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The synthesis of diazo, halo, and sulfoxy bile acid derivatives: Potential affinity labels
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THE SYNTHESIS OF DIAZO, HALO, AND SULFOXY BILE ACID DERIVATIVES: POTENTIAL AFFINITY LABELS Aaron A. Fried, Vladimir Petrow, and Leon Lack Department of Pharmacology, Duke University Medical Center Durham, North Carolina 27710
Received:
3-19-79 ABSTRACT
Bile acid derivatives, with and without C-3 sulfate groups, and having either the diazo- or halomethylketone moieties, have been synthesized in good yield and purity. The synthetic sequence, COOH-+COC1--+COCHN2-GOCH2X, was used with deoxycholic and cholic acids, which requires carefully controlled quench, work-up, and purification procedures, especially for the 3-sulfate esters (made from deoxycholic acid derivatives only). The pure title compounds are anticipated to be useful chemical probes (affinity labels), especially the completely water soluble sulfates, toward our studies of ileal active transport of bile salts. A new use for Sephadex LH-20 as a sulfate ester protecting group is reported. Also developed were the use of acetamide hydrochloride complex as a mild hydrochlorination reagent and a neutral desalting method for sulfate esters of deoxycholic acid derivatives. INTRODUCTION The active transport of bile salts has been observed at three anatomical sites: tubule.
the liver, the ileum, and the proximal renal
For a review of the topic see (1).
Recent -in vitro studies
of ileal bile salt transport using vesicles (2), suggest that the receptor macromolecule as well as the transport mechanism resides within the brush border membrane. In order to obtain further information on the nature of the ileal macromolecular receptor, we have turned to the concept of affinity labeling (3,4), which has been so successful in identifying structural components of enzyme site-receptor macromolecular
VoZwne 34, Number 2
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interactions.
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To this end we have replaced the carboxyl terminal
of deoxycholic and cholic acids with diazocarbonyl methylcarbonyl
(COCHN2) and halo-
(COCH2X) groups; we anticipate that these reactive bile
acid derivatives
(V-IX, XII, and XIII), will be compatible with the
transport mechanism and will be able to combine with an amino acid residue of the active site, thereby both inactivating and labeling it. EXPERIMENTAL All solvents and inorganic reagents were reagent grade, A.C.S. certified. Deoxycholic acid (MCB), cholic acid (Sigma), oxalyl chloride (Aldrich), acetamide (Fisher), pyridine:sulfur trioxide (Aldrich), anhydrous ammonia (Matheson), anhydrous hydrogen chloride (Matheson), and Sephadex LH-20 (Pharmacia) were commercial products used without further purification. Methylene chloride for use as a reaction solvent was treated with anhydrous magnesium sulfate and filtered (under nitrogen); for chromatography, it was distilled but not further dried. Benzene was distilled, the middle cut collected and stored over 4 I( molecular sieves (Davison). Pyridine was distilled from barium oxide. Methanol for use as a reaction solvent was distilled from freshly prepared magnesium methoxide. Granular sodium sulfate was used to dry solutions of the diazoketones (III, IV, and X). The use of powdered magnesium sulfate resulted in a much lower recovery of products. Drying agents were avoided with all of the halomethylketones. Chromatographic adsorbents used were Silica Gel G, 60 (E. Merck), silicic acid, 100-200 mesh ("SilicAR," CC-7, Mallinckrodt), silica gel, Activity III/30 mm (ICN), and 30-63 pm silica gel (ICN). TLC (thin layer chromatography) dipping slurry was prepared according to Peifer (5) from 35 g Silica Gel G and 100 ml chloroform:methanol (3:l) and stored in a ground glass stoppered bottle for at least five days before use. Adsorbent-coated glass microanalytical TLC slides (25 x 75 mm) were freshly prepared and air dried. The following TLC developing systems were used: A. CHC13:CH30H:HOAc:HOH (65:24:17:9) (6); B. ethyl acetate: methylene chloride (30:70); C. ethyl acetate:methylene chloride (9O:lO); D. ethyl acetate: methylene chloride (3:97); E. acetone:ethyl acetate (1:l); F. methanol:ethyl acetate (5:95). The TLC slides were visualized by spraying with 1N H2SO4, heating on a hot plate, and viewing under an ultraviolet light ("Mineralight" UVS-54). Dry columns (7) were prepared by packing silica gel (ICN) in nylon foil tubes (ICN). Separated sample bands were cut into l/8 - l/4" sections with scissors while the developed column was still moist with solvent. Isolation of precipitated sulfate ester fractions was done by a combination of centrifuging, decanting, air and vacuum drying.
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Glassware was flame-dried and cooled under nitrogen. Liquid transfers were made with dry syringe and needle or cannula. Infrared (ir) spectra were recorded on Perkin Elmer Models 337 and 297 spectophotometers. Specific optical rotation values (aD) were obtained on a Central Scientific Company (CENCO) visual polarimeter with a sodium D-line source. Proton magnetic resonance (pmr) spectra were recorded on Varian Associates Model EM 360-A and JEOL Model JNM-MH-100 spectrometers with chemical shift values (6) reported in parts per million downfield from the internal standard, tetramethylsilane. The following abbreviations describe the pmr data: s (singlet), d (doublet), b (broad singlet), m (multiplet), and 3 (coupling value). Melting points were determined on Fisher-Johns Combustion analyses melting point apparatus and were uncorrected. were performed by Atlantic Microlab, Inc., Atlanta, Ga. Acetamide hydrochloride complex (CHqCONH+HCl) was prepared from acetamide and dry HCl according to the procedure of Pinner and Klein (8). Crude product was stored over NaOH. Per cent hydrogen chloride in the complex was assayed before each use by titration with O.Ol5N NaOH with phenolphthalein indicator. Acetamide hydrobromide complex (CH$ONH+HBr) was prepared from acetamide and dry HBr according to the procedure of Werner (9). Per cent hydrogen bromide was assayed by titration as above. Deoxycholic acid diformate (3u,12a-diformyloxy-58_cholan-24-oic acid) (I) and cholic acid triformate (3a,7a,12e-triformyloxy-58cholan-24-oic acid) (II) were prepared by the method of Tserng and Klein (lob): (I), mp 195-197', lit. (lob) 197-198" (corrected); (II), mp 209.8-211.2", lit. 209-210' (lob), 210-211" (lid). 25-Diazo-3a,l2a-diformyloxy-26,27-dinor-5~-cho~estan-24-one (III). To a clear solution of the diformate (I, 1.00 g, 2.22 mmol) in dry benzene (20 ml) was added dropwise, with stirring and passage of dry nitrogen through the solution, oxalyl chloride (1.0 ml, 11.7 mmol) in 5 ml dry benzene during 10 min. The colorless solution was allowed to reach room temperature and after 18 min. the nitrogenbubbler tube was pulled above the solvent level. Stirring was continued under gentle nitrogen stream for 6.25 hr. Solvent and excess oxalyl chloride were removed by evaporating the solution to dryness in vacua. Residual reagent was removed by mixing the syrupy residue -with fresh dry benzene (25 ml) and evaporating. The mixing and evaporating were repeated and the residue redissolved in 25 ml benzene. To a cold ethereal solution of diazomethane [prepared (12, 13) from a mixture of 3.55 g N-nitroso-N-methylurea (14), 40% aqueous KOH (10.6 ml) and absolute ether (40 ml), dried over KOH pellets (10 hr) and decanted from the KOH (Note: particles of hydrated KOH decompose the product, III)] was added (15 min.) dropwise, and with stirring, a clear cold solution of the acid chloride: benzene solution. Controlled addition was effected via a stainless Stirring was steel cannula (19 G; blunt tip) and nitrogen pressure. continued at 0" (1 hr) and at room temperature (0.5 hr), when excess
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diazomethane was removed by evaporating the solution at 30° to 0.5 volume with dry nitrogen. The concentrated solution was taken to dryness on the rotary evaporator to give a yellow amorphous solid, 1.113 g. Rapid gradient elution chromatography (O-+3% ether in benzene) on a silicic acid column (58 g) packed in methylene chloride gave a yellow foam, 885 mg (1.87 mmol, 84% yield). Alternatively, flash chromatography (29) can be used; for example, 2.20 g of crude product on a 50 mm silica gel (30-63 pm) column with 10% EtOAc in methylene chloride as eluent (fraction volume = 25 ml) gave, in fractions 13-23, 1.80 g of compound III; mp 45-55"; TLC, Rf"0.70 (system B); ir (CH2C12) 2087 and 1644 (COCH=N2) (15), 1722 (ester C=O) cm-l; eg5 = 99.0' (absolute EtOH, c = 1.90); pmr (acetone-d6) SO.80 (CH -18), 0.97 (CIJ3-19), 0.50-2.67 (m, steroid nucleus + side chain), 4TgO (m, C-3E), 5.18 (m, C-12X), 5.53 and 5.66 (C-25& anti and syn), 8.00 (S, C-12CgO), 8.14 (S, C-3CEO). A sample suitable for combustion analysis was obtained by the use of dry column chromatography (silica gel:sample, 3OO:l; developing solvent:5 ethyl acetate:95 methylene chloride). Anal. Calcd. for C27H4ON205: C, 68.61, H, 8.53, N, 5.93; Found C, 68.55, H, 8.52, N, 5.91 (after vacuum drying at 30'). 25-Diazo-3cc,7ol,lZcL-triformyloxy-26,27-dinor-58-cholestan-24-one (Iv). Using the foregoing two-step procedure, triformate (II, 1.10 g, 2.23 mmol) gave 1.20 g crude product; TLC, Rf=0.21 (major spot) (system D). Purification in two equal batches by dry column chromatography (218:1, silica gel:sample; 3 ethyl acetate:97 methylene chloride) gave (IV) as a pale yellow viscous liquid, 880 mg (1.70 mmol, 76.4% yield). A crystalline solid, mp 131.0-132.0 dec, lit. (lle) 134.5-135.0' dec, was obtained from pentane-ether at 0'; TLC, Rf=O.Zl, one spot (system D), ir (CH2Cl2) 2097 and 1637 (COCH=N2), 1722 (broad, ester C=O) cm-l; pmr (CDC13) 60.77 (C%-18), 0.95 (CE3-19), 0.60-2.73 (m, steroid nucleus + side chain), 4.73 (m, C-3E), 5.10, 5.26, and 5.29 (overlapping C-12$ C-25& and C-7&), 8.05 (S, C-12CgO), 8.13 (S) and 8.20 (S) (C-7CgO) and C-3CgO). Anal. Calcd. for C28H40N207.1.5H20: C, 61.86, H, 7.97, N, 5.15; Found C, 61.35, H, 7.39, N, 4.80 (after vacuum drying at 25").
25-Diazo-3cL,12c(-dihydroxy-26,27-dinor-* Diformyldiazoketone (III, 838 mg, 1.77 mmol) was mixed with cold methanolic KOH (76 ml, 2.8% solution) and allowed to stir at room temperature for 92 hr. The bright yellow solution was poured with stirring into 300 ml cold saturated sodium bicarbonate and evaporated to dryness -in vacua at 35'. The solid residue was triturated with methylene chloride (50 ml, x2). Undissolved material was taken up with 60 ml water:methylene chloride (5:l) and partitioned with the combined organic extracts. An unidentified material was insoluble in both aqueous and organic phases. Dilution of the combined organic extract with an equal volume of methylene chloride, washing with water (80 ml, x2) and saturated NaCl (80 ml, xl), drying over anhydrous sodium sulfate and evaporation gave a pale yellow foam, 599 mg (1.44 mmol, 81% yield); mp 80.5" (dec with N2+86.0°); TLC, Rfz0.24 (system C); ir (CH2C12) 3600 and 3440 (OH), 2105 and 1638 (COCH=N2)
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cm-1 ; pmr (acetone-d6) 60.68 (C&-18), 0.90 (C&3-19), 0.50-2.60 (m, steroid nucleus + side chain), 3.47 (m, C-3~+C-3O_H+c-120~), 3.93 (m, C-12E), 5.53 and 5.66 (C-25X, anti and syn). Anal. Calcd. for C25H40N20 : Found C, 71.95, H, 9.73, N, 6.6 a .
C, 72.07, H, 9.68, N, 6.73;
25-Diazo-3a,7a,12a-trihydroxy-26,27-dinor-5B-cholestan-24-one (VI). Triformyldiazoketone (IV, 440 mg, 0.81 mmol) was treated as for diformyldiazoketone (III) with 55 ml methanolic KOH [5.56 ml aqueous KOH concentrate (25 g/27 ml) + 200 ml methanol] during 115 hr at room temperature. The usual work-up plus filtration through a glass frit funnel and evaporation gave a glassy yellow solid, 269.4 mg (0.60 mmol, 73.9% yield), mp 93" (dec, N2+); TLC, Rf=0.28 (s stem E); ir (CH2C12) 3600 and 3430 (OH), 2105 and 1638 (COCH=N2) cm-1 ; pmr (CDC13) 60.68 (Ca-18), 0.90 (C%-19), 0.37-2.80 (m, steroid nucleus + side chain), 3.72 (m, C-3,7,12IJ+C-3,7,12Oc + l/2 H20), 5.34 (C-2511+ l/2 H20). Anal. Calcd. for C25H4ON204.1 H20: C, 66.63, H, 9.40, N, 6.22; Found C, 66.54, H, 9.02, N, 5.89 (after vacuum drying at 40"). 25-Chloro-3a,12a-dihydroxy-26,27-dinor-5B-cholestan-24-one (VII). To a cold (0") solution of dihydroxydiazoketone (V, 50.7 mg, 0.122 mmol) in 6 ml THF (freshly distilled from lithium aluminum hydride) was added with stirring CH3CONH2+HCl (61.6 mg, 0.12 mm01 HCl) in one portion. Stirring was continued for 1 min at O", then the ice bath was removed and the suspension was allowed to reach room temperature (evolution of nitrogen was observed). After 19.3 hr stirring at room temperature, the clear, colorless solution was taken to dryness (30", in vacua), and the residue was redissolved in 50 ml THF:ether (3:97). -The new solution was washed with water (25 ml, x3) and the aqueous wash was extracted with wet ether (25 ml, xl). Finally the combined organic extracts were washed with saturated brine, filtered through a plug of glass wool, and evaporated to give a yellow liquid residue. Solidification to a white amorphous solid was effected by dilution with methylene chloride and evaporation. Gradient elution chromatography on dry-packed silicic acid (1.13 g) with 5-+40% ethyl acetate in methylene chloride gave a pure glassy solid, 39.5 mg (0.09 mmol, 76.2% yield); mp 71.5" (shrinks, 67.5"); TLC, Rf=0.42 (system C); ir (CH2C12) 3600 and 3450 (OH), 1733 and 1717 (C=O) cm-' (16); pmr (CDC13) 60.68 (C&-18), 0.92 (C&-19), 0.50-2.85 (m, steroid nucleus + C-12OIJ+ C-3Og), 3.60 (m, C-35), 4.05 (m, C-12@ overlapping with 4.11 (S, C-25&). Anal. Calcd. for C25H4lClO3: C, 70.64, H, 9.72, Cl, 8.34; Found C, 70.50, H, 9.75, Cl, 8.38 (after vacuum drying at 45"). 25-Bromo-3a,12a-dihydroxy-26,27-dinor-5B-cholestan-24-one (VIII). Dihydroxydiazoketone (V, 100 mg, 0.24 mmol) was treated with acetsmide+HBr (71.6 mg, 0.24 mm01 HBr) using the procedure indicated for the synthesis of chloromethylketone (VII). The reaction solution was concentrated -in vacua at room temperature, diluted with 100 ml absolute ether and minimum THF, then washed successively with cold saturated sodium bicarbonate (50 ml, x2), water (50 ml, x3), and
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saturated brine (50 ml, xl). Evaporation of the washed organic extracts and azeotropic drying with dry benzene gave 119.4 mg yellow liquid. Gradient elution chromatography on silicic acid (adsorbent: sample, 19.4:l) with ethyl acetate (5-40%) in methylene chloride gave a glassy colorless solid, 98.4 mg (0.21 mmol, 88% yield); mp 70-73'; TLC, Rf=O.42 (system C); ir (CH2C12) 3600 and 3450 (OH), 1733 and 1717 (C=O) cm-1 (16); pmr (CDC13) 60.67 (C&-18), 0.92 (C&-19) 0.97 (d, C%-21, J=6), 0.48-2.20 (m, steroid nucleus + side chain), 2.60 (m, C-233 + C-120! + C-30&), 3.6 (m, C-3g), 3.92 (S, C-253) overlapping with 3.96 (m, C-1211). Anal. Calcd. for C25H41Br03: C, 63.95, H, 8.80, Br, 17.02; Found C, 63.77, H, 8.83, Br, 17.16 (after vacuum drying at 35'). 25-Chloro-3a,7a,12a-trihydroxy-26,27-dinor-5$-cholestan-24-one (Ix). Applying the hydrohalogenation procedure to the trihydroxydiazoketone monohydrate (VI, 225.5 mg, 0.50 mmol) after 22.5 hr at room temperature and usual work-up gave a white solid, 212.7 mg (0.43 mmol, 94.5% yield as the hemihydrate); mp 195.5-196.5" (clear+opaque, 133'); TLC, Rf=0.46 (system F); ir (KBr) 3400 (OH), 1713 (C=O) cm-l; pmr (CD3OD) 60.70 (C&-18), 0.90 (C&-19), 0.57-2.30 (m, steroid nucleus + side chain), 2.30-4.09 (overlapping m, c-3, 7, 12~ + C-3, 7, 12OE), 4.27 (S, C-25%). Anal. Calcd. for C25H4lClO *;H20: C, 66.72, H, 9.41, Cl, 7.88; Found C, 67.27, H, 9.45, Cl, 7.$ 4 (after vacuum drying at 45'). 25-Diazo-12a-formyloxy-3a-hydroxy-26,27-dinor-5~-cholestan-24one (X). The following was adapted from the procedure of Tserng and Klein (lob). Diformyldiazoketone (III, 3.551 g, 7.51 mmol) was dissolved in anhydrous methanol (89 ml) and filtered. Anhydrous ammonia was bubbled into the cold (0') clear solution with stirring for 5 min. Stirring was continued at 0" for 1 min, then the ice bath was removed, and the solution allowed to stand at room temperature for 1.25 hr. The solution was concentrated to one-half volume at room temperature in vacua, then mixed with absolute ether (200 ml) and evaporated to a small volume. Treatment with ether and evaporation was repeated, x2 to give an amorphous yellow solid. Purification was effected by flash chromatography (30-63 pm silica gel) using a 50 mm column (29) and 1:l ethyl acetate:methylene chloride as eluent (fraction volume = 25 ml). Evaporation of combined fractions 21-44 gave a yellow foam, 3.05 g (6.72 mmol, 89.5% yield); mp 44.5-52.0'; TLC, Rf=0.33 (system B); ir (CC14) 3448 (OH), 2109 and 1651 (COCH=N2), 1725 (ester C=O> cm-l; pmr (CCl4) 60.76 (C&-18), 0.90 (Cg3-19), 0.50-3.00 (m, steroid nucleus + side chain), 3.00-4.00 (m, C-3~ + C-3Og), 5.16 and 5.20 (m, overlapping C-12g and C-25@, 8.04 (S, CEO). Anal. Calcd. for C26H4ON20 *l/2 H20: C, 68.84, H, 9.12, N, 6.18; Found C, 68.57, H, 9.49, N, 5.72 (after vacuum drying at 35'). Anal. Calcd. for C26H4ON20 : C, 70.23, H, 9.07, N, 6.30; Found C, 69.97, H, 9.51, N, 6.2 2 (after vacuum drying at 80").
25-Diazo-lZa-formyloxy-3a-sulfoxy-26,27-dinor-58-cholestan-24-one sodium salt (XI). The following was adapted from the procedure of Rajagopalan, et al. (17). Diazoketone (X, 3.00 g, 6.61 mmol) was mixed with pyridine-sulfur trioxide complex (7.17 g, 45.05 mmol) and anhydrous pyridine (80 ml) and stirred at room temperature under nitrogen for 50 min. The reaction mixture was added in portions with stirring to cold (0") saturated sodium bicarbonate (850 ml) to give a clear yellow solution. Pyridine was removed as the pyridinewater azeotrope -in vacua giving a copious pale yellow precipitate, which was collected by filtration on a medium porosity glass frit. Trituration of the solid with dry methylene chloride (a. 200 ml, 1 hr; b. 200 ml, 0.5 hr) and evaporation of the yellow filtrate gave a yellow electrostatic powder having a purity satisfactory for the subsequent reaction, 3.95 g (7.0 mmol, essentially quantitative); mp 199.0-2oo.oo; TLC, Rf (system A) = 0.44 (major), 0.16 and 0.28 (minor). An analytically pure sample was obtained by treating a portion of the partially purified product (50.9 mg) in methylene chloride (15 ml) with absolute ether (starting with 25 ml for the first precipitated fraction) to selectively precipitate the more polar impurities (Rf 0.16 and 0.28), though with some product loss. Concentrating the final supernate and adding it dropwise to ether (35 ml), precipitated pure (XI, 38.5 mg, 76% recovery); mp 200.0200.5' dec; ir (KBr)l3440 (H20), 2100 and 1635 (COCHN2), 1715 (ester C=O), 1218 @SOT) cm ; pmr (CDC13) 60.38-3.33 (m, steroid nucleus + side chain), 3.60-5.00 (m, C-3H + 2H20), 5.00-5.67 (C-12g + C-2511), 8.15 (b, CEO). Anal. Calcd. for C26H39N2NaC7S'l H20: C, 55.30, H, 7.32, N, 4.96, Na, 4.07, S, 5.68; Found C, 55.73, H, 6.91, N, 4.69, Na, 3.73, S, 5.30 (after vacuum drying at 35'). When in another experiment, the crude product precipitate was completely dissolved in saturated sodium bicarbonate, extracted with methylene chloride, and then treated as above, the dihydrate (mp 200.0-201.0" dec) was isolated: Anal. Calcd. for C26H3gN2NaC7S.2H20: C, 53.59, H, 7.44, N, 4.81, S, 5.50; Found C, 53.42, H, 6.92, N, 4.74, S, 5.36 (after vacuum drying at 35O). 25-Diazo-12a-hydroxy-3a-sulfoxy-26,27-dinor-5$-cholestan-24-one sodium salt (XII). Cold methanolic sodium hydroxide solution (59.0 ml, 21.91 mmol, 1.88 wt/wt X) was added to diazoketone (XI, 3.401 g, 6.00 mmol) with stirring at 0". The resultant suspension was allowed to stand at room temperature with occasional intermittent stirring for 6 hr. (Reaction seemed complete after 3 hr by TLC, but the longer reaction time was used to assure complete conversion). The dark redbrown mixture, undiluted, was centrifuged at 5" at 2300 rpm to give three distinct phases (in order of increasing density): yellow solution, dark brown semi-solid (decomposition material by TLC; discarded), and pale yellow solid. After decanting the cold yellow solution and quickly scraping out the semi-solid, the pale yellow solid was triturated with cold (0“) methanol and centrifuged. Three phases
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were again formed and treated as indicated above. This purification process was repeated, being careful to avoid solvent evaporation, until all of the yellow solid had dissolved. The combined methanol solutions (0") were acidified (pH 6) with glacial acetic acid (100 drops). A sharp color change to a lighter yellow was observed. Work-up was continued with rotary evaporation (room temperature) to dryness, redissolution in deionized water (193 ml), neutralization with saturated sodium bicarbonate (100 drops), and extraction with butanol (previously saturated with water), 480 ml, xl, and washing of the organic extract (bright yellow) with water (96 ml, xl). The aqueous wash was important in removing an unidentified impurity that could not be removed effectively by the subsequent fractional precipitation method (see belaw). The butanol extract was evaporated in vacua at 35-40" to a viscous gel and taken up with dry methylene -chloride (40 ml). Solvent was evaporated to give a concentrated, slightly viscous solution. Addition of the methylene chloride solution to cold (0") absolute ether and immediate washing of the resulting precipitate with more ether, gave a pale yellow powder in purity suitable for the next reaction: 2.58 g (4.86 nnnol, 81.0% yield); mp 154.5-154.9'. Further improvement in product (XII) yield was not effected with more butanol extractions; further extractions pulled out only more impurities. Fractional precipitation with methylene chloride:ether solutions as for (XI) gave an analytically pure white solid: mp 195.0-195.9', TLC, Rf"0.37 (~~;temlA4;=i;,(~) 3450 (OH), 2100 and 1635 (COCH-N2), 1230 (OSO?j)cm [water, c=Z.Ol; slow decomposition (N29) occurred during reading]; pmr (D20) 60.33-3.33 (m, steroid nucleus + side chain), 3.90 (m, C-3g), 4.33 (m, C-12g) overlapping with 4.57 (m, C-12Og), 6.13 (m, C-255). Anal. Calcd. for C25H38N2NaOgS'O.75 H20: C, 56.56, H, 7.45, N, 5.28, Na, 4.33, S, 6.04; Found C,56.76, H, 7.58, N, 5.08, Na, 4.20, S, 5.83 (after vacuum drying at 65"). C, 58.00, H, 7.40, N, 5.41, Na, Anal. Calcd. for C25H38N2NaCgS: 4.44, S, 6.19. Found C, 57.78, H, 7.50, N, 5.17, Na, 4.29, S, 5.90 (after vacuum drying at 95'; mp 194.5-194.9"). 25-Chloro-12a-hydroxy-3a-sulfoxy-26,27-dinor-5S-cholestan-24-one sodium salt (XIII). Diazoketone (XII, 41.3 rug, 0.08 mmol) of analytical grade, obtained by fractional precipitation as described for compound (XI), was mixed (3.5 hr, vigorous stirring) with 15 ml dry methylene chloride to obtain a milky solution. Sephadex LH-20 (XII), 42:1] was added and the resultant slurry L1.734 g; exchanger: stirred for 1.7 hr. Finally, CH3CONH2+HCl (43.8 mg, 0.08 mmol) was added and the mixture diluted with solvent to completely fill the reaction vessel (total volume = 30 ml). Stirring was continued at room temperature for 18.3 hr. The product mixture was filtered through a glass frit funnel (Kimex 40M) to remove most of the byproduct, CH3CONH2, and traces of desulfated material. The residue was washed with methylene chloride (10 ml, x9), wetted with 2 ml deionized water plus 1 drop saturated sodium bicarbonate, and extracted with dry butanol (50 ml, xl) then butanol:methanol (1:l; 40 ml, x2).
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After each extraction the phases were separated by centrifuging, followed by filtering the supernate through a medium porosity glass frit. The combined filtrates were evaporated (in -- vacua at 35") to a gel which was redissolved in 30 ml butanol (presaturated with water). The wet butanol solution was mixed with 6 ml water to give an emulsion which separated after 1 hr. Water was removed from the organic phase as the butanol-water azeotrope, and the resulting dry butanol concentrate diluted to 7 ml with more butanol, removed from insoluble material by centrifuging, and once again evaporated to a gel-like residue. Dropwise addition of the gel in minimum methylene chloride to 36 ml pentane:ether (2:l) gave a white solid (27.5 mg, 69.2% yield); TLC, Rf=0.58 (system A); Rf=0.52 and 0.70 (trace impurities); ir 1659 cm-l (by-product:acetamide). Repeating the trituration with 266 ml pentane-ether (2:l) removed virtually all of the acetamide (plus some product) yielding 17.5 mg (44% yield). Finally, a sample of analytical purity (minus the Rf 0.70 component) was obtained by triturating with 5 ml pure ethyl acetate. Centrifuging, decanting and air-drying gave 13.3 mg (33% yield); mp 16~iO-170.00; ir (XBr) 3419 (OH), 1728 (C=O), 1611 (H20), 1222 (OSO3) cm ; a$3 = 52.6' (methanol, c=1.99); pmr (CD30D) 60.70 (CQ-18), 0.93 (C%-19), 0.56-2.23 (m, steroid nucleus + side chain), 2.57 (m, C-23E+ C-120I-J, 3.95 (m, C-3g), 4.27 (m, C-12E), 4.78 (S, C-25fi). Anal. Calcd. for C25H3gClNaOgS*l H20: C, 55.18, H, 7.60, Cl, 6.52, Na, 4.23, S, 5.89; Found C, 55.42, H, 7.27, Cl, 6.05, Na, 3.91, S, 5.48 (after vacuum drying at either 65" or 95"). RESULTS AND DISCUSSION This paper reports further application of the diazoketone method (18) to the bile acid series with an adaptation of the procedures of Reber, et al. (lla) to the syntheses of halomethylketones and XIII; see Fig. 1).
(VII-IX,
This method was preferred since (i) it has
already been extensively studied (11, 18); (ii) the diazoketone moiety will permit conversion into photoaffinity labels (19) as well as conventional affinity labels(3,4). Though simple in theory, the diazoketone procedure required considerable attention to detail and development of mild selective conditions in order to obtain good yields of products.
Thus, for
example, we were led to develop novel applications of Sephadex LH-20 and acetamide hydrochloride complex, the former serving as a sulfate
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TRROXDLI
protecting agent and the latter as a mild stoicheometric hydrochlorination reagent. Figures 1A and 1B illustrate a simple strategy that takes advantage of the complementary properties of the diazoketone moiety, COCHN2 (ZO), and the formate protecting groups (lo), the latter being labile and the former, stable, in mild base. Preliminary biological study of products (VII, VIII, and IX) revealed inhibition of the transport mechanism in the ileal -in vitro assay (Zl), even though the test compounds were considerably less soluble in the aqueous testing medium than were the natural substrates.
Consequently, we undertook the synthesis of the correspond-
ing sulfate esters, which were expected to be much more water soluble and yet to retain some affinity for the bile salt active site (22). The required sulfation step was accommodated in the diazoketone method as shown in Figure 1B.
The deprotection step (X1--+X11)was practi-
cable since bile acid sulfates are stable to the mild basic conditions suitable for deacylation
(23, 24, 25).
However, in this case the
diazoketone moiety, not the sulfate group, is the limiting factor in determining the required reaction conditions which must be even milder than those recently reported for deformylation
(23).
above room temperature decomposed the side chain.
Temperatures
THF was not used
in the last reaction (X11-+X111) because it allowed complete acid catalyzed desulfation.
Desulfation was somewhat retarded in methyl-
ene chloride, but still important, and was prevented in the presence of Sephadex LH-20, a lipophilic anionic exchanger gel widely used for the chromatographic purification and separation of sulfate ester mixtures
(25, 26).
Sephadex LH-20 appears to be acting in a way
either to reduce the activity of the hydrogen chloride complex or as a weak sulfate protecting group.
It should be noted that it was
essential to carry out the reagent additions in the sequence described in the experimental section.
The resultant viscous slurry with
Sephadex LH-20 must be stirred vigorously and must fill the reaction vessel completely to permit a complete conversion.
Neat methanol or
aqueous solutions must not be used during the filtration and washing steps in order to avoid product contamination by Sephadex LH-20 fines. All of the purified products and intermediates were stable at room temperature, with the halomethylketones giving sharp melting points.
Nevertheless, storage in the dry cold and dark between use
is recommended, especially for the diazoketones.
These compounds,
with the exception of products (V, VII, and VIII), were isolated as hydrates.
Attempts to remove the water of hydration resulted in
decomposition, except where indicated in the experimental section. Unlike the products in Figure lA, the corresponding monosulfate derivatives in Figure 1B were soluble in water as expected.
Inter-
estingly, these sulfates (sodium salts) while in the form of viscous gels (from evaporated butanol solutions) were readily soluble in dry methylene chloride.
These sulfate gels which probably were some type
of bile acid derivative complex with butanol (27) allowed purification of the sulfates by fractional precipitation, where methylene chloride solutions were treated with either ether or pentane.
Also
this purification procedure provided a convenient neutral desalting method that does not require the use of derivatizing methods (23) that would likely decompose our multifunctional new impurities.
sulfates or introduce
Desalting with Amberlite XAD-2 resin (23, 25) was
S
-X!TEIICOIDb
attempted on the diasoketosulfate
183
(XI) but gave only low yields of
decomposition products. In the desalting method, once the sulfates were precipitated from methylene chloride-ether or methylene chloride-pentane-ether solutions and dried, they were only partially soluble in methylene chloride.
A methylene chloride-soluble sample was reconstituted by
treatment of the dry sulfate powder with wet butanol and evaporating to give the sulfate gel once again. The ability of bile acid derivatives, especially derivatives of deoxycholic acid, to form addition compounds with water or alcohols (27) occasionally makes it difficult to obtain unambiguous TLC data. Since it was desired to monitor all our reactions with TLC, methods were developed that would allow reproducibility section).
The cholylchloroketone
(see experimental
(IX) (Rf=0.46) originally appeared
to be contaminated by an unknown component (Rf=0.23). the component at Rf=0.46
was
However, when
scraped off and rechromatographed under
the same conditions, both spots (Rf=0.46 and 0.23) appeared again. Controlling the adsorbent activity and avoiding the use of water in developing solvents showed compound (IX) to be chromatographically pure. All the new compounds gave satisfactory combustion and spectral analyses.
The ir spectra of the halomethylketones
(VII, VIII, IX,
and XIII) gave unusually weak carbonyl bands, probably due to side chain interactions with the C-12 hydroxy group (28). The diazoketones
(III, IV, and X) decomposed on silicic acid or
silica gel and, therefore, satisfactory chromatographic purification was achieved only by using multiple small sample batches with the
dry column technique (7) as for compound (IV), or by using the Still "flash" chromatographymethod (29) as for compounds (III) and (X). Complete deformylation of compound (III) required 92 hours at room temperature; after 74 hours, deformylation was incomplete, and after 113 hours the product yield was 20 per cent lower due to decomposition of the side chain. The sulfation of compound (X) was better quenched with saturated sodium bicarbonate. Pure water formed an acidic solution that decomposed some of the product before the extraction step. The sulfate product (XI) to be used for the subsequent reaction (X1-+X11) should not be purified further than indicated in the experimental section. Precipitation from methylene chloride-ether gave a less methanolsoluble sample of compound (XI), and also adversely influenced the solubility of compound (XII) as it formed in methanolic sodium hydroxide. Reaction (X1+X11) under these conditions, and with stirring in an attempt to effect solution, gave a solid mass. Reaction (X1+X11) should be quenched with cold acetic acid before solvent evaporation; otherwise complete decomposition occurred even at room temperature under a dry nitrogen stream. Compound (XII) should be washed with absolute ether immediately after precipitation from methylene chloride-ether. Omitting the ether wash and then air or vacuum drying at room temperature gave a decomposed bright yellow amorphous mass. As with compound (XI), the method of purification affects the solubility of compound (XII). Although an extensively purified sample (moderatemethylene chloride solubility) of (XII) was used for reaction (X11-+X111),the more convenient readily soluble sample (before
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TDROXD=
185
fractional precipitation) was later found to be satisfactory. Compound (XIII) can be freed of acetamide by-product and other impurities by an alternate method that is more convenient for purification of larger samples.
The method consists simply of selectively
precipitating pure (XIII) by adding pentane to solutions of impure (XIII) in 1:3.3 dry butanol-ether. In summary, selected bile acid derivatives, with and without C-3 sulfate groups, and incorporating the diazo- and halomethylketone moieties, have been synthesized in good yield and purity. (V and VII-XIII) are new (30).
Diazoketones
Compounds
[III (lib), IV (lld, 31),
and VI (lid)] have been reported elsewhere, but except for compound (IV), were not fully characterized. The synthetic approaches shown in Figure 1 should be applicable to other bile acids besides deoxycholic acid and cholic acid, allowing entry into a series of useful water soluble affinity labels for bile salt transport studies. lithocholic
The formylated 25-diazo derivatives of
(llb), chenodeoxycholic
(llb), and hyodeoxycholic acid
(11~) which have already been reported, will clearly serve as useful starting materials. Also, from our synthesis efforts, three new methods were developed: 1.
The use of acetamide hydrochloride as a mild hydrochlorina-
tion reagent. 2.
The use of Sephadex LH-20 as an agent to prevent competing
acid catalyzed solvolysis of C-3 sulfate esters. 3.
A neutral desalting procedure for acid and base sensitive
sulfate esters of deoxycholic acid derivatives.
Further work is in progress to examine the scope of these methods, including other applications of Sephadex LH-20 in bile acid synthesis. Acknowledgments - This work was supported by research grants AM-09582 and T32-ES07002 from the National Institutes of Health. We thank the Department of Chemistry, Duke University, for use of the Perkin Elmer 297 infrared spectrophotometer and nuclear magnetic resonance spectrometers. We also thank Professor Ben Wittels, Department of Pathology, Duke University Medical Center, for loan of the CENCO polarimeter. REFERENCES
(1) Lack, L. and Weiner, I. M., in The Bile Acids (eds. P. P. Nair and D. Kritchevsky), Plenum Press, N.Y., pp 33-54 (1973). 1607 (1977). a. Shaw, E., in The Enzymes, 3rd ed., vol. 1 (P. D. Boyer, ed.), pp. 46-91. Academic Press, N.Y. (1970). b. Singer, S. J., Adv. Protein Chem., 2, 1 (1967). Baker, B. R., Design of Active-site-directed Irreversible Enzyme Inhibitors; The Organic Chemistry of the Enzymic Active-site, John Wiley and Sons, N.Y. (1967). Bobbitt, J. M., Thin-Layer Chromatography, Reinhold Publishing Corporation, N.Y., p. 46 (1963) Cass, 0. W., Cowen, A. E., Hofmann, A. F., and Coffin, S. B., J. Lipid Res., I&, 159 (1975). Chromatography Products, ICN Pharmaceuticals, Inc., Life Sciences Group, pp. 5, 6 (1977-78). Pinner, A., and Klein, B., Chem. Ber., 2, 1889 (1877). Werner, A., Chem. Ber., 36, 154 (1903). a. Fuchs, H., and Reichstein, T., Helv. Chim. Acta, 26, 511 (1943). b. Tserng, K. Y., and Klein, P. D., Steroids, 2, 635 (1977). a. Reber, F., Lardon, A., Reichstein, T., Helv. Chim. Acta, 37, 45 (1954). Cf: b. Lettrd, H., Greiner, J., Rutz, K., Hofmann, L., Liebigs Ann. Chem. 758, 89 (1972); c. Lettre, H., Egle, A., von Jena, J., and Mathes, K., Leibigs Ann. Chem. 708 224 (1967); d. Ruzicka, L., Plattner, A., Heusser, H., and Schlegel, W., Helv. Chim. Acta, 7, 186 (1944); e. Pearlman, W. H., J. Am. Chem. Sot., 2, 1475 (1947). Arndt, F., Org. Syntheses Coil. Vol. 2, 165 (1943). Use only scratch-free unused glassware when CAUTION/REMINDER: preparing and using diazomethane. Avoid ground glass joints; dry over smooth KOH pellets. See reference 12. Amstutz, E. D., and Myers, R. R., Organic Syntheses Coil. Vol. 2_, 462 (1943). Yates, P., Shapiro, B. L., Yoda, N., and Fugger, J., J. Am. Chem. Sot., 2, 5756 (1957). Avram, M. and Mateescu, GH.D., Infrared Spectroscopy, Applications in Organic Chemistry (Translated by L. Birladeanu), WileyInterscience, N.Y., pp. 370-373 (1972).
(2) Lack, L., Walker, J. T., Hsu, C-Y. H., Life Sci., 0, (3)
(4)
(5) (6) (7) (8) (9) (10)
(11)
(12) (13)
(14) (15) (16)
(17) Rajagopalan, M. S., Smith, D. S. H., and Turner, A. B., J. Chem. sot. (c), 646 (1971). (18) Oliveto, E. P., in Organic Reactions in Steroid Chemistry, Vol. 2 (eds. J. Fried and J. A. Edwards), van Nostrand Reinhold Company, N.Y., p. 174 (1972) (19) Katzenellenbogen, J. A., in Biochemical Actions of Hormones, Vol. 4 (ed. G. Litwack), Academic Press, N.Y., pp. 2-77 (1977). (20) a. Fridman, A. L., Ismagilova, G. S., Zalesov, V. S., Novikov, S. S., Russ. Chem. Rev., 41, 371 (1972). b. Weygand, F., and Bestmann, H. J., Angew. Chem., 72, 535 (1960). (21) Fried, A. A., Petrow, V., Lack, L., Unpublished results. (22) Cf. Dewitt, E. H. and Lack, L., Unpublished results. (23) Tserng, K-Y., and Klein, P. D., Lipids, 12, 479 (1978). (24) Haslewood, E. S., and Haslewood, G. A. D., Biochem. J., 155, 401 (1976). (25) Parmentier, G., and Eyssen, H., Steroids, a, 721 (1975). (26) Pharmacia Fine Chemicals booklet, Upplands Grafiska AB, Uppsala, Sweden (Dec. 1974-6). (27) Elsevier's Encyclopaedia of Organic Chemistry, Ser. III, s, Suppl. Part 4 (ed. F. Radt), Springer-Verlag, Berlin, 3229s (1962). (28) Fieser, L., and Fieser, M., Steroids, Reinhold Publishing Corporation, N.Y., p. 222 (1959). (29) Still, W. C., Kahn, M., and Mitra, A., J. Org. Chem. 43, 2923 (1978). Standard adsorbent column length is 5.5 in. (30) The 25-bromo analog of compound IX was indexed erroneously in Chemical Abstracts, Fifth Decennial Index, Formulas C2G-W2, Vol. 41-50, Chemical Abstracts Service, Columbus, Ohio, p. 2490 (1962). The actual compound reported was the corresponding triformyl-25-bromo compound: Kazuno, T., Moori, A., Sasaki, K., Kuroda, M., and Mizuguchi, M., Proc. Jpn. Acad., 2, 416 (1952). (31) For mass spectral analysis see: Dayal, B., Shefer, S., Tint, G. S., Salen, G., Mosbach, E. H., J. Lipid Res., 17, 74 (1976).
2463
THE SYNTHESIS OF DIAZO, HALO, AND SULFOXY BILE ACID DERIVATIVES: POTENTIAL AFFINITY LABELS Aaron A. Fried, Vladimir Petrow, and Leon Lack Department of Pharmacology, Duke University Medical Center Durham, North Carolina 27710
Received:
3-19-79 ABSTRACT
Bile acid derivatives, with and without C-3 sulfate groups, and having either the diazo- or halomethylketone moieties, have been synthesized in good yield and purity. The synthetic sequence, COOH-+COC1--+COCHN2-GOCH2X, was used with deoxycholic and cholic acids, which requires carefully controlled quench, work-up, and purification procedures, especially for the 3-sulfate esters (made from deoxycholic acid derivatives only). The pure title compounds are anticipated to be useful chemical probes (affinity labels), especially the completely water soluble sulfates, toward our studies of ileal active transport of bile salts. A new use for Sephadex LH-20 as a sulfate ester protecting group is reported. Also developed were the use of acetamide hydrochloride complex as a mild hydrochlorination reagent and a neutral desalting method for sulfate esters of deoxycholic acid derivatives. INTRODUCTION The active transport of bile salts has been observed at three anatomical sites: tubule.
the liver, the ileum, and the proximal renal
For a review of the topic see (1).
Recent -in vitro studies
of ileal bile salt transport using vesicles (2), suggest that the receptor macromolecule as well as the transport mechanism resides within the brush border membrane. In order to obtain further information on the nature of the ileal macromolecular receptor, we have turned to the concept of affinity labeling (3,4), which has been so successful in identifying structural components of enzyme site-receptor macromolecular
VoZwne 34, Number 2
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TEIROTDS
August, 1979
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172
interactions.
TIIEOXDl
To this end we have replaced the carboxyl terminal
of deoxycholic and cholic acids with diazocarbonyl methylcarbonyl
(COCHN2) and halo-
(COCH2X) groups; we anticipate that these reactive bile
acid derivatives
(V-IX, XII, and XIII), will be compatible with the
transport mechanism and will be able to combine with an amino acid residue of the active site, thereby both inactivating and labeling it. EXPERIMENTAL All solvents and inorganic reagents were reagent grade, A.C.S. certified. Deoxycholic acid (MCB), cholic acid (Sigma), oxalyl chloride (Aldrich), acetamide (Fisher), pyridine:sulfur trioxide (Aldrich), anhydrous ammonia (Matheson), anhydrous hydrogen chloride (Matheson), and Sephadex LH-20 (Pharmacia) were commercial products used without further purification. Methylene chloride for use as a reaction solvent was treated with anhydrous magnesium sulfate and filtered (under nitrogen); for chromatography, it was distilled but not further dried. Benzene was distilled, the middle cut collected and stored over 4 I( molecular sieves (Davison). Pyridine was distilled from barium oxide. Methanol for use as a reaction solvent was distilled from freshly prepared magnesium methoxide. Granular sodium sulfate was used to dry solutions of the diazoketones (III, IV, and X). The use of powdered magnesium sulfate resulted in a much lower recovery of products. Drying agents were avoided with all of the halomethylketones. Chromatographic adsorbents used were Silica Gel G, 60 (E. Merck), silicic acid, 100-200 mesh ("SilicAR," CC-7, Mallinckrodt), silica gel, Activity III/30 mm (ICN), and 30-63 pm silica gel (ICN). TLC (thin layer chromatography) dipping slurry was prepared according to Peifer (5) from 35 g Silica Gel G and 100 ml chloroform:methanol (3:l) and stored in a ground glass stoppered bottle for at least five days before use. Adsorbent-coated glass microanalytical TLC slides (25 x 75 mm) were freshly prepared and air dried. The following TLC developing systems were used: A. CHC13:CH30H:HOAc:HOH (65:24:17:9) (6); B. ethyl acetate: methylene chloride (30:70); C. ethyl acetate:methylene chloride (9O:lO); D. ethyl acetate: methylene chloride (3:97); E. acetone:ethyl acetate (1:l); F. methanol:ethyl acetate (5:95). The TLC slides were visualized by spraying with 1N H2SO4, heating on a hot plate, and viewing under an ultraviolet light ("Mineralight" UVS-54). Dry columns (7) were prepared by packing silica gel (ICN) in nylon foil tubes (ICN). Separated sample bands were cut into l/8 - l/4" sections with scissors while the developed column was still moist with solvent. Isolation of precipitated sulfate ester fractions was done by a combination of centrifuging, decanting, air and vacuum drying.
S
TpR&OXDtB
173
Glassware was flame-dried and cooled under nitrogen. Liquid transfers were made with dry syringe and needle or cannula. Infrared (ir) spectra were recorded on Perkin Elmer Models 337 and 297 spectophotometers. Specific optical rotation values (aD) were obtained on a Central Scientific Company (CENCO) visual polarimeter with a sodium D-line source. Proton magnetic resonance (pmr) spectra were recorded on Varian Associates Model EM 360-A and JEOL Model JNM-MH-100 spectrometers with chemical shift values (6) reported in parts per million downfield from the internal standard, tetramethylsilane. The following abbreviations describe the pmr data: s (singlet), d (doublet), b (broad singlet), m (multiplet), and 3 (coupling value). Melting points were determined on Fisher-Johns Combustion analyses melting point apparatus and were uncorrected. were performed by Atlantic Microlab, Inc., Atlanta, Ga. Acetamide hydrochloride complex (CHqCONH+HCl) was prepared from acetamide and dry HCl according to the procedure of Pinner and Klein (8). Crude product was stored over NaOH. Per cent hydrogen chloride in the complex was assayed before each use by titration with O.Ol5N NaOH with phenolphthalein indicator. Acetamide hydrobromide complex (CH$ONH+HBr) was prepared from acetamide and dry HBr according to the procedure of Werner (9). Per cent hydrogen bromide was assayed by titration as above. Deoxycholic acid diformate (3u,12a-diformyloxy-58_cholan-24-oic acid) (I) and cholic acid triformate (3a,7a,12e-triformyloxy-58cholan-24-oic acid) (II) were prepared by the method of Tserng and Klein (lob): (I), mp 195-197', lit. (lob) 197-198" (corrected); (II), mp 209.8-211.2", lit. 209-210' (lob), 210-211" (lid). 25-Diazo-3a,l2a-diformyloxy-26,27-dinor-5~-cho~estan-24-one (III). To a clear solution of the diformate (I, 1.00 g, 2.22 mmol) in dry benzene (20 ml) was added dropwise, with stirring and passage of dry nitrogen through the solution, oxalyl chloride (1.0 ml, 11.7 mmol) in 5 ml dry benzene during 10 min. The colorless solution was allowed to reach room temperature and after 18 min. the nitrogenbubbler tube was pulled above the solvent level. Stirring was continued under gentle nitrogen stream for 6.25 hr. Solvent and excess oxalyl chloride were removed by evaporating the solution to dryness in vacua. Residual reagent was removed by mixing the syrupy residue -with fresh dry benzene (25 ml) and evaporating. The mixing and evaporating were repeated and the residue redissolved in 25 ml benzene. To a cold ethereal solution of diazomethane [prepared (12, 13) from a mixture of 3.55 g N-nitroso-N-methylurea (14), 40% aqueous KOH (10.6 ml) and absolute ether (40 ml), dried over KOH pellets (10 hr) and decanted from the KOH (Note: particles of hydrated KOH decompose the product, III)] was added (15 min.) dropwise, and with stirring, a clear cold solution of the acid chloride: benzene solution. Controlled addition was effected via a stainless Stirring was steel cannula (19 G; blunt tip) and nitrogen pressure. continued at 0" (1 hr) and at room temperature (0.5 hr), when excess
174
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%?BROIDb
diazomethane was removed by evaporating the solution at 30° to 0.5 volume with dry nitrogen. The concentrated solution was taken to dryness on the rotary evaporator to give a yellow amorphous solid, 1.113 g. Rapid gradient elution chromatography (O-+3% ether in benzene) on a silicic acid column (58 g) packed in methylene chloride gave a yellow foam, 885 mg (1.87 mmol, 84% yield). Alternatively, flash chromatography (29) can be used; for example, 2.20 g of crude product on a 50 mm silica gel (30-63 pm) column with 10% EtOAc in methylene chloride as eluent (fraction volume = 25 ml) gave, in fractions 13-23, 1.80 g of compound III; mp 45-55"; TLC, Rf"0.70 (system B); ir (CH2C12) 2087 and 1644 (COCH=N2) (15), 1722 (ester C=O) cm-l; eg5 = 99.0' (absolute EtOH, c = 1.90); pmr (acetone-d6) SO.80 (CH -18), 0.97 (CIJ3-19), 0.50-2.67 (m, steroid nucleus + side chain), 4TgO (m, C-3E), 5.18 (m, C-12X), 5.53 and 5.66 (C-25& anti and syn), 8.00 (S, C-12CgO), 8.14 (S, C-3CEO). A sample suitable for combustion analysis was obtained by the use of dry column chromatography (silica gel:sample, 3OO:l; developing solvent:5 ethyl acetate:95 methylene chloride). Anal. Calcd. for C27H4ON205: C, 68.61, H, 8.53, N, 5.93; Found C, 68.55, H, 8.52, N, 5.91 (after vacuum drying at 30'). 25-Diazo-3cc,7ol,lZcL-triformyloxy-26,27-dinor-58-cholestan-24-one (Iv). Using the foregoing two-step procedure, triformate (II, 1.10 g, 2.23 mmol) gave 1.20 g crude product; TLC, Rf=0.21 (major spot) (system D). Purification in two equal batches by dry column chromatography (218:1, silica gel:sample; 3 ethyl acetate:97 methylene chloride) gave (IV) as a pale yellow viscous liquid, 880 mg (1.70 mmol, 76.4% yield). A crystalline solid, mp 131.0-132.0 dec, lit. (lle) 134.5-135.0' dec, was obtained from pentane-ether at 0'; TLC, Rf=O.Zl, one spot (system D), ir (CH2Cl2) 2097 and 1637 (COCH=N2), 1722 (broad, ester C=O) cm-l; pmr (CDC13) 60.77 (C%-18), 0.95 (CE3-19), 0.60-2.73 (m, steroid nucleus + side chain), 4.73 (m, C-3E), 5.10, 5.26, and 5.29 (overlapping C-12$ C-25& and C-7&), 8.05 (S, C-12CgO), 8.13 (S) and 8.20 (S) (C-7CgO) and C-3CgO). Anal. Calcd. for C28H40N207.1.5H20: C, 61.86, H, 7.97, N, 5.15; Found C, 61.35, H, 7.39, N, 4.80 (after vacuum drying at 25").
25-Diazo-3cL,12c(-dihydroxy-26,27-dinor-* Diformyldiazoketone (III, 838 mg, 1.77 mmol) was mixed with cold methanolic KOH (76 ml, 2.8% solution) and allowed to stir at room temperature for 92 hr. The bright yellow solution was poured with stirring into 300 ml cold saturated sodium bicarbonate and evaporated to dryness -in vacua at 35'. The solid residue was triturated with methylene chloride (50 ml, x2). Undissolved material was taken up with 60 ml water:methylene chloride (5:l) and partitioned with the combined organic extracts. An unidentified material was insoluble in both aqueous and organic phases. Dilution of the combined organic extract with an equal volume of methylene chloride, washing with water (80 ml, x2) and saturated NaCl (80 ml, xl), drying over anhydrous sodium sulfate and evaporation gave a pale yellow foam, 599 mg (1.44 mmol, 81% yield); mp 80.5" (dec with N2+86.0°); TLC, Rfz0.24 (system C); ir (CH2C12) 3600 and 3440 (OH), 2105 and 1638 (COCH=N2)
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TEIROXDM
175
cm-1 ; pmr (acetone-d6) 60.68 (C&-18), 0.90 (C&3-19), 0.50-2.60 (m, steroid nucleus + side chain), 3.47 (m, C-3~+C-3O_H+c-120~), 3.93 (m, C-12E), 5.53 and 5.66 (C-25X, anti and syn). Anal. Calcd. for C25H40N20 : Found C, 71.95, H, 9.73, N, 6.6 a .
C, 72.07, H, 9.68, N, 6.73;
25-Diazo-3a,7a,12a-trihydroxy-26,27-dinor-5B-cholestan-24-one (VI). Triformyldiazoketone (IV, 440 mg, 0.81 mmol) was treated as for diformyldiazoketone (III) with 55 ml methanolic KOH [5.56 ml aqueous KOH concentrate (25 g/27 ml) + 200 ml methanol] during 115 hr at room temperature. The usual work-up plus filtration through a glass frit funnel and evaporation gave a glassy yellow solid, 269.4 mg (0.60 mmol, 73.9% yield), mp 93" (dec, N2+); TLC, Rf=0.28 (s stem E); ir (CH2C12) 3600 and 3430 (OH), 2105 and 1638 (COCH=N2) cm-1 ; pmr (CDC13) 60.68 (Ca-18), 0.90 (C%-19), 0.37-2.80 (m, steroid nucleus + side chain), 3.72 (m, C-3,7,12IJ+C-3,7,12Oc + l/2 H20), 5.34 (C-2511+ l/2 H20). Anal. Calcd. for C25H4ON204.1 H20: C, 66.63, H, 9.40, N, 6.22; Found C, 66.54, H, 9.02, N, 5.89 (after vacuum drying at 40"). 25-Chloro-3a,12a-dihydroxy-26,27-dinor-5B-cholestan-24-one (VII). To a cold (0") solution of dihydroxydiazoketone (V, 50.7 mg, 0.122 mmol) in 6 ml THF (freshly distilled from lithium aluminum hydride) was added with stirring CH3CONH2+HCl (61.6 mg, 0.12 mm01 HCl) in one portion. Stirring was continued for 1 min at O", then the ice bath was removed and the suspension was allowed to reach room temperature (evolution of nitrogen was observed). After 19.3 hr stirring at room temperature, the clear, colorless solution was taken to dryness (30", in vacua), and the residue was redissolved in 50 ml THF:ether (3:97). -The new solution was washed with water (25 ml, x3) and the aqueous wash was extracted with wet ether (25 ml, xl). Finally the combined organic extracts were washed with saturated brine, filtered through a plug of glass wool, and evaporated to give a yellow liquid residue. Solidification to a white amorphous solid was effected by dilution with methylene chloride and evaporation. Gradient elution chromatography on dry-packed silicic acid (1.13 g) with 5-+40% ethyl acetate in methylene chloride gave a pure glassy solid, 39.5 mg (0.09 mmol, 76.2% yield); mp 71.5" (shrinks, 67.5"); TLC, Rf=0.42 (system C); ir (CH2C12) 3600 and 3450 (OH), 1733 and 1717 (C=O) cm-' (16); pmr (CDC13) 60.68 (C&-18), 0.92 (C&-19), 0.50-2.85 (m, steroid nucleus + C-12OIJ+ C-3Og), 3.60 (m, C-35), 4.05 (m, C-12@ overlapping with 4.11 (S, C-25&). Anal. Calcd. for C25H4lClO3: C, 70.64, H, 9.72, Cl, 8.34; Found C, 70.50, H, 9.75, Cl, 8.38 (after vacuum drying at 45"). 25-Bromo-3a,12a-dihydroxy-26,27-dinor-5B-cholestan-24-one (VIII). Dihydroxydiazoketone (V, 100 mg, 0.24 mmol) was treated with acetsmide+HBr (71.6 mg, 0.24 mm01 HBr) using the procedure indicated for the synthesis of chloromethylketone (VII). The reaction solution was concentrated -in vacua at room temperature, diluted with 100 ml absolute ether and minimum THF, then washed successively with cold saturated sodium bicarbonate (50 ml, x2), water (50 ml, x3), and
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TBROIDII
saturated brine (50 ml, xl). Evaporation of the washed organic extracts and azeotropic drying with dry benzene gave 119.4 mg yellow liquid. Gradient elution chromatography on silicic acid (adsorbent: sample, 19.4:l) with ethyl acetate (5-40%) in methylene chloride gave a glassy colorless solid, 98.4 mg (0.21 mmol, 88% yield); mp 70-73'; TLC, Rf=O.42 (system C); ir (CH2C12) 3600 and 3450 (OH), 1733 and 1717 (C=O) cm-1 (16); pmr (CDC13) 60.67 (C&-18), 0.92 (C&-19) 0.97 (d, C%-21, J=6), 0.48-2.20 (m, steroid nucleus + side chain), 2.60 (m, C-233 + C-120! + C-30&), 3.6 (m, C-3g), 3.92 (S, C-253) overlapping with 3.96 (m, C-1211). Anal. Calcd. for C25H41Br03: C, 63.95, H, 8.80, Br, 17.02; Found C, 63.77, H, 8.83, Br, 17.16 (after vacuum drying at 35'). 25-Chloro-3a,7a,12a-trihydroxy-26,27-dinor-5$-cholestan-24-one (Ix). Applying the hydrohalogenation procedure to the trihydroxydiazoketone monohydrate (VI, 225.5 mg, 0.50 mmol) after 22.5 hr at room temperature and usual work-up gave a white solid, 212.7 mg (0.43 mmol, 94.5% yield as the hemihydrate); mp 195.5-196.5" (clear+opaque, 133'); TLC, Rf=0.46 (system F); ir (KBr) 3400 (OH), 1713 (C=O) cm-l; pmr (CD3OD) 60.70 (C&-18), 0.90 (C&-19), 0.57-2.30 (m, steroid nucleus + side chain), 2.30-4.09 (overlapping m, c-3, 7, 12~ + C-3, 7, 12OE), 4.27 (S, C-25%). Anal. Calcd. for C25H4lClO *;H20: C, 66.72, H, 9.41, Cl, 7.88; Found C, 67.27, H, 9.45, Cl, 7.$ 4 (after vacuum drying at 45'). 25-Diazo-12a-formyloxy-3a-hydroxy-26,27-dinor-5~-cholestan-24one (X). The following was adapted from the procedure of Tserng and Klein (lob). Diformyldiazoketone (III, 3.551 g, 7.51 mmol) was dissolved in anhydrous methanol (89 ml) and filtered. Anhydrous ammonia was bubbled into the cold (0') clear solution with stirring for 5 min. Stirring was continued at 0" for 1 min, then the ice bath was removed, and the solution allowed to stand at room temperature for 1.25 hr. The solution was concentrated to one-half volume at room temperature in vacua, then mixed with absolute ether (200 ml) and evaporated to a small volume. Treatment with ether and evaporation was repeated, x2 to give an amorphous yellow solid. Purification was effected by flash chromatography (30-63 pm silica gel) using a 50 mm column (29) and 1:l ethyl acetate:methylene chloride as eluent (fraction volume = 25 ml). Evaporation of combined fractions 21-44 gave a yellow foam, 3.05 g (6.72 mmol, 89.5% yield); mp 44.5-52.0'; TLC, Rf=0.33 (system B); ir (CC14) 3448 (OH), 2109 and 1651 (COCH=N2), 1725 (ester C=O> cm-l; pmr (CCl4) 60.76 (C&-18), 0.90 (Cg3-19), 0.50-3.00 (m, steroid nucleus + side chain), 3.00-4.00 (m, C-3~ + C-3Og), 5.16 and 5.20 (m, overlapping C-12g and C-25@, 8.04 (S, CEO). Anal. Calcd. for C26H4ON20 *l/2 H20: C, 68.84, H, 9.12, N, 6.18; Found C, 68.57, H, 9.49, N, 5.72 (after vacuum drying at 35'). Anal. Calcd. for C26H4ON20 : C, 70.23, H, 9.07, N, 6.30; Found C, 69.97, H, 9.51, N, 6.2 2 (after vacuum drying at 80").
25-Diazo-lZa-formyloxy-3a-sulfoxy-26,27-dinor-58-cholestan-24-one sodium salt (XI). The following was adapted from the procedure of Rajagopalan, et al. (17). Diazoketone (X, 3.00 g, 6.61 mmol) was mixed with pyridine-sulfur trioxide complex (7.17 g, 45.05 mmol) and anhydrous pyridine (80 ml) and stirred at room temperature under nitrogen for 50 min. The reaction mixture was added in portions with stirring to cold (0") saturated sodium bicarbonate (850 ml) to give a clear yellow solution. Pyridine was removed as the pyridinewater azeotrope -in vacua giving a copious pale yellow precipitate, which was collected by filtration on a medium porosity glass frit. Trituration of the solid with dry methylene chloride (a. 200 ml, 1 hr; b. 200 ml, 0.5 hr) and evaporation of the yellow filtrate gave a yellow electrostatic powder having a purity satisfactory for the subsequent reaction, 3.95 g (7.0 mmol, essentially quantitative); mp 199.0-2oo.oo; TLC, Rf (system A) = 0.44 (major), 0.16 and 0.28 (minor). An analytically pure sample was obtained by treating a portion of the partially purified product (50.9 mg) in methylene chloride (15 ml) with absolute ether (starting with 25 ml for the first precipitated fraction) to selectively precipitate the more polar impurities (Rf 0.16 and 0.28), though with some product loss. Concentrating the final supernate and adding it dropwise to ether (35 ml), precipitated pure (XI, 38.5 mg, 76% recovery); mp 200.0200.5' dec; ir (KBr)l3440 (H20), 2100 and 1635 (COCHN2), 1715 (ester C=O), 1218 @SOT) cm ; pmr (CDC13) 60.38-3.33 (m, steroid nucleus + side chain), 3.60-5.00 (m, C-3H + 2H20), 5.00-5.67 (C-12g + C-2511), 8.15 (b, CEO). Anal. Calcd. for C26H39N2NaC7S'l H20: C, 55.30, H, 7.32, N, 4.96, Na, 4.07, S, 5.68; Found C, 55.73, H, 6.91, N, 4.69, Na, 3.73, S, 5.30 (after vacuum drying at 35'). When in another experiment, the crude product precipitate was completely dissolved in saturated sodium bicarbonate, extracted with methylene chloride, and then treated as above, the dihydrate (mp 200.0-201.0" dec) was isolated: Anal. Calcd. for C26H3gN2NaC7S.2H20: C, 53.59, H, 7.44, N, 4.81, S, 5.50; Found C, 53.42, H, 6.92, N, 4.74, S, 5.36 (after vacuum drying at 35O). 25-Diazo-12a-hydroxy-3a-sulfoxy-26,27-dinor-5$-cholestan-24-one sodium salt (XII). Cold methanolic sodium hydroxide solution (59.0 ml, 21.91 mmol, 1.88 wt/wt X) was added to diazoketone (XI, 3.401 g, 6.00 mmol) with stirring at 0". The resultant suspension was allowed to stand at room temperature with occasional intermittent stirring for 6 hr. (Reaction seemed complete after 3 hr by TLC, but the longer reaction time was used to assure complete conversion). The dark redbrown mixture, undiluted, was centrifuged at 5" at 2300 rpm to give three distinct phases (in order of increasing density): yellow solution, dark brown semi-solid (decomposition material by TLC; discarded), and pale yellow solid. After decanting the cold yellow solution and quickly scraping out the semi-solid, the pale yellow solid was triturated with cold (0“) methanol and centrifuged. Three phases
178
were again formed and treated as indicated above. This purification process was repeated, being careful to avoid solvent evaporation, until all of the yellow solid had dissolved. The combined methanol solutions (0") were acidified (pH 6) with glacial acetic acid (100 drops). A sharp color change to a lighter yellow was observed. Work-up was continued with rotary evaporation (room temperature) to dryness, redissolution in deionized water (193 ml), neutralization with saturated sodium bicarbonate (100 drops), and extraction with butanol (previously saturated with water), 480 ml, xl, and washing of the organic extract (bright yellow) with water (96 ml, xl). The aqueous wash was important in removing an unidentified impurity that could not be removed effectively by the subsequent fractional precipitation method (see belaw). The butanol extract was evaporated in vacua at 35-40" to a viscous gel and taken up with dry methylene -chloride (40 ml). Solvent was evaporated to give a concentrated, slightly viscous solution. Addition of the methylene chloride solution to cold (0") absolute ether and immediate washing of the resulting precipitate with more ether, gave a pale yellow powder in purity suitable for the next reaction: 2.58 g (4.86 nnnol, 81.0% yield); mp 154.5-154.9'. Further improvement in product (XII) yield was not effected with more butanol extractions; further extractions pulled out only more impurities. Fractional precipitation with methylene chloride:ether solutions as for (XI) gave an analytically pure white solid: mp 195.0-195.9', TLC, Rf"0.37 (~~;temlA4;=i;,(~) 3450 (OH), 2100 and 1635 (COCH-N2), 1230 (OSO?j)cm [water, c=Z.Ol; slow decomposition (N29) occurred during reading]; pmr (D20) 60.33-3.33 (m, steroid nucleus + side chain), 3.90 (m, C-3g), 4.33 (m, C-12g) overlapping with 4.57 (m, C-12Og), 6.13 (m, C-255). Anal. Calcd. for C25H38N2NaOgS'O.75 H20: C, 56.56, H, 7.45, N, 5.28, Na, 4.33, S, 6.04; Found C,56.76, H, 7.58, N, 5.08, Na, 4.20, S, 5.83 (after vacuum drying at 65"). C, 58.00, H, 7.40, N, 5.41, Na, Anal. Calcd. for C25H38N2NaCgS: 4.44, S, 6.19. Found C, 57.78, H, 7.50, N, 5.17, Na, 4.29, S, 5.90 (after vacuum drying at 95'; mp 194.5-194.9"). 25-Chloro-12a-hydroxy-3a-sulfoxy-26,27-dinor-5S-cholestan-24-one sodium salt (XIII). Diazoketone (XII, 41.3 rug, 0.08 mmol) of analytical grade, obtained by fractional precipitation as described for compound (XI), was mixed (3.5 hr, vigorous stirring) with 15 ml dry methylene chloride to obtain a milky solution. Sephadex LH-20 (XII), 42:1] was added and the resultant slurry L1.734 g; exchanger: stirred for 1.7 hr. Finally, CH3CONH2+HCl (43.8 mg, 0.08 mmol) was added and the mixture diluted with solvent to completely fill the reaction vessel (total volume = 30 ml). Stirring was continued at room temperature for 18.3 hr. The product mixture was filtered through a glass frit funnel (Kimex 40M) to remove most of the byproduct, CH3CONH2, and traces of desulfated material. The residue was washed with methylene chloride (10 ml, x9), wetted with 2 ml deionized water plus 1 drop saturated sodium bicarbonate, and extracted with dry butanol (50 ml, xl) then butanol:methanol (1:l; 40 ml, x2).
S
TDEOID=
179
After each extraction the phases were separated by centrifuging, followed by filtering the supernate through a medium porosity glass frit. The combined filtrates were evaporated (in -- vacua at 35") to a gel which was redissolved in 30 ml butanol (presaturated with water). The wet butanol solution was mixed with 6 ml water to give an emulsion which separated after 1 hr. Water was removed from the organic phase as the butanol-water azeotrope, and the resulting dry butanol concentrate diluted to 7 ml with more butanol, removed from insoluble material by centrifuging, and once again evaporated to a gel-like residue. Dropwise addition of the gel in minimum methylene chloride to 36 ml pentane:ether (2:l) gave a white solid (27.5 mg, 69.2% yield); TLC, Rf=0.58 (system A); Rf=0.52 and 0.70 (trace impurities); ir 1659 cm-l (by-product:acetamide). Repeating the trituration with 266 ml pentane-ether (2:l) removed virtually all of the acetamide (plus some product) yielding 17.5 mg (44% yield). Finally, a sample of analytical purity (minus the Rf 0.70 component) was obtained by triturating with 5 ml pure ethyl acetate. Centrifuging, decanting and air-drying gave 13.3 mg (33% yield); mp 16~iO-170.00; ir (XBr) 3419 (OH), 1728 (C=O), 1611 (H20), 1222 (OSO3) cm ; a$3 = 52.6' (methanol, c=1.99); pmr (CD30D) 60.70 (CQ-18), 0.93 (C%-19), 0.56-2.23 (m, steroid nucleus + side chain), 2.57 (m, C-23E+ C-120I-J, 3.95 (m, C-3g), 4.27 (m, C-12E), 4.78 (S, C-25fi). Anal. Calcd. for C25H3gClNaOgS*l H20: C, 55.18, H, 7.60, Cl, 6.52, Na, 4.23, S, 5.89; Found C, 55.42, H, 7.27, Cl, 6.05, Na, 3.91, S, 5.48 (after vacuum drying at either 65" or 95"). RESULTS AND DISCUSSION This paper reports further application of the diazoketone method (18) to the bile acid series with an adaptation of the procedures of Reber, et al. (lla) to the syntheses of halomethylketones and XIII; see Fig. 1).
(VII-IX,
This method was preferred since (i) it has
already been extensively studied (11, 18); (ii) the diazoketone moiety will permit conversion into photoaffinity labels (19) as well as conventional affinity labels(3,4). Though simple in theory, the diazoketone procedure required considerable attention to detail and development of mild selective conditions in order to obtain good yields of products.
Thus, for
example, we were led to develop novel applications of Sephadex LH-20 and acetamide hydrochloride complex, the former serving as a sulfate
180
S
181
TRROXDLI
protecting agent and the latter as a mild stoicheometric hydrochlorination reagent. Figures 1A and 1B illustrate a simple strategy that takes advantage of the complementary properties of the diazoketone moiety, COCHN2 (ZO), and the formate protecting groups (lo), the latter being labile and the former, stable, in mild base. Preliminary biological study of products (VII, VIII, and IX) revealed inhibition of the transport mechanism in the ileal -in vitro assay (Zl), even though the test compounds were considerably less soluble in the aqueous testing medium than were the natural substrates.
Consequently, we undertook the synthesis of the correspond-
ing sulfate esters, which were expected to be much more water soluble and yet to retain some affinity for the bile salt active site (22). The required sulfation step was accommodated in the diazoketone method as shown in Figure 1B.
The deprotection step (X1--+X11)was practi-
cable since bile acid sulfates are stable to the mild basic conditions suitable for deacylation
(23, 24, 25).
However, in this case the
diazoketone moiety, not the sulfate group, is the limiting factor in determining the required reaction conditions which must be even milder than those recently reported for deformylation
(23).
above room temperature decomposed the side chain.
Temperatures
THF was not used
in the last reaction (X11-+X111) because it allowed complete acid catalyzed desulfation.
Desulfation was somewhat retarded in methyl-
ene chloride, but still important, and was prevented in the presence of Sephadex LH-20, a lipophilic anionic exchanger gel widely used for the chromatographic purification and separation of sulfate ester mixtures
(25, 26).
Sephadex LH-20 appears to be acting in a way
either to reduce the activity of the hydrogen chloride complex or as a weak sulfate protecting group.
It should be noted that it was
essential to carry out the reagent additions in the sequence described in the experimental section.
The resultant viscous slurry with
Sephadex LH-20 must be stirred vigorously and must fill the reaction vessel completely to permit a complete conversion.
Neat methanol or
aqueous solutions must not be used during the filtration and washing steps in order to avoid product contamination by Sephadex LH-20 fines. All of the purified products and intermediates were stable at room temperature, with the halomethylketones giving sharp melting points.
Nevertheless, storage in the dry cold and dark between use
is recommended, especially for the diazoketones.
These compounds,
with the exception of products (V, VII, and VIII), were isolated as hydrates.
Attempts to remove the water of hydration resulted in
decomposition, except where indicated in the experimental section. Unlike the products in Figure lA, the corresponding monosulfate derivatives in Figure 1B were soluble in water as expected.
Inter-
estingly, these sulfates (sodium salts) while in the form of viscous gels (from evaporated butanol solutions) were readily soluble in dry methylene chloride.
These sulfate gels which probably were some type
of bile acid derivative complex with butanol (27) allowed purification of the sulfates by fractional precipitation, where methylene chloride solutions were treated with either ether or pentane.
Also
this purification procedure provided a convenient neutral desalting method that does not require the use of derivatizing methods (23) that would likely decompose our multifunctional new impurities.
sulfates or introduce
Desalting with Amberlite XAD-2 resin (23, 25) was
S
-X!TEIICOIDb
attempted on the diasoketosulfate
183
(XI) but gave only low yields of
decomposition products. In the desalting method, once the sulfates were precipitated from methylene chloride-ether or methylene chloride-pentane-ether solutions and dried, they were only partially soluble in methylene chloride.
A methylene chloride-soluble sample was reconstituted by
treatment of the dry sulfate powder with wet butanol and evaporating to give the sulfate gel once again. The ability of bile acid derivatives, especially derivatives of deoxycholic acid, to form addition compounds with water or alcohols (27) occasionally makes it difficult to obtain unambiguous TLC data. Since it was desired to monitor all our reactions with TLC, methods were developed that would allow reproducibility section).
The cholylchloroketone
(see experimental
(IX) (Rf=0.46) originally appeared
to be contaminated by an unknown component (Rf=0.23). the component at Rf=0.46
was
However, when
scraped off and rechromatographed under
the same conditions, both spots (Rf=0.46 and 0.23) appeared again. Controlling the adsorbent activity and avoiding the use of water in developing solvents showed compound (IX) to be chromatographically pure. All the new compounds gave satisfactory combustion and spectral analyses.
The ir spectra of the halomethylketones
(VII, VIII, IX,
and XIII) gave unusually weak carbonyl bands, probably due to side chain interactions with the C-12 hydroxy group (28). The diazoketones
(III, IV, and X) decomposed on silicic acid or
silica gel and, therefore, satisfactory chromatographic purification was achieved only by using multiple small sample batches with the
dry column technique (7) as for compound (IV), or by using the Still "flash" chromatographymethod (29) as for compounds (III) and (X). Complete deformylation of compound (III) required 92 hours at room temperature; after 74 hours, deformylation was incomplete, and after 113 hours the product yield was 20 per cent lower due to decomposition of the side chain. The sulfation of compound (X) was better quenched with saturated sodium bicarbonate. Pure water formed an acidic solution that decomposed some of the product before the extraction step. The sulfate product (XI) to be used for the subsequent reaction (X1-+X11) should not be purified further than indicated in the experimental section. Precipitation from methylene chloride-ether gave a less methanolsoluble sample of compound (XI), and also adversely influenced the solubility of compound (XII) as it formed in methanolic sodium hydroxide. Reaction (X1+X11) under these conditions, and with stirring in an attempt to effect solution, gave a solid mass. Reaction (X1+X11) should be quenched with cold acetic acid before solvent evaporation; otherwise complete decomposition occurred even at room temperature under a dry nitrogen stream. Compound (XII) should be washed with absolute ether immediately after precipitation from methylene chloride-ether. Omitting the ether wash and then air or vacuum drying at room temperature gave a decomposed bright yellow amorphous mass. As with compound (XI), the method of purification affects the solubility of compound (XII). Although an extensively purified sample (moderatemethylene chloride solubility) of (XII) was used for reaction (X11-+X111),the more convenient readily soluble sample (before
S
TDROXD=
185
fractional precipitation) was later found to be satisfactory. Compound (XIII) can be freed of acetamide by-product and other impurities by an alternate method that is more convenient for purification of larger samples.
The method consists simply of selectively
precipitating pure (XIII) by adding pentane to solutions of impure (XIII) in 1:3.3 dry butanol-ether. In summary, selected bile acid derivatives, with and without C-3 sulfate groups, and incorporating the diazo- and halomethylketone moieties, have been synthesized in good yield and purity. (V and VII-XIII) are new (30).
Diazoketones
Compounds
[III (lib), IV (lld, 31),
and VI (lid)] have been reported elsewhere, but except for compound (IV), were not fully characterized. The synthetic approaches shown in Figure 1 should be applicable to other bile acids besides deoxycholic acid and cholic acid, allowing entry into a series of useful water soluble affinity labels for bile salt transport studies. lithocholic
The formylated 25-diazo derivatives of
(llb), chenodeoxycholic
(llb), and hyodeoxycholic acid
(11~) which have already been reported, will clearly serve as useful starting materials. Also, from our synthesis efforts, three new methods were developed: 1.
The use of acetamide hydrochloride as a mild hydrochlorina-
tion reagent. 2.
The use of Sephadex LH-20 as an agent to prevent competing
acid catalyzed solvolysis of C-3 sulfate esters. 3.
A neutral desalting procedure for acid and base sensitive
sulfate esters of deoxycholic acid derivatives.
Further work is in progress to examine the scope of these methods, including other applications of Sephadex LH-20 in bile acid synthesis. Acknowledgments - This work was supported by research grants AM-09582 and T32-ES07002 from the National Institutes of Health. We thank the Department of Chemistry, Duke University, for use of the Perkin Elmer 297 infrared spectrophotometer and nuclear magnetic resonance spectrometers. We also thank Professor Ben Wittels, Department of Pathology, Duke University Medical Center, for loan of the CENCO polarimeter. REFERENCES
(1) Lack, L. and Weiner, I. M., in The Bile Acids (eds. P. P. Nair and D. Kritchevsky), Plenum Press, N.Y., pp 33-54 (1973). 1607 (1977). a. Shaw, E., in The Enzymes, 3rd ed., vol. 1 (P. D. Boyer, ed.), pp. 46-91. Academic Press, N.Y. (1970). b. Singer, S. J., Adv. Protein Chem., 2, 1 (1967). Baker, B. R., Design of Active-site-directed Irreversible Enzyme Inhibitors; The Organic Chemistry of the Enzymic Active-site, John Wiley and Sons, N.Y. (1967). Bobbitt, J. M., Thin-Layer Chromatography, Reinhold Publishing Corporation, N.Y., p. 46 (1963) Cass, 0. W., Cowen, A. E., Hofmann, A. F., and Coffin, S. B., J. Lipid Res., I&, 159 (1975). Chromatography Products, ICN Pharmaceuticals, Inc., Life Sciences Group, pp. 5, 6 (1977-78). Pinner, A., and Klein, B., Chem. Ber., 2, 1889 (1877). Werner, A., Chem. Ber., 36, 154 (1903). a. Fuchs, H., and Reichstein, T., Helv. Chim. Acta, 26, 511 (1943). b. Tserng, K. Y., and Klein, P. D., Steroids, 2, 635 (1977). a. Reber, F., Lardon, A., Reichstein, T., Helv. Chim. Acta, 37, 45 (1954). Cf: b. Lettrd, H., Greiner, J., Rutz, K., Hofmann, L., Liebigs Ann. Chem. 758, 89 (1972); c. Lettre, H., Egle, A., von Jena, J., and Mathes, K., Leibigs Ann. Chem. 708 224 (1967); d. Ruzicka, L., Plattner, A., Heusser, H., and Schlegel, W., Helv. Chim. Acta, 7, 186 (1944); e. Pearlman, W. H., J. Am. Chem. Sot., 2, 1475 (1947). Arndt, F., Org. Syntheses Coil. Vol. 2, 165 (1943). Use only scratch-free unused glassware when CAUTION/REMINDER: preparing and using diazomethane. Avoid ground glass joints; dry over smooth KOH pellets. See reference 12. Amstutz, E. D., and Myers, R. R., Organic Syntheses Coil. Vol. 2_, 462 (1943). Yates, P., Shapiro, B. L., Yoda, N., and Fugger, J., J. Am. Chem. Sot., 2, 5756 (1957). Avram, M. and Mateescu, GH.D., Infrared Spectroscopy, Applications in Organic Chemistry (Translated by L. Birladeanu), WileyInterscience, N.Y., pp. 370-373 (1972).
(2) Lack, L., Walker, J. T., Hsu, C-Y. H., Life Sci., 0, (3)
(4)
(5) (6) (7) (8) (9) (10)
(11)
(12) (13)
(14) (15) (16)
(17) Rajagopalan, M. S., Smith, D. S. H., and Turner, A. B., J. Chem. sot. (c), 646 (1971). (18) Oliveto, E. P., in Organic Reactions in Steroid Chemistry, Vol. 2 (eds. J. Fried and J. A. Edwards), van Nostrand Reinhold Company, N.Y., p. 174 (1972) (19) Katzenellenbogen, J. A., in Biochemical Actions of Hormones, Vol. 4 (ed. G. Litwack), Academic Press, N.Y., pp. 2-77 (1977). (20) a. Fridman, A. L., Ismagilova, G. S., Zalesov, V. S., Novikov, S. S., Russ. Chem. Rev., 41, 371 (1972). b. Weygand, F., and Bestmann, H. J., Angew. Chem., 72, 535 (1960). (21) Fried, A. A., Petrow, V., Lack, L., Unpublished results. (22) Cf. Dewitt, E. H. and Lack, L., Unpublished results. (23) Tserng, K-Y., and Klein, P. D., Lipids, 12, 479 (1978). (24) Haslewood, E. S., and Haslewood, G. A. D., Biochem. J., 155, 401 (1976). (25) Parmentier, G., and Eyssen, H., Steroids, a, 721 (1975). (26) Pharmacia Fine Chemicals booklet, Upplands Grafiska AB, Uppsala, Sweden (Dec. 1974-6). (27) Elsevier's Encyclopaedia of Organic Chemistry, Ser. III, s, Suppl. Part 4 (ed. F. Radt), Springer-Verlag, Berlin, 3229s (1962). (28) Fieser, L., and Fieser, M., Steroids, Reinhold Publishing Corporation, N.Y., p. 222 (1959). (29) Still, W. C., Kahn, M., and Mitra, A., J. Org. Chem. 43, 2923 (1978). Standard adsorbent column length is 5.5 in. (30) The 25-bromo analog of compound IX was indexed erroneously in Chemical Abstracts, Fifth Decennial Index, Formulas C2G-W2, Vol. 41-50, Chemical Abstracts Service, Columbus, Ohio, p. 2490 (1962). The actual compound reported was the corresponding triformyl-25-bromo compound: Kazuno, T., Moori, A., Sasaki, K., Kuroda, M., and Mizuguchi, M., Proc. Jpn. Acad., 2, 416 (1952). (31) For mass spectral analysis see: Dayal, B., Shefer, S., Tint, G. S., Salen, G., Mosbach, E. H., J. Lipid Res., 17, 74 (1976).