WO1988000194A1

WO1988000194A1 - Antitumor compositions and their methods of use

Info

Publication number: WO1988000194A1
Application number: PCT/US1987/001564
Authority: WO
Inventors: Tatsuo Higa; Shinichi Sakemi; Sue S. Cross
Original assignee: Harbor Branch Oceanographic Institution, Inc.
Priority date: 1986-06-26
Filing date: 1987-06-25
Publication date: 1988-01-14
Also published as: EP0271572A1; JPH01500519A

Abstract

Antiviral organic triterpene compositions and derivatives thereof; a process of producing the antiviral compositions; and a method for inhibiting viruses utilizing the compositions. More particularly, the compositions of formula (I), wherein R1 is a hydrogen, halogen, hydroxyl, acyloxy, or alkyl; R2 and R5 are a hydrogen, alkyl, or acyl; at least one R3 or R4 is hydrogen and the remaining R3 or R4 is hydrogen, hydroxyl, alkyl, halogen, alkoxy or an acyloxy, or R3 and R4 are in combination an oxygen (ketone), are derived from marine red alga, Laurencia venusta.

Description

t

ANTITUMOR COMPOSITIONS AND THEIR METHODS OF USE

₀ Field of the Invention

This invention relates to organic compounds which have useful antiviral activity. More particularly, this invention relates to organic - triterpene antiviral compositions derived from red alga Laurencia venusta.

Background of the Invention

Viral diseases inflict man, plants, insects, and animals. The prevention and control of viral diseases have important health and economic implications.

Viral diseases contribute to inflictions in - humans including common colds, herpes and cancer and the importance of their control is obvious. Also important is control of viral diseases in animals for economic reasons as well as the ability of such animals to become virus reservoirs or carriers which facilitate the spreading of viral diseases to humans. Viral plant diseases have been known to have a disruptive effect on the cultivation of fruit trees, tobacco, and various vegetables. Insect viral diseases are also of interest because of the insects' ability to transfer viral diseases to humans.

The prevention and control of viral diseases is thus of prime importance to man and considerable research has been devoted to antiviral measures. Certain methods and chemical compositions have been developed which aid in inhibiting, controlling or

5 Z destroying viruses but additional methods and antiviral chemical compositions are needed.

A potential source for antiviral compositions is marine plant and' animal life and of particular interest is the red alga.

Previous work has indicated that red alga genus Laurencia can be the source for useful bioactive compositions including the compositions disclosed in co-pending U.S. patent applications of T. Higa and K. M. Snader directed to new cyclohexadienone compositions and their methods of use, i.e., U.S.S.N. 744,620 and 772,330.

Other cyclic polyethers of marine origin have been reported, for example, brevetoxin derived from a dinoflagellate as reported by Y. Shimizu, H.N. Chou, H. Bando, G. Van Dayne, and J.C. Clardy, J_^ Am. Chem. Soc, IPS, 514 (1986); and norhalichondrin derived from a sponge as reported by D. Uemura, K. Takahashi, T. Yamamoto, C. Katayama, J. Tanaka, Y. Okumuru and Y. Hirata, J. Am. Chem. Soc, 107, 4796 (1985).

The following references describe triterpene and thyrsiferol derivatives which are analagous to some of those of the present invention: T. Suzuki, M. Suzuki, A. Furusaki, T. Matsumoto, A. Kato, Y. Imanaka, and E. Kurosawa, Tetrahedron Lett. , 26, 1329(1985); and J.W. Blunt, M.P. Hartshorn, T.J. McLennan, M.H. G. Munro,W.T. Robinson, and S.C. Yorke, Tetrahedron Lett. , 69 (1978). While the compositions described in these publications are shown to be cytotoxic no antiviral properties have been described or recognized for these compositions.

The entire disclosures of all of the above-noted patent applications and literature references are hereby incorporated herein by reference. While certain useful compositions of marine origin have been found, additional antiviral compositions are needed.

Summary of the Invention

It is therefore an object of the invention to provide novel compositions which are useful as antiviral agents; antiviral methods of using the compositions; and a process for producing such compositions.

Additional objects and advantages of the invention will be set forth, in part, in the description which follows and in part will be obvious from this description, or may be learned by the practice of the invention. The objects and advantages of the invention are realized and obtained by means of the compositions, processes, methods, and the combinations particularly pointed out in the appended claims.

To achieve the objects in accordance with the purposes of the invention, as embodied and fully described here, the invention comprises compositions of the general formula (I)

wherein R¹ is a hydrogen, halogen, hydroxyl, lower acyloxy, or lower alkyl group; R^ and R^ are a hydrogen, lower alkyl, or lower acyl group; at least one R^ or R^ is hydrogen and the remaining R^ or R^ is hydrogen, hydroxyl, lower alkyl, halogen, lower alkoxy, or a lower acyloxy group, or E and R^ are in combination an oxygen (ketone); with the proviso that, when R^ is bromine and R^ and R^ are hydrogens, is not hydroxyl or acetoxy and R^ is not hydrogen or an acetyl group.

In preferred embodiments of the invention, the composition is substantially pure. In further preferred embodiments the lower alkyl, alkoxy, acyl, and acyloxy groups have from 1 to 5 carbon atoms.

In more preferred embodiments of the invention, the invention comprises compositions of formula I wherein R^ is hydrogen or bromine; R^ is hydrogen; R^ is hydrogen or acetoxy; R⁴ is hydrogen, hydroxy or acetoxy; and R^ is hydroxy or acetoxy.

As embodied and fully described herein, the invention also comprises an antiviral composition comprising, as active ingredient, an effective antiviral or amount of one or more compositions according to formula I, with or without the proviso relating to R^~5 _anrj _a non-toxic pharmaceutically acceptable carrier or diluent. As defined herein compositions of formula I which are not subject to the proviso relating to R^1-5 will be described as compositions of unrestricted formula I.

As embodied and fully described herein, the invention also comprises a process to produce the compositions of unrestricted formula I. The process comprises the steps of: collecting red alga Laurencia venusta ; contacting the alga with a suitable organic solvent to obtain an extract of a composition of unrestricted formula I; and isolating

10 a composition according to unrestricted formula I from the extract.

In preferred embodiments of the invention the suitable organic solvent is selected from the group consisting of: acetone, ethyl acetate, ,e toluene, methanol, methyl ethyl ketone, ethanol, methyl isobutyl ketone and mixtures thereof. In preferred embodiments isolation of the compositions of the invention is accomplished by partition between aqueous and organic solvents and chromatography Λ,» methods.

As embodied and fully described herein, the invention further comprises a method for inhibiting viruses comprising contacting a virus with an effective antiviral amount of one or more oc compositions according to unrestricted formula I.

It is to be understood that both the foregoing general and the following detailed description are exemplary and explanatory only and are not intended to be restrictive of the invention

30 as claimed.

35 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Reference will now be made in detail to present preferred embodiments of the invention, examples of which are illustrated in the following example section.

In accordance with the invention as embodied and fully described herein, the invention comprises compositions of the general formula (I):

wherein R³- is a hydrogen, halogen, hydroxyl, lower acyloxy, or lower alkyl group; R^ and R^ are a hydrogen, lower alkyl, or lower acyl group; at least one R3 or R^ is hydrogen and the remaining R3 or R^ is hydrogen, hydroxyl, lower alkyl, halogen, lower alkoxy, or a lower acyloxy group, or R^ and R^ are in combination an oxygen (ketone); with the proviso that, when R is bromine and R^ and R^ are hydrogens, R^ is not hydroxyl or acetoxy and R^ is not hydrogen or an acetyl group.

In preferred embodiments of the invention, the composition is substantially pure. In further preferred embodiments the lower alkyl, alkoxy, acyl and acyloxy groups have from 1 to 5 carbon atoms.

In more preferred embodiments of the invention, the invention comprises compositions of formula I wherein R^ is hydrogen or bromine; R^ hydrogen; R³ is hydrogen or acetoxy; R⁴ is hydrogen, hydroxy or acetoxy; and R^ is hydroxy or acetyl.

In accordance with the invention an antiviral composition is provided comprising as active ingredient an effective antiviral amount of one or more of the compositions described above and identified by unrestricted formula I and a non-toxic pharmaceutically acceptable carrier or diluent.

While effective amounts may vary, as conditions in

10 which the antiviral compositions are used vary, a minimal dosage required for activity is generally between 50 and 200 micrograms against 25-80 plaque-forming unit of virus. The composition of the invention are active for inhibiting or killing a

■, - diverse range of viruses including, but not limited to, RNA viruses, vesicular stomatitis (herein "VSV") adeno-, corona- reo- and influenza viruses and the

DNA virus, Herpes Simplex - I and II (Herein "HSV-I" and "HSV-II") adeno- and papova- viruses. Useful

20 examples of non-toxic pharmaceutically acceptable carriers or diluents include, but are not limited to the following: ethanol; dimethyl sulfoxide; and glycerol.

In accordance with the present invention,

25 virus cells are inhibited or killed by a method comprising contacting a virus with an effective antiviral amount of one or more compositions according to unrestricted formula I. The minimal effective amount as stated above is generally from 50 to 100 micrograms against 25 to 80 plaque forming

35 β o units of virus cells. The composition of unrestricted formula I are active for inhibiting or killing a diverse range of viruses including, but not limited to, the RNA viruses, vesicular stomatitis (herein "VSV^,r), arenaviruses, coronaviruses, e rhinoviruses, influenza viruses and the DNA viruses, herpes simplex-I (herein "HSV-I"), other herpes viruses, adenoviruses, coxsackie viruses, polioviruses and papovaviruses.

The effectiveness of the compositions of the i_Q invention for inhibiting virus cells indicates that the compositions of unrestricted formula I should also be useful in controlling viral infections in host animals and plants which are caused by a virus which is thus inhibited or destroyed. Viral

•, e infections which may be controlled by utilizing compositions of the present invention include, but are not limited to, those caused by those RNA viruses and DNA viruses described above. The invention may also be useful in controlling common viral infections

2_Q of plants.

As embodied and fully described herein, the invention also comprises a new process to produce the compositions of unrestricted formula I. The process comprises the steps of: collecting red alga

2c Laurencia venusta; contacting the alga with a suitable organic solvent to obtain an extract of a composition of unrestricted ^"formula I; and isolating a composition according to unrestricted formula I from the extract.

30

35 ^<? A detailed description and explanation of a preferred embodiment of the process of the invention to produce the composition according to unrestricted formula I is as follows.

A quantity of red alga Laurencia venusta is collected by hand at an intertidal zone on the Coast at Onna, Okinawa. The alga is contacted with a first organic solvent and homogenized to form an extract. The organic solvent is removed by evaporation leaving an essentially aqueous suspension (the source of the

10 water is the natural water content of the alga). The suspension is then partitioned between water and a second organic solvent. The organic portion or extract, an oily residue which incorporates the desired compositions, is removed and subjected to

-, c chromatography to obtain and isolate the composition according to unrestricted formula I. The chromatography product is separated into fractions which contain the desired compositions. Specific compositions according to the invention are further

2_Q isolated by various chromatographic techniques from the fractions obtained.

Acetone is the presently preferred choice for the first solvent, however, other suitable solvents may be substituted for acetone. A suitable

2 first solvent should be capable of solubilizing the homogenized alga. Suitable first solvents which may be substituted for acetone include, but are not limited to, the following organic solvents: methyl ethyl ketone; methanol; ethanol; methyl isobutyl

30 ketone and mixtures thereof. Suitable second organic

35 solvents should be capable of extracting and separating into various fractions the various compounds of unrestricted formula I from other components that may be present in the aqueous residue. Suitable second solvents which may be substituted for ethyl acetate include, but are not limited to ether; tetrahydrofuran; toluene; methylene chloride; chloroform; trichloroethylene; hexane; lower alkanes; and mixtures thereof. Different ratios of solvent mixtures may be used or the irst and second solvents in the invention as would be known to those skilled in the art.

Any suitable fractionation and isolation techniques may be utilized in accordance with the process of the invention. Suitable fractionation techniques include various chromotography techniques such as, high pressure liquid chromatography with a suitable column as would be known to those skilled in the art (e.g., Merck Hibar RP-18 column) eluted with a suitable solvent such as, for example, 4:1, methanol: water.

It is therefore apparent that the compositions of the invention, the processes for producing the compositions of the invention and the methods for utilizing the compositions of the invention to inhibit viruses and tumors are effective for inhibiting or destroying viruses or tumors and therefore controlling diseases caused by or related to such viruses or tumors in fulfillment of the ob ects of the invention. 1\

EXAMPLES

The invention will now be illustrated by examples. The examples are not intended to be limiting of the scope of the present invention. In conjunction with the detailed and general description above, the examples provide further understanding of the present invention and outline a process for producing compositions of the invention.

The following examples represent preferred embodiments of the compositions, processes and methods of the invention for satisfying the stated objects of the invention. The starting materials and reagents in the examples whose method of preparation are not indicated are commercially available from sources known to the art such as chemical supply houses.

Preparation of Examples 1-7 Examples 1-3

Example 1

Preparation of Thyrsiferol:

1, 18S, 19R, R⁵=H (composition 1)

Example 2

Preparation of Thyrsiferyl 23-acetate:

2, 18S, 19R, R⁵=AC(composition 2)

Example 3

Preparation of Venustatriol:

3, 18R, 19S R⁵=H (composition 3)

A fresh sample (1.2 kg) of Laurencia venusta, collected at Onna, Okinawa, at an intertidal zone was extracted by contacting and steeping it with 2 liters of acetone for 24 hours. The extract is concentrated by evaporating acetone to leave an aqueous suspension which was extracted with ethyl acetate to yield 3 gms. of oil. The oil was chromatographed on a silica gel column by eluting with a heptane-acetone step gradient. Fractions 13 and 14 (total 240 mg) which showed antiviral activity against Vesicular stomatitis virus (VSV) and Herpes simplex virus type 1 (HSV-1) were further separated on a silica gel column (15:1 chlorofrom-acetone). Active fractions (107 mg) were combined and purified by HPLC (Dynamax Si column, 1:1 heptane-ethyl acetate) to yield 78 mg of thyrsiferyl 23-acetate (2).¹ Other active fractions (15 and 16) from the initial separation wera> separated on a silica gel column (8:1 00194

15 chloroform-ethyl acetate) to give 140 mg of a mixture of active omponents which was further separated by HPLC (Dynamax, 1:3 heptane-ethyl acetate) into five fractions. Fraction 3 (13 mg) was recrystallized from heptane to yield 9.7 mg of venustatriol (3) as colorless needles, mp 161.5 . Fraction 4 (87 mg) was purified by HPLC (Dynamax Si column, 12:1 chroloform-methanol) to give 85 mg of thyrsiferol

(D-2

Thyrsiferol (1). Recrystallization from heptane-methylene chloride gave colorless needles, mp 133-136 ; [oC3_D ²⁰ +6.4° (c 5, CHCI3); IR (KBr) 3400, 2960, 2860, 1450, 1367, 1170, 1114, 1090, 1050, and 1013 cm-¹; ^ NMR (CDCI3) 1.094 (3H, s), 1.128 (3H, s), 1.154 (3H, s), 1.184 (3H, s), 1.195 (3H, s), 1.210 (3H, s), 1.267 (3H, s), 1.396 (3H, s), 3.043 (IH, dd, J=2.1, 11.1 Hz), 3.451 (IH, dd, J=1.7, 10.1 Hz), 3.570 (IH, dd, 7.0, 11.0 Hz), 3.707 (IH, dd, J=2.7, 12.5 Hz), 3.758 (IH, dd, J=6.7, 9.3 Hz), and 3.891 (IH, dd, J=4.0, 12.2 Hz); ¹³C NMR (CDCI3) /20.06, 20.69, 21.15, 21.43, 22.87, 22.98, 23.39, 23.66, 23.93, 25.45, 26.60, 27.62, 28.22, 30.99, 32.36, wwwwwwwwww

1 The composition was confirmed by comparison to its previously reported physical and spectral characteristics or described in T. Suzuki, M. Suzuki, A. Furusaki, T. Matsumoto, A. Kato, Y. Imanaka, and E. Kurosawa, Tetrahedron Lett., 26, 1329 (1985).

2 The identity of the composition was confirmed by comparison to its previously reported physical and spectral characteristics as described in J.W. Blunt, M.P. Hartshorn, T.J. McLennan, M.H. G. Munro, W.T. Robinsono, and S.C. Yorke, Tetrahedron Lett. , 69 (1978). ι4

33.55, 37.00, 38.52, 58.96, 70.52, 71.93, 73.23, 74.37, 74.95, 76.06, 76.30, 77.63, 86.02, 86.52, and 87.42; EIMS m/z 588 (0.3), 586 (0.3, M⁺-H2θ), 570 (0.3), 568 (0.3), 552 (0.4), 550' (0.4), 534 (2.2), 506 (1.8), 491 (2.2), 489 (2,4), 470 (0.9), 445 (2.2), 443 (2.3), 429 (0.7), 409 (1.7), 363 (6), 227 (33), 209 (34), 207 (11), 205 (11), 143 (100), 125 (83), and 107 (20%).

Thyrsiferyl 23-acetate (2). Recrystallization from aqueous methanol gave colorless needles, mp 115-116 ; [CCJD²⁰ +2.64

(c. 5.7, CHC1₃); IR(film) 3420, 2950, 2920, 2860, 1732, 1455, 1368, 1248, 1236, 1157, 1117, 1096, 1055, 1040, and 1016 cm^-1; NMR (CDDl3) l.087 (3H, s), 1.153 (3H, s), 1.177 (3H, s), 1.191 (3H, s), 1.262 (3H, s), 1.392 (3H, s), 1.439 (3H, s), 1.474 (3H, s), 1.984 (3H, s), 3.041 (IH, dd, J=1.9, 11.4 Hz), 3.447 (IH, br d, J=9.7 Hz), 3.567 (IH, dd, J=7.4, 11.0 HZ), 3.705 (IH, dd, J=2.5, 12.6 Hz), 3.886 (IH, dd, J=3.8, 12.1 Hz), and 4.006 (IH, dd, J=6.0, 9.0 Hz); ¹³C NMR (CDCl3)/20.02, 20.64, 21.12, 21.39, 21.93, 22.03, 22.42, 22.81, 22.93, 23.19, 23.62, 25.32, 26.71, 28.17, 30.95, 31.76, 33.52, 36.97, 38.48, 58.93, 71.86, 73.15, 74.32, 74.89, 76.02, 76.26, 77.19, 82.46, 85.78, 86.28, 86.46, and 170.3; EIMS m/z 566 (1, M⁺-HBr), 506 (5), 488 (1.5), 421 (0.7), 403 (1), 385 (0.7), 363 (6), 345 (1),- 319 (1.7), 305 (0.6), 279^' (4), 269 (3), 227 (25), 209 (70), 185 (12), 165 (6), 155 (9), 153 (12), 151 (12), 137 (10), 125 (100), 111 (14), 109 (17), and 107 (17%). 15

Venustatriol (3). Mp 161.5 ; [αϋo^{20 +9}-⁴ (c 3.2, CHC1₃); IR (KBr) 3360, 2960, 2860, 1445, 1370, 1173, 1118, 1095, 1083, and 1015 cm^*"¹; !H NMR (CDCI3) Zl.099 (3H, s), 1.148 (3H, s), 1.185 (3H, s), 1.193 (3H, s), 1.206 (3H, s), 1.278 (3H, s), 1.282 (3H, s), 1.407 (3H, s), 3.054 (IH, dd, J=2.2, 11.2 Hz), 3.506 (IH, dd, J=7.0, 11.6 Hz), 3.536 (IH, dd, J=1.9, 10.3 Hz), 3.650 (IH, dd, J=2.8, 12.3 Hz), 3.769 (IH, dd, J=7.3, 7.3 Hz), and 3.831 (IH, dd, J=4.2, 12.2 Hz); ¹³C NMR (CDCI3) 20.05, 20.56, 21.12, 21.45, 22.99 (2C), 23.66, 23.79, 25.50, 25.90, 26.92, 27.53, 28.23, 30.99, 31.19, 33.16, 37.02, 38.57, 58.99, 71.91, 72.04, 73.23, 74.38, 74.94, 75.71, 76.33, 76.40, 77.55, 84.35, and 86.53; EIMS m/z 588 (1.2), 586 (1.1, M⁺ -H₂0), 570 (0.7), 568 (0.7), 552 (0.9), 550 (0.9), 529 (1.1), 527 (1.1), 524 (2.8), 506 (6), 491 (6), 486 (6), 470 (2.8), 445 (3.8), 443 (3.7), 409 (5), 363 (10), 227 (78), 209 (75), 207 (25), 205 (26), 193 (11), 165 (10), 127 (42), 125 (100), 111 (31), 109 (40), and 107 (40).

Example 4 debromothyrsiferol (composition 4)

A mixture of thyrsiferyl 23-acetate (10.5 mg) .and L1AIH4 (32 mg) in 0.5 ml dry tetrahydrofuran was heated at reflux for 1 hr. After cooling and addition of water (2 ml), the mixture was extracted with chloroform (2 ml X 3). The extract was passed through a short column of silica gel with acetone. The eluate was concentrated and purified by running on HPLC (Altex Ultrasphere-ODS, 30:1 MeOH-H2θ) to yield 7.8 mg (91%) of 4 as a colorless oil. ^XH NMR (CDCI3) /l.099 (3H, s), 1.126 (6H, s), 1.131 (3H, s), 1.156 (3H, s), 1.187 (6H, s), 1.212 (3H, s), 2.838 (IH, br s), 2.974 (IH, br s), 3.191 (IH, dd, J=2.0, 11.2 Hz), 3.451 (IH, dd, J=1.9, 10.0 Hz), 3.578 (IH, dd, J=7.4, 10.0 Hz), 3.719 (IH, dd, J=2.8, 13.5 Hz), and 3.756 (IH, dd, J=6.4, 9.5 Hz); ^l3C NMR (CDCI3) l6.10, 20.72, 21.18, 21.46, 21.73, 22.93, 23.11, 23.39, 23.93, 25.47, 26.57, 27.68, 29.21, 31.96, 32.34, 33.41, 33.47, 36.80, 38.72, 70.47, 71.18, 72.10, 73.23, 73.97, 76.02, 76.29, 77.64, 85.27, 86.01, and 87.39.

Example 5 Venustatriol 18-acetate (Composition 5)

Venustatriol (3) (4.5 mg) was treated with acetic anhydride (0.2 ml) and pyridine (0.2 ml) at room n temperature for 48 hr. An excess of the reagents was evaporated under vacuum, and the product was purified by HPLC (LiChrosorb Si-60, 1:1 heptane-ethyl acetate) to give 4.0 mg of the acetate as a colorless oil: ¹H NMR (CDCI3) l.062 (3H, s), 1.107 (3H, s), 1.154 (3H, s), 1.192 (3H, s), 1.215 (6H, s), 1.266 (3H, s), 1.396 (3H, s), 2.070 (3H, s), 3.036 (IH, dd, J=2.2, 11.5 Hz), 3.550 (IH, dd, J=7.4, 11.1 Hz), 3.672 (IH, dd, J=2.9, 12.8 Hz), 3.761 (IH, dd, J=7.5, 7.5 Hz), 3.892 (IH, dd, J=4.0, 12.3 Hz), and 4.956 (IH, dd, J=2.3, 9.7 Hz); ¹³C NMR (CDCI3) <^*20.05, 20.61, 21.06, 21.24, 21.40, 22.81, 23.02, 23.66, 24.09, 26.26, 27.56, 28.23, 31.00, 32.25, 34.14, 37.05, 38.49, 59.01, 70.94, 71.94, 73.20, 74.37, 74.95, 75.71, 76.26, 78.26, 84.29, 84.99, and 86.53.

Example 6 & 7

Preparation of Thyrsiferyl 18 acetate (composition 6)

Thyrsiferol (1) was treated with acetic anhydride and pyridine in the same manner as described in Example 5 to yield thyrsiferyl 18-acetate (6) as a colorless Oil. [0C] 20^{D +} 1-76 (C11.4, CH CI3); ^XH NMR (CDCI3) 1.071 (3H, s), 1.104 (3H, s), 1.144 (3H, s), 1.185 (6H, s), 1.191 (3H, s), 1.261 (3H, s), 1.391 (3H, s), 2.050 (3H, s), 3.037 (IH, dd, J=2.1, 11.2 Hz), 3.550 (IH, dd, J=7.3, 11.2 Hz), 3.689 (IH, dd, J=2.7, 13.0 Hz), 3.703 (IH, t, J=7.6 Hz), 4.894 (IH, m); ¹³C NMR (CDCI3) 20.05, 20.62, 21.11, 21.40, 22.96, 23.07, 23.66, 23.82, 24.06, 26.11, 27.49, 28.22, 30.99, 32.50, 34.81, 37.02, 38.55, 58.96, 70.38, 71.88, 72.95, 74.36, 74.95, 75 84, 76.26, 78.41, 84.11, 86.51, 87.00 and 171.00.

Preparation of Thyrsiferyl 18,23-diacetate (composition 7)

Thyrsiferyl 23-acetate (2) was treated with acetic anhydride and pyridine in the same manner as described in Example 5 to give thyrsiferyl 18,23-diacetate (7) as a colorless oil.

LO I Ό O [C 1.6 , CHCI3] NMR (CDC1₃) ^>1.072(3H, s), 1.142(3H, s), 1.188 (3H, s), 1.193 (3H, s), 1.262 (3H, s), 1.391 (3H, s), 1.418 (3H, s), 1.447 (3H, s), 1.972 (3H, s), 2.068 (3H, s), 3.034 (IH, dd, J=1.9, 11.4Hz), 3.548 (lHdd, J=7.3, 11.0Hz), 3.688 (IH, dd, J=2.8, 12.9Hz), 3.888 (IH, dd, J-4.1, 12.3Hz), 4.017 (IH, dd, J=6.0, 9.1Hz), and 4.893 (IH, dd, J=3.9, 9.2Hz); ¹³CNMR (CDCI3),^20.08, 20.63, 21.13 (2C), 21.41, 21.73, 22.44 (2C), 22.98 (2C), 23.12, 23.69, 23.85, 26.48, 28.25, 31.02, 32.21, 34.32, 37.05, 38.59, 58.99, 71.90, 72.98, 74.40, 74.98, 75.87, 76.30, 78.35, 82.61, 84.41, 84.97, 86.55, 170.41, and 170.81. ANTIVIRAL ACTIVITIES OF THE COMPOSITIONS OF THE INVENTION

The following assay methods were utilized to demonstrate the in vitro antiviral effectiveness of e compositions 1-7 as reported in Table 1.

Antiviral Disc Assay for HSV-1 and VSV

A. Maintenance of Cell Cultures

1. Virus

a. Both herpes simplex type 1 (HSV-1) and vesicular stomatitis virus (VSV) replicate in the CV-1 cell line. CV-1 is a fibroblast-like cell culture derived from primary African green monkey cells.

2. Growth of CV-1 Cells

a. Seed I50cm2 tissue culture flasks each with 10 x 10⁶ CV-1 cells in 40 ml of EMEM with 10% FBS (growth medium). b. Seven days after seeding the flasks cell numbers should be approximately 40-50 x 10⁶ cells. CV-1 cells have a doubling time of 72 hours based on these numbers. 3. Trypsinization a. Aseptically remove the medium. b. Rinse cell sheet with 10 ml of Ca⁺⁺ and Mg⁺⁺free

Dulbecco's phosphate buffered saline or Pucks G saline at least twice. c. Add 1.5 to 2.0 ml of trypsin -EDTA mixture. d. Incubate flask at room temperature or at 37 C with occasional rocking until the cells detach from the flask (about 15-30 min). Cells maintained on calf serum detach from the plastic at a faster rate than those held on fetal bovine serum (FBS). e. Shake flask. f. Add 10 ml EMEM growth medium and break up cell clumps with pipetting. g. Count cells.

B. Preparation of plates for viral assays

1. Cell Concentration

a. Dilute the cells with EMEM to 4 x 10³ cells/ml. b. Seed 24 well trays with 0.5 ml per well. Cell concentration per well is 2 x 10⁵ cells. c. Incubate at 37 C with 5% Cθ2- Zi o d. The wells can be used over the next several days beginning the day after seeding (preferably 2,3, or 4).

C. Assay of HSV-1 and VSV in CV-1 cells

1. Infection of CV-1 cells in plates with virus .

a. Remove medium from wells. b. Infect well with at least 25 and no more than 80 plaque forming units (PFU) of virus. c. Incubate infected cells at 37 ^{^} C for 1.5 hours. d. Pour off supernatant at end of incubation period. e. Add 0.5 ml of methylcellulose overlay medium (MCO).

1) MCO is a maintenance medium without phenol red made with 1% 4000 centipoise methylcellulose. FBS is used at 5% level.

2. Drug Evaluation

a. For drug evaluation wet filter paper discs (6 mm diameter) with approximately 0.02 ml of marine extract or test compound. 1) Allow solvent to evaporate for 20 to 30 minutes at room temperature. ^■2.2 2) Place discs in the well containing CV-1 cells, virus, and MCO. b. Incubate tissue culture plates for 48 hours at 37 C. c. After 48 hours place 0.5 ml NRMCO on each well.

1) NRMCO is a maintenance overlay medium without phenol red containing 0.1 mg neutral red dye per ml and 2% 15 centipoise methylcellulose.

10 d. Incubate plates at Zl° Q and read the following day.

1) Antiviral activity should be observed from two parameters. One is actual reduction in the number it of plaques and two is the diminution in plaque diameter. 3. Scoring Drug Activity

a) Antiviral activity is scored from

20 0 to +++.

+++ = complete inhibition of plaque formation

++ = partial inhibition

25 + = partial inhibition

0 = no protection b) Cytotoxicity

0 = no visual or microscopic cytotoxicity

30

35 16 = Complete cell destruction 8, 10, 12, 14 = partial cytotoxicity

Antiviral Assay for Mouse Coronavirus A-59

A. Cell culture

1. NCTC clone 1469 is a derivative of mouse liver.

2. ATCC No. CCL 9.I3 freeze 2518, passage no. 16, frozen November 1980 at 2.4 x 10³.

3. Received January, 1985, and reconstituted March, 1985.

4. Frozen in liquid nitrogen and reconstituted July, 1985.

5. Assigned log number HB/SP 16.

B. Maintenance of cell culture

1. Trypsinization a. Aseptically remove the medium. b. Rinse cell sheet with 10 ml of Ca⁺⁺ and Mg⁺⁺ free phosphate buffered saline. c. Add 4 ml of trypsin-EDTA mixture to a 150 cm flask. d. Leave for one minute or less and then shake flask. e. Add 10 ml of growth medium and break up cell clumps with 194

Z pipetting. f. Count cells.

Subcultures for.maintenance of cells for assays. a. Seed 150 cm tissue culture flasks with 10 x 10⁶ cells in 40 ml growth medium.

C. Virus

1. Mouse hepatitis virus strain MHV-A59 classified as a coronavirus.

2. ATCC No. 764.

3. Received January, 1985, passed in NCTC 1469 and assigned HB/SP 30 log number.

D. Preparation of plates for viral assays

Cell concentration a. Dilute the cells with growth medium between 5 x 10 and 7.5 x 10⁵ cells per ml. b. Seed 24 well trays with 1.0 ml per well.

E. Viral assay

Dilute drug or extract for test in the appropriate solvent.

2. Add 20 lambda to a 12 mm by 75 mm glass tube for a 16 mm test well. 3. Allow the solvent to evaporate under the laminar flow hood.

4. Dilute the MHV-A59 in Dulbecco's phosphate buffered saline with Ca⁺⁺ and Mg⁺⁺ to the appropriate predetermined dilution for the lot number currently in use. Normally the dilution of virus in a titration which gives a 3+ to 4+ CPE in 24 hours is the dilution used in this assay.

10 5. Remove medium from wells of 24 well plate containing NCTC 1469 cells seeded 24 earlier.

6. Add 200 lambda of diluted virus to each test well. Add PBS to control wells.

15 7. Incubate cells and virus for 1 hour at 37*C.

8. Pour off supernatant at end of incubation period.

9. To each glass tube add 10 lambda of

20 dimethyl sulfoxide (DMSO).

10. Add 1 ml of maintenance medium to each glass tube.

11. Pour the contents of the glass tube into the corresponding well of the

25 tissue culture plate.

12. Incubate infected cells at 37 C and read the following day.

13. At twelve hours areas of cell fusion are quite apparent and can be detected

30 both visually and microscopically.

35 2&

14. At 24 hours the CPE is extensive and on stained plates the difference between activity and none is apparent from visual examination.

15. To stain plates discard medium and to each 16 mm well add 200 lambda of methylene blue stain or other appropriate stain.

16. Leave the stain on the cell sheet for 30 minutes or more.

17. Pour off the stain and wash plates in

10 tap water until the water is clear.

18. Allow plates to dry. Plates can be kept as a permanent record for the experiment.

19. Scoring drug activity

15 a. Cytotoxicity scoring

100% = complete cell destruction 75% = partial cell destruction 50% = partial cell destruction

20 25% ~ partial cell destruction 0% = no cytotoxicity b. Antiviral activity is scored from 0 to +++. +++ = complete inhibition of cytopathic effects

25 and cell fusion

++- - partial inhibition

+ - partial inhibition

+/- = partial inhibtion

0 = no protection

30

35 7Λ Table 1

Antiviral results

Controls: Ribavirin (VSV) 100 ug +++, 10 ug +÷, 1 ug -; ARA A (HSV-1) 100 ug +++, 10 ug +, 1 ug -; Acyclovir (HSV-1) 100 ug +++, 10 ug +++,

1 ug ++, 0.1 ug ++

The results in Table 1 indicate that the compositions of the invention as represented by compositions 1-7 are effective for controlling VSV, HSV-1 and A-59 viruses, in vitro, in concentrations as little as 0.01 ug/well. The results are indicative of the utility of the compositions of the invention to control viruses in hosts both animal and plant.

The scope of the present invention is not limited by the description, examples, and suggested uses herein and modifications can be made without departing from the spirit of the invention. For example, it may be noted that other derivatives of the compositions of examples 1-7 such as a fluorinated derivative may possess antiviral activity analogous to those preferred embodiments described above. Further, the compositions described herein may have other useful applications such as, for example, analgesic applications. Application of the compositions of the present invention can be accomplished by any suitable therapeutic method and technique as is presently or prospectively known to those skilled in the art. Thus, it is intended that the present invention cover the modifications and variations of this invention provided that they come within the scope of the appended claims and their equivalents.

Claims

94

30

What is claimed is: 1. A composition according to the general formula:

wherein R¹ is a hydrogen, halogen, hydroxyl, acyloxy, or lower alkyl group; R² and R^ are a hydrogen, lower alkyl, or lower acyl group; at least one R³ or R⁴ is hydrogen and the remaining R³ or R⁴ is hydrogen, hydroxyl, lower alkyl, halogen, lower alkoxy or a lower acyloxy group, or R³ and R⁴ are in combination an oxygen (ketone); with the proviso that, when R¹ is bromine and R² and R³ are hydrogens, R⁴ is not hydroxyl or acetoxy and R^ is not hydrogen or an acetyl group.

2. A composition according to claim 1 wherein R¹ is hydrogen or bromine; R² and R³ are hydrogen; R⁴ is hydrogen, hydroxy or acetyl; and R⁵ is hydrogen or acetyl.

194

3. A composition according to claim 1 selected from the group consisting of:

4. A composition according to claim 1 wherein said composition is substantially pure.

5. The composition according to claim 2 wherein said composition is substantially pure.

6. The composition according to claim 3 wherein said composition is substantially pure.

7. An antiviral composition comprising, as active ingredient, an effective antiviral amount of one or more of the compositions of claim 1 and a non-toxic pharmaceutically acceptable carrier or diluent.

8. An antiviral composition comprising, as active ingredient, an effective antiviral amount of one or more of the compositions of claim 3 and a non-toxic pharmaceutically acceptable carrier or diluent.

9. A method for inhibiting viruses in a host comprising contacting said virus with an antiviral effective amount of a composition according to the formula:

wherein R¹ is a hydrogen, halogen, hydroxyl, acyloxy, or lower alkyl group; R and R^ are a hydrogen, lower alkyl, or lower acyl group; at least one R³ or R⁴ is hydrogen and the remaining R³ or R⁴ is hydrogen, hydroxyl, lower alkyl, halogen, lower alkoxy or a lower acyloxy group, or R³ and R⁴ are in combination an oxygen (ketone).

10. A method for inhibiting viruses in a host comprising contacting a virus with an effective antiviral amount of one or more compositions of claim 1.

11. A method for inhibiting viruses in a mammalian host comprising contacting a virus with an effective antiviral amount of one or more compositions of claim 3.

10 12. A process to produce a composition according to claim 1 comprising the steps of: collecting red alga Laurencia venusta; contacting said alga with an organic solvent to obtain an extract comprising a , - composition according to claim 1; and isolating a composition according to claim 1 from the extract.

13. A process to produce a composition _?n according to claim 9 comprising the steps of: collecting red alga Laurencia venusta; contacting said alga with an organic solvent to obtain an extract comprising a composition according to claim 9; and isolating a composition according to claim 9

25 from the extract.

30

35

14. A composition according to claim 1 of the formula:

15. A composition according to claim 1 of the formula:

16. A composition acording to c_laim 1 of the formula;