Tetracyclic inhibitors of acetylcholinesterase 353
with 1,3-diaminopropane (14.23 g, 16 mL, 192 mmol) in
2-methoxyethanol (12 mL). e crude product was re-
crystallized from 2-methoxyethanol to give 10 (125 mg,
11%): mp 202–207°C, conversion 193–197°C; 1H NMR
(DMSO-d6) δ 1.88–1.93 (m, 2H, 3-H), 2.70 (t, J= 5.7 Hz,
2H, 8-H), 2.79–2.81 (m, 2H, 7-H), 3.44 (s, 2H, 10-H), 3.65
(s, 2H, C6H5-CH2), 3.84 (t, J= 5.8 Hz, 2H, 2-H or 4-H), 7.30–
7.33 (m, 5H, C6H5), 7.76 (s, 1H, 12-H), one signal (2H, 2-H
or 4-H) is not visible due to the water signal (3.20–3.40);
1H NMR (CDCl3) δ 2.02–2.07 (m, 2H, 3-H), 2.83 (t, J= 5.8
Hz, 2H, 8-H), 3.03 (t, J= 5.8 Hz, 2H, 7-H), 3.44–3.48, 4.01
(m, t, J= 5.8 Hz, total 4H, 2-H, 4-H), 3.55 (s, 2H, 10-H),
3.72 (s, 2H, C6H5-CH2), 6.42 (s, 1H, 12-H), 7.27–7.37 (m,
5H, C6H5); 13C NMR (DMSO-d6) δ 19.79 (C-3), 25.80 (C-7),
38.63, 38.84 (C-2, C-4), 49.39, 51.06 (C-8, C-10), 61.13
(C6H5-CH2), 111.76 (C-6a), 121.47, 128.64 (C-6b, C-10a),
127.15 (C-4′), 128.37, 128.90 (C-2′, C-3′), 138.42 (C-1′),
150.91, 157.68 (C-11a, C-12a), 165.77 (C-6). Anal. found:
C, 61.99; H, 5.66; N., 15.03. C19H20N4OS × H2O requires: C,
61.60; H, 5.99; N, 15.12.
7.55 (s, 1H, 6-H); 13C NMR (DMSO-d6) δ 20.13 (C-3), 27.11
(C-12), 43.79 (C-2), 46.07 (C-4), 49.51 (C-11), 51.30 (C-9),
60.96 (C6H5-CH2), 120.98 (C-12b), 127.14 (C-4′), 128.37,
128.84 (C-2′, C-3′), 128.62, 129.86 (C-8a, C-12a), 138.34
(C-1′), 143.44, 154.77 (C-7a, C-12c), 147.37 (C-6).
Enzyme inhibition experiments
Cholinesterase inhibition was assayed spectrophoto-
metrically at 412 nm at 25°C19,29. Product formation
was monitored over 5 min. Assay buffer was 100 mM
sodium phosphate, 100 mM NaCl, pH 7.3. Enzyme
stock solutions were prepared with assay buffer in
the following concentrations ∼ 100 U/mL (EeAChE),
∼ 3 U/mL (hAChE), ∼ 10 U/mL (hBChE), and were
kept at 0°C. Appropriate dilutions of the EeAChE
(1:30) and hBChE (1:10) solutions were done imme-
diately before starting the measurements. Solutions
of ATCh (5, 10, 15, or 20 mM), BTCh (10 mM), and
DTNB (7 mM) were prepared in assay buffer and kept
at 0°C. Stock solutions of the inhibitors were prepared
in acetonitrile. EeAChE was assayed as follows. Into
a cuvette containing 830 µL assay buffer, 50 µL of the
DTNB solution, 50 µL acetonitrile, 10 µL of an inhibitor
solution, and 10 µL of an enzyme solution were added
and thoroughly mixed. After incubation for 15 min
at 25°C, the reaction was initiated by adding 50 µL of
the ATCh solution. The following final concentration
were used, ∼ 0.033 U/mL of EeAChE, 250, 500, 750, or
1000 µM of ATCh, 350 µM of DTNB, 6% acetonitrile.
Similarly, hAChE (final concentration ∼ 0.03 U/mL)
was assayed with 500 µM ATCh, and hBChE (final
concentration ∼ 0.01 U/mL) with 500 µM BTCh. The
rates of enzyme-catalyzed substrate hydrolysis were
corrected by those of the non-enzymatic hydrolysis of
ATCh or BTCh, respectively, as determined by using
10 µL of assay buffer, instead of the enzyme solution. A
2,3,8,9,10,11-Hexahydro-benzothieno[3,2-e]
imidazo[1,2-c]pyrimidine (11). 1H NMR (DMSO-d6) δ
1.67–1.80 (m, 4H, 9-H, 10-H), 2.65–2.69, 2.74–2.78 (each
m, total 4H, 8-H, 11-H), 3.83 (t, J= 9.9 Hz, 2H, 2-H), 4.00 (t,
J= 9.9 Hz, 2H, 3-H), 7.90 (s, 1H, 5-H). 13C NMR (DMSO-d6)
δ 22.01, 22.70 (C-9, C-10), 24.49, 25.44 (C-8, C-11), 45.27
(C-3), 53.17 (C-2), 117.22 (C-11b), 130.47, 131.36 (C-7a,
C-11a), 144.20 (C-5), 150.70, 157.56 (C-6a, C-11c).
3,4,9,10,11,12-Hexahydro-2H-benzothieno[3,2-e]
1
pyrimido[1,2-c]pyrimidine (12). H NMR (DMSO-d6) δ
1.67–1.77 (m, 4H, 10-H, 11-H), 1.86–1.92 (m, 2H, 3-H),
2.67–2.72, 2.84–2.88 (each m, total 4H, 9-H, 12-H), 3.43
(t, J= 5.7 Hz, 2H, 2-H), 3.95 (t, J= 5.4 Hz, 2H, 4-H), 7.73
1
(s, 1H, 6-H); H NMR (CDCl3) δ 1.69–1.78 (m, 4H, 10-H,
11-H), 1.89–1.94 (m, 2H, 3-H), 2.64–2.67, 2.87–2.90 (each
m, total 4H, 9-H, 12-H), 3.51 (t, J= 5.7 Hz, 2H, 2-H), 3.77 (t,
J= 5.8 Hz, 2H, 4-H), 7.16 (s, 1H, 6-H); 13C NMR (DMSO-d6)
δ 19.59 (C-3), 22.08, 22.40 (C-10, C-11), 24.87, 26.60 (C-9,
C-12), 43.04 (C-2), 46.47 (C-4), 120.05 (C-12b), 130.84,
132.27 (C-8a, C-12a), 144.70, 155.87 (C-7a, C-12c), 146.81
(C-6).
K
m value of 550 µM19 for the substrate ATCh used in the
EeAChE assay was taken for kinetic analyses.
Results and discussion
9-Benzyl-2,3,8,9,10,11-hexahydro-imidazo[1,2-c]
pyrido[4′,3′:4,5]thieno[3,2-e]pyrimidine (13). 1H NMR
(DMSO-d6) δ 2.72 (t, J= 5.8 Hz, 2H, 10-H), 2.82 (t, J= 5.8
Hz, 2H, 11-H), 3.55 (s, 2H, 8-H), 3.67 (s, 2H, C6H5-CH2),
3.85 (t, J= 10.0 Hz, 2H, 2-H), 4.00 (t, J= 10.0 Hz, 2H, 3-H),
7.23–7.35 (m, 5H, C6H5), 7.92 (s, 1H, 5-H); 13C NMR
(DMSO-d6) δ 25.67 (C-11), 45.33 (C-3), 49.23 (C-10),
51.06 (C-8), 53.18 (C-2), 60.9 (C6H5-CH2), 116.79 (C-11b),
127.19 (C-4′), 128.39, 128.91 (C-2′, C-3′), 128.85, 129.14
(C-7a, C-11a), 138.29 (C-1′), 144.46 (C-5), 150.57, 158.20
(C-6a, C-11c).
Synthesis
e preparation of the linearly fused compounds 7–10 is
depicted in Scheme 1. Ethyl 2-aminothiophenecarbox-
ylates 1 and 2 were prepared by Gewald synthesis, for a
review, see30, and reacted with phenylisothiocyanate to
afford thiourea derivatives 3 and 4. Cyclocondensation
and immediate S-alkylation31 gave tricyclic pyrimidines
5 and 6 bearing a nucleofuge at C-2. Indeed, upon re-
action with diaminoalkanes, methylmercaptane was
eliminated. e products, however, were formed by a
more complex mechanism, which can be envisaged as
follows. After the initial nucleophilic attack of the di-
aminoalkane and repulsion of the leaving group, a Dim-
roth rearrangement occurred. en, an intramolecular
nucleophilic attack of the terminal amino function,
again at C-2, led to the replacement of aniline and for-
mation of the fourth fused ring in the imidazo or pyrim-
1 0 - B e n z y l - 3 , 4 , 9 , 1 0 , 1 1 , 1 2 - h e x a h y d r o - 2 H -
pyrido[4′,3′:4,5]thieno[3,2-e]pyrimido[1,2-c]pyrimidin
1
(14). H NMR (DMSO-d6) δ 1.80–1.85 (m, 2H, 3-H), 2.68
(t, J= 5.8 Hz, 2H, 11-H), 2.89 (t, J= 5.8 Hz, 2H, 12-H), 3.39
(t, J= 5.5 Hz, 2H, 2-H), 3.52 (s, 2H, 9-H), 3.65 (s, 2H, C6H5-
CH2), 3.86 (t, J= 5.7 Hz, 2H, 4-H), 7.13–7.33 (m, 5H, C6H5),
© 2011 Informa UK, Ltd.