Potent antitumor 9-anilinoacridines and acridines bearing an alkylating N-mustard residue on the acridine chromophore: Synthesis and biological activity

Article abstract of DOI:10.1021/jm060197r

A series of 9-anilinoacridine and acridine derivatives bearing an alkylating N-mustard residue at C 4 of the acridine chromophore were synthesized. The N-mustard pharmacophore was linked to the C 4 of the acridine ring with an O-ethyl (O-C₂), O

Full text of DOI:10.1021/jm060197r

3710
J. Med. Chem. 2006, 49, 3710-3718
Potent Antitumor 9-Anilinoacridines and Acridines Bearing an Alkylating N-Mustard Residue
on the Acridine Chromophore: Synthesis and Biological Activity
Tsann-Long Su,*,† Yi-Wen Lin,† Ting-Chao Chou,‡ Xiuguo Zhang,‡ Valeriy A. Bacherikov,† Ching-Huang Chen,†
Leroy F. Liu,§ and Tsong-Jen Tsai†
Laboratory of Bioorganic Chemistry, Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, Molecular Pharmacology and
Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, and Department of Pharmacology, UniVersity of
Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854
ReceiVed February 20, 2006
A series of 9-anilinoacridine and acridine derivatives bearing an alkylating N-mustard residue at C4 of the
acridine chromophore were synthesized. The N-mustard pharmacophore was linked to the C4 of the acridine
ring with an O-ethyl (O-C2), O-propyl (O-C3), or O-butyl (O-C4) spacer. It revealed that all newly synthesized
compounds were very potent cytotoxic agents against human leukemia and various solid tumors in vitro.
These agents did not exhibit cross-resistance against vinblastine-resistant (CCRF-CEM/VBL) or taxol-resistant
(CCRF-CEM/taxol) cells. It also showed that these agents were DNA cross-linking agents rather than
topoisomerase II inhibitors. Of these agents, compounds 27a and 27c were shown to have potent antitumor
activity in nude mice bearing the human breast carcinoma MX-1 xenograft. The therapeutic efficacies of
these two agents are comparable to that of taxol.
Introduction
Our continued development of gene-targeting alkylating
agents have demonstrated that alkylating N-mustards (aliphatic
mustard) linked to the anilino ring of the 9-anilinoacridine
exhibited potent antitumor effects in inhibiting various human
tumor cell growth both in vitro and in vivo.21,22 The N-mustard
pharmacophore was linked to the C3′ or C4′ position of the
anilino ring with a short spacer: O-ethyl (O-C2), O-butyl (O-
C4), or methyl (C1) linker. The results showed that all
compounds exhibited potent in vitro cytotoxicity against human
lymphoblastic leukemia cells (CCRF-CEM) in culture.32 Studies
on the structure-activity relationships of these N-mustards
showed that their antitumor activity was slightly affected by
the length of the spacer and the location of the N-mustard
pharmacophore on the anilino ring. Among these agents,
compound 8 (BO-0742) exhibited significant cytotoxicity against
CCRF-CEM, with 107-fold higher potency than its parent
analogue, 3-(9-acridinylamino)-5-hydroxymethylaniline (AHMA,
9).23-25 Additionally, it also exhibited a significant cytotoxic
effect against drug-resistant sublines, such as those resistant to
vinblastine and taxol, CCRF-CEM/VBL and CCRF-CEM/taxol,
respectively. Remarkably, compound 8 at one-tenth of the
taxol’s therapeutic dose resulted in complete tumor remission
in nude mice bearing human breast carcinoma MX-1 xenografts.
Furthermore, 8 yielded xenograft tumor suppression of 81-
96% using human T-cell acute lymphoblastic leukemia CCRF-
CEM, colon carcinoma HCT-116, and ovarian adenocarcinoma
SK-OV-3 tumor models. Further studies suggested that the main
mechanism of action for compound 8 is primarily through its
DNA cross-linking activity rather than its inhibitory effect on
topoisomerases.
Gene-targeted alkylating agents formed by linking N-mustard
derivatives to DNA binding affinic molecules have been widely
applied in finding drugs with high sequence-selective binding
to the macromolecules and minimizing the side effects induced
by highly reactive N-mustards.1 The DNA binding affinic
molecules, such as DNA intercalators [e.g., heterocyclic com-
pounds,2,3 9-aminoacridine (e.g., 1, Chart1),4-6 4-anilinoquino-
line (2),7,8 9-anilinoacridine (3 and 4),9 anthraquinone (5),10
cyclopentanthraquinone (6)11] and DNA minor groove binders
[e.g., distamycin A and related analogues,12-20 such as talli-
mustine (7)] have been utilized for such purpose to improve
the antitumor efficacy of N-mustard derivatives. The targeting
N-mustards reported from these literatures were shown to be
more potent than the corresponding untargeted derivatives.
Despite the superior cytotoxicity of the targeting N-mustards,
only a limited number of compounds (e.g., compound 7) were
found to have potential clinical application.20
Among the DNA-targeting mustards, in which 9-anilino-
acridines were used as the DNA-affinic carrier, compounds 3
and 4 were synthesized by linking the alkylating aniline mustard
residue either to the acridine chromophore or to the aniline ring
of the clinical antileukemic amsacrine, respectively.9 However,
these agents were less cytotoxic than amsacrine in inhibiting
murine leukemic P388 or Chinese hamster ovary derived tumor
AA8 cell growth in culture and in animals, although they cross-
linked DNA with high sequence-selectivity and were consider-
ably more cytotoxic than their untargeted mustard counterparts.
The 4-linked analogues (3) showed slightly higher in vivo
antileukemic activity than their corresponding 1′-linked ana-
logues (4), suggesting that the N-mustard residue would prefer
to be linked to the acridine chromophore to have better
cytotoxicity.
These studies suggest that the type of N-mustard pharma-
cophore (aniline mustard or aliphatic mustard), the length of
the spacer between the carrier and N-mustard moiety, and the
location of the N-mustard residue on the 9-anilinoacridine
(linking to the anilino ring or to the acridine chromophore) may
affect their cytotoxicity and antitumor potency. In searching for
more powerful gene-targeting agents, we have synthesized a
series of 9-anilinoacridine and acridine analogues bearing the
N-mustard residue, which was linked to C4 of the acridine
* To whom correspondence should be addressed. E-mail:
tlsu@ibms.sinica.edu.tw. Phone: +886-2-27899045. Fax: +886-2-27827685.
† Institute of Biomedical Sciences, Academia Sinica.
‡ Memorial Sloan-Kettering Cancer Center.
§ University of Medicine and Dentistry of New Jersey - Robert Wood
Johnson Medical School.
10.1021/jm060197r CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/19/2006

Potent Antitumor 9-Anilinoacridines and Acridines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3711
Chart 1. N-Mustards Linked to DNA-Affinic Carriers and AHMA
Chart 2. Newly Synthesized 9-Anilinoacridines and Acridines Bearing N-Mustard Residuea
a a series: n ) 2. b series: n ) 3. c series: n ) 4.
chromophore by using O-C2 or O-C4 as the spacer. The studies
would provide more information for understanding of the effect
of the carrier (9-anilinoacridine or acridine) on the cytotoxicity
and the antitumor potency of the targeting N-mustards. The
results from both in vitro and in vivo models revealed that all
of these newly synthesized compounds exhibited significant
antitumor activity. A thorough description on the synthesis,
antitumor efficacy, and the mechanism of action of these
compounds is provided herein.
1,4-dibromobutane gave 4-(4-bromobutoxy)acridin-9-one (13),
which was then reacted with diethanolamine in diglyme at 115
°C with vigorous stirring for 30 min to yield 14. Prolongation
of the reaction of 14 with methanesulfonyl chloride in CH2Cl2
in the presence of triethylamine afforded 4-bis(2-chloroethyl)-
aminobutoxyacridin-9-one (15) in 70% yield. The N-mustard
substituted acridin-9-ones (11 and 15) were then reacted with
thionyl chloride to give the corresponding 9-chloroacridine
derivatives (12 and 16, respectively), which were used directly
for condensing with various substituted 1,3-phenylenediamines
(17, 19, 20, 21, and 22) and 3-hydroxy-5-hydroxymethylaniline
(18) to form the desired 9-anilinoacridines 23a,b, 25a,b, 26a,b,
27a,b, 28a,c, and 29a,b, which bear a N-mustard moiety at C4
of the acridine chromophore with O-C2 or O-C4 spacer (Chart
2) in moderate to good yield. Compound 23a was converted
into its ethyl carbamate derivative 24 (Chart 2) by reacting with
ethyl chloroformate in dry DMF in the presence of pyridine.
Acridine derivatives bearing a N-mustard pharmacophore at
C4 were prepared by following the same synthetic route for 12
Chemistry
All newly synthesized 9-anilinoacridines and acridines bearing
the N-mustard residue are shown in Chart 2. Acridin-9-one
having a N-mustard moiety was prepared starting from the
known 4-hydroxyacridin-9-one (10).26 Compound 10 was treated
with tris(2-chloroethyl)amine hydrochloride in dry DMF in the
presence of excess K2CO3 at room temperature for 20 h to give
acrdin-9-one (11) bearing a N-mustard residue with O-C2 as a
spacer in low yield (27%) (Scheme 1). Treatment of 10 with

3712 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Su et al.
Scheme 1. Synthesis Substituted 9-Anilinoacridines Having N-Mustard Moiety on Acridine Chromophorea
a Reagents and reaction conditions: (a) tris(2-chloroethyl)amine‚HCl/KF/K2CO3/DMF, room temperature, 20 h; (b) SOCl2/DMF, 80 °C, 40 min; (c)
Br(CH2)4Br/K2CO3/DMF, 40 °C, 2 h; (d) NH(CH2CH2OH)2, diglyme, 115 °C, 30 min; (e) MeSO2Cl/Et3N/CHCl3, room temperature, 3 d; (f)
4-methylmorpholine/CHCl3/EtOH, 0-25 °C, 4-8 h.
Scheme 2. Synthesis of Acridine Derivatives Bearing a
N-Mustard Pharmacophore at C4a
the new compounds showed only 3.1-20-fold resistance.
Another drug-resistant subline CCRF-CEM/VBL that expresses
the multidrug resistance (MDR1) gene product, P-glycoprotein
(Pgp),28 was also used. This cell line showed 1630-fold
resistance to taxol and 517-fold resistance to vinblastine.
However, the new N-mustard compounds showed only 2.9-
26.4-fold resistance. Thus, many of these new N-mustard
compounds have better antiproliferative activity than taxol or
vinblastine against drug-resistant human tumor cells. Table 1
also shows that many of these new N-mustard compounds
possess potent activities against the growth of human solid tumor
cells such as lung carcinoma A549, colon carcinoma HCT-116,
and mammary carcinoma MX-1. Overall, the human leukemic
CCRF-CEM cells seem more sensitive to the new compounds
than the human solid tumor cells (Table 1).
a Reagents and reaction conditions: (a) tris(2-chloroethyl)amine‚HCl/
KF/K2CO3/DMF, 40-50 °C, 8 h; (b) Br(CH2)3Br or Br(CH2)4Br/K2CO3/
DMF, 40-50 °C, 8 h; (c) NH(CH2CH2OH)2, diglyme, 115 °C, 4 h; (d)
MeSO2Cl/Et3N/CH2Cl2, room temperature, 3 d.
and 16 (Scheme 2). The known 4-hydroxyacridine (30)27 was
converted into the desired acridines containing bis(2-chloro-
ethyl)aminoethoxy moiety (31a) by treating with tris(2-chloro-
ethyl)amine hydrochloride in dry DMF in the presence of excess
K2CO3. Similarly, reaction of 30 with 1,3-dibromopropane or
1,4-dibromobutane gave 32b and 32c, respectively, which were
further reacted with diethanolamine to yield 33b,c. Treatment
of 33b,c with methanesulfonyl chloride/triethylamine afforded
4-bis(2-chloroethyl)aminopropoxy- (31b) or 4-bis(2-chloro-
ethyl)aminobutoxy- (31c) acridines, respectively (Chart 2).
Our previous study showed that AHMA ethylcarbamate was
more cytotoxic than AHMA.24 However, in contrast to the
previous studies, 24 was 3-times less cytotoxic than 23a,c,
indicating that compound 23a had increased lipophilicity by
converting to its ethylcarbamate derivative, which did not affect
the potency of the parent compound. In this experiment we also
found that the bioisosteric isomers, compounds 25a,c and 23a,c,
were equipotent against the same tumor cell line in culture.
To appreciate the role of the carrier (9-anilinoacridines vs
acridine) in the newly synthesized compounds, we linked the
N-mustard residue to the C4 of the acridine (i.e., compounds
31a,b,c) with O-ethyl, O-propyl, and O-butyl as the spacer. In
the same experiment, these agents were 3-10-fold less potent
than the corresponding 9-anilinoacridine derivatives. The length
of the spacer, however, did not markedly affect their potency.
The results suggested that the 9-anilinoacridines are a better
carrier than acridine for N-mustard alkylating agents to achieve
higher cytotoxicity.
Biological Results and Discussion
Cytotoxicity in Vitro. Table 1 shows that the new 9-anili-
noacridine derivatives bearing N-mustard residue on the acridine
chromophore (23-29, 31) are very potent cytotoxic agents
against human lymphoblastic leukemic CCRF-CEM cells. The
IC50 values are in the nanomolar ranges. These potencies are
comparable to taxol and are several 100-fold more potent than
the parent compound AHMA, which lacks N-mustard function-
ality. In general, the -(CH2)n- linker between mustard and the
acridine chromophore yields higher in vitro activity when n )
4 than n ) 2. We have also used the leukemic sub-cell-line
resistant to taxol, CCRF-CEM/taxol, which was 457-fold
resistant to taxol and 72-fold resistant to vinblastine. However,
Therapeutic Effects against Human Mammary Carcinoma
MX-1 Xenograft in Nude Mice. The pharmacological and
therapeutic properties of the selected new compounds in terms
of dose, route, and schedule of administration, toxicity, and
efficacy for 26a, 26c, 27a, 27c, and 28c were compared in Table

Potent Antitumor 9-Anilinoacridines and Acridines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3713
Table 1. Cytotoxicity of N-Mustard Derivatives against Human Lymphoblastic Leukemic Cells (CCRF-CEM) and Its Drug-Resistant Sublines
(CCRF-CEM/VBL and CCRF-CEM/Taxol) and Human Solid Tumor (A549, HCT-116, and MX-1 Cells) Cell Growth in Vitroa
IC50 (µM)
lymphoblastic leukemia
CCRF-CEM/taxol
solid tumors
HCT-116
compd
CCRF-CEM
CCRF-CEM/VBL
A549
MX-1
9
8
b
0.753
0.600[0.8×]
1.60[2.7×]
0.047
nd
0.0035
0.0035
0.0066
0.0555
0.0024
0.0580
0.0072
0.0219
0.0100
0.0281
0.0056
0.0071
0.0092
0.0241
0.0056
0.0708
0.0827
0.0820
0.0225
0.0020
0.0070
0.0067
0.0257
0.0042
0.0069
0.0059
0.0041
0.0037
0.0119
0.0042
0.0080
0.0043
0.0123
0.0059
0.0376
0.0353
0.0512
0.0011
0.0004
0.0340[4.9×]
0.0633[9.6×]
0.126[4.9×]
0.0075[1.1×]
0.126[18.8×]
0.231[9.0×]
0.111[26.4×]
0.152[22.0×]
0.338[57.3×]
0.0188[4.6×]
0.0545[14.7×]
0.0469[2.9×]
0.0513[12.3×]
0.0312[2.9×]
0.0423[9.8×]
0.0601[4.9×]
0.0865[14.7×]
0.163[4.3×]
0.253[7.2×]
0.308[6.0×]
1.731[1630×]
0.2066[517×]
0.0056
0.0324
0.0940
0.0053
0.1080
0.0251
0.0449
0.0089
0.0902
0.0190
0.0316
0.0154
0.1183
0.0190
0.1367
0.1663
0.2260
0.0021
0.0011
0.0055
0.0077
0.0595
0.0095
0.1250
0.0091
0.0232
0.0091
0.0538
0.0138
0.0090
0.0136
0.0482
0.0083
0.0949
0.1354
0.1175
0.0015
0.0007
23a
24
23c
25a
25c
26a
26c
27a
27c
28a
28c
29a
29c
31a
31b
31c
0.0405[9.6×]
0.139[20.1×]
0.117[19.7×]
0.0217[5.3×]
0.0611[16.6×]
0.0498[4.2×]
0.0439[10.5×]
0.0465[5.9×]
0.0578[13.4×]
0.0575[4.7×]
0.0899[15.3×]
0.117[3.1×]
0.150[4.2×]
0.393[7.7×]
taxol
vinblastine
0.4843[457×]
0.0287[71.8×]
a Cell growth inhibition for leukemic cells and solid tumor cells were measured by the XTT tetrazolium assay30 and by the sulforhodamine B method,31
respectively, after 72 h incubation for cell growth; nd ) not determined. IC50 values were determined from the dose-effect relationship at six or seven
concentrations of each drug in duplicate by using a mass-action law based computer program.32-34 b Each set of data have a linear correlation coefficient
(R value) of 0.960-0.995 on the median effect plots indicating the mass-action low.32,33 Numbers in the brackets are folds of resistance of the resistant cells
when compared with the IC50’s of the CCRF-CEM parent cells.
Table 2. Therapeutic Effects of 26a, 26c, 27a, 27c, and 28c against Human Mammary Carcinoma MX-1 Xenografts in Nude Micea
dose
schedulec
(iv injection)
antitumor effect
maximum body
weight loss (%)
compd
(mg/ kg)b
T/C (%)
26a
26c
27a
27c
28c
4.0
1.5
10.0
2.0
Q2D5
Q3D2
Q2D3 (on D7, 9, 11)
Q2D3 (on D8, 10, 12)
Q3D2
79 (D19)
6 (D14)
96 (D15)
77 (D14)
20 (D14)
15 (D17)
8 (D14)
15 (D13)
8 (D14)
21 (D12)
2.0
a MX-1 tissue (50 µg) was implanted subcutaneously in mice on day 0. Every other day iv treatments were given as indicated. b Vehicle was 20 µL of
DMSO + 180 µL of saline. c Mice were treated every 2 days (Q2D) or every 3 days (Q3D).
Figure 1. Therapeutic effects of 27a (10 mg/kg, Q2D×3, iv injection) in nude mice bearing human mammary carcinoma MX-1 xenograft (A) and
the body weight changes during treatments as indicated by arrows (B). Mice were treated every 2 days (Q2D).
2. Nude mice bearing human mammary MX-1 xenografts were
used. Among the new compounds, 27a and 27c showed more
impressive therapeutic results.
slightly recovered, but with a 10% decrease on days 17 and 19
(Figure 1B).
Intravenous injection of 27c at a dose as low as 2 mg/kg,
given every other day on days 8, 10, and 12 after tumor
implantation resulted in 77% tumor suppression on day 14
(Figure 2A). The maximal body weight decrease was 8% on
day 14, two days after the last dose (Figure 2B).
Under optimal therapeutic conditions, intravenous injection
of 27a, 10 mg/kg every other day (days 7, 9, 11) after tumor
implantation, yielded as much as 96% tumor suppression (day
15) against MX-1 xenografts in nude mice (Figure 1A). The
maximal toxicity as indicated by body weight decrease was a
15% drop from the initial pretreatment body weight (on day
13), two days after the last dose (Figure 1B). The body weight
Although 27a and 27c were slightly less potent than taxol in
vitro (Table 2), the optimal therapeutic doses of 10 mg/kg
(Figure 1) for 27a and 2 mg/kg (Figure 2) for 27c, respectively,

3714 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Su et al.
Figure 2. Therapeutic effects of 27c (10 mg/kg, Q2D×3, iv injection) in nude mice bearing human mammary carcinoma MX-1 xenograft (A) and
the body weight changes during treatments as indicated by arrows (B). Mice were treated every 2 days (Q2D).
Figure 3. Topoisomerase II-mediated DNA cleavage by VP-16 and N-mustard derivatives 26a,c, 27a,c, 28a,c, and 29a,c. The first lane is the
control without enzyme.
were considerably lower (i.e., more potent) than the optimal
therapeutic dose of taxol (20 mg/kg, Q2D×6) against the same
MX-1 xenograft tumor.21,22
proteins, thiols, or genes, lacking of intrinsic DNA binding
affinity of the core N,N-bis(2-chloroethyl)amine pharmacophore,
and a requirement for bifunctional cross-linking of DNA to be
fully cytotoxic, resulting in lower their potency and producing
a high ratio of genotoxic monoadducts to cross-linkers (up 20:
1).29 It has demonstrated that the targeting of mustards to DNA
by attaching to DNA-affinic carriers facilitates in finding
compounds of higher cytotoxicity and potency than the corre-
sponding untargeted N-mustard moiety.
Topoisomerase II-Mediated DNA Cleavage. Many anti-
tumor acridines are known to induce topoisomerase II-mediated
DNA cleavage. To test whether our newly synthesized com-
pounds act similarly, a topoisomerase II-mediated DNA cleavage
assay was performed. As shown in Figure 3, unlike VP-16,
which induced topoisomerase II-mediated DNA cleavage at 100
µM, none of the newly synthesized compounds induced any
detectable topoisomerase II-mediated DNA cleavage at 100-
200 µM. However, treatment of linear DNA with any of our
newly synthesized compounds resulted in dose-dependent
reduction of gel electrophoretic mobilities (Figure 3). Further-
more, similar reduction of gel electrophoretic mobility was
observed in the absence of topoisomerase II (data not shown).
This result suggests that our newly synthesized agents were able
to cause intramolecular cross-linking of the linear DNA resulting
in reduced electrophoretic mobilities. This result is also
consistent with results obtained from our previous studies which
showed that 9-anilinoacridine derivatives bearing an N-mustard
residue are DNA cross-linking agents.21,22 Together, these results
suggest that our new compounds are DNA cross-linking agents
but not topoisomerase II poisons. Consequently, they may exert
their cytotoxicity primarily through their DNA cross-linking
activity.
Among DNA-targeting mustards using 9-anilinoacridines as
a DNA-affinic carrier, as mentioned previously, compounds 3
and 4 were less cytotoxic than amsacrine, and the 4-linked
analogues (3) showed slightly higher in vivo antileukemic
activity than their corresponding 1′-linked analogues (4),
indicating that the N-mustard residue would prefer to be linked
to the acridone chromophore to have better cytotoxicity. In
contrast, compound 8 and the newly synthesized compounds
(Chart 2) were significantly more cytotoxic and potent than
9.21,22 In the present studies, we have synthesized a series of
N-mustard derivatives in which the aliphatic N-mustard residue
was linked to C4 of the acridine chromophore of 9-anilino-
acridines and clearly demonstrated that these new compounds
were significantly more potent than 9 and comparable to
compound 8 in inhibiting various human tumor cell growth in
vitro. It also revealed that the acridines bearing a N-mustard
residue were 3-10-fold less potent than the corresponding
9-anilinoacridine derivatives. These results demonstrated that
the length of the spacer between N-mustard moiety and carrier,
the type of the N-mustard (aniline mustard or aliphatic mustard),
the linking position of the N-mustard moiety to 9-anilinoacridine
(linking to the anilino ring or acridine chromophore), and the
sort of the carrier (9-anilinoacridine or acridine) affect the
cytotoxicity of the targeting mustards. Additionally, we have
Conclusions
Gene-targeting agents, such as DNA alkylators, have played
an important part in anticancer drug development. A drawback
of using DNA-alkylating agents includes their high reactivity
resulting in loss of the drug’s therapeutic activity against
malignancy by reacting with other cellular components such as

Potent Antitumor 9-Anilinoacridines and Acridines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3715
ArH), 7.92 (1H, m, ArH), 8.40 (1H, m, ArH), 9.45 (1H, brs,
exchangeable, NH). Anal. (C21H26N2O4) C, H, N.
found that the cytotoxicity of the newly synthesized compounds
was not affected by the substituent(s) (CH2OH, Me, or OMe)
on the anilino ring. Among these agents, compounds 27a and
27c exhibited potent in vivo therapeutic effect in nude mice
bearing human breast carcinoma MX-1 xenograft. To realize
the formation of DNA cross-linking or monoadduct and their
sequence-specific binding, the interaction of 27a,c and other
derivatives with DNA doubled strands are currently being
studied in our laboratories.
4-{4-[Bis(2-chloroethyl)amino]butoxy}-10H-acridin-9-one (15).
To a solution of 14 (1.11 g, 3.0 mmol) and triethylamine (1.25
mL, 9.0 mmol) in dry CHCl3 (25 mL) was added dropwise
methanesulfonyl chloride (0.6 mL, 7.5 mmol) at 0 °C. The reaction
mixture was stirred at room temperature for 3 days. The solution
was diluted with CHCl3 (50 mL), washed successively with water
(2 × 30 mL), ice cold aqueous NaHCO3 (30 mL), and ice water
(50 mL), dried over Na2SO4, and evaporated under reduced pressure
to dryness. The residue was recrystallized from EtOH, yield 0.859
g (70%): mp 119-120 °C; 1H NMR (DMSO-d6) δ 1.64 (2H, brs,
CH2), 1.93 (2H, t, J ) 6.7 Hz, CH2), 2.84 (2H, brs, NCH2), 3.34
(4H, brs, 2 × NCH2), 3.61 (4H, brs, 2 × CH2Cl), 4.28 (2H, t, J )
8.5 Hz, OCH2), 7.18 (1H, m, ArH), 7.27 (1H, m, ArH), 7.34 (1H,
m, ArH), 7.72 (1H, m, ArH), 7.80 (1H, m, ArH), 7.99 (1H, m,
ArH), 8.23 (1H, m, ArH), 10.90 (1H, brs, exchangeable, NH). Anal.
(C19H20Cl2N2O2) C, H, N.
Experimental Section
Melting points were determined on a Fargo melting point
apparatus and are uncorrected. Column chromatography was carried
out over silica gel G60 (70-230 mesh, ASTM; Merck). Thin-layer
chromatography was performed on silica gel G60 F254 (Merck)
plates with short wavelength UV light for visualization. Elemental
1
analyses were done on a Heraeus CHN-O Rapid instrument. H
[3-Amino-5-(4-{2-[bis(2-chloroethyl)amino]ethoxy}acridin-9-
yl-amino)phenyl]methanol Hydrochloride (23a). A mixture of
11 (1.52 g, 4.0 mmol) and SOCl2 (5.0 mL) containing 2 drops of
DMF was heated to 80 °C for 40 min. The reaction mixture was
evaporated under reduced pressure to dryness, and the residue was
coevaporated with CHCl3 (3 × 20 mL). The crude yellow product
12 was dissolved in 50 mL of CHCl3 and then filtered to remove
undissolved byproducts. The filtrate was then added dropwise to a
solution of 3,5-diaminobenzyl alcohol‚2HCl (17, 912 mg, 4.2 mmol)
and 4-N-methylmorpholine (2.3 mL, 21 mmol) in EtOH (60 mL)
in an ice bath. After being stirred at 0 °C for 3 h, the reaction
mixture was allowed to warm to room temperature and stirred
overnight. The reaction mixture was acidified with excess concen-
trated HCl (1 mL) in EtOH (10 mL). The solvent was evaporated
to dryness. The residue was chromatographed on a silica gel column
(6 × 25 cm) using CHCl3/MeOH (10:3 v/v) as the eluant. The
fractions containing the product were combined and evaporated in
vacuo to dryness. The solid residue was recrystallized from ethanol/
acetone to give 1.68 g (67%): mp 105-106 °C. 1H NMR (DMSO-
d6) δ 3.79 (4H, s, 2 × NCH2), 3.99 (2H, s, CH2N), 4.20 (4H, s, 2
× CH2Cl), 4.46 (2H, s, ArCH2), 4.74 (2H, s, OCH2), 7.18 (1H, s,
ArH), 7.26 (2H, s, ArH), 7.42-7.51 (2H, m, ArH), 7.60 (1H, m,
ArH), 7.95 (1H, m, ArH), 8.03 (1H, m, ArH), 8.35 (1H, m, ArH),
8.99 (1H, m, ArH), 11.96 (1H, brs, exchangeable NH). Anal.
(C26H28Cl2N4O2‚4HCl‚3H2O) C, H, N.
NMR spectra were recorded on a Bru¨cker DRX-600 spectrometer
with Me4Si as internal standard.
4-{2-[Bis(2-chloroethyl)amino]ethoxy}-10H-acridin-9-one (11).
A mixture of tris(2-chloroethyl)amine hydrochloride (9.64 g, 40
mmol), KF (1.16 g, 20 mmol), and dry powdered K2CO3 (6.91, 50
mmol) in dry DMF (15 mL) was stirred at room temperature for 1
h. A solution of 4-hydroxy-9-oxoacridine26 (10, 2.12 g, 10 mmol)
in dry DMF (5 mL) was added into the above mixture, and it was
stirred at room temperature for 20 h. The reaction mixture was
poured onto ice water (100 mL) and extracted with EtOAc (5 ×
100 mL). The organic extracts were combined, washed with ice
water, dried over Na2SO4, and evaporated in vacuo to dryness. The
residue was recrystallized from EtOH, yield 1.02 g (27%): mp
131-134 °C; 1H NMR (DMSO-d6) δ 3.01 (4H, t, J ) 6.8 Hz, 2 ×
NCH2), 3.20 (2H, t, J ) 5.6 Hz, CH2N), 3.64 (4H, t, J ) 6.7 Hz,
2×CH2Cl), 4.31 (2H, t, J ) 5.7 Hz, OCH2), 7.18 (1H, m, ArH),
7.27 (1H, m, ArH), 7.49 (1H, m, ArH), 7.72 (1H, m, ArH), 7.82
(1H, m, ArH), 7.92 (1H, m, ArH), 8.23 (1H, m, ArH), 10.78 (1H,
brs, exchangeable, NH). Anal. (C19H20Cl2N2O2) C, H, N.
4-(4-Bromobutoxy)-10H-acridin-9-one (13). A solution of
4-hydroxy-9-oxoacridine26 (10, 5.01 g, 24 mmol) and K2CO3 (6.64
g, 48 mmol) in DMF (35 mL) was stirred for 5 min. To the mixture
was added 1,4-dibromobutane (8.6 mL, 72 mmol), and the mixture
was then stirred at 40 °C for 2 h. The mixture was filtered through
a pad of Celite, and the filtrate was evaporated under reduced
pressure to remove DMF. The residue was diluted with water (30
mL) and extracted with CHCl3 (5 × 50 mL). The combined organic
extracts were washed successively with 1% NaOH (50 mL) and
water (30 mL), dried over Na2SO4, and evaporated in vacuo to
dryness. The residue was chromatographed on a silica gel column
(2 × 20 cm) using CHCl3 as the eluant. The fractions containing
the desired product were combined and evaporated, and the residue
was recrystallized from EtOH to give 13, 4.09 g (49%): mp 180-
181 °C; 1H NMR (DMSO-d6) δ 2.15 (4H, m, CH2CH2), 3.60 (2H,
t, J ) 6.0 Hz, CH2Br), 4.24 (2H, t, J ) 9.0 Hz, OCH2), 7.06 (1H,
m, ArH), 7.15 (1H, m, ArH), 7.41 (1H, m, ArH), 7.65 (1H, m,
ArH), 8.04 (1H, m, ArH), 8.48 (1H, m, ArH). Anal. (C17H16BrNO2)
C, H, N.
By following the same procedure as that for compound 23a, the
following N-mustard derivatives linked to the acridine chromophore
of the 9-anilinoacridine were prepared:
[3-Amino-5-(4-{4-[bis(2-chloroethyl)amino]butoxy}acridin-9-
yl-amino)phenyl]methanol Hydrochloride (23c). Compound 23c
was prepared from 15 (815 mg, 2.0 mmol) and 17 (317 mg, 1.5
mmol): yield 275 mg (26%); mp 247-248 °C; 1H NMR (DMSO-
d6) δ 2.04 (4H, m, 2 × CH2), 3.35 (2H, brs, CH2N), 3.57 (4H, t,
J ) 7.0 Hz, 2 × NCH2), 4.12 (4H, t, J ) 7.1, 2 × CH2Cl), 4.41
(2H, brs, OCH2), 4.46 (3H, brs, CH2OH and OH), 7.05 (1H, s,
ArH), 7.08 (1H, s, ArH), 7.11 (1H, s, ArH), 7.41 (1H, m, ArH),
7.49 (1H, m, ArH), 7.57 (1H, m, ArH), 7.86 (1H, m, ArH), 8.01
(1H, m, ArH), 8.32 (1H, m, ArH), 8.77 (1H, m, ArH), 11.66 (1H,
brs, exchangeable, NH). Anal. (C28H32Cl2N4O2‚3HCl‚1.5H2O) C,
H, N.
4-{4-[Bis(2-hydroxyethyl)amino]butoxy}-10H-acridin-9-one
(14). A mixture of 13 (2.77 g, 8.0 mmol) and diethanolamine (5.27
g, 50 mmol) in diglyme (10 mL) was heated at 115 °C with vigorous
stirring for 30 min. After cooling, the mixture was evaporated in
vacuo to remove excess diethanolamine, and the residue was
successively washed with hexane followed by ether. The oily
residue was dissolved in CHCl3 (200 mL), washed with water (6
× 80 mL), dried over Na2SO4, and evaporated under reduced
pressure to dryness. The residue was crystallized from EtOH/hexane
to give 14 as pale-yellow needles, 2.37 g, (80%): mp 124-126
°C; 1H NMR (DMSO-d6) δ 1.64 (2H, m, CH2), 1.99 (2H, m, CH2),
2.59 (2H, t, J ) 5.8 Hz, OCH2), 2.74 (4H, t, J ) 5.0 Hz, 2 ×
CH2OH), 3.76 (6H, m, 3 × NCH2), 6.62 (1H, m, ArH), 6.90 (1H,
m, ArH), 7.15 (1H, t, m, ArH), 7.31 (1H, m, ArH), 7.44 (1H, m,
3-(4-{2-[Bis(2-chloroethyl)amino]ethoxy}acridin-9-ylamino)-
5-hydroxymethylphenol Hydrochloride (25a). Compound 25a
was prepared from 11 (1.90 g, 5.0 mmol) and 3-amino-5-
hydroxybenzyl alcohol (18, 695 mg, 5.0 mmol): yield 1.73 g (73%);
1
mp 237-238 °C; H NMR (DMSO-d6 + D2O) δ 3.80-3.36 (6H,
m, 3 × NCH2), 4.09 (4H, s, 2 × CH2Cl), 4.57 (2H, s, ArCH2),
4.67 (2H, s, CH2), 6.71 (1H, s, ArH), 6.88 (1H, s, ArH), 6.90 (1H,
s, ArH), 7.42-7.40 (3H, m, 3 × ArH), 7.58 (1H, m, ArH), 7.85
(1H, m, ArH), 7.97 (1H, m, ArH), 8.20 (1H, m, ArH), 8.50 (1H,
brs, exchangeable, NH). Anal. (C26H27Cl2N3O3‚2HCl‚H2O) C, H,
N.
3-(4-{4-[Bis(2-chloroethyl)amino]butoxy}acridin-9-ylamino)-
5-hydroxymethylphenol Hydrochloride (25c). Compound 25c was

3716 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Su et al.
prepared from 15 (815 mg, 2.0 mmol) and 18 (278 mg, 2 mmol):
yield 244 mg (23%); mp 195-197 °C; H NMR (DMSO-d6) δ
CH2Cl), 4.39 (2H, t, J ) 8.7 Hz, OCH2), 7.07 (1H, m, ArH), 7.29
(1H, m, ArH), 7.40 (2H, m, ArH), 7.47 (1H, m, ArH), 7.55 (1H,
m, ArH), 7.87 (1H, m, ArH), 7.99 (1H, m, ArH), 8.33 (1H, m,
ArH), 8.80 (1H, m, ArH), 11.74 (2H, brs, exchangeable, NH2),
13.44 (1H, brs, exchangeable, NH). Anal. (C28H32Cl2N4O‚4HCl‚
4.8H2O) C, H, N.
N1-(4-{2-[Bis(2-chloroethyl)amino]ethoxy}acridin-9-yl)-4-
methoxybenzene-1,3-diamine Hydrochloride (29a). Compound
29a was prepared from 11 (759 mg, 2 mmol) and 4-methoxyphen-
ylene-1,3-diamine dihydrochloride (22, 422 mg, 2 mmol): yield
616 mg (62%); mp 195-196 °C; 1H NMR (DMSO-d6) δ 3.79 (4H,
t, J ) 8.7 Hz, 2 × NCH2), 3.91 (3H, s, OMe), 3.97 (2H, brs, CH2N),
4.20 (4H, t, J ) 8.2 Hz, 2 × CH2Cl), 4.72 (2H, t, J ) 8.2 Hz,
OCH2), 7.08-7.18 (2H, m, ArH), 7.35 (1H, m, ArH), 7.39-7.46
(2H, m, ArH), 7.57 (1H, m, ArH), 7.98 (2H, m, ArH), 8.32 (1H,
m, ArH), 8.94 (1H, m, ArH), 11.87 (1H, brs, exchangeable, NH),
13.55 (1H, brs, exchangeable, NH). Anal. (C26H28Cl2N4O2‚4HCl‚
1.5H2O) C, H, N.
N1-(4-{4-[Bis(2-chloroethyl)amino]butoxy}acridin-9-yl)-4-
methoxybenzene-1,3-diamine Hydrochloride (29c). Compound
29c was prepared from 15 (1.22 g, 3 mmol) and 22 (633 mg, 3.0
mmol): yield 499.5 mg (31.6%); mp 195-197 °C. 1H NMR
(DMSO-d6) δ 2.04 (4H, m, CH2CH2), 3.53 (2H, t, J ) 8.7 Hz,
CH2N), 3.58 (4H, t, J ) 8.7 Hz, 2 × NCH2), 3.89 (3H, s, OMe),
4.13 (4H, t, J ) 8.2 Hz, 2 × CH2Cl), 4.38 (2H, t, J ) 8.2 Hz,
OCH2), 7.04 (1H, m, ArH), 7.11 (1H, m, ArH), 7.24 (1H, m, ArH),
7.37 (1H, m, ArH), 7.44 (1H, m, ArH), 7.53 (1H, m, ArH), 7.88
(1H, m, ArH), 7.96 (1H, m, ArH), 8.31 (1H, m, ArH), 8.78 (1H,
m, ArH), 11.72 (2H, brs, exchangeable, NH2), 13.27 (1H, brs,
exchangeable, NH). Anal. (C28H32Cl2N4O2‚6.7HCl‚1.6H2O) C, H,
N.
1
2.03 (4H, m, 2 × CH2), 3.54 (6H, m, 2 × NCH2), 4.08 (4H, s, 2
× CH2Cl), 4.40 (2H, s, OCH2), 4.44 (2H, s, CH2OH), 6.70 (1H, s,
ArH), 6.79 (1H, s, ArH), 6.83 (1H, s, ArH), 7.39 (1H, m, ArH),
7.46 (1H, m, ArH), 7.55 (1H, m, ArH), 7.87 (1H, m, ArH), 7.97
(1H, m, ArH), 8.68 (1H, m, ArH), 9.90 (1H, brs, exchangeable,
NH), 11.49 (2H, brs, exchangeable, 2 × OH). Anal. (C28H31-
Cl2N3O3‚2.5HCl‚0.5H2O) C, H, N.
N1-(4-{2-[Bis(2-chloroethyl)amino]ethoxy}acridin-9-yl)-5-
methylbenzene-1,3-diamine Hydrochloride (26a). Compound 26a
was prepared from 11 (1.14 g, 3 mmol) and 5-methylphenylene-
1,3-diamine‚2HCl (19, 585 mg, 3 mmol): yield 756 mg (52%);
mp 247-278 °C; 1H NMR (DMSO-d6) δ 2.26 (3H, s, ArMe), 3.78
(4H, s, 2 × NCH2), 3.96 (2H, s, CH2N), 4.19 (4H, s, 2 × CH2Cl),
4.73 (2H, s, OCH2), 6.99 (2H, s, ArH), 7.02 (1H, s, ArH), 7.25
(1H, s, ArH), 7.44 (1H, m, ArH), 7.49 (1H, m, ArH), 7.60 (1H, m,
ArH), 7.98 (1H, m, ArH), 8.03 (1H, m, ArH), 8.38 (1H, m, ArH),
8.98 (1H, m, ArH), 11.94 (2H, s, exchangeable, NH2). Anal.
(C26H28Cl2N4O‚7HCl‚H2O) C, H, N.
N1-(4-{4-[Bis(2-chloroethyl)amino]butoxy}acridin-9-yl)-5-
methylbenzene-1,3-diamine Hydrochloride (26c). Compound 26c
was prepared from 15 (1.22 g, 3 mmol) and 19 (585 mg, 3.0
mmol): yield 698 mg (46%); mp 236-237 °C; 1H NMR (DMSO-
d6) δ 1.92 (4H, s, 2 × CH2), 2.25 (3H, s, ArCH3), 3.34 (2H, brs,
CH2N), 3.57 (4H, brs, 2 × NCH2), 4.12 (4H, m, 2 × CH2Cl), 4.41
(2H, m, OCH2), 6.86-6.91 (3H, m, ArH), 7.42 (1H, m, ArH), 7.50
(1H, m, ArH), 7.57 (1H, m, ArH), 7.89 (1H, m, ArH), 8.01 (1H,
m, ArH), 8.33 (1H, m, ArH), 8.75 (1H, m, ArH), 11.62 (2H, m,
exchangeable, NH2), 13.39 (1H, brs, exchangeable, NH). Anal.
(C28H32Cl2N4O‚6HCl) C, H, N.
N1-(4-{2-[Bis(2-chloroethyl)amino]ethoxy}acridin-9-yl)-5-
methoxybenzene-1,3-diamine Hydrochloride (27a). Compound
27a was prepared from 11 (1.14 g, 3.0 mmol) and 5-methoxyphen-
ylene-1,3-diamine‚2HCl (20, 633 mg, 3 mmol): yield 971 mg
(65%); mp 277-178 °C; 1H NMR (DMSO-d6) δ 3.66 (3H, s, OMe),
3.78 (4H, s, 2 × NCH2), 3.95 (2H, brs, CH2N), 4.19 (4H, brs, 2 ×
CH2Cl), 4.73 (2H, s, OCH2), 6.67 (2H, s, ArH), 6.75 (1H, s, ArH),
7.46 (1H, m, ArH), 7.51 (1H, m, ArH), 7.60 (1H, m, ArH), 7.99
(1H, m, ArH), 8.02 (1H, m, ArH), 8.36 (1H, m, ArH), 8.97 (1H,
m, ArH), 11.96 (2H, s, exchangeable, NH2). Anal. (C26H28Cl2N4O2‚
4HCl‚1.5H2O) C, H, N.
N1-(4-{4-[Bis(2-chloroethyl)amino]butoxy}acridin-9-yl)-5-
methoxybenzene-1,3-diamine Hydrochloride (27c). Compound
27c was prepared from 15 (1.22 g, 3 mmol) and 20 (665 mg, 3.15
mmol): yield 536 mg (34%); mp 260-261 °C; 1H NMR (DMSO-
d6) δ 2.04 (4H, s, 2 × CH2), 3.35 (2H, brs, CH2N), 3.58 (4H, brs,
2 × NCH2), 3.67 (3H, s, OMe), 4.11 (4H, m, 2 × CH2Cl), 4.42
(2H, m, OCH2), 6.45 (1H, brs, ArH), 6.47 (1H, brs, ArH), 6.51
(1H, brs, ArH), 7.44 (1H, m, ArH), 7.51 (1H, m, ArH), 7.58 (1H,
m, ArH), 7.92 (1H, m, ArH), 8.01 (1H, m, ArH), 8.34 (1H, m,
ArH), 8.70 (1H, m, ArH), 11.30-11.50 (2H, m, exchangeable,
NH2), 13.35 (1H, brs, exchangeable, NH). Anal. (C28H32Cl2N4O2‚
4HCl‚0.5H2O) C, H, N.
N3-(4-{2-[Bis(2-chloroethyl)amino]ethoxy}acridin-9-yl)-4-
methylbenzene-1,3-diamine Hydrochloride (28a). Compound 28a
was prepared from 11 (759 mg, 2 mmol) and 2,4-diaminotoluene
(21, 244 mg, 2 mmol): yield 508 mg (53%); mp 225-226 °C; 1H
NMR (DMSO-d6) δ 2.35 (3H, s, Me), 3.78 (4H, m, 2 × NCH2),
3.95 (2H, m, CH2N), 4.19 (4H, brs, 2 × CH2Cl), 4.72 (2H, brs,
OCH2), 7.05 (1H, m, ArH), 7.28 (1H, m, ArH), 7.38 (1H, m, ArH),
7.44 (1H, m, ArH), 7.48 (1H, m, ArH), 7.59 (1H, m, ArH), 7.95
(1H, m, ArH), 8.01 (1H, m, ArH), 8.32 (1H, m, ArH), 8.96 (1H,
m, ArH), 11.80 (2H, brs, exchangeable, NH2), 13.68 (1H, brs,
exchangeable, NH). Anal. (C26H28Cl2N4O‚4HCl‚2.2H2O) C, H, N.
N3-(4-{4-[Bis(2-chloroethyl)amino]butoxy}acridin-9-yl)-4-
methylbenzene-1,3-diamine (28c). Compound 28c was prepared
from 15 (1.22 g, 3.0 mmol) and 21 (366 mg, 3 mmol): yield 750
mg (49%); mp 216-218 °C. 1H NMR (DMSO-d6) δ 2.04 (4H, m,
CH2CH2), 2.37 (3H, s, CH3), 3.36 (2H, t, J ) 8.7 Hz, CH2N), 3.58
(4H, t, J ) 8.7 Hz, 2 × NCH2), 4.13 (4H, t, J ) 8.7 Hz, 2 ×
[3-(4-{2-[Bis(2-chloroethyl)amino]ethoxy}acridin-9-ylamino)-
5-hydroxymethylphenyl]carbamic Acid Ethyl Ester (24). To a
mixture of 23a (500 mg, 1.0 mmol) in DMF (15 mL) and pyridine
(0.096 mL, 1.2 mmol) was added dropwise ethyl chloroformate
(0.12 mL, 1.2 mmol) at 0 °C. After being stirred for 50 min in ice
bath, the reaction mixture was evaporated in vacuo to dryness, and
the product was purified by column chromatography (SiO2, CHCl3/
MeOH, 5:1 v/v). The product was recrystallized from ethanol/
hexane/acetone to give 497 mg (72%): mp 151-152 °C. 1H NMR
(DMSO-d6 + D2O) δ 1.20 (3H, s, CH3), 3.02 (4H, brs, 2 × NCH2),
3.17 (2H, brs, CH2N), 3.68 (4H, brs, 2 × CH2Cl), 4.06 (2H, brs,
OCH2), 4.24 (2H, s, CH2), 4.38 (2H, s, ArCH2), 6.37 (1H, s, ArH),
6.75 (1H, s, ArH), 6.9-7.2 (3H, m, ArH), 7.2-7.9 (4H, m, ArH),
8.15 (1H, brs, ArH), 9.49 (1H, s, NH). Anal. (C29H32Cl2N4O4) C,
H, N.
[2-(Acridin-4-yloxy)ethyl]-bis(2-chloroethyl)amine Hydro-
chloride (31a). By following the same procedure as that for 11,
compound 31a was prepared from 4-hydroxyacridine27 (30, 1.17
g, 6.0 mmol), tris(2-chloroethyl)amine hydrochloride (1.74 g, 7.2
mmol), KF (348 mg, 6 mmol), and K2CO3 (4.14 g, 30 mmol): yield
1
0.154 g (7.1%); mp 156-157 °C; H NMR (DMSO-d6) δ 3.84
(4H, t, J ) 8.1 Hz, 2 × NCH2), 3.99 (2H, t, J ) 6.1 Hz, NCH2),
4.29 (4H, t, J ) 8.2 Hz, 2×CH2Cl), 4.80 (2H, t, J ) 6.1 Hz, OCH2),
7.75 (1H, m, ArH), 7.85 (1H, m, ArH), 7.93 (1H, m, ArH), 8.09
(1H, m, ArH), 8.27 (1H, m, ArH), 8.50 (1H, m, ArH), 9.03 (1H,
m, ArH). Anal. (C19H20Cl2N2O‚HCl‚1.1H2O) C, H, N.
4-(3-Bromopropoxy)acridine (32b). By following the same
procedure as that for 13, compound 32b was prepared from 30
(2.92 g, 15 mmol), 1,3-dibromopropane (15.3 mL, 150 mmol), and
K2CO3 (4.14 g, 30 mmol) in DMF: yield 3.45 g (73%); mp 117-
1
119 °C; H NMR (CDCl3) δ 2.64 (2H, m, CH2), 3.79 (2H, t, J )
6.8 Hz, CH2Br), 4.47 (2H, t, J ) 6.8 Hz, OCH2), 7.10 (1H, m,
ArH), 7.44 (1H, m, ArH), 7.54 (1H, m, ArH), 7.60 (1H, m, ArH),
7.76 (1H, m, ArH), 7.98 (1H, m, ArH), 8.35 (1H, m, ArH), 8.73
(1H, m, ArH). Anal. (C16H14BrNO‚0.8H2O) C, H, N.
4-(4-Bromobutoxy)acridine (32c). By following the same
procedure as that for 13, compound 32c was prepared from 30 (1.17
g, 6.0 mmol), 1,3-dibromobutane (2.3 mL, 19 mmol), and K2CO3
(1.17 g, 6 mmol) in DMF: yield 0.95 g (48%); mp 82-83 °C; 1H
NMR (CDCl3) δ 2.23 (4H, m, 2 × CH2), 3.36 (2H, t, J ) 5.5 Hz,

Potent Antitumor 9-Anilinoacridines and Acridines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3717
OCH2), 4.36 (2H, t, J ) 5.2 Hz, CH2Br), 7.05 (1H, m, ArH), 7.43
(1H, m, ArH), 7.54 (1H, m, ArH), 7.58 (1H, m, ArH), 7.76 (1H,
m, ArH), 7.98 (1H, m, ArH), 8.35 (1H, m, ArH), 8.72 (1H, m,
ArH). Anal. (C17H16BrNO) C, H, N.
and Use of Animals and the protocol approved by the Memorial
Sloan-Kettering Cancer Center’s Institutional Animal Care and Use
Committee.
Topoisomerase II-Mediated DNA Cleavage Assay. Topo II-
mediated DNA cleavages were determined by following the
procedure described previously.35 The reaction mixture (20 µL each)
containing 40 mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2,
0.5 mM EDTA, 1 mM ATP, 30 µg/mL bovine serum albumin, 20
ng of 3′-end 32P-labeled YEpG DNA, 10 ng of purified hTopo IIR,
and a test compound was incubated at 37 °C for 30 min. The
reactions were terminated by addition of 5 µL of a solution
containing 5% SDS and 1 mg/mL proteinase K, followed by
incubation for an additional 60 min at 37 °C. DNA samples were
electrophoresed in 1% agarose gel containing 0.5× TPE buffer.
Gels were dried onto Whatman 3MM chromatographic paper and
autoradiographed at -80 °C using Kodak XAR-5 films.
2-[[3-(Acridin-4-yloxy)propyl]-(2-hydroxyethyl)amino]etha-
nol (33b). By following the same procedure as that for 14,
compound 33b was prepared from 32b (3.16 g, 10 mmol) and
diethanolamine (3.15 g, 30 mmol), yield 2.64 g (79%) as syrup:
1H NMR (DMSO-d6) δ 2.04 (2H, m, CH2), 2.59 (4H, brs, 2 ×
NCH2), 2.77 (2H, brs, NCH2), 3.48 (4H, brs, 2 × CH2OH), 4.29
(2H, brs, OCH2), 4.38 (2H, brs, exchangeable, 2 × OH), 7.21 (1H,
m, ArH), 7.51 (1H, m, ArH), 7.63 (1H, m, ArH), 7.67 (1H, m,
ArH), 7.84 (1H, m, ArH), 8.15 (1H, m, ArH), 8.21 (1H, m, ArH),
8.32 (1H, m, ArH). Anal. (C20H24N2O3) C, H, N.
2-[[4-(Acridin-4-yloxy)butyl]-(2-hydroxyethyl)amino]etha-
nol (33c). By following the same procedure as that for 14,
compound 33c was prepared from 32c (1.32 g, 4.0 mmol) and
diethanolamine (1.26 g, 13 mmol): yield 0.98 g (69%) as syrup.
1H NMR (DMSO-d6) δ 1.66 (2H, m, CH2), 1.94 (2H, brs, CH2),
2.59 (4H, brs, 2 × NCH2), 2.61 (2H, brs, NCH2), 3.48 (4H, brs, 2
× CH2OH), 4.24 (2H, brs, OCH2), 4.38 (2H, brs, exchangeable, 2
× OH), 7.21 (1H, m, ArH), 7.51 (1H, m, ArH), 7.63 (1H, m, ArH),
7.67 (1H, m, ArH), 7.84 (1H, m, ArH), 8.15 (1H, m, ArH), 8.21
(1H, m, ArH), 8.32 (1H, m, ArH). Anal. (C21H26N2O3) C, H, N.
Acknowledgment. This work was supported by the National
Science Council, Taiwan (Grant NSC93-2323-B-001-001), and
NIH RO1 grants CA39662 and CA102463 (to L.F.L.). The
NMR spectra of synthesized compounds were obtained at High-
Field Biomacromolecular NMR Core Facility supported by the
National Research Program for Genomic Medicine (Taiwan).
[3-(Acridin-4-yloxy)propyl]-bis(2-chloroethyl)amine Hydro-
chloride (31b). By following the same procedure as that for 15,
compound 31b was prepared from 33b (840 mg, 2.24 mmol),
methanesulfonyl chloride (0.4 mL, 5.2 mmol), and triethylamine
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
1
(0.86 mL, 6.2 mmol): yield 368 mg (40%); mp 148-149 °C; H
References
NMR (DMSO-d6) δ 2.48 (2H, m, CH2), 3.68 (6H, m, 3 × NCH2),
4.17 (4H, t, J ) 8.8 Hz, 2 × CH2Cl), 4.51 (2H, t, J ) 7.3 Hz,
OCH2), 7.67 (1H, m, ArH), 7.79 (1H, m, ArH), 7.89 (1H, m, ArH),
8.01 (1H, m, ArH), 8.22 (1H, m, ArH), 8.45 (1H, m, ArH), 9.09
(1H, m, ArH). Anal. (C20H22Cl2N2O‚1.8HCl‚1.5H2O) C, H, N.
(1) For review, see: Denny, W. A. DNA Minor Groove Alkylating
Agents. Curr. Med. Chem. 2001, 8, 533-544.
(2) Peck, R. M.; O’Connell, A. P.; Creech, H. J. Heterocyclic Derivatives
of 2-Chloroethyl Sulfide with Antitumor Activity. J. Med. Chem.
1966, 9, 217-221.
(3) Creech, H. J.; Preston, R. K.; Peck, R. M.; O’Connell, A. P. Antitumor
and Mutagenic Properties of a Variety of Heterocyclic Nitrogen and
Sulfur Mustards. J. Med. Chem. 1972, 15, 739-746.
[4-(Acridin-4-yloxy)butyl]-bis(2-chloroethyl)amine Hydro-
chloride (31c). By following the same procedure as that for 15,
compound 31c was prepared from 33c (3.19 g, 27 mmol),
methanesulfonyl chloride (1.74 mL, 22.5 mmol), and triethylamine
(4) Kohn, K. W.; Orr, A.; O’Connor, P. M.; Guziec, L. J.; Guziec, F. S.
Synthesis and DNA-Sequence Selectivity of a Series of Mono- and
Difunctional 9-Aminoacridine Nitrogen Mustards. J. Med. Chem.
1994, 37, 67-72.
1
(3.76 mL, 27 mmol): yield 1.76 g (50%); mp 136-137 °C. H
NMR (DMSO-d6) δ 2.08 (4H, m, 2×CH2), 3.40 (2H, m, NCH2),
3.59 (4H, t, J ) 8.4 Hz, 2 × NCH2), 4.12 (4H, t, J ) 8.4 Hz, 2 ×
CH2Cl), 4.45 (2H, t, J ) 7.3 Hz, OCH2), 7.68 (1H, m, ArH), 7.79
(1H, m, ArH), 7.89 (1H, m, ArH), 8.00 (1H, m, ArH), 8.21 (1H,
m, ArH), 8.45 (1H, m, ArH), 9.04 (1H, m, ArH). Anal. (C21H24-
Cl2N2O‚2HCl‚0.4H2O) C, H, N.
(5) Gourdie, T. A.; Valu, K. K.; Gravatt, G. L.; Boritzki, T. J.; Baguley,
B. C.; Wakelin, L. P. G.; Wilson, W. R.; Woodgate, P. D.; Denny,
W. A. DNA-Directed Alkylating Agents. 1. Structure-Activity
Relationships for Acridine-Linked Aniline Mustards: Consequences
of Varying the Reactivity of the Mustard. J. Med. Chem. 1990, 33,
1177-1186.
(6) Valu, K. K.; Gourdie, T. A.; Gravatt, G. L.; Boritzki, T. J.; Woodgate,
P. D.; Baguley, B. C.; Denny, W. A. DNA-Directed Alkylating
Agents. 3. Structure-Activity Relationships for Acridine-Linked
Aniline Mustards: Consequences of Varying the Length of the Linker
Chain. J. Med. Chem. 1990, 33, 3014-3019.
(7) Gravatt, G. L.; Baguley, B. C.; Wilson, W. R.; Denny, W. A. DNA-
Directed Alkylating Agents. 4. 4-Anilinoquinoline-Based Minor
Groove Directed Aniline Mustards. J. Med. Chem. 1991, 34, 1552-
1560.
(8) McClean, S.; Costelloe, C.; Denny, W. A.; Searcey, M.; Wakelin,
L. P. G. Sequence Selectivity, Cross-Linking Efficacy and Cytotox-
icity of DNA-Targeted 4-Anilinoquinoline Aniline Mustards. Anti-
Cancer Drug Des. 1999, 14, 187-204.
(9) Fan, J.-Y.; Valu, K. K.; Woodgate, P. D.; Baguley, B. C.; Denny,
W. A. Aniline Mustard Analogues of the DNA-Intercalating Agent
Amsacrine: DNA Intercalation and Biological Activity. Anti-Cancer
Drug Des. 1997, 12, 181-203.
(10) Koyama, M.; Takahashi, K.; Chou, T.-C.; Darzynkiewicz, Z.;
Kapuscinski, J.; Kelly, T. R.; Watanabe, K. A. Intercalating Agents
with Covalent Bond Forming Capability. A Novel Type of Potential
Anticancer Agents. 2. Derivatives of Chrysophanol and Emodin. J.
Med. Chem. 1989, 32, 1594-1599.
(11) Ko¨hler, B.; Su, T.-L.; Chou, T.-C.; Jiang, X.-J.; Watanabe, K. A.
Synthesis of Cyclopentanthraquinones: Analogues of Mitomycin C.
J. Org. Chem. 1993, 58, 1680-1686.
Biological Experiments. Cytotoxicity Assays. The effects of
the compounds on cell growth were determined in T-cell acute
lymphocytic leukemia CCRF-CEM) and human solid tumor cells
(i.e., lung adenocarcinoma A549, colon carcinoma HCT-116, and
breast carcinoma MX-1), in a 72 h incubation, by XTT-tetrazolium
assay30 and by sulforhodamine B method,31 respectively. After the
addition of phenazine methosulfate-XTT solution at 37 °C for 6 h,
absorbance at 450 and 630 nm was detected on a microplate reader
(EL 340; Bio-Tek Instruments Inc., Winooski, VT). Six to seven
concentrations of each compound were used. The IC50 and dose-
effect relationships of the compounds for antitumor activity were
calculated by a median-effect plot,32,33 using a computer program
on an IBM-PC workstation.34
In Vivo Studies. Athymic nude mice bearing the nu/nu gene
were used for human breast tumor MX-1, human T cell acute
lymphoblastic leukemia CCRF-CEM, human colon carcinoma
HCT-116, and human ovarian adenocarcinoma SK-OV-3 xe-
nografts. Outbred Swiss-background mice were obtained from
Charies River Breeding Laboratories. Male mice 8 weeks old or
older weighing 22 g or more were used for most experiments. Drug
was administrated via the tail vein by iv injection. Tumor volumes
were assessed by measuring length × width × height (or width)
by using caliper. Vehicle used was 20 µL of DMSO in 180 µL of
saline. All animal studies were conducted in accordance with the
guidelines of the National Institutes of Health Guide for the Care
(12) Wyatt, M. D.; Garbiras, B. J.; Haskell, M. K.; Lee, M.; Souhami, R.
L.; Hartley, J. A. Structure-Activity Relationship of a Series of
Nitrogen Mustard- and Pyrrole-Containing Minor Groove Binding
Agents Related to Distamycin. Anti-Cancer Drug Des. 1994, 9, 511-
525.

3718 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Su et al.
(13) Wyatt, M. D.; Lee, M.; Hartley, J. A. Alkylation Specificity for a
Series of Distamycin Analogues that Tether Chlorambucil. Anti-
Cancer Drug Des. 1997, 12, 49-60.
(14) Arcamone, F. M.; Animati, F.; Barbiery, B.; Configliacchi, E.;
D’Alessio, R.; Geroni, C.; Giuliani, F. C.; Lazzari, E.; Menozzi, M.;
Mongelli, N.; Penco, S.; Verini, M. A. Synthesis, DNA-Binding
Properties, and Antitumor Activity of Novel Distamycin Derivatives.
J. Med. Chem. 1989, 32, 774-778.
(15) Turner, P. R.; Ferguson, L. R.; Denny, W. A. Polybenzamide
Mustards: Structure-Activity Relationships for DNA Sequence-
Specific Alkylation. Anti-Cancer Drug Des. 1999, 14, 61-70.
(16) Baraldi, P. G.; Romagnoli, R.; Pavani, M. G.; Nunez, M. del C.;
Bingham, J. P.; Hartey, J. A. Benzoyl and Cinnamoyl Nitrogen
Mustard Derivatives of Benzoheterocyclic Analogues of Tallimus-
tine: Synthesis and Antitumor Activity. Bioorg. Med. Chem. 2002,
10, 1611-1618.
(17) Baraldi, P. G.; Balboni, G.; Romagnoli, R.; Spalluto, G.; Cozzi, P.;
Geroni, C.; Mongelli, N.; Rutigliano, C.; Bianchi, N.; Gambari, R.
PNU 157977: a New Potent Antitumor Agent Exhibiting Low In
Vivo Toxicity in Mice Injected with L1210 Leukemia Cells. Anti-
Cancer Drug Des. 1999, 14, 71-76.
(18) Cozzi, P.; Beria, I.; Caldarelli, M.; Capolongo, L.; Geroni, C.;
Mazzini, S.; Ragg, E. Phenyl Sulfur Mustard Derivatives of Dista-
mycin. Bioorg. Med. Chem. Lett. 2000, 10, 1653-1656.
(19) Baraldi, P. G.; Romagnoli, R.; Guadix, A. E.; Pineda des las Infantas,
M. J.; Gallo, M. A.; Espinosa, A.; Martinez, A.; Bingham, J. P.;
Hartley, J. A. Design, Synthesis, and Biological Activity of Hybrid
Compounds Between Uramustine and DNA Minor Groove Binder
Distamycin A. J. Med. Chem. 2002, 45, 3630-3638.
(20) Weiss, G. R.; Poggesi, I.; Rocchetti, M.; Demaria, D.; Mooneyham,
T.; Reilly, D.; Vitek, L. V.; Whaley, F.; Patricia, E.; von Hoff, D.
D.; O’Dwyer, P. A Phase I and Pharmacokinetic Study of Tallimus-
tine [PNU 152241 (FCE 24517)] in Patients with Advanced Cancer.
Clin. Cancer Res. 1998, 4, 53-59.
(24) Su, T.-L.; Chen, C.-H.; Huang, L.-F.; Chen, C.-H.; Basu, M. K.;
Zhang, X.-G.; Chou, T.-C. Synthesis and Structure-Activity Rela-
tionships of Potential Anticancer Agents: Alkylcarbamates of 3-(9-
Acridinylamino)-5-hydroxymethylaniline. J. Med. Chem. 1999, 42,
4741-4748.
(25) Su, T.-L. Development of DNA Topoisomerase II-Mediated Anti-
cancer Agents, 3-(9-Acridinylamino)-5-hydroxymethylaniline (AH-
MAs) and Related Compounds. Curr. Med. Chem. 2002, 9, 1677-
1688.
(26) Denny, W. A.; Atwell, G. J.; Cain, B. F. Potential Antitumor Agents.
32. Role of Agent Base Strength in the Quantitative Structure-
Antitumor Relationships for 4′-(9-Acridinylamino)methanesulfona-
nilide Analogues. J. Med. Chem. 1979, 22, 1453-1460.
(27) Knomu, M. The Synthesis of Certain Quinolone and Acridine
Compounds. J. Am. Chem. Soc. 1927, 49, 810-818.
(28) Chou, T.-C.; Depew, K. M.; Zheng. Y.-H.; Safer, M. L.; Chan, D.;
Helfrich, B.; Zatorska, D. A.; Bornmann, W.; Danishefsky, S. L.
Reversal of Anticancer Multidrug Resistance by the Ardeemins. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 8369-8374.
(29) Kaldor, J. M.; Day, N. E.; Hemminki, K. Quantifying the Carcino-
genicity of Antineoplastic Drugs. Eur. J. Cancer Clin. Oncol. 1988,
24, 703-711.
(30) Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.; Tierney,
S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R. Evaluation
of Soluble Tetrazolium/Formazan Assay for Cell Growth and Drug
Sensitivity in Culture Using Human and Other Tumor Cell Lines.
Cancer Res. 1988, 48, 4827-4833.
(31) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenny, S.; Boyd, M. R.
New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening.
J. Natl. Cancer Inst. 1990, 82, 1107-1112.
(32) Chou, T.-C.; Talalay, P. Quantitative Analysis of Dose-Effect
Relationships: the Combined Effects of Multiple Drugs or Enzyme
Inhibitors. AdV. Enzyme Regul. 1984, 22, 27-55.
(21) Bacherikov, V. A.; Chou, T.-C.; Dong, H.-J.; Chen, C.-H.; Lin, Y.-
W.; Tsai, T.-J.; Su, T.-L. Potent Antitumor N-Mustard Derivatives
of 9-Anilinoacridine, Synthesis and Antitumor Evaluation. Bioorg.
Med. Chem. Lett. 2004, 14, 4719-4722.
(22) Bacherikov, V. A.; Chou, T.-C.; Dong, H.-J.; Zhang, X.; Chen, C.-
H.; Lin, Y.-W.; Tsai, T.-J.; Lee, R.-Z.; Liu, L. F.; Su, T.-L. Potent
Antitumor 9-Anilinoacridines Bearing an Alkylating N-Mustard
Residue on the Anilino Ring: Synthesis and Biological Activity.
Bioorg. Med. Chem. 2005, 13, 3993-4006.
(33) Chou, T.-C. The Median-Effect Principle and the Combination Index
for Quantitation of Synergism and Antagonism. In Synergism and
Antagonism in Chemotherapy; Chou, T.-C., Rideout, D., Eds.;
Academic Press: New York, 1991; pp 61-102.
(34) Chou, T.-C.; Martin, N. CompuSyn for Dose-Effect Analysis and
for Quantitation of Synergism and Antagonism. Software and Manual.
ComboSyn, Inc.: NJ, 2005.
(23) Su, T.-L.; Chou, T.-C.; Kim, J. Y.; Huang, J.-T.; Ciszewska, G.; Ren,
W.-Y.; Otter, G. M.; Sirotnak, F. M.; Watanabe, K. A. 9-Substituted
Acridine Derivatives with Long Half-Life and Potent Antitumor
Activity: Synthesis and Structure-Activity Relationships. J. Med.
Chem. 1995, 38, 3226-3235.
(35) Rowe, T. C.; Chen, G. L.; Hsiang, Y.-H.; Liu, F. L. DNA Damage
by Antitumor Acridines Mediated by Mammalian DNA Topoi-
somerase II. Cancer Res. 1986, 46, 2021-2026.
JM060197R