Synthesis and cytotoxicity of potential anticancer derivatives of pyrazolo[3,4,5-kl]acridine and indolo[2,3-a]acridine

Article abstract of DOI:10.1016/S0040-4020(01)01119-X

Pyrazolo[3,4,5-kl]acridines were prepared by reaction of ethyl 1,9-dioxo-1,2,3,4,9,10-hexahydroacridine-4-carboxylate (4) with hydrazine and its methyl and 2-(dimethylamino)ethyl derivatives, followed by aromatization of the intermediate products with 1,4

Full text of DOI:10.1016/S0040-4020(01)01119-X

TETRAHEDRON
Pergamon
Tetrahedron 58 52002) 175±181
Synthesis and cytotoxicity of potential anticancer derivatives of
pyrazolo[3,4,5-kl]acridine and indolo[2,3-a]acridine
Xianyong Bu,a Junjie Chen,a Leslie W. Deadya,p and William A. Dennyb
aDepartment of Chemistry, La Trobe University, Vic. 3086, Australia
bAuckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019,
Auckland 1000, New Zealand
Received 29 August 2001; revised 16 October 2001; accepted 8 November 2001
AbstractÐPyrazolo[3,4,5-kl]acridines were prepared by reaction of ethyl 1,9-dioxo-1,2,3,4,9,10-hexahydroacridine-4-carboxylate 54) with
hydrazine and its methyl and 2-5dimethylamino)ethyl derivatives, followed by aromatization of the intermediate products with 1,4-benzo-
quinone. Conversion of the ester function to a carboxamide was also carried out and N-52-5dimethylamino)ethyl)-1-52-5dimethyl-
amino)ethyl)-1,2-dihydropyrazolo[3,4,5-kl]acridine-5-carboxamide 513c) was appreciably cytotoxic in a panel of cell lines. Reaction of 4
with 4-methoxyphenylhydrazine gave instead a novel indolo[2,3-a]acridine derivative. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
Many modi®cations have been made to the acridine skeleton
in the search for anticancer compounds. Among these are
the pyrazolo[3,4,5-kl]acridines, for example 1,1 a compound
in phase II clinical trial.2 The basic sidechain is essential for
activity and the nitro group provided activation for the
pyrazole ring forming reaction through displacement of a
chlorine at the 1-position of a precursor acridine.
The acridine-4-carboxamides are another class of anticancer
acridines, illustrated by 23 5also in clinical trial4), where the
basic side chain is linked through an amide function
speci®cally located peri to the ring nitrogen. The related
tetracyclic carboxamides 35 are also effective cytotoxins.
We have previously synthesized 46 5Scheme 1), which
contains an ester precursor of the desired carboxamide in
the key position, and 1,9-functionality which might allow a
pyrazole ring to be constructed. We were therefore
interested in this possibility and to make compounds
which combined some features of 1 and 2.
2. Results and discussion
2.1. Chemistry
Reaction of 4 with 1±2 mol equiv. of hydrazine or the
methyl and 2-5dimethylamino)ethyl derivatives in hot
ethanol gave the corresponding hydrazones 55a±c)
5Schemes 1 and 2); the target cyclization did not occur
Keywords: hydrazone; cyclization; 1,4-benzoquinone; carboxamide.
Corresponding author. Tel.: 161-3-9479-2561; fax: 161-3-9479-1399;
e-mail: l.deady@latrobe.edu.au
p
under these conditions, but neither did any reaction of the
ester group.
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020501)0 1119-X

176
X. Bu et al. / Tetrahedron 58 42002) 175±181
tri¯uoroacetic acid gave a satisfactory formation of the
pyrazolo compounds 6b,c, along with small amounts of
the des-ethoxycarbonyl analogues 7b,c 5Scheme 2). It was
essential that this reaction was done under nitrogen; without
this, 5b gave the unexpected and surprisingly stable tertiary
alcohol 8b.
It was subsequently found that the condensation and
cyclization could be achieved for all hydrazines in one
process, by re¯uxing 4 and a salt of the hydrazine
with sodium acetate in ethanol, though the yield from the
two-step sequence was better for the aminoalkyl derivative
6c.
1
Compounds 6 had complex H NMR spectra, especially in
the aliphatic region. The simplest was 6b where two
N-methyl peaks 5d 4.17, 4.38 ppm) and two sets of ethyl
signals in a 2:1 ratio could be distinguished. The explanation
is probably that 6 exist as a mixture of isomers, the form
drawn and some tautomeric form involving relocation of
H-5 5there is more than one possibility). Other results
support the lability of H-5; the hydroxylation of this position
58b) has been referred to, while bromination of 6b gave 9b.
In contrast to 6, these derivatives gave NMR spectra com-
patible with single species and standard DEPT and
HETCOR NMR experiments showed that C-5 had no
Scheme 1. 5i) NH2NH2/EtOH/re¯ux, 5ii) NH2NH31/NaOAc/EtOH/re¯ux,
5iii) 1,4-benzoquinone/dioxane/re¯ux, 5iv) Pd/C/Ph2O/N2/re¯ux.
1
hydrogen attached. The H NMR spectrum of 6a from the
one-step reaction was very complex and this crude sample
was used in the next aromatization step.
Initially, the dehydrative cyclization was attempted with
polyphosphoric acid but complex product mixtures were
the result. Various attempts at other acid catalyzed
cyclizations were also unsuccessful for the parent 5a, but
re¯ux of the substituted hydrazones in toluene containing
We have reported previously6 that palladium/charcoal in
boiling diphenyl ether was successful in dehydrogenation
Scheme 2. 5i) RNHNH2/EtOH/re¯ux, 5ii) CF3CO2H/toluene/N2/re¯ux, 5iii) CF3CO2H/toluene/re¯ux, 5iv) RNHNH31/NaOAc/EtOH/re¯ux, 5v) 1,4-benzo-
quinone/dioxane/re¯ux, 5vi) Br2/HOAc, 5vii) NaOH/H2O/MeOH, 5viii) CDI/dioxane, then NH2 5CH2)2NMe2.

X. Bu et al. / Tetrahedron 58 42002) 175±181
177
of 4 to 1-hydroxyacridones. However, aromatization of 6
proved to be a frustrating exercise.
of successful dehydrogenation was the loss of the complex
aliphatic signal pattern and appearance of the up®eld
aromatic doublet at d c 6.6 ppm for H-3. The stable 11c is
interesting since a des-ethoxycarbonyl analogue 5prepared
by a different route) was reported to be quite unstable.8
When compounds 6 were subjected to the boiling diphenyl
ether conditions, the product NMR spectra from the
1-substituted examples b and c were complex and no
identi®able compounds were isolated. Reaction of 6a was
surprisingly successful, given the impure nature of the start-
ing sample, except that the ester function was also removed
and 10 resulted 5Scheme 1). This occurred within 10min at
2608C and also in 2 h at 2008C. Unchanged 6a was present
after shorter reaction times at both temperatures and in
neither case was there any evidence for the target 11a.
Structure 10 is the preferred form, on the basis that the
Alkaline hydrolysis of esters 11 gave the intermediate acids
12, and these were converted to the carboxamides 13 by
reaction with 1,10-carbonyldiimidazole to generate an inter-
mediate imidazolide in situ, followed by amination with
N,N-dimethylethylenediamine 5Scheme 2).
In an attempt to extend the synthesis to arylhydrazines, 4
was reacted with 4-methoxyphenylhydrazine by the pro-
cedure described above for condensation/cyclization.
However, unlike with the alkylhydrazines, this reaction
only went as far as the hydrazone 5d 5Scheme 3) 5which
was better prepared as for the other 5). When subjected to
the TFA/toluene cyclization conditions, a preferential
Fischer indole synthesis occurred instead and 5d gave the
novel indolo[2,3-a]acridine system 14 in 78% yield. This
ester was converted by way of acid 15 to carboxamide 16 as
described earlier for 11 to 13.
1
two NH peaks in the H NMR spectrum had substantially
different chemical shifts, and follows from the structure for
1 proposed from a crystal structure determination.1 Much
effort was spent on variations to the above Pd/C conditions,
but with limited success, and quinone aromatization of 6
was then investigated as an alternative.
There are few references to the use of, for example,
dichlorodicyanobenzoquinone in the aromatization of
hydrazone related species.7 With the present compounds,
this reagent appeared to be too powerful and gave complex
unidenti®ed mixtures. However, the less reactive 1,4-benzo-
quinone ultimately gave general success and in boiling
dioxane did produce the target 11. The parent 11a 5Scheme
1) was quite readily isolated; the yield was only 29% but,
again, this was reasonable considering that 6a was impure.
However, 11a was dif®cult to work with, being insoluble in
most solvents, and was not completely puri®ed. It also
proved to be very unstable in base conditions and further
chemistry was abandoned. The N-substituted analogues
were more tractable 5Scheme 2); the methyl compound
511b) 544%), and the aminoalkyl compound 511c) 557%)
were each obtained after chromatography. A characteristic
2.2. Cytotoxicity results
Compounds 11c, 13b, 13c and 16 were evaluated for growth
inhibitory properties 5measured as IC50 values) in a series of
cell lines 5Table 1). P388 is a murine leukemia, LLTC is a
late-passage murine Lewis lung carcinoma, and the Jurkat
lines are human leukemias.5,9 JLC is the wild-type
5sensitive) line, while JLA is 85-fold resistant to the topo-
isomerase II poison amsacrine and similar agents by virtue
of a reduced level of topo II enzyme, and JLD is a
doxorubicin-resistant line, primarily by virtue of altered
levels of topo II, but probably also by additional
mechanisms.10,11 IC50 values are given for the P388,
LLTC and JLC lines, together with ratios of IC50 values
against JLC and the other two Jurkat lines 5JLAJLC and
JLD/JLC). Values of these ratios of less than about 2-fold
suggest a non-topo II mediated mechanism of action.
The three pyrazolo[3,4,5-kl]acridine derivatives 511c, 13b,
13c) showed signi®cant activity across the various cell lines.
Table 1. Growth inhibitory properties
Cpd
IC50 5nM)a
IC50 ratiob
P388c
LLTCd
JLC
JLAf/JLC
JLDg/JLC
e
11c
13b
13c
16
14
1700
170
2100
98
181
254
26
980
189
357
415
48
1190
5801.9
0.6
0.8
0.7
0.7
0.8
0.9
0.9
1.3
2h
2.3
a
IC50; concentration of drug 5nM) to reduce cell number to 50% of control
cultures 5see text). The value is the average of at least two independent
determinations; the coef®cient of variation was 13±18%.
Ratios of IC50s in the cell lines shown.
Murine P388 leukemia.
b
c
d
e
f
Murine Lewis lung carcinoma.
JLC: wild-type human Jurkat leukemia.
JLA: amsacrine-resistant Jurkat.
JLD: doxorubicin-resistant Jurkat.
g
h
Scheme 3. 5i) 4-MeOC6H4NHNH2/EtOH/re¯ux, 5ii) CF3CO2H/toluene/N2/
re¯ux, 5iii) NaOH/H2O/MeOH, 5iv) CDI/dioxane, then NH25CH2)2NMe2.
Data from Ref. 9.

178
X. Bu et al. / Tetrahedron 58 42002) 175±181
Analogues containing only the carboxamide 511c) or only
the N-1 sidechain amine 513b) had similar cytotoxicity to
DACA 52) while 13c, which contained both functions and
therefore combined structural features of 1 and 2, had
generally enhanced activity. The novel 16 was clearly
much less active in all lines.
Jˆ7.4 Hz, 1H), 7.58 5t, Jˆ7.8 Hz, 1H), 7.83 5d, Jˆ8.4 Hz,
1 H), 8.19 5d, Jˆ8.2 Hz, 1H), 14.67 5br s, NH). 13C NMR d
14.1 5CH3), 21.6 5CH2), 23.5 5CH2), 38.2 5CH), 48.4
5NCH3), 61.05OCH ), 106.8 5C), 120.9 5C), 122.6 5CH),
2
125.0 5CH), 128.2 5CH), 130.2 5CH), 147.5 5C), 150.0
5C), 155.1 5C), 163.1 5C), 172.3 5C). ESMS: m/z 314
5M11).
3.2.3. Ethyl 1-.2-.dimethylamino)ethyl)hydrazono-9-
oxo-1,2,3,4,9,10-hexahydroacridine-4-carboxylate .5c).
Reaction of 4 with 2-5dimethylamino)ethylhydrazine12
was for 2 h. The crude oil 595%) was used in this state in
further reaction. 1H NMR d 1.07 5t, Jˆ7.1 Hz, CH3), 2.12 5s,
N5CH3)2), 2.24±2.53 5m, 6H), 3.18 5br s, NCH2), 3.95 5t,
Jˆ4.9 Hz, H-4), 4.03 5q, Jˆ7.1 Hz, OCH2), 5.31 5br s, NH),
7.27 5t, Jˆ7.2 Hz, 1H), 7.46 5t, Jˆ7.0Hz, 1H), 7.73 5d,
Jˆ8.5 Hz, 1H), 8.09 5d, Jˆ7.9 Hz, 1H). 13C NMR d 13.8
3. Experimental
3.1. General
NMR spectra were recorded, in CDCl3 unless stated other-
wise, on a Bruker AM-300 spectrometer operating at
300.13 MHz 51H) and 75.47 MHz 513C). Various standard
techniques were used to identify proton-bound carbons in
13C NMR spectra. The electrospray mass spectra were
obtained on a VG Bio-Q triple quadrupole mass spectro-
meter using a water/methanol/acetic acid 550:50:1) mobile
phase. LSI 53-nitrobenzyl alcohol as liquid matrix) mode
high-resolution mass spectra 5obtained by Dr N. Davies,
University of Tasmania) or microanalyses 5Campbell
Microanalytical Laboratory, University of Otago, New
Zealand) are reported for the compounds submitted for
biological testing and for representative precursors. Electro-
spray mass spectra, and NMR spectra indicative of homo-
geneous samples were the criteria used for analogues.
5CH3), 21.5 5CH2), 23.2 5CH2), 45.1 52£CH3), 48.05CH ),
2
48.2 5CH), 57.3 5CH2), 60.6 5CH2), 106.6 5C), 120.6 5C),
122.3 5CH), 124.6 5CH), 128.05CH), 129.8 5CH), 147.2 5C),
150.1 5C), 154.9 5C), 162.8 5C), 171.9 5C). ESMS: m/z 371
5M11).
3.3. Pyrazole ring formation
3.3.1. Ethyl 1-methyl-1,3,4,5-tetrahydropyrazolo[3,4,5-
kl]acridine-5-carboxylate .6b). Method A. Tri¯uoroacetic
acid 5100 mg) was added to a solution of hydrazone 5b
50.31 g, 1 mmol) in toluene 570 mL). The apparatus was
¯ushed with nitrogen and the mixture was re¯uxed under
Dean±Stark conditions for 16 h, also under nitrogen. The
cooled solution was washed with water 530mL £2), dried
5MgSO4), and the toluene was removed under reduced pres-
sure. The residue was puri®ed by column chromatography
5silica; ethyl acetate) to give an inseparable mixture of
3.2. Reaction of acridinediones with hydrazines in
ethanol
A solution of acridinedione 46 50.25 g) and the appropriate
hydrazine 52 mol equiv.) in ethanol 510mL) was heated
under re¯ux for 2 h 5except 5a, 1 h), with stirring, and
then evaporated to dryness under reduced pressure. The
residue was dissolved in chloroform, washed with water,
and the chloroform was evaporated. The product in this
form was generally suf®ciently pure for further reaction.
Representative examples were puri®ed further and identi-
®ed. In this way the following compounds were prepared:
1
isomers 5c 2:1) 50.2 g, 68%) as an oil, Rf 0.51. H NMR d
1.22 5t, Jˆ6.0Hz, CH 3), 1.31 5t, Jˆ6.0Hz, CH 3), 2.3±3.3
5multiple complex signals), 4.1±4.25 5m, OCH2£2), 4.17 5s,
NCH3), 4.38 5s, NCH3), 7.03±7.12 5m), 7.24±7.32 5m), 7.54
5t, Jˆ7.9 Hz), 7.64±7.71 5m), 8.21 5d, Jˆ8.4 Hz). ESMS:
m/z 296 5M11). This sample was used directly in the next
reaction.
3.2.1. Ethyl 1-hydrazono-9-oxo-1,2,3,4,9,10-hexahydro-
acridine-4-carboxylate .5a). The crude solid 594%) was
recrystallized from ethanol to give the product as a yellow
The chromatography also gave a 9% yield of 1-methyl-
1,3,4,5-tetrahydropyrazolo[3,4,5-kl]acridine 57b) as an
1
solid, mp 254±2568C. H NMR d 1.23 5t, Jˆ7.1 Hz, 3H,
1
oil, Rf 0.14. H NMR d 2.305m, Jˆ6.1 Hz, CH2), 3.01 5t,
CH3), 2.20±2.74 5m, 4H, 2£CH2), 4.11 5t, Jˆ4.7 Hz, H-4),
4.17 5q, Jˆ7.1 Hz, 2H, OCH2), 5.23 5br s, 3H, NH1NH2),
7.43 5t, Jˆ7.3 Hz, 1H), 7.63 5t, Jˆ8.1 Hz, 1H), 7.87 5d,
Jˆ8.6 Hz, 1H), 8.24 5d, Jˆ8.6 Hz, 1H), 14.58 5s, 1H, NH-
10). 13C NMR d 14.2 5CH3), 21.1 5CH2), 23.5 5CH2), 48.4
5CH), 61.2 5CH2), 106.7 5C), 120.6 5C), 122.8 5CH), 125.1
5CH), 128.4 5CH), 130.5 5CH), 147.8 5C), 153.5 5C), 155.3
5C), 163.4 5C), 172.3 5C). ESMS: m/z 300 5M11). Anal.
calcd for C16H17N3O3: C, 64.2; H, 5.7; N, 14.0. Found: C,
64.0; H, 5.6; N, 13.9%.
Jˆ6.1 Hz, CH2), 3.08 5t, Jˆ6.1 Hz, CH2), 4.38 5s, NCH3),
7.53 5t, Jˆ7.4 Hz, 1H), 7.66 5t, Jˆ7.7 Hz, 1H), 8.16 5d,
Jˆ8.4 Hz, 1H), 8.205d, Jˆ8.1 Hz, 1H). 13C NMR d 22.7
5CH2), 25.05CH 2), 30.4 5CH2), 38.9 5CH3), 116.7 5C), 117.0
5C), 121.6 5CH), 125.4 5CH), 128.5 5CH), 130.0 5CH), 137.7
5C), 147.3 5C), 147.6 5C), 158.5 5C). ESMS: m/z 224
5M11).
When the above reaction was carried out for 20h without
the nitrogen atmosphere, chromatography of the crude
product 5silica; ethyl acetate/hexane, 4:1) gave a 59%
yield of ethyl 5-hydroxy-1-methyl-1,3,4,5-tetrahydropyra-
zolo[3,4,5-kl]acridine-5-carboxylate 58b) as an oil, Rf
3.2.2. Ethyl 1-methylhydrazono-9-oxo-1,2,3,4,9,10-hexa-
hydroacridine-4-carboxylate .5b). The crude material
585%) could be used in the next step, but a sample was
puri®ed by column chromatography 5silica; ethyl acetate)
to give an oil, Rf 0.51. 1H NMR d 1.19 5t, Jˆ7.1 Hz, CH3),
2.10±2.60 5m, 4H), 2.99 5s, NCH3), 4.05 5t, Jˆ4.7 Hz, H-4),
4.13 5q, Jˆ7.1 Hz, OCH2), 4.72 5br s, NH), 7.39 5t,
1
0.37. H NMR d 1.19 5t, Jˆ7.1 Hz, CH3), 2.48 5m, 1H,
H-4a), 2.69 5m, 1H, H-4b), 3.23 5m, 2H, H-3), 4.17±4.30
5m, OCH2), 4.405s, NCH 3), 4.53 5br s, OH), 7.57 5t,
Jˆ7.9 Hz, 1H), 7.65 5t, Jˆ7.2 Hz, 1H), 8.205d, Jˆ7.9 Hz,

X. Bu et al. / Tetrahedron 58 42002) 175±181
179
1H), 8.23 5d, Jˆ7.8 Hz, 1H). 13C NMR d 14.1 5CH3), 20.3
as 9b. 1H NMR d 1.28 5t, Jˆ7.1 Hz, CH3), 2.76±2.82 5m, 1
H, H-4a), 3.05±3.13 5m, 1 H, H-4b), 3.22 5m, 2 H, H-3),
4.33 5q, Jˆ7.1 Hz, OCH2), 4.38 5s, NCH3), 7.49 5t,
Jˆ8.1 Hz, 1H), 7.63 5t, Jˆ8.3 Hz, 1H), 8.13 5d, Jˆ7.9 Hz,
5CH2), 36.2 5CH2), 39.05NCH ), 62.3 5OCH2), 74.9 5C,
3
C-5), 116.05C), 117.3 5C), 121.6 5CH), 126.3 5CH), 128.5
5CH), 131.05CH), 138.3 5C), 146.2 5C), 146.9 5C), 155.5
5C), 173.4 5C). ESMS: m/z 312 5M11).
1H), 8.22 5d, Jˆ8.4 Hz, 1H). 13C NMR d 14.05CH ), 21.5
3
5CH2), 39.05NCH ), 39.5 5CH2), 61.1 5C), 63.1 5OCH2),
3
Method B. A mixture of dione 4 50.57 g, 2.0 mmol), methyl
hydrazine sulfate 50.57 g, 4.0 mmol) and sodium acetate
52.0g) in ethanol 550mL) was heated under re¯ux for
24 h. The ethanol was removed under reduced pressure
and the residue was extracted with chloroform 530mL £2).
The combined extracts were washed with water, dried
5Na2SO4).and the solvent was removed to give the crude
6b as an orange oil 50.59 g, 98%), which was suf®ciently
pure for use in the next step.
114.3 5C), 117.1 5C), 121.3 5CH), 126.6 5CH), 128.5
5CH), 131.3 5CH), 138.5 5C), 144.5 5C), 146.9 5C), 153.8
5C), 168.4 5C). ESMS: m/z 374 5100%), 375 520), 376 5100),
377 522), all 5M11) for C17H16BrN3O2.
3.5. Aromatization
3.5.1. 2,6-Dihydropyrazolo[3,4,5-kl]acridine .10).
A
mixture of crude 6a 50.1 g) and 10% Pd/C 50.05 g) in
diphenyl ether 55 mL) was re¯uxed for 10min, then passed
through a Celite bed and hexane was added to the ®ltrate.
The solid which separated 50.06 g) was ®ltered off and
recrystallized from toluene to give the product as a pale
3.3.2. Ethyl 1-.2-.dimethylamino)ethyl)-1,3,4,5-tetra-
hydropyrazolo[3,4,5-kl]-acridine-5-carboxylate
.6c).
From 5c by method A. Reaction was for 16 h and the
crude product was puri®ed by column chromatography
5silica; chloroform/methanol, 4:1) to give a mixture of
1
yellow solid 50.04 g), mp.3008C. H NMR 5DMSO-d6) d
6.02 5d, Jˆ7.2 Hz, 1 H), 6.49 5d, Jˆ8.2 Hz, 1H), 6.93 5t,
Jˆ7.5 Hz, 1H), 7.00±7.05 5m, 2H), 7.22 5t, Jˆ7.3 Hz, 1H),
7.67 5d, Jˆ7.9 Hz, 1H), 9.86 5s, NH), 12.2 5s, NH). 13C
NMR 5DMSO-d6) d 95.7 5CH), 97.3 5CH), 116.1 5CH),
116.9 5C), 117.4 5C), 120.2 5CH), 122.5 5CH), 128.7
5CH), 129.9 5CH), 137.3 5C), 141.1 5C), 141.2 5C), 141.9
5C). ESMS: m/z 208 5M11). Anal. calcd for
C13H9N3´0.25H2O: C 73.8, H, 4.5, N 19.9. Found: C 73.9;
H 4.5; N 19.8%.
1
isomers 5c 3:1, 53%) as an oil, Rf 0.65. H NMR d 1.2 5t,
Jˆ6.0Hz, CH 3), 1.3 5t, Jˆ6.0Hz, CH 2), 2.33 5s, N5CH3) 2),
2.36 5s, N5CH3) 2), 2.4±3.2 5multiple complex signals), 4.1±
4.25 5m, OCH2£2), 4.6 5t, Jˆ7.4 Hz, NCH2), 4.8 5t,
Jˆ7.7 Hz, NCH2), 7.1±8.22 5m£5). ESMS: m/z 353 5M11).
The chromatography also gave 8% of 1-42-4dimethyl-
amino)ethyl)-1,3,4,5-tetrahydropyrazolo[3,4,5-kl]acridine
57c) as an oil, Rf 0.46. H NMR d 2.32 5m, CH2), 2.37 5s,
1
N5CH3) ), 2.91 5t, Jˆ7.6 Hz, CH2), 3.05 5t, Jˆ6.3 Hz,
2
3.5.2. Ethyl 1,2-dihydropyrazolo[3,4,5-kl]acridine-5-
carboxylate .11a). A mixture of crude 6a 50.28 g) and
1,4-benzoquinone 50.43 g) in 1,4-dioxan 520 mL) was
heated under re¯ux for 2 h. The solvent was evaporated
and the residue was extracted with hot ethanol 52£15 mL),
then with hot ethyl acetate 52£15 mL). The insoluble
material was extracted into chloroform 580mL) and the
solvent was evaporated to give the product 50.08 g) as a
dark brown solid, which still contained minor impurities.
1H NMR d 1.38 5t, Jˆ7.2 Hz, CH3), 4.36 5q, Jˆ7.2 Hz,
OCH2), 6.63 5d, Jˆ9.0Hz, H-3), 7.30 5t, Jˆ7.5 Hz, 1H),
7.39 5d, Jˆ8.3 Hz, 1H), 7.66 5t, Jˆ7.3 Hz, 1H), 8.12 5d,
Jˆ9.0Hz, H-4), 8.38 5d, Jˆ7.3 Hz, 1H), 12.32 5s, NH),
12.51 5br s, NH).
CH2), 3.11 5t, Jˆ6.3 Hz, CH2), 4.83 5t, Jˆ7.6 Hz, CH2),
7.58 5t, Jˆ7.0Hz, 1H), 7.70 5t, Jˆ8.4 Hz, 1H), 8.18 5d,
Jˆ8.6 Hz, 1H), 8.21 5d, Jˆ7.7 Hz, 1H). 13C NMR d 22.8
5CH2), 24.9 5CH2), 30.4 5CH2), 45.8 5CH3), 50.1 5CH2), 58.8
5CH2), 116.8 52£C), 121.6 5CH), 125.6 5CH), 128.5 5CH),
130.2 5CH), 137.4 5C), 147.8 5C), 148.0 5C), 158.6 5C).
ESMS: m/z 281 5M11).
3.3.3. Ethyl 1,3,4,5-tetrahydropyrazolo[3,4,5-kl]acri-
dine-5-carboxylate .6a). Method B. Reaction was as for
6b. After the ethanol was removed the residue was boiled
with water 550mL), cooled to room temperature and the
solid was collected by ®ltration, washed with water and
dried. The yellow solid 50.30 g from 0.57 g 4), mp 248±
1
2498C, had a very complex H NMR spectrum. ESMS m/z
282 578%; M11, 6a), 300 530%; M11, 5a), 354 535%;
unknown), 595 530%, unknown).
3.5.3. Ethyl 1-methyl-1,2-dihydropyrazolo[3,4,5-kl]acri-
dine-5-carboxylate .11b). A mixture of 6b 50.30 g,
1.0mmol) and 1,4-benzoquinone 50.43 g, 4.0mmol) in
dioxane 530mL) was heated under re¯ux for 16 h. The
solvent was removed under reduced pressure and the residue
was extracted with chloroform 53£20mL). The combined
organic extracts were washed with 2% sodium hydroxide
52£15 mL), water, dried, and the chloroform was removed.
Chromatography of the residue 50.21 g) 5alumina; ethyl
acetate containing 2% diethylamine) gave the the product
50.13 g, 44%) as a dull yellow, blue-tinged solid, Rf 0.7, mp
3.4. Bromination
3.4.1. Ethyl 5-Bromo-1-methyl-1,3,4,5-tetrahydropyra-
zolo[3,4,5-kl]acridine-5-carboxylate .9b). A solution of
bromine in acetic acid 53 mL£0.1 M, 0.3 mmol) was
added dropwise, with stirring, to a solution of 6b 590mg,
0.3 mmol) in acetic acid 55 mL). The solution became
green. After the addition was complete, water 525 mL)
was added immediately and the mixture was extracted
with chloroform 520mL £3). The combined chloroform
extracts were washed with saturated sodium bicarbonate
510mL), water 515 mL £2), dried 5MgSO4) and the chloro-
form was removed under reduced pressure to give an oil
580mg), the predominant component of which was assigned
1
169±1718C. H NMR d 1.39 5t, Jˆ7.1 Hz, CH3), 4.28 5s,
NCH3), 4.32 5q, Jˆ7.1 Hz, OCH2), 6.61 5d, Jˆ9.2 Hz, H-3),
7.08±7.14 5m, 2H), 7.33 5t, Jˆ8.4 Hz, 1H), 7.52 5d,
Jˆ9.2 Hz, H-4), 7.71 5d, Jˆ7.9 Hz, 1 H), 10.10 5br s,
NH). 13C NMR d 14.5 5CH3), 39.3 5NCH3), 59.8 5OCH2),
94.6 5C), 104.05CH), 114.7 5C), 118.05CH), 122.4 5CH),
122.5 5CH), 129.6 5CH), 129.8 5CH), 134.05C), 139.1

180
X. Bu et al. / Tetrahedron 58 42002) 175±181
52£C), 141.8 5C), 148.5 5C), 168.3 5C). ESMS: m/z 294
5M11). Anal. calcd for C17H15N3O2: C, 69.6; H, 5.1; N,
14.3. Found: C, 69.7; H, 5.2; N, 14.5%.
water and dried to give the intermediate acid 15 as a yellow
solid 50.18 g, 97%), mp.3008C. 5some sublimes .2548C).
This acid 50.18 g, 0.50 mmol), with 1,10-carbonyldiimida-
zole 50.12 g, 0.75 mmol) in dioxane 530 mL) was re¯uxed
with stirring for 6 h. N,N-Dimethylethylenediamine 570mg,
0.75 mmol) was added and the solution was re¯uxed for
24 h. The solvent was removed at reduced pressure and
the residue was dissolved in chloroform, washed with
10% sodium carbonate, then with warm water, and dried
5MgSO4). Evaporation of the solvent gave the amide as a
3.5.4. Ethyl 1-.2-.dimethylamino)ethyl)-1,2-dihydro-
pyrazolo[3,4,5-kl]acridine-5-carboxylate .11c). This was
prepared from 6c, as for 11b, and obtained after column
chromatography 5alumina; ethyl acetate), Rf 0.63, as a
bluish yellow oil 557%) which gradually solidi®ed on stand-
ing, mp 92±938C. 1H NMR d 1.39 5t, Jˆ6.9 Hz, CH3), 2.34
5s, N5CH3)2), 2.89 5t, Jˆ7.6 Hz, CH2), 4.33 5q, Jˆ7.0Hz,
OCH2), 4.705t, Jˆ7.5 Hz, CH2), 6.64 5d, Jˆ9.2 Hz, 1H),
7.12±7.21 5m, 2H), 7.36 5t, Jˆ7.5 Hz, 1H), 7.55 5d,
Jˆ9.2 Hz, 1H), 7.75 5d, Jˆ7.9 Hz, 1H), 10.23 5s, NH).
13C NMR d 14.53 5CH3), 45.8 5N5CH3)2), 50.7 5CH2),
58.6 5CH2), 59.8 5CH2), 94.5 5C), 104.2 5CH), 114.6 5C),
118.05C), 118.2 5CH), 122.5 5CH), 122.7 5CH),129.6 5CH),
129.8 5CH), 133.7 5C), 139.2 5C), 142.05C), 148.8 5C),
168.3 5C). LSIMS: Found 351.1825 5M1H1). C20H23N4O2
requires 351.1816.
yellow solid 50.19 g, 88%), mp 239±2408C 5from aceto-
1
nitrile). H NMR d 2.42 5s, N5CH3) ), 2.76 5t, Jˆ5.7 Hz,
2
CH2), 3.705q, Jˆ5.7 Hz, CH2), 3.83 5s, OCH3), 7.01 5dd,
Jˆ8.7, 2.3 Hz), 7.26 5s, CONH), 7.30±7.37 5m, 2H), 7.42±
7.46 5m, 2 H), 7.65 5t, Jˆ7.2 Hz, 1H), 8.42 5s), 8.48 5d,
Jˆ7.9 Hz, 1H), 11.38 5s, NH), 12.76 5s, NH). 13C NMR d
37.4 5CH2), 45.5 5N5CH3)2), 55.8 5OCH3), 58.05CH 2), 102.1
5CH), 108.1 5C), 112.3 5CH), 114.1 5CH), 114.4 5C), 118.1
5CH), 121.5 5C), 121.9 5CH), 123.1 5C), 125.3 5CH), 125.7
5CH), 133.05CH), 134.05C), 139.5 5C), 140.6 5C), 141.3
5C), 154.8 5C), 169.6 5C), 178.9 5C). ESMS: m/z 429
5M11). Anal. calcd for C25H24N4O3´H2O: C 67.3; H 5.9;
N 12.6. Found: C 67.6; H 5.7; N 12.2%.
3.6. Indoloacridine formation
3.6.1. Ethyl 4-methoxy-13-oxo-8,13-dihydro-1H-indolo-
[2,3-a]acridine-7-carboxylate .14).
A mixture of 4
50.18 g, 0.63 mmol) and 4-methoxyphenylhydrazine hydro-
chloride 50.13 g, 0.74 mmol) in ethanol 515 mL) was heated
under re¯ux for 16 h, then cooled on ice. The yellow hydra-
zone 5d which separated 50.21 g, 82%) was ®ltered off and
washed with a little ethanol. 1H NMR d 1.22 5t, Jˆ7.0Hz,
3.7.2. N-.2-.Dimethylamino)ethyl)-1-methyl-1,2-dihydro-
pyrazolo[3,4,5-kl]acridine-5-carboxamide
.13b).
A
mixture of ester 11b 50.18 g, 0.61 mmol), 10% NaOH
510mL) and methanol 55 mL) was heated under re¯ux for
1 h. After being cooled, most of the methanol was removed
at reduced pressure, the remainder was ®ltered and the
®ltrate was taken to pH 2 with concentrated HCl. The pre-
cipitate which separated was ®ltered off, washed with water
and dried to give the intermediate acid 12b as a yellow solid
50.14 g, 86%).
CH3), 2.1±3.05m, 4H), 3.77 5s, OCH ), 4.16 5q, Jˆ7.0Hz,
3
CH2), 4.35 5br t, 1H), 6.87 5d, Jˆ8.5 Hz, 2H), 7.04 5d,
Jˆ8.5 Hz, 2H), 7.45 5t, Jˆ7.3 Hz, 1H), 7.65 5t, Jˆ7.6 Hz,
1H), 8.01 5d, Jˆ8.4 Hz, 1H), 8.26 5d, Jˆ8.2 Hz, 1H).
ESMS: m/z 406 5M11).
This was dissolved in toluene 515 mL), tri¯uoroacetic acid
540mg) was added and the mixture was heated under re¯ux
for 16 h, then evaporated to dryness at reduced pressure to
give the product as a yellow solid 50.19 g, 78%), mp 2728C
5from ethanol). 1H NMR 5assigned from additional HMQC,
HMBC and 1H±1H COSY experiments) d 1.53 5t,
Jˆ7.1 Hz, CH3), 3.94 5s, OCH3), 4.505q, Jˆ7.1 Hz CH2),
7.07 5dd, Jˆ8.6, 2.3 Hz, H-3), 7.35 5t, Jˆ7.5 Hz, H-11),
7.47 5d, Jˆ8.7 Hz, H-2), 7.49 5d, Jˆ8.0Hz, H-9), 7.54 5d,
Jˆ2.0Hz, H-5), 7.70 5t, Jˆ7.6 Hz, H-10), 8.48 5d,
Jˆ8.0Hz, H-12), 9.02 5s, H-6), 11.46 5s, NH), 12.45 5s,
Amidation as for the preparation of 16 gave the crude
product 579%). A sample was puri®ed by column chromato-
graphy 5alumina; ethyl acetate with 4% diethylamine)
followed by recrystallization from benzene/light petroleum
5bp 60±908C) to give a pale yellow solid, mp 157±1598C,
which gained a blue tinge as it gradually absorbed water. 1H
NMR d 2.34 5s, N5CH3)2), 2.61 5t, Jˆ5.7 Hz, CH2), 3.53 5q,
Jˆ5.7 Hz, CH2), 4.31 5s, NCH3), 6.65 5d, Jˆ9.0Hz, 1H),
6.78 5br t, NH), 7.08 5t, Jˆ7.6 Hz, 1H), 7.13±7.205m, 3H),
7.31 5t, Jˆ7.4 Hz, 1H), 7.73 5d, Jˆ7.9 Hz, 1H), 10.92 5s,
NH). 13C NMR d 36.3 5CH2), 39.4 5CH3), 45.052 £CH3),
58.1 5CH2), 96.7 5C), 103.8 5CH), 114.7 5C), 118.0
5CH), 118.5 5C), 122.05CH), 122.3 5CH), 126.7 5CH),
129.8 5CH), 134.1 5C), 139.6 5C), 141.05C), 148.05C),
166.9 5C). ESMS: m/z 3365M11). Anal. calcd for
C19H21N5O´0.5H2O: C, 66.3; H, 6.4; N, 20.3. Found: C,
66.0; H, 6.4; N, 20.0%.
NH). 13C NMR d 14.5 5CH3), 56.05CH ), 61.2 5CH2),
3
102.7 5CH), 104.0 5C), 106.8 5C), 112.5 5CH), 114.7
5CH), 115.3 5C), 117.9 5CH), 121.8 5C), 122.4 5CH),
123.6 5C), 126.05CH), 13.02 5CH), 133.3 5CH), 134.2
5C), 139.4 5C), 141.6 5C), 143.05C), 155.2 5C), 168.9 5C),
178.9 5C). Anal. calcd for C23H18N2O4: C 71.5; H 4.7; N 7.3.
Found: C 71.3; H 4.6; N 7.1%.
3.7.3. N-.2-.Dimethylamino)ethyl)-1-.2-.dimethylamino)-
ethyl)-1,2-dihydropyrazolo[3,4,5-kl]acridine-5-carboxa-
mide .13c). To a solution of ester 11c 50.25 g, 0.71 mmol)
in methanol 510mL) was added 10% NaOH 58 mL) and the
mixture was re¯uxed with stirring for 2 h. Most of the
methanol was removed, water 510mL) was added and the
solution was washed with chloroform 52£10mL), then care-
fully acidi®ed with concentrated HCl to pH 3±4. The solu-
tion was saturated with potassium chloride and a blue solid
3.7. Preparation of amides
3.7.1. N-.2-.Dimethylamino)ethyl)-4-methoxy-13-oxo-8,13-
dihydro-1H-indole[2,3-a]acridine-7-carboxamide .16).
A mixture of ester 14 50.20 g, 0.52 mmol), methanol
58 mL) and 10% NaOH 510 mL) was heated under re¯ux
for 24 h, then cooled and acidi®ed with concentrated HCl.
The precipitate which formed was ®ltered off, washed with

X. Bu et al. / Tetrahedron 58 42002) 175±181
181
separated on standing. This was ®ltered off and dried over-
night in a vacuum oven at 508C to give the intermediate acid
12c 50.21 g, 91%), mp 1778C 5dec.). 1H NMR 5DMSO-d6) d
2.86 5s, N5CH3)2), 3.69 5t, Jˆ6.2 Hz, CH2), 5.13 5t,
Jˆ6.2 Hz, CH2), 6.58 5d, Jˆ9.1 Hz, 1H), 7.24 5t,
Jˆ7.7 Hz, 1H), 7.46±7.505m, 2H), 7.79 5d, Jˆ8.2 Hz,
1H), 8.12 5d, Jˆ7.9 Hz, 1H), 10.50 5s, NH), 10.83 5br s,
CO2H).
inhibition data given in Table 1. The work was supported
by an Australian Research Council Small Grant and X. B.
and J. C. are grateful for the award of La Trobe University
and Overseas Postgraduate Research Scholarships.
References
1. Capps, D. B.; Dunbar, J.; Kesten, S. R.; Shillis, J.; Werbel,
L. M.; Polwman, J.; Ward, D. L. J. Med. Chem. 1992, 35,
4770±4778.
Amidation as for the preparation of 16 gave the amide 13c
as a blue semi-solid 590%).1H NMR d 2.26 5s, N5CH3)2),
2.34 5s, N5CH3)2), 2.51 5t, Jˆ5.9 Hz, CH2), 2.87 5t,
Jˆ7.6 Hz, CH2), 3.47 5q, Jˆ5.4 Hz, CH2), 4.69 5t,
Jˆ7.6 Hz, CH2), 6.63±6.66 5m, 2H, H-3 and CONH),
7.06±7.17 5m, 3 H), 7.31 5t, Jˆ7.6 Hz, 1H), 7.71 5d,
Jˆ8.0Hz, 1H), 10.99 5s, NH). 13C NMR d 36.6 5CH2),
45.2 5N5CH3)2), 45.8 5N5CH3)2), 50.6 5CH2), 57.9 5CH2),
58.6 5CH2), 96.7 5C), 103.8 5CH), 114.4 5C), 118.1 5CH),
118.5 5C), 122.1 5CH), 122.3 5CH), 126.7 5CH), 129.8 5CH),
133.8 5C), 139.6 5C), 141.15C), 148.2 5C), 168.9 5C).
LSIMS: Found 393.2399 5M1H1). C22H29N6O requires
393.2397.
2. Adjei, A. A. Invest. New Drugs 1999, 17, 43±48.
3. Atwell, G. J.; Rewcastle, G. W.; Baguley, B. C.; Denny, W. A.
J. Med. Chem. 1987, 30, 664±669.
4. McCrystal, M. R.; Evans, B. D.; Harvey, V. J.; Thompson,
P. I.; Porter, D. J.; Baguley, B. C. Cancer Chemother.
Pharmacol. 1999, 44, 39±44.
5. Deady, L. W.; Kaye, A. J.; Finlay, G. J.; Baguley, B. C.;
Denny, W. A. J. Med. Chem. 1997, 40, 2040±2046.
6. Chen, J.-J.; Deady, L. W. Synth. Commun. 1997, 27, 95±106.
7. Turner, A. B. Synthetic Reagents; Pizey, J. S., Ed.; Ellis
Horwood: Chichester, 1977; Vol. 3 Chapter 2.
8. Cholody, W. M.; Lhomme, M. F.; Lhomme, J. Tetrahedron
Lett. 1987, 28, 5029±5032.
3.8. In vitro growth delay assays
9. Spicer, J. A.; Gamage, S. A.; Atwell, G. J.; Finlay, G. J.;
Baguley, B. C.; Denny, W. A. J. Med. Chem. 1997, 40,
1919±1929.
These were carried out as reported previously.10,11 Inde-
pendent assays were performed in duplicate, and
coef®cients of variation were from 13 to 18%.
10. Finlay, G. J.; Baguley, B. C.; Snow, K.; Judd, W. J. Natl.
Cancer Inst. 1990, 82, 662±667.
11. Finlay, G. J.; Holdaway, K. M.; Baguley, B. C. Oncol. Res.
1994, 6, 33±37.
Acknowledgements
12. Elslager, E. F.; Weinstein, E. A.; Worth, D. F. J. Med. Chem.
1964, 7, 493±500.
We thank Mr I. Thomas for recording the electrospray mass
spectra and Dr G. Finlay for the tumour cell growth