Use for topsentin compounds and pharmaceutical compositions containing same

A novel use for the class of biologically active bis-indole alkaloid compounds, which have been named topsentins, nortopsentins, or dragmacidins, pharmaceutical compositions containing them, methods of producing the compounds, and methods of using the compounds are disclosed. Specifically, the novel utility pertains to the anti-inflammatory properties exhibited by the bis-indole compounds and their analogs.

DESCRIPTION 
1. Field of the Invention 
This application relates to bis-indole alkaloid compounds and compositions 
containing such compounds as active ingredients. More particularly, the 
invention concerns a novel use of biologically active bis-indole 
compounds, pharmaceutical compositions containing these compounds, methods 
of producing the compounds, and methods of using the compounds. The novel 
use of the compounds relates to the anti-inflammatory properties of the 
disclosed bis-indole compounds, which include topsentins, nortopsentins, 
dragmacidins, and their analogs. 
2. Background of the Invention 
The prevention and control of inflammation is of prime importance to man, 
and much research has been devoted to development of compounds having 
anti-inflammatory properties. Certain methods and chemical compositions 
have been developed which aid in inhibiting or controlling inflammation, 
but additional methods and anti-inflammatory compositions are needed. 
It has been found that some natural products and organisms are potential 
sources for chemical molecules having useful biological activity of great 
diversity. Marine sponges have proved to be such a source, and a number of 
publications have issued disclosing organic compounds derived from marine 
sponges. Such publications include Scheuer, P. J., Ed. (1978-1983) Marine 
Natural Products, Chemical and Biological Perspectives, Academic Press, 
New York; Faulkner, D. J. (1984) Natural Products Reports 1:551-598; 
Faulkner, D. J. (1986) Natural Products Reports 3:1-33; Faulkner, D. J. 
(1987) Natural Products Reports 4:539-576; Uemura, D., K. Takahashi, T. 
Yamamoto, C. Katayama, J. Tanaka, Y. Okumura, Y. Hirata (1985) J. Am. 
Chem. Soc. 107:4796-4798. 
Indole compounds of marine origin have also been described in Moquin, C., 
M. Guyot (1984) Tetrahedron Letters 25(44):5047-5048 and Norton, R. S., R. 
J. Wells (1982) J. Am. Chem. Soc. 104(13):3628-3635. 
Utilizing sponges as a source material and supplemented by novel synthetic 
production methods, new classes of biologically active compounds and new 
pharmaceutical compositions useful as antitumor and antiviral agents have 
been provided to the art. See U.S. Pat. Nos. 4,866,084, 4,895,844, and 
4,970,266. The present invention provides a novel utility for these 
compounds, namely as anti-inflammatory compositions. 
Other advantages and further scope of applicability of the present 
invention will become apparent from the detailed descriptions given 
herein; it should be understood, however, that the detailed descriptions, 
while indicating preferred embodiments of the invention, are given by way 
of illustration only, since various changes and modifications within the 
spirit and scope of the invention will become apparent from such 
descriptions. 
BRIEF SUMMARY OF THE INVENTION 
The objects of the invention are accomplished by the provision of a novel 
utility for the class of biologically active bis-indole compounds that 
have been named topsentins, nortopsentins, or dragmacidins, and have a 
structure according to the formula: 
##STR1## 
R.sub.1-8 are the same or different selected from --H, --OH, halogen, --R, 
--OR, --OCOR, or --OA; 
Y is the single group .dbd.O, or two groups, same or different, selected 
from --H, --OH, --OR, or --OCOR; 
Z is selected from --H, --R, or --COR; 
R is C1-5 alkyl and A is --R-phenyl. 
A preferred group of compounds of the invention are those of the formula: 
##STR2## 
and wherein R.sub.1 and R.sub.3 are --H while R.sub.2 and R.sub.4 are --H, 
--OH, halogen, --R, --OR, --OCOR, or --OA; or R.sub.2 and R.sub.4 are --H 
while R.sub.1 and R.sub.3 are --H, --OH, halogen, --R, --OR, --OCOR, or 
--OA; R is C1-5 alkyl, and A is --R-phenyl. 
Particularly preferred compounds of the invention are those of the 
formulae: 
##STR3## 
wherein: 
__________________________________________________________________________ 
I(a): 
R.sub.1, R.sub.2, R.sub.3 = H; R.sub.4 = OH 
(Topsentin) 
I(b): 
R.sub.1, R.sub.3 = H; R.sub.2 = Br, R.sub.4 = OH 
(Bromotopsentin) 
I(c): 
R.sub.1, R.sub.3, R.sub.4 = H; R.sub.2 = OH 
(Isotopsentin) 
I(d): 
R.sub.1, R.sub.3 = H; R.sub.2, R.sub.4 = OH 
(Hydroxytopsentin) 
I(e): 
R.sub.1, R.sub.2, R.sub.3, R.sub.4 = H 
(Deoxytopsentin) 
I(f): 
R.sub.1, R.sub.2, R.sub.4 = H; R.sub.3 = OH 
(Neotopsentin) 
I(g): 
R.sub.2, R.sub.3, R.sub.4 = H; R.sub.1 = OH 
(Neoisotopsentin) 
I(h): 
R.sub.1, R.sub.3 = OH; R.sub.2, R.sub.4 = H 
(Neohydroxytopsentin) 
II(j): 
R.sub.1, R.sub.2, R.sub.3, R.sub.5 = H; R.sub.4 = OCOCH.sub.3 
(Topsentin monoacetate) 
II(k): 
R.sub.1, R.sub.2, R.sub.3 = H; R.sub.4 = OCOCH.sub.3 ; R.sub.5 = 
COCH.sub.3 (Topsentin diacetate) 
II(l): 
R.sub.1, R.sub.3 = H; R.sub.2, R.sub.4 = Br 
(Nortopsentin A) 
III(m): 
R.sub.1, R.sub.2, R.sub.3 = H; R.sub.4 = Br 
(Nortopsentin B) 
III(n): 
R.sub.1, R.sub.3, R.sub.4 = H; R.sub.2 = Br 
(Nortopsentin C) 
IV(o): 
R.sub.1, R.sub.3 = H; R.sub.2, R.sub.4, R.sub.5 = Br 
(Dragmacidin) 
__________________________________________________________________________ 
These bis-indole compounds have been previously described as having 
antimicrobial, antitumor, or antiviral activity. The topsentins are 
disclosed in U.S. Pat. No. 4,866,084. Dragmacidin and its related 
compounds isolated from the marine sponge of the Dragmacidon sp. are 
disclosed in U.S. Pat. No. 4,895,844. Similarly, the nortopsentins have 
been disclosed in U.S. Pat. No. 4,970,226. These patents are herein 
incorporated by reference. 
Skilled chemists will be able to use procedures as disclosed herein and 
others to synthesize these compounds from available stock substances. In 
carrying out such operations, any suitable filtration, chromatographic, 
and other purification techniques include reversed phase, medium pressure, 
and high pressure liquid chromatography (RPLC, MPLC, and HPLC, 
respectively) with a suitable column as would be known to those skilled in 
the art, including silica gel, Sephadex LH-20, ammonia-treated silica gel, 
and LiChrosorb NH.sub.2 columns. Such columns are eluted with suitable 
elements such as heptane, ethyl acetate, methylene chloride, methanol, 
isopropyl alcohol, and various combinations and ratios thereof. 
As embodied and fully described herein, the invention also comprises 
pharmaceutical compositions, e.g., anti-inflammatory compositions, 
containing as active ingredient an effective amount, preferably between 
about 0.1 to 45%, especially 1 to 25%, by weight based on the total weight 
of the composition, of one or more compounds according to the formulas 
expressed above and a non-toxic, pharmaceutically acceptable carrier or 
diluent. In addition, a pharmaceutical composition can comprise at least 
one of the subject bis-indole compounds and a second component comprising 
at least one other anti-inflammatory compounds. Such other 
anti-inflammatory compounds include, but are not limited to, steroidal 
compounds, e.g., hydrocortisone and the like; non-steroidal 
anti-inflammatories, e.g., acetylsalicylic acid (aspirin), ibuprofen, 
acetominophen, indomethacin, and the like. 
As embodied and fully described herein, the invention comprises processes 
for the production of compounds and compositions of the invention and 
novel methods of use thereof, e.g., methods of inhibiting an inflammatory 
response in an animal. 
In accordance with the invention, methods for inhibiting inflammation 
comprise administering to the animal in need of such treatment an 
effective amount of the pharmaceutical compositions of the invention.

DETAILED DISCLOSURE OF THE INVENTION 
A more complete understanding of the invention can be obtained by reference 
to preferred embodiments of the invention, which are illustrated by the 
following specific examples of compounds, compositions, and methods of the 
invention. It will be apparent to those skilled in the art that the 
examples involve use of materials and reagents that are commercially 
available from known sources, e.g., chemical supply houses, so no details 
are given respecting them. 
One method of preparation for the compounds used for the subject invention 
involves extraction from marine sponges of the order Halichondrida (Phylum 
Porifera, Class Demospongiae) which is a problematic taxonomic group, with 
generic distinctions not clearly defined. Four samples used in connection 
with this invention have been assigned to the genus Spongosorites, Topsent 
1896, a genus characterized by: a distinct and thick (up to 1 mm) dermal 
layer of smaller spicules arranged tangentially to the surface; a confused 
choanosomal arrangement of spicules with sporadic vague spicule tracts 
running parallel to the surface; bright-yellow color when alive, turning 
brown or black when preserved in alcohol; and two or three size categories 
of straight or crooked oxea. Spongosorites sp.1 (4-XII-84-1-22, black in 
alcohol) has crooked oxea and is distinguished in association with 
vermetids (Phylum Mollusca, Class Gastropoda); Spongosorites sp.3 
(4-XII-84-1-23 and 23-VIII-85-1-39, tan-brown in alcohol) has fusiform 
straight oxea. Voucher samples are deposited in the Indian River Coastal 
Zone Museum of Harbor Branch Oceanographic Institution at Fort Pierce, 
Fla. 
A current taxonomic identification of the sponge from which the compound 
dragmacidin was extracted is: Phylum Porifera, Class Demospongiae, Order 
Axinellida, Family Axinellidae, Genus Dragmacidon, as disclosed in U.S. 
Pat. No. 4,895,844. 
The sponge Dragmacidon sp. is dark brown, both alive and preserved in 
ethanol. The consistency is brittle and non-compressible. The ectosome is 
a heavy organic skin with foreign material. The choanosome is fibrous, 
with sparse dendriticplumose spicule tracts. Spicules are trichodragmata 
and styles, 400-520 .mu.m in length by 7-10 .mu.m in width. 
Identification of the sponge to the family Axinellidae and genus 
Dragmacidon is based on microscopic examination of a taxonomic voucher 
specimen. A similar voucher specimen is deposited at the Indian River 
Coastal Zone Museum (Catalog No. 003:00039), Harbor Branch Oceanographic 
Institution, Inc., Fort Pierce, Fla. (HBOI/DBMR number 2-VI-84-3-15). The 
sponge in the voucher specimen is preserved in 70% ethanol with an 
expected shelf life of at least 30 years and is accessible to those 
skilled in the art for identification purposes. 
The sponge Dragmacidon sp. was collected from southeast Grand Bahama Island 
(latitude 26.degree.28.75' N, longitude 77.degree.53.50' W) at a depth of 
480 ft. on a rock and sand slope. 
As disclosed herein, a novel use for the described compounds is their use 
as an agent in the control of an inflammatory response. For purposes of 
the subject invention, it will be understood by those of ordinary skill in 
the art that the terms "inflammation" and "inflammatory response" refer to 
any and all such inflammatory cellular and tissue reactions including, but 
not limited to, immune-related responses and/or allergic reactions to a 
physical, chemical, or biological stimulus. "Anti-inflammatory activity," 
as used herein, will be understood by those of ordinary skill in the art 
to mean biological activity inhibiting or controlling any inflammatory 
response. Anti-inflammatory activity can occur by modes of action which 
can include, but are not limited to, lipid-mediated inflammatory 
responses, e.g., (i) suppression of cellular activation of phospholipase 
A2, either directly (as is known for the anti-inflammatory compound, 
monoalide) or indirectly (as is known for the anti-inflammatory compound, 
hydrocortisone); (ii) by inhibiting, or controlling, cyclooxygenation of 
arachidonic acid, similar to the action of non-steroidal anti-inflammatory 
drugs; or (iii) by affecting lipooxygenase products of peroxidase 
reactions to arachidonic acid, or by non-lipid-mediated inflammatory 
responses, e.g., protease-induced inflammatory responses, and the like. 
Following are examples which illustrate procedures, including the best 
mode, for practicing the invention. These examples should not be construed 
as limiting. All percentages are by weight and all solvent mixture 
proportions are by volume unless otherwise noted. 
EXAMPLE 1 
Preparation of Topsentin and Bromotopsentin 
The frozen sample (264 g) of marine sponge, Spongosorites ruetzleri (Van 
Soest and Stentoft 1988), collected at a depth of 1149 feet at Goulding's 
Cay, Bahamas, was extracted twice with methanol-toluene (3:1). The 
combined extracts on concentration on a water bath at 30.degree. C. in 
vacuo gave as a residue (11.32 g) of crude extract which was partitioned 
between pentane and 10% aqueous methanol. The alcohol layer was then 
diluted to 30% water and extracted with CH.sub.2 Cl.sub.2. The aqueous 
methanol layer was concentrated and partitioned between butanol and water. 
A portion (200 mg) of the Herpes simplex virus type 1 (HSV-1)-active, 
butanol-soluble fraction was dissolved in 20% aqueous methanol (1 ml) and 
chromatographed on a column (ID=22 mm; height=40 mm) packed with 
reversed-phase material (Amicon silica C8, 20-45 .mu.m). The active 
fraction (123 mg) was eluted with 20% aqueous methanol and purified by 
reversed-phase HPLC (IBM 5.mu. C18, 10 mm.times.250 mm, 20% aqueous 
methanol) to yield pure topsentin I(a), 20 mg, and bromotopsentin I(b), 67 
mg as yellow powder. 
Topsentin, amorphous, bright-yellow solid, mp&gt;250.degree. C. when analyzed 
by conventional methods and apparatus, produced the following spectral 
data: 
UV absorption, .lambda..sub.max (MeOH) 208 nm (.epsilon.12,000), 246 sh 
(5100), 285 (4500), and 375 (4600); 
IR (KBr) 3395, 3275, 1635, 1590, 1530, 1455, 1270, 1165, 1115, 1095, 1005, 
and 876 cm.sup.-1 ; 
.sup.1 H NMR (360 MHz, DMSO-d.sub.6 +1% TFA-H) 6.841 (1H, dd, J=8.6, 1.8 
Hz), 6.997 (1H, d, J=1.8 Hz), 7.201 (2H, m), 7.523 (1H, d, J=7.9 Hz), 
7.990 (1H, d, J=7.6 Hz), 8.041 (1H, d, J=8.6 Hz), 8.155 (1H, d, J=2.8 Hz), 
8.159 (1H, s), 8.487 (1H, d, J=3.2 Hz), 11.762 (1H, s), 12.355 (1H, d, 
J=2.2 Hz); 
.sup.13 C NMR (90 MHz, DMSO+1% TFA-H) 98.11 (d), 102.72 (s), 113.12 (d), 
113.95 (s), 116.00 (d), 118.67 (s), 119.46 (d), 120.50 (d), 122.02 (d), 
122.44 (d), 124.27 (s), 125.74 (d), 131.11 (s), 136.53 (s), 137.78 (d), 
138.33 (s), 141.23 (s), 155.25 (s), 171.5 (s); 
EIMS 342 (100%, C.sub.20 H.sub.14 N14O.sub.2, M.sup.+), 209 (39, C.sub.12 
H.sub.7 N.sub.2 O), 183 (28, C.sub.11 H.sub.9 N.sub.3), 171 (17, C.sub.10 
H.sub.7 N.sub.2 O), 160 (145, C.sub.9 H.sub.7 N.sub.2 O), 133 (65, C.sub.8 
H.sub.7 NO), and 105 (15). 
Bromotopsentin, yellow crystals, m.p. 296.degree.-7.degree. C., when 
analyzed by conventional methods and apparatus, produced the following 
spectral data: 
UV absorption, .lambda..sub.max (MeOH) 209 nm (.epsilon.13,000), 236 
(9700), 287 (5000), and 374 (5800); 
IR (KBr) 3400-3100, 2255, 2120, 1635, 1590, 1520, 1445, 1265, 1230, 1165, 
1028, 1005, and 875 cm.sup.-1 ; 
.sup.1 H NMR (360 MHz, CDCl.sub.3 :CF.sub.3 COOH: 1:1) 7.098 (1H, dd, 
J=8.6, 2.4 Hz), 7.193 (1H, d, J=2.4 Hz), 7.227 (1H, dd, J=8.6, 1.8 Hz), 
7.558 (1H, dd, J=8.6 Hz), 7.668 (1H, d, J=1.8 Hz), 7.824 (1, s), 7.927 
(1H, d, J=3 Hz), 8.202 (1H, d, J=8.6 Hz), 8.371 (1H, d, J=3 Hz), 9.272 
(1H, brs), 10.409 (1H, brs); 
.sup.13 C NMR (90 MHz, CDCl.sub.3 :CF.sub.3 COOH: 1:1) 101.6 (d), 103.7 
(s), 116.7 (d), 117.0 (s), 117.5 (d), 118.2 (d), 119.6 (s), 121.5 (d), 
122.6 (s), 125.2 (s), 125.5 (d), 127.7 (d), 128.0 (d), 135.0 (s), 139.7 
(s), 140.5 (s), 140.8 (d), 141.7 (s), 155.0 (s), 172.4 (s); 
EIMS 422/420 (40%, C.sub.20 H.sub.13 BrN.sub.4 O.sub.2, M.sup.+), 394/392 
(1.3, C.sub.19 H.sub.11 BrN.sub.3 O.sub.2), 342 (13, M.sup.+ -Br), 289/287 
(6%, C.sub.12 H.sub.7 BrN.sub.3 O), 263/261 (100, C.sub.11 H.sub.8 
BrN.sub.3), 223/221 (13, C.sub.9 H.sub.6 BrN.sub.2), 209/207 (9.5, C.sub.9 
H.sub.6 BrNO), 182 (15, 261-Br), and 133 (94, C.sub.8 H.sub.7 NO). 
EXAMPLE 2 
Frozen sponge sample of Spongosorites sp.3 collected at Goulding's Cay, 
Bahamas at -229 m was homogenized and steeped repeatedly in methanol and 
10% toluene followed by methanol. The alcohol layer was concentrated and 
re-partitioned between 1-butanol and water, and the butanol-soluble 
fraction was vacuum chromatographed over RP material (Amicon, silica gel 
C18, 20-45 .mu.m) using 20% aqueous methanol. The yellow fraction was then 
subjected twice to RP-HPLC (C18, 5 .mu.m, 20% water in MeOH) to give 
bromotopsentin I(b) and 4,5-dihydro-6"-deoxybromotopsentin. 
4,5-dihydro-6"-deoxybromotopsentin is a yellow powder with the following 
spectral data: 
[.alpha.].sup.24 D 198.degree. (c 2.0, MeOH); 
UV (MeOH) .lambda..sub.max nm 328 (.epsilon.5700), 274 (8800), 214 
(34,000), 198 (29,500); 
IR (KBr) 3630, 3390, 3280, 2920, 2860, 1664, 1570, 1450, 1420, 1332, 1240, 
1160, 1120, 1100, 1020, 950, 805, and 750 cm.sup.-1 ; 
LREIMS m/z (rel. intensity) 406 (95), 404 (100), 378 (41), 376 (39), 326 
(10), 298 (6), 297 (7), 291 (10), 289 (9), 235 (6), 233 (6), 210 (12), 208 
(10), 197 (10), 195 (10), 189 (5), 156 (12), 155 (19), 144 (28), 130 (14). 
.sup.1 H and .sup.13 C NMR collected data also supported the structure 
given above. 
Calcd for C.sub.20 H.sub.13.sup.79 BrN.sub.4 O: 404.0272 (M--2H). Found 
404.0300 (HREIMS). The conversion of bromotopsentin to topsentin; the 
preparation of 3-(hydroxyacetyl)indole, 
3-chloroacetyl-6-(benzyloxy)indole, and 
3-hydroxyacetyl-6-(benzyloxy)indole as synthons; the synthesis directly 
from (hydroxylacetyl)indoles of O-benzyltopsentin, O-benzylisotopsentin, 
O,O'-dibenzylhydroxytopsentin, and deoxytopsentin I(e); the preparation of 
compounds I(e) and O-benzyltopsentin, O-benzylisotopsentin, and 
O,O'-dibenzylhydroxytopsentin from isolated glyoxal intermediates; the 
conversion of O-benzyltopsentin to topsentin I(a); the conversion of 
O-benzylisotopsentin to isotopsentin I(c); the synthesis of 
hydroxytopsentin I(d) from 3-hydroxy-acetyl-6-(benzyloxy)-indole; 
preparation of 3-chloroacetyl-5-benzyl(oxy)indole; the preparation of 
3-hydroxyacetyl-5-(benzyloxy)indole; synthesis of neohydroxytopsentin 
I(h); and synthesis of neotopsentin I(f) and neoisotopsentin I(g) are 
described in U.S. Pat. No. 4,866,084, which has been incorporated herein 
by reference. Topsentin monoacetate II(j) and topsentin diacetate II(k) 
can also be converted from topsentin or its analogs by methods well known 
and readily available to those skilled in the art. 
EXAMPLE 3 
Isolation of Nortopsentin A, B, & C 
The sponge Spongosorites sp. (80 g), collected at the depth of 630 ft. off 
Chub Cay, Bahamas, on Aug. 26, 1985, was lyophilized and extracted with 
methanol-toluene (3:1). The extract was evaporated to dryness and 
partitioned between ethyl acetate and water. The water soluble fraction 
was further partitioned with butanol. The combined ethyl acetate and 
butanol fractions were chromatographed on a Hibar LiChrosorb NH.sub.2 
column using HPLC with CHCl.sub.3 -MeOH (5:1) as elution solvent to yield 
a semi-purified compound, nortopsentin B (3 mg). 
Sponge of the genus Halichondria (830 g) was collected at the depth of 1512 
ft. off Nassau, Bahamas, on Mar. 15, 1987. The frozen sponge was extracted 
with 1.5 l of methanol four times. The extracts were combined and 
concentrated under reduced pressure to give a 400 ml of water suspension, 
which was then extracted with ethyl acetate (300 ml.times.3). The 
resulting ethyl acetate fraction was evaporated to dryness to yield a 
crude fraction (12.02 g). It was found that the majority of the components 
in this fraction was topsentin and bromotopsentin. 
A two-phase solvent system was generated by mixing heptane, ethyl acetate, 
methanol, and water in a ratio of 4:7:4:3. The crude fraction (12.00 g) 
was partitioned between 150 ml of the upper phase solvent and 300 ml of 
the lower phase solvent. The resulting lower layer fraction was extracted 
with 150 ml of the upper phase solvent three more times. The combined 
upper layer fractions were evaporated to dryness (5.75 g) and dissolved in 
50 ml of the upper phase solvent. The solids were filtered off and the 
eluant was evaporated to dryness (4.46 g). The residue was dissolved again 
in 30 ml of the upper phase solvent. After removal of the insoluble 
material and evaporation of the solvent, 2.75 g of a solid was obtained. 
This solid was further fractionated by using centrifugal countercurrent 
chromatography with two different solvent systems consisting of 
heptane/ethyl acetate/methanol/water in ratios of 4:7:4:3 and 5:7:4:3. A 
fraction containing nortopsentin A and a mixture of nortopsentin B and C 
along with topsentin (400 mg) and bromotopsentin (540 mg) were obtained. 
Nortopsentin A (250 mg) was purified by HPLC on a Hibar NH.sub.2 column 
(10.times.250 mm), using 5:1 chloroform/methanol as eluant. Preparative 
TLC (Kieselgel 60F.sub.264, 2 mm thickness, ethyl acetate) afforded a pure 
nortopsentin C (200 mg) and a fraction containing nortopsentin B. Pure 
nortopsentin B (250 mg) was finally recrystallized from ethyl 
acetate/chloroform. 
EXAMPLE 4 
Anti-Inflammatory Properties of the Bis-Indole Compounds 
The anti-inflammatory activity of a particular compound can be demonstrated 
by two standard assays, one in vivo and one in vitro. These assays are 
commonly employed by those skilled in the art and are accepted as 
indicative of anti-inflammatory activity in humans. These assays are 
described below. 
A. Mouse ear anti-inflammatory assay. The test compound and a known 
inflammatory agent, phorbol myristate acetate (PMA), are topically applied 
simultaneously to the left ears of mice. Three hours and 20 minutes 
following application, the mice are sacrificed. Both left and right ears 
are removed and standard-sized bores taken. Edema (inflammation) is 
measured as the difference in weight between left and right ears (Van 
Arman, C. G. [1974] Clin. Pharmacol. Ther. 16:900-904). 
B. In vitro inactivation of bee venom phospholipase A2. Phosphatidylcholine 
dipalmitoyl (labeled and unlabeled) is used as the substrate in 
monomolecular form. Test compounds are preincubated with the enzyme (25 
units/0.5 ml) for 1 hour at 41.degree. C. The reaction is initiated by the 
addition of an aliquot of the drug-enzyme mixture to the substrate (0.68 
.mu.moles/0.5 ml) and allowed to continue for 15 seconds. The reaction is 
terminated and the hydrolysis product is measured via scintillation 
counting. For screening, the test compounds (in methanol or DMSO) are 
added to the enzyme at a standard bench concentration of 5 mg/ml, for an 
enzyme-incubation concentration of 80 .mu.g/ml, and a final concentration 
of 0.8 .mu.g/ml. Assays are done in triplicate and results are averaged 
and compared to a vehicle control rate of hydrolysis. 
The bis-indole compounds of the subject invention show significant 
anti-inflammatory properties. When screened for the ability to reduce 
edema in mouse ears caused by application of a known inflammatory agent, 
phorbol myristate acetate, topsentin was found to have greater potency 
than to the known anti-inflammatories hydrocortisone, indomethacin, and 
manoalide (see Table 1). 
TABLE 1 
______________________________________ 
Relative potency of topsentin, manoalide, 
Indomethacin, and hydrocortisone in the topical inhibition of 
PMA-induced mouse ear edema 
Compound ED.sub.50 (.mu.g/ear) 
______________________________________ 
Hydrocortisone 20 
Indomethacin 250 
Manoalide 100 
Topsentin 15 
______________________________________ 
In addition, the bis-indole compounds were tested for percent inhibition of 
PMA-induced edema. Topsentin, bromotopsentin, dragmacidin, nortopsentin A, 
and nortopsentin C displayed significant potency (Table 2). Topsentin 
monoacetate and diacetate showed moderate activity in this assay. 
TABLE 2 
__________________________________________________________________________ 
Percent inhibition of PMA induced edema in mouse ears 
by topsentin and analogs 
Compound Name 
Compound Number 
Dose % Inhibition of edema 
__________________________________________________________________________ 
Topsentin HB 18 50 .mu.g/ear 
70.6 
Bromotopsentin 
HB 19 50 .mu.g/ear 
75.4 
Topsentin monoacetate 50 .mu.g/ear 
45.8 
Topsentin diacetate 50 .mu.g/ear 
42.6 
Dragmacidin HB 6 50 .mu.g/ear 
64.0 
Nortopsentin A 
HB 127 50 .mu.g/ear 
98.1 
Nortopsentin B 
HB 128 50 .mu.g/ear 
38.2 
Nortopsentin C 
HB 129 50 .mu.g/ear 
70.1 
__________________________________________________________________________ 
Topsentin also proved to be capable of the inactivation of bee venom 
phospholipase A2 (Table 3). Hydrocortisone and indomethacin were inactive 
at concentrations up to 1 mM. 
TABLE 3 
______________________________________ 
Relative potency of topsentin, manoalide, 
indomethacin, and hydrocortisone in the inactivation of 
bee venom phospholipase A2 
Compound IC.sub.50 
______________________________________ 
Hydrocortisone &gt;1 mM 
Indomethacin &gt;1 mM 
Manoalide 0.05 .mu.M 
Topsentin 0.5 .mu.M 
______________________________________ 
Bis-indole compounds of the subject invention were tested at a final 
concentration of 1 .mu.M for their percent inactivation of the bee venom 
phospholipase A2. The results of these tests are shown in Table 4. 
TABLE 4 
__________________________________________________________________________ 
Percent inactivation of bee venom phospholipase A2 
by topsentin and analogs 
Compound Name 
Compound Number 
Final Concentration 
% Inactivation 
__________________________________________________________________________ 
Topsentin HB 18 1 .mu.M 67 
Bromotopsentin 
HB 19 1 .mu.M 33 
Topsentin monoacetate 1 .mu.M 42 
Topsentin diacetate 1 .mu.M 32 
Dragmacidin HB 6 1 .mu.M 27 
Nortopsentin A 
HB 127 1 .mu.M 30 
Nortopsentin B 
HB 128 1 .mu.M 27 
Nortopsentin C 
HB 129 1 .mu.M 26 
__________________________________________________________________________ 
In consideration of the data presented, the subject bis-indole compounds 
have been shown to have potent anti-inflammatory characteristics with 
unique clinical applications. Their mechanism of action appears to be the 
consequence of inactivation of phospholipase A2. 
Dose-response curves for topsentin and bromotopsentin were generated and 
are shown as FIGS. 1, 2, and 3. Specifically, the dose-response curve for 
topsentin, measured as percent inactivation of bee venom phospholipase A2 
(FIG. 1) shows up to 80% inactivation at a final concentration of 
approximately 2 .mu.M. In the mouse ear edema inhibition assay, a dose of 
about 12 .mu.g/ear of topsentin achieved nearly 50% inhibition of edema, 
and doses of 100 .mu.g/ear showed more than 90% inhibition of edema (FIG. 
2). Similarly, percent inhibition of mouse ear edema by bromotopsentin 
ranged from about 20% inhibition for a dose of approximately 25 .mu.g/ear 
to about 75% inhibition at a dose of 50 .mu.g/ear. 
EXAMPLE 5 
Formulation and Administration 
The compounds of the invention are useful for various non-therapeutic and 
therapeutic purposes. It is apparent from the testing that the compounds 
of the invention are effective for immunomodulation, antiviral activity, 
and for controlling tumor growth. The compounds can be used to inhibit 
unwanted viral growth in the work areas of virology labs. Also, the 
compounds can be used as ultraviolet screeners in the plastics industry 
since they effectively absorb UV rays. As disclosed herein, they can be 
used therapeutically for treating tumors, or as immunomodulatory or 
antiviral agents in animals and humans. 
Therapeutic application of the new compounds and compositions containing 
them can be contemplated to be accomplished by any suitable therapeutic 
method and technique presently or prospectively known to those skilled in 
the art. Further, the compounds of the invention have use as starting 
materials or intermediates for the preparation of other useful compounds 
and compositions. 
The dosage administration to a host in the above indications will be 
dependent upon the identity of the infection, the type of host involved, 
its age, weight, health, kind of concurrent treatment, if any, frequency 
of treatment, and therapeutic ratio. 
The compounds of the subject invention can be formulated according to known 
methods for preparing pharmaceutically useful compositions. Formulations 
are described in detail in a number of sources which are well known and 
readily available to those skilled in the art. For example, Remington's 
Pharmaceutical Science by E. W. Martin describes formulations which can be 
used in connection with the subject invention. In general, the 
compositions of the subject invention will be formulated such that an 
effective amount of the bioactive compound(s) is combined with a suitable 
carrier in order to facilitate effective administration of the 
composition. 
In accordance with the invention, pharmaceutical compositions comprising, 
as active ingredient, an effective amount of one or more of the new 
compounds and one or more non-toxic, pharmaceutically acceptable carriers 
or diluents. In addition, the pharmaceutical composition can comprise one 
or more of the bis-indole compounds as a first active ingredient plus a 
second active ingredient comprising an anti-inflammatory compound known in 
the art. Such known anti-inflammatory drugs include, but are not limited 
to, the steroidal anti-inflammatory drugs (SAIDs) and the non-steroidal 
anti-inflammatory drugs (NSAIDs). 
In accordance with this invention, pharmaceutically effective amounts of a 
known anti-inflammatory agent and the bis-indole compounds are 
administered sequentially or concurrently to the patient. The most 
effective mode of administration and dosage regimen of bis-indole 
compounds and anti-inflammatory agent will depend upon the type of disease 
to be treated, the severity and course of that disease, previous therapy, 
the patient's health status, and response to bis-indoles and the judgment 
of the treating physician. Bis-indole compositions may be administered to 
the patient at one time or over a series of treatments. 
Preferably, the bis-indole composition and the second anti-inflammatory 
agent are administered sequentially to the patient, with the 
anti-inflammatory agent being administered before, after, or both before 
and after treatment with the bis-indole compound. Sequential 
administration involves treatment with the anti-inflammatory agent at 
least on the same day (within 24 hours) of treatment with bis-indole and 
may involve continued treatment with the anti-inflammatory agent on days 
that the bis-indole is not administered. Conventional modes of 
administration and standard dosage regimens of anti-inflammatory agents 
may be used (see Gilman, A.G. et al. [eds.] The Pharmacological Basis of 
Therapeutics, pp. 697-713, 1482, 1489-91 [1980]; Physicians Desk 
Reference, 1986 Edition). For example, indomethacin may be administered 
orally at a dosage of about 25-50 mg, three times a day. Higher doses may 
also be used. Alternatively, aspirin (about 1500-2000 mg/day), ibuprofen 
(about 1200-3200 mg/day), or conventional therapeutic doses of other 
anti-inflammatory agents may be used. Dosages of anti-inflammatory agents 
may be titrated to the individual patient. 
According to one embodiment of this invention, the patient may receive 
concurrent treatments with the anti-inflammatory agent and compositions 
comprising bis-indoles. Local, intralesional, or intravenous injection of 
bis-indoles is preferred (see Gilman et al., supra at pp. 1290-91). The 
anti-inflammatory agent should preferably be administered by subcutaneous 
injection, subcutaneous slow-release implant, or orally. 
Alternatively, the patient may receive a composition comprising a 
combination of one or more bis-indole compounds and an anti-inflammatory 
agent according to conventional modes of administration of agents which 
exhibit anticancer, antitumor, or anti-inflammatory activity. These 
include, for example, parenteral, subcutaneous, intravenous, or 
intralesional routes of administration. 
The compositions used in these therapies may also be in a variety of forms. 
These include, for example, solid, semi-solid, and liquid dosage forms, 
such as tablets, pills, powders, liquid solutions or suspension, 
suppositories, injectable and infusible solutions. The preferred form 
depends on the intended mode of administration and therapeutic 
application. The compositions also preferably include conventional 
pharmaceutically acceptable carriers and adjuvants which are known to 
those of skill in the art. Preferably, the compositions of the invention 
are in the form of a unit dose and will usually be administered to the 
patient one or more times a day. 
The compounds of the subject invention may also be administered utilizing 
liposome technology, slow release capsules, implantable pumps, and 
biodegradable containers. These delivery methods can, advantageously, 
provide a uniform dosage over an extended period of time. 
Examples of such carriers or diluents include ethanol, dimethyl sulfoxide, 
glycerol, silica, alumina, starch, and equivalent carriers and diluents. 
While effective amounts may vary, as conditions in which compositions are 
used vary, a minimal dosage required for anti-inflammatory activity is 
generally between 0.01 and 100 .mu.g of the compound. To provide for the 
administration of such dosages for the desired therapeutic treatment, new 
pharmaceutical compositions of the invention will advantageously comprise 
between about 0.1% and 45%, and especially, 1 and 15% by weight of the 
total of one or more of the new compounds based on the weight of the total 
composition including carrier or diluent. 
Illustratively, dosage levels of the administered active ingredients can 
be: intravenous, 0.01 to about 20 mg/kg; intraperitoneal, 0.01 to about 
100 mg/kg; subcutaneous, 0.01 to about 100 mg/kg; intramuscular, 0.01 to 
about 100 mg/kg; orally 0.01 to about 200 mg/kg, and preferably about 1 to 
100 mg/kg; intranasal instillation, 0.01 to about 20 mg/kg; and aerosol, 
0.01 to about 20 mg/kg of animal (body) weight. 
Once improvement of the patient's condition has occurred, a maintenance 
dose is administered if necessary. Subsequently, the dosage or the 
frequency of administration, or both, may be reduced, as a function of the 
symptoms, to a level at which the improved condition is retained. When the 
symptoms have been alleviated to the desired level, treatment should 
cease. Patients may, however, require intermittent treatment on a 
long-term basis upon any recurrence of disease symptoms. 
It should be understood that the examples and embodiments described herein 
are for illustrative purposes only and that various modifications or 
changes in light thereof will be suggested to persons skilled in the art 
and are to be included within the spirit and purview of this application 
and the scope of the appended claims.