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We have synthesized and studied the ability of a series of seven novel 1 alpha,25(OH)2 vitamin D3 analogues to inhibit clonal growth of prostate cancer cells (LNCaP, PC-3 and DU-145). Addition of double and triple bonds to the C/D ring (C-16) and side chain (C-22 and C-23) as well as lengthening of the side chain were important for enhanced activity against LNCaP and PC-3. Reorientation of the side chain in the 20-epi configuration resulted in analogues that were extremely potent only against LNCaP (ED50 approximately 5 x 10(-11) M). Compounds with six fluorines on the end of the side chain were very active against both PC-3 and LNCaP (ED50 approximately 2 x 10(-8) M). DU-145 cells were relatively resistant to compounds with all of these modifications, but removal of C-19 (e.g. 1,25(OH)2-16-ene-23-yne-26,27-F6-19-nor-D3) resulted in an analogue that was inhibitory against all three prostate cell lines. Further analysis showed that pulse exposure (3 days, 10(-7) M) to this analogue was enough to inhibit clonal growth of PC-3 cells by 50%. The same exposure also induced cell cycle arrest of all three cell lines, accompanied by upregulated protein expression of the cyclin-dependent kinase inhibitor (CDKI) known as p21waf1 in all three cell lines, and the CDKI known as p27kip1 in LNCaP cells. Associated with upregulation of these CDKIs, partial differentiation occurred as measured by increased expression of both prostate-specific antigen by LNCaP cells and E-cadherin, a cell adhesion protein that may act as a putative tumour suppressor (LNCaP and PC-3 cells). In summary, this is the first report of a potent series of 19-nor-vitamin D3 analogues with the ability to inhibit proliferation of LNCaP, PC-3 and DU-145 prostate cancer cell lines. These compounds may mediate their potent anti-proliferative activities through a cell cycle arrest pathway.

Dendritic cells (DCs) not only induce but also modulate T cell activation. 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induces DCs with a tolerogenic phenotype, characterized by decreased expression of CD40, CD80, and CD86 costimulatory molecules, low IL-12 and enhanced IL-10 secretion. We have found that a short treatment with 1,25(OH)(2)D(3) induces tolerance to fully mismatched mouse islet allografts that is stable to challenge with donor-type spleen cells and allows acceptance of donor-type vascularized heart grafts. This effect is enhanced by co-administration of mycophenolate mofetil (MMF), a selective inhibitor of T and B cell proliferation that has also effects similar to 1,25(OH)(2)D(3) on DCs. Graft acceptance is associated with an increased percentage of CD4(+)CD25(+) regulatory cells in the spleen and in the draining lymph node that can protect 100% of syngeneic recipients from islet allograft rejection. CD4(+)CD25(+) cells, able to inhibit the T cell response to a pancreatic autoantigen and to significantly delay disease transfer by pathogenic CD4(+)CD25(-) cells, are also induced by treatment of adult nonobese diabetic (NOD) mice with 1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor vitamin D(3) (BXL-698). This treatment arrests progression of insulitis and Th1 cell infiltration, and inhibits diabetes development at non-hypercalcemic doses. The enhancement of CD4(+)CD25(+) regulatory T cells, able to mediate transplantation tolerance and to arrest type 1 diabetes development by a short oral treatment with VDR ligands, suggests possible clinical applications of this approach.

0.39 mmol) and lithium borohydride (0.2 mL of a 2 M solution in tetrahydrofuran, 0.4 mmol) in 1.8 mL of tetrahydrofuran was stirred for 1.5 h at -78 O C . The reaction was quenched with acetic acid and methanol; the mixture was then allowed to warm to room temperature. The volatiles were removed in vacuo, and the residue was separated by two successive stages of TLC: the first plate was developed with acetone/chloroform (3:2) to remove unreacted 5; the second plate was developed with chloroform/methanol(91) to yield 40 mg (40%) of l l d and 21 mg (21%) of 10d, which exhibited spectrometric properties indistinguishable from those previously reported.'s (b) 1Od by Desilylation of loa. To a cooled (ice bath) solution of 10a (114 mg, 0.28 mmol) in 25 mL of tetrahydrofuran was added 0.28 mL of a 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran. The reaction was complete (TLC) in 30 min. The volatiles were removed, and the resulting residue was purified by preparative TLC using acetone/chloroform (3:2) (R, 0.26) for development to yield 67 mg (94%) of 10d.(c) 1 Id by Desilylation of 1 la. Desilylation of 1 la (125 mg, 0.30 "01) was accomplished according to the procedure described above for desilylation of 10a to afford 66 mg (86%) of Ild: R, 0.49 (9:l chloroform/methanol); MS, m / z (relative intensity) 257 la,25Dihydroxycholecalciferol(4) and la,25-dihydroxyergocalciferol(7), the hormonally active forms of vitamin D3(1) and vitamin D2 (5), were synthesized by a Horner-Wittig reaction of the phosphine oxide 11 with the ketones 10 and 12, respectively. The synthon 11 was obtained by a sequence that involves the stereospecific opening of epoxide 15, with sodium acetate in acetic acid, followed by oxidative degradation of the isopropenyl side chain and dehydration of the intermediate 22. Photoisomerization of the resulting 23 gave 24, which was finally converted to 11. The hydroxylated ketone 10 was obtained from the known intermediate 28. The introduction of the 25-hydroxy side chain was achieved by reaction of the lithium derivative of 30 with the tosylate 29 to give 31, which was catalytically hydrogenated to 32 and then converted to 10. The ketone 12 was prepared by a stereocontrolled route that involves as the key step, the [3 + 21 dipolar cycloaddition of nitrone 35 with methyl 3,3-dimethylacrylate (36) to give a 1:l mixture of isoxazolidines 37 and 38. Stereochemical control was achieved by taking advantage of the thermal reversibility of the cycloaddition, which allows the reequilibration of undesired 37. Isoxazolidine 38 was readily transformed to 43 by reduction, followed by elimination of the nitrogen function, and finally oxidation to 12.In the past two decades, extensive investigations of the vitamin D, (cholecalciferol, 1) metabolism have led to the discovery of a number of transformations which this essential vitamin undergoes in biological systems.2 The most fundamental of these transformations is the sequence of hydroxylations of 1, which starts in the liver to give 25hydroxycholecalciferol...

Prostate cancer is a major cause of male cancer death. In vitro and in vivo data support a role for 1a,25 Dihydroxyvitamin D 3 (1a,25(OH) 2 D 3 ) in regulating the growth and di erentiation of the normal prostate gland yet prostate cancer cells appear signi®cantly less sensitive to this action. Vitamin D 3 receptor (VDR) content or mutational status do not correlate clearly with the antiproliferative e ects of 1a,25(OH) 2 D 3 and therefore it is unclear why prostate cancer cell lines are signi®cantly less sensitive to this action. We hypothesized that the antiproliferative responses of prostate cancer cells to 1a,25(OH) 2 D 3 are suppressed by a process involving histone deacetylation. Sodium butyrate (NaB) and trichostatin A (TSA) are inhibitors of histone deacetylase (HDAC) activity. Low doses of NaB or TSA (300 mM and 15 nM respectively), which alone were relatively inactive, synergized with 1a,25(OH) 2 D 3 in liquid and semi-solid agar to inhibit the growth of LNCaP, PC-3 and DU-145 prostate cancer cells. Still greater synergy was observed between vitamin D 3 hexa¯uoride analogs and either NaB or TSA. The mechanism appeared to involve neither the cyclindependent kinase inhibitor, p21 (waf1/cip1) nor cell cycle arrest, but rather induction of apoptosis. These data suggest that cells dysregulate the normal pro-apoptotic signals of 1a,25(OH) 2 D 3 during prostate cancer development by a mechanism involving histone deacetylation. Combination therapy with potent vitamin D 3 analogs and clinically approved HDAC inhibitors may overcome this lesion and improve the treatment of both androgendependent and independent prostate cancer. Oncogene (2001) 20, 1860 ± 1872.