Synthesis and properties of hypericins substituted with acidic and basic residues: Hypericin tetrasulfonic acid - A water soluble hypericin derivative

Article abstract of DOI:10.1007/PL00000089

Sulfonation of hypericin leads to its di-, tri-, and tetrasulfonic acid derivatives. The latter is soluble in water up to millimolar solutions. Homoaggregate formation (J-aggregates) was observed only above 5 · 10^-4 mol/1. In water solutions, the tetrasulfonic acid hypericin derivative was found to be present as its bay-phenolate with most of the sulphonic acids dissociated. Thus, the first water soluble hypericin derivative, which in contrast to hypericin is not prone to homoassociation, is presented. Hypericin tetrtasulfonic acid forms heteroassociates with serum albumin, DNA, and γ-cyclodextrin. Hypericin derivatives with primary and tertiary amino group appendages at the hypericin methyl groups were synthesized. However, upon salt formation or quaternization these derivatives became virtually insoluble in all common solvents including water.

Full text of DOI:10.1007/PL00000089

Monatshefte fuÈr Chemie 129, 309±318 (1998)
Synthesis and Properties of Hypericins
Substituted with Acidic and Basic Residues:
Hypericin Tetrasulfonic Acid ± A Water Soluble
Hypericin Derivative
Ã
and Robert Altmann
Heinz Falk , Abd-El-Wareth A. O. Sarhan, Huyen T. N. Tran,
È
Institut fuÈr Chemie, Johannes Kepler Universitat, A-4040 Linz, Austria
Summary. Sulfonation of hypericin leads to its di-, tri-, and tetrasulfonic acid derivatives. The latter
is soluble in water up to millimolar solutions. Homoaggregate formation (J-aggregates) was
observed only above 5 Á 10ꢀ4 mol/l. In water solutions, the tetrasulfonic acid hypericin derivative was
found to be present as its bay-phenolate with most of the sulphonic acids dissociated. Thus, the ®rst
water soluble hypericin derivative, which in contrast to hypericin is not prone to homoassociation, is
presented. Hypericin tetrtasulfonic acid forms heteroassociates with serum albumin, DNA, and ꢀ-
cyclodextrin. Hypericin derivatives with primary and tertiary amino group appendages at the
hypericin methyl groups were synthesized. However, upon salt formation or quaternization these
derivatives became virtually insoluble in all common solvents including water.
Keywords. Hypericin sulfonic acids; Appended primary and tertiary amino hypericins; J-
Aggregates; Heteroassociates.
Synthese und Eigenschaften von mit sauren und basischen Resten substituierten Hypericinen:
È È
Hypericintetrasulfonsaure ± ein wasserlosliches Hypericinderivat
È
Zusammenfassung. Sulfonierung von Hypericin fuÈhrt zu seinen Di-, Tri- und Tetrasulfonsaure-
È
derivaten. Letzteres ist in Wasser bis zu millimolaren Konzentrationen loslich. Homoaggregatbildung
(J-Aggregate) wurde erst ab Konzentrationen von 5 Á 10ꢀ4 Mol/l beobachtet. Die Tetrasulfonsaure
È
È È È
liegt in waûriger Losung als bay-Phenolat vor, wobei die meisten Sulfonsauregruppen dissoziiert
È
sind. Damit wurde das erste wasserlosliche Hypericinderivat, das im Gegensatz zum Hypericin nicht
È
zur Homoassoziation neigt, gefunden. Hypericintetrasulfonsaure bildet Heteroassociate mit
Serumalbumin, DNA und ꢀ-Cyclodextrin. Es wurden weiters Hypericinderivate synthetisiert, die
È
È È
uÈber primare und tertiare Aminogruppen im Bereich der Methylgruppen des Hypericins verfugen.
È
Bei Salzbildung oder Quarternierung werden diese Derivate jedoch in allen uÈblichen Losungsmit-
È
teln, inklusive Wasser, praktisch unloslich.
* Corresponding author

310
H. Falk et al.
Introduction
Hypericin (1) and most of its derivatives and structural relatives are known for their
intrinsically low solubility in most common solvents, especially in water. Since the
latter solvent is fundamental with respect to physiological and therapeutic
conditions, the search for hypericin derivatives of enhanced water solubility is an
important objective. Recently we have been able to enhance the solubility of
hypericin in water by attaching long chains of polyethylene glycols to its two
methyl groups [1]. However, although the solubility of the bis-!,!0-polyethylene-
glycol-1000 hypericin derivative in water has been enhanced to about 5Á 10ꢀ5 mol/l,
it started to homoassociate into H-aggregates at concentrations as low as 10ꢀ6 mol/l
[1]. Therefore, the quest for water soluble derivatives, preferable compounds
without association tendency, was pursued again using derivatives with acidic and
basic functional groups. Following the lead of color chemistry [2] to enhance water
solubility, the preparation of hypericin sulfonic acids (which allow the formation of
sulfonic acid salts) and amines (which allow the formation of ammonium salts and
quaternary ammonium salts) was set as a goal and will be described in this paper.
Results and Discussion
Hypericin sulfonic acids
Since it has been established recently that hypericin (1) could be substituted
stepwise by means of electrophilic bromination to yield regioselectively 2,5-
dibromohypericin, 2,5,9-tribromohypericin, and 2,5,9,12-tetrabromohypericin [3],
electrophilic sulfonation seemed to be promising in this context.
2 : R1 ˆ R2 ˆ H
3 : R1 ˆ SO3H; R2 ˆ H
4 : R1 ˆ R2 ˆ SO3H
Unfortunately, the photochemical sulfonation of 1 using Na2SO3 in aqueous
pyridine gave only unreacted 1. However, upon sulfonation of 1 with oleum at
room temperature it turned out (monitoring by NMR) that the sulfonation
proceeded regioselectively and stepwise into the aromatic positions 2/5 and 2/5/9,
thus providing the corresponding disulfonic and trisulfonic hypericin derivatives 2
and 3 in 27% yield. The rest to the reaction mixture proved to be unreacted 1.
Therefore, the reaction temperature was raised to 78ꢁC in the next experiment and

Hypericin Tetrasulfonic Acid
311
Fig. 1. Concentration dependence of the absorp-
tion spectrum of 4 in water; c from 5 Á 10ꢀ7 to
2 Á 10ꢀ4 mol/l (±), c ˆ 1 Á 10ꢀ3 mol/l (- - -)
kept for one hour. This resulted in a mixture of 2, 3 and tetrasulfonic acid 4, with 2
and 4 as the minor components. The major product 3 could be isolated by
chromatography on Sephadex in 58% yield and was characterized. The
tetrasulfonic acid 4 was then prepared in 86% yield by prolonging the reaction
time to about 1.5 hours under the conditions given above. Although the water
solubility of 2 and 3 was somewhat enhanced in relation to 1, the tetrasulfonic acid
4 displayed by far the best water solubility. Therefore we focused upon preparation,
characterization, and properties of 4.
The tetrasulfonic acid 4 proved to be soluble in water up to concentrations of
about 10ꢀ3 mol/l. As demonstrated in Fig. 1, the absorption spectrum of 4 in water
at concentrations between 5 Á 10ꢀ7 to 2 Á 10ꢀ4 displays the characteristic features of
a monomolecularly dissolved hypericinate species mol/l [4]. Compared to 1, the
long wavelength band was bathochromically shifted to 615 nm. For concentrations
above 5 Á 10ꢀ4 mol/l and up to the solubility limit of about 10ꢀ3 mol/l, a
bathochromically shifted shoulder developed at 660 nm and became gradually
more intense on the expense of the band at 615 nm. In contrast to the hypericin-
water system which is characterized by a H-aggregate [4], this species could
constitute a J-aggregate. However, it could not be ruled out that it is simply an
È
undissociated species since addition of Hunig's base to a concentrated solution
recovered the low concentration species. Nevertheless, the rather broad long
wavelength absorption band together with its low intensity and a hypericinate type
short wavelength band system favor the ®rst explanation. The ¯uorescence
spectrum of 4 in water peaked at 625 nm, exhibiting a slightly larger Stokes shift
(10 nm) than that observed for the parent system 1 dissolved in ethanol (which,
exactly speaking, is that of the bay-hypericinate ion in both cases). The
¯uorescence quantum yield of 0.8 was found to be considerably enhanced
compared to that of 1 dissolved in DMSO [4]. It should be noted that dissolving 1
in water by means of sonication or solubilization with DMSO renders the
compound to be non-¯uorescent [4].
Of course, the protonation-deprotonation equilibria of 4 are rather complex due
to the presence of the rather acidic bay-hydroxylic groups (the corresponding pKa
of 1 has been deduced to amount to 1.8 [1]) and the four sulfonic acid groups (the
pKa of benzene sulfonic acid has been determined to amount to 0.7 [5]).
Nevertheless, with respect to the hypericin chromophore the C=O-protonated, the

312
H. Falk et al.
Fig. 2. Absorption spectra of 4 dissolved in water
; arbitrary E values displaying correct
(c ˆ 2 Á 10ꢀ5
relative band intensities) at Hꢀ ˆ ꢀ6.5 (a), pH ˆ 0 (b),
pH ˆ 5 (c), and pH ˆ 10 (d); ¯uorescence peaks in these
cases were observed at 668 (a), 610 (b), 625 (c), and 625
(d) nm
undissociated, the bay-hypericinate, and the bay-peri-hypericindiate species could
be detected upon spectrophotometric titration (Fig. 2: traces a±d). For the
corresponding steps, pKa values of ꢀ6, 3.8, and 8.8 were estimated. A
potentiometric titration yielded only one very broad cumulative step up to pH 7.
Although the dissociation of the sulfonic acid groups play a negligible role for the
spectroscopic shifts of the various species, these results point to a decrease of the
bay-hydroxyl acidity induced by the adjacent dissociated sulfonic acid group(s).
The absorption spectrum of 4 in dimethylsulfoxide was found to be
superimposable on that of its water solution at low concentrations (Fig. 1).
However, in other common organic solvents, like methanol, ethanol, acetonitrile,
acetone, and tetrahydrofuran, the solubility of 4 was exceptionally low, and the
absorption spectra of such only faintly colored solutions displayed severely
broadened and bathochromically shifted long wavelength bands resembling the
high concentration species of Fig. 1. An exception was the pyridine solutions of 4
which displayed the characteristic hypericinate type spectrum.
The tetrasulfonic acid 4 formed heteroassociates with bovine and human serum
albumin (BSA and HSA), DNA, and ꢀ-cyclodextrine as indicated by a sigmoidal
shift vs. concentration dependence of the long wavelength absorption band upon
gradual addition of these compounds. Thus, this band was shifted from 615 nm to
621 and 619 nm with BSA and HSA displaying saturation behaviour above the
concentration threshold of 4: (B)HSA ˆ 1:2. However, in contrast to the HSA
complex of 1 [4], no chiroptical signal of the hypericin chromophore could be
detected. Bilirubin and warfarin were not displaced by 4 from their HSA
complexes. Although the HSA complex of 4 could be chromatographed it seemed
to be less stable than the corresponding hypericin complex, and the complexation
seemed to be non-speci®c. In the case of DNA, a continuous shift to about 625 nm
was observed; however, no chiroptical signal for the hypericin chromophore could
be detected. Complexation with ꢀ-cyclodextrin seemed to be rather limited since a
shift of the long wavelength band of 4 to 618 nm was observed only upon addition
of a 270 fold excess. Again, no chiroptical signal could be detected.

Hypericin Tetrasulfonic Acid
313
Amino derivatives of hypericin
To attach an amino group to the hypericin moiety, it was decided to prepare a
primary and a tertiary amine functionality somewhat spaced from the strongly
acidic bay region. Thus, we resorted to the strategy applied to the synthesis of a
variety of !,!0-appended hypericin derivatives [6].
5 : R ˆ CH3CO; X ˆ OCH2CH2NHCOCH3
6 : R ˆ H; X ˆ OCH2CH2NHCOCH3
10 : R ˆ H; X ˆ OCH2OCH2Cl
7 : X ˆ OCH2CH2NHCOCH3
11 : X ˆ OCH2OCH2Cl
8 : X ˆ OCH2CH2NHCOCH3
9 : X ˆ OCH2OCH2NH2
12 : X ˆ OCH2CH2NHꢂCH2†
5
Accordingly, 5 was prepared from the recently described !-iodo-tri-O-acetoxy
emodin [7] by means of a nucleophilic displacement with N-(2-hydroxyethyl)
acetamide in presence of AgTf in 37% yield. Acidic saponi®cation (90%) to yield 6
and reduction (90%) afforded the corresponding anthron 7 which was then coupled
and photocyclized to the appended hypericin derivative 8 in only 10% yield. Upon
acidic saponi®cation, the primary amino derivative 9 was produced. However, it
proved to be virtually insoluble in common solvents. Experiments to quarternize 9
with CH3J also failed to yield any soluble material.
To prepare a tertiary amino derivative of 1, !-bromo-tri-O-acetoxy emodin [6]
was substituted by means of 2-chloroethanol in presence of silver perchlorate to
yield 10 (95%). Upon reduction, the anthron 11 was obtained (90%) which was
then dimerized in presence of an excess of piperidine to yield 7% of the bis-
piperidinyl derivative 12. This compound displayed the usual characteristic
spectroscopic features of a hypericinate derivative. It was obviously present as its
zwitterion with respect to the bay hydroxyl group. Judged from its absorption
spectrum, the second amino group was insuf®cient, however, to ionize an
additional peri positioned hydroxyl group. It displayed marginal water solubility,
and upon protonation or quaternization of the nitrogen atoms it became virtually
insoluble in all common solvents. This phenomenon might be due to the intrinsic
insolubility of nonionized hypericin.
Conclusions
Two routes to enhance the water solubility of hypericin without changing its
functional groups were pursued. On the one hand, tetrasulfonation of hypericin
provided the ideal means to enhance the water solubility of hypericin even into the

314
H. Falk et al.
millimolar region. This should be suf®cient for any biological purposes. The
interactions of this material with certain biopolymers was shown to result in
heteroassociation complexes. On the other hand, although amino derivatives of
hypericin are obtainable in principle, they yield virtually insoluble salts upon
protonation or quaternization and are thus unsuited for further physiological
studies.
Experimental
1
Melting points were taken by means of a Ko¯er hot stage microscope (Reichert, Vienna). H, 13C,
IR, UV/Vis, ¯uorescence, CD, and mass spectra were recorded using Bruker DPX 200 and 500,
Biorad FT-IR-45, Perkin-Elmer IR-710B, Hitachi-U-3210 and F-4010, ISA-mark-V, and API-5989
instruments. Spectrophotometric titrations (dist. water) were carried out as described in Ref. [1]. For
the determination of the ¯uorescence quantum yields, Rhodamine B ¯uorescence (Èf ˆ 0.69;
ethanol) was used as the standard. Hypericin (1) was prepared according to Ref. [8] Bovine and
human serum albumin, calf thymus DNA, and ꢀ-cyclodextrin were of commercial origin (Sigma).
Heteroassociation was studied spectrophotometrically in phosphate buffer solutions of pH 7 as
described in Ref. [4].
Sulfonation procedure
41 mg 1 and 125 mg oleum were kept at the temperature and time given. The reaction mixture was
gently diluted with ice water and saturated with NaCl. After centrifugation, the precipitated green
crystals were washed with cold water and thoroughly dried in vacuum. The material in question was
isolated by column chromatography on Sephadex-LH20 with methanol/water (4/1) as the eluent.
Hypericin-2,5-disulfonic acid (2; C30H16O14S2)
Proceeding with the solfonation procedure given above with 32 mg 1 and 80 mg oleum at room
temperature for 1.5 h, 12 mg 2‡3 which could not be separated properly were obtained. From this
1
1
mixture, the H NMR data of 2 could be derived H NMR (DMSO-d6, ꢁ, 500 MHz): 15.00 (s, 2H),
14.36 (s, 2H), 13.51 (s, 2H) 7.48 (s, CH-9, 12), 2.74 (s, 6H, CH3-10, 11) ppm.
Hypericin-2,5,9-trisulfonic acid (3; C30H16O17S3)
Proceeding with the sulfonation procedure given above with 41 mg 1 and 125 mg oleum at 78ꢁC for
1 h, 35 mg (58%) 3 were obtained.
M.p.: not below 300ꢁC; 1H NMR (DMSO-d6, ꢁ, 500 MHz): 17.38 (s, 1H), 15.04 (s, 1H), 14.34 (s,
1H), 14.12 (s, 1H), 13.96 (s, 1H), 13.47 (s, 1H), 7.44 (s, CH-9), 2.98 (s, CH3-11), 2.71 (s, CH3-10)
ppm; 13C NMR (50.3 MHz): due to its low solubility, the spectrum was not properly resolved;
however, two C=O signals at 197 and 195 ppm were signi®cant; IR (KBr): ꢂ ˆ 3426, 1595, 1447,
1359, 1235, 1036, 823 cmꢀ1; MS (e-spray ionization from water/acetonitrile ˆ 1/1): m/z ˆ 744 (10;
‡
M ), 663 (30), 474 (8), 457 (7), 371 (76), 331 (1000), 247 (91), 220 (30), 169 (8), 117 (24); UV/Vis
(H2O, c ˆ 3 Á 10ꢀ6 mol/l): ꢃmax ˆ 605 (22000), 570 (15000), 449 (15000), 318 (30000) nm (");
¯uorescence (H2O, c ˆ 3 Á 10ꢀ6): ꢃem ˆ 617 (1), 660 (0.4, sh) nm (rel. intensity).
Hypericin-2,5,9,12-tetrasulfonic acid (4; C30H16O20S4)
Proceeding with the sulfonation procedure given above with 50 mg 1 and 250 mg oleum at 75ꢁC
for 1.3 h, 78 mg (95%) 4 were obtained. Elemental analysis showed, however, that due to the salting

Hypericin Tetrasulfonic Acid
315
out procedure the material could be contaminated even after chromatography by as much as 75%
with NaCl.
M.p.: not below 300ꢁC; 1H NMR (DMSO-d6, ꢁ, 500 MHz): 17.34 (s, 2H), 14.05 (s, 2H), 14.03 (s,
2H), 2.96 (s, CH3-10, 11) ppm; 13C NMR (DMSO-d6, ꢁ, 50.3 MHz): 184.9 (C=O-7, 14), 168.1 (C-3,
4), 162.0 (C-1, 6), 158.3 (C-8, 13), 144.5 (C-10, 11), 128.2 (C-3a, 3b), 125.7 (C-6b, 14b), 122.9 (C-
7c, 14c), 120.8 (C-10a, 10b), 117.8 (C-7b, 13b), 112.0 (C-9, 12), 110.9 (C-6a, 14a), 91.9 (C, 2, 5),
90.0 (C-7a, 13a), 23.3 (CH3-10, 11) ppm; IR (KBr): ꢂ ˆ 3420, 1638, 1619, 1580, 1461, 1367, 1239,
1204, 1132, 1040, 782 cmꢀ1; MS (e-spray ionization from water/acetonitrile ˆ 1/1): m/z ˆ 822 (2;
‡
M ), 743 (5), 669 (2), 564 (4), 473 (6), 411 (17), 371 (20), 323 (19), 268 (100), 241 (45), 220 (35),
169 (6), 117 (13); UV/Vis (H2O, c ˆ 7 Á 10ꢀ6 mol/l): ꢃmax ˆ 615 (20800), 569 (8800), 470 (6000),
395 (14000), 340 (16800) nm ("); ¯uorescence (H2O, c ˆ 7 Á 10ꢀ6 mol/l): ꢃem ˆ 625 (1), 665 (0.3)
nm (rel. intensity), Èfluo ˆ 0.8; UV/Vis (DMSO, c ˆ 8 Á 10ꢀ6 mol/l): ꢃmax ˆ 614 (20800), 569 (8800),
470 (6000), 395 (14000), 340 (16800) nm ("); UV/Vis (pyridine, c ˆ 1 Á 10ꢀ5 mol/l): ꢃmax ˆ 601 (1),
569 (0.4), 470 (0.7) nm (rel. intensities); UV/Vis (acetone): ꢃmax ˆ 648 nm; UV/Vis (ethanol):
ꢃmax ˆ 645 nm; UV/Vis (methanol): ꢃmax ˆ 625, 450 nm; UV/Vis (acetonitrile): ꢃmax ˆ 645 nm.
N-(2-Hydroxyethyl) acetamide
In analogy to Ref. [9], however, improved by changing equivalents, a mixture of 55 mg (0.9 mmol) of
ethanolamine and 580 mg (2.5 mmol) of penta¯uorophenyl acetate (prepared according to Ref. [9])
in 1.5 ml of dimethylformamide was allowed to stand overnight and was then evaporated to dryness
under vacuum. The residue was dissolved in 15 ml of water, the excess of penta¯uorophenyl acetate
was separated, and the solution was evaporated and dried under vacuum again to get an advanced
yield of 93 mg of N-(2-hydroxyethyl) acetamide (98%).
1H NMR (CDCl3, ꢁ, 200 MHz): 6.15 (bs, 1H, -NH), 3.72 (t, J ˆ 4.8 Hz, 2H, CH2NH), 3.4 (q, 2H,
CH2OH), 2.01 (s, 1H, CH3CO) ppm; 13C NMR (CDCl3, ꢁ, 200 MHz): 171.6 (C=O), 61.9 (CH2OH),
42.3 (CH2NH), 23.0 (CH3CO) ppm.
1,3,8-Tri-O-acetoxy-6-(!-2-acetamino-ethoxy-methyl)-anthracene-9,10-dione (5; C25H23NO10)
A mixture of 60 mg (0.11 mmol) 1,6,8-tri-O-acetoxy-3-iodomethyl-anthraquinone (prepared from
emodin according to Ref. [7]), 120 mg (1.1 mmol) of N-(2-hydroxyethyl)-acetamide (prepared
according to Ref. [9]), and 40 mg (0.15 mmol) of silver tri¯uoromethane sulfonate in 7 ml of CH2Cl2
was re¯uxed for 20 h. The reaction mixture was cooled to room temperature, diluted with 50 ml
CH2Cl2, and ®ltered to remove AgI. The ®ltrate was washed 3 times with water, dried over Na2SO4,
and evaporated. Column chromatography of the product mixture on silica using ®rst a mixture of
CHCl3/EtOAc ˆ 1/1 to remove the by-product, which was identi®ed as 1,6,8-tri-O-acetoxy-3-
hydroxymethyl-anthraquinone, and then CHCl3/MeOH ˆ 20/1 gave 20 mg of 3 (37%).
1H NMR (CDCl3, ꢁ, 200 MHz): 8.14 (s, 1H, ar-H), 7.95 (d, J ˆ 2.5 Hz, 1H, ar-H), 7.38 (s, 1H, ar-
H), 7.24 (d, J ˆ 2.5 Hz, 1H, ar-H), 6.0 (bs, 1H, NH-), 4.62 (s, 2H, ar-CH2-O), 3.62 (t, J ˆ 4.8 Hz, 2H,
CH2-NH), 3.53 (t, J ˆ 4.8 Hz, 2H, CH2-O), 2.44 (s, 6H, 2-CH3COO), 2.35 (s, 3H, CH3COO), 2.07 (s,
3H, CH3CONH) ppm; 13C NMR (CDCl3, ꢁ, 50.3 MHz): 181.1 (C=O of quinone), 179.5 (C=O of
quinone), 170.3 (C=O of NHCOCH3), 169.4 (C=O of CH3COO), 169.0 (C=O of CH3COO), 167.9
(C=O of CH3COO), 154.7 (Car), 151.5 (Car), 150.4 (Car), 146.0 (Car), 135.5 (Car), 134.3 (CHar),
128.4 (CHar), 128.3 (Car), 124.5 (Car), 123.5 (CHar), 123.1 (Car), 118.3 (CHar), 71.3 (ar-CH2-O), 69.9
(R-CH2-O), 39.3 (CH2-NH), 23.3 (CH3 of NHCOCH3), 21.1 (CH3 of 3CH3COO) ppm.
1,3,8-Trihydroxy-6-(!-2-acetamino-ethoxy-methyl)-anthracene-9,10-dione (6; C19H17NO7)
A solution of 55 mg (0.11 mmol) of 5 in 5 ml acetone and 2.5 ml of 3 N HCl was re¯uxed for 30 min;
afterwards, another 2.5 ml of HCl were added and re¯uxed for further 2 h. Then the reaction mixture

316
H. Falk et al.
was evaporated to remove acetone and extracted with ethylacetate. The organic phase was washed
twice brine and water, dried over Na2SO4, and chromatographed on a silica column using a mixture
of CHCl3/MeOH ˆ 10/1 as eluent to give 36 mg (90%) 6.
1H NMR (DMSO-d6, ꢁ, 200 MHz): 12.09 (s, 1H, OH), 12.08 (s, 1H, OH), 11.43 (s, 1H, OH), 8.03
(bt, 1H, NH), 7.62 (s, 1H, ar-H), 7.30 (s, 1H, ar-H), 7.14 (d, J ˆ 2 Hz, ar-H), 6.61 (d, J ˆ 2 Hz, ar-H),
4.59 (s, 2H, ar-CH2-O), 3.48 (t, J ˆ 5.4 Hz, 2H, CH2-NH), 3.27 (t, J ˆ 5.4 Hz, 2H, CH2-O), 1.81 (s,
3H, CH3CONH) ppm.
1,3,8-Trihydroxy-6-(!-2-acetamino-ethoxy-methyl)-10H-anthracen-9-one (7; C19H19NO6)
To a solution of 65 mg (0.17 mmol) of 6 in 16 ml of glacial acetic acid, 350 mg of SnCl2 Á 2H2O
(1.54 mmol) in 1.8 ml of conc. HCl were added under heating and re¯uxed for additional 10 min.
After cooling, the reaction mixture was poured into 150 ml of cold brine. The precipitate was
centrifuged, washed with water, and dried under vacuum at 60ꢁC to yield 55 mg (90%) 7.
1H NMR (DMSO-d6, 200 MHz): 12.32 (s, 1H, OH), 12.36 (s, 1H, OH), 10.87 (s, 1H, OH), 8.0
(bs, 1H, NH), 6.88 (s, 1H, ar-H), 6.79 (s, 1H, ar-H), 6.41 (s, 1H, ar-H), 6.22 (d, J ˆ 1.8 Hz, ar-H),
4.48 (s, 2H, ar-CH2-O), 4.31 (s, 2H, ar-CH2-ar), 3.45 (t, J ˆ 5.6 Hz, 2H, CH2-NH), 3.26 (t,
J ˆ 5.5 Hz, 2H, CH2-O), 1.81 (s, 3H, CH3CONH) ppm; 13C NMR (CDCl3, ꢁ, 50.3 MHz): 191.1
(C=O quinone), 169.3 (C=O of NHCOCH3), 165.1 (Car), 164.6 (Car), 161.7 (Car), 147.3 (Car), 145.0
(Car), 142.1 (Car), 117.1 (CHar), 113.9 (Car), 112.7 (CHar), 108.4 (Car), 107.4 (CHar), 101.1 (CHar),
70.9 (ar-CH2-O), 68.9 (O-CH2), 39.0 (overlap with DMSO peak, CH2-NH), 32.4 (ar-CH2-ar), 22.5
(CH3CONH) ppm.
1,3,4,6,8,13-Hexahydroxy-10,11-bis-(!-2-acetamino-ethoxy-methyl)-phenanthro[1,10,9,8-o,p,q,r,a]-
perylene-7,14-dione (8; C38H30N2O12)
A mixture of 80 mg (0.22 mmol) anthrone 7, 130 mg (1.44 mmol) pyridine-N-oxide, 100 ml
piperidine, and 9 mg FeSO4 Á 7H2O dissolved in 1 ml of pyridine was stirred in the dark at 110ꢁC for
1.5 h, cooled to room temperature, and quenched with a solution of 1.5 ml conc. HCl in 15 ml H2O.
After standing for 1 h, the precipitate was isolated by centrifugation and washed once with water.
The precipitate was suspended in 600 ml of acetone and irradiated with a 500 W photo ¯ood lamp for
1.5 h. The resulting suspension was ®ltered and the ®ltrate was evaporated. Preparative silica thin
layer chromatography of the residue using a mixture of CHCl3/MeOH ˆ 4/1 as the eluent gave 9 mg
(10%) 8.
1
M.p.: not below 300ꢁC; H NMR (acetone-d6, ꢁ, 200 MHz): 18.84 (bs, OH-3), 14.83 and 14.27
(2s, OH-1, 6 and OH-8, 13), 7.66 (s, H-9, 12), 7.08 (bs, 2NH), 6.62 (s, H-2, 5), 5.16 and 4.70 (AB-
system, J ˆ 12 Hz, ar-CH2O), 3.5 (m, 2CH2N), 3.2 (m, 2CH2O), 1.76 (s, 2CH3) ppm; UV/Vis
(ethanol, 6 Á 10ꢀ6 mol/l): ꢃmax ˆ 594 (43333), 550 (20666), 512 (7333), 480 (10666), 386 (10000),
328 (26500), 286 (39300) nm (").
1,3,4,6,8,13-Hexahydroxy-10,11-bis-(!-2-amino-ethoxy-methyl)-phenanthro[1,10,9,8-
o,p,q,r,a]perylene-7,14-dione (9; C34H26N2O10)
The acetamino derivative 8 could be saponi®ed by means of boiling 6 N HCl; however, neither the
obtained hydrochloride nor the free base could be dissolved in any solvent. IR (KBr): ꢂ ˆ 3448,
2923, 1617, 1465, 1247, 1112, 951 cmꢀ1
.
1,3,8-Trihydroxy-6-(!-2-chloro-ethoxy-methyl)-anthracene-9,10-dione (10; C17H13O6Cl)
A mixture of 160 mg (0.33 mmol) 1,6,8-tri-O-acetoxy-3-bromomethyl-anthraquinone (prepared from
emodin according to Ref. [6]), 4.5 g (55 mmol) of 2-chloroethanol, and 150 mg (0.58 mmol) AgClO4

Hypericin Tetrasulfonic Acid
317
was heated at 90ꢁC for 3 h. the reaction mixture was cooled to room temperature, diluted with 150 ml
CH2Cl2, and ®ltered. The ®ltrate was washed with brine and H2O, dried over Na2SO4, and
evaporated. Column chromatography of the product mixture on silica using a mixture of CHCl3/
MeOH ˆ 20/1 gave 110 mg of 10 (95%).
1H NMR (DMSO-d6, ꢁ, 200 MHz): 12.08 (bs, 2H, 2OH), 11.45 (bs, 1H, OH), 7.65 (s, 1H, ar-H),
7.29 (s, 1H, ar-H), 7.14 (d, J ˆ 2.3 Hz, 1H, ar-H), 6.62 (d, J ˆ 2.3 Hz, 1H, ar-H), 4.68 (s, 2H, ar-CH2-
1
O), 3.89±3.76 (m, 4H, CH2-O and CH2-Cl) ppm; H NMR (acetone-d6, ꢁ, 200 MHz): 12.16 (s, 1H,
OH), 12.13 (s, 1H, OH), 10.19 (bs, 1H, OH), 7.72 (s, 1H, ar-H), 7.32 (s, 1H, ar-H), 7.27 (d,
J ˆ 2.3 Hz, 1H, ar-H), 6.68 (d, J ˆ 2.3 Hz, 1H, ar-H), 4.76 (s, 2H, ar-CH2-O), 389±3.76 (m, 4H,
CH2-O and CH2-Cl) ppm; 13C NMR (DMSO-d6, ꢁ, 50.3 MHz): 190.5 (C=O), 182.1 (C=O), 166.5
(Car), 165.4 (Car), 162.2 (Car), 149.1 (Car), 135.9 (Car), 134.0 (Car), 122.5 (CHar), 118.4 (CHar),
115.5 (Car), 109.3 (Car), 109.7 (CHar), 108.3 (CHar), 71.3 (ar-CH2-O), 71.1 (R-CH2-O), 44.5
(CH2-Cl) ppm.
1,3,8-Trihydroxy-6-(!-2-chloro-ethoxy-methyl)-10H-anthracen-9-one (11; C17H15O5Cl)
To a solution of 110 mg (0.31 mmol) of 10 in 24 ml glacial acetic acid, 400 mg of SnCl2 Á 2H2O
(1.76 mmol) dissolved in 2.8 ml of conc. HCl were added under heating and re¯uxed for additional
10 min. After cooling, the reaction mixture was poured into 150 ml of cooled brine. The precipitate
was centrifuged, washed with water, and dried under vacuum at 60ꢁC to yield 93 mg (90%)
of 11.
1H NMR (acetone-d6, ꢁ, 200 MHz): 12.47 (s, 1H, OH), 12.37 (s, 1H, OH), 9.8 (bs, 1H, OH), 6.99
(s, 1H, ar-H), 6.88 (s, 1H, ar-H), 6.55 (s, 1H, ar-H), 6.35 (d, J ˆ 1.8 Hz, ar-H), 4.66 (s, 2H, ar-CH2-
1
O), 4.38 (s, 2H, ar-CH2-ar), 3.83±3.76 (m, 4H, CH2-O and CH2-Cl) ppm; H NMR (DMSO-d6, ꢁ,
200 MHz): 12.37 (s, 1H, OH), 12.31 (s, 1H, OH), 10.9 (bs, 1H, OH), 6.94 (s, 1H, ar-H), 6.83 (s, 1H,
ar-H), 6.45 (s, 1H, ar-H), 6.25 (d, J ˆ 2.2 Hz, ar-H), 4.66 (s, 2H, ar-CH2-O), 4.38 (s, 2H, ar-CH2-ar),
3.83±3.76 (m, 4H, CH2-O and CH2-Cl) ppm.
1,3,4,6,8,13-Hexahydroxy-10,11-bis-(!-2-piperidinyl-ethoxy-methyl)-phenanthro-
[1,10,9,8-o,p,q,r,a]perylene-7,14-dione (12; C44H42O10N2)
A mixture of 115 mg (0.34 mmol) 11, 200 mg (2.2 mmol) pyridine-N-oxide, 200 ml piperidine, and
12 mg FeSO4 Á 7H2O dissolved in 2 ml of pyridine was stirred in the dark at 110ꢁC for 1 h, cooled to
room temperature, and quenched with a solution of 2 ml conc. HCl in 20 ml H2O. After standing for
1 h, the precipitate was isolated by centrifugation and washed once with water. The precipitate was
suspended in 1000 ml acetone and irradiated with a 500 W photo ¯ood lamp for 1.5 h. The resulting
suspension was ®ltered and the ®ltrate was evaporated. Column chromatography of the residue on
Sephadex LH-20 using MeOH as the eluent gave 10 mg (7%) of 11.
1H NMR (DMSO-d6, ꢁ, 200 MHz): 14.83 and 14.32 (2s, OH-1, 6 and OH-8, 13), 7.52 (s, H-9,
12), 6.53 (s, H-2, 5), 5.1 and 4.6 (AB-system, J ˆ 12 Hz, ar-CH2O), 3.5 (m, 4H, 2CH2N), 3.3 (m, 4H,
2CH2O), 2.8 (m, 8H, NCH2 of piperidinyl group), 1.5 (m, 12H, CH2 of piperidinyl group) ppm; 13
C
NMR (DMSO-d6, 50.3 MHz): 184.5 (C=O), 176.1 (Car), 169.1 (Car), 162.3 (Car), 145.1 (Car), 128.2
(Car), 122.3 (Car), 120.2 (Car), 119.8 (CHar), 118.5 (CHar), 110.8 (Car), 106.2 (CHar), 102.8 (CHar),
72.5 (ar-CH2-O), (R-CH2-O), 71.4 (R-CH2-O), 50.9 (CH2N of piperidinyl), 42 (overlap with DMSO
peak; CH2N), 25.1, 23.4 (CH2 of piperidinyl) ppm; UV/Vis (ethanol, 6 Á 10ꢀ6 mol/l): ꢃ ˆ 591
(41130), 547 (20833), 510 (9167), 474 (12983), 380 (11600), 327 (29620) nm ("); UV/Vis (DMSO,
6 Á 10ꢀ6 mol/l): ꢃ ˆ 601 (43300), 556 (22275), 517 (13500), 485 (13562), 388 (17125), 330 (32550)
nm ("); UV/Vis (acetone, 6l Á 10ꢀ6 mol/l): ꢃ ˆ 598 (43625), 553 (21125), 512 (4762), 460 (7975),
382 (6137) nm (").

318
H. Falk et al.: Hypericin Tetrasulfonic Acid
Acknowledgements
È
This work was sponsored by the Fonds zur Forderung der Wissenschaftlichen Forschung (Project N.
È
P10680-CHE); A.A.O.S. gratefully acknowledges a grant of the Osterreichischer Akademischer
Austauschdienst.
References
[1] Altmann R, Falk H (1997) Monatsh Chem 128: 571
[2] Zollinger H (1987) Color Chemistry. VCh, Weinheim
[3] Falk H, Schmitzberger W (1993) Monatsh Chem 124: 77
[4] Falk H, Meyer J (1994) Monatsh Chem 125: 753
[5] Handbook of Chemistry and Physics, 70th edn (1990) Tab D-163, CRC Press
[6] Falk H, Vaisburg AF, Amer AM (1995) Monatsh Chem 126: 993; Falk H, Tran TNH (1996)
Monatsh Chem 127: 717; Thiem J, Wessel HP (1985) Z Naturforsch 40b: 422
[7] Altmann R, Etzlstorfer C, Falk H (1997) Monatsh Chem 128: 361
[8] Mazur Y, Block H, Lavie D (1991) Can Pat Appl, CA 2, 029, 993
[9] Kisfaludy L, Mohacsi T, Low M, Drexler F (1979) J Org Chem 44: 654
Received October 22, 1997. Accepted November 13, 1997