Synthesis and antibacterial activity of C-12 pyrazolinyl spiro ketolides

Article abstract of DOI:10.1016/j.ejmech.2010.09.060

A series of C-12 pyrazolinyl spiro ketolide derivatives were designed and synthesized. The C-12 modifications involved replacing the natural C-12 methyl group in clarithromycin core with different pyrazolinyl spiros via chemical synthesis. Potential anti-

Full text of DOI:10.1016/j.ejmech.2010.09.060

European Journal of Medicinal Chemistry 45 (2010) 5943e5949
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antibacterial activity of C-12 pyrazolinyl spiro ketolides
Lei Hu a, Ping Lan a, Qiu-Ling Song a, Zhi-Jian Huang a, Ping-Hua Sun a, Chao Zhuo b, Ying Wang a,
,
Shunian Xiao b, Wei-Min Chen a
*
a Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, PR China
b State Key Laboratory of Respiratory Diseases, Guangzhou Medical College, Guangzhou 510230, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of C-12 pyrazolinyl spiro ketolide derivatives were designed and synthesized. The C-12 modi-
fications involved replacing the natural C-12 methyl group in clarithromycin core with different
pyrazolinyl spiros via chemical synthesis. Potential anti-bacterial activities against both erythromycin-
susceptible and erythromycin-resistant bacteria were reported.
Received 12 May 2010
Received in revised form
27 September 2010
Accepted 28 September 2010
Available online 7 October 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Dedicated to Professor Henry N.C. Wong on
the occasion of his 60th birthday
Keywords:
Ketolide
Anti-bacterial activity
Pyrazolinyl
1. Introduction
2. Results and discussion
The first macrolide antibiotic erythromycin A has been widely
used as an agent to treat upper and lower respiratory tract infec-
tions for about six decades [1]. Due to its chemical instability and
gastro-intestinal side effects [2], erythromycin A had been gradu-
ally modified to produce the second-generation macrolides such as
clarithromycin, azithromycin and roxithromycin in the 1990s [3,4].
Recently the growing emergence of bacterial resistance, however,
has compromised the utility of macrolides [5]. The discovery of
ketolides, in which the C-3 cladinosyl is modified as a keto group,
significantly improved the activities against macrolide-resistant
bacteria [6]. Two of the most efficient ketolides are telithromycin
and cethromycin (ABT-773) [7,8] (Fig. 1). In our previous work [9],
C-12 pyrazolinyl intermediate was firstly synthesized from clari-
thromycin according to the bioisostere principle by replacing
C-11,12 cyclic carbamate of telithromycin with C-12 pyrazolinyl
ring. In this paper, we report herein on the preparation of a novel
C-12 pyrazolinyl spiro macrolide core, conversion of the core to
ketolide derivatives, and in vitro anti-bacterial activities of the
resulting ketolides.
The purpose of this paper was to synthesize some novel keto-
lides bearing different pyrazolinyl moieties via [2 þ 3] cycloaddi-
tion on C-12,21 double bond. The synthetic routes for accessing
such C-12 modified macrolides and ketolides were outlined in
Schemes 1e3. The key step of this strategy was the formation of
a C-12,21 double bond, which offered a site for reacting with diazo
compounds.
Intermediate 5 was prepared by methods described previously
[9]. Briefly, clarithromycin was used as a starting material to react
with benzoic anhydride in the presence of triethylamine and
4-dimenthylaminopyridine (DMAP) to give C-20, 400 dibenzoyl ester
macrolide. Subsequent reduction of C-9 carbonyl group using
NaBH4 led to C-9 hydroxyl macrolide 1. Treatment of compound 1
with 2,2-dimethoxypropane and pyridinium p-toluenesulfonate
(PPTS) for protection of C-9 and C-11 hydroxyl groups, generated
C-9, C-11 dimethylketal macrolide. Then C-12 hydroxyl group of
this C-9,11 dimethylketal macrolide was eliminated by SOCl2 to
form a C-12,21 exocyclic alkenyl macrolide 2 [10]. In the next two
steps, C-3 cladinose was hydrolyzed and C-9,11 dihydroxyl groups
were converted to dimethylketal group again. Under these reaction
conditions [11], oxidation of C-3 hydroxyl of compound 4 afforded
a C-12,21 exocyclic alkenyl ketolide derivative 5.
C-12 pyrazolinyl spiro ketolide derivatives 6 were then synthe-
sized via [2 þ 3] cycloaddition [12] of compound 5 with series of
* Corresponding author. Tel.: þ86 2085224497; fax: þ86 2085224766.
E-mail address: twmchen@jnu.edu.cn (W.-M. Chen).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.060

5944
L. Hu et al. / European Journal of Medicinal Chemistry 45 (2010) 5943e5949
N
N
N
O
N
BzO
O
BzO
N
N
O
N
O
OMe
OMe
N
O
HO
O
R
O
O
O
O
O
viii
HO
O
OMe
N
H
N
N
O
O
O
N
O
O
O
O
O
O
O
O
O
O
O
5
O
6
O
O
O
O
Scheme 2. Reagents and conditions: (viii) RCHN2, ether, 0e25 ꢀC or 1,2-dichloro-
Cethromycin (ABT-773)
Telithromycin
ethane, 84 ꢀC, yields range from 69.7% to 98.2%.
N
HO
HO
carbene in the light, which led to the generation of many byprod-
ucts. Unfortunately, compound 6 was then identified as a pair of
stereoisomers which could not be separated by general gel column
chromatography. This means that there is no stereoselectivity in
these cycloadditions. The diazo compounds could attack to the
C-12,21 double bond from both anti and syn faces.
Removal of C-9,11 hydroxyl protecting group gave compound 7.
In this step, N]N of the pyrazolinyl rearranged to C]N in the
presence of a catalytic amount of PPTS. C-9 hydroxyl group of
compound 7 was selectively oxidized by using Dess-Martin peri-
odinane [16] to form C-9 carbonyl C-12 pyrazolinyl spiro ketolide
derivatives, and successive deprotection of the C-20 benzoyl group
[17] yielded final compounds 8a-d.
The anti-bacterial activities of the final compounds in vitro were
assessed against both erythromycin-susceptible and erythromycin-
resistant bacteria, with particular emphasis on respiratory patho-
gens. Broth microdilution MIC (Lowest concentration of compound
inhibiting visible growth) determinations were performed accord-
ing to procedures established by the national committee for Clinical
Laboratory Standards[18]. ResultsarepresentedinTable 1for twelve
selected bacterial strains, including erythromycin-susceptible
Staphylococcus. aureus ATCC 29213, S. aureus ATCC 6538p, S. aureus
ATCC 26001 Staphylococcus pneumonia 2860, Haemophilus. influenza
5096 and erythromycin-resistant S. aureus 5776, S. aureus 5677,
S. aureus AD-08, S. pneumonia 1210, S. pneumonia 5158, S. pneumonia
OMe
HO
O
O
N
HN
O
O
O
C-12 pyrazonlinly spiro macrolide core
Fig. 1. Structures of novel ketolides.
diazo compounds. The mechanism of the formation of pyrazolines
on C-12,21 double bond could be illustrated as Scheme 4 [13], and it
accorded with the frontier molecular orbital (FMO) theory.
According to this concept, cycloadditions of simple diazoalkanes
and diazoacetic esters are highest occupied molecular orbital
(HOMO, dipole)elowest unoccupied molecular orbital (LUMO,
dipolarophile) controlled. Two reaction conditions were used in this
step: The newly prepared diazomethane [14] and diazoethane were
dissolved in ether, and the reactions proceeded in room tempera-
ture; The newly prepared ethyl diazoacetate and methyl diazo-
acetate [15], due to their lower reaction activities, were dissolved in
1,2-dichloroethane, so that the reactions could proceed at a higher
temperature (84 ꢀC). Dark condition was a very important factor for
this kind of reaction, for the diazo compounds would decompose to
N
N
N
HO
BzO
BzO
OMe
O
HO
HO
HO
O
OMe
OMe
HO
HO
O
O
O
O
O
i, ii
O
iii, iv
O
O
O
O
O
O
O
O
O
O
O
O
OBz
OMe
OBz
OMe
OH
OMe
O
O
2
O
1
Clarithromycin
N
N
O
N
O
BzO
BzO
BzO
OMe
HO
HO
O
OMe
OMe
O
O
O
O
O
vii
vi
v
OH
O
OH
O
O
O
O
3
O
4
O
5
Scheme 1. Reagents and conditions: (i) (Bz)2O, DMAP, Et3N, EtOAc; (ii) NaBH4, THF; (iii) 2,2-dimethoxypropane, PPTS, acetone; (iv) SOCl2, Et3N, EtOAc, 0 ꢀC; (v) diluted HCl,
acetonitrile/H2O; (vi) 2,2-dimethoxypropane, PPTS, acetone; (vii) Dess-Martin periodinane, CH2Cl2, r.t.; 40.7% for seven steps.

L. Hu et al. / European Journal of Medicinal Chemistry 45 (2010) 5943e5949
5945
N
O
N
N
O
BzO
OMe
BzO
HO
O
HO
HO
O
OMe
OMe
O
R
HO
O
O
O
O
x, xi
ix
N
N
N
HN
N
HN
O
O
O
O
O
O
R
R
8a: R=H
8b: R=CH3
O
6
O
7
O
8
8c: R=COOC2H5
8d: R=COOCH3
Scheme 3. Reagents and conditions: (ix) PPTS, acetonitrile/H2O; (x) Dess-Martin periodinane, CH2Cl2, 0 ꢀC; (xi) CH3OH, reflux, yields range from 32.9% to 38.3% for two steps.
Overall yields range from 12.0% to 16.0%.
clarithromycin in both erythromycin-resistant S. pneumonia 1210 as
well as H. influenza 2412 and erythromycin-susceptible S. pneumonia
2860 strains. All of the compounds exhibited the same MIC values as
erythromycin A and clarithromycin towards erythromycin-suscep-
tible H. influenza 5096 strain.
N
O
N
O
BzO
BzO
O
O
OMe
OMe
O
O
R
O
O
N
N
N
N
O
O
O
O
3. Conclusion
R
O
5
O
6
A series of C-12 pyrazolinyl spiro ketolides were designed and
synthesized for the first time. Compounds with various alkane and
ester groups at pyrazolinyl spiros were investigated for their anti-
bacterial activities against both erythromycin-susceptible and
erythromycin-resistant bacteria, with particular emphasis on
respiratory pathogens. All derivatives were found to have better
anti-bacterial activities than erythromycin A and clarithromycin
against S. aureus strains, and with almost equivalent bioactivities
against S. pneumonia and H. influenza strains. Among the C-12
pyrazolinyl spiro ketolides, compounds (8c and 8d) with ester
substituents displayed better anti-bacterial activities than those of
compounds (8a and 8b) with alkyl substituents. This suggests that
ketolides possessing spiro C-12 pyrazolinyl moiety may have pre-
sented improved anti-bacterial properties. The result can be useful
to aid the designing of new ketolides with better anti-bacterial
activities.
Scheme 4. Illustration of the formation of pyrazolines on C-12,21 double bond reacted
with diazo compounds.
673, H. influenza 2412. Twofold differences in the MIC value are
within the error of the method.
Table 1 summarizes the anti-bacterial activities of the target
compounds. All C-12 pyrazolinyl spiro analogs exhibited distinct
activities against S. aureus erythromycin-susceptible strains and
improved activities against S. aureus erythromycin-resistant strains,
and with almost comparative bioactivities against S. pneumonia and
H. influenza strains compared with erythromycin A and clari-
thromycin. In general, among the C-12 pyrazolinyl spiro derivatives,
compounds (8c and 8d) with ester substituents possessed better
anti-bacterial activities than those of compounds (8a and 8b) with
alkyl substituents. For S. aureus ATCC 29213 and S. aureus ATCC 6538p
strains, both compounds 8c and 8d exhibited slightly better activities
4. Experimental
than 8a and 8b with the MIC value of 0.12 mg/mL. Compound 8d
was found to be more potent than other compounds against S. aureus
26001. All of the designed molecules showed significantly increased
inhibitory profiles than erythromycin A and clarithromycin against
erythromycin-resistant S. aureus 5676 and S. aureus 5677 strains.
Compounds 8c and 8d displayed slightly improved potencies than
erythromycin A and clarithromycin against erythromycin-resistant
S. pneumonia 5158 and S. pneumonia 673 strains. Compounds 8a-d
All reagents and solvents were reagent grade. Further purifica-
tion and drying by standard methods were employed when
necessary. Clarithromycin and its derivatives were pre-dried
azeotropically from benzene. CH2Cl2 and EtOAc were distilled from
CaH2. All organic solvents were evaporated under reduced pressure
with a rotary evaporator. The plates used for thin-layer chroma-
tography (TLC) were Qingdao-haiyang silica gel GF254 (0.1 mm
thickness) precoated on glass plates, and they were visualized
were found to be less potential than erythromycin
A and
Table 1
MIC (mg/mL) for C-12 pyrazolinyl spiro ketolides.
Compound
Erythromycin A
Clarithromycin
8a
8b
8c
8d
S. aureus ATCC 29213
S. aureus ATCC 6538p
S. aureus 26001
S. aureus 5676
S. aureus 5677
0.5
0.5
0.25
0.5
0.25
0.25
0.12
4
8
512
>64
0.5
>64
>64
>8
0.5
0.25
0.12
8
8
512
>64
0.5
>64
>64
>8
0.12
0.12
0.12
8
8
512
>64
0.5
8
8
>8
0.12
0.12
0.03
4
4
256
>64
0.5
8
8
>8
ꢁ 0.063
>1024
>1024
>1024
>32
0.25
>32
>32
4
ꢁ0.063
>1024
>1024
>1024
>16
0.125
>16
>16
S. aureus AD-08
S. pneumonia 1210
S. pneumonia 2860
S. pneumonia 5158
S. pneumonia 673
H. influenza 2412
H. influenza 5096
8
0.06
0.06
0.06
0.06
0.06
0.06

5946
L. Hu et al. / European Journal of Medicinal Chemistry 45 (2010) 5943e5949
under both long (365 nm) and short (254 nm) UV light. Column
chromatography was performed using Qingdao-haiyang silica gel
(200e300 mesh). NMR spectra were recorded on Bruker-
AV400 MHz spectrometer (400 MHz for 1H and 100 MHz for 13C
NMR). All NMR measurements were carried out at 300 K in
deuterated chloroform solution unless otherwise stated. Chemical
shifts are reported as parts per million (ppm) in d unit in the scale
relative to the resonance of CDCl3 (7.26 ppm in the 1H, 77.00 ppm
for the central line of the triplet in the 13C modes, respectively).
Coupling constants (J) are reported in Hz. Splitting patterns are
described by using the following abbreviations: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet. 1H NMR data is reported
in this order: chemical shift; multiplicity; coupling constant(s),
number of proton. Mass spectra (ERMS) were obtained with
a Thermofinnigan MAT95XL spectrometer. Relevant data were
tabulated as m/z. High-resolution mass spectra (HRMS) were
obtained on a Agilent 6410 TOF LC/MSD in ESIþ or ESIꢂ mode.
Compound 2 was prepared as the method of literature [10].
4.95e5.07 (m, 2H), 5.26 (s, 1H), 5.32 (s, 1H), 7.42 (t, J ¼ 7.8 Hz, 2H),
7.51 (t, J ¼ 6.2 Hz,1H), 8.03 (d, J ¼ 7.1 Hz, 2H). MS (ESI): 719.0 (MHþ).
4.3. (2S,3R,4S,6R)-4-(dimethylamino)-2-((1R,3R,6R,8R,9R,10R,12R,
13S,17S)-3-ethyl-10-methoxy-6,8,10,12,15,15,17-heptamethyl-2-
methylene-5,7-dioxo-4,14,16-trioxabicyclo[11.3.1]heptadecan-9-
yloxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate (5)
To a solution of 4 (14.4 g, 20 mmol) in CH2Cl2 (110 mL) at room
temperature was added Dess-Martin periodinane (12.7 g, 30 mmol).
The solution was stirred for 1 h and then was diluted with EtOAc
(380 mL). After washing with 1:1 saturated NaHCO3/10% Na2S2O3
(120 mL), the aqueous layer was extracted with EtOAc (2 ꢃ 110 mL)
and the combined organic layer was washed with brine (50 mL),
dried over Na2SO4, filtered, and concentrated. The residue was
purified on a silica gel (3:1 petroleum ether-acetone with 1% Et3N) to
give 5 (13.6 g, 95.6%) as a white foam. 1H NMR (CDCl3, ppm)
d
0.84e0.87 (m, 7H), 0.92 (d, J ¼ 7.2 Hz, 3H), 1.04 (d, J ¼ 6.8 Hz, 3H),
1.11e1.16 (dd, J ¼ 19.6, 7.2 Hz, 1H), 1.27 (d, J ¼ 1.6 Hz, 3H), 1.28 (d,
J ¼ 2.8 Hz, 3H),1.31 (s, 3H),1.32 (s, 3H),1.36 (s, 3H),1.41e1.47 (m, 2H),
1.74e1.87 (m, 4H), 2.04e2.09(m,1H), 2.26 (s, 6H), 2.74e2.87 (m, 2H),
3.01 (s, 3H), 3.48e3.55 (m,1H), 3.56e3.62 (m,1H), 4.11e4.15 (m,1H),
4.18 (d, J ¼ 2.4 Hz,1H), 4.52 (d, J ¼ 7.2 Hz,1H), 4.93 (t, J ¼ 13.6 Hz,1H),
5.01e5.05(m,1H), 5.33(s,1H), 5.46(s,1H), 7.42 (t, J¼ 7.4 Hz, 2H), 7.54
4.1. (2S,3R,4S,6R)-4-(dimethylamino)-2-((3R,4S,5S,6R,7R,9R,10S,11S,
12R,14R)-14-ethyl-4,10,12-trihydroxy-7-methoxy-3,5,7,9,11-
pentamethyl-13-methylene-2-oxooxacyclotetradecan-6-yloxy)-6-
methyltetrahydro-2H-pyran-3-yl benzoate (3)
To a solution of 2 (39.2 g, 40 mmol) in acetonitrile (145.6 mL)
was added HCl (3.3 mol/L, 145.6 mL) dropwise at room tempera-
ture. The solution was then stirred at room temperature for 24 h.
The reaction mixture was diluted by CH2Cl2 (400 mL), washed with
saturated NaHCO3 (2 ꢃ 120 mL). The organic layer was separated
and the aqueous layer was extracted by CH2Cl2 (2 ꢃ 200 mL). The
combined organic layer was washed with brine (120 mL), dried
over Na2SO4, filtered, and concentrated. The residue was purified
on a silica gel column (4:1 petroleum ether-acetone with 1% Et3N)
to give 3 (23.2 g, 85.2%) as a white foam. 1H NMR (CDCl3, ppm)
(t, J ¼ 7.4 Hz,1H), 8.03 (d, J ¼ 7.0 Hz, 2H).13C NMR (CDCl3, ppm)
d 9.5,
14.0, 14.9, 16.3, 19.9, 20.5, 21.1, 23.5, 23.9, 26.7, 31.6, 33.8, 34.6, 40.3,
48.5, 49.9, 50.8, 63.4, 68.7, 71.3, 75.8, 77.9, 80.0, 80.4, 99.8, 101.9,
114.3, 127.8, 129.3, 130.2, 132.2, 141.7, 164.8, 168.6, 203.8. MS (ESI):
716.8 (MHþ). HRMS (ESI) m/z (MHþ). Calcd for C40H62NO10
:
716.4368, Found: 716.4315.
4.4. (2S,3R,4S,6R)-4-(dimethylamino)-2-((1R,3R,6R,8R,9R,10R,12R,
13S,17S)-3-ethyl-10-methoxy-6,8,10,12,15,15,17-heptamethyl-5,7-
dioxo-30,50-dihydro-4,14,16-trioxaspiro[bicyclo[11.3.1]heptadecane-
2,40-pyrazole]-9-yloxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate
(6a)
d
0.60 (d, J ¼ 7.2 Hz, 3H), 0.84 (t, J ¼ 7.3 Hz, 3H), 0.90 (d, J ¼ 7.3 Hz,
3H), 0.96 (d, J ¼ 7.1 Hz, 3H), 1.13 (s, 1H), 1.18 (d, J ¼ 6.6 Hz, 3H),
1.30e1.32 (m, 6H), 1.33e1.90 (m, 10H), 2.01 (s, 1H), 2.26 (s, 6H), 3.03
(s, 1H), 3.22 (s, 3H), 3.30e3.34 (m, 1H), 3.47 (dd, J ¼ 9.8, 7.2 Hz, 1H),
3.57e3.61 (m, 1H), 3.89 (d, J ¼ 2.6 Hz, 1H), 4.4 (s, 1H), 4.78 (d,
J ¼ 7.6 Hz, 1H), 5.0 (m, 1H), 5.06 (m, 1H), 5.16 (s, 1H), 5.30 (s, 1H),
5.61 (t, J ¼ 7.2 Hz, 1H), 7.44 (t, J ¼ 7.6 Hz, 2H), 7.56 (t, J ¼ 7.3 Hz, 1H),
8.02 (d, J ¼ 8.4 Hz, 2H). MS (ESI): 679.1 (MHþ).
Newly prepared CH2N2-ether solution (30 mL, CH2N2 15 mmol
approx) was added to 5 (4.29 g, 6 mmol) in a tinfoil wrapped three-
neck flask at 0 ꢀC. The solution was stirred for 5 h and then warmed
to 25 ꢀC. After stirring for 44 h, the solution was dried over Na2SO4,
filtered, and concentrated. The residue was purified on a silica gel
(8:1 petroleum ether-acetone with 1% Et3N) to give 6a (4.08 g,
4.2. (2S,3R,4S,6R)-4-(dimethylamino)-2-((1R,3R,6R,7S,8S,9R,10R,12R,
13S,17S)-3-ethyl-7-hydroxy-10-methoxy-6,8,10,12,15,15,17-
heptamethyl-2-methylene-5-oxo-4,14,16-trioxabicyclo[11.3.1]
heptadecan-9-yloxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate (4)
89.0%) as a white foam. 1H NMR (CDCl3, ppm)
d
0.81 (t, J ¼ 7.5 Hz,
3H), 0.85e0.88 (m, 2H), 0.96 (d, J ¼ 7.2 Hz, 3H), 1.01 (d, J ¼ 6.6 Hz,
3H), 1.10e1.11 (m, 4H), 1.14 (s, 3H), 1.19 (s, 3H), 1.26e1.30 (m, 4H),
1.32 (s, 3H), 1.35 (d, J ¼ 6.8 Hz, 3H), 1.41e1.51 (m, 3H), 1.76e1.80 (m,
2H), 1.88e1.94 (m, 1H), 3.55e3.60 (m, 1H), 4.09 (d, J ¼ 6.8 Hz, 1H),
4.41e4.62 (m, 4H), 5.00e5.05 (m, 1H), 5.37e5.40 (m, 1H), 7.44 (t,
J ¼ 8.3 Hz, 2H), 7.56 (t, J ¼ 7.3 Hz, 1H), 8.03 (d, J ¼ 6.8 Hz, 2H). 13C
To a solution of 3 (20.4 g, 30 mmol) in acetone (400 mL) was
added 2,2-dimethoxypropane (90 mL, 730 mmol) and PPTS (25.5 g,
101.1 mmol) at room temperature. The solution was stirred and
heated at 60 ꢀC for 18 h. Upon cooling, the solution was added Et3N
(25.5 mL 176.7 mmol) and stirred for another 1 h. The solution was
concentrated in vacuo and the residue was dissolved in CH2Cl2
(390 mL), washed with 10% KH2PO4 (2 ꢃ 210 mL). The aqueous
layer was back extracted with EtOAc (2 ꢃ 70 mL). The combined
organic layer was washed with brine (135 mL), dried over Na2SO4
and concentrated. The residue was purified on a silica gel (4:1
petroleum ether-acetone with 1% Et3N) to give 4 (19.5 g, 91.3%) as
NMR (CDCl3, ppm)
d 9.6, 11.1, 15.5, 17.8, 19.0, 20.1, 21.6, 23.9, 24.7,
27.0, 32.1, 32.6, 35.6, 40.2, 41.2, 44.9, 50.7, 64.1, 69.4, 70.8, 72.5, 75.3,
75.7, 79.6, 80.7, 81.1, 83.4, 100.5, 101.2, 102.1, 128.6, 130.3, 131.0,
133.1, 165.9, 175.7. MS (ESI): 758.8 (MHþ). HRMS (ESI) m/z (MHþ).
Calcd for C41H63N3O10: 757.4513, Found: 757.4504.
4.5. (2S,3R,4S,6R)-4-(dimethylamino)-2-((1R,3R,6R,8R,9R,10R,12R,
13S,17S)-3-ethyl-10-methoxy-30,6,8,10,12,15,15,17-octamethyl-5,7-
dioxo-30,50-dihydro-4,14,16-trioxaspiro[bicyclo[11.3.1]heptadecane-
2,40-pyrazole]-9-yloxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate
(6b)
a white foam. 1H NMR (CDCl3, ppm)
d 0.75 (m, 9H), 0.87 (d,
J ¼ 6.9 Hz, 3H), 1.01 (d, J ¼ 6.6 Hz, 3H), 1.19e1.31 (m, 14H), 1.35e1.63
(m, 4H), 1.74e1.82 (m, 2H), 1.84e1.95 (m, 2H), 2.26 (s, 6H),
2.32e2.39 (m, 1H), 2.94 (d, J ¼ 4.1 Hz, 1H), 2.96 (d, J ¼ 4.4 Hz, 1H),
3.19 (s, 3H), 3.61 (m, 1H), 3.68 (dd, J ¼ 3.7, 10.0 Hz, 1H), 3.75 (d,
J ¼ 4.4 Hz, 1H), 4.38 (d, J ¼ 1.8 Hz, 1H), 4.74 (d, J ¼ 7.7 Hz, 1H),
Following the synthesis of compound 6a using compound 5
(4.29 g, 6 mmol) and newly prepared diazoethane-ether solution
(120 mL, CH3CHN2 60 mmol approx) yielded compound 6b (4.53 g,

L. Hu et al. / European Journal of Medicinal Chemistry 45 (2010) 5943e5949
5947
98.2%) as a white solid. 1H NMR (CDCl3, ppm)
d
0.76 (t, J ¼ 4.9 Hz,
4.8. (2S,3R,4S,6R)-4-(dimethylamino)-2-((6R,9R,11R,12R,13R,15R,
16S,17S,18R)-6-ethyl-16,18-dihydroxy-13-methoxy-9,11,13,15,17-
pentamethyl-8,10-dioxo-7-oxa-2,3-diazaspiro[4.13]octadeC-1-en-
3H), 0.82e1.16 (m, 10H), 1.19 (s, 3H), 1.25 (s, 6H), 1.27e1.53 (m,
15H), 1.70e1.85 (m, 3H), 2.13 (s, 3H), 2.26 (s, 6H), 2.71e2.75 (m,
1H), 2.82e2.87 (m, 1H), 2.90 (s, 3H), 3.44e3.49 (m, 1H), 3.54e3.59
(m, 1H), 4.05e4.09 (m, 1H), 4.47e4.59 (m, 2H), 5.01 (t, J ¼ 7.4 Hz,
1H), 7.42(t, J ¼ 8.3 Hz, 2H), 7.54 (t, J ¼ 7.3 Hz, 1H), 8.00 (d,
12-yloxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate (7a)
To a solution of 6a (1.14 g, 1.50 mmol) in 2:1 acetonitrile/H2O
(15 mL) was added PPTS (1.89 g, 8.34 mmol), and the reaction was
stirred for 20 h at 82 ꢀC. Upon cooling, the solution was diluted with
EtOAc (30 mL), washed with saturated NaHCO3 (2 ꢃ 70 mL). The
aqueous layer was extracted with EtOAc (2 ꢃ 30 mL), and the
combined organic layer was washed with brine (70 mL), dried over
Na2SO4 and concentrated in vacuo. The residue was purified on
a silica gel (3:1 petroleum ether-acetone with 1% Et3N) to give
compound 7a (0.96 g, 90%) as a white foam. 1H NMR (CDCl3, ppm)
J ¼ 6.8 Hz, 2H). 13C NMR (CDCl3, ppm)
d 10.6, 14.4, 17.8, 18.0, 18.5,
20.9, 21.6, 23.3, 24.0, 26.6, 29.6, 30.5, 32.4, 34.1, 35.2, 40.6, 49.3,
50.2, 50.9, 63.7, 69.0, 69.9, 75.2, 78.0, 80.4, 81.2, 88.8, 100.4, 102.0,
102.2, 128.2, 129.7, 130.4, 132.7, 165.1, 168.8, 203.7. MS (ESI): 772.9
(MHþ). HRMS (ESI) m/z (MHþ). Calcd for C42H66N3O10: 772.4743,
Found: 772.4705.
d
0.82 (t, J ¼ 7.2 Hz, 3H), 0.93 (d, J ¼ 7.2 Hz, 3H), 0.97e1.02 (m, 5H),
4.6. (1R,3R,6R,8R,9R,10R,12R,13S,17S)-ethyl-9-((2S,3R,4S,6R)-3-
(benzoyloxy)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-2-
yloxy)-3-ethyl-10-methoxy-6,8,10,12,15,15,17-heptamethyl-5,7-
dioxo-30,50-dihydro-4,14,16-trioxaspiro[bicyclo[11.3.1]heptadecane-
2,40-pyrazole]-30-carboxylate (6c)
1.06e1.1.08 (m, 1H), 1.22e1.31 (m, 9H), 1.34 (s, 2H), 1.38e1.1.48 (m,
2H), 1.75e1.84 (m, 2H), 1.92e1.97 (m,1H), 2.07e2.10 (m,1H), 2.17 (s,
1H), 2.27 (s, 6H), 2.43 (q, J ¼ 1.4 Hz, 1H), 2.88e2.90 (m, 1H), 2.95 (s,
3H), 2.99e3.04 (m, 1H), 3.25e3.26 (m, 1H), 3.62e3.74 (m, 3H), 4.41
(d, J ¼ 6.8 Hz, 1H), 4.55 (d, J ¼ 7.6 Hz, 1H), 4.94 (d, J ¼ 9.6 Hz, 1H),
5.02e5.06 (m, 1H), 5.50 (dd, J ¼ 7.8, 2.6 Hz, 1H), 6.07 (s, 1H), 6.56 (s,
1H), 7.46 (t, J ¼ 8.3 Hz, 2H), 7.58 (t, J ¼ 7.3 Hz, 1H), 8.03 (d, J ¼ 6.8 Hz,
Newly prepared ethyl diazoacetate-1,2- dichloroethane solution
(18 mL, ethyl diazoacetate 10 mmol approx) was added to
compound 5 (0.72 g, 1.01 mmol) in a tinfoil wrapped three-neck
flask. Then the solution was heated at 84 ꢀC for 25 h. The solution
was dried over Na2SO4, filtered, and concentrated. The residue was
purified by silica gel (8:1 petroleum ether-acetone with 1% Et3N) to
give compound 6c (0.53 g, 64%) as a white solid. 1H NMR (CDCl3,
2H). 13C NMR (CDCl3, ppm)
d 10.4, 13.2, 14.8, 15.6, 20.1, 20.7, 21.0,
22.9, 31.4, 32.3, 33.9, 35.5, 37.2, 40.7, 45.4, 50.2, 51.2, 63.6, 69.3, 69.5,
70.2, 76.8, 78.6, 79.3, 82.6, 101.4, 128.3, 128.5, 129.7, 130.5, 132.9,
141.0, 165.1, 170.0, 205.0. MS (ESI): 718.7 (MHþ). HRMS (ESI) m/z
(MHꢂ). Calcd for C38H58N3O10: 716.4128 Found: 716.4090.
ppm)
d 0.77e0.81 (m, 1H), 0.82e0.85 (m, 1H), 0.85e0.92 (m, 6H)
4.9. (2S,3R,4S,6R)-4-(dimethylamino)-2-((6R,9R,11R,12R,13R,15R,
16S,17S,18R)-6-ethyl-16,18-dihydroxy-13-methoxy-4,9,11,13,15,17-
hexamethyl-8,10-dioxo-7-oxa-2,3-diazaspiro[4.13]octadeC-1-en-
12-yloxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate (7b)
0.94 (d, J ¼ 8.2 Hz, 2H), 0.97 (d, J ¼ 7.0 Hz, 3H), 1.01e1.07 (m, 4H),
1.25 (s, 3H), 1.28 (t, J ¼ 5.5 Hz, 9H), 1.33 (s, 6H),1.34e1.37 (m, 4H),
1.93 (s, 1H), 2.30 (s, 6H), 2.65e2.74 (m, 1H), 2.89 (s, 3H), 2.96e3.13
(m, 4H), 3.52 (dd, J ¼ 13, 2, 6.0 Hz, 1H), 3.58e3.73 (m, 2H), 4.13 (d,
J ¼ 8.5 Hz, 1H), 4.29 (dd, J ¼ 14.5, 7.5 Hz, 2H), 4.58e4.67 (m, 2H),
7.45 (t, J ¼ 8.3 Hz, 2H), 7.58 (t, J ¼ 7.3 Hz, 1H), 8.05 (d, J ¼ 6.8 Hz,
Following the synthesis of compound 7a using compound 6b
(2.28 g, 3.01 mmol) yielded compound 7b (2.16 g, 83.2%) as a white
2H). 13C NMR (CDCl3, ppm)
d 9.8, 10.7, 14.0, 14.2, 15.2, 16.3, 18.8, 19.1,
solid. 1H NMR (CDCl3, ppm)
d
0.82 (t, J ¼ 7.2 Hz, 3H), 0.99 (d,
20.5, 22.6, 24.7, 26.4, 29.2, 29.5, 29.6, 31.8, 39.5, 41.5, 44.2, 47.1,
49.5, 51.8, 63.4, 68.5, 70.3, 70.9, 77.7, 78.2, 79.6, 92.1, 100.8, 114.5,
128.5, 129.9, 130.7, 133.7, 145.3, 163.5, 164.9, 169.1, 204.0. MS (ESI):
J ¼ 6.2 Hz, 2H), 1.07 (d, J ¼ 6.7 Hz, 3H), 1.20e1.24 (m, 11H), 1.36 (s,
3H), 1.41e1.49 (m, 3H), 1.69e1.78 (m, 3H), 1.91e2.07 (m, 3H), 2.26
(s, 6H), 2.46 (s, 1H), 2.79 (d, J ¼ 10.4 Hz, 1H), 2.96 (s, 3H), 2.81e3.05
(m, 2H), 3.22 (s, 1H), 3.60e3.75 (m, 2H), 3.79 (s, 1H), 3.86e4.05 (m,
1H), 4.36 (d, J ¼ 7.0 Hz, 1H), 4.60 (d, J ¼ 7.4 Hz, 1H), 4.95 (s, 1H), 5.04
(t, J ¼ 8.3 Hz, 1H), 5.45 (d, J ¼ 10.7 Hz, 1H), 6.10 (s, 1H), 6.47 (s, 1H),
7.44 (t, J ¼ 8.2 Hz, 2H),7.61 (t, J ¼ 7.3 Hz, 1H), 8.03 (d, J ¼ 6.8 Hz, 2H).
13C NMR (CDCl3, ppm) 13.2, 14.8, 15.3, 16.2, 16.9, 18.5, 19.6, 20.0,
21.4, 26.7, 29.2, 30.8, 33.6, 35.9, 37.9, 40.1, 42.5, 49.8, 50.3, 61.7, 68.9,
70.8, 74.4, 79.3, 80.5, 82.6, 85.1, 103.7, 127.3, 128.7, 128.8, 131.9,
150.2, 164.8, 168.9, 204.8. MS (ESI): 732.9 (MHþ). HRMS (ESI) m/z
(MHꢂ). Calcd for C39H60N3O10: 730.4284, Found: 730.4249.
831.0(MHþ). HRMS (ESI) m/z (MHþ). Calcd for C44H68N3O12
:
830.4798, Found: 830.4790.
4.7. (1R,3R,6R,8R,9R,10R,12R,13S,17S)-methyl-9-((2S,3R,4S,6R)-3-
(benzoyloxy)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-2-
yloxy)-3-ethyl-10-methoxy-6,8,10,12,15,15,17-heptamethyl-5,7-
dioxo-30,50-dihydro-4,14,16-trioxaspiro[bicyclo[11.3.1]heptadecane-
2,40-pyrazole]-30-carboxylate (6d)
Following the synthesis of compound 6c using compound 5 and
newly prepared methyl diazoacetate-1,2- dichloroethane solution
(18 mL, methyl diazoacetate 10 mmol approx) yielded compound
4.10. (6R,9R,11R,12R,13R,15R,16S,17S,18R)-ethyl-12-((2S,3R,4S,6R)-
3-(benzoyloxy)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-
2-yloxy)-6-ethyl-16,18-dihydroxy-13-methoxy-9,11,13,15,17-
pentamethyl-8,10-dioxo-7-oxa-2,3-diazaspiro[4.13]octadeC-3-ene-
1-carboxylate (7c)
6d (1.64 g, 69.7%) as a white solid. 1H NMR (CDCl3, ppm)
d 0.74 (t,
J ¼ 7.5 Hz, 3H), 0.85 (d, J ¼ 7.0 Hz, 3H), 0.98 (t, J ¼ 6.7 Hz, 4H), 1.05
(d, J ¼ 6.6 Hz, 4H), 1.10 (s, 3H), 1.15 (s, 3H), 1.25 (m, 11H), 1.44 (m,
3H), 1.80 (m, 3H), 2.13 (s, 1H), 2.24 (s, 6H), 2.71 (t, J ¼ 8.3 Hz, 1H),
2.83 (m, 2H), 2.89 (s, 3H), 3.46 (q, J ¼ 7.0 Hz, 1H), 3.57 (m, 1H), 4.05
(d, J ¼ 9.9 Hz, 1H), 4.51 (m, 3H), 5.01 (m, 1H), 5.36 (m, 1H), 7.41 (t,
J ¼ 8.3 Hz, 2H), 7.53 (t, J ¼ 7.3 Hz, 1H), 7.99 (d, J ¼ 6.8 Hz, 2H). 13C
Following the synthesis of compound 7a using compound 6c
(1.0 g, 1.20 mmol) yielded compound 7c (0.82 g, 85.6%) as a white
solid. 1H NMR (CDCl3, ppm)
d
0.80 (t, J ¼ 4.9 Hz, 3H), 0.84e0.87 (m,
2H), 0.91 (d, J ¼ 7.2 Hz, 3H), 0.96 (dd, J ¼ 13.5, 7.1 Hz, 5H), 1.28 (s,
3H), 1.30 (t, J ¼ 2.7 Hz, 6H), 1.33e1.36 (m, 7H), 1.39e1.48 (m, 2H),
1.79e1.85 (m, 3H), 2.27 (s, 6H), 2.81e2.89 (m, 1H), 2.95 (s, 3H), 3.00
(t, J ¼ 7.2 Hz, 1H), 3.27e3.32 (m, 1H), 3.62e3.68 (m, 1H), 3.73 (q,
J ¼ 6.7 Hz, 1H), 3.80 (s, 1H), 4.26e4.31 (m, 2H), 4.44 (d, J ¼ 7.0 Hz,
1H), 4.54 (d, J ¼ 7.6 Hz, 1H), 5.03 (dd, J ¼ 10.4, 7.6 Hz, 1H), 5.32 (d,
J ¼ 10.2 Hz, 1H), 5.49 (dd, J ¼ 11.0, 2.6 Hz, 1H), 6.86 (s, 1H), 7.45 (t,
NMR (CDCl3, ppm)
d 11.2, 13.6, 14.0, 16.9, 17.1, 19.1, 20.3, 20.8, 23.4,
23.6, 27.3, 29.5, 30.2, 32.5, 33.7, 34.0, 35.2, 41.3, 48.8, 50.0, 51.0,
52.0, 63.7, 65.5, 69.6, 71.4, 71.9, 77.9, 78.0, 80.1, 100.8, 101.4, 128.4,
129.7, 130.8, 133.1, 163.1, 165.1, 169.0, 204.2. MS (ESI): 816.9 (MHþ).
HRMS (ESI) m/z (MHþ). Calcd for C43H66N3O12: 816.4641, Found:
816.4641.

5948
L. Hu et al. / European Journal of Medicinal Chemistry 45 (2010) 5943e5949
J ¼ 8.3 Hz, 2H), 7.55 (t, J ¼ 7.3 Hz, 1H), 8.04 (d, J ¼ 6.8 Hz, 2H). 13
C
solid. 1H NMR (CDCl3, ppm)
d
0.76 (t, J ¼ 7.7 Hz, 3H), 0.79e0.88 (m,
NMR (CDCl3, ppm)
d
10.2, 13.8, 14.3, 14.8, 15.8, 19.8, 20.7, 21.1, 22.6,
2H), 0.91 (d, J ¼ 7.2 Hz, 3H), 0.94e1.00 (m, 2H), 1.11 (t, J ¼ 6.9 Hz,
1H), 1.15 (d, J ¼ 7.5 Hz, 3H), 1.22 (d, J ¼ 6.9 Hz, 3H), 1.26e1.33 (m,
5H),1.37e1.45 (m, 4H),1.53e1.66 (m, 2H),1.67 (s, 3H),1.79e1.82 (m,
1H), 2.01e2.12 (m, 1H), 2.28 (s, 6H), 2.62e2.65 (m, 1H), 2.68 (s, 1H),
2.85 (s, 3H), 2.87e3.04 (m, 2H), 3.15 (t, J ¼ 8.0 Hz, 1H), 3.44 (q,
J ¼ 6.9 Hz, 1H), 3.54e3.61 (m, 1H), 4.06 (d, J ¼ 10.1 Hz, 1H), 4.50 (d,
J ¼ 7.6 Hz, 1H), 5.02e5.08 (m, 1H), 6.90 (s, 1H). 13C NMR (CDCl3,
30.4, 32.5, 33.6, 34.2, 34.8, 40.8, 45.8, 49.9, 51.2, 61.1, 63.6, 69.3, 70.5,
72.1, 73.2, 75.5, 79.7, 82.6, 101.4, 128.4, 129.7, 130.4, 132.9, 138.8,
163.0, 165.2, 169.6, 205.1. MS (ESI): 791.0 (MHþ). HRMS (ESI) m/z
(MHþ). Calcd for C41H64N3O12: 790.4485, Found: 790.4482.
4.11. (6R,9R,11R,12R,13R,15R,16S,17S,18R)-methyl-12-((2S,3R,4S,6R)-
3-(benzoyloxy)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-
2-yloxy)-6-ethyl-16,18-dihydroxy-13-methoxy-9,11,13,15,17-
pentamethyl-8,10-dioxo-7-oxa-2,3-diazaspiro[4.13]octadeC-3-ene-1-
carboxylate (7d)
ppm)
d 8.1, 10.4, 11.2, 11.7, 15.2, 18.6, 19.0, 20.7, 20.8, 21.9, 29.1, 30.4,
31.7, 39.4, 41.1, 43.0, 50.6, 50.9, 52.6, 65.2, 66.7, 70.3, 71.2, 73.0, 78.7,
83.9, 116.8, 149.1, 159.7, 204.6, 206.1. MS (ESI): 626.9 (MHþ). HRMS
(ESI) m/z (MHþ). Calcd for C32H56N3O9: 626.3972, Found: 626.4007.
Following the synthesis of compound 7a using compound 6d
(1.44 g, 1.76 mmol) yielded compound 7d (1.12 g, 81.9%) as a white
4.14. (6R,9R,11R,12R,13R,15R,17R,18R)-ethyl-12-((2S,3R,4S,6R)-4-
(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-
yloxy)-6-ethyl-18-hydroxy-13-methoxy-9,11,13,15,17-pentamethyl-
8,10,16-trioxo-7-oxa-2,3-diazaspiro[4.13]octadeC-3-ene-1-
carboxylate (8c)
solid. 1H NMR (CDCl3, ppm)
d
0.74 (t, J ¼ 4.7 Hz, 3H), 0.76e0.81 (m,
2H), 0.83 (d, J ¼ 7.2 Hz, 3H), 0.89 (dd, J ¼ 13.3, 6.9 Hz, 5H), 1.15 (t,
J ¼ 2.6 Hz, 2H), 1.17e1.39 (m, 12H), 1.72e1.75 (m, 3H), 2.06e2.11 (m,
1H), 2.26 (s, 6H), 2.75e2.84 (m, 2H), 2.88 (s, 3H), 3.22e3.25 (m, 1H),
3.58e3.73 (m, 3H), 3.76 (s, 3H), 4.04 (s, 1H), 4.35 (d, J ¼ 6.8 Hz, 1H),
4.47 (d, J ¼ 7.5 Hz, 1H), 4.97 (dd, J ¼ 10.4, 7.5 Hz, 1H), 5.29 (d,
J ¼ 9.8 Hz, 1H), 5.42 (dd, J ¼ 10.8, 2.4 Hz, 1H), 6.86 (s, 1H), 7.39 (t,
J ¼ 8.2 Hz, 2H), 7.51 (t, J ¼ 7.2 Hz, 1H), 7.96 (d, J ¼ 6.7 Hz, 2H). 13C
Following the synthesis of compound 8a using compound 7c
(0.79 g, 1.00 mmol) yielded compound 8c (0.33 g, 48.1%) as a white
solid. 1H NMR (CDCl3, ppm)
d
0.83 (t, J ¼ 7.5 Hz, 3H), 0.84e0.86 (m,
2H), 0.88 (d, J ¼ 7.2 Hz, 3H), 1.18 (d, J ¼ 7.5 Hz, 4H), 1.20e1.25 (m,
10H), 1.34 (t, J ¼ 7.0 Hz, 5H), 1.40 (s, 3H), 1.64e1.70 (m, 2H), 1.84 (s,
2H), 2.27 (s, 6H), 2.36e2.46 (m, 2H), 2.82 (s, 3H), 2.88e2.94 (m, 2H),
3.17e3.27 (m, 3H), 3.57 (q, J ¼ 6.9 Hz, 2H), 3.68 (s, 1H), 4.05 (d,
J ¼ 10.2 Hz, 1H), 4.32 (t, J ¼ 7.5 Hz, 1H), 5.08e5.11 (m, 1H), 6.95 (s,
NMR (CDCl3, ppm)
d 10.2, 13.8, 14.8, 15.8, 19.8, 20.7, 21.0, 22.6, 31.6,
32.5, 33.7, 34.2, 35.1, 40.8, 45.8, 50.0, 51.1, 52.0, 63.6, 69.3, 70.5, 71.9,
73.3, 75.5, 77.3, 79.7, 82.6, 101.5, 128.4, 129.7, 130.4, 132.9, 138.4,
163.7, 165.1, 169.6, 205.0. MS (ESI): 777.0 (MHþ). HRMS (ESI) m/z
(MHꢂ). Calcd for C40H60N3O12: 774.4182, Found: 774.4147.
1H). 13C NMR (CDCl3, ppm)
d 10.1, 10.2, 14.6, 15.4, 18.3, 18.8, 20.0,
21.2, 22.4, 27.6, 28.2, 29.7, 39.0, 40.3, 42.5, 47.3, 47.7, 50.9, 51.1, 65.8,
69.3, 69.6, 70.3, 74.7, 78.8, 79.8, 85.7, 104.0, 154.9, 168.5, 169.4,
203.9, 217.4. MS (ESI): 684.9 (MHþ). HRMS (ESI) m/z (MHþ). Calcd
for C34H58N3O11: 684.4027 Found: 684.4010.
4.12. (2S,3R,4S,6R)-4-(dimethylamino)-2-((6R,9R,11R,12R,13R,15R,
17R,18R)-6-ethyl-18-hydroxy-13-methoxy-9,11,13,15,17-pentamethyl-
8,10,16-trioxo-7-oxa-2,3-diazaspiro[4.13]octadeC-1-en-12-yloxy)-6-
methyltetrahydro-2H-pyran-3-yl benzoate (8a)
4.15. (6R,9R,11R,12R,13R,15R,17R,18R)-methyl-12-((2S,3R,4S,6R)-4-
(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-
yloxy)-6-ethyl-18-hydroxy-13-methoxy-9,11,13,15,17-pentamethyl-
8,10,16-trioxo-7-oxa-2,3-diazaspiro[4.13]octadeC-3-ene-1-
carboxylate (8d)
To a solution of 7a (0.54 g, 0.75 mmol) in CH2Cl2 (8 mL) at ꢂ5 ꢀC
was added Dess-Martin periodinane (0.38 g, 0.90 mmol). The solu-
tion was stirred for 20 h, and more Dess-Martin periodinane (22 mg,
0.04 mmol) was added. After stirring for 8 h, the solution was diluted
with EtOAc (170 mL) and washed with 1:1 saturated NaHCO3/10%
Na2S2O3 (2 ꢃ 35 mL). The combined aqueous layer was back
extracted with EtOAc (4 ꢃ 35 mL) and the combined organic layer
were then washed with brine (35 mL), dried over Na2SO4, filtered,
and concentrated. To the crude material was added CH3OH (2 mL)
and the solutionwas stirred for 15 h at 65 ꢀC. Upon concentration, the
residue was purified on a silica gel (3:1 petroleum ether-acetone with
1% Et3N) to give compound 8a (0.20 g, 42.5%) as a white solid.1H NMR
(CDCl3, ppm) 0.78 (t, J ¼ 7.4 Hz, 3H), 1.13 (d, J ¼ 7.6 Hz, 3H), 1.18e1.26
(m,11H),1.29 (d, J ¼ 6.9 Hz, 3H),1.35 (s, 3H),1.58e1.67 (m, 3H),1.77 (s,
3H), 2.01e2.09 (m, 1H), 2.12 (s,1H), 2.31e2.36 (m, 1H), 2.41e2.47 (m,
1H), 2.64 (d, J ¼ 18.8 Hz, 1H), 2.81 (s, 3H), 2.86e3.00 (m, 2H),
3.13e3.18 (m, 2H), 3.12e3.21 (m, 2H), 3.40e3.57 (m, 2H), 4.01 (d,
J ¼ 10.2 Hz, 1H), 4.26 (d, J ¼ 7.3 Hz, 1H), 5.02 (dd, J ¼ 11.2, 2.1 Hz, 1H),
Following the synthesis of compound 8a using compound 7d
(0.78 g, 1.03 mmol) yielded compound 8d (0.30 g, 44.7%) as a white
solid. 1H NMR (CDCl3, ppm)
d
0.83e0.91 (m, 4H), 0.95 (d, J ¼ 7.0 Hz,
2H), 1.14 (t, J ¼ 5.75 Hz, 3H), 1.23e1.27 (m, 3H), 1.30e1.37 (m, 3H),
1.40 (d, J ¼ 8.5 Hz, 2H), 1.61 (d, J ¼ 13.5 Hz, 2H), 1.66e1.68 (m, 2H),
1.76 (s, 3H), 1.87e1.93 (m, 2H), 2.06e2.14 (m, 2H), 2.27 (s, 6H),
2.43e2.49 (m, 1H), 2.69 (s, 1H), 2.77 (s, 3H), 2.85e2.89 (m, 2H),
2.98e3.01 (m, 2H), 3.18e3.21 (m, 1H), 3.51e3.58 (m, 2H), 3.84 (s,
3H), 4.00 (d, J ¼ 10.5 Hz, 1H), 4.31 (t, J ¼ 9.0 Hz, 2H), 4.80 (d,
J ¼ 11.5 Hz, 1H), 4.95 (dd. J ¼ 10.2, 3.0 Hz, 1H), 6.97 (s, 1H). 13C NMR
(CDCl3, ppm)
d 9.9, 11.4, 14.8, 18.1, 19.6, 21.1, 25.2, 27.2, 29.7, 38.2,
40.2, 42.1, 43.9, 45.3, 47.6, 49.7, 51.2, 52.3, 65.9, 71.3, 74.7, 78.2, 81.1,
83.5, 88.1, 104.6, 151.6, 162.8, 168.2, 203.3, 218.5. MS (ESI): 670.8
(MHþ). HRMS (ESI) m/z (MHþ). Calcd for C33H56N3O11: 670.3909,
Found: 670.3927.
5.88 (s,1H). (CDCl3) d. d 7.8,10.1,10.4,11.8,13.5,
13C NMR (CDCl3, ppm)
19.2, 20.0, 20.3, 29.2, 30.7, 38.1, 39.4, 40.2, 40.8, 42.3, 42.9, 49.3, 49.9,
50.4, 62.0, 68.9, 69.9, 70.8, 79.1, 81.2, 101.3, 148.9, 162.4, 201.8, 202.7.
MS (ESI): 612.9 (MHþ). HRMS (ESI) m/z (MHꢂ). Calcd for C31H52N3O9:
610.3849, Found: 610.3844.
Acknowledgement
This work was financially supported by the Science Foundation
of Guangdong Province (06025150).
4.13. (6R,9R,11R,12R,13R,15R,17R,18R)-12-((2S,3R,4S,6R)-4-
(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-
yloxy)-6-ethyl-18-hydroxy-13-methoxy-4,9,11,13,15,17-hexamethyl-
7-oxa-2,3-diazaspiro[4.13]octadeC-1-ene-8,10,16-trione (8b)
Appendix. Supplementary data
Following the synthesis of compound 8a using compound 7b
(0.73 g, 1.01 mmol) yielded compound 8b (0.25 g, 39.6%) as a white
Supplementary data associated with this article can be found in
the on-line version, at doi:10.1016/j.ejmech.2010.09.060.

L. Hu et al. / European Journal of Medicinal Chemistry 45 (2010) 5943e5949
5949
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