EP0695294A1 - Michellamines useful as antiviral agents - Google Patents
Michellamines useful as antiviral agentsInfo
- Publication number
- EP0695294A1 EP0695294A1 EP93912320A EP93912320A EP0695294A1 EP 0695294 A1 EP0695294 A1 EP 0695294A1 EP 93912320 A EP93912320 A EP 93912320A EP 93912320 A EP93912320 A EP 93912320A EP 0695294 A1 EP0695294 A1 EP 0695294A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- antiviral
- effective amount
- michellamines
- hiv
- michellamine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
- C07D217/02—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with only hydrogen atoms or radicals containing only carbon and hydrogen atoms, directly attached to carbon atoms of the nitrogen-containing ring; Alkylene-bis-isoquinolines
- C07D217/04—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with only hydrogen atoms or radicals containing only carbon and hydrogen atoms, directly attached to carbon atoms of the nitrogen-containing ring; Alkylene-bis-isoquinolines with hydrocarbon or substituted hydrocarbon radicals attached to the ring nitrogen atom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
Definitions
- the present invention relates to compounds which exhibit antiviral activity, compositions containing the compounds, methods for isolating the compounds from plants, and methods for using the compounds.
- the compounds of the present invention exhibit advantageous pharmacological, toxicological, and antiviral properties, such as, for example, the inhibition of the cytopathic effects of the human immunodeficiency virus (HIV) , which is implicated as a causative agent of Acquired Immune Deficiency Syndrome
- AZT is the first commercially available, known clinically active agent currently used widely in the therapy of AIDS. While extremely useful in antiviral therapy, AZT is limited in its use due to its toxicity and an insufficient therapeutic index to make it adequate for therapy. Thus, new classes of antiviral agents to be used alone or in combination with AZT and other agents are needed urgently for effective antiviral therapy against HIV. It is also especially important to have new agents which have antiviral activity against HIV-1 as well as HIV-2.
- ichellamines in particular michellamines A, B, and C, respectively.
- the present invention is also directed more generally to a substantially pure michellamine or derivative thereof having the formula:
- R 1 and R 6 are the same or different and are each H, Ci-Cg alkyl, R 11 CO-, or R n S ⁇ 2- wherein R 11 is C 1 -C 6 alkyl or aryl;
- R 2 , R 3 , R 4 , R 7 , R 8 and R 9 are the same or different and are each H, C ⁇ Cg alkyl, R 1:L CO-, R u S0 2 - wherein R 11 is defined above;
- R 5 and R 10 are the same or different and are each H
- R 12 is Ci-C alkyl or R 13 CO- or R 13 S0 2 -, wherein R 13 is Ci-Cg alkyl or aryl;
- R 14 , R 15 , R 16 , and R 17 are the same or different and are each "*CH 3 or "ii CH 3 . and wherein one or more of the ring H positions at 1' , 3' , 7' , 4, 7, 1", 3", 7", 4" and 7" can be substituted with a halogen, nitro, amino, hydroxyl, thiol, or cyano group; or a pharmacologically acceptable salt thereof.
- the present invention further provides a method of isolating the aforementioned michellamines from a new species of the plant genus Ancistrocladus. tentatively named Ancistrocladus sp. novum (DT 6889) , which comprises the steps of:
- the present invention also provides a method for the interconversion of either of michellamines A or B, into a mixture of michellamines A, B, and C, which comprises:
- the present invention includes the aforementioned michellamines, particularly michellamines A, B, and C, their derivatives, and pharmacologically acceptable salts thereof in substantially pure form, as well as antiviral compositions which comprise an antiviral effective amount of at least one of these michellamines, or derivatives or pharmacologically acceptable salts thereof, and a pharmacologically acceptable carrier.
- the antiviral compositions can further include an antiviral effective amount of AZT and/or other known antiviral agents.
- the present invention also encompasses a method of treating a viral infection which comprises administering to a patient in need thereof an antiviral effective amount of at least one of these michellamines, particularly michellamines A, B, or C, or a derivative or pharmacologically acceptable salt thereof.
- the method of the present invention may also involve co-administering an antiviral effective amount of AZT and/or other known antiviral agents with at least one of these michellamines or a derivative or pharmacologically acceptable salt thereof.
- Figure 1 illustrates the structures of michellamines A, B, and C.
- the ring-position numbering scheme is shown only for michellamine A, but is the same for michellamines B and C.
- Figures 2A-D show the anti-HIV-1 activity of michellamine A (free base) .
- Figures 2A, 2B, and 2C show the effects of a range of concentrations of michellamine A upon uninfected CEM-SS cells (o) and upon CEM-SS cells infected with HIV-1 (•) , as determined after 6 days in culture.
- Fig. 2A depicts the relative numbers of viable CEM-SS cells, as assessed by the BCECF assay.
- Fig. 2B depicts the relative DNA content of the respective cultures.
- Fig. 2C depicts the relative numbers of viable CEM-SS cells, as assessed by the XTT assay.
- the data points are represented as the percent of the uninfected, non-drug treated control values.
- Fig. 2D shows the effects of a range of concentrations of michellamine A upon indices of infectious virus or viral replication. These indices include viral reverse transcriptase activity (A) , production of viral core protein p24 ( ⁇ ) , and syncytium-forming units ( ⁇ ) .
- A viral reverse transcriptase activity
- ⁇ production of viral core protein p24
- ⁇ syncytium-forming units
- the data points are represented as the percent of the infected, non-drug treated control values.
- Figures 3A-D show the anti-HIV-1 activity of michellamine A (HBr salt) .
- Figures 3A, 3B, and 3C show the effects of a range of concentrations of michellamine A (HBr salt) upon uninfected CEM-SS cells (o) and upon CEM-SS cells infected with HIV-1 (•) , as determined after 6 days in culture.
- Fig. 3A depicts the relative numbers of viable CEM-SS cells, as assessed by the BCECF assay.
- Fig. 3B depicts the relative DNA content of the respective cultures.
- Fig. 3C depicts the relative numbers of viable CEM-SS cells, as assessed by the XTT assay.
- the data points are represented as the percent of the uninfected, non-drug treated control values.
- Fig. 3D shows the effects of a range of concentrations of michellamine A (HBr salt) upon indices of infectious virus or viral replication. These indices include viral reverse transcriptase activity (A) , production of viral core protein p24 ( ⁇ ) , and syncytium-forming units ( ⁇ ) .
- the data points are represented as the percent of the infected, non-drug treated control values.
- Figures 4A-D show the anti HIV-1 activity of michellamine B (free base) . Figs.
- FIG. 4A, 4B, and 4C show the effects upon a range of concentrations of michellamine B upon uninfected CEM-SS cells (o) and upon CEM-SS cells infected with HIV-1 (•) as determined after 6 days in culture.
- Fig. 4A depicts the relative numbers of viable CEM-SS cells, as assessed by the BCECF assay.
- Fig. 4B depicts the relative DNA content of the respective cultures.
- Fig. 4C depicts the relative numbers of viable CEM-SS cells, as assessed by the XTT assay.
- Fig. 4D shows the effects of a range of concentrations of michellamine B upon indices of infectious virus or viral replication.
- indices include viral reverse transcriptase activity (A) , production of viral core protein p24 ( ⁇ ) and syncytium-forming units ( ⁇ ) .
- A viral reverse transcriptase activity
- ⁇ production of viral core protein p24
- ⁇ syncytium-forming units
- Figures 5A-D show the anti-HIV-1 activity of michellamine B (HBr salt) .
- Figs. 5A, 5B, and 5C show the effects of a range of concentrations of michellamine B (HBr salt) upon uninfected CEM-SS cells (o) and upon CEM-SS cells infected with HIV-1 (•) , as determined after 6 days in culture.
- Fig. 5A depicts the relative numbers of viable CEM-SS cells, as assessed by the BCECF assay.
- Fig. 5B depicts the relative DNA content of the respective cultures.
- Fig. 5C depicts the relative numbers of viable CEM-SS cells, as assessed by the XTT assay.
- 5D shows the effects of a range of concentrations of michellamine B (HBr salt) upon indices of infectious virus or viral replication. These indices include viral reverse transcriptase activity (A) , production of viral core protein p24 ( ⁇ ) , and syncytium-forming units ( ⁇ ) .
- A viral reverse transcriptase activity
- ⁇ production of viral core protein p24
- ⁇ syncytium-forming units
- Figures 6A and 6B show the anti-HIV-2 activity of michellamine A (free base and HBr salt) .
- Fig. 6A shows the effects of a range of concentrations of michellamine A (free base) upon uninfected MT-2 cells (o) and upon MT-2 cells infected with HIV-2 (•) as determined using the XTT assay after 6 days in culture. The open bars show the corresponding supernatant reverse transcriptase activities.
- Fig. 6B shows the effects of a range of michellamine A (HBr salt) concentrations upon uninfected MT-2 cells (o) and upon MT-2 cells infected with HIV-2 (•) as determined using the XTT assay after 6 days in culture. The open bars show the corresponding reverse transcriptase activities. In both graphs, all data points are represented graphically as the percent of their respective controls.
- Figures 7A and 7B show the anti-HIV-2 activity of michellamine B (free base and HBr salt) .
- Fig. 7A shows the effects of a range of concentrations of michellamine B (free base) upon uninfected MT-2 cells (o) and upon MT-2 cells infected with the NIH-DZ strain of HIV-2 (•) as determined using the XTT assay after 6 days in culture. The open bars show the corresponding supernatant reverse transcriptase activities.
- Fig. 7B shows the effects of a range of michellamine B (HBr salt) concentrations upon uninfected MT-2 cells (o) and upon MT-2 cells infected with HIV-2 (•) as determined using the XTT assay after 6 days in culture. The open bars show the corresponding supernatant reverse transcriptase activities. In both graphs, all data points are represented graphically as the percent of their respective controls.
- Figures 8A, 8B, and 8C show the XTT anti-HIV assay results of comparative testing of the acetate salts of michellamines A, B, and C, respectively, upon uninfected (o) CEM-SS cells and upon CEM-SS cells infected (•) with the CBL20 strain of HIV-2.
- the horizontal axis scaling units are ⁇ M, and the data points are represented as the percent of the uninfected, non-drug treated control values.
- the present invention is predicated on the discovery that compounds isolated from a previously unknown plant species of the genus Ancistrocladus. tentatively named Ancistrocladus sp. novum (DT 6889) , have antiviral properties and are useful in antiviral treatments.
- the present invention provides michellamines in substantially pure form and derivatives thereof which exhibit antiviral activity, methods of isolating such michellamines from native plants, pharmaceutical compositions containing such michellamines, and methods of treating viral infections through the administration of such michellamines.
- the specific michellamine of interest has the formula:
- the present invention more generally provides a substantially pure michellamine or michellamine derivative having the formula:
- R 1 and R 6 are the same or different and are each H, Ci-Cg alkyl, R 1:L CO-, or R 1:L S0 2 - wherein R 11 is Ci-C 6 alkyl or aryl;
- R 2 , R 3 , R 4 , R 7 , R 8 and R 9 are the same or different and are each H, Ci-C 6 alkyl, R n CO-, R 1:L S0 2 - wherein R 11 is defined above;
- R 5 and R 10 are the same or different and are each H
- R 12 is C ⁇ C alkyl or R 13 CO- or R 13 S0 2 -, wherein R 13 is Ci ⁇ Cg alkyl or aryl;
- R 14 , R 15 , R 16 and R 17 are the same or different and are each " ⁇ CH 3 or «m CH 3 . and wherein one or more of the ring H positions at 1' , 3' , 7' , 4, 7, 1", 3", 7", 4" and 7" can be substituted with a halogen, nitro, amino, hydroxyl, thiol or cyano group, or pharmacologically acceptable salt thereof.
- the present inventive method of isolating one of the aforementioned michellamines, particularly michellamine A, B, or C, from Ancistrocladus SP. novum comprises (a) extracting dried plant material with an organic solvent to obtain a crude extract, (b) acid-base partitioning the crude extract to obtain a crude organic base fraction, (c) subjecting the crude organic base fraction to centrifugal partition chromatography, and (d) isolating the michellamines with an amino-bonded phase HPLC column.
- the present inventive method of interconverting michellamines A or B into a mixture of michellamines A, B, and C comprises (a) dissolving michellamines A or B in an organic solvent and (b) reacting the michellamines A or B with a base.
- the organic solvent is preferably an alcohol such as methanol, and the base is preferably sodium hydroxide.
- the present inventive composition is an antiviral composition which comprises a pharmaceutically acceptable carrier and an antiviral effective amount of at least. one of the aforementioned michellamines, particularly michellamines A, B, or C, derivatives thereof, or pharmacologically acceptable salts thereof.
- the present inventive composition may include other active or inactive components, in particular, other antiviral agents such as an antiviral effective amount of AZT or other known effective antiviral agent.
- the present inventive method of treating a viral infection comprises administering to a patient in need thereof an antiviral effective amount of at least one of the aforementioned michellamines, particularly michellamines A, B, or C, derivatives thereof, or pharmacologically acceptable salts thereof.
- the treatment method may involve the use of the aforementioned antiviral compositions, and, thus, the treatment method may involve the use of pharmaceutically acceptable carriers and the coadministration of other active or inactive components, in particular, other antiviral agents such as an antiviral effective amount of AZT or other known effective antiviral agent.
- the particular infecting virus may be any suitable virus, particularly a retrovirus such as human immunodeficiency virus (HIV) , including HIV-1 and HIV-2.
- HIV human immunodeficiency virus
- the pharmacologically acceptable salts may be any such suitable salts.
- Examples of pharmacologically acceptable salts include HBr, HC1, oxalate, citrate, acetate, tartrate salt, and the like.
- C -C alkyl is meant straight or branched chain i-C alkyl groups. Examples include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tertiary-butyl, n-pentyl, iso-pentyl and n-hexyl.
- aryl an organic radical derived from an aromatic hydrocarbon.
- An example of an aryl group is phenyl.
- aliphatic organic radical derived from an open hydrocarbon chain. Examples of aliphatic radicals include alkanes, alkenes and alkynes. Specific examples of aliphatic radicals which can be used in the present invention include, but are not limited to, Ci-C 6 alkyl radicals, straight or branched.
- examples include, but are not limited to, Type C and Type D retroviruses, HTLV-1, HTLV-2, HIV-1, HIV-2, feline leukemia virus, simian immunodeficiency virus, murine leukemia virus, bovine leukemia virus, equine infections, anemia virus, avian sarcoma viruses, such as rous sarcoma virus and the like, hepatitis type A, B, non A/non B, herpes viruses type 1 and 2, cytomegaloviruses, influenza viruses, arboviruses, varicella viruses, measles, mumps, and rubella viruses.
- Type C and Type D retroviruses HTLV-1, HTLV-2, HIV-1, HIV-2, feline leukemia virus, simian immunodeficiency virus, murine leukemia virus, bovine leukemia virus, equine infections, anemia virus, avian sarcoma viruses, such as rous sarcoma virus and
- the present inventive compounds are isolated from a newly identified plant species of the genus Ancistrocladus. now tentatively named Ancistrocladus sp. novum (DT 6889) .
- a preliminary communication (Manfredi et al., J ⁇ Med. Chem.. 34. 3402-3405 (1991)) named the source plant of michellamines as Ancistrocladus abbreviatus. See also U.S. patent application Serial No. 07/684,197 (filed April 12, 1991) and PCT/US92/02805.
- a ⁇ . abbreviatus is actually devoid of michellamines and, that the true michellamine-containing plant species, while having many similarities to A. abbreviatus. is an Ancistrocladus species previously unknown to science.
- Ancistrocladaceae is a small paleotropical family in the order Theales, with about 20 species known from Asia and tropical Africa. So far, ten species have been described from Africa.
- Ancistrocladus sp. novum (DT 6889) , presently the only known source of michellamines, differs from all previously described African species of Ancistrocladus in having petals slightly shorter than the sepals; the petals are about twice as long as the sepals in other species.
- the new species is unique in Africa for the short petals described above, it can also be distinguished by other characteristics. Some of the other species in the genus have a pseudo-petiole, where the leaf blade narrows abruptly towards the base of the leaf, and the basal portion is very narrow. In the new species, the leaf blade narrows gradually to the base. This characteristic distinguishes Ancistrocladus sp. novum from A ⁇ . abbreviatus, A. barteri. A. elalensis. A. letestui. A. robertsoniorum. and A_ s . uncinatus.
- Ancistrocladus differ from the new species (DT 6889) in the following ways:
- A. barter i (Sc. Elliot) : p s e u d o - p e t i o l e , n o intramarginal nerve
- A. uncinatus (Hutch & Dalz) : leaves small, pseudo- petiole
- Ancistrocladus sp. novum (DT 6889) is not the only undescribed species in the genus from Cameroon.
- Ancistrocladus sp. novum (D. Thomas 9016, sterile; Cheek and Ndumbe 3915, flowers) , found near Douala and Limbe, has large rather thin leaves, short inflorescences, and white flowers with long petals; it does not appear to contain michellamines.
- Cameroon collections have been made from a small area of forest west of Mundemba Town, partly inside the Korup National Park. This area has high rainfall (about 5,000 mm per year) most of which occurs in the single, long, wet season from April to November.
- the soil on which the plants have been found in Cameroon is a leached, nutrient poor, sandy clay.
- One old (sterile) collection has been made in the Oban area of southeastern Nigeria, about 45 km west of the original Cameroonian collection sites.
- the forest in which Ancistrocladus sp. novum (DT 6889) occurs is part of an ancient refuge area - that is, an area thought to have been continuously forested during the pleistocene period.
- This refuge very rich in rare and endemic species, extends from the Cross River in southeastern Nigeria through Cameroon and to the Mayombe forests of the Congo Republic.
- the refuge extends up to about 200 km inland; it is still largely covered with species-rich lowland rain forest.
- the new species could conceivably be found anywhere within this refuge area, but may be unlikely to be found outside it. It is most likely to be discovered in areas close to its present known localities.
- the forest outside the park has no known clear ownership at present and is defined as "National Land", belonging to the Cameroon Government.
- the forest is threatened in the long term both by commercial logging and by conversion to agricultural land. It is anticipated that the areas of unprotected forest in which the vine occurs will be protected by the Cameroon Government through the creation of one or more Forest Reserves.
- the leaves of the plant are richest in michellamine content.
- a survey is in progress to ascertain the effects of leaf harvesting on the continued viability of the vines. So far, none of the vines from which leaves were harvested has died, but the vine's rate of recovery is not yet known.
- Other ongoing research is investigating the distribution and size of the wild population of the plant, as well as methods of propagation and cultivation of the plant. This research will show the quantities of michellamine-containing Ancistrocladus leaves which can be expected from the wild population, and how this might be augmented or replaced by cultivated material. This research will critically set the stage for a rapid increase in the quantity of Ancistrocladus leaves available, should demand for the michellamines increase rapidly.
- a variety of methods can be used to isolate the michellamines. Among these methods are extraction, solvent-solvent partitioning, centrifugal partition chromatography, gel permeation chromatography and HPLC with a variety of bonded phases. The isolation of the compounds can be monitored by UV, TLC, and anti-HIV bioassay.
- the procedure described herein is of a scale to accommodate an initial starting amount of approximately 1/2 kilogram of the air-dried plant material consisting of leaves, stems, and twigs.
- This plant material is first ground to a coarse powder and extracted with 1:1 MeOH:CH 2 Cl 2 , followed by a second extraction with methanol.
- These initial crude organic extracts typically amount to a total of approximately 8-10% of the mass of the original dried plant material.
- This crude extract then is dissolved in 5% aqueous HCl and extracted with CHC1 3 .
- the aqueous layer is then made basic with concentrated NH OH to a pH of 10-11; it is then extracted with 4:1 CHCl 3 :MeOH and then with 1:1 MeOH:CHCl 3 to give a total of about 0.5-1.0 g of basic extract after removal of the solvent.
- the extract is then dissolved in the lower phase of a 5:5:3 (CHCl 3 :MeOH:0.5% aqueous HBr) biphasic solvent system and placed on a Sanki CPC operating in the descending mode.
- the effluent is monitored at 270 nm.
- the final peak to elute in descending mode contains the HBr salts of both michellamines A and B plus a trace of C.
- this mixture typically comprises a total mass of about 200-300 mg.
- the mixture is further separated with amino-bonded phase HPLC using 43:7 CHC1 3 :MeOH/0.075% (NH 4 ) 2 C0 3 as the solvent.
- the overall yield of michellamines from crude organic extract is about 0.5-2% for michellamine A and 2-10% for michellamine B.
- Michellamine C is isolated in trace amounts following the same procedure.
- Example 1 This example illustrates the isolation of michellamines from the plant species Ancistrocladus sp. novum (DT 6889) .
- the remaining aqueous layer was treated with concentrated NH 4 0H until the pH of the solution was between 10 and 11.
- the basic aqueous phase was extracted with five 100 ml aliquots of 4:1 CHCl 3 :MeOH.
- the extracts were combined and the solvent removed at reduced pressure to give 0.3195 g of extract.
- An anti-HIV assay was run according to the procedure set forth in Weislow et al., supra, and the material was found to be active.
- the remaining aqueous layer was extracted further with three 100 ml aliquots of 1:1 MeOH:CHCl 3 .
- the extracts were combined and the solvent removed at reduced pressure to give 0.2534 g of extract, which was again tested according to the same assay (Weislow et al., supra). The material was found to be active.
- Fraction H (45.4 mg) was dissolved in 500 ⁇ l of CHCl 3 :MeOH (43:7) and injected onto a Waters Delta Prep HPLC using a Rainin Dynamax NH 2 column (21.4 mm x 250 mm equipped with a guard column) . The sample was eluted with CHCl 3 :MeOH/0.075% (NH 4 ) 2 C0 3 (43:7) at a flow rate of 13 ml/min and monitored at 260 nm. Six fractions were collected and tested for HIV-inhibitory activity.
- Their chemical and spectral characteristics are set forth below.
- Fraction G was treated in a similar manner, except that it was dissolved in 1.5 ml of solvent and placed on the column in three 500 ⁇ l injections. From this sample, 5.0 mg of michellamine A and 39.5 mg of michellamine B were obtained. 3.0 mg of an inactive, unidentified compound were also collected.
- Fraction F (18.8 mg) afforded 2.8 mg of michellamine A and 8.1 mg of michellamine B along with two minor inactive compounds ( ⁇ 2 mg) .
- Fraction G (18.2 mg) afforded 10.1 mg of michellamine A and 2.1 mg of an unknown inactive substance.
- a third, minor compound, michellamine C was isolated on one occasion as a shoulder on the michellamine B chromatographic peak. It has not been encountered in subsequent, more rapidly processed material.
- the overall yield of the active fractions from starting crude extract was 1.4% michellamine A and 5.0% michellamine B.
- This example sets forth information on the chemical structures of the michellamines isolated in accordance with Example 1.
- Mass spectral analyses via plasma desorption mass spectrometry ( 252 Cf PDMS) , demonstrated that the two compounds had identical molecular weights (m/z 756) .
- the molecular formula was established as C 46 H 48 N 2 0 8 by accurate-mass, fast atom bombardment mass spectrometry.
- Ancistrocladaceae is well known as a source of naphthalene-tetrahydroisoquinoline alkaloids (Bringmann, supra; Ruangrungsi et al., J. Nat. Prod. _ 48. 529-534 (1989) , and references cited therein) .
- the mass spectral data and the complex NMR spectra of the isolated compounds suggested that these antiviral compounds were dimeric relatives of the known Ancistrocladaceae alkaloids.
- the NMR data for michellamine B are provided in Table 2.
- a pseudoaxial position on the ring was evident from its couplings to the H-4/H-4" protons (11.8, 4.3 Hz); a moderate to strong nOe response to the methyl group attached to C-l/C-1'" established the 1,3 diaxial relationship between the two and therefore the trans relationship between the methyl groups attached to C-l/C-l'" and C-3/C-3" * .
- the composition of one ring in the naphthalene system was established through HMQC, HMBC, and difference nOe experiments as a pair of meta-disposed protons, with an intervening methyl group and a flanking methoxyl. The remaining ring had a single proton, one hydroxyl group and linkages to two other aryl systems.
- HMBC and HMQC data suggested a 1,3 relationship of the proton and hydroxyl substituents.
- the complete substitution of that ring and the relative stereochemistry and conformation of the naphthalene/tetrahydroisoquinoline connection were secured from difference nOe data.
- Each of the benzylic methylene protons (C-4/C-4'") of the tetrahydroisoquinoline system exhibited an nOe relationship to different naphthalene protons, H-4e/H-4e'" to H-7' /H-7" and H-4a/H-4a'" to H-l' /H-l" .
- the tetrahydroisoquinoline was linked to the naphthalene by a bond from C-5/C-5'" to C-8'/C-8".
- the naphthalenes therefore, had to be connected at C-6' /C-6".
- Michellamine C appears to have the opposite configuration from michellamine A about the C-5/C-8' and C-5 w /C-8" bonds. Variable temperature NMR experiments failed to show evidence of spontaneous interconversion.
- the michellamines are unique molecules in several regards. They are the first dimeric alkaloids of this class to be discovered, and they possess an unusual C-5/C-8' (and C-5"7c-8") linkage between the two ring systems. Further, they are the most polar compounds in the class, containing more free phenols per monomeric unit than any of the known compounds.
- the originally-depicted (Manfredi et al., supra) absolute stereochemistry of the michellamines was arbitrarily assigned based upon literature precedents (e.g., Bringmann, supra) .
- the FID-GC data were obtained on an OVl-column (0.33 mm x 30 m) ; temperature program: from 140°C (1 min) to 155°C (1 min) at l°C/min, from 155°C (1 min) to 160°C (1 min) at 0.5°C/min.
- michellamine B was unambiguously established to have R-configurations at C-l and C-3 of both "molecular halves". Given the relative configuration at the two stereogenic biaryl axes (at C-5/C-8' and C-5"7c-8") of the two molecular halves) vs. the stereocenters, as deduced from the NOE experiments (see above) , the complete absolute stereostructure of michellamine B therefore was established as 1R, 3R, 5R (or W) , l ⁇ R, 3 ⁇ , 5"'S (or P) , depicted in Figure 1.
- Michellamine A likewise was subjected to the same degradation analysis, and its absolute stereostructure established similarly as 1R, 3R, 5S (or P) , 1 W R, 3" ⁇ S (or P) , as depicted in Figure 1. Since the NMR data had indicated opposite configurations at the C-5/C-8' (and C-5'"/C-8") linkages in michellamine A versus C, the absolute stereostructure in michellamine C was deduced to be 1R, 3R, 5R (or M) , l'"R, 3'"R, 5"'R (or M) , as depicted in Figure 1. Thus, differing from many other Ancistrocladaceae-alkaloids (Bringmann, supra) , the michellamines have an oxygen function at C-6, but R-, not S-configuration at C-3.
- CD-curve was essentially opposite to that of ancistrobrevine B [CD: 25°C; ⁇ e 2 oo ⁇ 64 ' ⁇ e 209 +101, ⁇ e 15 +88, ⁇ e 226 +191, ⁇ e 239 -134 (EtOH; ⁇ 0.088)] which is structurally closely related to the molecular half of michellamine A, but has the opposite (M) configuration at the axis. This underscores further the correct assignment of the absolute configuration of both biaryl axes of michellamine A as P.
- CD-curve had a distinctly less pronounced character [CD:25°C; ⁇ e 2 oo +15, ⁇ e 20 ⁇ -34, ⁇ e 201 5 +24, ⁇ e 202 . 5 -68, ⁇ e 203#5 -31, ⁇ e 205 -92, ⁇ € 205#5 -40, ⁇ e 208 -116, ⁇ e 2 ⁇ 0 -116, ⁇ e 2 ⁇ 0 ⁇ 5 -130, ⁇ e 2 ⁇ 8 -158, ⁇ e 219 . 5 +1,
- Example 3 This example illustrates a procedure for the preparation of HBr salts of the michellamines as obtained in Example 1.
- a solution of michellamine B in MeOH was treated dropwise with 9 M HBr (2.2 mole equivalents). After addition was complete, the solvents were evaporated, providing the HBr salt.
- Other salts of the michellamines have been prepared in a similar manner.
- This example illustrates a procedure for the interconversion of the michellamines as obtained in Example 1.
- Example 5 This example illustrates a procedure for the preparation of derivatives of the michellamines as obtained in Example 1. Using standard organic chemical methodology, a number of structural modifications of the michellamines can be made for purposes of preparing derivatives of the michellamines which express antiviral activity.
- the michellamines can be substituted at one, some, or all of the respective positions.
- acetate can be substituted at one, some, or all of R 2 , R 3 , R 4 , R 7 , R 8 , and R 9 .
- R 1 and R 6 can form benzene sulfonamide derivatives.
- Examples of these include, but are not limited to: 1. Preparation of ester, sulfonate ester, and ether derivatives at one or more of the six phenolic hydroxyl positions in the michellamines (C-5' , C-6, C-8) .
- michellamine A or B can be reacted with an acid (RC0 2 H or RS0 3 H wherein R is an aliphatic or aromatic radical) and dicyclohexylcarbodiimide in triethylamine to prepare the ester or sulfonate ester.
- an acid RC0 2 H or RS0 3 H wherein R is an aliphatic or aromatic radical
- RX alkyl halide
- michellamine A or B is reacted with BBr 3 or BX 3 - (CH 3 ) 2 S in CH 2 C1 2 (where X - F, Cl or Br) .
- the resulting phenol can be converted to esters, sulfonate esters or ethers as described above (in 1) .
- RX alkyl halide
- michellamine A or B is reacted with an aldehyde and the resulting product reduced with NaBH 4 .
- michellamine A or B is reacted with Br 2 in H 2 0.
- michellamine A or B is treated with HN0 3 /H0Ac to provide nitro-substituted (-N0 2 ) derivatives.
- the nitro derivative can be reduced to the amino derivative.
- the amino-derivative is the point of origin of the chloro, iodo, cyano, thiol, and hydroxyl substitution via well known and practiced diazonium substitution reactions. For example:
- Example 6 This example illustrates the antiviral activity of the compounds of the present invention.
- a battery of interrelated assays on individual wells from 96-well microtiter plates was performed to show antiviral activity. Measurements of cellular viability, in the presence and absence of the compounds in uninfected and virus-infected cells, by an adaptation of the procedure set forth in Weislow et al., J. Natl. Cancer Inst.. 81. 577-586 (1989) , as well as by an adaptation of a method using the fluorescent probe 2' -7' -biscar-boxyethyl-5 (6) - carboxyfluorescein acetoxymethyl ester (BCECF) as set forth in Rink et al., J. Cell Biol..
- BCECF is a nonfluorescent molecule which readily enters viable cells where it is hydrolyzed by cellular esterases to a fluorescent molecule.
- Total cellular DNA content was measured with the dye, 2-diamidino-phenylindole (DAPI) , which fluoresces when intercalated at A-T specific sites in chromatin, according to the procedure set forth in McCaffrey et al.. In Vitro Cell. Develop. Biol.. 24., 247-252 (1988) .
- DAPI 2-diamidino-phenylindole
- PCFIA Particle Concentration Fluorescent Immunoassay technology
- Screen Machine specifically the Screen MachineTM available from Baxter Healthcare Corporation (Mundelein, IL) .
- the Screen Machine is a semiautomated fluorescent plate reader capable of adding reagents and/or wash buffers to filter-bottomed, 96-well plates with the subsequent evacuation of fluid and concentration of fluorescently-stained cells on the cellulose acetate filter. Fluorescence is detected via epifluorescence.
- the human lymphocytic target cell lines, CEM-SS and MT-2, used in the antiviral assays were maintained in RPMI 1640 medium (Gibco, Grand Island, NY) without phenol red and supplemented with 5% fetal bovine serum (FBS) (Gibco) , 2 mM L-glutamine, and 50 ⁇ g/ml gentamicin (Gibco) (complete medium) .
- Exponen ⁇ tially-growing CEM-SS or MT-2 cells were pelleted and resuspended at a concentration of 2.0 x 10 5 cells/ml in complete medium.
- the Haitian variant of HIV, HTLV-III RF was used.
- the NIH-DZ strain or the CBL20 strain was used. Frozen virus stock solutions were thawed immediately before use and resuspended in complete medium to yield 1.2 x 10 5 SFU/ml. Reagents. The tetrazolium reagent, XTT, was obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute. Biscarboxyethyl-5(6)-carboxy- fluorescein acetoxymethyl ester (BCECF) was purchased from Molecular Probes, Inc. (Eugene, OR) and dissolved immediately before use in DMSO (1 mg/ml).
- BCECF Biscarboxyethyl-5(6)-carboxy- fluorescein acetoxymethyl ester
- DAPI Dulbecco's phosphate-buffered saline
- PBS Dulbecco's phosphate-buffered saline
- DAPI 4' ,6-diamidino-2-phenylindole
- Stock solutions of DAPI were prepared at 100 ⁇ g/ml in distilled water by sonication, passed through a 0.45 ⁇ m filter, and stored at -20°C.
- Working solutions of DAPI were prepared at 10 ⁇ g/ml in PBS containing 0.5% nonidet P-40 (NP-40) (Sigma).
- XTT was prepared at a concentration of 1 mg/ml in serum-free RPMI 1640.
- Phenazine methosulfate (PMS) (Sigma) was prepared at 0.153 mg/ml in PBS and stored at -20°C. Immediately before use, XTT was dissolved at 37°C, and PMS was added to yield a final concentration of 20 ⁇ M.
- the Biomek was used to perform eight serial dilutions of each drug and to transfer a 100 ⁇ l aliquot of each dilution to the test plate.
- Uninfected CEM-SS or MT-2 cells were plated at a density of 1 x 10 4 cells in 50 ⁇ l of complete medium. Diluted HIV-1 or HIV-2 virus was then added to appropriate wells in a volume of 50 ⁇ l to yield a multiplicity of infection of 0.6. Appropriate cell, virus, and drug controls were used, with the final volume in each well being 200 ⁇ l. Uninfected, untreated cell controls, and untreated virus infected cell controls were placed on both sides of the 96-well test plates; drug blanks were placed along the top and bottom of the plates.
- Biomek was used to disperse evenly the contents of each well of the test plate and transfer 50 ⁇ l aliquots to each of two new microtiter plates. These plates subsequently were used to measure either cellular viability using BCECF or total DNA content using DAPI.
- XTT Assay As an estimate of cellular viability, the metabolic reduction of the tetrazolium salt, XTT, to the soluble, colored formazan was carried out by adding 50 ⁇ l of the XTT/PMS solution to each well of the original test plate and incubating for 4 hrs at 37 ⁇ C. After incubation the plates were covered with adhesive plate sealers (Dynatech, Alexandria, VA) and shaken, and optical densities were determined using a V-max photometer (Molecular Devices, Inc., Menlo Park, CA) at a test wavelength of 450 nm.
- BCECF Assay Cellular viability also was measured using BCECF. Freshly prepared BCECF solution (25 ⁇ l) was added to each well of the microtiter plate, and the plates incubated at 37°C for 30 min. Subsequently, 25 ⁇ l of a 2% solution of paraformaldehyde was added to each well and incubated a further 30 min to inactivate the virus. The contents of each well were mixed, and a 75 ⁇ l aliquot was then transferred to a filter-bottomed, 96-well plate (Baxter Healthcare Corp.).
- DAPI Assay Assay.
- Total DNA content of each well was determined by the following modifications to the method described in McCaffrey et al.. In Vitro Cell. Develop. Biol.. 24., 247-252 (1988) .
- the contents of each well were fixed by adding 25 ⁇ l of a 2% paraformaldehyde solution and incubating the plate at 37 °C for 30 min.
- 25 ⁇ l of the DAPI/NP-40 solution was added to each well and incubated for 2 hrs.
- the contents of each well were mixed, and a 75 ⁇ l aliquot was transferred to a filter-bottomed 96-well plate (Baxter Healthcare Corp.).
- the DAPI plate was placed in the Screen Machine and processed by the same protocol as the BCECF plate above with the signal channel set at an excitation of 400 nm and an emission of 450 nm.
- p24 Assay The production of the HIV-l internal core p24 antigen was measured using a p24 antigen-capture assay (Coulter Immunology, Hialeah, FL) . Supernatants from test plates were diluted 1:100 in 10% Triton X-100 and stored frozen at -20°C until needed. Two hundred microliter aliquots of Triton X-treated samples were added to microtiter wells previously coated with a murine monoclonal anti-HIV-l p24 antigen.
- the plates were sealed and incubated at 37°C for 1 hr. Plate washings were carried out using an automated Denley Wellwash 4 (Coulter Immunology) plate washer. After washing and blotting dry the plates, 200 ⁇ l of a biotinylated human monoclonal anti-HIV-1 p24 was added to appropriate wells, and the plates were reincubated for 1 hr at 37°C. After additional washing, 200 ⁇ l of a streptavidin-horseradish peroxidase solution was added, and the plates were then incubated for 30 min at 37°C. A tetramethylbenzene solution was added to each well and incubated at room temperature for 30 min.
- Syncytium Assay The syncytium assay described in Nara et al., AIDS Res. Hum. Retroviruses. 3. * 283-302 (1987), was used for quantitation of infectious virus. Supernatants from test plates were examined in CEM-SS cell monolayers at multiple dilutions to obtain countable numbers syncytia (50-200 per well) in 2-4 days. Reverse Transcriptase Assay. A 30 ⁇ l aliquot of supernatant was added to 30 ⁇ l of a virus disruption buffer containing 50 mM Tris pH 7.8, 0.15 mg/ml dithiothreitol (DTT) , and 0.1% Triton X-100.
- DTT dithiothreitol
- a 10 ⁇ l sample of lysed virus was added to 30 ⁇ l of a cocktail containing 2 ⁇ l of 1 M Tris, pH 7.8, 1 ⁇ l of 3 M KCl, 5 ⁇ l of 3 mg/ml DTT, 5 ⁇ l of 0.1 M magnesium acetate, 10 ⁇ l of Poly(rA)»p(dT) 10 (2 units/ml) (Pharmacia, Piscataway, NJ) , 6.5 ⁇ l of distilled H 2 0, 0.5 ⁇ l of 10% Triton X-100, and 10 ⁇ l of [ 3 H]dTTP (16.56 Ci/mmol) (Amersham Corp., Arlington Heights, IL) .
- Samples were incubated for 30 min at 37°C, harvested onto DE81 ion exchange paper, and allowed to absorb for 15 min. Sample pads first were rinsed six times with 5% Na 2 HP0 , then twice with distilled H 2 0. Pads were dried and counted in a liquid scintillation counter. Samples were counted in triplicate.
- Figures 2 and 3 illustrate the antiviral activity of michellamine A, as the free base or the HBr salt, respectively.
- Figures 4 and 5 illustrate the antiviral activity of michellamine B, as the free base or the HBr salt, respectively. Both compounds gave very similar activity profiles.
- Figs. 2A and 2C, 3A and 3C, 4A and 4C, and 5A and 5C describe the relative numbers of viable human CEM-SS lymphoblastoid target cells, either uninfected (o) or infected (•) with the HIV-l virus, remaining in the culture wells 6 days after introduction of a range of concentrations of michellamines in the form of their free bases or their HBr salts. The results are represented as the percent of the appropriate uninfected, non-drug treated controls. At michellamine concentrations between approximately 20 to 200 ⁇ M, both the BCECF and the XTT viability assays showed essentially complete protection of the target cells from the killing effects of the virus.
- FIGs. 2B, 3B, 4B, and 5B show indices of viral replication in cultures of human CEM-SS lymphoblastoid target cells infected with HIV-1 and assayed 6 days after introduction of various concentrations of michellamines in the form of their free bases or HBr salts.
- In vitro cytoprotective effects such as the above are known to predict for antiviral activity in humans.
- AZT similarly was selected initially for evaluation in human patients on the basis of its .in vitro cytoprotective effects against the HIV-1 form of the AIDS virus in cultured human lymphoblastoid cell lines (Yarchoan et al., Lancet, 1 , 575-580 (1986)).
- Figure 6 describes the relative numbers of viable human lymphoblastoid MT-2 cells, either uninfected (o) or infected with the NIH-DZ strain of the HIV-2 virus (•) , remaining in the culture wells 6 days after introduction of a range of concentrations of michellamine A in the form of its free base (Fig. 6A) or in the form of its HBr salt (Fig. 6B) .
- Figure 7 describes the relative numbers of viable human lymphoblastoid MT-2 cells, either uninfected (o) or infected with the NIH-DZ strain of the HIV-2 virus (•) , remaining in the culture wells 6 days after introduction of a range of concentrations of michellamine B in the form of its free base (Fig.
- FIG. 8A, 8B, and 8C show the results for various concentrations of michellamines A, B, and C, respectively, tested against uninfected (o) cells and upon cells infected (•) with the HIV-2.
- michellamines B and C showed similar potencies against HIV-2 (EC 50 ⁇ 2 ⁇ M)
- michellamine A appeared somewhat less potent (EC 50 - 10 ⁇ M) .
- the michellamines inhibit at least two types of HIV retrovirus.
- the michellamines and compositions thereof will likely inhibit other retroviruses and other pathogenic viruses.
- Example 7 This example illustrates various possible pharmaceutical compositions which include the compounds of the present invention.
- the compounds of the present invention may be made into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols in the usual ways for their respective route of administration.
- the compounds can be used singularly alone, in combination with each other, or in combination with other antiviral agents.
- at least one compound of the present invention can be co-administered with AZT.
- the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts and also may be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
- the compounds of the present invention may be used alone or in combination with appropriate additives to make tablets, powders, granules, or capsules, e.g., with conventional additives such as lactose, mannitol, corn starch, or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatins; with disintegrators such as corn starch, potato starch, or sodium carboxymethylcellulose; with lubricants such as talc or magnesium stearate; and, if desired, with diluents, buffering agents, moistening agents, preservatives, and flavoring agents.
- conventional additives such as lactose, mannitol, corn starch, or potato starch
- binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatins
- disintegrators such as corn starch, potato starch, or sodium carboxymethylcellulose
- lubricants such as tal
- the compounds of the present invention may be formulated into preparations for injections by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives.
- the compounds of the present invention can be utilized in aerosol formulation to be administered via inhalation.
- the compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.
- the compounds of the present invention may be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
- bases such as emulsifying bases or water-soluble bases.
- the compounds of the present invention can be administered rectally via a suppository.
- the suppository can include vehicles such as cocoa butter, carbowaxes, and polyethylene glycols, which melt at body temperature, yet are solid at room temperature.
- Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, e.g., teaspoonful, tablespoonful, tablet, or suppository contains a predetermined amount of the composition containing at least one compound of the present invention; similarly, unit dosage forms for injection or intravenous administration may comprise a michellamine composition as a solution in sterile water, normal saline, or other pharmaceutically acceptably carrier.
- unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of at least one compound of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable, diluent, carrier, or vehicle.
- the specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.
- the pharmaceutically acceptable excipients for example, vehicles, adjuvants, carriers, or diluents, are readily available to the public. One skilled in the art can determine easily the appropriate method of administration for the precise formulation of the composition being used. Any necessary adjustments in dose can be made readily to meet the nature or severity of the infection and adjusted accordingly by the skilled practitioner.
- Example 8 This example illustrates various possible uses of the michellamines of the present invention in the treatment of viral infections.
- the present invention relates further to a method of treating viral infections comprising the administration of an antiviral effective amount of at least one compound of the present invention.
- Antiviral effective amount is defined as that amount of compound required to be administered to an individual patient to achieve an antiviral effective blood and/or tissue level to inhibit the virus.
- the antiviral effective blood level might be chosen, for example, to inhibit a virus in a screening assay.
- An example of such an amount would be 20-200 ⁇ M, e.g., from Figures 2-7.
- the antiviral effective blood level can also be defined as that concentration which inhibits markers (e.g., p24) of the virus in the patient's blood, or which renders the patient asymptomatic to the particular viral infection. Since a fixed antiviral effective blood level is used as the preferred endpoint for dosing, the actual dose and schedule for drug administration for each patient will vary depending upon interindividual differences in pharma- cokinetics, drug disposition, and metabolism. Moreover, the dose may vary when the compounds are used prophylactically or when used in combination with other drugs.
- markers e.g., p24
- the dosage for humans can range from about between 0.01 mg/kg body weight to 200 mg/kg body weight.
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Abstract
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US5639761A (en) | 1994-02-14 | 1997-06-17 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Antimalarial naphthylisoquinoline alkaloids and pharmaceutical compositions and medical uses thereof |
US6140339A (en) | 1994-02-14 | 2000-10-31 | The United States Of America As Represented By The Department Of Health And Human Services | Monomeric and dimeric arylisoquinoline alkaloids and derivatives thereof |
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