Journal of Medicinal Chemistry
Article
chromatography (silica gel, CH2Cl2 → 8:2, CH2Cl2/MeOH). The
isolated product, in free base form, was dissolved in CH2Cl2/MeOH
(9:1) and an excess of 2 M HCl in Et2O was added, and the mixture
2H), 3.06 (br s, 2H), 3.01 (br s, 2H), 1.60−1.54 (m, 2H), 1.36−1.22
(m, 4H), 1.05 (s, 6H), 0.90 (t, J = 7.2 Hz, 3H). ESI MS for [M + H]+
= 241.8 Da.
1
was evaporated to give a yellow oil (0.46 g, 19%). H NMR (300
1-Butyl-5,5-dimethyl-1,4,5,6-tetrahydropyrimidine-2-carbalde-
hyde Oxime Hydrochloride (15a). The title compound was obtained
MHz, d6-DMSO) δ = 13.50 (s, 1H), 10.13 (br s, 1H), 8.27 (s, 1H),
4.00−3.76 (m, 4H), 3.56 (t, J = 7.2 Hz, 2H), 1.58−1.48 (m, 2H),
1.32−1.19 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H). ESI MS for [M + H]+ =
169.9 Da.
1
in a manner similar to that used for 12a, as a yellow solid (37%). H
NMR (500 MHz, d6-DMSO) δ = 13.22 (br s, 1H), 9.98 (br s, 1H),
8.29 (s, 1H), 3.54−3.51 (m, 2H), 3.29 (br s, 2H), 3.06 (br s, 2H),
1.57−1.51 (m, 2H), 1.31−1.22 (m, 2H), 0.97 (s, 6H), 0.88 (t, J = 7.2
Hz, 3H). 13C NMR (125 MHz, d6-DMSO) δ = 152.3, 138.2, 56.7,
51.2, 48.9, 29.6, 25.9, 23.1, 18.9, 13.5. ESI MS for [M + H]+ = 212.1
Da.
1-(2,4-Dichlorobenzyl)-4,5-dihydro-1H-imidazole-2-carbalde-
hyde Oxime Hydrochloride (12c). The title compound was obtained
1
in a manner similar to that used for 12a, as a yellow solid (30%). H
NMR (300 MHz, d6-DMSO) δ = 13.59 (s, 1H), 10.65 (br s, 1H), 8.40
(s, 1H), 7.72 (d, J = 1.8 Hz, 1H), 7.55−7.47 (m, 2H), 4.93 (s, 2H),
3.88−3.76 (m, 4H). 13C NMR (125 MHz, d6-DMSO) δ = 159.0,
135.1, 133.8, 133.7, 131.4, 130.8, 129.2, 127.8, 49.6, 47.3, 42.8. ESI MS
for [M + H]+ = 271.9 Da.
1-(3,5-bis(trifluoromethyl)benzyl)-4,5-dihydro-1H-imidazole-2-
carbaldehyde oxime hydrochloride (12d). The title compound was
obtained in a manner similar to that used for 12a, as a yellow solid
(41%). 1H NMR (300 MHz, d6-DMSO) δ = 13.53 (s, 1H), 10.58 (br
s, 1H), 8.42 (s, 1H), 8.18 (br s, 2H), 8.12 (br s, 1H), 5.00 (s, 2H), 3.82
(br s, 4H). 13C NMR (125 MHz, d6-DMSO) δ = 159.2, 137.9, 135.4,
130.5 (q, J = 32.9 Hz), 129.2, 123.2 (q, J = 271.2 Hz), 121.9, 49.7,
48.8, 42.8. ESI MS for [M + H]+ = 340.0 Da.
5,5-Dimethyl-1-pentyl-1,4,5,6-tetrahydropyrimidine-2-carbalde-
hyde Oxime Hydrochloride (15b). The title compound was obtained
1
in a manner similar to that used for 12a, as a yellow solid (35%). H
NMR (500 MHz, d6-DMSO) δ = 13.22 (br s, 1H), 9.98 (br s, 1H),
8.29 (s, 1H), 3.54−3.50 (m, 2H), 3.27 (br s, 2H), 3.06 (br s, 2H),
1.59−1.52 (m, 2H), 1.31−1.20 (m, 4H), 0.98 (s, 6H), 0.86 (t,J = 7.2
Hz, 3H). 13C NMR (125 MHz, d6-DMSO) δ = 152.2, 138.2, 56.7,
51.4, 48.9, 27.7, 27.2, 25.9, 23.1, 21.6, 13.7. ESI MS for [M + H]+ =
226.2 Da.
Enzyme Studies. Recombinant AChE, β-lactoglobulin from
bovine milk (BLG), acetylthiocholine (ATC), butyrylthiocholine
(BTC), and 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) were pur-
chased from Sigma Aldrich Chemical Co. (St. Louis, MO).
Recombinant monomeric hBChE was expressed and purified from
mammalian cells as previously described.18 Syntheses and purification
methods of OP model compounds were previously reported.15,16
Reactivation studies were conducted using the same method for both
AChE and BChE enzymes. ChE enzyme was diluted in PBS/BLG (pH
7.4), and a sample was taken and set aside for noninhibitor-treated
controls. The remaining portion was incubated with a nerve agent
model compound or ETP (Figure 7) for sufficient time to achieve 90%
or greater inhibition (i.e., ca. 15 min). The reaction of the enzyme with
nerve agent model compounds afforded covalent modification of the
enzyme identical to that obtained with authentic nerve agents.15,16 An
excess of OP was removed from the inhibited enzyme by filtration
through a 10 kDa MWCO filter with a modified PES membrane
(VWR Scientific, Inc., San Diego, CA), followed by two washes prior
to the final resuspension in PBS/BLG (pH 7.4). A 1000-fold excess of
dilution of the OP was thus achieved. ChE enzyme was then added to
PBS/BLG (pH 7.4) containing amidine-oxime, MINA, or 2-PAM (100
μM oxime with 2% DMSO after addition of enzyme) or vehicle.
Oxime samples were equilibrated at 37 °C prior to the addition of
enzyme. Enzyme was allowed to reactivate at 37 °C for 20 min
(BChE) or 1 h (AChE), at which time the catalytic activity was
determined using a modified Ellman’s assay19 in the presence of 1 mM
substrate in PBS/BLG (pH 7.4). The Ellman assay utilized a 20-fold
dilution of enzyme from the oxime incubation, yielding incubation
concentrations in the range of 10 to 50 units L−1 (where 1 unit cleaves
1 μmol of substrate per min in PBS pH 7.4, room temperature) and 5
μM oxime. Two reactivation samples were prepared per oxime. The
reported data show the average esterase activity for each oxime divided
by the average functional activity of noninhibited control samples, and
the propagated errors of the average functional activities.
1-Butyl-1,4,5,6-tetrahydropyrimidine-2-carbaldehyde Oxime Hy-
drochloride (12e). The title compound was obtained in a manner
1
similar to that used for 12a, as a pink solid (28%). H NMR (300
MHz, d6-DMSO) δ = 13.06 (s, 1H), 9.69 (br s, 1H), 8.27 (s, 1H),
3.56−3.48 (m, 4H), 3.30−3.36 (m, 2H), 1.96−1.88 (m, 2H), 1.58−
1.48 (m, 2H), 1.31−1.19 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H). 13C NMR
(125 MHz, d6-DMSO) δ = 153.0, 138.5, 51.3, 46.3, 38.1, 29.6, 18.9,
18.3, 13.5. ESI MS for [M + H]+ = 184.1 Da.
1-Pentyl-1,4,5,6-tetrahydropyrimidine-2-carbaldehyde Oxime
Hydrochloride (12f). The title compound was obtained in a manner
1
similar to that used for 12a, as a brown oil (42%). H NMR (500
MHz, d6-DMSO) δ = 13.20 (s, 1H), 9.90 (br s, 1H), 8.27 (s, 1H),
3.553.50 (m, 4H), 3.35−3.30 (m, 2H), 1.96−1.90 (m, 2H), 1.59−1.53
(m, 2H), 1.31−1.17 (m, 4H), 0.86 (t, J = 7.2 Hz, 3H). 13C NMR (125
MHz, d6-DMSO) δ = 152.9, 138.4, 51.5, 46.3, 38.1, 27.7, 27.2, 21.7,
18.3, 13.7. ESI MS for [M + H]+ = 198.0 Da.
1-(2,4-Dichlorobenzyl)-1,4,5,6-tetrahydropyrimidine-2-carbalde-
hyde Oxime Hydrochloride (12h). The title compound was obtained
1
in a manner similar to that used for 12a, as a yellow solid (21%). H
NMR (300 MHz, d6-DMSO) δ = 13.10 (s, 1H), 10.19 (br s, 1H), 8.26
(s, 1H), 7.72 (d, J = 2.1 Hz, 1H), 7.52−7.40 (m, 2H), 4.90 (s, 2H),
3.48−3.35 (m, 4H), 2.02−1.92 (m, 2H). 13C NMR (125 MHz, d6-
DMSO) δ = 154.2, 138.8, 133.5, 133.3, 131.1, 130.4, 129.2, 127.8,
52.5, 46.6, 38.3, 18.1. ESI MS for [M + H]+ = 285.9 Da.
1-(3,5-Bis(trifluoromethyl)benzyl)-1,4,5,6-tetrahydropyrimidine-
2-carbaldehyde Oxime Hydrochloride (12i). The title compound was
obtained in a manner similar to that used for 12a, as a yellow solid
(63%). 1H NMR (300 MHz, d6-DMSO) δ = 13.05 (s, 1H), 10.11 (br
s, 1H), 8.35 (s, 1H), 8.11−8.06 (m, 3H), 5.02 (s, 2H), 3.49−3.32 (m,
4H), 1.99−1.85 (m, 2H). 13C NMR (125 MHz, d6-DMSO) δ = 154.3,
139.2, 138.5, 130.6 (q, J = 32.9 Hz), 128.8, 123.2 (q, J = 271.3 Hz),
121.8, 53.7, 46.5, 38.3, 18.1. ESI MS for [M + H]+ = 354.0 Da.
5,5-Dimethyl-2-(nitromethylene)hexahydropyrimidine (13). The
title compound was obtained in a manner similar to that used for 9
In Vivo Studies. Adult Swiss Webster female mice (from Taconic)
were housed in groups of four and maintained in a temperature-
controlled environment on a 12 h/12 h light cycle (0600 h on−1800 h
off) upon arrival to the laboratory. Animals were given free access to
food and water during a one-week habituation period to the
laboratory. Animals used in the research studies were handled, housed,
and euthanized in accordance with a current HBRI IACUC protocol
and NIH guidelines regarding the use and care of laboratory animals,
and all applicable local, state, and federal regulations and guidelines.
Each test compound, MINA, and 2-PAM were dissolved in isotonic
saline and administered to separate groups of mice in a volume of 0.25
mL/mouse. The survival rate was recorded after 24 h from inititating
an experiment.
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using 2,2-dimethylpropane-1,3-diamine, as a yellow solid (92%). H
NMR (500 MHz, d6-DMSO) δ = 8.91 (br s, 2H), 6.27 (s, 1H), 2.99−
2.98 (m, 4H), 0.95 (s, 6H). ESI MS for [M + H]+ = 171.7 Da.
1-Butyl-5,5-dimethyl-2-(nitromethylene)hexahydropyrimidine
(14a). The title compound was obtained in a manner similar to that
used for 11a, as an orange solid (47%). 1H NMR (500 MHz, CDCl3)
δ = 10.80 (br s, 1H), 6.66 (s, 1H), 3.15−3.11 (m, 2H), 3.07−3.06 (m,
2H), 3.02 (br s, 2H), 1.59−1.53 (m, 2H), 1.36−1.27 (m, 2H), 1.06 (s,
6H), 0.94 (t, J = 7.3 Hz, 3H). ESI MS for [M + H]+ = 227.7 Da.
5,5-Dimethyl-2-(nitromethylene)-1-pentylhexahydropyrimidine
(14b). The title compound was obtained in a manner similar to that
1
used for 11a using 1-iodopentane, as a yellow solid (71%). H NMR
Sp-GB-Am Studies. At the 0 min time point, mice received Sp-
(500 MHz, CDCl3) δ = 10.79 (br s, 1H), 6.65 (s, 1H), 3.14−3.11 (m,
GB-Am hydrochloride (0.08 mg/kg in 0.25 mL/mouse in saline, i.p.).
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dx.doi.org/10.1021/jm201364d | J. Med. Chem. 2012, 55, 465−474