Month 2014
Design, Synthesis, and Biological Evaluations of Several Y-26732 Analogues
NMR (CDCl3, 600 MHz) δ: 10.02 (s, 1H), 8.56 (d, J = 6.2 Hz,
2H), 7.63 (d, J = 6.2 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 1.38
(t, J = 7.1 Hz, 3H). 13C NMR (CDCl3, 150 MHz) δ: 154.3, 150.2,
147.3, 113.3, 61.8, 14.8.
As seen in Figure 9, BV2 cells were chosen for
further testing, and the cytotoxicity of these compounds
(1, 5, 15, and 50 μg/mL) was tested. Compound 4 has
the lowest cytotoxicity, and Fasudil has the highest.
The rest of the compounds show no obvious toxicity.
Compounds 3 and 4 in high concentration (50 μg/mL)
could reduce the cytotoxicity.
The results of the above study provide evidence of the
neuroprotective effects of these Rho kinase inhibitors.
Compounds 3 and 4 modulate the neurite growth and
protect neurons from cytotoxicity-induced cell death.
Compound (1a) was added into a solution of L-prolinol in
1,4-dioxane with N-methylpyrrolidine as catalyst. The mixture
was reflux for 24h, and then the residue was concentrated. The
residue was purified by silica gel column (ethyl acetate/methanol=
5/1, v/v) to afford (1) 2.23g, 40.7% yield. Mp: 154.5–155.1°C, 1H
NMR (CDCl3, 600 MHz) δ: 8.16 (d, J = 6.0 Hz, 2H), 7.28
(d, J= 6.0 Hz, 2H), 3.97 (s, 1H), 3.58–3.69 (m, J= 121.5 Hz, 5H),
1.77–2.02 (m, 4H), 1.57 (s, 1H). 13C NMR (CDCl3, 150 MHz) δ:
155.77, 149.20, 147.99, 113.07, 66.77, 60.11, 47.16, 29.18, 23.55.
HR-MS (ESI), calcd C11H15N3O2: [M+ H]+ m/z: 221.1243; found:
221.1246.
CONCLUSION
Compounds 2 and 3 were synthesized by the same method.
In summary, we successfully synthesized four pyridine
derivatives. Their biological activities were evaluated,
including Rho kinase inhibitory activity, cell regeneration
and synapse formation, cell viability, and cytotoxicity by the
MTT and LDH methods. Compound 4 exhibited higher inhib-
itory activities of Rho kinase than Fasudil and modulated
neurite growth and protect neurons from cytotoxicity-induced
cell death, and compound 3 was less effective than compound
4. Observing the structures of compounds 1, 2, 3, and 4, if the
amino group is a binding site, compounds 3 and 4 is less
stereo-hindrance to facilitate a combination with the Rho
kinase and the nucleophilic ability of amino group is higher
than the hydroxyl group. Thus, it is inferred that electron
effects or steric effects may be the crucial factors to affect
the results. The activity of compounds 1 and 2 are different
possibly because of the chirality of binding sites, and
compound 1 is better than 2 possibly because majority of
the proteins in the body is in L-configuration. Further studies
are necessary to clarify the inference. Compound 4 will be
under further study as a drug candidate.
(R)-2-(Hydroxymethyl)-N-(pyridin-4-yl)pyrrolidine-1-
carboxamide (2).
40.6% yield, mp: 154.1–155.7°C, 1H NMR
(CDCl3, 600 MHz) δ: 8.13 (d, J= 6.1 Hz, 2H), 7.26 (d, J=6.1Hz,
2H), 3.94 (m, 1H), 3.71–3.47 (m, 4H), 3.28 (s, 1H), 1.95–1.74 (m,
4H), 1.54 (s, 1H). 13C NMR (CDCl3, 150 MHz) δ: 155.71, 149.03,
148.15, 113.07, 66.73, 60.11, 47.14, 29.19, 23.52. HR-MS (ESI),
calcd C11H15N3O2: [M+H]+ m/z: 221.1243; found: 221.1246.
N-(Pyridin-4-yl)piperazine-1-carboxamide (3).
51.1%
yield, mp: 196.3–197.1°C, 1H NMR (CD3OD, 600 MHz) δ:
8.24 (d, J = 6.0 Hz, 2H), 7.48 (d, J = 6.0 Hz, 2H), 3.51 (s, 4H),
2.83 (s, 4H). 13C NMR (CD3OD, 150 MHz) δ: 156.47, 150.28,
149.94, 114.96, 46.31, 45.85. HR-MS (ESI), calcd
C10H15ClN4O: [M + H]+ m/z: 207.1246; found: 207.1252.
(R)-N-(Pyridin-4-yl)pyrrolidine-2-carboxamide (4).
The
solution of di-tert-butyl dicarbonate (9.09 g, 41.69 mmol) in THF
(30 mL) was added to a mixture of L-proline (4.00 g, 34.74 mmol)
and sodium hydroxide solution (2 mol/L, 30 mL) at 0°C. The
mixture was stirred at room temperature for 12 h and then
concentrated. The rest of the reaction mixture was adjusted to
pH = 2–3 by 5% hydrochloric acid solution. Then, the aqueous
phase was extracted by ethyl acetate (50 mL × 3). The ethyl acetate
phases were combined, dried over MgSO4, and concentrated to
give Boc-L-proline 7.10 g, 95.0% yield.
The solution of Boc-L-proline (7.10 g, 33.00 mmol) and
triethylamine (3.34 g, 33.00 mmol) in dry THF (100 mL) was
cooled to 0°C. Under stirring, ethyl chloroformate (3.58 g,
33.00 mmol) was introduced into the solution in 15 min. After
the mixture had been stirred for a further 30 min at 0°C,
4-aminopyridine (3.10 g, 33.00 mmol) was added slowly over
15 min. This reaction mixture was stirred for 1 h at 0°C, 16 h at
room temperature, and heated at reflux for 3 h. The mixture was
subsequently diluted with ethyl acetate and triethylamine and then
filtrated. The resulting residue was dissolved in ethyl acetate,
washed with saturated aqueous ammonium chloride, dried over
MgSO4, filtered, and concentrated to give the compound 8.17 g
yellow liquid (4c), 72.9% yield. Trifluoroacetic acid (10 mL)
was added dropwise to the compound (4c) in dichloromethane
(20 mL) at 0°C. The mixture was stirred at room temperature for
12h, then adjusting to pH = 12–13 by 2 mol/L sodium hydroxide
solution. Then, the aqueous phase was extracted by
dichloromethane (50mL × 3). The dichloromethane phases were
combined, dried over MgSO4, and concentrated to give 5.14 g
yellow solid (4), 95.7% yield. mp: 209.1–209.5°C, 1H NMR
(CD3OD, 600 MHz) δ: 8.39 (d, J= 6.6Hz, 2H), 7.68 (d, J=6.6Hz,
EXPERIMENTAL
All reagents were used as purchased from commercial suppliers
without further purification unless otherwise noted. Solvents were
dried and purified according to standard procedures before use. The
course of reactions was monitored by TLC (silica gel GF254s). Flash
1
chromatography was performed using 200–300-mesh silica gel. H
and 13C NMR spectra were recorded on an INOVA 400/600-Hz
spectrometer with TMS as an internal standard. HR-MS was recorded
on MicrOTOF-Q II (Bucker Daltonics Inc., Billerica, MA).
(S)-2-(Hydroxymethyl)-N-(pyridin-4-yl)pyrrolidine-1-
carboxamide (1).
To a solution of 4-aminopyridine (3.76 g,
39.95 mmol) in CH2Cl2 (50 mL) at 0°C was added triethylamine
(4.85 g, 47.94 mmol) and ethyl chloroformate (4.34 g,
39.95 mmol). The resulting mixture was allowed to warm to room
temperature overnight and then was concentrated. The solid
products were slurried with water. The reaction mixture was
adjusted to pH= 5–6 by 5% hydrochloric acid. Then, the aqueous
phase was extracted by ethyl acetate (50 mL × 3). The ethyl
acetate phases were combined, dried over MgSO4, and
concentrated to afford 5.80g white solid (1a), 87.3% yield. 1H
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet