrected. NMR spectra were
taken with an Avance 400 Bruker spectrometer (Bruker BioSpin
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Neat commercial 2,3-dichloro-1,4-naphtoquinone (0.057 g,
0.25 mmol), methylacetoacetate (0.04 mL, 0.37 mmol) and 3methylpyridine (0.15 mL, 1.5 mmol) or methyl nicotinate
(0.20 g, 1.5 mmol), or methyl isonicotinate (0.20 g, 1.5 mmol),
were irradiated in a closed vessel at 540 W for 4 min. The crude
reaction mixture was partitioned between dichloromethane
and water, extracted (36) and the combined organic phases
washed with a cold 1 M aqueous solution HCl and then with
water, dried over anhydrous Na2SO4 and evaporated. The residue
was subjected to FC (hexane / AcOEt gradient elution) to obtain
in each case: a mixture of 5a / 5b in 72 : 28 ratio, as deduced by
1
H-NMR spectrum (0.065 g, 81%), compound 6 (0.07 g, 76%), and
7 (0.07 g, 78%).
A solution of 6,7-dichloroquinoline-5,8-dione (0.057 g,
0.25 mmol) methylacetoacetate (0.04 mL, 0.37 mmol) and pyridine (0.12 mL, 1.5 mmol) in t-BuOH (0.5 mL) was irradiated in a
closed vessel at 540 W for 4 min. Solvent was evaporated and the
resultant residue was subjected to the same procedure as above,
obtaining pure 8 (0.056 g, 74%, starting from a 82 : 18 syn /anti
crude mixture). A solution of 6,7-dichloroquinoline-5,8-dione
(0.057 g, 0.25 mmol) in anhydrous CH3CN (2.0 mL) was stirred
with solid MgCl2 (0.031 g, 0.33 mmol) for 1 h, then evaporated to
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Arch. Pharm. Chem. Life Sci. 2009, 342, 80 – 86
leave a metal complex which was treated with methylacetoacetate and pyridine as above, to give 9 as a single regio-isomer
(0.048 g, 64%).
3-Methyl-6,11-dioxo-6,11-dihydro-benzo[f]pyrido[1,2a]indole-12-carboxylate 5a and 1-methyl-6,11-dioxo6,11-dihydro-benzo[f]pyrido[1,2-a]indole-12-carboxylate
5b
Red-brown solid; 1H-NMR (CDCl3, with the not overlapping signals for 5b in brackets) d: 2.48 [2.55] (s, 3H, CH3-3), 4.06 [4.10] (s,
3H, COOCH3), 7.33 (d, J = 7.3 Hz, 1H, H-2), [7.09, m, 2H, 2H and
3H], 7.74 (m, 2H, H-7 and H-8), 8.31 [8.20] (m, 3H, H-1, H-6 and H9), 9.73 (s, 1H, H-4); EI-MS m/z (%): 319 [M+9] (52), 288 (70), 261(15);
HR(EI)MS: 319.0835 l 0.0030 (C19H13NO4, calc. 319.0845).
Dimethyl-6,11-dioxo-6,11-dihydro-benzo[f]pyrido[1,2a]indole-3,12-dicarboxylate 6
Crystalline orange solid; m.p. 255 – 2568C (ethanol); 1H-NMR
(CDCl3) d: 4.00 and 4.07 (two s, 3H each, COOCH3), 7.74 (m, 2H, H7 and H-8), 7.69 (dd, J = 7.3, 1.7 Hz, 1H, H-3), 8.24 (m, 2H, H-6 and
H-9), 8.95 (s, 1H, H-1), 9.84 (d, J = 7.3 Hz, 1H, H-4); EI-MS m/z (%):
363 [M+9] (16), 332 (21); HR (EI)MS: 363.0733 l 0.0030 (C20H13NO6,
calc. 363.0743).
Dimethyl-6,11-dioxo-6,11-dihydro-benzo[f]pyrido[1,2a]indole-2,12-dicarboxylate 7
Pale orange powder; m.p. 2648C (ethanol); 1H-NMR (CDCl3) d: 4.02
and 4.05 (two s, 3H each, COOCH3), 7.74 (m, 2H, H-7 and H-8),
7.94 (d, J = 9.3 Hz, 1H, H-3), 8.24 (d, J = 9.3 Hz, 2H, H-6 and H-9),
8.33 (d, J = 9.3 Hz, 1H, H-1), 10.50 (s, 1H, H-4); EI-MS m/z (%): 363
[M+9] (17), 332 (24); HR(EI)MS: 363.0736 l 0.0030 (C20H13NO6, calc.
363.0743).
Methyl-5,10-dioxo-5,10-dihydro-4,6-diazabenzo[b]fluorene-11-carboxylate 8 and Methyl-5,10-dioxo-5,10dihydro-4,9a-diazabenzo[b]fluorene-11-carboxylate 9
For both compounds: Data in agreement with the ones reported
in Defant et al. [2].
Conversion of esters to amides 10 – 14
Each methyl ester was stirred in the presence of a molar excess
of N,N-dimethylaminoethylamine (0.5 mL) at 808C for 3 h. The
crude mixture was evaporated in vacuo, water (5 mL) was added
to the residue and extracted with dichloromethane (36), to give
a combined organic phase which was washed with brine. After
drying over anhydrous Na2SO4 and evaporation, the residue was
subjected to preparative TLC eluted with CH2Cl2 / MeOH, 95 : 5,
to give pure amides as verified by NMR analysis (98% yields). In
the case of ester 5a / 5b, the 72 : 28 mixture was used in the
amide preparation, but only isomer 5a was reactive giving
amide 10, easily separated through FC from ester 5b.
3-Methyl-6,11-dioxo-6,11-dihydro-benzo[f]pyrido[1,2-a]indole-(2-dimethylamino-ethyl)-12-carboxamide 10
Crystalline red solid; m.p. 220 – 2218C (dichloromethane / methanol); 1H-NMR (CDCl3) d: 2.38 (s, 6H, N(CH3)2), 2.40 (s, 3H, CH3-3),
2.68 (t, J = 6.7 Hz, 2H, CH2N(CH3)2), 3.66 (pseudo q, J = 6.7 Hz, 2H,
CONHCH2), 7.26 (br d, J = 9.6 Hz, 1H, H-2), 7.67 and 7.73 (two br t,
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Synthesis / Evaluation of Naphtindolizinedione Derivatives
85
J = 7.4, Hz, 1H each, H-8 and H-9), 8.19 and 8.21 (two br d, J =
7.4 Hz, 1H each, H-7 and H-10), 8.95 (d, J = 9.6 Hz, 1H, H-1), 9.70 (s,
1H, H-4), 10.50 (br s, 1H, CONH); 13C-NMR (CDCl3) d: 19.74 (CH3-2),
37.51 (CONHCH2), 44.82 (N(CH3)2), 61.20 (CH2N(CH3)2), 125.77 (C1), 126.88 and 127.06 (C-7 and C-10), 129.60 and 130.94 (C-2 and
C-4), 133.05 (C-8), 133.39 and 133.85 (C-6a and C-10a), 134.30 (C9), 139.66 (C-12a), 162.15 (CONH), 174.68 (C-6), 184.29 (C-11); EIMS m/z (%): 375 [M+9] (2), 288 (19), 261 (11); HR(EI)MS:
375.1573 l 0.0030 (C22H21N3O3, calc.375.1583).
N,N-Bis[2-(dimethylamino)ethyl]-6,11-dioxo-6,11dihydro-benzo[f]pyrido[1,2-a]indole-3,12-dicarboxamide
11
Red solid; m.p. 235-2368C (dichloromethane / methanol); 1HNMR (CDCl3) d: 2.31 and 2.37 (two s, 6H each, N(CH3)2), 2.58 and
2.67 (two t, J = 6.7 Hz, 2H each, CH2N(CH3)2),3.58 and 3.65 (two q, J
= 6.7 Hz, 2H each, CONHCH2), 7.73 (m, 3H, H-3, H-8 and H-9), 8.24
(m, 2H, H-7 and H-10), 9.11(d, J = 9.3 Hz, 1H, H-1), 10.33 (s, 1H, H4), 10.42 (br s, 2H, CONH); 13C-NMR (CDCl3) d: 37.52 (two
CONHCH2), 44.94 (two N(CH3)2), 57.44 (two CH2N(CH3)2), 123.05 (C1), 125.77 (C-3), 126.96 (C-7 and C-10), 127.35 (C-4), 132.43, 132.62
and 133.01 (C-6a, C-10a and C12a), 133.59 (C-8 and C-9), 140.24 (C5a) 164.32 (CONH), 174.41 (C-6), 183.59 (C-11); ESI (+)-MS m/z: 476
[M + H]+; MS/MS (476) fi 431; (MS)3 (431) fi 386.
N,N-Bis[2-(dimethylamino)ethyl]-6,11-dioxo-6,11dihydro-benzo[f]pyrido[1,2-a]indole-2,12-dicarboxamide
12
Red solid; m.p. 241 – 2438C(dichloromethane / methanol); 1HNMR (CDCl3) d: 2.31 and 2.37 (two s, 6H each, N(CH3)2), 2.58 and
2.67 (two t, J = 6.7 Hz, 2H each, CH2N(CH3)2), 3.58 and 3.65 (two q,
J = 6.7 Hz, 2H each, CONHCH2), 7.70 (m, 3H, H-3, H-8 and H-9),
8.19 (m, 2H, H-7 and H-10), 9.35(s, H-1), 9.81 (d, J = 7.6 Hz, 1H, H4), 10.48 (br s, 2H, CONH); 13C-NMR (CDCl3) d: 37.52 (two
CONHCH2), 45.33 (two N(CH3)2), 57.83 (two CH2N(CH3)2), 117.38 (C3), 121.06 (C-1), 126.87 (C-4), 127.15 and 127.54 (C-7 and C-10),
133.60 (C-8 and C-9), 165.04 (CONH), 174.80 (C-6), 184.50 (C-11);
ESI (+)-MS m/z: 476 [M + H]+; MS/MS (476) fi 431; (MS)3 (431) fi
386.
N-[2-(Dimethylamino)ethyl]-5,10-dioxo-5,10-dihydro-4,6diazabenzo[b]fluorene-11-carboxamide 13
Crystalline deep red solid; m.p. 217 – 2188C(dichloromethane /
methanol); 1H-NMR (CDCl3) d: 2.44 (s, 6H, N(CH3)2), 2.76 (br t, J =
6.7 Hz, 2H, CH2N(CH3)2), 3.66 (pseudo q, J = 6.7 Hz, 2H, CONHCH2),
7.27 (br t, J = 7.1 Hz, 1 H, H-3), 7.50 (m, 1H, H-2), 7.66 (dd, J = 7.8,
4.6 Hz, 1H, H-8), 8.59 (dd, J = 7.8, 1.9 Hz, 1H, H-9), 9.05 (dd, J = 4.6,
1.5 Hz, 1H, H-7), 9.13 (d, J =9.1Hz, 1H, H-1), 10.10 (d, J = 7.1 Hz, 1H,
H-4), 10.31 (br s, 1H, CONH); 13C-NMR (CDCl3) d: 34.79 (CONHCH2),
43.38 (N(CH3)2), 56.46 (CH2N(CH3)2), 119.34 (C-3), 123.05 (C-1),
128.13 (C-4), 129.11 (C-2), 130.08 (C-10a), 135.94 (C-12a), 141.02
(C-6a), 141.21 (C-5a), 155.08 (C-8), 164.06 (CONH), 183.58 (C-11); EIMS m/z (%): 362 [M+9] (3), 319 (4), 275(7); HR(EI)MS:
362.1377 l 0.0030 (C20H18N4O3, calc. 362.1379).
N-[2-(Dimethylamino)ethyl]-5,10-dioxo-5,10-dihydro4,9a-diazabenzo[b]fluorene-11-carboxamide 14
Crystalline deep red solid; m.p. 190–1918C(dichloromethane /
methanol); 1H-NMR (CDCl3) d: 2.39 (s, 6H, N(CH3)2), 2.74 (br t, J =
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86
A. Defant et al.
6.7 Hz, 2H, CH2N(CH3)2), 3.70 (pseudo q, J = 6.7 Hz, 2H, CONHCH2),
7.28 (m, 1H, H-3), 7.49 (m, 1H, H-2), 7.72 (m, 1H, H-8), 8.59 (dd, J =
7.8, 1.5 Hz, 1H, H-7), 9.02 (dd, J = 4.6, 1.7 Hz, 1H, H-1), 9.14 (d, J =
9.2 Hz, 1H, H-9), 9.89 (d, J =7.0 Hz, 1H, H-4), 10.36 (br s, 1H,
CONH); 13C-NMR (CDCl3) d: 34.40 (CONHCH2), 44.94 (N(CH3)2),
58.03 (CH2N(CH3)2), 119.14 (C-3), 122.85 (C-1), 127.93 (C-4), 128.32
(C-8), 128.33 (C-2), 131.05 (C-6a), 135.55 (C-7), 141.02 (C-12a),
148.05 (C-11a), 152.93 (C-9), 163.47 (CONH), 172.45 (C-6), 184.22
(C-11); EI-MS m/z (%): 362 [M+9] (10), 275 (7); HR(EI)MS:
362.1374 l 0.0030 (C20H18N4O3, calc. 362.1379).
Biology
Compounds 10 – 14 were evaluated for their in-vitro activity
against cancer cell lines by the National Cancer Institute (NCI)
following its anticancer drug development program based on
automated sulforhodamine blue (SRB) cytotoxicity assay [9].
Arch. Pharm. Chem. Life Sci. 2009, 342, 80 – 86
[2] A. Defant, G. Guella, I. Mancini, Eur. J. Org. Chem. 2006,
4201 – 4210.
[3] A. Defant, G. Guella, I. Mancini, Synth. Commun. 2008, 38,
3003 – 3016.
[4] Y. Cheng, L.-K. An, N. Wu, X.-D. Wang, et al., Bioorg. Med.
Chem. 2008, 16, 4617 – 4625.
[5] J.-H. Tan, Q.-X. Zhang, Z.-S. Huang, Y. Cheng, et al., Eur. J.
Med. Chem. 2006, 41, 1041 – 1047.
[6] L. W. Deady, M. L. Rogers, L. Zhuang, B. C. Baguley, W. A.
Denny, Bioorg. Med. Chem. 2005, 13, 1341 – 1355.
[7] A. Defant, G. Guella, I. Mancini, Arch. Pharm.Chem. Life Sci.
2007, 340, 147 – 153.
[8] E. F. Pratt, R. G. Rice, R. W. Luckenbaugh, J. Am. Chem. Soc.
1957, 79, 1212 – 1217.
[9] http://dtp.nci.nih.gov/branches/btb/ivclsp.html (assessed in
April 2008).
References
[1] H. J. Park, H.-J. Lee, E.-J. Lee, H. J. Hwang, et al., Biosci. Biotechnol. Biochem. 2003, 67, 1944 – 1949.
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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synthesis, part, naphtindolizinedione, evaluation, iiimproved, activity, novem, cytotoxicity, vitro, aza, derivatives, analogues
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