Highly potent cell differentiation-inducing analogues of 1alpha,25-dihydroxyvitamin D3: synthesis and biological activity of 2-methyl-1,25-dihydroxyvitamin D3 with side-chain modifications

Eight 2-methyl substituted analogues of 20-epi-22R-methyl-1alpha,25-dihydroxyvitamin D3 (5) and 20-epi-24,26,27-trihomo-22-oxa-1alpha,25-dihydroxyvitamin D3 (6: KH-1060) were convergently synthesized. Preparation of the CD-ring portions with modified side chains of 5 and 6, followed by palladium-catalyzed cross-coupling with the A-ring enyne synthons (20a-d), (3S,4S,5R)-, (3S,4R,5R)-, (3S,4S,5S)- and (3R,4R,5S)-3,5-bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-1-en-7-yne, afforded two sets of four A-ring stereoisomers of 20-epi-2,22-dimethyl-1,25-dihydroxyvitamin D3 (7a-d) and 20-epi-24,26,27-trihomo-2-methyl-22-oxa-1,25-dihydroxyvitamin D3 (8a-d). The biological profiles of the hybrid analogues were assessed in terms of affinity for vitamin D receptor (VDR) and HL-60 cell differentiation-inducing activity in comparison with the natural hormone. The combined modifications of the A-ring at the 2-position and the side chain yielded analogues with high potency.     

The antagonism between 2-methyl-1,25-dihydroxyvitamin D3 and 2-methyl-20-epi-1,25-dihydroxyvitamin D3 in non-genomic pathway-mediated biological responses induced by 1alpha,25-dihydroxyvitamin D3 assessed by NB4 cell differentiation

We synthesized all eight possible A-ring diastereomers of 2-methyl substituted analogs of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and also all eight A-ring diastereomers of 2-methyl-20-epi-1alpha,25(OH)2D3. Their biological activities, especially the antagonistic effect on non-genomic pathway-mediated responses induced by 1alpha,25(OH)2D3 or its 6-s-cis-conformer analog, 1alpha,25(OH)2-lumisterol3, were assessed using an NB4 cell differentiation system. Antagonistic activity was observed for the 1beta-hydroxyl diastereomers, including 2beta-methyl-1beta,25(OH)2D3 and 2beta-methyl-3-epi-1beta,25(OH)2D3. Very interestingly, 2beta-methyl-3-epi-1alpha,25(OH)2D3 also antagonized the non-genomic pathway, despite its 1alpha-hydroxyl group. Other 1alpha-hydroxyl diastereomers did not show antagonistic activity. 20-epimerization diminished the antagonistic effect of all of these analogs on the non-genomic pathway. These findings suggested that the combination of the 2-methyl substitution of the A-ring and 20-epimerization of the side chain could alter the biological activities in terms of antagonism of non-genomic pathway-mediated biological response. Based on a previous report, 2-methyl substitution alters the equilibrium of the A-ring conformation between the alpha- and beta-chair conformers. The 2beta-methyl diastereomers, which exhibited antagonism on non-genomic pathway-mediated response, were considered to prefer the beta-conformer. Further examination to elucidate the relationship between the altered ligand shape and receptors interaction will be important for molecular level understanding of the mechanism of antagonism of the non-genomic pathway.     

Synthesis and biological evaluation of new 6-s-cis locked 1,2,25-trihydroxyprevitamin D3 analogues

An efficient synthesis of several diastereomers of 2-hydroxy substituted 1alpha,25-dihydroxyprevitamin D3 derivatives was accomplished utilizing a practical route to the A-ring synthon. The biological activity of the analogues was evaluated in vitro. All the synthesized derivatives demonstrated low affinity for the vitamin D receptor and vitamin D-binding protein compared with 1alpha,25-dihydroxyvitamin D3, the natural hormone. 1alpha,2beta,25-trihydroxy-19-nor-pre-D3 was the most potent of the analogues in inhibiting proliferation of MCF-7 cells but requires higher EC50 concentrations than 1alpha,25-dihydroxyvitamin D3.     

2,2-Functionalized analogues of 1alpha,25-dihydroxyvitamin D3, the potent inducers of cell differentiation

All four possible A-ring stereoisomers of 2,2-dimethyl-1,25-dihydroxyvitamin D(3) (4) were designed and convergently synthesized. Nine-step conversion of methyl hydroxypivalate 6 provided the desired A-ring enyne synthon (13a,b) in good overall yield. Cross-coupling reaction of the A-ring synthon 13a,b with the CD-ring portion in the presence of palladium catalyst, followed by deprotection, gave the vitamin analogues (4a-d). We also synthesized four stereoisomers of 2,2-ethano-1,25-dihydroxyvitamin D(3) (5), as novel spiro-ring analogues having cyclopropane fused at the C2 position. Biological potencies of the synthesized compounds were assessed in terms of the vitamin D receptor (VDR) binding affinity, as well as the HL-60 cell differentiation-inducing activity. The 2,2-ethano analogue 5a showed a comparable activity to the natural hormone 1, while the 2,2-dimethyl analogue 4a exhibited one-third of the activity of 1 in cell differentiation, with the reduced VDR binding affinity.     

Concise synthesis and biological activities of 2alpha-alkyl- and 2alpha-(omega-hydroxyalkyl)-20-epi-1alpha,25-dihydroxyvitamin D3

We found a concise route to the Trost A-ring precursor enyne for synthesizing 2alpha-alkylated 1alpha,25-dihydroxyvitamin D(3) (1) from D-glucose. The enynes were coupled with the 20-epi-CD ring part to study the effect of the double modification of 2alpha-substitution and 20-epimerization upon biological activities of 1. The novel three analogues of 2alpha-alkyl- and four analogues of 2alpha-(omega-hydroxyalkyl)-20-epi-1alpha,25-dihydroxyvitamin D(3) (5b-d and 6a-d) showed higher binding affinity for vitamin D receptor (VDR) and more potent activity in induction of HL-60 cell differentiation than those of the natural hormone 1.     

Synthesis and evaluation of a 3-position diastereomer of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71)

A 3-position diastereomer of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71, 2), 3-epi-ED-71 (4), was synthesized by the convergent method coupling the A-ring fragment (5) with the C/D-ring fragment (6). As the results of preliminary in vitro biological evaluation of 3-epi-ED-71 (4), the inhibition of parathyroid hormone secretion in bovine parathyroid cells and binding affinity to human recombinant vitamin D receptor and to human vitamin D binding protein in comparison with ED-71 (2), 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3, 1), and 3-epi-1,25(OH)2D3 (3) are described.     

New highly calcemic 1 alpha,25-dihydroxy-19-norvitamin D(3) compounds with modified side chain: 26,27-dihomo- and 26,27-dimethylene analogs in 20S-series

New highly potent 2-substituted (20S)-1 alpha,25-dihydroxy-19-norvitamin D(3) analogs with elongated side chain were prepared by Wittig-Horner coupling of A-ring phosphine oxide with the corresponding protected (20S)-25-hydroxy Grundmann's ketones. Biologic evaluation in vitro and in vivo of the synthesized compounds was accomplished. All the synthesized vitamins possessing a 25-hydroxylated saturated side chain were slightly less active (3-5X) than 1 alpha,25-dihydroxyvitamin D(3) in binding to the porcine intestinal vitamin D receptor and significantly more potent (12-150X) in causing differentiation of HL-60 cells. In vivo, 2-methylene-26,27-dihomo and 2 alpha-methyl-26,27-dimethylene analogs were at least 10 times more active, and 2 alpha-methyl-26,27-dihomo compound at least 5 times more active than the vitamin D hormone both in stimulating intestinal calcium transport and bone calcium mobilization (serum calcium increase). It was also established that a 260 pmol dose of the corresponding 2 beta-methyl analogs had a similar effect on intestinal calcium transport and a much more pronounced effect on bone calcium mobilization as the same dose of 1 alpha,25-dihydroxyvitamin D(3).     

Metabolic pathways from 1 alpha,25-dihydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3-26,23-lactone. Stereo-retained and stereo-selective lactonization

The present study was carried out in order to elucidate the metabolic pathway from 1 alpha,25-(OH)2D3 to 1 alpha,25-(OH)2D3-26,23-lactone. For that purpose, we stereospecifically synthesized the vitamin D3 derivatives 1 alpha,23(S),25-(OH)3D3, 1 alpha,23(S),25(R),26-tetrahydroxyvitamin D3, and 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-lactol. The in vitro metabolism of these compounds was examined in kidney homogenates and intestinal mucosa homogenates from 1 alpha,25-(OH)2D3-supplemented chicks. The naturally occurring 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone was produced (in increasing amounts) from 1 alpha,25-(OH)2D3, 1 alpha,25(R),26-(OH)3D3, 1 alpha,23(S),25-(OH),D3, 1 alpha,23(S),25(R),26-(OH)4D3, and 23(S),25(R)-1 alpha,25-(OH)2D3-26,23-lactol. These results indicated that there are two possible metabolic pathways from 1 alpha,25-(OH)2D3 to 1 alpha,23(S),25(R),26-(OH)4D3: the major one is by way of 1 alpha,23(S),25-(OH)3D3 and the minor one is by way of 1 alpha,25(R),26-(OH)3D3. 1 alpha,23(S),25(R),26-Tetrahydroxyvitamin D3 is further metabolized to 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone via 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactol. In the course of our studies, a new biosynthetic vitamin D3 metabolite was isolated in pure form. This metabolite was identified as 23(S),25(R)-1 alpha,25-(OH)2D3-26,23-lactol by UV spectrophotometry and mass spectrometry. Furthermore, we establish in this report that the lactonization of 1 alpha,23,25,26-(OH)4D3 and 1 alpha,25-(OH)2D3-26,23-lactol occurs in a stereo-retained and stereo-selective fashion.     

Biological activity of 1 alpha, 25-dihydroxyvitamin D derivatives--24-epi-1 alpha, 25-dihydroxyvitamin D-2 and 1 alpha,25-dihydroxyvitamin D-7

Biological activity of 24-epi-1 alpha,25-dihydroxyvitamin D-2 (24-epi-1,25(OH)2D2) and 1 alpha,25-dihydroxyvitamin D-7 (1,25(OH)2D7), the 22,23-dihydro derivative of the former compound, was investigated. Both of the vitamin D derivatives stimulated intestinal calcium transport and calcium mobilization from bones in rats; however, the effect was about 50% of that of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)2D3). On the other hand, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 inducement of HL-60 human leukemia cell differentiation was comparable to that of 1,25(OH)2D3. Accordingly, the differentiation-inducing activity of 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 was much greater than their ability to stimulate calcium metabolism. In contrast to 1,25(OH)2D3, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 exerted little hypercalcemic activity in mice. These results suggest that both vitamin D derivatives will be useful as anti-tumor agents.     

Versatile synthesis and biological evaluation of 1,3-diamino-substituted 1alpha,25-dihydroxyvitamin D3 analogues

A concise route to 1alpha,3beta-diamino-25-hydroxy-3-deoxyvitamin D(3) (5) and 1beta,3alpha-diamino-25-hydroxy-3-deoxyvitamin D(3) (6) has been developed starting from (R)- or (S)-carvone for the construction of the modified A-ring fragments. The conversion of the hydroxyl group to amine function with complete inversion of the configuration was efficiently accomplished by Mitsunobu reaction using phthalimide as nucleophile or activation of the hydroxyl group as mesylate followed by reaction with NaN(3). Diamino 5 and 6 as well as monoamino 3, 4, 30, and 31 vitamin D(3) derivatives have shown poor binding to VDR compared with 1alpha,25-dihydroxyvitamin D(3). The most active compound in the inhibition of MCF-7 cell proliferation and HL 60 cell differentiation was 1alpha-amino analogue 3. Also, very low in vivo calcemic effects of derivatives 3 and 4 were found.     

1alpha,25-dihydroxyvitamin D(3)-26,23-lactone analogs antagonize differentiation of human leukemia cells (HL-60 cells) but not of human acute promyelocytic leukemia cells (NB4 cells).

We examined the effects of two novel 1alpha,25-dihydroxyvitamin D(3)-26,23-lactone (1alpha,25-(OH)(2)D(3)-26,23-lactone) analogs on 1alpha,25(OH)(2)D(3)-induced differentiation of human leukemia HL-60 cells thought to be mediated by the genomic action of 1alpha, 25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) and of acute promyelocytic leukemia NB4 cells thought to be mediated by non-genomic actions of 1alpha,25-(OH)(2)D(3). We found that the 1alpha,25-(OH)(2)D(3)-26,23-lactone analogs, (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9648), inhibited differentiation of HL-60 cells induced by 1alpha,25-(OH)(2)D(3). However, 1beta-hydroxyl diastereomers of these analogs, i.e. (23S)-25-dehydro-1beta-hydroxyvitamin D(3)-26, 23-lactone (1beta-TEI-9647) and (23R)-25-dehydro-1beta-hydroxyvitamin D(3)-26,23-lactone (1beta-TEI-9648), did not inhibit differentiation of HL-60 cells caused by 1alpha,25-(OH)(2)D(3). A separate study showed that the nuclear vitamin D receptor (VDR) binding affinities of the 1-hydroxyl diastereomers were about 200 and 90 times weaker than that of 1alpha-hydroxyl diastereomers, respectively. Moreover, none of these lactone analogs inhibited NB4 cell differentiation induced by 1alpha,25-(OH)(2)D(3). In contrast, 1beta,25-dihydroxyvitamin D(3) (1beta,25-(OH)(2)D(3)) and 1beta,24R-dihydroxyvitamin D(3) (1beta,24R-(OH)(2)D(3)) inhibited NB4 cell differentiation but not HL-60 cell differentiation. Collectively, the results suggested that 1-hydroxyl lactone analogs, i.e. TEI-9647 and TEI-9648, are antagonists of 1alpha,25-(OH)(2)D(3), specifically for the nuclear VDR-mediated genomic actions, but not for non-genomic actions.     

The stereochemical configuration of the natural 1 alpha,25-dihydroxyvitamin D3-26,23-lactone

Four possible diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone were chemically synthesized and compared with the natural metabolite by high-pressure liquid chromatography. The four synthetic diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone could be separated into three peaks by high-pressure liquid chromatography. The naturally occurring 1 alpha,25-dihydroxyvitamin D3-26,23-lactone isolated from dog serum and in vitro incubation of chick kidney homogenates comigrated with 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone. The four diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone were tested against naturally occurring 1 alpha,25-dihydroxyvitamin D3-26,23-lactone to determine their relative competition in the 1 alpha,25-dihydroxyvitamin D3-specific cytosol receptor binding assay for 1 alpha,25-dihydroxyvitamin D3. 23(S)25(S)-1 alpha,25-Dihydroxyvitamin D3-26,23-lactone was the best competitor followed by 23(R)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone and 23(R)25(S)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone, and 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone was the poorest competitor. Natural 1 alpha,25-dihydroxyvitamin D3-26,23-lactone isolated from dog serum had almost the same binding affinity as that of 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone. These data unequivocally demonstrate that the stereochemistry of the natural 1 alpha,25-dihydroxyvitamin D3-26,23-lactone has the 23(S) and 25(R) configuration.     

Design and synthesis of potent vitamin D receptor antagonists with A-ring modifications: remarkable effects of 2alpha-methyl introduction on antagonistic activity

Novel A-ring analogues of the vitamin D receptor (VDR) antagonist (3a), ZK-159222, and its 24-epimer (3b) were convergently synthesized. Preparation of the CD-ring portions with the side chains of 3a,b, followed by palladium-catalyzed cross-coupling with the A-ring enyne precursors (15a,b), (3S,4S,5R)- and (3S,4S,5S)-bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-1-en-7-yne, afforded the 2alpha-methyl-introduced analogues (4a,b) and their 3-epimers (5a,b). The biological profiles of the hybrid analogues were assessed in terms of affinity for VDR, and antagonistic activity to inhibit HL-60 cell differentiation induced by the natural hormone, 1alpha,25-dihydroxyvitamin D(3). The analogue 4a showed an approximately fivefold higher antagonistic activity compared with 3a. The 2alpha-methyl introduction into 3a increased the receptor affinity, thereby enhancing VDR antagonism. This approach to design potent antagonists based on hybridization of structural motifs in the A-ring and in the side chain may prove to be valuable.     

Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1alpha,25-dihydroxyvitamin D3

Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1alpha-hydroxyvitamin D3 compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by 1H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3 was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 and 22-iodo-1alpha,25-dihydroxyvitamin D3 isomers were ca. ten times less potent than the natural hormone 1alpha,25-(OH)2D3 both in intestinal calcium transport and bone calcium mobilization.     

Studies on the mechanism of action of 1 alpha, 24-dihydroxyvitamin D3. II. Specific binding of alpha, 24-dihydroxyvitamin D3 to chick intestinal receptor

The binding of vitamin D3 analogues to the chick intestinal cytosol receptor was studied. In intestinal cytosol fraction, receptor proteins having the sedimentation constant of 2.5 S and 3.7 S to which 1 alpha,25-dihydroxyvitamin D3 binds were present, and the latter was specific for the compound. The binding of 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3 to the receptor was also observed, while very weak binding was seen in the case of 24(R)25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3. The binding affinity of 1 alpha,24(R)-dihydroxyvitamin D3 to the 3.7 S receptor was 1.3 times as high as that of 1 alpha,25-dihydroxyvitamin D3, whereas those of 1 alpha,24(S)-dihydroxyvitamin D3, 1 alpha-hydroxyvitamin D3 and 25-hydroxyvitamin D3 were 10, 304 and 652 times lower than 1 alpha,25-dihydroxyvitamin D3, respectively. The dissociation constant of the receptor-1 alpha,25-dihydroxyvitamin D3 complex at 0 degrees C was 3.0 x 10(-11) M, and the dissociation constants were calculated to be 2.4 x 10(-11) M and 2.7 x 10(-10) M for the complexes with 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3, respectively.     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) in vitamin D receptor-ablated mice in vivo

The metabolism of 1alpha,25-dihydroxyvitamin D(3) was studied in vitamin D receptor-ablated mice following the administration of a physiological dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). The degradation of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) in the vitamin D receptor null mutant mice was substantially reduced compared to the wild-type control mice. At 24 h postadministration of radiolabeled 1alpha,25-dihydroxyvitamin D(3) more than 50% of the radioactivity was recovered unmetabolized, whereas in wild-type mice nearly all of the 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) was degraded. In wild-type mice three polar metabolites other than 1alpha,25-dihydroxyvitamin D(3) were detected and identified on straight-phase and reverse-phase high-performance liquid chromatography as 1alpha,24(R),25-trihydroxy-[26,27-(3)H]vitamin D(3), 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3), and (23S, 25R)-1alpha,25-dihydroxy-[(3)H]vitamin D(3)-26,23-lactone. Only one metabolite was detected in the plasma and kidneys of vitamin D receptor null mutant mice at 3 h following an intrajugular dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). This metabolite was clearly identified as 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3) by comigration on two HPLC systems and periodate cleavage reaction. At 6, 12, and 24 h postinjection 1alpha,24(R), 25-trihydroxy-[26,27-(3)H]vitamin D(3) was also detected at low levels in plasma, kidneys, and liver of some but not all mutant mice. The presence of 25-hydroxyvitamin D(3)-24-hydroxylase mRNA in the kidneys of these vitamin D receptor null mutant mice was confirmed by ribonuclease protection assay.     

Novel vitamin D3 derivatives, 26-homo-delta 22-dehydro-1 alpha,25(S)-dihydroxyvitamin D3 and 26-homo-delta 22-dehydro-1 alpha,25(R)-dihydroxyvitamin D3: preferential activity in c-myc mRNA production and in induction of phenotypic differentiation of HL-60 cells

Novel vitamin D3 derivatives, 26-homo-delta 22-1 alpha,25(S)-dihydroxyvitamin D3 and 26-homo-delta 22-1 alpha,25(R)-dihydroxyvitamin D3 were compared with the native hormone, 1,25-dihydroxyvitamin D3, and with other vitamin D3 derivatives, in inhibition of cell growth, induction of phenotypic differentiation, and c-myc mRNA reduction of HL-60 cells. The degree of inhibition in cell growth caused by 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 was the greatest followed by 26-homo-delta 22-1 alpha,25(R)-(OH)2D3. The ability to reduce NBT was parallel to that to inhibit cellular proliferation. 26-homo-delta 22-1 alpha,25(S)-(OH)2D3, 26-homo-delta 22-1 alpha,25(R)-(OH)2D3, 24-homo-24-F2-1 alpha,25-(OH)2D3, and 1 alpha,24(R)-(OH)2-26-Cl-D3 were more active than 1 alpha,25-(OH)2D3 in the induction of OK-M1+ and OK-Mo-2+ HL-60 cells. Using two color flow cytometric analysis, the percentages of OK-M5(+)- and OK-DR(+)-HL-60 cells were 33% in the treatment with 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 plus interferon-gamma (IFN-gamma) but 14% in the treatment with 1 alpha,25-(OH)2D3 plus IFN-gamma. 26-Homo-delta 22-1 alpha,25(S)-(OH)2D3 has an inhibitory effect on c-myc reduction in treated HL-60 cells. These results suggest that the novel vitamin D3 derivatives, 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 and 26-homo-delta 22-1 alpha,25(R)-(OH)2D3, have preferential activity in inducing phenotypic differentiation and in inducing cell proliferation related c-myc mRNA activity.