Synthesis and photodynamic therapy properties of a water-soluble hypocrellin modified by cyclodextrin

Article abstract of DOI:10.1246/cl.2001.838

For improving water solubility of hypocrellin B (HB), a cyclodextrin modified hypocrellin B (HBCD) was designed and synthesized. Electron spin resonance (ESR) measurement indicated that this HB derivative remained photodynamically active in terms of type I and type II mechanisms. HBCD is water-soluble and possesses stronger photosensitized damage ability to calf thymus DNA than hypocrellin B.

Full text of DOI:10.1246/cl.2001.838

8
38
Chemistry Letters 2001
Synthesis and Photodynamic Therapy Properties of
a Water-Soluble Hypocrellin Modified by Cyclodextrin
Zhi-Ze Ou, Jing-Rong Chen, Xue-Song Wang, Bao-Wen Zhang,*and Yi Cao
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100101, P.R. China
(Received May 15, 2001; CL-010447)
For improving water solubility of hypocrellin B (HB), a
cyclodextrin modified hypocrellin B (HBCD) was designed and
synthesized. Electron spin resonance (ESR) measurement indi-
cated that this HB derivative remained photodynamically active
in terms of type I and type II mechanisms. HBCD is water-solu-
ble and possesses stronger photosensitized damage ability to
calf thymus DNA than hypocrellin B.
Hypocrellins, including hypocrellin A (HA) and hypocre-
llin B (HB), are new photodynamic agents isolated from the
fungus of Hypocrella bambuase. These lipid–soluble agents
exhibit strong absorption in the phototherapeutic window
(600–900 nm). They have several advantages over the present
used phototherapy agents, e.g. ready preparation and purifica-
tion, low aggregation tendency, and significantly reduced pho-
1
tosensitivity on normal tissue because of its rapid metabolism.
As a result, HA has been successfully employed in the photody-
2
namic therapy (PDT) treatment of certain skin diseases. The
poor solubility in water limited their further use in the photody-
namic therapy, which is an impetus to design and synthesize
water-soluble hypocrellins. In this letter, a covalently linked
Compound 5 was prepared by treating compound 4 with
ethylenediamine. The compound 6 (HBCD) was synthesized
by amidation of compound 2 with 1.5 equiv of compound 5 in
dry DMF at room temperature using dicyclohexylcarbodi-
imide/1-hydroxybenzotriazole hydrate (DCC/HOBT, 1 equiv/
hypocrellin B of β-cyclodextrin was synthesized for improving
water solubility.3 The amino group was selected as the spacer
for superior water solubility as well as for an affinity increase to
4
the negative charged groove of double-strand DNA.
The compound 2 (MAHB) can be prepared in a yield of
1
.4 equiv) as coupling reagents, and was purified on Sephadex
1
2% by photoreaction of mercaptoacetic acid with hypocrellin
8
1
5
C-25 (30% yield). HBCD was characterized by H NMR and
MALDI-TOF ([M + Na ]: 1780).
B (HB) in the presence of O2. The mechanism for this pho-
toreaction has proven to be a nucleophilic addition of mercap-
toacetic acid anion to triplet HB followed by oxidation. The
+
Irradiation of argon-gassed DMSO-buffer solution (1/1,v/v,
pH = 7) of HBCD (0.1 mM) generated an ESR spectrum
role of O in this reaction, however, is complex. On the one
2
–
ascribed to the semiquinone anion radical of HBCD (HBCD· )
hand, O is indispensable; on the other hand, too high concen-
2
–
(
Figure 1, spectrum A). It has the same position as that of HB·.
tration of O will either quench HB triplet or speed up the oxi-
2
–
The HBCD· was produced by self-electron transfer between a
ground and an excited HBCD. When the oxygen was bubbled
dation of mercaptoacetic acid anion, and consequently reduce
the yield of mono- or di- mercaptoacetic acid substituted HB.
9
through the HBCD solution in the presence of 5,5-dimethyl-1-
1-pyrroline N-oxide (DMPO) and the HBCD solution was irra-
diated for 3 min, the ESR signal of DMPO-superoxide anion
radical adduct was observed (Figure 1, spectrum B).
Superoxide anion radical was generated via electron transfer
Therefore it is necessary to optimize the concentration of O for
achieving high yield. In our experiments, this factor was con-
trolled carefully, and a collection yield as high as 67% was
2
6
obtained when O was bubbled into the reaction system at the
2
rate of 5 mL/min and the molar ratio of mercaptoacetic acid to
HB was enhanced from 50 to 100 (Scheme 1). This mixture of
–
from HBCD· to oxygen. When the oxygen saturated DMSO-
buffer solution (1/1, v/v pH = 7) of HBCD (0.1 mM) was irradi-
ated for 40 s while 2,2,6,6-tetramethylpiperridine (TEMP) was
used as a spin trap, an ESR signal of 2,2,6,6-tetramethyl-1-
piperridinyloxy radical (TEMPO) was observed which could be
generated from the reacting of TEMP with singlet oxygen. The
singlet oxygen was formed via the energy transfer from triplet
HBCD to oxygen. All finds above indicates that HBCD was
photodynamic active in terms of type I and type II mechanism.
It has been reported that hypocrellins and their derivatives
5
- or 8– monomercaptoacetic acid substituted hypocrellin B
(MAHB) is able to meet the PDT requirement because both iso-
mers have the same photochemical and photophysical proper-
ties. Compound 4 was prepared by reaction of β-cyclodextrin
(
β-CD) with tosyl chloride in dry pyridine. A low yield (ca.
7
3
1%) was often obtained in this reaction because the β-
cyclodextrin was difficult to be dehydrated completely. When
we added the equal molar dicyclohexylcarbodiimide (DCC) to
serve as dehydrating agent, the yield increased to 42%.
Copyright © 2001 The Chemical Society of Japan

Chemistry Letters 2001
839
DNA. Further photophysical, photochemical and phototherapy
study on this promising phototherapy agent HBCD is under way.
This research was supported by NNSFC (No. 29971031,
19733100, 20073050).
References and Notes
1
2
J. Z. Diwu and W. Lown Photochem. Photobio., 52, 609
1990).
N.W. Fu, Y.X. Chu, J.Y. An, Acta. Pharm. Sin., 10, 371
1989).
(
(
3
4
S. Samal and K.E. Geckler, Chem. Commun., 2000, 1101.
T. Ikeda, K. Yoshida and H.J. Schneider, J. Am .Chem.
Soc., 117, 1453 (1995).
5
6
Y.Y. He, J.Y. An, W. Zou, L.J. Jiang, J. Photochem.
Photobiol. B: Biol., 44, 45 (1998).
Synthesis of 2, 500 mL of methanol-buffer (1:3 vol/vol, pH
=
11) solution of a mixture of HB (2.8 mM) and mercapto-
acetic acid (0.2 M) was put into a three necked round bot-
tomed flask. The reaction mixture was bubbled with air (5
mL/min) and irradiated (> 470 nm) for 10 h. The mixture
was separated by TLC as ref 7. Mono-substituted HB
1
(
0.63 g) was obtained (yield: 67%). H NMR(400 MHz,
CDCl , δ): 15.97 (s, 2H, exchangeable with D O, phenolic
3
2
OH), 6.56 (s,1H), 3.75–4.2 (s,14H), 2.38 (s,3H), 1.93
–
exhibited photosensitized cleavage and damage to isolated and
cellular DNA, whether under aerobic or anaerobic condition.
(s,3H). FAB-MS ([M-H] ): 617.
1
0
7
8
Y. Matsui and A. Okimoto, Bull. Chem. Soc. Jpn., 51, 3030
(1978).
Herein, calf thymus DNA was used as phototherapeutic target
to examine the photoinduced damage ability of HBCD accord-
Synthesis of 6: compound 2 (10 mmol) and compound 5
(15 mmol) was dissolved in dry DMF (20 ml), and then
DCC (10 mmol), HOBT (14 mmol) was added. After stir-
ring 48 h at room temperature, 200 mL of acetone was
poured into the reaction mixture. The final product, 6
(HBCD), was purified on Sephadex C-25 (30% yield).
1
0
ing to a literature procedure. We compared the capability of
photosensitized damage to calf thymus DNA with HB and
MAHB under the same condition. The binding site remaining
was 62.5%, 59.1%, 35.6%, respectively, when HB, MAHB,
HBCD was added into calf thymus DNA solution separately,
and irradiation was carried out for 30 min with a medium-pres-
sure sodium lamp (λ > 470 nm) under aerobic condition.
Clearly, HBCD exhibited stronger damage to calf thymus DNA
than HB and MAHB under aerobic condition.
1
MALDI-TOF: 1780. H NMR (400 MHz, DMSO-d δ):
6
8.04 (s,1H), 6.42 (s,1H), 5.74 (s,14H), 4.81 (s,7H), 4.55
(s,6H), 3.0–4.1 (62H), 2.77 (s,1H), 2.07 (s,3H), 1.89
(s,3H).
In conclusion, a water-soluble derivative of hypocrellin B
was synthesized by modification with β-cyclodextrin, which
enhanced the PDT ability of hypocrellin B due to the affinity
increase of HBCD to the negatively groove of double-strand
9
Y. Z. Hu, J. Y. An, L. J. Jiang and D. W. Chen, J.
Photochem. Photobiol. A:Chem., 89, 45 (1995).
10 Y. Y. He and L. J. Jiang, Biochim. Biophys. Acta, 1532, 29
(2000).