Synthesis and biological evaluation of desmethylveramiline, a micromolar Hedgehog inhibitor

Article abstract of DOI:10.1016/j.bmcl.2011.04.103

Desmethylveramiline (1), an aza steroid analogue of veramiline was designed as a surrogate for cyclopamine, a reference antagonist of the Sonic Hedgehog (Shh) pathway. Desmethyveramiline (1) was prepared in seven steps from commercially available Fernholt

Full text of DOI:10.1016/j.bmcl.2011.04.103

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3608–3612
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of desmethylveramiline, a micromolar
Hedgehog inhibitor
Guillaume Guerlet a, Thomas Spangenberg a, André Mann a, , Hélène Faure b, Martial Ruat b
⇑
a Laboratoire d’Innovation Thérapeutique, UMR 7200 CNRS-Université de Strasbourg Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France
b Signal Transduction and Developmental Neuropharmacology group, Neurobiology and Development, CNRS UPR-3294, 1 Avenue de la Terrasse, 91198 Gif-Yvette, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Desmethylveramiline (1), an aza steroid analogue of veramiline was designed as a surrogate for cyclop-
amine, a reference antagonist of the Sonic Hedgehog (Shh) pathway. Desmethyveramiline (1) was pre-
pared in seven steps from commercially available Fernholtz acid using the hydroformylation of a
terminal olefine as the key step for the construction of the piperidine appendage. In two assays (i) the
inhibition of the Shh-induced Gli-dependent luciferase activity in Shh-light2 cells, (ii) the inhibition of
the SAG-induced differentiation of the mesenchymal C3H10T1/2 cells, desmethylveramiline (1) is an
Received 23 February 2011
Revised 21 April 2011
Accepted 22 April 2011
Available online 29 April 2011
Keywords:
Sonic Hedgehog pathway
Cyclopamine
inhibitor in the lM range comparable to cyclopamine.
Ó 2011 Elsevier Ltd. All rights reserved.
Hydroformylation
Cyclohydrocarbonylation
The Sonic Hedgehog (Shh) signaling pathway plays a pivotal role
in embryogenesis and in adult tissues, including brain.1–3 The acti-
vation of Shh signaling is initiated by the binding of the Shh ligand
to the membrane receptor Patched (Ptc), which relieves the Ptc-
mediated inhibition of the transmembrane protein Smoothened
(Smo). Aberrant Shh signals have been reported to be involved in
numerous cancers. Therefore it is commonly accepted that antago-
nists for Shh signaling have great potential in cancer therapy.4–8
Cyclopamine, an alkaloid with a C-nor-D-homo steroid skeleton is
the reference antagonist of the Shh signaling pathway. However
cyclopamine has a poor biodisponibility and chemical stability. Fur-
thermore, cyclopamine is readily converted to inactive veratramine
in acidic media,9 that precludes any oral administration as a drug.
Interestingly, Tremblay’s group have performed extensive transfor-
mations on the core structure of cyclopamine culminating in the dis-
covery of IPI-926, a compound with improved pharmacokinetic
properties.10 In the past, other steroidal alkaloids such as solanidine
and veramiline were mentioned for their teratogenic profile, but un-
til now few data were available for their efficiency on the Shh signal-
ing.11 In our effort to discover new ligands interfering with Shh
signaling,12 we found of interest to prepare azasteroid 1, a seco-ana-
logue of cyclopamine, having a steroidal ring system and bearing at
C22 a piperidine ring. Compound 1, desmethylveramiline, was se-
lected as target with the following rationales: (i) 1 is readily ob-
tained from the Fernholtz acid13 via an original approach (vide
infra); (ii) 1 has a simple steroid frame in respect to the C-nor-D-
homo steroid skeleton, but orients the nitrogen lone pair present
in the piperidinyl ring, in similar spatial areas14; (iii) azasteroid of
type 1 can be a surrogate for structurally more complex cyclopamine
or jervine. In this Letter, we report the synthesis and the biological
activity on the Shh signaling pathway of desmethylveramiline (1)
(Fig. 1). The chemical strategyused is based on the hydroformylation
of a terminal olefine.
Esterification of commercially available Fernholtz acid in meth-
anol/H2SO4 provided ester 2 in 89% yield. Protection of the second-
ary alcohol at C3 with TBSOTf furnished 3 and subsequent LAH
reduction of the ester function gave 4 in 95% (two steps). The de-
sired aldehyde 5 was obtained as a white solid which matched re-
ported analytical data, either by a Swern oxidation protocol (91%)
or by using IBX (quant) (Scheme 1).15 Next, 5 was subjected to a
three components aza-Sakurai–Hosomi reaction under optimized
conditions, a convenient route for the synthesis homallylamines
from aldehydes, using a carbamate and allylsilane as partners.16
If diastereoselectivity is a concern it has been shown that a sub-
strate control is operating.17 Indeed the reaction of Fernhotlz alde-
hyde (5), benzylcarbamate and allylsilane in presence of BF3ꢀEt2O
proceeded rapidly with an excellent diastereoselectivity at C22
(dr 95/5) and homoallylamine 6 was obtained with a good yield
(87%). As the reaction mixture was allowed to reach room temper-
ature, the TBS group on the secondary alcohol at C3 was cleaved in
the meantime.
The formation of a water molecule during the amino-allylation
and the presence of BF3ꢀEt2O are accounting for this observed
desilylation (fluoride ions are produced). The stereochemistry at
C22, expected to be S (see below), could be explained by a modified
⇑
Corresponding author.
E-mail address: andre.mann@unistra.fr (A. Mann).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.103

G. Guerlet et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3608–3612
3609
HN
HO
H
F
22
22
H+
E
H
O
N
H
N
H
H
N
D
C
A
B
HO
HO
HO
HO
cyclopamine
veramiline
desmethylveramiline (1)
veratramine
Figure 1. Steroidal alkaloids with a piperidine ring.
O
O
OMe
TBSOTf,
2,6-lutidine,
OH
MeOH,
DCM,
- 40 °C to rt,
quant.
H2SO4,
reflux,
89%
HO
HO
2
Fernholtz acid
OH
O
OMe
LiAlH4,
THF, 0 °C to reflux,
95%
TBSO
TBSO
3
4
O
DMSO, oxalyl chloride,
NEt3, DCM,
- 78 °C, 91%
or
IBX,
EtOAc, reflux,
2 h, quant
TBSO
5
Scheme 1. Preparation of Fernholtz aldehyde 5.
SiMe3
22
Cbz NH2 1 equiv
TMS
S
1 equiv
NHCbz
H
Me
N
BF3-Et2O 1 equiv
LA
5
H
DCM, 0 °C to rt, 1 h,
Cbz
3
87% dr = 95/5
HO
5
one pot
6
6
T
Scheme 2. 1,2-syn-diastereoselective aza-Sakurai–Hosomi reaction on Fernholtz aldehyde 5.
Felkin–Anh model (the transition state T in the box, Si attack of the
allylsilane, Scheme 2).18
phosphites as the ligands.21,22 Therefore the chemoselective hydro-
formylation of the terminal olefine in allylamine 6 was designed:
the formation of the linear aldehyde followed by its intramolecular
collapse with the resident Cbz carbamate to a piperidine ring were
expected. An other hurdle may be anticipated with allylamine 6
when submitted to hydroformylation. As syngas is a mixture of
H2/CO: 1:1, the inertia of the C5–C6 double bound towards hydro-
genation was questionable, but precedents show that even under
Homoallylamines are attractive substrates for the construction
of piperidines, indeed the missing carbon to build in the six mem-
bered heterocycle may be introduced by hydroformylation on the
terminal olefine.19,20 The control of the troublesome linear/
branched ratio for the resulting aldehydes is now manageable by
using Rh(I) as the metal and bulky bidendate phosphines or

3610
G. Guerlet et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3608–3612
Rh(CO)2(acac) 2 mol%
Biphephos 4 mol%
H2/CO (1:1) 5 bar,
CHO
S
N
Cbz
NHCbz
6
pTSA 10 mol%
THF, 65 °C, 18 h, 86%
7
HO
MeO
OMe
22
H2 1 bar
t-Bu
O
O
t-Bu
N
H
Pd/C 10%
P
P
O
O
O
O
EtOAc
2 d, 85%
3
1
Biphephos
HO
5
6
Scheme 3. Cyclohydrocarbonylation towards desmethylveramiline 1.
harsh conditions (120 bar, 120 °C), the internal double bound may
remain intact (Scheme 3).23 The unprotected secondary alcohol at
C3 on homoallylamine 6 was also a possible chelating group for
rhodium(I) present in the catalyst. In the past, we had some expe-
rience for linear hydroformylation and therefore we tested a stan-
mesenchymal C3H10T1/2 cells stimulated by SAG (100 nM), a
synthetic Smo agonist.30 The cell-based bioassays were performed
as described.12 Compared to Cur61414, another reference Smo
Table 1
dard catalytic mixture. Indeed when the allylamine
6 was
Comparison of the inhibition induced by desmethylveramiline (1), Cur61414 and
cyclopamine on ShhN-induced luciferase activity in Shh-light2 cells and SAG-induced
differentiation of C3H10T1/2 cells
submitted to hydroformylation in THF with a mixture of Rh(CO)a-
cac2, biphephos as ligand and 10 mol % of p-TSA as additive, the de-
sired enamide 7 was obtained uneventfully as the only adduct in
86% yield. The isomeric branched aldehyde was not detected in
our conditions. Indeed, in one step compound 7 is formed by the
internal reaction of the homologated aldehyde with the NCbz car-
bamate (via a transient immonium). This sequence described as a
cyclohydrocarbonylation (CHC) is now well documented. Recently
our group has reported several examples using this strategy for the
synthesis of natural compounds or biomolecules.17,24–26
% of inhibition
Shh-light2
C3H10T1/2
10
85
Compounds
1
Cur61414
Cyclopamine
0.3
5
89
58
l
M
3
lM
10
87
92
98
l
M
1
1
1
l
M
lM
4
2
1
4
7
7
47
97
96
4
3
1
13
78
71
4
1
3
99
105
2
In the next step, we planned to reduce in one pot the enamide
function and hydrogenolyze the Cbz N-protection in 7. Indeed, the
use Pd/C (10% on C) as catalyst in presence of a blanket of hydrogen
delivered desmethylveramiline 1 (85% yield) (Scheme 3). The rela-
tive stereochemistry at carbon C22 was definitively secured by sin-
gle crystal X-ray crystallography,27 and confirmed the rationale
proposed for aza-Sakurai–Hosomi reaction of allylsilane and the
Fernholtz aldehyde 5 (Scheme 4). Interestingly a recent report of
the Giannis’ group reported the syntheses of aminosteroids bearing
piperidone at C20,28 combining chiral sulfinylimine chemistry and
metathesis. In this regard the present route to 1 is complementary
in considering step and atom economy.
N
O
O
N
N
NH
O
Cl
N
N
S
O
O
O
HN
SAG
CURIS 61414
The biological evaluation of 1 on the Shh pathway was per-
formed using the two following assays: (i) the Gli-dependent lucif-
erase reporter assay in Shh-light2 cells incubated for 40 h with the
active aminoterminal fragment of Shh (ShhN, 5 nM),29 (ii) the alka-
line phosphatase (AP) assaythat reflects the differentiation of the
A
B
100
80
60
40
20
0
100
80
60
40
20
0
-8
-7
-6
-5
-8
-7
-6
-5
Log[compound], M
Log[compound], M
Figure 2. Dose dependent activity of 1 (open squares), Cur61414 (open triangle)
and cyclopamine (stars): (A) ShhN-induced luciferase activity in Shh-light2 cells;
Cur61414: IC50 = 0.1
l
M; cyclopamine: IC50 = 0.3
M. (B) SAG-induced differentiation of C3H10T1/2 cells; Cur61414:
M; cyclopamine: IC50 = 0.6 M; desmethylveramiline (1): IC50 = 3.2 M.
lM; desmethylveramiline (1):
IC50 = 1.1
IC50 = 0.3
l
l
l
l
Scheme 4. Crystal structure of 1 displayed with Mercury™ (one molecule of CDCl3
The data shown are representative of independent experiments and are the
means SEM of triplicates. The values are expressed as % of the maximal response.
and most hydrogens were omitted for clarity).27

G. Guerlet et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3608–3612
3611
antagonist31 and cyclopamine, desmethylveramiline (1) is a micro-
molar inhibitor of the Shh signaling pathway as measured in the
luciferase-based and the AP assays (Table 1). Interestingly aza-ste-
roid (1) reveals an inhibitory activity towards Shh signaling in the
same range than cyclopamine. In the Shh-light2 assay, des-
12. (a) Manetti, F.; Faure, H.; Roudaut, H.; Gorojankina, T.; Traiffort, E.;
Schoenfelder, A.; Mann, A.; Solinas, A.; Taddei, M.; Ruat, M. Mol. Pharmacol.
2010, 78, 658; (b) Roudaut, H.; Traiffort, E.; Gorojankina, T.; Vincent, L.; Faure,
H.; Schoenfelder, A.; Mann, A.; Manetti, F.; Solinas, A.; Taddei, M.; Ruat, M.
Mol. Pharmacol. 2011, 79, 453.
13. Fernholtz acid was purchased from Steraloids Inc. (USA).
14. Winkler, J. D.; Isaacs, A.; Laura Holderbaum, L.; Tatard, V.; Nadia Dahmane, N.
Org. Lett. 2009, 11, 2824.
methylveramiline (1) (IC50 = 1.1
lM) is three to four times less po-
15. Atot, M.; Schmidt, S. J.; Adams, J. L.; Dolle, R. E.; Kruse, L. I.; Frey, C. L.; Barone,
J. M. J. Med. Chem. 1992, 35, 100.
16. Veenstra, S. J.; Schmid, P. Tetrahedron Lett. 1997, 38, 997.
17. Spangenberg, T.; Airiau, E.; Thuong, M. B. T.; Donnard, M.; Billet, M.; Mann, A.
Synlett 2008, 2859.
18. Loh, T.-P.; Xu, J.; Hu, Q.-Y.; Vittal, J. J. Tetrahedron: Asymmetry 1565, 2000, 11.
19. Ojima, I.; Tsai, C.-Y.; Tzamarioudaki, M.; Bonafoux, D. Org. React. 2000, 56, 1.
20. Breit, B.; Seiche, W. Synthesis 2001, 1.
21. Billig, E.; Abatjoglou, A. G.; Bryant, D.R. US patent 4,668,651 1987; Chem. Abstr.
1987, 107, 7392.
22. Cuny, G. D.; Buchwald, S. L. J. Am. Chem. Soc. 1993, 115, 2066.
23. Toros, S.; Gemes-Pesci, I.; Heil, B.; Maho, S.; Tuba, Z. J. Chem. Soc., Chem.
Commun. 1992, 11, 858.
24. Chiou, W.-H.; Schoenfelder, A.; Sun, L.; Mann, A.; Ojima, I. J. Org. Chem. 2007,
72, 9418.
25. Airiau, E.; Chemin, C.; Girard, N.; Lonzi, G.; Mann, A.; Petricci, E.; Salvadori, J.;
Taddei, M. Synthesis 2010, 17, 2901.
26. Airiau, E.; Spangenberg, T.; Girard, N.; Schoenfelder, A.; Salvadori, J.; Taddei,
M.; Mann, A. Chem. Eur. J. 2009, 14, 10938.
tent than cyclopamine itself (IC50 = 0.3
l
M) (Fig. 2). These results
demonstrate that aza-steroid 1, missing some structural elements
in respect to cyclopamine such as the C-nor-D-Homo substructure
and the E-furane ring, can operate as a micromolar inhibitor of Shh
signaling in the two distinct assays. Those results are in line with
recent results reported by Winckler’s group for a steroidal ana-
logue of cyclopamine (Table 1).14
In conclusion we have shown that the combination of an aza-
Sakurai-Hosomi with a hydroformylation reaction is a powerful
tactic for the preparation of 25-desmethyl-veramiline (1), a steroi-
dal alkaloid, in seven steps from Fernholtz acid (57% overall yield);
from a biological point of view that 1 is a micromolar inhibitor on
Shh signaling. It comforts the assumption that a well designed aza-
steroid may match the activity of cyclopamine. Further studies on
this topic are currently undertaken in our laboratory.32
27. Crystallographic data for 1: formula: C26H43NO, CHCl3; space group: P 21 21 21;
cell lengths: a: 7.3450(2); b: 14.1260(4); c: 26.1680(8); cell angles:
a: 90.00; b:
90.00;
c
: 90.00; cell volume: 2715.0 Z: 4 Z0: 0; R-factor (%): 6.0. The data have
Acknowledgements
been registered at the Cambridge Crystallographic Data Bank with the
following number CCDC 801379.
The authors thankDr. L. Brelot for the crystallographic analysis, P.
Wehrung and P. Buisine for providing HR-MS support and
C. Antheaume for NMR expertise. T.S. gratefully acknowledges the
‘Ministère de l’Enseignement Supérieur et de la Recherche’ for a fel-
lowship. This work was supported by a grant from La Ligue contre le
Cancer (Comité des Yvelines) to M.R.
28. Fousteris, M. A.; Schubert, U.; Roell, D.; Roediger, J.; Bailis, N.; Nikolaropoulos,
S. S.; Baniahmad, A.; Giannis, A. Bioorg. Med. Chem. 2010, 18, 6960.
29. Pascual, O.; Traiffort, E.; Baker, D. P.; Galdes, A.; Ruat, M.; Champagnat, J. Eur. J.
Neurosci. 2005, 22, 389.
30. Chen, J. K.; Taipale, J.; Young, K. E.; Maiti, T.; Beachy, P. A. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 14071.
31. Williams, J. A.; Guicherit, O. M.; Zaharian, B. I.; Xu, Y.; Chai, L.; Wichterle, H.;
Kon, C.; Gatchalian, C.; Porter, J. A.; Rubin, L. L.; Frank, Y.; Wang, F. Y. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 4616.
References and notes
32. Experimental for selected compounds
(3b,20S)-pregn-5-ene-22S-benzylbut-24-enylcarbamate (6). In a flame dried
flask under argon was introduced 21 (300 mg, 0.675 mmol) and anhydrous
DCM (2.25 mL). Benzylcarbamate (102 mg, 0.675 mmol) was added and the
1. Scales, S. J.; de Sauvage, F. J. Trends Pharmacol. Sci. 2009, 30, 303.
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mixture was cooled to 0 °C. Trimethylallylsilane (107
added followed by the dropwise addition of freshly distilled trifluoroboron
etherate (87 L, 0.675 mmol). The mixture turns pink after 1 h at 0 °C then blue
lL, 0.675 mmol) was
l
after allowing the temperature to reach room temperature for 1.5 h. Saturated
aqueous NaHCO3 was then added and the mixture was extracted with DCM
(three times). The organic layer was dried over Na2SO4 and the solvent was
removed under reduced pressure. The residue was purified by silica gel
chromatography (heptane/EtOAc 7:3) to yield a white solid (296 mg, 87%).
Rf = 0.20 (heptane/EtOAc 7:3). Mp: 151–152° C. ½a D20
ꢁ
ꢂ39.2 (c 1, CHCl3). 1H NMR
(200 MHz, CDCl3): d 7.33 (m, 5H), 5.72 (m, 1H), 5.33 (m, 1H), 5.07 (m, 2H), 4.97
(m, 1H), 4.47 (m, 1H), 3.86 (m, 1H), 3.50 (m, 1H), 0.98 (s, 3H), 0.86 (d, J = 6,8 Hz,
3H), 0.66 (s, 3H). 13C NMR (75 MHz, CDCl3): d 157.0 (CO), 140.9 (Cquat), 136.7
(Cquat), 135.3 (CH), 128.6 (CHAr), 128.1 (CHAr), 121.6 (CH), 117.1 (CH2), 71.6
(CH), 66.9 (CH), 66.7 (CH2), 56.7 (CH), 53.0 (CH), 50.2 (CH), 42.3 (CH2–CH), 39.8
(CH2), 39.0 (CH2), 38.7 (CH), 37.4 (CH2), 36.5 (Cquat), 32.0 (CH), 31.9 (CH2), 31.6
(CH2), 28.3 (CH2), 24.3 (CH2), 21.2 (CH2), 19.5 (CH3), 12.5 (CH3), 11.8 (CH3). HR-
MS (ESI positive): calculated (M+1) 506.3629, found (M+1) 506.3615.
(3b,20S)-pregn-5-ene-22S-benzyl-25,26-dehydropiperidincarboxylate (7). In a
dry autoclave under argon was introduced para-toluenesulfonic acid
monohydrate (17 mg, 0.089 mmol). In a flame dried schlenk under argon
was introduced Rh(CO)2(acac) (4 mg, 0.019 mmol), anhydrous and degassed
THF followed by biphephos (23 mg, 0.036 mmol). CO evolution was observed.
Compound 22 (100 mg, 0.196 mmol) was then added and the mixture was
transferred in the autoclave and the schlenk was rinsed three times with THF
(Vtot = 11 mL). After three H2/CO (1:1) flushing cycles, the pressure was set at
5 bar and the autoclave was heated at 65 °C (internal temperature) for 18 h.
After cooling, the mixture was transferred into a flask and the solvent was
removed under reduced pressure. The residue was purified by silica gel
chromatography (heptane/EtOAc 7:3) to yield the desired enamide 23 as a
8. Cooper, M. K.; Porter, J. A.; Young, K. E.; Beachy, P. A. Science 1998, 280, 1603.
9. Keeler, R. F. Teratology 1970, 3, 169.
10. Tremblay, M. R.; Lescarbeau, A.; Grogan, M. J.; Tan, E.; Lin, G.; Austad, B. C.; Yu,
L.-C.; Behnke, M. L.; Nair, S. J.; Hagel, M.; White, K.; Conley, J.; Manna, L. D.;
Alvarez-Diez, T. M.; Hoyt, J.; Woodward, C. N.; Sydor, J. R.; Pink, M.;
MacDougall, J.; Campbell, M. J.; Cushing, J.; Ferguson, J.; Curtis, M. S.;
McGovern, K.; Read, M. A.; Palombella, V. J.; Adams, J.; Castro, A. C. J. Med.
Chem 2009, 52, 4400.
white solid (296 mg, 87%). Mp: 128–129 °C. ½a D20
ꢁ
ꢂ56.1 (c 1, CHCl3). 1H NMR
(200 MHz CDCl3): d 7.29 (m, 5H), 6.88 (m, 1H), 5.33 (m, 1H), 5.17 (m, 2H), 4.88
(m, 1H), 4.19 (m, 1H), 3.52 (m, 1H), 1.25 (d, J = 6.8 Hz, 3H), 1.24 (s, 3H), 0.97 (s,
3H).13C NMR (50 MHz CDCl3): d 154.1 (CO), 141.0 (Cquat), 136.6 (Cquat), 129.2
(C36), 128.7 (CHAr), 126.0 (CHAr), 121.8 (CH), 106.6 (CH), 71.9 (CH), 67.7 (CH2),
56.3 (CH), 55.7 (CH), 53.7 (CH), 50.3 (CH), 42.5 (CH2), 39.8 (CH2), 37.5 (CH2),
36.7 (Cquat), 32.0 (CH CH2), 31.8 (CH2), 29.9 (CH2), 28.0 (CH2), 24.5 (CH2), 21.2
(CH2), 19.6 (CH3), 16.3 (CH3), 11.8 (CH3).
25-desmethylveramiline (1)
In a flame dried flask under argon was introduced 23 (77 mg, 0.149 mmol)
in anhydrous EtOAc (3 mL) and Pd/C 10% (15 mg). A hydrogen balloon was
bubbled in the mixture for 1 min and the mixture was stirred for 2 days
11. Gaffield, W.; Keeler, R. F. Pure Appl. Chem. 1994, 66, 2407.

3612
G. Guerlet et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3608–3612
under 1 atm of H2. A filtration over a celite pad was performed and MeOH
1.61–1.25 (m, 16H), 1.18 (td, J = 12.2, 4.5 Hz, 1H), 1.07 (m, 3H), 0.99 (s, 3H),
0.93 (d, J = 6.1 Hz, 3H), 0.67 (s, 3H). 13C NMR (50 MHz, CDCl3):
141.2
was used to rinse the solid waste. Solvents were removed under reduced
pressure and the residue was purified by silica gel chromatography (DCM/
MeOH 95:5 then 8:2 with 5% NEt3) to yield the desired product as a white
d
(Cquat), 121,6 (CH), 71.7 (CH), 59.8 (CH), 56.9 (CH), 52.7 (CH), 50.4 (CH),
47.6 (CH2), 42.4 (CH2), 40.9 (CH), 39.9 (CH2), 37.5 (CH2), 36.6 (CH), 32.0
(CH–CH2), 31.9 (CH2), 31.0 (CH2), 28.0 (CH2), 26.5 (CH2), 25.3 (CH2), 24.3
(CH2), 21.3 (CH2), 19.6 (CH3), 13.5 (CH3), 11.8 (CH3). HR-MS (ESI positive):
calculated (M+1) 386.3417, found (M+1) 386.3425.
solid (44 mg, 85%). Mp: decomposition (>200 °C). ½a D20
ꢁ
ꢂ48.6 (c 1, CHCl3). 1H
NMR (400 MHz CDCl3): d 5.32 (br d, J = 5.3 Hz, 1H), 3.51 (m, 1H), 3.15 (br d,
J = 12.2 Hz, 1H), 2.57 (m, 2H), 2.26 (m, 2H), 1.97 (m, 2H), 1.83 (m, 4H),