The synthesis of spermine analogs of the shark aminosterol squalamine

Steroids 67 (2002) 291–304

Youheng Shu^a, Stephen R. Jones^a,b, William A. Kinney^b,1, Barry S. Selinsky^a,*

^aChemistry Department, Villanova University, Villanova, PA 19085, USA

^bGenaera Corporation, 5110 Campus Drive, Plymouth Meeting, PA 19462, USA

Received 17 October 2000; received in revised form 13 July 2001; accepted 24 July 2001

Abstract

Aminosterols isolated from the dogﬁsh shark Squalus acanthias are promising therapeutic agents in the treatment of infection and cancer.

One of these, MSI-1436, has been shown to possess antimicrobial activity slightly better than squalamine. In this study, a series of analogs

of MSI-1436 have been synthesized from stigmasterol. The 7␣-hydroxy substituent of MSI-1436 was either omitted or the stereochemistry

modiﬁed to the 7␤ position. Also, analogs of MSI-1436 with 24-sulfate, 24-amino, and 24-hydroxy substituents were synthesized in order

to assess the importance of the side chain functional group on antimicrobial activity. All of the analogs possess signiﬁcant antimicrobial

Keywords: Aminosterol; Antimicrobial; Sterol; Squalamine; Synthesis

1. Introduction

sterols isolated from the shark exhibit antimicrobial activity,

but MSI-1436 displays the greatest efﬁcacy against a variety

of microorganisms [5].

Squalamine is a novel aminosterol isolated from tissues

of the dogﬁsh shark Squalas acanthias [1]. The chemical

structure of squalamine was demonstrated to be 3␤-N-1-

The synthesis of a variety of aminosterols has been

reported. Simple aminosterols have been prepared by the

addition of an amine to a 3-oxo bile acid or methyl ester by

reductive amination [6–9]. Several squalamine analogs con-

taining a trans-AB ring junction have been synthesized and

characterized from hyodeoxycholic acid [10]. Others were

prepared by making the amide of a bile acid, with sulfation

of the 3-hydroxyl group [11].

Over the past several years, we and others have devel-

oped and reﬁned synthetic approaches to squalamine and

MSI-1436 [3,12–15]. We now apply and adapt this meth-

odology to the synthesis of related analogs. In particular, we

would like to explore the relationship between the func-

tional groups on MSI-1436 and antimicrobial activity.

In this report, the synthesis of a series of MSI-1436

analogs, all of which contain a C-3 spermine substituent, is

described. To determine the role of the 7␣-hydroxy group of

squalamine, analogs with a 7␤-hydroxy group and analogs

with no 7-hydroxy group were synthesized. The contribu-

tion of the 24-sulfate functional group was explored by the

preparation of compounds with three different functional

groups at C24: [1] a sulfate group, which is identical to

squalamine and is negatively charged at neutral pH; [2] an

amino group, which is positively charged at neutral pH; and

[N(3-[4-aminobutyl])-1,3

diaminopropane]-7␣-hydroxy-

5␣-cholestan-24R-yl sulfate by mass spectroscopy, two di-

mensional NMR spectroscopy, and by comparison to the

synthetic compound [2,3]. Squalamine exhibits potent bac-

tericidal activity against both Gram-negative and Gram-

positive bacteria [1]. More recent studies show that

squalamine possesses antiangiogenic activity [4], and may

have clinical utility in the chemotherapy of malignant tu-

mors.

Squalamine is only one of a number of aminosterols

isolated from the dogﬁsh shark [5]. Another of these, MSI-

1436 (3␤-spermine-7␣-hydroxy-5␣-cholestan-24R-yl sul-

fate; see Fig. 1 for structure), is structurally identical to

squalamine except for the polyamine at C3. Squalamine

possesses a spermidine moiety while MSI-1436 is linked to

the symmetrical polyamine spermine. All of the amino-

* Corresponding author. Tel.: ϩ1-610-519-4840; fax: ϩ1-610-519-

7167.

E-mail address: barry.selinsky@villanova.edu (B.S. Selinsky).

¹Current address: R.W. Johnson Pharmaceutical Research Institute,

Welsh and McKean Rds., Spring House, PA 19477.

PII: S0039-128X(01)00161-1

292

Y. Shu et al. / Steroids 67 (2002) 291–304

tography (TLC) was performed using Whatman K6 glass-

backed plates. Developed plates were air dried and visual-

ized by Hahnessian dip (2% ammonium molybdate and 1%

cerium sulfate in 10% sulfuric acid), or by ninhydrin for

amines. Column chromatography was performed using sil-

ica gel (J.T. Baker). Reverse phase HPLC experiments were

carried out using a Whatman Partisil 10 ODS-2 column.

Melting points were determined using a Thomas Hoover

melting point apparatus and are uncorrected. Low resolution

ﬁeld desorption (FD) and fast atom bombardment (ϩFAB)

mass spectra were acquired by the Mass Spectrometry Lab-

oratory in University of Illinois at Urbana-Campaign. Nu-

clear magnetic resonance spectra were recorded on either a

Varian XL-200 or a Bruker AM 400 spectrometer. Deuter-

ated solvents were used for frequency lock, and residual

solvent was used as the chemical shift reference. Elemental

analyses were performed by Robertson Microlit Lab Inc.

(Madison, NJ, USA). Minimum inhibitory concentrations

(MICs) for bacteria and yeast were determined by incubat-

ing logarithmic-phase organisms (0.9–1.1 ϫ 10⁴colony-

forming units/ml) in trypticase soy broth (Difco) at 37°C for

18–24 h [17]. The organisms chosen for testing were Staph-

ylococcus aureus, Escherichia coli (D31 strain), Pseudomo-

nas aeruginosa, and Candida albicans.

2.2. Preparation of 3-methyl-1-

triphenylphosphoranylidene-2-butanone [18]

Triphenylphosphine (38 g, 0.145 moles) was dissolved in

benzene (100 ml) in a 500 ml round bottom ﬂask. 1-Bromo-

3-methyl-2-butanone [19] (20.0 g, 0.12 moles) was added

and the mixture was stirred at room temperature for 24 h.

The solid was collected by ﬁltration, taken up in fresh benzene,

reﬁltered and dried under vacuum to give the phosphonium

salt. The product was treated with a saturated sodium carbon-

ate aqueous solution at room temperature for 12 h. The solid

was collected, washed with distilled water and dried for 12 h

under vacuum. The product was recrystallized from ethyl ac-

etate/hexane to afford the ﬁnal product (35.0 g, 83%). ¹H NMR

(200 MHz, CDCl₃): ␦ 1.07, 1.10 (d, J ϭ 6.5 Hz, 6H), 2.43–

2.56 (m, 1H), 3.61, 3.75 (d, 1H), 7.35–7.75 (m, 15H).

Fig. 1. The structures of the ﬁnal aminosterol products, with their corre-

sponding identifying numbers. The products are divided into two classes:

mixed isomers at C-24 that could not be separated, and products isolated as

a single stereoisomer at C-24. The structures of squalamine (with an

attached spermidine polyamine) and MSI-1436 (with an attached spermine

polyamine) are shown for comparison.

[3] a hydroxy group which is uncharged at neutral pH. The

stereochemistry at C24 was also varied with the different

functional groups. Analogs possessing a 24-sulfate were

stereoselectively synthesized as the 24-R diastereomer,

which is identical to MSI-1436. The analogs with a 24-

amino group were synthesized as mixed diastereomers, as

were the 24-hydroxy analogs without a 7-hydroxyl. All of

the analogs were synthesized from the natural steroid stig-

masterol, using a synthetic route optimized previously [15,

16]. Modiﬁcation of all of these structural elements gener-

ated compounds that were effective antibiotics.

2.2.1. Synthesis of MSI-1436 analogs without a 7-

hydroxyl substituent

2.2.1.1. Stigmasta-4,22-dien-3-one (2). Stigmasterol 1 (18.0

g, 43.6 mmoles) and aluminum isopropoxide (15.0 g, 73.4

mmoles) were added to a 500 ml round bottom ﬂask

equipped with a condenser, magnetic stir bar and heating

mantle. Toluene (200 ml) and 4-methyl-2-pentanone (100

ml) were also added to the ﬂask. The mixture was reﬂuxed

overnight, then cooled and washed with dilute hydrochloric

acid. The organic layer was dried over Na₂SO₄and the

solvent was removed on a rotary evaporator. The residue

was recrystallized from dichloromethane-methanol to yield

2. Experimental

2.1. General methods

Solvents and reagents were purchased as reagent grade

and used without further puriﬁcation. Thin-layer chroma-

the desired product 2 (14.7 g; 82%). The ¹H NMR and ¹³

C

Y. Shu et al. / Steroids 67 (2002) 291–304

293

NMR data [20] and melting point [21] are consistent with

previous results. 2: mp. 120–122°C; H NMR (200 MHz,

MHz, CDCl₃): ␦ 0.67 (s, 3 H), 0.80–0.86 (m, 12 H), 1.01

(d, J ϭ 6.1 Hz, 3 H), 3.92 (s, 4 H), 4.94–5.20 (m, 2 H); ¹³

1

C

CDCl₃): ␦ 0.73 (s, 3 H), 0.77–0.85 (m, 9 H), 1.01 (d, J ϭ

6.5 Hz, 3 H), 1.18 (s, 3 H), 4.95–5.21 (m, 2 H), 5.72 (s, 1

H); ¹³C NMR (50.3 MHz, CDCl₃): ␦ 12.0, 12.1, 17.2, 18.8,

20.6, 20.8, 20.9, 24.1, 25.2, 28.7, 31.7, 31.9, 32.8, 33.8,

35.4, 35.5, 38.4, 39.3, 40.3, 42.1, 51.1, 53.7, 55.7, 55.8,

123.6, 129.3, 138.0, 171.4, 199.4; MS (FD), m/z 410; Anal-

ysis calculated for C₂₉H₄₆O: C, 84.81; H, 11.29; Found: C,

84.75; H, 11.31.

NMR (50.3 MHz, CDCl₃): ␦ 11.3, 12.1, 18.9, 21.0, 21.1,

24.2, 25.3, 28.5, 28.8, 31.0, 31.8, 35.4, 35.9, 37.9, 39.8,

40.4, 42.4, 43.6, 51.1, 53.9, 55.9, 56.5, 64.0, 109.3, 129.1,

138.3; MS (FD) m/z 456; Analysis calculated for C₃₁H₅₂O₂:

C, 81.52; H, 11.48; Found: C, 81.47; H, 11.58.

2.2.1.4. 3,3-(Ethylenedioxy)-23,24-bisnor-5␣-cholan-22-al

(5). A solution of 4 (9.0 g, 19.7 mmoles) in 300 ml of 50/50

dichloromethane/ethanol was ozonolyzed at Ϫ48°C in a

Welsbach ozonolyzer. The ozonolyzer was set at 90 V and

switched on. Ozone was bubbled into the magnetically

stirred solution at 7 psi, 1 ml/min, until a light blue colora-

tion was observed. Oxygen was then bubbled into the ﬂask

until the blue color dissipated. Trimethyl phosphite (10 ml)

was added to the ﬂask, which was allowed to warm to room

temperature. The solvent was removed on a rotary evapo-

rator and the residue was placed under high vacuum over-

night. The residue was further puriﬁed on silica gel, eluting

with 15% ethyl acetate in toluene, to yield the white solid 5

(7.0 g, 95%). The ﬁnal product was identiﬁed using ¹H and

¹³C NMR and carried onto the next step without further

characterization. 5: ¹H NMR (200 MHz, CDCl₃): ␦ 0.69 (s,

3 H), 0.80 (s, 3 H), 1.10 (d, J ϭ 6.5 Hz, 3 H), 3.92 (s, 4 H)

9.55 (d, J ϭ 3.5 Hz, 1 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦

11.3, 12.3, 13.3, 21.1, 24.5, 26.9, 28.4, 31.0, 31.8, 35.3,

35.9, 37.9, 39.6, 43.1, 43.5, 49.3, 51.0, 53.9, 55.6, 63.6,

64.0, 109.1, 204.6.

2.2.1.2. 5␣-Stigmast-22-en-3-one (3). Tetrahydrofuran (200

ml) was added to a 1000 ml 3-neck round bottom ﬂask

equipped with a magnetic stir bar, 250 ml addition funnel

and dry ice condenser and cooled to –78°C. Ammonia was

collected to a total solution volume of approximately 500

ml. Lithium wire (1.0 g, 0.14 moles) was cut into small

pieces and added to the ﬂask with vigorous stirring. A deep

blue solution was obtained after the lithium was completely

dissolved. Stigmasta-4,22-dien-3-one 2 (12.0 g, 29.2

mmoles) dissolved in 100 ml tetrahydrofuran was added to

the ﬂask in a steady stream from an addition funnel. The

solution was stirred for 1 h, and then quenched by the

addition of a large excess of NH₄Cl. The ammonia was

allowed to evaporate at room temperature after the dry ice

condenser and bath were removed. The residue was trans-

ferred to a separatory funnel by dissolving in 80/20 diethyl

ether/tetrahydrofuran (600 ml) and dilute hydrochloric acid

(100 ml). The aqueous layer was removed and the organic

layer was washed with distilled water followed by brine.

The organic layer was dried over Na₂SO₄and the solvent

was removed on a rotary evaporator. The residue was pu-

riﬁed on silica gel, eluting with 90/10 hexanes/ethyl acetate,

to give the desired product 3 (9.2 g, 76%) as a white solid.

3: mp. 166–167°C, consistent with previous results [22,23];

¹H NMR (200 MHz, CDCl₃): ␦ 0.70 (s, 3 H), 0.78–0.86 (m,

9 H), 1.01 (d, J ϭ 6.5 Hz, 3 H), 1.18 (s, 3 H), 4.95–5.21 (m,

2 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦ 11.3, 12.1, 18.9,

21.1, 21.3, 24.2, 25.3, 28.8, 31.6, 31.8, 35.3, 35.5, 38.1,

38.4, 39.7, 40.4, 42.3, 44.6, 46.6, 51.1, 53.7, 55.9, 56.3,

129.2, 138.2, 212.1; MS (FD), m/z 412; Analysis calculated

for C₂₉H₄₈O: C, 84.40; H, 11.72; Found: C, 84.46; H, 11.82.

2.2.1.5. 3,3-(Ethylenedioxy)-5␣-cholest-22-en-24-one (6).

Aldehyde 5 (2.0 g, 5.3 mmoles) and 3-methyl-1-tri-

phenylphosphoranylidene-2-butanone (2.8 g, 8.1 mmoles)

were dissolved in 100 ml toluene in a 500 ml round bottom

ﬂask. The solution was reﬂuxed overnight and then cooled

to room temperature. The solvent was removed on a rotary

evaporator. The residue was chromatographed on silica gel,

eluting with 15% ethyl acetate in toluene, to give 6 (2.2 g,

93%) as a white solid. The spectral data of 6 agree with

those reported [16]. 6: mp. 153–154°C (lit. 168–169°C)

[16]; ¹H NMR (200 MHz, CDCl₃): ␦ 0.69 (s, 3 H), 0.81 (s,

3 H), 1.09 (d, J ϭ 6.5 Hz, 3 H), 2.82 (hept, J ϭ 6.9 Hz, 1

H), 3.93 (s, 4 H), 6.05 (d, J ϭ 15.8 Hz, 1 H), 6.71 (d of d,

J ϭ 15.8 and 9 Hz, 1 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦

11.2, 12.2, 18.3, 18.4, 19.1, 21.0, 24.0, 28.0, 28.3, 31.0,

31.7, 35.3, 35.8, 37.8, 38.0, 39.7, 39.8, 42.8, 43.4, 53.8,

54.9, 56.1, 63.9, 109.2, 126.0, 152.4, 204.2; MS (FD) m/z

442; Analysis calculated for C₂₉H₄₆O₃: C, 78.68; H, 10.47;

Found: C, 78.46; H, 10.47.

2.2.1.3 3,3-(Ethylenedioxy)-5␣-stigmast-22-ene (4). To a

solution of 3 (8.6 g, 20.8 mmoles) in 350 ml toluene was

added ethylene glycol (25 ml) and p-toluenesulfonic acid

(500 mg). The resulting solution was reﬂuxed overnight

with the use of a Dean-Stark trap to remove water, and then

cooled to room temperature. The solution was transferred to

a separatory funnel. Approximately 2 g of anhydrous

Na₂CO₃was carefully added to the funnel, followed by

distilled water. The organic layer was washed with sodium

bicarbonate solution, distilled water and brine. The organic

layer was dried over Na₂SO₄, then ﬁltered and the solvent

was removed in vacuo to yield the desired product 4 (9.0 g,

2.2.1.6. 3,3-(Ethylenedioxy)-5␣-cholestan-24-one (7). A so-

lution of 6 (4.27 g, 9.7 mmoles) in 100 ml 80/20 ethyl

acetate/tetrahydrofuran was placed in a 400 ml hydrogena-

tion ﬂask. The Parr apparatus was charged with hydrogen at

40 psi. Palladium on carbon (5%, 200 mg) was added to the

ﬂask. The reaction was carried out at room temperature for

1

95%) as a white solid. 4: mp. 134–135°C; H NMR (200

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Y. Shu et al. / Steroids 67 (2002) 291–304

4 h. The palladium on carbon was removed by ﬁltration and

2.2.1.9. Spermine conjugates of 24-amino-5␣-cholestan-3-

one (10 and 11). To a solution of 9 (600 mg, 1.5 mmoles)

in 100 ml of 50/50 methanol/tetrahydrofuran was added

spermine (1.0 g, 4.9 mmoles), and activated molecular

sieves (2.0 g). The mixture was stirred for 5 h, then 500 mg

sodium borohydride (13.2 mmoles) was added. The mixed

slurry was stirred for 1 h, then ﬁltered and washed with

chloroform. The ﬁltrate was basidiﬁed with 20% sodium

hydroxide solution and extracted with chloroform ﬁve

times. The organic layer was dried over Na₂SO₄. The sol-

vent was removed on a rotary evaporator to give 550 mg

(61%) of the crude product, which was a mixture of the

3␣-spermine addition product 10 and the 3␤ addition prod-

uct 11. The crude product was dissolved in 1.5 l of an

aqueous solution containing 5% (v/v) acetonitrile and 0.2%

(v/v) triﬂuoroacetic acid. The crude solution was puriﬁed

using reverse phase HPLC (Whatman C-18 column, eluting

with a gradient of 0–50% acetonitrile containing 0.2% (v/v)

TFA in aqueous solution containing 0.2% (v/v) TFA), to

afford the less polar ␣-isomer 10 and the more polar ␤-iso-

mer 11 as the triﬂuoroacetate salts.

the solvent was removed on a rotary evaporator to give the

1

white solid 7 (4.27 g, 99%). 7: mp. 123–124°C; H NMR

(200 MHz, CDCl₃): ␦ 0.65 (s, 3 H), 0.81 (s, 3 H), 0.88, 0.91

(d, J ϭ 6.5 Hz, 3 H), 1.07 (s, 3 H), 1.10 (s, 3 H), 2.34–2.47

(m, 1 H), 2.54–2.67 (m, 1 H), 3.92 (s, 4 H); ¹³C NMR (50.3

MHz, CDCl₃): ␦ 11.2, 11.9, 18.1, 18.2, 21.0, 24.0, 27.9,

28.4, 29.6, 30.9, 31.7, 35.1, 35.2, 35.8, 36.9, 37.7, 39.8,

40.6, 42.4, 43.4, 53.8, 55.7, 56.2, 63.9, 109.2, 215.1; MS

(FD) m/z 444; Analysis calculated for C₂₉H₄₈O₃: C, 78.33;

H, 10.88; Found: C, 78.11; H, 10.81.

2.2.1.7. 3,3-(Ethylenedioxy)-5␣-cholestan-24-amine (8). To

a solution of 7 (1.0 g, 2.2 mmoles) in 50 ml of 50/50

methanol/tetrahydrofuran was added ammonium acetate

(2.0 g) and activated molecular sieves (3 Å, 2.0 g). The

mixture was stirred for 5 h, then 500 mg sodium cyanoboro-

hydride (8.0 mmoles) was added and the pH was adjusted to

6 with acetic acid. The mixed slurry was stirred for 1 h,

ﬁltered, and washed with chloroform. The ﬁltrate was

washed with dilute hydrochloric acid, distilled water and

brine. The organic layer was dried over Na₂SO₄. The sol-

vent was removed on a rotary evaporator to give the desired

product 8 (700 mg, 69%) as a white solid. 8: mp. 126–

10: ¹H NMR (200 MHz, CD₃OD): ␦ 0.71 (s, 3 H),

0.85–0.95 (m, 12 H), 3.02–3.17 (m, 10 H); MS (ϩFAB) m/z

589.4 (Mϩ1); Analysis calculated for C₃₇H₇₂N₄-4TFA: C,

51.72; H, 7.30; N, 5.36; F, 21.81; Found: C, 49.82; H, 6.58;

N, 4.85; F, 22.84.

1

127°C; H NMR (200 MHz, CDCl₃): ␦ 0.59 (s, 3 H), 0.74

11: ¹H NMR (200 MHz, CD₃OD): ␦ 0.70 (s, 3 H),

0.87–0.96 (m, 12 H), 3.01–3.17 (m, 10 H); MS (ϩFAB) m/z

589.5 (Mϩ1); Analysis calculated for C₃₇H₇₂N₄-4TFA: C,

51.72; H, 7.30; N, 5.36; F, 21.81; Found: C, 51.43; H, 7.08;

N, 5.16; F, 22.06.

(s, 3 H), 0.83 (d, J ϭ 7.6 Hz, 6 H), 2.32–2.43 (broad m, 1

H), 3.85 (s, 4 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦ 11.1,

11.8, 16.4, 17.0, 18.4, 19.0, 19.2, 20.9, 23.9, 28.0, 28.3,

30.8, 31.6, 32.4, 33.1, 35.1, 35.7, 37.7, 39.7, 42.3, 43.3,

53.7, 55.7, 56.1, 56.6, 56.8, 63.8, 109.0; MS (ϩFAD): m/z

446.5 (Mϩ1); Analysis calculated for C₂₉H₅₁NO₂: C,

78.14; H, 11.53; N, 3.09; Found: C, 77.69; H, 11.35; N,

3.09.

2.2.1.10. 3,3-(Ethylenedioxy)-5␣-cholestan-24-ol (12). So-

dium borohydride (1.0 g, 26.4 mmoles) was added to a

solution of 7 (3.0 g, 6.8 mmoles) in 100 ml of 50/50

methanol/tetrahydrofuran. The solution was stirred for 1 h,

and then 100 ml distilled water was slowly added. The

solution was transferred to a 500 ml separatory funnel and

extracted with diethyl ether. The organic layer was dried

over Na₂SO₄and the solvent removed by rotary evaporation

to give the desired product 12 (2.9 g, 96%) as a white solid.

The ﬁnal product was carried onto the deprotection step

directly without characterization.

2.2.1.8. 24-Amino-5␣-cholestan-3-one (9). Ketal 8 (700

mg, 1.6 mmoles) was dissolved in 100 ml acetone in a 250

ml round bottom ﬂask equipped with magnetic stirring.

Concentrated hydrochloric acid (4 ml) was added dropwise

to the ﬂask. The solution was stirred at room temperature for

2 h, after which 100 ml of distilled water was added to the

solution. The majority of the acetone was evaporated and

the solution was extracted with chloroform and washed with

distilled water followed by brine. The organic layer was

dried over Na₂SO₄and the solvent removed on a rotary

evaporator to give 600 mg (95%) of the desired product as

a white solid. The ﬁnal product was identiﬁed using ¹H and

¹³C NMR and carried onto the next step without further

characterization. 9: ¹H NMR (200 MHz, CDCl₃): ␦ 0.61 (s,

3 H), 0.78–0.87 (m, 9 H), 0.94 (s, 3 H), 2.93–3.07 (broad

m, 1 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦ 10.9, 11.6, 16.2,

16.8, 18.2, 18.3, 18.8, 19.1, 20.9, 23.7, 27.8, 28.5, 29.9,

30.6, 30.8, 30.9, 31.2, 32.2, 33.0, 34.9, 35.1, 35.3, 35.5,

37.6, 38.0, 39.4, 42.1, 44.2, 46.2, 53.3, 55.6, 55.7, 56.3,

56.6, 211.0.

2.2.1.11. 24-Hydroxy-5␣-cholestan-3-one (13). The 24-al-

cohol 12 (2.9 g, 6.5 mmoles) was deprotected in the same

manner as 8 to yield 2.58 g (98%) of the desired product as

1

a white solid. 13: mp. 161–162°C; H NMR (200 MHz,

CDCl₃): ␦ 0.61 (s, 3 H), 0.82–0.87 (m, 9 H), 0.94 (s, 3 H),

3.18–3.26 (broad m, 1 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦

11.1, 11.8, 16.5, 17.0, 18.3, 18.5, 18.6, 18.8, 21.1, 23.9,

27.8, 27.9, 28.6, 30.3, 30.4, 31.4, 31.7, 31.9, 32.8, 33.3,

34.8, 35.0, 35.3, 35.4, 35.6, 37.8, 38.2, 39.6, 42.3, 44.3,

46.4, 53.5, 55.8, 55.9, 77.5, 211.8; MS (FD) m/z 426;

Analysis calculated for C₂₉H₄₆O₂: C, 80.54; H, 11.51;

Found: C, 80.49; H 11.67.

Y. Shu et al. / Steroids 67 (2002) 291–304

295

2.2.1.12. Spermine conjugates of 24-hydroxy-5␣-cholestan-

3-one (14 and 15). Spermine (2.5 g, 7.2 mmoles) was added

to 13 (800 mg, 2.0 mmoles) in the same manner as de-

scribed in the synthesis of 10 and 11 to give 980 mg (82%)

of crude product (14 and 15). The crude product was dis-

solved in 4 l of an aqueous solution containing 5% (v/v)

acetonitrile and 0.2% (v/v) triﬂuoroacetic acid and puriﬁed

using reverse phase HPLC to afford the less polar ␣-isomer

14 and the more polar ␤-isomer 15 as the triﬂuoroacetate

salts.

tilled water and brine. The organic layer was dried over

Na₂SO₄and the solvent was removed on a rotary evapora-

tor. The residue was recrystallized from hexanes/diethyl

ether to give the desired product 17 (16.0 g, 88%) as a white

1

solid. 17: mp. 176–178°C; H NMR (200 MHz, CDCl₃): ␦

0.70 (s, 3 H), 0.78–0.86 (m, 9 H), 1.02 (d, J ϭ 6.5 Hz, 3 H),

3.30–3.42 (m, 1 H), 3.51–3.65 (m, 1 H), 4.95–5.22 (m, 2

H); ¹³C NMR (50.3 MHz, CDCl₃): ␦ 12.1, 12.2, 18.8, 20.9,

21.2, 25.2, 26.9, 29.2, 31.3, 31.7, 34.8, 36.7, 37.6, 37.8,

39.7, 40.2, 41.9, 43.3, 51.1, 52.3, 54.9, 55.6, 70.9, 75.0,

129.3, 138.0; MS (FD) m/z 430; Analysis calculated for

C₂₉H₅₀O₂: C, 80.87; H, 11.70; Found: C, 80.64; H, 11.50.

1

14: H NMR (200 MHz, CD₃OD): ␦ 0.72 (s, 3 H), 0.86

(s, 3 H), 0.97–1.02 (m, 9 H), 3.00–3.17 (m, 10 H); MS

(ϩFAB) m/z 588.5 (Mϩ1); Analysis calculated for

C₃₇H₇₃N₅-5TFA: C, 48.74; H, 6.79; N, 6.05; F, 24.61;

Found: C, 47.83; H, 6.30; N, 5.81; F, 27.54.

2.2.2.3. 7␤-Hydroxy-5␣-stigmast-22-en-3-one (18). Silver

carbonate on celite was prepared by dissolving AgNO₃

(11.0 g, 65.0 mmoles) in 200 ml deionized water and adding

celite (10.0 g) with vigorous stirring. A large excess of a pH

11 carbonate buffer was slowly added to the slurry. Silver

carbonate precipitated out onto the celite as a yellow-green

solid. The solid was ﬁltered, washed with deionized water,

and then dried in a foil covered vacuum desiccator over-

night.

1

15: H NMR (200 MHz, CD₃OD): ␦ 0.72 (s, 3 H), 0.88

(s, 3 H), 0.97–1.03 (m, 9 H), 3.01–3.17 (m, 10 H); MS

(ϩFAB) m/z 588.5 (Mϩ1); Analysis calculated for

C₃₇H₇₃N₅-5TFA: C, 48.74; H, 6.79; N, 6.05; F, 24.61;

Found: C, 47.61; H, 6.35; N, 5.61; F, 24.67.

2.2.2. Synthesis of MSI-1436 analogs with a 7␤-hydroxyl

substituent

The silver carbonate was added to a solution of 17 (7.0 g,

16.3 mmoles) in 350 ml toluene in a 500 ml round ﬂask

equipped with a Dean-Stark trap and reﬂuxed overnight.

The reaction was cooled to room temperature and ﬁltered

through a short column of ﬂorisil eluting with ethyl acetate.

After the solvent was removed on a rotary evaporator, the

crude product was chromatographed on a silica gel column,

eluting with 40/60 ethyl acetate/hexanes. A trace of silver

impurity was removed by a second short column of ﬂorisil.

The desired product 18 (5.6 g, 80%) was obtained as a white

2.2.2.1. 3␤-Hydroxystigmasta-5,22-dien-7-one (16). 3␤-

Hydroxystigmasta-5,22-dien-7-one was synthesized as de-

scribed previously [15]. The crude product was recrystal-

lized from methanol (2X) to yield the desired product 16

(81%) as a white solid. The analytical data for 16 agree with

those reported [15]. 16: mp. 144°C; ¹H (200 MHz, CDCl₃):

␦ 0.70 (s, 3 H), 0.78–0.86 (cm, 9 H), 1.02 (d, J ϭ 6.5 Hz,

3 H), 1.20 (s, 3 H), 3.69 (m, 1 H), 4.95–5.26 (m, 2 H), 5.71

(s, 1 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦ 12.0, 12.1, 17.1,

18.8, 20.9, 21.2, 25.2, 26.2, 28.9, 30.8, 31.7, 36.2, 38.1,

38.4, 40.1, 41.6, 42.8, 45.2, 49.7, 49.8, 51.0, 54.5, 70.0,

125.6, 129.3, 137.9, 165.8, 202.5; MS (FD) m/z 426; Anal-

ysis calculated for C₂₉H₄₆O₂: C, 80.63; H, 10.87; Found: C,

81.77; H, 11.04.

1

solid. 18: mp. 153°C; H (200 MHz, CDCl₃): ␦ 0.69 (s, 3

H), 0.82–0.93 (cm, 9 H), 1.02 (d, J ϭ 6.5 Hz, 3 H), 1.00 (s,

3 H), 3.84 (sharp m, 1 H), 4.92–5.23 (m, 2 H); ¹³C NMR

(50.3 MHz, CDCl₃): ␦ 11.4, 12.1, 12.2, 18.8, 20.9, 21.2,

21.3, 21.5, 25.2, 26.8, 29.1, 31.7, 34.9, 37.9, 38.1, 39.5,

40.1, 42.9, 43.3, 43.7, 43.9, 51.0, 51.6, 54.8, 55.5, 74.3,

129.3, 137.9, 211.3; MS (FD) m/z 428; Analysis calculated

for C₂₉H₄₈O₂: C, 81.25; H, 11.29; Found: C, 80.98; H,

11.19.

2.2.2.2. 3␤,7␤-Dihydroxy-5␣-stigmast-22-ene (17). Tetra-

hydrofuran (400 ml) was cooled to –78°C with dry ice

acetone, and then ammonia was collected in the same ﬂask

to a total volume of approximately 1000 ml. Lithium wire

(2.0 g, 0.29 moles) was cut into small pieces and added to

the ﬂask with vigorous stirring. A deep blue solution was

obtained after the lithium was completely dissolved. 3␤-

Hydroxystigmasta-5,22-dien-7-one 16 (18.0 g, 42.2

mmoles) dissolved in 150 ml tetrahydrofuran was added to

the ﬂask in a steady stream from an addition funnel. The

solution was stirred for 0.5 h, and then quenched by the

addition of methanol until the blue coloration dissipated.

The ammonia was allowed to evaporate at room tempera-

ture, and the residue was transferred to a separatory funnel

by dissolving in 50/50 toluene/ethyl acetate and dilute hy-

drochloric acid. The aqueous layer was removed and the

organic layer washed repeatedly with 0.1 N HCl, then dis-

2.2.2.4. 3,3-(Ethylenedioxy)-5␣-stigmast-22-en-7␤-ol (19).

The 3-oxo group of 18 (5.0 g, 11.7 mmoles) was protected

with ethylene glycol as described in the preparation of 4.

The desired product 19 (5.3 g, 96%) was isolated as a white

1

solid. 19: mp. 138°C; H NMR (200 MHz, CDCl₃): ␦ 0.70

(s, 3 H), 0.77–0.84 (m, 12 H), 1.02 (d, J ϭ 6.1 Hz, 3 H),

3.31–3.44 (m, 1 H), 3.93 (s, 4 H), 4.94–5.20 (m, 2 H); ¹³

C

NMR (50.3 MHz, CDCl₃): ␦ 10.6, 11.4, 11.5, 18.1, 20.2,

20.5, 24.5, 26.1, 28.5, 30.3, 31.0, 34.0, 35.0, 36.7, 37.1,

39.0, 39.5, 40.0, 42.6, 50.4, 51.3, 54.2, 55.0, 63.3, 74.0,

108.3, 128.5, 137.4; MS (FD) m/z 473; Analysis calculated

for C₃₁H₅₂O₃: C, 78.76; H, 11.09; Found: C, 78.79; H,

11.05.

296

Y. Shu et al. / Steroids 67 (2002) 291–304

2.2.2.5. 3,3-(Ethylenedioxy)-7␤-hydroxy-23,24-bisnor-5␣-

cholan-22-al (20). The side chain of 3,3-(ethylenedioxy)-

5␣-stigmast-22-en-7␤-ol 19 (5.3 g, 11.2 mmoles) was con-

verted to the 22-aldehyde as described in the preparation of

5. The desired product 20 (4.2 g, 96%) was isolated as a

56.9, 63.9, 74.5, 108.9; MS (FD) m/z 462; Analysis calcu-

lated for C₂₉H₅₁NO₃: C, 75.44; H, 11.13; N, 3.03; Found: C,

75.50; H, 11.46; N, 3.27.

2.2.2.9. 24-Amino-7␤-hydroxy-5␣-cholestan-3-one (24).

3,3-(Ethylenedioxy)-7␤-hydroxy-5␣-cholestan-24-amine

23 (500 mg, 1.1 mmoles) was deprotected with HCl in

acetone as described in the preparation of 9. The product 24

(420 mg, 93%) was carried onto the next step directly

without characterization.

1

white solid. The ﬁnal product was identiﬁed using H and

¹³C NMR and carried onto the next step without further

characterization. 20: ¹H NMR (200 MHz, CDCl₃): ␦ 0.69 (s,

3 H), 0.81 (s, 3 H), 1.09 (d, J ϭ 6.5 Hz, 3 H), 2.27–2.37 (m,

1 H), 3.28–3.41 (m, 1 H), 3.90 (s, 4 H), 9.54 (d, J ϭ 3.2 Hz,

1 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦ 11.1, 12.2, 13.1,

20.9, 26.8, 26.9, 30.6, 34.4, 35.4, 37.1, 38.0, 39.2, 40.4,

42.7, 43.6, 48.8, 49.9, 51.7, 54.6, 63.6, 74.1, 108.6, 204.6.

2.2.2.10. Spermine conjugates of 24-amino-7␤-hydroxy-5␣-

cholestan-3-one (25 and 26). Spermine was added to 24-

amino-7␤-hydroxy-5␣-cholestan-3-one 24 (400 mg, 1.0

mmoles) in the same manner as described in the synthesis of

10 and 11. The crude addition product (480 mg, 83%) was

puriﬁed using reverse phase HPLC to afford the less polar

␣-isomer 25 and the more polar ␤-isomer 26 as their tri-

ﬂuoroacetate salts.

2.2.2.6. 3,3-(Ethylenedioxy)-7␤-hydroxy-5␣-cholest-22-en-

24-one (21). 3-Methyl-1-triphenylphosphoranylidene-2-bu-

tanone (4.66 g, 13.4 mmoles) was added to aldehyde 20 (3.5

g, 9.0 mmoles) in the same manner as in the preparation of

6. The desired product 21 (3.7 g, 92%) was isolated as a

white solid. 21: mp. 129°C; ¹H NMR (200 MHz, CDCl₃): ␦

0.67 (s, 3 H), 0.79 (s, 3 H), 1.04 (d, J ϭ 6.5 Hz, 9 H),

2.71–2.85 (m, 1 H), 3.24–3.36 (m, 1 H), 3.87 (s, 4 H), 6.02

(d, J ϭ 15.8 Hz, 1 H), 6.67 (d of d, J ϭ 15.8 and 9 Hz, 1 H);

¹³C NMR (50.3 MHz, CDCl₃): ␦ 11.2, 12.2, 18.2, 18.3,

19.2, 21.1, 26.6, 28.2, 30.8, 34.6, 35.6, 37.3, 37.9, 39.5,

40.6, 42.9, 43.6, 51.9, 53.9, 55.3, 63.8, 74.4, 108.8, 125.9,

152.2, 204.2; MS (FD) m/z 458; Analysis calculated for

C₂₉H₄₆O₄: C, 75.94; H, 10.11; Found: C, 75.69; H, 10.28.

1

25: H NMR (200 MHz, CD₃OD): ␦ 0.74 (s, 3 H), 0.89

(s, 3 H), 0.99–1.04 (m, 9 H), 3.07–3.18 (m, 10 H); MS

(ϩFAB) m/z 604.4 (Mϩ1); Analysis calculated for

C₃₇H₇₃N₅O-5TFA: C, 48.08; H, 6.70; N, 5.96; F, 24.27;

Found: C, 48.21; H, 6.66; N, 5.82; F, 24.61.

1

26: H NMR (200 MHz, CD₃OD): ␦ 0.74 (s, 3 H), 0.88

(s, 3 H), 0.98–1.04 (m, 9 H), 3.06–3.15 (m, 10 H); MS

(ϩFAB) m/z 604.4 (Mϩ1); Analysis calculated for

C₃₇H₇₃N₅O-5TFA: C, 48.08; H, 6.70; N, 5.96; F, 24.27;

Found: C, 47.79; H, 6.53; N, 5.77; F, 24.67.

2.2.2.7. 3,3-(Ethylenedioxy)-7␤-hydroxy-5␣-cholestan-24-

one (22). The double bond of 3,3-(ethylenedioxy)-7␤-hy-

droxy-5␣-cholest-22-en-24-one 21 (3.5 g, 7.6 mmoles) was

reduced by catalytic hydrogenation using the same proce-

dure described in the preparation of 7. The desired product

22 (3.3 g, 94%) was isolated as a white solid. 22: mp.

2.2.2.11. 3,3-(Ethylenedioxy)-5␣-cholest-22-en-7␤, 24R-

diol (27). A solution of 1.0 M Corey’s (R)-methyl ox-

azaborolidine reagent (6.0 ml, 6.0 mmoles) in toluene and

1.0 M borane (15.0 ml, 15.0 mmoles) were added to a 500

ml 3-neck round bottom ﬂask under argon. The mixture was

stirred at room temperature for 2 h, then cooled to Ϫ20°C in

a dry ice/carbon tetrachloride bath. A solution of 21 (2.3 g,

5.0 mmoles) in 100 ml toluene was slowly added to the ﬂask

by the addition funnel. The reaction mixture was stirred at

Ϫ20°C for 1.5 h, then warmed to room temperature. Satu-

rated ammonium chloride solution (50 ml) and 1.0 N hy-

drochloric acid (150 ml) were added to the ﬂask. The aque-

ous phase was extracted with toluene (3X). The organic

layer was washed with saturated sodium bicarbonate solu-

tion and brine, and dried over Na₂SO₄. The solvent was

removed on a rotary evaporator. The residue was puriﬁed on

a silica gel column, eluting with 50/50 ethyl acetate/toluene

to give the desired product 27 (2.1 g, 91%) as a white solid.

1

143–144°C; H NMR (200 MHz, CDCl₃): ␦ 0.72 (s, 3 H),

0.87 (s, 3 H), 0.95 (d, J ϭ 6.5 Hz, 3 H), 1.13 (d, J ϭ 6.5 Hz,

6 H), 2.58–2.72 (m, 1 H), 3.33–3.45 (m, 1 H), 3.96 (s, 4 H);

¹³C NMR (50.3 MHz, CDCl₃): ␦ 11.2, 11.9, 18.0, 18.2,

20.9, 21.1, 26.5, 28.3, 29.5, 30.8, 34.5, 34.9, 35.5, 36.9,

37.2, 37.9, 39.7, 40.4, 40.6, 42.9, 43.2, 51.9, 54.7, 55.5,

63.8, 74.4, 108.8, 215.0; MS (FD) m/z 460; Analysis cal-

culated for C₂₉H₄₈O₄: C, 75.61; H, 10.50; Found: C, 75.68;

H, 10.65.

2.2.2.8. 3,3-(Ethylenedioxy)-7␤-hydroxy-5␣-cholestan-24-

amine (23). The 24-oxo group of 22 (800 mg; 1.7 mmoles)

was converted to the 24-amine in the same manner as

described in the preparation of 8. The desired product 23

(520 mg, 71%) was isolated as a white solid. 23: mp.

1

27: mp. 139–140°C; H NMR (200 MHz, CDCl₃): ␦ 0.70

1

124–125°C; H NMR (200 MHz, CDCl₃): ␦ 0.68 (s, 3 H),

(s, 3 H), 0.85–0.94 (m, 9 H), 1.04 (d, J ϭ 6.5 Hz, 3 H),

3.31–3.44 (m, 1 H), 3.70–3.76 (m, 1 H), 3.95 (s, 4 H),

5.30–5.52 (m, 2 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦ 11.4,

12.3, 18.0, 18.2, 20.5, 21.2, 26.8, 29.1, 31.0, 33.8, 34.8,

35.8, 37.4, 37.8, 39.8, 40.8, 43.2, 43.5, 52.0, 54.5, 55.6,

64.1, 74.8, 78.4, 109.0, 128.6, 139.4; MS (FD) m/z 461;

0.83 (s, 3 H), 0.89–0.94 (m, 9 H), 2.48–2.52 (broad m, 1

H), 3.31–3.43 (m, 1 H), 3.93 (s, 4 H); ¹³C NMR (50.3 MHz,

CDCl₃): ␦ 11.2, 11.9, 16.5, 17.1, 18.5, 18.6, 18.9, 19.2,

21.1, 26.6, 28.5, 30.4, 30.8, 32.1, 32.4, 32.8, 34.6, 35.6,

37.3, 37.9, 39.7, 40.6, 43.0, 43.3, 51.9, 54.8, 55.5, 56.7,

Y. Shu et al. / Steroids 67 (2002) 291–304

297

Analysis calculated for C₂₉H₄₆O₄: C, 75.61; H, 10.50;

Found: C, 75.41; H, 10.45.

100 ml pyridine was cooled to 4°C in an ice water bath.

Benzoyl chloride (5.0 ml, 43.6 mmoles) was added drop-

wise to the solution, followed by 4-dimethylaminopyridine

(200 mg). The reaction was allowed to warm to room

temperature and stirred for approximately 8 h. The solution

was poured over ice and allowed to stand overnight. The

resulting mixture was ﬁltered. The solid was collected and

redissolved in toluene. The solution was washed with 1.0 N

HCl (2X), and then sodium bicarbonate solution and brine.

The organic layer was dried over Na₂SO₄and the solvent

removed on a rotary evaporator. The residue was chromato-

graphed on silica gel eluting with a gradient of ethyl acetate

in toluene. The desired product 32 (4.2 g, 85%) was isolated

2.2.2.12. 3,3-(Ethylenedioxy)-5␣-cholestan-7␤, 24R-diol

(28). A solution of 27 (2.5 g, 5.6 mmoles) in 100 ml of

70/30 ethyl acetate/tetrahydrofuran was placed in a 400 ml

hydrogenation ﬂask. The Parr apparatus was charged with

hydrogen at 50 psi. Platinum (IV) oxide (60 mg) was added

to the ﬂask. The ﬂask was charged with hydrogen and the

Parr apparatus switched on for 4 h at room temperature. The

catalyst was ﬁltered off, and the solvent removed on a rotary

evaporator. The residue was puriﬁed on a silica gel column

eluting with a gradient of 40% ethyl acetate in toluene, to

give the desired product 28 (1.7 g, 85%) as a white solid. 28:

mp. 165°C; ¹H NMR (200 MHz, CDCl₃): ␦ 0.62 (s, 3 H), 0.76

(s, 3 H), 3.18–3.36 (m, 2 H), 3.86 (s, 4 H); ¹³C NMR (50.3

MHz, CDCl₃): ␦ 11.5, 12.2, 17.3, 18.7, 18.9, 21.4, 26.9, 28.8,

30.6, 31.1, 32.1, 33.6, 34.9, 35.6, 35.9, 37.5, 38.0, 40.0, 40.9,

43.3, 43.6, 52.2, 55.1, 55.7, 64.1, 74.9, 76.9, 109.2; MS (FD)

m/z 463; Analysis calculated for C₂₉H₄₆O₄: C, 75.28; H, 10.89;

Found: C, 75.24; H, 10.92.

1

as a white solid. 32: mp. 203–204°C; H NMR (200 MHz,

CDCl₃): ␦ 0.76 (s, 3 H), 0.91 (s, 3 H), 1.04–1.10 (m, 9 H),

2.73–2.87 (m, 1 H), 3.93 (s, 4 H), 4.75–4.89 (m, 1 H), 6.01

(d, J ϭ 16 Hz, 1 H), 6.66 (d of d, J ϭ 16 and 9 Hz, 1 H),

7.39–7.54 (m, 4 H), 8.02 (d, J ϭ 9 Hz, 1 H); ¹³C NMR (50.3

MHz, CDCl₃): ␦ 11.4, 12.4, 18.3, 18.4, 19.3, 21.2, 26.1,

28.2, 31.1, 33.9, 34.7, 35.6, 37.0, 37.9, 39.5, 39.6, 40.5,

43.6, 52.1, 54.0, 54.9, 64.0, 64.1, 77.2, 108.8, 126.1, 128.2,

129.4, 130.7, 132.6, 152.2, 165.9, 204.4; MS (FD) m/z 562;

Analysis calculated for C₃₆H₄₉O₅: C, 76.97; H, 8.79;

Found: C, 76.46; H, 8.93.

2.2.2.13. 7␤, 24R-Dihydroxy-5␣-cholestan-3-one (29). 3,3-

(Ethylenedioxy)-5␣-cholestan-7␤, 24R-diol 28 (1.2 g, 2.6

mmoles) was deprotected with HCl in acetone in the same

manner as described in the preparation of 9. The desired

2.2.2.16. 3,3-(Ethylenedioxy)-24R-hydroxy-5␣-cholest-22-

en-7␤-yl benzoate (33). The 24-ketone of 32 (2.9 g, 5.2

mmoles) was reduced to the R-alcohol in the same manner

as described in the preparation of 27. The desired product 33

(2.7 g, 93%) was isolated as a white solid. 33: mp. 151–

1

product 29 (1.0 g, 92%) was identiﬁed using H and ¹³C

NMR and carried onto the next step without further char-

1

acterization. 29: H NMR (200 MHz, CDCl₃): ␦ 0.72 (s, 3

H), 0.90–0.95 (m, 9 H), 1.04 (s, 3 H), 3.27–3.45 (broad m,

2 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦ 11.5, 12.1, 17.3,

18.7, 18.9, 21.7, 26.8, 28.7, 30.5, 32.0, 33.5, 35.0, 35.5,

38.1, 38.2, 39.8, 43.1, 43.5, 43.8, 44.1, 51.7, 55.0, 55.5,

74.5, 76.9, 211.6.

1

152°C; H NMR (200 MHz, CDCl₃): ␦ 0.65 (s, 3 H), 0.69

(s, 3 H), 0.74 (d, J ϭ 6.5 Hz, 3 H), 0.80 (d, J ϭ 6.5 Hz, 6

H), 3.54–3.60 (m, 1 H), 3.90 (s, 4 H), 4.67–4.79 (m, 1 H),

5.21–5.26 (m, 2 H), 7.28–7.43 (m, 4 H), 7.93 (d, J ϭ 9 Hz,

1 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦ 11.2, 12.1, 17.8,

18.1, 20.3, 21.1, 25.9, 28.7, 30.9, 33.6, 33.7, 34.5, 35.5,

36.9, 39.4, 39.5, 39.7, 40.3, 43.1, 52.0, 54.3, 55.1, 63.9,

77.0, 78.0, 108.6, 128.0, 128.5, 129.3, 130.6, 132.4, 138.8,

165.6; MS (FD) m/z 564; Analysis calculated for C₃₆H₅₁O₅:

C, 76.69; H, 9.12; Found: C, 76.61; H, 9.34.

2.2.2.14. Spermine conjugates of 7␤, 24R-dihydroxy-5␣-

cholestan-3-one (30 and 31). Spermine was added to 7␤,

24R-dihydroxy-5␣-cholestan-3-one 29 (500 mg, 1.2

mmoles) in the same manner as described in the synthesis of

10 and 11. The crude addition product (480 mg, 67%) was

puriﬁed using reverse phase HPLC to afford the less polar

␣-isomer 30 and the more polar ␤-isomer 31 as their trif-

luoroacetate salts.

2.2.2.17. 3,3-(Ethylenedioxy)-24R-hydroxy-5␣-cholestan-

7␤-yl benzoate (34). The double bond at position 22 of 33

(2.7 g, 4.8 mmoles) was reduced by catalytic hydrogenation

in the same manner as described in the preparation of 28.

The desired product 34 (2.4 g, 88%) was isolated as a white

30: ¹H NMR (400 MHz, CD₃OD): ␦ 0.69 (s, 3 H),

0.83–0.88 (m, 9 H), 0.94 (d, J ϭ 6.5 Hz, 3 H), 3.01–3.15

(m, 10 H), 3.40 (broad s, 1 H); MS (ϩFAB) m/z 605.5

(Mϩ1); Analysis calculated for C₃₇H₇₂N₄O₂-4TFA: C,

50.94; H, 7.22; N, 5.28; Found: C, 50.25; H, 6.83; N, 5.20.

31: ¹H NMR (200 MHz, CD₃OD): ␦ 0.74 (s, 3 H),

0.89–0.97 (m, 12 H), 3.07–3.18 (m, 10 H); MS (ϩFAB)

m/z 605.4 (Mϩ1); Analysis calculated for C₃₇H₇₂N₄O₂-

4TFA: C, 50.94; H, 7.22; N, 5.28; F, 21.49; Found: C,

49.88; H, 7.00; N, 5.25; F, 22.15.

1

solid. 34: mp. 135–136°C; H NMR (200 MHz, CDCl₃): ␦

0.73 (s, 3 H), 0.86–0.93 (m, 12 H), 3.21–3.29 (broad m, 1

H), 3.91 (s, 4 H), 4.75–4.89 (m, 1 H), 7.37–7.52 (m, 4 H),

8.02 (d, J ϭ 9 Hz, 1 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦

11.2, 11.9, 17.0, 18.5, 18.6, 21.2, 25.9, 28.3, 30.3, 31.0,

31.7, 33.2, 33.8, 34.5, 35.3, 35.5, 36.9, 39.6, 40.3, 43.1,

52.0, 54.8, 55.0, 63.9, 76.5, 77.2, 108.7, 128.0, 129.3, 130.6,

132.4, 165.7; MS (FD) m/z 566; Analysis calculated for

C₃₆H₅₃O₅: C, 76.42; H, 9.44; Found: C, 76.16; H, 9.57.

2.2.2.15.

3,3-(Ethylenedioxy)-24-oxo-5␣-cholest-22-en-

7␤-yl benzoate (32). A solution of 21 (4.0 g, 8.7 mmoles) in

298

Y. Shu et al. / Steroids 67 (2002) 291–304

2.2.2.18. 3-Oxo-24R-hydroxy-5␣-cholestan-7␤-yl benzoate

(35). Ketal 34 (2.2 g, 3.9 mmoles) was deprotected with

HCl in acetone as described in the preparation of 9. The

desired product 35 (1.9 g, 94%) was identiﬁed using ¹H and

¹³C NMR and carried onto the next step without further

characterization. 35: ¹H NMR (200 MHz, CDCl₃): ␦ 0.76 (s,

3 H), 0.87–0.94 (m, 9 H), 1.10 (s, 3 H), 3.23–3.31 (broad m,

1 H), 4.76–4.89 (m, 1 H), 7.40–7.56 (m, 4 H), 8.02 (d, J ϭ

9 Hz, 1 H); ¹³C NMR (50.3 MHz, CDCl₃): ␦ 11.1, 11.9,

17.0, 18.4, 18.6, 21.4, 25.8, 28.2, 30.2, 31.6, 33.2, 33.9,

34.6, 35.2, 37.5, 39.4, 43.0, 43.4, 51.4, 54.8, 76.3, 76.4,

128.0, 129.3, 130.3, 132.5, 165.6, 210.5.

(s, 3 H), 0.92–0.95 (m, 9 H), 3.01–3.15 (m, 10 H), 4.08

(broad m, 1 H); MS (ϩFAB) m/z 685.5 (Mϩ1); Analysis

calcd. for C₃₇H₇₂N₄O₅S-3TFA: C, 50.28; H, 7.36; N, 5.45;

F, 16.65; S, 3.12; Found: C, 50.11; H, 7.34; N, 5.36; F,

16.65; S, 2.94.

3. Results and discussion

3.1. Overview of aminosterol synthesis from stigmasterol

The structures of the aminosterols synthesized in this

report are shown in Fig. 1. Stigmasterol was chosen as the

starting material in the synthesis of MSI-1436 analogs both

with and without a 7-hydroxyl group. Stigmasterol is a

relatively inexpensive sterol, critical for the preparation of

signiﬁcant quantities of a multistep synthetic product. The

5-oleﬁn of stigmasterol provides a convenient method to

introduce the 7␤-hydroxy group via allylic oxidation. Fi-

nally, the 22-oleﬁn of stigmasterol provides access to the

22-aldehyde via ozonolysis. The aldehyde can be reacted

with the ylide 3-methyl-1-triphenylphosphoranylidene-bu-

tanone to generate the unsaturated 24-ketone, which can be

manipulated into the desired side chain structures.

2.2.2.19. 3-Oxo-5␣-cholestan-7␤-24R-diyl 7-benzoate 24-

sulfate (36). Compound 35 (1.0 g, 1.9 mmoles) was dis-

solved in 15 ml pyridine in a 250 ml round bottom ﬂask.

Sulfur trioxide pyridine complex (500 mg, 3.14 mmoles)

was added to the ﬂask. The mixed slurry was stirred at room

temperature for 1 h. Saturated NaCl solution (200 ml) was

added to the slurry. The solution was extracted with chlo-

roform (3X). The organic layer was dried over Na₂SO₄. The

solvent was removed in vacuo to afford 1.1 g (85%) of the

desired product 36 as a white solid. The ﬁnal product was

1

identiﬁed using H and ¹³C NMR and carried onto the next

step without further characterization. 36: ¹H NMR (200 MHz,

CDCl₃): ␦ 0.66 (s, 3 H), 0.75–0.83 (m, 9 H), 1.03 (s, 3 H),

4.05–4.12 (broad m, 1 H), 4.68–4.80 (m, 1 H), 7.34–7.50 (m,

4 H), 7.61–7.68 (m, 2 H), 7.96 (d, J ϭ 9 Hz, 1 H), 8.02–8.10

(m, 2 H), 8.77 (d, J ϭ 6 Hz, 1 H); ¹³C NMR (50.3 MHz,

CDCl₃): ␦ 11.4, 12.0, 17.7, 17.8, 18.6, 21.6, 26.0, 26.6, 28.3,

30.2, 30.4, 30.7, 34.2, 34.8, 35.3, 37.8, 39.5, 43.2, 43.7, 51.7,

54.7, 55.0, 76.6, 84.7, 125.3, 126.2, 128.3, 129.4, 130.6, 132.8,

143.4, 143.9, 165.8, 210.7.

3.2. The synthetic route to MSI-1436 analogs without a

7-hydroxyl

The synthetic route for MSI-1436 analogs lacking a

7-hydroxyl substituent is shown in Figs. 2 and 3. Stigma-

sterol 1 was ﬁrst oxidized to stigmasta-4,22-dien-3-one 2

via the Oppenauer oxidation [21]. A large excess of 4-meth-

yl-2-pentanone (b.p. 117–118°C) was used as the hydrogen

acceptor to allow the reaction to be run at elevated temper-

ature. The conjugate reduction of stigmasta-4,22-dien-3-one

2 to the saturated ketone 3 was achieved by lithium ammo-

nia reduction; the more thermodynamically stable A/B trans

ring junction was stereoselectively obtained. The reduction

was quenched with solid ammonium chloride to yield the

saturated ketone. After protection of the 3-ketone, ozonol-

ysis was used to convert the 22 oleﬁn 4 to the 22 aldehyde

5. The reaction was quenched by the addition of trimethyl

phosphite while still chilled in a dry ice-ethanol bath. The

time chosen to stop the reaction was critical, since a small

amount of overoxidized product was obtained if excess

ozone stayed in the solution for a small period of time. The

overoxidized product was isolated and determined to be the

22 carboxylic acid by NMR. The ﬁnal product can be either

recrystallized from cyclohexane-methylene chloride or pu-

riﬁed on a silica gel column. Since the 22 aldehyde can

easily be oxidized to the 22 carboxylic acid even by air,

column chromatography was the preferred choice for puri-

ﬁcation. Column chromatography will also remove traces of

the 20-R 22-aldehyde generated by epimerization during

ozonolysis.

2.2.2.20. Spermine conjugates of 3-oxo-24R-hydroxy-5␣-

cholestan-7␤-yl sulfate (37 and 38). Spermine was added to

36 (600 mg, 0.88 mmoles) in the same manner as described

in the synthesis of 10 and 11. The crude addition product

(630 mg, 83%) was dissolved in 300 ml of deionized water

in a 500 ml round bottom ﬂask equipped with a heating

mantle, condenser and magnetic stirring bar. Lithium hy-

droxide (500 mg, 20.8 mmoles) was added to the ﬂask. The

solution was reﬂuxed overnight. After cooling to room

temperature, the solution was acidiﬁed with a 1% triﬂuoro-

acetic acid solution to pH 5, then puriﬁed using reverse

phase HPLC (Whatman C-18 column, eluting with 0.2%

TFA and a gradient of 40% acetonitrile containing 0.2%

(v/v) TFA), to afford the less polar ␣-isomer 37 and the

more polar ␤-isomer 38 as the triﬂuoroacetate salts.

1

37: H NMR (400 MHz, CD₃OD): ␦ 0.70 (s, 3 H), 0.85

(s, 3 H), 0.92–0.95 (m, 9 H), 3.01–3.15 (m, 10 H), 3.37

(broad s, 1 H), 4.08 (broad m, 1 H); MS (ϩFAB) m/z 685.5

(Mϩ1); Analysis calculated for C₃₇H₇₂N₄O₅S-3TFA: C,

50.28; H, 7.36; N, 5.45; F, 16.65; S, 3.12; Found: C, 50.11;

H, 7.47; N, 5.39; F, 16.56; S, 3.42.

1

38: H NMR (400 MHz, CD₃OD): ␦ 0.70 (s, 3 H), 0.86

The 3,3-(ethylenedioxy)-5␣-cholest-22-en-24-one 6 was

Y. Shu et al. / Steroids 67 (2002) 291–304

299

Fig. 2. The synthetic scheme describing the preparation of 3,3-(ethylenedioxy)-5␣-cholestan-24-one 7, an intermediate in the synthesis of MSI-1436 analogs

without a 7-hydroxyl substituent. Compound numbers and synthetic yields are indicated in the scheme adjacent to the compounds.

prepared from the 22 aldehyde 5 via a Wittig addition of

3-methyl-1-triphenylphosphoranylidene-2-butanone, fol-

lowed by hydrogenation catalyzed by palladium on carbon

to get the intermediate 7.

3.3. The synthetic route to 7␤-hydroxyl MSI-1436 analogs

The synthetic route describing the preparation of MSI-

1436 analogs with a 7␤-hydroxyl substituent is shown in

Figs. 4 and 5. The synthesis begins with the allylic oxidation

of stigmasterol by O₂oxidation to the 7-hydroperoxide in

the presence of N-hydroxyphthalimide and benzoyl perox-

ide as a radical initiator [15,24]. The hydroperoxide was

dehydrated to the 7-oxosterol using copper chloride. Lith-

ium ammonia reduction of the 5-enone 16 gave the satu-

rated 7␤-alcohol 17; methanol was used as the quenching

reagent to yield the desired alcohol. The 5␣-stigmast-22-

en-3␤,7␤-diol 17 was then selectively oxidized with silver

carbonate on celite to the 7␤-hydroxy-3-one 18. Protection

of the 3-ketone 18 with ethylene glycol, ozonolysis to the 22

aldehyde 20, and a Wittig reaction generated the ␣,␤ unsat-

urated ketone 21.

Three intermediates were prepared from 3,3-(ethyl-

enedioxy)-7␤-hydroxy-5␣-cholest-22-en-24-one 21 (Fig.

5). First, 3,3-(ethylenedioxy)-7␤-hydroxy-5␣-cholestan-24-

one 22 was prepared via hydrogenation with palladium on

carbon. Second, 3,3-(ethylenedioxy)-5␣-cholest-22-en-

7␤,24R-diol 27 was prepared via chiral reduction using

Corey’s oxazaborolidine methodology as described previ-

ously in our laboratory [16,25]. Third, the 7-hydroxyl group

was protected as the 7␤-benzoate 32.

The protected 3,24-dione 7 was used to generate four

different aminosterols (Fig. 3). First, the 24-ketone was

converted either into the 24-amino mixed diasteriomers 8 by

reductive amination, or converted into the 24-ol 12 by

reduction with sodium borohydride. After deprotection of

the 3-ketone, the last step for all the analogs was reductive

amination with the symmetrical polyamine spermine. Mo-

lecular sieves (3 Å) were used to remove the water and

make the formation of imine favored in the equilibrium.

Sodium cyanoborohydride or sodium borohydride was used

to reduce the imine. After workup, a mixture of 3␣- and

3␤-spermine isomers were obtained, which were separated

by reverse phase HPLC. Both the 24-amino analogs and

24-hydroxy analogs have poor solubility in water. In order

to dissolve the crude product, 5% acetonitrile was added to

a large amount of 0.2% triﬂuoroacetic acid in water to

improve the solubility. The crude solution was ﬁltered with

a membrane ﬁlter (pore size 0.45 ␮m) and then loaded on a

C-18 RP-HPLC column. There was signiﬁcant loss of prod-

uct during this process due to poor solubility. The ﬁnal

products were eluted from the HPLC column using an

acetonitrile gradient in the presence of triﬂuoroacetic acid.

Fractions were collected and lyophilized to give the white

crystalline products as their triﬂuoroacetate salts.

From 22, the 24-amine 23 was prepared as the mixed

diasteriomers via reductive amination. After deprotection of

the 3-ketone, the 24-amino-3-ketone was coupled with

300

Y. Shu et al. / Steroids 67 (2002) 291–304

Fig. 3. The synthetic scheme describing the synthesis of aminosterols without a 7-hydroxyl substituent from intermediate 7 (see Fig. 2). Compound numbers

and synthetic yields are indicated in the scheme adjacent to the compounds. The C24 substituents are synthesized as mixed isomers and cannot be separated.

spermine to make the 7␤-hydroxy-24-amino analogs as the

3␣- and 3␤-spermine mixed isomers. The mixed isomers

were puriﬁed using reverse phase HPLC to yield the ﬁnal

products 25 and 26.

From 27, the allylic alcohol was hydrogenated to the

saturated alcohol 28 with platinum oxide in ethyl acetate.

After deprotection of the 3-ketone, spermine was added by

reductive amination, followed by HPLC puriﬁcation, to

yield the 7␤,24R-dihydroxy analogs 30 and 31.

ate the 24 allylic alcohol 33, followed by hydrogenation to

the 24 saturated alcohol 34. After the deprotection of the

3-ketone, the next step in the scheme was the sulfation of

the 24-hydroxyl while the 7␤-hydroxyl was protected as the

benzoate ester. A large excess of SO₃/pyridine complex was

used to ensure complete sulfation. Due to the relative insta-

bility of the sulfate product, the crude product was identiﬁed

1

by H NMR and ¹³C NMR spectra and then carried on

without further puriﬁcation. After reductive amination of

From the 32, chiral reduction was ﬁrst applied to gener-

the sulfate product, the crude product was deprotected by

Y. Shu et al. / Steroids 67 (2002) 291–304

301

Fig. 4. Synthetic scheme describing the preparation of 3,3-(ethylenedioxy)-7␤-hydroxy-5␣-cholest-22-en-24-one 21, an intermediate in the synthesis of

MSI-1436 analogs with a 7␤-hydroxyl substituent. Compound numbers and synthetic yields are indicated in the scheme adjacent to the compounds.

the lithium hydroxide-catalyzed hydrolysis of the benzoate

ester. Without work up, the solution was acidiﬁed with a 1%

triﬂuoroacetic acid solution to pH 5. Reverse phase HPLC

was applied to separate and purify the 3␣- and 3␤-spermine

isomers (37 and 38) as their triﬂuoroacetate salts.

It is interesting to note that the 7␤-hydroxy group dra-

matically improves the solubility of all the analogs in water.

The reason for this is not entirely clear, but one possible

explanation is that analogs with a 7-hydroxy substituent in

either conﬁguration do not aggregate in water, as do analogs

without a 7-hydroxy group.

reported antimicrobial activities for both squalamine [1] and

MSI-1436 [5] shown for comparison. Table 1 shows that all

the analogs without the 7-hydroxy have similar antimicro-

bial activities, but are somewhat less potent than MSI-1436.

The 24-amino analogs are somewhat more effective than the

24-hydroxy analogs. The 7-hydroxy substituent may not be

important, since all the analogs without the 7-hydroxyl have

antimicrobial activities almost equal to that of MSI-1436.

Also, the 24-sulfate moiety on the side chain of squalamine

and MSI-1436 may not signiﬁcantly contribute to antimi-

crobial potency, as the 24-amino, 7-dihydro analogs have

antimicrobial activity similar to MSI-1436 on P. aerugi-

nosa. Simple carboxylic acids or carboxylic acid methyl

esters on the side chain also possess signiﬁcant antimicro-

bial activity [10].

3.4. Antimicrobial activities for MSI-1436 analogs

Antimicrobial activities for the MSI-1436 analogs syn-

thesized in this report are shown in Table 1, with the

Squalamine and MSI-1436 have similar antimicrobial

302

Y. Shu et al. / Steroids 67 (2002) 291–304

Fig. 5. Synthetic scheme describing the synthesis of MSI-1436 analogs with a 7␤-hydroxyl and 24R-sulfate substituent from intermediate 21 (see Fig. 4).

Compound numbers and synthetic yields are indicated in the scheme adjacent to the compounds. The 24-amino compounds are synthesized as mixed isomers

and cannot be separated.

Y. Shu et al. / Steroids 67 (2002) 291–304

303

Table 1

Antimicrobial activities for MSI-1436 analogs. The antimicrobial activities of the naturally occurring aminosterols squalamine [1] and MSI-1436 [5] are

included for comparison. Compound structures are provided in Fig. 1

Squalamine analog tested

Minimum inhibitory concentration (␮g/ml)

S. aureus

E. coli

P. aeruginosa

C. albicans

Squalamine

MSI-1436

1

4

1

16

4

16

4

Analogs without a 7-hydroxyl

24-Amino-3␣-spermine (10)

24-Amino-3␤-spermine (11)

24-Hydroxy-3␣-spermine (14)

24-Hydroxy-3␤-spermine (15)

Analogs with a 7␤-hydroxyl

1–2

2–4

4–8

4

8

16

8

4

32

2

4

64

7␤-Hydroxy-24-amino-3␣-spermine (25)

7␤-Hydroxy-24-amino-3␤-spermine (26)

7␤-Hydroxy-24R-hydroxy-3␣-spermine (30)

7␤-Hydroxy-24R-hydroxy-3␤-spermine (31)

7␤-Hydroxy-24R-sulfate-3␣-spermine (37)

7␤-Hydroxy-24R-sulfate-3␤-spermine (38)

2–4

4

4–8

16

8

64–128

32

64

256

128

32

128

8

64

16

4

16

64

16

2–4

8–16

activities; the spermidine moiety of squalamine can appar-

ently be replaced with spermine with little affect upon

antimicrobial activity. The stereochemistry at the polyamine

addition site seems to have a small affect on antimicrobial

activity. In general, the 3␤-analogs have slightly better

antimicrobial activity than the corresponding 3␣-analogs.

The analogs with a 7-hydroxy substituent appear to be the

exception; however, the differences between the two iso-

mers are not signiﬁcant. It is interesting to note that the

7␤-hydroxy-24R-sulfate-3␣-spermine analog is one of the

least active compounds, while the 7␤-hydroxy-24R-sulfate-

3␤-spermine analog shows almost equal activity to that of

MSI-1436.

The wide structural variety of aminosterols with antimi-

crobial activity supports a nonspeciﬁc mechanism for anti-

microbial activity. We have proposed that the positively

charged polyamine of the aminosterol binds to membrane

phosphate groups. After membrane binding, holes are gen-

erated, resulting in cell death [26]. Apparently, any mole-

cule with a polyamine and a cholestane ring has antibacte-

rial activity. Antimicrobial potency and selectivity arises

from the chemical nature and stereochemistry of substitu-

tions on the sterol ring and side chain.

In conclusion, a number of aminosterols with spermine

attached at C3 were successfully synthesized from stigma-

sterol. The synthesis of the analogs without a 7-hydroxyl

was accomplished in 9 steps. Four analogs were obtained,

including 24-amino-3␣-spermine-5␣-cholestane, 24-amino-

3␤-spermine-5␣-cholestane, 24-hydroxy-3␣-spermine-5␣-

cholestane and 24-hydroxy-3␤-spermine-5␣-cholestane. All

four analogs were prepared as mixed diasteriomers, since

the introduction of functionality at C24 was not stereose-

lective. The synthesis of the analogs with a 7␤-hydroxyl

substituent was accomplished in 9–13 steps. Six analogs

were obtained, including 7␤-hydroxy-24-amino-3␣-sperm-

ine-5␣-cholestane, 7␤-hydroxy-24-amino-3␤-spermine-5␣-

cholestane, 7␤,24R-dihydroxy-3␣-spermine-5␣-cholestane,

7␤,24-dihydroxy-3␤-spermine-5␣-cholestane, 7␤-hydroxy-

3␣-spermine-5␣-cholestan-24-yl sulfate and 7␤-hydroxy-

3␤-spermine-5␣-cholestan-24-yl sulfate. The 7␤-hydroxy-

24-amino analogs were prepared as mixed diasteriomers.

The others were prepared as a single diasteriomer and the

stereochemistry at C24 is the same as that for MSI-1436.

All the analogs exhibit potent, broad-spectrum antimi-

crobial activity, with some approaching the activity exhib-

ited by MSI-1436. Changing the functionality on C24 at the

side chain has little effect upon antimicrobial activity. A

7-hydroxyl substituent does not seem essential to the activ-

ity of squalamine. The stereochemistry on C7, however,

does affect the activity, especially with a simultaneous

change of stereochemistry at C3. All the analogs are syn-

thesized with high yield from an inexpensive starting ma-

terial and are promising alternatives to squalamine as po-

tential antibiotics.

Acknowledgments

The authors would like to thank the Genaera Corporation

for ﬁnancial support of this research, including graduate

fellowships to Y.S. and S.J. We would also like to Lisa M.

Jones of Genaera Corporation for performing the antimicro-

bial assays on the aminosterols.

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Article Doi

The synthesis of spermine analogs of the shark aminosterol squalamine

DOI: 10.1016/S0039-128X(01)00161-1

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Article abstract of DOI:10.1016/S0039-128X(01)00161-1

Full text of DOI:10.1016/S0039-128X(01)00161-1

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