Note: Descriptions are shown in the official language in which they were submitted.
~WO 94/24108 216 0 8 6 9 PCT~S93103682
MICHELLAMINES USEFUL AS ANTIVIRAL AGENTS
TECHNICAL FIELD OF THE l~v~NllON
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
(AIDs).
BACKGROUND OF THE lN v~llON
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.
It is an object of the present invention to provide
such new antiviral agents. It is a further object of the
present invention to provide methods of obt~i~;ng such
antiviral agents, pharmaceutical compositions containing
30 such antiviral agents, and methods of using such antiviral
agents.
These and other objects and advantages of the present
invention, as well as additional inventive features, will
be apparent from the description of the invention provided
herein.
W094/24108 . PCT~S93/03682 ~
2160869
BRIEF SUMMARY OF THE INVENTION
The present invention is directed specifically to a
substantially pure compound having the formula:
Me OH
H ~ 7
Me~ ~ OH
M ~
MeO HO 7~ ~ Me
H~ ~ ~ ~ ~ Me
7 ~ NH
OH Me
or a pharmacologically acceptable salt thereof,
particularly a substantially pure compound having the
formula:
Me OH Me OH
M ~ OH Me ~ OH
M ~ 5' 4' M~ ~ OH OMe
7J
MeO HO 7~ ~ MeO HO ~ Me
HO ~ Me HO ~ Me
OH Me OH Me
SUB~ I 11 ~JTE SHEET
WO 94/24108 s, ?~ 1 fi ~ 8 6 9 PCT/US93/03682
Me OH
Me~- OH
Me` ~h OH OMe
or ~- ~ ` ~
MeO HO ~)~M
HO ~j~Me
OH Me
or a pharmacologically acceptable salt thereof. These
compounds are hereinafter referred to as michellamines, 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 OR
R ~ ~ ,-
R'4~oR3
M ~ ,. OR OR
3'~ =3'
R o RO '~ ~ Me
R ~ R ~ 6
, ~N~R6
OR R
WO94124108 . ~i PCT~S93/03682 ~
216086~ _
or a pharmacologically acceptable salt thereof,
particularly a substantially pure compound having the
formula:
15 2 15 2
~ ~OR ~ ~OR
M~ / q7, OR OR M~ 5
3.~"1~3, ~ ~ ~ t
R OR O 7'b~`Me R O R O ~Me
R~ ~RI6 R'r Rl6
7~N~R6 ~N~R6
OR R OR R
R OR
oR3
M~ OR OR
5 or ~
R O RO ~Me
8 16
R O~ ~R
~7
OR R
SUBS ~ )TE SHEET
~wo g4,~l08 2~ 8 ~ ~ PCT~S93/03682
wherein R1 and R6 are the same or different and are each H,
C1-C6 alkyl, R11C0-, or R11S02- wherein R11 is C1-C6 alkyl or
aryl;
R2, R3, R4, R7, R8 and R9 are the same or different and
are each H, C1-C6 alkyl, R11C0-, R11S02- wherein Rl1 is
defined above;
RS and R10 are the same or different and are each H,
C1-C6 alkyl, ~ or ~OH
wherein R12 is C1-C6 alkyl or R13Co- or R13So2-, wherein R13
is C1-C6 alkyl or aryl;
R14, R15, R16, and R17 are the same or different and are
each - C H3 or ~ CH3;
and wherein one or more of the ring H positions at 1',
3', 7', 4, 7, ln, 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:
(a) extracting dried plant material with an organic
solvent to obtain a crude extract;
(b) acid-base partitioning said crude extract to
obtain a crude organic base fraction;
(c) subjecting said crude organic base fraction to
centrifugal partition chromatography; and
(d) isolating said michellamines with an amino-bonded
phase HPLC column.
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:
(a) dissolving either of michellamines A or B in an
organic solvent; and
WO941~108 21~ 0 8 6 9 PCT~S93/03682 ~
(b) reacting said michellamines A or B with a base.
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.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure l 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-l 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-l (-), as determined after 6 days in
culture. Fig. 2A depicts t~e 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
~ WO94/~108 21~ ~ 8 ~ 9 PCT~S93/03682
CEM-SS cells, as assessed by the XTT assay. In Figs. 2A,
2B, and 2C, 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 (-), production of viral core
protein p24 (-), and syncytium-forming units (-). In Fig.
2D, the data points are represented as the percent of the
infected, non-drug treated control values.
Figures 3A-D show the anti-HIV-l 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-l (-), 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. In Figs. 3A,
3B, and 3C, 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 conc~ntrations of
michellamine A (HBr salt) upon indices of infectious virus
or viral replication. These indices include viral reverse
transcriptase activity (-), production of viral core
protein p24 (~), and syncytium-forming units (-). In Fig.
3D, the data points are represented as the percent of the
infected, non-drug treated control values.
Figures 4A-D show the anti HIV-l activity of
michellamine B (free base). Figs. 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-l (-) 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
W094/~108 ~18 0 ~ ~ 9 PCT~S93/03682 ~
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.
These indices include viral reverse transcriptase activity
(-), production of viral core protein p24 (-) and
syncytium-forming units (-). In Figures 4A, 4B, and 4C,
the data points are represented as the percent of the
uninfected, non-drug treated control values. In Fig. 4D
the data points are represented as the percent of the
infected, non-drug treated control values.
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. SA 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. ~ig. 5C depicts the relative numbers of viable
CEM-SS cells, as assessed by the XTT assay. Fig. 5D shows
the effects of a ran~e of concentrations of michellamine B
(HBr salt) upon indices of infectious virus or viral
replication. These indices include viral reverse
transcriptase activity ~-), production of viral core
protein p24 (-), and syncytium-forming units (-). In Figs.
5A, 5B, and 5C, the data points are represented as the
percent of the uninfected, non-drug treated control values.
In Fig. 5D, the data points are represented as the percent
of the infected, non-drug treated control values.
~ igures 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.
WO94/~108 ,~ 6 ~ PCT~S93/03682
.
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 ~e~Lesented graphically as the percent of their
respective ~u~LLLols.
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.
DESCRIPTION OF THE PREFERRED EMBODIMBNTS
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. In
particular, the present invention provides michellamines in
WO 94/~108 2 ~ ~ 8 ~ 9 PCT~S93/03682 ~
substantially pure form and derivatives thereof which
exhibit antiviral activity, methods of isolating such
michellamines from native plants, pharmaceutical
compositions cont~ining such michellamines, and methods of
treating viral infections through the administration of
such michellamines.
The specific michellamine of interest has the formula:
Me OH
HN~ ''
Me/~ ~OH
Me ~ ". OH OMe
3~/~3'
MeO HO 7 ~Me
H~Me
7 ~i/ \j~NH
OH Me
or is a pharmacologically acceptable salt thereof, and
particularly is a compound of formula:
~WO 94/24108 " _2 ~.6 0 8 6 9 PCT/US93/03682
11
Me O8,H Me OH
HN ~' ~
Me ~OH Me~~ OH
Me~ OH OMe Me~ OH OMe
3~ 3~
MeO HO 7~1 ~Me MeO HO ~Me
Ho~,f ~Me HO~ Me
7~,NH I~NH
OH Me OH Me
Me OH
Me ~ OH
\~ ~ OH OMe
or ~
MeO HO ~Me
HO ~Me
OH Me
or a pharmacologically acceptable salt thereof. The
present invention provides such compounds in substantially
SLJE~S 111 ~JTE SHEET
WO94/~108 ~1 6 0 8 6 ~ PCT~S93/03682
12
pure form. These specific michellamines are referred to
herein as michellamines A, B, and C, respectively, as
depicted in Figure 1. Michellamines are members of the
naphthylisoquinoline alkaloid class of compounds.
Literature precedents (e.g., Bringmann, The Naphthyl
Isoquinoline Alkaloids, in The Alkaloids, Vol. 29, Brossi,
ed., Academic Presc, New York, 1986, pp. 141-184;
Bringmann, et al., Planta Med., 58 (Suppl. 1), 703-704
(1992)) from other plant-derived compounds of this general
class support that michellamines or michellamine
derivatives, having different absolute configurations about
one or more of the stereocenters at C-l, C-3, C-lm or C-3'~,
might be isolated from natural sources or be synthesized
chemically.
Therefore, as one skilled in the art will readily
appreciate, the present invention more generally provides
a substantially pure michellamine or michellamine
derivative having the formula:
R OR
R ~ ~ ,-
Rl4~J~oR3
, .J ~ ~ 3
R O R O ' ~ Me
R 0~6
OR R
WO94/24108 / ; PCT~S93/03682
or a pharmacologically acceptable salt thereof,
particularly a substantially pure compound having the
formula:
R ~R ~\OR
~ 1~7- oR4 oR5 Me~ ,J~ OR OR
3'~ 3~
R O R O 7'~Me R O R O ~ ~Me
R~RI6
7~N~R6 ~;,N~R6
15 oR2
A ~OR
or ~ e
OR R
8~JB~ ~ E Sl IE~
WO941~108 2 1 6 ~ 8 ~ 9 PCT~S93/03682 ~
14
wherein R1 and R6 are the same or different and are each H,
Cl-C6 alkyl, R11CO-, or R11SO2- wherein R11 is Cl-C6 alkyl or
aryl;
R2, R3, R4, R7, R8 and R9 are the same or different and
are each H, Cl-C6 alkyl, R11CO-, R11SO2- wherein Rll is
defined above;
R5 and R10 are the same or different and are each H,
Cl-C6 alkyl, ~ or ~OH
wherein R12 is C1-C6 alkyl or Rl3Co- or R13So2-, wherein R13
is Cl-C6 alkyl or aryl;
R14, R15, R16 and R17 are the same or different and are
each - cH3 or ~ CH3;
and wherein one or more of the ring H positions at 1',
3', 7', 4, 7, 1~, 3n, 7~, 4~ and 7~ can be su~stituted 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 ~e. novum (DT 6889) comprises
- 20 (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 ~ase 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. While any suitable organic solvent and base may be
used, 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
~ WO94/~108 2~16 0 8 ~ 3 PCT~Sg3/036~2
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-l and HIV-2.
Definitions
The pharmacologically acceptable salts may be any such
suitable salts. Examples of pharmacologically acceptable
salts include HBr, HCl, oxalate, citrate, acetate, tartrate
salt, and the like.
By Cl-C6 alkyl is meant straight or branched chain
Cl-C6 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.
By aryl is meant an organic radical derived from an
aromatic hydrocarbon. An example of an aryl group is
phenyl.
=~
WO94/~108 2 I ~ o 8 ~ 9 PCT~S93/03682
16
By aliphatic is meant 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, Cl-C6 alkyl
radicals, straight or branched.
Of the viral infections that can be treated, examples
include, but are not limited to, Type C and Type D
retroviruses, HTLV-l, HTLV-2, HIV-l, 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 l and 2, cytomegaloviruses, influenza viruses,
arboviruses, varicella viruses, measles, mumps, and rubella
viruses.
Anci strc~cladus sp . novum (DT 6889)
The present inventive compounds are isolated from a
newly identified plant species of the genus Ancis~rocladus,
now tentatively named Ancistrocladus sp. novum (DT 6889).
A preliminary communication (Manfredi et al., J. Med.
Chem., 34, 3402-3405 (l99l)) named the source plant of
michellamines as Ancistrocladus abbreviatus. See also U.S.
patent application Serial No. 07/684,197 (filed April 12,
l99l) and PCT/US92/02805. However, it subsequently has
become clear that ~. 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 previouæly
unknown to science.
The 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
~WO g41~108 ~I 6 ~8 6 g PCT~S93/03682
-
17
Ancistrocladus in having petals slightly shorter than the
sepals; the petals are about twice as long as the sepals in
other species. The original specimen of the plant from
which antiviral activity first was detected was collected
(collection #6889) on March 25, 1987 by Duncan Thomas (DT)
in the Korup National Park, west of Mundemba Town in
Cameroon's Southwest Province (5Ol'N; 851'E, 60 m
elevation above sea level). Because as yet it has no
validly published name, the plant is referred to herein as
"species novum" or SD. novum (i.e., new species), followed
by the plant collector's name or initials and the
collection number that refers to a herbarium specimen. A
specimen of the new species (DT 6889) is preserved in the
herbarium of the Missouri Botanical Garden, where it is
available for viewing by the public.
Although the new species is unique in Africa for the
short petals described above, it can also be distinglliche~
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
disting~ hec Ancistrocladus sp. novum from A abbreviatus,
~ barteri, A. elalensis, A. letestui, A. robertsoniorum,
and A. uncin~tus.
Besides having longer petals, other African species of
Ancistrocladus differ from the new species (DT 6889) in the
following ways:
A. abbreviatus (Airy-Shaw): pseudo-petiole, much
shorter inflorescences
A. barteri (Sc. Elliot): pseudo-petiole, no
intramarginal nerve
A. conqensis (J. Léonard): pseudo-petiole, smaller
leaves
35 A. ealensis (J. Léonard): very slender inflores-
cences
A. ~uineenis (Oliver): leaf margin involute at
the base, blade thinner
WO941~108 2 16 0 8 6 9 rcT~s93lo368z ~
A. letestui (Pellegr.): small narrow leaves with a
pronounced pseudo-petiole
A. likolo (J. Léonard): nerves prominent below
A. PachYrrhachis (Airy-Shaw): inflorescence very narrow
with thick rachis
A. robertsoniorum (J. Léonard): slight pseudo-petiole,
smaller leaves
A. uncinatus (Hutch & Dalz): leaves small, pseudo-
petiole
Ancistrocladus sp. novum (DT 6889) is not the only
undescribed species in the genus from Cameroon. For
example, 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.
It will be proposed that the new michellamine-bearing
species ~ncistrocladus ~e. novum (DT 6889) be formally
named Ancistrocladus korupensis. However, this name is not
considered official until it has been published in a
r~og~;zed botanical journal; it is anticipated that
publication will take place during 1993-94.
No detailed comparisons have been made between the new
species and those described from Asia. However, it appears
unlikely that a narrowly endemic species from lowland
evergreen forest in western Africa would be conspecific
with a plant from Asia.
~istribution of Ancistrocladus sp. novum (DT 6889)
As ~ar as is known, the new species occurs only in a
small area in the Southwest Province of the Republic of
Cameroon and possibly in ad~acent parts of Cross River
State, Nigeria. However, the area is poorly known
botanically, and in the future the species may be found
elsewhere.
~ W094/~108 2 ~ 6 0 ~ 6 9 PCT~S93/03682
-
19
The only known 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 s~. 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 i nl A~; 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.
Availability of Ancistro~ladus sp . novum (DT 6889) Leaves
The current known population is small and partly
situated in a National Park (501'N; 847'E-852'E), where
collecting should not be carried out. The rem~;~ing
population grows in a small area of forest north of the
PAMOL oil palm plantation at Ndian and east of the Korup
National Park boundary, which is formed by the Mana (Ndian)
river at that point (501~'N; 852~'E)~
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
WO94/~108 ~ `8 ~ ~ PCT~S93/03682
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.
At present, the known resource appears ~o be
sufficient for initial drug development. 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-cont~; n; ng
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.
Isolation of the Michellamines from Plant ~Ytracts
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 ~onded phases. The isolation o~ the compounds
can be monitored by W, TLC, and anti-HIV bioassay.
The procedure described herein is of a scale to
accommodate an initial starting amount of approximately l/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 l:l
MeOH:CH2Cl2, 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
~ WO94/~108 ; ~ 1 6 0 8 6 9 PCT~S93t03682
-
21
in 5% aqueous HCl and extracted with CHCl3. The aqueous
layer is then made basic with concentrated NH40H to a pH of
10-11; it is then extracted with 4:1 CHCl3:MeOH and then
with l:l MeOH:CHCl3 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
(CHCl3:MeOH:0.5% aqueous HBr) biphasic solvent system and
placed on a Sanki CPC operating in the descen~ing mode.
The effluent is monitored at 270 nm. The final peak to
elute in desc~n~;ng mode contains the HBr salts of both
michellamines A and B plus a trace of C. After removal of
the solvent, 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 CHCl3:MeOH/0.075%
(NH4)2CO3 as the solvent. Using this general procedure, 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.
~m~les
The following examples further illustrate the present
invention but, of course, should not be construed as in any
way limiting its scope.
~xAmple 1
This example illustrates the isolation of
michellamines from the plant species ~n~istrocladus ~.
novum (DT 6889).
The leaves and stems of dried Ancistrocladus sP. novum
(DT 6889) (449 g) were ground in a Wiley mill and extracted
with 1:1 MeOH-CH2Cl2 in a Kimax percolator. The ground
material was allowed to steep in the solvent overnight.
The solvent was removed by filtration and evaporated at
reduced pressure to give 36.62 g of crude organic extract.
A portion (2.107 g) of this extract was
suspended/dissolved in 330 ml of 5% aqueous HCl and
W094/~l08 2 1 6 ~ PCT~S93/03682
extracted with four 100 ml aliquots of CHC13. The extracts
were combined and the solvent removed at reduced pressure
to give 0.657 g of extract. A primary anti-HIV assay was
performed according to the procedure set forth in Weislow
et al., J. Natl. Cancer Inst., 81, 557-586 (~989), and the
material was found to be inactive.
The remaining aqueous layer was treated with
concentrated NH40H 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 CHC13: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:CHC13. The extracts were
com~ined 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.
NMR and TLC analyses of the two active extracts
indicated that both samples con~in~ the same compounds.
An aliquot of extract from the 4:1 CHC13:MeOH procedure
(264.1 mg) was dissolved in a small amount of the lower
phase of a 5:5:3 MeOH:CHC13:0.5% aqueous HBr biphasic
system. This sample was injected into a Sanki centrifugal
partition chromatograph (CPC) operating in the desc~n~;ng
mode with 12 analytical cartridges (400 rpm, 3.0 ml/min).
The effluent was monitored at 270 nm using a Linear W/~is
200 monitor. Eight fractions were collected (A-H) while
the instrument was operating in the descending mode, and a
ninth fraction was collected (I) when the instrument
operation was reversed to the ascending mode. Fractions A,
C, E, and F were inactive in the anti-HIV assay (Weislow et
al., supra). Fractions B (14.4 mg), D (9.0 mg), and I
(31.8 mg) showed relatively little activity in the anti-HIV
~ WO94/~108 21 ~ O ~ 6 9 PCT~S93/03682
assay. The majority of the anti-HIV activity was found in
fractions G (72.7 mg) and H (45.4 mg).
Fraction H (45.4 mg) was dissolved in 500 ~1 of
CHC13:MeOH (43:7) and injected onto a Waters Delta Prep HPLC
using a Rainin Dynamax NH2 column (21.4 mm x 250 mm equipped
with a guard column). The sample was eluted with
CHC13:MeOH/0.075% (NH4)2CO3 (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.
Fractions 1 (retention time = 10 min., 1.1 mg), 2
(retention time = 19 min., 4.3 mg), 4 (retention time = 26
min., 4.6 mg), and 5 (retention time = 31.5 min., 1.0 mg)
were found to be inactive. Fraction 3 proved to be
michellamine A (retention time = 22 min., 10 mg), and
fraction 6 proved to be michellamine B (retention time - 36
min., 14.4 mg). 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 ~1 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.
The sample obtained from the MeOH:CHC13 (1:1) extract
described above (251 mg) was placed on the Sanki CPC under
the same conditions as the 4:1 extract. In this case,
seven fractions were collected while the instrument was
operated in the desc~;ng mode (A-G) and one fraction
collected during the asc~n~ing mode (H).
Fractions A, B, C, D, and H were inactive in the
anti-HIV assay, while E, F, and G were all active.
Preparative HPLC of Fraction E (72.4 mg), under the
identical conditions as above, afforded 0.8 mg of
michellamine A, 44.5 mg of michellamine B, and 6.3 mg of an
inactive tetrahydroisoquinoline compound. 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 (<
W094/~108 ~-1 6 Q ~ 6 9 PCT~S93/03682
24
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.
~mple 2
This example sets forth information on the chemical
structures of the michellamines isolated in accordance with
Example 1.
An n vitro anti-HIV screening assay, according to the
procedure set forth in Weislow et al., supra, initially
disclosed AIDS-antiviral acti~ity in the CH30H-CH2Cl2 ~
extracts of Ançistrocladus ~. novum (DT 6889).
Preliminary fractionation established that the active
constituents were basic alkaloids. The crude alkàloid
fraction, obtained by acid-base partitioning, was subjected
to centrifugal partition chromatography (CHCl3-CH30H-0.5%
HBr/H20, 5:5:3), and elution with the lower phase gave four
fractions. Fraction 4 yielded two active compounds,
related as atropisomers to which were given the names
michellamines A and B (Figure 1), upon HPLC on an
amino-hon~ phase semi-preparative column [CHC13-0.075%
(NH4)2C03/CH30H, 43:7].
Mass spectral analyses, via plasma desorption mass
spectrometry (252Cf PDMS), demonstrated that the two
compounds had identical molecular weights (m/z 756). The
molecular formula was established as C46H48N208 by
accurate-mass, fast atom bombardment mass spectrometry.
The family 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
~ wo 94~108 2~1~6 0 ~ ~ 9 PCT~S93/03682
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 A are provided in Table
1.
TABLE 1. NMR DATA FOR M~T~MINE A
Carbon # ~ ~# att~he~ H) lH ~ (Multiplicity) J t~z)
1/1'~ 49.5 (1) 4.64 g 6.5
3/3~O 4s.2 (1) 3.54 ddq 11.8,4.3,6.5
4/4"' 33.1 (2) (e)2.69 dd 18.6,4.3;
(a)2.05 dd 18.6,11.8
4a/4a'N133.1 (0)
5/5"'120.3 (o)
6/6~ 156.9 (0)
7/7'~102.0 (1) 6.40 s
8/8~ 155.4 (0)
8a/8a~113.1 (0)
1'/1~llg.1 (1) 6.75 s
2'/2~137.6 (0)
3'/3~108.0 (0) 6.84 s
4'/4~158.1 (0)
4a'/4a~115.2 (0)
5'/5~152.2 (0)
6'/6~119.0 (0)
7'/7~134.8 (1) 7.30 s
8'/8~124.1 (0)
8a'/8a~136.6 (0)
OMe/OMe57.1 (3) 4.10 s
Me-3/Me-3~19.4 (3) 1.16 d 6.5
Me-l/Me-lm18.4 (3) 1.57 d 6.5
Me-2'/Me-2~22.1 ~3) 2.33 s
13C (125 MHz) and lH (500 MHz) NMR spectra of the HBr
salt were recorded in d4-methanol. # attached H determ-
ined from DEPT experiments. The designations "a" and "e~'
refer to axial and equatorial, respectively.
Other spectral data and other characteristics for
michellamine A are as follows: MP=220C (dec)i [a]D
= -10.5, [a]365 = +65.7 (c=0.38, MeOH); FAB-MS: m/z
WO94/~108 ~1 ~0 8 6 ~ PCT~S93/03682
26
757.342 (MH+, calc'd for C46H49N2O8 757.3487); AmaX (MeOH)
230 nm (log ~=4.4), 262(4.1), 287(3.8), 312(3.8), 331(3.8),
344(3-8); vmaX (neat) 3380, 1617, 1584 cm~1.
The NMR data for michellamine B are provided in Table
2.
TAB~ 2. NMR DATA FOR MIC~rr~MT~ B
Carbon # ~ (# att~hed H) 1~ ~ tMUltipliCity) J ~HZ)
1/1'~ 49.6, 49.3 (1) 4.44, 4.26 q 6.5
3/31n 45 3' 4~.2 (1) 3.27, 3.21 dd~ 11.4, 4.5,
6.S
4/4lN 33.g, 33-1 (2) (eR) 2.49 dd 17.5, 4.5;
(aR) 1.86 dd 17.5, 11.0;
(aS) 2.22 dd 17.5, 11.0;
(eS) 2.08 dd 17.5, 4.5
4a/4a'~ 133.1, 133.0 (0)
5/5'~ 120.0, 120.2 (0)
6/6 156.90, 156.88 (O)
7/~ 102.0, 102.1 (1) 6.34 s
8/8~ 155.54, 155.51 (0)
8a/8a~ 113.0, 113.2 (0)
1'/1~ 119.2, 119.2 (1) 6.77, 6.86 s
2 /2 137.60, 137.56 (0)
3'/3~ 108.12, 108.11 (1) 6.84, 6.82 s
~ 20 4'/4~ 158.0, 158.1 (0)
4a'/4a~ 115.22, 115.17 (0)
5'/~" 152.2, 153.3 (0)
6'/6~ 119.0, 119.1 (0)
7'/7~ 136.7, 135.5 (1) 7.28, 7.24 s
8'/8~ 124.12, 124.10 (0)
8a'/8a~ 135.2, 134.7 (0)
OMe/OMe 57.04, 57.05 (3) 4.08, 4.09 s
Me-3/Me-3~' 19.3, 19.3 (3) 1.05, 1.01 d 6.5
Me-1/Me-lm 18.42, 18.40 (3) 1.52, 1.48 d 6.5
Me-2'/Me-2~ 22.1, 22.2 (3) 2.36, 2.31 s
3C (125 MHz) and lH (500 MHz) NMR spectra were recorded
in d4-methanol. 13C chemical shifts are reported ~or the
HBr salt. lH chemical shifts are reported for the free
base. The designations (eS,aS) and (aR,eR) refer to the
methylene signals on the isoguinoline systems with the
'S' and 'R' stereochemistry at the 5-8' and 5~-8"' ring
junctures; "a" and "e" refer to axial and equatorial,
respectively. # attached H were determined from DEPT
experiments.
~ WO94/~108 21 6 0 8 6 9 PCT~S93/03682
Other spectral data and other characteristics for
michellamine B are as follows: MP=230C (dec); ta]D =
-14-8, t~]36s = -23.4O (c=0.74, MeOH); FAB-MS; m/z 757.350
(MH+, calc'd for C46H49N2O8 757.3487); W and IR were
identical to those reported for michellamine A.
The presence of only 23 resonances in the 13C-NMR
spectrum of michellamine A indicated that the two
naphthalene-isoquinoline components were equivalent. The
structure and relative stereochemistry of the
tetrahydroisoquinoline subunit could be discerned readily
from 1H-1H coupling constant analysis and difference nOe
experiments. The H-3/H-3"' protons served as linchpins in
the analysis (the ring-numbering scheme follows the same
scheme as in the Bringmann reference cited above). 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 ~L~v~y nOe response to the methyl group
attached to C-1/C-1~ established the 1,3 diaxial
relationship between the two and therefore the trans
relationship between the methyl ylOU~3 attached to C-l/C-lN'
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 flAnk;ng
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 stereochçm;Stry
and conformation of the naphthalene/tetrahydroisoquinoline
connection were secured from difference nOe data. Each of
the benzylic methylene protons (C-4/C-41N) of the
tetrahydroisoquinoline system exhibited an nOe relationship
to different naphthalene protons, H-4e/H-4e'N to H-7' /H-7N
and H-4a/H-4a'N to H-1'/H-1". Thus, the
tetrahydroisoquinoline was linked to the naphthalene by a
WO94/~108 ~ - 2 ~ 6 0 8 6 ~ PCT~S93/03682 ~
28
bond from C-5/C-5"' to C-8'/C-8". The naphthalenes,
therefore, had to be connected at C-6'/C-6~.
In contrast, the 13C-NMR spectrum of michellamine B was
comprised of 46 signals. A similar series of NMR
experiments provided the same gross structure found for
michellamine A. The d~fferences between the two compounds
lay in the relative configuration of the ring connections.
In michellamine B the C-4 methylene signals appeared as
four discreet resonances, and each produced an nOe
enhancement of an aromatic proton signal upon irradiation.
In one set, the relationships were the same as those for
michellamine A: H-4e and H-7', H-4a and H-l'. The
relationships were reversed in the other half of the
molecule: H-4e~ and H-l", H-4am and H-7~. As before, the
assignments of the protons in the tetrahydroisoquinoline
system were established clearly from coupling constants and
the nOe data.
A trace amount of a third atropisomer, to which has
been given the trivial name michellamine C (Figure l), also
was ~n~o~ntered. The NNR data for michellamine C are
provided in Table 3.
WO941~10N ~1 6~0 8 6 9 PCT~593/0365
TAB~B 3. NMR DATA FOR ~Ir~T~MINE C
Carbon # ~ (# attached E) 1H ~ ~Multiplicity) J (Hz)
1/1'~ 49.1 (1) 4 73 q 7.0
3/3'~ 45.0 (1) 3.65 ddq 11.5, 5.0, 6.5
4/4'~ 34.3 (2) (a) 2.62 dd 17.5, 11.5;
(e) 2-35 dd 17.5, 5.0
4a/4a'~ 133.5 (0)
5/5'~ 120.3 (0)
6/6 156.6 (0)
7/7'~ 102.0 (1) 6.43 s
8/8' 155.6 (0)
8a/8a'N 113.9 (0)
1'/1~ 119.3 (1) 6.84 s
2 /2 137.4 (0)
3'/3~ 108.0 (1) 6.85 s
4 /4 158.0 (0)
4a'/4a~ 115.2 (0)
5'/5~ 152.2 (0)
6'~6~ 119.0 (0)
7'/7~ 135.3 (1) 7.28 8
8'/8~ 124.3 (0)
8a'/8a~ 136.4 (0)
OMe/OMe 57.0 (3) 4.09 s
Me-3/Me-3~ 19.6 (3) 1.30 d 6.5
Me-1/Me-1~ 18.6 (3) 1.68 d 7.0
Me-2'/Me-2~ 22.2 (3) 2.36 s
13C (125 MHz) and lH (500 MHz) NMR spectra of the free
base were recorded in d4-methanol. The designations "a"
and 'le" refer to axial and equatorial, respectively. #
attached H were determined from DEPT experiments.
Michellamine C appears to have the opposite configuration
from michellamine A about the C-5/C-8' and C-5~/C-8~ bonds.
Variable temperature NMR experiments failed to show
evidence of spontaneous interconversion.
Molecular modeling calculations determined the barrier
to rotation about the C-5/C-8' (and C-5'~/C-8") bond in the
michellamines to be 81 KJ/mole; in contrast, the calculated
barrier for rotation about the C-6'/C-6~ bond (51 KJ/mole)
was within the range for available thermal energy to enable
WO94/~108 2 1 fi 0 8 ~ 9 PCT~S93/03682 ~
rotation past the barrier (Still et al., Macromodel, ~ 2.5,
Dept. of Chemistry, Columbia University, NY).
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~/C-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., su~ra) absolute stereochemistry of the
michellamines was arbitrarily assigned based upon
literature precedents (e.g., Bringmann, su~ra). Until
recently, none of the known "monomeric" alkaloids contained
such a C-5/C-8' linkage; however, the structure of a
1~ monomeric naphthylisoquinoline alkaloid, ancistrobrevine B,
which contains the C-5/C-8' linkage, was reported
(Bringmann et al., PhYtochemistrY, 31, 4011-4014 (1992)).
For definitive determination of the absolute
configurations of the michellamines, an efficient
= 20 ruthenium-mediated oxidative degradation procedure,
recently introduced in the field of ~Imonomeric~
naphthylisoquinoline alkaloids (Bringmann et al., supra),
was extended to the dimeric compounds. This analysis is
based upon the configurations of resulting amino acids,
3-amino~utyric acid derived from C-3, and alanine derived
from C-1. A specific example follows.
Michellamine B (9.3 mg, 12.4 ~mol) was added to 4 mL
of a mixture of MeCN/CC14/aqueous phosphate buffer
(pH=6)(1:1:2) under stirring at room temperature, followed
by RuC13 3H20 (0.1 mg) and NaIO4 (99 mg). After 2.5 h, the
aqueous phase was separated and lyophilized. The residue
was extracted with dry MeOH. The resulting solution, which
contained the product amino acids, was saturated with
gaseous HCl at 0C and stirred at room temperature for 3 h
to yield the corresponding methyl esters. After
evaporation of the solvent, the residue was suspended in
dry CH2Cl2 tl mL). Subse~uently, NEt3 (0.2 mL) and
~ WO 94/~108 ' ~ `2 ~1~6 0 8 G 9 PCT~S93/0368t
(R) --methoxy-a-trifluoromethylphenylacetic acid chloride
(MTPA-Cl) (46 ~mol, prepared from the corresponding
(S)-acid) were added to give the Mosher derivatives of the
esters after stirring at room temperature for 1 h. These
were analyzed by FID-GC and found to be derived from
- D-alanine (tR = 15.6 min) and (R) -~-aminobutyric acid (tR =
21.2 min). These assignments were confirmed by
co-injection with the corresponding racemic as well as
enantiomerically pure amino acid stAn~rds. The FID-GC
data were obtained on an OV1-column (0.33 mm x 30 m);
temperature program: from 140C (1 min) to 155C (1 min) at
1C/min, from 155C (1 min) to 160C (1 min) at 0.5C/min.
From the exclusive formation of both amino acids in
their R-configurations, 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~/C-8N)
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 lR, 3R, 5R (or M), l~R, 3~R, 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 lR, 3R,
5S (or P), l~R, 3~R, 5~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 lR, 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.
The complete structural assignments, as deduced from
the elucidation of the relative configurations o~ axial vs.
central chirality within the "halves", combined with the
knowledge of the absolute configuration at the
WO94/~108 21 fi 0 8 6 9 PCT~S93/03C82 ~
32
stereocenters, as evident from the oxidative degradation
reaction, was confirmed further by the circular dichroism
(CD) of michellamines A, B, and C. As for other
naphthylisoquinoline alkaloids, the CD-behavior is greatly
dominated by the element of axial chirality.
Thus, michellamine A, in which both axes have
P-configurations, exhibited very strong Cotton effects tCD:
25C; A~200 -17, ~200.5 -118~ A~202 ~40~ 210.5 --521~ 233.5
+210, ~242.5 +84, A~255,5 +129, Q~281.s ~7~ A~296 +24~ ~318
-48~ 326 ~44~ ~333 52~ 350 -14 (EtOH; c 0.02)J, due to
the identical stereochemistry at the two axes. The
CD-curve was essentially opposite to that of
ancistrobrevine B ~CD: 25C; /~200 -64~ ~209 +lOl, A~215
+88~ ~226 +191~ 239 -134 (EtOH; c 0.088)] which is
structurally closely related to the mol~c~ r 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.
In michellamine B, the CD-curve had a distinctly less
pronounced character tCD:25C; ~200 +15, l~201 ~34~ ~201.5
+24, ~202 5 --68,~203.5 --31~ 205 92~ 205-5 ' 208
--116,~210 --116,~210.5 --130~ 218.5 158, ~219.5 1,
~231-5 +~ ~242 ~75~ ~276 +~ ~299 +76, A~338 -31, Q~350
-22 (EtOH; c 0.021)]. This logically was due to the
opposite contributions of the two axes to the molar
ellipticities, the CD-graph mainly arising from the
contributions of the stereocenters (and possible chiral
interactions between the molecular halves).
In further confirmation of the absolute configuration
of michellamine C, the 200-240 nm region of the CD-curve
was found to be nearly opposite to that of michellamine A
~CD: 25C; ~200 +57r A~200.5 +116~ ~201 +7~ 201.5 +115
~202 +85, ~207 5 +373~ ~238 -238, ~271.5 ~4~ ~272 +6,
~295.5 +156, ~311 +156, ~345 ~ 350 --18(EtOH; c
0. 011) ] .
~ W094/~108 t 2 1 fi ~ 8 6 ~ PCT~S93/03682
..
33
Exam~le 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.
Example 4
This example illustrates a procedure for the
interconversion of the michellamines as obtained in Example
1.
To a solution of michellamine A (1 mg in 1 ml MeOH-d4)
was added 0.5 ml of 0.5 ~ NaOD/D20. lH-NMR analysis
indicated a slow conversion of michellamine A to a mixture
of michellamines A, B, and C (- 3:3:1) over a period of 7
days. Likewise, michellamine B was converted to the same
mixture under identical conditions. HPLC analyses
confirmed these results.
Examle 5
This example illustrates a procedure for the
preparation of derivatives of the michellamines as obtained
in Example 1.
Using st~n~rd organic chemical methodology, a number
of structural modifications of the michellamines can be
made for PUL~O-eS of preparing derivatives of the
michellamines which express antiviral activity.
Depending on the stoichiometric amount of the
particular reactant, the michellamines can be substituted
at one, some, or all of the respective positions. For
example, when one of the michellamines A, B, or C is
reacted with a certain amount of CH3COCl, acetate can be
substituted at one, some, or all of R2, R3, R4, R7, R8, and
R9. Likewise, when one of the michellamines A, B, or C is
WO94/~108 ~Se PCT~S93~03682
34
reacted with a certain amount of benzene sulfonyl
chloride, one or both of Rl and R6 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).
For preparation of esters or sulfonate ester,
michellamine A or B can be reacted with an acid halide
(RCOX or RSO2X, where X 2 Cl, Br, or I and R is an C1-C6
aliphatic or an aromatic radical) in anhydrous pyridine or
triethylamine.
Alternatively, michellamine A or B can be reacted
with an acid (RCO2H or RS03H wherein R is an aliphatic or
aromatic radical) and dicyclohexylcarbodiimide in
triethylamine to prepare the ester or sulfonate ester.
For preparation of ethers, michellamine A or B
can be reacted with an alkyl halide (RX, where X = Cl, Br,
or I and R is an Cl-C6 aliphatic or aromatic radical) in
anhydrous acetone with anhydrous potassium carbonate.
For example:
CH3COCl
michellamine A ~"michellamine A acetate
pyridine
CH3I
michellamine B ~ "O-methyl michellamine B"
K2C03
acetone
2. Removal of the ether methyl group at C-4' to
provide a phenolic hydroxyl functionality and/or conversion
of that moiety to an ester, sulfonate, or other ether.
For cleavage of the ether methyl and conversion
to phenolic hydroxyl, michellamine A or B is reacted with
BBr3 or BX3-(CH3)2S in CH2C12 (where X = F, Cl or Br). The
resulting phenol can be converted to esters, sulfonate
esters or ethers as described above (in 1).
For example:
W094/~108 ~1 6 Q g 6 9 PCT~S93/03682
michellamine A ~ "O-demethyl michellamine A"
CH2C12
3. Preparation of amide or sulfonamide derivatives
at one or both amine sites in the michellamines.
For preparation of amide or sulfonamide
derivatives, the same procedures described above (in 1)
apply. In either case (1 or 3), an appropriate functional
group protection strategy (blocking/deblocking of selected
groups) is applied.
For example:
benzenesulfonyl chloride
michellamine C ~ "michellamine
Et3N C benzenesul-
fonamide"
4. Conversion of the sero~ry amine functionality
to a tertiary amine or tetraalkyl quaternary ammonium salt.
For preparation of tertiary amines or tetraalkyl
ammonium salts, michellamine A or B is reacted with one or
two equivalents of alkyl halide (RX, where X = Cl, Br or I
and R is an Cl-C6 aliphatic radical) in anhydrous aprotic
solvent.
Alternatively, michellamine A or B is reacted
with an aldehyde and the resulting product reduced with
25 NaBH4.
For example:
CH3I
michellamine B ~ "michellamine B dimethylammonium
CH2Cl2 iodide"
5. Substitution of one or more of the hydrogen
substituents on the aryl systems (C-7/C-7"', C-1'/C-lN,
C-3'/C-3", C-7'/C-7~) by halogen, nitro, amino, hydroxyl,
thiol, or cyano groups.
For preparation of bromine substituted
derivatives, michellamine A or B is reacted with Br2 in H2O.
For preparation of other substituted derivatives,
michellamine A or B is treated with HNO3/HOAC to provide
WO94/~l08 2 i g ~ 8 6 3 PCT~Sg3/03682 ~
36
nitro-substituted (-NO2) derivatives. In turn, 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: -
Br2
michellamine A ~ "bromo michellamine A"
~2
HN03 ~H]
michellamine A "nitro michellamine A" - -
HOAc
NaN02
"aminomichellamine A" ~ "diazomichellamine A"
HCl
CuCl
"diazomichellamine A" "chloromichellamine A"
CuCN
~ "diazomichellamine A" "cyano michellamine A"
H2O
- "diazomichellamine A" "hydroxymichellamine A"
~YAmple 6
This example illustrates the antiviral activity of the
compounds o~ the present invention.
25A 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
30forth 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., 95, 189-196 (1982), were
performed as described herein below. BCECF is a
~ WOg4n~l08 ~ ~ 21 6 0 8 6 9 PCT~S931~365~
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). These dyes are used in combination with
Particle Concentration Fluorescent Immunoassay technology
(PCFIA), 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.
Also concl~rrent with the above, confirmatory assays of
p24 antigen production, reverse transcriptase activity, and
synthesis of infectious virions were performed. These and
other details of the procedures and results are described
in further detail as follows.
Cells and virus. 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 105 cells/ml in
complete medium. For the HIV-l studies, the Haitian
variant of HIV, HTLV-IIIRF was used. For the HIV-2 studies,
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 105
SFU/ml.
W094/~108 2 1 ~ ~ 8 6 ~ PCT~S93/03682 ~
38
Reaqents. The tetrazolium reagent, XTT, was obtained
from the Drug Synthesis and Chemistry Branch, Developmental
Therapeutics ~Lo~Lam, Division of Cancer Treatment,
National Cancer Institute. Biscarboxyethyl-5(6)-carboxy-
fluorescein acetoxymethyl ester (BCECF) was purchased fromMolecular Probes, Inc. (Eugene, OR) and dissolved
immediately before use in DMSO (1 mg/ml). A working
solution of 2 ~g/ml was prepared in Dulbecco's
phosphate-buffered saline (PBS) (Gibco).
4',6-diamidino-2-phenylindole (DAPI) was purchased from
Sigma Chemical Co. (St. Louis, MO). 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
-20C. 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 -20C.
Immediately before use, XTT was dissolved at 37C, and PMS
was added to yield a final concentration of 20 ~M.
Protocol ~or Definitive Anti-HIV ~valuations. The
appLupriate amounts of the pure compounds for anti-HIV
evaluations were dissolved in 100~ dimethylsulfoxide (DMSO)
and then diluted in complete medium to the desired initial
concentration (with final DMSO content not exc~e~i~g 1%).
All serial dilutions of the michellamines A, B, and C,
reagent additions, and plate-to-plate transfers were
carried out using an automated Biomek 1000 Workstation
(Beckman Instruments, Palo Alto, CA). Each compound was
diluted initially in complete medium and added to a single
column of a 96-well microtiter plate (dilution plate). The
Biomek was used to perform eight serial dilutions of each
drug and to transfer a 100 ~1 aliquot of each dilution to
the test plate. Uninfected CEM-SS or MT-2 cells were
plated at a density of 1 x 104 cells in 50 ~1 of complete
medium. Diluted HIV-1 or HIV-2 virus was then added to
appropriate wells in a volume of 50 ~1 to yield a
WO941~108 -2 ~ ~ O ~ 6 9 PCT~S93/036~2
39
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. Cells that
received test compounds were included in quadruplicate
virus-infected wells and duplicate uninfected wells.
Plates were incubated at 37C in an atmosphere containing
5% C02 for 6 days. Subsequently, aliquots of cell-free
supernatant were removed from each well using the Biomek,
and analyzed for reverse transcriptase activity, p24
antigen production, and synthesis of infectious virions
(see further below). A 25 ~l sample of .002% (w/v)
Fluoricon reference particles (590/620 nm) (Baxter
Healthcare Corp.) was added to each well of the test plate
to be used as an internal stAndArd for fluorescence assays.
The 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 37C. After incubation
the plates were covered with adhesive plate sealers
(Dynatech, AleyAn~ria~ VA) and shaken, and optical
densities were determined using a V-max photometer
(Mol~cul~r Devices, Inc., Menlo Park, CA) at a test
wavelength of 450 nm.
BCECF Assav. 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 37C for 30 min. Subse~uently, 25 ~l of a 2~
solution of paraformaldehyde was added to each well and
WO94/~108 216 0 8 6 9 PCT~S93103682 ~
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.). The plate was placed in the
Screen Machine programmed to execute the following
protocol: (l) add 20 ~l of 0.25% w/v suspension of 3.2 ~m
polystyrene beads (Baxter Healthcare Corp.) in PBS as a
filtration support matrix; (2) filter away the liquid phase
using a vacuum pressure of 15 mm Hg for l~ min; (3) wash
the cell-bead cake in each well with PBS using a vacuum
pressure of 20 mm Hg for l min; and (4) read fluorescence
of each well (signal channel = excitation at 485 nm,
emission at 535 nm, and reference channel 5 excitation at
590 nm, emission at 620 nm).
DAPI Assay. Total DNA content of each well was
determined by the following modifications to the method
described in McCaffrey et al., Ia_Vitro Ce~ evelo~.
Biol., 24, 247-252 (1988). The contents of each well were
fixed by ~ 25 ~l of a 2% paraformaldehyde æolution and
i~cl~h~ting the plate at 37C 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 o~ 400 nm and an emission of 450 nm.
D24 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 l:lO0 in lO~ Triton X-lO0 and stored
frozen at -20C 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 37C for l hr. Plate washings were carried
out using an automated Denley Wellwash 4 (Coulter
~ WO 94/~108 ' ' t ~ 6 0 8 6 9 PCT~S93/03682
41
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 37C. After additional
washing, 200 ~l of a streptavidin-horseradish peroxidase
solution was added, and the plates were then incubated for
30 min at 37C. A tetramethylbenzene solution was added to
each well and incubated at room temperature for 30 min.
Following incubation, an acidic stopping reagent was added
to each well, and the absorbance was read at 450 nm within
30 min using a Vmax photometer (Molecular Devices). The
concentration of p24 was determined by comparison with a
st~n~rd curve of known p24 concentrations.
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. A 10 ~l sample of lysed
virus was added to 30 ~l of a cocktail cont~ining 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)l0 (2
units/ml) (Pharmacia, Piscataway, NJ), 6.5 ~l of distilled
H20, 0.5 ~l of 10% Triton X-100, and 10 ~l of [3H~dTTP
(16.56 Ci/mmol) (Amersham Corp., Arlington Heights, IL).
Samples were inc~lh~ted for 30 min at 37C, harvested onto
DE81 ion ~ch~nge paper, and allowed to absorb for 15 min.
Sample pads first were rinsed six times with 5~ Na2HP04,
then twice with distilled H20. Pads were dried and counted
in a liquid scintillation counter. Samples were counted in
triplicate.
Linearity of AssaY End~oint to Cell Number.
Exponentially-growing CEM-SS cells were harvested, washed
WO94/~108 A '' 2~ ~ ~ 8 ~ ~ PCT~S93/03682 ~
42
and plated in 96-well microtiter wells at varying cell
concentrations. Following the cell inoculation, the cells
were treated with either XTT, BCECF, or DAPI according to
the above protocols. The fluorescence assays, using BCECF
and DAPI, showed excellent linearity over a wide range of
cell concentrations. Reproducible results could be
obtained from both assays with cell numbers below lO00
cells/assay. The colorimetric XTT assay also showed good
linearity but with a higher detection limit of 5,000-lO,000
cells/assay.
Antiviral ActivitY of the Michellamines. 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,
lS 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 to) or
infected (-) with the HIV-l virus, remaining in the culture
wells 6 days after in~Lo~uction 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.
There was little or no direct cytotoxicity of the
michellamines to the target cells with drug concentrations
below approximately lO0 ~M. The results of the DNA assay
(Figs. 2B, 3B, 4B, and 5B) were consistent with the
viability assays, i.e., the results provided further
indication of the antiviral activity of the michellamines.
The DNA measuremen~s, as expected, paralleled the cell
numbers present.
~ W094/~108 2 ~ 6 0 8 6 9 rCT~S93/03682
43
Figures 2D, 3D, 4D, and 5D 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. The results are
represented as the percent of the appropriate HIV-infected,
non-drug treated controls. At michellamine concentrations
within the same range giving essentially complete
cytoprotection (see above), there was a dramatic,
essentially complete inhibition of p24 viral core antigen
production (-) and viral reverse transcriptase activity
(-), which are indicators of viral replication; there was
a similarly complete inhibition of SFU, further indicating
a loss of infectious virus.
In another study, the acetate salts of michellamines
A, B, and C were evaluated in a side-by-side comparison for
anti-HIV-1 activity using the XTT assay. The results are
summarized in Table 4. All three c~,u~ounds exhibited
similar anti-HIV-l activity.
TABI.B ~. . RE8ULT8 OF COMPARI~ON 8T~DY OF ANTI--HIV--1
A~ vl-~ ~ OF MIr~T T-~INE8 A, B, and C
Michellamine EC50 (~M) IC50 (~M)
A 9.6 168
B 9.6 >240
C 12.8 193
~ a vitro cytoprotective effects such as the above are
known to predict for antiviral activity in humans. For
example, 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 (-),
wo 94/~108 2 1 ~ 0 8 ~ 9 pCT~S93l03682 ~
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. 6A) or its HBr salt
(Fig. 6B). The results are represented both in Figure 6
and in Figure 7 as the percent of the appropriate controls.
Both michellamines A and B, either as their free bases or
as their HBr salts, showed antiviral effects (Figures 6 and
7) against HIV-2. However, michellamine B consistently was
more potent than michellamine A against HIV-2. With
concentrations of michellamine B typically between 30-100
~M, essentially complete protection was obtained against
the killing effects of HIV-2 upon MT-2 cells. In contrast,
with concentrations of michellamine A as high as 250 ~M
there was only partial protection (20-40%) of the MT-2
cells against HIV-2.
A side-by-side comparative analysis was performed of
the anti-HIV-2 activities of the acetate salts of
michellamines A, B, and C using the CBL20 strain of HIV-2
with CEM-SS target cells and the XTT assay. Figures 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. In this study, michellamines B and C showed similar
potencies against HIV-2 (EC50 < 2 ~M), while michellamine A
appeared somewhat less potent (EC50 ~ 10 ~M).
As described above, the michellamines inhibit at least
two types of HIV retrovirus. As one skilled in the art
will appreciate, the michellamines and compositions thereof
will likely inhibit other retroviruses and other pathogenic
viruses.
WO94/~l08 ~ i21- 6 0 8 6 ~ PCT~S93/03682
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. When patients infected with HIV-1 and/or
HIV-2 are being treated, at least one compound of the
present invention can be co-administered with AZT.
The following methods and excipients are merely
exemplary and are in no way limiting.
In pharmaceutical dosage forms, 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 a~Iopriate association, as well as in
combination, with other pharmaceutically active compounds.
In the case of oral preparations, the compounds of the
present invention may be used alone or in combination with
a~Lo~liate 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.
WO94/~108 ~ 1 ~ 0 8 ~ 3 PCT~S93/03682 ~
46
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.
Furthermore, 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.
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.
The term "unit dosage form" as used herein refers to
physically discrete units suitable as unitary dosages for
human and animal subjects, each unit contAin;n~ a
predetermined quantity of at least one compound of the
present invention calculated in an amount sufficient to
~ W094/~lO~ 21 6 0 8 6 9 PCT~593/03682
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
S 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.
Exam~le 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. Alternatively, 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
Wog4/~108 ~ 6 ~ 8 6 9 PCT~S93/03682
48
for drug administration for each patient will vary
dep~inq 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.
Such dosage amounts can be readily ascertained without
undue burden and experimentation by those skilled in the
art.
As an example of an antiviral effective amount, the
dosage for humans can range from about between 0.01 mg/kg
body weight to 200 mg/kg body weight.
All of the references cited herein are hereby
incorporated in their entireties by reference.
While this invention has been described with an
emphasis upon preferred embodiments, it will be obvious to
those of ordinary skill in the art that variations of the
preferred products and methods may be used and that it is
int~n~e~ that the inve~tion may be practiced otherwise than
as specifically described herein. Accordingly, this
invention includes all modifications encompassed within the
spirit and scope of the invention as defined by the
following claims.
