Synthesis and antitumor properties of N-[2- (dimethylamino)ethyl]carboxamide derivatives of fused tetracyclic quinolines and quinoxalines: A new class of putative topoisomerase inhibitors

Article abstract of DOI:10.1021/jm970044r

A series of tetracyclic quinoline- and quinoxalinecarboxamides were prepared, and their cytotoxicities were evaluated in a series of murine human tumor cell lines. Most of the quinoline derivatives were prepared by an adaptation of the Pfitzinger synthesis, followed by thermal decarboxylation and coupling with N,N-dimethylethylenediamine via a mixed anhydride method using isobutyl chloroformate. The quinoline analogues showed cytotoxicities broadly similar to those of the known tricyclic acridine-4-carboxamide mixed topoI/II inhibitor DACA, with thieno and indeno analogues being the most active. They showed little decrease in potencies against the Jurkat human leukemia topo II-resistant lines JL(A) and JL(C), suggesting their cytotoxicity does not result primarily from inhibition of topo II. The quinoxaline analogues had more varied IC₅₀ values, being on average less cytotoxic than the quinoline derivatives, but appeared to have a similar mode of action. Overall, this new class of compounds appear to be mixed topo I/II inhibitors, up to 3-fold more cytotoxic than DACA in the human leukemia cell lines studied, with in vivo activity in colon 38 comparable to that of DACA and doxorubicin.

Full text of DOI:10.1021/jm970044r

2040
J . Med. Chem. 1997, 40, 2040-2046
Syn th esis a n d An titu m or P r op er ties of N-[2-(Dim eth yla m in o)eth yl]ca r boxa m id e
Der iva tives of F u sed Tetr a cyclic Qu in olin es a n d Qu in oxa lin es: A New Cla ss of
P u ta tive Top oisom er a se In h ibitor s
Leslie W. Deady,*,† Anthony J . Kaye,† Graeme J . Finlay,‡ Bruce C. Baguley,‡ and William A. Denny‡
School of Chemistry, La Trobe University, Bundoora, Victoria, Australia 3083, and Cancer Research Laboratory, Faculty of
Medicine and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand
Received J anuary 21, 1997X
A series of tetracyclic quinoline- and quinoxalinecarboxamides were prepared, and their
cytotoxicities were evaluated in a series of murine human tumor cell lines. Most of the quinoline
derivatives were prepared by an adaptation of the Pfitzinger synthesis, followed by thermal
decarboxylation and coupling with N,N-dimethylethylenediamine via a mixed anhydride method
using isobutyl chloroformate. The quinoline analogues showed cytotoxicities broadly similar
to those of the known tricyclic acridine-4-carboxamide mixed topoI/II inhibitor DACA, with
thieno and indeno analogues being the most active. They showed little decrease in potencies
against the J urkat human leukemia topo II-resistant lines J LA and J LC, suggesting their
cytotoxicity does not result primarily from inhibition of topo II. The quinoxaline analogues
had more varied IC50 values, being on average less cytotoxic than the quinoline derivatives,
but appeared to have a similar mode of action. Overall, this new class of compounds appear
to be mixed topo I/II inhibitors, up to 3-fold more cytotoxic than DACA in the human leukemia
cell lines studied, with in vivo activity in colon 38 comparable to that of DACA and doxorubicin.
Following the clinical success of the DNA-intercalat-
ing topo II inhibitors doxorubicin, mitoxantrone, and
analogues as anticancer drugs, a great deal of work has
been devoted toward other classes of compounds with
similar overall topology (polycyclic chromophores bear-
ing a flexible cationic side chain) as topo II inhibitors.
Among the more successful examples are the benzoiso-
quinolinediones such as mitonafide (1),1 the anthrapy-
razoles such as losoxantrone (2),2 and the phenazine-
1-carboxamides (e.g., 3).3 More recently, interest has
focused on compounds with the ability to inhibit both
topo I and topo II enzymes. Examples of such “mixed”
inhibitors that show broad-spectrum activity against
solid tumors and are in clinical trial include the acri-
dine-4-carboxamide DACA (4),4,5 the imidazoacridanone
(5),6 and various tetracyclic chromophores (e.g., 6).7
defined, and because of the potential utility of such
compounds, the discovery of further classes of such
agents remains an area of active interest. Within the
broad subclass of polycyclic heterocyclic carboxamides,
the nature and positioning of the carboxamide side
chain was shown to be critical, with attachment to a
terminal ring peri to an electron-withdrawing atom in
the central ring being required for biological activity;
the acridine-4-carboxamides (e.g., 4) and phenazine-1-
carboxamides (e.g., 3) are the most biologically active
of the subclass.8,9 Prompted by the above, and by recent
reports of the effectiveness of various tetracyclic com-
pounds as anticancer drugs,6,10,11 we report in this paper
the synthesis and evaluation of a series of tetracyclic
quinoline- and quinoxalinecarboxamides (7-23) as ana-
logues of the acridine- and phenazinecarboxamides,
respectively.
Ch em istr y
The basic strategy for synthesis of the quinoline-based
compounds (7-13 and 19) of Table 1 involved an
adaptation of the Pfitzinger synthesis, in which a
7-substituted isatin (24 or 25) was reacted with a ketone
to give the tetracycles 30-35 (Scheme 1). Most work
utilized isatin 24, with the final acid function already
in place. This was prepared by a modification of a
literature method,12 and reactions with 26-29 gave 30-
33. Two reactions in this series were also carried out
with 7-methylisatin (25),13 condensing this with 28 and
29 to give the tetracycles 34 and 35. Many variations
of base catalysis have been employed in the Pfitzinger
reaction.14 A number were tried in the present work,
and 10% aqueous sodium hydroxide at 90-100 °C15 was
found satisfactory for all except one reaction. For 26,
the literature procedure16 for a related compound (20%
potassium hydroxide at room temperature for 10 days
in the dark) proved superior, although the yield of 30
Because the relationships between structure and the
ability to inhibit topoisomerases are still not well
X Abstract published in Advance ACS Abstracts, J une 1, 1997.
S0022-2623(97)00044-7 CCC: $14.00 © 1997 American Chemical Society

Putative Topoisomerase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2041
Sch em e 1a
Sch em e 2a
a
(i) Aqueous NaOH/90 °C/45 min; (ii) 300 °C/5 min; (iii)
Na2Cr2O7/3 M H2SO4/reflux/3 h.
Sch em e 3
a
(i) Aqueous OH-/50-100 °C; (ii) heat (250-300 °C); (iii) CrO3/
H2SO4/AcOH/20 °C.
a
(i) PPA/110 °C/5 h.
was only 21% and was accompanied by much indigo
formation. Workup of the reactions was quite individual
with respect to the species (salt or free acid) which
separated at particular acidities (see the Experimental
Section for details). The high-melting acids were gener-
ally difficult to completely purify, but the final amides
were obtained clean (>96%) as judged by HPLC. How-
ever, a small number of these formed hydrated species
for which satisfactory analytical data could not be
obtained.
The initial condensation products were then decar-
boxylated to give compounds 36-43. Attention to detail
was required in the decarboxylation process in order to
obtain good yields. With one exception, the compounds
were heated approximately to their melting points while
being observed under mild magnification, and heating
was discontinued after a few minutes when the obvious
reaction had ceased. In this way, 30-33 and 35 gave
37-41. For 34, the solid phase decarboxylation was not
successful and reaction was better achieved in boiling
sulfolane over 30 min to give 36. Oxidation of the
methyl substituent in 36 and 37 with CrO3/H2SO4 was
accompanied by oxidation in the five-membered ring to
give the required monoacids 42 and 43.
There is no general method of synthesis of the
isomeric quinoline acids in which the orientation of the
five-membered ring is reversed. One member of this
series was prepared by the same reaction sequence as
above (Scheme 2). 7-Methylisatin (25) was reacted with
2-indanone (44) to give 45, which was again decarboxy-
lated in boiling sulfolane to give 46 and oxidized to the
oxo compound 47.
Synthesis of the new quinoxaline-based acids was by
condensation of 2,3-diaminobenzoic acid (48) (or the
5-chloro analogue) with the appropriate R,â-dicarbonyl
compound (see Scheme 3 for a representative example).
Details of the synthesis and assignment of the isomeric
acids of this series is reported elsewhere.17
line-based amides (14-18, 20-23) follow from those
determined for the acids.17 For those acids containing
an NH group in the five-membered ring, some reaction
also occurred at this nitrogen. The quinoline-based
compound 14 was initially formed by way of the
intermediate carbonyl chloride. However, this method
gave low yields of impure compounds with the quinoxa-
line-based analogues, and the mixed anhydride method
was modified (Scheme 4b). For example, reaction of 50
with 2 equiv of acylating agent gave the amide/carbam-
ate 51, by reaction of both the CO2H and NH with the
isobutyl chloroformate, followed by selective reaction of
the anhydride with the amine. Selective hydrolysis of
the carbamate group of 51 with mild base then gave
20. The isomeric amide mixture 14/21 was prepared
similarly.
Resu lts a n d Discu ssion
The structures of the two series of tetracyclic quino-
line- and quinoxalinecarboxamides are given in Table
1. They were all evaluated for growth inhibitory
properties, measured as IC50 values for continuous (72
h) drug exposure, against the murine leukemia P388
and the late-passage murine Lewis lung carcinoma line
LLTC (as examples of murine leukemias and carcino-
mas, respectively). They were also assessed against a
wild-type human leukemia line (J urkat; J LC), and two
sublines (J LA and J LD). These lines have previously
been described in detail.18,19 The J LA line is resistant
to the DNA intercalator amsacrine and similar agents
by virtue of a reduced level of topo II enzyme. The J LD
line is resistant to doxorubicin primarily also by a lower
level of topo II, and probably also by resistance to oxygen
radical damage. The ratios of the IC50 values of a drug
in the parent line compared with one of the sublines
(IC50[J LA]/IC50[J LC] and (IC50[J LD]/IC50[J LC]) therefore
provide some indication of the mechanism of cytotoxic-
ity. Classical topo II inhibitors such as amsacrine,
doxorubicin, and etoposide have large ratios (10-90-
fold), whereas topo I inhibitors such as camptothecin
and mixed topo I/II inhibitors such as DACA (4) have
ratios of only about 2-fold (Table 1). Values of these
Most of the amides of Table 1 were prepared from the
corresponding acids by a mixed anhydride method,9
using isobutyl chloroformate as the initial reagent,
followed by reaction with N,N-dimethylethylenediamine
(Scheme 4a). The structures of the isomeric quinoxa-

2042 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Deady et al.
Ta ble 1. Biological Data for Tetracyclic Quinoline- and Quinoxalinecarboxamides
IC50 (nM)a
IC50 ratios
P388b
LLTCc
J LC
J LA/J LC
J LD/J LC
d
no.
form
X
Y
Z
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
NH
O
S
CH
H
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
Cl
H
Cl
370
170
46
25
88
290
210
66
30
190
91
450
430
170
34
320
180
860
980
1.0
1.2
1.5
0.5
1.8
1.2
3.2
0.8
NDf
1.0
1.0
1.9
0.9
0.9
1.2
1.0
1.2
1.9
85
1.1
1.2
1.6
0.9
1.9
0.9
4.2
0.9
NDf
1.0
1.1
2.7
0.9
1.0
1.3
1.0
>1.3
2.3
74
CH
CH
C-Re
CH
CH
CH
N
N
N
N
N
CH
N
N
N
N
S
CH2
CO
SO2
NH
O
CH2
CO
S
CO
NH
NH
O
130
1800
1600
5800
360
700
100
85
670
15000
2200
6800
71
430
700
>2000
390
880
110
150
990
360
1700
1400
190
12
>2000
550
1100
150
370
1400
300
2600
1500
580
23
4
B
O
DACA
amsacrine
20
37
doxorubicin
etoposide
camptothecin
15
25
22
180
33
9.6
160
5.6
4.4
13
2.0
13
90
1.4
a
b
IC50: concentration of drug to reduce cell number to 50% of control cultures (see text). Murine P388 leukemia. c Murine Lewis lung
carcinoma. Human J urkat leukemia. e R ) CONH(CH2)2NMe2. f Not determinable (both IC50s >2000 nM).
d
Sch em e 4a
J LA/J LC and J LD/J LC ratios, suggesting that cytotoxicity
does not result primarily from inhibition of topo II. The
resistance properties of these compounds resemble those
of 7-chloroDACA, which has been shown to stimulate
sequence-selective cleavage of DNA in response to topo
I.19 In contrast, the thienodioxide analogue 13 was
much less cytotoxic, possibly because of a non-coplanar
ring system that could compromise intercalative bind-
ing, and had larger IC50 ratios (ca. 3).
The quinoxaline analogues 14-18 had considerably
varying IC50 values (e.g., from 130 to 1500 nM against
J LC), but were on average somewhat less cytotoxic than
the quinoline derivatives. However, their IC50 ratios in
the J urkat cell lines were also generally around unity,
suggesting a similar mode of action. The effect of the
chloro group in 14 and 15 could not be determined
precisely, since the corresponding unsubstituted deriva-
tives were not available, but the IC50 results suggest it
is probably not advantageous.
a
(i)Isobutyl chloroformate (1.2 equiv)/CH2Cl2/Et3N/1.5 h, then
N,N-dimethylethylenediamine/CH2Cl2/0-20 °C/2.5 h; (ii) as for (i),
2.4 equiv of isobutyl chloroformate; (iii) aqueous NaOH/dioxane/
20 °C/16 h.
In the isomeric quinoline series, only the indeno
analogue (19) was available, and this compound showed
a pattern of activity comparable to that of its isomer
12. A larger range of isomeric quinoxalines was avail-
able (20-23), but these were found to be less cytotoxic
on average than their counterparts. In this series there
were two sets of compounds (20/21 and 22/23) available
to evaluate the effects of a chloro substituent, and an
analysis of these results suggests it does increase
potency, at least in the human lines. This position
might therefore be available to manipulate other phys-
icochemical aspects (such as lipophilicity) which might
affect pharmacology and metabolism.
ratios of less than about 1.5-2 therefore suggest cyto-
toxicity by a non-topo II mediated mechanism.
The monocationic quinoline analogues 7-9, 11, and
12 showed broadly similar cytotoxicities to DACA (IC50s
of 46-370 nM against P388, 66-290 nM against Lewis
lung, and 170-450 nM against the wild-type human
line J LC). This compares with IC50s of 98, 200, and 580
nM, respectively, for DACA (Table 1). The thieno and
indeno analogues 9 and 12 were the most active, being
about 2-fold more cytotoxic than DACA against J LC. The
dicationic thieno derivative 10 was more cytotoxic again
(IC50 34 nM in J LC). All of these compounds had low
Compound 12, which was one of the most effective
against J LC cells in culture, was evaluated against

Putative Topoisomerase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2043
and triplets with J ) 6-8 Hz, except the pyrido ring proton,
a singlet. In addition to the peaks listed, all carboxamides
had a common pattern for the side chain: δ 2.4 (s, 6 H,
N(CH3)2), 2.7 (t, J ) 6 Hz, 2 H, CH2N), 3.75 (q, J ) 6 Hz, 2 H,
NHCH2).
Isa tin -7-ca r boxylic Acid (24). A solution of methyl an-
thranilate (31.5 g), chloral hydrate (35 g), and hydroxylamine
hydrochloride (28.6 g) in concentrated H2SO4 (25 g) and water
(1.4 L) was heated at 95 °C for 10 min and then kept at 4 °C
for 16 h. The cream-colored isonitroso intermediate (29.8 g,
64%) was filtered off, washed with water, and dried. This
compound (15.0 g) was added, with stirring, in portions over
30 min to concentrated H2SO4 (75 g) maintained at 60-65 °C.
The mixture was then heated at 95 °C for 1 h and poured onto
ice (600 g). The resulting brown solid was filtered, dissolved
in 1 M NaOH solution, and filtered, and the filtrate was taken
to pH 2 with concentrated HCl to give 24 (8.4 g, 65%), mp
274-275 °C (H2O) (lit.12 mp 276-277 °C).
P r ep a r a tion of 6-Meth yl-11H-in d en o[1,2-b]qu in olin e-
10-ca r boxylic Acid (34): Exa m p le of th e Gen er a l P fitz-
in ger Rea ction . 7-Methylisatin (25) (1.54 g, 9.56 mmol) was
added with stirring to 10% NaOH solution (30 mL), at 90 °C.
To this was added 1-indanone (29) (0.8 g, 6.06 mmol) in small
portions, and the solution was heated and stirred for a further
45 min and then filtered while hot. The filtrate was cooled
and the sodium salt of the product separated. This was filtered
off and dissolved in hot water and the solution taken to pH 5
with AcOH to give 34 as a yellow solid (1.1 g, 70%), mp 262
-265 °C; 1H NMR δ 2.84 (s, 3 H, CH3), 4.20 (s, 2 H, CH2),
7.48-7.57 (m, 3 H) 7.64 (d), 7.70 (d), 8.15 (d), 8.19 (d).
The following acids were prepared in this manner:
4-Meth ylben zoth ien o[3,2-b]qu in olin e-11-car boxylic acid
(35) by reaction of 25 and benzo[b]thiophen-3(2H)-one (28) at
100 °C, under N2, for 6 h. A blue/mauve sodium salt separated
on cooling (the filtrate gave some 28 at pH 6 and some 25 at
pH 2), and this, dissolved in hot water, gave the free acid 35
at pH 5 (concentrated HCl) in 41% yield: mp 294-296 °C (with
decarboxylation); 1H NMR δ 2.87 (s, 3 H, CH3), 7.55-7.68 (m,
4 H), 8.03 (d), 8.47 (d), 8.76 (d).
F igu r e 1. Growth of subcutaneous colon 38 tumors in
C57BL/6 mice either untreated (b) or treated with 12 at a dose
of 60 mg/kg (9) or 90 mg/kg (4). See ref 5 for details.
murine colon 38 tumors implanted subcutaneously in
C57BL/6 mice. This advanced colon 38 tumor model5
is fairly refractory to standard clinical topo II agents,
with doxorubicin and etoposide providing growth delays
of 8 and 1.5 days, respectively. Drug treatment with
12 was initiated at a time when the tumors were
approximately 4 mm in diameter and was given as a
split dose to minimize acute toxicity.5 At the maximum
tolerated dose of 90 mg/kg, 12 provided a growth delay
of about 7 days (Figure 1). By comparison, the mixed
topo I/II inhibitor DACA provided a growth delay of 8.8
days for this administration schedule and up to 22 days
for extended schedules.5
Overall, this new class of compounds have a mecha-
nism of cytotoxicity similar to that of the acridine-4-
carboxamide DACA, which is a mixed topo I/II inhibi-
tor.5 While insufficient analogues have been studied to
draw firm conclusions about SAR in this series, the
chloro substituent is well-tolerated, and the isomer
pattern of compounds 7-18 is somewhat favored over
that of compounds 19-23. If the cationic side chains
of these compounds are assumed to lie in the DNA
minor groove as suggested previously for other tricyclic
carboxamides,20,21 simple overlay modeling suggests the
geometry of the former does result in better overlap in
the DNA base pair intercalation site. Some of the
analogues are up to 3-fold more cytotoxic than DACA
in the human leukemia cell lines studied here, and the
one derivative (12) evaluated in vivo shows a growth
delay comparable to that of the clinical topo II agent
doxorubicin and superior to that of etoposide. The
results suggest this is an interesting new class of mixed
topo I/II inhibiting anticancer drugs, and more detailed
studies of their mechanism of action are in progress.
11H-In d en o[1,2-b]qu in olin e-6,10-d ica r boxylic a cid (33)
by reaction of isatin-7-carboxylic acid (24) and 29. A sodium
salt separated when the pH was taken to 6 with AcOH (which
left unreacted 24 still in solution). This was filtered off and
dissolved in hot water and the pH taken to 2 with concentrated
HCl to give free acid 33 as a pale yellow solid (62% yield): mp
1
295-298 °C (with decarboxylation); H NMR δ 4.29 (s, 2 H,
CH2), 7.55-7.67 (m, 2 H), 7.74-7.84 (m, 2 H), 8.08 (d), 8.56
(d), 8.67 (d).
Ben zoth ien o[3,2-b]qu in olin e-4,11-dicar boxylic acid (32)
by reaction of 24 and 28 at 100 °C, under N2, for 8 h. A sodium
salt separated at pH 6 (concentrated HCl), and this, dissolved
in hot water, gave free acid 32 at pH 2, in 55% yield: mp 282-
283 °C (with decarboxylation); 1H NMR δ 7.60 (t), 7.72 (t), 7.84
(t), 8.03 (d), 8.25 (d), 8.48 (d), 9.12 (d).
Ben zofu r o[3,2-b]qu in olin e-4,11-d ica r boxylic a cid (31)
by reaction of 24 and benzofuran-3(2H)-one (27) under reflux
for 6 h. The mixture was then cooled and filtered to remove
a base insoluble byproduct. The sodium salt of the product
was largely soluble, and acidification of the filtrate to pH 2
with concentrated HCl gave a 5:2 mixture of the free acid (31)
along with some unreacted 24 which could not be separated
(0.19 g from 0.3 g of 27). This was treated further by
dissolution in 6% Na2CO3 solution and reacidified to give a
solid mixture which was decarboxylated as detailed below: 1H
NMR δ 7.60 (t), 7.80-7.93 (m, 3 H), 8.36 (d), 8.47 (d), 8.63 (d).
10H-Qu in olin e-4,11-d ica r boxylic a cid (30). A cooled
solution of 24 (1.6 g, 8.4 mmol) in KOH (28.8 g) and water
(120 mL) was run into a flask containing 3-acetoxy-1-acetyl-
indole (0.8 g, 3.9 mmol) (which generates 26 in situ) under an
atmosphere of nitrogen, and the mixture was shaken until
solution was complete. The flask was sealed and stored in
the dark for 10 days. Water (60 mL) was added, the green-
yellow solution was heated, and oxygen was passed through
for 20 min. The solution was filtered while hot to remove
indigo, and the filtrate was acidified to pH 4 with concentrated
HCl to give 0.80 g of crude product. This was dissolved in 6%
Exp er im en ta l Section
Analyses indicated by symbols of the elements were within
(0.4% of the theoretical. 7-Methylisatin,13 benzothiophen-
3(2H)-one,22 and 3-acetoxy-1-acetylindole23 were prepared as
reported. Ethyl salicylate and ethyl bromoacetate were re-
acted as reported24 and the reaction worked up by an alterna-
tive procedure25 to give ethyl O-carbethoxymethylsalicylate.
This was then converted to benzofuran-3(2H)-one (27) as
reported.24 1H NMR spectra were obtained at 300 MHz, in
(CD3)2SO unless stated otherwise, and are referenced to Me4-
Si. In the listings, proton counts for aromatic protons (which
have not been assigned) are given only for unresolved mul-
tiplets; the other aromatic signals are single proton doublets

2044 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Deady et al.
Na2CO3 solution and filtered, and the filtrate was taken to
pH 6 to give the free acid 30 as a red solid (0.24 g, 21.5%):
mp softens at 280 °C and decarboxylates 290-295 °C; 1H NMR
δ 7.33 (t), 7.69-7.79 (m, 3 H), 8.23 (d), 8.44 (d), 9,23 (d), 11.63
(s, NH).
temperature for 1 h and then added to 100 mL of ice/water
and taken to pH 2 with 50% NaOH solution. The resultant
solid was filtered off, washed with cold Me2CO, recrystallized
from ethane-1,2-diol, and washed with EtOH to give 42 as
cream-colored needles (0.24 g, 22%): mp 358-359 °C; 1H NMR
δ 7.73 (t), 7.81 (t), 7.85-7.90 (m, 2 H), 8.08 (d), 8.41 (d), 8.52
(d), 8.83 (s).
Ben zoth ien o[3,2-b]qu in olin e-4-ca r boxylic Acid 10,10-
Dioxid e (43). This was prepared similarly by oxidation of
37 with CrO3 and was obtained as a cream-colored solid in
35% yield, mp >310 °C, when the aqueous acid solution was
taken to pH 4 with concentrated aqueous NaOH.
4-Meth yl-6H-in den o[2,1-b]qu in olin e-11-car boxylic acid
(45) by reaction of 25 and 2-indanone (44) as for the prepara-
tion of 34. The sodium salt separated from the cold reaction
mixture. This was filtered off and dissolved in water, and the
solution was then taken to pH 4 with AcOH. The dark brown
solid which formed was filtered off and extracted with 6% Na2-
CO3 solution. The extract was acidified with concentrated HCl
to give the free acid (45) as a pale brown solid (49% yield):
6-Oxo-6H-in den o[2,1-b]qu in olin e-4-car boxylic Acid (47).
A solution of 46 (1.1 g) in 3 M H2SO4 (50 mL) was heated to
reflux. To this mixture was added, in small portions over 1
h, a solution of Na2Cr2O7 (1.65 g) in 3 M H2SO4 (25 mL). The
mixture was then heated under reflux for a further 3 h, cooled,
and added to 150 mL of water, and the resulting tan precipi-
tate of 4-methyl-6-oxo-6H-indeno[2,1-b]quinoline (0.9 g), mp
215-220 °C, was filtered off. This was added to concentrated
H2SO4 (7 M, 100 mL), and the mixture was heated to reflux.
To this was added, in small portions over 1 h, a solution of
Na2Cr2O7 (3.6 g) in 3 M H2SO4 (50 mL). The mixture was
heated under reflux for a further 2 h, cooled, and added to
800 mL of ice-water and kept at 4 °C for 16 h. The product
was collected by filtration and dissolved in 1 M sodium
hydroxide, insoluble material was removed by filtration, and
the filtrate was acidified with concentrated HCl to give the
1
mp 280-282 °C dec; H NMR δ 2.77 (s, 3 H, CH3), 4.21 (s, 2
H, CH2), 7.35-7.42 (m, 3 H), 7.53 (d), 7.61 (d), 7.75 (d), 7.99
(d).
P r ep a r a tion of Ben zoth ien o[3,2-b]qu in olin e-4-ca r box-
ylic a cid (40): Exa m p le of th e Gen er a l Deca r boxyla tion
p r oced u r e. Finely ground diacid 32 (0.11 g) was heated on
a hot-stage apparatus until it decarboxylated and liquefied (ca.
280 °C). After 5 min the vessel was cooled, the residue was
extracted into 1 M NaOH solution and filtered, and the filtrate
was acidified (pH 2, concentrated HCl) to give the free acid
(40) as a brown solid (0.09 g, 95%): mp 281-283 °C; 1H NMR
δ 7.61 (t), 7.70-7.80 (m, 2 H), 8.08 (d), 8.27-8.32 (m, 2 H),
8.53 (d), 9.24 (s).
The following acids were prepared in this manner:
4-Meth ylben zoth ien o[3,2-b]qu in olin e (37). Finely ground
acid 35 was heated at 300 °C at 20 mmHg pressure for 5 min.
The residue was extracted three times with hot Me2CO and
the solvent evaporated to give 37 as a reddish brown solid (96%
yield), mp 124-126 °C. The product can be further purified
to a yellow crystalline solid by vacuum sublimation: 1H NMR
δ 2.86 (s, 3 H, CH3), 7.53 (t), 7.59-7.68 (m, 3 H), 7.86 (d), 8.06
(d), 8.52 (d), 8.90 (s).
11H-In den o[1,2-b]qu in olin e-6-car boxylic Acid (41). Acid
33 was heated at 295-300 °C for 5 min, and the product 41
(60% yield) was recrystallized from CH2Cl2/petroleum ether
(bp 90-110 °C): 1H NMR δ 4.16 (s, 2 H, CH2), 7.58-7.64 (m,
2 H), 7.73-7.78 (m, 2 H), 8.06 (d), 8.33 (d), 8.52 (d), 8.72 (s).
Ben zofu r o[3,2-b]qu in olin e-4-ca r boxylic Acid (39). The
5:2 mixture of 31 and 24 obtained above (0.19 g) was heated
at 245 °C for ca. 5 min. The residue was stirred with warm
EtOH which dissolved 24, and the insoluble product 39 (0.07
g), mp 250-255 °C, was filtered off: 1H NMR δ 7.60 (t), 7.82-
7.92 (m, 3 H), 8.41 (d), 8.47 (d), 8.54 (d), 8.96 (s).
10H-Qu in olin e-4-ca r boxylic Acid (38). Diacid 30 was
heated to 310 °C until gas evolution ceased and the product
(38) was left as yellow needles in 93% yield: mp >310 °C; 1H
NMR δ 7.33 (t), 7.62-7.71 (m, 3-H), 8.31 (d), 8.40 (d), 8.49 (d),
8.63 (s), 11.90 (s, NH).
1
free acid 47 (0.46 g, 30%) as a brown solid: mp >300 °C; H
NMR (75 °C) δ 7.55 (t), 7.77-7.90 (m, 3 H), 8.03 (d), 8.29 (d),
8.40 (d), 8.87 (s).
P r ep a r a t ion of N-[2-(Dim et h yla m in o)et h yl]-11-oxo-
11H -in d en o[1,2-b]q u in olin e-6-ca r b oxa m id e (12): E x-
a m p le of th e Gen er a l Am id a tion Rea ction . Freshly
distilled Et3N (0.13 g, 1.3 mmol) was added to a stirred
suspension of the acid 42 (0.29 g, 1.1 mmol) in CH2Cl2 (20 mL)
in a nitrogen atmosphere. The resulting solution was taken
to <-10 °C, and isobutyl chloroformate (0.18 g, 1.3 mmol) in
CH2Cl2 (15 mL) was added dropwise over 0.75 h. After a
further 0.5 h at this temperature, a solution of N,N-dimeth-
ylethylenediamine (0.12 g, 1.3 mmol) in CH2Cl2 (15 mL) was
added dropwise over 0.5 h. The solution was stirred at <-5
°C for 0.5 h, 0 °C for 1 h, and room temperature for 1 h and
then filtered, and the filtrate was washed with a saturated
solution of NaHCO3 (3 × 30 mL) and then with brine and
water. The organic layer was dried (MgSO4), the solvent was
evaporated, and the crude product was recrystallized from
EtOH to give 12 as white needles (0.25 g, 65%): mp 222-223
°C; 1H NMR δ 7.65-7.72 (m, 2 H), 7.79-7.84 (m, 2 H), 8.22-
8.27 (m, 2 H), 8.62 (d), 8.68 (s), 10.71 (br s, NH). Anal.
(C21H19N3O2) C, H, N.
The following N-[2-(dimethylamino)ethyl]carboxamides were
prepared in this manner:
The following compounds were prepared by heating the
appropriate acid in boiling sulfolane for 30 min and then
diluting the dark mixture with water:
N-[2-(Dim eth ylam in o)eth yl]ben zofu r o[3,2-b]qu in olin e-
4-ca r boxa m id e (8): pale yellow solid (24%); mp 105-107 °C
(from petroleum ether, bp 90-110 °C); 1H NMR δ 7.49 (t)
7.61-7.71(m, 3 H), 8.06 (dd, J ) 7.9, 1.5 Hz), 8.23 (s), 8.45
(d), 8.88 (dd, J ) 7.3, 1.5 Hz), 11.60 (s, NH). Anal. (C20H19N3O2)
C, H, N.
6-Meth yl-11H-in d en o[1,2-b]qu in olin e (36). The aqueous
mixture was extracted three times with Et2O, the combined
organic extracts were washed with water and dried (MgSO4),
and the solvent was removed to give a black residue. This
was extracted with hot MeOH, the mixture filtered while hot,
and the filtrate concentrated and cooled on ice to give orange
crystals with some black residue. Recrystallization of the
orange material again from MeOH gave 36 (72% yield): mp
91.5 -91.8 °C; 1H NMR δ 2.82 (s, 3 H, CH3), 4.09 (s, 2 H, CH2),
7.43 (t), 7.50-7.57 (m, 2 H), 7.58 (d), 7.68 (d), 7.80 (d), 8.14
(d), 8.39 (s).
4-Meth yl-6H-in den o[2,1-b]qu in olin e (46). The solid which
separated was filtered off and washed with 6% Na2CO3 and a
copious amount of warm water to give 46 as a black solid (94%
yield): mp 120-122 °C; 1H NMR δ 2.73 (s, 3 H, CH3), 4.14 (s,
2 H, CH2), 7.38-7.51 (m, 3 H), 7.56 (d), 7.63 (d), 7.83 (d), 8.02
(d), 8.66 (s).
N-[2-(Dim eth yla m in o)eth yl]ben zoth ien o[3,2-b]qu in o-
lin e-4-ca r boxa m id e (9): red solid (51%) which could not be
freed of trace impurities; mp 137-139 °C (from CH2Cl2/
1
petroleum ether, bp 90-110 °C), H NMR δ 7.64-7.78 (m, 3
H), 8.12 (d), 8.23 (d) 8.70 (d), 8.94 (d), 9.19 (s), 11.18 (br s,
NH). A satisfactory elemental analysis could not be obtained.
N,N′-Bis[2-(d im eth yla m in o)eth yl]ben zoth ien o[3,2-b]-
qu in olin e-4,11-d ica r boxa m id e (10): red solid (30%); mp
177-179 °C (from MeCN); 1H NMR δ 7.49-7.64 (m, 3 H), 7.83
(d), 8.30 (d) 8.72 (d), 8.82 (d), 11.22 (br s, NH). Anal.
(C25H29N5O2S) C, H, N.
N-[2-(Dim eth yla m in o)eth yl]-11H-in d en o[1,2-b]qu in o-
lin e-6-ca r b oxa m id e (11): yellow needles (52%); mp 138-
140 °C (from toluene/petroleum ether, bp 90-110 °C); 1H NMR
δ 4.14 (s, 2 H, CH2), 7.57-7.60 (m, 2 H), 7.67 (t), 7.71 (d), 8.18
(d), 8.50 (d), 8.58 (s), 8.62 (d), 11.45 (br s, NH). Anal.
(C21H21N3O) C, H, N.
P r ep a r a tion of 11-Oxo-11H-in d en o[1,2-b]qu in olin e-6-
ca r boxylic Acid (42). Concentrated H2SO4 (2.7 mL) was
added dropwise with stirring to a cooled solution of 36 (0.90
g, 3.89 mmol) in AcOH (90 mL) and Ac2O (27 mL). CrO3 (9.0
g) was then added, and the mixture was stirred at room

Putative Topoisomerase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2045
N-[2-(Dim eth yla m in o)eth yl]ben zoth ien o[3,2-b]qu in o-
lin e-4-ca r boxa m id e 10,10-d ioxid e (13): red needles (41%);
mp 248-255 °C (from EtOH); H NMR δ 7.82-7.91 (m, 2 H),
8.03 (t), 8.17 (d), 8.33 (d), 8.72 (d), 8.74 (d), 9.37 (s), 10.45 (br
s, NH). Anal. (C20H18N3O3S) C, H, N.
N-[2-(Dim eth yla m in o)eth yl]-11H-in d en o[1,2-b]qu in ox-
a lin e-6-ca r boxa m id e (16). 11-Oxo-11H-indeno[1,2-b]qui-
noxaline-6-carboxylic acid26 (0.2 g), ethylene glycol (40 mL),
KOH (0.88 g), and hydrazine hydrate (0.64 g) were heated at
140 °C with stirring for 2 h. The condenser was removed, and
the temperature was increased gradually to 180 °C over 1 h.
The condenser was replaced, and the solution was heated
under reflux for 4 h. Water (40 mL) was added to the cooled
solution, which was then taken to pH 2 with concentrated HCl.
The resulting precipitate was extracted into CHCl3, the
solution was dried (MgSO4), and the solvent was removed
under reduced pressure to give 11H-indeno[1,2-b]quinoxaline-
6-carboxylic acid (0.12 g, 63%), sufficiently pure for amidation.
A sample recrystallized from EtOH had mp >250 °C (slow
decomposition): 1H NMR δ 4.24 (s, 2 H, CH2), 7.56-7.70 (m,
2H), 7.78 (d) 7.90 (t) 8.14 (d), 8.29-8.33 (m, 2 H). This was
reacted by the standard method to give the amide 16 as yellow
needles (57%): mp 188-190 °C [from petroleum ether (bp 90-
110 °C)]; 1H NMR (CDCl3) δ 4.16 (s, 2 H, CH2), 7.53-7.58 (m,
2 H), 7.67 (d), 7.78 (t), 8.20 (d), 8.43 (d), 8.85 (d), 11.14 (s, NH).
Anal. (C20H20N4O) H, N; C: calcd, 72.3; found, 71.8.
N-[2-(Dim eth yla m in o)eth yl]-10H-qu in olin e-4-ca r box-
a m id e (7). 10H-Quinoline-4-carboxylic acid 38 (0.22 g) in
SOCl2 (3 mL) was heated at 80 °C for 1 h, and the excess
reagent was removed at 20 mmHg. The residue was washed
by decantation with dry CH2Cl2 (2 × 3 mL), and fresh CH2Cl2
(3 mL) was added. The mixture was cooled to 0 °C and stirred,
and N,N-dimethylethylene-1,2-diamine (0.10 g) was added.
After being stirred at room temperature for 1 h, the solution
was filtered, the filtrate was washed with 10% Na2CO3 solution
and water and dried (MgSO4), and the solvent was removed
to give 7 (0.17 g, 63%), mp 251-254 °C (from MeCN), which
could not be freed from a trace impurity: 1H NMR δ 7.06 (t),
7.20-7.32 (m, 2 H), 7.41 (t), 7.73 (d), 7.80 (s), 8.14(d), 8.64 (d),
9.72 (s, ring NH), 11.92 (br t, amide NH). A satisfactory
elemental analysis could not be obtained.
N-[2-(Dim eth yla m in o)eth yl]-8-ch lor oben zofu r o[2,3-b]-
qu in oxa lin e-10-ca r boxa m id e (15) a n d N-[2-(Dim eth yl-
a m in o)eth yl]-9-ch lor oben zofu r o[2,3-b]qu in oxa lin e-7-ca r -
boxa m id e (23). The precursor isomeric acid mixture17 was
reacted with SOCl2 and then N,N-dimethylethylene-1,2-di-
amine as for the preparation of 7. The crude product was first
washed through a short alumina column with CHCl3 and the
solvent removed to give the carboxamide mixture as a yellow
solid (56%). This (0.18 g) was recrystallized from MeCN to
give a sample of 23 (containing <10% 15 by NMR) (0.04 g):
mp 205-212 °C; 1H NMR (CDCl3) δ 7.54 (t), 7.70 (d), 7.75 (t),
8.24 (s), 8.34 (d), 8.80 (s), 10.82 (s, NH). The recrystallization
filtrate was evaporated to dryness, and the residue was stirred
with cold MeCN (2 × 2 mL) and filtered each time. Evapora-
tion of the solvent from the combined filtrates gave 15
(containing <5% 23 by NMR) (0.07 g): mp 128-133 °C; 1H
NMR (CDCl3) δ 7.53 (t), 7.69 (d), 7.77 (t), 8.32 (d), 8.37 (s),
8.80 (s), 10.19 (s, NH). Alkaline hydrolysis gave the corre-
sponding carboxylic acid. Anal. (on a sample of the isomeric
amide mixture, recrystallized from MeCN) (C19H17ClN4O2) H,
N; C: calcd, 61.9; found, 61.4.
In Vitr o Gr ow th Dela y Assa ys. Murine P388 leukemia
cells, Lewis lung carcinoma cells (LLTC), and human J urkat
leukemia cells (J LC), together with their amsacrine and
doxorubicin-resistant derivatives (J LA and J LD respectively),
were obtained and cultured as described.18,19 Growth inhibi-
tion assays were performed by culturing cells at 4.5 × 103
(P388), 103 (LLTC), and 3.75 × 103 (J urkat lines) per well in
microculture plates (150 mL/well) for 3 (P388) or 4 days in
the presence of drug. Cell growth was determined by [3H]TdR
uptake (P388)27 or the sulforhodamine assay.28 Independent
assays were performed in duplicate, and coefficients of varia-
tion were 12% (P388), 12% (LLTC), 6.3% (J LC), 9.3% (J LA),
and 5.7% (J LD).
1
N-[2-(Dim eth yla m in o)eth yl]-11-oxo-11H-in d en o[1,2-b]-
qu in oxa lin e-6-ca r boxa m id e (17): yellow needles (61%); mp
220-221 °C (from EtOH); 1H NMR δ 7.77 (t), 7.90-8.05 (m, 3
H), 8.27 (d), 8.34 (d), 8.64 (d), 10.55 (br s, NH). Anal.
(C20H18N4O2) C, H, N.
N-[2-(Dim et h yla m in o)et h yl]-6-oxo-6H -in d en o[2,1-b]-
qu in olin e-4-ca r boxa m id e (19): yellow needles (59%); mp
163-165 °C (from toluene); 1H NMR δ 7.46 (t), 7.62-7.74 (m,
3 H), 7.81 (d), 7.94 (d), 8.26 (s), 8.76 (d), 10.87 (br t, NH). Anal.
(C21H19N3O2) C, H, N.
N-[2-(Dim eth yla m in o)eth yl]ben zofu r o[2,3-b]qu in oxa -
lin e-7-ca r boxa m id e (22): pale tan solid (61%), after tritu-
ration with hexane, but which formed a sticky hydrate on
standing; 1H NMR (CDCl3) δ 7.45 (t), 7.57-7.68 (m, 2 H), 7.79
(t), 8.21 (d), 8.29 (d), 8.79 (d), 10.14 (s, NH). The compound
was >96% pure by HPLC, but a satisfactory elemental analysis
could not be obtained.
N-[2-(Dim eth yla m in o)eth yl]ben zoth ien o[3,2-b]qu in ox-
a lin e-10-ca r boxa m id e (18): pale yellow needles (66%); mp
208-210 °C (from MeCN); 1H NMR (CDCl3) δ 7.47 (t), 7.58
(t), 7.75 (d), 7.82 (t), 8.16 (d), 8.73 (d), 8.84 (d), 11.02 (s, NH).
Anal. (C19H18N4OS) C, H, N.
N-[2-(Dim eth yla m in o)eth yl]-6H-in d olo[2,3-b]qu in oxa -
lin e-4-ca r boxa m id e (20). 6H-Indolo[2,3-b]quinoxaline-4-car-
boxylic acid17 (50) was reacted with twice the molar ratio of
other reagents described in the general method above to give
the intermediate carbamate 51 as a tan solid (43%, >97% pure
by NMR): mp >128 °C (slow decomposition) after trituration
1
of the crude oil with hexane; H NMR (CDCl3) δ 1.12 (d, J )
6.7 Hz, 6 H, CH(CH3)2), 2.27 (m, 1 H, CH(CH3)2), 2.44 (s, 6 H,
N(CH3)2), 2.89 (t, J ) 6 Hz, 2 H, CH2N), 3.85 (q, J ) 6 Hz, 2
H, NHCH2), 4.41 (d, J ) 6.6 Hz, 2 H, OCH2), 7.53 (t), 7.72 (t),
7.84 (t), 8.19 (d), 8.34 (d), 8.40 (d), 8.87 (d), 11.17 (br s, 1 H,
NH).
A solution of aqueous NaOH (6 mL, 0.25 M) was added with
stirring to a solution of 51 (0.1 g) in dioxane (20 mL), causing
the solution to turn deep red. Stirring was continued for a
further 16 h, when the solution was neutralized with HCl and
the mixture was concentrated under reduced pressure to 5 mL.
This was extracted with CH2Cl2 (3 × 10 mL), the combined
extracts were dried (MgSO4), and the solvent was removed to
give 20 as a viscous yellow semisolid (0.06 g, 76%, >95% pure
by NMR): 1H NMR (CDCl3) δ 7.05 (t), 7.21 (t), 7.29 (d), 7.54
(t), 7.91 (d), 7.94 (d), 8.02 (d), 11.13 (s, NH), 12.50 (s, NH).
Attempts to purify this material for microanalysis were not
satisfactory, and various salts were very hygroscopic.
N-[2-(Dim eth yla m in o)eth yl]-6H-3-ch lor oin d olo[2,3-b]-
qu in oxa lin e-1-ca r boxa m id e (14) a n d N-[2-(Dim eth yla m i-
n o)eth yl]-6H-2-ch lor oin d olo[2,3-b]qu in oxa lin e-4-ca r box-
a m id e (21). An isomeric mixture of precursor acids17 was
reacted as for 20 to give the intermediate carbamate mixture
as a pale yellow solid (40%) after trituration of the crude oil
with hexane. This mixture was hydrolyzed as for 20. In this
case, all solvents were removed from the neutralized reaction
mixture, water was added, and the crude mixture of carbox-
amide isomers was separated as an orange solid. This (0.1 g)
was stirred with ice-cold CHCl3 (1 mL) and filtered. Evapora-
tion of the filtrate gave a sample of 14 (free of 21) (0.03 g):
mp 218-220 °C; 1H NMR (CDCl3) δ 7.29 (d), 7.31 (t), 7.62 (t),
7.94 (s), 7.95 (s), 8.08 (d), 11.10 (s, NH), 12.48 (s, NH).
Alkaline hydrolysis gave the corresponding carboxylic acid.
The solid from the first filtration was stirred with CHCl3 (3 ×
1 mL) and filtered each time, and the final insoluble solid was
a sample of 20 (free of 14) (0.028 g): mp 294-296 °C; 1H NMR
[CDCl3/(CD3)2SO] δ 7.32 (t), 7.50 (d), 7.62 (t), 8.12 (s), 8.38 (d),
8.49 (s), 11.15 (s, NH), 11.99 (s, NH). For microanalysis, a
monoperchlorate salt of the amide mixture was prepared and
had mp >270 °C (slow decomposition) after recrystallization
from water. Satisfactory figures were not obtained for this
hydrated species although the C:N ratio was acceptable.
In Vivo Colon 38 tu m or Assa y of 12. Colon 38 tumors5
were grown subcutaneously from 1 mm3 fragments implanted
in one flank of mice (anesthetized with pentobarbitone 90 mg/
kg). When tumors reached a diameter of approximately 4 mm
(7-8 days), mice were divided into control and drug treatment
groups (5 mice/group), with similar average tumor volumes

2046 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Deady et al.
dimethyl)-10h-pyrido [2,3-b] carbazoles and 4-amino-substituted
11-methyl (and 5,11-dimethyl)-10h-pyrido[3′,4′:4,5] pyrrolo [3,2-
g] quinolines, two new series related to antitumor ellipticine
derivatives. Anti-Cancer Drug Des. 1993 8, 399-416.
(12) Waldmann, H. Isatincarboxylic acid. J . Prakt. Chem. 1937, 147,
338-343.
(13) Chen, Q.; Deady, L. W. Synthesis of some benzo[b][1,6]-
naphthyridines and benzo[b][1,7]naphthyridines. Aust. J . Chem.
1993, 46, 987-993.
in each group. A solution of 12 in distilled water was injected
in a volume of 0.01 mL/g body weight in two equal injections
administered 1 h apart. The mice were monitored closely, and
tumor diameters were measured with callipers three times a
week. Tumor volumes were calculated as 0.52a2b, where a
and b are the minor and major tumor axes and data plotted
on a semilogarithmic plot as mean tumor volumes ((SEM)
versus time after treatment. The growth delay was calculated
as the time taken for tumors to reach a mean volume 4-fold
higher than their pretreatment volume.
(14) J ones, G. In The Chemistry of Heterocyclic Compounds; Weiss-
berger, A., Taylor, E. C., Eds.; Wiley-Interscience: London, 1977;
Vol. 32, The Quinolines, Part I, Chapter 2.
(15) Noelting, E.; Herzbaum, A. Condensation products of isatic acid
and hydroxythionaphthene, indanedione and indanone. Ber.
1911, 44, 2585-2590.
(16) Holt, S. J .; Petrow, V. Carbazoles, carbolines, and related
compounds. Part 1. Quindoline derivatives. J . Chem. Soc. 1947,
607-611.
(17) Deady, L. W.; Kaye, A. J . Fused tetracyclic quinoxalines from
reactions of o-phenylenediamines in polyphosphoric acid. Aust.
J . Chem., in press.
(18) Finlay, G. J .; Holdaway, K. M.; Baguley, B. C. Comparison of
the effects of genistein and amsacrine on leukemia cell prolifera-
tion. Oncol. Res. 1994, 6, 33-37.
(19) Finlay, G. J .; Riou, J .-F.; Baguley, B. C. From amsacrine to
DACA (N-[2-(dimethylamino)ethyl]acridine-4-carboxamide): se-
lectivity for topoisomerases I and II among acridine derivatives.
Eur. J . Cancer 1996, 32A, 708-714.
(20) Atwell, G. J .; Bos, C. D.; Baguley, B. C.; Denny, W. A. Potential
Antitumor Agents. 56. “Minimal” DNA-intercalating ligands as
antitumor drugs: phenylquinoline-8-carboxamides. J . Med.
Chem. 1988, 31, 1048-1052.
Ack n ow led gm en t. We thank Mr. J . Desneves, who
first prepared compound 17, Mrs. Debra Greenhalgh for
help with some of the in vitro assays, and Dr. Li Zhuang
and Miss Tristan Gregory for performing the in vivo
assays. This work was supported in part by the Auck-
land Division of the Cancer Society of New Zealand.
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