U.S. patent application number 14/348750 was filed with the patent office on 2014-08-28 for method of isolating ingenol.
The applicant listed for this patent is INDENA S.P.A.. Invention is credited to Giovanni Appendino, Maria Luz Bellido Cabello De Alba, Eduardo Munoz Blanco, Alberto Pagani.
Application Number | 20140243551 14/348750 |
Document ID | / |
Family ID | 47080451 |
Filed Date | 2014-08-28 |
United States Patent
Application |
20140243551 |
Kind Code |
A1 |
Bellido Cabello De Alba; Maria Luz
; et al. |
August 28, 2014 |
METHOD OF ISOLATING INGENOL
Abstract
The present invention relates to a new method for isolating
ingenol (C.sub.20H.sub.28O.sub.5) from mixtures of diterpenoid
esters and ingenol esters in a single step. Ingenol isolated by
means of this method can be used as a precursor for the synthesis
of biologically active ingenol derivatives, such as
ingenol-3-angelate and ingenol-3-tigliate.
Inventors: |
Bellido Cabello De Alba; Maria
Luz; (Cordoba, ES) ; Appendino; Giovanni;
(Cordoba, ES) ; Pagani; Alberto; (Cordoba, ES)
; Munoz Blanco; Eduardo; (Cordoba, ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INDENA S.P.A. |
Milano |
|
IT |
|
|
Family ID: |
47080451 |
Appl. No.: |
14/348750 |
Filed: |
October 2, 2012 |
PCT Filed: |
October 2, 2012 |
PCT NO: |
PCT/EP2012/069452 |
371 Date: |
March 31, 2014 |
Current U.S.
Class: |
560/218 |
Current CPC
Class: |
C07C 45/65 20130101;
C07C 45/65 20130101; C07C 45/80 20130101; C07C 45/78 20130101; C07C
49/727 20130101; C07C 49/727 20130101; C07D 319/08 20130101; C07C
49/727 20130101; C07C 67/08 20130101; C07C 67/60 20130101; C07C
45/80 20130101; B01D 15/08 20130101; C07C 45/78 20130101; C07C
45/85 20130101 |
Class at
Publication: |
560/218 |
International
Class: |
C07C 67/60 20060101
C07C067/60 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 4, 2011 |
ES |
P201131601 |
Claims
1. A method of isolating ingenol from a mixture of diterpenoid
esters and ingenol esters said method comprising: combining
diterpenoid esters and ingenol esters to obtain a mixture;
combining phases of hydrolyzing ingenol present in said mixture and
isolating ingenol from said mixture in a single step.
2. The method according to claim 1, wherein the mixture of
diterpenoid esters and ingenol esters is subjected to a single step
of combined treatment with an acidified water and organic solvent
solution.
3. The method according to claim 2, wherein the acidified water to
organic solvent ratio is 1:1.
4. The method according to claim 2, wherein the organic solvent is
THF.
5. The method according to claim 2, wherein the acidified water
contains inorganic salts.
6. The method according to claim 2 wherein the acidified water
contains NaCl.
7. The method according to claim 2 wherein the acidified water and
organic solvent solution consists of a combination of 2N
H.sub.2SO.sub.4 and H.sub.2O plus NaCl at 35% weight/volume at a
ratio of 1:1.
8. The method according to claim 1, further comprising an
additional phase of purifying isolated ingenol.
9. The method according to claim 8, wherein the phase of purifying
isolated ingenol is performed by means of gravity column
chromatography.
10. The method according to claim 8, wherein the purified isolated
ingenol is subjected to an additional process of partial chemical
synthesis to produce a derivative at position 3 selected from:
ingenol-3-tigliate, ingenol-3-angelate or mixtures thereof.
11. The method according to claim 10, wherein the additional
process of partial chemical synthesis for obtaining ingenol
derivatives at position 3 to which the purified isolated ingenol is
subjected has ingenol-5,20-acetonide as the common intermediate
compound.
12. A method of isolating ingenol from plant material comprising
the phases of: a) Subjecting the plant material to mechanical
stirring in a solution of sodium methylate in methanol. b)
Neutralizing the preceding reaction with a solution of a glacial
acetic acid or perchloric acid. c) Filtering or suctioning the
preceding solution to obtain a pellet. d) Washing the pellet
obtained in the previous phase with methanol. e) Concentrating the
pellet by means of vacuum techniques. f) Extracting from the pellet
the fraction containing ingenol esters with petroleum ether. g)
Isolating ingenol from the remaining compounds contained in the
fraction obtained in preceding phase f) by means of treating with
acidified water and THF. h) Optionally purifying the isolated
ingenol
13. The method according to claim 12, wherein the plant material
are Euphorbia lathyris seeds.
14. The method according to claim 12, wherein the concentration of
sodium methylate in methanol is 0.20 N and the time for which the
seeds are subjected to mechanical stirring is 4 hours.
15. The method according to claim 12, wherein the filtration or
suction of step c) is performed on celite.
16. The method according to claim 12 wherein the acidified water
solution preferably consists of a combination of 2N H.sub.2SO.sub.4
and H.sub.2O plus NaCl at 35% weight/volume at a ratio of 1:1.
17. The method according to claim 12 wherein the phase of purifying
ingenol is performed by means of gravity column chromatography.
18. The method according to claim 17, wherein a silica gel column
is used as stationary phase with a petroleum ether-ethyl acetate
mobile phase.
19. The method according to claim 12, wherein the purified isolated
ingenol is subjected to an additional process of partial chemical
synthesis to produce a derivative at position 3 selected from:
ingenol-3-tigliate, ingenol-3-angelate or mixtures thereof.
20. The method according to claim 19, wherein the additional
process of partial chemical synthesis for obtaining ingenol
derivatives at position 3 to which the purified isolated ingenol is
subjected has ingenol-5,20-acetonide as the common intermediate
compound.
Description
FIELD OF THE INVENTION
[0001] The present invention pertains the field of biology,
pharmacy and medicine. More specifically, the present invention
relates to a method of isolating ingenol from mixtures of
diterpenoid esters and ingenol esters, more particularly when said
mixtures come from plants, specifically from seeds of the Euphorbia
genus and, more specifically from seeds of the E. lathyris
species.
STATE OF THE ART
[0002] Ingenols are compounds which, even though they can be
obtained by chemical synthesis, they are also present fundamentally
in plants of the Euphorbiaceae family, especially in those
varieties with purgative activity [Evans F J and Kinghorn A D J.
Linn. Soc. Bot. (London) 1977; 74:23-35; Evans F J and Taylor S E.
In Progress in the Chemistry of Organic Natural Products; Herz W,
Grisebach H, Kirby G W, Eds.; Springer: New York, 1983; 44:1-99].
Specifically ingenol (C.sub.20H.sub.28O.sub.5) (CAS registry number
30220-46-3) (Formula 1) has been isolated from the seeds of plaints
of the Euphorbia lathyris L. species, commonly known as spurge.
Spurge seed oil is marketed in different countries worldwide and is
an important raw material for the chemical industry because it is
used in the composition of a number of products such as paints,
varnishes, cosmetics, lubricants, plastics, biodiesel, etc.
##STR00001##
[0003] Me: CH Throughout the Present Specification
[0004] Enormous interest in the ingenol compound and its
derivatives has been raised in recent years due to its important
biological activities. Certain ingenols and especially the
derivatives esterified at position 3', whether they are natural or
obtained by partial synthesis, present potent anticarcinogenic and
antiviral activity [Ogbourne S M et al. Cancer Res 2004; 64:2833-9;
Benhadji K A at al. Br J Cancer. 2008; 99(11):1808-15; Kedei N at
al. Cancer Res. 2004; 64(9):3243-55; Warrilow D at al., AIDS Res
Hum Retroviruses. 2006; 22(9):854-64; Fujiwara M at Antimicrob
Agents Chemother. 1996; 40(1):271-3]. Partial synthesis is
understood as the isolation of ingenol from plants containing it
and the substitution in its molecular structure by suitable
chemical reactions in each case of groups leading to the different
derivatives of interest. Said derivatives esterified at position 3'
which present biological activity and can be used in clinical
practice are preferably ingenol-3-tigliate (Formula II) and
ingenol-3-angelate (Formula III).
##STR00002##
[0005] Ingenol is present in the form of esters in mixtures with
diterpenoid esters, in plants and in plant extracts. However,
spurge seeds are the only commercial source for isolating ingenol
from which it is possible to obtain the biologically active ingenol
derivatives used in clinical practice by means of different
chemical methods. Said seeds have a complex matrix containing, in
addition to fats (40-47%) and proteins (15%) [Duke, J. D. Handbook
of Energy Crops http://www.hort.purdue.edu/newcrop)], a series of
diterpenoid esters known as L.sub.1-L.sub.9 Euphorbia Factors
comprising a small but relatively constant amount of ingenol
monoesters (L.sub.4, L.sub.5 and L.sub.6) [Adolf W and Hecker E Z.
Krebsforsch. 1975, 84, 325-344], the fraction containing ingenol
being minor compared to the fraction containing the other types of
diterpenoids. Ingenol esters are highly toxic, constituting a
highly irritating oil, and even though ingenol alone isolated in
free form is inoffensive, isolating ingenol from said esters is
technically complex and not free of risk for the health of the
operators involved in said isolation operations due to the
aforementioned toxicity of the ingenol ester fraction.
[0006] A method for isolating ingenol from E. lathyris seeds was
published in 1991 [Bagavathi R. et al., Naturforsch. 1991; 46b:
1425-1433]. Said method consisted of separating the fraction
containing ingenol esters by means of a series of extractions with
solvents, such as methanol, petroleum ether and chloroform,
followed by selective adsorption in silica gels. The fraction
containing ingenol esters was then subjected to hydrolysis and the
ingenol obtained was finally purified by means of column
chromatography techniques. Said method of isolating ingenol
required a great deal of time and work due to the large number of
steps or phases, its yield therefore being low and thereby
complicating the development of new semi-synthetic derivatives that
could potentially be used as drugs.
[0007] To solve the problem of the low efficiency and low yield of
the method previously described in the state of the art for
isolating ingenol, Appendino et al. [Appendino G et al., J Nat
Prod. 1999; 62(1):76-9] developed a shorter and less toxic protocol
for extracting and isolating ingenol because the time in contact
with the irritating oil extracted from E. lathyris seeds containing
ingenol esters and also containing macrocyclic diterpenoid
compounds was less than that previously described. In said method,
isolating ingenol from E. lathyris seed oil was carried out in
three basic steps. Briefly, (1) separating a crude diterpenoid
fraction, (2) hydrolyzing the L.sub.1-L.sub.9 Euphorbia factors,
and (3) separating ingenol from the polyols resulting from
hydrolyzing macrocyclic diterpenoid esters (latirol, epoxylatirol,
7-hydroxylatirol, isolatirol, jolkinol etc.). Both separations
involve purifying a small concentration of ingenol from a complex
mixture of natural compounds which have a similar polarity, which
translates into obtaining an ingenol which, due to the complexity
of the sample in winch it is included and due to said similarity in
the polarity of the compounds forming the aforementioned sample,
can have impurities as it is contaminated with other substances
present in the mentioned mixture, primarily non-hydrolyzed ingenol
esters or esters of other macrocyclic diterpenoids.
[0008] The technical problem solved by the present invention is a
method that is an alternative to those described in the state of
the art of isolating ingenol from mixtures of diterpenoid esters
and ingenol esters. These mixtures can have any origin, i.e., they
can be natural (from plants) or generated in a chemical synthesis
process. The invention preferably isolates ingenol from plants of
the Euphorbia genus, and more specifically from E. lathyris
seeds.
[0009] To solve the described problems existing in the state of the
art for isolating ingenol, the present invention describes a method
of isolating said compound from mixtures of diterpenoid esters and
ingenol esters, preferably produced when processing E. lathyris
seeds, with a higher yield than those known in the state of the art
by simplifying the number of steps necessary for said isolation,
reducing it to a single step (with an optional step of
purification) and obtaining an ingenol with fewer impurities
(purity >99%).
DESCRIPTION OF THE INVENTION
BRIEF DESCRIPTION OF THE INVENTION
[0010] The method described in the present invention combines
hydrolyzing ingenol present in the mixture of diterpenoid esters
and ingenol esters and separating said ingenol from said mixture in
a single step. To that end, said mixture of diterpenoid esters and
ingenol esters is treated with a solution of an organic solvent,
preferably tetrahydrofuran (THE), and acidified water, more
preferably acidified brine (water+salt). The method described in
the present invention prevents isolating toxic or irritating
intermediates, such as the ingenol ester fraction, simplifies the
process and times for purifying ingenol and furthermore, the
production yield of said compound is much greater than those known
until now in the state of the art. Ingenol with a lower
concentration of impurities is further obtained, which makes the
ingenol thus isolated more suitable for being the starting compound
in processes for obtaining derivatives thereof, with a higher yield
and purity that can be used as drugs.
[0011] Ingenol obtained by means of the method described in the
present invention can be used for obtaining esterified derivatives
of said ingenol which are biologically active, such as
ingenol-3-tigliate (Formula II) and ingenol-3-angelate (Formula ID)
for example. The method of obtaining said biologically active
derivatives includes an intermediate step of synthesizing the
compound ingenol-5,20-acetonide (Formula IV) and subsequently its
esterification (Diagram I), to yield the aforementioned
biologically active compounds according to techniques known in the
state of the art for said purpose.
[0012] Diagram 1. Method of obtaining biologically active ingenol
derivatives by means of methods known in the state of the art from
ingenol obtained according to the method described in the present
invention.
##STR00003##
[0013] The biologically active derivatives obtained from ingenol by
means of method described in the present invention presented purity
greater than 99%. For the particular case of obtaining
ingehol-3-angelate from ingenol obtained by means of the method
described in the present invention, the production yield thereof
significantly improves the yield of raw material per kg and is much
more efficient and industrially scalable than the methods described
in the state of the art for obtaining said product (Hohmann, et a
Planta Med. 2000, 66: 291-294). Therefore, the production yield of
ingenol-3-angelate from Euphorbia peplus known in the state of the
art is 1.1 mg per kg of plant, whereas with the method of the
present invention, by combining the extraction of ingenol from
Euphorbia Lathyris seed powder followed by a process for the
partial chemical synthesis of the ingenol-3-angelate derivative, up
to 190 mg per kg of Euphorbia Lathyris seed powder can be
obtained.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The object of the present invention relates to a method of
isolating ingenol from mixtures comprising diterpenoid esters
(including ingenol esters) combining the phases of hydrolyzing and
isolating/extracting said ingenol iii a single step and optionally
further comprising an additional phase of purifying isolated
ingenol. The terms isolating and extracting in relation to ingenol
must be interpreted as being equivalents throughout the present
invention.
[0015] To that end, the method of the invention subjects the
mixture of diterpenoid esters and ingenol esters to a single step
of combined treatment with an acidified water and organic solvent
solution, preferably the ratio of acidified water to organic
solvent is 1:1.
[0016] The preferred organic solvent is THF, and the acidified
water also preferably contains inorganic salts, more preferably,
the acidified water is an acidified brine containing NaCl. The
concentration of NaCl present in the acidified water or brine is to
saturation, i.e., approximately 35% weight/volume. For the purpose
of the present invention, the term acidic brine refers to acidified
water in which a specific concentration of inorganic salt or salts,
specifically NaCl, is present.
[0017] In a preferred embodiment, the acidified water and organic
solvent solution used in the method of the invention consists of a
combination of H.sub.2SO.sub.4 at a concentration of 2N and
H.sub.2O+NaCl at 35% weight/volume (w/v), at a ratio of 1:1.
[0018] Optionally, the method of the invention further comprises an
additional phase of purifying isolated ingenol. Said phase of
purifying ingenol is preferably performed by means of gravity
column chromatography.
[0019] In a preferred manner of carrying out the method of the
invention, the single step of hydrolyzing and isolating/extracting
ingenol is carried out in a process starting from plant material
and comprising the phases of:
[0020] a) Subjecting the plant material, preferably 100 g of ground
and homogenized seeds, to mechanical stirring for preferably 4
hours at room temperature in a methanol solution containing sodium
methylate, preferably at a concentration of 0.20 N.
[0021] b) Neutralizing the preceding reaction with a solution of a
glacial acetic acid or perchloric acid at a concentration of 0.03
M.
[0022] c) Filtering or suctioning the preceding solution preferably
through celite.
[0023] d) Washing the pellet obtained in the previous phase with
70% methanol.
[0024] e) Concentrating the pellet preferably in a rotavapor.
[0025] f) Extracting from the previous pellet the fraction
containing ingenol esters with petroleum ether.
[0026] g) Isolating ingenol from the remaining compounds comprised
in the fraction obtained in the preceding phase f) by means of
combined treatment with acidified water and THE in a single
step.
[0027] h) Optionally purifying the ingenol isolated in the
preceding phase.
[0028] In a preferred embodiment of the invention the plant
material is Euphorbia lathyris seeds.
[0029] The mechanical stirring is preferably carried out by means
of a rotor driven by an electric motor. In a preferred embodiment
of the invention, the seeds are subjected to this mechanical
stirring after having been previously ground, and more preferably
ground to seed powder.
[0030] In another preferred embodiment of the method of isolating
ingenol from a starting plant material used, the acidified water
solution preferably consists of a combination of H.sub.2SO.sub.4 at
a concentration of 2N and H.sub.2O+NaCl at 35% w/v at a ratio of
1:1.
[0031] In another preferred embodiment of the method of isolating
ingenol from a starting plant material used, the phase of purifying
ingenol is performed by means of gravity column chromatography,
preferably using a silica gel column as stationary phase with a
petroleum ether-ethyl acetate mobile phase.
[0032] As repeatedly mentioned, ingenol produced by means of the
method of the invention can be used as a precursor for obtaining
biologically active derivatives thereof, preferably for obtaining
ingenol-3-tigliate (Formula II) and ingenol-3-angelate (Formula
III), which can be used in clinical practice for the treatment of
different pathologies. Said derivatives as well as the ingenol
itself isolated according to the method of the invention, are
obtained with degrees of purity >99%.
[0033] To that end, in a preferred embodiment of the invention, the
optionally purified isolated ingenol, whether it is obtained from a
mixture of ingenol esters and diterpenoid esters of any origin or
said mixture is generated from a plant material source, preferably
E. lathyris seeds, said ingenol is subjected to an additional
process of partial chemical synthesis to produce a derivative at
position 3 selected front: ingenol-3-tigliate (Formula II),
ingenol-3-angelate (Formula III), or mixtures thereof. Preferably,
said additional process of partial chemical synthesis for obtaining
ingenol derivatives at position 3 to which the optionally purified
isolated ingenol is subjected has ingenol-5,20-acetonide as the
common intermediate compound (Formula IV).
[0034] The object of the examples described below is to illustrate
the invention without limiting the scope thereof.
EXAMPLE 1
Method of Isolating Ingenol (Formula I) from Euphorbia Lathyris L
(Spurge) Seeds
[0035] The Euphorbia lathyris L seed powder (1 kg) was mechanically
stirred in a solution of 2 l of sodium methylate in 0.20 N methanol
for 4 hours. After said time, the solution was neutralized with
glacial acetic acid (or perchloric acid) and filtered and/or
suctioned through a bed of celite. The pasta obtained of said
filtration was washed several times with methanol (100 ml every
time), and the filtered materials were concentrated by means of
vacuum techniques to a volume of 1.5 l and were subsequently
extracted with petroleum ether. The methanol phase obtained from
said extraction was evaporated with a rotary evaporator and the
resulting residue was subjected to solution of acidic brine (2N
H.sub.2SO.sub.4+brine 1:1, 0.5 l) and THF (0.2 l), at room
temperature. The upper phase obtained was evaporated at 80.degree.
C., and the residue was purified by gravity silica gel column
chromatography (150 ml) using a petroleum ether-EtOAc (ethyl
acetate) gradient until obtaining, 0.750 g of ingenol (Formula 1)
with a purity >99%.
EXAMPLE 2
Producing Ingenol-5,20-Acetonide (Formula IV)
[0036] Pyridinium p-toluenesulfonate (50 mg) was added to a
solution of ingenol (100 mg, 0.29 mmol) dissolved in acetone (5
ml). The solution was stirred at room temperature for 12 hours and
was then subjected to evaporation. The residue was purified by
gravity column chromatography until reaching 62 mg (55% yield) of
ingenol-5,20-acetonide (Formula IV). For physical and spectroscopic
data, see Bangavathi R. et al. On the Chemistry of Ingenol IV. Z.
Naturforsch. 1991, 46b, 1425-1433.
EXAMPLE 3
Producing Ingenol Tigliate (Formula II)
[0037] A solution of ingenol-5,20-acetonide (100 mg, 0.26 mmol),
tiglic acid (39 mg, 0.39 mmol, 1.5 mol. equiv.) and DMAP (48 mg,
0.39 mmol, 1.5 mol. equiv.) in toluene (4 ml), was stirred at room
temperature for 2 hours and was then filtered through a bed of
Celite and evaporated. The resulting material was filtered in
silica gel (about 5 g) and evaporated. The residue was purified by
means of silica gel gravity column chromatography (5 g, petroleum
ether:EtOAc 85:15) until obtaining over 80 mg of ingenol-3-tigliate
with a purity >99%). For physical and spectroscopic data, see
Hohmann J et al. Planta Medica 2000, 66, 291-294.
EXAMPLE 4
Producing Ingenol Tigliate (Formula II)
[0038] A solution of ingenol-5,20-acetonide (100 mg, 0.26 mmol),
angelic acid (39 mg, 0,39 mmol, 1.5 mol. equiv.) and DMAP (48 mg,
0.39 mmol, 1.5 mol. equiv.) in toluene (4 ml), was stirred at room
temperature for 2 hours and was then filtered through a bed of
celite and evaporated. The resulting material was filtered in
silica gel (ca. 5 g) and evaporated. The residue was purified by
means of silica gel gravity column chromatography (5 g, petroleum
ether:EtOAc 85:15) until obtaining over 80 mg of ingenol-3-angelate
with a purity >99%. For physical and spectroscopic data, see
Hohmann J et al. Planta Medica 2000, 66, 291-294.
LITERATURE
[0039] Adolf W and Hecker E Z. Krebsforsch. 1975, 84, 325-344.
[0040] Appendino O et al., An expeditious procedure for the
isolation of Ingenol from the Euphorbia lathyris seeds. J Nat Prod.
1999 January; 62(1):76-9, [0041] Bangavathi R. et al. On the
Chemistry of Ingenol IV. Z. Naturforsch. 1991, 46b, 14251433.
[0042] Benhadji K A et al. Antiproliferative activity of PEP005, a
novel ingenol angelate that modulates PKC functions, alone and in
combination with cytotoxic agents in human colon cancer cells. Br J
Cancer. 2008 Dec. 2; 99(11):1808-15. [0043] Duke, J. D. Handbook of
Energy Crops (Available on the Internet at
http://www.hort.purdue.edu/newcrop). [0044] Evans F J and Kinghorn
A D J. Linn. Soco Bot. (London) 1977, 74, 23-35. [0045] Evans, F.
J. and Taylor, S. E. in Progress in the Chemistry of Organic
Natural Products, Vol. 44, ed. W. Herz, H. Grisebach and G. W.
Kirby. Springer, Vienna, 1983, p. I. [0046] Fujiwara M et al.,
Mechanism of selective inhibition of human immunodeficiency virus
by ingenol triacetate. Antimicrob Agents Chemother. 1996 January;
40(1):271-3. [0047] Herz W, Fujiwara M et al. Upregulation of HIV-1
replication in chronically infected cells by ingenol derivatives.
Arch Virol. 1998; 143(10):2003-10. [0048] Hohmann, J. et al.
Diterpenoids from Euphorbia peplus. Planta Med. 66 (2000); 291:
294, [0049] Kedei N et al. Characterization of the interaction of
ingenol-3-angelate with protein kinase C. Cancer Res. 2004;
64(9):3243-55. [0050] Le T I et al. Immunostimulatory cancer
chemotherapy using local ingenol-3-angelate and synergy with
immunotherapies. Vaccine. 2009 May 18; 27(23):3053-62. [0051]
Mainieri F et al. Synthesis of Sapintoxin D and
N-Methylanthranilate-based Fluorescent Bioprobes. Nat. Prod.
Commun. 2007, 2, 375-379, [0052] Ogbourne S M et al. Antitumor
activity of 3-ingenyl angelate: plasma membrane and mitochondrial
disruption and necrotic cell death. Cancer Res 2004; 64:2833-9.
[0053] Olsnes A M et al. The protein kinase C agonist PEP005
increases NF-kappaB expression, induces differentiation and
increases constitutive chemokine release by primary acute myeloid.
leukaemia cells. Br J Haematol. 2009 June; 145(6):761-74. [0054]
Warrilow D et al., HIV type I inhibition by protein kinase C
modulatory compounds. AIDS Res Hum Retroviruses. 2006 September;
22(9):854-64.
* * * * *
References