Esters of 2-(1-hydroxyalkyl)-1,4-dihydroxy-9,10-anthraquinones with melphalan as multifunctional anticancer agents

Article abstract of DOI:10.1016/S0960-894X(01)00260-8

Eight esters of 2-(1-hydroxyalkyl)-1,4-dihydroxy-9,10-anthraquinone with melphalan were prepared and tested for their antitumor activity (S-180) and cytotoxicity. 2-{1-[4-(p-Bis(2-chloroethyl)-aminophenyl)-butanoyloxy]methyl}-1,4- dihydroxy-9,10-anthraquinone and 2-{1-[4-(p-bis(2-chloroethyl)-aminophenyl)-butanoyloxy]ethyl}-1,4-dihydroxy- 9,10-anthraquinone showed remarkable antitumor activity (T/C, 265 and 272%).

Full text of DOI:10.1016/S0960-894X(01)00260-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1473–1476
Esters of 2-(1-Hydroxyalkyl)-1,4-dihydroxy-9,10-anthraquinones
with Melphalan as Multifunctional Anticancer Agents
Guang-Zhu Jin,a Young-Jae Youb and Byung-Zun Ahnb,*
aCollege of Pharmacy, Yanbian University, Yanji, Jilin 133000, China
bCollege of Pharmacy, Chungnam National University, Taejon 305-764, Republic of Korea
Received 8 February 2001; accepted 16 April 2001
Abstract—Eight esters of 2-(1-hydroxyalkyl)-1,4-dihydroxy-9,10-anthraquinone with melphalan were prepared and tested for their
antitumor activity (S-180) and cytotoxicity. 2-{1-[4-(p-Bis(2-chloroethyl)-aminophenyl)-butanoyloxy]methyl}-1,4-dihydroxy-9,10-
anthraquinone and 2-{1-[4-(p-bis(2-chloroethyl)-aminophenyl)-butanoyloxy]ethyl}-1,4-dihydroxy-9,10-anthraquinone showed
remarkable antitumor activity (T/C, 265 and 272%). # 2001 Elsevier Science Ltd. All rights reserved.
1,4-Dihydroxy-9,10-anthraquinone (DHAQ) is
t
he L1210 cells and increased antitumor activity in mice
bearing S-180 ascitic cells.12 The enhanced activity
could be attributed to the synergistic action of DHAQ
and chlorambucil: facilitated transportation of chlor-
ambucil to cell nucleus and subsequent alkylation of the
DNA nucleophiles by chlorambucil moiety. These
results prompted us to prepare esters of 2-(1-hydroxy-
alkyl)-DHAQs with melphalan and investigate their
antitumor activity (Scheme 1).
common structural moiety of anthracycline anticancer
agents1,2 and mitoxanthron.3 The mechanisms involving
DNA intercalation and sequential inhibition of DNA
topoisomerase II are well known.4,5 Anthraquinones
generally bind to DNA by insertion and stacking
between the base pairs of the DNA double helix.6 Our
recent study showed that derivatives of 2-(1-hydroxy-
alkyl)-DHAQ showed antitumor activity.7
Melphalan did not undergo esterification with the 10-
OH of 2-(1-hydroxyalkyl)-DHAQ derivatives under the
presence of DCC (dicyclohexylcarbodiimide) and
DMAP (4-dimethylaminopyridine) due to the presence
of the amino group. Therefore, the amino group was
formylated to produce formylmelphalan according to
the general N-formylation method.13 The DHAQ deri-
vative (0.66 mmol) was added to a solution of for-
mylmelphalan (0.55 mmol), DCC (0.605 mmol) and
DMAP (0.275 mmol) in CH2Cl2 (30 mL) in an ice
bath. After stirring for 3 h at room temperature,
hexane (30 mL) was added to the reaction mixture.
The filtrate was purified over a silica gel column
(hexane/ethyl acetate, 3:1). The esters were obtained
in 30–50% overall yield. The chemical shiftof 1.0
ppm of 10-H in the 1H NMR spectrum verified the
esterification. The multiplet at 3.62 ppm, equivalent
to the eight protons of the bis(2-chloroethyl)amino
group in the 1H NMR spectra of the synthesized
esters confirmed the presence of the chloroethyl moi-
eties of the esters, which are capable of alkylating the
DNA nucleophiles. The racemic 2-(1-hydroxyalkyl)-
DHAQ derivatives were used without resolution for the
esterification.
In contrast, the tumor cell cytotoxicity of melphalan is
believed to be the result of DNA alkylation.8 Similar to
other alkylating agents, the dose limiting factor of mel-
phalan is hematologic suppression.9 Thus, some deriva-
tives of melphalan were synthesized to enhance the
selective accumulation to cancer cell or to increase the
antitumor activity.10,11
In our recent study, esters of 2-(1-hydroxyalkyl)-DHAQs
with chlorambucil showed enhanced cytotoxicity against
*Corresponding author. Tel.: +82-42-821-5923; fax: +82-42-823-
6566; e-mail: ahnbj@cnu.ac.kr
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00260-8

1474
G.-Z. Jin et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1473–1476
Scheme 1. (a) HCOOH, CO(COCH3)2; (b) DCC, DMAP, 0 ꢀC.
with a shorter alkyl chain, expected to offer a relatively
higher hydrophilicity, possessed higher cytotoxicity.
Table 1. Antitumor effect of 2-{1-[4-(p-bis(2-chloroethyl)-aminophe-
nyl)-butanoyloxy]alkyl}-DHAQsa
Consistent with cytotoxicity, the antitumor activity of
the esters tended to be dependent on the size of the alkyl
chains. The esters with shorter alkyl chains displayed
better antitumor activity than the ones with longer
chains. With the exception of Eg and Eh, the other
esters expressed greater antitumor activity than either 2-
(1-hydroxyalkyl)-DHAQs or melphalan itself, suggest-
ing that the anthraquinone and melphalan moieties of
the esters exerted the antitumor action in a synergistic
manner. In other words, in addition to the alkylating
capability of the melphalan component, the DHAQ
moiety should contribute to prolonging the life span of
the mice with their own mechanisms. Survival ratio
(SR) expressing the number of mice that survived after
50 days among the experimental mice must be impor-
tant for evaluation of the antitumor activity. Among the
esters, Ea, Eb and Ec, all of which showed high T/C
values, displayed high SR values. Six of the eight
experimental mice survived more than 50 days after
administration of Eb (R=methyl).
R
R1=mpl
R1=H
d
ED50
(mg/mL)
T/Cb
(%)
SRc
ED50
(mg/mL)
T/Cd
(%)
Ea
Eb
Ec
Ed
Ee
Ef
H
Methyl
Ethyl
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Melphalan
0.086
0.12
0.2
0.97
0.4
3.1
4.5
5.9
0.89
265
272
245
240
230
230
210
205
225
3/8
6/8
3/8
3/8
2/8
1/8
0/8
0/8
3/8
15
1.9
7.2
10.2
23.7
58.0
60
125
139
135
125
110
108
115
103
Eg
Eh
>80
aR=alkyl group; mpl=(p-bis(2-chloroethyl)-aminophenyl)-butanoyl-
oxy group of melphalan.
bMice were injected via ip with 1ꢁ107 S-180 cells on day 0. Com-
pounds were administered ip on days 1, 2, 3, 4, 5, 6 and 7 with a dose
of 16 mmol/kg. Melphalan was administered at 16 mmol/kg.
cSR=survival ratio, no. of mice that survived after 50 days among
eightmice.
dThe data was adopted from ref 7.
The cytotoxicity of the esters against L1210 cells and the
antitumor activity in mice bearing S-180 cells are shown
in Table 1. All of the esters showed an enhanced cyto-
toxic activity when compared to those of 2-(1-hydroxy-
alkyl)-DHAQs. Furthermore, four of them (Ea–c,e,
R0=H, methyl, ethyl, butyl) showed an even higher
cytotoxic activity than melphalan (ED50=0.89 mg/mL).
The derivatives, of which the alkyl chain was H (Ea,
ED50=0.086 mg/mL) or methyl (Eb, ED50=0.12 mg/
mL), exhibited increased cytotoxic activity by factors of
174 and 16 times that of their 2-(1-hydroxyalkyl)-
DHAQs, respectively. The hydrophilicity of the mole-
cule mightbe one facotr responsible for hte enhance-
ment of cytotoxicity. As shown in Table 1, the esters
The intercalating capability of the DHAQ moiety4,5
could be expected to speed up alkylation of the mel-
phalan moiety as an intramolecular reaction. The bio-
reductive alkylation12 and the oxidative stress14,15 may
be also important action mechanisms for the antitumor
activity and were summarized in Figure 1. According to
the proposed mechanism of bioreductive alkylation, the
ester accepts an electron from the redox system of the
cell and converts to an anion radical (II), which trans-
forms to a quinone methide (IV) and melphalan mole-
cule (VI). The reaction II!IV and VI could be
accelerated since the melphalan moiety in II is a good
leaving group. Both IV and VI are capable of alkylating

G.-Z. Jin et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1473–1476
1475
Figure 1. Bioreductive alkylation as a proposed action mechanism of the esters.
nucleophiles in DNA, enzymes, and other molecules.16,17
Furthermore, the radical anion transforms molecular
oxygen to a superoxide, which triggers an oxidative stress.18
2. Budavari, S.; O’Neil, M. J.; Smith, A.; Heckelman, P. E. In
The Merck Index, 11th ed.; Merck & Co.: Rahway, NJ, 1989;
p 2828.
3. Budavari, S.; O’Neil, M. J.; Smith, A.; Heckelman, P. E. In
The Merck Index, 11th ed.; Merck & Co.: Rahway, NJ, 1989;
p 6136.
4. Smith, P. J.; Soues, S. Br. J. Cancer. Suppl. 1994, 23, S47.
5. Corbett, A. H.; Osheroff, N. Chem. Res. Toxicol. 1993, 6,
585.
6. Silverman, R. B. In The Organic Chemistry of Drug
Design and Drug Action; Acdemic: San Diego, CA, 1992; p
236.
7. Jin, G. Z.; Song, G. Y.; Zheng, X. G.; Kim, Y.; Sok, D. E.;
Ahn, B. Z. Arch. Pharm. Res. 1998, 21, 198.
8. Foye, W. O. In Cancer Chemotherapeutic Agent; American
Chemical Society: Washington, DC, 1995; pp 113–114.
9. Mascret, B.; Maraninchi, D.; Gastaut, J. A. Eur. J. Cancer
Clin. Oncol. 1986, 22, 461.
In conclusion, we report that esters of melphalan with 2-
substituted-DHAQs show increased antitumor activity
as well as increased cytotoxicity, with both 2-{1-[4-(p-
bis(2-chloroethyl)-aminophenyl)-butanoyloxy]methyl}-
DHAQ (Ea) and 2-{1-[4-(p-bis(2-chloroethyl)-amino-
phenyl)-butanoyloxy]ethyl}-DHAQ (Eb) displaying the
greatest activity. The results of this study support our
belief that DHAQ can play a multifunctional role.
Acknowledgements
10. Kupczyk-Subotkowska, L.; Tamura, K.; Pal, D.;
Sakaeda, T.; Siahaan, T. J.; Stella, V. J.; Borchardt, R. T. J.
Drug. Target. 1997, 4, 359.
This study was supported by the Korea Science and
Engineering Foundation (KOSEF).
11. Catsoulacos, P.; Papageorgiou, A.; Margarity, E.; Mour-
elatos, D.; Mioglou, E. Oncology 1994, 51, 74.
12. Jin, G. Z.; You, Y. J.; Kim, Y.; Nam, H. N.; Ahn, B. Z.
Eur. J. Med. Chem., in press.
References and Notes
13. Sheehan, J. C.; Yang, D.-D. H. J. Am. Chem. Soc. 1958,
80, 1154.
14. Pan, S.-S.; Pedersen, L.; Bachur, N. R. Mol. Pharmacol.
1981, 19, 184.
1. Budavari, S.; O’Neil, M. J.; Smith, A.; Heckelman, P. E. In
The Merck Index, 11th ed.; Merck & Co.: Rahway, NJ, 1989;
pp 3426–3427.

1476
G.-Z. Jin et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1473–1476
15. Hertzberg, R. P.; Dervan, P. B. Biochemistry 1984, 23,
3934.
17. Osborne, M. R.; Lawley, P. D.; Crofton-Sleigh, C.; War-
ren, W. Chem. Biol. Interact. 1995, 18, 287.
16. Lin, A. J.; Pardini, R. S.; Cosby, L. A.; Lillis, B. J.;
Shansky, C. W.; Sartorelli, A. C. J. Med. Chem. 1973, 16,
1268.
18. Liwo, A.; Jeziorek, D.; Ossowski, T.; Dyl, D.; Tempczyk,
A.; Tarasiuk, J.; Nowacka, M.; Borowski, E.; Woznicki, W.
Acta Biochim. Pol. 1995, 42, 445.