AU616323B2 - Creation of Inventions and Patents with Artificial Intelligence

AU616323B2 – Steroidal 5,7-dienes
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AU616323B2 – Steroidal 5,7-dienes
– Google Patents
Steroidal 5,7-dienes

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Publication number
AU616323B2

AU616323B2
AU45373/89A
AU4537389A
AU616323B2
AU 616323 B2
AU616323 B2
AU 616323B2
AU 45373/89 A
AU45373/89 A
AU 45373/89A
AU 4537389 A
AU4537389 A
AU 4537389A
AU 616323 B2
AU616323 B2
AU 616323B2
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AU
Australia
Prior art keywords
hexane
ethyl acetate
ether
mixture
washed
Prior art date
1985-04-23
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AU45373/89A
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AU4537389A
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Inventor
Hector F. Deluca
Wang Fang Lau
Heinrich K. Schnoes
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Wisconsin Alumni Research Foundation

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Wisconsin Alumni Research Foundation
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1985-04-23
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1989-11-22
Publication date
1991-10-24

1989-11-22
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1990-03-08
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patent/AU4537389A/en

1991-10-24
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1991-10-24
Publication of AU616323B2
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patent/AU616323B2/en

2006-04-21
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Classifications

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS

C07C401/00—Irradiation products of cholesterol or its derivatives; Vitamin D derivatives, 9,10-seco cyclopenta[a]phenanthrene or analogues obtained by chemical preparation without irradiation

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES

A61K31/00—Medicinal preparations containing organic active ingredients

A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07J—STEROIDS

C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane

Description

lan .A Assocition. Registered Patent Attorney To: THE COMMISSIONER OF PATENTS.
WATERMARK PATENT TRADEMAR i-SDtONAL
APPLICATION
:i 😡
I
B
_I Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION
(ORIGINAL)
616323 Int. Class
I
Class Application Number: Lodged: a a Complete Specification Lodged: Accepted: Published: Priority Related Art N’r me of Applicant ‘AUdress of Applicant S Actualnventor Actual Inventor WISCONSIN ALUMNI RESEARCH FOUNDATION 614 North Walnut Street, Madison, Wisconsin 53705, United States of America HECTOR F. DeLUCA and HEINRICH K. SCHNOES Address for Service WATERMARK PATENT TRADEMARK ATTORNEYS.
290 Burwood Road, Hawthorn, Victoria, Australia Complete Specification for the invention entitled: STEROIDAL 5,7-DIENES The following statement is a full description of this invention, including the best method of performing it known to US i 1. i f by. 4 lThe basic application… referred to in paragraphs 3 of this Declaration was were the first applikatiori….made in a Con~vention country in respect of the invention the subject of the application.
Insert c’.ice mid kt~ite of signature. D~eclared at Madison WI this C-V day of O 7- 1 8 Sieti o l.’llmrant(s) (noBy 4 ;~qokker.Managing Director Edward Waters Son, Queen StreeL, Mel bourne 3000, Vi ctoriat, AIrrl a -2- This invention relates to a group of steroidal 5,7-dienes which are useful in the preparation of compounds that are effective in inducing the differentiation of malignant cells, such as the differentiaion of leukemic cells to normal macrophages.
A useful therapeutic method for the treatment of malignancies is the administration of compounds that 0 stimulate the differentiation of malignant cells to normal 0 cells, thereby inhibiting and/or reversing the malignant transformation. Thus, it has been shown by Suda et al.
Patent 4,391,802) that Th-hydroxyvitamin D compounds specifically 16,25-dihydroxyvitamin D 3 and la,- #443 *hydroxyvitamin D 3 possess, for example, potent antileukernic activity by virtue of inducing the dif-ferentiation of malignant cells (specifically leukemia *cells) to non-malignant macrophages (monocytes) Bence,.
04 these compounds are useful for the treatment of certain malignancies, specifically for the treatment of leukemia.
When used for such treatment, however, these known lahydroxyvitamin D compounds have the disadvantage that they are also very potent calcemic agents, i.e. they cause elevated blood calcium levels by stimulating intestinal.
calcium j: fK<. 61 RPMROFF ~lilil~~~-i- i-~ 3 0 0 0 0 o e 0 4 o o 00 0 0 0 0 o a er* absorption and bone calcium resorption. This calcemic activity represents, indeed, the well-known, classical function of these compounds. Furthermore, the cell differentiation activity (and, hence, antileukemic activity) of these compounds correlates with their calcemic activity. For example, 1,25-dihydroxyvitamin D 3 the most potent compound in inducing the differentiation of malignant cells to macrophages, is also the most potent vitamin D metabolite in stimulating calcium transport or raising serum calcium levels. For practical use as cell-differentiating agents, this potent calcemic activity is, of course, an undesired side effect, since the doses required for efficacy in differentiating malignant cells can lead to excessively high and non-physiological serum calcium levels in the treated subjects. It has now been found that effective cell differentiation reversal of malignant transformation) can be achieved with a novel class of seco-sterols, that do not have the undesired side-effects (potent calcemic action) mentioned above. This selectivity and specificity of action makes the secosterols of this invention useful and preferred agents for achieving malignant cell differentiation. Purely structurally, this class of secosterols has. similarity with some of the known vitamin D compounds. Unlike the rknown vitamin D compounds, however, the secosterols of the present invention do not express the classic vitamin D activities in vivo, i.e. stimulation of intestinal calcium transport, or the mobilization of bone calcium, and hence they cannot be classified as vitamin D derivatives from the functional point of view. In light of the prior art, it was all the more surprising and unexpected to find that these secosterols are remarkably effective in inducing the differentiation of leukemia cells to normal (non-malignant) macrophages, since, as mentioned above, potent cell 0000 0 0 0 00oo 0 o 0 a 4 0 0. 0 0 0 0 0 00 0 00 0 00 (Ir "e: ".B 1 ii ii I j jr i i:;1 44 difi-'erentiation activity among the known vitamin D-related compounds was always closely correlated with patent calcemic activity. Thus, the secosterols of the present invention overcome the shortcomings of the known vitamin D-related antileukemic, agents mentioned above, and can be considered '4 preferred agents for the control and treatment of malignant diseases such as leukemia. This finding provIdes an effective method for the treatment of malignancies, sintm these seco.atexcls can be adminis! ter-ed to subjects in doses 0 0 sufficient to cause differentiation of malignant cells to S normal cels,, -without producing simultaneously 0 unphysiologically high and deleterious blood calcium levels. 00 These secosterols which are described in Application No. 000 0 57727/86 are characterized by the general structures I and II 0400 0.00 shown below: 0 G 4 4V4 44 A A where R is hydrogen, methyl, ethyl or propyl and vhnere each of Xand Xrepresent, independently, 'hydrogen or an acyl group. As used herein, an acyl group is an alkanoyl group of 1, to 6 carbons in all its isomeric forms, or an aroyl group, such as benzoyl, or halo-, nitro- or alk-yi-substituted benzoyl groups, or a dicarboxy-lic acy. group such as oxalyl, maloriyl, succinoyl, glutaroyl, or adipoyl. An alkyl group is a hydrocarbon radical of 1 to 6 carbons in all isomeric forms. cells thus provides a measure of their differentiation to noni-malignant monacyces. The assay was performed according to the procedure given by Yen et al., J. Cellular Physiol. 118, 277 (1984). The NBT reagent was prepared by dissolving 50 mg li3T and 5 pg of 4A-phorbol 12 myristate-13-acetate in 50 ml of phosphate-buffered saline. This reagent (200 p1) was added ta 200 p1l of the harvested cells (ca. 10 6cells/m1 phosphate-buffered saline). The mixture was incubated in a water bath at 37*C for 30 mini. The cells were then counted o and the percentage of cells that reduced NBT to formazan blue 0 was recorded. Results are given in Table 1, below. 0 Assay for rosette formation. o 0 0 This assay is based on rosette formation betweren 0. 0 d-ifferentiated monocytes and sheep erythrocytes coatod with 000 rabbit antibody and mouse complement. The assay was done according to the procedure of Lotem and Sachs, Internat. J. Cancer 15, 731 (1975). Sheep erytzhrocytes were washed three 00 tizies with rbsphae-buf fered saline and resuspended to a 000: 0(v/v) suspension. Equal volumes of erythrocyes and a 1:1500 dilution of rabbit autiserum to sheep erythrocytes were mixed 000000aad incubated at 3700 for 30 min. The antibody-coated erythrocy7tes (EA) were washed three times with #=sphate-buffered saline, pH 7.0 and resuspended at 0 (v/v) 0 Fresh mouse blood was .spun down and the serum was 0 collected.. Equal volumes of EKA and a 1:10 dilution of the =ouse were mixed and incubated at 37*C for 30 min, then vashed three times with phosphate-buffered saline and resuspended in RPM-I medium, 1% to give the erythrocytes coated with antibody and complement (EAC), 100 p1. of EAC was 6 mixed with an aliquot of IIL-60 cells (about 10 cells in 100 p1 RPMI), incubated for 30 min at 37*C, and theni centrifuged for 3 min at SO0xg. The pellet was dispersed and the cells with attached EAC the number of rosettes)were determined, and expressed as a percent of the total cells pr~esent. The rosette formation", indicative of the 6 differentiation of HL-60 cells to monocytes, is listed in Table 1, below. Assay for a-na-nhthyl acetate esterase activity. 4 a-Naphthyl acetate esterase is an enzyme charcteristic of monocytes. The presence of the enzymn thus indicates differentiation of HiL-60 cells to monocytes. The assay is based or, the enzymatic hydrolysis of the a-naphthyl acetate to liberate free naphthol which couples with a diazonium salt to form highly colored deposits at the sites of enzyme activity. The assay was carried out as described in Technical Bulletin~ 0 0No. 90 (Sigma Chemical Co., St. Louis, MO 63178). Cells were fixed on slides in -a citrate-acetone-mtao iaiefr3 0 se atroo teperaure Th slde5were then washed with deinied ;aer ndair-dried at least,20 min. The slides wer thn saind i a taiingsolution prepared by disolvng 5 m Fat Bue R sltin 50 ml of a 1:10 dilution of RIZAL M bffe cocenrat pH7.6, followed by the "0 00 aditon o 20mg f a-aphhylacetate (in 2 ml ethylene 6 0. glycol monomethyl ether). The slides were incubated at 370C for 30 mi~n (protected from light). They were then washed, ccunter-stained for 5-10 min in Mayer's hematoxylin solution, washed and then air-dried. The percentage of cells with 'black granulation, indicative of ix-naphthyl esterase activity, was determined. Results are listed in Table 1, below. f i" "i-~t .i :liF ;ild i i IEIl 00 00 0 0 0 o O ii 00 0 00 00 00. PO° 0D 00 QoQ e o o 0 00 (00 0 000 0 tO 4 0*40 Table 1 Percent differentiation of HL-60 cells induced by seco-sterols or by known la-hydroxyvitamin D compounds administered at various concentrations as measured by NBT-reduction, rosette formation and esterase activity assays Compound NlT Rosette Esterase Administered Concentration Reduction Formation Activity (z) EtO Control 4.5 9 Secosterol I 1 x 10 7 9 9 2 XIX2=H) x 10 6 14 23 11 -6 x 10 6 59 68 69 -7 Secosterol I 1 x 10 7 15 23 (RPCH 2 CH 3 X =X 1 x 10 6 28 30 77 I x 10 5 69 70 91 -7 I-OH-D 3 1 x 10 7 10 44 12 -6 Sx 10 6 39 61 90 1 x 10 5 85 79 100 la,25-(OH)2 3 1 x 10 8 39 44 65 1 x 10-7 83 76 90 lxlO 83 76 90 oo s o r 44 0 •O t 0 00 0 0 0000 0 00 0 i4 444 0 0 0 0 04 1 1 1 i; 1-1 8 The above results illustrate the efficacy of the seco-sterols of general structure I as agents for the differentiation of human leukemia cells to macrophages (monocytes). The compounds show highly significant activity in all three of the differentiation assays used; 50% differentiation is achieved -6 at concentrations of about 10 M. For comparative purposes, the table above also includes the cell differentiation activity exhibited by la-hydroxyvitamin D3 (l-OH-D 3 and D 3 (1,25-(OH) 2 D 3 two known vitamin D o0 derivatives with potent antileukemic action. The tabulated 0o data show that the level of activity of the seco sterols (I) is lower than that shown by 1,25-(0H)2D3 (the most potent Svitamin D-derived agent for differentiation of leukemia 09 0 cells), but is approximately equivalent to that shown by l-hydroxyvitamin D 3 a compound known to be effective in the treatment of human leukemoid diseases (Suda et al., U.S. Patent 4,391,802). Assay of secosterols of structure I for bone calcium mobilization and calcium transport. o Male weanling rats, purchased from the Holtzman Co., Madison, WI, were fed the low calcium, vitamin D-deficient 0 diet described by Suda et al. Nutr. 100, 1049 (1970)] ad libitum for 3 weeks. The rats were then divided into 4 groups of 6 animals each. The first group (control group) received S0.05 ml of 95% EtOH by intrajugular injection. The second and third groups were dosed by the same route with 625 picomoles and 6250 picomoles, respectively, of secosterol I (R-CH 3 4 132 X =X 2H) dissolved in 0.05 ml of EtOH, and the fourth group received an intrajugular injection of 625 picomole of D 3 (in 0.05 ml of EtOH). Seven hours after dosing, the rats were killed by decapitation and their blood was collected and centrifuged to obtain serum. Serum calcium concentration was determined with an atomic absorption spectrometer according to the conventional protocol. Results are listed in Table 2 below. 1 f t i o a 04 0 0 0O 0 0 40>0 0 Q 000 a 9 s; a 4 4 t at t1 t 4 a 4 a 0 4′ *0 4 0 4 *o 4i The small intestines of these same rats were removed, rinsed and everted for measurement of calcium transport activity according to the technique of Martin and DeLuca [Am.
J. Physiol. 216, 1351 (1969)1. The measured intestinal calcium transport activity data, expressed as the ratio of serosal/mucosal calcium concentration, are also listed in Table 2.
Table 2 Serum Calcium Intestinal Compound Administered Amount Concentration Ca-transport (pmole) (mg/100 ml) [Ca-serosal]/ mean S.D. [Ca-mucosal] mean S.D.
Et0H (control) 2.6 0.1 3M6 0.1 Secosterol I 625 2.9 0.1 3.4 0.1 (R=CH 3, X=X2=H) Secosterol I 6250 3.0 0.1 3.4 0.1
(R=CH
3
X=X
2
=H)
1,25-(OH) D 3 625 3.8 0.2 6.7 0.8 The above results show that secosterol I X I= 1 2
=H)
expresses no significant calcemic activity even at high doses.
The compound does not elevate serum calcium levels ant! thus is devoid of significant bone calcium mobilization activity.
Further, the compound does not stimulate calcium transport in the intestine at a dose level of 6250 picomole per animal.
Under the same conditions, the known active vitamin D metabollte, 1,25-(OH) 2
D
3 is, as expected, 2ully active at times lower dose levels.
V I UP^- t Assay of the seco-sterol homologue of structure I (where R=H, and X =X under conditions analogous to those described above (except that response was measured 12 hr after injection of test compound) gave very similar results, as i shown in Table 3, below.
i Table 3 090 095 0 0 0 0 0 0 9 0 0 o 000 09 0 o o o o o’ 0 0 0 00* 9 0 00 a 0 a0 0 o 0 0? 00 0 06 0 0 00 0 0 0 0 0 0 0 0 4 SSerum Calcium Intestinal Compound Administered Amount Concentration Ca-transport (pmole) (mg/100 [Ca-serosal]/ mean S.D. [Ca-cucosal mean S.D.
EtOH (control) 4.4 0.3 1.7 0.2 Secosterol I 6250 4.4 0.1 1.3 0.1 X=x2=H) Again the data in Table 3 de=onstrateo that the seco-sterol of structure I(R-H; X 1 elicits no respars In vtvo vith respect to intestinal calcium transport or calcium mobilization from bone, even when administered at high doses.
It can be concluded, therefore, that these seco steroids of general structure I (where R is hydrogen, methyl, ethyl, propyl) do not carry out the classical vitamin D functions in vivo, since they elicit no significant in vivo biological response with respect to bone mineral mobilization, and intestinal calcium transport activation.
The above data establish that the seco-sterols of this invention possess an unusual and unexpected spectrum of i activities. They exhibit highly significant cell differentiation activity, like some of the known vitamin D-related compounds, but do not express the calcemic activity i “tynical of vitamin D-derviatives. Thus, in being devoid of li 1 l^ 1A1 Secosterol I (-4here R-1H) can be prepared according to the method of Lam et [Steroids 26, 422 (1975)]. The secosterols of Stz-qctI es and II where R is methyl, etyl or propyl, are new cocpounds which cav be prepared according to the general process illustrated in Process Scheme 1. Suitable starting materials are the i-ether steroids of general strcture 1 where, depending on the final product desired, R may be methyl, ethyl or propyl. Conversion of a compounds of structure 1 to the 5-ene steroid of structure 2 is accomplished by solvol7sis in glacial acetic acid, according to known procedures. The 5-ene steroid is then dehydrogenated to the 5,7-diene steroid using the sequence of allylic a a bromination at C-7 followed by dehydrobromination, and the acetate group of compound 3 is saponified to obtain the ato at f, a 0 a 00 t a a tO a a at o a 12 corresponding alcohol of structure 4. Irradiation of a solution of alcohol 4 with ultraviolet light open ring B of the steroid to provide the initial secosterol derivative (the 5(10),6,8-triene), which can be purified and isolated if desired by standard chromatographic methods, but can also, and generally most advantageously, be directly thermally isomerized to the 5,7,10(19)-triene compound of structure The further conversion of this intermediate to the desired la-hydroxylated analog can be accomplished according to the general procedures given in U.S. Patents 4,195,027 and 4,260,549. Intermediate 5 (Process Scheme I) is first tosylated to obtain the 3p-tosylate and this tosylate is Sthen solvolyzed in buffered methanol to produce the o 6-methoxy-3,5-cyclo-derivative represented by structure 7.
o Solvolysis of the tosylate in other alcoholic solvents (e.g.
ethanol, propanol, butanol produces the-analogous 6-0-alkyl-3,5-cyclo intermediates, where the alkyl group derives from the alkyl portion ethyl, propyl, butyl, etc.) of the alcohol used. Any of these 6-0-alkyl-homologs of A0 compound 7 can be used for the subsequent reactions of this process. Oxidation of intermediate 7 with selenium dioxide in o the presence of a hyd.-peroxide introduces the desired lI-hydroxy function and provides compound 8. This intermediate is then solvolyzed in a medium containing a 0 low-molecular weight organic acid to yield the 3-acylate, in s 1 2 having structure 9 (X =acyl. X2=H) and the corresponding 1 2 5,6-trans-isomer of structure 10 (X 1acyl. X (where the Sacylate groups in each case derive from the organic acid used in the solvolysis reaction). These 5,6-cis and 5,6-trans (compounds 9 and 10) are advantageously separated at this stage so as to obtain each compound in pure form.
They can then be subjected to saponification base in methanol) to obtain the corresponding free hydroxy compounds 9 (X=X 2=H) and 10 (X Alternatively, the monoacylates of 9 and 10, or the free hydroxy compounds can be subjected to 13 acylation under conventional and known conditions acid anhydride or acyl halide in a nitrogenous base solvent) to produce any desired C-i-mono acyl, C-3-mono acyl or A C-l,3-di-acyl derivative, e.g. the compounds1 of structure 9 or 10, where X -acy! and X or where X =H 2. 2 and 2 acyl, or where X acyl and X 2 ‘acyl, where the acyl groups may be the samae or different.
It is evident from the above description and the Process Schemv that the nature of the side-chain in the starting material deter-mines the side-chain structure in the final products. Thus, using as starting material the sterol of structure 1, where R is methyl, provides the products of structures 9a and 1 Ca. where R is methyl. Compound 1, where P.
*is, ethyl, as. starting material, gives products 9a and v.here R is ethyl, and upon processing of steroid 1, where R. is propyl, through the above described process, there is obtained the product 9c and 10,where R. represents propyl.
The 5,6-t-rans-compounds of structure 10 in Process Scheme 0 I have utility as biologically active analogs of the 4 4 corresponding 5,6-cis-compounds of structure 9, or they may be converted to the 5,6-cis products of structure 93, by the trans to cis isomerization processes well-known in the art.
Prevaration of starting matezials. The starting materials of structure I (where R-CH 3 CH2 CH3 or C 2 CH 3 CH 3 can be prepared by conventional methods from stigmasterol. Thus 3 by conversion of stigmasterol to its i-ether derivative, followed by ozonolytic cleavage of the side-chain double bond, and subsequent hydride reduction, there is obtained the known 22-alcohol, having the structure: OcOH 3C 49:
H
4, 14 Tosylation of this alcohol, followed by hydride reduction of this 22-6osylate provides directly the compound of structure 1, where R=CH 3 Treatment of the above 22-tosyl intermediate with sodium cyanide gives the corresponding 2 3 -nitrile derivative. By t-wo-step hydride reduction of this nit-rile, there is obtained the 23-alcohol which, after tosylation, and another hydride reduction of this 23-tosyloxy intermediate, provides the stcarting compoundc of structure where R-ethyl- Similarly, the above 2 3-tosyloxy intermediate, by trearment with sodiuxn cyanide, givez the correaspondin.- 2 1 -nirrlle derivative, which after hydride reduction (to obtain the 24-alcohol) and another :05y1atian and hydride reduction sequence, prov-ides the compound of structure’ 1, where R represent: n-provyl- The compounds 3 and 4 from the subject of the present
I
I I i 1*11 II a a a a a 4~ a 4 II, 4 4 II 4~.
1141 a 11
II
invention.
te preparation of the novel compounds of this invention is more specifically described by the following Ex amples. In these Eamples, the identification of products by Arabic ny-erais =–pounds 1, 2, 3, etc.) refers to the structnras so nuno ered -in the Process Scheme.
EXamnie 1.
Prevaration of Coinnounds 9a and 10a. 38-ace tox-213 24-dinorchol-5-ene (2) A solution of compound I. (R=CH (1.26 g, 3,8 mmol) in glac~.a-. acetic acid (35 mL) was heated at 70*C for 4.5 h. The reaction mixture was cooled and poured into ice water, neutralized wi-th 1.0: aqueous sodium hydroxide and extracted w-ith chloroform (3 x 100 the chloroform extracts were washed with water (2 x 50 mL), saturated sodium chloride solution (2 x 50 dried over anhydrous magnesium sulfate, filtered and concentrated to dryness to give 0.86 g (63Z yield) of compound 2 (R-CH M{ass spectrum: m/e (relative intensity), 358 (M 298 283 255 190 177 ‘H-NMR (CDCl 3 0.66 s,18-H), 0.81 J=7 Hz, 22-H), 0.95 JI=7.0 Hz, 9 0 1 I- 0 The following statement is a full description of this invention, including the best method of performing it known to us 21-H), 1.02 19-H), 2.06 3-OCOCH 3 4.59 5.38 6-H).
3p-acetoxv-23 ,24-dinorchola-5 ,7-diene CR=CH 3 A stirred solution of 2 (R=CH (0.6 g, 8 mmol) in dry bexane (50 mL.) containing finely divided sodium bicarbonate (0.7 g, 8 maol) was heated to 80’C at reflux under nitrogen ‘1 before 1,3-dibromo-5,5-dimethylhydantoin (0.24 g, 0.85 mmol) was added. The reaction was allowed to proceed f or 20 min.
The mixture was cooled, then filtered and concentrated under reduced pressure.
0The residue was immediately dissolved in 15 mL. dry xyrlene 00 and added dropwise to a mixture of xylene (25 mL.) and s-collidine (0.4 g, 3.3 mmcl). The mixture was flushed with nitrogen and was then cooled, diluted with benzene (50 mL), washed with 3% aqueous hydrochloric acid (3 x 20 mL.), saturated sodium bicarbonate solution (1 x 25 water (I x saturated aqueous sodium chloride (2 x 25 mL.) ,dried over anhydrous magnesium sulfate and filtered. The solvent-.
was evaporated under reduced pressure.
The oily residue was dissolved in dry dioxane (40i mL.) and 000 0 p-toluenesulfonic acid (0.076 g, 0.4 mmol) was added. The mixture was flushed with nitrogen and refluxed at 70*C for 0 0 Ih. After cooling, it was diluted with water (100 mL.) and O extracted with ethyl acetate (1 x 70 mL., 2 x 60 the 0:0 organic extract was washed with saturated sodium bicarbonate solution (I x 30 mQ., saturated sodium chloride (2 x 30 mL) O dried over anhydrous magnesium sulfate, filtered and the solvent was evaporated under reduced pressure to give an oi’ K;’containing the desired the 5,7-diene CRf 0.29 in 10% ethyl acetate-hexane) and the 2,,4,6-triene CRf 0.5 in 10% ethyl acetate:hexane) Preparative TLC using 10% ethyl acetate-hexane gave 0.26 g of 3 (R=CH in ca. 43% yield.
UV (C H OH) X 282, 293, 272, 262 nm; mass spectrum: uile (relative intensity), 356 296 (100), 281 253 211. 158 143 3-H-NZR (CDCl 3 0.62 Cs, 16 1 04 00 o 0 0 a a a o 0 0 0 CC C 0 C CC 0 04 0 0a 0 C C a 000 4 04 o 4 0000
I
44 18-H), 0.88 J=7.0 Hz, 22-H), 0.95 0.96 (d, Hz, 21-H), 2.04 3-OCOCH3), 4.7 5.4 (m, 5.58 6-H).
3 -Hydroxy-23,24-dinorchola-5,7-diene (R=CH 3 A 10% sodium hydroxide in methanol solution was added dropwise to a stirred solution of 3 (R=CH 3 259 mg; Rf 0.56 on ethyl acetate-hexane) in ether (15 mL) over a 5 min period under nitrogen. The reaction proceeded at 23°C and was monitored on TLC. It was completed within 35 min. The mixture was diluted with ether (100 mL) and water (30 mL) was added. The layers were separated and the aqueous layer was extracted with ether (2 x 50 mL). The combined ether fractions were washed with water (2 x 30 mL), saturated sodium chloride solution (2 x 30 mL), dried over anhydrous magnesium sulfate, filtered and evaporated to dryness under reduced pressure to give 200 mg of 4 (77% yield) after preparative TLC using 25% ethyl acetate/hexane.
UV (EtOH): X 282, 293, 272, 262 nm; mass spectrum: m/e (relative intensity), 314 85), 296 281 (100), 261 255 211 171 143 H-NHR (CDC1 3 8 0.62 18-H), 0.87 J=7.0 Hz, 22-H), 0.95 19-H), 0.97 J=7.0 Hz, 21-H), 3.63 (sh, 5.38 Cm, 5.58 (m, 6-H).
Seco-sterol 5” (R=CH 3 The 5,7-diene (36 mg) dissolved in a mixture of benzene-ether (100 mL) was placed in a jacket around a double-walled, water-cooled quartz immersion well equipped with a Hanovia 608A quartz-medium pressure mercury vapor ultraviolet lamp with a vycor filter. The mixture was irradiated for 4.5 min. The system was purged continuously with nitrogen throughout irradiation. The solvent was then removed under reduced pressure and the residue redissolved in dryethanol. It was flushed with nitrogen and then heated to at reflux under nitrogen for 3 h. It was then cooled and i concentrated under reduced pressure. Purification by TLC V i mnese secoscerols are remarkably etlective In inducing tne differentiation of leukemia cells to normal (non-malignant) nacrophages, since, as mentioned above, potent cell i i 1
I
17 :i I 4 Sa o o a O 4 0 4 o5 0 0 000 01eo
I
4 4 i i using 20% ethyl acetate-hexane afforded 5 (R=CH 3 in 31.7% yield (11.4 mg).
UV (EtOH) X 264 nm; mass spectrum: m/e (relative intensity) max 314 14), 296 281 271 253 136 (82), 118 (100); 1 -NMR (CDC13): 6 0.54 18-H), 0.85 Hz, 22-H), 0.94 J=7.0 Hz, 21-H), 3.93 4.82 (m (sharp), 5.04 (m (sharp), 6.02 J=12.0 Hz, 6.22 J-12.0 Hz, 6-H).
Tosylate 6 (R-CH 3 A solution of 5 (15 mg, 47 mmol, Rf 0.28 in 30% ethyl acetate-hexane) in dry pyridine (0.5 mL) was treated with p-toluenesulfonyl chloride (22 mg, 117 mmol) at 5°C under nitrogen for 24 h. The reaction was quenched with ice water and the mixture extracted with ether (3 x 30 mL). The combined extracts were washed with 3% aqueous hydrochloric acid (2 x 30 mL), saturated aqueous sodium bicarbonate (1 x mL), saturated sodium chloride solution (1 x 50 MnL), dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give 20 mg of tosylate 6 (R-CH 3 (Rf 0.6 in 30% ethyl acetate-hexane).
3,5-Cyclo-derivative 7 (R=CH 3 The crude tosylate 6 (R=CH 3 (20 mg, 0.41 imuol, Rf 0.5 in ethyl acetate-hexane) was added to a stirred solution of finely divided sodium bicarbonate (200 mg, 2.4 mmol) in anhydrous methanol (20 mL). The mixture was heated to 55’C at reflux under nitrogen for 8 h, cooled, diluted with ether (100 mL) .and washed with water (3 x 30 mL), saturated aqueous sodium chloride solution (1 x 30 mL), dried over anhydrous magnesium sulfate, filtered and the solvent was evaporated under reduced pressure. The crude mixture was chromatographed on preparative TLC using 10% ethyl acetate-hexane to obtain 7
(R=CH
3 (7 mg, Rf 0.66 in 10% ethyl acetate-hexdne) in 50% yield.
Mass spectrum: m/e (relative intensity), 328 (M 14), 296 281 253 159 135 145 1
H-NMR
d a :3 i
;:W
:I
;:r it a:: is i :9
B-
such as oxalyl, malonyl, succinoYl, glutaroyl, or adipoyl- An alkyl group is a hydrocarbon radical of 1 to 6 carbons in all isomeric formns.
II
K
“FORT, 00 09 00 0 o 0 0 004 04 00 00 0 0 0 0 00 0 0 0 000 0 0400 0 0444 0*00 0 0 0 00 0 0 00 0 0 0 0 00 0 4 4 0 I 004004 0* 0 00 04 I 0 0 1 0 01 18 (CDC1 3 6 0.55 18-H), 0.85 3=7.0 Hz, 22-H), 0.93 (d, Hz, 21-H), 3.26 6(R)-OCH 3 4.18 J=10 Hz, 6-H), 4.89 (mn (sharp), 5.0 J=10O Hz, 5.06 (mn sharp) la-Hydroxy-r-ampound 8 (R7-CR) t-Butyl hydroperoxide (7 p1, 0.05 mmnol) was added to a suspension of selenium dioxide (SeO 2 1.1 mg, 10 jumole) in 1% dry pyridine-nethylene chloride (5 niL) under nitrogen. The mixture was stirred at 23*C for 0.5 h, then diluted with another 10 niL of 1Z pyridine-siethylene chloride solution. Trhe mixture was cooled on an ice bath and intermediate 7 (R=CH 3 (7 mng, 21 pinole; Rf 0.62 in 25% ethyl acetate-hexane) in dry =ethylene chloride was introduced. The reaction was monitored on TLC*. It proceeded at 23%C for 16 min before 10% sodium hydroxide solution (20 aL) was added to quench the reaction.
The mixture was diluted with ether (100 mQL, phases were separated and the ether phase was washed with 10% aqueous sodium hydroxide (2 x 25 mQL, water (2 x 20 mQL, saturated aqueous sodium chloride (2 x 20 mQL and dried ever anhydrous miagnesium sulfate. It was filtered and the solvent vas evaporated under reduced pressure. Preparative TLC of the residue using 251 ethyl acetate-hexane save the 1ca-hydroxy-derivative 8 (RoCH 3 (3 mg,, Rf 0.15 in 25% ethyl acetate-hexane) in 41% yield.
Hass spectrum: nile (relative intensity): 344 (M 34), 312 271 177 (567), 135 (100), 1 R-NM~R (CDdl 3 6 0.55 ’18-H) 0.85 J=7.0 Hz, 22-H), 0.94 Cd, J=7.0 Hz, 21-H), 3.26 6(R)-OC 3 4.17 (m(sharp), 4.22 (mn (sharp), 4.94 J-9 Hz, 5.16 J-2.2 Hz, 5.24.
3=2.2 Hz, 19(E)-H).
5,6-cis and trans secosterol 30-acetates 9a and (X =Ac, X 2=H) A solution of 8 (R=CH (3 mig) in glacial acetic acid niL was heated to 55*C under nitrogen for 20 min, cooled and poured over ice cold sodium bicarbonate solution (15 niL) with attached EAC the number of rosettes)vere determined, and expressed as a percent of the total cells i present. The rosette formation”, indicative of the 19 and extracted with ether (3 x 30 mL). The combined ether extracts were washed with saturated aqueous sodium bicarbonate (1 x 20 mL), water (2 x 20 mL), saturated sodium chloride (1 x mL) and dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was chromatographed in 35% ethyl acetate-hexane (multiple elutions, 4 times) to yield 0.8 mg of the 5,6-cis-30-acetate S(9a) and .45 mg of the 5,6-trans-3p-acetate (lOb), respectively.
1 2 Acetate 9a (X -Ac, X UV (EtOH): X 264 ni, X max min 228 nm.; mass spectrum: m/e (relative intensity) 37Z (M 7), 312 269 (10),-189 134 (100); 1 H-NMR (CDC1 3 6 0.54 18-H), 0.85 J-7.0 Hz, 22-H), 0.92 J=7.0 Hz, 21-H), 2.02 3-0COCH 3 4.4 (broad, 5.01 (m(sharp), 5.21 5.33 (m (sharp), 6.0 (d, SJ=12.0 Hz, 6.34 J=12.0 Hz, 6-H).
Acetate 10a (X 1 =Ac, X2=H): UV (EtOH): X 273 nm, Xin 228 nm; mass spectrum: m/e (relative intensity) 372 (M 3), 328 312 269 177 149 135 (100); o 1 BH-iR (CDC1.): 6 0.54 18-H), 0.85 J=7.0 Hz, 22-H), 0.92 J=7.0 Hz, 21-H), 2.02 3-OCOCH 3 4.4 (broad, 4.99 (m(sharp), 5.13 (m (sharp), 5.8 id, J=12.0 Hz, 6.58 J=12.0 Hz, 6-H).
1 2 l-Hydroxy-secosterol 9a (X =X An ether solution (10 ml) of the 30-acetate 9a as obtained in the preceding experiment was hydrolyzed using sodium hydroxide in methanol at 23C under nitrogen for 0.5 h.
The mixture was diluted with water (10 mL) and extracted with ether (3 x 50 mL). The combined ether extracts were washed with water (2 x 10 mL), saturated sodium chloride solution (2 x 10 mL), dried over anhydrous magnesium sulfate, filtered and evaporated to dryness under reduced pressure. -j The residue was purified by HPLC on a Zorbax-Sil analytical column (4.6 mm x 25 cm) using 5% isopropanol-hexane at 265 nm to give 9a (X =X High resolution mass spectral analysis: calc. for C H22 3402, 330.2559; found 330,2541; UV (EtOH): X 264 nm i 227 nm; mass spectrum,: max min m/e (relative intensity) 330 (M 55), 312 287 269 251 189 152 134 (100); 1B-NMR (CDC13 6 0.54 18-H), 0.85 J=7.0 Hz, 22-H), 0.93 J=7.0 Hz, 21-H), 4.23 4.42 5.0 5.34 6.02 J=12.0 Hz, 6.39 J=12.0 Hz, 6-H).
5,6-trans-analog 10a (X =X =H) The 3p-acetate (O0a) was hydrolyzed in a similar manner and purified on HPLC using 5% isopropanol-hexane at 273 nm to ,give 10a (X High resolution mass spectral analysis: Scalc. for C 2 2 3 4 0 2 330.2559; found, 330.2532; UV (EtOH): Xma x 273 nm, Xi 227 r; mass spectrum: m/e (relative intensity), min 330 (M 69), 312 287 269 251 189 152 134 (100).
Example 2 Preparation of Compounds 9b and O10b (X 1
=X
2
H)
38-Acetoxy-24-norahol-5-ene R=ethyl) A solution of 1 (R=Et) (0.5 g; Rf 0.8 in 25% ethyl acetate-hexane) in glacial acetic acid (7 mL) was heated at for 4.5 The reaction mixture was cooled, poured into ice water, neutralized with 10% aqueous sodium hydroxide and extracted with chloroform (3 x 70 mL). The organic extract 24 was washed with water (2 x 25 mL) and saturated sodium chloride (2 x 25 mL) and then dried over anhydrous magnesium sulfate. It was filtered and concentrated to dryness under reducea pressure to give 0.5 g of residue containing 2 (R=Et) (Rf 0.18 in 25% ethyl acetate-hexane) in ca. 90% yield. This material was used without further purification.
Mass spectrum: m/e (relative intensity) 372 (M 312 (100), 298 283 255 191 1 H-NMR (CDC 3 6 0.69 18-H), 0.82 J=7.5 Hz, 23-H), 0.9 J=7.0 Hz, 21-H), 1.05 19-H), 2.04 3-COCH 3 4.58 5.38 J=4 Hz, 6-H).
1x 10- 83 76 11 21 3P-Acetoxy-24-norchola-5,7-diene (R-Et) :1 A stirred solution of 2 (R=Et) (270 mg, 0.72 mmol) in dry hexane (50 mL) containing finely divided sodium bicarbonate (600 mg, 7.14 mmol) was heated to 80’C at reflux under nitrogen and 1,3-dibromo-5,5-dimethylhydantoin (125 mg, 0.43 =ol) was then added. The reaction was allowed to proceed for min at 80°C, then cooled and filtered. The filtrate was evaporated to dryness in vacuo. The residue was immediately dissolved in dry xylene (5 mL) and added dropwise to a mixture of xylene (30 mL) and s-collidine (174 mg, 1.44 mmol). The S ixture was then flushed with nitrogen and refluxed at 145 0
C
4. for 1.5 h. It was then cooled, diluted with benzene (100 mL), washed with 3% aqueous hydrochloric acid (3 x 20 mL), .0 saturated aqueous sodium bicarbonate (1 x 20 mL), saturated sodium chloride solution (2 x 20 mL), dried over anhydrous nagnesium sulfate, filtered and evaporated to dryness under reduced pressure.
The residue was dissolved in dioxane (20 mL) and S>p-toluenesulfonic acid (60 mg, 0.32 mmol) was added. The -mixture was flushed with nitrogen and refluxed at 70°C for Smin, cooled, diluted with water (10 mL) and then extracted with ethyl acetate (1 x 100 mL, 2 x 50 mL). The organic phase was washed with saturated aqueous sodium bicarbonate (1 x mL), water (1 x 25 mL), saturated aqueous sodium chloride (2 x 30 mL) and then dried over anhydrous magnesium sulfate. It 4 t was filtered and evaporated under reduced pressure to dryness.
The residue was chromatographed on preparative TLC using 4 ethyl acetate-hexane to yield 148 mg of 3 (R-Et).
UV (EtOH): A 282 nm, 293, 272, 262. mass spectrum: m/e max (relative intensity) 370 (M 328 310 (100), 296 j 253 158 H-NMR (CDC13): 6 0.63 18-H), 0.84 J=7.5 Hz, 23-H), 0.95 J-7.0 Hz, 21-H), 0.96 r 19-H), 2.04 3-OCOCH 3 4.71 5.4 5.5,1 6-H).
3 -Hydroxy-24-norchola-5.7-diene (R-Et) i? calcium concentration was determined with an atomic absorption spectrometer according to the conventional protocol. Results are listed in Table 2 below.
22 Sodium hydroxide in methanol (10% solution) was added dropwise to a stirred solution of 3 (130 mg, 0.35 mmol, Rf 0.4 in 15% ethyl acetate-hexane) in ether (20 mL) under nitrogen.
The reaction was allowed to proceed at 23″C for 40 min. It was then diluted with ether (100 mL), water (20 mL) was added and the phases were separated. The aqueous layer was further extracted with ether (2 x 60 mL) and the ether extracts were combined, washed with water (2 x 30 mL), saturated aqueous sodium chloride (2 x 30 mL), dried over anhydrous magnesium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was. chromatographed on 4 preparative TLC in 30% ethyl acetate-hexane to yield 103 mg of 4 (R=Et) (Rf 0.09 in 15% ethyl acetate-hexame).
UV (EtOH): A 282, 293, 272, 262 nm;. mass spectrum: m/e max (relative intensity), 328 (M 100), 3i4 310 295 281 269 255 1 -NlMR (CDC1 3 6 0.63 (s, 18-H), 0.84 J=7.5 Hz, 23-H), 0.94 J=7.0 Hz, 21-H), 0.96 19-H), 3.64 5.38 5.57 6-H).
o Secosterol analog 5 (R=Et) The 5,7-diene 4 (R-Et) (125 mg) in 1:4 dry benzene-ether (150 mL) was irradiated in a manner similar to that described in Example 1, above, for 25 min. The solvent was evaporated under reduced pressure. The crude residue was immediately 0 dissolved in dry ethanol (30 mL) saturated with nitrogen. The t* solution was refluxed at 70°C under nitrogen for 3 h, then cooled and concentrated under reduced pressure. Purification by preparative TLC using silica gel plates in 30Z ethyl acetate-hexane afforded 5 (R=Et) (30 mg) in ca. 24% yield.
UV (EtOH): X 264 nm, A 227 nm; mass spectrum: m/e M4 max m~* (relative intensity) 328 (M 23), 310 295 271 253 136 118 (100); 1 H-NMR (CDC1 3 6 0.55 V 18-H), 0.83 J=7.5 Hz, 23-H), 0.92 J=7.0 Hz, 21-H), 3.93 (broad, 4.81 (m (sharp), 5.01 (m (sharp), 6.02 J=12.5 Hz, 6.22 J=12.5 Hz, Tosylate 6 (R=Et) metabolite, 1,25-(01fl 2
D
3 is, as expected, fully active at 2~ 31 times lower dose levels.
23I
J’
J;;
Asolution o Rt (15×2 ,te drie 0o045 anhydous Rfa0.23in25 suhlaeadecohenae indr preduned (1ress trateld withg aod the mturlae extracted ith geather (3a 950 puri.Thorai 0 Yphasespwecrm omied (eati iashednwity) 4ate 8 30(7) go aqeu hyrohlrO acid8, 9 (0,8 203 (3)156), saurte sodiu11 o fThe oslate 6 (RE) in2 gmgte than45%o;R puit.54i5 finsec m divde sorebiarveonatensi6y 484 0.1, 8o) 31in 2) 296e (18)oge 295 (2)81 (10)ole, 253te (39) 158(6)er4 (47 )18 chlori y (2ox d mrvativ 7a rie vr nyrosmgnsu Thel aosclte rdc6 (R-Et) g004 l;Rf 0.54 in ehacetate-hexane) as addyed.t tre upnino finel dividedm som brlaiarbonteniy 340 0,7 10o), in8 anhdruy mehanoeoid (20. ml) The mixtur was ae atde 5o 0 a udensiton fo8 eenu oole dilute with eth2 r (150 mndr)1 chlride-m(2 30 hlorid (1t wa arie over e nhrons aniesiumh yhela ccrude pronc (REt) (m,004Rl f 0.6 in et acetate-hexane) in d 68% hyyield choie4. m) a addetd. Thdrtonexd (14. moniore 0.1 TLC was adceded for a0 airreretiatiofl actIvIty, J1iice some of the known vitamin D-related compounds, but do not express the calcemic activity *typical of vitamin D-derviatives. Thus, in being devoid of 4 24 min before 10% sodium hydroxide (10 mL) was added to quench the reaction. The mixture was diluted with ether (150 mL), phases were separated and the ether phase was washed with sodium hydroxide solution (2 x 30 mL), water (2 x 30 mL)j, A sodium chloride (2 x: 30 mL) and dried over anhydrous magnesium 4 sulfate. It was then filtered and concentrated under reduced pressure. Preparative TLC using 25Z ethyl acetate-hexane gave 8 (R=Et) (8 mg, R~f 0.18 ini 25% ethyl acetate-hexane) in ca.
yield.
IYass spectrum: m/e (relative intensity) 358 21), 326 4(48), 285 269 191 135 (100); -NR(C1) 6 0.55 18-H), 0.85 Ct, J=7.5 Hz, 23-H), 0.92 J-~7.0 Rz, 21-nH), 3,27 6R–OCH 3 4.18 J=10 Hz, 4.22 (m, :.00 141), 4.95 J-10 Hz, 5.16 Cd, J=2 .0 Hz, 19(Z)-H), 5.26 J=2.2 H2:, 19(E)-H).
5,6-cis and trans secosterol 30-acetates 9b and Cx 1 Ac,, X 2=H) A solution of 8 (R=Et) (8 mg) in glacial acetic acid was heated to 55’C under nitrogen for 15 mini, cooled and poured over ice.-cold sodium bicarbonate solution (15 rnL). The mixture was ex.tracted with ether (3 x 30 mL), and the ether extract was washed with saturated sodium lr~tcarbonate solution (x 20 ml), water (2 x 20 mL), saturated aqueous sodium chloride~ (1 x: 20 mL) dried over anhydrous magnesium sulfate and f~l1tered. The filtrate was concentrated under reduced pressure and chromatographed by preparative TLC on 30Z ethyl acetate-hexane Cx 2) to give the 5,6-cis-30-acetate 9b (X 1 c X 2 (Rf 0.13 an 25% ethyl acetate-hexane) and the 5,6-trans–3g-acetate 10b (X =Ac, X inH) CRf 0.11 on 25% ethyl acetate-hexane).
The products were further purified on HPLC on a Zorbax-Sil colun (4.6 mm x 25 cm) in 1% isopropanol-hexane to give the acetates of 9b and 10b in 28.9% and 10.4% yields, respectively (retention volumes of 39 mL and 46.5 mL) 1 2= 9b (X =Ac, X H) mm- :0 o 0 0 00 00 o o o,.o 00 00 00 0 0 0 o 00 0 0 0 004 0 0000 08,0 TJV (EcOH) X mx264 nm, x i 226 nm; mass spectrum: m/e (relative intensity) 386 326 308 269 203 134 (100), ‘H-NMfR (C DC 1 3 6 0.55 Cs, 18-H), 0.84 Ct, J-W7.5 Hz, 23-H), 0.92 J-7.0 Hz, 21-H), 2.04 (s, 3-.OCOCEI 3 4.41 5.02 (m (sharp), 5.22 (mn, 5.35 (m (sharp), 19(E)-H) 6.03 Cd, J-12.5 Hz, 6.35 J1=12.5 Hz, 6-H).
10b (X I =Ac’ X2=1) U7 (Er0H) X 1 2 73 um, Xmn226 mass spectrum: m/e (relative intensity): 386 12), 326 312 297 279 269 2031 134 (100); 1 H-NMR (CDC1 3 6 0.56 18-H), 0.Bp J1=7.5 Hz, 23-H), 0.93 J1=7.0 Hiz, 21-H) 2.30 3-OCOCH 3 4.49 1-H) 5.0 (m (sharp), 19(Z-H), 5.14 Cm (.Aarp), 19 5.26 5.82 (di, J1=12.5 Hz, 6.58 .1-12.5 Hz, 6-H).
la-Hydroxy-secosterols 9b, and l0b (X I X 2 H) The 30-acetate 9b (X =Ac, X (1.5 mng, Rf 0.31 in ethyl acetate-hexane) was hydrolyzed using 10% sodium hydroxide In methanol (2 mL) at 23’C under nitrogen for 0.5 h.
The mixture was diluted with ether (50 mL) and water (5 ml) vas added. The phasen were separated and the aqueous layer was extracted with ether (2 x 30 The ether eaxtractS were combined, washed with water (2 x 10 saturated sodium chloride solution (2 x 10 mQL, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to give 9b CX I=XH) CRf 0.06 in 20% ethyl acetate-hexane).
Similarly, acetate l0b (X I=Ac, X 2 H) (0.75 mg. Rf 0.26 in ethyl acetate-hexane) was hydrolyzed to give 5,6-trans-isomer 10b CR=Et, XI=X 2 CRf 0.06 in 40Z ethyl acetate-hexane).
Each of the products was chrontatographed on preparative TLC in 60% ethyl acetate-hexane. followed by HPLC on a Zorbax-Sil analytical colutm (4.6 =m x 25 cm) in 7% isopropanol-hexane and then reverse phase HPLC CPEPLC) on a 0000 0 0 0 00 0 0 00 o 0 0 0 00 008004 O 0 000000 O 1 00 0 O S I
II
00 I 0 II .u o o corresponding tree hydroxy compounds (X =X 2=H) and 10 (X =X Alternatively, the monoacylates of 9 and 10, or the free hydroxy compounds can be subjected to 26 Zorbax-ODS analytical column (4.6 mm x 25 cm) in nethanol-water.
Coumound 9b (X 2X=H) UV (EtOH): Xm 264 nm, X 227 n; high resolution mass max min analysis: calc. for C 23
H
36 0 2 344.2715; found, 344.2707; mass spectrum: m/e (relative intensity) 344 23), 326 J 287 269 251 203 152 134 (100); 1B-NMR (CDC13): 6 0.52 18-H), 0.82 J=7.5 Hz, 23-H), 0.90 J-7.0 Hz, 21-H), 4.29 4.42 4.99 (m (sharp), 5.31 (m (sharp), 6.0 Cd, 04 00 0J=12.5 Hz, 6.37 J12.5 Hz, 6-H).
5,6-trans-compound 10b (X X 2
=H)
0 UV (EtOH): x 273 nm, X 226 um; high resolution mass max min 0 0 analysis: calc. for C23 3602, 344.2715; found, 344.2705; mass 0 spectrum: m/e (relative intensity) 344 (b 12), 326 287 0000 269 251 203 152 134 (100); 1
H-NR
(CDC13): 6 0.56 18-H), 0.84 J-7.5 Hz, 23-H), 0.91 (d, Hz, 21-H), 4.22 (m (sharp), 4,48 (m (sharp), 1-4), tot* 4.96 5.12 (m (sharp), 5.88 J=12.5 Ez, 6.57 J=12.5 Hz, 6-H).
SoExarole 3 Preparation of compounds 9c and From 6-uethaxy-3a,5-cyclo-5a-cholane (compound 1, where R-propyl), processed through all the reaction steps given in Example 2 above under analogous experimental conditions, there is obtained the la-hydroxy-secosterol analog of structure 9c (X=X 2=H) and the corresponding 5,6-trans-compound of structure 10C (Xl =X 2
H).
Example 4 Synthesis of Starting Material, Compound 1 (R=Me) (22E)-6 -Methoxy-3a,5-cyclo-5a-stigmast-22-ene (Stigmasteryl i–methylether) Freshly crystallized p-toluenesulfonyl chloride (20 g, 0.10 mole) was added to a solution of stigmasterol (25 g, 0.06 4) 4
A
*1 0i 0~ o o 0 O 0 04 o 0 o o44 04 04 00 4 o 4 o 44 0 004 0 0004 0000 0 4 0 0 00 00 0 0 00 0 000*00 C00040 0 le; Rf 0.26 in 30X ethyl acetate-hexane) in dry pyridine (230 mQ.. The reaction mixture was stirred at 23%C for 24 h, after which it was slowly poured into saturated aqueous sodium bicarbonate solution. The precipitate was collected by filtration, washed with water several times until, neutral and dried utnder reduced pressure overnight to yield stigmastery’l-3,9-tosylate (32.06 g) in 93.2% yield.
The tosy2.ate ‘was converted to the i-ether without further Vurification. A solution of the tosylate (32 S, 0.06 mole) in chloroform (100 mL.) was added slowly to a refluxed solution of finely divided sodium bicarbonate (30 g, 0.36 mole) in metbanol (400 mQ.. The mixturd was stirred at reflux for 14 h, cooled and concentrated to ca. 100 mL.. Hexane was added (300 mL.) and the resulting mixture was ‘washed with water (100 The phases were separated atid the aqueous layer was back extracted with hexane (2 x 200 mL., 1 x 100 The organic layers were combined and washed with water (2 x 100 mL.) saturated aqueous sodium chloride (2 x 100 dried over 2–hydrous magnesium sulfate, filtered and concentrated under reduced pressure to give 24 g of a crude oil containing the desired i-methyl ether (Rf. 0.52 in 107 ethyl acetate-hexane).
Mass spectrum: m/e (relative intensity) 426- (M 87), 411 394 371 368 351 255 (61) 83 0 4 4 0 00 00 0 0 0 4 00 6p-Metboxy-3a,5-ecyclo-23 ,24-dinor-Sa-cholan-22-Ol.
A stirred solution of stigmasteryl i-ether as obtained above (5.0 g, 11.7 mmol, Rf 9.64 in 30% ethyl acetate-hexan~e) in 1% pyridine-methylene chlori~de (100 mQL was cooled to -69*C o= a dry ice-acetone bath and treated with ozone generated bv a V’elsbach model T816 ozonator, until a pale blue color~ persisted due to excess ozone. The mixture was purged wi t b oxylgen for 5 min and allowed to warm to 230C. Sodifum borohydride (0.7 g, 18.4 mmol) in ethanol was tdded, After 2 h, the reaction mixture was diluted with ether (200 r1.).
it yield) of compound 2 (R=CH3).
Mass spectrum: m/e (relative intensity), 358 298 (100) 283 255 190 177 1 H-NMR (CDC1 3 0.66 18-H), 0.81 J=7 Hz, 22-H), 0.95 J=7.0 Hz, 11 p 4
A
44 44 4 4 4 4 44 4 4 4 444 14 14 4 4 4 41.4 4 44±4 4144 4 44~ 114 44 4 Water (100 mL) was added, the phases were separated and the aqueous layer was extracted with ether (2 x 200 mL). The conbined organic fractions were washed with saturated aqueous sodium chloride (2 x 50 mL) and dried over anhydrous magnesium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was applied on a silica gel column and eluted using 20% ethyl acetate-hexane to afford 2.6 g of the desired 22-alcohol derivative in 65% yield. Mass spectrum: m/e (relative i1 ensity) 346 (M 75), 331 314 291 (100), 288 H-NMiR (CDC1 3 6 0.44 0.65 (m, 0.75 18-H), 1.04 19-H), 1.08 J=7.0 Ez, 21-H), 2.77 sha 3.32 6-OCH 3 6a-Methoxy-3a,5-cyclo-23,24-dinor-5a-cholan-22-yl tosylate p-Toluenesulfonyl chloride (2.0 g, 11 mmol) was added to a solution of the 22-alcohol obtained in the previous experiment (1.9 g; 5.5 mmol) in dry pyridine (35 mL). The reaction mixture was stirred at 23°C for 18 h, then poured into ice-cold saturated aqueous sodium bicarbonate (50 mL) and extracted with ethyl acetate (2 x 150 mL, 1 x 100 mL). The organic extract was washed with water (3 x 50 mL), saturated sodium chloride (2 x 50 mL) and dried over anhydrous magnesium sulfate. It was then filtered and evaporated to dryness under reduced pressure. The residue was dried under reduced pressure overnight to give 2.2 g of the 22-tosylate in 91.0% yield. Mass spectrum: m/e (relative intensity) 500 (M 69), 485 468 (100), 445 442 296 273 (24).
6 -Methoxy-3a,5-cyclo-23,24-dinor-5a-cholane (compound I, R=Me) To the 22-tosylate (2.15 g, 4 mmol) in anhydrous ether (100 mL) was added 0.22 g lithium alumnium hydride (liAlH, 0.22 g, 6 mmol). The reaction mixture was refluxed 10 h, cooled and excess reagent was decomposed by saturated aqueous sodium chloride. The mixture was filtered and the layers 1 1 29 separated. The aqueous fraction was back-extracted with ether (2 x 100 mL). Ether fractions were combined, washed with water (1 x 50 mL), saturated sodium chloride solution (2 x rL), dried over anhydrous magnesium sulfate, evaporated to dryness and dried under vacuum to give compound 1 (R=Me) in 96.0% yield. Mass spectrum: m/e (relative intensity) 330 C( 41), 315 298 (100), 283 275 272 (18), 177 1 -NMR (CDC1 3 6 0.40 0.64 0.72 18-H), 0.85 J=7.0 Hz, 22-H), 0.92 J-7.0 Hz, 21-H), 2.77 sharp), 3.35 6-OCH 3 Exaile 0o Preparation of Starting Material, Comoound 1 (R=Et) S.a 6-Methoxy-3a,5-cyclo-24-nor-5a-cholane-23-nitrile.
To the 22-tosylate obtained in Example 4(c) above (10 g, 20 mmol, Rf 0.53 in 25% ethyl acetate-hexane) dissolved in 0oo diethylsulfoxide (200 mL) was added sod-um cyanide (1.95 g, mmol). The mixture was stirred at 80 0 C under nitrogen for 2 h, then cooled and stirred at room temperature for 1 h. It was then poured over ice-saturated ammonium chloride solution So (250 mL) and extracted with ether (3 x 450 mL). The combined ether fractions were washed with water (3 x 200 mL), saturated sodium chloride (2 x 200 mL), dried over anhydrous magnesium.
sulfate, filtered and concentrated under reduced pressure to S give the desired 23-nitrile derivative (7.05 g crude, Rf 0.57 In 25% ethyl acetate-hexane). The crude mixture was used in Sthe next step without further purification. Mass spectrum: ‘3 m/e. (relative intensity) 355 33), 340 323 308 297 300 (100), 218 149 1-NMR (CDC 3 6 0.43 0.65 0.74 18-H), 1.02 (s, 19-H), 1.15 J=7.0 Hz, 21-H), 2.77 J=2.5 Hz, 6-H).
6a-Methoxy-3a,5-cyclo-24-nor-5a-cholan-23-ol.
To the nitrile derivative as obtained in above g, 19.7 mmol, Rf 0.27 in 10% ethyl acetate-hexane) dissolved in dry benzene (150 mL) and cooled on ice under nitrogen was :r .t dryethanol. It was flushed with nitrogen and then heated to at reflux under nitrogen for 3 h. It was then cooled and concentrated under reduced pressure. Purification by TLC I. 530 ad’ ed slowly diisobutylaluminum hydride [DIBAL-H, 1.5 mole solution in toluene (20 mL., 30 mnol)]. The ice bath was renoved after addition was complete and the reaction was allowed to proceed at 23*C for 3 h. Methanol (150 mL) was added to decompose the aluminum salt complex and the mixture was poured over ice water (200 The mixture w-as filtered an.d the aqueous layer was extracted with ether (3 x 150 niL).
The organic phases were combined, washed with saturated sodium ch-loride (2 x 60 dried over anhydrous maguesium sulfate and concentrated under reduced pressure to give 6.25 g of product. This product was a mixture of the original 23-nitrile and the expected 23-aldehyde. This mixture was 4 again treated with the samu reducing agent. Thus, the crude product was dissolved in dry benzene and treated ‘vith TJIBXL-U 0 (17.5 mL., 26.25 mmol). Work-up as before and chromatography the -residue on silica gel, eluted with 107% ethyl acetate-hexane yielded 0.85 g of the desired 23-alcohol derivative (Rf 0.03 in 10i ethyl acetate-hexame). Mass spectrum: m/e (relative intensity) 360 (H4k, 75), 345 328 4 305 (100), 302 281 25-5 1 H-Nb a (CDCl 3 b 0.44 (mo, 3-H1), 0.65 0.72 18-H), 0.93 Ez, 21-H1), 1.02 19-H1). 2.77 nharp, 3.3 (s, 6-OCH 3 1 The remainder of the product (2 g) was the corresponding 23-aldehyde, which, if desired, can be further’reduced using the above conditions to yield additional quantities of the 23-alcohol product.
I(c) 23-Hydroxy-6 O-me thoxy-3a 5 -cyclo-2 4-no r-5a-cho lane 23-tosylate.
p-Toluenesulfonyl chloride (0.95 g, 5.0 rm 1) was added to a solution of the 23-alcohol obtained in above (0.85 g 2.36 imol) in dry pyxidize (10 mQ.. The reaction mixture was kept at 0* 0 C for 20 h, then poured into ice-cold saturated aqueous sodium bicarbonate (25 mQ., extracted with ethyl Mass spectrum: m/e (relative intensity), 328 14), 296 281 253 159 135 145 ‘H-NHR 31 acetate (3 x 100 mL), washed with water (3 x 40 mL), saturated sodium chloride (2 x 40 mL), dried over anhydrous magnesium sulfate, filtered and evaporated to dryness under reduced pressure to give the 23-tosylate (1.15 g) in ca. 95% yield.
Mass spectrum: m/e (relative intensity) 514 (M 42), 499 482 (100), 468 459 456 361 255 (29).
60-Methoxy-3,5-cyclo-24-nor-5a-cholane (compound 1, R=Et).
To the above tosylate (1.15 g, 2.08 =mol, Rf 0.7 in ethyl acetate-hexane) in anhydrous ether (100 mL) was added LiAlH 4 (0.12 g, 3 miol). The reaction mixture was refluxed h, cooled and excess reagent was decomposed by saturated sodium chloride solution. The mixture was filtered, the phases were separated and the aqueous fraction was extracted o with ether (2 x 100 mL). The ether extracts were combined, washed with water (1 x 50 mL), saturated sodium chloride (2 .x 4 II 50 mL), dried over anhydrous magnesium sulfate, evaporated to dryness under reduced pressure and then dried under vacuum to give the desired 24-nor-cholane derivative (compound 1, R=Et) (0.59 g, Rf 0.64 in 10% ethyl acetate-hexane) in ca. 82% 2 yield. Mass spectrum: m/e (relative intensity) 344 (M 9), 329 312 289 286 255 191 69 1 H-NMR (CDC1 6 0.44 0.65 0.72 (s, 18-H), 0.82 J=7.5 Hz, 23-H), 0.9 J=7.0 Hz, 21-H), 1.02 19-H), 2.77 sharp, 3.32 6-OCH).
S*Examole 6 Preparation of Starting Material, Compound 1 (R=propyl) Treatment of the 23-tosylate obtained in Example above, with sodium cyanide under conditions analogous to those described in Example provides 6 -methoxy-3a,5-cyclo-5a-cholan-24-nitrile. By reduction of this nitrile, using the procedure of Example 5(b) above, there is obtained 60-methoxy-3a,5-cyclo-5a-cholan-24-ol. This A solution of 8 3 (3 mng) in glacial acetic acid nl) was heated to 55*C under nitrogen for 20 mini, cooled and poured over ice cold sodium bicarbonate solution (15 niL) 32 alcohol is converted to the corresponding 24-tosylate derivative by a procedure analogous to that described in Example 5(c) above, and the 24-tosylate. subjected to hydride reduction as described in Example 5(d) above, then provides the desired compound, 6P -metho xy-3a. 5-cyclo-5a-cho lane (compound 1, where Rpropyl).
00 0 0 0 0 00 00. 0 0 00 00 0 The residue was purified by HPLC on a Zorbax-Sil analytical column (4.6 mm x 25 cm) using 5% isopropanol-hexane at 265 nm to give 9a (X IX 2H). High resolution mass 0* V 33 Process Scheme I AcO0 X0 OCH 3 09 00 0 o 0 00 0 000 #0 #0 00 0 ~M e~O 0 #0 00 4 00~ 0 6000 6 X=Tcsyl 0 41 4 4 4 #0 0 #0 4 4 0 0 40 4#0I0~ 444640 0 0 #0 4 4 00 0 #4 04 0 O 4 0 #0
C
*OX
2
R=CH
3
R=CH
2
CH
3
R=CH
2
CH
2
H
3 10C
R=CH
3
R=CH
2
H
£C R=CH?%CH CH 3

Claims (1)

19-H), 2.04 3-OCOCH 3 4.71 5.4 5. 3~-Hd rxy-2-nochoa-5,7-dene(4) (R-Et) 0 -4 34 THE CLAIMS DEFINING THE INVENTION ARE~ AS FOLLOWS: 1. A compound having the formula: X() where in or acyl. R is methyl, ethyl or propyl and wherein X is hydrogen 2. 00 0 0 3. *or acetyl. 4444 P 4 0 DAT Wis 0 A compound according to claim 1 wherein A compound according to claim 1 wherein 3P-Acetoxy-23 ,24-dinorchola-5, 7-diene. 3P-Hydroxy-23 ,24-dinorchola-5, 7-diene. R is methyl. is hydrogen ED this 21st day of November 1989. CONSIN ALUMNI RESEARCH FOUNDATION 0 0 0 0 4 WATERMARK PATENT TRADEMAARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN. VIC. 3122. V.

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