U.S. patent application number 10/872616 was filed with the patent office on 2004-11-18 for dihydroartemisinin and dihydroartemisitene dimers as anti-cancer and anti-infective agents.
Invention is credited to ElSohly, Mahmoud A., Galal, Ahmed M., Ross, Samir A..
Application Number | 20040229938 10/872616 |
Document ID | / |
Family ID | 32069214 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040229938 |
Kind Code |
A1 |
ElSohly, Mahmoud A. ; et
al. |
November 18, 2004 |
Dihydroartemisinin and dihydroartemisitene dimers as anti-cancer
and anti-infective agents
Abstract
This invention comprises compositions containing
dihydroartemisinin and dihydroartemisitene dimers with activity as
anticancer agents and anti-protozal, including anti-malarial and
anti-leishmanial properties. This invention also describes methods
of preparation of these compositions and methods of use of such
compositions for the treatment of cancer, and protozoal infections,
including malaria, or leishmaniasis. The compounds of this
invention represent a potential new class of anti-tumor agents, one
that has shown promising activity against solid tumors, and with a
pattern of selectivity that suggests a possible new mechanism of
action.
Inventors: |
ElSohly, Mahmoud A.;
(Oxford, MS) ; Ross, Samir A.; (Oxford, MS)
; Galal, Ahmed M.; (Nasr City, EG) |
Correspondence
Address: |
GREENBERG TRAURIG, LLP
885 3RD AVENUE
NEW YORK
NY
10022
US
|
Family ID: |
32069214 |
Appl. No.: |
10/872616 |
Filed: |
June 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10872616 |
Jun 21, 2004 |
|
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|
10271960 |
Oct 15, 2002 |
|
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6790863 |
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Current U.S.
Class: |
514/452 |
Current CPC
Class: |
C07D 519/00 20130101;
A61P 33/02 20180101; A61K 31/335 20130101; Y02A 50/409 20180101;
Y02A 50/411 20180101; Y02A 50/30 20180101; A61P 35/00 20180101 |
Class at
Publication: |
514/452 |
International
Class: |
A61K 031/335 |
Claims
1. A method of treating cancer comprising administering to a
subject suffering from cancer an effective amount of at least one
compound of the formula: 7where R is 8or a compound of the formula
9where R is selected from one of the substituents described above
or a simple (C.sub.2-C.sub.4) alkyl residue.
2. A method of treating a protozoal infection comprising
administering to a subject suffering from an infection an effective
amount of at least one compound of the formula: 10where R is 11or a
compound of the formula 12where R is selected from one of the
substituents described above or a simple (C.sub.2-C.sub.4) alkyl
residue.
3. A compound of the formula: 13where R is 14or a compound of the
formula 15where R is selected from one of the substituents
described above or a simple (C.sub.2-C.sub.4) alkyl residue.
4. A pharmaceutical composition comprising at least of one compound
according to claim 3 and pharmaceutically acceptable carrier and/or
excipient.
5. A method of preparing compounds of the formulas: 16where R is
17or a compound of the formula 18where R is selected from one of
the substituents described above or a simple (C.sub.2-C.sub.4)
alkyl residue; comprising reacting dihydroartemisin or
dihydroartemistene with an appropriate optionally substituted 1, 2
or 1, 3 or 1, 4 glycol under acidic conditions such as
borontrifluoride etherate followed by additional functionallization
of the resulting dimer as necessary.
6. The method of claim 5 where R is a glycerol residue comprising
reaction of dihydroartemisinin with glycerol in the presence of an
acid catalyst such as boron trifluoride etherate followed by
purification of the reaction mixture.
7. The method of claim 5 where R is a cyclohexane diol residue
comprising the reaction of dihydroartemisinin with cis- or
trans-cycloheane diols or a mixture thereof in the presence of an
acid catalyst such as boron trifluoride etherate followed by
purification of the reaction mixture and separation of the
appropriate isomer.
8. The method of claim 5 where R is a dihydroxy acetone residue in
the presence of an acid such as boron trifluoride etherate followed
by purification of the reaction mixture.
9. The method of claim 6 by sodium borohydride reduction of the
compound of claim 8 followed by purification of the reaction
mixture.
10. The method of preparing the hemisuccinate ester of the compound
of claim 6 by reacting the compound of claim 6 with succinic
anhydride in the presence of a base catalyst such as a mixture of
dimethylaminopyridine and triethylamine followed by the
purification of the reaction mixture.
11. The method of preparing compounds of claim 5 by reacting
dihydroartemisitene with the appropriate 1, 2 or 1, 3 or 1, 4
glycol in the presence of an acid catalyst such as borontrifluride
etherate followed by the purification of the reaction mixture.
12. The method of claim 11 where the 1, 2 glycol is ethylene
glycol.
13. The method of claim 11 where the 1, 2 glycol is 1, 2
propane-diol.
14. The method of claim 11 where the 1, 3 glycol is glycerol.
15. The method of claim 11 where the 1, 3 glycol is dihydroxy
acetone.
16. The method of claim 11 where the 1,4 glycol is 1,
4-butane-diol.
17. The method of preparing a compound of claim 11 where the 1, 4
glycol is selected from 1, 4-cis-cyclohexanediol, 1,
4-trans-cyclohexanediol or a mixture thereof, followed by the
purification of the reaction mixture and separation of the desired
product.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of U.S. Ser.
No. 10/271,960, filed on Oct. 15, 2002. The disclosure of that
application is expressly incorporated herein by reference.
FIELD OF INVENTION
[0002] The present invention relates to dihydroartemisinin and
dihydroartemisitene dimers and their use in the treatment of cancer
and as antiprotzoal agents.
BACKGROUND OF THE INVENTION
[0003] Cancer deaths in the U.S. alone were over 500,000 in 2001,
and in spite of many advances, cancer remains one of the leading
killers (1). There is a critical need for the development of new
anti-cancer agents, especially those with novel and selective
mechanisms of action. Although some of the promise of non-cytotoxic
therapies is beginning to be realized (e.g. immunostimulants,
growth factor antagonists, anti-sense therapy), the mainstay of the
treatment of most cancers remains with cytotoxic drugs. In view of
the limited success rates, incidence of toxicities, and development
of resistance to such agents, there is a dire need for new classes
of these drugs, especially those that may act by new mechanisms or
exhibit exploitable selectivity. There is also a need for a better
understanding of dosing, scheduling, and concomitant therapies in
order to optimize treatment protocols.
[0004] Natural products have historically been a rich source of
new, successful prototype classes of lead compounds from which
analogs have been developed. According to a recent review, 60% of
the anti-infective and anti-cancer drugs that have successfully
advanced to the clinic are derived from natural products (2).
Examples of these among currently used anti-cancer agents include
the anthracycline class (e.g., doxorubicin), the Catharanthus
(Vinca) alkaloids, paclitaxel, and derivatives of podophyllotoxin
and camptothecin. A recently published tabulation of natural
product-based anti-tumor drugs shows more than 25 agents currently
in Phase I or II (3). This and other recent reviews are important
reminders of the critical role of natural products as a resource
for the discovery of new anti-tumor agents (4,5).
[0005] The natural product artemisinin (1) is a sesquiterpene
endoperoxide first isolated in 1971 from the Chinese plant
Artemisia annua (6). The compounds as numbered herein are depicted
in FIG. 1. The compound was shown to have anti-malarial activity
against both chloroquine-sensitive and chloroquine-resistant
strains of Plasmodium falciparum.
[0006] Because of the importance of the clinical effects of
artemisinin in treating malaria, many derivatives were prepared in
order to develop the most effective and least toxic anti-malarial
agent. Initially, simple derivatives were prepared--e.g.,
dihydroartemisinin (DHA, in which the lactone carbonyl is reduced
resulting in a hemiacetal), artemether (the methyl ether of DHA)
and several other ether and ester analogs. The sodium salt of the
hemisuccinate ester (sodium artesunate) was one of these
derivatives that showed more activity and less toxicity than
artemether, the latter being more active than artemisinin itself.
Continued interest in the activity of artemisinin and DHA analogs
later resulted in the preparation of artemisinin acetal dimers
through reaction of dihydroartemisinin with
borontrifluoride-etherate.
[0007] In addition to its anti-malarial activity, artemisinin had
been reported to have cytotoxic effects against EN-2 tumor cells
(7), P-388, A549, HT-29, MCF-7, and KB-tumor cells (8). As more
analogs were evaluated for anti-tumor activity, it was reported
that the unsymmetrical dimer (2) showed strong cytotoxic activity
and was more potent than cisplatin (9). The symmetrical dimer (3)
also showed pronounced cytotoxic activity (10).
[0008] This finding stimulated interest in other types of DHA
dimers. Posner et al. (11) prepared dimers linked with a
polyethylene glycol spacer (3 units of ethylene glycol), an eight
carbon glycol, and a dithio-derivative. The authors also prepared
simpler trioxane dimers. Posner et al. also prepared several dimers
of DHA where the linking units between the two molecules of
dihydroartemisinin were dicarboxylic acids of different types (12).
Zhang and Darbie (13,14) also proposed several dihydroartemisinin
dimers to be linked via different coupling agents. Some of these
artemisinin dimers and some of the simpler trioxanes had
anti-malarial effects, anti-cancer activity, and anti-proliferative
effects with some compounds being as active as calcitriol in an
anti-proliferative assay in murine keratinocytes.
SUMMARY OF THE INVENTION
[0009] This invention comprises compositions containing
dihydroartemisinin and dihydroartemisitene dimers with activity as
anticancer agents and anti-protozal, including anti-malarial and
anti-leishmanial properties. This invention also describes methods
of preparation of these compositions and methods of use of such
compositions for the treatment of cancer, and protozoal infections,
including malaria, or leishmaniasis. The compositions of this
invention have not been previously described.
[0010] The compounds of this invention represent a potential new
class of anti-tumor agents, one that has shown promising activity
against solid tumors, and with a pattern of selectivity that
suggests a possible new mechanism of action.
DESCRIPTION OF THE INVENTION
[0011] In the interest of development of new chemotherapeutic
agents, artemisinin dimers were prepared in this invention by
condensation of DHA with a variety of vicinal and non-vicinal
glycols. These dimers have been evaluated in the NCI anti-tumor
screening program, and all passed to the 60-cell line screen (4-9).
Several of these compounds have been advanced into further testing
by the NCI into the Hollow Fiber Assay (HFA) protocol (Compounds 4,
5, and 6). Additional supplies of Compounds 5 and 6 have been
recently prepared and provided to the NCI for testing in xenograft
tumor models.
[0012] The present invention relates to a method of treating cancer
comprising administering to a subject suffering from cancer an
effective amount of at least one compound of the formula: 1
[0013] where R is 2
[0014] or a compound of the formula 3
[0015] where R is selected from one of the substituents described
above or a simple (C.sub.2-C.sub.4) alkyl residue.
[0016] Furthermore the invention encompasses a method of treating a
protozoal infection comprising administering to a subject suffering
from an infection an effective amount of at least one compound of
the formulas given hereinabove.
[0017] Compounds within the scope of the invention are compounds of
the formula: 4
[0018] where R is 5
[0019] or a compound of the formula 6
[0020] where R is selected from one of the substituents described
above or a simple (C.sub.2-C.sub.4) alkyl residue.
[0021] A pharmaceutical composition can be prepared which comprise
at least of one compound of this invention and pharmaceutically
acceptable carrier and/or excipient.
[0022] Compounds of the invention can be prepared by reacting
dihydroartemisin or dihydroartemistene with an appropriate
optionally substituted 1, 2 or 1, 3 or 1, 4 glycol under acidic
conditions such as borontrifluoride etherate followed by additional
functionallization of the resulting dimer as necessary.
[0023] In the case where R is a glycerol residue the reaction
comprises reaction of dihydroartemisinin with glycerol in the
presence of an acid catalyst such as boron trifluoride etherate
followed by purification of the reaction mixture.
[0024] Where R is a cyclohexane diol residue the reaction can
conprise of dihydroartemisinin with cis- or trans-cycloheane diols
or a mixture thereof in the presence of an acid catalyst such as
boron trifluoride etherate followed by purification of the reaction
mixture and separation of the appropriate isomer.
[0025] Where R is a dihydroxy acetone residue the reaction can be
carried out in the presence of an acid such as boron trifluoride
etherate followed by purification of the reaction mixture followed
by sodium borohydride reduction and purification of the reaction
mixture.
[0026] The hemisuccinate esters can be prepared by reacting the
appropriate precursor with succinic anhydride in the presence of a
base catalyst such as a mixture of dimethylaminopyridine and
triethylamine followed by the purification of the reaction
mixture.
[0027] Dihydroartemisitene dimers can be prepared with the
appropriate 1, 2 or 1, 3 or 1, 4 glycol in the presence of an acid
catalyst such as borontrifluride etherate followed by the
purification of the reaction mixture.
[0028] Illustrative glycols include, for example, ethylene glycol,
1, 2 propane-diol, glycerol, dihydroxy acetone, or 1,
4-butane-diol, 1, 4-cis-cyclohexanediol, 1, 4-trans-cyclohexanediol
or a mixture thereof.
[0029] Although the mechanism of action of these DHA dimers remains
to be determined, some clues regarding possible molecular targets
are suggested. Use of the NCI COMPARE analysis revealed that the
cell sensitivity profile of these compounds in the 60-cell line
assay was similar to platinum compounds. These compounds inhibit
cell replication by forming DNA intrastrand cross-links.
Correlations on micro-array data for the 60 cell lines also
indicate that cells most sensitive to these dimers contain lower
levels of the mRNAs encoding proteins involved in integrin and
hypoxia signaling. Lower levels of expression of these proteins may
result in enhanced sensitivity either because these proteins are
direct targets, or because their reduced expression reflects a
condition within the cell (e.g., redox potential) that augments
sensitivity.
[0030] Administration of the instant dimers may be by any of the
conventional routes of administration, for example, oral,
subcutaneous, intraperitoneal, intramuscular, intravenous or
rectally. In the preferred embodiment, the compound is administered
in combination with a pharmaceutically-acceptable carrier which may
be solid or liquid, dependent upon choice and route of
administration. Examples of acceptable carriers include, but are
not limited to, starch, dextrose, sucrose, lactose, gelatin, agar,
stearic acid, magnesium stearate, acacia, and similar carriers.
Examples of liquids include saline, water, edible oils, e.g. peanut
and corn.
[0031] When administered in solid form, the compound and diluent
carrier may be in the form of tablets, capsules, powders, lozenges,
suppositories prepared by any of the well known methods. When given
as a liquid preparation, the mixture of active compound and liquid
diluent carrier may be in the form of a suspension administered as
such. The compound is administered in a non-toxic dosage
concentration sufficient to inhibit the growth and/or destroy
cancer or to destroy protozoal organisms such as malaria and
leishmania. The actual dosage unit will be determined by the well
recognized factors as body weight of the patient and/or severity
and type of pathological condition the patient might be suffering
with. With these considerations in mind, the dosage unit for a
particular patient can be readily determined by the medical
practitioner in accordance with the techniques known in the medical
arts.
[0032] The compounds of this invention have been prepared by
reaction of dihydroartemisinin or dihydroartemistene with a variety
of optionally substituted 1,2-, 1-3- or 1,4 glycols under acidic
conditions (borontrifluoride etherate) in dry ether followed by
chromatography of the reaction mixture to isolate the desired
product. Optional substitutients include, for example, alkoxy or
acyloxy groups. The dimers of the present invention can also be
prepared by the reaction of dihydroxy ketones such as, for example,
dihydroxyacetone, with DHA or dihydroartemistene followed by
reduction of the keto-group and reaction of the hydroxy group
formed in the reduction of the ketone with hydroxy reactive
compounds such as mono or dicarboxylic acids as their acid halides
and acid anhydrides. The starting material (dihydroartemisinin) is
prepared by sodium borohydrite reduction of the natural product
artemisinin (1). The latter compound is isolated from the leaves of
Artemisia annua following the procedures previously described (15,
16). The compounds of the invention were tested in the NCI
anti-tumor screen and in the anti-malarial and anti-Leishmanial
screens. The activities are shown in Tables 1-8 as shown in FIGS.
2A, 2B, 3A, 3B, 4A, 4B and FIGS. 5 to 9.
EXAMPLES
[0033] Reactions were run in oven dried round-bottomed flasks.
Diethyl ether (ether) was distilled from sodium benzophenone ketyl
prior to use under an atmosphere of argon. All chemicals were
purchased from Sigma-Aldrich and used without further purification,
except the diols, which were dried over grade I alumina prior to
use. Artemisinin (1) was isolated from locally grown Artemisia
annua L. plants, using a literature procedure (15,16), and was
reduced to dihydroartemisinin as previously reported (17).
[0034] Column chromatography was performed using flash
chromatography, using silica gel purchased from Merck (particle
size 230-400 mesh). Analytical thin-layer chromatography (TLC) was
performed with silica gel 60 F.sub.254 plates (250 .mu.m thickness;
Merck), using n-hexane-EtOAc mixtures as solvent systems.
Visualization was accomplished by spraying with p-anisaldehyde
spray reagent followed by heating using a hot-air gun (18).
[0035] Mp's were recorded on an Electrothermal 9100 instrument. IR
spectra were obtained using AATI Mattson Genesis Series FTIR.
Optical rotations were recorded at ambient temperature using JASCO,
DIP-370, digital polarimeter. 1D and 2D NMR spectra were obtained
on Bruker Avance DRX 500 spectrometers at 500 MHz (.sup.1H) and 125
MHz (.sup.13C) or Bruker DRX 400 spectrometer using the solvent
peak as the internal standard. HRESIFTMS were obtained using a
Bruker Bioapex FT-MS in ESI mode. Low resolution MS were measured
on a ThermoQuest aQa LC/MS.
[0036] Preparation of the 1,3-.beta.,.beta.-Dihyroartemisinin Dimer
with Glycerol (Compound 4)
Example 1
[0037] To a stirred solution of dihydroartemisinin (160 mg, 0.56
mmol) in a round bottomed flask (50 mL) in dry ether (10 mL), was
added dry glycerol (26 mg) and BF.sub.3.OEt.sub.2 (267 .mu.L) using
a hypodermic syringe. The mixture was stirred under argon for 70
min. then quenched and worked up as usual to leave a gummy residue
(199 mg). Upon crystallization from ether, it yielded 4 (52 mg) as
cubic crystals, 28.7%); [.alpha.].sub.D+173.degree. (c 0.022,
CHCl.sub.3); IR (film) .nu..sub.max: 352 (OH), 2953, 2933, 2881,
1449, 1376, 1194, 1176, 1134, 1107, 1027, 991 cm.sup.-1; .sup.1H
NMR (CDCl.sub.3, 500 MHz, for one of the two identical monomeric
units): .delta. 5.40 (1H, s, H-5,), 4.79 (1H, d, J J=3.9, H-12),
4.78 (1H, d, J=3.5 Hz, H-12'), 3.87 (1H, m, H-16, H-16', H-17),
3.49 (1H, dd, J=5.9, 4.3 Hz, H-18), 3.42 (1H, q, J=5.4 Hz, H-18'),
2.63 (1H, m, H-11), 2.34 (1H, ddd, J=14.0, 4.0, 3.9 Hz, H-3), 2.00
(1H, m, H-3'), 1.85 (1H, m, H-2), 1.68 (3H, m, H-2', H-8, H-9),
1.46 (2H, m, H-7, H-8'), 1.39 (3H, s, Me-15), 1.34 (1H, m, H-10),
1.21 (1H, m, H-1), 0.92 (3H, d, J=6.4 Hz, Me-14), 0.90 (3H, d,
J=7.4 Hz, Me-13), 0.89 (3H, d, J=7.3 Hz, H-13'), 0.87 (1H, m,
H-9'); .sup.13C NMR (CDCl.sub.3, 125 MHz): .delta. 104.5 (s, C-4),
103.13 (d, C-12), 103.06 (s, C-12'), 88.3 (d, C-5), 81.4 (s, C-6),
70.3 (t, C-16), 70.1 (t, C-18), 70.0 (d, C-17), 52.9 (d, C-1), 44.7
(d, C-7, C-7'), 37.7 (d, C-10), 36.8 (t, C-3), 35.0 (t, C-9), 31.27
(d, C-11), 31.25 (d, C-11'), 26.5 (q, C-15), 25.0 (t, C-2), 25.0
(t, C-8), 20.7 (q, C-14), 13.4 (q, C-13); HRESIFTMS [m/z] 625.351
[M+H].sup.+ (calcd for C.sub.33H.sub.53O.sub.11, 625.3582).
[0038] Preferred Procedure for Preparation of Compound 4.
Example 2
[0039] The preferred method of preparing Compound 4 was to first
prepare the ketone precursor through condensation of
dihydroxyacetone with dihydroartemisinin in the presence of boron
trifluoride-etherate followed by sodium borohydride reduction of
the resulting ketone to give Compound 4. This is detailed in the
following examples.
[0040] Preparation of the .beta.,.beta.-Dihydroartemisinin Dimer
with Dihydroxyacetone (Compound 7)
Example 3
[0041] Dihydroartemisinin (284 mg, 1 mmol) and 1,3-dihydroxyacetone
dimer (45.05 mg, 0.25 mmol) were suspended in diethylether (10 mL).
To the mixture (cooled to 5.degree. C. under argon) was then added
35.5 mg BF.sub.3.Et.sub.2O (0.25 mmol, 31 .mu.L) and the mixture
stirred at 5.degree. C. for 20 minutes then at room temperature for
1 hr. Workup as usual gave 319 mg of residue.
[0042] The residue was chromatographed on silica gel column (30 g)
and eluted with hexane:EtOAC (95:5) with polarity increasing to
80:20. Fractions were collected and pooled according to TLC
similarities to give four major fractions. The most polar fraction
(140.2 mg) was identified as Compound 7 (converts to Compound 4
upon NaBH.sub.4 reduction):
[0043] .sup.1H-NMR in CDCl.sub.3 at 500 MHz: .delta. 5.44 (2H, s,
H-5 & H-5'), 4.805 (2H, d, J=3.39 Hz, H-12 & H-12'), two
broad doublets (2H each J=17.59) centered at .delta. 4.46 &
4.285 (H-16 & H-18), 2.665 (2H, m, H-11 & H-11'), 2.355
(2H, ddd, H-3), 2.025 (2H, m, H-3'), 1.88 (2H, m, H-2), 1.81 (4H,
m, H-9 & H-9'), 1.675 (2H, m, H-8), 1.475 (4H, H-7, H-7', H-10
& H-10'), 1.41 (6H, s, Me-15 & Me-15'), 1.255 (2H, m, H-1
& H-1'), 0.99-0.95 (12H, Me-14, Me-14', Me-13 &
Me-13').
[0044] .sup.13C-NMR in CDCl.sub.3 at 124 MHz: .delta. 204.8 (s,
C=0), 104.53 (s, C-4), 102.87 (d, C-12), 88.51 (d, C-5), 81.36 (s,
C-6), 72.06 (t, C-16 & C-17), 52.89 (d, C-1), 44.67 (d, C-7),
37.75 (d, C-10), 36.77 (t, C-3), 34.99 (5, C-9), 31.13 (d, C-11),
26.47 (q, C-15), 25.03 (t, C-2), 24.74 (t, C-8), 20.69 (q, C-14),
13.43 (q, C-13).
Example 4
[0045] To a suspension of 2.84 g dihydroartemisinin (10 mmol) and
450 mg (2.4 mmol) of 1,3-dihydroxyacetone dimer in ether (100 mL)
was added 127 .mu.L of BF.sub.3.Et.sub.2O (142 mg, 1 mmol) at
5.degree. C. The mixture was stirred at room temperature for 30
minutes, then a second portion (127 .mu.L) of BF.sub.3.Et.sub.2O
was added. A third portion and a fourth portion (254 .mu.L) of
BF.sub.3.Et.sub.2O were added at 15-minute intervals making up a
total of 4 mmols. Stirring was continued for 1.5 hr. Workup as
usual provided an oily residue which was chromatographed in a
manner similar to that described under Example 3 and fractions were
combined based on their TLC similarities.
[0046] The fractions with R.sub.f values corresponding to the dimer
prepared in Example 3 were combined and the solvent evaporated to
produce 2.05 g of an oily residue which foamed in vacuum. This
material was identical to that prepared under Example 3 (Compound
7).
Example 5
[0047] To a suspension of 3.3 g dihydroartemisinin (11.6 mmols) and
522 mg, 2.9 mmols) of 1,3-dihydroxyacetone dimer (0.25 equivalent)
in ether (100 mL) was added 0.88 mL of BF.sub.3.Et.sub.2O (0.986 g,
6.9 mmol, 0.6 equivalent) at 5.degree. C. The mixture was then
stirred at room temperature for 3 hr, then worked up as usual to
provide an oily residue. The residue was chromatographed over
silica gel column (130 g) and eluted with mixtures of hexane-EtOAC
ranging from 95:5 to 85:15 to give several fractions which were
combined according to TLC similarities. Fractions containing the
desired product (identical to that prepared under Example 3) were
combined to give 1.628 g of Compound 7.
[0048] Preparation of the 1,3-.beta.,.beta.-Dihydroartemisinin
Dimer of Glycerol (Compound 4) Starting from Compound 7.
Example 6
[0049] The ketone intermediate (Compound 7), (1.94 g, 312 mmols)
was dissolved in 225 mL of a mixture of THF and water (2:1). The
solution was stirred and NBH.sub.4 (474 mg, 4 molar equivalent) was
then added in portions at room temperature over a 15 minute period.
The mixture was then neutralized with 2N HCl. The THF was then
evaporated under vacuum. The precipitate was filtered and washed
with water and air dried to give 1.8 g (92.5% yield) of Compound 4,
identical to that prepared under Example 1).
Example 7
[0050] The same procedure described under Example 6 was repeated
using 0.778 g of Compound 7 to yield 0.73 g of Compound 4.
[0051] Preparation of the .beta.,.beta.-Dihydroartemisinin Dimer
with Cyclohexanediol (Compounds 5 and 6).
Example 8
[0052] In a round-bottomed flask (100 mL) was introduced
dihydroartemisinin (850 mg, 3.0 mmol) and dry ether (25 mL) then
the mixture was stirred at room temperature with
cyclohexane-1,4-diol (mixture of cis and trans) (170 mg). To the
stirred solution, BF.sub.3.OEt.sub.2 (570 .mu.L) was then added
using a hypodermic syringe. The stirring was continued for 80 min.,
then the reaction was quenched and worked up as usual to leave a
gummy residue (1.13 g). The residue was loaded on Si gel column
(170 g) and eluted with increasing amounts of EtOAc in n-hexane
(15.fwdarw.50%). Fractions of 5 mL were collected and similar
fractions were pooled by guidance of TLC to afford Compound 5 (238
mg, oil). Earlier fractions were pooled and re-chromatographed on a
silica gel column to yield 6 (70 mg, white amorphous solid).
Compound 5; [.alpha.].sub.D+142.degree.(c 0.036, MeOH); IR (film)
.nu..sub.max: 2938, 2872, 1448, 1375, 1227, 1194, 1122, 1099, 1029
cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 500 MHz, for one of the
identical monomereric units): .delta. 5.42 (1H, s, H-5), 4.90 (1H,
d, J=3.3, H-12), 3.78 (1H, brs, H-16), 2.62 (1H, m, H-11), 2.36
(1H, ddd, J=14.0, 12.4, 3.8 Hz, H-3), 2.04 (1H, m, H-3'), 1.88 (2H,
m, H-2, H-8), 1.77 (2H, m, H-2', H-8'), 1.71 (2H, m, H-17a, H-17b),
1.65-1.56 (4H, m, H-9, H-9', H-17'a, H-17'b), 1.48 (1H, m, H-7),
1.43 (3H, s, Me-15), 1.32 (1H, m, H-10), 1.25 (1H, m, H-1), 0.95
(3H, d, J=6.2 Hz, Me-14), 0.90 (3H, d, J=7.2 Hz, Me-13); .sup.13C
NMR (CDCl.sub.3, 125 MHz): .delta. 104.4 (s, C-4), 100.0 (d, C-12),
88.4 (d, C-5), 81.5 (s, C-6), 72.7 (d, C-16), 53.0 (d, C-1), 44.9
(d, C-7), 37.9 (d, C-10), 36.9 (t, C-3), 35.2 (t, C-9), 31.2 (d,
C-11), 26.6 (q, C-15), 24.9 (t, C-2), 25.1 (t, C-8), 20.7 (q,
C-14), 13.5 (q, C-13); HRESIFTMS [m/z] 647.3445[M-1].sup.-(calcd
for C.sub.36H.sub.55O.sub.10, 647.3510).
[0053] Compound 6; R.sub.f 0.42 (n-hexane:EtOAc, 8:2),
[.alpha.].sub.D+114.degree. (c. 0.042, CHCl.sub.3); IR (film) no OH
absorption; .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 5.41 (1H, s,
H-5), 4.88(1H, d, J=3.3 Hz, H-12), 3.75 (1H, brs, H-16), 1.47 (1H,
m, H-7), 1.43 (3H, s, Me-15), 1.26 (2H, m, H-1, H-10), 0.95 (3H, d,
J=6.0 Hz, Me-14), 0.87 (3H, d, J=7.3 Hz, Me-13); .sup.13C NMR
(CDCl.sub.3, 100 MHz): .delta. 104.4 (s, C-4), 100.4 (d, C-12),
88.4 (d, C-5), 81.6 (s, C-6), 73.9 (t, C-16), 53.0 (d, C-1), 44.9
(m, C-7), 37.9 (d, C-10), 36.9 (t, C-3), 35.1 (t, C-9), 31.2 (d,
C-11), 30.4 (t, C-17'), 27.5 (t, C-17), 26.6 (q, C-15), 24.9 (t,
C-2), 25.1 (t, C-8), 20.8 (q, C-14), 13.5 (q, C-13); HRESIFTMS
[m/z] 671.3772 [M+Na].sup.+(calcd for C.sub.36H.sub.56O.sub.10Na,
671.3765).
Example 9
[0054] A mixture of dihydroartemisinin (372 mg 1.31 mmol) and
1,4-cyclohexanediol (cis and trans mixture) (74.4 mg, 0.64 mmol)
were suspended in 10 mL dry ether and 0.25 mL of BF.sub.3.Et.sub.2O
(280 mg, 1.97 mmol) was added at 0.degree. C. The mixture was then
stirred at room temperature for 80 minutes followed by workup by
shaking with a sodium bicarbonate solution and separation of the
ether layer. The aqueous layer was washed with ether (2.times.10
mL). The ether layers were combined and washed with water and brine
and the ether layer was dried over anhydrous sodium sulfate.
Evaporation of the ether resulted in an oily residue (429 mg) which
was chromatographed on silica gel column (30 g) and eluted with
hexane ether mixtures ranging from 97:3 to 80:20 and fractions were
pooled together according to their TLC similarities. Compound 6 (56
mg) was isolated as white solid and Compound 5 (24 mg) was isolated
as an oil which foamed under vacuum. These were found to be
identical to Compounds 5 and 6 prepared under Example 8.
Example 10
[0055] A mixture of dihydroartemisinin (744 mg, 2.62 mmol) and
1,4-cyclohexanediol (149 mg, 1.28 mmol) of the cis and trans
mixture were stirred in 20 mL dry ether at 5.degree. C. To the
mixture was added BF.sub.3.Et.sub.2O (83 .mu.L, 0.655 mmol) and
stirring was continued at room temperature for 1 hr. A second
portion of BF.sub.3.Et.sub.2O (83 .mu.L) was added, and the mixture
continued to stir for 1 hr. The reaction mixture was then worked up
as usual to give 890 mg of an oily residue. Column chromatography
of the residue using silica gel (32 g) and elution with
hexane:ether 92:2, 96:4, 94:6, and then 90:10 (200 mL each) yielded
several fractions pooled according to TLC similarities. Compound 6
was isolated as cubic crystals (238 mg, melting point
146-148.degree. C. Compound 5 was isolated from later fractions as
an amorphous foam (184 mg, melting point 93-97.degree. C.). These
were found to be identical to those previously prepared under
Examples 8 and 9.
Example 11
[0056] A mixture of dihydroartemisinin (10 g, 35.2 mmol) and
1,4-cyclohexanediol (cis and trans mixture) (2 g, 17.2 mmol) were
suspended in 260 mL dry ether and 1.11 mL of BF.sub.3.Et.sub.2O was
added at 0.degree. C. under argon. Two additional portions of
BF.sub.3.Et.sub.2O (1.11 mL each) were added after 1 hr intervals.
The mixture was then stirred at room temperature for 1 hr after the
last addition of BF.sub.3.Et.sub.2O followed by workup as usual to
give 12 g of an amorphous residue which was chromatographed on
silica gel column to produce 2.69 g of Compound 5 and 2.78 g of
Compound 6. These were found to be identical to Compounds 5 and 6
prepared under Examples 8-10.
[0057] It must be mentioned that should only Compound 5 or only
Compound 6 be desired that only the cis or trans
1,4-Cyclohexanediol be used as the starting material to produce the
desired product.
[0058] Preparation of the Hemisuccinate Ester of Compound 4
(Compound 8)
Example 12
[0059] To a stirred solution of Compound 4 (200 mg, 0.32 mmol) in
dry methylene chloride (4 mL) were added triethylamine (0.14 mL,
1.3 equiv), dimethylaminopyridine (16 mg, 0.4 equiv) and succinic
anhydride (92 mg, 3 equiv). The resulting solution was slowly
stirred at room temperature for 16 hr. Following evaporation of the
solvent under reduced pressure, the residue was purified over a
silica gel column using hexane:acetone (6:4) as the eluent. The
product of the reaction was isolated as white amorphous solid (156
mg) with R.sub.f value of 0.68 (hexane:acetone, 1:1) identified as
the hemisuccinate ester of Compound 4 (Compound 8), based on
spectral data (see details under Example 13).
Example 13
[0060] The reaction of Example 12 was repeated on a larger scale
(starting with 550 mg of Compound 4) where all reactants were
scaled up proportionally. Purification of the reaction product in
the same manner produced 355 mg of Compound 8 as amorphous white
powder with the following spectral characteristics:
[0061] .sup.1H-NMR (acetone-d.sub.6, 500 MHz): .delta. 5.442 (1H,
s, H-5), 5.396 (IH, s, H-5'), 5.181 (IH, t, J=4.9 Hz, H-17), 4.755
(IH, d, J=3.4 Hz, H-12), 4.740 (IH, d, J=3.4 Hz, H-12'), 3.990 (2H,
m, H-16), 3.608 (IH, dd, J=4.5 Hz & 4.5 Hz, H-18), 3.560 (IH,
q, J=5.19 Hz, H-18), 2.652 (4H, m, CO--CH.sub.2--CH.sub.2--CO--),
2.548 (2H, m, H-11 & H-11'), 2.304 (2H, ddd, J=3.0, 3.89 &
3.0 Hz, H-3), 2.065 (2H, m, H-3'), 1.886 (2H, m, H-2), 1.786 (2H,
m, H-2), 1.786 (2H, m, H-9), 1.686 (2H, m, H-8), 1.534 (2H, m,
H-2'), 1.489 (2H, m, H-7 & H-10), 1.332 (6H, s, Me-15 &
Me-15'), 1.213 (2H, m, H-1 & H-1'), 0.980-0.937 (12H, Me-14,
Me-13, Me-14' & Me-13').
[0062] .sup.13CNMR (acetone-d.sub.6, 125 MHz): .delta. 172.99 (s,
CO--OH), 171.7 (s, --CO--O--), 103.9 (s, C-4), 102.46 (d, C-12),
102.32 (d, C-12'), 88.02 (d, C-5), 87.99 (d, C-5'), 80.97 (s, C-6),
71.92 (d, C-17), 66.74 (t, C-16), 66.59 (t, C-18), 53.09 (d, C-1),
44.89 (d, C-7), 37.63 (d, C-10), 36.70 (t, C-3), 34.98 (t, C-8),
31.33 (d, C-11), 31.29 (d, C-11'), 29.48 & 28.78 (t, methylenes
of CO--CH.sub.2--CH.sub.2--CO), 25.72 (q, C-15), 24.98 (t, C-2),
24.7 (t, C-9), 20.23 (q, C-14), 12.81 (q, C-13). HRESIFTMS m/z)
723.362 [M-H].sup.+, (Calcd. for C.sub.37H.sub.55O.sub.14).
[0063] Preparation of the .beta.,.beta.-Dihydroartemisitene Dimer
with Ethylene Glycol (Compound 9)
Example 14
[0064] To a stirred solution of dihydroartemisitene (prepared from
artemisinin as previously described (19) (75 mg) in dry ether (15
mL) and ethylene glycol (52 mg), was added BF.sub.3.OEt.sub.2 (18
.mu.L) and the reaction mixture was allowed to stand for 24 hr,
then quenched and worked up as usual. Column chromatography of the
crude reaction mixture using a gradient of EtOAc in n-hexane
(20%.fwdarw.50%) afforded Compound 9 as a gum (7 mg),
[.alpha.].sub.D+181.degree. (c 0.022, MeOH); IR (film)
.nu..sub.max: 2937, 2875, 1681, 1449, 1376, 1191, 1102, 987
cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. 5.89 (1H, s,
H-5), 5.38 (1H, s, H-12), 5.08 (1H, s, H-13a), 4.94 (1H, s, H-13b),
3.89 (1H, d, J=7.5 Hz, H-16a), 3.57 (1H, d, J=7.6 Hz, H-16b), 2.31
(1H, m, H-7), 1.44 (3H, s, Me-15), 1.25 (2H, m, H-1, H-10), 0.7
(3H, d, J=6.2 Hz, Me-14); .sup.13C NMR (CDCl.sub.3, 100 MHz):
.delta. 143.1 (s, C-11), 114.5 (t, C-13), 103.5 (s, C-4), 101.2 (d,
C-12), 88.1 (d, C-5), 80.8 (s, C-6), 66.6 (t, C-16), 51.9 (d, C-1),
48.3 (d, C-7), 37.0 (d, C-10), 36.5 (t, C-3), 34.4 (t, C-9), 31.4
(t, C-8), 24.6 (t, C-2), 25.8 (q, C-15), 20.0 (q, C-13). HRESIFTMS
[m/z] 613.2943 [M+Na].sup.+ (calcd for C.sub.32H.sub.46O.sub.10Na,
613.2983).
Example 15
[0065] Compounds of this invention were subjected to anti-cancer
activity screen carried out by the National Cancer Institute (NCI)
following their standard protocol against 60 different cancer cell
lines. The activity of the compounds of this invention against
selected cell lines fro leukemia, non-small cell lung cancer, colon
cancer, CNS cancer, melanoma, ovarian cancer, renal cancer,
prostate cancer, and breast cancer were determined in terms of GI50
(Table 1), TGI (Table 2), and LC50 (Table 3) concentrations. G150
is the concentration which inhibits 50% of the growth of the cells,
TGI is the concentration causing total growth inhibition, and LC50
is the concentration which kills 50% of the cells.
Example 16
[0066] Compounds of this invention were further tested under the
NCI's Hollow Fiber Assay Standard Protocol which assesses the in
vivo activity. Compounds are considered to have enough activity to
progress into further testing if the combined IP and SC sores were
.gtoreq.20 or if the SC score was .gtoreq.8 or if there was a net
cell kill of one or more cell lines. Table 4 (FIG. 5) shows the
results of the testing of compounds of this inversion in this
assay.
Example 17
[0067] Compounds of this invention were subjected to in vitro
assays to assess their anti-angiogenic activity. These assays are
carried out by the NCI according to their standard protocol for
HUVEC assays for initial in vitro testing. The three assays are the
Growth Inhibition Assay, the Cord Formation Assay, and the Cell
Migration Assay. Compounds are considered for further testing if
activity is shown in at least one of the above assays. Table 5
(FIG. 6) shows the activity of compounds of this invention in these
assays.
Example 18
[0068] Compounds of this invention were subjected to anti-protozoal
screens at the National Center for Natural Products Research
(NCNPR) at the University of Mississippi following standard
protocols for assessing anti-malarial and anti-Leishmanial
activity. Compounds' activities against these two organisms were
compared to the activity of standard medications for each
organism.
[0069] Table 6 (FIG. 7) shows the activity of compounds of this
invention against two strains of the malaria parasite (Plasmodium
falciparum), one is chloroquine sensitive (D6 clone) and one is
chloroquine resistant (W2 clone). The cytotoxicity of the compounds
was also assessed using Vero cells. The data show that compounds of
this invention are more active than chloroquine or artemisinin as
anti-malarial drugs.
[0070] Table 7 (FIG. 8) shows the activity of a selected group of
compounds of this invention against the malaria parasite. These are
from different synthetic lots than those tested in Table 6 (FIG.
7). This confirms the activity of compounds of this invention as
anti-malarial agents.
[0071] Table 8 (FIG. 9) shows the activity of compounds of this
invention against the leishmania parasite with activity comparable
to that of pentamidine.
* * * * *