Stability studies of C-4′,6′ acetal benzylmaltosides synthesized as inhibitors of smooth muscle cell proliferation

Article abstract of DOI:10.1016/j.bmcl.2004.03.049

In our investigations to synthesize inhibitors of smooth muscle cell (SMC) proliferation, compound 6a displayed submicromolar activity in in vitro antiproliferative assays and reduced intimal thickening using a rat balloon angioplasty model via iv administration. In order to identify analogs that could be administered orally, the chemical instability of the C-4′,6′ acetal of compound 6a was addressed. Several novel variants with increased acid stability and comparable in vitro activity were prepared.

Full text of DOI:10.1016/j.bmcl.2004.03.049

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2829–2833
Stability studies of C-40,60 acetal benzylmaltosides synthesized as
inhibitors of smooth muscle cell proliferation
Scott C. Mayer,a,* William Gallaway,a John Kulishoff,a,z Maisheng Yin,b
Vidya Gadamasettib and Robert Mitchellb
aChemical and Screening Sciences Department, Wyeth Research, CN 8000, Princeton, NJ 08543-8000, USA
bCardiovascular Discovery Department, Wyeth Research, Collegeville, PA 19426, USA
Received 23 January 2004; revised 16 March 2004; accepted 18 March 2004
Abstract—In our investigations to synthesize inhibitors of smooth muscle cell (SMC) proliferation, compound 6a displayed sub-
micromolar activity in in vitro antiproliferative assays and reduced intimal thickening using a rat balloon angioplasty model via iv
administration. In order to identify analogs that could be administered orally, the chemical instability of the C-40,60 acetal of
compound 6a was addressed. Several novel variants with increased acid stability and comparable in vitro activity were prepared.
Ó 2004 Elsevier Ltd. All rights reserved.
Restenosis, a complication of vascular wounding, is
often caused by injuries from surgical procedures such
as PTCA (balloon angioplasty).1 Roughly 30–50% of
the patients receiving PTCA undergo a re-narrowing of
the affected vascular segment within 6 months of surgery
and may require additional surgical intervention.2 Vas-
cular restenosis occurs when appropriate healing
mechanisms become over productive and generate more
than the required permanent tissue, resulting in an
inadvertent re-thickening of the vascular wall. One
approach3 to controlling this problem is to selectively
inhibit the growth and migration of smooth muscle cells
(SMC) without affecting those of endothelial cells (EC).
In the course of synthesizing a series of disaccharides
that exhibit selective SMC antiproliferative activity, a
tetrahydroxy carbohydrate lead (6a) was generated that
possessed submicromolar inhibitory activity (0.020 lM,
Table 1) in vitro with no apparent cytotoxicity.4 Com-
pound 6a displayed 30-fold selectivity over EC prolif-
eration through a mechanism, which may result from
blocking production of lactosylceramide (LacCer).
LacCer is a ceramide linked disaccharide that is thought
to be linked to both proliferation and intercellular
adhesion molecule expression by acting as a precursor
signal to an obligatory superoxide production.5 Thus, by
either preventing the production of LacCer, or by
Table 1. SARof C-40, 60, acetal derivatives
Compound
First step for 5a–h: see below for reagents.
Second step for 6a–h: acylation with BzCl
Yield, % (acetal and R4 (see Scheme 1 for SMC assaya
EC assayb
acylation steps)
core structure)
[IC50 (lM)]
[IC50 (lM)]
6a
6b
6c
6d
6e
6f
Benzaldehyde dimethyl acetal, TsOHÆH2O
Ph-acetaldehyde dimethyl acetal, CSA
Acetaldehyde dimethyl acetal, TsOHÆH2O
Isobutyraldehyde diethyl acetal, CSA
Propionaldehyde diethyl acetal, TsOHÆH2O
4-Cl-benzaldehyde dimethyl acetal, CSA
4-NO2-benzaldehyde dimethyl acetal, CSA
78/80
35/63
62/54
45/49
57/47
51/50
42/35
Ph
Bn
0.020
0.035
0.103
0.003
0.037
0.023
0.003
0.003
0.923
––
CH3
Isopropyl
Et
0.257
––
0.235
0.324
0.368
––
4-Cl-Ph
4-NO2-Ph
4-Pyridinyl
6g
6h
4-Pyridinecarboxaldehyde, DMF, H2SO4, 110 °C 20/46
a Concentration inducing 50% inhibition of smooth muscle cell proliferation.
b Concentration inducing 50% inhibition of endothelial cell proliferation.
* Corresponding author. Tel.: +1-732-274-4457; fax: +1-732-274-4505; e-mail: mayers@wyeth.com
z
Deceased: 9/11/01.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.049

2830
S. C. Mayer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2829–2833
inhibiting LacCer’s ability to bind to its downstream
target, an inhibitory analog should have a profound
effect on both proliferative and pro-inflammatory pro-
cesses. To date, no other nonsulfated disaccharide has
been linked to cellular events in such a manner. There-
fore, the link between the proliferative pathway in SMC
and the structural similarity of our disaccharides to
LacCer, although speculative, could be one explanation
to the inhibitory activity of these analogs and is con-
sistent with our ongoing studies.6 Future work in this
area should help to further elucidate this mechanism.
chemical instability of the C-40,60 acetal precluded its
advancement as a lead. This functionality was identified
to be a potential source of acid induced degradation
when 6a was dosed orally. Under conditions that mimic
the stomach (pH 1), compound 6a showed substantial
degradation to 7 (Scheme 1 and Table 2). The antipro-
liferative activity of 7 was significantly reduced com-
pared to parent (34.9 vs 0.020 lM). Therefore, we
focused our attention on the synthesis of a library of
acetals with the view of improving stability while
maintaining SMC inhibitory activity.
When tested in vivo at doses of 100 mg/kg/h iv, com-
pound 6a also caused a statistically significant reduction
of intimal thickening, ranging from 18–39%.7 Although
compound 6a showed promising in vivo activity, the
Aromatic and aliphatic C-40,60 acetals of 6a were syn-
thesized according to the chemistry illustrated in Scheme
1 (6b–h and Table 1). Hepta-O-acetyl-a-maltosyl bro-
mide was coupled with 3-nitro-4-chloro-benzyl alcohol
in the presence of mercuric bromide and mercuric cya-
nide to yield glycoside 1. Reduction of the nitro group of
1 with stannous chloride afforded the anilino compound
2. Acylation of the amine with acetyl chloride in the
presence of Et3N afforded intermediate 3, which was
converted to the heptahydroxy compound 4 by reaction
with catalytic sodium methoxide in methanol. In most
cases, the substituted C-40,60 acetal (5a–g) was formed by
use of a substituted aldehyde dimethyl or diethyl acetal
and a catalytic acid source (TsOHÆH2O or CSA).
Selective acylation of the 6-position primary alcohol
using benzoyl chloride in a 1:1 mixture of tetrahydro-
furan and 2,4,6-collidine afforded compounds 6a–g.8;9
To prepare compound 6h, intermediate 4 was condensed
with 4-pyridinecarboxaldehyde using concentrated
H2SO4 followed by similar acylation conditions with
2,4,6-collidine.
1) 3-NO2-4-Cl-
BnOH, HgBr2,
Hg(CN)2,
OAc
O
OAc
O
AcO
AcO
OAc
AcO
AcO
CH3CN, 90%
AcO
OAc
O
O
Cl
R1
AcO
O
2) SnCl2 2H2O,
EtOAc, 96%
AcO
O
O
AcO
AcO
AcO
1: R1 = NO2
Br
2: R1 = NH2
OR2
1) AcCl, Et3N,
THF, 75%
R2O
O
R2O
OR2
R2O
O
2) NaOMe (cat.),
MeOH, 65 °C,
99%
O
Cl
O
R2O
O
R2O
N
H
3: R2 = Ac
4: R2 = H
1) Acetal, TsOH H2O,
or CSA, DMF, 65 °C
(except 5h: aldehyde,
DMF, H2SO4, 110 °C)
*See Table 1 for details.
R4
O
O
O
OR3
O
HO
HO
The chemical stability of each acetal (6b–h) was evalu-
ated using 6a as a benchmark. Replacing the phenyl
group of compound 6a with a heterocycle (6h) or an
aliphatic acetal (6b–e) generated more stable analogs
without loss of in vitro activity (Tables 1 and 2). Analog
6f had only a modest improvement in stability over 6a in
the pH range 1.4–2.5. Acid stability of compound 6g
could not be determined due to its precipitation under
conditions of the assay. When the benzylidene at the
40,60-position was replaced with a methyl acetal (6c), the
compound exhibited 5-fold less potency (0.103 vs
0.020 lM for 6a) suggesting the need for a substituent
larger than Me to retain biological activity. However, 6c
had slightly improved chemical stability compared to 6a
(32% vs 100% degradation over 18 h period at pH 1).
O
HO
Cl
O
O
HO
2) BzCl, collidine:THF
(1:1), -40 °C to rt
N
H
5a-5h: R3 = H; R4 = see Table 1
6a-6h: R3 = Bz; R4 = see Table 1
acetal
degradation
(acidic conditions)
OH
O
O
O
HO
HO
HO
O
O
Cl
HO
O
O
HO
N
H
7
Scheme 1.
Table 2. Stability studies of C-40,60 acetal derivatives
Compound R4 (see
Scheme 1
for core
pH
% Remaining pH with
at 18–20 h
(pH 1)
ꢀ90% at
1
1.4
1.9
2.5
3
3.5
4
4.5
18–20 h
t1=2 (h)
structure)
6a
6b
6c
6d
6e
6f
Ph
Bn
1.9
2.3
4.2
13
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
0
94
68
53
52
0
3.5
1
1.4–1.9
>20
>20
>20
>20
>20
>20
>20
>20
4
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
CH3
Isopropyl
Et
4-Cl-Ph
2
1.9–2.5
1.7
13.3
>20
3
1
6h
4-Pyridinyl >20
>20
97

S. C. Mayer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2829–2833
2831
ated with the addition of compound, 3H thymidine and
serum/growth factor to serum deprived synchronized cells
and results are reported in this paper accordingly.
Compounds were added to each well at 50-fold dilution
(20 lL/ well) and the plates were incubated for 24–36 h at
37 °C in 5% CO2. Compounds were initially dissolved in
50% ethanol and serially diluted into media. Compounds
were routinely assayed at concentrations from 1 to 100
lM. As a control, grade II porcine intestinal mucosal
heparin (sodium salt) from Sigma (H-7005) was routinely
assayed in all cell preparations at concentrations from 0.1
to 100 lg/mL.
At the completion of the experiment, plates were placed on
ice, washed three times with ice cold phosphate buffered
saline (PBS), and incubated in ice cold 10% trichloroacetic
acid (TCA) for 30 min to remove acid soluble proteins.
Solution was transferred to scintillation vials containing
0.4 N HCl (500 lL/vial to neutralize NaOH) and each well
was rinsed two times with water (500 lL) for a total volume
of 2 mL/vial.
When compound 6c was tested in vivo (iv) in the intimal
thickening assay, no activity was observed. Incorporat-
ing a nicotinoyl group into the acetal functionality re-
sulted in a compound possessing excellent acid stability
without loss of in vitro activity (6h, 0.003 lM). Over the
18 h period at pH 1, only 3% degradation to compound
7 was detected.
In conclusion, analogs of disaccharide 6a with improved
chemical stability have been prepared. Specifically, by
modifying the C-40,60 benzylidene acetal with aromatic
and aliphatic variants provided more stable compounds
while maintaining SMC antiproliferative activity.
Incorporating a nicotinoyl acetal in this position afford-
ed an analog (6h) with excellent acid stability and good
in vitro activity (0.003 lM). Addressing this chemical
stability issue has provided a series of stable acetal
derivatives available for future in vivo studies.
Data was obtained, in triplicate, for both control and
experimental samples. Control (100%) data was obtained
from maximally stimulated cells, as the result of growth
factor or serum stimulation. Experimental data was
obtained from cells maximally stimulated with growth
factor or serum and treated with compound. Data was
expressed as a percent of control from which a percent
inhibition or IC50 could be determined.
C. Cytotoxicity: Visually, all cells were found to tolerate
high levels of all compounds quite well, however to insure
that no toxicity was present, cytotoxicity of compounds
was examined using a commercial modification of the
Acknowledgements
We are grateful to the Discovery Analytical Chemistry
department of Wyeth Research for elemental analyses,
300 and 400 MHz 1H NMR, mass spectroscopy, and
FTIRand to Dr. Robert McDevitt, Mr. Paul Dollings,
and Ms. Folake Adebayo for scientific discussions.
MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-
lium bromide) assay. Briefly, cells were again grown in
24 well plates to 70–80% confluency and, as before, serum
deprived for 24–48 h prior to initiation of the experimental
protocol. To insure that the MTT assay monitored
toxicity rather than proliferation, cells were incubated
with 100 lg/mL of drug in fresh medium without serum for
24 h at 37 °C in a humidified CO2 incubator. Upon
completion of the compound treatment, MTT indicator
dye was added for 4 h at 37 °C . Cells were then lysed and
aliquots from each well were transferred to a 96 well plate
for analysis. Absorbance at 570 nm wavelength with a
reference wavelength of 630 nm was recorded using an
ELISA plate reader. Results were determined as percent
viable using no drug (100% viable) and pre-solubilization
(0% viable) standards. All compounds (6a–h) exhibited no
toxicity up to 100 lg/mL.
References and notes
1. Clowes, A. W.; R eidy, M. A.J. Vasc. Surg. 1991, 13, 885–891.
2. Herrman, J. P. R.; Hermans, W. R. M.; Vos, J.; Serruys,
P. W. Drugs 1993, 46, 18–52, and 249–262.
3. Weissberg, P. L.; Grainger, D. J.; Shanahan, C. M.;
Metcalfe, J. C. Cardiovasc. Res. 1993, 27, 1191–1198.
4. Procedures for SMC and EC proliferation assays:
A. Human SMC and EC culture conditions: Human aortic
smooth muscle (HASMC) and endothelial (HAEC) cells
were obtained from Clonetics (San Diego, CA) and were
routinely maintained in T-75 tissue culture flasks in either
SMC or EC media supplied by Clonetics and supplemented
with fetal bovine serum (10% for HASMC, 5% FBS for
HAEC) at 37 °C in a humidified 5% CO2 atmosphere. The
culture medium was changed into fresh medium every 2–3
days. Both cell types were routinely sub-cultured into 24
well plates after trypsinization for use in critical growth
assays. Cells used for this study were from 3rd through 6th
passage.
5. Bhunia, A. K.; Han, H.; Snowden, A.; Chatterjee, S.
J. Biol. Chem. 1997, 272, 15642–15649.
6. Wyeth internal report, unpublished results.
7. Procedure for in vivo rat balloon injury model: Vascular
injuries were produced by introducing and inflating a
balloon catheter into the left common carotid artery of
anesthetized male Sprague–Dawley rats (Charles River
Breeding Laboratories). This type of injury generally
damages roughly 25–30 mm of vascular wall. One hour
prior to sacrificing, the animals were intravenously
injected with 0.9% Evans Blue. At the time of sacrifice,
the animals were further perfused with isotonic saline for
5 min, followed by 3.5% glutaraldehye. Arteries were then
excised and evaluated to determine intima to media (I=M)
ratio (intimal thickening).
8. Ishihara, K.; Kurihara, H.; Yamamoto, H. J. Org. Chem.
1993, 58, 3791–3793.
9. Experimental: Step 1: 4-Chloro-3-nitrobenzyl hepta-O-acet-
yl-b-maltoside (1). To a stirred solution of 4-chloro-3-
nitrobenzyl alcohol (6.70 g, 35.7 mmol) and HgBr2 (14.2 g,
B. Examination of the effects of compounds on cell
proliferation using 3H thymidine incorporation: Cell lines
were grown as described above in 24 well plates and all
were assayed using an identical protocol. When cell
density reached approximately 70–80% confluency, culture
medium was exchanged with fresh DMEM, M199, or
AIM-V (GibCo BRL) lacking any serum or growth
factors, and cell growth was continued for an additional
24–48 h to induce a quiescent state. Cells were then
re-stimulated with either serum or the appropriate individ-
ual growth factors for a period of 24 h in the presence
3
of 0.5 lCi/mL H thymidine before harvest.
Although compounds were found to be more effective with
longer pre-incubations, in general, experiments were initi-

2832
S. C. Mayer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2829–2833
39.3 mmol) in freshly distilled CH3CN (239 mL) was added
8.32 (s, 1H); IR(KBr) 3400, 2950, 1750, 1690, 1600, 1540,
1425, 1375, 1230, and 1050 cmÀ1; mass spectrum [(+) ESI],
m=z 818/820 (M + H)þ, 840 (M + Na)þ; Anal. Calcd for
C35H44ClNO19: C, 51.38; H, 5.42; N, 1.71. Found: C, 51.03;
H, 5.36; N, 1.59.
in one portion Hg(CN)2 (9.02 g, 35.7 mmol). After 0.5 h,
hepta-O-acetyl-a-maltosyl bromide (25.0 g, 35.7 mmol) was
added, and the mixture stirred for 18 h at rt. The reaction
was then quenched with a mixture of H2O/brine (1:1,
100 mL) and extracted with 10% CH2Cl2/EtOAc. The
combined organic extracts were dried (MgSO4) and con-
centrated. Purification by flash chromatography (10:90–
80:20, EtOAc/petroleum ether gradient) gave 51.9 g (90%)
of the title compound as a glassy oil, which was recrystal-
lized from Et2O/petroleum ether to afford a glassy white
solid, mp 107–111 °C; 1H NMR(CDCl 3) d 2.00 (s, 3H),
2.02 (s, 3H), 2.03, (s, 3H), 2.04 (s, 6H), 2.11 (s, 3H), 2.15 (s,
3H), 3.70 (ddd, J ¼ 2:9, 4.2, 9.7 Hz, 1H), 3.94–3.98 (m, 1H),
4.01–4.07 (m, 2H), 4.20–4.28 (m, 2H), 4.54 (dd, J ¼ 2:9,
12.3 Hz, 1H), 4.63–4.68 (m, 2H), 4.84–4.94 (m, 3H), 5.06 (t,
J ¼ 10:1 Hz, 1H), 5.26 (t, J ¼ 9:2 Hz, 1H), 5.36 (dd,
J ¼ 9:7, 10.3 Hz, 1H), 5.42 (d, J ¼ 4:2 Hz, 1H), 7.43 (dd,
J ¼ 2:2, 8.3 Hz, 1H), 7.53 (d, J ¼ 8:3 Hz, 1H), 7.83 (d,
J ¼ 2:0 Hz, 1H); IR(KBr) 3450, 2950, 1755, 1550, 1375,
1230, and 1050 cmÀ1; mass spectrum [(+) ESI], m=z 823/825
(M + NH4þ), 828/830 (M + Na)þ; Anal. Calcd for
C33H40ClNO20: C, 49.17; H, 5.00; N, 1.74. Found: C,
49.16; H, 4.88; N, 1.71.
Step 4: 3-Acetamido-4-chlorobenzyl-b-maltoside (4). A solu-
tion containing 3-acetamido-4-chlorobenzyl hepta-O-ace-
tyl-b-maltoside (0.945 g, 1.12 mmol) and 25 wt % NaOMe in
MeOH (19.2 lL, 0.336 mmol) in MeOH (27.6 mL) was
refluxed for 2.5 h. The reaction was cooled to room
temperature and concentrated, and the resulting residue
was triturated with Et2O to afford the product (0.583 g,
99%) as a foam; 1H NMR(DMSO- d6) d 2.07 (s, 3H), 3.03–
3.16 (m, 2H), 3.19–3.49 (m, 7H), 3.55–3.62 (m, 2H), 3.67–
3.73 (m, 1H), 4.28 (d, J ¼ 7:7 Hz, 1H), 4.33–5.76 (br s, 7H),
4.67 (ABq, J ¼ 12:5 Hz, Dd ¼ 0:22, 2H), 5.01 (d,
J ¼ 3:7 Hz, 1H), 7.21 (dd, J ¼ 1:8, 8.1 Hz, 1H), 7.44 (d,
J ¼ 8:1 Hz, 1H), 7.64 (d, J ¼ 1:5 Hz, 1H), 9.33–9.69 (br s,
1H); IR(KBr) 3400, 2900, 1680, 1600, 1540, 1430, 1375,
1310, 1150, and 1035 cmÀ1, mass spectrum [(+) ESI], m=z
524/526 (M + H)þ, 546 (M + Na)þ; Anal. Calcd for
C21H30ClNO12Æ1.0 MeOH: C, 47.53; H, 6.16; N, 2.52.
Found: C, 47.94; H, 6.34; N, 2.42.
Step 5: 3-Acetamido-4-chlorobenzyl 40,60-O-benzylidene-b-
maltoside (5). To a stirred solution of 3-acetamido-4-
chlorobenzyl b-maltoside (14.15 g, 27.0 mmol) in DMF
(325 mL) at rt was added benzaldehyde dimethyl acetal
(8.11 mL, 54.0 mmol) dropwise followed by TsOHÆH2O
(2.57 g, 13.5 mmol). The reaction mixture was heated to
60 °C for 6 h and then quenched with K2CO3 (1.87 g,
13.5 mmol) with an additional 0.5 h heating at this temper-
ature. At this point, the solution was filtered hot, and the
solvent was distilled off using the high vac. The residue was
purified by flash chromatography (80:2:1–20:2:1, EtOAc/
EtOH/H2O gradient) to afford the product (10.8 g, 65%) as
a white solid, mp 143–147 °C; 1H NMR(DMSO- d6) d 2.08
(s, 3H), 3.07–3.12 (m, 1H), 3.28–3.50 (m, 5H), 3.51–3.60 (m,
2H), 3.64–3.75 (m, 3H), 4.10–4.12 (m, 1H), 4.30 (d,
J ¼ 7:9 Hz, 1H), 4.67 (t, J ¼ 5:9 Hz, 1H), 4.68 (ABq,
J ¼ 12:5 Hz, Dd ¼ 0:22, 2H), 5.14 (d, J ¼ 4:0 Hz, 1H),
5.25 (d, J ¼ 5:1 Hz, 1H), 5.30 (d, J ¼ 5:3 Hz, 1H), 5.51 (d,
J ¼ 3:3 Hz, 1H), 5.57 (s, 1H), 5.63 (d, J ¼ 6:8 Hz, 1H), 7.22
(dd, J ¼ 1:5, 8.3 Hz, 1H), 7.35–7.38 (m, 3H), 7.42–7.46 (m,
3H), 7.66 (s, 1H), 9.53 (s, 1H); IR(KBr) 3500, 3410, 2910,
2850, 1700, 1600, 1550, 1440, 1425, 1375, 1310, 1230, 1150,
1070, and 1030 cmÀ1; mass spectrum [(+) FAB], m=z 634
(M + Na)þ; Anal. Calcd for C28H34ClNO12Æ1.0 H2O: C,
53.38; H, 5.76; N. 2.22, Found: C, 53.58; H, 5.62; N, 2.25.
Step 6: 3-Acetamido-4-chlorobenzyl 6-O-benzoyl-40,60-O-
benzylidene-b-maltoside (6a). To a stirred solution of 3-
acetamido-4-chlorobenzyl 40,60-O-benzylidene-b-maltoside
(5.00 g, 8.17 mmol) in THF (80 mL) at À40 °C was added
collidine (80 mL, 605 mmol) dropwise followed by dropwise
addition of BzCl (1.14 mL, 9.80 mmol). After 2 h at this
temperature, it was warmed to rt and stirred an additional
48 h. At this point, the solvent was distilled off using the
high vac, and the residue was diluted with EtOAc (700 mL).
This layer was washed with 1 N HCl (70 mL), satd
NaHCO3 (70 mL), and brine (70 mL) and then dried
(MgSO4). After concentration, the oily residue was purified
by flash chromatography (1–11%, MeOH/CHCl3 gradient)
and recrystallization (EtOAc/hexane) to afford the product
(4.04 g, 69%) as a white solid, mp 185–187 °C; 1H NMR
(DMSO-d6) d 2.05 (s, 3H), 3.16–3.22 (m, 1H), 3.32–3.42 (m,
2H), 3.48–3.64 (m, 4H), 3.71 (dd, J ¼ 4:8, 9.7 Hz, 1H),
3.74–3.79 (m, 1H), 4.05 (dd, J ¼ 4:8, 10.3 Hz, 1H), 4.35 (dd,
J ¼ 5:3, 12.3 Hz, 1H), 4.39 (d, J ¼ 7:7 Hz, 1H), 4.58–4.63
(m, 1H), 4.65 (ABq, J ¼ 12:5 Hz, Dd ¼ 0:14, 2H), 5.14 (d,
4.0 Hz, 1H), 5.34 (t, J ¼ 5:1 Hz, 2H), 5.52 (s, 1H), 5.57 (d,
Step 2: 3-Amino-4-chlorobenzyl hepta-O-acetyl-b-maltoside
(2). A solution containing 4-chloro-3-nitrobenzyl hepta-O-
acetyl-b-maltoside (19.3 g, 23.9 mmol) and tin(II) chloride
dihydrate (37.7 g, 167 mmol) in EtOAc (479 mL) was
refluxed for 2 h. The reaction was cooled to rt, carefully
quenched with satd aq NaHCO3 (until basic), diluted with
EtOAc (250 mL), stirred for 0.5 h, and filtered. The biphasic
filtrate was separated and the aqueous phase extracted with
EtOAc. The combined organic extracts were dried
(Na2SO4) and concentrated. Purification by flash chroma-
tography (0–12% acetone/CHCl3 gradient) gave 17.8 g
(96%) the title compound as a glassy solid, mp 78–79 °C;
1H NMR(CDCl ) d 2.00 (s, 9H), 2.026 (s, 3H), 2.032 (s,
3
3H), 2.11 (s, 3H), 2.16 (s, 3H), 3.00–5.00 (br s, 2H), 3.64–
3.68 (m, 1H), 3.97 (ddd, J ¼ 2:4, 4.2, 10.1 Hz, 1H), 4.02–
4.07 (m, 2H), 4.24 (dd, J ¼ 2:2, 3.7 Hz, 1H), 4.27 (dd,
J ¼ 2:6, 4.0 Hz, 1H), 4.50–4.57 (m, 3H), 4.74 (d,
J ¼ 12:1 Hz, 1H), 4.83–4.90 (m, 2H), 5.05 (t, J ¼ 10:1 Hz,
1H), 5.22 (t, J ¼ 9:2 Hz, 1H), 5.35 (dd, J ¼ 9:7, 10.5 Hz,
1H), 5.42 (d, J ¼ 4:0 Hz, 1H), 6.62 (dd, J ¼ 2:0, 8.1 Hz,
1H), 6.76 (d, J ¼ 2:0 Hz, 1H), 7.21 (d, J ¼ 8:1, 1H); IR
(KBr) 3450, 3350, 2950, 1755, 1650, 1425, 1375, 1230, and
1050 cmÀ1; mass spectrum [(+) ESI], m=z 776/778 (M + H)þ,
798/800 (M + Na)þ; Anal. Calcd for C33H42ClNO18: C,
51.07; H, 5.45; N, 1.80. Found: C, 50.94; H, 5.52; N, 1.60.
Step 3: 3-Acetamido-4-chlorobenzyl hepta-O-acetyl-b-malto-
side (3). To a stirred solution of 3-amino-4-chlorobenzyl
hepta-O-acetyl-b-maltoside (20.6 g, 26.5 mmol) and trieth-
ylamine (8.13 mL, 58.3 mmol) in THF (265 mL) at 0 °C was
added dropwise acetyl chloride (2.26 mL, 31.8 mmol). After
0.5 h at this temperature, it was warmed to rt, and stirred an
additional 6 h. At this point, the reaction was concentrated
and taken up in EtOAc (700 mL). This organic solution was
washed with 1 N HCl (70 mL), satd aq NaHCO3 (70 mL),
and brine (70 mL) and then dried (MgSO4). After concen-
tration, the residue was purified by flash chromatography
(20:80–100:0, EtOAc/petroleum ether gradient) to afford
the product (16.2 g, 75%) as a glassy solid, mp 84–86 °C; 1H
NMR(CDCl ) d 2.00 (s, 6H), 2.020 (s, 3H), 2.027 (s, 3H),
3
2.03 (s, 3H), 2.11 (s, 3H), 2.16 (s, 3H), 2.24 (s, 3H), 3.66–
3.69 (m, 1H), 3.94–3.98 (m, 1H), 4.00–4.06 (m, 2H), 4.22–
4.28 (m, 2H), 4.50–4.61 (m, 3H), 4.80–4.91 (m, 3H), 5.05 (t,
J ¼ 10:1 Hz, 1H), 5.22 (t, J ¼ 9:2 Hz, 1H), 5.35 (dd,
J ¼ 9:4, 10.5 Hz, 1H), 5.41 (d, J ¼ 4:0 Hz, 1H), 6.99 (dd,
J ¼ 2:0, 8.1 Hz, 1H), 7.34 (d, J ¼ 8:1 Hz, 1H), 7.62 (s, 1H),

S. C. Mayer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2829–2833
2833
J ¼ 3:1 Hz, 1H), 5.79 (d, J ¼ 6:2 Hz, 1H), 7.19 (dd, J ¼ 2:0,
8.3 Hz, 1H), 7.32–7.38 (m, 3H), 7.38–7.45 (m, 3H), 7.51–
7.55 (m, 2H), 7.63–7.68 (m, 2H), 7.98–8.01 (m, 2H), 9.49 (s,
1H); IR(KBr) 3380, 3290, 2890, 2870, 1730, 1670, 1600,
1540, 1440, 1420, 1375, 1275, 1070, 1050, 1025, 975, and
710 cmÀ1; mass spectrum [(+) FAB], m=z 716/718 (M + H)þ,
738/740 (M + Na)þ; Anal. Calcd for C35H38ClNO13: C,
58.70; H, 5.35; N, 1.96. Found: C, 58.53; H, 5.36; N, 1.94.
Compounds 6b–h were prepared using similar procedures
to steps 5–6 of footnote 9 with the one exception that
intermediate 5h was prepared with 4-pyridinecarboxalde-
hyde using concentrated H2SO4 in DMF. All intermediates
(5b–h) and final compounds (6b–h) were verified by 1H
NMR, IR, MS, and CHN.