Synthesis and structure-activity relationships of potential anticancer agents: Alkylcarbamates of 3-(9-acridinylamino)-5-hydroxymethylaniline

Article abstract of DOI:10.1021/jm9901226

A series of potential 9-anilinoacridine antitumor agents, 3-(9- acridinylamino)-5-hydroxymethylaniline (AHMA) derivatives with monosubstituent at C4' and disubstituents at C4' and C5' of the acridine ring and their alkylcarbamates, were synthesized for ev

Full text of DOI:10.1021/jm9901226

J . Med. Chem. 1999, 42, 4741-4748
4741
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of P oten tia l An tica n cer
Agen ts: Alk ylca r ba m a tes of 3-(9-Acr id in yla m in o)-5-h yd r oxym eth yla n ilin e
Tsann-Long Su,*,† Ching-Huang Chen,† Lin-Fei Huang,† Chao-Hao Chen,† Manas K. Basu,† Xiu-Guo Zhang,‡ and
Ting-Chao Chou‡
Laboratory of Bioorganic Chemistry, Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, R.O.C., and
Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Received J uly 14, 1999
A series of potential 9-anilinoacridine antitumor agents, 3-(9-acridinylamino)-5-hydroxy-
methylaniline (AHMA) derivatives with monosubstituent at C4′ and disubstituents at C4′ and
C5′ of the acridine ring and their alkylcarbamates, were synthesized for evaluation of their
antitumor activity. A structure-activity relationship (SAR) study showed that the AHMA-
alkylcarbamates were more potent than their corresponding parent AHMA compounds. In
addition, the cytotoxicity of the AHMA-alkylcarbamate decreased with increasing length and
size of the alkyl function. Among these compounds, AHMA-ethylcarbamate (18) and 4′-methyl-
5′-dimethylaminoethylcarboxamido-AHMA-ethylcarbamate (34) possess potent cytotoxicity on
the inhibition of human leukemic HL-60 cell growth in culture. Further in vivo studies of these
compounds displayed significant anticancer therapeutic effects in mice bearing sarcoma 180,
Lewis lung carcinoma, and P388 leukemia.
Ch a r t 1
In tr od u ction
Members of the class of 9-anilinoacridine derivatives
have been synthesized as potential topoisomerase II
(Topo II)-mediated anticancer agents.1-6 Structure-
activity relationships (SAR) including the position of
substituent(s), chemical and physical properties, lipo-
philicity-hydrophilicity balance, etc., have also been
investigated by Atwell and Baguley et al.1-6 The first
9-anilinoacridine derivative to achieve clinical use as
an anticancer agent was amsacrine (m-AMSA, 1; Chart
1) to treat leukemia and lymphoma.7-9 However, m-
AMSA lacks broad-spectrum clinical activity and has a
low therapeutic index. In addition, this agent has been
difficult to formulate because of its low aqueous solubil-
ity. Subsequently, the 4-methyl-5-methylcarboxamide-
substituted m-AMSA (CI-921, 2; Chart 1) was synthe-
sized and was found to have superior antileukemic
activity and a broad spectrum of antitumor activity.10,11
The chemical structures of m-AMSA and CI-921
indicate that both possess a methanesulfonyl function
at the para-position of the 9-acridinylamino group and
readily undergo reversible oxidation either chemically12
or microsomally,12,13 giving the reactive species qui-
nonediimine (m-AQDI). The intermediate m-AQDI fur-
ther forms glutathione conjugates and is excreted. The
half-life of m-AMSA is about 30 min in the presence of
fresh mouse blood at 37 °C.14,15 Therefore, the antitumor
potency of m-AMSA derivatives suggests a correlation
with the derivatives’ redox potential. However, a clear
quantitative relationship between redox potential and
antitumor activity could not be discerned since the
isomer, o-AMSA, was found to be biologically inactive.
To determine whether the oxidized m-AQDI is required
for antitumor activity, we previously synthesized a
series of 9-anilinoacridine analogues in which the amino
was replaced by an O or S atom or compounds lacking
the substituent at the para-position to the 9-amino
function of acridine.16-18 Among these compounds, 3-(9-
acridinylamino)-5-hydroxymethylaniline (AHMA, 3;
Chart 1) bearing an NH2 and CH2OH at the meta-
position to the 9-amino function was found to have
greater antitumor efficacy against murine leukemia and
solid tumors than m-AMSA or VP-16 and was also less
toxic to the host. We also found that AHMA possesses
a longer plasma half-life (1.5 h) than m-AMSA.
Previously, we synthesized a series of AHMA deriva-
tives without substituents on the acridine ring. Our SAR
study was mainly focused on the modification of the
substituent(s) on the amino and/or hydroxymethyl
group(s).16 However, AHMA without a substituent at
5-CH2OH exhibited slightly better in vitro antitumor
activity against human leukemic HL-60 and L1210 cell
lines, while a substituent or substituents at NH2 and/
or CH2OH, in general, did not greatly affect the cyto-
toxicity of AHMA. Therefore, we concluded that the
antitumor activity of 9-anilinoacridine derivatives is
mainly attributed to Topo II-mediated cleavage of
double-stranded DNA, common to DNA-intercalating
agents.19,20 Formation of stable ternary complexes be-
tween drugs (such as AHMA), DNA, and Topo II is
* To whom all correspondence should be addressed. Fax: 886-2-
2782 5573. E-mail: tlsu@ibms.sinica.edu.tw.
† Academia Sinica.
‡ Memorial Sloan-Kettering Cancer Center.
10.1021/jm9901226 CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/21/1999

4742 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Su et al.
Sch em e 1
essential for pharmacologically based antitumor activ-
ity.19,20 Other factors such as water solubility, alterna-
tion of lipophilicity-hydrophilicity balance, and stereo
effect for drug binding with DNA by substituent(s) at
NH2 and/or CH2OH of AHMA may also affect AHMA’s
antitumor activity.
alkyl, phenyl, or benzyl chloroformates (Scheme 1) in
pyridine. AHMA-alkylcarbamates with a monosubstitu-
ent at C4′ or with disubstituents at C4′ and C5′ of the
acridine ring were prepared starting from the requisite
known 9(10H)-acridones (I, R1 ) Me, OMe, F, Cl, Br,
or NO2, R2 ) H)25,26 and 9-oxoacridan-4-carboxylic acids
(R1 ) COOH, R2 ) H, Me, or OMe).6,27-29 The mono-
substituted 9(10H)-acridones were treated with thionyl
chloride to yield the corresponding 9-chloroacridines (II).
The disubstituted 9-oxoacridan-4-carboxylic acids were
converted to 9-chloroacridan-4-carboxamides (II, where
R1 ) CONHMe, R2 ) OMe or Me; R1 ) CONH(CH2)2-
NMe2, R2 ) OMe or Me) treating with thionyl chloride
followed by addition of methylamine or N,N-dimethyl-
ethylenediamine.4,6 Condensation of 9-chloroacridines
(II) with 3,5-diaminobenzyl alcohol dihydrochloride (III)
in a mixture of CHCl3/MeOH in the presence of 4-meth-
ylmorpholine gave various AHMA derivatives (IV) by
following our previous method.16 In a similar manner,
AHMA derivatives with substituent(s) (III) on the
acridine ring were treated with ethyl chloroformate in
pyridine to give the desired AHMA-alkylcarbamates (V).
During the course of our earlier SAR studies of AHMA
derivatives, we found that one of the intermediates, tert-
butylcarbamate of AHMA (4; Chart 1), was more cyto-
toxic than the parent compound AHMA.21 This finding
prompted us to synthesize a series of alkylcarbamates
of AHMA for evaluation of their antitumor activity.
A study of the DNA binding kinetics of 9-anilino-
acridines suggested that, at equilibrium, amsacrine-4-
caboxamides bind with the acridine chromophore in a
position of (1) maximum overlap with the DNA base
pairs; (2) the carboxamide side chain lying in the DNA
minor groove; and (3) the bulky anilino chain lying in
the major groove.22,23 Both equilibrium and kinetic
measurements show that amsacrine-4-carboxamide binds
selectively to poly(dG-dC)‚poly(dG-dC) compared to poly-
(dA-dT)‚poly(dA-dT). These findings suggest that the
highest affinity sites in the natural DNA sequence (calf
thymus DNA) for the antitumor compound are GC-
rich.22-24 The substituent on the acridine ring may affect
the drug binding in terms of their sequence-specific
binding sites and binding constants. In the present
study, a series of AHMA derivatives with mono- or
disubstituent(s) at C4′ or/and C5′ of the acridine moiety
and their alkylcarbamates were synthesized to observe
the effect of these substituent(s) on the derivatives’
cytotoxicity. Since our preliminary studies indicated
that ethylcarbamate of AHMA was shown to be the most
potent among the alkylcarbamates of AHMA in inhibit-
ing HL-60 cell growth in cell culture, a series of
ethylcarbamates of AHMA with substituent(s) on the
acridine ring were also synthesized for SAR study. In
this paper we describe the synthesis, SAR, and anti-
tumor effects of the AHMA-alkylcarbamates in vitro and
in vivo.
Biologica l Resu lts a n d Discu ssion
DNA Topo II has emerged as the chemotherapeutic
target for a diverse group of antitumor agents.30 It was
suggested that the interaction of drug with DNA and
DNA Topo II to produce a ternary complex is essential
for the antitumor activity of drug. In a ternary drug-
DNA-Topo II complex, the drug association site could
reside a priori on either the enzyme or DNA exclusively,
or could be at a DNA-enzyme interface in which the
drug exhibits close contact with both components.
However, the nature of drug-DNA-Topo II ternary
complex is not fully clear. To design an effective
anticancer drug, such as 9-anilinoacridine derivatives,
one has to take account of the drug-DNA and drug-
Topo II associations for consideration. In addition, other
factors such as pKa value and lipophilicity-hydrophi-
licity associated with drug distribution affect the drug’s
bioavailability.
Ch em istr y
The derivatives of AHMA-alkylcarbamates were pre-
pared in good yield by reacting AHMA16 with various
To find AHMA derivatives with superior antitumor
activity as compared to the parent compound, we

Synthesis and SAR of Potential Anticancer Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4743
Ta ble 1. In Vitro Cytotoxicity of Derivatives of AHMA and Their Ethylcarbamates on the Inhibition of Human Leukemic HL-60 Cell
Growth
inhibn of human
leukemic HL-60
IC50 (µM)a
inhibn of human
leukemic HL-60
IC50 (µM)
AHMA
derivatives
AHMA-
ethylcarbamates
R1
R2
H
OMe
Me
H
H
H
H
H
H
H
H
H
3
5
6
7
8
0.027
0.011
0.0076
0.29
0.13
14.0
0.15
0.37
1.5
0.12
18
23
24
25
26
27
28
29
30
31
32
33
34
0.0044
0.010
0.0047
0.088
0.0057
3.8
0.031
0.067
0.13
0.032
0.0097
0.011
0.0023
CONHMe
CONH(CH2)2NMe2
NO2
F
Cl
9
10
11
12
13
14
15
16
Br
OMe
OMe
Me
Me
CONHMe
CONH(CH2)2NMe2
CONHMe
0.038
0.093
0.0074
CONH(CH2)2NMe2
a
The IC50 values were determined by computer fit of 5-6 data points from a dose-response range.
synthesized a series of AHMA derivatives with mono-
and disubstituents on the acridine ring and carried out
SAR studies. Table 1 shows the in vitro cytotoxicity of
the parent compound AHMA (3) and its derivatives with
mono- or disubstituent(s) on the acridine ring (5-16)
against the growth of human leukemic HL-60 cell line
in culture. In the series of AHMA derivatives with a
monosubstituent at C4′ of the acridine ring, AHMA with
a C4′-OMe or C4′-Me substituent (5 and 6) or with a
chargeable side chain C4′-CONH(CH2)2NMe2 (8) were
more cytotoxic than AHMA by 2-4 times, while AHMA
with C4′-CONHMe, C4′-NO2, or C4′-halogen substitu-
ents (compounds 7, 9, and 10-12, respectively) were
less potent than the parent compound AHMA. Com-
pound 9 bearing a strong electron-withdrawing nitro
function decreased the cytotoxicity of AHMA dramati-
cally. In the series of AHMA derivatives with disub-
stituents at C4′ and C5′, the compound was approxi-
mately 3 times more potent than AHMA. The cytotoxicity
of compound 5 or 6 is decreased by introducing the
CONHMe group to the C5′-position.
In contrast to m-AMSA derivatives,6 introduction of
the monosubstituent OMe or Me to the C4′-position
enhanced the in vitro antitumor activity of AHMA,
while introduction of disubstituents OMe or Me and
CONHMe to C4′- and C5′-positions, respectively, de-
creased the activity. Unlike N-[2-(N,N-dimethylamino)-
ethyl]-9-aminoacridine-4-carboxamide,4 the in vitro an-
titumor activity of N-[2-(N,N-dimethylamino)ethyl]-
AHMA-4-carboxamide (8) was not parallelyl increased
by adding OMe or Me to the C5′-position, since com-
pound 14 was about 3 times less cytotoxic than 5, while
compound 16 was 2 times more potent than the parent
compound.
showed that monosubstituted derivatives of m-AMSA
with an electron-withdrawing group (such as halogen
and NO2) having smaller pKa values and similar or
smaller DNA-binding constants were found to be less
active against leukemia L1210 in cell culture.26 Intro-
duction of Me or OMe group to the C4′-position of
m-AMSA provides a considerable increase in binding to
poly(dA-dT) over that of the parent compound, but the
carbamoyl or methylcarbamoyl group at the same
position gives no such increase as they bind selectively
to poly(dG-dC). The effects on the inhibition of L1210
cell growth in culture showed that 4′-Me- or 4′-OMe-m-
AMSA was almost as potent as or a little more potent
than the parent m-AMSA, while the in vivo antitumor
activity against mice bearing leukemic P388 revealed
enhanced activity. In the same studies, the carbamoyl
derivative showed a large decrease in its in vitro
antitumor activity but showed an increase in the case
of in vivo studies. The disubstituted 5′-Me- or 5′-OMe-
4′-CONHMe-m-AMSA showed enhancement of tight-
ness of drug-DNA binding as shown by increase of the
binding constant. The in vitro activity of these two
compounds is as potent as or more potent than that of
m-AMSA, while their in vivo activities are higher.
Among these compounds, CI-921 (2) is the most active.
Earlier QSAR studies suggested a significant correla-
tion between antitumor activity of 9-anilinoacridines
and 9-aminoacridine and DNA association constants.
The drug-DNA binding constant is affected by the
substituent(s) at the acridine chromorphore. However,
our SAR studies indicate that the effects of the substit-
uent(s) at the acridine ring on the in vitro cytotoxicity
of AHMA are somewhat different from previous obser-
vations of the QSAR studies of m-AMSA31 and 9-amino-
acridine derivatives4 as exemplified by the increase of
cytotoxicity of 5, 6, and 8. The difference may attribute
to the different tumor cells tested and the different
strength of binding with Topo II. We previously showed
So far the most significant influence on the antitumor
activity of these classes of drug was found to be the
steric effects of groups placed at various positions on
the 9-anilinoacridine skeleton.31 Previous QSAR studies

4744 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Su et al.
Ta ble 2. In Vitro Cytotoxicity of AHMA-alkylcarbamates on the Inhibition of Human Leukemic HL-60 Cell Growth
AHMA (3)
17
Me
0.0085
18
Et
0.0044
19
20
4
21
22
R
n-Pro
0.0080
i-Bu
0.0090
t-Bu
0.024
C6H5
0.161
CH2C6H5
0.029
inhibn of HL-60 IC50 (µM)
0.027
Ta ble 3. Anticancer Activity of AHMA-ethylcarbamate (18) in B6D2F1 Mice Bearing Sarcoma 180 and Lewis Lung Carcinomaa
average wt change (g) average tumor vol (treated control)
tumor cell
Sarcoma 180
dose (mg/kg)
day 7
day 10
day 14
day 7
day 10
day 14
control
3.0
5.0
7.0
control
3.0
-0.5
-2.0
-2.6
-3.4
-1.0
-2.3
-3.0
+0.7
-0.7
+1.0
-0.1
-0.1
-1.3
-2.6
+1.0
-0.8
+1.2
-0.4
+0.5
-0.8
-0.5
1.00
1.24
0.06
0.22
1.00
0.80
0.44
1.00
1.21
0.63
<0.01
1.00
1.00
0.74
0.80
<0.01
1.00
Lewis lung carcinoma
0.63
0.37
0.68
0.52
6.0
a
Sarcoma 180 (3 × 106 cells) was inoculated sc on day 0. Treatment started on day 1, ip, QDx5. Control had 4 mice, and each dose had
2 mice. Lewis lung carcinoma (4 × 106 cells) was inoculated sc on day -2. Treatment started on day 1, ip, QDx5. Control had 4 mice, and
each dose had 3 mice. Tumor sizes were evaluated on days 7, 10, and 14.
that AHMA exhibited superior in vitro and in vivo
antitumor activity as compared to m-AMSA. Aside from
the drug-DNA binding, one can envisage that the
3-amino-5-hydroxymethylaniline moiety of AHMA may
engage in a critical hydrogen bond interaction in the
drug-Topo II binding site which may effect better
binding than that of m-AMSA. Our molecular modeling
study of AHMA-DNA binding also reveals that this
amino functionality, which lies outside the drug-DNA
complex, may be in favor of forming a hydrogen bond
with Topo II at the active binding site. It was shown30
that DNA intercalators (9-aminoacridine phenols and
amsacridine derivatives) possess good activity for Topo
II-mediated DNA strand cleavage, while the activity
was decreased or diminished after methoxylation at the
ortho-position of the phenol or methanesulfonyl func-
tions. It can be seen that o-AMSA is biologically inactive,
suggesting the methoxy group may abolish or inhibit
the drug-enzyme interaction probably due to its electron-
donating properties or steric encumbrance. Therefore,
the antitumor activity of 9-anilinoacridine derivatives
is greatly altered by the substituent(s) on the anilino
ring.
Taking the consideration of the drug-enzyme binding
ability as an important account for drug activity, our
previous SAR studies of AHMA derivatives demon-
strated that AHMA lacking a substituent at 5-CH2OH
exhibited slightly better in vitro antitumor activity
against human leukemic HL-60 and L1210 cell lines in
culture, while the substituent(s) at NH2 and/or CH2OH,
in general, had no significant effect on the cytotoxicity
of AHMA. We also showed that AHMA bearing the
â-acetylpropyl side chain at NH2 only or at both NH2
and CH2OH revealed to be as potent as or slightly more
potent than the parent compound AHMA.16 In the
present studies, we found that AHMA-alkylcarbamates
in general are more potent than their corresponding
parent AHMA derivatives against human leukemic HL-
60 cell growth in culture.
Table 2 shows the in vitro cytotoxicity of AHMA with
various alkylcarbamates that results from altering the
alkylcarbamate chain length and size. This clearly
demonstrates that the cytotoxicity of AHMA-alkylcar-
bamate decreases with increasing length and size of the
alkyl group. Since AHMA-ethylcarbamate was shown
to be the most potent among AHMA-alkylcarbamate
derivatives, AHMA derivatives with substituent(s) at
acridine ring were converted into their ethylcarbamate
derivatives for SAR studies (Table 1). It is also impor-
tant to note that all AHMA-ethylcarbamates (18 and
23-34) were more potent than their corresponding
AHMA derivatives (5-16); among these compounds,
derivative 34 was the most potent with an IC50 value
of 0.0023 µM (10 times more potent than AHMA). Our
present results suggest that introduction of an alkyl-
formate function to the NH2 group of AHMA forming
AHMA-alkylcarbamates may alter the lipophilicity-
hydrophilicity balance as well as the interaction of the
compound with the active site of Topo II enzyme,
thereby possibly affecting the antitumor activity of
9-anilinoacridine derivatives.
Results from the present study involving the search
for a leading compound in the AHMA analogues series,
such as AHMA-ethylcarbamate (18) and its derivative
4′-methyl-5′-N-(dimethylamino)carboxamido-AHMA-eth-
ylcarbamate (34), formed a basis for further in vivo
antitumor evaluation. The antitumor efficacies of AHMA-
ethylcarbamate (18) in B6D2F1 mice bearing sarcoma
and Lewis lung carcinoma are shown in Table 3. Tumor
sizes were reduced >99% and 63% by AHMA-ethylcar-
bamate at 7 and 6 mg/kg, respectively, by intravenous
injection (iv), QDx5. The antileukemic effects of AHMA
(3) and its derivatives, AHMA-ethylcarbamate (18) and
AHMA-tert-butylcarbamate (4), along with m-AMSA (1)
and adriamycin are compared and shown in Table 4.
Percent increase in lifespan (%ILS) was measured in
B6D2F1 mice bearing P388 leukemia. Among these
compounds, adriamycin showed better potency. How-

Synthesis and SAR of Potential Anticancer Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4745
Ta ble 4. Therapeutic Effects of AHMA Hydrochloride, AHMA-ethylcarbamate (18), and AHMA-tert-butylcarbamate (4) in B6D2F1
Mice Bearing P388 Leukemiaa
average wt change (g)
dose (mg/kg) &
schedule, iv
average survival
increase in
lifespan (%)
drug
day 1
day 7
day 9
day 11
timeb (day ( SD)
toxicity death
control
3
0
21.8
23.3
23.8
22.6
22.9
23.0
23.1
23.9
22.3
22.8
22.7
24.5
23.0
23.6
0
133
230
240
188
106
181
245
228
91
0/5
0/5
0/5
0/4
0/5
4/5
0/5
0/5
2/4
0/5
1/5
5/5
0/5
0/5
16 (Q2Dx5)
22 (Q2Dx5)
30 (Q2Dx5)
10 (Q2Dx5)
15 (Q2Dx5)
12 (Q2Dx5)
18 (Q2Dx5)
24 (Q2Dx5)
8 (Q2Dx5)
12 (Q2Dx5)
18 (Q2Dx5)
3 (Q2Dx5)
4 (Q2Dx5)
+0.8
-1.3
+0.2
-0.2
-1.6
-0.10
-1.3
-1.4
+0.1
-0.7
-4.3
-0.4
-1.4
-0.5
-1.5
-0.1
-0.4
-2.0
-0.7
-1.2
-1.4
-2.1
-1.4
- -
-2.1
-2.8
-0.2
-0.1
-3.7
+0.3
-1.6
-1.8
-3.8
-3.2
- -
14.9 ( 1.1
21.1 ( 2.2
21.8 ( 4.0
18.4 ( 1.2
13.2 ( 4.2
18.0 ( 1.3
22.1 ( 2.0
21.0 ( 4.0
12.2 ( 0.5
14.1 ( 1.6
7.6 ( 0.8
18
4
m-AMSA
120
19
adriamycin
-03.2
-1.6
-3.6
-3.2
14.2 ( 1.1
27.4 ( 6.7
122
328c
a
P388/0 ascites cell (0.2 mL) (ascites:saline ) 1.10) was implanted iv on day 0. Tumor grew in liver in mice; Q2D, iv treatment was
given on days 1, 3, 5, 7, and 9. Survival time was recorded based on following death times: 8 a.m. (0.0), 1 p.m. (0.25), 5 p.m. (0.5). c 1
b
of 5 mice died of tumor on day 40.
(4.29 mL, 39 mmol) was added dropwise into a suspension of
III (2.74 g, 13 mmol) in CHCl3/EtOH (1:3 v/v) in an ice bath.
After stirring for 1 h, the crude 9-chloro-4-methoxyacridine
(prepared from 4-methoxy-9(10H)-acridone,26 2.83 g, 13 mmol)
in CHCl3 (15 mL) was added dropwise into the mixture at 0
°C and stirred vigorously for 4 h. The crude solid product was
collected by filtration, washed with EtOH, and then recrystal-
ever, 1 of 5 mice died of tumor on day 40 at 4 mg/kg,
Q2Dx5. Of AHMA derivatives, AHMA-tert-butylcar-
bamate (4) is as potent as AHMA-ethylcarbamate (18)
but has less toxicity to the host. Although compound
18 revealed better antitumor efficacy than AHMA at 10
mg/kg, Q2Dx5, 18 is more toxic than AHMA in the
testing system. From this study, it demonstrats that
AHMA derivatives are more potent than m-AMSA and
yet have less toxicity to the host.
The in vivo therapeutic studies presented in this
paper are preliminary results. More elaborate experi-
ments such as the antitumor efficacy of AHMA-alkyl-
carbamate against human xenografts in nude mice will
be carried out. Actually, some compounds are shown to
have promising antitumor effects in this testing system.
The pKa values and DNA-binding measurements as well
as the computational modeling calculation of AHMA and
AHMA-alkylcarbamate derivatives are in progress at
our laboratory. We hope these studies will lead to a clear
QSAR for new anticancer drug design.
1
lized from MeOH to give 5: 2.02 g (70%); mp 247-8 °C; H
NMR (DMSO-d6) δ 4.16 (1H, s, OMe), 4.38 (2H, s, CH2), 5.20
(1H, br, OH), 5.82 (2H, br, NH2), 6.46, 6.51 and 6.64 (each 1H,
s, 3 × ArH), 7.41 (1H, t, J ) 8.7 Hz, ArH), 7.46 (1H, t, J ) 8.7
Hz, ArH), 7.54 (1H, d, J ) 8.0 Hz, ArH), 7.88 (1H, d, J ) 8.7
Hz, ArH), 7.96 (1H, t, J ) 8.0 Hz, ArH), 8.27 and 8.37 (each
1H, d, J ) 8.6 Hz, 2 × ArH), 11.33 (1H, brs, NH). Anal.
(C21H19N3O2‚2HCl) C, H, N.
Using the same procedure as that for the synthesis of 5,
the following AHMA derivatives were prepared.
4′-Meth yl-AHMA h yd r och lor id e (6) was synthesized
from 4-methyl-9(10H)-acridinone (1.20 g, 5.70 mmol)26 and III
(1.21 g, 5.70 mmol): yield 1.67 g (88%); mp 264-5 °C; 1H NMR
(DMSO-d6) δ 2.78 (1H, s, Me), 4.37 (2H, s, CH2), 5.18 (1H, br,
OH), 5.50 (2H, br, NH2), 6.39, 6.46 and 6.59 (each 1H, s, 3 ×
ArH), 7.37 (1H, t, J ) 8.0 Hz, ArH), 7.43 (1H, t, J ) 8.0 Hz,
ArH), 7.86 (1H, d, J ) 8.0 Hz, ArH), 7.96 (1H, t, J ) 8.0 Hz,
ArH), 8.23 and 8.26 (each 1H, d, J ) 8.8 Hz, 2 × ArH), 8.41
(1H, d, J ) 8.0 Hz, ArH), 11.33 (1H, brs, NH). Anal.
(C21H19N3O‚HCl‚H2O) C, H, N.
4′-Meth ylca r boxa m id o-AHMA (7) was synthesized from
crude 9-chloroacridan-4-methylcarboxamide (3.64 g, 13 mmol)6
and III (2.744 g, 13 mmol): yield 2.86 g (59%); mp 275-6
°C; 1H NMR (DMSO-d6) δ 2.96 (3H, s, NHMe), 4.38 (2H, s,
CH2), 5.21 (1H, br, OH), 5.80 (2H, br, NH2), 6.46, 6.52 and
6.64 (each 1H, s, 3 × ArH), 6.19 (2H, s, 2 × ArH), 7.44 (1H,
m, ArH), 7.51 (1H, m, ArH), 7.96 (1H, m, ArH), 8.11 (1H, m,
ArH), 8.26 (1H, m, ArH), 8.49 (2H, br, ArH), 9.34 and 11.63
(each 1H, br, 2 × NH). Anal. (C22H20N4O2‚HCl‚1/2H2O) C, H,
N.
Exp er im en ta l Section
Melting points were determined on a Fargo melting point
apparatus and are uncorrected. Column chromatography was
carried out on silica gel G60 (70-230 mesh, ASTM; Merck).
Thin-layer chromatography was performed on silica gel G60
F
254 (Merck) with short-wavelength UV light for visualization.
Elemental analyses were done on a Heraeus CHN-O Rapid
instrument. 1H NMR spectra were recorded on a Brucker-400
spectrometer with Me4Si as the internal standard. Chemical
shifts are reported in ppm (δ), and the signals are described
as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
br (broad), and brs (broad singlet). Values reported for coupling
constants are first-order.
4′-Dim eth ylam in oeth ylcar boxam ido-AHMA (8) was syn-
thesized from crude 9-chloroacridan-4-dimethylaminoethyl-
carboxamide (2.05 g, 6.57 mmol)4 and III (1.39 g, 6.57 mmol):
The synthesis of the requisite C4-monosubstituted 9(10H)-
acridones (I, R1 ) 4-OMe, 4-Me, 4-NO2, 4-F, 4-Cl, and
4-NO2)25,26 and 9-oxoacridan-4-carboxylic acids (I , R1
)
1
COOH)27 and the C5-substituted 9-oxoacridan-4-carboxylic
acids (I, R1 ) COOH, R2 ) OMe or Me)27-29 were prepared by
following the literature procedures. Conversion of the C4-
monosubstituted acridones to their 9-chloroacridines (II) or the
C5-substituted 9-oxoacridan-4-carboxylic acids to their meth-
ylcarbamoyl or N,N-dimethylethylenecarbamoyl derivatives
was performed by the known methods.4,6 All 9-chloroacridines
were then used directly to condense with 3,5-diaminobenzyl
alcohol (III) without further purification.
yield 1.83 g (65%); mp 82-3 °C; H NMR (DMSO-d6) δ 2.35
(6H, s, NMe2), 3.47 and 3.62 (each 2H, brs, 2 × CH2) 4.29 (2H,
s, CH2), 5.21 (1H, br, OH), 5.52 (2H, br, NH2), 5.90 (1H, s, ArH),
6.19 (2H, s, 2 × ArH), 7.45 (1H, t, J ) 8.0 Hz, ArH), 7.51 (1H,
t, J ) 8.0 Hz, ArH), 7.87 (1H, d, J ) 8.0 Hz, ArH), 8.14 (1H,
d, J ) 8.8 Hz, ArH), 8.20 (1H, d, J ) 8.8 Hz, ArH), 8.37 (1H,
d, J ) 8.8 Hz, ArH), 8.66 (1H, d, J ) 8.0 Hz, ArH), 9.23 (1H,
br, NH), 12.23 (1H, brs, NH). Anal. (C25H27N5O2‚HCl‚H2O) C,
H, N.
Syn th esis of AHMA Der iva tives (IV) in Ta ble 1. 4′-
Meth oxy-AHMA Hyd r och lor id e (5). 4-Methoxymorpholine
4′-Nitr o-AHMA h yd r och lor id e (9) was synthesized from
4-nitro-9(10H)-acridinone26 (4.8 g, 20.0 mmol) and III (4.22 g,

4746 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Su et al.
20.0). The TLC (SiO2, CHCl3/MeOH, 10:1 v/v) of the crude
products (5.63 g, 81%) showed a mixture of two main com-
pounds (Rf ) 0.6 and 0.3), which were not separated due to
their poor solubility in organic solvent. The mixture was
directly used for the next reaction without further purification.
A small amount of the compound with low Rf value was
isolated by column chromatography (SiO2, CHCl3/MeOH, 10:1
v/v) and then treated with 1 N HCl to form the hydrochloride
2.83 (3H, s, Me), 3.11 (3H, s, NHMe), 4.54 (2H, s, CH2), 5.50
(1H, br, OH), 6.65 (2H, br, NH2), 6.64, 6.67 and 6.82 (each 1H,
s, 3 × ArH), 7.52 (1H, t, J ) 8.0 Hz, ArH), 7.67 (1H, t, J ) 8.0
Hz, ArH), 8.02, 8.26 and 8.67 (each 1H, d, J ) 8.0 Hz, ArH),
9.78 and 11.92 (each 1H, br, NH). Anal. (C23H22N4O2‚HCl‚23/
4H2O) C, H, N.
5 ′-M e t h y l -4 ′-d i m e t h y l a m i n o e t h y l c a r b o x a m i d o -
AHMA (16) was synthesized from crude 5′-methyl-9-chloro-
acridan-5-dimethylaminoethylcarboxamide (1.80 g, 6.0 mmol)4
and III (1.26 g, 6.0 mmol): yield 0.847 g (55%); mp 210-3 °C
dec; 1H NMR (DMSO-d6) δ 2.78 (3H, s, Me), 2.87 (6H, s, NMe2),
3.42 and 3.84 (each 2H, brs, 2 × CH2), 4.46 (2H, s, CH2), 5.20
(1H, br, OH), 7.35, 7.37 and 7.40 (each 1H, s, 3 × ArH), 7.62
(1H, t, J ) 8.0 Hz, ArH), 7.98 (1H, d, J ) 8.0 Hz, ArH), 8.19
(1H, d, J ) 8.0 Hz, ArH), 8.65 (1H, d, J ) 8.0 Hz, ArH), 8.95
(1H, d, J ) 8.0 Hz, ArH), 10.10 (1H, br, NH), 10.08 (1H, brs,
NH), 10.80 (1H, br, NH). Anal. (C26H29N4O2‚HCl) C, H, N.
1
salt and recrystallized from EtOH: mp 219-20 °C; H NMR
(DMSO-d6) δ 4.50 (2H, s, CH2), 5.21 (1H, br, OH), 6.09 (2H,
br, NH2), 7.36 (1H, s, ArH), 7.39 (2H, s, 2 × ArH), 7.50 (1H, t,
J ) 8.0 Hz, ArH), 7.58 (1H, t, J ) 8.0 Hz, ArH), 8.02 (1H, d,
J ) 8.0 Hz, ArH), 8.25 (1H, d, J ) 8.0 Hz, ArH), 8.37 (1H, d,
J ) 8.8 Hz, ArH), 8.81 (1H, d, J ) 8.8 Hz, ArH), 8.86 (1H, d,
J ) 8.0 Hz, ArH), 9.36 (1H, br, NH). Anal. (C20H16N4O3‚HCl‚
H2O) C, H, N.
In a similar manner, compounds 10-12 were synthesized
and purified as described for 9.
Syn th esis of AHMA-a lk ylca r ba m a te Der iva tives (V) in
Ta bles 1 a n d 2. AHMA-m eth ylca r ba m a te (17). Methyl
chloroformate (0.58 g, 5.4 mmol) was added dropwise to a
solution of 3 (1.87 g, 4.50 mmol) in dry DMF (50 mL)
containing pyridine (1.42 mL, 17.6 mmol) at - 20 °C. The
mixture was then stirred at room temperature for 15 min and
evaporated in vacuo to dryness. The residue was coevaporated
several times with EtOH and was chromatographed on a silica
gel column (5 × 50 cm). The impurities were eluted with
CHCl3, while the product was eluted by CHCl3/MeOH (10:1
v/v). The fractions containing the product were collected,
concentrated, and recrystallized twice from DMF/EtOH to give
4′-Flu or o-AHMA (10) was synthesized from 4-fluoro-9(10H)-
acridinone (2.13 g, 10.0 mmol)26 and III (2.11 g, 10.0 mmol).
The crude product yielded 2.40 g, 76%. A small amount of
crude product was purified by silica gel column chromatogra-
phy (CHCl3/MeOH, 20:1 v/v) to give pure 10 for analysis: mp
>280 °C; 1H NMR (DMSO-d6) δ 4.31 (2H, d, J ) 4.2 Hz, CH2),
5.01 (1H, t, J ) 4.2 Hz, OH), 5.98, 6.14, 6.27 (each 1H, s, 3 ×
ArH), 7.12 (2H, m, 2 × ArH), 7.24 (2H, m, 2 × ArH), 7.37 (2H,
brs, NH2), 7.56, 7.71, 7.94 and 8.10 (each 1H, m, 4 × ArH).
Anal. (C20H16FN3O‚H2O) C, H, N.
4′-Ch lor o-AHMA h yd r och lor id e (11) was synthesized
from 4-chloro-9(10H)-acridinone (2.17 g, 10.0 mmol)26 and III
(2.11 g, 10.0 mmol). The crude product yielded 2.42 g 72%.
1
17: 1.08 g (64%); mp 273-4 °C; H NMR (DMSO-d6) δ 3.64
(3H, s, Me), 4.45 (2H, s, CH2), 5.32 (1H, br, OH), 7.44 (2H, s,
2 × ArH), 7.45 (2H, d, J ) 7.3 Hz, Ar), 7.49 (2H, d, J ) 5.6
Hz, 2 × ArH), 8.00 (2H, t, J ) 7.8 Hz, 2 × ArH), 8.09 (2H, d,
J ) 8.4 Hz, 2 × ArH), 8.26 (2H, d, J ) 8.8 Hz, 2 × ArH), 9.91
and 11.49 (each 1H, brs, 2 × NH). Anal. (C22H19N3O3‚HCl‚
H2O) C, H, N.
1
11-HCl salt: mp 232-4 °C; H NMR (DMSO-d6) δ 4.49 (2H,
s, CH2), 5.21 (1H, br, OH), 6.62 (2H, br, NH2), 7.48 (3H, s, 3 ×
ArH), 7.51 (1H, t, J ) 8.0 Hz, ArH), 7.55 (1H, t, J ) 8.0 Hz,
ArH), 8.03 (1H, d, J ) 8.0 Hz, ArH), 8.21 (1H, d, J ) 8.0 Hz,
ArH), 8.47 (1H, d, J ) 8.8 Hz, ArH), 8.55 (1H, d, J ) 8.8 Hz,
ArH), 8.60 (1H, d, J ) 8.0 Hz, ArH), 9.16 (1H, br, NH). Anal.
(C20H16ClN3O‚HCl‚H2O) C, H, N.
Following the same procedure as that for the synthesis of
17, the following AHMA-alkylcarbamates were prepared.
4′-Br om o-AHMA h yd r och lor id e (12) was synthesized
from 4-bromo-9(10H)-acridinone (2.74 g, 10.0 mmol)26 and III
(2.11 g, 10.0 mmol). The crude product yielded 2.97 g 78%.
12-HCl salt: mp >280 °C; 1H NMR (DMSO-d6) δ 4.46 (2H, s,
CH2), 5.18 (1H, br, OH), 6.62 (2H, br, NH2), 7.10 (1H, s, ArH),
7.22 (2H, s, 2 × ArH), 7.39 (1H, t, J ) 8.0 Hz, ArH), 7.50 (1H,
t, J ) 8.0 Hz, ArH), 7.96 (1H, d, J ) 8.0 Hz, ArH), 8.30 (1H,
d, J ) 8.0 Hz, ArH), 8.32 (1H, d, J ) 8.8 Hz, ArH), 8.47-8.50
(2H, m, 2 × ArH), 8.98 (1H, br, NH). Anal. (C20H16BrN3O‚HCl‚
2H2O) C, H, N.
4′-Meth oxy-5′-m eth ylca r boxa m id o-AHMA h yd r och lo-
r id e (13) was synthesized from crude 4-methoxy-9-chloro-
acridan-5-methylcarboxamide (5.55 g, 18.5 mmol)6 and III
(3.91 g, 18.5 mmol): yield 4.40 g (60%); mp 222-3 °C; 1H NMR
(DMSO-d6) δ 2.82 (3H, s, NHMe), 4.13 (3H, s, OMe), 4.38 (2H,
s, CH2), 5.20 (1H, br, OH), 5.80 (2H, br, NH2), 6.45, 6.49 and
6.63 (each 1H, s, 3 × ArH), 7.36 (1H, t, J ) 8.0 Hz, ArH), 7.51
(2H, m, 2 × ArH), 7.80 (1H, d, J ) 8.0 Hz, ArH), 8.51 (1H, d,
J ) 8.0 Hz, ArH), 8.56 (1H, d, J ) 8.0 Hz, ArH), 9.43 (1H, br,
NH). Anal. (C23H22N4O3‚HCl‚1/2H2O) C, H, N.
AHMA-eth ylca r ba m a te (18) was synthesized from 3 (6.30
g, 20 mmol) with ethyl chlorofomate (3.24 g, 30.0 mmol): yield
6.32 g (82%); mp 226-8 °C dec; 1H NMR (DMSO-d6) δ 1.06
(3H, t, J ) 7.0 Hz, Me), 4.10 (2H, q, J ) 7.0 Hz, CH2), 4.45
(2H, s, CH2), 5.35 (1H, br, OH), 6.97 (1H, s, ArH), 7.46 (2H, s,
2 × ArH), 7.48-7.51 (2H, m, 2 × ArH), 8.01, 8.03, 8.05 and
8.07 (each 1H, d, J ) 8.0 Hz, 4 × ArH), 8.26 (1H, d, J ) 8.8
Hz, ArH), 9.91 (1H, s, NH), 11.47 (1H, brs, NH). Anal.
(C23H21N3O3‚HCl‚11/2H2O) C, H, N.
AHMA-p r op ylca r ba m a te (19) was synthesized from 3
(0.630 g, 2.0 mmol) with propyl chlorofomate (0.30 g, 2.4
mmol): yield 0.62 g (78%); mp 208-9 °C; 1H NMR (DMSO-d6)
δ 0.91 (3H, t, J ) 7.1 Hz, Me), 1.59-1.62 (2H, m, CH2), 4.02
(2H, t, J ) 6.4 Hz, CH2), 4.46 (2H, s, CH2), 5.25 (1H, br, OH),
6.93 (1H, s, ArH), 7.46-7.52 (4H, m, 4 × ArH), 7.99-8.03 (2H,
m, 2 × ArH), 8.11-8.13(2H, m, 2 × ArH), 8.28 (1H, d, J ) 8.8
Hz, ArH), 9.89 (1H, s, NH), 11.82 (1H, brs, NH). Anal.
(C24H23N3O3‚HCl‚H2O) C, H, N.
AHMA-isobu tylca r ba m a te h yd r och lor id e (20) was syn-
thesized from 3 (0.63 g, 2.0 mmol) with isobutyl chlorofomate
(0.41 g, 3.0 mmol): yield 0.729 g (88%); mp 255-6 °C; 1H NMR
(DMSO-d6) δ 0.90 (6H, d, J ) 7.0 Hz, 2 × Me), 1.84-1.91 (2H,
m, CH), 3.84 (2H, d, J ) 6.4 Hz, CH2), 4.45 (2H, s, CH2), 5.35
(1H, br, OH), 6.98 (1H, s, ArH), 7.44-7.50 (4H, m, 4 × ArH),
8.00 (2H, t, J ) 8.8 Hz, 2 × ArH), 8.14 (2H, d, J ) 8.8 Hz, 2
× ArH), 8.28 (2H, d, J ) 8.8 Hz, 2 × ArH), 9.89 (1H, s, NH),
11.82 (1H, brs, NH). Anal. (C25H25N3O3‚HCl‚11/2H2O) C, H, N.
AHMA-p h en ylca r ba m a te h yd r och lor id e (21) was syn-
thesized from 3 (0.63 g, 2.0 mmol) with phenyl chlorofomate
(0.468 g, 3.0 mmol): yield 0.787 g (90%); mp 228-9 °C; 1H
NMR (DMSO-d6) δ 4.48 (2H, s, CH2), 5.40 (1H, br, OH), 7.02
(1H, s, ArH), 7.22 (2H, s, 2 × ArH), 7.42-7.52 (6H, m, 6 ×
ArH), 8.00 (2H, m, 2 × ArH), 8.07 (2H, m, 2 × ArH), 8.29 (2H,
m, 2 × ArH), 10.49 (1H, brs, NH), 11.55 (1H, br, NH). Anal.
(C27H21N3O3‚HCl‚11/2H2O) C, H, N.
4′-Me t h o x y -5′-d im e t h y la m in o e t h y lc a r b o x a m id o -
AHMA (14) was synthesized from crude 4-methoxy-9-chloro-
acridan-5-dimethylaminoethylcarboxamide (1.21 g, 3.71 mmol)4
and III (0.783 g, 3.71 mmol): yield 0.904 g (52%); mp >280
1
°C; H NMR (DMSO-d6) δ 2.87 (6H, s, NMe2), 3.41 and 3.82
(each 2H, brs, 2 × CH2), 4.15 (3H, s, OMe), 4.46 (2H, s, CH2),
5.20 (1H, br, OH), 7.13 (1H, s, ArH), 7.19 (2H, s, 2 × ArH),
7.43 (1H, t, J ) 8.0 Hz, ArH), 7.60 (2H, m, 2 × ArH), 7.83
(1H, d, J ) 8.0 Hz, ArH), 8.60 (1H, d, J ) 8.0 Hz, ArH), 8.94
(1H, d, J ) 8.0 Hz, ArH), 10.00 (1H, br, NH), 10.08 (1H, brs,
NH), 12.09 (1H, br, NH). Anal. (C26H29N5O3‚2HCl‚101/2H2O)
C, H, N.
4′-Met h yl-5′-m et h ylca r b oxa m id o-AH MA h yd r och lo-
r id e (15) was synthesized from crude 4-methyl-9-chloroacri-
dan-5-methylcarboxamide (3.58 g, 13 mmol)6 and III (2.74 g,
13 mmol), 4.80 g (95%), mp 275-7 °C; 1H NMR (DMSO-d6): δ

Synthesis and SAR of Potential Anticancer Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23 4747
AHMA-ben zylca r ba m a te h yd r och lor id e (22) was syn-
thesized from 3 (0.65 g, 2.0 mmol) with benzyl chlorofomate
(0.51 g, 3.0 mmol): yield 0.75 g (78%); mp 214-5 °C; 1H NMR
(DMSO-d6) δ 4.50 (2H, s, CH2), 5.51 (2H, s, CH2), 5.31 (1H,
br, OH), 7.02 (1H, s, ArH), 7.37-7.77 (4H, m, 4 × ArH), 8.03
(2H, t, J ) 8.0 Hz, 2 × ArH), 8.21 (2H, d, J ) 8.0 Hz, 2 ×
ArH), 8.33 (2H, d, J ) 8.8 Hz, ArH), 10.11 (1H, s, NH), 11.64
(1H, brs, NH). Anal. (C28H23N3O3‚HCl‚11/2H2O) C, H, N.
4′-Meth oxy-AHMA-eth ylca r ba m a te (23) was synthesized
from 5 (0.345 g, 1.0 mmol) and ethyl chloroformate (0.130 g,
1.2 mmol): yield 195 mg (47%); mp 210-2 °C; 1H NMR
(DMSO-d6) δ 1.23 (3H, t, J ) 6.7 Hz, Me), 4.10 (2H, q, J ) 6.7
Hz, CH2), 4.16 (1H, s, OMe), 4.44 (2H, s, CH2), 5.28 (1H, br,
OH), 6.93 (1H, s, ArH), 7.39-7.53 (4H, m, 4 × ArH), 7.54 (1H,
d, J ) 8.0 Hz, ArH), 7.82 (1H, d, J ) 8.7 Hz, ArH), 7.92 (1H,
t, J ) 8.0 Hz, ArH), 8.25 and 8.36 (each 1H, d, J ) 8.6 Hz, 2
× ArH), 9.87 and 12.10 (each 1H, brs, NH). Anal. (C24H23N3O4‚
HCl‚H2O) C, H, N.
4′-Meth yl-AHMA-eth ylca r ba m a te (24) was synthesized
from 6 (0.584 g, 1.5 mmol) and ethyl chloroformate (0.195 g,
1.8 mmol): yield 0.506 g (73%); mp >280 °C; 1H NMR (DMSO-
d6) δ 1.22 (3H, t, J ) 6.8 Hz, Me), 2.81 (3H, s, Me), 4.09 (2H,
q, J ) 6.8 Hz, CH2), 4.43 4.44 (2H, s, CH2), 6.92 (1H, s, ArH),
7.38-7.49 (4H, m, 4 × ArH), 7.87 (1H, d, J ) 6.8 Hz, ArH),
8.01 (1H, t, J ) 6.8 Hz, ArH), 8.19-8.23 (2H, m, 2 × ArH),
8.46 (1H, d, J ) 8.4 Hz, ArH), 9.88 (1H, brs, NH), 11.48 (1H,
br, NH). Anal. (C24H23N3O3‚HCl‚11/2H2O) C, H, N.
d, J ) 8.8 Hz, ArH), 9.91 (1H, br, NH), 12.00 (1H, brs, NH).
Anal. (C23H20ClN3O3‚HCl‚7H2O) C, H, N.
4′-Br om o-AHMA-eth ylca r ba m a te (30) was synthesized
from crude 4′-bromo-AHMA (12; 1.92 g, 4.5 mmol) and ethyl
chloroformate (0.585 g, 5.4 mmol): yield 1.42 g (68%); mp 200-
1
203 °C; H NMR (DMSO-d6) δ 1.25 (3H, t, J ) 7.0 Hz, Me),
4.11 (2H, q, J ) 7.0 Hz, CH2), 4.44 (2H, s, CH2), 5.20 (1H, br,
OH), 6.95 (1H, s, ArH), 7.34-7.48 (4H, m, 4 × ArH), 8.02 (1H,
d, J ) 8.6 Hz, ArH), 8.24 (1H, d, J ) 8.6 Hz, ArH), 8.35 (1H,
d, J ) 8.8 Hz, ArH), 8.38 (1H, d, J ) 8.8 Hz, ArH), 8.53 (1H,
d, J ) 8.8 Hz, ArH), 9.90 (1H, br, NH), 12.05 (1H, brs, NH).
Anal. (C23H20BrN3O3‚HCl‚11/2H2O) C, H, N.
4′-Meth oxy-5′-m eth ylcar boxam ido-AHMA-eth ylcar bam -
a te h yd r och lor id e (31) was synthesized from 13 (325 mg,
0.805 mmol) and ethyl chloroformate (110 mg, 0.96 mmol):
yield 178 mg (46%); mp 214-5 °C; 1H NMR (DMSO-d6) δ 1.23
(3H, t, J ) 7.4 Hz, Me), 2.94 (3H, s, NHMe), 4.1 (2H, q, J )
7.2 Hz, CH2), 4.15 (3H, s, OMe), 4.44 (2H, s, CH2), 5.52 (1H,
br, OH), 6.96 (1H, s, ArH), 7.42 (1H, t, J ) 8.0 Hz, ArH), 7.46
(2H, s, 2 × ArH), 7.52-7.58 (2H, m, 4 × ArH), 7.73 (1H, d, J
) 8.8 Hz, ArH), 8.46 (1H, d, J ) 8.8 Hz, ArH), 8.55 (1H, d, J
) 7.2 Hz, ArH), 9.37 and 9.9210.11 and 11.71 (each 1H, br,
NH). Anal. (C26H26N4O5‚HCl) C, H, N.
4′-Meth oxy-5′-dim eth ylam in oeth ylcar boxam ido-AHMA-
eth ylca r ba m a te (32) was synthesized from 14 (200 mg, 0.43
mmol) and ethyl chloroformate (56 mg, 0.54 mmol): yield 103
mg (45%); mp 220-1 °C; 1H NMR (DMSO-d6) δ 1.23 (3H, t, J
) 7.1 Hz, Me), 2.88 (6H, s, NMe2), 3.40 and 3.81 (each 2H,
brs, 2 × CH2), 4.11 (2H, q, J ) 7.0 Hz, CH2), 4.15 (3H, s, OMe),
4.44 (2H, s, CH2), 5.20 (1H, br, OH), 6.95 (1H, s, ArH), 7.41-
7.46 (3H, m, 3 × ArH), 7.58-7.60 (2H, m, 2 × ArH), 7.79 (1H,
d, J ) 8.0 Hz, ArH), 8.54 (1H, d, J ) 8.0 Hz, ArH), 8.86 (1H,
d, J ) 8.0 Hz, ArH), 9.88, 9.97 and 10.62 (each 1H, br, NH).
Anal. Calcd for C29H33N5O5‚HCl‚11/2H2O: C, 58.53; H, 6.27; N,
11.78. Found: C, H, N.
4′-Meth yl-5′-m eth ylca r boxa m id o-AHMA-eth ylca r ba m -
a te h yd r och lor id e (33) was synthesized from 15 (732 mg,
2.0 mmol) and ethyl chloroformate (239 mg, 2.2 mmol): yield
596 g (65%); mp 252-3 °C; 1H NMR (DMSO-d6) δ 1.43 (3H, t,
J ) 7.2 Hz, Me), 2.93 (3H, s, Me), 3.17 (3H, s, NHMe), 4.31
(2H, q, J ) 7.2 Hz, CH2), 4.64 (2H, s, CH2), 5.52 (1H, br, OH),
7.14 (1H, s, ArH), 7.55-7.78 (4H, m, 4 × ArH), 8.10, 8.33, 8.68
and 8.86 (each 1H, d, J ) 8.0 Hz, ArH), 9.80, 10.11 and 11.57
(each 1H, br, NH). Anal. (C26H26N4O5‚HCl‚13/4H2O) C, H, N.
4′-Meth yl-5′-d im eth yla m in oeth ylca r boxa m id o-AHMA-
eth ylca r ba m a te (34) was synthesized from 16 (310 mg, 0.75
mmol) and ethyl chloroformate (106 mg, 0.98 mmol): yield 105
mg (30%); mp 240-3 °C; 1H NMR (DMSO-d6) δ 1.21 (3H, t, J
) 7.0 Hz, Me), 2.74 (3H, s, Me), 2.86 (6H, s, NMe2), 3.46 and
3.82 (each 2H, brs, 2 × CH2), 4.09 (2H, q, J ) 7.0 Hz, CH2),
4.44 (2H, s, CH2), 5.20 (1H, br, OH), 6.97 (1H, s, ArH), 7.38
(1H, t, J ) 8.0 Hz, ArH), 7.42 and 7.48 (each 1H, s, 2 × ArH),
7.58 (1H, t, J ) 8.0 Hz, ArH), 7.94, 8.24, 8.59 and 8.93 (each
1H, d, J ) 8.0 Hz, ArH), 9.93, 10.02 and 10.82 (each 1H, brs,
3 × NH). Anal. (C29H33N5O4‚HCl‚5H2O) C, H, N.
4′-Meth ylcar boxam ido-AHMA-eth ylcar bam ate (25) was
synthesized from 7 (0.63 g, 2.0 mmol) and ethyl chloroformate
(0.324 g, 3.0 mmol): yield 0.506 g (73%); mp 232-3 °C; 1H
NMR (DMSO-d6) δ 1.28 (3H, t, J ) 6.4 Hz, Me), 2.60 (3H, s,
NHMe), 4.16 (2H, q, J ) 6.7 Hz, CH2), 4.53 (2H, s, CH2), 5.28
(1H, br, OH), 7.01 (1H, s, ArH), 7.51-7.60 (4H, m, 4 × ArH),
8.08 (1H, t, J ) 8.0 Hz, ArH), 8.15 (1H, d, J ) 8.0 Hz, ArH),
8.24 (1H, d, J ) 8.0 Hz, ArH), 8.48 (2H, d, J ) 8.0 Hz, 2 ×
ArH), 8.93 and 9.93 (each 1H, brs, NH). Anal. (C25H24N4O4‚
HCl‚31/4H2O) C, H, N.
4′-Dim eth yla m in oeth ylca r boxa m id o-AHMA-eth ylca r -
ba m a te (26) was synthesized from 8 (0.215 mg, 0.5 mmol)
and ethyl chloroformate (0.081 g, 0.75 mmol): yield 0.174 g
1
(69%); mp 115-7 °C; H NMR (DMSO-d6) δ 1.23 (3H, t, J )
7.0 Hz, Me), 2.88 (6H, s, NMe2), 3.34 and 3.81 (each 2H, brs,
2 × CH2), 4.10 (2H, q, J ) 7.0 Hz, CH2), 4.44 (2H, s, CH2),
5.38 (1H, br, OH), 6.96 (1H, s, ArH), 7.48-7,55 (4H, m, 4 ×
ArH), 8.00 (1H, d, J ) 8.0 Hz, ArH), 8.19 (1H, d, J ) 8.8 Hz,
ArH), 8.26 (1H, d, J ) 8.8 Hz, ArH), 8.52 (1H, d, J ) 8.8 Hz,
ArH), 8.69 (1H, d, J ) 8.0 Hz, ArH), 9.91 (1H, br, NH), 10.65
(1H, brs, NH). Anal. (C28H31N5O4‚HCl‚H2O) C, H, N.
4′-Nitr o-AHMA-eth ylca r ba m a te (27) was synthesized
from crude 4′-nitro-AHMA (9; 3.60 g, 10 mmol) and ethyl
chloroformate (1.30 g, 12.0 mmol): yield 3.24 g (75%); mp
180-2 °C; 1H NMR (DMSO-d6) δ 1.22 (3H, t, J ) 7.2 Hz, Me),
4.08 (2H, q, J ) 7.2 Hz, CH2), 4.41 (2H, s, CH2), 5.48 (1H, br,
OH), 6.81 (1H, s, ArH), 7.30-7.38 (4H, m, 4 × ArH), 7.50 (1H,
m, ArH), 8.07 (1H, m, ArH), 8.19 (1H, m, ArH), 8.65-8.17 (2H,
m, 2 × ArH), 9.52 and 9.60 (each 1H, brs, NH), 10.85 (1H, br,
NH). Anal. (C23H20N4O5‚HCl‚H2O) C, H, N.
4′-F lu or o-AHMA-eth ylca r ba m a te (28) was synthesized
from crude 4′-fluoro-AHMA (10; 2.19 g, 6.00 mmol) and ethyl
chloroformate (0.78 g, 7.19 mmol): yield 2.19 g (90%); mp
238-9 °C; 1H NMR (DMSO-d6) δ 1.23 (3H, t, J ) 7.2 Hz, Me),
4.11 (2H, q, J ) 7.2 Hz, CH2), 4.46 (2H, s, CH2), 5.23 (1H, br,
OH), 6.99 (1H, s, ArH), 7.36-7.52 (4H, m, 4 × ArH), 7.89-
8.01 (2H, m, 2 × ArH), 8.12 (1H, d, J ) 8.8 Hz, ArH), 8.27-
8.29 (2H, m, 2 × ArH), 9.91 (1H, br, NH), 12.00 (1H, brs, NH).
Anal. (C23H20N3O3‚HCl‚11/4H2O) C, H, N.
Cytoxicity Assa ys. The HL-60 human promyelocytic leu-
kemic cells were cultured at an initial density of 5 × 104 cells/
mL. The cells were maintained in a 5% CO2-humidified
atmosphere at 37 °C in RPMI medium 1640 (GIBCO/BRL)
containing penicillin (100 units/mL), streptomycin (100 µg/mL)
(GIBCO/BRL), and 10% heat-inactivated fetal bovine serum.
For HL-cells in suspension, cytotoxicity was measured by
XTT-tetrazolium microculture assay32 in duplicate in 96-well
microtiter plates following 72-h incubation. The absorbance
of each well was measured with a microplate reader (EK-340,
Bio-Tek, Burlington, VT) as previously discribed.33
Each run
included six to seven concentrations of the tested drugs. Dose-
effect relations data were analyzed for IC50 (concentration for
50% inhibition) with the media-effect plot34 by using a previ-
ously described computer program.35
An im a ls. B6D2F1 mice were obtained from Toconic Farms.
Eight-week-old male mice weighing 20-25 g were used. For
iv injection, the drug was administrated via the tail vein.
Typically, P388 leukemia-bearing mice following 106 cell
4′-Ch lor o-AHMA-eth ylca r ba m a te (29) was synthesized
from crude 4′-chloro-AHMA (11; 1.72 g, 4.5 mmol) and ethyl
chloroformate (0.586 g, 5.4 mmol): yield 1.06 g (56%); mp
174-5 °C; 1H NMR (DMSO-d6) δ 1.24 (3H, t, J ) 7.0 Hz, Me),
4.11 (2H, q, J ) 7.0 Hz, CH2), 4.44 (2H, s, CH2), 5.22 (1H, br,
OH), 6.96 (1H, s, ArH), 7.41-7.49 (4H, m, 4 × ArH), 8.00 (1H,
d, J ) 8.8 Hz, ArH), 8.19 (1H, d, J ) 8.8 Hz, ArH), 8.25 (1H,
d, J ) 8.8 Hz, ArH), 8.35 (1H, d, J ) 8.8 Hz, ArH), 8.87 (1H,

4748 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 23
Su et al.
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implantation by ip injection survived 6-7 days in the control
group. The percentage increases in lifespan (%ILS) in the
treatment group were measured and averaged. All animal
studies were conducted in accordance with the guidelines of
the National Institutes of Health: A Guide for the Care and
Use of Animals.
Ack n ow led gm en t. This work was supported by the
National Health Research Institute, Taiwan, R.O.C.
(Grant No. HDO87-HR-715), and the Elsa U. Pardee
Foundation, MSKCC Preclinical Pharmacology Core
Facility.
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