Vitamin D3 analogs

Details Vitamin D3 analogs Vitamin D3 analogs

2000-05-24

2001-12-18

Qazi, Sabiha (Department: 1616)

Organic compounds -- part of the class 532-570 series

Organic compounds

9,10-seco-cyclopentanohydrophenanthrene ring system or...

C514S167000

Reexamination Certificate

active

06331642

ABSTRACT:

SUMMARY OF THE INVENTION
The invention relates to a compound of the formula
wherein
R
1
is hydrogen or an alkyl group;
R
2
is hydrogen or an alkyl group; or
R
1
, R
2
and C
20
together are cyclopropyl;
R
3
is alkyl, hydroxy-alkyl or fluoroalkyl; and
R
4
is alkyl, hydroxy-alkyl or fluoroalkyl.
It has been found that the compounds of formula I induce inhibition of proliferation in prostate, breast and myeloid leukemic cancer cell lines. Accordingly, the compounds of formula I are useful as agents for the treatment of prostate cancer, breast cancer, and for the treatment of leukemia.
It has also been found that the compounds of formula I have antiandrogenic activity. Accordingly, the compounds of formula I are useful for treating benign prostate growth, baldness and cancer of the prostate.
It has also been found that the compounds of formula I have activity making them useful for the treatment of sebaceous gland diseases such as acne or seborheic dermatitis.
It has also been found that the compounds of formula I have activity making the compounds useful for treating osteoporosis.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term “alkyl group” denotes a straight or branched chain alkyl group having from 1-4 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, t-butyl and the like. The term “hydroxy-alkyl” denotes an alkyl group having a hydroxy substituent on any of the carbon atoms of the alkyl group. The term “fluoro-alkyl” denotes an alkyl group having one, two or three fluorine atoms substituted on any of the carbon atoms of the alkyl group.
In the formulas presented herein, the various substituents are illustrated as joined to the nucleus by one of the following notations: a wedged solid line
indicating a substituent which is above the plane of the molecule; and a wedged dotted line
indicating a substituent which is below the plane of the molecule.
Preferably, R
1
is hydrogen and R
2
is alkyl, or R
1
is alkyl and R
2
is hydrogen. More preferably, R
1
is hydrogen and R
2
is methyl or R
1
is methyl and R
2
is hydrogen.
Preferably, R
3
and R
4
are independently, alkyl, hydroxy-alkyl or trifluoro-alkyl. More preferably, R
3
and R
4
are, independently, methyl, hydroxy-methyl or trifluoro-methyl.
Preferably, A is
The most preferred compound of formula I is 1,25-dihydroxy-16-ene-5,6-trans-cholecalciferol.
The compounds of formula I are prepared as hereafter described, with particular reference the Formula Scheme below.
R
5
is hydrogen or trimethylsilyl.
In the above Formula Scheme, wherein Ph is phenyl, the compound of formula II, which is the compound [3S-(1E,3&bgr;,5&agr;)-2-[3,5-bis(dimethylethyl)dimethylsilyl]oxy]-2-methylene-cyclohexylidene]ethyl]diphenylphosphine oxide, is converted to a compound of formula IA by reaction with a compound of formula III.
The reaction is carried out at −60° C. to −90° C., in a polar aprotic, organic solvent, such as dry ether or more preferably dry tetrahydrofuran, in the presence of a strong base such as an alkyl lithium like butyl lithium.
The protecting groups of compounds of formula IA are removed by reaction with a flouride salt, such as tetrabutyl-ammonium fluoride in a polar, organic solvent such as ether, or more preferably tetrahydrofuran, to yield the corresponding compound of formula I.
The compound of formula II is prepared as hereinafter described in Examples 1-6, or alternatively, as described in Examples 7-8.
Compounds of formula III are known (for example, in U.S. Pat. No. 5,087,619) or can be prepared according to known methods.
The useful activity of compounds of formula I as agents for the treatment of prostate cancer, breast cancer, and for the treatment of leukemia can be demonstrated by the following test processes which are known in the art.
Materials selected for use herein and procedures employed were as follows:
Cell lines. The breast cancer cell line (MCF-7), prostate cancer cell line (LNCaP) and myeloid leukemia cell line (HL-60) were maintained as follows: MCF-7 cells were maintained in Dulbecco's Modified Eagle Media (DMEM) with 10% fetal calf serum (FCS); LNCaP and HL-60 were cultured in RPMI1640 with 10% FCS; All three cell lines were maintained in a 37° C. incubator containing 5% CO
2
.
Vitamin D
3
Compounds. The vitamin D
3
compounds were dissolved in absolute ethanol at 10
−3
M as stock solution, which were stored at −20° C. and protected from light. For in vitro use, compounds were diluted in DMEM or RPMI medium. For in vivo use, compounds were diluted with phosphate buffered saline (PBS). An aliquot was used only once and LNCaP cells were trypsinized. Washed single-cell suspensions of cells were enumerated and plated into 24-well flat-bottomed plates with a total of 1×10
−3
cells/well in a volume of 400 &mgr;l/well. The feeder layer was prepared with agar that had been equilibrated at 42° C. Prior to this step, compounds were pipetted into wells. After incubation, the colonies were counted. All experiments were done at least three times using triplicate plates per experimental point.
Serum Calcium Levels in vivo. Twenty-eight male Balb/c mice at 8 to 9 weeks of age were maintained in pathogen-free conditions and fed a standard laboratory diet. Four mice per group were injected intraperitoneally every other day (except Saturday and Sunday) with either vitamin D
3
compound or diluant (100 &mgr;l/mouse) for 3 weeks. Doses of 1,25(OH)
2
-16-ene-5,6-trans-D
3
were: 0.1, 0.5, 1.0, and 2.0 &mgr;g. Doses of 1,25(OH)
2
D
3
were 0.1 &mgr;g/mouse. Control mice were injected with 100 &mgr;l of PBS. Serum calcium values were measured every week by the quantitative, colorimetric detection assay.
Pulse-Exposure Experiments. The MCF-7 cells were incubated in liquid culture with 10
−7
of 1,25(OH)
2
D
3
or 1,25(OH)
2
-16-ene-5,6-trans-D
3
for various duration. After incubation, these cells were carefully washed twice with PBS and viable cells were counted and plated into 24-well plates for soft agar colony assay, as previously described.
Cell Cycle Analysis by Flow Cytometry. Cell cycle analysis was performed on MCF-7 cells incubated for 4 days with either 1,25(OH)
2
D
3
or 1,25(OH)
2
-16-ene-5,6-trans-D
3
at 10
−7
M. The cells were fixed in chilled methanol overnight before staining with 50 &mgr;g/ml propidium iodide, 1 mg/ml Rnase (100 units/ml) and 0.1% NP40. Analysis was performed immediately after staining. All experiments were done at least three times independently. All data were statistically analyzed by Student's test.
Western Blot Analysis. Cells were washed twice with PBS, suspended in Lysis buffer (50 mM Tris Ph 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, 100 &mgr;g/ml phenylmethylsulfonyl fluoride, 2 &mgr;g/ml aprotinin, 1 &mgr;g/ml pepstatin and 10 &mgr;g/ml leupeptin), and placed on ice for 30 minutes. After centrifugation at 15,000 g for 20 minutes at 4° C., the supernatant was collected. Protein concentrations were quantitated. Whole lysates (15 &mgr;g) were resolved by 15% sodium dodecyl sulphate polyacrylamide gel, were transferred to an immobilon polyvinylidene difuride membrane and were probed with anti-p
27
kip1
rabbit polyclonal antibody and anti-Actin murine monoclonal antibody. Blots were developed.
Telomerase Activity. To detect the relative telomerase activity, telomeric repeat amplification protocol (TRAP) assays were performed. For human telomerase reverse transcriptase (hTERT), total RNAs were isolated from HL-cells which were treated with either 1,25(OH)
2
D
3
or 1,25(OH)
2
-16-ene-5,6-trans-D
3
(10
−
9 10
−8
, and 10
−7
M) for 4 days with a monophasic solution of phenol and guanidine isothiocyanate. RT-PCR was performed with 1 &mgr;g of total RNA and random hexamer primers. cDNA was amplified using primers specific for the hTERT gene or the GAPDH gene, which was used as a control. The primers used for hTERT were: 5′-CGGAAGAGTGTCTGGAGCAA-3′ (sense) (SEQ ID NO: 1), and 5′-GGATGAAGCGGAG

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