U.S. patent application number 12/723912 was filed with the patent office on 2010-07-15 for synthesis of sodium narcistatin and related compounds.
This patent application is currently assigned to AZ Board of Regents, a body corporate of the State of AZ, Acting for & on Behalf of AZ State Univ.. Invention is credited to Noeleen Melody, George R. Pettit.
Application Number | 20100179108 12/723912 |
Document ID | / |
Family ID | 36677977 |
Filed Date | 2010-07-15 |
United States Patent
Application |
20100179108 |
Kind Code |
A1 |
Pettit; George R. ; et
al. |
July 15, 2010 |
Synthesis of Sodium Narcistatin and Related Compounds
Abstract
The present invention involves use of the compounds narciclasine
(2a) and 7-deoxy-narciclasine (2c), which are obtained via
isolation from the medicinal plant species Narcissus
(Amaryllidaceae), as precursors in a novel synthesis method in
which each of these compounds are selectively hydrogenated to
produce trans-dihydronarciclasine (1a) and
7-deoxy-trans-dihydronarciclasine (1c). Also described herein is a
novel synthesis method for producing sodium narcistatin (11) from
narciclasine (2a). Further described herein are certain novel
3,4-cyclic phosphate prodrugs, including sodium-7-deoxynarcistatin
(8), sodium-7-deoxy-transdihydronarcistatin (9), and sodium
transdihydronarcistatin (10).
Inventors: |
Pettit; George R.; (Paradise
Valley, AZ) ; Melody; Noeleen; (Mesa, AZ) |
Correspondence
Address: |
MCANDREWS HELD & MALLOY, LTD
500 WEST MADISON STREET, SUITE 3400
CHICAGO
IL
60661
US
|
Assignee: |
AZ Board of Regents, a body
corporate of the State of AZ, Acting for & on Behalf of AZ
State Univ.
Scottsdale
AZ
|
Family ID: |
36677977 |
Appl. No.: |
12/723912 |
Filed: |
March 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11813657 |
Jan 21, 2009 |
7709643 |
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PCT/US06/01658 |
Jan 17, 2006 |
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12723912 |
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60644397 |
Jan 14, 2005 |
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Current U.S.
Class: |
514/81 |
Current CPC
Class: |
A61P 31/02 20180101;
A61P 35/02 20180101; C07F 9/65744 20130101; C07D 491/04 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
514/81 |
International
Class: |
A61K 31/675 20060101
A61K031/675; A61P 35/02 20060101 A61P035/02; A61P 35/00 20060101
A61P035/00 |
Goverment Interests
STATEMENT OF FEDERALLY SPONSORED RESEARCH
[0002] Financial assistance for this invention was provided by the
United States Government, Division of Cancer Treatment and
Diagnosis, National Cancer Institute, Department of Health and
Human Services Outstanding Investigator Grant Numbers
CA-44344-01A1-01-12; CA 44344-01-12 and CA 90441-01-03; the Arizona
Disease Control Research Commission; and private contributions.
Thus, the United States Government has certain rights in this
invention.
Claims
1-10. (canceled)
11. A method of inhibiting the growth of a cancer cell comprising
contacting said cancer cell with a compound having the following
structure: ##STR00001##
12. The method of claim 11, wherein said cell is selected from the
group consisting of a leukemia cell, a pancreatic cancer cell, a
breast cancer cell, a central nervous system cancer cell, a lung
cancer cell, a colon cancer cell and a prostate cancer cell.
13. A method of inhibiting the growth of a cancer cell comprising
contacting said cancer cell with a compound having the following
structure: ##STR00002##
14. The method of claim 13, wherein said cell is selected from the
group consisting of a leukemia cell, a pancreatic cancer cell, a
breast cancer cell, a central nervous system cancer cell, a lung
cancer cell, a colon cancer cell and a prostate cancer cell.
15. A method of inhibiting the growth of a cancer cell comprising
contacting said cancer cell with a compound having the following
structure: ##STR00003##
16. The method of claim 15, wherein said cell is selected from the
group consisting of a leukemia cell, a pancreatic cancer cell, a
breast cancer cell, a central nervous system cancer cell, a lung
cancer cell, a colon cancer cell and a prostate cancer cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is based on and claims the priority to U.S.
Provisional Patent Application No. 60/644,397 filed on Jan. 14,
2005, the disclosure of which is incorporated herein in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates certain compounds, and methods
for synthesis of certain compounds, wherein the compounds have
shown anti-neoplastic activity against cancerous cell lines, and
therefore are anticipated to be useful in the treatment of various
forms of cancer in animals and humans.
SUMMARY OF THE INVENTION
[0004] The present invention involves use of the compounds
narciclasine (2a) and 7-deoxy-narciclasine (2c), which are obtained
via isolation from the medicinal plant species Narcissus
(Amaryllidaceae), as precursors in a novel synthesis method in
which each of these compounds are selectively hydrogenated to
produce trans-dihydronarciclasine (1a) and
7-deoxy-trans-dihydronarciclasine (1c). Also described herein is a
novel synthesis method for producing sodium narcistatin (11) from
narciclasine (2a). Further described herein are certain novel
3,4-cyclic phosphate prodrugs, including sodium-7-deoxynarcistatin
(8), sodium-7-deoxy-trans-dihydronarcistatin (9), and sodium
transdihydronarcistatin (10).
[0005] The present invention involves use of the compounds
narciclasine (2a) and 7-deoxy-narciclasine (2c) which are obtained
via isolation from certain Narcissus (Amaryllidaceae) as precursors
in a novel and unobvious process by which each of these compounds
may be selectively hydrogenated to produce
trans-dihydronarciclasine (1a) and
7-deoxy-trans-dihydronarciclasine (1c).
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 shows structural formulas for the compounds described
herein
[0007] FIG. 2 shows reaction schemes for synthesizing some of the
compounds of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Materials and Methods
[0008] The isolation of new compounds from substances found in
nature, such as plants, and the creation of derivatives of these
compounds, is an active area of research for compounds having
pharmaceutical utility. Described herein are methods for the
synthesis of, and for the use of such "natural" compounds in the
synthesis of other compounds, many of which show promise as
anti-neoplastic drugs.
[0009] From a 1982 collection (bulbs) of the Chinese medicinal
plant Zephyranthes candido (Amaryllidaceae) was isolated the strong
(ED.sub.50 0.0032 .mu.g/ml) P388 lymphocytic leukemia cell growth
inhibitor trans-dihydronarciclasine (1a). (Pettit G. R., et al.,
Antineoplastic 162. Zephyranthes candida. J. Nat. Prod. 53:176-178;
1990.) The structure was established by detailed spectral analyses
of its peracetate derivative (1b) and confirmed by comparison with
the product from catalytic hydrogenation of narciclasine (2a).
(Mondon, A., et al., Zur Kenntnis des Narciclasins. Chem. Ber.
108:445-463; 1975.) Hydrogenation afforded as the major product the
expected cis-dihydronarciclasine (3a), accompanied by the trans
isomer (1a) and iso-narciclasine (4a). More recently,
trans-dihydronarciclasine (1a) was found to exhibit strong cancer
cell growth inhibition (mean panel GI.sub.50 12.6 nM) against the
U.S. National Cancer Institute (NCl) panel of cancer cell lines,
whereas its cis isomer (3a) was only very weakly active (mean panel
GI.sub.50 3800 nM). (Pettit, G. R., et al., Antineoplastic agents
256. Cell growth inhibitory isocarbostyrils from Hymenocallis. J.
Nat. Prod. 56: 1682-1687; 1993.) Importantly, the trans isomer (1a)
gave an active Compare correlation coefficient of 0.92 in respect
to (+)-pancratistatin (5) equals 1.00. (Pettit, G. R., et al.,
Antineoplastic agents 256. Cell growth inhibitory isocarbostyrils
from Hymenocallis. J. Nat. Prod. 56: 1682-1687; 1993.) The trans
isomer (1a) also showed strong activity against a range of RNA
viruses while the synthetic cis isomer (3a) was completely
inactive. (Gabrielsen, B., et al., Antiviral (RNA) activity of
selected Amaryllidaceae isoquinoline constituents and synthesis of
related substances. J. Nat. Prod. 55:1569-1581; 1992.)
[0010] Narciclasine (2a) was isolated from the bulbs of Narcissus
imcomparabilus, and 7-deoxynarciclasine (2c) and
7-deoxy-transdihydronarciclasine (1c) were isolated from the bulbs
of Hymenocallis littoralis grown by the ASU-CRI research group in
Tempe, Ariz., now part of the Arizona Biodesign Institute. (Piozzi,
F., et al., Narciclasine and Narciprimine. Tetrahedron.
24:1119-1131; 1968), (Pettit, G. R., et al., Antineoplastic agents
256. Cell growth inhibitory isocarbostyrils from Hymenocallis. J.
Nat. Prod. 56: 1682-1687; 1993). All solvents were redistilled, and
reagents were purchased from Lancaster, Sigma-Aldrich Co. and Aeros
Chemical Co. Reaction progress was ascertained by thin-layer
chromatography using Analtech silica gel GHLF Uniplates visualized
under long- and short-wave UV irradiation and developed in an
ethanolic solution of phosphomolybdic acid reagent (Sigma-Aldrich
Co.). Column chromatography was performed with silica gel 60
(230-400 mesh) from E. Merck. Dowex 50WX8-400 cation exchange resin
(H.sup.+) form) was first eluted with methanol, 1N hydrochloric
acid and deionized water. The cation forms of the resin were
prepared by eluting with a 1N solution of the appropriate base
followed by deionized water. All reaction products were colorless
solids unless otherwise noted. All melting points were determined
with an Electrothermal digital melting point apparatus model IA9200
and are uncorrected.
[0011] Methods for Synthesis of trans-dihydronarciclasine (1a)
[0012] 2,3,4,7-O-Tetraacetoxy-narciclasine (2b)--Compound 2b is
produced as follows. To a stirred solution of narciclasine (1.00 g,
3.25 mmol) in pyridine (3 ml under nitrogen), add acetic anhydride
(6 ml). Stir for 16 hours at room temperature, add ice (50 ml) to
the mixture, and extract with dichloromethane (3.times.20 ml). The
combined extract is dried over MgSO.sub.4, filtered and evaporated
in vacuo to afford 2,3,4,7-O-Tetraacetoxy-narciclasine (2b) as a
light brown powder (1.4 g, 90% yield).
[0013] 2,3,4,7-Tetraacetoxy-trans-dihydronarciciasine (1b).
[0014] Method 1 for producing 1b. To a solution of narciclasine
tetraacetate (2b) (0.97 g, 20.42 mmol) in glacial acetic acid (120
ml), add 5% Pd/C catalyst (0.56 g, 26 mol %). Stir the mixture
under an atmosphere of hydrogen at room temperature for 3 hours and
then filter the solution, such as through fluted filter paper. Dry
the filtrate over MgSO.sub.4, again filter and evaporate in vacuo.
Purify the residue by column chromatography on silica gel eluting
with 0.5% methanol in dichloromethane to afford the product (1b) as
a powder (0.290 g, 30%) along with the cis-dihydro-peracetate (3b)
as a solid (0.60 g, 62%). Analysis of 1b by comparison of NMR data
found it to be identical with an authentic sample. (Pettit G. R.,
et al., Antineoplastic 162. Zephyranthes candida. J. Nat. Prod.
53:176-178; 1990.)
[0015] Method 2 for producing 1b. To a solution of narciclasine
tetracetate (2b) (0.200 g, 0.42 mmol) in a 1:1 mixture of
ethanol/dichloromethane was added 10% Pd/C catalyst (0.004 g, 0.042
mmol). The mixture was stirred under 1 atm. of hydrogen at room
temperature for 4 hours. The reaction mixture was then filtered
through a pad of silica and the solvent removed in vacuo. The
residue was then purified by column chromatography (flash silica;
eluant 45:55 n-hexane-EtOAc) to afford the trans-dihydro-peracetate
(1b) as a solid (0.131 g, 65%), along with the
cis-dihydro-peracetate (3b) as a solid (0.050 g, 25%).
[0016] Trans-Dihydronarciclasine (1a). Dissolve
2,3,4,7-O-tetraacetoxy-trans-dihydronarciclasine (1b) (0.512 g,
1.07 mmol) in methanol:water (9:1) (20 ml), and add dichloromethane
(12 ml) to aid in solubility. Add Potassium carbonate (0.009 g,
0.06 mmol) and stir the reaction at room temperature for three
days. TLC(CH.sub.2Cl.sub.2:CH.sub.3OH 4%) shows complete conversion
to the product.
[0017] The reaction mixture is concentrated and the residue
purified by column chromatography on silica gel to give
(CH.sub.2Cl.sub.2:CH.sub.3OH 4%) (1a) as an amorphous solid (0.134
g, 40%); mp 260.degree. C. (dec), 285.degree. C. (melts).
[0018] 3,4-isopropylidene-7-deoxynarciclasine (2e)--Initially,
7-deoxynarciclasine (2c) (0.205 g, 0.704 mmol) and TsOH (0.133 g,
0.704 mmol) are dissolved in DMF (10 ml) and 2',2'-dimethoxypropane
(0.864 ml, 7.04 mmol) is added. The resulting solution is stirred
for 16 hours and then poured into water (50 ml) and extracted with
ethyl acetate (4.times.30 ml). The combined organic phase is dried
(MgSO.sub.4), filtered and concentrated in vacuo to yield a pale
yellow solid which is separated by column chromatography (flash
silica; eluant 3:7 n-hexane-EtOAc) to afford the product 2e as a
solid (0.215 g, 92%); Recrystallized from methanol as needles.
[0019]
2-[Cert-Butyl-1,1-dimethylsilyl]oxy-3,4-isopropylidene-7-deoxy-narc-
iclasine (2f)--To 3,4-isopropylidene-7-deoxy-narciclasine (2e,
0.024 g, 0.0725 mmol) in DMF (3 ml) is added TBDMSCI (0.016 g,
0.109 mmol) and imidazole (0.007 g, 0.109 mmol). Stir the resulting
solution for 5 hours and remove the DMF in vacuo to afford a pale
yellow oil. The residue is separated by column chromatography
(flash silica; eluant 3:2/n-hexane-EtOAc) to afford the silyl ether
as a solid (0.028 g, 87%): m.p. 269.degree. C.
[0020]
2-[tert-Butyl-1,1-dimethylsilyl]oxy-3,4-isopropylidene-7-deoxy-tran-
s-dihydro-narciclasine (Id)--To a solution of
2-(tert-Butyl-1,1-dimethylsilyl]oxy-3,4-isopropylidene-7-deoxy-narciclasi-
ne (2f, 0.050 g, 0.112 mmol), in a 1:1 mixture of ethanol and
dichloromethane (8 ml) add 10% Pd/C (1.2 mg, 0.0112 mmol). Stir the
resulting mixture was stirred under 1 atm. of hydrogen for 4 hours
and then pass through a short column of silica gel, eluting with
ethyl acetate. Remove solvent in vacuo to afford a solid. Separate
the residue by column chromatography (gravity, silica gel; eluant
7:3/n-hexane EtOAc) to yield a solid (0.028 g, 56%): m.p.
181.5-182.5.degree. C.
[0021] Also isolated was
2-[tert-Butyl-1,1-dimethylsilyl]oxy-3,4-isopropylidene-7-deoxy-cis-dihydr-
o-narciclasine (3c) as a solid (0.013 g, 26%): m.p.
237.5-238.5.degree. C.
[0022] The third minor component isolated was
2-[tert-Butyl-1,1-dimethylsilyl]oxy-3,4-isopropylidene-7-deoxy-iso-dihydr-
o-narciclasine (4c) as a solid (0.005 g, 10%): m.p.
246.5-248.0.degree. C.
[0023] 7-deoxy-trans-dihydronarciclasine (1c).
2-[tert-Butyl-1,1-dimethylsilyl]oxy-3,4-isopropylidene-7-deoxy-trans-dihy-
dro-narciclasine (0.02 g, 0.045 mmol) is dissolved in
tetrahydrofuran (2 ml) and formic acid (60%) (2 ml) is added at
room temperature. The reaction mixture is heated to 60.degree. C.
for three hours. TLC (ethylacetate 15%:hexane) shows complete
conversion to a slower moving product. The reaction is concentrated
to a white residue which is purified by silica gel flash column
chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH 10%) to yield a white
solid (13.1 mg, 71.4%) mp 230.degree. C. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) showed the silyl ether still present, which was
confirmed by HRMS, APCI.sup.+calcd. for C.sub.20H.sub.30NO.sub.6Si
(M+H).sup.+=408.1842, found m/z=408.1845. This material is taken
without further purification to the silyl ether deprotection
step.
[0024]
2-[tert-Butyl-1,1-dimethylsilyl]oxy-7-deoxy-trans-dihydronarciclasi-
ne (0.037 g, 0.09 mmol) is dissolved in tetrahydrofuran (5 ml), and
tetrabutylammoniumfloride (TBAF) (0.01 ml, 0.01 mmol) is added and
the reaction stirred at room temperature under argon.
TLC(CH3.sub.3OH 10%; CH.sub.2Cl.sub.2) after 6 hours shows
incomplete conversion starting material to product, and therefore
TBAF (0.1 ml, 0.1 mmol) is added and the reaction continued for 24
hours. Additional TBAF (0.1 ml, 0.1 mmol) was added after 24 hours.
The reaction is stirred for 5 days. Ethyl acetate (35 ml) is added
and the organic phase washed with brine (25 ml), dried MgSO.sub.4,
filtered and concentrated in vacuo to a yellow oil. The oil is
taken up in tetrahydrofuran and eluted on a column of silica gel
with a gradient elution using CH.sub.2Cl.sub.2:CH.sub.3OH
10%-CH.sub.2Cl.sub.2:CH.sub.3OH 30%. The product is isolated as a
white solid, 13.4 mg, 50% and was identical by .sup.1H NMR with a
natural sample of 7-deoxy-trans-dihydronarciclasine. (Gabrielsen,
B., et al., Antiviral (RNA) activity of selected Ammyllidaceae
isoquinoline constituents and synthesis of related substances. J.
Nat. Prod. 55:1569-1581; 1992.)
[0025] Functional groups such as hydroxyl, ester and amide often
direct the stereochemistry of hydrogenation. Homogenous
hydrogenation of allylic alcohols usually occurs with high
stereoselectivity. Catalysts used in such hydroxy-directed
hydrogenation often include Wilkinson's catalyst (6)
[RhCI(PPh.sub.3).sub.3] and Crabtree's catalyst (7)
[Ir(COD)(Pcy.sub.3)(py)]PF.sub.6 (8). Consequently, narciclasine,
protected as its acetonide (2d), was treated with Crabtree's
catalyst (7) in dichloromethane, but failed to yield any
hydrogenation product. (Mondon, A., et al., Zur Kenntnis des
Narciclasins. Chem. Ber. 108:445-463; 1975.) The reaction was also
attempted with Wilkinson's catalyst (6) in toluene and again
narciclasine acetonide (2d) resisted hydrogenation. With a related
trisubstituted styrene that proved unreactive towards hydrogenation
with Wilkinson's catalyst (6) even under forcing conditions, it was
successfully hydrogenated when first converted to its alkoxide.
That led exclusively to the cis isomer. (Thompson, H. W., et al.,
Stereochemical control of reductions. IV. Control of hydrogenation
stereochemistry by intramolecular anionic coordination to
homogeneous catalysts. J. Amer. Chem. Soc. 96:6232-6233; 1974.)
This approach was unsuccessful when using narciclasine acetonide
(2d). Without intending to be bound by this theory, because this
reaction is believed to be associated with the olefin's ability to
donate unshared electron pairs to unfilled surface orbitals of the
catalyst metal, the double bond in narciclasine is probably too
hindered to allow this type of hydroxy-directed hydrogenation.
(Thompson, H. W. Stereochemical control of reductions. The
directive effect of carbomethoxy vs. hydroxymethyl groups in
catalytic hydrogenation. J. Org. Chem. 36:2577-2581; 1971.) So,
attention was next directed to ionic hydrogenation. Interestingly,
in our experiments narciclasine (2a) and derivatives (2b) and (2d)
resisted hydrogenation with triethylsilane/trifluoroacetic acid in
dichloromethane at -75.degree. C. and at 25.degree. C.
[0026] Hydrogentation of narciclasine (2a) using Adam's catalyst in
ethanol results in the following: 28% of the trans isomer (1a) was
usually obtained, along with 58% of the cis isomer 3a and 13% of
iso-narciclasine (4a). (Mondon, A., et al., Zur Kenntnis des
Narciclasins. Chem. Ber. 108:445-463; 1975.) The hydrogenation of
narciclasine peracetate (2b) was conducted in the presence of 5%
Pd/C (20 mol %) at 1 atm and a variety of solvents: ethyl acetate,
ethanol, acetic acid, hexane, tetrahydrofuran, pyridine and
dimethylformamide. (Thompson, H. W., et al., Stereochemical control
of reductions. 5. Effects of electron density and solvent on group
haptophilicity. J. Org. Chem. 41:2903-2906; 1976), (Okamoto, T., et
al., Lycoricidinol and lycoricidine. New plant growth regulators in
the bulbs of Lycoris radiata Herb. Chem. Pharm. Bull. 16:1860-1864;
1968), (Immirzi, A., et al., The crystal and molecular structure of
narciclasine tetra-acetate. J. Amer. Chem. Soc. 240-240; 1972.) The
results are shown in Table I.
TABLE-US-00001 TABLE I Effect of solvent on hydrogenation of
narciclasine acetate (2b) with 5% Pd/C (20 mol %) at 1 atm.,
25.degree. C. for 2 hours. Products Wt. g Sm % Sm.sup.a %
trans.sup.a % cis.sup.a % iso.sup.a Solvent 2b 2b 1b 3b 4b hexane
0.026 100 -- -- -- pyridine 0.022 100 -- -- -- tetrahydrofuran
0.025 70 15 15 -- ethyl acetate 0.025 -- 47 40 13 ethanol 0.219 --
36 54 10 ethanol:DCM (1:1) 0.025 22 25 53 -- ethanol:DCM (1:1)*
0.200 -- 65 25 -- acetic acid 0.024 -- .sup. 51.sup.b 47 2
Dimethylformamide 0.019 -- 38 42 20 .sup.aValues determined by
.sup.1H NMR; .sup.bIncreased to 57% with 55 mol % of Pd/C, but
dropped to 49% with 100 mol % of Pd/C. *Isolated yield from
reaction with 10% Pd/C (10% mol).
[0027] The trans:cis:iso ratios were determined by .sup.1H-NMR and
were based on a 100% conversion of starting material to product.
The best ratio observed was 51:47:2 respectively with acetic acid
as the solvent and the reaction carried out on a small scale
(approx. 0.020 g of narciclasine peracetate). Scaleup of this
reaction showed a wide variation in results. When 5 g of
narciclasine was hydrogenated in acetic acid in the presence of 5%
Pd/C (8 mol %) for 20 hr only starting material was recovered. When
the hydrogenation was carried out on narciclasine peracetate 1 g
using 10% Pd/C (25.8 mol %) the trans and cis products were
isolated in 30% and 62% yield respectively following chromatography
on silica gel. The solvent system dichloromethane:ethanol (1:1)
gave good results when narciclasine tetraacetate (2b) (200 mg) was
hydrogenated in the presence of 10% Pd/C (10 mol %). The yield of
trans was 65% following chromatography. The peracetylated isomers
(1b), (3b), and (4b) were separated by column chromatography on
silica gel. The structure of the synthetic trans isomer (1b) was
established by detailed spectral data comparison with an authentic
sample. (Pettit G. R., et al., Antineoplastic 162. Zephyranthes
candida. J. Nat. Prod. 53:176-178; 1990.)
[0028] Method for Synthesis of 7-deoxy-trans-dihydronarciclasine
(1c)
[0029] Having developed a method for the hydrogenation of 2b to 1b,
a similar method was sought for the interconversion of
7-deoxynarciclasine (2c) to 7-deoxy-trans-dihydronarciclasine (1c).
This was achieved by initially protecting the cis diol unit as its
acetonide (2d) in good yield (92%). The remaining hydroxyl group
was protected as its silyl ether (2f) to avoid the potential
problems which had already been encountered when attempting the
hydrogenation of narciclasine acetonide (2d). With the silyl ether
in hand, the hydrogenation of the olefin was attempted using the
conditions which had been most successful in the reduction of
peracetate 2b to the trans-dihydroperacetate (1b). Hydrogenation
with 10% Pd/C (10 mol %) at 1 atm in ethanol/dichloromethane (1:1)
of (2f)(0.05 g) gave a separable mixture of trans:cis:iso in
56%:26%:10% yields, respectively. However, this reaction suffered
upon scaleup and yields of trans were reduced to 27% when the
reaction was carried out on a 2 g scale. The synthetic trans-isomer
(1d) was obtained by deprotection of the acetonide using formic
acid (60%) followed by deprotection of the silyl ether with TBAF to
yield 1c which was found to be identical with an authentic sample
of 7-deoxy-trans-dihydronarciclasine (1d). (Gabrielsen, B., et al.,
Antiviral (RNA) activity of selected Amaryllidaceae isoquinoline
constituents and synthesis of related substances. J. Nat. Prod.
55:1569-1581; 1992.)
[0030] As described below, the synthesis of sodium narcistatin (11)
was improved (88% overall yield) and the modified reaction sequence
was utilized to synthesize sodium 7-deoxy-narcistatin (8), sodium
7-deoxy-trans-dihydronarcistatin (9) and sodium
trans-dihydro-narcistatin (10). The human cancer cell line
inhibitory isocarbostyril precursors were isolated from the bulbs
of Hymenocallis littoralis obtained by horticultural production or
reduction of narciclasine (2a) from the same source. Solvents were
distilled prior to use and pyridine was dried over potassium
hydroxide and distilled. The three new 3,4-cyclic phosphate
prodrugs (8, 9, and 10), whose synthesis is discussed in further
detail below, are being evaluated for further development as
anticancer drugs.
[0031] Method for Synthesis of Sodium Narcistatin (11). Synthesis
of 3,4-cyclic phosphate 11 from narciclasine (2a) (0.113 g, 0.368
mmol) was carried out in pyridine (4 ml) using tetrabutylammonium
dihydrogen phosphate (0.075 g, 0.22 mmol) and
dicyclohexylcarbodiimide (0.4 g, 1.94 mmol), with additional
amounts of tetrabutylammonium dihydrogen phosphate (0.185 g) and
dicyclohexylcarbodiimide (0.4 g) added after about the first 24
hours stirring at about 80.degree. C. The reaction is stirred for
about 96 hours, cooled and filtered to remove precipitated
dicyclohexylurea (DCU). Water (100 ml) is added and the mixture
refiltered to remove any residual DCU. The mother liquor is
concentrated to minimum volume. The aqueous fraction is eluted
through an ion exchange column (sodium form). The UV active
fractions are combined and lypholized to yield the phosphate 11 as
a cream solid (88% yield). Rather than eluting the aqueous fraction
via a sodium form of an ion exchange column, another suitable salt
form (such as potassium or lithium) could be used to produce a
compound such as potassium narcistatin or lithium narcistatin.
[0032] Method for Synthesis of 3,4-cyclic phosphates sodium
7-deoxynarcistatin (8), sodium 7-deoxy-trans-dihydronarcistatin (9)
and sodium trans-dihydronarcistatin (10).
[0033] The 7-deoxynarciclasine (2c) and
7-deoxy-trans-dihydronarciclasine (1c) mixture separated from H.
littoralis was acetylated by dissolving in pyridine (20 mL) and
adding acetic anhydride (20 mL, 2.4 equiv.) The mixture slowly
becomes a solution with stirring overnight at room temperature, and
TLC(CH.sub.2Cl.sub.2--CH.sub.3OH, 2%) showed no starting material.
Ice water (200 ml) is added to the reaction mixture with vigorous
stirring. A cream colored precipitate develops and is collected
following being stirred for two hours to provide 13.7 g.
Thereafter, the peracetate mixture (acetylation product) is
separated by elution using 7:3 toluene-ethyl acetate, via silica
gel column chromatography, to yield the following isocarbostyrils
12 (60% recovery) and 13 (19% recovery). Both 2, 3,
4-triacetoxy-7-deoxynarciclasine (12) and
2,3,4-triacetoxy-7-deoxy-trans-dihydronarciclasine (13) were then
deprotected with potassium carbonate in aqueous methanol to afford
the corresponding triols 14 and 15 in 72% yields.
[0034] The 3,4-cyclic phosphates 8, 9 and 10 were synthesized
employing an improvement in the procedure we developed for
synthesis of sodium narcistatin (11) using tetrabutylammonium
dihydrogen phosphate and an excess of dicyclocarbodiimide in dry
pyridine under argon at 80.degree. C. for 48 hours. (Mondon, A., et
al., Chem. Ber. 1975, 94, 617), (Pettit, G. R., et al., J. Nat.
Prod. 1986, 49, 995-1002), (Pettit, G. R., et al., J. Nat. Prod.
2003, 66, 92-96.)
[0035] In each case, .sup.1H-NMR of the crude product showed the
reaction to be only 50% complete following a 24 hour period.
Additional reagents were added at this stage and the reaction
allowed to proceed to completion (a further 24 hours). Increasing
the amount of tetrabutylammonium dihydrogen phosphate from 0.65
equivalents to 1 equivalent in the first 24 hours did not increase
the reaction rate. Water was added to the reaction mixture to
precipitate the dicyclohexylurea (DCU) and the pyridine/water
filtrate was concentrated to remove the pyridine. An aqueous
extract of the residue was passed through a Dowex 50WX8-400 ion
exchange column (sodium form). The UV responsive fractions were
combined and lyophilized to afford the new 3,4-cyclic phosphates
designated sodium 7-deoxy-narcistatin (8, 88% yield), sodium
7-deoxy-trans-dihydro-narcistatin (9, 65% yield) and sodium
trans-dihydro-narcistatin (10, 94% yield).
[0036] In a more preferred embodiment, deletion of the p-toluene
sulfonic acid component increased the yield of phosphate 11 to 88%
versus the original 50%. That was one of the major improvements
that allowed new phosphates 8, 9 and 10 to be obtained in very good
yields.
[0037] The cyclic phosphate prodrugs, along with the parent
compounds were evaluated against a minipanel of human cancer cell
lines and murine P388 lymphocytic leukemia. See Table 2.
TABLE-US-00002 TABLE 2 Aqueous Solubility, Human Cancer Cell Line
and Murine P-388 Lymphocytic Inhibitory Activities GI.sub.50
(.mu.g/ml) Aqueous ED.sub.50 Lung- Solubility (.mu.g/ml) NSC
25.degree. C. Leukemia Pancreasa Breast CNS NCI- Colon Prostate
Isocarbostyril (mg/ml) P388 BXPC-3 MCF-7 SF268 H460 KM20L2 DU-145
2c <1 0.019 0.070 0.046 0.120 0.053 0.084 0.051 1c <1 0.029
0.046 0.034 0.059 0.043 0.051 0.040 1a <1 0.0024 0.012 0.0053
0.020 0.0092 0.015 0.0066 10 >190 1.7 5.3 4.0 6.3 4.7 5.6 3.9
10a >10 0.42 >1 >1 >1 >1 >1 >1 10b >10 1.4
>1 >1 >1 >1 >1 >1 11 >10 1.6 >10 7.2 >10
>10 >10 >10 11a >5 0.39 >1 >1 >1 >1 >1
>1 11b >5 1.7 >1 >1 >1 >1 >1 >1 12 >10
0.88 5.6 4.6 8.8 7.8 9.6 5.2 12a >1 0.26 >1 >1 >1 >1
>1 >1 12b >1 0.35 >1 0.64 >1 >1 >1 0.54
[0038] Results of the cancer cell line evaluations reconfirmed the
strong cancer cell growth inhibitory activity of
7-deoxy-trans-dihydronarciclasine (1c) and
trans-dihydronarciclasine (1a). The corresponding 3,5-cyclic
phosphates were less inhibitory under the experimental conditions
employed. However, cleavage of the phosphate groups is expected to
be very effective in vivo and such anticancer evaluations are now
underway as part of the further preclinical development of these
new anticancer drug candidates. (Pettit, G. R., et al., J. Nat.
Prod. 2003, 66, 92-96), (Dowlati, A., et al., Cancer Research 2002,
62, 3408-3416), (Dziba, J. M., et al., Thyroid 2002, 12,
1063-1070), (Eikesdal, H. P., et al., Cancer Lett. 2002, 178,
209-217), (Prise, V., et al., Int. J. Oncology 2002, 21, 717-726),
(Hill, S. A., et al., Int. J. Cancer 2002, 102, 70-74.)
[0039] 7-Deoxy-narciclasine (2c). To obtain 7-deoxy-narciclasine
(2c), a solution of 2,3,4-triacetoxy-7-deoxy-narciclasine (12, 4.36
g) in CH.sub.3OH-(99 ml) H.sub.2O-(1 ml) CH.sub.3OH (30 ml) is
added potassium carbonate (0.124 g), with stirring continued for 16
hours at room temperature while a white precipitate separated. The
mixture is neutralized with acetic acid (2 ml), stirred for 15
minutes and concentrated to minimum volume. The colorless product
is collected (2.18 g, 72%), recrystallization from acetic
acid-methanol afforded fine needles: mp 205-210.degree. C.
(dec).
[0040] Sodium 7-Deoxy-narcistatin (8). A solution of
7-deoxy-narciclasine (2c, 0.2 g, 0.69 mmol) in pyridine (8 ml) was
heated to 80.degree. C. and tetrabutylammonium dihydrogen phosphate
(0.15 g, 0.45 mmol, 0.65 equiv) followed by
dicyclohexylcarbodiimide (0.8 g, 5.6 equiv) were added. The
reaction was allowed to proceed at 80.degree. C. for 24 hours. An
.sup.1H NMR analysis of the reaction mixture composition indicated
a 50:50 mixture of starting material to product. Tetrabutylammonium
dihydrogen phosphate (0.15 g) was added followed by DCCl (0.8 g)
and the reaction continued for a further 24 hours. At this point,
.sup.1H NMR analysis of a sample from the reaction mixture showed
reaction was complete. The reaction mixture was cooled and water
(100 ml) was added. The precipitated dicyclohexylurea (DCU) was
collected and the pyridine-water mother liquor was concentrated to
minimum volume. The aqueous fraction was then passed through an ion
exchange column (DOWER 50W8-400) in the sodium form. The UV
responsive fractions were combined and lypholized to yield
phosphate 8 as a colorless solid: 227 mg (88%); mp 255.degree. C.
(dec.).
[0041] Methods for Synthesis of 7-Deoxy-narcistatin prodrugs 8a and
8b.
[0042] Sodium 7-deoxy-narcistatin (8, 52 mg) is dissolved in water
(1 ml) and the solution is passed through a column of Dowex
50WX8-400, bearing the respective cation. For example, a column
containing lithium or potassium cations, respectfully, may be used.
The UV-active fractions are then combined and freeze-dried to give
the corresponding narcistatin salt as a white solid, as
follows:
[0043] Lithium 7-Deoxy-narcistatin (10a). 34 mg, mp 250.degree. C.
(dec).
[0044] Potassium 7-Deoxy-narcistatin (10b). 43 mg, mp
230-235.degree. C. (dec).
[0045] 7-Deoxy-trans-dihydro-narciclasine (1c). This compound is
produced as follows. 2,3,4-triacetoxy-7-deoxy-trans-dihydro
narciclasine (13, 0.14 g) is saponified in 9:1 aqueous methanol and
with potassium carbonate (0.003 g), and is conducted as described
above for obtaining alcohol 2c to yield triol 1c as a colorless
solid; 89 mg (91% yield); mp>300.degree. C. (dec.).
[0046] Sodium 7-Deoxy-trans-dihydro-narcistatin (9). The conversion
of 7-deoxy-trans-dihydro-narciclasine (1c, 0.15 g, 0.51 mmol) to
narcistatin 9 in pyridine (6 ml) with tetrabutylammonium dihydrogen
phosphate (0.21 g, 0.62 mmol, 1.2 equiv), and
dicyclohexylcarbodiimide (0.54 g, 2.62 mmol, 5.13 equiv) is
conducted and the phosphate isolated as summarized for synthesis of
narcistatin 8 (cf, above) including the additional
tetrabutylammonium dihydrogen phosphate (0.21 g, 1.2 equiv) and
DCCl (0.54 g). The aqueous extract of product is subjected to ion
exchange column of Dowex 50WX8-400 (sodium form) and the UV
fluorescing fractions combined and lyophilized as noted above (cf,
8). A solution of the sodium salt is prepared in methanol (15 ml
with heating), the insoluble material is collected, and the
filtrate concentrated to yield (0.124 g, 65%); mp 297.degree. C.
(dec.)
[0047] Method for Synthesis of 7-Deoxy-trans-dihydro-narcistatin
prodrugs 11a and 11b. Sodium 7-deoxy-trans-dihydro-narcistatin (9,
30 mg) was dissolved in water (1 ml) and the solution passed
through a column of Dowex 50WX8-400, bearing the respective cation.
The UV-active fractions were combined and freeze-dried to give the
corresponding narcistatin salt as a white solid.
[0048] Lithium 7-deoxy-trans-dihydro-narcistatin (9a). 25.4 mg, mp
253.degree. C. (dec).
[0049] Potassium 7-deoxy-trans-dihydro-narcistatin (9b). 23.2 mg,
mp 287.degree. C. (dec).
[0050] Sodium trans-dihydro-narcistatin (10). Synthesis of
3,4-cyclic-phosphate 10 from trans-dihydronarciclasine (1a, 57 mg,
0.184 mmol) is accomplished in pyridine (2 ml) employing
tetrabutylammonium dihydrogen phosphate (60 mg, 0.176 mmol) and 60
mg for the delayed addition) and dicyclohexylcarbodiimide (0.18 g,
0.87 mmol) and 0.18 g for the second addition as described for
preparation of phosphate 9 (refer above).
(Trans-dihydro-narciclasine was synthesized by our group in 1992
from narciclasine according to procedures described by Mondon and
Krohn.19), (Mondon, A., et al., Chem. Ber., 1975, 108, 445-463.)
The aqueous fraction eluted from the ion exchange column (Dowex
50WX8-400, sodium form) provided sodium trans-dihydro narcistatin
(10) as a colorless solid (86 mg, 94% yield), mp>300.degree.
C.
[0051] Method for Synthesis of Trans-dihydro-narcistatin Prodrugs
10a and 10b.
[0052] Sodium trans-dihydro-narcistatin (10, 0.010 g) was dissolved
in water (1 ml) and the solution passed through a column of Dowex
50WX8-200, bearing the respective cation. The UV-active fractions
are then combined and freeze-dried to give the corresponding
trans-dihydro-narcistatin salt as a white solid.
[0053] Lithium trans-dihydro-narcistatin (10a). 8 mg, mp
275.degree. C. (dec).
[0054] Potassium trans-dihydro-narcistatin (10b). 7.1 mg, mp
230-235.degree. C. (dec).
* * * * *