ORIGINAL ARTICLES
3.6.2. Cis-(2S, 5S)-2-(tert-butyl)-5-phenyl-5-cyclopentyl-1,3-dioxolan-4-one,
11
The crude product was dissolved in a small volume of methylene chloride
and poured into 100 ml of dry ethyl ether to obtain a precipitate. This pro-
cedure was repeated three times and gave the product 16 (0.81 g, 89 %,
white powder). 1H NMR (CDCl3, 300 MHz): 1.30–1.70 (m, 8 H), 1.82–
1.95 (brs, 1 H), 2.10–2.20 (m, 1 H), 2.75–2.90 (m, 2 H), 3.25, 3.60 (2s,
total 3 H, N-CH3), 3.75, 3.79 (2s, total 3 H, O-Me), 4.10–4.60 (m, 4 H),
4.92, 5.35 (2AB, total 2 H, N-CH2-COOMe), 5.52–5.58 (m, 1 H), 7.23–
7.38 (m, 3 H), 7.56–7.60 (m, 2H). 13C NMR (CDCl3, 300 MHz): 25.8,
25.9; 26.3, 26.4; 26.4, 26.5; 27.0, 27.0; 29.8, 30.1; 45.9, 46.8; 50.2, 51.4;
53.2, 53.2; 62.2, 63.2; 64.6, 64.7; 69.6, 69.7; 72.8, 73.1; 79.4, 79.6; 125.7,
125.7; 127.6, 127.9; 128.2, 128.4; 141.0, 141.2; 165.3, 165.5; 173.9,
174.2.
At ꢃ78 ꢁC, a lithium bis-(trimethylsilyl)amide in hexane solution (120 ml,
120 mmol, 1.0 M in hexane) was added to compound 10 (25g,
113.5 mmol, dissolved in 100 ml of dried THF), stirred for 1 h, followed
by addition of cyclopentyl bromide (25 g, 168 mmol). This reaction was
kept at ꢃ78 ꢁC for 4 h, then slowly warmed up to room temperature and
continued for overnight. The completion of the reaction was followed by
TLC. With stirring, a solution of 10% of NH4Cl (25 ml) was added to the
mixture. Then, the mixture was poured into a separation funnel containing
10% NH4Cl solution (200 ml). The aqueous layer was discarded, and the
organic layer was dried over Na2SO4. The solvent was removed to give a
crude product, which was then re-crystallized in hexane to give a pure pro-
duct, 11 (20.36 g, yield 63%, white crystal). 1H NMR (CDCl3, 300 MHz):
1.15 (s, 9 H), 1.55–1.95 (m, 8 H), 2.74 (m, 1 H), 5.62 (s, 1 H), 7.44–7.56
(m, 3 H), 7.88–7.91 (n, 2 H) ppm. 13C NMR (CDCl3, 300 MHz): 23.5,
24.5, 25.3, 26.6, 35.6, 50.9, 83.2, 110.6, 124.9, 127.5, 127.9, 138.9, 173.7.
3.6.8. 30(S)-[S-Cyclopentylphenylhydroxyacetoxy]-10-methyl-10-methoxycar-
bonylpyrrolidinium bromide, 17 (2S30S-SGM)
Following the same procedure as for 16, except compound 15 was used
instead of compound 14, the product of 17 (0.8 g, 88 %, white powder)
was obtained. 1H NMR (CDCl3, 300 MHz): 1.30–1.75 (m, 8H), 1.80–1.90
(brs, 1H), 2.15–2.30 (m, 1H), 2.78–2.95 (m, 2H), 3.10, 3.65 (2s, total 3H,
N-CH3), 3.75, 3.78 (2s, total 3H, O-Me), 4.15–4.52 (m, 4H), 4.85, 5.38
(2AB, total 2H, N-CH2-COOMe), 5.50–5.58 (m, 1H), 7.23–7.38 (m, 3H),
7.56–7.66 (m, 2H). 13C NMR (CDCl3, 300 MHz): 25.8, 25.9; 26.2, 26.3;
26.3, 26.4; 26.8, 26.9; 29.4, 29.6; 45.6, 46.9; 50.1, 51.4; 53.1, 53.1; 62.2,
63.3; 64.8, 64.8; 69.5, 69.8; 72.8, 73.2; 79.4, 79.6; 125.6, 125.9; 127.6,
127.9; 128.2, 128.4; 140.7, 141.1; 165.2, 165.5; 173.9, 174.2.
3.6.3. S(þ)-Cyclopentylmandelic acid, 12
To a solution of cis-(2S, 5S)-2-(tert-butyl)-5-cyclopentyl-5-phenyl-1,3-diox-
olan-4-one (14.35 g, 50 mmol) in 100 ml methanol and 50 ml water, 15 g
of KOH was added slowly. The mixture was stirred and heated (65 ꢁC) to
reflux for 3–4 h, then cooled down to the room temperature, and methanol
was removed. To the aqueous solution, 100 ml of ethyl acetate was added
then acidified to pH 1 with 3N HCl. The mixture was poured to a separa-
tion funnel, and the organic layer was separated. The aqueous layer was
extracted two times with ethyl acetate (50 ml). The combined organic layers
were dried over Na2SO4, filtered, and the solvent was removed to provide
13.44 g of yellowish crude product, which was re-crystallized to give a pure
product of S(þ)-cyclopentylmandelic acid, 12 (6.89 g, yield 62%, white
crystal). 1H NMR (CDCl3, 300 MHz): 1.28–1.75 (m, 8 H), 2.94 (m, 1 H),
7.24–7.34 (m, 3 H), 7.62–7.68 (m, 2 H). 13C NMR (CDCl3, 300 MHz):
25.9, 26.3, 26.4, 26.9, 47.1, 79.2, 125.8, 127.7, 128.2, 140.8, 180.9.
3.7. Hydrolysis of esters and HPLC separations
The procedures used for obtaining the 2S-isomers 18a, 18b, 19a and 19b
(white powder) were the same as for 2R-isomers 8a, 8b, 9a and 9b.
10(R),30(R)-[2(S)-Cyclopentyl-2-phenyl-2-hydroxyacetoxy]-10-methyl-10-car-
boxymethyl pyrrolidinium inner salt, 18a (2S30R10R-SGa): 1H NMR
(CDCl3, 300 MHz): 1.30–1.65 (m, 8 H), 2.02–2.45 (m, 2 H), 2.82–2.90
(m, 1 H), 3.10–3.18 (m, 1 H), 3.25 (s, 3 H), 3.50–4.05 (m, 6 H), 5.34–
5.40 (m, 1 H), 7.23–7.38 (m, 3 H), 7.50–7.68 (m, 2 H).
10(S),30(R)-[2(S)-Cyclopentyl-2-phenyl-2-hydroxyacetoxy]-10-methyl-10-car-
boxymethyl pyrrolidinium inner salt, 18b (2S30R10S-SGa): 1H NMR
(CDCl3, 300 MHz): 1.45–1.85 (m, 9 H), 2.05–2.15 (m, 1 H), 2.80–2.90
(m, 1 H), 3.00–3.10 (m, 1 H), 3.35 (s, 3 H), 3.70–3.80 (m, 1 H), 3.90–
4.10 (m, 4 H), 4.22–4.35 (m, 1 H), 5.50–5.60 (m, 1 H), 7.36–7.55 (m,
3 H), 7.72–7.80 (m, 2 H).
3.6.4. Methyl S(þ)-cyclopentylmandelate, 13
S(þ)-Cyclopentylmandelic acid, 12 (5.5 g, 25 mmol), and potassium carbo-
nate (8.61 g, 63 mmol) in DMF (60 ml) solution was mixed with methyl
iodide (10.6 g, 75 mmol). The mixture was stirred at room temperature for
3 h, poured to water, and extracted with hexane for three times. Evaporation
of dried hexane extract gave a pure product of S(þ)-cyclopentylmandelate,
10(R),30(S)-[2(S)-Cyclopentyl-2-phenyl-2-hydroxyacetoxy]-10-methyl-10-car-
boxymethyl pyrrolidinium inner salt, 19a (2S30S10R-SGa): 1H NMR
(CDCl3, 300 MHz): 1.20–1.65 (m, 8 H), 1.95–2.10 (m, 1 H), 2.20 (brs,
1 H), 2.40–2.50 (m, 1 H), 2.78–2.90 (m, 1 H), 3.15 (s, 3 H), 3.70–3.90
(m, 2 H), 3.96–4.20 (m, 4 H), 5.38–5.50 (m, 1 H), 7.20–7.38 (m, 3 H),
7.55–7.65 (m, 2 H).
1
13 (5.85 g, 100 %, clear oil). H NMR (CDCl3, 300 MHz): 1.32–1.61 [8 H,
m, (CH2)4], 2.90 [1 H, p, CHC(OH)], 3.76 (s, 3 H), 3.78 (s, 1 H), 7.25–7.35
(m, 3H), 7.63–7.65 (m, 2 H). 13C NMR (CDCl3, 300 MHz): 25.9, 26.2,
26.3, 26.8, 47.1, 53.2, 79.1, 125.8, 127.3, 128.0, 141.6, 176.0.
3.6.5. (30R)-N-Methyl-30-pyrrolidinyl-(S)-cyclopentylmandelate, 14
10(S),30(S)-[2(S)-cyclopentyl-2-phenyl-2-hydroxyacetoxy]-10-methyl-10-carb-
oxymethyl pyrrolidinium inner salt, 19b (2S30S10S-SGa): 1H NMR (CDCl3,
300 MHz): 1.35–1.70 (m, 8 H), 2.00–2.15 (m, 1 H), 2.70–2.90 (m, 2 H),
3.00 (s, 3 H), 3.42 (brs, 1 H), 3.58–3.68 (m, 2 H), 3.80–3.95 (m, 3 H),
4.08–4.18 (m, 1 H), 5.38–5.48 (m, 1 H), 7.20–7.40 (m, 3 H), 7.55–7.62
(m, 2 H).
In a 250 ml 3-neck flask equipped with Dean-Stark condenser, the mixture
of methyl S(þ)-cyclopentylmandelate, 13 (2 g, 8.8 mmol), (R)-3-hydroxy-
N-methyl pyrrolidine, (R)-3 (2 g, 20 mmol), and 100 ml of heptane was
stirred and heated (110 ꢁC) until 20 ml of heptane had been distilled. The
temperature was reduced to 25 ꢁC, and approximately 0.003 g of sodium
was added. The mixture was stirred and heated to 110 ꢁC again for 3 h as
the distillation was continued. An additional piece of sodium (0.002 g) was
added at the 1 h point. More heptane was added at such a rate as to keep
the reaction volume constant. The mixture was cooled to 0 ꢁC, mixed with
5 ml of water, and the organic layer was separated. The organic layer was
extracted with 3N HCl. The acid extract was made alkaline (pH 10) with
5N NaOH and extracted three times with ether. Removal of dried ether so-
lution (over Na2SO4) gave a clear, oily product 14 (1.6 g, 61.5%).1H NMR
(CDCl3, 300 MHz): 1.28–1.80 [m, 9 H], 2.15–2.25 (m, 1 H), 2.30–2.40
(m, 1 H), 2.37 (s, 3 H),2.65–2.80 (m, 3 H), 2.90–3.00 (m, 1 H), 3.85 (1 H,
brs, OH), 5.22 (m, 1 H), 7.20–7.35 (m, 3 H), 7.64–7.70 (m, 2 H).
13C NMR (CDCl3, 300 MHz):26.0, 26.4, 26.5, 26.7, 32.1, 42.0, 47.1, 54.8,
62.0, 76.5, 79.1, 125.8, 127.3, 128.0, 141.7, 175.3.
3.8. Receptor binding affinity
Receptor binding studies on soft anticholinergics isomers and their zwitter-
ionic metabolite isomers, as well as glycopyrrolate, and N-methylscopol-
amine were performed with N-[3H]-methyl-scopolamine (NMS) in assay
buffer (phosphate-buffered saline, PBS, without Caþþ or Mgþþ, pH 7.4),
following the protocol from Applied Cell Science Inc. (Rockville, MD). A
10 mM NaF solution was added to the buffer as an esterase inhibitor. The
assay mixture (0.2 ml) contained 20 ml diluted receptor membranes (recep-
tor proteins: M1, 38 mg/ml; M2, 55 mg/ml; M3, 27 mg/ml; M4, 84 mg/ml).
The final concentration of NMS for the binding studies was 0.5 nM. Spe-
cific binding was defined as the difference in [3H]NMS binding in the ab-
sence and presence of 5 mM atropine for M1 and M2 or 1 mM atropine for
M3 and M4. Incubation was carried out at room temperature for 2 h. The
assay was terminated by filtration through a Whatman GF/C filter (pre-
soaked overnight with 0.5% polyethyleneimine). The filter was then
washed six times with 1 ml ice cold buffer (50 mM Tris-HCl, pH 7.8,
0.9% NaCl), transferred to vials, and 5 ml of Scintiverse was added. Detec-
tion was performed on a Packard 31800 liquid scintillation analyzer (Pack-
ard Instrument Inc., Downer Grove, IL). Data obtained from the binding
3.6.6. (30S)-N-Methyl-30-pyrrolidinyl-(S)-cyclopentylmandelate, 15
Following the same procedure as for 14, except (S)-3 was used instead of
(R)-3, a clear, oily product of 15 (2.33 g, 89.6%) was obtained. 1H NMR
(CDCl3, 300 MHz): 1.24–1.70 (m, 9 H), 1.80–1.88 (m, 1 H), 2.25–2.40
(m, 2 H), 2.35 (s, 3 H), 2.55–2.70 (m, 2 H), 2.75–2.82 (m, 1 H), 2.90–3.00
(m, 1 H), 3.95 (1 H, bs, OH), 5.22 (m, 1 H), 7.24–7.40 (m, 2 H), 7.64–
7.69 (m, 5 H). 13C NMR (CDCl3, 300 MHz): 26.0, 26.3, 26.4, 26.7, 32.6,
42.0, 47.1, 54.9, 61.6, 76.4, 79.2, 125.8, 127.3, 128.0, 141.7, 175.2.
experiments were fitted to the equation %[3H] NMS bound ¼ 100
–
[100xn/k/(1 þ xn/k)], to obtain the Hill coefficient n, and then to the equa-
tion %[3H] NMS bound ¼ 100 – [100xn/IC50/(1 þ xn/IC50)], to obtain the
IC50 values (x being the concentration of the tested compound). Based on
the method of Cheng and Prusoff (Cheng and Prusoff 1973), Ki was de-
rived from the equation Ki ¼ IC50/(1 þ L/Kd), where L is the concentration
of the radioligand. IC50 represents the concentration of the drug causing
50% inhibition of specific radioligand binding, and Kd represents the dis-
sociation constant of the radioligand receptor complex. Data were analyzed
3.6.7. 30(R)-[S-Cyclopentylphenylhydroxyacetoxy]-10-methyl-10-methoxycar-
bonylpyrrolidinium bromide, 16 (2S30R-SGM)
To compound 14 (0.6 g, 1.96 mmol) in 30 ml of dry acetonitrile, methyl
bromoacetate (1.0 g, 6.4 mmol) was added at room temperature. The mix-
ture was stirred for 3 h. Evaporation of acetonitrile gave a crude product.
208
Pharmazie 63 (2008) 3