Toward hypericin-derived potential photodynamic therapy agents

Article abstract of DOI:10.1562/0031-8655(2001)074<0211:THDPPT>2.0.CO;2

To optimize a hypericin derivative as a potential photodynamic therapy agent its light-induced singlet oxygen/superoxide radical formation capability should be enhanced and its long-wavelength absorption band should be bathochromically shifted to better match medicinal lasers. A heavy-atom-substituted derivative was realized by electrophilic iodination of hypericin to yield 2,5-diiodo-hypericin. Using photodestruction of bilirubin IXα this derivative was demonstrated to exhibit an enhanced light-induced singlet oxygen/superoxide radical formation capability as compared to hypericin. With respect to a bathochromically shifted derivative styryl residues were attached to the methyl groups of hypericin by de novo ring synthesis. Although the long-wavelength absorption band of this derivative displayed a bathochromic shift of nearly 40 nm it unfortunately immediately underwent an intramolecular [2 + 2] cycloaddition to yield the corresponding cyclobutane derivative in which the added conjugation system became interrupted.

Full text of DOI:10.1562/0031-8655(2001)074<0211:THDPPT>2.0.CO;2

Toward Hypericin-derived Potential Photodynamic Therapy Agents
Author(s): Roland A. Obermüller, Karin Hohenthanner, Heinz Falk
Source: Photochemistry and Photobiology, 74(2):211-215.
Published By: American Society for Photobiology
https://doi.org/10.1562/0031-8655(2001)074<0211:THDPPT>2.0.CO;2
URL: http://www.bioone.org/doi/full/10.1562/0031-8655%282001%29074%3C0211%3ATHDPPT
%3E2.0.CO%3B2
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Photochemistry and Photobiology, 2001, 74(2): 211–215
Toward Hypericin-derived Potential Photodynamic Therapy Agents¶
Roland A. Obermu¨ller, Karin Hohenthanner and Heinz Falk*
Institute of Chemistry, Johannes Kepler University, Linz, Austria
Received 2 November 2000; accepted 6 May 2001
ABSTRACT
prerequisite, derivatives of this kind should retain the main
functional groups of 1. Accordingly, synthesis efforts should
be restricted to a derivatization of the carbon atoms of 1.
In principle, the first goal could be reached by heavy-atom
substitution of the aromatic protons of 1 because it is known
that such substitution would enhance intersystem crossing
and concomitantly also enhance, to a certain degree, singlet
oxygen sensitization (2). The second goal could be attained
by either substituting the aromatic protons by conjugation
extending substituents or by enlarging conjugation via the
two methyl groups in positions 10 and 11.
To optimize a hypericin derivative as a potential photo-
dynamic therapy agent its light-induced singlet oxygen/
superoxide radical formation capability should be en-
hanced and its long-wavelength absorption band should
be bathochromically shifted to better match medicinal
lasers. A heavy-atom–substituted derivative was realized
by electrophilic iodination of hypericin to yield 2,5-diio-
do-hypericin. Using photodestruction of bilirubin IX
␣
this derivative was demonstrated to exhibit an enhanced
light-induced singlet oxygen/superoxide radical forma-
tion capability as compared to hypericin. With respect to
a bathochromically shifted derivative styryl residues
were attached to the methyl groups of hypericin by de
novo ring synthesis. Although the long-wavelength ab-
sorption band of this derivative displayed a bathochrom-
ic shift of nearly 40 nm it unfortunately immediately un-
It might be mentioned that chelation of the peri-hydroxyl/
carbonyl region of 1 should also result in a bathochromic
shift; however, such chelates would be rather unstable under
the mainly hydrolytic physiological conditions as experi-
ϩ
ϩ
ϩ
ϩ
ϩ
enced for the Al3 , Fe3 , Cu2 , Gd3 and Tb3 chelates of 1
(7). Following a suggestion of Peter de Witte we prepared
the mono- and bis-diphenyl-boron chelates of 1 (displaying
derwent an intramolecular [2
the corresponding cyclobutane derivative in which the
added conjugation system became interrupted.
؉ 2] cycloaddition to yield
electrospray mass peaks at m/z
ϭ 667 and 831 corresponding
to the mono- and bis-diphenylboryl complexes), which
might have constituted one of the utmost stable chelates to
be thought of. Although the main absorption bands of these
complexes were found to be shifted to 645 nm they proved
to be extremely sensitive with respect to hydrolysis being
stable only in aprotic solvents. Accordingly, they were found
to be unsuitable as photodynamic therapy agents.
On the one hand we report here attempts to synthesize a
heavy-atom–substituted derivative of 1 to check whether this
leads to enhanced singlet oxygen production, and on the oth-
er hand the preparation of a conjugation-enhanced derivative
to test to what extension the absorption band of 1 could be
bathochromically shifted by this means.
INTRODUCTION
Hypericin (1) the photosensitizing pigment from the ubiq-
uitous proliferating weed, St. John’s wort (Hypericum per-
foratum L.), constitutes a promising agent for the photody-
namic therapy of certain viral or tumoral ailments (for re-
views see Kraus et al. [1] and Falk [2]). Besides its capa-
bility to acidify its surroundings following excitation (3–6)
this is thought to be mainly due to its pronounced tendency
to sensitize the formation of singlet oxygen and to a smaller
extent of superoxide radicals (2). However, the efficiency of
1 for this application might be improved with respect to two
issues. Firstly, it might be desirable to enhance the quantum
efficiency of the singlet oxygen/superoxide radical forma-
tion, and secondly, shift the main absorption band wave-
length of hypericin, or more precisely of the 3-hypericinate
ion (2) which is slightly below 600 nm, to higher wave-
lengths in order to match it better with the emission wave-
length of common medicinal lasers. It is thought that, as a
¶Posted on the website on 8 June 2001.
*To whom correspondence should be addressed at: Institute of Chem-
istry, Johannes Kepler University, Altenbergerstrasse 69, A 4040
Linz, Austria. Fax: 732-2468-8747; e-mail: hfalk@soft.uni-linz.ac.at
†Abbreviations: NMR, nuclear magnetic resonance.
MATERIALS AND METHODS
Chemicals and syntheses. Hypericin was prepared and purified ac-
᭧
2001 American Society for Photobiology 0031-8655/01
$5.00
ϩ0.00
cording to Falk et al. (8) and Kapinus et al. (9). Bilirubin IX␣ was
211

212 Roland A. Obermu¨ller et al.
of commercial origin (Sigma Chemical Co., Vienna, Austria). Sol-
vents and reagents of highest quality were obtained from commercial
suppliers. 1,6,8-Trimethoxy-anthraquinon-3-yl-methyl-triphenyl-
phosphonium bromide (3) was prepared according to Falk and Tran
(10).
Pyridinium-2,5-diiodo-1,4,6,8,13-pentahydroxy-10,11-dimethyl-
phenanthro[1,10,9,8-opqra]perylene-7,14-dione-3-olate (2·pyridine;
C35H19I2NO8). To a solution of 35 mg 1 (0.105 mmol) in 12 cm3
absolute pyridine, 1.2 cm3 of a solution of I2 in absolute pyridine (c
ϭ
0.4 mol·dmϪ3) was added under an argon atmosphere and under
protection from light. After stirring for 2 h pyridine was distilled off
on a rotavapor and the residue was washed twice with a 20% aque-
ous solution of Na2S2O3, three times with H2O and dried overnight
at high vacuum over P4O10. It should be noted that the pyridinium
ion could not be removed by means of acidification of a solution of
2·pyridine because as deduced by electrospray mass spectrum and
1H nuclear magnetic resonance (1H NMR†) spectra 2 became par-
tially deiodinated by this operation. Yield: 76.4 mg (87%); m.p.:
1
Ͼ300
Њ
C; H NMR (200 MHz,
␦
, dimethyl sulfoxide-d6): 15.76 (s,
OH-1,6), 13.71 (s, OH-8,13), 8.88 (m, Hpyr-2,6), 8.50 (m, Hpyr-4),
7.98 (m, Hpyr-3,5), 7.33 (s, H-9,12), 2.69 (s, CH3-10,11) ppm; 13C
NMR (50 MHz, ␦, dimethyl sulfoxide-d6): 182.7 (OϭC-1,14), 170.4,
Figure 1. Normalized absorption changes (A/A0) at ␭ ϭ 457 nm
with time of aerated solutions of disodium bilirubinate IX (1
10Ϫ mol·dm3) in 80%
166.0, 161.3, 145.6, 143.8, 142.7, 127.0, 125.7, 125.14, 120.6,
120.2, 118.8, 115.6, 107.4, 101.5, 82.4 (Car), 23.9 (CH3-10,11) ppm;
␣
ϫ
5
6
10Ϫ mol·dmϪ3) containing 1Ϫ or 2Ϫ (3
ϫ
electrospray mass spectrum: m/z
idine; c
10Ϫ5 mol·dmϪ3): ␭ ϭ 604 (50 900), 559 (14 000), 390
(12 080), 345 (33 950), 303 (37 000) nm ( ); fluorescence (pyridine;
615 (1), 656 (0.6)
ϭ 755 ([M Ϫ
H]Ϫ); UV–Vis (pyr-
aqueous ethanol upon irradiation at ␭ Ͼ 570 nm.
ϭ
1 ϫ
⑀
7
3
c
ϭ
1
ϫ
10Ϫ mol·dmϪ
,
␭
ϭ
550 nm):
␭
ϭ
em
exc
room temperature in the dark. The precipitate was centrifuged,
washed three times with 3% HCl, three times with H2O and dried
over P4O10 under high vacuum. Yield: 106.5 mg (100%); due to its
sensitivity it could be characterized only by its electrospray mass
nm (relative intensity); ⌽f ϭ 0.02.
(E)-1,3,8-Trimethoxy-6-styryl-anthraquinone (4; C25H20O5). A
mixture of 512 mg 3 (0.78 mmol), 222 mg dry and freshly powdered
K2CO3 (1.61 mmol), 149 mg 18-crown-6 (0.56 mmol) and 30 cm3
CH2Cl2 (p.a., absolute) was refluxed for 15 min. To the resulting
dark blue ylide solution 835 mg freshly distilled benzaldehyde (7.87
mmol) dissolved in 30 cm3 CH2Cl2 (p.a., absolute) was added drop-
wise in three portions whereby after each addition the mixture was
refluxed for 40 min. After refluxing the reaction mixture for an ad-
ditional 30 min it was cooled to room temperature, diluted with 350
cm3 CH2Cl2, filtered and extracted with saturated NaCl solution. The
organic phase was dried with anhydrous Na2SO4, filtered and the
solvent evaporated on a rotavapor. The resulting brown oil was trit-
urated with 150 cm3 ether, the yellow product was filtered, thor-
spectrum: m/z
ϭ 681 ([M Ϫ
H]Ϫ) and its qualitative UV–Vis: ␭ ϭ
599 (33), 565 (38), 403 (59), 334 (100) nm (relative intensity).
(E,E)-1,3,4,6,8,13-Hexahydroxy-10,11-distyryl-phenanthro[1,10,9,
8-opqra]perylene-7,14-dione (7; C44H24O8). When a solution of 6
in acetone was irradiated under bubbling with air using the usual
method of preparation of 1 (8) a primary, but intermediate, product
absorbing at 632 nm could be observed. However, because of its
pronounced photosensibility we were not able to isolate it in a pure
form. Therefore, the final product 8 from the intramolecular two-
step photoreaction comprising 7 as its intermediate was prepared
and characterized as described below.
(E,E)-1,3,4,6,8,13-Hexahydroxy-10,11-diphenyl-9b,11a-dihydro-di-
benzo[bcef]cyclobuta[m]coronene-7,14-dione (8; C44H24O8). A solu-
tion of 72 mg 6 in 1.8 dm3 acetone (p.a.) (6 was dissolved by means
of sonication) was bubbled with air and then irradiated for 1.5 h by
means of a 700 W tungsten lamp. The solvent of the resulting red
solution was evaporated on a rotavapor and the residue separated by
oughly washed with ether and dried on high vacuum. Yield: 306 mg
1
(98%); m.p.: 183–188
6.86 (m, 9 Har CH
(s, OCH3) ppm; 13C NMR (50 MHz,
181.7 (C O), 164.0, 162.0, 160.4 (3 Car–O), 142.9, 136.5, 133.7,
136.5, 129.1, 128.84, 128.80, 128.6, 127.2, 122.8, 120.2, 118.7,
Њ
ϭ
C; H NMR (200 MHz,
␦
, acetone-d6): 7.93–
ϩ
CH), 4.02 (s, OCH3), 3.99 (s, OCH3), 3.96
, acetone-d6): 184.5 (C O),
␦
ϭ
ϭ
117.1, 105.6, 102.2, (C
UV–Vis (methanol; c
ϭ
ϭ
C
1
ϩ
ϫ
Car), 56.7 (OCH3), 56.1 (OCH3) ppm;
means of preparative thin layer chromatography (silica, 20
0.2 cm) using tetrahydrofuran/acetic acid 10/1 as the eluent. Yield:
, dimethyl sulfoxide-d6): 18.2
ϫ 20 ϫ
5
10Ϫ mol·dmϪ3): ␭ ϭ 419 (7100), 293
ϭ
(18 700), 225 (30 900) 201 (47 400) nm (
⑀).
1
14.2 mg (20%); H NMR (500 MHz,
␦
(E)-1,3,8-Trihydroxy-6-styryl-10H-anthracene-9-one (5; C22H16O4).
A mixture of 104 mg 4 (0.26 mmol), 2.11 g SnCl2·2H2O, 100 cm3 HBr
(47%) and 400 cm3 glacial acetic acid (p.a.) was refluxed for 4 h. The
reaction mixture was then cooled to room temperature, poured into 1500
cm3 H2O and centrifuged. The residue was washed twice with H2O
(br. s, OH), 14.5 (br. s, 4 OH), 7.7–6.6 (m, 14 Har), 4.13 (m, AB-part
of ABCD-system, 2 CHcyclobutane), 3.55 (m, CD-part of ABCD-system,
2 CHcyclobutane) ppm; 13C NMR (50 MHz,
␦, dimethyl sulfoxide-d6):
186.1 (CϭO), 174.2 (Cbay–O), 167.0, 155.0, 145.7, 142.7, 141.1,
141.0, 140.6, 131.7, 131.6, 128.7, 127.4, 127.3, 125.5, 120.4, 114.6,
110.3, 67.4 (2 CHcyclobutane), 38.4 (2 CHcyclobutane) ppm; 2D C–H cor-
and dried over P4O10 in high vacuum. Yield: 67 mg (75%); m.p.:
1
1
relation cross peak between the 13C and H signals at 67.4 and 4.13
225–229
Њ
C; H NMR (500 MHz,
␦
, acetone-d6): 12.45 (s, OH-1),
-part of the AA MM X-
C), 7.40 (M,M -part,
C–H), 7.16
12.32 (s, OH-8), 9.85 (s, OH-3), 7.65 (AA
system, Hph-2,6), 7.44 (d, J 16.3 Hz, H–C
Hph-3,5), 7.32 (X-part, Hph-4), 7.21 (d, J
Ј
Ј
Ј
ppm and 38.4 and 3.55 ppm, respectively, electrospray mass spectrum:
m/z
505 (21), 470 (29), 342 (79) nm (relative intensity); fluorescence
(acetone, 550 nm): 590.4 (100), 637 (32) nm (relative
intensity).
Photodestruction of bilirubin IX
Falk (11) irradiation of the thermostated (20.0
lutions of sodium bilirubinate, together with 1Ϫ or 2Ϫ in 80% aque-
ous ethanol contained in SiO2 cuvettes (d 1 cm), were performed
ϭ 679 ([M Ϫ
H]Ϫ); UV–Vis (acetone): ␭ ϭ 585 (100), 543 (49),
ϭ
ϭ
Ј
ϭ
16.3 Hz, Cϭ
(s, H-5), 7.02 (s, H-7), 6.49 (m, H-4), 6.30 (m, H-2), 4.37 (s, CH2)
ppm; 13C NMR (50 MHz,
, acetone-d6): 192.6 (C O), 166.6, 165.8,
163.9 (3 Car–O), 146.1, 145.8, 143.4, 137.8, 133.6, 129.8, 129.4,
128.6, 128.3, 115.6, 113.4, 110.3, 110.1, 108.3, 102.2 (C Car),
10Ϫ mol·dmϪ3): ␭ ϭ
␭
ϭ
␭
ϭ
em
exc
␦
ϭ
␣
. Following Hagenbuchner and
0.1 C) aerated so-
ϭC ϩ
Ϯ
Њ
5
33.6 (CH2) ppm; UV–Vis (methanol; c
388 (32 500) nm ( ).
ϭ 1 ϫ
⑀
ϭ
(E,E)-1,3,4,6,8,15-Hexahydroxy-10,13-distyryl-dibenzo[a,o]pery-
lene-7,16-dione (6; C44H26O8). A mixture of 108.1 mg 5 (0.314
mmol), 4.4 mg FeSO4·7H2O (0.016 mmol), 162.2 mg pyridine-N-ox-
ide (1.71 mmol), 1.6 cm3 pyridine and 160 mm3 piperidine were
by means of a 300 W tungsten lamp and a cut-off filter blocking
light below 570 nm and measuring the UV–Vis spectra at intervals
of 20 s (Fig. 1).
General. Melting points were measured by means of a Kofler hot
stage microscope (Reichert, Vienna, Austria)—they are uncorrected.
1H and 13C NMR spectra were measured on the Bruker DPX 200
stirred under argon with protection from light for 1 h at 105
cooling down to room temperature the dark blue reaction mixture was
poured into 12 cm3 HCl (c 2 mol·dmϪ3) and stirred for 30 min at
ЊC. After
ϭ
and DRX 500 spectrometers at 20ЊC using the solvent proton signal

Photochemistry and Photobiology, 2001, 74(2) 213
Extension of conjugation
as internal reference. Two-dimensional C–H correlation was mea-
sured using the pulse program INVIEAGSSI, Bruker XWINNMR
2.60, of the spectrometers standard software. UV–Vis spectra were
recorded by means of a Hewlett–Packard 8453 instrument. Fluores-
cence was recorded by means of a Hitachi F-4010 spectrometer the
quantum yield of 2Ϫ was derived using a fluorescence quantum yield
Extension of conjugation for derivatives of 1 with retention
of the oxo and hydroxyl functionalities could be reached in
principle by either attaching substituents to the free ring po-
sitions or by extending the conjugation of the hypericin moi-
ety at the two methyl groups. In the first case, the additional
substituent would become sterically congested by its neigh-
boring functional groups, and therefore the resulting conju-
gation extension would become greatly diminished by tor-
sional deformation unless the conjugation was achieved via
an acetylene bond. In this case no problems from an inter-
action with the neighboring groups could occur. However,
our attempts to prepare such derivatives by means of cou-
pling of acetylenes with halo-hypericin or halo-emodin de-
rivatives were so far in vain.
estimate of 1Ϫ of
⌽ ϭ 0.2 (2) as comparison. Electrospray mass
f
spectra were measured with a Hewlett–Packard 89987 quadrupole
instrument.
RESULTS AND DISCUSSION
Heavy-atom substitution
Because it is commonly known that heavy-atom substitution
enhances intersystem crossing and thus also enhances singlet
oxygen/superoxide radical formation, bromine has been hith-
erto introduced by means of electrophilic substitution to the
hypericin nucleus to yield the 2,5-di-, 2,5,9-tri- and 2,5,9,12-
tetrabromo-hypericins (12). In addition to lowered fluores-
cence quantum yields as compared to 1 these derivatives also
showed enhanced light-mediated antiviral properties (13). Fol-
lowing this lead we now explored iodine substitution of 1.
It turned out that upon reaction of 1 with I2 in pyridine
the electrophilic disubstitution product 2 was obtained as its
pyridinium salt 2·pyridine in high yield. To explore other
substitution degrees iodination under changed conditions
was applied. However, by using one equivalent of I2 only a
mixture of the mono- and di-iodination product of the re-
action was obtained, which could not be separated satisfac-
torily. In contrast, a high molecular excess (1:40) of iodine
did not result in any tri- or tetra-substituted derivatives.
The constitution of 2 as the 2,5-diiodo-derivative of 1 fol-
1
lowed immediately from its H NMR and mass spectra. The
UV–Vis main absorption band of the pyridinium salt of 2
became only slightly bathochromically shifted to 604 nm as
compared to the absorption band of the pyridinium salt of 1
at 602 nm (14). Compared to 1 the fluorescence quantum
yield of 2 dropped to about 0.02.
To test for an enhanced photochemical singlet oxygen/
superoxide radical formation of 2 in comparison to 1 we
used the recently introduced photosensitized bilirubin IX
␣
destruction method (11). As shown in Fig. 1 this experiment
proved that indeed there is enhanced formation of the de-
structive oxygen species since after less than 3 min bilirubin
Therefore, we set out to prepare a stilbene analogous hy-
pericin derivative in which the styryl groups were attached
to the two ␻,␻Ј-sites of the methyl groups. They were chosen
IX␣ was completely photooxidized upon sensitization by 2,
because molecular modeling predicted only small torsional
deformation at the styryl residues thus providing effective
conjugation between the styryl and hypericin moieties. To
achieve this goal we started with the emodin-derived phos-
phonium salt 3, which recently had allowed for the prepa-
whereas in the case of 1 about 70% were still remaining.
According to these experiments electrophilic iodination of
the most activated nucleus positions, 2 and 5, of 1 will result
in small bathochromic shifts only; however, this substitution
introduces a dramatic enhancement of the photosensitized
singlet oxygen/superoxide radical formation.
ration of an ␻,␻Ј-appended 18 carbon chains hypericin de-
rivative (10). Wittig reaction of the ylid derived from 3 with
benzaldehyde provided the styryl-quinone 4 in nearly quan-
titative yield. Compound 4 was then regioselectively reduced
and deprotected in an one-pot reaction using SnCl2–acetic
acid–HBr to yield about 75% of the styryl-anthrone 5. Fol-
lowing the preparation of 1 described in detail by Falk et al.
(8) the anthrone 5 was oxidatively dimerized to yield the
protohypericin analogous compound 6. The configurations
(E) of 4, 5 and 6 at the exocyclic double bonds were inferred
1
from the analysis of the high-resolution H NMR spectrum
of 5 in which the vinyl and aromatic proton signals could

214 Roland A. Obermu¨ller et al.
Figure 3. Valence tautomerism between the cyclobuta-cyclohex-
adiene tautomer 8 and the cyclooctatriene tautomer 8
Ј.
trans,trans,cis-diastereomer comparable to the configuration
of the main product I of stilbene dimerization. Photochem-
ical [2
ϩ 2] cycloaddition in the second case would yield
the trans,cis,trans-diastereomer 9 (Fig. 2) corresponding to
the configuration of the minor product II of stilbene dimer-
ization.
Figure 2. Intramolecular photochemical [2
ϩ 2] cycloaddition of
the two conformers 7a and 7b to yield the cyclobutane
trans,trans,cis-8 and trans,cis,trans-9 diastereomers (arbitrary en-
antiomers drawn).
Now, the constitutional possibility 8 is consistent with the
two carbon signals at 67.4 and 38.4 ppm in the 13C NMR
spectrum. The chemical shifts of the cyclobutane protons of
our final product were observed at about 4.13 and 3.55 ppm
as the AB and CD parts of an ABCD system, and they were
found to be correlated to the carbon signals at 67.4 and 38.4
ppm. Correlations and symmetry thus proved the constitu-
tion 8 for the final product. This structural assignment is
further corroborated by the earlier finding (18) that for stil-
bene dimerization the diastereomer I, which corresponds to
the stereochemistry of 8, is the one that is mainly formed.
The unusual high chemical shift value for two cyclobutane
carbon atoms at 67.4 ppm might be explained by a fast va-
lence tautomerism involving the cyclobuta-cyclohexadiene
be unequivocally separated. Thus, the vinylic protons dis-
played a coupling constant of the H–CϭC–H fragment of
about 16 Hz, which is characteristic of an (E)-configured
diastereomer.
The photocyclization of 6 to yield eventually the desired
conjugation extended hypericin 7 provided an unexpected
result. Upon irradiation of an aerated acetone solution of 6
an intermediate absorption at 632 nm developed in the ab-
sorption spectrum of the withdrawn samples but this was
synchronously superseded by an absorption at 585 nm. At-
tempts to recover or isolate this intermediate to which we
assign the constitution 7 and the configuration (E,E) failed
because of its pronounced photosensitivity. Therefore, the
final product of the two-step photoreaction was isolated and
its constitution derived by mass and NMR spectroscopy was
found to be that of a hypericin with a diphenyl-substituted
unit of 8 and the cyclooctatriene system of 8
in Fig. 3. Such a fast equilibrium between 8 and 8
Ј
as displayed
would
Ј
lead to the signal of two carbon atoms bearing the phenyl
substituents within the normal shift region of cyclobutanes
at about 40 ppm. The chemical shift of the other two carbon
atoms at nearly 70 ppm would then be the result of averaging
the shift of an aromatic (120 ppm) and a cyclobutane (40
ppm) carbon type atom. Interestingly enough, molecular
modeling showed these two tautomers to be of similar en-
ergy.
cyclobutane ring attached at the
groups. A cyclobutane ring was formed by a photochemi-
cally allowed intramolecular [2 2] cycloaddition of the
two vinyl groups (15). Nevertheless, this result was surpris-
ing because the analogous photochemical [2 2] cycload-
␻,␻Ј-sites of the two methyl
ϩ
ϩ
CONCLUSIONS
dition of (E)-stilbene to yield mainly the trans,trans,cis-di-
astereomer I and to a small extent the trans,cis,trans-diaste-
reomer II of 1,2,3,4-tetraphenyl-cyclobutane proceeded very
sluggishly to yield only about 15% of the first and 2–5% of
the latter isomer upon irradiation of stilbene solutions in ben-
zene for up to 2 months (16–18).
With respect to the reaction mechanism, the constitution
and stereochemistry of the final product molecular modeling
(MM2 force field of ChemDraw) of the educt distyryl–hy-
pericin 7 revealed that, in principle, there are two low-energy
conformations, which might serve as starting points of the
photoreaction. In the first one the two styryl groups are
somewhat twisted against the twisted hypericin moiety as
shown in Fig. 2 (7a). The other one (7b) is characterized by
an overlap of the two double bonds within the conjugated
The results showed that heavy-atom substitution, e.g. by io-
dine, of hypericin (1) at the ring sites provides a good means
to enhance the desired photosensitized singlet oxygen/super-
oxide radical formation. Extending the conjugation of the
hypericin system by attaching styryl moieties to the methyl
groups of 1 failed in effect because of the very high pho-
toreactivity of the resulting double bonds to undergo a [2
2] cycloaddition.
ϩ
Interestingly enough, this reaction constitutes one of the
rare exceptions of hypericin derivatives exhibiting intramo-
lecular photochemistry as exemplified with 3-O-benzyl-hy-
pericin and 3,4-di-O-benzyl-hypericin. The first derivative
resulted in the formation of the blepharismin type compound
10 when irradiated in presence of 1,8-bis-dimethylamino-
naphthalene (19). The second compound produced, under
the same conditions, a stable radical ion pair (20).
system (Fig. 2). Photochemical [2
ϩ 2] cycloaddition in the
first case yields 8, which would be formed as the

Photochemistry and Photobiology, 2001, 74(2) 215
deprotonation of the photoxidized 3-hypericinate ion. Monatsh.
Chem. 130, 1333–1339.
6. Immitzer, B., C. Etzlstorfer, R. A. Obermu¨ller, M. Sonnleitner,
G. J. Schu¨tz and H. Falk (2000) On the photochemical proton
expulsion capability of fringelite D—a model of the protist pho-
tosensory pigments of the stentorin and blepharismin types.
Monatsh. Chem. 131, 1039–1045.
7. Nafir, M. and P. Jardon (1994) Proprie´te´s spectroscopiques et
photophysiques de complexes me´talliques de l’hypericine en re-
lation avec leur activite´ photodynamiques. J. Chim. Phys. 91,
99–112.
Nevertheless, the experiments described above proved that
the long-wavelength absorption band can be considerably
bathochromically shifted by conjugation extension with two
styryl residues. Attempts are now under way in our labora-
tory to extend hypericin conjugation by means of condensing
additional benzene rings to the methyl bearing sides of the
hypericin nucleus or by attaching conjugation extension
groups to one methyl group of hypericin only.
8. Falk, H., J. Meyer and M. Oberreiter (1993) A convenient semi-
synthetic route to hypericin. Monatsh. Chem. 124, 339–341.
9. Kapinus, E. I., H. Falk and T. N. H. Tran (1999) Spectroscopic
investigations of the molecular structure of hypericin and its
salts. Monatsh. Chem. 130, 623–635.
10. Falk, H. and T. N. H. Tran (1996) Synthesis and properties of
an ␻,␻Ј-appended eighteen carbon chains hypericin derivative.
Monatsh. Chem. 127, 717–723.
11. Hagenbuchner, K. and H. Falk (1999) Concerning the hypericin
sensitized photooxidation of bilirubin IX␣. Monatsh. Chem.
130, 1075–1081.
12. Falk, H. and W. Schmitzberger (1993) On the bromination of
hypericin: the gymnochrome chromophores. Monatsh. Chem.
124, 77–81.
13. Hudson, J. B., E. Delaey and P. A. de Witte (1999) Bromohy-
pericins are potent photoactive antiviral agents. Photochem.
Photobiol. 70, 820–822.
Acknowledgement Advice of Prof. Dr. N. Mu¨ller with regard to
NMR interpretations is gratefully acknowledged.
14. Falk, H. and J. Meyer (1994) On the homo- and heteroassocia-
tion of hypericin. Monatsh. Chem. 125, 753–762.
15. Woodward, R. B. and R. Hoffmann (1970) Die Erhaltung der
Orbitalsymmetrie. Verlag Chemie, Weinheim.
16. Pailer, M. and J. Mu¨ller (1948) Die Konstitution des photodi-
meren stilbens. Monatsh. Chem. 79, 615–619.
17. Dunitz, J. D. (1949) The structure of the centrosymmetric iso-
mer of 1:2:3:4-tetraphenylcyclobutane. Acta Crystallogr. 2, 1–
13.
18. Shechter, H., W. J. Link and G. V. D. Tiers (1963) Halogena-
tion, dehydrohalogenation and dehalogenation of stilbene photo-
dimers (1,2,3,4-tetraphenylcyclobutanes). J. Am. Chem. Soc. 85,
1601–1605.
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