C-3β-tethered functional cholesterol conjugates by nitrile oxide alkyne cycloaddition (NOAC)

Article abstract of DOI:10.1002/ejoc.201301822

Covalent cholesterol conjugates have been prepared by isoxazole-generating [3+2] nitrile oxide alkyne cycloaddition (NOAC) chemistry. Steroidal building blocks functionalised with either an alkyne or a nitrile oxide precursor were evaluated. The reaction has been demonstrated for the tethering to cholesterol of groups capable of bioreporting, and for the formation of cholesterol- biomolecule conjugates. Nitrile oxide alkyne cycloaddition (NOAC) chemistry - a tool for attaching cholesterol to biomolecules or groups capable of bioreporting. Copyright

Full text of DOI:10.1002/ejoc.201301822

FULL PAPER
DOI: 10.1002/ejoc.201301822
C-3β-Tethered Functional Cholesterol Conjugates by Nitrile Oxide Alkyne
Cycloaddition (NOAC)
Virginie Algay,[a] Justine O’Sullivan,[a] and Frances Heaney*[a]
Keywords: Steroids / Lipids / Cycloaddition / Alkynes / Heterocycles / Conjugation
Covalent cholesterol conjugates have been prepared by iso-
xazole-generating [3+2] nitrile oxide alkyne cycloaddition
(NOAC) chemistry. Steroidal building blocks functionalised
with either an alkyne or a nitrile oxide precursor were evalu-
ated. The reaction has been demonstrated for the tethering
to cholesterol of groups capable of bioreporting, and for the
formation of cholesterol–biomolecule conjugates.
chemistry[8] for the formation of cholesterol conjugates an-
chored by way of a polar, aromatic, metabolically stable
isoxazole nucleus.
Introduction
Cholesterol is one of the most common natural lipids,
and due to its bioavailability and structural rigidity, func-
tionalised cholesterol building blocks have attracted interest
in areas ranging from materials to medicinal chemistry.
There have been a number of reports on triazole-mediated
conjugation by copper(I)-promoted dipolar cycloaddition
using both azido lipids and cholesterol–alkynes. This
powerful click chemistry approach[1] has opened access to
new cholesterol pharmacophores,[2a,2b] and has allowed the
creation of specialist materials, including cholesterol-incor-
porating organogels,[2c,2d] nanoparticles,[2e] liquid crys-
tals,[2f] and orthopaedic medical devices.[2g] It has also facili-
tated access to cholesterol–dye conjugates for biomem-
brane[2h] or metabolic studies,[2i] to steroidal glycosides,[2j]
and to lipid–oligonucleotide conjugates for drug delivery
applications.[2k–2m] In addition, catalyst-free routes to tri-
azole-linked cholesterol–oligonucleotide conjugates have
been reported.[3] However, aside from exploitation of the
masked functionality of the isoxazole nucleus to access
polyhydroxylated steroids, cholesterol conjugates with this
heterocyclic linker are relatively unknown. Thermally pro-
moted cycloadditions to C-17-anchored steroidal alkynes
have yielded important reaction intermediates.[4] In recent
years, the regioselectivity of such cycloadditions has been
enhanced through the use of copper[5a–5c] or ruthenium ca-
talysis.[5d] Transient nitrile oxide dipoles have been gener-
ated by the action of PhNCO on steroidal nitroalkane pre-
cursors,[6] or from C-22 aldoximes by the NCS/NEt3 proto-
col.[7] The work described in this paper demonstrates the
versatility of nitrile oxide alkyne cycloaddition (NOAC)
Results and Discussion
To maximize structural diversity, and to avoid potential
stereochemical complications, we required the alkyne or
nitrile oxide precursors to be anchored to the C-3β oxygen
atom rather than directly to the tetracyclic core.[6b] The
Montmorillonite K-10 (MK-10)[9] promoted route to steroi-
dal ethers 1–3 is shown in Scheme 1. The reported pro-
cedure for the formation of propargyl ether 1, which re-
quires a solution of cholesterol and the corresponding
alcohol in CHCl3 to be stirred for 7 d at 55 °C,[10] was
modified so that the product could be obtained with mini-
mal compromise in yield after just 17 h at 90 °C in a micro-
wave (MW) reactor. Following the modified procedure, ho-
mologous alkynyl ethers 2 and 3 were obtained in 62 and
70% yields, respectively.
Model NOAC reactions were explored between 1–3 and
phenyl nitrile oxide, which was generated in situ from benz-
aldehyde oxime on exposure to an ethanolic solution of
chloramine-T (Ch-T). While there was no evidence for a
compromise in regiochemical integrity, the yields of 3,5-di-
substituted isoxazoles 4a–6a were greatly influenced by the
spacing between the bulky lipid and the reacting alkyne.
Thus, compound 6a, with a 4-carbon linker, was obtained
in 90% yield after 1 h at room temperature. Compound 5a,
with a shorter ethylene spacer, was obtained in only 44%
after the same reaction time. Significantly, compound 4a,
with a methylene spacer, was formed in a mere 19% yield,
even after 17 h. The same reactivity trend was seen with the
putative dipoles generated from 1-naphthyl and 1-pyrenyl
oximes. Gratifingly, we found that the sluggish reactivity of
propargylated steroid 1a could be circumvented by con-
ducting the reaction in a microwave reactor. In this way,
[a] Department of Chemistry, National University of Ireland,
Maynooth, Co. Kildare, Republic of Ireland
E-mail: mary.f.heaney@nuim.ie
http://www.nuim.ie/chemistry
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301822.
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C-3β-Tethered Functional Cholesterol Conjugates
Scheme 1. Steroidal alkynes and conjugates formed by NOAC chemistry.
the product yields for 4a–4c increased to 78, 96, and 71%,
respectively.
The potential of the NOAC reaction to access biolo-
gically relevant cholesterol probes is illustrated by the prep-
aration of steroid–coumarin 7 and steroid–azobenzene 8
conjugates. Cholesterol is implicated in a vast array of bio-
logical processes, and fluorescent derivatives are useful tools
that can be used for imaging in cells and tissues;[11] azo-
derivatised molecules are attractive for the construction of
thermal and photoresponsive materials,[12a,12b] including
phototriggered drug-delivery vehicles.[12c] Fluorescent cou-
marin conjugate 7 was formed in 75% yield by the cycload-
dition reaction between 3 and the nitrile oxide generated
from coumarin 6-aldoxime.[13a] Photoresponsive azo-conju-
gated steroid 8 was obtained by a parallel reaction sequence
involving 4-(phenylazo)benzaldehyde oxime[13b] as the di-
pole precursor (Figure 1). Initially obtained almost exclu-
sively as the trans-isomer, 8 was transformed into an 84:16
Figure 1. Structures of fluorescent and photoresponsive cholesterol
conjugates 7 and 8 (n = 4).
mixture of trans and cis isomers after exposure to sunlight trapped with both propargyl alcohol and propargyl phenyl
for 2 h. When the sample was returned to darkness, the ether, however, the yields of the cholesterol conjugates (i.e.,
original exclusively trans geometry was restored.
14a and 14b) were modest (21 and 37%). The low yields of
To increase the flexibility of the NOAC approach to the cycloadducts may be a consequence of solubility issues
cholesterol conjugation, steroidal building blocks bearing arising from a suboptimal balance of hydrophilic and
nitrile oxide precursors were pursued. The reported suc- hydrophobic character of the microenvironments of the
cesses of cholesterol chloroformate as a handle for the in- lipid–amidocarbamate–isoxazole conjugate. Stable long-
troduction of alkyne[14a] or azido[14b] functionalities sug- chain hydrophilic linkers are attractive for bioconjugation,
gested that it would be an appropriate building block for and for this reason, amidocarbamate substrates were aban-
oxime introduction by way of an amidocarbamate linker. doned in favour of ether-linked analogues 16 (Scheme 3).
However, after sequential acetal deprotection and oxime Regioisomeric steroidal oxime ethers 16 were prepared in
formation, as outlined in Scheme 2, the target oxime (i.e., two steps, as shown in Scheme 3. MK-10-mediated conden-
12) was obtained in just 2% overall yield, primarily as a sation[9] between cholesterol and the relevant hydroxyeth-
result of a low-yielding deprotection step. Fortunately, we oxybenzaldehyde gave aldehydes 15 in 35–58% yield after 8 h
discovered that aldehyde regeneration and oximation could in a microwave reactor (Pmax 300 W). As noted for p-15, no
be achieved in a one-pot procedure. Thus, direct exposure significant increase in yield was observed when the reaction
of acetal 10 to NH2OH·HCl in ethanolic pyridine in a mi- time was doubled to 17 h. Oximation proceeded quantita-
crowave reactor (125 °C, 1 h, Pmax 300 W) yielded steroidal tively, and the latent reactivity of 16 as a nitrile oxide pre-
oxime 12 in 91% yield. Putative steroidal nitrile oxide 13, cursor was demonstrated by the formation of cholesterol
derived from 12 upon treatment with chloramine-T, was conjugates 18. Thus, following overnight stirring of an eth-
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V. Algay, J. O’Sullivan, F. Heaney
FULL PAPER
Scheme 2. Amidocarbamate steroidal oximes and conjugates formed by NOAC chemistry. DCC = N,NЈ-dicyclohexylcarbodiimide; DMAP
= 4-(dimethylamino)pyridine.
Scheme 3. Steroidal oximes and conjugates formed by NOAC chemistry.
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C-3β-Tethered Functional Cholesterol Conjugates
Scheme 4. Aryl steroidal ethers and conjugate formation by NOAC chemistry.
anolic solution of oxime 16 and chloramine-T in the pres- thetic oligonucleotides with tuneable amphiphilic proper-
ence of phenyl propargyl ether, 18a–18c were isolated in 29– ties.
58% yield. It is likely that the variation in yield of the o-,
m-, and p-substituted isomeric cycloadducts reflects the ef-
fect of the bulky steroidal core on the transition state lead-
ing to isoxazole formation. Thus, the steric demand is great-
est for the alignment leading to o-18, and the yield of the
cycloadduct is lowest (29%); the bulk of the steroidal core
has a minimal impact on the transition state leading to p-
18, which results in a significantly improved yield of the
cycloadduct (58%). A further improvement in the yield of
p-18 to an attractive 80% was achieved by adding the steroi-
dal oxime and chloramine-T portionwise to a solution of
the dipolarophile in ethanol.
Aryl ethers of natural alcohols are inherently interesting
both as end products and as intermediates en route to func-
tional materials. Their synthesis is non-trivial and has at-
tracted significant interest.[15] To validate NOAC chemistry
as a vehicle for the assembly of cholesterol conjugates like
21, aldehyde-functionalised aryl ether 19 was required. It is
known that MK-10-promoted aryl etherification of choles-
terol in CHCl3 at 50–70 °C is suited to electron-rich, but
not to electron-deficient phenols.[9] However, we are pleased
to report that the use of microwave irradiation in the reac-
tion between cholesterol and p-hydroxybenzaldehyde
(90 °C, 8 h, Pmax 300 W) resulted in the formation of 19
(62%). With the key intermediate to hand, subsequent ox-
imation and cycloaddition resulted in the formation of isox-
azole-linked aryl cholesterol ether 21 (Scheme 4).
Covalently functionalised cholesterol bioconjugates in-
Scheme 5. Cholesterol–biomolecule conjugates formed by NOAC
cluding sugar–cholesterol,[16a] amphiphilic DNA–cholester-
chemistry.
ol,[16b] and nucleoside–cholesterol[16c] conjugates have valu-
The structures of all the isoxazole-linked cholesterol con-
able roles as components of model biological mem-
jugates are supported by spectroscopic data. In particular,
branes,[16d] and as specialist materials for drug-delivery ap-
diagnostic NMR spectral signals consistent with the regio-
plications. Thus, the potential of NOAC chemistry to de-
specific formation of 3,5-disubstituted isoxazoles include a
liver cholesterol bioconjugates is an effective marker of its
singlet resonance in the region 6.30–6.63 ppm of the 1H
utility. Steroidal glycoconjugate formation is demonstrated
spectrum, characteristic of the 4-H ring proton,[5d] and a
by the regioselective formation of 23 (85%) by the reaction
resonance in the of the 13C spectrum in the range 99–
of putative nitrile oxide p-17 with protected d-galactose 22
104 ppm due to the corresponding carbon atom.
as a model sugar dipolarophile.[17] The selective tethering
of one or two cholesterol units to a thymidine skeleton was
demonstrated by trapping of the same dipole by 5Ј-pro-
Conclusions
tected mono- or bis-propargylated thymidines 24 or 26[18] to
give conjugates 25 and 27 in 81 and 68% yields, respectively
In conclusion, NOAC chemistry has been verified as a
(Scheme 5). The mono- and bis-steroidal conjugates are po- straightforward and convenient method for the formation
tentially useful building blocks for the formation of syn- of diverse cholesterol conjugates including those of poten-
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V. Algay, J. O’Sullivan, F. Heaney
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3
4
(td, J = 2.6, 7.1 Hz, 2 H, OCH2CH2), 1.97 (t, J = 2.6 Hz, 1 H,
CHCCH2), 1.88–0.80 (m, 40 H), 0.68 (s, 3 H, Me) ppm.
tial value as biochemical probes. The NOAC tethering
method avoids catalysts and optimisation of numerous rea-
gents as well as the potentially problematic task of separat-
(3β)-3-(5-Hexyn-1-yloxy)cholest-5-ene (3): Compound 3 (423 mg,
ing such reagents from the crude reaction products. The 70%) was isolated as a white solid. Rf = 0.80 (hexane/EtOAc, 9:1),
m.p. 112–114 °C. 1H NMR: δ = 5.34 (br. d, 3J = 5.2 Hz, 1 H,
reaction is flexible in that either of the two functionalities
needed for the [3+2] cycloaddition can be introduced at the
3β position of the steroidal core. This facilitates the intro-
duction of the conjugating partner as either the dipole or
CH=C), 3.47 (t, 3J = 7.1 Hz, 2 H, OCH2), 3.19–3.05 (m, 1 H,
OCH), 2.45 (td, 3J = 7.1, 2.6 Hz, 2 H, OCH2CH2), 1.97 (t, 4J =
2.6 Hz, 1 H, CHCCH2), 1.88–0.80 (m, 40 H), 0.68 (s, 3 H, Me)
ppm. 13C NMR: δ = 141.1 (C=CH), 121.5 (C=CH), 84.4 (CHϵC),
the dipolarophile, without incurring any stereochemical
79.0 (OCH), 68.3 (OCH2), 67.4 (CHϵC), 56.8, 56.2, 50.2, 42.3,
penalties. The hydrophobic/hydrophilic balance of the
39.8, 39.5, 39.2, 37.3, 36.9, 36.2, 35.8, 32.0, 31.9, 29.2, 28.5, 28.3,
steroidal reactants and products is important for the success
28.0, 25.3, 24.3, 23.8, 22.8, 19.4, 18.8, 18.3, 11.9 ppm. HRMS
(ESI): calcd. for C33H55O [M + H]+ 467.4247; found 467.4262
(3.2 ppm).
of the reaction, as is the length of the linker that separates
the rigid cholesterol core from the cycloaddition partner. In
a future communication, we will describe the use of NOAC
on the solid phase for the synthesis of oligonucleotide–
cholesterol conjugates.
General Procedures for the Cycloaddition Reaction to Alkyne
Cholesterol Ethers
Method 1: The oxime (2 equiv.), Ch-T (2.5 equiv.), and EtOH
(1 mL per 0.1 mmol oxime) were put into a round-bottomed flask,
and alkyne 1, 2, or 3 (1 equiv.) was added. The mixture was stirred
at room temp. To overcome solubility issues with pyrene oxime, the
mixture was sonicated for 10 min before the reaction. The mixture
was stirred for 1 h, then the solvent was removed. The residue was
dissolved in chloroform, and this solution was washed with H2O.
The organic layer was dried with anhydrous Na2SO4, and the sol-
vent was removed. The crude reaction products were purified by
flash column chromatography (hexane/EtOAc 0–5%).
Experimental Section
General Remarks: Analytical TLC was carried out on precoated
(250 mm) silica gel 60 F-254 plates from Merck. Plates were visual-
ized by UV irradiation, and/or staining with H2SO4 (5% in eth-
anol) followed by heating. Flash-chromatography-grade silica gel
60 (230–400 mesh) was obtained from Merck. High-resolution
mass spectra were recorded with a Bruker BioApex 70 eV spec-
trometer. IR spectra were recorded on disposable polyethylene
cards (19 mm, type 61, 3M) with a Perkin–Elmer Spectrum 100
RX-I series FTIR spectrophotometer. Reactions using microwave
(MW) irradiation were carried out with a Discover SP (CEM, Mat-
thews North Carolina, USA) apparatus, with an Explorer, con-
trolled by the SynergyTM software. Melting points were determined
with a Stuart Equipment SMP 11 apparatus. NMR spectra were
obtained for 1H at 300 MHz and for 13C at 75 MHz with a Bruker
instrument at 25 °C. All samples for NMR spectroscopy were pre-
pared in CDCl3. Chemical shifts are reported in ppm downfield
from tetramethylsilane, and coupling constants are reported in
Hertz. Anhydrous solvents were distilled from calcium chloride,
calcium hydride, potassium hydroxide, or sodium as appropriate.
Chemical reagents were purchased from Aldrich Chemical Com-
pany unless otherwise noted, and were used without further purifi-
cation.
Method 2 (MW Activation): The oxime (2 equiv.), Ch-T (2.5 equiv.),
and EtOH (3 mL per 0.5 mmol oxime) were put into a 10 mL mi-
crowave vessel, and cholesteryl propargyl ether 1 (1 equiv.) was
added. The mixture was heated (100 °C for 1 h with benzaldehyde
oxime; 60 °C for 30 min for 1-naphthaldehyde or 1-pyrene oxime)
in a microwave reactor (Pmax = 300 W). The solvent was removed,
and the residue was dissolved in chloroform and washed with H2O.
The organic layer was dried with anhydrous Na2SO4, and the sol-
vent was removed under reduced pressure. The crude reaction prod-
ucts were purified by flash column chromatography (hexane/EtOAc
0–5%).
Method 3: The oxime (2 equiv.) was put into a round-bottomed
flask, and Ch-T (2.5 equiv.) in EtOH (25% aq.; 1 mL per 0.1 mmol
oxime) was added. The resulting solution was allowed to stir for
10 min at room temp., after which the alkyne (1 equiv.) was added.
The mixture was stirred for 1 h, then the solvent was removed. The
residue was dissolved in chloroform and washed with H2O. The
organic layer was dried with anhydrous Na2SO4, and the solvent
was removed. The crude reaction products were purified by flash
column chromatography (petroleum ether/EtOAc 0–10%).
General Etherification Procedure: A mixture of cholesterol (500 mg,
1.29 mmol), alkynyl alcohol (6.47 mmol, 5 equiv.), and MK-10
(1.00 g, activated at 120 °C overnight before use) in chloroform
(5 mL) was heated to 90 °C for 17 h in a microwave reactor (10 mL
vessel, Pmax = 300 W). The solvent was removed under reduced
pressure, hexane (20 mL) was added, and the catalyst was removed
by filtration and then washed with hexane. The filtrate and wash-
ings were evaporated, and the residue was purified by flash column
chromatography (hexane/EtOAc; 0–5%) to give the title com-
pounds as white solids.
5-[(Cholest-5-en-3β-yloxy)methyl]-3-(phenyl)isoxazole (4a): Com-
pound 4a (Method 1: 25 mg, 19%; Method 2: 103 mg, 78%) was
isolated as a white solid. Rf = 0.54 (hexane/EtOAc, 9:1), m.p. 123–
1
125 °C. H NMR: δ = 7.85–7.76 (m, 2 H, Ar-H), 7.48–7.40 (m, 3
H, Ar-H), 6.56 (s, 1 H, isoxazole-H), 5.37 (br. s, 1 H, CH=C), 4.69
(s, 2 H, OCH2), 3.42–3.27 (m, 1 H, OCH), 2.47–0.79 (m, 40 H),
0.68 (s, 3 H, Me) ppm. 13C NMR: δ = 170.7 (C=N), 162.4 (isox-
azole-CO), 140.4 (C=CH), 130.0, 129.1, 128.9, 126.8 (Ar-C and 5
Ar-CH), 122.1 (C=CH), 100.7 (isoxazole-CH), 79.6 (OCH), 61.1
(OCH2), 56.8, 56.2, 50.2, 42.3, 39.8, 39.5, 38.9, 37.1, 36.8, 36.2,
35.8, 32.0, 31.9, 28.3, 28.2, 28.0, 24.3, 23.8, 22.8, 22.6, 21.1, 19.4,
18.7, 11.9 ppm. HRMS (ESI): calcd. for C37H54NO2 [M + H]+
544.4149; found 544.4165 (2.6 ppm).
(3β)-3-(2-Propynyloxy)cholest-5-ene (1):[10] Compound 1 (373 mg,
68%) was isolated as a white solid. The data agreed with the re-
1
2
ported data. H NMR: δ = 5.37 (br. d, J = 5.2 Hz, 1 H, CH=C),
4.19 (d, 4J = 2.4 Hz, 2 H, OCH2), 3.47–3.31 (m, 1 H, OCH), 2.42–
0.80 (m, 41 H), 0.68 (s, 3 H, Me) ppm.
(3β)-3-(Butyn-1-yloxy)cholest-5-ene (2):[10] Compound 2 (353 mg,
62%) was isolated as a white solid. The data agreed with the re-
1
2
ported data. H NMR: δ = 5.35 (br. d, J = 5.2 Hz, 1 H, CH=C),
5-[(2-Cholest-5-en-3β-yloxy)ethyl]-3-(phenyl)isoxazole (5a): Follow-
3.60 (t, J = 7.1 Hz, 2 H, OCH2), 3.19–3.03 (m, 1 H, OCH), 2.45 ing Method 1, compound 5a (57 mg, 44%) was isolated as a white
3
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C-3β-Tethered Functional Cholesterol Conjugates
1
3
solid. Rf = 0.60 (hexane/EtOAc, 9:1), m.p. 112–114 °C. H NMR:
δ = 7.83–7.74 (m, 2 H, Ar-H), 7.49–7.42 (m, 3 H, Ar-H), 6.39 (s, 1
H, isoxazole-H), 5.35 (br. s, 1 H, CH=C), 3.86 (t, 3J = 6.6 Hz, 2
H, OCH2), 3.31–3.08 (m, 1 H, OCH), 3.11 (t, 3J = 6.6 Hz, 2 H,
isoxazole-CH2), 2.45–0.80 (m, 40 H), 0.67 (s, 3 H, Me) ppm. 13C
NMR: δ = 170.6 (C=N), 162.2 (isoxazole-CO), 140.1 (C=CH),
130.0, 129.2, 128.8, 126.9 (Ar-C and 5 Ar-CH), 122.0 (C=CH),
OCH2), 3.27–3.08 (m, 1 H, OCH), 2.90 (t, J = 6.3 Hz, 2 H, isox-
azole-CH2), 2.44–0.80 (m, 44 H), 0.67 (s, 3 H, Me) ppm. 13C NMR:
δ = 172.2 (C=N), 161.4 (isoxazole-CO), 139.9 (C=CH), 132.7,
130.0, 129.0, 127.4, 126.6, 126.2, 125.9, 125.2, 124.7, 124.1 (3 Ar-
C and 7 Ar-CH), 120.5 (C=CH), 101.4 (isoxazole-CH), 78.1
(OCH), 66.3 (OCH2), 55.7, 55.1, 49.1, 41.3, 38.7, 38.5, 38.1, 36.2,
35.8, 35.2, 34.8, 30.9, 30.8, 28.6, 27.4, 27.2, 27.0, 25.6, 23.4, 23.3,
103.4 (isoxazole-CH), 79.6 (OCH), 65.0 (OCH2), 56.8, 56.2, 50.2, 22.8, 21.5, 20.0, 18.4, 17.7, 10.8 ppm. HRMS (ESI): calcd. for
42.4, 39.8, 39.5, 39.2, 37.2, 36.9, 36.2, 35.8, 32.1, 31.9, 28.4, 28.2,
28.0, 24.3, 23.8, 22.8, 22.6, 21.1, 19.4, 18.8, 11.9 ppm. HRMS
(ESI): calcd. for C38H56NO2 [M + H]+ 558.4306; found 558.4306
(0 ppm).
C44H62NO2 [M + H]+ 636.4775; found 636.4812 (5.8 ppm).
5-[(Cholest-5-en-3β-yloxy)methyl]-3-(1-pyrenyl)isoxazole (4c): Com-
pound 4c (Method 1: 27 mg, 17%; Method 2: 113 mg, 71%) was
isolated as a white solid. Rf = 0.68 (hexane/EtOAc, 9:1), m.p. 162–
164 °C. 1H NMR: δ = 8.57 (d, 3J = 9.3 Hz, 1 H, Ar-H), 8.36 (d, 3J
= 8.1 Hz, 1 H, Ar-H), 8.27–7.98 (m, 7 H, Ar-H), 6.59 (s, 1 H,
isoxazole-H), 5.34 (br. s, 1 H, CH=C), 4.63 (s, 2 H, OCH2), 3.44–
5-[(4-Cholest-5-en-3β-yloxy)-1-butyl]-3-(phenyl)isoxazole (6a): Fol-
lowing Method 1, compound 6a (115 mg, 90%) was isolated as a
white solid. Rf = 0.60 (hexane/EtOAc, 9:1), m.p. 128–130 °C. 1H
NMR: δ = 7.83–7.74 (m, 2 H, Ar-H), 7.49–7.42 (m, 3 H, Ar-H), 3.27 (m, 1 H, OCH), 2.53–0.80 (m, 40 H), 0.67 (s, 3 H, Me) ppm.
3
6.32 (s, 1 H, isoxazole-H), 5.35 (br. s, 1 H, CH=C), 3.53 (t, J =
6.3 Hz, 2 H, OCH2), 3.27–3.08 (m, 1 H, OCH), 2.90 (t, 3J = 6.3 Hz,
2 H, isoxazole-CH2), 2.44–0.80 (m, 44 H), 0.67 (s, 3 H, Me) ppm.
13C NMR: δ = 174.0 (C=N), 162.4 (isoxazole-CO), 141.0 (C=CH),
129.9, 129.4, 128.9, 126.8 (Ar-C and 5 Ar-CH), 121.6 (C=CH), 99.0
(isoxazole-CH), 79.1 (OCH), 67.3 (OCH2), 56.8, 56.2, 50.2, 42.3,
39.8, 39.5, 39.2, 38.7, 37.3, 36.9, 36.2, 31.9, 31.0, 30.3, 29.6, 28.9,
28.5, 26.7, 24.4, 24.3, 23.8, 22.8, 22.6, 21.1, 18.8, 11.9 ppm. HRMS
(ESI): calcd. for C40H60NO2 [M + H]+ 586.4619; found 586.4651
(5.5 ppm).
13C NMR: δ = 170.0 (C=N), 162.5 (isoxazole-CO), 140.7 (C=CH),
132.5, 131.3, 130.6, 128.7, 128.4, 126.2, 125.9, 125.6, 125.1, 125.0,
124.9, 122.7 (7 Ar-C and 9 Ar-CH), 121.8 (C=CH), 104.3 (isox-
azole-CH), 82.0 (OCH), 62.0 (OCH2), 56.8, 56.2, 50.9, 50.2, 42.3,
42.3, 39.8, 39.5, 37.2, 36.5, 36.2, 35.8, 31.9, 31.6, 28.2, 28.0, 24.2,
23.8, 22.8, 22.6, 21.1, 19.4, 18.8, 11.9 ppm. HRMS (ESI): calcd. for
C47H58NO2 [M + H]+ 668.4462; found 668.4437 (–3.7 ppm).
5-[(2-Cholest-5-en-3β-yloxy)ethyl]-3-(1-pyrenyl)isoxazole (5c): Fol-
lowing Method 1, compound 5c (57 mg, 35%) was isolated as a
white solid. Rf = 0.70 (hexane/EtOAc, 9:1), m.p. 160–162 °C. 1H
3
3
5-[(Cholest-5-en-3β-yloxy)methyl]-3-(1-naphthyl)isoxazole
(4b): NMR: δ = 8.57 (d, J = 9.3 Hz, 1 H, Ar-H), 8.32 (d, J = 8.1 Hz,
Compound 4b (Method 1: 61 mg, 43%; Method 2: 138 mg, 96%)
was isolated as a white solid. Rf = 0.62 (hexane/EtOAc, 9:1), m.p.
146–148 °C. 1H NMR: δ = 8.42–8.33 (m, 1 H, Ar-H), 7.98–7.85
1 H, Ar-H), 8.20–7.93 (m, 7 H, Ar-H), 6.44 (s, 1 H, isoxazole-H),
5.37 (br. s, 1 H, CH=C), 3.79 (t, J = 6.7 Hz, 2 H, OCH2), 3.32–
3
3.09 (m, 3 H, OCH, isoxazole-CH2), 2.48–0.80 (m, 40 H), 0.67 (s,
(m, 2 H, Ar-H), 7.73–7.65 (m, 1 H, Ar-H), 7.53–7.47 (m, 3 H, Ar- 3 H, Me) ppm. 13C NMR: δ = 170.6 (C=N), 162.6 (isoxazole-CO),
H), 6.57 (s, 1 H, isoxazole-H), 5.38 (br. s, 1 H, CH=C), 4.76 (s, 2
H, OCH2), 3.48–3.32 (m, 1 H, OCH), 2.53–2.24 (m, 2 H), 2.10–
140.7 (C=CH), 132.4, 131.3, 130.5, 128.6, 128.4, 126.2, 125.9,
125.7, 125.1, 125.0, 124.9, 122.7 (7 Ar-C and 9 Ar-CH), 121.7
1.74 (m, 6 H), 1.66–0.80 (m, 32 H), 0.68 (s, 3 H, Me) ppm. 13C (C=CH), 103.3 (isoxazole-CH), 81.9 (OCH), 65.1 (OCH2), 56.8,
NMR: δ = 170.0 (C=N), 162.5 (isoxazole-CO), 140.4 (C=CH),
56.2, 50.3, 50.2, 42.3, 42.1, 39.8, 39.5, 37.2, 36.4, 36.2, 35.8, 32.0,
31.9, 28.3, 28.0, 24.3, 23.8, 22.8, 22.6, 21.1, 19.4, 18.7, 11.9 ppm.
133.9, 131.0, 130.2, 128.5, 127.8, 127.0, 126.9, 126.3, 125.7, 125.2
(3 Ar-C and 7 Ar-CH), 122.2 (C=CH), 104.1 (isoxazole-CH), 79.9 HRMS (ESI): calcd. for C48H60NO2 [M + H]+ 682.4619; found
(OCH), 61.2 (OCH2), 56.8, 56.2, 50.2, 42.3, 39.8, 39.6, 39.0, 37.2,
36.9, 36.2, 35.8, 32.0, 31.9, 28.3, 28.2, 28.0, 24.3, 23.8, 22.8, 22.6,
21.1, 19.4, 18.8, 11.9 ppm. HRMS (ESI): calcd. for C41H56NO2 [M
+ H]+ 594.4306; found 594.4319 (2.2 ppm).
682.4638 (2.8 ppm).
5-[(4-Cholest-5-en-3β-yloxy)-1-butyl]-3-(1-pyrenyl)isoxazole
Following Method 1, compound 6c (135 mg, 80%) was isolated as
a white solid. Rf = 0.79 (hexane/EtOAc, 9:1), m.p. 155–157 °C. H
NMR: δ = 8.57 (d, J = 9.3 Hz, 1 H, Ar-H), 8.36 (d, J = 8.1 Hz,
1 H, Ar-H), 8.24–7.98 (m, 7 H, Ar-H), 6.30 (s, 1 H, isoxazole-H),
5.34 (br. s, 1 H, CH=C), 3.55 (t, J = 6.4 Hz, 2 H, OCH2), 3.30–
(6c):
1
3
3
5-[(2-Cholest-5-en-3β-yloxy)ethyl]-3-(1-naphthyl)isoxazole (5b): Fol-
lowing Method 1, compound 5b (76 mg, 54%) was isolated as a
white solid. Rf = 0.65 (hexane/EtOAc, 9:1), m.p. 134–136 °C. 1H
3
NMR: δ = 8.41–8.34 (m, 1 H, Ar-H), 7.99–7.87 (m, 2 H, Ar-H), 3.11 (m, 1 H, OCH), 2.91 (t, 3J = 6.4 Hz, 2 H, isoxazole-CH2),
7.73–7.65 (m, 1 H, Ar-H), 7.57–7.46 (m, 3 H, Ar-H), 6.42 (s, 1 H,
2.51–0.80 (m, 44 H), 0.67 (s, 3 H, Me) ppm. 13C NMR: δ = 172.7
(C=N), 161.9 (isoxazole-CO), 140.8 (C=CH), 132.5, 131.3, 130.6,
128.7, 128.4, 126.2, 125.9, 125.6, 125.1, 125.0, 124.9, 122.7 (7 Ar-
3
isoxazole-H), 5.35 (br. s, 1 H, CH=C), 3.87 (t, J = 6.6 Hz, 2 H,
3
OCH2), 3.30–3.10 (m, 1 H, OCH), 3.14 (t, J = 6.6 Hz, 2 H, isox-
azole-CH2), 2.45–2.14 (m, 2 H), 2.10–0.80 (m, 38 H), 0.67 (s, 3 H, C and 9 Ar-CH), 121.5 (C=CH), 101.6 (isoxazole-CH), 79.9
Me) ppm. 13C NMR: δ = 170.8 (C=N), 162.6 (isoxazole-CO), 140.7
(C=CH), 133.8, 131.1, 130.0, 128.4, 127.6, 127.3, 126.9, 126.2,
125.7, 125.2 (3 Ar-C and 7 Ar-CH), 121.9 (C=CH), 103.4 (isox-
azole-CH), 79.5 (OCH), 65.1 (OCH2), 56.8, 56.2, 50.2, 42.3, 39.8,
39.5, 39.1, 37.2, 36.9, 36.2, 35.8, 32.0, 31.9, 28.4, 28.2, 28.0, 24.3,
23.8, 22.8, 22.6, 21.1, 19.4, 18.7, 11.9 ppm. HRMS (ESI): calcd. for
C42H58NO2 [M + H]+ 608.4462; found 608.4487 (4.1 ppm).
(OCH), 66.4 (OCH2), 56.4, 55.3, 49.1, 41.3, 38.7, 38.5, 38.1, 36.2,
35.8, 35.2, 34.8, 30.9, 30.8, 28.6, 27.4, 27.2, 27.0, 25.6, 23.4, 23.3,
22.8, 21.5, 21.1, 19.4, 18.8, 11.9 ppm. HRMS (ESI): calcd. for
C50H64NO2 [M + H]+ 710.4932; found 710.4968 (5.1 ppm).
5-[(4-Cholest-5-en-3β-yloxy)-1-butyl]-3-[6-(2H-1-benzopyran-2-one)-
isoxazole] (7): Following Method 1, compound 7 (26 mg, 75%) was
isolated as a white solid. Rf = 0.53 (hexane/EtOAc, 8:2), m.p. 161–
5-[(4-Cholest-5-en-3β-yloxy)-1-butyl]-3-(1-naphthyl)isoxazole (6b): 163 °C. 1H NMR: δ = 8.15–8.00 (m, 2 H, Ar-H), 7.81 (d, 3J =
3
Following Method 1, compound 6b (125 mg, 90%) was isolated as
8.0 Hz, 1 H, Ar-H), 7.49 (d, J = 7.6 Hz, 1 H, Ar-H), 6.63 (s, 1 H,
1
a white solid. Rf = 0.69 (hexane/EtOAc, 9:1), m.p. 130–132 °C. H
isoxazole-H), 6.54 (d, 3J = 7.6 Hz, 1 H, Ar-H), 5.35 (br. s, 1 H,
NMR: δ = 8.41–8.33 (m, 1 H, Ar-H), 7.98–7.87 (m, 2 H, Ar-H), CH=C), 3.54 (t, 3J = 6.2 Hz, 2 H, OCH2), 3.28–3.11 (m, 1 H,
3
7.72–7.66 (m, 1 H, Ar-H), 7.59–7.49 (m, 3 H, Ar-H), 6.32 (s, 1 H,
OCH), 2.94 (t, J = 6.2 Hz, 2 H, isoxazole-CH2), 2.38–0.80 (m, 44
3
isoxazole-H), 5.35 (br. s, 1 H, CH=C), 3.53 (t, J = 6.3 Hz, 2 H,
H), 0.68 (s, 3 H, Me) ppm. 13C NMR: δ = 169.8 (C=N), 163.8
Eur. J. Org. Chem. 2014, 2522–2532
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2527

V. Algay, J. O’Sullivan, F. Heaney
FULL PAPER
(isoxazole-CO), 160.2 (coumarin-CO), 159.2 (coumarin-CO-
OC=C), 143.9 (coumarin-COC=C), 140.8 (C=CH), 133.5, 132.5,
130.1, 120.1, 119.0, 119.0 (2 Ar-C and 4 Ar-CH), 121.7 (C=CH),
was extracted with toluene. The organic phase was washed with
water and dried with anhydrous Na2SO4, and the solvent was re-
moved under reduced pressure. The residue was purified by flash
101.6 (isoxazole-CH), 79.3 (OCH), 69.6 (OCH2), 56.8, 56.2, 50.1, column chromatography (hexane/EtOAc 30–60 %) to give com-
42.3, 42.3, 39.8, 39.5, 37.3, 36.5, 36.2, 35.8, 31.9, 31.6, 28.2, 28.0,
pound 11 (50 mg, 10% over the two steps from 9 to 11) as an off-
24.3, 23.8, 22.8, 22.6, 21.5, 20.8, 19.4, 18.7, 11.8 ppm. HRMS white solid. Rf = 0.58 (hexane/EtOAc, 8:2), m.p. 152–154 °C. 1H
(ESI): calcd. for C43H60NO4 [M + H]+ 654.4517 found: 654.4535 NMR: δ = 10.08 (s, 1 H, CHO), 7.96 (s, 4 H, Ar-H), 6.70 (br. s, 1
(2.8 ppm).
H, NH), 5.37 (br. s, 1 H, CH=C), 4.76 (br. s, 1 H, NH), 4.56–4.42
(m, 1 H, OCH), 3.58–3.48 (m, 2 H, CONHCH2), 3.28–3.17 (m, 2
5-[(4-Cholest-5-en-3βyloxy)-1-butyl]-3-[4-(2-phenyldiazenyl)phenyl]-
isoxazole (8): Following Method 3, compound 8 (24 mg, 56%) was
isolated as a bright orange solid. Rf = 0.42 (petroleum ether/
EtOAc, 9:1), m.p. 103–105 °C. 1H NMR: δ = 8.05–7.91 (m, 6 H,
Ar-H), 7.58–7.45 (m, 3 H, Ar-H), 6.38 (s, 1 H, isoxazole-H), 5.34
H, OCONHCH2), 2.41–0.81 (m, 44 H), 0.67 (s, 3 H, Me) ppm. 13
C
NMR: δ = 191.6 (C=O), 167.8 (CONH), 157.4 (OCONH), 139.8
(C=CH), 138.1, 132.5 (2 Ar-C), 130.9, 129.8, 128.8, 127.7 (4 Ar-
CH), 122.6 (C=CH), 76.6 (OCH), 56.7, 56.2, 50.0, 42.3, 39.8, 39.6,
39.5, 38.7, 38.6, 37.0, 36.6, 36.2, 35.8, 31.9, 30.4, 28.9, 28.2, 28.0,
27.9, 24.3, 23.8, 23.0, 22.8, 22.6, 21.1, 19.3, 18.7, 11.0 ppm.
3
(br. d, 1 H, CH=C), 3.51 (t, J = 6.2 Hz, 2 H, OCH2), 3.22–3.06
(m, 1 H, OCH), 2.85 (t, 3J = 7.4 Hz, 2 H, isoxazole-CH2), 2.47–
0.77 (m, 44 H), 0.67 (s, 3 H, Me) ppm. 13C NMR: δ = 174.4 (C=N),
153.3 (isoxazole-CO), 152.6 (Ar-C), 141.0 (C=CH), 131.7 (Ar-C),
131.3 (Ar-CH), 129.2 (Ar-CH), 128.9 (Ar-C), 127.6 (Ar-CH),
127.2, 123.4 (Ar-CH), 123.0 (Ar-CH), 121.6 (C=CH), 99.1 (isox-
azole-CH), 79.1 (OCH), 67.3 (OCH2), 56.8, 56.2, 50.2, 42.3 (q-C),
39.8, 39.5, 39.2, 37.3, 36.9 (q-C), 36.2, 35.8, 32.0, 31.9, 29.6, 28.5,
28.2, 28.0, 26.7 (isoxazole-CH2), 24.4, 24.3, 23.8, 22.8, 22.6, 21.1,
19.4, 18.7, 11.9 ppm. HRMS (ESI): calcd. for C46H64N3O2 [M +
H]+ 690.4993; found 690.5018 (3.6 ppm).
3β-(N-{4-[(4-Hydroxyiminoformylbenzoyl)amino]butyl}carbamoyl)-
cholesterol (12)
Method A Ϫ from Free Aldehyde 11: A mixture of aldehyde 11
(80 mg, 0.13 mmol), hydroxylamine hydrochloride (14 mg,
0.19 mmol, 1.5 equiv.), and pyridine (15 mg, 0.19 mmol, 1.5 equiv.)
in EtOH (3 mL) was heated to 125 °C for 1 h in a microwave reac-
tor (10 mL vessel, Pmax = 300 W). The solvent was removed under
reduced pressure. The residue was dissolved in chloroform, and this
solution was washed with H2O. The organic layer was dried with
anhydrous Na2SO4, and the solvent was removed under reduced
pressure to give oxime 12 (42 mg, 48%) as an off-white solid.
3β-[N-(4-Aminobutyl)carbamoyl]cholesterol (9):[19] In a modifica-
tion of the literature procedure, a solution of cholesterol chloro-
formate (2.25 g, 5 mmol) in dry toluene (100 mL) was added drop-
wise over 30 min to a solution of ethylenediamine (16.7 mL,
250 mmol) in dry toluene (150 mL) at 0 °C under a nitrogen atmo-
sphere. The resulting solution was stirred at room temperature for
17 h. The reaction mixture was washed with water, the organic ex-
tract was dried with anhydrous magnesium sulfate, and the solvents
were evaporated to dryness in vacuo to give compound 9 (490 mg,
88%) as a white solid.
Method B Ϫ from Protected Aldehyde 10: A mixture of 10 (80 mg,
0.12 mmol), hydroxylamine hydrochloride (52 mg, 0.71 mmol,
6 equiv.), and pyridine (28 mg, 0.35 mmol, 3 equiv.) in EtOH
(3 mL) was heated to 125 °C for 1 h in a microwave reactor (10 mL
vessel, Pmax = 300 W). The solvent was removed under reduced
pressure. The residue was dissolved in chloroform, and this solution
was washed with satd. aq. NaHCO3 and H2O. The organic layer
was dried with anhydrous Na2SO4, and the solvent was removed
under reduced pressure to give pure oxime 12 (75 mg, 91%) as an
off-white solid. Rf = 0.60 (hexane/EtOAc, 8:2), m.p. 156–158 °C.
3β-(N-{4-[(4-Formylbenzoyl)amino]butyl}carbamoyl)cholesterol (11):
DCC (99 mg, 0.48 mmol, 1.2 equiv.) and DMAP (59 mg,
0.48 mmol, 1.2 equiv.) were added to a suspension of aminocarb-
amate cholesterol 9 (200 mg, 0.40 mmol) and 4-(dimethoxymethyl)-
benzoic acid[20] (94 mg, 0.48 mmol, 1.2 equiv.) in dry CH2Cl2
(5 mL) and dry toluene (5 mL) at 0 °C under argon. The mixture
was stirred at room temperature for 17 h, then the solvent was re-
moved. The residue was purified by flash column chromatography
(hexane/EtOAc 35%) to give 3β-cholest-5-en-3-yl N-{3-[(4-dimeth-
oxylbenzoyl)amino]butyl}carbamate (10) (144 mg, 53%) as an off-
white solid. Rf = 0.79 (hexane/EtOAc, 7:3), m.p. 158–160 °C. 1H
3
1H NMR: δ = 8.15 (s, 1 H, CHNOH), 7.80 (d, J = 8.1 Hz, 2 H,
Ar-H), 7.63 (d, 3J = 8.1 Hz, 2 H, Ar-H), 6.47 (br. s, 1 H, NH), 5.36
(br. s, 1 H, CH=C), 4.73 (br. s, 1 H, NH), 4.58–4.42 (m, 1 H,
OCH), 3.56–3.45 (m, 2 H, CONHCH2), 3.31–3.17 (m, 2 H,
OCONHCH2), 2.42–0.82 (m, 44 H), 0.68 (s, 3 H, Me) ppm. 13C
NMR: δ = 166.9 (CONH), 156.5 (OCONH), 149.9 (C=N), 139.8
(C=CH), 135.6, 135.0 (2 Ar-C), 127.4, 127.4, 127.1, 127.1 (4 Ar-
CH), 122.5 (C=CH), 76.6 (OCH), 56.7, 56.2, 50.0, 42.3, 39.8, 39.5,
38.6, 37.0, 36.6, 36.2, 35.8, 31.9, 28.2, 28.2, 27.7, 26.5, 24.3, 23.8,
3
3
22.8, 22.6, 21.1, 19.3, 18.7, 11.9 ppm. IR: ν = 3280, 1765, 1620,
˜
NMR: δ = 7.78 (d, J = 8.3 Hz, 2 H, Ar-H), 7.52 (d, J = 8.3 Hz,
2 H, Ar-H), 6.38 (br. s, 1 H, NH), 5.52 [s, 1 H, CH(OMe)2], 5.43
(br. s, 1 H, CH=C), 4.70 (br. s, 1 H, NH), 4.55–4.41 (m, 1 H,
OCH), 3.55–3.45 (m, 2 H, CONHCH2), 3.32 (s, 3 H, OMe), 3.31
(s, 3 H, OMe), 3.28–3.17 (m, 2 H, OCONHCH2), 2.41–0.96 (m, 44
H), 0.68 (s, 3 H, Me) ppm. 13C NMR: δ = 167.3 (CONH), 156.4
(OCONH), 141.4 (C=CH), 139.8, 134.7 (2 Ar-C), 126.9, 126.9,
126.9, 126.8 (4 Ar-CH), 122.5 (C=CH), 102.4 [CH(OMe)2], 76.6
(OCH), 56.7, 56.2, 52.6, 50.0, 49.1, 42.3, 40.4, 39.8, 39.7, 39.5, 38.6,
37.0, 36.6, 36.2, 35.8, 34.0, 31.9, 28.2, 28.0, 27.7, 25.6, 25.2, 24.2,
23.8, 22.8, 22.6, 21.1, 19.4, 18.8, 11.9 ppm. HRMS (ESI): calcd. for
C42H67N2O5 [M + H]+ 679.5044; found 679.5085 (6.0 ppm).
1461, 1405, 1382, 1211, 1041, 870, 692 cm–1. HRMS (ESI): calcd.
for C40H62N3O4 [M + H]+ 648.4735; found 648.4772 (5.7 ppm).
General Procedure for the Formation of Amidocarbamate-Linked
Steroidal Conjugates 14a,b: The alkyne (1 equiv.), propargyl
alcohol (leading to 14a), or phenyl propargyl ether (leading to 14b)
was added to a round-bottomed flask containing oxime 12
(2 equiv.) and Ch-T (2.5 equiv.) in EtOH (1 mL per 0.1 mmol ox-
ime). The mixture was stirred for 17 h at room temp. The mixture
was extracted with toluene, and the organic phase was washed with
H2O. The organic layer was dried with anhydrous Na2SO4, and
the solvent was removed under reduced pressure. The residue was
purified by flash column chromatography (hexane/EtOAc 0–15%)
to give the title compounds.
A solution of HCl (2 n; 10 mL) was added to a solution of crude
protected aldehyde 10 (512 mg) in toluene (10 mL). After a few
minutes in a sonication bath, the emulsion was stirred for 2 h at
room temp. The reaction was carefully quenched with satd. aque-
ous NaHCO3 until the aqueous layer became neutral. The product
3β-[N-(4-{[4-(5-Hydroxymethyl)isoxazol-3-yl]benzoylaminobutyl})-
carbamoyl] cholesterol (14a): Compound 14a was isolated (11 mg,
21%) as an off-white solid. Rf = 0.26 (hexane/EtOAc, 8:2), m.p.
2528
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2522–2532

C-3β-Tethered Functional Cholesterol Conjugates
1
3
120–122 °C. H NMR: δ = 7.84 (d, J = 8.0 Hz, 2 H, Ar-H), 7.26
(OCH), 75.6, 67.0, 65.3, 55.8, 55.2, 49.2, 41.3, 38.8, 38.5, 38.0, 36.2,
35.9, 35.2, 34.77, 30.94, 30.9, 27.4, 27.2, 27.0, 23.3, 22.8, 21.8, 21.6,
3
(d, J = 8.0 Hz, 2 H, Ar-H), 6.58 (s, 1 H, isoxazole-H), 5.39 (br. s,
1 H, CH=C), 4.81 (s, 2 H, OCH2), 4.74 (br. s, 1 H, NH), 4.57–4.41 20.1, 18.4, 17.7, 10.9 ppm. IR: ν = 2753, 1708, 1460, 1371, 1232,
˜
(m, 1 H, OCH), 3.63–3.55 (m, 2 H, CONHCH2), 3.26–3.11 (m, 2 1027, 691 cm–1. HRMS (ESI): calcd. for C36H55O3 [M + H]+
H, OCONHCH2), 2.62–0.75 (m, 44 H), 0.67 (s, 3 H, Me) ppm. 13
C
535.4146; found 535.4135 (2.0 ppm).
NMR: δ = 170.7 (C=N), 166.9 (CONH), 160.5 (isoxazole-CO),
156.5 (OCONH), 140.4 (C=CH), 134.6, 134.1 (2 Ar-C), 129.7,
128.7, 128.7, 128.1 (4 Ar-CH), 122.3 (C=CH), 99.8 (isoxazole-CH),
79.6 (OCH), 56.7, 56.2, 53.0, 50.1, 42.3, 39.8, 39.5, 38.6, 37.0, 36.6,
36.2, 35.9, 31.9, 28.2, 27.8, 26.5, 24.3, 23.8, 22.7, 22.6, 21.1, 19.3,
4-[2-(Cholest-5-en-3β-yloxy)ethoxy]benzaldehyde (p-15): An off-
white solid (802 mg, 58%). Rf = 0.60 (hexane/EtOAc, 8:2), m.p.
1
3
90–92 °C. H NMR: δ = 9.88 (s, 1 H, CHO), 7.84 (d, J = 8.7 Hz,
3
2 H, Ar-H), 7.03 (d, J = 8.7 Hz, 2 H, Ar-H), 5.39–5.30 (m, 1 H,
3
3
CH=C), 4.19 (t, J = 4.9 Hz, 2 H, OCH2), 3.86 (t, J = 4.9 Hz, 2
H, OCH2), 3.31–3.19 (m, 1 H, OCH), 2.45–0.75 (m, 40 H), 0.67 (s,
3 H, Me) ppm. 13C NMR: δ = 190.8 (C=O), 164.0 (Ar-C), 140.7
(C=CH), 131.9, 130.0 (2 Ar-H), 121.8 (Ar-C), 121.8 (C=CH), 114.9
(2 Ar-H), 79.9 (OCH), 76.6, 68.1, 66.2, 65.9, 56.8, 56.2, 50.2, 42.3,
39.8, 39.5, 39.0, 37.2, 36.9, 36.2, 35.8, 32.0, 31.9, 31.6, 28.4, 24.3,
18.7, 11.9 ppm. IR: ν = 3354, 3293, 1770, 1632, 1565, 1480, 1441,
˜
1375, 1035, 1021, 890 cm–1. HRMS (ESI): calcd. for C43H64N3O5
[M + H]+ 702.4840; found 702.4867 (3.8 ppm).
3β-[N-(4-{[4-(5-Phenoxymethyl)isoxazol-3-yl]benzoylaminobutyl})-
carbamoyl] cholesterol (14b): Compound 14b was isolated (21 mg,
37%) as an off-white solid. Rf = 0.45 (hexane/EtOAc, 8:2), m.p.
107–109 °C. 1H NMR: δ = 7.88–7.71 (m, 4 H, Ar-H), 7.50–7.41
(m, 3 H, Ar-H), 7.25 (d, 3J = 8.0 Hz, 2 H, Ar-H), 6.56 (s, 1 H,
isoxazole-H), 5.37 (br. s, 1 H, CH=C), 4.72 (br. s, 1 H, NH), 4.69
(s, 2 H, OCH2), 4.55–4.40 (m, 1 H, OCH), 3.65–3.55 (m, 2 H,
CONHCH2), 3.24–3.10 (m, 2 H, OCONHCH2), 2.60–0.77 (m, 44
H), 0.68 (s, 3 H, Me) ppm. 13C NMR: δ = 170.7 (C=N), 167.0
(CONH), 162.4 (isoxazole-CO), 156.5 (OCONH), 140.5 (C=CH),
134.6, 134.0, 130.0 (3 Ar-C), 129.8, 129.7, 129.6, 129.6, 128.7,
128.2, 127.8 (9 Ar-CH), 122.2 (C=CH), 100.7 (isoxazole-CH), 79.7
(OCH), 61.1 (OCH2), 56.7, 56.3, 50.1, 42.3, 39.8, 39.6, 38.6, 37.1,
36.6, 36.3, 35.9, 31.9, 28.2, 27.8, 26.6, 24.3, 23.8, 22.8, 21.1, 19.3,
23.8, 22.8, 22.7, 22.6, 21.1, 19.4, 18.7, 11.9 ppm. IR: ν = 2761,
˜
1705, 1461, 1382, 1225, 1028, 832 cm–1. HRMS (ESI): calcd. for
C36H55O3 [M + H]+ 535.4146; found 535.4168 (4.1 ppm).
General Procedure for the Preparation of Oximes 16: A mixture
of aldehyde 15 (500 mg, 0.94 mmol), hydroxylamine hydrochloride
(97 mg, 1.40 mmol, 1.5 equiv.), and pyridine (110 mg, 1.40 mmol,
1.5 equiv.) in EtOH was heated to 125 °C for 1 h in a microwave
reactor (10 mL vessel, Pmax = 300 W). The solvent was removed
under reduced pressure, the residue was dissolved in chloroform,
and this solution was washed with H2O. The organic layer was
dried with anhydrous Na2SO4, and the solvent was removed to give
the pure oximes as off-white solids in quantitative yields.
18.7, 11.9 ppm. IR: ν = 3288, 1768, 1644, 1570, 1450, 1383, 1022,
˜
2-[2-(Cholest-5-en-3β-yloxy)ethoxy]benzaldehyde oxime (o-16): An
off-white solid (511 mg). Rf = 0.57 (hexane/EtOAc, 8:2), m.p. 112–
114 °C. 1H NMR: δ = 8.55 (s, 1 H, CH=NOH), 8.20 (s, 1 H,
888 cm–1. HRMS (ESI): calcd. for C49H68N3O5 [M + H]+ 778.5153;
found 778.5184 (4.0 ppm).
3
General Procedure for the Preparation of Aldehydes 15: A mixture
of cholesterol (1.00 g, 2.59 mmol), 2-, 3-, or 4-(2-hydroxyethoxy)-
benzaldehyde (purchased from TCI Europe; 2.15 g, 12.93 mmol,
5 equiv.), and MK-10 (activated at 120 °C overnight before use;
2.00 g) in chloroform (5 mL) was heated to 90 °C for 8 h in a micro-
wave reactor (10 mL vessel, Pmax = 300 W). The solvent was re-
moved under reduced pressure, and hexane (20 mL) was added.
The catalyst was removed by filtration and washed with hexane.
The solvent was evaporated, and the residue was purified by flash
column chromatography (hexane/EtOAc 0–10%) to give the title
compounds.
CH=NOH), 7.72 (d, J = 7.8 Hz, 1 H, Ar-H), 7.36–7.27 (m, 2 H,
Ar-H), 7.01–6.86 (m, 1 H, Ar-H), 5.40–5.25 (m, 1 H, CH=C), 4.15
(t, 3J = 5.1 Hz, 2 H, OCH2), 3.85 (t, 3J = 5.0 Hz, 2 H, OCH2),
3.35–3.20 (m, 1 H, OCH), 2.46–0.77 (m, 40 H), 0.68 (s, 3 H, Me)
ppm. 13C NMR: δ = 157.0 (Ar-C), 146.5 (C=N), 140.8 (C=CH),
131.1, 129.2 (2 Ar-H), 123.4 (Ar-C), 121.8 (C=CH), 112.6 (2 Ar-
H), 79.8 (OCH), 76.6, 68.5, 66.3, 56.8, 56.2, 50.2, 42.3, 39.8, 39.5,
39.1, 37.2, 36.9, 36.2, 35.8, 32.0, 31.9, 28.4, 28.3, 28.0, 24.3, 23.9,
22.8, 22.6, 21.1, 19.4, 18.7, 11.9 ppm. IR: ν = 3230, 1452, 1381,
˜
1225, 1029, 765 cm–1. HRMS (ESI): calcd. for C36H56NO3 [M +
H]+ 550.4255; found 550.4285 (5.4 ppm).
2-[2-(Cholest-5-en-3β-yloxy)ethoxy]benzaldehyde (o-15): An off-
white solid (485 mg, 35%). Rf = 0.62 (hexane/EtOAc, 8:2), m.p.
86–88 °C. H NMR: δ = 10.53 (s, 1 H, CHO), 7.88–7.80 (m, 1 H,
3-[2-(Cholest-5-en-3β-yloxy)ethoxy]benzaldehyde oxime (m-16): An
off-white solid (512 mg). Rf = 0.59 (hexane/EtOAc, 8:2), m.p. 110–
112 °C. 1H NMR: δ = 8.08 (s, 1 H, CH=NOH), 7.35–7.07 (m, 4
H, Ar-H, CH=NOH), 7.03–6.90 (m, 1 H, Ar-H), 5.40–5.25 (m, 1
H, CH=C), 4.13 (t, J = 5.1 Hz, 2 H, OCH2), 3.84 (t, J = 5.1 Hz,
2 H, OCH2), 3.35–3.20 (m, 1 H, OCH), 2.48–0.77 (m, 40 H), 0.68
(s, 3 H, Me) ppm. 13C NMR: δ = 158.1 (Ar-C), 149.2 (C=N), 139.8
(C=CH), 132.4, 128.7 (2 Ar-H), 124.8 (Ar-C), 120.7 (C=CH), 115.9
(2 Ar-H), 78.8 (OCH), 75.6, 66.8, 65.4, 55.8, 55.1, 49.2, 41.3, 38.8,
38.5, 38.0, 36.2, 35.9, 35.2, 34.8, 30.9, 30.9, 27.2, 27.0, 23.3, 22.8,
1
Ar-H), 7.59–7.47 (m, 1 H, Ar-H), 7.11–6.95 (m, 2 H, Ar-H), 5.41–
3
5.29 (m, 1 H, CH=C), 4.23 (t, 3J = 5.0 Hz, 2 H, OCH2), 3.89 (t, J
3
3
= 5.0 Hz, 2 H, OCH2), 3.35–3.19 (m, 1 H, OCH), 2.46–0.77 (m, 40
H), 0.68 (s, 3 H, Me) ppm. 13C NMR: δ = 189.9 (C=O), 161.4
(Ar-C), 140.6 (C=CH), 135.8, 128.2 (2 Ar-H), 125.1 (Ar-C), 121.9
(C=CH), 112.9 (2 Ar-H), 79.9 (OCH), 76.6, 68.5, 65.9, 56.8, 56.2,
50.2, 42.3, 39.8, 39.5, 39.1, 37.2, 36.9, 36.2, 35.8, 31.9, 28.4, 28.2,
28.0, 24.3, 23.8, 22.8, 22.6, 21.1, 19.4, 18.7, 11.9 ppm. IR: ν = 2765,
˜
21.8, 21.6, 20.1, 18.4, 17.7, 10.8 ppm. IR: ν = 3221, 1465, 1409,
˜
1702, 1450, 1378, 1227, 1034, 760 cm–1. HRMS (ESI): calcd. for
C36H55O3 [M + H]+ 535.4146; found 535.4131 (2.8 ppm).
1384, 1227, 1025, 702 cm–1. HRMS (ESI): calcd. for C36H56NO3
[M + H]+ 550.4255; found 550.4284 (5.2 ppm).
3-[2-(Cholest-5-en-3β-yloxy)ethoxy]benzaldehyde (m-15): An off-
white solid (803 mg, 58%). Rf = 0.62 (hexane/EtOAc, 8:2), m.p. 80–
4-[2-(Cholest-5-en-3β-yloxy)ethoxy]benzaldehyde oxime (p-16): An
off-white solid (516 mg). Rf = 0.60 (hexane/EtOAc, 8:2), m.p. 120–
122 °C. 1H NMR: δ = 8.07 (s, 1 H, CH=NOH), 7.49 (d, 3J =
1
82 °C. H NMR: δ = 9.96 (s, 1 H, CHO), 7.64–7.38 (m, 3 H, Ar-
H), 7.24–7.17 (m, 1 H, Ar-H), 5.49–5.32 (m, 1 H, CH=C), 4.17 (t, 9.0 Hz, 2 H, Ar-H), 7.35 (br. s, 1 H, CH=NOH), 6.92 (d, 3J =
3
3J = 4.8 Hz, 2 H, OCH2), 3.86 (t, J = 4.8 Hz, 2 H, OCH2), 3.35– 9.0 Hz, 2 H, Ar-H), 5.42–5.29 (m, 1 H, CH=C), 4.13 (t, 3J =
3
3.19 (m, 1 H, OCH), 2.57–0.80 (m, 40 H), 0.68 (s, 3 H, Me) ppm. 5.1 Hz, 2 H, OCH2), 3.84 (t, J = 5.1 Hz, 2 H, OCH2), 3.35–3.19
13C NMR: δ = 191.1 (C=O), 158.5 (Ar-C), 139.8 (C=CH), 136.7,
129.0 (2 Ar-H), 122.5 (Ar-C), 120.7 (C=CH), 112.1 (2 Ar-H), 78.8
(m, 1 H, OCH), 2.47–0.78 (m, 40 H), 0.68 (s, 3 H, Me) ppm. 13C
NMR: δ = 160.4 (Ar-C), 150.0 (C=N), 140.8 (C=CH), 128.4 (2 Ar-
Eur. J. Org. Chem. 2014, 2522–2532
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2529

V. Algay, J. O’Sullivan, F. Heaney
FULL PAPER
H), 124.8 (Ar-C), 121.7 (C=CH), 115.4 (2 Ar-H), 79.8 (OCH), 76.6,
67.8, 66.4, 56.8, 56.2, 50.2, 42.3, 39.8, 39.5, 39.1, 37.2, 36.9, 36.2,
35.8, 32.0, 31.9, 28.4, 28.2, 28.0, 24.3, 23.8, 22.8, 22.6, 21.1, 19.4,
dried with anhydrous Na2SO4, and the solvent was removed under
reduced pressure. The crude reaction product was purified by flash
column chromatography (hexane/EtOAc 0–10%) to give compound
18.7, 11.9 ppm. IR: ν = 3222, 1431, 1379, 1228, 1024, 842 cm–1. p-18 (65 mg, 80%) as an off-white solid. Rf = 0.45 (hexane/EtOAc,
˜
HRMS (ESI): calcd. for C36H56NO3 [M + H]+ 550.4255; found
550.4272 (3.1 ppm).
8:2). H NMR: δ = 7.82–7.70 (m, 2 H, Ar-H), 7.37–7.22 (m, 2 H,
Ar-H), 7.10–6.94 (m, 5 H, Ar-H), 6.58 (s, 1 H, isoxazole-H), 5.35
(br. s, 1 H, CH=C), 5.19 (s, 2 H, OCH2), 4.15 (t, 3J = 5.0 Hz, 2 H,
OCH2), 3.85 (t, 3J = 4.8 Hz, 2 H, OCH2), 3.32–3.18 (m, 1 H,
OCH), 2.50–0.78 (m, 40 H), 0.68 (s, 3 H, Me) ppm. 13C NMR: δ
= 168.3 (C=N), 162.1 (C=CH), 160.4 (Ar-C), 157.8 (Ar-C), 140.8
(isoxazole-CO), 133.9, 129.8, 129.7, 128.2, 127.9, 121.9, 121.7,
121.4, 115.4, 115.0, 114.8 (Ar-C, Ar-CH, and C=CH), 101.1 (isox-
azole-CH), 79.8 (OCH), 67.9 (OCH2), 66.4 (OCH2), 61.5
(CH2OPh), 56.8, 56.2, 50.2, 42.3, 39.8, 39.5, 39.1, 37.2, 36.8, 36.2,
35.8, 32.0, 31.9, 28.4, 28.2, 28.0, 24.3, 23.8, 22.8, 22.6, 21.1, 19.4,
18.7, 11.9 ppm. HRMS (ESI): calcd. for C45H62NO4 [M + H]+
680.4673; found 680.4672 (–1.5 ppm).
1
General Procedure for Cycloaddition Between (Prop-2-yn-1-yloxy)-
benzene and Putative Nitrile Oxides 17, derived from 16: (Prop-2-
yn-1-yloxy)benzene (16 mg, 0.12 mmol) was put into a round-bot-
tomed flask, a solution of oxime 16 (100 mg, 0.18 mmol, 1.5 equiv.)
and Ch-T (54 mg, 0.24 mmol, 2 equiv.) in EtOH (1 mL per
0.1 mmol oxime) was added. The mixture was stirred for 17 h at
room temp. The solvent was removed under reduced pressure, the
residue was dissolved in toluene, and this solution was washed with
H2O. The organic layer was dried with anhydrous Na2SO4, and the
solvent was removed under reduced pressure. The crude reaction
product was purified by flash column chromatography (hexane/
EtOAc 0–10%) to give the title compounds as off-white solids.
(3β)-3-[(4-Formyl)phenyloxy]cholest-5-ene (19): Compound 19, pre-
pared in an analogous manner to the hydroxyethoxy analogues 15,
was isolated (788 mg, 62%) as an off-white solid. Rf = 0.71 (hexane/
EtOAc, 8:2). 1H NMR: δ = 9.86 (s, 1 H, CHO), 7.81 (d, 3J =
8.7 Hz, 2 H, Ar-H), 6.97 (d, 3J = 8.7 Hz, 2 H, Ar-H), 5.49–5.37
(m, 1 H, CH=C), 4.33–4.17 (m, 1 H, OCH), 2.56–0.77 (m, 40 H),
0.69 (s, 3 H, Me) ppm. 13C NMR: δ = 189.7 (C=O), 163.1 (Ar-C),
139.8 (C=CH), 132.1, 129.6 (2 Ar-H), 122.9 (Ar-C), 122.8 (C=CH),
115.6 (2 Ar-H), 77.0 (OCH), 76.6, 56.8, 56.2, 50.2, 42.3, 39.8, 39.5,
38.4, 37.1, 36.8, 36.2, 35.8, 32.0, 31.9, 29.7, 28.2, 28.0, 24.3, 23.8,
22.8, 22.6, 21.1, 19.4, 18.7, 11.9 ppm. HRMS (ESI): calcd. for
C34H51O2 [M + H]+ 491.3884; found 491.3912 (5.7 ppm).
3-{[2-(2-Cholest-5-en-3β-yloxy)ethoxy]phenyl}-5-(phenoxymethyl)-
isoxazole (o-18): Compound o-18 (20 mg, 25%) was isolated. Rf =
0.40 (hexane/EtOAc, 8:2). 1H NMR: δ = 7.72 (d, 3J = 7.9 Hz, 1 H,
Ar-H), 7.38–7.20 (m, 3 H, Ar-H), 7.09–6.84 (m, 5 H, Ar-H), 6.57
(s, 1 H, isoxazole-H), 5.35 (br. s, 1 H, CH=C), 5.18 (s, 2 H, OCH2),
4.16 (t, 3J = 5.1 Hz, 2 H, OCH2), 3.85 (t, 3J = 4.9 Hz, 2 H, OCH2),
3.30–3.18 (m, 1 H, OCH), 2.50–0.79 (m, 40 H), 0.68 (s, 3 H, Me)
ppm. 13C NMR: δ = 164.2 (C=N), 162.0 (C=CH), 160.4 (Ar-C),
157.8 (Ar-C), 140.7 (isoxazole-CO), 134.8, 130.6, 130.6, 129.2,
126.8, 122.0, 121.8, 120.4, 116.0, 114.9, 114.8 (Ar-C, Ar-CH, and
C=CH), 101.2 (isoxazole-CH), 79.8 (OCH), 67.9 (OCH2), 66.4
(OCH2), 61.5 (CH2OPh), 56.8, 56.2, 50.2, 42.2, 39.8, 39.5, 39.1,
37.2, 36.8, 36.2, 35.8, 32.0, 31.9, 28.4, 28.2, 28.0, 24.4, 23.8, 22.8,
22.6, 21.1, 19.4, 18.7, 11.9 ppm. HRMS (ESI): calcd. for
C45H62NO4 [M + H]+ 680.4673; found 680.4712 (5.7 ppm).
(3β)-3-[(4-Hydroxyiminoformyl)phenyloxy]cholest-5-ene (20): Com-
pound 20, prepared in an analogous manner to the hydroxyethoxy
analogues 16, was isolated (510 mg, quantitative) as an off-white
1
solid. Rf = 0.69 (hexane/EtOAc, 8:2). H NMR: δ = 8.08 (s, 1 H,
3
CH=NOH), 7.85 (br. s, 1 H, CH=NOH), 7.48 (d, J = 8.7 Hz, 2
3-{[3-(2-Cholest-5-en-3β-yloxy)ethoxy]phenyl}-5-(phenoxymethyl)-
isoxazole (m-18): Compound m-18 (31 mg, 39%) was isolated. Rf
H, Ar-H), 6.88 (d, 3J = 8.7 Hz, 2 H, Ar-H), 5.48–5.33 (m, 1 H,
CH=C), 4.25–4.06 (m, 1 H, OCH), 2.55–0.78 (m, 40 H), 0.69 (s, 3
H, Me) ppm. 13C NMR: δ = 159.3 (Ar-C), 150.0 (C=N), 140.1
(C=CH), 128.5 (2 Ar-H), 124.4 (Ar-C), 122.5 (C=CH), 115.9 (2
Ar-H), 77.0 (OCH), 76.6, 56.8, 56.2, 50.2, 42.4, 39.8, 39.5, 38.6,
37.2, 36.8, 36.2, 35.8, 32.0, 31.9, 28.2, 28.2, 28.0, 24.3, 23.8, 22.8,
22.6, 21.1, 19.4, 18.7, 11.9 ppm. HRMS (ESI): calcd. for
C34H52NO2 [M + H]+ 506.3993; found 506.4021 (5.5 ppm).
1
= 0.44 (hexane/EtOAc, 8:2). H NMR: δ = 7.39–7.10 (m, 5 H, Ar-
H), 7.09–6.84 (m, 4 H, Ar-H), 6.57 (s, 1 H, isoxazole-H), 5.34 (br.
s, 1 H, CH=C), 5.17 (s, 2 H, OCH2), 4.15 (t, 3J = 5.1 Hz, 2 H,
OCH2), 3.85 (t, 3J = 5.0 Hz, 2 H, OCH2), 3.32–3.19 (m, 1 H,
OCH), 2.50–0.79 (m, 40 H), 0.68 (s, 3 H, Me) ppm. 13C NMR: δ
= 168.2 (C=N), 162.1 (C=CH), 159.8 (Ar-C), 159.6 (Ar-C), 140.7
(isoxazole-CO), 133.7, 130.5, 130.4, 129.7, 129.8, 122.1, 121.5,
118.9, 114.7, 114.6, 113.6 (Ar-C, Ar-CH, and C=CH), 101.1 (isox-
azole-CH), 79.8 (OCH), 67.9 (OCH2), 66.4 (OCH2), 61.6
(CH2OPh), 56.8, 56.3, 50.2, 42.2, 39.8, 39.5, 39.0, 37.2, 36.8, 36.2,
35.8, 32.0, 31.9, 28.5, 28.2, 28.0, 24.4, 23.8, 22.8, 22.6, 21.1, 19.4,
18.7, 11.9 ppm. HRMS (ESI): calcd. for C45H62NO4 [M + H]+
680.4673; found 680.4706 (4.9 ppm).
3-[(4-Cholest-5-en-3β-yloxy)phenyloxy]-5-(phenoxymethyl)isoxazole
(21): Compound 21, prepared following the modified procedure de-
scribed above for p-18, was isolated (46 mg, 60%) as an off-white
1
3
solid. Rf = 0.60 (hexane/EtOAc, 8:2). H NMR: δ = 7.80 (d, J =
8.4 Hz, 2 H, Ar-H), 7.71 (d, 3J = 8.4 Hz, 2 H, Ar-H), 7.37–7.23
(m, 2 H, Ar-H), 7.05–6.91 (m, 3 H, Ar-H), 6.58 (s, 1 H, isoxazole-
H), 5.33–5.48 (m, 1 H, CH=C), 5.19 (s, 2 H, OCH2), 4.25–4.08 (m,
1 H, OCH), 2.50–0.78 (m, 40 H), 0.69 (s, 3 H, Me) ppm. 13C NMR:
δ = 168.2 (C=N), 162.1 (C=CH), 159.7, 159.6 (2 Ar-C), 140.6 (isox-
azole-CH), 133.6, 130.5, 130.4, 129.8, 129.8, 122.1, 121.5, 118.9,
114.7, 114.6, 113.6 (Ar-C, 9 Ar-CH, and C=CH), 101.1 (isoxazole-
CH), 79.8 (OCH), 61.5 (CH2OPh), 56.8, 56.2, 50.2, 42.2, 39.8, 39.5,
39.0, 37.3, 36.8, 36.2, 35.8, 32.0, 31.9, 28.4, 28.2, 28.0, 24.4, 23.8,
22.8, 22.6, 21.1, 19.4, 18.7, 11.9 ppm. HRMS (ESI): calcd. for
C43H58NO3 [M + H]+ 636.4411; found 636.4449 (6.0 ppm).
3-{[4-(2-Cholest-5-en-3β-yloxy)ethoxy]phenyl}-5-(phenoxymethyl)-
isoxazole (p-18)
Method A Ϫ Following the General Procedure: Compound p-18 was
isolated in 58% yield (47 mg).
Method B Ϫ Following a Modified Procedure: (Prop-2-yn-1-yloxy)-
benzene (16 mg, 0.12 mmol) was put into a round-bottomed flask,
and a solution of oxime p-16 (50 mg, 0.09 mmol, 0.75 equiv.) and
Ch-T (27 mg, 0.12 mmol, 1 equiv.) in EtOH (1 mL per 0.1 mmol
oxime) was added. The mixture was stirred for 4 h at room temp.
The same dose of oxime/Ch-T was added again, and then the mix-
ture was stirred for a further 17 h at room temp. The solvent was
removed under reduced pressure, the residue was dissolved in tolu-
ene, and this solution was washed with H2O. The organic layer was
General Procedure for the Nitrile Oxide–Alkyne Cycloaddition with
Sugar and Nucleoside Substrates: Alkylated sugar 22[2 1]
(0.75 equiv.), thymidine mono-alkyne 24 (0.75 equiv.), or thymidine
bis-alkyne 26[18] (1.5 equiv.) was put into a round-bottomed flask,
and a solution of oxime p-16 and Ch-T (1.5 equiv. for 22 and 24,
2530
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 2522–2532

C-3β-Tethered Functional Cholesterol Conjugates
and 3.0 equiv. for 26) in EtOH (2 mL per 0.1 mmol oxime) was
added. The mixture was stirred for 4 h at room temp. The same
dose of oxime/Ch-T was added again, and then the mixture was
stirred for a further 17 h at room temp. The solvent was removed
under reduced pressure. The residue was dissolved in CH2Cl2, and
this solution was washed with H2O. The organic layer was dried
with anhydrous sodium sulfate, and the solvent was removed under
reduced pressure. The crude product was purified as described be-
low.
H), 6.42 (t, 3J = 6.9 Hz, 1 H, 1Ј-H), 5.47–5.28 (m, 3 H, CH=C,
NCH2), 4.58–4.42 (m, 3 H, 3Ј-H, OCH2), 4.23–4.15 (m, 4 H, 2
OCH2), 4.07–4.02 (m, 1 H, 4Ј-H), 3.90–3.82 (m, 4 H, 2 OCH2) 3.79
(m, 6 H, 2 OMe), 3.52–3.21 (m, 4 H, 2 OCH, 2 5Ј-H), 2.46–0.78
(m, 85 H), 0.68 (s, 6 H, 2 Me) ppm. 13C NMR: δ = 169.8 (C=N),
168.1 (C=N), 163.0 (C-2), 161.9 (2 C=CH), 159.7 (2 isoxazole-CO),
150.1 (C-4), 142.8 (Ar-C), 135.0 (C-6), 132.0, 130.2, 130.1, 129.2,
128.7, 128.6, 128.1, 127.5, 126.9, 121.6, 114.8 (Ar-C, Ar-CH, 2
C=CH), 111.2 (C-5), 102.3 (2 isoxazole-CH), 87.6 (CPh3), 85.1 (C-
1Ј), 83.8 (C-4Ј), 80.1 (C-3Ј), 79.6 (2 OCH), 74.5 (2 isoxazole-
OCH2), 69.6 (2 OCH2), 68.9 (2 OCH2), 63.8 (C-5Ј), 62.3 (OCH2),
58.5, 56.5, 56.1, 50.1, 42.5, 39.8, 39.6, 37.9, 37.3, 36.5, 36.2, 35.9,
31.8, 31.6, 28.2, 26.1, 24.8, 24.4, 24.0, 23.7, 22.8, 22.7, 21.1, 19.4,
3-{4-[2-(Cholest-5-en-3β-yloxy)ethoxy]phenyl}-5-(6-O-{[1,2:3,4-bis-
O-(1-methylethylidene)-α-D-galactopyranose]methoxymethyl}-
isoxazole) (23): The crude reaction product was purified by flash
column chromatography (hexane/EtOAc 10–35 %) to give com-
pound 23 (118 mg, 85%) as a white sticky solid. Rf = 0.35 (hexane/
EtOAc, 7:3). 1H NMR: δ = 7.92–7.80 (m, 2 H, Ar-H), 7.07–6.99
18.7, 11.9 ppm. IR: ν = 1699, 1580, 1411, 1335, 1223, 1021,
˜
838 cm–1. HRMS (ESI): calcd. for C109H143N4O13 [M + H]+
1716.0646; found 1716.0741 (5.5 ppm).
3
(m, 2 H, Ar-H), 6.54 (s, 1 H, isoxazole-H), 5.54 (d, J = 5.7 Hz, 1
3
Supporting Information (see footnote on the first page of this arti-
H, 1Ј-H), 5.34 (br. s, 1 H, CH=C), 5.10 (s, 2 H, OCH2), 4.61 (d, J
1
cle): Copies of H and 13C NMR spectra of new compounds.
= 5.7 Hz, 1 H, 2Ј-H), 4.31–4.14 (m, 4 H, 3Ј-H, 4Ј-H, OCH2), 4.07–
3.96 (m, 3 H, 5Ј-H, OCH2), 3.82–3.73 (m, 1 H, 6Ј-H), 3.73–3.63
(m, 1 H, 6Ј-H), 3.32–3.15 (m, 1 H, OCH), 2.36–0.81 (m, 52 H),
0.68 (s, 3 H, Me) ppm. 13C NMR: δ = 167.3 (C=N), 162.0 (C=CH),
159.8 (isoxazole-CO), 140.8 (Ar-C), 132.0 (Ar-CH), 130.2 (C=CH),
121.6 (Ar-C), 114.8 (Ar-CH), 109.3, 108.6 [C(CH3)2], 100.9 (isox-
azole-CH), 96.3 (C-1Ј), 79.6 (OCH), 74.6 (isoxazole-OCH2), 71.7,
71.2, 70.7, 70.5 (C-2Ј, C-3Ј, C-4Ј, C-5Ј), 69.6 (OCH2), 68.7 (OCH2),
66.7 (C-6Ј), 61.0 (OCH2), 58.5, 56.7, 56.1, 50.1, 42.3, 39.8, 39.5,
37.2, 36.5, 36.2, 35.8, 31.9, 31.6, 28.2, 28.0, 26.0, 24.9, 24.5, 24.3,
Acknowledgments
The work was supported by the Science Foundation of Ireland
(programme code 05/PICA/B838/NSs) and the Irish Research
Council of Science and Engineering (Embark Postgraduate Re-
search Scholarship to JOS, programme code RS/2012/128)
24.0, 23.8, 22.8, 22.6, 21.1, 19.4, 18.7, 11.9 ppm. IR: ν = 1562,
˜
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1409, 1330, 1245, 1160, 1019, 832 cm–1. HRMS (ESI): calcd. for
C51H76NO9 [M + H]+ 846.5515; found 846.5558 (5.1 ppm).
3Ј-O-{(3)-4-Ethoxy-[2-(Cholest-5-en-3β-yloxy)phenyl]isoxazol-5-
yl}methyl-5Ј-O-[bis(4-methoxyphenyl)phenylmethyl]thymidine (25):
The crude reaction product was purified by flash column
chromatography (hexane/EtOAc 20–40 %) to give compound 25
(53 mg, 81%) as an off-white gum. Rf = 0.30 (hexane/EtOAc, 7:3).
1H NMR: δ = 8.32 (br. s, 1 H, NH), 7.91–7.83 (m, 2 H, Ar-H),
7.63–7.58 (m, 1 H, Ar-H), 7.47–7.23 (m, 2 H, Ar-H), 7.10–7.03 (m,
2 H, Ar-H), 6.92–6.83 (m, 4 H, Ar-H), 6.56 (s, 1 H, isoxazole-H),
6.44 (t, 3J = 6.9 Hz, 1 H, 1Ј-H), 5.39 (br. s, 1 H, CH=C), 4.66–4.58
(m, 1 H, 3Ј-H), 4.52 (s, 2 H, OCH2), 4.23 (t, 3J = 13.9 Hz, 2 H,
OCH2), 4.12–4.06 (m, 1 H, 4Ј-H), 3.90 (t, 3J = 13.9 Hz, 2 H,
OCH2), 3.83 (m, 6 H, 2 OMe), 3.57–3.24 (m, 3 H, OCH, 2 5Ј-H),
2.50–0.82 (m, 45 H), 0.72 (s, 3 H, Me) ppm. 13C NMR: δ = 170.0
(C=N), 163.4 (C-2), 161.9 (C=CH), 159.7 (isoxazole-CO), 150.1
(C-4), 140.2 (Ar-C), 135.3 (C-6), 132.0, 130.2, 130.1, 129.1, 128.6,
128.4, 128.1, 127.4, 126.9, 121.1, 113.8 (Ar-C, Ar-CH, C=CH),
111.2 (C-5), 102.3 (isoxazole-CH), 86.5 (CPh3), 83.9 (C-1Ј), 83.8
(C-4Ј), 80.1 (C-3Ј), 79.6 (OCH), 74.5 (isoxazole-OCH2), 69.6
(OCH2), 68.9 (OCH2), 63.8 (C-5Ј), 62.3 (OCH2), 58.8, 56.5, 56.1,
50.1, 42.4, 39.8, 39.6, 37.8, 37.3, 36.5, 35.8, 31.8, 31.6, 28.2, 28.1,
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˜
= 3310, 1709, 1570, 1411, 1350, 1229, 1021, 848 cm–1. HRMS
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Eur. J. Org. Chem. 2014, 2522–2532
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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Received: December 6, 2013
Published Online: February 24, 2014
2532
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Eur. J. Org. Chem. 2014, 2522–2532