CHEMICAL SYNTHESIS OF SQUALAMINE

Background of the Invention

Sgualamlne is a novel aminosterol recently isolated from the dogfish shark _/ Sgualus acanthias (K. Moore, et al., Proc. Nat. Acad. Sci. USA £0:1354-1358,1993. This water soluble steroid exhibits potent bactericidal activity against both Gram-positive and Gram-negative bacteria. In addition, sgualamlne is fungicidal and exhibits lytic activity against protozoa. The molecule was initially recovered as a natural product through extraction of several tissues of the Dogfish shark, including stomach, liver, gallbladder, and spleen. Its structure was determined by fast atom bombardment mass spectroscopy and NMR. The chemical structure of sgualamlne is that of a cationic steroid characterized by a condensation of an anionic bile salt intermediate with spermidine. A review of the chen\ical database revealed that sgualamlne represented a new chemical entity, and a member of a novel class of antibiotics.

Summary of the Invention

The present invention provides a method of preparing sgualamlne which comprises alkylating the 3-position of a 3-oxo-7α-hydroxy-24S-protecting group ether-5α-cholestane with a spermidino moiety to form a 3β-spermidino-7α-hydroxy-24.τ-protecting group ether-5α-cholestane; deprotecting the 24^-position of the 3β-spermidino-7α-hydroxy-24i-protecting group ether-5-x-cholestane to the free hydroxyl; and sulfating the 24Ϊ-position of the 3β-spermidino-7α, 244-

-dihydroxy-5α-cholestane. The method can optionally include protecting the 7α-hydroxy group and the spermidino amino groups during sulfating of the 24-position.

Preferably, formation of the 3β-spermidino-7α-hydroxy-24'-protecting group ether-5σC-cholestane comprises the steps of forming a 3β-amino-7α-hydroxγ-24$-protecting group ether-5α-cholestane from the 3-oxo-7α-hydroxy-24$-protecting group ether-5α-cholestane; alkylating the 3β-amino-7α-hydroxy-24C -protecting group ether-5α-cholestane with a spermidino precursor moiety at the 3-position; and reducing the spermidino precursor moiety to form a 3β-spermidino-7α-hydroxy-24(-protecting group ether-5α-cholestane.

Brlef Description of the Drawings

Figure la - If illustrate the pathway used for the chemical synthesis of sgualamlne described in Example 1.

Figure 2 illustrates the synthesis of 3-oxo-7α-hydroxy-24 £tert-butyldimethylsiloxy-5α-cholestane (compound 13) from chenodeoxycholic acid described in Example 2.

Figure 3 illustrates the synthesis of compound 13 from chenodeoxycholic acid described in Example 3.

Figures 4a and 4b illustrate the synthesis of compound 13 from fucosterol acetate described in Example 4 and the synthesis of sgualamlne.

Figure 5 illustrates the synthesis of compound 13 from dehydroepiandrosterone acetate described in Example 5.

Figure 6 illustrates the synthesis of compound 13 through microbial conversion of 3,

24-diketocholestane-24f-ethylenedioxyketal to the 7σ-hydroxy derivative described in Example 6.

Figure 7 illustrates the synthesis of compound 13 from pregnenolone acetate described in Example 7.

Detailed Description of Preferred Embodiments

A principal aspect of the invention provides a method for synthesizing squalamine which comprises:

modifying the 3-position of a 3-oxo-7α-hydroxy-24"ζ -ether protected alcohol group-5α-cholestane with a spermidino moiety to form a 3β-spermidino-7α-hydroxy-protecting group ether-5α-cholestane; deprotecting the 24*-position of the 3β-spermidino-7α-hydroxy-24 -ether protected alcohol group -5α-cholestane to the free hydroxyl; and sulfating the 24 > -position of the 3β-spermidino-7α, 24v-dihydroxy-5α-cholestane. This can further comprise the step of protecting the 7α-hydroxy group and the spermidino amino groups during sulfation of the 24 -position and deprotecting such positions after sulfation.

In a preferred embodiment of this aspect, formation of the 3β-spermidino-7α-hydroxy-24ζ-ether protected alcohol group -5α-cholestane comprises the steps of: forming a 3β-amino-7α-hydroxy-24Ϊ-ether protected alcohol group -5α-cholestane from the 3-oxo-7α-hydroxy-.24.T -ether protected alcohol group-5α-cholestane; alkylating the 3β-amino-7σ-hydroxy-24 -ether protected alcohol group-5α-cholestane with a spermidino precursor moiety at the 3-position; and reducing the spermidino precursor moiety to form a 3β-spermidino-7of-hydroxy-24 C -ether protected alcohol group -5α-cholestane.

The 3-oxo-7α-hydroxy-24 » -ether protected alcohol group -5α-cholestane can be formed from 3β-hydroxy

5-cholenic acid. Preferably, this comprises the steps of protecting the hydroxyl moieties of 3β-hydroxy-5-cholenic acid to form a 3β-protecting group ether-24 -protecting group ester of-5-cholenic acid;

homologating the 24* -protected position to the 3β,24 ^~ ζ -diprotected cholesterol;

allylically oxidizing the 7-position of the diprotected cholesterol;

reducing the cholestene and C ₇ carbonyl groups;

deprotecting the 3-position to form 3β,7α-24 -trihydroxy cholestane 24ζ -ether protected alcohol; and

oxidizing to the 3-keto form.

In another preferred embodiment of this aspect, forming the 3-oxo-7α-hydroxy-24 i -ether protected alcohol group -5α-cholestane comprises the steps of: oxidizing 3-acetoxychenodeoxycholic acid into the- 1,4-dienone; reducing to 3-keto-7α-hydroxy-5α-cholanic acid; protecting the C ₃ keto and 7-hydroxy functionalities; side-chain homologating; and deprotecting yielding 3-keto-7α,24 dihydroxy-5α-cholestane 24-ether protected alcohol.

Alternatively, this can be achieved by oxidizing chenodeoxycholic acid to the 3-one-4-ene and reducing the double bond to 3-keto-7α-hydroxy-5α-cholanic acid.

The following detailed description identifies compounds named in the examples by their reference numbers and should be read in conjunction with the drawings.

In accordance with the principal aspect of the invention, sgualamlne (25) is prepared from its cognate sodium sulfate salt (24) or other suitable hydrolyzable alkali metal salts by acid hydrolysis in the presence of a Cχ-C_» alkyl alcohol or other water soluble organic solvent. This final step consists primarily in neutralization of the salt form of the title compound.

The cognate alkali metal salt is prepared from the pyridinium sulfate (23) by mixture with a Cα.-C _* alkyl alcohol and a strong alkali metal base such as an alkali metal hydroxide, e.g., NaOH, under conditions where the pyridine moiety is cleaved to form the sulfate salt with the metal ion of the alkali metal base and pyridinium halide by¬ products are formed. Appropriate solvents for this step are also the C _x -C« alcohols.

The pyridinium intermediate (23) is formed by sulfur trioxide pyridine sulfation of compound (22) with anhydrous pyridine. Other sulfating agents that could be used include chlorosulfonic acid, trimethylsilyl chylchlorosulfonic acid, sulfur trioxide trimethylamine complex and sulfur trioxide triethylamine complex.

Compound (22) is formed by removing the carbobenzoxy (CBZ) protective groups used to protect the amine groups on the spermidine side chain. The CBZ protective groups are removed by catalytic hydrogenation using 10% palladium-charcoal and hydrogen gas. Also in this deprotection step, the tertiary butyldimethylsilyl (TBDMS)

is removed by acid cleavage using, for example, hydrochloric acid in ethanol. Other strong acids in suitable water soluble alcohol solvents can also be used. Compound (21) is prepared by acetylation of the hydroxyl at the C-, of the steroid ring nucleus. Acetylation of this site protects it from subsequent reactions in the synthesis. Other known acylating agents can also be used to provide the derived alkyl or aryl esters. Thus, the C _y hydroxyl is protected from sulfation at the time that the C ₂ _» carbon is sulfated during preparation of compound (23).

Compound (20) is prepared by protection with CBZ. Other protecting groups which could be used for protection of these amino sites on the side chain are CBZ derivatives or sulfonylating agents, e.g., p-toluenesulfonyl chloride or p-bromophenylsulfonyl chloride. Compound (19) is prepared by treating compound (18) with lithium aluminum hydride (LAH) in diethyl ether, which reduces the terminal cyano (C N) group to a primary amine. Other chemical reducing agents which could be used include, for example, alkali metal lithiohydride derivatives, as well as catalytic hydrogenation methods, such as use of rhodium plated on alumina. Compound (18) is prepared from compound (17) by cleavage of the tosyl protective group from the tosylamide to yield the free secondary amine. Other protecting groups which could be used in this step are N-carbobenzoxy and p-bromosulfonoxy.

Preparation of compound (17) from compounds (16) and (15) constitutes attachment of the spermidine side chain precursor by displacement of the iodine from N-3 (cyanopropyl)N-3-propyl iodide. The spermidine side chain precursor is attached, once the iodine has been displaced.

to the C ₃ β-amino group of the steroid nucleus using K ₂ C0 ₃ or any related alkali metal base.

Compound (16) is formed by a three-step reaction. The bromine of 3-cyanopropyl bromide is displaced by the amino group of 3-hydroxypropylamine, the thus formed hydrobromic acid being neutralized by K _a C0 _3/ to form the spermidine side chain precursor. Then, this spermidine side chain precursor is tosylated at the secondary amine and the hydroxyl moiety, such that the free secondary amine becomes a tosyl protected group and the hydroxyl becomes a tosyl ester. This protected intermediate is then reacted with sodium iodide or other suitable alkali metal halide to provide compound (16).

Compound (15) is formed from compound (14) by reductive formation of the 3β-amino group from the benzyloxyimino compound (14) using LAH as one of the appropriate reducing agents, others of which can be used are sodium borohydride and catalytic hydrogenation.

Compound (14) can also be obtained from compound (13) using alkoxyamines or other oxyimine groups as well as the oxime itself.

Compound (13) is prepared by an Oppenauer selective oxidation of compound (12) using aluminum trl-tert-butoxlde. Other aluminum alkoxides that could be used include aluminum tri-isopropoxide. Another ketone which can be used is acetone.

Compound (12) is prepared from compound (11) by the cleavage of the C ₃ acetoxy group using sodium cyanide in methanol. Other methods which could be used in this step

include sodium carbonate in methanol and lithium hydroxide in methanol.

Compound (11) is formed by reduction of the corresponding 7-one, compound (10), such as with K-selectride (potassium tri-sec-butylborohydride, Aldrich Chemical Co., Milwaukee, WI), which reduces the carbonyl stereoselectively and does not cleave the acetyl group at position 3 of compound 10. Compound 10 is formed by saturation of the 5-cholestene (compound 9) stereoselectively to the corresponding αβ-trans cholestane.

Compound (9) is made by allylic oxidation of the 5-cholestene (compound 8) at the 7-position using chromium hexacarbonyl and tert-butyl hydroperoxide. Other known oxidative methods for this transformation are: tert-butyl chomate; Jones reagent; N-bromosuccinimide, H _a O, Mn0 ₃ .

Compound (8) is formed by acetylation of compound (7). Compound (7) is made by deprotection of the C ₃ tetrahydropyranol ether using pyridinium paratoluene sulfonate, or other mild acids which do not cleave the C ₂ _» protective group. Compound (6) is formed by protecting compound (5) at the C _a * with tert-butyl-dimethysilyl chloride (TBDMS) to yield the derived C _a* TBDMS ether (6). Other trialkyl such as trimethyl and triethyl or diaryl alkyl, such as diphenylmethyl silyl groups.

Compound (5) is a Grignard addition of isopropyl magnesium halide, e.g., bromide, to the C _a* aldehyde (4). Compound (4) is formed by Swern oxidation of the C _a _» hydroxy (3) to the derived aldehyde. Compound (3) is formed by reduction of the C _a<» tetrahydropyranyl ester (2), e.g., using lithium aluminum hydride. Compound (2) is formed by

reaction of 5-cholenic acid-3β-ol (Steroids, Ltd., Chicago, IL) with 2, 3-dihydropyrane with pyridinium para-toluene sulfonate as a catalyst. Another protecting group that can be used for protection of the C ₃ and C _a „ groups is the tetrahydrofuranyl moiety.

Example 1

Chemical Synthesis of Sgualamlne

The title compound, 3β-N-l-[N(3-[4-aminobutyl])- l,3-diaminopropaneJ-7α, 24 -dihydroxy-5α-cholestane- 24-sulfate, which has been given the common name sgualamine, has now been chemically synthesized. The preferred method of chemical synthesis of the title compound is described in this example with reference to the synthesis pathway and numbered compounds illustrated in Figures la-lf as follows.

3fl-J 2-Tetrahydropyranoxy) -5-cholenlc acid tetrahydropyranyl ester (2)

To a stirred solution of 3β-hydroxy-5-cholenic acid (5-cholenic acid-3β-ol) (1) (100 g, 270 mmole) in anhydrous CH _a Cl ₂ (600 ml) was added pyridinium p-toluenesulfonate (PPTS) (3.40 g, 10 mmole) and 2,3-dihydropyran (67.3 g, 800 mmole) at room temperature. The reaction mixture was stirred for 8 hours, then it was diluted with CH _a Cl _a (600 ml), washed with saturated NaHC0 ₃ solution (2 x 50 ml), brine (1 x 50 ml), dried (MgSO«), filtered and concentrated in vacuo to get compound (2) (117.0 g, 80%) as a viscous oil. The crude compound was used in the step without further purification. ^X H NMR (400 MHz, CDCl ₃ )δ: 0.68 (3H, s, 18-CH), 0.94 (3H, d, 21-CH ₃ ), 1.01 (3H, s, 19-CH ₃ ), 2.36 (2H, m, CH _a CO _a ), 3.50 (2H, m, OCH _a ), 3.68 (IH, m, 3α-H) , 3.90 (2H, m, OCH _a ), 4.72 (IH, t, OCHCH _a ), 5.35 (IH, t, C=CH), and 5.96 (IH, t, OCHCH _a ).

3β-(2-Tetrahydropyranoxy)-5-cholen-24-ol (3)

To a stirred suspension of lithium aluminium hydride (8.20 g, 216 mmole) in anhydrous tetrahydrofuran (500 ml) was added dropwise a solution of compound (2) (117 g, 216 mmole) in anhydrous tetrahydrofuran (200 ml) at 0°C (ice bath). Then the reaction mixture was stirred at room temperature for 2 hours and decomposed by pouring onto the crushed ice and dilute hydrochloric acid. The agueous solution was extracted with ether (3 x 500 ml). The combined ether extracts were washed with brine (1 x 70 ml), dried (MgSO _« ), filtered and concentrated in vacuo to get almost pure compound (3) (81.6 g, 85%). ^H NMR (400 MHz, CDCl ₃ )ό: 0.69 (3H, s, 18-CH ₃ ), 0.96 (3H, d, 21-CH ₃ ), 1.01 (3H, merged s, 19-CH ₃ ), 3.50 (2H, m, OCH _a ) , 3.61 (2H, merged t, 24-CH _a ), 3.93 (IH, m, 3α-H), 4.72 (IH, m, OCHO) , 5.35 (IH, t, C=CH-CH _a ). The compound was used in the next reaction step.

3fl-(2-Tetrahydropyranoxy)-5-cholen-24-al (4)

To a stirred solution of anhydrous dimethylsulfoxide (DMSO) (51 ml, 720 mmole) in anhydrous CH _a Cl _a (400 ml) was added dropwise a solution of oxalyl chloride (31.4 ml, 360 mmole) in anhydrous CH _a Cl _a (200 ml) at -78°C under argon. After stirring the mixture for 20 minutes at the same temperature, a solution of compound (3) (81.6 g, 183 mmole) in anhydrous CH ₂ Cl _a (200 ml) was added dropwise. The reaction mixture was stirred for 1 hour at -78°C, and then anhydrous triethylamine (100 ml, 717 mmole) was added slowly. The mixture was allowed to warm to room temperature, and quenched with saturated NH C1 solution. The organic phase was separated from the agueous, and agueous phase was extracted with CH ₂ Cla (2 x 500 ml).

The combined organic extracts were washed with saturated NaHC0 ₃ solution (2 x 75 ml), brine (1 x 70 ml), dried (MgSO _* ), filtered and concentrated ^n vacuo to get crude compound (4) (40.1 g, 49%). The compound was used in the next step without further purification. ^X H NMR (200 MHz, CDC1 ₃ ) δ: 0-68 (3H, s, 18-CH ₃ ), 0.91 (3H, d, 21-CH ₃ ), 1.00 (3H, s, 19-CH ₃ ), 2.36 (2H, m, 23-CH _a ), 3.49 (2H, m, OCH _a ) , 3.88 (IH, m, 3α-H) , 4.70 (IH, m, 0CHCH _a ), 5.32 (IH, m, C=CH), 9.75 (IH, t, 24-CHO). CIMS (m/e): 443 (M*+l, 1%), 425 (1.2%), 383 (1.1%), 375 (3%), 357 (11%), 341 (100%), 323 (10%), 273 (4%), 255 (4%).

3β-(2-Tetrahydropyranoxy)-5-choleatene-24 -ol (5)

To a stirred solution of compound (4) (40.0 g, 90 mmole) in anhydrous tetrahydrofuran (500 ml) was added a solution of isopropylmagnesium bromide in tetrahydrofuran (2M in THF, 135 ml, 270 mmole) dropwise at 0°C (ice bath). After complete addition, the reaction mixture was stirred at room temperature for 4 hours and then the reaction was guenched by slow addition of 2N HC1 solution. The agueous solution was extracted with ether (3 x 400 ml). The combined ether extracts were washed with water (2 x 100 ml), brine (1 x 70 ml), dried (MgSO _* ), filtered and concentrated in vacuo to get crude product which was purified by flash chromatography on silica gel to get pure compound (4) (37.2 g, 85%). ^X H NMR (400 MHz, CDC1 ₃ ) δ: 0.69 (3H, s, 18~CH ₃ ), 1.02 (3H, s, 19-CH ₃ ), 3.32 (IH, m, 24δ-H) , 3.51 (2H, m, OCH _a ), 3.92 (IH, m, 3 _/ -H) , 4.72 (IH, t, OCHCH _a ), and 5.35 (IH, t, C=CH-CH ₂ ).

3β-(2-Tetrahvdropyranoxy)-24_ -tert-butyldlmethylsiloxy-5-ch- olestene (6)

A solution of compound (5) (37.2 g, 76.5 mmole), tert-butyldimethylsllyl chloride (17.3 g, 114.8 mmole) and imidazole (39.0 g, 572.8 mmole) in anhydrous CH ₂ C1 ₂ (500 ml) was stirred at room temperature for 14 hours. The reaction mixture was diluted with CH _a Cl _a (500 ml), washed with water (1 x 250 ml), 2N HCl (2 x 100 ml), brine (1 x 100 ml), dried (MgSO_ _» ), filtered and concentrated iji vacuo to get crude compound (6) (41.4 g, 90%). The crude compound was used in the next step without purification. ¹ H NMR (400 MHz, CDC1 ₃ ) δ: 0.00 & 0.01 (2 x 3H, two s, Si(CH ₃ ) _a ), 0.68 (3H, s, 18-CH ₃ ), 0.91 (9H merged s, SiC(CH ₃ ) ₃ ), 1.01 (3H, s, 19-CH ₃ ), 3.36 (IH, M, 24δ-H) , 3.52 (2H, m, OCH _a ) , 3.83 (IH, m, 3(α-H), 4.72 (IH, t, OCHCH _a ), and 5.36 (IH, t, C=CH-CH ₂ ) •

3fl-Hydroxy-24 -tert-butyldimethylBiloxy-5-cholestene (7) A solution of compound (6) (41.4 g, 68.8 mmole) and pyridinium p-toluenesulfonate (PPTS) (0.86 g, 3.42 mmole) in methanol (600 ml) was refluxed for 4 hours. The reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was dissolved in ether (800 ml), washed with 2N HCl (2 x 100 ml), brine (1 x 100 ml), dried (MgSO,), filtered and concentrated _, in vacuo to get compound (7) (30.2 g, 85%). The crude compound was used directly in the next step.

3β-Acetoxy-24h-tert-butyldimethylslloxy-5-cholestene (8)

A solution of compound (7) (30.0 g, 58 mmole), dry pyridine (200 ml, 2473 mmole) and acetic anhydride (30 ml, 318 mmole) was heated at 80°C (oil bath temperature) for 1 hour. Then the cold reaction mixture was poured into a crushed ice and saturated NaHC0 ₃ solution. The agueous solution was extracted with ether (3 x 300 ml). The combined ether extracts were washed with saturated NaHC0 ₃ solution (2 x 100 ml), water (2 x 150 ml), 2NHC1 (3 x 75 ml), brine (1 x 100 ml), dried (MgS0 ₄ ), filtered and concentrated ij vacuo to afford the crude product which was purified by flash chromatography on silica gel to give pure compound (8) (28.6 g, 88%). ^X H NMR (400 MHz, CDC1 ₃ ) δ: 0.01 (6H, br s, Si(CH ₃ ) _a ), 0.68 (3H, s, 18-CH ₃ ), 0.90 (9H, merged s, S1C(CH ₃ ) ₃ ), 1.02 (3H, s, 19-CH ₃ ), 3.37 (IH, m, 24^-H) , 4.60 (IH, m, 3(α-H), and 5.38 (IH, m, C=CH-CH _a ).

3β-Acβtoxy-24 -tert-butyldimethylBiloxy-5-choleatene-7-one

SH

A solution of compound (8) (28.0 g, 50.1 mmole), chromium hexacarbonyl (11.6 g, 52.7 mmole) and tert-butyl hydroperoxide (100 ml, 94 g, 1043 mmole) in anhydrous acetonitrlle (500 ml) was refluxed under argon for 12 hours. Acetonitrlle was removed JLn vacuo, and the residue was dissolved in ether (500 ml). The ether extract was washed with saturated NaHC0 ₃ (3 x 150 ml), brine (1 x 100 ml), dried (MgSO _* ), filtered and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel to give pure compound (9) (13.2 g, 46%). IR (neat): 1674 (C=CHCO), 1736 (OCOCH ₃ ) cm- ¹ ; ^: H NMR (400 MHz, CDC1 ₃ ), δ:0.00 & 0.01 (2 x 3H, two s, Si(CH ₃ ) _a ), 0.68 (3H, s, 18-CH ₃ ), 0.90 (9H, merged s,. SiC(CH ₃ ) ₃ ) , 1.20 (3H, s, 19-CH ₃ ), 2.04 (3H, s, OCOCH ₃ ), 3.38 (IH, m, 24^-H), 4.72 (IH, m, 3 -H), and 5.69 (IH, s, C=CH) .

3β-Acβtoxy-24 -tert-butyidimethylalloxy-5α-cholestane-7-one (10)

To a solution of compound (9) (13.0 g, 22.7 mmole) in dry ether (50 ml) was distilled in liguid ammonia (300 ml) at -78°C. Lithium (0.5 g, 72.1 mmole) was added in small portions until a blue coloration persisted for 10 minutes, after which the solution was guenched with solid NH _* C1 (50 g) . Ammonia was evaporated, and the resulting residue was partitioned between water (500 ml) and ether (300 ml). The aqueous solution was extracted further with ether (3 x 200 ml). The combined ether extracts were washed with brine (1 x 100 ml), dried (MgSO*), filtered and concentrated i i vacuo to get the crude product. Flash chromatography on silica gel of the crude product gave pure compound (10) (10.6 g, 81%). IR (neat): 1711 (CO), 1736 (OCOCH ₃ ) CM ^" - ¹ ; ^X H NMR (400 MHz, CDC1 ₃ ) δ:0.01 (6H, s, Si(CH ₃ ) ₃ ), 0.61 (3H, s, 18-CH ₃ ), 0.82 (3H, merged s, 19-CH ₃ ), 0.89 (9H, merged s, SiC(CH ₃ ) ₃ ), 2.00 (3H, s, OCOCH ₃ ), 3.35 (IH, m, 24 ^" f-H) , and 4.66 (IH, m, 3α-H).

3β-Acetoxy-7α-hydroxy-24 -tert-butyldimethylsiloxy-5α-chol- estane (11)

To a stirred solution of compound (10) (10.6 g, 18.4 mmole) in anhydrous tetrahydrofuran (300 ml) was added dropwise a solution of K-selectride (1.0 M in THF, 75.6 ml, 75.6 mmole) (Aldrich Chemical Co., Milwaukee, WI) at -50 ^e C. The reaction mixture was stirred at that temperature for 5 hours, and then guenched with 30% hydrogen peroxide solution (20 ml) and saturated NH_»C1. The agueous solution was extracted with ether (3 x 100 ml). The combined ether extracts were washed with saturated NaHC0 ₃ (2 x 70 ml), water (2 x 100 ml), brine (1 x 70 ml), dried (MgSO _* ), filtered and concentrated ^n vacuo to get the crude product. The product was purified by flash chromatography on silica gel to give pure compound (11) (8.5 g, 80%). IR (neat): 1732 (OCOCH ₃ ), 3456 (OH) cm-- ¹ ; ^X H NMR (400 MHz, CDC1 ₃ ) δ: 0.01, 0.02 (6H, two s, 2 x SiCH ₃ ), 0.63 (3H, s, 18-CH ₃ ), 0.80 (3H, s, 19-CH ₃ ), 0.88 (9H, s, SiC(CH ₃ ) ₃ ), 2.01 (3H, s, COCH ₃ ), 3.36 (IH, m, 24f-H) , 3.82 (IH, m, 7β-H) and 4.70 (IH, m, 3α-H).

3 ^β ,7α-Dihydroxy-24l-tert-butyldimethylBlloxy-5α-choles tane (12)

A solution of compound (11) (2.20 g, 3.81 mmole) and sodium cyanide (0.20 g, 4.08 mmole) in methanol (70 ml) was refluxed for 8 hours (monitored by tic) . After completion of reaction, methanol was evaporated ij vacuo, and the residue was extracted with CH _a Cl _a (3 x 30 ml). The combined CH _a Cl ₂ extracts were concentrated in vacuo to get a pure white solid compound (12) (1.85 g, 88%). ^X H NMR (400 MHz, CDC1 ₃ ) ₃ ) δ:0.00,0.01 (2 x 3H, two s, Si(CH ₃ ) ₃ ), 0.66 (3H, s, 18-CH ₃ ), 0.80 (3H, s, 19-CH ₃ ) ₃ ), 0.85 (IH, d, 21-CH ₃ ), 0.91 (9H, merged s, SiC(CH ₃ ) ₃ ), 3.36 (IH, m, 244-H) , 3.64 (IH, m, 3α-H), and 3.84 (IH, m, 7β-H) . CIMS (m/e): 534 [(M ^* +l)-l, 3.7%], 533 (8.6%), 499 (16.5%), 473 (20%), 385 (97%), 367 (100%), 301 (17%), and 273 (16%).

3-Oxo-7α-hydroxyr--22441>--1tert-butyldlmethylsiloxy- 5α-cholestane 1131

A solution of compound (12) (1.27 g, 2.37 mmole), aluminum tri-tert-butoxide (4.75 g, 19.28 mmole) and cyclohexanone (30 ml) in anhydrous toluene (90 ml) was stirred and heated to 120°C (oil bath) for 72 hours. The reaction mixture was then cooled to room temperature, diluted with benzene (100 ml) and acidified with 2N HCl (50 ml). The organic layer was then separated from the agueous layer. The agueous layer was extracted with benzene (3 x 50 ml). The combined organic extracts were washed with water (1 x 50 ml), saturated NaHC0 ₃ (2 x 50 ml), water (1 x 50 ml), brine (1 x 50 ml), dried (MgSO_»)/ filtered and evaporated iji vacuo to get the crude product along with cyclohexanone. Flash chromatography of the crude product using benzene followed by a gradient of EtOAc/hexane (5, 10, 20 and 40%) solvent system gave a pure white solid compound (13) (0.743 g, 59%). ^H NMR (400 MHz, CDC1 ₃ ) δ:0.00, 0.01 (2 x 3H, two s, Si(CH ₃ ) _a ), 0.66 (3H, s, 18-CH ₃ ), 0.82 (3H, merged d, 21CH ₃ ), 0.89 (9H, merged s, SiC(CH ₃ ) ₃ ), 0.99 (3H, s, 19-CH ₃ ), 2.31 (4H, m, CH _a COCH _a ), 3.36 (IH, m, 24if-H) , and 3.85 (IH, m, 7β-H) . CIMS (m/e): 533 (M ^* +I, 61%), 401 (34%), 383 (100%), 327 (3%), 299 (11%).

3-Benzyloxyimino-7α-hydroxy-24a-tert-butyldlmethylailoxy -5α- choleatane (14)

A solution of compound (13) (0.74 g, 1.39 mmole), benzyloxyamine hydrochloride (0.49 g, 3.07 mmole) and anhydrous pyridine (20 ml) in anhydrous ethanol (40 ml) was gently refluxed for 16 hours. Excess pyridine and ethanol was removed JLn vacuo, and the residue was dissolved in EtOAc (100 ml) and water (100 ml). The agueous layer was

separated, and the EtOAc layer was washed with 2N HCl (3 x 20 ml), water (2 x 20 ml), brine (1 x 20 ml), dried (MgSO..), filtered and evaporated Ij vacuo to get a white solid compound (14) (0.86g, 97%). ^H NMR (400 MHz, CDC1 ₃ ) δ:0.00, 0.01 (2 x 3H, two s, Si(CH ₃ ) _a ), 0.69 (3H, s, 18-CH ₃ ), 0.89 (9H, merged s, SiC(CH ₃ ) ₃ ), 2.98, 3.27 (m, due to syn and anti oxime next to α-CH _a Of A-ring), 3.37 (IH, m, 24 -H) , 3.86 (IH, m, 7β-H), 5.06 (2H, br s, OCH _a C ₆ H _s ), and 7.26-7.42 (5H, m, C ₆ H _S ): CIMS (m/e): 638 (M ^* +I, 100%), 512 (3.4%), 488 (13%), 380 (21%).

3β-Amlno-7α-hydroxy-24l--tert-butyldimethylBiloxy-5α-c hole- stane (15)

To a well stirred suspension of lithium aluminum hydride (LAH) (0.252 g, 6.66 mmole) in dry ether (100 ml) was added dropwise a solution of compound (14) (0.85 g, 1.33 mmole) in dry ether (50 ml) at room temperature. After complete addition, the reaction mixture was refluxed gently for 16 hours. The reaction mixture was cooled to 0°C (ice bath), then a solution of 2N NaOH was added dropwise until white solid granulates were formed. Ether was decanted and the residue further extracted with benzene (3 x 50 ml), and the combined organic extracts were dried (K _a C0 ₃ ), filtered and evaporated Iji vacuo to give compound (15) along with traces of benzyl alcohol as by-product (0.89 g total, quantitative yield) . The crude product was used as such in the next step. ¹ H NMR (400 MHz, CDC1 ₃ ) δ: 0.01, 0.02 (2 x 3H, two s, Si(CH ₃ ) _a ), 0.66 (3H, S, 18-CH ₃ ), 0.79 (3H, s, 19-CH ₃ ), 0.91 (9H, merged s, SiC(CH ₃ ) ₃ ), 2.69 (IH, m, 3α-H), 3.39 (IH, m, 24$-H), and 3.84 (IH, m, 7β-H) . CIMS (m/e): 534 (M ^* +l, 60%), 518 (14%), 476 (7%), 384 (100%), 367 (6.3%), 300 (5%).

N-(3-Cyanopropyl)-N-(3-iodopropyl)p-toluenesulfonylamine 1161

To the stirred and refluxed mixture of 3-amino-l-propanol (3.0 g, 39.94 mmole), anhydrous potassium carbonate (6.10 g, 44.16 mmole) and anhydrous sodium iodide (0.60 g, 4.00 mmole) in anhydrous acetonitrlle (150 ml) was added a solution of 3-cyanopropyl bromide (4-bromobutyronitrile) (5.91 g, 39.67 mmole) in anhydrous acetonitrlle (50 ml) dropwise over a period of 6 hours. After complete addition, reflux of the reaction mixture was continued for 20 hours. The cold reaction mixture was concentrated ^n vacuo, then the residue was extracted with CHC1 ₃ (3 x 60 ml). Evaporation of CHC1 ₃ afforded almost pure colorless oil N-(3-cyanopropyl)-N-(3-hydroxypropyl)amine (5.65 g, 100%). ¹ H NMR (400 MHz, CDC1 ₃ ) δ: 1.71 (2H, m, CH _a ) , 1.83 (2H, m, CH _a ), 2.44 (2H, t, CH _a CN), 2.58 (IH, m, NH) , 2.77 (2H, t, CH _a N), 2.89 (2H, t, CH _a N) , 3.80 (2H, t, CH _a 0). This compound (5.60 g, 39.38 mmole) was N,0-bis-tosylated by treating with p-toluenesulfonyl chloride (18.4 g, 96.51 mmole), anhydrous triethylamine (19.6 g, 193.71 mmole) and

N,N-dimethylaminopyridine (DMAP) (2.35 g, 19.23 mmole) in dry CH _a Cl _a at 0°C for 2 hours followed by 0°C to room temperature for 16 hours. After complete reaction, CH _a Cl ₂ was removed in vacuo and then the residue was dissolved in EtOAc (150 ml), washed with water (2 x 50 ml), 2N HCl (3 x 50 ml), water (1 x 50 ml), saturated NaHC0 ₃ (2 x 50 ml), brine (1 x 50 ml), dried (MgSO«), filtered and evaporated in vacuo to afford almost pure solid N-(3-cyanopropyl)-N-(3- toluenesulfonyl-oxypropyl)p-toluenesulfonylamine (14.55 g, 82%). ^X H NMR (400 MHz, CDC1 ₃ ) δ: 1.87-2.01 (4H, m, 2 x CH ₂ ) , 2.42 (2H, t, CH _a ,CN), 2.46 (3H, s, CH ₃ Ar) , 2.48 (3H, s, CH ₃ Ar), 3.15 (4H, m, 2 x CH _a N) , 4.08 (2H, t, CH _a 0), 7.33, 7.38, 7.67, 7.79 (4 x 2H, four d, 2 x - C _β H _* -). Without

further purification, the crude N,0-bis-tosylated compound (14.50 g, 32.18 mmole) was treated with anhydrous sodium iodide (9.65 g, 64.38 mmole) in refluxing anhydrous acetone (250 ml) for 16 hours. Acetone was removed by evaporating in vacuo, and the residue was extracted with CHC1 ₃ (3 x 70 ml). The combined CHC1 ₃ extracts were evaporated iji vacuo to afford a pale yellow solid compound (12.95 g, 99%.). Flash chromatography on silica gel of the crude compound using EtOAc/hexane (4:6) as eluant gave pure N-(3-cyanopropyl)-N-(3-iodopropyl)p-toluenesulfonylamine (16) (11.62 g, 89%) as crystalline solid. IR (neat) 1338, 1454, (SO _a ), 1597 (aromatic), 2245 (CN) cm ^-1 . ^X H NMR (400 MHz, CDC1 ₃ ) δ: 1.95 (2H, m, CH _a ), 2.05 (2H, m, CH _a ), 2.43 (3H, merged s, CH ₃ Ar), 2.44 (2H, merged t, CH _a CN), 3.13-3.20 (6H, m, 2 x CH _a N x CH _a I,), 7.32, 7.68 (2 x 2H, two d, -CH*-). ¹³ C NMR (100 MHz, CDC1 ₃ ) δ: 1.88, 14.52, 21.52, 25.11, 32.52, 47.96, 49.90, 118.82, 127.15, 129.87, 135.59, and 143.79. CIMS (m/e): 407 (M ^* +I, 100%), 315 (5.7%), 279 (7.6%), 251 (7.3%), 135 (11%).

3fl-N-l-[N(3-Cyanopropyl)-N(p-toluβneaulfonyl)-1,3-diami nopr- opanβ1-7α-hydroxy-24^-tert-butyldimethylalloxy-5α- choleatane (17)

To a stirred and refluxed mixture of compound (15) (0.70 g, 1.31 mmole) and anhydrous potassium carbonate (0.20 g, 1.45 mmole) in anhydrous acetonitrlle (200 ml) was added a solution of N-(3-cyanopropyl)-N-(3-iodopropyl)p- toluenesulfonylamine (16) (0.54 g, 1.33 mmole) in anhydrous acetonitrlle (50 ml) dropwise over a period of 6 hours. After complete addition, the reaction mixture was continued to reflux for 16 hours. The cold reaction mixture was concentrated in vacuo, and the residue was extracted with CHC1 ₃ (3 x 60 ml). The combined CHC1 ₃ extracts were evaporated iji vacuo to get almost pure compound (17) (1.06

g, 100%) as viscous oil. ^X H NMR (400 MHz, CDC1 ₃ ) δ : 0.02, 0.03 (2 x 3H, two s, Si(CH ₃ ) ₃ )/ 0.66 (3H, s, 18-CH ₃ ), 0.78 (3H, s, 19-CH ₃ ), 0.90 (9H, merged s, SiC(CH ₃ ) ₃ ), 1.96 (4H, merged m, 2 x CH _a ), 2.42 (4H, merged m, CH ₂ N x CH ₂ CN) , 2.43 (3H, merged s, CH ₃ Ph), 2.62 (IH, m, 3α-H), 3.18 (4H, m, 2 x CH _a N), 3.38 (IH, m, 24 -H) , 3.84 (IH, m, 7β-H) , 7.32, 7.19 (2 x 2H, two d, ArH). CIMS (m/e): 813 (M*+I, 44%), 754 (32%), 656 (52%), 575 (48%), 547 (54%), 418 (43%), 384 (26%), 334 (35%), 296 (55%), 251 (60%), 239 (87%), 155 (100%).

3A-N-1-TN(3-Cyanopropyl)-1,3-diaminopropane ]-7α-hydroxy- 24S-tertbutyldimethylailoxy-5α-choleatane (18)

To a solution of compound (17) (1.0 g, 1.23 mmole) in dry tetrahydrofuran (THF) (20 ml) was distilled liquid ammonia (80 ml) at -78 ^P C. Sodium (1.5g, 65.25 r.nole) was added in small portions until a blue coloration persisted for 1 hour, and the reaction was allowed to warm to room temperature overnight. After 16 hours, the reaction mixture was guenched with ethanol. THF and ethanol from the reaction mixture were removed by evaporation iri vacuo, and the residue was extracted with benzene (3 x 50 ml), dried (K _a C0 ₃ ), filtered and evaporated .in vacuo to get compound (18) (0.74 g, 91%) as viscous oil. ^X H NMR (400 MHz, CDC1 ₃ ) δ: 0.01, 0.02 (2 x 3H, two s, Si(CH ₃ ) _a ), 0.66 (3H, s, 18-CH ₃ ), 0.79 (3H, s, 19-CH ₃ ), 0.91 (9H, merged s, SiC(CH ₃ ) ₃ ), 2.41-2.74 (9H, m, CH _a CN, 3 x CH _a N & 3αH) , 3.37 (IH, m, 24 ^* f-H) and 3.83 (IH, m, 7β-H) . This crude product was used in the next step without further purification.

3β-N-l- Nr3-(4-Aminobutyl) 1-1,3-diamlnopropane - 7α-hydroxy-24l-tertbutyldlmethylalloxy-5α-choleBtane (19)

To a stirred suspension of lithium aluminium hydride (1.0 g, 26.35 mmole) in anhydrous ether (150 ml) was added a solution of compound (18) (0.72 g, 1.09 mmole) in anhydrous ether (50 ml) dropwise at room temperature over a period of 1 hour. After completion of the addition, the reaction mixture was refluxed for 5 hours. Then at 0°C (ice-bath) the reaction mixture was treated with a solution of 2N NaOH until white solid granulates were formed. Ether was decanted and the residue further extracted with benzene (3 x 60 ml), and the combined organic extracts were dried (KOH and K _a C0 ₃ ), filtered and evaporated in vacuo to get compound (19) (0.67 g, 93%) as colorless viscous oil. ^X H NMR (400 MHz, CDC1 ₃ ) δ: 0.00, 0.01 (2 x 3H, two s, Si(CH ₃ ) ₃ ), 0.66 (3H, s, 18-CH ₃ ), 0.81 (3H, merged s, 19-CH ₃ ), 0.91 (9H, merged s, SiC(CH ₃ ) ₃ ), 2.38-2.78 (9H, m, 4 x CH _a N & 3α-H), 3.38 (IH, m, 24 -H) , and 3.84 (IH, m, 7β-H) . CIMS (m/e): 662 (M ^* +I, 31%), 604 (16%), 560 (13%), 548 (100%), 530 (31%), 432 (25%), 410 (28%), 384 (12%), 321 (8%), 188 (10%), 144 (29%). Compound (19) was used in the next step without further purification.

3A-N-l-{Nf3-(4-Benzyloxycarbonyl)amlnobutyl) 1-1,3- (dibenzyloxycarbonyl)diaminopropane> -7-α-hydroxy-24 ^' f-tert-butyldlmβthylBiloxy-5α-choleatane (20)

To a cold (5°C) stirred solution of compound (19) (0.67 g, 1.01 mmole) in tetrahydrofuran (20 ml) was added separately but simultaneously a solution of benzyloxycarbonyl chloride (benzyl chloroformate) (CBZ CI) (1.31 g, 7.70 mmole) in THF (30 ml) and 2N NaOH in 30 minutes. After complete addition, the reaction mixture was allowed to warm to room

temperature, and stirred overnight. After 16 hours, THF was removed JLn vacuo from the reaction mixture, and the agueous layer was extracted with benzene (3 x 50 ml). The combined benzene extracts were dried (K _a C0 ₃ ), filtered and evaporated in vacuo to get compound (20) (1.06 g, 99%) as a colorless viscous oil. ^X H NMR (400 MHz, CDC1 ₃ ) δ:0.01 (6H, merged two s, Si(CH ₃ ) ₃ ), 0.68 (3H, s, 18-CH ₃ ), 0.87 (3H, merged s, 19-CH ₃ ), 0.91 (9H, merged s, SiC(CH ₃ ) ₃ ), 2.94-3.58 (9H, m, 4 x CH _a N & 3α-H), 3.48 (IH, merged m, 24 ^" f-H) , 3.81 (IH, m, 7β-H), 5.10 (6H, br s, 3 x OCH _a Ph) , and 7.20-7.60 (15H, m, 3 X C ₆ H _S ). CIMS (m/e): 1064 (M~+I, 0.1%), 1017 (0.3%), 926 (2%), 518 (10%), 367 (13%), 225 (11%), 181 (23%), 163 (100%). This compound (20) was used in next step without further purification.

3β-N-{N-T3-(4-Benzyloxycarbonyl)aminobutyl1-1,3- (dlbenzyloxycarbonyl)diamlnopropane}-7a-acetoxy-24r-tert- butyldlmethylalloxy-Sα-choleatane (21)

To a stirred solution of compound (20) (1.0 g, 0.94 mmole) and 4-dimethylaminopyridine (DMAP) (1.20 g, 9.82 mmole) in anhydrous CH _a Cl _a (10 ml) was added acetic anhydride (Ac ₂ 0) (0.78 g, 7.61 mmole) at room temperature. The reaction mixture was monitored by tic. After 4 hours, methanol was added to the reaction mixture, then the organic solvents were evaporated iri vacuo to get oily residue. The residue was dissolved in EtOAc (100 ml), washed with 2NHC1 (3 x 25 ml), water (1 x 50 ml), saturated NaHC0 ₃ (3 x 25 ml), brine (1 x 25 ml), dried (MgSO«), filtered and evaporated in vacuo to give compound (21) (0.89 g, 86%) as a viscous oil. ^X H NMR (400 MHz, CDC1 ₃ ) δ: 0.02, 0.03 (2 x 3H, two s, Si(CH ₃ ) _a ), 0.66 (3H, s, 18-CH ₃ ), 0.87 (3H, merged s, 19-CH ₃ ), 0.90 (9H, merged s, SiC(CH ₃ ) ₃ ), 2.11 (3H, merged s, OCOCH ₃ ), 2.92-3.58 (9H, m, 4 x CH _a N & 3α-H), 3.38 (IH, merged m, 24 -H) , 4.88 (IH, m, 7β-H), 5.11 (6H, br s, 3 x CH _a Ph), 7.21-7.44 (15H, m, 3 x C _e H _s ). CIMS (m/e): 1107 (M ^* +l, 0.2%), 1059 (0.2%), 984 (0.5%), 968 (2%), 650 (5%), 519 (7%), 410 (8%),223 (44%),163 (100%).

3fl-N-l-{N-T3-(4-Aminobutyl) 1-1,3-diaminopropane-7α-acetoxy- -24 ^" f-hydroχy-5α-cholβatane trlhydrochloride (22)

A mixture of compound (21) (0.46 g, 0.42 mmole), 10% palladium-charcoal (0.2g) and anhydrous ethanol saturated with HCl gas (3.0 ml) in anhydrous ethanol (30 ml) was stirred in hydrogen atmosphere (filled in a balloon) at room temperature for 20 hours. The reaction mixture was filtered through celite using a sintered glass funnel and the residue was washed with ethanol (3 x 40 ml). Combined filtrates were evaporated in vacuo to get a hygroscopic solid compound (22) (0.26 g, 90%). ^X H NMR (400 MHz, MeOH-«) 6:0 (3H, s, 18-CH ₃ ), 1.96 (3H, merged s, C0CH ₃ ), 2.95-3.85 (m, spermidine chain), 3.31 (IH, merged s, 241-H) , and 4.90 (IH, m, 7β-H).This compound was used in the next step without further purification.

3fl-N-l-{Nf3-(4-Aminobutyl) 1-1,3-diamlnopropanel -7α-acetoxy-24*-hydroxy-5ct-cholβatane 24-pyridlum aulfate trlhydrochloride (23)

A solution of compound (22) (0.137 g, 0.196 mmole) and sulfur trioxide pyridine complex (0.187 g, 1.175 mmole) in anhydrous pyridine (2 ml) was heated at 60-80°C (oil bath temperature) for 2 hours. Then pyridine was removed in vacuo to get a crude 24-sulfated compound (23) along with an excess of sulfur trioxide pyridine complex. The crude product was used directly without isolation in the next step.

3β-N-l- Nr3-(4-Aminobutyl) 1-l,3-diaminopropane -7α, 24f-di- hydroxy-5α-choleatane 24-sodium aulfate (24).

The crude compound (23) from the previous reaction was dissolved in methanol (15 ml) and to that solution was added 2N NaOH solution (10 ml). Then the reaction mixture was heated at 80°C (oil bath temperature) for 20 hours. The cold reaction mixture which contained compound (24) was taken in the next step without isolation.

3fl-N-l--fNT3-(4-Aminobutyl) }-1,3-dlaminoproρane}-7α,24&- dlhydroxy-5α-choleatane 24-aulfate trlhydrochloride (Sgualamlne) (25) .

The above reaction mixture containing compound (24) was acidified to pH 1 with cold 2NHC1 (20 ml) and then water removed Iji vacuo. The light grey solid compound obtained was dried in a vacuum desiccator at room temperature to provide crude sgualamlne hydrochloride (25) which was purified by reversed phase liguid chromatography. ^X H NMR (400 MHz, MeOH-d*) δ:0.60 (3H, s, 18-CH ₃ ), 0.80 (3H, merged s, 19-CH ₃ ), 2.88-3.57 (m, spermidine chain), 3.72 (IH, m, 7β-H), 4.08 (IH, m, 24 -H) . ^X H NMR (400 MHz, DMSO-d ₆ ) δ: 0.51 (3H, s, 18-CH ₃ ), 0.68 (3H, merged s, 19-CH ₃ ), 2.61-3.25 (m, spermidine chain), 3.55 (IH, m, 7β-H) and 3.80 (IH, m, 24 ^" f-H) . This material was identical with an authentic sample of squalamine.

Considerable flexibility appears to exist in the selection of the starting sterodial compound onto which the sulfated side chain and the spermidine moiety must be condensed. The critical intermediate in the pathway. Compound 13, characterized by the 3-keto group, trans AB ring structure, and 7α-OH group, can be reached by several

routes. To accommodate the different starting steroids, additional variations in the choice of the chemical blocking groups reguired along the route are also introduced.

Example 2 Syntheaia of Compound 13 from Chenodeoxycholic Acid

The following description is illustrated by Figure 2. In this synthesis chenodeoxycholic acid (26) is diacetylated at the two secondary hydroxyl groups to yield diacetate (27) which is selectively hydrolyzed to (28) which in turn is oxidized to dienone (29) following the procedure of K. Ochi, I. Matsunga, M. Shindo, and C. Kaneko, J.Chem.Soc, Perkin Trans. , 1979, 161. Lithium in ammonia reduction of (29) according to the procedure of A. Kallner, Acta Chem.Scand., 1967, ^:322 yields (30) which is protected at the C- ₇ -dL-hydroxyl group using methoxymethyl chloride to yield (31). Further reactions are formation of the C ₃ ketal (32), elongation of the side chain (COOH -CH(OH)-C(CH ₃ ) _a ) as was done in Example 1 and finally deprotection at C ₇ and protection at C _a<4 to yield (13).

Example 3 Alternate Synthesis of Compound 13 from Chenodeoxycholic Acid

Compound (33) formed by Oppenauer oxidation of chenodeoxycholic acid (26) is oxidized using selenium dioxide to the conjugate ketone (34) which is reduced using lithium in liguid ammonia to the saturated ketone (35). Selective oxidation of (35) using isopropyl alcohol, (CH ₃ ) ₃ PO and Ir(IV)Cl _* yields (36) following the procedure of A. Kallner, Acta Chem.Scand, 1967, 2_l ^s _322. ⁱ ^e compound referenced as (35) with respect to this example is the same molecule as (30) in Example 2. It is converted to (13) using the same steps as those of Example 2.

Example 4 Synthesis of Compound 13 from Fucosterol Acetate and the Conversion of Fucoaterol Acetate to Squalmlne

In Example 4 the naturally ocurring marine sterol fucosterol is acetylated at C ₃ to yield (37) which is converted to keto compound (38) by ozonolysis following the procedure of Takeshita et al., U.S. Patent 4,022,891. Protection of the C _a* keto group by ketalization yields (39) which is allylically oxidized as done in Example 1 to yield the conjugated ketone (40). Lithium in liquid ammonia reduction yields (41) which is stereoselectively reduced using K-selectride as done in Example 1 to yi.eid the C ₇ alcohol (42) which is converted to (13) by deprotection at C _a« using acid-catalyzed hydrolysis, follwed by calcium borohydride reduction of the C _a _» keto group to the alcohol and protection with TBDMS-C1. Cleavage of the C ₃ acetate and Collin's reagent oxidation yields (13).

Compound (42) is converted to squalamine by cleavage of the acetate and Collin's reagent oxidation to yield (43) followed by benzylation of the C ₇ alcohol using benzylchloride and sodium iodide in dimethylformamide to yield (44). The spermidine chain is introduced using a diprotected precursor, namely NH _a (CH _a ) ₃ N(BOC) (CH _a ) _* NHBOC. The C-, O-benzyl derivative of the (43), namely (44), is condensed with the diprotected precursor of the spermidine side-chain followed by reduction with sodium cyanoborohydride to yield (45). Deprotection of the C ₂ « ketal yields the C _a * ketone (46) which is reduced with sodium borohydride to the C _a* alcohol (47). Sulfation of (47) with Py/S0 ₃ yields (48). Catalytic reduction of (48) leads to deprotection of the two BOC groups on the nitrogen

atoms (49) as well as the C ₇ -0-benzyl group (50) to yiel _d sgualamlne (51).

Example 5 Alternative Route to Compound 13 Starting From Dehydroepiandroaterone to Yield 24-Oxo- Cholesteryl Acetate

This route proceeds from dehydroepiandrosterone (52) which is converted to the C _i7 ethylidine derivative using the Wittig reaction to yield (53) which in turn is treated with (CH ₃ ) _a A1C1 and isopropyl vinyl ketone followed by catalytic hydrogenation to yield 24-oxocholesteryl acetate following the known procedure (B.S. Snider and E.A. Deutsch, J. Orq.Chem., 1982, 47, 745). 24-Oxo cholesteryl acetate (38) can be used as in Example 4.

Example 6 A Microbiological Route to Compound 13

Compound (39) is reduced via catalytic hydrogenation to (55). Chromic acid oxidation of alcohol (56) obtained by saponification of (55) is subjected to C ₇ hydroxylation using Neurospora sp (M714), vegetable inoculum of penicillium sp ATCC 11598, Phycomyces blakesleanus, Peziza species, Cephalosporium species (Lederle Culture 2164), Diplodia Anatalensis ATCC 9055, or Helminthosporium culture to yield (43) of Example 4.

Example 7 Alternate Route to Compound 13 Starting From Pregnenolone Acetate

Starting with pregnenolone acetate (3β-acetoxy-5-pregnen-20-one) (58) _; ketalization at C _ao yields (59) followed by allylic oxidation, lithium in liquid ammonia reduction, K-selectride reduction as done in Example 1, followed by formation of the methoxymethyl protected C ₇ -alcohol product (62) and selective deprotection of the C _ao ketal to yield (63). Wittig coupling of the protected side-chain yields (64) which upon catalytic reduction yields (65). Deprotection at C ₇ yields (13). The side-chain is formed as follows:

R = TBDMS