Highly active analogs of 1alpha,25-dihydroxyvitamin D(3) that resist metabolism through C-24 oxidation and C-3 epimerization pathways

The secosteroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized in its target tissues through modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway is initiated by hydroxylation at C-24 of the side chain and leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways are initiated by hydroxylations at C-23 and C-26 of the side chain and lead to the formation of the end product, calcitriol lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 of the A-ring to form 1alpha,25(OH)(2)-3-epi-D(3). A rational design for the synthesis of potent analogs of 1alpha,25(OH)(2)D(3) is developed based on the knowledge of the various metabolic pathways of 1alpha,25(OH)(2)D(3). Structural modifications around the C-20 position, such as C-20 epimerization or introduction of the 16-double bond affect the configuration of the side chain. This results in the arrest of the C-24 hydroxylation initiated cascade of side chain modifications at the C-24 oxo stage, thus producing the stable C-24 oxo metabolites which are as active as their parent analogs. To prevent C-23 and C-24 hydroxylations, cis or trans double bonds, or a triple bond are incorporated in between C-23 and C-24. To prevent C-26 hydroxylation, the hydrogens on these carbons are replaced with fluorines. Furthermore, testing the metabolic fate of the various analogs with modifications of the A-ring, it was found that the rate of C-3 epimerization of 5,6-trans or 19-nor analogs is decreased to a significant extent. Assembly of all these protective structural modifications in single molecules has then produced the most active vitamin D(3) analogs 1alpha,25(OH)(2)-16,23-E-diene-26,27-hexafluoro-19-nor-D(3) (Ro 25-9022), 1alpha,25(OH)(2)-16,23-Z-diene-26,27-hexafluoro-19-nor-D(3) (Ro 26-2198), and 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor-D(3) (Ro 25-6760), as indicated by their antiproliferative activities.     

Calcitriol derivatives with two different side chains at C-20 III. An epimeric pair of the gemini family with unprecedented antiproliferative effects on tumor cells and renin mRNA expression inhibition

The searches for drugs that exhibit antineoplastic activity and regulate blood pressure are among the most prevalent and compelling research activities today. Amazingly, there is ample precedence for the antiproliferative action of vitamin-D-related compounds and their role as endocrine suppressors of renin biosynthesis. We have recently synthesized a number of novel calcitriol analogs of the gemini family and originally selected for further studies an epimeric pair related to 19-nor-calcitriol whose 21-methyl group was replaced by a 5,5,5-trifluoro-4-hydroxy-4-(trifluoromethyl)-2-pentynyl group. While maintaining the acceptable calcemic responses, the IC50 concentrations of interferon-γ release were reduced and the antiproliferative activity and inhibition of renin mRNA expression enhanced. Replacing the geminal methyl groups on the calcitriol-related side chain of these gemini compounds with trideuteriomethyl moieties further boosted the potency in the colon cancer model in mice some 10-fold, reduced NMU-induced breast cancer carcinogenesis in rats and decreased the IC₅₀ values for renin mRNA inhibition into the pM range.     

Synthesis of argentatin A derivatives as growth inhibitors of human cancer cell lines in vitro

The syntheses of nine argentatin A analogs are described. These compounds were assessed for their ability to inhibit growth in vitro in four human cancer cell lines. Our results showed that the presence of either a double bond at C-1/C-2, or a bromine atom or formyl moiety at C-2 as well as the presence of an isoxazol ring in argentatin A enhanced its potency in all cell lines tested. In addition, an X-ray study of (16S,17R,20S,24R)-3-oxime-20,24-epoxy-16,25-dihydroxy-cycloartan-3-one led to the determination of the correct stereochemistry of argentatin A.     

Structure-function study of gemini derivatives with two different side chains at C-20, Gemini-0072 and Gemini-0097

Derivatives of vitamin D(3) containing a second side-chain emanating at C-20 are known as gemini and act as vitamin D receptor agonists. Recently, two of these, namely Gemini-0072 and the epimeric Gemini-0097, were selected for further studies in view of their high biological activities and lack of hypercalcemic effects. We now show that the two analogs recruit coactivator SRC-1 better than the parental gemini and act as VDR superagonists. The crystal structures of complexes of zVDR with Gemini-0072 and Gemini-0097 indicate that these ligands induce an extra cavity within the ligand-binding pocket similar to gemini and that their superagonistic activity is due to an increased stabilization of helix H12.     

Enhanced biological activity of 1alpha,25-dihydroxy-20-epi-vitamin D3, the C-20 epimer of 1alpha,25-dihydroxyvitamin D3, is in part due to its metabolism into stable intermediary metabolites with significant biological activity

1alpha,25-dihydroxy-20-epi-vitamin D3 (1alpha,25(OH)2-20-epi-D3), the C-20 epimer of the natural hormone 1alpha,25(OH)2D3, is several fold more potent than the natural hormone in inhibiting cell growth and inducing cell differentiation. At present, the various mechanisms responsible for the enhanced biological activities of this unique vitamin D3 analog are not fully understood. In our present study we compared the target tissue metabolism of 1alpha,25(OH)2D3 with that of 1alpha,25(OH)2-20-epi-D3 using the technique of isolated perfused rat kidney. The results indicated that the C-24 oxidation pathway plays a major role in the metabolism of both compounds in the rat kidney. However, it was noted that the concentrations of two of the intermediary metabolites of 1alpha,25(OH)2-20-epi-D3, namely, 1alpha,24(R),25(OH)3-20-epi-D3 and 1alpha,25(OH)2-24-oxo-20-epi-D3 in the kidney perfusate, exceeded the concentrations of the corresponding intermediary metabolites of 1alpha,25(OH)2D3. Furthermore, 1alpha,25(OH)2-24-oxo-20-epi-D3 induces the conformation of the vitamin D receptor similar to that induced by its parent analog and is nearly as potent as its parent in inducing transactivation of a gene construct containing the human osteocalcin vitamin D-responsive element. We conclude that 1alpha,25(OH)2-20-epi-D3 by itself is not metabolically stable when compared to 1alpha,25(OH)2D3, but it acquires its metabolic stability because of the reduced rate of catabolism of its intermediary metabolites. Furthermore, 1alpha,25(OH)2-24-oxo-20-epi-D3, the stable bioactive intermediary metabolite plays a significant role in generating the enhanced biological activities ascribed to 1alpha,25(OH)2-20-epi-D3.     

The Chemical Constituents of Astraglus Adsurgens

Fifteen comounds have been isolated from Astragalus adsurgens Pall. Structures of eleven compounds were identified by means of spectral and chemical methods and compared with known compounds. Among all the compounds, 9 and l0 are new triterpene compounds, their structures have been elucidated as follows; (20R, 24S)-3, 16- dicorbonyl-6 α, 25-dihydroxy-20, 24-epoxy-9, 19-cyclpadsurgenin, (20R, 24S)-3,16-dicorbonyl-6α,25-dihydroxy-20, 24-epoxy-23-nitrogen-9, 19-cycloadsurgenin.     

Stable isotope studies on steroid metabolism and kinetics: sulfates of 3 alpha-hydroxy-5 alpha-pregnane derivatives in human pregnancy

The metabolism and production rates of 3 alpha-hydroxy-5 alpha-pregnan-20-one sulfate and the 3-sulfate and 3,20-disulfate of 5 alpha-pregnane-3 alpha,20 alpha-diol in pregnant women were studied. The steroid sulfates were labeled with deuterium in the 3 beta,11,11- or 3 beta,11,11,20 beta-positions and were injected intravenously. The deuterium content of steroids in the monosulfate and disulfate fraction of plasma collected at different times after the injection was determined by capillary column gas chromatography/mass spectrometry. The injected steroid sulfates underwent oxidoreduction at C-20 and 16 alpha-hydroxylation. In addition, the 3-sulfate of 5 alpha-pregnane-3 alpha,20 alpha-diol became hydroxylated at C-21. The pregnanediol and pregnanetriol monosulfates were also converted to disulfates. No evidence was obtained for a metabolic sequence involving hydrolysis, oxidoreduction, and resulfation at the C-3 position. Production rates and rates of metabolic transformations were determined using different one- and two-pool models. The production rate of the pregnanolone/pregnanediol monosulfate couple was 0.08 to 0.5 mmol/24 h, the variability probably depending both on individual factors and stage of pregnancy. The half-life time for oxidation and reduction at C-20 was 0.1 to 0.4 hours, reduction being the faster process. The half-life time for the turnover of the steroid skeleton was 1.3 to 3.3 hours. The injected steroid monosulfates were 16 alpha-hydroxylated at a rate of 1 to 8 mumol/24 h. A significant fraction of these 16 alpha-hydroxylated steroid sulfates, 0.5 to 25 mumol/24 h, was formed from other, probably unconjugated, precursors. The 16 alpha-hydroxylated steroid monosulfates underwent rapid oxidoreduction at C-20. The 3-sulfate of 5 alpha-pregnane-3 alpha,20 alpha-diol was hydroxylated at C-21. The production rate of 5 alpha-pregnane-3 alpha,20 alpha,21-triol 3-sulfate was 8 to 36 mumol/24 h in four women and 180 mumol/24 h in one woman, and this steroid was not formed from other precursors to a significant extent. 5 alpha-Pregnane-3 alpha,20 alpha-diol disulfate was a metabolic end product accounting for a major part of the elimination of the steroids injected. Its half-life time was 1.4 to 2.8 hours. The results show that the formation of sulfated steroids with a 3 alpha-hydroxy-5 alpha configuration may account for 50% of the metabolism of progesterone in late pregnancy.     

Characterization of Vitamin D insensitive prostate cancer cells

The antitumor effects of 1,25-dihydroxyvitamin D₃ (calcitriol) are being exploited for prevention and treatment of prostate cancer (CaP). These studies examined the antiproliferative effects of calcitriol in primary cell cultures derived from transgenic adenocarcinoma of mouse prostate (TRAMP) mice chronically treated with calcitriol (20 μg/kg) or vehicle 3×/week from 4 weeks-of-age until palpable tumors developed. This is a report on the response of two representative control (Vitamin D naïve, naïve) and calcitriol-treated (Vitamin D insensitive, VDI) cells to calcitriol. VDI cells were less sensitive to calcitriol based on less cell growth inhibition and less inhibition of DNA synthesis as measured by MTT and BrdU incorporation assays. Similarly, VDI cells were less sensitive to growth inhibition by the vitamin analog, 19-nor-1,25-dihydroxyvitamin D₂ (paricalcitol). There was no change in apoptosis following treatment of naïve and VDI cells with calcitriol. Vitamin D receptor (VDR) expression was up-regulated by calcitriol in both naïve and VDI cells. In addition, calcitriol induced the Vitamin D metabolizing enzyme, 24-hydroxylase (cyp24) mRNA and enzyme activity similarly in naïve and VDI cells as measured by RT-PCR and HPLC, respectively. In summary, VDI cells are less responsive to the antiproliferative effects of calcitriol. Understanding Vitamin D insensitivity will further clinical development of Vitamin D compounds for prevention and treatment of CaP.     

Identification of 17-methyl-18-norandrosta-5,13(17-dien-3beta-ol, the C19 fragment formed by adrenal side chain cleavage of a 20-aryl analog of (20S)-20-hydroxycholesterol.

Incubation of (20R)-20-phenyl-5-pregnene-3beta,20-diol, an aromatic analog of (23S)-20-hydroxycholesterol, with an adrenal mitochondrial preparation leads to the formation of four compounds: pregnenolone, phenol, a C8 ketone, acetophenone, and a nonpolar C19 compound. This latter compound has now been identified by reverse isotope dilution analysis and by gas chromatography/mass spectrometry as 17-methyl-18-norandrosta-5,13(17)-dien-3beta-ol. From these results it is evident that enzymatic fission of the C-17,20 bond of this synthetic derivative occurs. On the other hand, when (20S)-20-hydroxy[21-14C]cholesterol was used as substrate, the analogous cleavage did not take place. Thus, substitution of an aromatic group on C-20 facilitates side chain cleavage between that carbon atom and the nucleus whereas neither of the naturally occuring precursors, cholesterol or its 20-hydroxylated counterpart, are metabolized to a C8 fragment.     

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.     

Synthesis and bioactivity evaluation of brassinosteroid analogs

Four new analogs of 28-homocastasterone have been synthesized and completely characterized for the first time from stigmasterol. (22R, 23R,24S)-3beta-acetoxy-22,23-dihydroxy-5alpha-stigmastan+ ++-6-one (17), (22R,23R,24S)-3beta-bromo-22,23-dihydroxy-5alpha-stigmast an-6-one (18), (22R,23R,24S)-3beta-acetoxy-5,22, 23-trihydroxy-5alpha-stigmastan-6-one (20), and (22R,23R, 24S)-3beta-bromo-5,22,23-trihydroxy-5alpha-stigmastan-6-one (21), were obtained through a synthetic route based on regioselective Delta(5) epoxidation. Compounds 17 and 18, bearing a 5alphaH moiety, were prepared through a reductive opening of the 5beta,6beta epoxy precursor, and compounds 20 and 21, analogs with a 5alphaOH moiety were obtained by hydrolytic opening of a mixture of 5alpha,6alpha and 5beta,6beta epoxy precursors. Known compounds 19 and 22 were also obtained following the described synthetic routes, respectively. The new compounds were tested with the traditional auxin-like bioassay for brassinosteroids with 19 and 22 as standards. All compounds were comparatively evaluated for their inhibitory effect on the replication of DNA (HSV-1) virus.     

New ursolic and betulinic derivatives as potential cytotoxic agents

Fifteen new ursolic and betulinic triterpenoids, bearing various functionalities at C-3 and C-28 were synthesized as potential cytotoxic agents. All compounds were obtained by a hemisynthetic route via ursolic and betulinic acids. Preliminary screening of these compounds on human HT 29 colon cancer cells revealed inhibitory activity for three of them. Beta-D-Glucopyranosyl-3beta-hydroxyurs-12(13)-en-28-oate 1c, 3beta-3-(3-pyridyl)-prop-2-enoyloxyurs-12(13)-en-28-oic acid 1i and the potassium salt of 3beta-cinnamoyloxylup-20(29)-en-28-oic acid 2d demonstrated cytotoxic activity in the micromolar range: 8.0, 45.0 and 8.0 microM, respectively.     

Bioactivity of 25-hydroxy-, 26-hydroxy, 25,26-dihydroxy- and 25,26-epoxybrassinolide.

The bioactivity of 25-hydroxybrassinolide, (25S)- and (25R)-26-hydroxybrassinolide, (25S)- and (25R)-25,26-dihydroxybrassinolide, and of (25R)-25,26-epoxybrassinolide was tested in the rice leaf lamina inclination assay. The 25- and (25S)-26-hydroxy derivatives are known metabolites of the naturally-occurring phytohormone brassinolide, whereas the other compounds are novel, but closely related, congeners. When tested alone, all showed either no activity or only weak activity at relatively high doses. When coapplied with indole-3-acetic acid (IAA), an auxin that synergizes the effects of brassinosteroids, enhanced bioactivity was observed for each compound. However, even when applied together with IAA, none of the compounds proved more bioactive than brassinolide with or without IAA. We conclude from these results that enzymatic hydroxylation of endogenous brassinolide at C-25 and/or C-26 does not enhance brassinosteroid activity, and so does not comprise an activation pathway in brassinolide biosynthesis. Instead, these hydroxylations result in modest to appreciable metabolic deactivation.     

Evaluation of the antiviral activity of natural sulfated polyhydroxysteroids and their synthetic derivatives and analogs.

Disodium 3beta,21-dihydroxypregn-5-en-20-one disulfate (2), sodium 3beta,21-dihydroxypregn-5-en-20-one 3-sulfate (3), sodium 3beta,21-dihydroxypregn-5-en-20-one 21-sulfate (4), and disodium 3beta,6alpha-dihydroxy-5alpha-pregnan-20-one disulfate (6) have been synthesized and completely characterized for the first time from readily available materials. Sulfation was performed using triethylamine-sulfur trioxide complex in dimethylformamide as the sulfating agent. Selective sulfation of 3beta,21-dihydroxypregn-5-en-20-one rendered sodium 3beta,21-dihydroxypregn-5-en-20-one 3-sulfate (3) as the major compound. The synthetic sulfated steroids as well as natural disulfated polyhydroxysteroids (7-9) isolated by us from the antarctic ophiuroid Astrotoma agassizii and the synthetic derivatives disodium 2beta,3alpha,21-trihydroxy-(20R)-cholesta-5,24-diene 3-acetate, 2,21-disulfate (7a) and 2beta,3alpha,21-trihydroxy-(20R)-cholesta-5,24-diene (7b) were comparatively evaluated for their inhibitory effect on the replication of one DNA (HSV-2) and two RNA (PV-3, JV) viruses. In general, steroids with sulfate groups at C-21 and C-2 or C-3 were the most effective in their inhibitory action against HSV-2 and also proved to be active against PV-3 and JV.     

Influence of electron transport proteins on the reactions catalyzed by Fusarium fujikuroi gibberellin monooxygenases

The multifunctional cytochrome P450 monooxygenases P450-1 and P450-2 from Fusarium fujikuroi catalyze the formation of GA14 and GA4, respectively, in the gibberellin (GA)-biosynthetic pathway. However, the activity of these enzymes is qualitatively and quantitatively different in mutants lacking the NADPH:cytochrome P450 oxidoreductase (CPR) compared to CPR-containing strains. 3beta-Hydroxylation, a major P450-1 activity in wild-type strains, was strongly decreased in the mutants relative to oxidation at C-6 and C-7, while synthesis of C19-GAs as a result of oxidative cleavage of C-20 by P450-2 was almost absent whereas the C-20 alcohol, aldehyde and carboxylic acid derivatives accumulated. Interaction of the monooxygenases with alternative electron transport proteins could account for these different product distributions. In the absence of CPR, P450-1 activities were NADH-dependent, and stimulated by cytochrome b5 or by added FAD. These properties as well as the decreased efficiency of P450-1 and P450-2 in the mutants are consistent with the participation of cytochrome b5:NADH cytochrome b5 reductase as redox partner of the gibberellin monooxygenases in the absence of CPR. We provide evidence, from either incubations of GA12 (C-20 methyl) with cultures of the mutant suspended in [18O]H2O or maintained under an atmosphere of [18O]O2:N2 (20:80), that GA15 (C-20 alcohol) and GA24 (C-20 aldehyde) are formed directly from dioxygen and not from hydrolysis of covalently enzyme-bound intermediates. Thus these partially oxidized GAs correspond to intermediates of the sequential oxidation of C-20 catalyzed by P450-2.     

The novel pyrimidine and purine derivatives of l-ascorbic acid: synthesis, one- and two-dimensional 1H and 13C NMR study, cytostatic and antiviral evaluation

The syntheses of the novel C-5 substituted pyrimidine derivatives of l-ascorbic acid containing free hydroxy groups at C-2' (6-10) or C-2' and C-3' (11-15) positions of the lactone ring are described. Debenzylation of the 6-chloro- and 6-(N-pyrrolyl)purine derivatives of 2,3-O,O-dibenzyl-l-ascorbic acid (16 and 17) gave the new compounds containing hydroxy groups at C-2' (18) and C-2' and C-3' (19 and 20). Z- and E-configuration of the C4'C5' double bond and position of the lactone ring of the compounds 6-9 were deduced from their one- and two-dimensional (1)H and (13)C NMR spectra and connectivities in NOESY and HMBC spectra. Compounds 15 and 18 showed the best inhibitory activities of all evaluated compounds in the series. The compound 15 containing 5-(trifluoromethyl)uracil showed marked inhibitory activity against all human malignant cell lines (IC(50): 5.6-12.8 microM) except on human T-lymphocytes. Besides, this compound influenced the cell cycle by increasing the cell population in G2/M phase and induced apoptosis in SW 620 and MiaPaCa-2 cells. The compound 18 containing 6-chloropurine ring expressed the most pronounced inhibitory activities against HeLa (IC(50): 6.8 microM) and MiaPaCa-2 cells (IC(50): 6.5 microM). The compound 20 with 6-(N-pyrrolyl)purine moiety showed the best differential inhibitory effect against MCF-7 cells (IC(50): 35.9 microM).     

23-carboxy-24,25,26,27-tetranorvitamin D3 (calcioic acid) and 24-carboxy-25,26,27-trinorvitamin D3 (cholacalcioic acid): end products of 25-hydroxyvitamin D3 metabolism in rat kidney through C-24 oxidation pathway

During the past two and half decades the elucidation of the metabolic pathways of 25OHD(3) and its active metabolite 1alpha,25(OH)(2)D(3) progressed in parallel. In spite of many advances in this area of vitamin D research, the unequivocal identification of the end products of 25OHD(3) metabolism through C-24 oxidation pathway has not been achieved. It is now well established that both 25OHD(3) and 1alpha,25(OH)(2)D(3) are metabolized through the same C-24 oxidation pathway initiated by the enzyme 24-hydroxylase (CYP24A1). Based on the information that the end product of 1alpha,25(OH)(2)D(3) metabolism through C-24 oxidation pathway is 1alpha-OH-23- COOH-24,25,26,27-tetranor D(3) or calcitroic acid; the metabolism of 25OHD(3) into 23-COOH-24,25,26,27-tetranor D(3) has been assumed. Furthermore, a previous study indicated 24-COOH-25,26,27-trinor D(3) as a water soluble metabolite of 24R,25(OH)(2)D(3) produced in rat kidney homogenates. Therefore, 24-COOH-25,26,27-trinor D(3) was also assumed as another end product of 25OHD(3) metabolism through C-24 oxidation pathway. We embarked on our present study to provide unequivocal proof for these assumptions. We first studied the metabolism of 25OHD(3) at low substrate concentration (3x10(-10)M) using [1,2-(3)H]25OHD(3) as the substrate in the perfused rat kidneys isolated from both normal and vitamin D(3) intoxicated rats. A highly polar water soluble metabolite, labeled as metabolite X was isolated from the kidney perfusate. The amount of metabolite X produced in the kidney of a vitamin D intoxicated rat was about seven times higher than that produced in the kidney of a normal rat. We then produced metabolite X in a quantity sufficient for its structure identification by perfusing kidneys isolated from vitamin D intoxicated rats with high substrate concentration of 25OHD(3) (5x10(-6)M). Using the techniques of electron impact and thermospray mass spectrometry, we established that the metabolite X contained both 23-COOH-24,25,26,27-tetranor D(3) and 24-COOH-25,26,27-trinor D(3) in a ratio of 4:1. The same metabolite X containing both acids in the same ratio of 4:1 was also produced when 24R,25(OH)(2)D(3) was used as the starting substrate. Previously, the trivial name of cholacalcioic acid was assigned to 24-COOH-25,26,27-trinorvitamin D(3). Using the same guidelines, we now assign the trivial name of calcioic acid to 23-COOH-24,25,26,27-tetranor D(3). In summary, for the first time our study provides unequivocal evidence to indicate that both calcioic and cholacalcioic acids as the end products of 25OHD(3) metabolism in rat kidney through C-24 oxidation pathway.