U.S. patent application number 11/690799 was filed with the patent office on 2007-07-19 for halocombstatins and methods of synthesis thereof.
This patent application is currently assigned to ARIZONA BOARD OF REGENTS. Invention is credited to Mathew D. Minardi, George R. Pettit, Heidi J. Rosenberg.
Application Number | 20070167412 11/690799 |
Document ID | / |
Family ID | 34830347 |
Filed Date | 2007-07-19 |
United States Patent
Application |
20070167412 |
Kind Code |
A1 |
Pettit; George R. ; et
al. |
July 19, 2007 |
HALOCOMBSTATINS AND METHODS OF SYNTHESIS THEREOF
Abstract
The invention relates to novel compounds denominated
halocombstatins. The halocombstatins are derivatives of
combretastatin A-3, and include compounds that exhibit cancer
growth cell inhibition against a panel of human cancer cell lines
and the murine P388 leukemia, as well as activity as inhibitors of
tubulin polymerization and inhibitors of the binding of colchicine
to tubulin.
Inventors: |
Pettit; George R.; (Paradise
Valley, AZ) ; Minardi; Mathew D.; (Mather, CA)
; Rosenberg; Heidi J.; (Tempe, AZ) |
Correspondence
Address: |
FENNEMORE CRAIG
3003 NORTH CENTRAL AVENUE
SUITE 2600
PHOENIX
AZ
85012
US
|
Assignee: |
ARIZONA BOARD OF REGENTS
699 S. Mill Avenue Brickyard Suite 601, Room 691AA
Tempe
AZ
85281
|
Family ID: |
34830347 |
Appl. No.: |
11/690799 |
Filed: |
March 23, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
10948926 |
Sep 24, 2004 |
7223747 |
|
|
11690799 |
Mar 23, 2007 |
|
|
|
60505935 |
Sep 24, 2003 |
|
|
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Current U.S.
Class: |
514/129 ; 514/89;
514/90; 558/197 |
Current CPC
Class: |
C07F 9/12 20130101; C07C
43/23 20130101 |
Class at
Publication: |
514/129 ;
558/197; 514/089; 514/090 |
International
Class: |
A61K 31/66 20060101
A61K031/66; C07F 9/02 20060101 C07F009/02; A61K 31/675 20060101
A61K031/675 |
Goverment Interests
INTRODUCTION
[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
CA44344-05-12; R01-CA90441-01; and R01 CA090441-03-041; the Arizona
Disease Control Research Commission contract Number 9815; and
private contributions. Thus, the United States Government has
certain rights in this invention.
Claims
1. A compound having a structure as follows: ##STR4## wherein X is
F, Cl, Br or I.
2. A compound having a structure as follows: ##STR5## wherein X is
F, Cl, Br or L and R is a metal cation such as Na, Li, K, Cs, Rb,
Ca, Mg or is morpholine, pipenidine, glycine-OCH.sub.3,
tryptophan-OCH.sub.3 or NH(CH.sub.2OH).sub.3.
3. A compound having a stucture as follows: ##STR6## wherein X is
F, Cl, Br or I, and Z is a metal cation such as Na, Li, K, Cs, Rb,
Ca, Mg or is morpholine, piperidine, glycine-OCH.sub.3,
typtophan-OCH.sub.3 or NH(CH.sub.2OH).sub.3.
4. The compound of claim 1 wherein X.dbd.F and the compound is in
the Z configuration.
5. The compound of claim 1 wherein X.dbd.I and the compound is in
the Z configuration.
6. The compound of claim 2 wherein X.dbd.F.
7. The compound of claim 2 wherein X.dbd.I.
8. The compound of claim 3 wherein X.dbd.I.
9. A method for treating cancer comprising administering to a human
or mammal an effective amount of a compound of claim 1.
10. A method for treating cancer comprising administering to a
human or mammal an effective amount of a compound of claim 2.
11. A method for treating cancer comprismg administering to a human
or mammal an effective amount of a compound of claim 3.
12. The method of claim 9 wherein the cancer is thyroid cancer and
in the compound X.dbd.I.
13. The method of claim 10 wherein tihe cancer is thyroid cancer
and in the compound X.dbd.I.
14. The method of claim 11 wherein tihe ecacer is thyroid cancer
and in the compound X.dbd.I.
15. A pharmaceutical composition comprsing a compound of claim 1
and a pharmaceutically acceptable carrier therefor.
16. A pharmaceutical composition comprising a compound of claim 2
and a pharmaceutically acceptable carrier therefor.
17. A pharmaceutical composition comprising a compound of claim 3
and a pharmaceutically acceptable carrier therefor.
Description
RELATED APPLICATION DATA
[0001] This application is based on and claims the benefit of U.S.
Provisional Patent Application No. 60/505,935 filed on Sep. 24,
2003, the disclosure of which is incorporated herein in its
entirety by this reference.
TITLE OF INVENTION
[0003] Halocombstatins and Methods of Synthesis Thereof.
FIELD OF THE INVENTION
[0004] This invention relates to novel compounds having utility in
the treatment of cancer and/or as antimicrobials.
BACKGROUND OF THE INVENTION
[0005] Pharmaceutical agents to treat cancer and/or tumors are
widely sought. Antiangiogenesis agents are being pursued as a
promising antitwnor therapeutic agents. Combretastatin A-4 is one
such antiangiogenesis agent. Studies have demonstrated that
combretastatin A-4 disrupts the microtubules of human umbilical
vein endothelial cells (HUVEC) in culture. It has also been shown
that the tubulin-binding properties shown in cell-free systems are
retained when the compound enters cells, and that tubulin binding
is a significant component of biological acitivity.
[0006] The African Bush Willow Combretum caffrum has proved to be a
very important source of cancer cell growth inhibitory constituents
named combretastatins. The most potent of these constituents is
combretastatin A-4 (1a, "CA-4"), and its sodium phosphate
derivative (1b, "CA-4P") was advanced to Phase I human cancer
clinical trials in 1998. (Remick, S. C., et al., (1999) Phase I
Pharmacokitictics Study of Single Dose Intravenous (IV)
Combretastatin A-4 Prodrug (CA4P) in Patients (pts) with Advanced
Cancer, Molecular Targets and Cancer Therapeutics Discovery
Discovery, Development, and Clinical Validation, Proceedings of the
AACR-NCI-EORTC International Congress, Washington, D.C., #16, p.
4.) Overall results continue to be promising, and human cancer
Phase II and combination Ib trials are currently underway.
[0007] Antivascular, antiangiogenesis and general antimetastatic
activities of CA4P as well as its synergistic utility in
combination with other anticancer drugs, radioimmunotherapy and
hyperthermia are all areas of active research interest. (see
Griggs, J., et al, Combretastatin A-4 Disrupts Neovascular
Development in Non-Neoplastic Tissue, British J. of Cancer 2001,
84, 832-835; Folkman, J., Angiogenesis-Dependent Diseases, Seminars
in Oncology 2001, 28, 536-542; Kruger, E. A. et al., Approaches to
Preclinical Screening of Antiangiogenic Agents, Seminars in
Oncology 2001, 28, 570-576; Jin, X., et al., Evaluation of
Endostatin Antiangiogenesis Gene Therapy in vitro and in vivo,
Cancer Gene Therapy 2001, 8, 982-989; Vacca, A., et al., Bone
Marrow Angiogenesis in Patients with Active Multiple Myeloma,
Seminars in Oncology 2001, 28, 543-550; Rajkumar, S. V., et al.,
Angiogenesis in Multiple Myeloma, Seminars in Oncology 2001, 28,
560-564, Griggs, J., et al., Potent Anti-metastatic Activity of
Combretastatin AX, Int J Oncol. 2001, 821-825; Pedley, R. B. et
al., Eradication of Colorectal Xenografts by Combined
Radioirnunotherapy and Combretastatin A-4 3-O-Phosphate, Cancer
Research 2001, 61, 4716-4722; Eikesdal, H. P., et al., Tumor
Vasculature is Targeted by the Combination of Combretastatin A-4
and Hyperthermia, Radiotherapy and Oncology 2001, 61, 313-320.)
[0008] Several of the compounds of the present invention are
particularly concerned with treatment of thyroid gland cancer. By
2002, some 20,000 people in the United States were diagnosed with
carcinoma of the thyroid gland; of these the distribution was about
80% papillary and 14% follicular differentiated carcinomas derived
from follicular epithelial cells producing thyroid hormone. Of the
remaining thyroid malignancies, about 4% were medullary carcinoma
(neuroendocrine) and 2% of the exceptionally aggrwsive anaplastic
carcinoma (median survival 4-5 months and a near 100% lethal
outcome). Significantly, the incidence of both follicular and
anaplastic carcinomas are elevated in geographic areas of iodine
deficiency. Radiation exposure represents the most general risk
factor for thyroid cancer. In addition, excess production of the
pituitary homone thyroid-stimulating hormone (THS), which is very
important m regulating thyroid gland growth and fimction, may be
important in the etiology of thyroid cancer. Previously used
clinical treatments for thyroid cancer include surgery, suppression
of THS, .sup.131I-radiotherapy, and anticancer dmgs. But in 2002,
another 1,300 victims of thyroid cancer in the U.S. died,
emphasizing the great need for more routinely effective anticancer
drugs.
SUMMARY OF THE INVENTION
[0009] The present invention relates to novel compounds
constituting modifications of combretastatin A-3 (3a) and its
phosphate prodrug (3b), wherein the 3-hydroxy group or the
3-hydroxy and 5-hydroxy groups are replaced with a halide.
Representative halides are fluorine, chlorine, bromine and iodine.
Salts of the novel compounds are also disclosed herein. Also
described herein are phosphate ester derivatives of the 3-fluoro,
3-chloro, 3-bromo and 3-iodo-stilbenes. Compounds of the Invention
Comprise: ##STR1## [0010] Wherein X is F, Cl Br or I ##STR2##
[0011] Werein X is F, Cl, Br or I, and R is a metal cation such as
Na, Li, K, Cs, Rb, Ca, Mg or is morpholine, piperidine,
glycine-OCH.sub.3, tryptophan-OCH.sub.3 or NH(CH.sub.2OH).sub.3.
##STR3## [0012] Wherin X is F, Cl, Br or I, and Z is a metal cation
such as Na, Li, K, Cs, Rb, Ca, Mg or is morpholine, piperidine,
glycine-OCH.sub.3, trytophan-OCH.sub.3 or NH(CH.sub.2OH).sub.3.
[0013] Several of the compounds of the invention exhibit greatly
enhanced (>10-100x) cancer cell growth ibihbition, as compared
to pnor art combretastin compounds such as CA-4 and CA-3, against a
panel of human cancer cell lines and the muoine P388 leukemia. The
iodo compounds appear to show particular promise in the treatment
of thyroid cancers. The compounds of the present invention exhibit
inhibiting of tubua polymerization and binding of colchicine to
tablulin. In addition, several of the compounds exhibit
antimicrobial properties.
DESCRIPION OF THE DRAWINGS
[0014] FIG. 1 shows the structural formulas of several prior art
compounds.
[0015] FIG. 2 shows the reaction scheme for synthesizing some of
the compounds of the present invention, including strucbual
formulas for the compounds of the invention.
[0016] FIG. 3 shows a continuation of the reaction scheme of FIG.
2.
[0017] FIG. 4 shows the reaction scheme for synthesizing some of
the compounds of the present invention, including structural
formulas for the compounds of the invention.
[0018] FIG. 5 shows photographs of results of the cord formation
assay.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The concept of antiangiogenesis as a therapeutic approach
for the treatment of cancer, particularly tumors, is being actively
pursued as a promising strategy. The compound combretastatin A-4
has previously been demonstrated to disrupt the microtubules of
hurnan umbilical vein endothelial cells (HUVEC) in culture. Those
stes confirmed that the tubulin-binding properties shown in
cell-free systems are retained when the compound enters cells, and
that tubulin binding is a significant component of the biological
activity.
[0020] Thus, an object of the present invention is to provide new
compounds that may be useful as tubulin binding agents.
[0021] A further object of the invention is to provide compounds
that possess antiangiogenesis properties.
[0022] Yet another object of the invention is to provide compounds
for use as therapeutic agents for the treatment of mammals,
including humans, afflicted with cancer, particularly tumors.
[0023] Still a further object of the invention is to provide
compounds for use as antimicrobials.
[0024] Results and Discussion
[0025] Preparation of the stilbenes of the present invention was
accomplished as described in detail herein. The reaction sequence
was initiated by protection of isovanillin as the
tert-butyldiphenylsilyl ether 4. Benzaldehyde 4 was reduced using
sodium borohydride to benzyl alcohol 5, followed by conversion to
phosphonium bromide 6. Condensation of Wittig intermediate 6 with
the respective halo-aldehyde using n-butyllithium in THF led to
silyl group protected stilbenes 7-10. Subsequent deprotection
(Scheme 2) with tetrabutylammonium fluoride afforded
3-halo-stilbenes 11-14. The Z isomers 11a, 13a and 14a were
phosphorylated using dibenzylphosphite, diisopropylethyl-amine,
N,N-dimethylamino-pyridine and carbon tetrachloride in acetonitrile
to provide bisbenzyl phosphates 15-17. Debenzylation of phosphate
esters 15-17 was achieved using trirnethylsilybromide followed by
the corresponding base to produce phosphates 18-20. (See Pettit, G.
R., et al., Antineoplastic Agents 440. Asymmetric Synthesis and
Evaluation of the Combretastatin A-1 SAR Probes (1S,2S) and
(1R,2R)-1-2-Dihydroxy-1-(2',3'-dihydroxy-4'-methoxyphenyl)-2-(3'',4'',5''-
-timethoxyphenyl)-ethane, J. Nat. Prod. 2000, 63, 969-974; Pettit,
G. R., et al., Antineopiastic Agents 460. Synthesis of
Combretastatin A-2 Prodrugs, Anticancer Drug Design 2001, 16,
185-194; Pettit, G. R., et al. Antineoplastic Agents 463. Synthesis
of Combretastatin A-3 Diphosphates, Anticancer Drug Design 2000,
15, 397404.; Ladd, D. L., et al.; A New Synthesis of
3-Fluoroveratrole and Z-Fluoro-3,4 Dimethoxy Benzaldahyde, Synth.
Commun. 1985, 15, 61.)
[0026] Compared to the related combretastatins, the new
halo-stilbenes or halocombstatins shown in Table I as compounds 11a
through 20a, all exhibited very strong inhibition of cancer cell
growth. The three stilbenes (11a, 13a, 14a) converted to phosphate
salts all retained strong activity and demonstrated markedly better
aqueous solubility than their 3-halo-stilbene precursors. The E
geometrical isomers evaluated appeared in vitro to be much less
effective as inhibitors of cancer cell growth.
[0027] Because of their potent cytotoxicity, the four
halocombstatins (11a, 12a, 13a, and 14a) were compared to
combretastain A-4 (1a) for inhibitory effects on tubulin
polymerization and on the binding of [.sup.3H]colchicine to
tubulin. The results of this comparison are shown in Table II.
These experiments demonstrate that the five compounds are
essentially identical in their apparent interactions with tubulin.
The four halocombretastatins inhibited the polymerization reaction
with IC.sub.50 values of 1.5-1.6 .mu.M:M, versus an IC.sub.50 value
of 1.8 .mu.M:M for CA4 (1a). The minor differences between the
compounds were within experimental error as indicated by the
standard deviations.
[0028] Similarly, all four cis-stilbenes were highly potent
inhibitors of the colchicine binding assay. When present at a
concentration one fifth of that of [.sup.3]colchicine but equimolar
to the tubulin concentration, binding of the radio labeled ligand
was inhibited by 75-89% (note that the lowest and highest
inhibitory effects were observed with stilbenes 11a and 13a, which
were the two compounds that displayed the greatest inhibitory
effects in the polymerization assay). In an earlier study,
combretastatin A-3 (3a), with a hydroxyl substituent instead of the
methoxy group or a halogen at position C-3 in the A ring, was found
to be about half as active as CA4 (1a) as an inhibitor of tubulin
assembly, about one fifth as active as an inhibitor of colchicine
binding to tubulin, and about one seventh as active as an inhibitor
of cell growth. (See Lin, C. M., et al, Interactions of Tubulin
with Potent Natural and Synthetic Analogs of the Antimnitotic Agent
Combretastatin: A Structure-activity Study, Mol. Pharmacol., 1988,
34, 200-208). A related finding is that elimination of the C-3
substituent entirely, by replacing it with a hydrogen atom, results
in about a 7-fold reduction in inhibitory effect on polymerization
and complete loss of cytotoxic activity. (See Cusliman, M., et al.,
Synthesis and Evaluation of
(Z)-1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethane as
Potential Cytotoxic and Antimitotic Agents, J. Med. Chem. 1992, 35,
2293-2306.)
[0029] Thus, while not intending to be bound by this theory, it
appears the optimal activity observed with CA4 (1a) and the novel
halocombstatins of the present invention requires a C-3 substituent
of some size, where the fluorine atom may represent a minimum.
Therefore, it seems unlikely that the predominant effect of the
substituent results from direct enhancement of the interaction of
ligand with protein. The A-ring substituents most likely cause the
active cis-stilbenes to assume with greater probability a
conformation that favors the drug-tubulin interaction. (See Hamel,
E.; Evaluation of Antimitotic Agents by Quantitative Comparisons of
Their Effects on the Polymerization of Purified Tubulin, Cell
Biochem. Biophys., In Press.)
[0030] Tubulin polymerization was evaluated by turbidimetry at 350
nm using Beckman DU7400/7500 spectrophotometers as described in
detail elsewhere. (See National Committee for Clinical Laboratory
Standards. Reference Method for Broth Dilution Antifungal
Susceptibility Testing of Yeasts. Approved Standard M27-A. Wayne, P
A: NCCLS, 1997.) Varying concentrations of drug were preincubated
with 10 .mu.M:M (10 mg/mL) purified tubulin (See Hamel, E., et al.,
Separation of Active Tubulin and Microtubule-associated Proteins by
Ultracentrifiigation and Isolation of a Component Causing the
Formation of Microtubule Bundles, Biochemistry 1984, 23,
4173-4184). Samples were chilled on ice, GTP (0.4 mM) was added,
and polymerization was followed at 30.degree. C. The parameter
measured was extent of the reaction after 20 minutes. Coichicine
binding was measured as described in detail previously. Reaction
mixtures contained 1.0 .mu.M :M tubulin, 5.0:M, [.sup.3H]colchicine
(from Dupont), and inhibitor at 1.0 .mu.M:M. Incubation was for 10
minutes at 37.degree. C.
[0031] The inventors have also demonstrated the ability of
halocombstatins 11a and 12a to disrupt microtubules in human
umbilical vein endothelial cells (HUVEC). HUVECs were isolated
according to methods know to one of skill in the art (see Jaffe, E.
A. et al., Culture of Human Endothelial Cells Derived From
Umbilical Veins. Identification by Morphologic and Immunologic
Criteria, J. Clin. Invest. 1973, 52, 2754-2756.)
[0032] In a further detailed series of experiments, compound 11a
(flurocombstatin) was further evaluated against HUVECs in vitro.
These cells showed significant sensitivity to the fluorocombstatin
(11a): ED.sub.50 0.00025 .mu.g/mL. Cords length as well as junction
numbers were markedly reduced at both 0.01 and 0.001 .mu.g/mL
compared to untreated controls. Such activity against endothelial
cells is significant, as endothelial cells are known to play a
central role in the angiogenic process.
[0033] The halocombstatins of the present invention appear to also
have antimicrobial properties. More specifically, they appear to
have antifungal and/or antibacterial properties. Antimicrobial
evaluation of the halocombstatins involved susceptibility testing
performed by the reference broth microdilution assay. The
antimicrobial activities of the halocombstatins were very similar,
targeting Gram-positive bacteria and the pathogenic fungi
Cryptococcus neoformans, and results are shown in Table III. The
sodium phosphate dervative (16a) of fluorocombstatin (11a) did not
retain significant antimicrobial activity.
[0034] Similarly, the inventors have previously shown that
combretastatin A-3 but not its sodium phosphate prodrug inhibited
growth of the pathogenic fungus Cryptococcus neoformans. (See
Pettit, G. R., et al., Antineoplastic Agents 463. Synthesis of
Combretastatin A-3 Diphosphates, Anticancer Drug Design 2000, 15,
397-404.) To determine the antimicrobial activity of the present
compounds, susceptibility testing was performed by the reference
broth microdilution assay. (See National Committee for Clinical
Laboratory Standards. Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria that Grow Aerobically. Approved
Standard M7-A5. Wayne, P A: NCCLS, 2000. National Committee for
Clinical Laboratory Standards. Reference Method for Broth Dilution
Antifungal Susceptibility Testing of Yeasts. Aproved Standard
M27-A. Wayne, P A: NCCLS, 1997.) The antimicrobial activities of
the halocombretastatins of the present invention were very similar,
targeting Gram-positive bacteria and Cryptococcus neoformans. This
is illustrated in further detail in Table III. Thus, several of the
novel compounds of the present invention appear to have potential
as antimicrobial agents, such as antifingals and
antibacterials.
Experimental Section
[0035] Materials and Methods. All solvents (ether refers to diethyl
ether) and reagents were obtained from commercial sources (Acros
Organics, Sigma-Aldrich Co., Alfa Aesar, City Chemicals or
Lancaster Synthesis, Inc.). The 3-iodo-4,5-dimethoxybenzaldehyde
was purchased from Lancaster Synthesis, Solvents were redistilled.
Solvent extracts of aqueous solutions were dried over anhydrous
magnesium sulfate. Gravity column chromatography was performed
using silica gel from VWR Scientific 70-230 mesh) or from Merck
(230-400 mesh). Analtech silica gel GHLF plates were employed for
TLC.
[0036] All melting points were determined with an electrochemical
digital melting point apparatus, Model 9100 or IA-9200, and are
uncorrected. NMR spectra were recorded employing Varian Gemini 300
or Varian Unity 400 instruments. Chemical shifts are reported in
ppm downfield from tramethylsilane as an internal standard in
CDCl.sub.3 or where noted in D.sub.2O. High resolution mass spectra
were obtained with a Kratos Ms-50 instrument (Midwest Center for
Mass Spectroscopy, University of Nebraska-Lincoln) or in the Cancer
Research Institute at Arizona State University with a Jeol LCmate
instrument. Elemental analyses were determined by Galbraith
Laboratories, Inc., Knoxville, Tenn.
[0037] General Procedure for Synthesis of
Dimethoxyhalobenzaldehydes.
[0038] 3-Fluoro-4,5-ditnethoxybenzaldehyde. To a stirred solution
prepared from 100 mL of DMF and 5-fluorovanillin (lit 1.0 g, 5.88
mmol). After 15 minutes, iodomethane was added, and stirring at
room temperature continued for 16 hours. The reaction was
terminated by the. addition of water, the mixture was extracted
with hexane (3.times.100 ML), and solvents were removed in vacuo.
Purification by flash chromatography on a column of silica gel
using hexane-ethyl acetate (4:1) as eluent afforded a colorless
solid (1 g, 93% yield); mp 51-53.degree. C. (Lit.sup.17 mp
52-53.degree. C.) .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 3.94
(s, 3H), 4.05 (s, 3H), 7.24 (s, 1H), 7.26 (s, 1H), 9.82 (s,
1H).
[0039] 3-Chloro-4,5-dimethoxybenzaldehyde. The preceding reaction
was repeated with 5-chlorovanillin (10 g, 54 mmol) to give this
compound, which was isolated as set forth in the preceding
experiment to afford a colorless solid (10.4 g, 97% yield); mp
88-90.degree. C. (Lit.sup.17 mp 87-89.degree. C.); .sup.1H-NMR (300
MHz, CDCl.sub.3) .delta. 3.95 (s, 3H), 3.96 (s, 3H), 7.36 (d, J=1.5
Hz, 1H), 7.50 (d, J=1.5 Hz, 1H), 9.85 (s, 1H).
[0040] 3-Bromo-4,5-dimethoxybenzaldehyde. The experiment was
repeated with 5-bromovanillin (10 g, 43.3 mmol) as described for
the preceding aldehydes to give compound (6) which was separated by
flash chromatography on a column of silica gel using hexane-ethyl
acetate (9:1) as eluent to afford a colorless solid (8 g, 75%
yield); mp 64-65.degree. C.; .sup.1H-NMR (300 MHz, CDCl.sub.3)
.delta. 3.94 (s, 3H), 3.95 (s, 3H), 7.40 (d, 1H, J=1.8 Hz), 7.65
(d, 1H, J=1.8 Hz), 9.85 (s, 1H).
[0041] 3,5-diiodo-4-methoxybenzaldehyde
[0042] 3-Iodo-4,5-dimethoxybenzaldehyde was obtained from
Sigma-Aldrich Chemical Company.
[0043]
3-O-tert-Butlydiphenylsiloxy-imethoxybenzytriphenylphosphonium
bromide (6). To 400 mL of dry dichloromethane was added benzyl
alcohol 5 (84 g, 214 mmol) (Pettit, G. et al., Antineoplastic
Agents 463, Synthesis of Combretastatin A-3 Diphosphates.
Anticancer Drug Design 2000, 15, 397-404) and phosphorous
tribromide (10 mL, 106 mmol, 0.5 eq). The reaction mixture was
allowed to stir for 16 hours, and was terminated by the addition of
10% NaRCO.sub.3, and the product was extracted with
dichloromethane. The solvent was removed (in vacuo), the resulting
benzyl bromide was dissolved in 500 mL of toluene, and
triphenylphosphine (62 g, 236 mmol, 1.1 eq) was added. The mixture
was heated at reflux for 1 hour and stirred at RT for 15 hours. The
precipitate was collected and triturated with ether to afford 132 g
of phosphonium salt, in 86% yield; .sup.1H-NMR (300 MHz CD.sub.3OD)
.delta.1.00 (s,9H), 3.51 (s, 3H), 4.69 (d, 2H, J=17.4 Hz), 6.34
(dt, 1H, J=2.4, 8.1 Hz), 6.59 34 (d, 1H, J=8.1 Hz), 6.65 34 (t, 1H,
J=2.4 Hz); and .sup.13C NMR (75 MHz CD.sub.3OD) .delta. 20.47,
27.07, 55.60, 102.20, 113.15, 118.48, 119.60, 123.43, 126.56,
126.85, 128.17, 128.74, 13107, 131.12, 133.91, 135.10, 135.23,
136.17, 136.55, 146.55, 152.76.
[0044] General Procedure for the Stilbene Syntheses.
[0045]
3-Fluoro-4,4',5-trimethoxy-3'-O-tert-butyldiphenylsilyl-Z-stilbene
(7a). To a mixture of phosphonium salt 6 (4.7 g, 6.5 mmol) and
tetrahydrofuiran (25 ml, cooled to -78.degree. C.) was added n-BuLi
(2.6 mL, 2.5 M, 6.5 mmol, over 5 minutes), followed by stirring for
one hour. Next, 3-fluoro-4,5-dimethoxybenzaldehyde (1 g, 5.4 mmol)
in tetrahydrofiiran (10 ml) was added (dropwise) over 30 minutes.
The mixture was allowed to warm. to room temperature, and stirring
continued for 16 hours. The reaction was terminated by the addition
of water (50 mL), the product was extracted with ethyl acetate,
solvents were removed in vacuo, and the residue (1:1 F/Z, 75%
yield) obtained was subjected to flash chromatography on silica gel
using hexanesthyl acetate (9:1) as eluent to afford Z-stilbene 7a
(1 g, 34%/o) as a clear oil; .sup.1H-NMR (300 MHz, CDCl.sub.3)
.delta. 1.07 (s, 9H), 3.46 (s, 3H), 3.65 (s, 3H), 3.90 (s, 3H),
6.24 (d, 1H, J=12 Hz), 6.33 (d, 1H, J=12 Hz), 6.56 (m, 2H), 6.72
(m, 3H), 7.35 (m, 6H), 7.70 (m, 4H); and .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 19.75, 26.65, 55.07, 55.99, 61.43, 108,26,
108.28, 109.40, 109.55, 111.74, 120.83, 122.42, 127.36, 127.46,
127.48, 127.70, 129.37, 129.50, 129.63, 130.32, 132.59, 132.66,
133.57, 134.77, 135.27, 135.83, 135.95, 144.74, 149.88, 152.91,
152.95, 154.59, 156.53; HRMS (caled for C.sub.33H.sub.36FO.sub.4Si)
[M+H]+ 543.2368, found 543.2372.
[0046] Further elution gave the E-isomer 7b (1.2 g, 41% yield):
.sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 1.19 (s, 9H), 3.58 (s,
3H), 3.92 (s, 3H), 3.95 (s, 3H), 6.48 (d, 1H, J=15.9 Hz), 6.76 (d,
1H, J=8.7 Hz), 6.77 (d, 1H, J=16.5 Hz), 6.8 (d, 1H, J=1.5 Hz), 6.92
(d, 1H, J=2.1 Hz), 6.97 (dd, 1H, J=1.8, 8.4 Hz), 7.42 (m, 6H), 7.78
(m, 4H). .sup.13C NMR (75 MHz, CDCl.sub.3).delta. 19.76, 26.62,
55.20, 56.13, 61.39, 105.40, 106.45, 106.75 112.62, 117.62, 120.57,
125.26, 127.48, 128.52, 129.58, 139.68, 133.15, 133.28, 133.59,
135.35, 145.14, 150.52, 153.52. HRMS calcd for.
C.sub.33H.sub.36FO.sub.4Si [M+H].sup.+ 543.2368, found
543.2392.
[0047]
3Chloro-4,4',5-trimethoxy-3'-O-tert-butyl-dipbenylsilyl-Z-stilbene
(8a). The experimental procedure noted above for 7a was repeated
with 3-Chloro-4,5-dimethoxybenzaldehyde (2.8 g, 14 mmol) to yield
the Z-isomer 8a (1.6 g, 21%) as a clear oil: .sup.1H-NMR (300 MHz,
CDCl.sub.3) .delta. 1.07 (s, 9H), 3.46 (s, 3H), 3.60 (s, 3H), 3.84
(s, 3H), 6.24 (d, 1H, J=12 Hz), 6.34 (d, 1H, J=12 Hz), 6.59 (s, 1H,
J=7.5 Hz), 6.66 (s, 1H), 6.73 (s, 1H), 6.73 (d, 1H, J=9 Hz) 6.81
(s, 1H), 7.33 (m, 6H), 7.65 (dd, 4H, J=6.67, 1.2 Hz), and .sup.13C
NMR (125 MHz, CDCl.sub.3) .delta. 19.76, 26.66, 55.11, 55.82,
60.71, 111.35, 111.75, 120.86, 122.40, 122.46, 127.13, 127.38,
127.85, 129.32, 129.51, 130.27, 133.60, 133.81, 135.27, 144.22,
144.75, 149.91, 153.12; HRMS calcd for.
C.sub.33H.sub.36ClO.sub.4SiCl 561.2042 [M+H].sup.+, found 561.2449,
Cl 559.2071 [M+H].sup.+; found 559.1996.
[0048] Continued elution of the chromatographic column led to the
isolation of E-stilbene 8b (4.9 g, 62% yield) as a clear oil;
.sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 1.14 (s, 9H), 3.56 (s,
3H), 3.86 (s, 3H), 3.90 (s, 3H), 6.44 (d, 1H, J=16.5 Hz), 6.74 (d,
1H, J=16.5 Hz), 6.74 (s, 1H), 6.82 (d, 1H, J=1.5 Hz), 6.87 (d, 1H,
J=1.8 Hz), 6.94 (dd, 1H, J=8.1, 2.1 Hz), 6.99 (d, 1H, J=1.5 Hz),
7.40 (m, 6H), 7.75 (m, 4H); and .sup.13C NMR (75 Mz, CDCl.sub.3)
.delta. 19.82, 26.67, 55.30, 55.09, 60.78, 108.43, 112.13, 117.71,
119.78, 120.59, 124.97, 127.53, 128.78, 129.61, 129.73, 133.65,
134.31, 135.41, 144.55, 150.61, 153.74.
[0049] 3-Bromo-4,4
,5-trimethoxy-3'-O-tert-butyl-diphenylsilyl-Z-stilbene (9a). To 100
mL of THF was added phosphonium salt 6 (25.7 g, 36 mmol) and the
solution cooled to -78.degree. C. Once the temperature reached
-78.degree. C., n-BuLi (14.4 mL, 2.5 M, 36 mmol) was added over 5
minutes followed by stirring for one hour. Then the
bromo-benzaldehyde (8 g, 33 mmol, in 100 mL THF) was added dropwise
over 30 minutes. The mixture was allowed to warm to room
temperature and stfiing continued for 16 hours. The reaction was
then terminated by the addition of water (50 mL), product was
extracted with ethyl acetate, solvents were removed in vacuo, and
the residue was separated by column chromatography to yield 4.2 g
9a (Z-stilbene), 2:1, E:Z (65% overall yield); HRMS
(M+Na)+625.1364, (M+Na)+2 627.1338; IR 2962, 1730, 1510, 1267, 908,
735, 650 cm.sup.-1; .sup.1HNMR (300 MHz, CDCl.sub.3) .delta. 1.07
(s, 9H), 3.45 (s, 3H), 3.58 (s, 3H), 3.82 (s, 3H), 6.97 (d, 1H,
J=1.5 Hz), 6.23 (d, 1H, J=12 Hz), 6.32 (d, 1H, J=12 Hz), 6.52 (d,
1H, J=8.1 Hz), 6.71 (dd, 1H, J=1.5 Hz, J=8.1 Hz), 7.57 (d, 1H,
J=1.5 Hz), 7.32 (m, 6H), 7.65 (dd, 4H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 152.87, 149.79, 145.16, 144.66, 135.17, 134.37,
133.52, 130.40, 129.43, 129.24, 127.30, 126.90, 125.16, 122.40,
120.79, 117.18, 112.01, 111.71, 94.38, 60.61, 55.81, 55.15,
21.10.
[0050] Further elution of the chromatogram led to isolation of 8.1
g of the E-isomer 9b: IR 2934, 2859, 1710, 1510, 1275, 908, 732,
650 cms.sup.-1; .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 1.14 (s,
9H), 3.54 (s, 3H), 3.84 (s, 3H), 3.88 (s, 3H),), 6.46 (d, 1H, J=12
Hz), 6.76 (d, 1H, J=12 Hz), 6.71 (d, 1H, J=8.1 Hz), 6.85 (d, 1H,
J=2.1 Hz), 6.87 (d 1H, J=2.1), 6.94 (dd, 1H, J=8.4 Hz, J=2.4 Hz),
7.15 (d, 1H, J=2.4) 7.38 (m, 6H), 7.74 (dd, 4H); and .sup.13C-NMR
(75 MHz, CDCl.sub.3) .delta. 19.76, 26.65, 55.25, 56.03, 60.59,
109.18, 109.85, 112.13, 117.70, 120.57, 122.59, 124.79, 127.48,
128.80, 129.58, 133.64, 134.91, 135.35, 145.16, 150.57, 153.58.
[0051] 3-Iodo-4,4',5-trimethoxy-3'O-tert-butyl-diphenyl-Z-stilbene
(10a). A gradient column chromatogram from 0-3% ethyl acetate in
hexane afforded Z-stilbene 10a (1.4 g) in 21% yield mp
122-124.degree. C.: HRMS, found: [+H].sup.+ 651.1474.
C.sub.33H.sub.36O.sub.4Si requires [M+H].sup.+, 651.1427;
.sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 1.07 (s, 9H), 3.45 (s,
3H), 3.55 (s, 3H), 3.79 (s, 3H), 6.21 (d, 1H, J =12 Hz), 6.31 (d,
1H, J=12 Hz), 6.59 (d, 1H, J=7.8 Hz), 6.72 (s, 2H), 6.77 (dd, 1H,
J=7.8, 1.5 Hz), 7.19 (d, 1H, J=1.8 Hz), 7.32 (m, 6H), 7.64 (d, 4H,
J=7.5 Hz); and .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 19.68,
26.62, 55.05, 55.56, 60.33, 91.94, 111.72, 113.09, 120.78, 122.43,
126.73, 127.33, 129.32, 130.28, 130.93, 133.54, 135.17, 144.70,
149.82, 151.82.
[0052] General Procedure for Cleavage of the Silyl Ether Protecting
Group.
[0053] 3-Fluoro-4,4',5-trimethoxy-3'-hydroxy-Z-stilbene(11a,
Fluorcombstatin). A solution prepared from Z-isomer 7a (2.4 g, 4.4
mmol), tetrahydrofuran (50 ml) and 1M tetrabutylammonium fluoride
(4.5 ml, 4.5 mnmol) was stirred fro 3 hours. The reaction was
terminated by the addition of water (50 ml), the mixture was
extracted with ethyl acetate and the solvents were removed in
vacuo. Separation by flash chromatography using: 1:4 ethyl
acetate-hexane as eluent provided Z-stilbene (11a) (1.12 g, 83%) as
a colorless solid, which was recrystallized from ethyl
acetate-hexane: mp 93-94.degree. C.; (300 MHz, CDCI.sub.3) .delta.
3.67 (s, 3H), 3.87 (s, 3H, 3.90 (s, 31), 5.30 (bs, 1H), 6.35 (d, J
=12 Hz, 1H), 6.48 (d, J=12 Hz, 1H), 6.61 (d, J=2.4 Hz, 1H), 6.64
(d, J=1.8 Hz, 1H), 6.72 (d, J=8.4 Hz, 1H), 6.75 (dd, J=1.5, 8.4 Hz,
2H), 6.86 (d, J=1.5 Hz, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. 55.87, 56.01, 61.39, 108.24, 109.37, 109.57, 110.32,
114.83, 120.88, 127.72, 130.03, 130.14, 132.38, 132.48, 135.81,
135.95, 145.15, 145.78, 152.85, 152.88, 154.22, 156.65; and
.sup.19F NMR (CDCl.sub.3) .delta. -11.32 (d, J=12.8 Hz, 1F). HRMS
calcd for C.sub.17H.sub.18FO.sub.4 305.1189 [M+H].sup.+.
[0054] 3-Fluoro-4,4',5-trimethoy-3'-hydroxy-E-stilhene (11b).
Cleavage of silyl ester 7b (150 mg, 0.27 mmol) was performed as
described for the synthesis of 11a. Separation by flash
chromatography on silica using ethyl acetate-hexane (3:7) afforded
a colorless solid 11b (75 mg, 88% yield): mp 86-87.degree. C.,
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 3.87 (s, 3H), 3.90 (s,
3H), 3.93 (s, 3H), 5.75 (bs, 1H), 6.77-6.86 (m, 5H), 6.93 (m, 1H),
6.86 (d, J=1.8 Hz, 1H). .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
55.87, 56.18, 61.39, 100.64, 105.68, 106.51, 106.79, 107.55,
110.63, 111.76, 119.30, 125.80, 127.61, 128.62, 129.53, 130.56,
133.13, 133.23, 134.73, 136.41, 145.78, 146.59, 153.57, 154.40,
157.64.
[0055] 3-Cloro-4,4',5-trimethoxy-3'-hydroxy-Z-stilbene (12a).
Deprotection of silyl ester 8a (1.5 g, 2.7 mmol) was conducted as
summarized for the synthesis of 11a. Separation by flash
chromatography on silica using ethyl acetate-hexane (3:7) gave
compound 12a (754 mg, 89%). Recrystallization from hexane gave a
white solid; mp 105-106.degree. C.; .sup.1H-NMR (300 MHz,
CDCl.sub.3) .delta. 3.65 (s, 3H), 3.86 (s, 3H), 3.88 (s, 3H), 5.52
(s, 1H), 6.36 (d, 1H, J=12.3 Hz), 6.49 (d, 1H, J=12 Hz), 6.75 (m,
3H), 6.88 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 55.86,
55.94, 60.76, 110.37, 111.40, 114.86, 114.94, 121.05, 122.51,
127.60, 127.92, 130.15, 130.43, 133.74, 144.36, 145.31, 145.92,
153.21.
[0056] 3-Chloro-4,4',5-trimethoxy-3'-hydroxy-E-stilbene 5 (12b).
Column chromatography (elution with 7:3 hexane-ethyl acetate)
afforded a colorless solid, E-isomer 12b, mp 138-140.degree. C., in
79% yield: .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 3.87 (s, 3H),
3.88 (s, 3H), 3.90 (s, 3H), 5.69 (bs, 1H), 6.79 (d, 1H, J=15.9 Hz),
6.81 (d, 1H, J=8.4 Hz), 6.90 (d, 1H, J=1.5 Hz), 6.90 (d, 1H, J=15.9
Hz), 6.94 (dd, 1H, J=8.1, 1.5 Hz), 7.08 (dd, 1H, J=1.8 Hz), 7.11
(d, 1H, J=2.1 Hz); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 55.93,
56.06, 60.74, 100.66, 108.66, 110.65, 111.77, 119.38, 119.79,
125.49, 128.41, 128.85, 130.59, 134.24, 144.65, 145.79, 146.61,
153.78. HRMS calcd for C.sub.17H.sub.17ClO.sub.4 321.0894
[M+H].sup.+, found 321.0893. Anal. Calcd for
C.sub.17H.sub.17ClO.sub.4 C, H.
[0057] 3-Bromo-4,4',5-trimethoxy-3'-hydroxy-Z-stilbene (13a). The
silyl ester cleavage reaction for 9a (4 g, 6.6 mmol) was completed
as described for the synthesis of phenol 11a. Isolation by flash
chromatography on silica gel using ethyl acetate-hexane (1:4) gave
compound 13a (2.22 g of 92%). Recrystallization from hexane
afforded a colorless solid: mp 108-109.degree. C.; HRMS calcd for
C.sub.17H.sub.17BrO.sub.4 364.0303, found [M.sup.+2] 366.0287;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 3.63 (s, 3H) 3.84 (s,
3H), 3.86 (s, 3H), 6.34 (d, 1H, J=12 Hz), 6.49 (d, 1H, J=12 Hz),
6.73 (d, 1H, J=8.4 Hz), 6.77 (dd, 1H, J=8.7, 1.8 Hz), 6.79 (d, 1H,
J=1.8 Hz), 6.86 (d, 1H, J=1.5 Hz), 7.04 (d, 1H, J=1.5 Hz). .sup.13C
NMR (75 MHz, CDCl.sub.3) .delta. 55.77, 55.88, 60.56, 110.45,
112.13, 114.98, 117.18, 121.01, 125.28, 127.33, 130.07, 130.43,
134.37, 145.32, 146,02, 153.03. IR 3539, 3441, 3011, 2939, 2839,
1554, 1510, 1273, 1047, 908, 732 cm.sup.-1. HRMS calcd for
C.sub.17H.sub.17O.sub.4.sup.81Br. 366.0287.
[0058] 3-Brombo-4,4',5-trimethoxy-3'-hydroxy-E-stilbene (13b). By
the same procedure used to obtain phenol 13a, silyl ester 9b was
converted to E phenol 13b, and isolated by flash chromatography on
silica gel with ethyl acetate-hexane (3:7) to give E-isomer 13b
(0.14 g, 81%). Recrystallization from hexane gave colorless solid.
mp 152-154.degree. C.; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
3.86 (s, 3H), 3.89 (s, 3H), 3.90 (s, 3H), 6.80 (d, 1H, J=15.9 Hz),
7.38(d, 1H, J=15.9 Hz), 6.82 (d, 1H, J=8.4 Hz), 6.96 (dd, 1H,
J=8.4, 2.4 Hz), 6.88 (s, 1H), 1H), 7.11 (d, 1H, J=1.8 Hz), 7.25 (d,
1H, J=1.5 Hz); and .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 56.00,
56.11, 60.58, 94.36, 109.32, 110.60, 11.72, 117.81, 119.32, 122.58,
125.29, 128.82, 130.53, 134.81, 145.60, 145.70, 146.50, 153.53.
[0059] 3-lodo-4,4',5trimethoxy-3'-hydroxy-Z-stilbene (14a). The
silyl ester cleavage reaction for 10a was completed as described
for the phenol 11a. The crude product was separated by column
chromatography using 1:4 ethyl acetate-hexane as eluent to give
1.38 g of Z-isomer 14a in 81% yield: mp 92-94.degree. C: HRMS calc
for C.sub.17H.sub.18O.sub.4Si found (M+H).sup.+ 413.0250. .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 3.61 (s, 3H), 3.81 (s, 3H), 3.84
(s, 3H), 6.32 (d, 1H, J=12 Hz), 6.34 (s, 1H), 6.56 (d, 1H, J=12
Hz), 6.75 (s, 1H), 6.83 (d, 1H, J=1.8 Hz), 6.85 (s, 3H), 7.25 (d,
1H, J=1.5 Hz); and .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 55.56,
55.82, 60.33, 91.78, 110.50, 113.11, 115.00, 120.91, 126.96,
129,94, 130.28, 135.93 145.29, 146.10, 147.67, 151.79. IR 3543,
3011, 2937, 2841, 1510, 1273, 1001, 908, 732 cm.sup.-1.
[0060] 3-Iodo-4,4',5-trimethoxy-3'-hydroxy-E-stilbene (14b).
Separation by column chromatography (30% ethyl acetate-hexane as
eluent) gave 0.29 g of E-isomer 14b in 98% yield: mp
111-113.degree. C.; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 3.84
(s, 3H), 3.87 (s, 3H), 3.88 (s, 3H), 5.85 (bs, 1H), 6.77 (d, 1H,
J=16.5 Hz), 6.89 (d, 1H, J=16.5 Hz), 6.82 (s, 1H), 6.96 (s, 1H),
6.93 (d, 1H, J =2.4 Hz), 7.11 (d, 1H, J=1.5 Hz), 7.46 (d, 1H, J
=1.5 Hz); and .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 55.85,
60.41, 92.56, 110.40, 110.63, 111.77, 119.28, 124.97, 128.36,
128.70, 130.15, 135.71, 145.71, 146.56, 148.11, 152.44.
[0061] Dibenzyl (Z)-3-fluoro-4,4',5-trimethoxy-Z-stilbene
3'-O-phosphate (15). Z-stilbene 11a (1.1 g, 3.6 mmol) in 20 mL of
acetonitrile (20 mL) and 3.5 mL (36 mmol) of carbon tetrachloride
was cooled to -10.degree. C., and stirred for 10 minutes. Then
DIPEA (1.3 mL, 7.4 mmol), immediately followed by DMAP (44 mg, 0.36
mmol), were added. After 1 minute dibeiizyl phosphite (1.2 mL, 5.4
mmol) was added over 5 minutes, and the mixture was stirred for an
additional 3 hours at -10.degree. C. The reaction was terminated by
the addition of 0.5 M KH.sub.2PO.sub.4, the mixture was extracted
with ethyl acetate, solvents were removed in vacuo, and the product
was isolated by column chromatography (1;1 elution with ethyl
acetate-hexane) to yield 1.5 g of phosphate in 74% yield: b.p. dec.
280.degree. C. (0.01 mmHg); .sup.1H-NMR (500 MHz, CDCl.sub.3)
.delta. 3.65 (s, 3H), 3.77 (s, 3H), 3.87 (s, 3H), 5.12 (s, 2H),
5.14 (s, 2H), 6.38 (d, 1H, J=12 Hz), 6.43 (d, 1H, J=12 Hz), 6.57
(s, 1H) 6.62 (dd, 1H, J=1.5, 11.5 Hz), 6.78 (d, 1H, J=8.5 Hz), 7.03
(d, 1H, J=8.5 Hz), 7.12 (s, 1H), 7.82 (m, 10H); .sup.13C NMR (125
MHz, CDCl.sub.3) .delta.; and .sup.31P NMR (162 MHz CDCl.sub.3)
.delta. -7.84 (s).
[0062] Dibenzyl (3-bromo-4,4',5-trimethoxy-Z-stilbene
3'-O-phosphate (16). The preceding reaction (see Compound 15) was
repeated with Z-stilbene 13a (1 g, 2.7 mmol) to afford 1.6 g of
phosphate 16 in 94% yield: b.p. dec. 271.degree. C. (0.01 mmHg);
.sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 3.59 (s, 3H), 3.76 (s,
3H), 3.79 (s, 3H), 5.11 (s, 2H), 5.13 (s, 2H), 6.37 (d, 1H, J=12.4
Hz), 6.43 (d, 1H, J=12 Hz), 6.72 (d, 1H, J=1.5 Hz), 6.78 (d, 1H,
J=8.4 Hz), 7.02 (d, 1H, J=8.2 Hz), 7.03 (d, 1H, J=2.4 Hz), 7.10 (d,
1H, J=1.8 Hz), 7.28 (m, 10H); .sub.13C NMR (75 MHz, CDCl.sub.3)
.delta. 55.77, 55.88, 60.51, 65.59, 69.67, 69.73, 111.83, 112.22,
117.23, 121.96, 121.99, 125.04, 126.32, 126.75, 127.30, 127.69,
127.77, 127.88, 128.31, 128.35, 129.46, 129.47, 133.93, 135.44,
135.51, 139.30, 139.38, 145.30, 149.77, 149.82, 152.99; and
.sup.31P NMR (162 MHz CDCl.sub.3) .delta. -7.84 (s).
[0063] Dibenzyl 3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate
(17). The phosphorylation reaction used to obtain phosphate 15 was
repeated with Z-stilbene 14a (2.39 g, 0.95 mmol) to obtain 0.55 g
of Z-stilbene 17 in 86% yield as a colorless oil. b.p. dec.
274.degree. C. (0.01 mmHg); HRMS calc for C.sub.31H.sub.31PO.sub.7
[M+H].sup.+ 673.0852; found [+H].sup.+, 673.0808. .sup.1H-NMR (300
MHz, CDCl.sub.3) .delta. 3.51 (s, 3H), 3.65 (s, 3H), 3.72 (s, 3H),
5.04 (s, 2H), 5.06 (s, 2H), 6.36 (d, 1H, J=9 Hz), 6.42 (d, 1H, J=9
Hz), 6.77 (d, 1H, J=1.2 Hz), 6.89 (d, 1H, J=6 Hz), 7.02 (d, 1H, J=6
Hz), 7.01 (s, 1H), 7.19 (d, 1H, J=1.2 Hz), 7.28 (m, 10H); and
.sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. 56.20, 56.50, 60.73,
71.18, 71.24, 92.73, 113.72, 114.23, 122.58, 122.61, 128.10,
128.93, 129.50, 129.56, 130.18, 130.85, 130.86, 131.87, 136.26,
136.66, 136.73, 140.32, 140.3, 149.12, 151.21, 151.25, 153.26.
[0064] General Procedure for Synthesis of Phosphate Cation
Deriatives
[0065] Method A. Each of the metal cation containing salts were
obtained by the procedure outlined below for preparing sodium salt
19a. The metal counter ions were introduced by treatment of the
phosphoric acid with either the corresponding hydroxide (e.g.,
potassium, lithium) or acetate (e.g. magnesium).
[0066] Sodium 3-bromo-4,4',-5-trimethoxy-Z-stilbene 3'-O-phosphate
(19a). To a solution of dibenzyl phosphate 16 (0.28 g, 0.45 mnmol)
in dry dichloromethane (10 mL) was added trimethylsilylbromide (125
.mu.L, 0.95 mmol). The reaction mixture was stirred for 30 minutes
under argon, and the reaction was terminated by the addition of
methanol (20 mL). Following removal of solvents (in vacuo), the
free phosphoric acid was dissolved in ethanol (10 mL) and sodium
methoxide (49 mg, 0.9 mmol) were added to the residue. After the
reaction mixture was stirred for 30 minutes, the precipitate was
collected and washed with ether to provide sodium salt 19a (0.17 g)
as a colorless solid: m.p. 196-197.degree. C.; .sup.1H-NMR (300
MHz, D.sub.2O) .delta. 3.53 (s, 3H), 3.68 (s, 3H), 3.70 (s, 3H),
6.52 (d, 1H, J=12 Hz), 6.72 (d, 1H, J=12 Hz), 6.75 (s, 1H), 6.77
(s, 1H), 6.79 (s, 1H), 7.01 (s, 1H), 7.15 (s, 1H).
[0067] Sodium 3-fluoro-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate
(18a). m.p. 200-202.degree. C.; .sup.1H-NMR, (300 MHz, D.sub.2O)
.delta. 3.52 (s, 3H), 3.67 (s, 3H), 3.68 (s, 3H), 6.52 (d, 1H, J=12
Hz), 6.71 (d, 1H, J=12 Hz), 6.72 (s, 1H), 6.78 (s, 1H), 6.79 (s,
1H), 6.79 (s, 1H), 7.03 (s, 1H), 7.16 (s, 1H).
[0068] Lithium 3-bromo-4,4',5-trbnethoxy-Z-stilbene 3'-O-phosphate
(19b). m.p. 265-268.degree. C. (dec); .sup.1H NMR (300 MHz,
D.sub.2O) .delta. 3.53 (s, 3H), 3.66 (s, 3H), 3.69 (s, 3H), 6.35
(d, 1H, J=12 Hz), 6.52 (d, 1H, J=12 Hz), 6.70 (s, 2H), 6.81 (d, 1H,
J=1.5 Hz), 7.01 (d, 1H, J=1.5 Hz), 7.23 (s, 1H).
[0069] Potassium 3-bromo-4,4',5-trimethoxy-Z-stilbene
3'-O-phosphate (19c). m.p. 230-233.degree. C. (dec); .sup.1H-NMR
(300 MHz, D.sub.2O) .delta. 3.53 (s, 3H), 3.66 (s, 3H), 3.69 (s,
3H), 6.35 (d, 1H, J=12 Hz), 6.52 (d, 1H, J=12 Hz), 6.70 (s, 2H),
6.81 (d, 1H, J=1.5 Hz), 7.01 (d, 1H, J=1.5 Hz), 7.23 (s, 1H).
[0070] Cesium 3-bromo-4,4',5-trimethoiy-phenyl-Z-stilbene
3'-O-phosphate (19d). m.p. 233-235.degree. C.; .sup.1H-NMR (300
MHz, DMSO) .delta. 3.51 (s, 3H), 3.62 (s, 3H), 3.65 (s, 3H), 6.38
(d, 1H, J=12 Hz), 6.50 (d, 1H, J=12 Hz), 6.71 (s, 1H), 6.83 (d, 1H,
J=1.5 Hz), 7.03 (d, 2H, J=1.5 Hz), 7.23 (s, 1H).
[0071] Rubidium 3-bromo-4,4',5-trimetboxy-Z-stilbene 3'-O-phosphate
(19e). m.p. 204-206.degree. C.; .sup.1H-NMR (300 MHz, DMSO) .delta.
3.50 (s, 3H), 3.64 (s, 3H), 3.66 (s, 3H), 6.35 (d, 1H, J=12 Hz),
6.52 (d, 1H, J=12 Hz), 6.68 (s, 2H), 6.80 (d, 2H, J=1.5 Hz), 7.00
(d, 2H, J=1.5 Hz), 7.25 (s, 1H).
[0072] Calcium 3-bromo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate
(19f). m.p. 245-248.degree. C. (dec); .sup.1H-NMR (300 MHz, DMSO)
.delta. 3.53 (s, 3H), 3.69 (s, 3H), 3.70 (s, 3H), 6.33 (d, 1H, J=12
Hz), 6.50 (d, 1H, J=12 Hz), 6.71 (s, 2H), 6.81 (d, 2H, J=1.5 Hz),
7.99 (d, 2H, J=1.5 Hz), 7.23 (s, 1H).
[0073] Magnesium 3-bromg-4,4',5-trimethoxy-Z-stilbene
3'-O-phosphate (19 g). m.p. 290-285.degree. C. (dec); .sup.1H-NMR
(300 MHz, DMSO) .delta. 3.50 (s, 3H) 3.60 (s, 3H), 3.65 (s, 3H),
6.33 (d, 1H, J=12Hz), 6.50 (d, 1H, J=12 Hz), 6.68 (s, 2H), 6.79 (d,
2H, J=1.5 Hz), 7.00 (d, 2H, J=1.5 Hz), 7.21 (s, 1H).
[0074] Sodium 3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-0-phosphate
(20a). m.p. 194-195.degree. C., .sup.1H-NMR (300 MHz, D.sub.2O)
.delta. 3.50 (s, 3H), 3.67 (s, 3H), 3.68 (s, 3H), 6.50 (d, 1H, J=12
Hz), 6.70 (d, 1H, J=12 Hz), 6.72 (s, 1H), 6.77 (s, 1H), 6.79 (s,
1H), 7.01 (s, 1H), 7.13 (s, 1H).
[0075] Method B The potassium salt 18c (approximately 30 mg) was
dissolved in de-ionized water (1 mL) and applied to a Dowex-50w
(HCR-W2) resin column (amine or amino acid) and developed by water.
The eluent was concentrated by freeze drying to give the required
compound.
[0076]
3-Iodo-4,4',5-trimethoxy-3-O-tert-butyldiphenylsilyl-z-stilbene
(10a) and
3-Iodo-4,4',5-trimethoxy-3'-O-tert-butyldiphenylsilyl-E-stdlben- e
(10b).
[0077] Method A. Phosphonium bromide 6 (3.67 g, 5.13 mmol) was
dissolved in DCM at 0.degree. C. Sodium hydride (60% dispersion in
mineral oil, 0.41 g, 10.2 mmol) was added and the mixtture turned
orange. Next, 3-iodo-4,5-dimethoxybenzaldehyde (1 g, 3.42 mmol) was
added and stirring was continued for 21 hrs. The reaction was
terminated by adding water (50 mL) and extracted with DCM
(3.times.50 mL), which was dried, filtered and concentrated. The
oil obtained was subjected to flash chromatography on silica gel
with the eluent 0-3% ethyl acetate in hexane to afford z-stilbene
10a (0.86 g, 39%) which crystallized as a colorless solid from
hexane: mp 122-124.degree. C.: .sup.1H-NMR (300 MHz, CDCl.sub.3)
.delta. 1.07 (s, 9H, 3.45 (s, 3H), 3.55 (s, 3H), 3.79 (s, 3H), 6.21
(d, 1H, J=12 Hz), 6.31 (d, 1H, J 12 Hz), 6.59 (d, 1H, J=7.8 Hz),
6.72 (s, 2H), 6.77 (dd, 1H, J=7.8, 1.5 Hz), 7.19 (d, 1H, J=1.8 Hz),
7.40-7.20 (m, 6H), 7.64 (d, 4H, J=7.5 Hz); .sup.13C-NMR (75 MHz,
CDCl.sub.3) .delta. 19.68, 26.62, 55.05, 55.56, 60.33, 91.94,
111.72, 113.09, 120.78, 122.43, 126.73, 127.33, 129.32, 130.28,
130.93, 133.54, 135.17, 144.70, 149.82, 151.82; HRMS calcd for
C.sub.33H.sub.36IO.sub.4Si 651.1428 [M+H].sup.+, found 651.1474;
Anal. caled for C.sub.33H.sub.35I0.sub.4Si C, 60.92; H, 5.45.
Found, C, 60.79; H, 5.67%.
[0078] Further elution gave E-stiibene 10b (0.96 g, 43%) that
crystallized from hexane as a colorless solid; mp 98-99.degree. C.;
.sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 1.14 (s, 9H), 3.55 (s,
3H), 3.82 (s, 3H), 3.82 (s, 3H), 3.89 (s, 3H), 6.43 (d, 1H, I =15.9
Hz), 6.71-6.76 (m, 2H), 6.86-6.95 (m, 3H), 7.33-7.42 (m, 6H);
.sup.13C-NMR (100 MHz, CDCl.sub.3) .delta. 19.81, 26.67, 55.28,
60.50, 92.65, 110.22, 112.11, 117.70, 120.56, 124.58, 127.52,
128.45, 128.68, 129.60,129.77, 133.64, 135.38, 135.82, 145.18,
148.13, 150.56, 152.49; HRMS calcd for C.sub.33H.sub.36IO.sub.4Si
651.1428 [M+H].sup.+, found 651.1400; Anal. calcd for
C.sub.33H.sub.35I0.sub.4Si, C, 60.92; H, 5.42, found C, 60.88; H,
5.63%.
[0079] Method B. Butyllithium (4.5 mL, 11.3 mmol) was added to a
stirred and cooled (-70.degree. C.) suspension of phosphoniuin
bromide 6 in dry THF (100 mL). The solution was stirred for 30 min
at -70.degree. C. then 6 hours at room temperature. Water (50 mL)
was added and the reaction mixture was extracted with EtOAc
(3.times.100 mL), the extract dried, filtered and concentrated. The
oil obtained was subjected to flash chromatography on silica eluent
0-3% ethyl acetate in hexane to afford Z-stilbene 10a (1.4 g, 21%)
as a colorless solid: mp 122-124.degree. C.,
[0080]
3,5-diiodo-4,4'-dimethoxy-3'-O-tert-butyl-diphenylsilyl-z-stilbene
and
3,5-diiodo-4,4'-dimethoxy-3'-O-tert-butyl-diphenylsuyl-E-stilbene
[0081] Method A. Phosphonium bromide 6 (2.77 g, 3.87 mmol) (8) was
dissolved in DCM at 0.degree. C. When sodium hydride (60%
dispersion in mineral oil, 0.31 g, 7.7 mmol) was added, the mixture
turned orange. Aldehyde (1.0 g, 2.57 mmol) was added and stirring
was continued for 7.5 hrs. The reaction was terminated by adding
water (50 mL) and extracted with DCM (3.times.50 mL). The organic
extract was dried, filtered and concentrated. The oily residue was
subjected to flash chromatography on silica gel using hexane as
eluent to give an isomeric mixture of the title compounds (71%
yield, 1.35 g). Further elution gave E-isomer (0.10 g, 5%) as a
colorless oil in pure form: .sup.1H-NMR (300 MHz, CDCl.sub.3)
.delta. 1.14 (s, 9H), 3.56 (s, 3H), 3.84 (s, 3H), 6.33(d, 1H, J
15.9 Hz), 6.72 (d, 1H, J 8.4 Hz), 6.73 (d, 1H, J 15.9 Hz), 6.72 (d,
1H, J 8.4 Hz, ArH), 6.85 (d, 1H, J 2.1 Hz), 6.92 (dd, 1H, J 1.8 Hz
and J 8.4 Hz), 7.34-7.46 (m, 6H) and 7.72-7.75 (m, 6H);
.sup.13C-NMR (100 MHz, CDCl.sub.3) .delta. 19.82, 26.69, 55.30,
60.77, 90.59, 112.09, 117.73, 120.83, 122.47, 127.55, 129,38,
129.65, 129.99, 133.58, 135.40, 137.15, 137.73, 145.22, 150.84 and
157.55; and HRMS caled for C.sub.32H.sub.33I.sub.2O.sub.3Si
747.0289 [M+H].sup.+, found 747.0442.
[0082] Method B. Butyllithium (0.6 mL, 1.47 mmol) was added to a
stirred and cooled (-10.degree. C.) suspension of phosphoniurn
bromide 6 (1.01 g, 1.4 mmol) in dry THF (80 mL). The orange-red
solution was stirred for 10 minutes at room temperature. Aldehyde
(0.50 g, 1.33 mmol) was added and the reaction mixture color
changed from red to yellow. Stirring was continued at room
temperature for 10 minutes, ice water (100 mL) was added and the
mixture extracted with EtOAc (3.times.100 mL). The extract was
washed with water (100 mL), dried, filtered and concentrated. The
resulting oil was partially separated by flash chromatography on
silica gel using hexane-EtOAc (100:1) as eluent to give an isomeric
mixture in a ratio approximately 1:1.9, (cis:trans, 0.90 g,
90%).
[0083] 3-Iodo-4,4',5-trimethoxy-3'-hydroxy-z-stilbene (14a). To a
solution of silyl ether 7a (1.30 g, 1.99 mmol) in THF was added
tetrabutylammomum flouride (2.2 mL, 2.2 mmol). The mixture was
stirred under Ar in the dark for 10 min. and the reaction was
terminated by the addition of water (5 mL), the product was
extracted with EtOAc (3.times.15 mL), and the extract dried,
filtered and concentrated. The crude product was separated by
silica gel column chromatography using 1:4 ethyl acetate-hexane as
eluent to give stilbene 14a (0.70 g, 85%) as colorless solid: mp
92-94.degree. C., IR 3543, 3011, 2937, 2841, 1510, 1273, 1001, 908,
732 cm.sup.-1; .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 3.61 (s,
3H), 3.81 (s, 3H), 3.84 (s, 3H), 6.32 (d, 1H, J=12 Hz), 6.34 (s,
1H), 6.56 (d, 1H, J=12 Hz), 6.75 (s, 1H), 6.83 (d, 1H, J=1.8 Hz),
6.85 (s, 3H), 7.25 (d, 1H, J=1.5 Hz); .sup.13C-NMR (75 MHz,
CDCl.sub.3) .delta. 55.56, 55.82, 60.33, 91.78, 110.50, 113.11,
115.00, 120.91, 126.96, 129.94, 130.28, 135.93 145.29, 146.10,
147.67, 151.79; HRMS calcd for C.sub.17H.sub.18IO.sub.4 413.0259
[M+H].sup.+, found 413.0250. Anal. calcd for
C.sub.17H.sub.17IO.sub.4 C, 49.53; H, 4.16. Found C, 49.38; H,
4.24%.
[0084] 3-Iodo-4,4',5-trimethoxy-3'-hydroxy-E-stilbene (9b). The
trans isomer 14b (0.29 g, 98%) was obtained from silyl ether 10b
(0.46 g, 0.7 mmol) as described above for the synthesis of the cis
isomer 14a. Separation by column chromatography (7:3 hexane-ethyl
acetate as eluent) gave E-isomer 14b (0.29 g, 98%) as colorless
solid: mp 111-113.degree. C.; .sup.1H-NMR (300 MHz, CDCl.sub.3)
.delta. 3.84 (s, 3H), 3.87 (s, 3H), 3.88 (s, 3H, 5.85 (bs, 1H),
6.77 (d, 1H, J=16.5 Hz), 6.89 (d, 1H, J=16.5 Hz), 6.82 (s, 1H),
6.96 (s, 1H), 6.93 (d, 1H, J=2.4 Hz), 7.11 (d, 1H, J=1.5 Hz), 7.46
(d, 1H, J=1.5 Hz); .sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. 55.85,
60.41, 92.56, 110.40, 110.63, 111.77, 119.28, 124.97, 128.36,
128.70, 130.15, 135.71, 145.71, 146.56, 148.11, 152.44; HRMS calcd
for C.sub.17H.sub.18IO.sub.4 413.0257 [M+H].sup.+, found 413.0250.
Anal. calcd for C.sub.17H.sub.17IO.sub.4 C, 49.53; H, 4.16. Found,
C, 49.38; H, 4.24%.
[0085] 3,5-diiodo-4,4'-dimethoxy-3'-hydroxy-z-stilbene 22a and
3,5-diiodo-4,4'-dimethoxy-3'-hydroxy-E-stilbene 22b
[0086] These stilbenes were obtained from the z and E silyl ether
mixture 21ab (1.35 g, 1.81 mmol) as described above for the
synthesis of cis-isomer 14a. The oily mixture was separated by
column chromatography with 2:1 hexane-EtOAc as eluent to provide
cis-isomer 22a as an oil (0.45 g, 49%): .sup.1H-NMR (300 MHz,
CDCl.sub.3) .delta. 3.85 (s, 3H), 3.89 (s, 3H), 5.54 (s, 1H), 6.26
(d, 1H, J=12 Hz), 6.49 (d, 1H, J=12 Hz), 6.74 (s, 2H), 6.82 (s, 1H)
and 7.67 (s, 2H); .sup.13C-NMR (125 z, CDCl.sub.3) .delta. 55.98,
60.73, 89.98, 110.46, 114.87, 120.98, 125.08, 29.47, 131.57,
137.37, 139.96, 145.42, 146.21, 157.50. HRMS calcd for
C.sub.16H.sub.15I.sub.2O.sub.3 508.9113 [M+H].sup.+, found
508.9111. Anal. calcd for C.sub.16H.sub.14I.sub.2O.sub.3 C, 37.82;
H, 2.78. Found, C, 37.80; H, 2.83.
[0087] Further elution led to the E-stilbene 22b (0.46 g, 50%
yield) as a colorless solid which was crystallized from hexane: mp
127-129.degree. C.; .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 3.86
(s, 3H), 3.91 (s, 3H), 5.62 (s, 1H), 6.71 (d, 1H, J=16.5 Hz), 6.83
(d, 1H, J=8.1 Hz), 6.90 (d, 1H, J=17.1 Hz), 6.95 (d, 1H, J=8.4Hz),
7.10 (d, 1H, J=2.4 Hz) and 7.85 (s, 2H, H-2); .sup.13C-NMR (75 MHz,
CDCl.sub.3) .delta. 55.51, 60.30, 90.17, 100.17, 100.21, 110.18,
111.35, 119.17, 122.56, 129.63, 129.81, 136.82, 137.19, 146.34 and
157.25; HRMS calcd for C.sub.16H.sub.15I.sub.2O.sub.3 508.9113
[M+H].sup.+, found 508.9119. Anal. calcd for
C.sub.16H.sub.14I.sub.2O.sub.3 C, 37,82; H, 2.78. Found, C, 38.01;
H, 2.91.
[0088] 3,5-diiodo-4,4'-dimethoxy-3'-acetyl-z-stilbene (22c)
[0089] An appropriate phenol 22a (0.45 g) was dissolved in pyridine
(3 mL) acetic anhydride (170 .mu.L) and stirred for 2 hrs. The
mixture was concentrated under reduced pressure from toluene
(3.times.10 mL). The residue was diluted with EtOAc (30 mL), washed
successively with water (10 mL), NaHCO.sub.3 (10% aq. sol., 10 mL),
dried, and the solution filtered and concentrated. The acetate was
ftirther purified by flash chromatography on silica using 1:24
hexane-EtOAc:hexane as eluent to afford acetate 22c (0.20 g, 41%)
as a colorless solid: recrystallized from hexane mp 121-122.degree.
C.; .sup.1H-NMR (300 MHz, CDCl.sub.3) 3 2.29 (s, 3H), 3.83 (s, 3H),
3.85 (s, 3H), 6.29 (d, 1H, J=12 Hz), 6.48 (d, 1H, J=12 Hz), 6.85
(d, 1H, J=8.7 Hz), 6.93 (d, 1H, J=2.43), 7.06 (d, 1H, J=1.5), 7.09
(d, 1H, J=2.4 Hz) and 7.67 (s, 2H); .sup.13C-NMR (125 MHz,
CDCl.sub.3) .delta. 20.66, 55.94, 60.72, 90.11, 112.16, 123.25,
125.41, 127.47, 128.85, 130.64, 137.10, 139.54, 139.89, 150.69,
157.67 and 168.79; HRMS caled for C.sub.19H.sub.20I.sub.2O.sub.5
582.9479 [M+CH.sub.3OH].sup.+, found 582.9482; Anal. calcd for
C.sub.18H.sub.16I.sub.2O.sub.4 C, 39.30; H, 2.93. Found C, 39.30;
H, 3.13%.
[0090] 3-iodo-4,4',5-trimethoxy-3'-acetyl-Z-stilbene
[0091] An appropriate phenol (0.1 g, 0.24 mmol) was dissolved in 3
mL anhydrous pyridine. Acetic anhydride (50 .mu.L, 0.51 mmol) was
added with cat DMAP. The mixture was stirred for 90 minutes. The
reaction was terminated by the addition of 5 mL CH.sub.3OH. The
mixture was diluted with toluene and concentrated under reduced
pressure. It was purified on flash chromatography on silica gel
using EtOAc:hexane (1:9) as eluent to give a white solid (0.1 mg,
91%). The solid was crystallized from hexane: mp 103-104.degree.
C.; .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 2.27 (s, 3H), 3.61
(s, 3H), 3.81 (s, 6H), 6.38 (d, 1H, J=12 Hz), 6.48 (d, 1H, J=12
Hz), 6.77 (d, 1H, J=1.8 Hz), 6.83 (d, 1H, J=8.4 Hz), 6.96 (d, 1H,
J=1.5 Hz), 7.09 (dd, 1H, J=8.4 Hz, J=2.4 Hz), and 7.26 (s, 1H);
.sup.13C-NMR (125 MHz, CDCl.sub.3) .delta. 20.61, 55.67, 55.93,
60.44, 92.07, 112.07, 112.92, 123.17, 127.63, 127.74, 129.39,
129.65, 103.97, 134.96, 139.49, 147.99, 150.39, 152.05 and 168.81;
HRMS calcd for C.sub.19H.sub.20IO.sub.5 455.0355 [M+H].sup.+, found
455.0356. Anal. calcd for C.sub.19H.sub.19IO.sub.5 C, 50.24; H,
4.22. Found, C, 49,67; H, 4.18%.
[0092] Dibenzyl 3,5-diiodo-4,4'-dimethoy-z-stilbene 3'-O-phosphate
(23)
[0093] An appropriate dibenzyl phosphate (0.38 g, 55% yield) was
obtained (0.46 g, 0.91 mmol) as described above for the synthesis
of iodide 10a. Colorless oil: bp dec 220.degree. C.; .sup.1H-NMR
(300 MHz, CDCl.sub.3) .delta. 3.78 (s, 3H), 3.81 (s, 6H), 5.13 (s,
2H), 5.16 (s, 2H), 6.28 (d, 1H, J=12 Hz), 6.42 (d, 1H, J 12 Hz),
6.78 (d, 1H, J 9 Hz), 7.00 (d, 1H, J 8.7 Hz), 7.07 (s, 1H), 7.33
(s, 10H) and 7.64 (s, 2H); .sup.13C-NMR (100 MHz, CDCl.sub.3)
.delta. 55.96, 60.71, 69.83, 69.89, 90.15, 112.40, 122.23, 122.26,
125.60, 126.20, 126.21, 127.93, 128.49, 128.55, 130.66, 137.12,
139.92 and 157.68; HRMS calcd for C.sub.30H.sub.28I.sub.2O.sub.6P
768.9713 [M+H].sup.+, found 768.9699; .sup.31P-NMR (162 Mz,
CDCl.sub.3) .delta. -5.51.
[0094] General procedures for syntheses of the phosphoric acids and
derivatives.
[0095] Method A. Each of the metal cation phosphate salts was
obtained by the procedure outlined herein for preparing the
potassium salt 20c, except for the metal counterions introduced by
treatment of the phosphoric acid using either lithium hydroxide or
sodium methoxide.
[0096] Method B. Dowex-50W (2 g) (HCR-W2) was placed in a column
and washed successively with CH.sub.3OH (50 mL), 1 N HCl (until pH
1), water (until pH 7), base/amine/amino acid (until pH 7-14) and
water (until pH 7). The column was recycled. The potassium salt or
its corresponding diiodo phosphate salt (about 25 mg) was dissolved
in de-ionized water (1 mL) and applied to a Dowex-50W (HCR-W2)
resin column (bearing the appropriate amine or amino acid methyl
ester) and developed with approximately 40 mL of water. The eluent
was concentrated by freeze drying to give the required cation
derivative.
[0097] Method C. Amino Acid Methyl Esters. The amino acid methyl
ester hydrochloride was neutralized in CH.sub.3OH solution by
adding potassium carbonate. Ether was added to precipitate the
potassium chloride and the solution was filtered and concentrated.
The amino acid methyl ester residue was then applied to the
Dowex-50W (HCR-W2) resin column as described in Method B.
[0098] Potassium 3-iodo-4,4',5-trimethoxy-z-stilbene 3'-O-phosphate
(20c).
[0099] Trimethylbromosilane (277 .mu.L, 1.8 mmol) was added to a
cooled (0.degree. C.) solution of phosphate 9a in DCM (40 mL).
After stirring for 90 minutes, sodium thiosulfate (10% aq., 10 mL)
was added and the mixture was stirred for an additional 1 minute.
The phases were separated and the aqueous phase extracted with DCM
(20 mL), followed by EtOAc (2.times.20 mL). The combined organic
extracts were dried, filtered and concentrated to afford the
phosphoric acid intermediate as a clear oil. After drying (high
vacuum) for 1 hour, the oil was dissolved in CH.sub.3OH (10 mL),
cooled to 0.degree. C., and KOH (1.8 mL, 1 M sol. in CH.sub.3OH)
was added. The mixture was stirred for 20 minutes, the precipitate
was collected and triturated with ether to afford the potassium
salt as a colorless solid: mp 197-198.degree. C. (dec); .sup.1H-NMR
(300 MHz, D.sub.2O) .delta. 3.51 (s, 3H), 3.64 (s, 3H), 3,71 (s,
3H), 6.33 (d, 1H, J=12 Hz), 6.51 (d, 1H, J=12 Hz), 6.70 (s, 2H),
6.84 (s, 1H) and 7.22 (s, 2H); and .sup.31P-NMR (162 MHz, D.sub.2O)
.delta. 0.94.
[0100] Sodium 3-iodo-4,4',5-trimethoxy-z-stilbene 3'-O-phosphate
(20a).
[0101] Isolated as a colorless solid: mp 194-195.degree. C. (dec);
.sup.1H-NMR (300 MHz, D.sub.2O) .delta. 3.50 (s, 3H), 3.67 (s, 3H),
3.68 (s, 3H), 6.50 (d, 1H, J=12 Hz), 6.70 (d, 1H, J=12 Hz), 6.72
(s, 1H), 6.77 (s, 1H), 6.79 (s, 1H), 7.01 (s, 1H) and 7.13 (s,
1H),
[0102] Lithium 3-iodo-4,4',5-trimethoxy-z-stilbene 3'-O-phosphate
(11c).
[0103] Discovered as a colorless solid: mp 245-275.degree. C.
(dec); .sup.1H-NMR (400 MHz, D.sub.2O) .delta. 3.50 (s, 3H), 3.62
(s, 3H), 3.66 (s, 3H), 6.33 (d, 1H, J=12 Hz), 6.49 (d, 1H, J=12
Hz), 6.70 (s, 2H), 6.83 (s, 1H), 7.20 (s, 1H) and 7.22 (s, 1H).
[0104] Morpholine 3-iodo-4,4',5-trimethoxy-z-stilbene
3'-O-phosphate.
[0105] Another colorless oil: .sup.1H-NMR (300 MHz, D.sub.2O)
.delta. 3.11-3.15 (m, 8H), 3.50 (s, 3H), 3.63 (s, 3H), 3.68 (s,
3H), 3.77-3.81 (m, 8H), 6.33 (d, 1H, J 12 Hz), 6.50 (d, 1H, J 12
Hz), 6.73 (s, 2H), 6.82 (s, 1H), 7.18 (s, 1H) and 7.20 (s, 1H).
[0106] Piperidene 3-iodo-4,4',5-trimethoxy-z-stilbene
3'-O-phosphate.
[0107] Colorless oil: .sup.1H-NMR (300 MHz, D.sub.2O) .delta. 1.51
(m, 4H), 1.62 (m, 8H), 3.00 (t, 8H, J=6 Hz), 3.51 (s, 3H), 3.63 (s,
3H), 3.67 (s, 3H), 6.34 (d, 1H, J=12.6 Hz), 6.51 (d, 1H, J=12.6
Hz), 6.72 (s, 2H), 6.83 (s, 1H) and 7.21 (s, 1H).
[0108] Glycine-OMe 3-iodo-4,4',5-trimethoxy-z-stilbene
3'-O-phosphate.
[0109] Obtained as a colorless solid: mp 74-78.degree. C.;
.sup.1H-NMR (300 MHz , D.sub.2O) .delta. 3.48 (s, 3H), 3.61 (s,
3H), 3.67 (s, 3H), 3.68 (s, 3H), 3.76 (s, 2H), 6.30 (d, 1H, J=12
Hz), 6.46 (d, 1H, J=12 Hz), 6.69-6.77 (m, 3H), 7.10 (s, 1H) and
7.16 (s, 1H).
[0110] Tryptophan-OMe 3-iodo-4,4',5-trimethoxy-z-stilbene
3'-O-phosphate.
[0111] Colorless solid: mp 108-112.degree. C.; .sup.1H-NMR (300
MHz, DMSO) .delta. 3.19 (d, 2H, J=6.3 Hz), 3.56 (s, 3H), 3.61 (s,
3H), 3.66 (s, 3H), 3.70 (s, 3H), 4.09 (t, 1H, J=6 Hz), 6.35 (d, 1H,
J=12 Hz), 6.47 (d, 1H, J=12 Hz), 6.81-6.85 (m, 2H), 6.98 (t, 1H,
J=7.2 Hz), 7.07 (t, 1H, J=8.1 Hz), 7.18 (s, 1H), 7.22 (s, 1H), 7.34
(d, 1H, J=8.1 Hz), 7.40 (s, 1H) and 7.46(d, 1H,J=7.2 Hz).
[0112] Tris 3-iodo-4,4',5-trimethoxy-z-stilbene 3'-O-phosphate.
[0113] Colorless solid: mp 75-81.degree. C.; .sup.1H-NMR (300 MHz,
DMSO) .delta. 3.42 (s, 9 H),3.57 (s, 3H), 3.67 (s, 3H), 3.70 (s,
3H), 6.35 (d, 1H, J=12 Hz), 6.48 (d, 1H, J=12 Hz), 6.76 (d, 1H,
J=8.4 Hz), 6.81 (d, 1H, J=8.7 Hz), 6.92 (s, 1H), 7.22 (s, 1H) and
7.42 (s, 1H).
[0114] Potassium 3,5-dilodo-4,4'dimethoxy-z-stilbene
3'-O-phosphate.
[0115] Phosphate (0.20 g, 80%) was obtained from appropriate ester
9c (0.29 g, 0.38 mmol) as described above for the synthesis of 20c,
except the phosphoric acid was insoluble in EtOAc and DCM, so the
aqueous phase was extracted with butyl alcohol (3.times.25 mL). The
potassium salt was a colorless solid: mp 210-215.degree. C. (dec);
.sup.1H-NMR (300 MHz, D.sub.2O) .delta. 3.69 (s, 6H), 6.27 (d, 1H,
J=12 Hz), 6.49 (d, 1H, J=12 Hz), 6.64 (s, 2H), 7.20 (s, 1H) and
7.62 (s, 2H); .sup.31P-NMR (162 MHz, D.sub.2O) .delta. 0.973.
[0116] Sodium 3,5-diiodo-4,4'dimethoxy-z-stilbene
3'-O-phosphate,
[0117] Obtained as a colorless solid: mp 215-234.degree. C. (dec);
.sup.1H-NMR (300 MHz, D.sub.2O) .delta. 3.69 (s, 3H), 3.72 (s, 3H),
6.29 (d, 1H, J=12 Hz), 6.49 (d, 1H, J=12 Hz), 6.69 (s, 2H), 7.20
(s, 1H) and 7.64 (s, 2H).
[0118] Lithium 3,5-diiodo-4,4'dimethoxy-z-stilbene
3'-O-phosphate.
[0119] A colorless solid melting at 250-270.degree. C. (dec);
.sup.1H-NMR (300 MHz, D.sub.2O) .delta. 3.68 (s, 3H), 3.71 (s, 3H),
6.28 (d, 1H, J=12 Hz), 6.49 (d, 1H, J=12 Hz), 6.68 (s, 2H), 7.19
(s, 1H) and 7.64 (s, 2H). .sup.31P NMR (162 MHz, D.sub.2O) .delta.
0.96.
[0120] Morpholine 3,5diiodo-4,4'dimethoxy-z-stilbene
3'-O-phosphate.
[0121] Colorless waxy solid; mp 75-80.degree. C.; .sup.1H-NMR (300
MHz, DMSO) .delta. 2.96-2.99 (m, 8H), 3.74-3.77 (m, 8H), 3.82 (s,
3H), 3.83 (s, 3H), 6.43 (d, 1H, J=12.5 Hz), 6.60 (d, 1H, J=12.5
Hz), 6.86 (d, 1H, J=8.2Hz), 6.93 (d, 1H, J=8.2 Hz), 7.49 (s, 1H)
and 7.78 (s, 2H).
[0122] Piperidine 3,5-diiodo-4,4'dimethoxy-z-stilbene
3'-O-phosphate.
[0123] Isolated as a colorless oil; .sup.1H-NMR (300 MHz, DMSO)
.delta. 1.51 (br s, 12 H), 2.79-2.81 (m, 8H), 3.70 (s, 3H), 3.72
(s, 3H), 6.31 (d, 1H, J=12 Hz), 6.49 (d, 1H, J=12 Hz), 6.73 (d, 1H,
J=8.4 Hz), 6.80 (d, 1H, J=8.4 Hz), 7.40 (s, 1H) and 7.61 (s,
1H).
[0124] Glycine-OMe 3,5-diiodo-4,4'dimethoxy-z-stilbene
3'-O-phosphate.
[0125] Colorless solid; mp 90-97.degree. C.; .sup.1H-NMR (300 MHz,
DMSO) .delta. 3.61 (s, 4 H), 3.68 (s, 6H), 3.70 (s, 3H), 3.72 (s,
3H), 6.31 (d, 1H, J=12 Hz), 6.49 (d, 1H, J=12 Hz), 6.72 (d, 1H,
J=9.6 Hz), 6.80 (d, 1H, J=8.1 Hz), 7.37 (s, 1H) and 7.67 (s,
1H).
[0126] Tryptophan-OMe 3,5-diiodo-4,4'dimethoxy-z-stilbene
3'-O-phosphate.
[0127] Collected as a colorless solid; melting at 125-130.degree.
C.; .sup.1H-NMR (300 MHz, DMSO) .delta. 3.34 (d, 1H, J=6.5 Hz),
3.36 (d, 1H, J=6.5 Hz), 3.66 (s, 3H), 3.70 (s, 3H), 3.72 (s, 3H),
4.32 (t, 1H, J=6.5 Hz), 6.31 (d, 1H, J=12 Hz), 6.48 (d, 1H, J=12
Hz), 6.78-6.81 (m, 2H), 7.01 (s, 1H), 7.05 (t, 1H, J=7 Hz), 7.13
(t, 1H, J=7 Hz), 7.39 (d, 1H, J=7.5 Hz), 7.47 (d, 1H, J=8 Hz) and
7.60 (s, 1H).
[0128] Tris 3,5-diiodo-4,4'dimethoxy-z-stilbene 3'-O-phosphate.
[0129] Colorless solid; mp 115-120.degree. C.; .sup.1H-NMR (300
MHz, DMSO) .delta. 3.34 (s, 18H), 3.69 (s, 3H), 3.71 (s, 3H), 6.30
(d, 1H, J=12 Hz), 6.47 (d, 1H, J=12 Hz), 6.70 (d, 1H, J=8.1 Hz),
6.78 (d, 1H, J=8.1 Hz), 7.37 (s, 1H) and 7.67 (s, 2H).
[0130] Cancer Cell Line Procedures
[0131] Inhibition of human cancer cell growth was assessed using
the National Cancer Institute's standard sulforhodamine B assay.
After 48 hours, the plates were fixed with trichloracetic acid,
stained with sulforhodamine B and read with an automated microplate
reader. A growth inhibition of 50% (GI.sub.50 or the drug
concentration causing a 50% reduction in the net protein increase)
was calculated from optical density data with Inmunosoft software.
Inhibition of the mouse leukemia P388 cells was assessed in a 10%
horse serum/Fisher medium soution for 24 hours, followed by a 48
hour incubation with serial dilutions of the compounds. Cell growth
inhibition (ED.sub.50) was then calculated using a Z1
Becleman/Coulter particle counter.
[0132] Tubulin Evaluations: Tubulin polymerization was evaluated by
turbidimetry at 35 nm using Beckman DU7400/7500 spectrophotometers
as known to one of skill in the art. Varying concentrations of the
compound were preincubated with 10 .mu.M. Incubation was for 10
minutes at 37.degree. C.
[0133] Antiangiogenesis
[0134] HUVEC Procedures
[0135] In vitro Matrigel antiangiogenesis assays were implemented
according to the Developmental Therapeutics Program NCI/NIH
protocols known to one of skill in the art. Matrigel, a basement
membrane matrix, was obtained from BD Biosciences. Growth
inhibition and cord formation assays were Conducted using human
umbilical vein endothelial cells obtained from GlycoTeCh. HUVEC
cells were grown in EGM-2 medium.
[0136] Cord Formation Assay
[0137] An aliquot of sixty microliters was placed in each well of
an ice-cold 96-well plate. The plates were then left for 15 minutes
at room temperature, then incubated for 30 minutes at 37.degree. C.
to permit the matrigel to polymerize. Meanwhile, HUVEC cells were
harvested and diluted to a concentration of 2.times.10.sup.5
cells/ml. A solution of 100 .mu.L containing the compounds to be
tested was added next. After 24 hours incubation, pictures were
taken for each concentration using an inverted Nikon Diaphot
microscope and D100 digital camera. Drug effect was assessed,
compared to untreated controls, by measuring the length of cords
formed and number ofjunctions.
[0138] The standard sulforhodamine B assay (see Cancer Cell Line
Procedures above) was used to evaluate results using HUVEC cells.
IC.sub.50 or ED.sub.50 (drug concentration causing 50% inhibition)
was calculated from the plotted data.
Administration
[0139] Dosages
[0140] The dosage to be administered to humans and other animals
requiring treatment will depend upon the identity of the neoplastic
disease or microbial infection; the tpe of host involved, including
its age, health and weight; the kind of concurrent treatment, if
any; the frequency of treatment and therapeutic ratio. Hereinafter
are described various possible dosages and methods of
administration, with the understanding that the following are
intended to be illustrative only, and that the actual dosages to be
administered, and methods of administration or delivery may vary
therefrom. The proper dosages and administration forms and methods
may be determined by one of skill in the art.
[0141] Illustratively, anticipated dosage levels of the
administered active ingredients may be in the following ranges:
intravenous, 0.1 to about 200 mg/kg; intramuscular, 1 to about 500
mg/kg; orally, 5 to about 1000 mg/kg; intranasal instillation, 5 to
about 1000 mg/kg; and aerosol, 5 to about 1000 mg/k of host body
weight.
[0142] Expressed in terms of concentration, an active ingredient
can be present in the compositions of the present invention for
localized use about the cutis, intranasally, pharyngolaryngeally,
bronchially, intravaginally, rectally, or ocularly in concentration
of from about 0.01 to about 50% w/w of the composition; preferably
about 1 to about 20% w/w of the composition; and for parenteral use
in a concentration of from about 0.05 to about 50% w/v of the
composition and preferably from about 5 to about 20% w/v.
[0143] The compositions of the present invention are intended to be
presented for administration to humans and animals in unit dosage
forms, such as tablets, capsules, pills, powders, granules,
suppositories, sterile parenteral solutions or suspensions, sterile
non-parenteral solutions of suspensions, and oral solutions or
suspensions and the like, containing suitable quantities of an
active ingredient. Other dosage forms known in the art may be
used.
[0144] For oral administration either solid or fluid unit dosage
forms may be prepared.
[0145] Powders may be prepared by comminuting the active ingredient
to a suitably fine size and mixing with a similarly comminuted
diluent. The diluent can be an edible carbohydrate material such as
lactose or starch. Advantageously, a sweetening agent or sugar is
present as well as a flavoring oil.
[0146] Capsules may be produced by preparing a powder mixture as
here inbefore described and filling into formed gelatin sheaths.
Advantageously, as an adjuvant to the filling operation, a
lubricant such as talc, magnesium stearate, calcium stearate and
the like is added to the powder mixture before the filling
operation.
[0147] Soft gelatin capsules may be prepared by machine
encapsulation of a slurry of active ingredients with an acceptable
vegetable oil, light liquid petrolatum or other inert oil or
triglyceride or other pharmaceutically acceptable carrier.
[0148] Tablets may be made by preparing a powder mixture,
granulating or slugging, adding a lubricant and pressing into
tablets. The powder mixture may be prepared by mixing an active
ingredient, suitably comminuted, with a diluent or base such as
starch, lactose, kaolin, dicalcium phosphate and the like. The
powder mixture can be granulated by wetting with a binder such as
corn syrup, gelatin solution, methylcellulose solution or acacia
mucilage and forcing through a screen. As an alternative to
granulating, the powder mixture may be slugged, i.e., run through
the tablet machine and the resulting imperfectly formed tablets
broken into pieces (slugs). The slugs can be lubricated to prevent
sticking to the tablet-forming dies by means of the addition of
stearic acid, a stearic salt, talc or mineral oil. The lubricated
mixture is then compressed into tablets.
[0149] Advantageously, for protection of the tablet itself and/or
to ease swallowing, the tablet can be provided with a
pharmaceutically acceptable coating such as a sealing coat or
enteric coat of shellac, a coating of sugar and methylcellulose and
polish coating of camauba wax.
[0150] Fluid unit dosage forms for oral administration such as in
syrups, elixirs and suspensions may be prepared wherein each
teaspoonfuil of composition contains a predetermined amount of an
active ingredient for administration.
[0151] The water-soluble forms may be dissolved in an aqueous
vehicle together with sugar, flavoring agents and preservatives to
form a syrup. An elixir is prepared by using a hydroalcoholic
vehicle with suitable sweeteners together with a flavoring agent.
Suspensions may be prepared of the insoluble forms with a suitable
vehicle with the aid of a pharmaceutically acceptable suspending
agent such as acacia, tragacanth, methylcellulose and the like.
[0152] For parenteral administration, fluid unit dosage forms may
be prepared utilizing an active ingredient and a sterile vehicle,
for examples water. The active ingredient, depending on the form
and concentration used, can be either suspended or dissolved in the
vehicle. In preparing solutions the water-soluble active ingredient
can be dissolved in water for injection and filter sterilized
before filling into a suitable vial or ampule and sealing.
Advantageously, adjuvants such as a local anesthetic, preservative
and buffering agents can be dissolved in the vehicle. Parenteral
suspensions may be prepared in substantially the same manner except
that an active ingredient is suspended in the vehicle instead of
being dissolved and sterilization cannot be accomplished by
filtration. The active ingredient may be sterilized by exposure to
ethylene oxide before suspending in the sterile vehicle.
Advantageously, a pharmaceutically acceptable surfactant or wetting
agent may be included in the composition to facilitate uniform
distribution of the active ingredient.
[0153] In addition to oral and parenteral administration, the
rectal and vaginal routes can be utilized. An active ingredient can
be administered by means of a suppository. A vehicle which has a
melting point at about body temperature or one that is readily
soluble can be utilized. For example, cocoa butter and various
polyethylene glycols (Carbowaxes) can serve as the vehicle.
[0154] For intranasal installation, a fluid unit dosage form may be
prepared utilizing an active ingredient and a suitable
pharmaceutical vehicle, such as purified water, a dry powder, can
be formulated when insuffilation is the administration of
choice.
[0155] For use as aerosols, the active ingredients may be packaged
in a pressurized aerosal container together with a gaseous or
liquefied propellant, for example, dichlorodifluoromethane, carbon
dioxide, nitrogen, propane, and the like, with the usual adjuvants
such as cosolvents and wetting agents, as may be necessary or
desirable.
[0156] The term "unit dosage form" as used in the specification and
claims refers to physically discrete units suitable as unitary
dosages for human and animal subjects, each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical diluent, carrier or vehicle. The specifications for
the novel unit dosage forms of this invention are dictated by and
are directly dependent on (a) the unique characteristics of the
active material and the particular therapeutic effect to be
achieved, and (b) the limitation inherent in the art of compounding
such an active material for therapeutic use in humans, as disclosed
in this specification, these being features of the present
invention. Examples of suitable unit dosage forms in accord with
this invention are tablets, capsules, troches, suppositories,
powder packets, wafers, cachets, teaspoonfuls, tablespoonfuls,
dropperfuls, ampules, vials, segregated multiples of any of the
foregoing, and other forms as herein described.
[0157] The active ingredients to be employed as antineoplastic
agents may be prepared in such unit dosage form with the employment
of pharmaceutical materials which themselves are available in the
art and can be prepared by established procedures. The following
preparations are illustrative of the preparation of the unit dosage
forms of the present invention, and not as a limitation thereof.
Shown in the following are examples of dosage forms for the
compounds of the present invention, in which the notation "active
ingredient" signifies the compounds described herein.
Composition "A"
Hard-Gelatin Capsules
[0158] One thousand two-piece hard gelatin capsules for oral use,
each capsule containing 200 mg of an active ingredient may be
prepared from the following types and amounts of ingredients:
TABLE-US-00001 Active ingredient, micronized 200 g Corn Starch 20 g
Talc 20 g Magnesium stearate 2 g
[0159] The active ingredient, finely divided by means of an air
microrizer, is added to the other finely powdered ingredients,
mixed thoroughly and then encapsulated in the usual manner.
[0160] Using the procedure above, capsules may be similarly
prepared containing an active ingredient in 50, 250 and 500 mg
amounts by substituting 50 g, 250 g and 500 g of an active
ingredient for the 200 g used above.
Composition "B"
Soft Gelatin Capsules
[0161] One-piece soft gelatin capsules for oral use, each
containing 200 mg of an active ingredient, finely divided by means
of an air micronizer, may prepared by first suspending the compound
in 0.5 ml of corn oil to render the material capsulatable and then
encapsulating in the above manner.
Composition "C"
Tablets
[0162] One thousand tablets, each containing 200 mg of an active
ingredient, may be prepared from the following types and amounts of
ingredients: TABLE-US-00002 Active ingredient, micronized 200 g
Lactose 300 g Corn starch 50 g Magnesium stearate 4 g Light liquid
petrolatum 5 g
[0163] The active ingredient, finely divided by means of an air
micronizer, is added to the other ingredients and then thoroughly
mixed and slugged. The slugs are broken down by forcing them
through a Number Sixteen screen. The resulting granules are then
compressed into tablets, each tablet containing 200 mg of the
active ingredient.
[0164] Using the procedure above, tablets may similarly prepared
containing an active ingredient in 250 mg and 100 mg amounts by
substituting 250 g and 100 g of an active ingredient for the 200 g
used above.
Composition "D"
Oral Suspension
[0165] One liter of an aqueous suspension for oral use, containing
in each teaspoonfuil (5 ml) dose, 50 mg of an active ingredient,
may be prepared from the following types and amounts of
ingredients: TABLE-US-00003 Active ingredient, micronized 10 g
Citric acid 2 g Benzoic acid 1 g Sucrose 790 g Tragacanth 5 g Lemon
Oil 2 g Deionized water, q.s. 1000 ml
[0166] The citric acid, benzoic acid, sucrose, tragacanth and lemon
oil are dispersed in sufficient water to make 850 ml of suspension.
The active ingredient, finely divided by means of an air
micronizer, is stirred into the syrup unit uniformly distributed.
Sufficient water is added to make 1000 ml
Composition "E"
Parenteral Product
[0167] A sterile aqueous suspension for parenteral injection,
containing 30 mg of an active ingredient in each milliliter for
treating a neoplastic disease, may be prepared from the following
types and amounts of ingredients: TABLE-US-00004 Active ingredient,
micronized 30 g POLYSORBATE 80 5 g Methylparaben 2.5 g
Propylparaben 0.17 g Water for injection, q.s. 1000 ml.
[0168] All the ingredients, except the active ingredient, are
dissolved in the water and the solution sterilized by filtration.
To the sterile solution is added the sterilized active ingredient,
finely divided by means of an air micronizer, and the final
suspension is filled into sterile vials and the vials sealed.
Composition "F"
Suppository, Rectal and Vaginal
[0169] One thousand suppositories, each weighing 2.5 g and
containing 200 mg of an active ingredient may be prepared from the
following types and amounts of ingredients: TABLE-US-00005 Active
ingredient, micronized 15 g Propylene glycol 150 g Polyethylene
glycol #4000, q.s. 2,500 g
[0170] The active ingredient is finely divided by means of an air
micronizer and added to the propylene glycol and the mixture passed
through a colloid mill until uniformly dispersed. The polyethylene
glycol is melted and the propylene glycol dispersion is added
slowly with stirring. The suspension is poured into unchilled molds
at 40.degree. C. The composition is allowed to cool and solidify
and then removed from the mold and each suppository foil
wrapped.
Composition "G"
Intranasal Suspension
[0171] One liter of a sterile aqueous suspension for intranasal
instillation, containing 20 mg of an active ingredient in each
milliliter, may be prepared from the following types and amounts of
ingredients: TABLE-US-00006 Active ingredient, micronized 15 g
POLYSORBATE 80 5 g Methylparaben 2.5 g Propylparaben 0.17 g
Deionized water, q.s. 1000 ml.
[0172] All the ingredients, except the active ingredient, are
dissolved in the water and the solution sterilized by filtration.
To the sterile solution is added the sterilized active ingredient,
finely divided by means of an air micronizer, and the final
suspension is aseptically filled into sterile containers.
Composition "H"
Powder
[0173] Five grams of active ingredient in bulk form is finely
divided by means of an air micronizer. The micronized powder is
placed in a shaker-type container.
Composition "I"
Oral Powder
[0174] One hundred grams of an active ingredient in bulk form may
be finely divided by means of an air micronizer. The micronized
powder is divided into individual doses of 200 mg and packaged.
Composition "J"
Insulation
[0175] One hundred grams of an active ingredient in bulk form is
finely divided by means of an air micronizer.
[0176] It is of course understood that such modifications,
alterations and adaptations as will readily occur to the artisan
confronted with this disclosure are intended within the spirit of
the present invention. TABLE-US-00007 TABLE I Human cancer cell
line inhibition (GI.sub.50 .mu.g/mL) and murine P388 lymphocytic
leukemia inhibitory activity (ED.sub.50 .mu.g/ml) of
halocombstatins and other compounds. Leukemia Pancreas- Breast adn
CNS Lung-NSC Colon Prostate Compound P388 a BXPC-3 MCF-7 SF268
NCI-H460 KM20L2 DU-145 1a 0.0003 0.39 -- <0.001 0.0006 0.061
0.0008 1b 0.0004 -- -- 0.036 0.029 0.034 -- 2a 0.251 4.4 -- -- 0.74
0.061 0.17 2b <0.01 1.5 0.024 0.036 0.038 0.53 0.034 3a 0.257
2.3 0.49 0.0083 0.19 1.2 0.0043 3b 0.305 2.8 0.92 0.052 0.45 3.5
0.048 11a <0.01 0.016 <0.01 <0.01 <0.01 1.1 <0.01
11b 0.253 2.2 0.051 0.35 0.18 0.53 0.18 12a <0.01 0.043
<0.001 <0.001 <0.001 0.15 <0.001 12b 0.027 0.59 0.041
0.048 0.034 1.4 0.038 13a <0.01 0.16 <0.001 <0.001
<0.001 0.086 <0.001 13b 0.0174 1.6 0.14 0.18 0.15 1.2 0.13
14a <0.01 0.11 0.00022 0.00035 0.00019 0.15 0.00052 14b 0.189
2.7 0.18 0.55 0.21 1.7 0.27 18a 0.0298 0.59 0.0044 0.0051 0.0094
1.5 0.0036 19a <0.01 0.093 0.0041 0.0034 0.0028 0.23 0.0046 19b
<0.01 0.13 0.0039 0.0030 0.0026 0.11 0.0066 19c <0.01 0.20
0.0035 0.0032 0.0029 0.24 0.0028 19d <0.01 0.15 0.0044 0.0064
0.0066 0.48 0.0079 19e <0.01 0.56 0.043 0.023 0.041 2.6 0.042
19f 0.288 <0.001 0.0022 0.0022 0.0068 0.37 0.0063 19g <0.01
0.074 0.0045 0.0053 0.0039 0.27 0.0045 19h <0.01 0.17 0.0049
0.0067 0.0047 0.45 0.0049 19i 2.22 >10 3.2 4.1 2.9 >10 2.8
20a <0.01 0.47 0.012 0.0052 0.0031 0.37 0.0078
[0177] TABLE-US-00008 TABLE Ia Solubilities of some of the
synthetic modifications, human cancer cell line growth inhibition
(GI.sub.50 .mu.g/mL) and murine P388 lymphocytic leukemia
inhibitory activity (ED.sub.50 .mu.g/ml). Solubility.sup.a Leukemia
Pancreas Breast CNS Lung-NSC Colon Prostate Compound (mg/mL) P388
BXPC-3 MCF-7 SF268 NCI-H460 KM20L2 DU-145 A -- 0.0003 0.39 --
<0.001 0.0006 0.061 0.0048 B -- 0.0004 -- -- 0.036 0.029 0.034
-- C -- 0.26 2.3 0.49 0.0083 0.19 1.2 0.0043 D -- 0.0020 0.745
0.0027 0.0016 0.0032 >1 0.019 E -- 0.0020 0.048 0.00022 0.00018
0.00029 0.328 0.00018 F -- 0.189 2.7 0.18 0.55 0.21 1.7 0.27 G --
0.0028 0.038 0.0027 0.0036 0.0034 0.15 0.0021 H -- >10 3.0 0.94
3.3 3.4 >10 5.8 I -- 0.0089 0.040 0.00053 0.0023 0.0032 0.075
0.0020 J -- 0.022 0.080 <0.0001 0.0002 0.00031 0.16 0.00026 K 14
0.0021 0.381 0.0064 0.0057 0.0043 >1 0.0038 L 2 0.0020 0.469
0.018 0.018 0.017 >1 0.011 M .gtoreq.2.4 0.017 0.490 0.0038
0.0040 0.0039 >1 0.0043 N -- 0.0032 0.21 0.0047 0.0037 0.0036
0.24 0.0026 O .gtoreq.4 0.0026 0.32 0.0065 0.0044 0.0036 0.51
0.0029 P .gtoreq.2 0.0026 0.16 0.0044 0.0033 0.0031 0.32 0.0021 Q
-- 0.0022 0.26 0.035 0.0097 0.0034 0.59 0.0030 R -- 0.0029 0.37
0.0048 0.0043 0.0040 0.40 0.0047 S 22 0.0034 0.44 0.050 0.053 0.046
>1 0.028 T 2 0.030 >1 0.066 0.051 0.327 >1 0.242 U
.gtoreq.4 0.021 0.37 0.051 0.050 0.050 >1 0.032 V -- 0.014 0.35
0.066 0.054 0.033 >1 0.028 W -- 0.011 0.33 0.070 0.041 0.025
>1 0.025 X -- 0.011 0.36 0.10 0.054 0.030 >1 0.023 Y -- 0.017
0.37 0.22 0.086 0.033 >1 0.026 Z -- 0.026 0.33 0.047 0.040 0.025
0.94 0.021 .sup.aSolubility values were obtained using 1 mL
D.sub.2O at 25.degree. C. Key to Table Ia A = combretastatin A-4 B
= sodium combretastatin A-4 phosphate C = combretastatin A3 D =
fluorocombstatin E = 3-Iodo-4,4',5-trimethoxy-3'-hydroxy-Z-stilbene
F = 3-Iodo-4,4',5-trimethoxy-3'-hydroxy-E-stilbene G =
3,5-diiodo-4,4'-dimethoxy-3'-hydroxy-Z-stilbene H =
3,5-diiodo-4,4'-dimethoxy-3'-hydroxy-E-stilbene I =
3,5-diiodo-4,4'-dimethoxy-3'-acetyl-Z-stilbene J =
3-iodo-4,4',5-trimethoxy-3'acetyl-Z-stilbene K = Potassium
3-iodo,4,4',5 trimethoxy-Z-stilbene 3'-O-phosphate L = Sodium
3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate M = Lithium 3
iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate N = Morpholine
3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate O = Piperidine
3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate P =
Glycine-O-Me-3-iodo,4,4',5-trimethoxy-Z-stilbene-3'-O-phosphate Q =
Tryptophan-O-Me-3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate
R = Tris-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate S =
Potassium 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate T =
Sodium 3,5-diiodo-4,4'-dimethoxy-Z-stilbene 3'-O-phosphate U =
Lithium 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate V =
Morpholine 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate W =
Piperidine 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate X =
Glycine-O-Me 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate Y
= Tryptophan-OMe-3,5-diiodo-4,4' dimethoxy-Z-stilbene
3'-O-phosphate Z = Tris 3,5 diiodo-4,4' dimethoxy-Z-stilbene
3'-O-phosphate
[0178] TABLE-US-00009 TABLE II Inhibition of tubulin polymerization
and binding of [.sup.3H] colchicine to tubulin by halocombstatins
Com- Inhibition of polymerization Inhibition of colchicine binding
pound IC.sub.50 (.mu.M) .+-. S.D. % inhibition .+-. S.D. 1a 1.8
.+-. 0.2 81 .+-. 3 11a 1.5 .+-. 0.2 75 .+-. 6 12a 1.6 .+-. 0.3 85
.+-. 4 13a 1.5 .+-. 0.2 89 .+-. 2 14a 1.6 .+-. 0.2 84 .+-. 7
[0179] TABLE-US-00010 TABLE III Antimicrobial activities of
halocombstatins and other compounds Range of minimum inhibitory
concentration (.mu.g/ml) Compound Microorganism 11a 11b 12a 14a 14b
13a 13b 18a 20a Cryptococcus neoformans 64 64 64 32-64 64 * * * *
Candida albicans * * * * * * * * * Staphylococcus aureus * 32-64 *
* 8-64 * * * * Streptococcus pneumoniae 64 64 32-64 64 * * * * *
Enterococcus faecalis * * * * * * * * * Micrococcus luteus 32-64
16-32 32 16-32 4-8 32-64 * * * Escherichia coli * * * * * * * * *
Enterobacter cloacae * * * * * * * * * Stenotrophomonas * * * * * *
* * * maltophilia Neisseria gonorrhoeae 32 8-16 16 16-32 4-16 32-64
* 16 16-32 * = no inhibition at 64 .mu.g/ml
[0180] TABLE-US-00011 TABLE IV Human Anaplastic Thyroid Carcinoma
Cell Line Inhibition Values (GI.sub.50) expressed in .mu.g/mL.
Compound KAT-4 SW1736
3-Iodo-4,4',5-trimethoxy-3'-hydroxy-Z-stilbene 0.089-0.14 2.2
3,5-diiodo-4,4'-dimethoxy-3'-hydroxy-Z-stilbene 0.039-0.063 1.2
Potassium 3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate
0.37-0.43 >10 Potassium 3,5-diiodo-4,4'dimethoxy-Z-stilbene
3'-O-phosphate 0.38-0.44 >10
[0181] TABLE-US-00012 TABLE V Human Umbilical Vein Endothelial Cell
(HUVEC) Inhibition Values (GI.sub.50) expressed in .mu.g/mL.
Compound HUVEC 3-Iodo-4,4',5-trimethoxy-3'hydroxyl-Z-stilbene
0.000040 3,5-diiodo-4,4'-dimethoxy-3'-hydroxy-Z-stilbene 0.00028
Potassium 3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate
0.00025 Sodium 3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate
0.00035 Potassium 3,5-diiodo-4,4'dimethoxy-Z-stilbene 3'-O- 0.0049
phosphate Sodium 3,5-diiodo-4,4'dimethoxy-Z-stilbenes
3'-O-phosphate 0.051
[0182] TABLE-US-00013 TABLE VI Length of Cords Formed, Number of
Junctions and Relative Percent Growth Drug Lengths of Number of
Relative % Concentration Cords junctions Growth
3-Iodo-4,4',5-trimethoxy-3'-hydroxy-Z-stilbene 0.01 .mu.g/ml - - 14
0.001 .mu.g/ml + + 14 0.0001 .mu.g/ml ++(+) ++(+) 18 0.00001
.mu.g/ml 90 3,5-diiodo-4,4' dimethoxy-3'-hydroxy-Z-stilbene 0.01
.mu.g/ml - - 4 0.001 .mu.g/ml + (+) 8 0.0001 .mu.g/ml +++ +++ 84
0.00001 .mu.g/ml 87 Potassium
3-iodo-4,4',5-trimethoxy-Z-stilbene-3'-O-phosphate 0.01 .mu.g/ml 1
0.001 .mu.g/ml ++ ++(+) 10 0.0001 .mu.g/ml +++ +++ 77 0.00001
.mu.g/ml +++ +++ 95 Sodium
3-iodo-4,4',5-trimethoxy-Z-stilbene-3'-O-phosphate 0.1 .mu.g/ml - -
7 0.01 .mu.g/ml - - 14 0.001 .mu.g/ml + + 5 0.0001 .mu.g/ml 104
Potassium 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate 0.1
.mu.g/ml - - 15 0.01 .mu.g/ml ++(+) ++(+) 33 0.001 .mu.g/ml +++ +++
88 0.0001 .mu.g/ml 96 Sodium 3,5-diiodo-4,4'-dimethoxy-Z-stilbene
3'-O-phosphate 1 .mu.g/ml - - -7 0.1 .mu.g/ml + (+) -2 0.01
.mu.g/ml ++(+) ++(+) >100 0.0001 .mu.g/ml >100 Lengths of
Number of Legend Cords junctions - No Cords No Junctions + Small
Few ++ .about.50% of .about.50% of Control Control +++ Same as Same
as Control Control
[0183] TABLE-US-00014 TABLE VII Antimicrobial activities of
iodocomstatins Range of MIC (.mu.g/ml) ATCC or (Presque Compound
Microorganism Isle) # A B C D E F G H I J K L M N 0 P Q R S T U
Cryptococcus 90112 * 64 * * * * * * * * * * * * * * * * * * *
neoformans Candida 90028 * * * * * * * * * * * * * * * * * * * * *
albicans Staphylococcus 29213 * * * * * * * * * * * * * * * * * * *
* * aureus Streptococcus 6303 * * * * * * * * * * * * * * * * * * *
* * pneumoniae Enterococcus 29212 * * * * * * * * * * * * * * * * *
* * * * faecalis Micrococcus (456) * * 4-16 2-4 * * * * * * * * * *
* * * * * * * luteus Escherichia 25922 * * * * * * * * * * * * * *
* * * * * * * coli Enterobacter 13047 * * * * * * * * * * * * * * *
* * * * * * cloacae Stenotrophomonas 13637 * * * * * * * * * * * *
* * * * * * * * * maltophilia Neisseria 49226 64 * * * * * * *
16-32 * * 4-8 32-64 <0.5-4 32-64 <0.5-2 <0.5 <0.5
<0.5-1 <0.5 <0.5-2 gonorrhoeae Key for Table VII B
3-iodo-4,4'5-trimethoxy-3'-hydroxy-Z-stilbene C
3,5-diiodo-4,4'-dimethoxy-3' hydroxy-Z-stilbene D
3,5-diiodo-4,4'-dimethoxy-3' hydroxy-E-stilbene E
3,5-diiodo-4,4'-dimethoxy-3'-acetyl-Z-stilbene F Potassium 3
iodo-4,4'5-trimethoxy-Z-stilbene 3'-O-phosphate G Sodium 3
iodo-4,4',5 trimethoxy-Z-stilbene-3'-O-phosphate H
Lithium-3-iodo-4,4'5 trimethoxy-Z-stilbene 3'O-phosphate I
Morpholine 3 iodo-4,4'5-trimethoxy-Z-stilbene-3'-O phosphate J
Piperidene 3-iodo-4,4',5-trimethoxy-Z-stilbene-3'O phosphate K
Glycine-O-Me-3-iodo-4,4',5-trimethoxy-Z-stilbene-3'-O-phosphate L
Tryptophan-O-Me-3'-iodo-4,4',5 trimethoxy-Z-stilbene 3'-O-phosphate
M Tris-3-iodo-4,4',5-trimethoxy-Z-stilbene 3'-O-phosphate N
Potassium 3,5 diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate 0
Sodium 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'-O-phosphate P
Lithium 3,5-diiodo-4,4' dimethoxy-Z-stilbene 3'O phosphate Q
Morpholine 3,5 diiodo-4,4' dimethoxy-Z-stilbene 3-O-phosphate R
Piperdine 3,5 diiodo-4,4' dimethoxy-Z-stilbene 3'O-phosphate S
Glycine O Me 3,5-diiodo-4,4' dimethoxy-Z-stilbene-3'-O-phosphate T
Tryptophan-O Me 3,5 diiodo 4,4' dimethoxy-Z-stilbene-3'-O-phosphate
U Tris 3,5-diodo-4,4' methoxy-Z-stilbene 3'O-phosphate
* * * * *