On the chemistry of a dibenzohypericin derivative

Article abstract of DOI:10.1007/s007060270009

A hypericin derivative was synthesized in which instead of the methyl groups two benzene rings were condensed to the chromophoric system in order to extend its conjugation. This derivative showed lowered fluorescence and concomitantly enhanced sensitized production of active oxygen species as compared to hypericin. However, in contrast to intuition its long wavelength band remained unshifted in comparison to its parent compound hypericin. Geometry and absorption properties were also investigated by means of semiempirical calculations.

Full text of DOI:10.1007/s007060270009

Monatshefte fuÈr Chemie 133, 89±96 (2002)
On the Chemistry of a Dibenzohypericin
Derivative
Ã
È
Roland A. Obermuller, Christoph Etzlstorfer, and Heinz Falk


Institut fur Chemie, Johannes Kepler Universitat, A-4040 Linz, Austria
Summary. A hypericin derivative was synthesized in which instead of the methylgroups two
benzene rings were condensed to the chromophoric system in order to extend its conjugation. This
derivative showed lowered ¯uorescence and concomitantly enhanced sensitized production of active
oxygen species as compared to hypericin. However, in contrast to intuition its long wavelength band
remained unshifted in comparison to its parent compound hypericin. Geometry and absorption prop-
erties were also investigated by means of semiempirical calculations.
Keywords. Photodynamic therapy; Bilirubin photooxidation; Fluorescence; Hypericin.
Introduction
In the search for better suited photodynamic agents derived from hypericin (1), the
photosensitizing pigment isolated from the weed St. John's wort (Hypericum
perforatum L.) [1], a series of derivatives has been synthesized so far to overcome a
serious drawback for its use. This has been thought to be necessary because the
main absorption band of 1 is situated slightly below 600 nm, which is somewhat
outside of the 640±660 nm wavelength of commercial medicinal lasers.
The efforts to shift the long wavelength absorption band of 1 to longer
wavelengths, but concomitantly retaining its main functional groups, centered
therefore on a derivatization of 1 with respect to its carbon atoms. Derivatives like
2,5-diodo-hypericin or enhanced conjugation derivatives comprising stilbenoid
extension have been synthesized [2, 3]. However, upon irradiation with visible light
Ã
Corresponding author. E-mail: hfalk@soft.uni-linz.ac.at

90
R. A. ObermuÈller et al.
one of the latter rapidly has underwent an intramolecular photochemical [2 ‡ 2]
cycloaddition, and upon irradiation the other one did not yield the active oxygen
species thought to be essentialfor its use in photodynamic therapy. This effect has
been attributed to interactions of the excited state with its auxochromic amino
group. Therefore it was envisaged that extension of the conjugated system and
thereby a bathochromic shift of the long wavelength absorption band might
be achieved by condensing additionalbenzene rings to the parent compound 1. Such
a derivative 2 was chosen as the target compound of the present communication.
Results and Discussions
Synthesis
To achieve the goalstated above, the naphthoic acid amide 3 was lithiated with
sec-butyllithium, the use of tert-butyl lithium having recently been shown to lead to
an unprecedented methoxy ± tert-butylexchange [4].

Dibenzohypericin
91
Quenching the lithium derivative with 3,5-dimethoxybenzaldehyde yielded 4.
Because of the acid sensitivity of 4 it was immediately converted in about 25%
overall yield to the lactone 5 by means of 4-toluenesulfonic acid. Reductive ring
opening of 5 with Zn/NaOH provided the naphthoic acid 6 in 47% yield. It should
be mentioned that hydrogenolysis on Pd/C resulted in intractable product mixtures
in this case. Ring closure of 6 to the tetracene derivative 7 was achieved in high
yield by means of tri¯uoroacetic anhydride in tri¯uoroacetic acid. Interestingly
enough it turned out that this tetracene 7 was present as the carbonyltautomer
instead of the phenolic tautomer as observed in the case of the corresponding
anthrone derivative [5]. AM1 calculations revealed that the oxo tautomer 7 was
stabilized by 21.5 kJ Á molÀ1, whereas in the anthrone case the stabilization
amounted to 3.5 kJ Á molÀ1 only. The rather weak stabilization of the latter would
be small enough to be overwhelmed by interactions with the solvent and thus
account for the observation that in this case the phenoltautomer was the predomi-
nant one. Because of the air-sensitivity of compound 7 it was demethylated
under reducing conditions by HBr/SnCl2 to give the stable trihydroxytetracenone
8 in high yield. The tetracene derivative 8 was then dimerized in the con-
ventionalway of hypericin synthesis [6, 7] to the protohypericin derivative 9.
However, according to an ES-MS and UV/Vis spectrum of the reaction product,
most of 9 was already converted to 2 due to its extreme light sensitivity. Com-
pletion of the photochemical oxidative ring closure of 9 eventually provided 2 in
rather low yield.
Properties
It came as a surprise that the dibenzo-condensed hypericin derivative 2, which in
solution was shown (only one 1H NMR signalof a phenoilc proton at position 3/4
at about 18 ppm was observed) to be present as its 3/4 phenolate ion, displayed a
long wavelength band at approximately the same wavelength as that observed for
the phenolate ion of hypericin (1). The overall absorption spectra of the two ions
were very similar and, accordingly, the anticipated bathochromic shift of the long
wavelength band induced by the conjugation extension of 1 could not be observed.
Figure 1 displays the observed spectra of 1À and 2À together with the results of the
semiempirical PPP-PEP calculations, which recently had been used extensively
and successfully on hypericin derivatives [8]. These calculations also showed that,
contrary to intuition, one could not assume a signi®cant difference between the
absorption spectra of the two compounds. As AM1 calculations showed that the
dihedraldeformation of the skeeltons of 1 in the most stable double butter¯y
conformations (ꢀ3,3a,3b,4 ˆ 23.1ꢀ, ꢀ10,10a,10b,11 ˆ 32.7ꢀ) and 2 (ꢀ3,3a,3b,4 ˆ 24.6ꢀ,
ꢀ12,12a,12b,12c,12d ˆ 33.1ꢀ) are virtually identical, the observed and calculated
absorption behaviour had to be attributed to the compensation of the loss of the
bathochromic effect from the two methylgroups on the one hand and to the smal
bathochromic effect of the two additionalcondensed benzene rings on the other
hand. Nevertheless, 1 and 2 differed in another aspect relevant to their potential use
as photodynamic agents. Thus, 2À showed a very diminished ¯uorescence yield
(Èf ˆ 0.03) as compared to the about ten times higher one of 1À, pointing to an
ef®cient intersystem crossing. Indeed the active oxygen producing ef®ciency of 2,

92
R. A. ObermuÈller et al.
Fig. 1. Absorption spectra of 1À (grey) and 2À (black) in MeOH and PPP-PEP calculation results on
the same species; the polarization of the absorption bands at ca. 600 nm was found to be identical
along the C2-axis of the molecules
Fig. 2. Hypericin derivative sensitized photooxidation of bilirubin IXꢁ: normalized absorption
changes (A/A0) with time of solutions of disodium bilirubinate IXꢁ together with (3À)1 and (3À)2 in
aereated 80% EtOH upon irradiation at ꢂ > 570 nm

Dibenzohypericin
93
as estimated from the photosensitized destruction of bilirubinate IXꢁ [9], was
observed (Fig. 2) to be much more pronounced than that of hypericinate ion (1À).
With respect to the tautomeric equilibria, AM1 calculations revealed that in
accordance with the results for 1 [13] the Q7,14 tautomer of 2 is more stable than
the most stable Q1,6 tautomer of the remaining tautomers by 90 kJ Á molÀ1.
In conclusion, it turned out that the dibenzo-condensed derivative of
hypericin 2 might well serve as a photodynamic agent of enhanced active
oxygen species producing ef®ciency. Unfortunately, however, it did not yield
the envisaged bathochromic shift of its long wavelength absorption band as
compared to its parent compound 1 and thus does not convincingly improve
the situation with respect to the desired wavelength corresponding to that of
medicinalalsers.
Experimental
Solvents were of p.a. quality. 1H and 13C NMR, UV/Vis, ¯uorescence, and mass spectra were
recorded using Bruker DRX 500 and DPX 200, Hewlett Packard 8453 UV/Vis, Hitachi 4010F, and
Hewlett Packard 59987 quadrupole instruments. The ¯uorescence quantum yield was determined as
described previously [9]. Hypericin sensitized photooxidation of bilirubinate IXꢁ was executed as
described in Ref. [10]. AM1 calculations [11] were performed at the SGI Origin 2000 of the LIZENS
using the MOPAC package [12, 13]. The PPP-PEP calculations were executed using the PEP
program [14]. Substrate 3 was prepared according to Ref. [4]; 3,5-dimethoxy-benzaldehyde was of
commercialorigin (Adl rich).
1,3,4,6,8,17-Hexahydroxy-benzo[fg]benzo[10,20 : 4,5]phenaleno[3,2,1,9-rstuv]
pentaphene-7,18-dione (2; C36H16O8)
To 67.7 mg 8 (0.23 mmol), 13.5 mg FeSO4 Á 7H2O (p.a.; 0.05 mmol), 155 mg pyridin-N-oxide
(1.63 mmol), 1.2 cm3 absolute pyridine, and 120 mm3 piperidine were added, and the mixture was
heated to 100±110ꢀC under Ar, stirring, and protection from light for 1 h. The reaction mixture was
then cooled and poured on 20 cm3 HCl( c ˆ 2 mol Á dmÀ3). After standing for 30 min, the dark-blue
precipitate was centrifuged and dried over silica. The dry residue consisting mainly of 9 was
dissolved in 2.5 dm3 acetone and irradiated with a 700 W day light lamp (Philips) under stirring and
air admission. The bordeaux-red solution was evaporated; the residue was dissolved under sonication
in THF, centrifuged, and the supernatant was chromatographed on a preparative TLC plate using
THF : petrolether : gal cialacetic acid ˆ 35 : 5 : 5. The red band was eluted with THF : MeOH ˆ 1 : 1.
To get rid of the suspended silica, the solution was evaporated and distributed between CHCl3 and
H2O, and the organic phase was evaporated.
1
Yield: 3.7 mg (2.8%); H NMR (200 MHz, ꢃ, DMSO-d6): 18.06 (s, 1H, OH-3 or OH-4), 16.20
(s, OH-8,17), 14.14 (s, OH-1,6), 8.65 (d, J ˆ 6.9 Hz, H-12,13), 8.18 (X-part of ABCX-system,
J ˆ 7.5 Hz, H-9,16), 7.90±7.65 (AB or C-part of ABCX-system, H-10,15 or H-11,14), 7.65±7.45
(C or AB-part of ABCX-system, H-11,14 or H-10,15), 6.58 (s, H-2,5) ppm; 13C NMR
(125 MHz, ꢃ, DMSO-d6): 184.4 (2CO), 166.5 (2COH), 158.0 (2COH), 154.0 (2COH), 133.0, 132.7,
131.6, 129.6, 126.7, 124.3, 124.0, 123.3, 121.1, 117.4, 111.1, 102.2 (28Car) ppm; ES-MS
(i-PrOH : H2O ˆ 80 : 20 ‡ 1% NH3, negative mode): m/z ˆ 575 ((M±H)À); UV/Vis (MeOH,
c ˆ 1 Á 10À5 mol Á dmÀ3): ꢂmax(") ˆ 587 (17850), 545 (12800), 469 (13300), 342 (19500), 290
(36300), 263 (41400), 219 (50400) nm; ¯uorescence (MeOH, c ˆ 1 Á 10À5 mol Á dmÀ3, ꢂexc ˆ 550 nm):
ꢂ
em (rel. intensity) ˆ 596 (100), 637 (34) nm, Èf ˆ 0.03.

94
R. A. ObermuÈller et al.
(R,S)-N,N-Diethyl-3-((3,5-dimethoxyphenyl)-hydroxymethyl)-1-methoxy-
naphthalin-2-carboxamide (4; C25H29NO5)
Into a solution of 1.4 cm3 abs. N,N,N0N0-tetramethylethylenediamine (9.3 mmol) and 1.03 g 3
(4 mmol) in 36 cm3 absolute. THF a solution of sec-butyllithium (c ˆ 1.3 mol Á dmÀ3 in cyclohexane)
was added dropwise under Ar at À80 to À85ꢀC during 15 min. To this mixture a solution of 0.73 g
3,5-dimethoxybenzaldehyde (4.4 mmol) in 4 cm3 absolute. THF was added dropwise during 10 min
under stirring, and the reaction mixture was stirred for additional20 min below À80ꢀC. The reaction
mixture was then brought to room temperature during 30 min, quenched with 4 cm3 H2O, and
acidi®ed with 17 cm3 HCl( c ˆ 2 mol Á dmÀ3). The organic part of the solvent was evaporated, the
residualphase extracted with CHCl , the organic phase washed with brine, dried over Na2SO4, and
3
evaporated. The resulting light-brown oil (2.17 g) was used without further puri®cation for the next
step. Column chromatography on Kieselgel 60 using CHCl3 : MeOH ˆ 40 : 1 as the eluent yielded
the analytical sample.
Rf (silica, CHCl3 : MeOH ˆ 40 : 1) ˆ 0.36; 1H NMR (500 MHz, ꢃ, CDCl3): 8.09 (d, J ˆ 8.0 Hz,
Hn-8), 7.75 (d, J ˆ 7.6 Hz, Hn-5), 7.55±7.49 (m, Hn-6,7), 7.46 (s, Hn-4), 6.61 (d, J ˆ 2.2 Hz, Hb-2 ‡
Hb-6), 6.44 (t, J ˆ 2.2 Hz, Hb-4), 5.92 (s, CHOH), 3.99 (s, OCH3), 3.95±3.83 (m, NCHH), 3.80
(s, 2OCH3), 3.45±3.38 (m, NCHH), 3.14 (q, J ˆ 7.2 Hz, NCH2), 1.34 (t, J ˆ 7.1 Hz, NCH2CH3) ppm;
13C NMR (50 MHz, ꢃ, CDCl3): 169.1 (CON), 160.9 (Cn-OMe), 160.8 (2Cb-OMe), 146.4 (Cn), 143.8
(Cn), 140.0 (Cn), 139.6 (Cn), 134.8 (Cn), 128.2 (Cn), 128.7 (Cn), 127.8 (Cn), 126.8 (Cn),
122.6 (Cn), 105.6 (Cb-2,6), 99.3 (Cb-4), 73.1 (CHOH), 62.9 (OCH3), 55.6 (2OCH3), 44.0 (NCH2),
39.1 (NCH2), 13.1 (NCH2CH3), 12.8 (NCH2CH3) ppm.
(R,S)-3-(3,5-Dimethoxyphenyl)-9-methoxy-3H-naphtho[2,3-c]furan-1-one (5; C21H18O5)
To a solution of 2.17 g crude 4 in 30 cm3 toluene 10 mg 4-toluenesulfonic acid were added, and the
mixture was re¯uxed for 20 h. After cooling to room temperature the organic layer was washed twice
with 5% Na2CO3 solution, H2O, HCl( c ˆ 2 mol Á dmÀ3), and H2O. After drying over Na2SO4 the
solvent was evaporated and the remaining oil crystallized from diethyl ether.
Yield: 0.41 g (25%); m.p.: 135±140ꢀC; Rf (silica, CHCl3) ˆ 0.48; 1H NMR (200 MHz, ꢃ, CDCl3):
8.44 (d, J ˆ 8.0 Hz, Hn-8), 7.80 (d, J ˆ 8.0 Hz, Hn-5), 7.70±7.45 (m, Hn-6,7), 7.40 (s, Hn-4), 6.51
(d, J ˆ 2.1 Hz, Hb-2,6), 6.46 (t, J ˆ 2.1 Hz, Hb-4), 6.40 (s, OCH), 4.46 (s, OCH3), 3.78 (s, 2OCH3)
ppm; 13C NMR (50 MHz, ꢃ, CDCl3): 168.3 (CO), 161.4 (COMe), 158.0 (COMe), 144.8 (COMe),
140.1, 138.1, 129.8, 128.2, 128.1, 126.7, 124.5, 116.2, 110.5, 105.0, 101.0 (13Car), 82.0 (OCH), 64.2
(OCH3), 55.7 (2OCH3) ppm; IR (KBr): ꢄ ˆ 3004, 2946, 2841, 1763, 1633, 1605, 1473, 1462, 1428,
1370, 1346, 1335, 1305, 1286, 1272, 1206, 1164, 1147, 1123, 1089, 1046, 1022, 835 cmÀ1; UV/Vis
(CHCl3, c ˆ 1 Á 10À5 mol Á dmÀ3): ꢂmax(") ˆ 348 (3840), 303 (4560), 290 (7150), 281 (6720), 246
(50880) nm.
3-(3,5-Dimethoxyphenylmethyl)-1-methoxynaphthalin-2-carboxylic acid (6; C21H20O5)
To a solution of 980 mg 5 (2.8 mmol) in 79 cm3 10% NaOH, 9.95 g activated Zn dust
(152 mmol) were added, and the mixture was re¯uxed for 19 h, cooled on ice, and acidi®ed with
30 cm3 conc. HCl. After extraction with CH2Cl2, washing with brine, and drying over Na2SO4
the solvent was evaporated. The residual oil was crystallized from diethyl ether to yield 467 mg
(47%) 6.
M.p.: 147±149ꢀC; 1H NMR (200 MHz, ꢃ, CDCl3): 9.61 (br s, COOH), 8.17±8.09 (m, Hn-8),
7.90±7.65 (m, Hn-5), 7.60±7.48 (Hn-6,7), 7.40 (s, Hn-4), 6.54 (s, Hb-2,6), 6.40 (s, Hb-4), 4.24
(s, CH2), 4.06 (s, OCH3), 3.75 (s, 2OCH3) ppm; 13C NMR (50 MHz, ꢃ, CDCl3): 170.3 (COOH),
161.0, 155.3, 142.4, 136.5, 135.6, 128.2, 126.3, 125.6, 122.8, 121.0, 107.7, 98.5 (16Car), 64.1
(OCH3), 55.2 (2OCH3), 39.9 (CH2) ppm; IR (KBr): ꢄ ˆ 3444, 2999, 2937, 2841, 2673, 2580, 1686,

Dibenzohypericin
95
1630, 1606, 1595, 1573, 1499, 1454, 1430, 1370, 1353, 1339, 1306, 1292, 1206, 1157, 1144, 1093,
1067, 1056, 954, 946, 922, 843 cmÀ1; UV/Vis (CHCl3, c ˆ 1 Á 10À5 mol Á dmÀ3): ꢂmax(") ˆ 327
(1700), 283 (7320), 242 (3950) nm; ES-MS (MeOH : H2O ˆ 80 : 20 ‡ 1% conc. NH3, negative
mode): m/z ˆ 351 ((M±H)À).
5,12-Dihydro-1,3,11-trimethoxytetracen-12-one (7; C21H18O4)
Under Ar protection, 27 mg 6 (0.077 mmol) were dissolved in 1 cm3 tri¯uoroacetic acid. The solution
was cooled to À10ꢀC, and 150 mm3 tri¯uoroacetic acid anhydride were added dropwise during
5 min. After stirring for 2 h at À10ꢀC the solution was brought to room temperature and evaporated.
Yield: 23 mg (89%); Rf (silica, CHCl3) ˆ 0.46; 1H NMR (200 MHz, ꢃ, CDCl3): 8.28 (d, J ˆ 8 Hz,
H-10), 7.80 (d, J ˆ 7.9 Hz, H-7), 7.75±7.35 (m, H-6,8,9), 6.58 (s, H-4), 6.47 (s, H-2), 4.34 (CH2),
4.17 (s, OCH3), 3.96 (s, OCH3), 3.93 (s, OCH3) ppm; no 13C NMR spectrum could be obtained due
to the sensitivity of 7 towards oxidation.
5,12-Dihydro-1,3,11-trihydroxytetracen-12-one (8; C18H12O4)
To a solution of 25 mg 7 (0.075 mmol) in 10 cm3 glacial acetic acid, 0.1 g SnCl2 Á 2H2O and 1.5 cm3
47% HBr were added, and the mixture was re¯uxed for 2 h. The reaction mixture was then poured
into 30 cm3 H2O and centrifuged. The pellet was washed three times with H2O and dried over P4O10.
Yield: 15.3 mg (85%); m.p.: > 320ꢀC; 1H NMR (200 MHz, ꢃ, DMSO-d6): 13.87 (s, OH-11),
12.39 (s, OH-1), 10.91 (s, OH-3), 8.29 (d, J ˆ 8.3 Hz, C-10), 7.85 (d, J ˆ 8.1 Hz, H-7), 7.69 (m, H-9),
7.53 (m, H-8), 6.46 (s, H-4), 6.25 (s, H-2), 4.43 (s, CH2) ppm; 13C NMR (50 MHz, ꢃ, CDCl3): 191.4
(CO), 165.4 (COH), 164.8 (COH), 161.2 (COH), 145.0, 136.5, 134.8, 130.5, 127.0, 125.4, 123.3,
122.7, 116.5, 109.5, 108.5, 107.6, 101.0 (13Car), 32.3 (CH2) ppm; IR (KBr): ꢄ ˆ 1631, 1597, 1452,
1414, 1398, 1335, 1300, 1161, 1070, 973, 925 cmÀ1; UV/Vis (DMSO, c ˆ 6 Á 10À5 mol Á dmÀ3):
ꢂ
max(") ˆ 403 (11300), 358 (14900), 312 (10000), 262 (20700) nm.
Acknowledgments
We gratefully acknowledge to Dr. C. Klamp¯ for recording mass spectra and to B. Hager for running
IR spectra.
References
[1] For a recent overview see: Falk H (1999) Angew Chem 111: 3306; Angew Chem Int Ed 38: 3116

[2] Obermuller RA, Hohenthanner K, Falk H (2001) Photochem Photobiol 74: 211
[3] Obermuller RA, Falk H (2001) Monatsh Chem 132: 1519
[4] Obermuller RA, Dax TG, Falk H (2001) Monatsh Chem 132: 1057
[5] Falk H, Schoppel G (1991) Monatsh Chem 122: 739
[6] Mazur Y, Bock H, Lavie D (1992) Can Pat ApplCA 2.029,993
[7] Falk H, Meyer J, Oberreiter M (1993) Monatsh Chem 124: 339

[8] Etzlstorfer C, Falk H, Muller N, Tran TNH (1996) Monatsh Chem 127: 659
[9] Falk H, Meyer J (1994) Monatsh Chem 125: 753
[10] Hagenbuchner K, Falk H (1999) Monatsh Chem 130: 1075
[11] Dewar MJS, Zoebisch EG, Healy EF, Stewart JJP (1985) JAm Chem Soc 107: 3902; Dewar MJS,
Dieter KM (1986) J Am Chem Soc 108: 8075; Stewart JJP (1990) J Comp Aided MolDesign 4: 1
[12] MOPAC 6.0 DEC-3100 Ed 1990, FJ Seiler Res Lab, USAF Acad CO80840
[13] For AM1 calculations on the geometrical and energetic realms of phenanthroperylene quinones
see: Gutman I, Markovic Z, Solujic S, Sukdolak S (1998) Monatsh Chem 129: 481; Etzlstorfer C,

96
R. A. ObermuÈller et al.: Dibenzohypericin
Falk H (1998) Monatsh Chem 129: 855; Gutman I, Markovic Z (1998) Monatsh Chem 129: 1019;
Etzlstorfer C, Gutman I, Falk H (1999) Monatsh Chem 130: 1333
[14] Westerhoff UM, QCPE program 11,167 based upon Brearly N, Bloor JE (1965) Can J Chem 43:
1761; Pariser R (1955) J Chem Phys 24: 250; Pariser R (1956) J Chem Phys 25: 1112; Pople JA
(1954) J Chem Phys 23: 81, and an adaption for torsionalstates as given in [8]
Received July 27, 2001. Accepted August 9, 2001