Synthesis of 14′,15′-dehydro-ritterazine y via reductive and oxidative functionalizations of hecogenin acetate

Article abstract of DOI:10.1016/j.steroids.2012.10.021

An analog of ritterazine Y was synthesized from hecogenin acetate in 23 steps via functional group manipulations of hecogenin acetate. Preparation of the north G and south Y units and the late stage Guo-Fuchs asymmetric coupling of the both units afforded

Full text of DOI:10.1016/j.steroids.2012.10.021

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Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of 140,150-dehydro-ritterazine Y via reductive and oxidative
functionalizations of hecogenin acetate
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Yi Kou, Young Cheun, Myong Chul Koag, Seongmin Lee
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The Division of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin, Austin, TX 78712, USA
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a r t i c l e i n f o
a b s t r a c t
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Article history:
Received 4 July 2012
Received in revised form 5 October 2012
Accepted 29 October 2012
Available online xxxx
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An analog of ritterazine Y was synthesized from hecogenin acetate in 23 steps via functional group
manipulations of hecogenin acetate. Preparation of the north G and south Y units and the late stage
Guo–Fuchs asymmetric coupling of the both units afforded the ritterazine Y analog.
Ó 2012 Elsevier Inc. All rights reserved.
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Keywords:
Cephalostatin
Ritterazine
Steroidal alkaloid
Anticancer steroids
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1. Introduction
1 and OSW-1 inhibit novel anticancer targets called oxysterol-
binding protein (OSBP) and OSBP-related proteins (ORP)
[12,18,19]. These highly oxygenated steroidal anticancer agents
tightly bind OSBP and reduce OSBP level via proteasome-dependent
degradation [12].
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The cephalosatin/ritterazine family comprises of 45 members of
bissteroidal pyrazine marine natural products; 19 cephalostains (1–
19) from the marine tube worm Cephalodiscus gilchristi [1–4] and 26
ritteazines (A–Z) from the tunicate Ritterella tokioka [5,6]. They are
structurally related, featuring two C27 sterodial units (referred to
as north and south, Fig. 1) and a central pyrazine ring. Cephalostatin
1 1 and ritterazine B 2, the two most potent members of the family,
are among the most powerful anticancer agents ever tested by the
NCI with avg. GI50 1.8 and 3.2 nM, respectively, in the NCI 60 cancer
cell lines. The cytotoxicity profiles of these anticancer agents had no
significant correlation to any molecule of known mechanism of ac-
tion. Cephalostatin 1 1 has shown to induce apoptosis in a novel
mitochondrial pathway [7–11]. Interestingly, cephalostatin 1 1
and ritterazine B 2 are about ꢀ50- to 250-fold more cytotoxic to cells
with mutations in p21 tumor suppressor gene [12]. A cephalostatin
analog has also shown selective cytotoxicity to cells with mutations
in ras tumor suppressor genes [13], which is important in the etiol-
ogy of many cancers, implicating cephalostatin/ritterazine as poten-
tial ‘‘synthetic lethal’’ anticancer agents [12]. Another class of
anticancer steroids exhibiting cephalostatin-like activity is OSW-1
3, a mono-steroidal glycoside from the bulb of Ornithogalum
saundersiae [14–17]. Surprisingly, the cytotoxicity profile of OSW-
1 3 was very similar to that of cephalostatins with correlation coef-
ficient of 0.60–0.83, suggesting that they share the same mechanism
of action. Indeed, very recently, it has shown that both cephalostatin
Because of medical significance of cephalostatins and ritterazines,
many research groups including us reported the synthesis of the nat-
ural bissteroidal pyrazines (e.g., cephalostatin 1 and 7, ritterazine K
and M) and their analogs [20–28]. Ritterazine Y 4 is an interesting
molecule, because, although it lacks 70-OH and 170-OH functionalities
of ritteazine B 2 (Fig. 2), it has been shown to exhibit considerable
cytotoxicity against P388 murine cancer cell line [5]. In conjunction
with efforts to develop efficient synthetic routes for cephalostatins
and ritterazines, the Fuchs laboratory has embarked on a ‘‘reduc-
tion/oxidation’’ strategy [29,30] where they seek to prepare target
compounds using multiple reductive/oxidative functionalizations of
commercially available hecogenin acetate 6 with retention of all the
27 carbon atoms in the starting material (Fig. 3). Herein, we report
the synthesis of 140,150-dehydro-ritterazine Y 5 via reductive/oxida-
tive functionalizations of hecogenin acetate, which involves the
synthesis of the north G and the south Y hemispheres and an
asymmetric coupling of the both hemispheres.
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2. Experimental
2.1. General methods
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Boron trifluoride etherate (BF3OEt2), triethylsilane (Et3SiH),
imidazole, iodine, iodobenzene diacetate (PhI(OAc)2), potassium
carbonate, and N,N-dimethyl formamide (DMF) were purchased
⇑
Corresponding author. Tel.: +1 512 471 1785; fax: +1 512 471 4726.
E-mail address: SeongminLee@mail.utexas.edu (S. Lee).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.10.021
Please cite this article in press as: Kou Y et al. Synthesis of 140,150-dehydro-ritterazine Y via reductive and oxidative functionalizations of hecogenin ace-
tate. Steroids (2012), http://dx.doi.org/10.1016/j.steroids.2012.10.021

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Fig. 1. Anticancer steroids. Cephalostatin 1, ritterazines B and Y, and OSW-1.
Q4
Fig. 2. Anticancer steroids. Cephalostatin 1, ritterazines B and Y, and OSW-1.
to purify the products. 1H and 13C NMR spectra were generated
by Varian MERCURY 400 (400 MHz). CDCl3 was used as the NMR
standard. Peak multiplicates in 1H NMR spectra, when reported,
were abbreviated as s (singlet), d (doublet), t (triplet), m (multi-
plet), ap (apparent), and br (broad). High-resolution mass
spectrometry data were generated by Agilent 6530 Accurate-Mass
Q-TOF LC/MS.
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2.2. Chemical synthesis
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2.2.1. 3b-Acetoxy-12b-benzyloxy-5a-spirostan-14-ene (10)
To a solution of ketone 9 (12.7 g, 27.0 mmol) in THF/MeOH (1:1,
135 mL/135 mL) was added cerium chloride heptahydrate (7.03 g,
18.9 mmol) at 0 °C and the mixture was stirred for 30 min at the
same temperature. Sodium borohydride (1.53 g, 40.5 mmol) was
added portionwise over 1 h and the resulting mixture was stirred
for additional four hours. The reaction was quenched by adding
water (250 mL) to give precipitates, which were removed by filtra-
tion. The filtrate was concentrated under reduced pressure, and par-
titionedbetween ethylacetate(200 mL) and water. The organic layer
was washed with brine, dried over Na2SO4, concentrated, and sub-
Fig. 3. Strategy for synthesis of 140,150-dehydro-ritterazine
acetate.
Y form hecogenin
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from Acros Organics (Geel, Belgium). Methylene chloride (dichlo-
romethane or DCM), tetrahydrofuran (THF), and methanol were
purchased from Fisher Chemical (Farlawn, NJ). Triphenylphosphine
(PPh3) was purchased from Alfa Aesar (Ward Hill, MA). Sodium
azide was purchase from MP Biomedicals, LLC (Solon, OH). All reac-
tions were performed under positive pressure of argon in anhy-
drous solvents. Each reaction progress was monitored by thin
layer chromatography (TLC). TLC Silica gel 60 F254 glass plates from
EMD Chemicals Inc. (Darmstadt, Germany) and appropriate solvent
systems were used for TLC development. TLC plates were visual-
ized by ultraviolet illumination (254 nm) and p-anisaldehyde
solution (4 mL of concentrated sulfuric acid, 800 mL of ethanol,
1.2 mL of acetic acid, and 1.6 mL of p-anisaldehyde). Analytical
samples were prepared via flash silica gel chromatography. 60 Å
silica from Bonna-Agela technologies (Wilmington, DE) was used
jected to sgc to give C12-b alcohol (11.0 g, 87%) and C12-a alcohol
(1.04 g, 8%). To a pyridine (120 mL) solution of the C12-b alcohol
(11.0 g, 23.3 mmol) was added benzoyl chloride (4.89 g, 35.0 mmol)
and the resulting mixture was stirred for 6 h. The reaction was
quenched by adding saturated aqueous sodium bicarbonate. The
resulting mixture was concentrated in vacuo, and subjected to silica
gel column chromatography to provide benzoate 10 (12.6 g, 94%) as
white solids (mp, 171–173 °C).
1H NMR (300 MHz, CDCl3) d 7.39–8.05 (5H, m), 5.46 (1H, s, C14-
H), 4.86 (1H, d, C12-H), 4.59–4.74 (2H, m, C3-H, C16-H), 3.32–3.50
(2H, m, C26-H), 2.43 (1H, t), 2.12 (1H,), 1.99 (3H, s, C3-OAc), 1.22
(3H, s), 0.86 (3H, d), 0.84 (3H, s), 0.74 (3H, d); 13C NMR (75 MHz,
Please cite this article in press as: Kou Y et al. Synthesis of 140,150-dehydro-ritterazine Y via reductive and oxidative functionalizations of hecogenin ace-
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CDCl3) d 170.6, 165.7, 156.5, 132.4, 130.4, 129.4, 128.1, 120.2,
106.6, 84.3, 81.6, 73.0, 66.2, 55.7, 52.0, 51.2, 44.2, 36.6, 36.0, 31.0,
30.0, 29.2, 28.7, 27.2, 26.5, 21.0, 17.0, 14.8, 13.7, 12.0. 1H and 13C
NMR spectral data are consistent with known values [25].
and dried by vacuum to give tertiary alcohol 13 (381 mg, 66%) as
a clear oil.
1H NMR (300 MHz, CDCl3) d 8.00–7.97 (2H, m), 7.52–7.48 (1H,
m), 7.41–7.28 (1H, m), 5.40 (1H, s), 4.78 (1H, d, J = 5.1 Hz), 4.19
(1H, d, J = 7.8 Hz), 4.64 (1H, m), 4.55 (1H, m), 2.20–2.11 (1H, m),
2.09–1.97 (1H, m), 1.94 (3H, s), 1.18 (3H, s), 1.13 (6H, s), 0.83
(3H, s), 0.80 (3H, s); 13C NMR (300 MHz, CDCl3) d 170.4, 170.3,
165. 7, 165.6, 157.0, 153.9, 132.9, 132.8, 130.3, 130.1, 129.3,
128.4, 128.3, 120.5, 119.9, 87.4, 86.8, 86.2, 85.9, 81.5, 78.5, 73.1,
69.9, 69.9, 59.3, 55.8, 51.8, 51.6, 50.4, 50.3, 44.2, 44.0, 41.1, 40.8,
40.5, 36.4, 36.3, 35.7, 35.5, 34.5, 34.3, 34.0, 33.6, 31.4, 29.3, 29.2,
29.2, 27.9, 27.4, 27.2; HRMS for C30H50O6 (M+H) calcd: 579.3680,
found: 579.3683.
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2.2.2. 3b-Acetoxy-12b-benzyloxy-5a-furostan-26-hydroxy-14-ene
(11)
To a CH2Cl2 solution of benzoate 10 (5.76 g, 10 mmol) and tri-
ethylsilane (3.19 mL, 20 mmol) was added dropwise CH2Cl2
(100 mL) solution of boron trifluroide diethyletherate (2.13 g,
15 mmol) over a period of 1 h at 0 °C, and the resulting mixture
was stirred for 18 h at 25 °C. The reaction mixture was quenched
by slowly adding saturated aqueous sodium bicarbonate, extracted
with CH2Cl2, dried over anhydrous Na2SO4, concentrated under re-
duced pressure, and subjected to silica gel chromatography to yield
a primary alcohol 11 (5.40 g, 94%) as a clear oil.
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2.2.5. 3b-Acetoxy-12b-benzyloxy-5a-spirostan-14-ene (14)
To a CH2Cl2 solution of PhI(OAc)2 (423.5 mg, 1.3 mmol) and io-
dine (333.5 mg, 1.3 mmol) was added tertiary alcohol 13
(380.5 mg, 0.66 mmol) in CH2Cl2 dropwise at 0 °C, and then the
resulting mixture was stirred for 3 h. The reaction mixture was
quenched by aqueous saturated Na2S2O3, extracted with dichloro-
methane (3ꢁ30 ml), washed by the brine, dried over sodium sul-
fate, and subjected to silica gel column chromatography to give
5/5 spiroketal 14 (262 mg, 69%) as a clear oil.
1H NMR (300 MHz, CDCl3) d 7.39–8.05 (5H, m), 5.44 (1H, s, C14-
H), 4.74 (1H, d, C12-H), 4.61–4.72 (2H, m, C3-H, C16-H), 3.42 (2H,
m, C26-H), 3.21 (1H, m, C22-H), 2.21 (1H, t), 2.09 (1H, m), 1.99 (3H,
s, C3-OAc), 1.24 (3H, s), 0.86 (3H, d), 0.84 (3H, s), 0.79 (3H, d); 13C
NMR (75 MHz, CDCl3) d 170.6, 165.7, 157.0, 132.8, 130.2, 129.0,
128.2, 119.9, 87.1, 85.7, 81.6, 73.0, 67.7, 59.2, 51.6, 44.1, 40.8,
36.2, 35.6, 33.9, 33.6, 30.0, 29.8, 29.2, 28.7, 27.9, 27.0, 26.5, 20.8,
16.2, 15.8, 11.8. 1H and 13C NMR spectral data are consistent with
known values [25].
1H NMR (300 MHz, CDCl3) d 8.03–8.00 (2H, m), 7.56–7.51 (1H,
m), 7.44–7.39 (2H, m), 5.43 (1H, s), 4.93 (1H, d, J = 6.6 Hz), 4.66–
4.64 (1H, m), 4.62–4.57 (1H, m), 2.46–2.41 (1H, m), 1.99 (3H, s),
1.32 (3H, s), 1.22 (6H, s), 1.09 (3H, s), 0.87 (3H, s); 13C NMR
(75 MHz, CDCl3) d 170.4, 165.7, 156.0, 132.7, 130.5, 129.3, 128.2,
120.5, 117.2, 84.1, 81.8, 81.4, 73.2, 55.9, 52.1, 51.4, 44.1, 41.1,
37.1, 36.4, 35.9, 33.9, 33.7, 33.1, 29.9, 29.4, 28.3, 28.0, 27.1, 26.4,
21.3, 15.0, 14.0, 11.9. HRMS for C30H48O6 (M+H) calcd: 577.3524,
found: 577.3522.
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2.2.3. 3b-Acetoxy-12b-benzyloxy-5a-furostan-14,26-diene (12)
A primary alcohol 11 (5.28 g, 9.13 mmol), triphenyl phosphine
(4.78 g, 18.3 mmol), and imidazole (3.13 g, 45.7 mmol) were dis-
solved in THF (90 mL), iodine (4.60 g, 18.3 mmol) was added over
a period of 30 min, and the resulting mixture was stirred for 2 h
at an ambient temperature. The reaction mixture was quenched
by adding saturated sodium thiosulfate solution, extracted with
ethyl acetate, washed with brine, dried over anhydrous sodium
thiosulfate, concentrated under reduced pressure to give a crude
mixture of the corresponding primary iodide. To a DMF (45 mL)
solution of the iodide was added DBU (2.78 mL, 18.3 mmol), and
the resulting mixture was stirred at 25 °C. After 12 h, the reaction
mixture was partitioned between ethylacetate (450 mL) and water
(450 mL). The organic layer was washed with brine and saturated
lithium chloride solution, dried over anhydrous sodium sulfate,
and concentrated in vacuo, and subjected to a silica gel chromatog-
raphy to give diene 12 (3.96 g, 78%) as white solids (mp, 108–
110 °C).
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2.2.6. 3b-Hydroxy-12b-benzyloxy-5a-spirostan-14-ene (15)
To acetate 14 (250 mg, 0.43 mmol) in 10 ml MeOH was added
K2CO3 (120 mg, 0.87 mmol), and the mixture was stirred at room
temperature for 2 h. The reaction was quenched by adding satu-
rated aqueous ammonium chloride, extracted with ethyl acetate
(3ꢁ30 ml), and washed with brine. The combined organic layer
was dried over sodium sulfate, concentrated under reduced pres-
sure, and subjected to silica gel column chromatography to give
alcohol 15 (185 mg, 80%) as a clear oil.
1H NMR (300 MHz, CDCl3) d 8.02–7.99 (2H, m), 7.58–7.50 (1H,
m), 7.43–7.38 (2H, m), 5.42 (1H, s), 4.94–4.91 (1H, m), 4.60–4.55
(1H, m), 3.61–3.50 (1H, m), 2.42 (1H, t, J = 9.0 Hz), 2.19–1.97 (2H,
m), 1.95 (1H, s), 1.31 (3H, s), 1.22 (6H, s), 1.08 (3H, s), 0.84 (3H,
s); 13C NMR (75 MHz, CDCl3) d 165.9 156.2, 132.8, 130.6, 129.4,
128,3, 120.4, 117.3, 84.2, 81.9, 81.6, 70.9, 55.9, 52.3, 51.5, 44.4,
41.2, 37.8, 37.1, 36.7, 36.0, 34.6, 34.1, 33.1, 31.5, 31.2, 29.9, 28.3,
29.6, 28.2, 29.5, 26.5, 25.2, 22.6, 20.6, 15.0, 14.1, 14.0, 12.0; HRMS
for C34H46O5 (M+Na) calcd: 557.3238, found: 557.3237.
1H NMR (300 MHz, CDCl3) d 7.41–8.02 (5H, m), 5.52 (1H, s),
4.65–4.82 (4H, m), 3.27 (1H, m), 1.99 (3H, s), 1.70 (3H, s), 1.24
(3H, s), 0.95 (3H, s), 0.80 (3H, d, J = 7.1); 13C NMR (75 MHz, CDCl3)
d 170.6, 165.7, 163.5, 145.7, 133.0, 130.4, 129.4, 128.5, 120.2, 86.3,
85.6, 81.7, 73.3, 59.2, 51.7, 44.2, 41.0, 36.6, 36.4, 35.7, 33.7, 33.1,
31.2, 29.2, 28.7, 27.2, 26.3, 22.4, 21.0, 16.9, 15.8, 12.0. 1H and 13C
NMR spectral data are consistent with known values [25].
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2.2.7. 12b-Benzyloxy-3-keto-5a-spirostan-14-ene (16)
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2.2.4. 3b-Acetoxy-12b-benzyloxy-5
(13)
a
-furostan-25-hydroxy-14-ene
To the secondary alcohol 15 (180 mg, 0.34 mmol) in acetone
was added Jones reagent (0.23 ml, 0.31 mmol) dropwise at 0 °C.
The reaction was quenched after 10 min with saturated Na2S2O3
solution, and then extracted with ethyl acetate (3ꢁ30 ml). The or-
ganic layer was washed with brine, dried over anhydrous sodium
sulfate, and purified by silica gel column chromatography to give
ketone 16 (157 mg, 87%) as a clear oil.
To terminal olefin 12 (560 mg, 1 mmol) in 6 ml of THF wrapped
with aluminum foil, was added PhI(OAc)2 (350.5 mg, 1.1 mmol) in
4 ml of H2O and 6 ml of THF. The reaction proceeded at room tem-
perature overnight in dark, and was added NaBH4 (370 mg,
10 mmol) afterwards. After 2 more hours, the reaction was
quenched with 5 N ammonium chloride solution. Then mixture
was extracted with 30 ml ethyl acetate for 3 times and organic
layer was washed with brine. After drying over sodium sulfate,
the organic layer was concentrated and taken to silica gel column
chromatography using a gradient of 4:1 to 1:1 of ethyl acetate to
hexane to elute. The final product was collected, concentrated
1H NMR (300 MHz, CDCl3) d 8.02–7.99 (2H, m), 7.56–7.52 (1H,
m), 7.44–7.39 (2H, m), 5.44 (1H, s), 4.95–4.91 (1H, m), 4.62–4.57
(1H, m), 2.47–2.41 (1H, m), 2.33–2.19 (2H, m), 2.16–1.95 (3H,
m), 1.30 (3H, s), 1.23 (6H, d, J = 6.9 Hz), 1.08 (3H, s), 1.03 (3H, s);
13C NMR (75 MHz, CDCl3) d 211.1, 165.8, 155.5, 132.9, 130.5,
129.4, 128.3, 121.0, 117.3, 84.1, 82.0, 81.3, 55.9, 51.7, 51.5, 46.0,
Please cite this article in press as: Kou Y et al. Synthesis of 140,150-dehydro-ritterazine Y via reductive and oxidative functionalizations of hecogenin ace-
tate. Steroids (2012), http://dx.doi.org/10.1016/j.steroids.2012.10.021

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44.3, 41.2, 38.0, 37.8, 37.1, 36.0, 34.6, 33.9, 33.1, 31.5, 29.9, 29.6,
29.2, 28.4 26.64 25.2, 22.6, 15.0, 14.0, 11.2; HRMS for C34H44O5
(M+Na) calcd: 555.3081, found: 555.3081.
2.2.11. 2
ene (20)
To azido methoxime 19 (40 mg, 0.07 mmol) in 1.5 ml THF and
l of water was added PPh3 (52 mg, 0.2 mmol), and the mixture
was stirred at room temperature for 2 h. The reaction mixture was
quenched with water and extracted with ethyl acetate (3ꢁ20 ml).
The combined organic layer was washed with brine, dried over so-
dium sulfate, concentrated in vacuo, and the subjected to silica gel
column chromatography using to give aminomethoxime 20
(38 mg, 99%) as a white foam.
a-Amino-3-(methoxyimino)-12b-benzyloxy-5a-spirostan-14-
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2.2.8. 2a-Bromo-3-keto-12b-benzyloxy-5a-spirostan-14-ene (17)
To the ketone 16 (142 mg, 0.27 mmol) in 5 ml of THF was added
PTAB (91.0 mg, 0.24 mmol) at 0 °C. After 30 min, the reaction was
quenched with saturated Na2S2O3 solution and extracted with
ethyl acetate (3ꢁ30 ml). The combined organic layer was washed
with brine, dried over sodium sulfate, concentrated under reduced
pressure, and subjected to silica gel column chromatography to
give bromoketone 17 (125 mg, 77%) as a clear oil.
1H NMR (300 MHz, CDCl3) d 8.02–8.00 (2H, m), 7.57–7.52 (1H, t,
J = 7.5 Hz), 7.45–7.40 (2H, m), 4.94 (1H, d, J = 7.2 Hz), 4.62–4.57
(1H, dd, J = 4.5 and 11.1 Hz), 3.95–3.88 (1H, dd, J = 6.3 Hz), 2.98
(1H, s), 2.46–2.40 (1H, m), 2.35–2.24 (1H, m), 1.95 (2H, s), 1.31
(3H, s), 1.23 (6H, d, J = 6.3 Hz), 1.08 (3H, s), 0.96 (3H, s). 13C NMR
(75 MHz, CDCl3) d 204.5, 165.8, 154.9, 132.9, 130.3, 129.4, 128.3,
121.3, 117.3, 83.96, 82.0, 80.9, 63.5, 55.9, 51.5, 51.4, 46.9, 44.9,
43.4, 41.1, 37.2, 37.1, 33.3, 33.1, 31.5, 29.9, 29.6, 29.0, 28.3, 27.8,
26.7, 22.6, 15.0, 14.1, 14.1, 12.2; HRMS for C35H49N2O5 (M+H)
calcd: 577.3636, found: 577.3635.
1H NMR (300 MHz, CDCl3) d 8.02–7.99 (2H, m), 7.56–7.51 (1H,
m), 7.44–7.39 (2H, m), 5.45 (1H, s), 4.94–4.91 (1H, m) 4.70–4.63
(1H, m), 4.62–4.56 (1H, m), 2.60–2.50 (1H, m), 2.61–2.38 (5H,
m), 2.16–2.05 (1H, m), 1.30 (3H, s), 1.23 (6H, d, J = 6 .0 Hz), 1.11
(3H, s), 1.08 (3H, s); 13C NMR (75 MHz, CDCl3) d 200.4, 165.8,
154.8, 133.0, 130.3, 129.34, 128.3, 121.9, 117.3, 84.0, 82.0, 80.9,
55.9, 53.5, 51.7, 51.5, 51.3, 50.9, 46.8, 43.5, 41.1, 39.1, 38.0, 37.1,
36.0, 34.6, 34.4, 33.4, 33.1, 31.5, 29.9, 29.6, 28.9, 28.3, 27.8, 26.7,
25.2, 22.6, 20.6, 15.0, 14.1, 14.0, 11.8, 11.4; HRMS for C34H43BrO5
(M+Na) calcd: 633.2186, found: 633.2184.
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
2.2.12. 3b-Acetoxy-12b,25-dibenzyloxy-5a-26-OTBS-furostan-14-ene
(22)
To a co-solvent of t-BuOH (5 mL) and H2O (5 mL), were added
K3Fe(CN)6 (987 mg, 3 mmol), (DHQ)2PHAL (78 mg, 10 mol%),
K2OsO4 (7 mg, 2 mol%), and K2CO3 (414 mg, 3 mmol), and the
resulting mixture was vigorously stirred for 10 min at 0 °C. Termi-
nal olefin 12 was added to the mixture and then the mixture was
stirred at the same temperature. After 18 h, the reaction mixture
was quenched by adding saturated aqueous sodium thiosulfate
(10 mL). The aqueous layer was extracted with EtOAc (20 mLꢁ3),
washed with brine (10 mL), dried over anhydrous sodium sulfate,
and concentrated under reduced pressure to give diol 21, which
was subjected to the next reaction without further purification.
To the diol 21 in dichloromethane (10 mL) were added TBSCl
(225 mg), imidazole (391 mg), and DMAP (14 mg), and the result-
ing mixture was stirred at 25 °C for an hour. The reaction mixture
was partitioned between CH2Cl2 (20 mL) and water, and the organ-
ic layer was extracted with CH2Cl2 (3ꢁ20 mL). The combined or-
ganic solution was dried over anhydrous sodium sulfate, and
concentrated to give the monosilylated compound, which was sub-
jected to the next reaction without further purification. To CH2Cl2
(10 mL) solution of the mono-protected compound, were added
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
2.2.9. 2a-Azido-3-keto-12b-benzyloxy-5a-spirostan-14-ene (18)
To bromoketone 17 (120 mg, 0.20 mmol) in 20 ml CH3NO2 was
added TMGN3 (128 mg, 0.82 mmol) at 0 °C. The mixture was stir-
red overnight, quenched by addition of water, and extracted with
ethyl acetate (3ꢁ20 ml). The combined organic layer was washed
with brine, dried over anhydrous sodium sulfate, concentrated,
and subjected to silica gel column chromatography using a gradi-
ent of 4:1 to 1:1 of hexane to ethyl acetate to give azido ketone
18 (105 mg, 93%) as a clear oil.
1H NMR (300 MHz, CDCl3) d 8.02–7.99 (2H, m), 7.56–7.51 (1H,
m), 7.44–7.39 (2H, m), 5.45 (1H, s), 4.92 (1H, d, J = 8.2 Hz) 4.68
(1H, dd, J = 6.9 and 13.9 Hz), 3.91 (1H, dd, J = 6.1 and 13.0 Hz),
2.60–2.50 (1H, m), 2.98 (1H, s), 2.61–2.38 (5H, m), 1.95 (3H, s),
1.30 (3H, s), 1.24 (3H, s), 1.22 (3H, s), 1.08 (3H, s), 0.89 (3H, d,
J = 6.9 Hz); 13C NMR (75 MHz, CDCl3) d 204.5, 165.8, 154.8, 132.9,
130.3, 129.38, 128.3, 121.3, 117.3, 84.0, 82.0, 80.9, 63.5, 55.9,
51.5, 51.4, 46.9, 44.9, 43.4, 41.1, 39.1, 37.2, 37.0, 33.3, 33.0, 31.5,
29.9, 29.6, 28.9, 28.3, 27.8, 26.7, 22.6, 15.0, 14.1, 14.0, 13.9, 12.2.
triethylamine (340
l
L), MgBr2ꢂ2Et2O (423 mg), and benzoic anhy-
dride (555 mg), and the resulting mixture was stirred at 25 °C for
2 h. The reaction was quenched by adding saturated aqueous so-
dium bicarbonate, extracted with CH2Cl2, dried over anhydrous so-
dium sulfate, concentrated in vacuo, and subjected to silica gel
chromatography to give tertiary benzoate 22 as a clear oil.
1H NMR (300 MHz, CDCl3) d 8.10–7.92 (4H, m), 7.59–7.32 (6H,
m), 5.47 (1H, s), 4.77 (2H, d, J = 8.1 Hz), 4.71–4.63 (2H, m), 3.92–
3.85 (1H, m), 3.30–3.28 (1H, m), 2.25–2.20 (1H, m), 2.09 (1H, s),
1.98 (3H, s), 1.51 (3H, s), 1.21 (3H, s), 0.86 (3H, s), 0.81 (9H, s),
0.0 (6H, s); 13C NMR (75 MHz, CDCl3) d 171.1, 170.6, 165.9, 165.5,
157.1, 133.5, 132.9, 132.3, 131.6, 130.4, 130.0, 129.4, 129.3,
128.3, 128.0, 120.1, 87.4, 86.0, 84.7, 81.7, 73.2, 66.1, 59.9, 51.9,
51.7, 44.1, 40.9, 36.4, 35.8, 34.1, 33.7, 32.9, 31.5, 29.4, 28.0, 27.2,
27.1, 26.6, 25.7, 21.3, 21.3, 18.0, 17.1, 16.0, 14.0, 11.9.
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
2.2.10. 2a-Azido-3-(methoxyimino)-12b-benzyloxy-5a-spirostan-14-
ene (19)
To azidoketone 18 (95 mg, 0.17 mmol) in dichloromethane
(24 ml) and pyridine (1.6 ml) was added methoxy amine (67 mg,
0.8 mmol), and the mixture was stirred for 4.5 h at room tempera-
ture. The mixture was quenched by adding water, extracted with
ethyl acetate (3ꢁ20 ml), and the combined organic layer was
washed with brine. After drying over sodium sulfate and concen-
tration, the residue was subjected to silica gel column chromatog-
raphy (hexane/EtOAc = 4:1) to give azido methoxime 19 (44 mg,
44%) as a clear oil.
1H NMR (300 MHz, CDCl3) d 8.07–8.04 (2H, m), 7.58 (1H, t,
J = 7.2 Hz), 7.49–7.44 (2H, m), 5.48 (1H, s), 4.979–4.96 (1H, m),
4.68–4.62 (1H, m), 3.93 (3H, s), 3.91 (1H, s), 3.10–3.04 (1H, m),
2.51–2.45 (1H, t, J = 8.7 Hz), 2.18 (1H, s), 2.16–2.10 (1H, m), 2.01–
1.98 (2H, m), 1.35 (3H, s), 1.27 (6H, s), 1.13 (3H, s), 0.98 (3H, s);
13C NMR (75 MHz, CDCl3) d 165.8, 155.3, 154.9, 132.9, 130.5,
129.4, 128.4, 121.1, 117.3, 84.05, 82.00, 81.1, 62.0, 57.6, 55.9,
51.7, 51.5, 44.5, 44.2, 41.2, 37.1, 36.9, 34.6, 33.5, 33.12 30.9, 30.0,
29.7, 29.0, 28.4, 27.6, 27.3, 26.5, 25.2, 15.0, 14.0, 12.2; HRMS for
C35H46N4O5 (M+H) calcd: 603.3541, found: 603.3539.
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372
373
374
375
2.2.13. 3b-Acetoxy-12b,26-dibenzyloxy-5a-furostan-26-hydroxy-14-
ene (23)
To 22 (388 mg, 0.48 mmol) in CH2Cl2 (10 mL) was added drop-
wise boron trifluoride diethyletherate and the mixture was stirred
at °C for an hour. The reaction was quenched by adding aqueous
saturated sodium bicarbonate, extracted with CH2Cl2 (3ꢁ20 mL),
concentrated under reduced pressure, and subjected to silica gel
Please cite this article in press as: Kou Y et al. Synthesis of 140,150-dehydro-ritterazine Y via reductive and oxidative functionalizations of hecogenin ace-
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chromatography to give primary alcohol 23 (238 mg, 71%) as a
clear oil.
1H NMR (300 MHz, CDCl3) d 8.01–7.95 (4H, m), 7.56–7.33 (6H,
m), 5.48 (1H, s), 4.92 (1H, d, J = 7.8 Hz), 4.65 (1H, dd, J = 5.2 and
10.8 Hz), 4.05 (1H, d, J = 9.9 Hz), 3.64 (1H, d, J = 12.0 Hz), 2.49–
2.44 (3H, m), 2.38–2.11 (5H, m), 2.02–2.00 (2H, m), 1.49 (3H, s),
1.24 (3H, s), 0.87 (3H, s), 0.85 (3H, s); 13C NMR (75 MHz, CDCl3) d
211.1, 165.9, 165.6, 156.1, 133.0, 132.5, 131.6, 130.4, 129.4,
129.3, 128.38, 128.2, 120.5, 106.4, 84.8, 81.1, 78.1, 66.4, 55.9,
51.8, 51.5, 46.0, 44.3, 44.2, 38.1, 37.8, 36.1, 34.0, 30.3, 29.2, 28.4,
26.7, 21.6, 15.0, 13.6, 11.2.
1H NMR (300 MHz, CDCl3) d 8.03–7.94 (4H, m), 7.58–7.36 (6H,
m), 5.45 (1H, s), 4.75–4.63 (4H, m), 3.94 (1H, s), 3.84–3.79(1H,
m) 2.22 (1H, t, J = 7.8 Hz), 2.20–2.09 (1H, m), 1.99 (4H, s), 1.48
(3H, s), 1.21 (3H, s), 0.89 (3H, s), 0.83 (3H, s); 13C NMR (75 MHz,
CDCl3) d 170.6, 166.9, 165.9, 157.2, 133.0, 130.8, 130.4, 129.6,
129.4, 128.4, 128.3, 120.0, 87.2, 87.0, 86.1, 81.7, 73.2, 68.2, 59.6,
51.9, 51.8, 44.2, 40.8, 36.5, 35.9, 34.1, 33.7, 33.6, 31.5, 29.5, 28.0,
27.2, 26.7, 26.6, 25.2, 22.6, 21.4, 20.7, 16.8, 16.0, 14.1, 11.9; HRMS
for C43H55O8(M+H) calcd: 699.3892, found: 699.3883.
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
2.2.17. 2a-Azido-3-keto-12b,26-dibenzyloxy-5a-furostan-14-ene (28)
To a solution of ketone 26 (45 mg, 0.067 mmol) in THF (1 mL)
was added phenyltrimethylammonium tribromide (28 mg,
0.078 mmol) at 0 °C. After stirring for 20 min at the same temper-
ature, the reaction mixture was quenched with saturated aqueous
sodium thiosulfate and extracted with ethyl acetate. The combined
extract was washed with brine, dried over anhydrous sodium sul-
fate, concentrated, and subjected to silica gel chromatography to
give the corresponding bromoketone 27. A solution of bromoke-
tone 27 in freshly dried CH3NO2 was cooled to 0 °C and TMGN3
was added portionwise for 2 min. The reaction was allowed to
warm slowly to 25 °C during 6 h of stirring and partitioned be-
tween EtOAc and brine, and organic layer was dried over anhy-
drous Na2SO4 and concentrated to give crude mixture, which was
subjected to silica gel chromatography (Hexane/EtOAc = 4:1) to
give azidoketone 28 (14 mg, 30% over two steps) as a clear oil.
1H NMR (300 MHz, CDCl3) d 8.01–7.95 (4H, m), 7.68–7.62 (2H,
m), 7.56–7.34 (4H, m), 5.50 (1H, s), 4.06–4.02 (3H, m), 3.68 (1H,
d, J = 12.0 Hz) 2.47 (1H, t, J = 8.7 Hz), 2.37 (1H, s), 2.32–2.30 (1H,
m), 2.02 (3H, s), 1.49 (3H, s) 1.23 (3H, s), 1.13(3H, s), 0.87 (3H,
s); 13C NMR (75 MHz, CDCl3) d 211.1, 165.9, 165.6, 156.1, 133.0,
132.5, 131.6, 130.4, 129. 4, 129.3, 128.38, 128.2, 120.5, 106.4,
84.8, 81.1, 78.2, 66.4, 55.9, 51.8, 51.5, 46.0, 44.3, 44.2, 38.1, 37.8,
36.1, 34.0, 30.3, 29.2, 28.4, 26.7, 21.6, 15.0, 13.6, 11.2; HRMS for
C41H47N3O7(M+Na) calcd: 716.3306, found: 716.3301.
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
2.2.14. 3b-Acetoxy-12b,26-dibenzyloxy-5a-furostan-14-ene (24)
To a dichloromethane/cyclohexane (1 mL/3 mL) solution of
iodobenzene diacetate (128 mg, 0.40 mmol) and iodine (101 mg,
0.40 mmol), was added the primary alcohol 23 (140 mg,
0.20 mmol) in dichloromethane (3 mL), and the resulting mixture
was stirred at 0 °C for 6 h. The reaction was quenched by adding
saturated sodium thiosulfate, extracted with dichloromethane
(3ꢁ20 mL), concentrated in vacuo, and subjected to silica gel chro-
matography (hexane/EtOAc = 4:1) to give 5/6 spiroketal 24 (89 mg,
64%) as a white foam.
1H NMR (300 MHz, CDCl3) d 8.01–7.95 (4H, m), 7.55–7.24 (6H,
m), 5.46 (1H, s), 4.91 (1H, d, J = 8.1 Hz), 4.63 (1H, dd, J = 5.2 and
11.4 Hz), 4.07–4.02 (1H, m), 3.69 (1H, d, J = 12.3 Hz), 2.48 (1H, s),
2.44 (1H, d, J = 8.4 Hz), 2.15–2.08 (1H, m), 1.99 (3H, s), 1.49 (3H,
s), 1.20 (6H, s), 0.87 (3H, s); 13C NMR (75 MHz, CDCl3) d 170.6,
165.6, 156.7, 132.9, 132.5, 131.6, 129.4, 129.4, 128.4, 128, 2,
120.1, 107.7, 106.3, 84.9, 81.4, 78.1, 73.2, 66.4, 55.9, 52.2, 51.5,
44.2, 36.5, 36.0, 34.1, 33.8, 30.3, 29.4, 28.1, 27.2, 26.6, 21.6, 21.4,
15.0, 13.6, 12.0; HRMS for C43H55O8(M+H) calcd: 697.3735, found:
697.3728.
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
2.2.15. 3b,12b,26-Trihydroxy-5a-furostan-14-ene (25)
1H NMR (300 MHz, Pyr-d5) d 5.59 (1H, s), 5.21 (1H, d), 4.05 (1H,
d), 3.72 (1H, d), 3.47 (1H, m), 3.13 (1H, t), 2.52 (1H, dt),1.32 (3H, d),
1.28 (3H, s), 1.24 (3H, s), 0.82 (3H, s); 13C NMR (75 MHz, Pyr-d5) d
158.1 (140), 119.6 (150), 107.1 (220), 85.5 (160), 79.0 (120), 70.5 (260),
70.1 (260), 66.0 (250), 56.6 (170), 53.5 (90), 53.4 (130), 45.2 (200), 44.9,
39.0, 37.3, 36.2 (100), 34.3, 33.8 (240), 32.4 (80), 30.9 (110), 30.1, 28.8
(70), 27.7 (60), 27.0 (230), 14.4 (210), 14.1 (180), 12.3 (190).
2.2.18. 140,150-dehydro-ritterazine Y (5)
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
To a solution of azidoketone 28 (14 mg, 0.020 mmol) and alpha-
aminomethoxime 20 (15 mg) in 10 mL benzene was added dichloro-
dibutylstannane (cat.) and polyvinyl pyridine (20 mg). The reaction
flask was equipped with a Dean–Stark trap, and the mixture was
heated at reflux for 3 h (2 mL of fresh benzene was added twice to
maintain the solvent level in the reaction vessel), at which time
TLC indicated no remaining azidoketone 28. The reaction mixture
was cooled and filtered, and the solids were washed with CH2Cl2.
Evaporation of the filtrate and silica gel chromatography of the res-
idue gave the protected ritterazine Y analog, which was subjected to
NMR spectra of the south unit of ritterazine Y: 1H NMR (Pyr-d5) d
5.23 (1H, s), 4.24 (1H, d), 3.72 (1H, d), 3.50 (1H, m), 3.12 (1H, t),
2.56 (1H, dt), 1.35 (3H, d), 1.25 (3H, s), 1.18 (3H, s), 1.78 (3H, s);
13C NMR (75 MHz, Pyr-d5) d 157.7 (140). 120.5 (150), 107.1 (220),
85.4 (160), 78.7 (120), 70.2 (260), 66.0 (250), 56.4 (170), 52.7 (90),
52.6 (130), 46.2 (10), 45.2 (200), 33.8 (240), 33.1 (80), 30.9 (110),
29.7 (70), 27.0 (270), 14.5 (210), 14.0 (180), 11.8 (190).
a global deprotection with KOH to give the desired
D
14-ritterazine Y
5 (4.2 mg, 24%) as a white foam after chromatography (EtOAc/
MeOH = 10:1).
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
2.2.16. 3-Keto-12b,26-dibenzyloxy-5a-furostan-14-ene (26)
1H NMR (400 MHz, CDCl3) d 5.41 (1H, s), 5.39 (1H, s), 5.14 (1H,
br), 5.09 (1H, br), 4.93 (1H, d, J = 8.5 Hz), 4.85 (1H, d, J = 8.5 Hz),
3.79 (1H, s), 3.31 (2H, m), 2.90–2.76 (4H, m), 2.62–2.49 (3H, m),
2.46 (1H, s), 2.38 (1H, s), 1.25 (3H, s), 1.22 (3H, s), 1.18 (3H, s),
1.11 (3H, s), 1.09 (3H, d, J = 6.8 Hz), 1.02 (6H, s), 0.86 (3H, d,
J = 5.5 Hz), 0.84 (3H, s); HRMS for (M+H) calcd: 863.5574, found:
863.5575.
To a well stirred solution of steroidal acetate 24 (88 mg,
0.127 mmol) in 5:1 MeOH/H2O at 25 °C was added potassium car-
bonate (17 mg, 0.127 mmol). After stirring for 12 h, the reaction
mixture was quenched with saturated NH4Cl and the solvent was
evaporated under reduced pressure to afford crude product.
Extraction with EtOAc, washing with brine, drying over anhydrous
sodium sulfate, and evaporation under reduced pressure afforded
the crude product mixture, which was subjected to silica gel chro-
matography to give the corresponding alcohol. To acetone solution
of the alcohol was added dropwise Jones reagent, and the mixture
was stirred for 20 min at 0 °C. The reaction mixture was quenched
with saturated aqueous sodium thiosulfate, extracted with ethyl
acetate, concentrated in vacuo, and subjected to silica gel chroma-
tography (hexane/EtOAc = 2:1) to give ketone 26 (45 mg, 53% over
two steps) as a clear oil.
493
3. Results and discussion
To prepare 140,150-dehydro-ritterazine Y, we first synthesized
terminal olefin 12 from hecogenin acetate 6 by using synthetic pro-
cedures similar to those for ritterazine M (Scheme 1) [30]. Conver-
sion of hecogenin acetate 6 into lumihecogenin acetate 7 via the
Welzel photolysis [23,30,31] followed by functional group
494
495
Q2 496
497
498
Please cite this article in press as: Kou Y et al. Synthesis of 140,150-dehydro-ritterazine Y via reductive and oxidative functionalizations of hecogenin ace-
tate. Steroids (2012), http://dx.doi.org/10.1016/j.steroids.2012.10.021

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manipulations of 7 afforded diene 12 [30], which was then trans-
formed into 140,150-dehydro-ritterazine Y north hemisphere 20
(Scheme 2) by adopting procedures developed by the Fuchs and
Shair groups [25,26,33].
26-alcohol 23. Hypoiodite-mediated alkoxy radical cyclization of
the primary alcohol 23 stereoselectively provided 24 with establish-
ment of 5/6 spiroketal. The stereochemistry of 5/6 spiroketal 24 was
assessed by comparison of 1H and 13C NMR data of triol 25 and the
south unit of ritterazine Y (see Fig. 4 and Section 2.2.15). The carbon
NMR chemical shifts of C16, 22, and 25 of triol 25 matched well with
C160, 220, and 250 of the south unit of ritterazine Y, suggesting triol 25
has the same spiroketal stereochemistry as that of the ritterazine Y
south hemisphere. Having established the desired spiroketal
stereochemistry, we then converted 3-acetate 24 into azidoketone
28, the south Y unit, employing the similar procedures that were
used for the preparation of the north G unit (Scheme 2).
The synthesis of the 140,150-dehydro-ritterazine Y south hemi-
sphere 28 began with oxidative modification of the terminal olefin
12 that was used in the synthesis of 140,150-dehydro-ritterazine Y
north hemisphere 20 (Scheme 3). Subjection of the terminal olefin
12 into sharpless asymmetric dihydroxylation produced C25,26-
diol 21 without affecting
D
14 olefin moiety. For the reestablishment
of the 5/6 spiroketal (1,6-dioxaspiro[4,5]decane), it was necessary to
protect the tertiary alcohol in 21 to prevent oxidative cleavage of the
1,2-diol. Sequential protection of the primary alcohol with TBDMS
group and the tertiary alcohol with benzoyl group and removal of
TBDMS group with boron trifluoride diethyletherate provided
The final stages of 140,150-dehydro-ritterazine Y 5 involve the
Guo–Fuchs asymmetric pyrazine coupling [34] of the north G
and south Y units and the global removal of the protecting groups,
Scheme 1. Preparation of terminal olefin 12: (i) hv (450 W), CH2Cl2, (ii) ZnCl2, CH2Cl2, 25 °C, 60% two steps, (iii) Jones reagent, acetone, 0 °C 10 min, 89% (iv) CeCl3, NaBH4,
THF/MeOH, 0 °C 5 h; BzCl, pyridine, 25 °C 6 h, 82% (v) Et3SiH, BF3 OEt2, CH2Cl2, 18 h, 94%, (vi) PPh3, I2, imidazole, THF; DBU, DMF, 25 °C 12 h, 78%.
Scheme 2. Synthesis of the 140,150-dehydro-ritterazine Y north hemisphere 20. (i) Hg(OAc)2, THF/H2O; NaBH4, 63% (ii) PhI(OAc)2, I2, CH2Cl2, 69% (iv) K2CO3, MeOH, 80% (v)
CrO3, H2SO4, acetone, 87% (v) phenyltrimethylammonium tribromide, THF, 77% (vi) tetramethylguanidinium azide, nitromethane, 93% (vii) MeONH2, 44% (viii)
triphenylphosphine, THF, H2O, 99%.
Scheme 3. Synthesis of the 140,150-dehydro-ritterazine Y south hemisphere 28. (i) AD-mix
a (ii) TBSCl, imidazole, CH2Cl2; MgBr2 Et2O, TEA, CH2Cl2 (iii) BF3 OEt2, CH2Cl2, 71%
(iv) PhI(OAc)2, I2, CH2Cl2, 64% (v) K2CO3, MeOH; CrO3, H2SO4, acetone, 53% (vi) phenyltrimethylammonium tribromide, THF (vii) tetramethylguanidinium azide, nitromethane,
30% over two steps.
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Fig. 4. Comparison of carbon NMR chemical shifts of the south unit of ritterazine Y and triol 25. Chemical shifts for C16, 22, and 25 of the ritterazine Y south unit and C160, 220,
and 250 of the triol 25 are shown in ppm.
Fig. 5. Synthesis of 140,150-dehydro-ritterazine Y 5 via Guo–Fuchs asymmetric pyrazine coupling.
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which provided the desired ritterazine Y analog 5 (Fig. 5). In sum-
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mary, we have prepared 140,150-dehydro-ritterazine Y 5 in 23 steps
via reductive/oxidative modifications of commercially available
hecogenin acetate. The synthesis of the north G and south Y units
involve the use of a common intermediate 12, which enabled the
facile construction of the both units. The bioactivity test of this
novel bissteroidal pyrazine 5 and synthesis of derivatives of
140,150-dehydro-ritterazine Y are in progress and their results will
be reported elsewhere in due course.
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tate. Steroids (2012), http://dx.doi.org/10.1016/j.steroids.2012.10.021