P. Bissel et al. / Bioorg. Med. Chem. 10 (2002) 3031–3041
3039
112.5, 114.9, 126.7, 128.1, 132.1. Anal. calcd for
C12H16ClN3 (237.73): C, 60.63; H, 6.78; N, 17.68.
Found: C, 60.36; H, 6.95; N, 17.45.
1-Isopropyl-4-(1-methylpyrrol-2-yl)pyridinium bromide
.
(43 Br). A mixture of the free base 38 (1 g, 6.3 mmol)
and isopropyl bromide (5 mL) was stirred for 24 h
under reflux. The resulting solid was collected and
.
1-Allyl-4-(1-methylpyrrol-2-yl)pyridinium bromide (41 Br).
.
washed with ether to give analytically pure 43 Br as
Treatment of the free base 38 (1 g, 6.3 mmol) with allyl
bromide (2.2 mL, 25 mmol) according to the previous
description gave 1.65 g (94%) of the corresponding
yellow crystals (0.63 g, 42%): mp 155–157 ꢁC; UV
1
(H2O) lmax 368 nm (e 25,500); H NMR (CD3OD) d
1.66 (d, J=6.8 Hz, 6H), 3.99 (s, 3H), 4.86 (m, 1H), 6.33
(m, 1H), 7.10 (m, 1H), 7.20 (m, 1H), 8.06 (m, 2H), 8.72
(m, 2H); 13C NMR (CD3OD) d 23.4, 38.2, 64.5, 111.8,
119.7, 123.5, 129.6, 135.0, 143.1, 148.4. Anal. calcd for
C13H17BrN2 (281.18): C, 55.53; H, 6.09; N, 9.96. Found:
C, 55.72; H, 6.12; N, 9.91.
.
allylpyridinium bromide 41 Br as yellow needles from
EtOH: mp 206–208 ꢁC; UV (H2O) lmax 372 nm (e
1
28,768); H NMR (CD3OD) d 3.99 (s, 3H), 5.10 (m,
2H), 5.51 (m, 2H), 6.18 (m, 1H), 6.34 (m, 1H), 7.13 (m,
1H), 7.21 (m, 1H), 8.08 (m, 2H), 8.61 (m, 2H); 13C
NMR (CD3OD) d 37.9, 62.9, 111.9, 120.0, 123.1, 123.2,
129.7, 132.4, 135.3, 144.8, 148.8. Anal. calcd for
C13H15BrN2 (279.17): C, 55.93; H, 5.42; N, 10.03.
Found: C, 55.96; H, 5.53; N, 9.88.
Oxalate salt of 1-isopropyl-4-(1-methylpyrrol-2-yl)-
.
1,2,3,6-tetrahydropyridine [29 (COOH)2]. An ether
solution of the free base 29, prepared as described above
for the propargyl analogue 25, was filtrated through
basic alumina. The filtrate was treated with an etheral
solution of oxalic acid (1.0 equiv) to yield the analy-
tically pure oxalate salt of 29 (43%) as a light yellow
solid: mp 138–140 ꢁC; UV (H2O) lmax 266 nm (e
1-Allyl-4-(1-methylpyrrol-2-yl)-1,2,3,6-tetrahydropyridine
.
hydrochloride (27 HCl). The free base of this compound
was prepared according to the procedure described
previously for the corresponding free base of the pro-
pargyl derivative 25. A methanolic solution of HCl (1.5
equiv) was added at 0 ꢁC to a solution of the free base in
MeOH (2 mL). After standing overnight in the ꢁfreezer,
1
10,350); H NMR (CD3OD) d 1.41 (d, J=6.5 Hz, 6H),
1.80 (m, 2H), 3.49 (m, 2H), 3.63 (m, 1H), 3.69 (s, 3H),
3.91 (m, 2H), 5.77 (m, 1H), 6.03 (m, 1H), 6.15 (m, 1H),
6.68 (m, 1H); 13C NMR (CD3OD) d 17.2, 28.0, 36.2,
47.4, 47.8, 58.9, 108.4, 109.2, 116.0, 126.5, 129.9, 132.8,
166.8. Anal. calcd for C15H22N2O4 (294.35): C, 61.21;
H, 7.53; N, 9.52. Found: C, 60.88; H, 7.53; N, 9.35.
.
pure 27 HCl (59%) was obtained: mp 196–198 C; UV
1
(H2O) lmax 268 nm (e 11,376); H NMR (DMSO-d6) d
2.55–2.74 (m, 2H), 3.15 (m, 1H), 3.52 (m, 1H), 3.64 (s,
3H), 3.71 (m, 1H), 3.78–3.82 (m, 3H), 5.54 (m, 2H), 5.76
(m, 1H), 5.98–6.13 (m, 3H) 6.77 (m, 1H), 10.97 (bs, 1H);
13C NMR (CD3OD) d 27.3, 36.3, 50.0, 51.3, 59.4, 108.4,
110.1, 115.3, 126.6, 127.0, 127.8; 129.9. Anal. calcd for
C13H19ClN2 (238.75): C, 65.40; H, 8.02; N, 11.73.
Found: C, 65.37; H, 8.08; N, 11.58.
Enzymology
General methods. All collections of human placenta
were approved by the Internal Review Boards of Mon-
tgomery County Hospital and Virginia Tech, and the
collection of beef liver was approved by the Animal
Care Committee of Virginia Tech. Human placenta and
beef liver mitochondrial homogenates were prepared
using the methodology reported earlier by Salach with
minor modifications and were stored atꢀ70 ꢁC prior to
use.48 Phosphate buffer was prepared using Na2HPO4
and NaH2PO4. For determinations of the protein con-
centrations (triplicate samples) using the Bradford
assay, the mitochondrial preparations initially were
diluted approximately 1:2 with phosphate buffer con-
taining 50% (w/v) glycerol.49 A UV–vis background
subtraction was performed for each sample as follows:
Background samples were prepared following the same
procedures as those for the corresponding samples used
for the analysis of substrate and inhibitor properties of
the test compounds. At t=0 min 70% aq HClO4 was
added to each background sample and the resulting
mixture was centrifuged. The UV–vis signal from the
supernatant fraction was subtracted from each corres-
ponding sample signal.
4 - (1 - Methylpyrrol - 2 - yl) - 1 - propylpyridinium bromide
.
(41 Br). A mixture of the free base 38 (1 g, 6.3 mmol)
and propyl bromide (2.3 mL, 25 mmol) was stirred for
48 h under reflux. After cooling, the precipitate was
collected and recrystallized from EtOH to give pure
.
41 Br as light-yellow crystals (1.7 g, 96%): mp 222–
224 ꢁC; UV (H2O) lmax 370 nm (e 25,352); H NMR
1
(CD3OD) d 1.02 (t, J=7.6 Hz, 3H), 2.03 (sext, J=7.6
Hz, 2H), 3.99 (s, 3H), 4.42 (t, J=7.6 Hz, 2H), 6.33 (m,
1H), 7.13 (m, 1H), 7.20 (m, 1H), 8.06 (m, 2H), 8.65 (m,
2H); 13C NMR (CD3OD) d 11.0, 25.7, 37.8, 62.7, 111.8,
119.8, 123.2, 129.7, 135.0, 144.8, 148.6. Anal. calcd for
C13H17BrN2 (281.18): C, 55.53; H, 6.09; N, 9.96. Found:
C, 55.44; H, 6.09; N, 9.96.
4-(1-Methylpyrrol-2-yl)-1-propyl-1,2,3,6-tetrahydropyridine
.
hydrochloride (28 HCl). The free base 28, prepared as
described for the propargyl analogue 25, was converted
to its HCl salt in methanol at 0 ꢁC. Crystallization from
.
EtOH gave pure 28 HCl in 43% overall yield: mp 205–
208 ꢁC (dec.); UV (H2O) lmax 267 nm (e 10,093); H
1
NMR (CD3OD) d 1.05 (t, J=7.2 Hz, 3H), 1.84 (m, 2H),
2.78 (m, 2H), 3.19 (m, 2H), 3.35–3.65 (m, 2H), 3.69 (s,
3H), 3.94 (m, 2H), 5.75 (m, 1H), 6.03 (m, 1H), 6.17 (m,
1H), 6.69 (m, 1H); 13C NMR (CD3OD) d 11.4, 18.9, 27.3,
36.3, 50.7, 51.7, 58.9, 108.4, 110.1, 115.2, 126.6, 129.7,
128.6. Anal. calcd for C13H21ClN2 (240.77): C, 64.85; H,
8.79; N, 11.93. Found: C, 64.70; H, 8.77; N, 11.44.
Enzyme concentration determinations. Studies to esti-
mate enzyme concentrations (nmol enzyme/mg mito-
chondrial protein) were conducted as follows: The rate
of the MAO-A catalyzed oxidation of 1-methyl-4-phen-
oxy-1,2,3,6-tetrahydropyridine (35) was determined
using a 4 mM stock solution of the substrate in 0.1 M
phosphate buffer (pH 7.2–7.4, 475 mL) preincubated for