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56.3 ppm; HRMS (ESI) m/z calcd for C18H16N5O+: 318.1349 [M+H]+,
found: 318.1351; IR (neat): n˜ =3104, 1684, 1653, 1559, 1517, 1457,
1254, 1201, 1130 cmꢀ1. Note: The formation of the intermediate
27a was confirmed based on ESI-MS of the reaction mixtures: m/z
calcd for C12H14N3O2+: 232.1 [M+H]+, found: 232.1.
tives of ageladine A. Out of the 20 compounds synthesized
using this facile cascade, several of them notably modulated
the neural-cell differentiation to neurons even at 10 nm, with-
out exhibiting an astrocyte-differentiation activity. Consequent-
ly, we have successfully transformed ageladine A into a new
class of compounds that are potent and selective in modulat-
ing the neural differentiation. We believe that our new com-
pounds could contribute to the mechanistic understanding of
the neural differentiation and for the therapeutic development
aimed at neurologically related diseases.
One-pot synthesis of ageladine A (1): To a solution of 2,4-dinitro-
phenylguanidine (28, 30.0 mg, 0.133 mmol) obtained above in
DMF (670 mL), the dialdehyde 9 (56.0 mg, 0.666 mmol) was added.
After stirring at room temperature for 10 min, NH4OAc (30.8 mg,
0.666 mmol), AcOH (150 mL), and 3,4-dibromopyrrole-2-carboalde-
hyde (13b, 33.7 mg, 0.133 mmol) were added to the mixture,
which was stirred at 758C for 30 min. After the mixture was cooled
down to room temperature, saturated NaHCO3 aq. (1.0 mL) and 2-
mercaptehanol (330 mL) were added to the solution and the result-
ing mixture was stirred for additional 1 h at room temperature.
After the mixture was concentrated in vacuo, the residue was puri-
fied by reversed-phase HPLC (20–60% of MeCN containing 0.1%
TFA over 40 min) to give 1 as a white solid. Yield: 2.3 mg (5%);
1H NMR (500 MHz, CD3OD, 258C): d=8.05 (d, J=6.5 Hz, 1H), 7.42
(d, J=6.5 Hz, 1H), 7.17 ppm (s, 1H); 13C NMR (125 MHz, CD3OD,
258C): d=160.8, 147.1, 136.6, 133.0, 128.5, 125.6, 115.2, 107.8,
105.4, 102.3 ppm; HRMS (ESI) m/z calcd for C10H8Br2N5+: 355.9141
[M+H]+, found: 355.9142; IR (neat): n˜ =3350, 1653, 1559, 1436,
1200, 1139, 840 cmꢀ1. Note: Before the one-pot thiolysis of the di-
nitrophenyl protecting group, the successful generation of the in-
termediate 30 from the reaction mixture was analyzed and identi-
fied by reversed-phase HPLC (20–60% of MeCN containing 0.1%
Experimental Section
General procedure for the one-pot synthesis of N1-aryl-2-amino-
imidazoles: Exemplary shown for the preparation of 2-(2-amino-1-
phenyl-1H-imidazol-5-yl)acetic acid (24a (=20b), Table 1, meth-
od B; for further details see the Supporting Information). To a solu-
tion of aniline (21a, 46 mL, 0.50 mmol) and cyanamide (63.1 mg,
1.50 mmol) in MeOH (0.50 mL), conc. HCl (125 mL, 1.50 mmol) was
added at room temperature. After the solution was stirred under
reflux for 12 h, the resulting mixture was cooled down to room
temperature. The crude guanidine 22a was then neutralized by
NaOMe (81.0 mg, 1.50 mmol) in MeOH (2.0 mL) and treated with
methyl fumaraldehydate (18, 57.1 mg, 0.500 mmol). After the solu-
tion was stirred at room temperature for 24 h, NaOH aq. (1.0 mL,
1.0m) was added to the solution that was subsequently stirred for
1 h. The resulting mixture was washed with CHCl3 (3ꢁ30 mL). After
the aqueous layer was neutralized by HCl aq. (1.0m) and washed
again with CHCl3 (3ꢁ30 mL), the aqueous layer was directly passed
through a column filled with a positive ion-exchange resin that
was eluted with HCl aq. (0.1m) and concentrated in vacuo. After
the HCl salt of 24a was dissolved in water, the solution was again
passed through a column filled with a negative ion-exchange resin
that was eluted with a 3% ammonium solution, to give 24a as
a yellow solid. Yield: 97 mg (90%); 1H NMR (400 MHz, CD3OD,
258C): d=7.61–7.58 (m, 3H), 7.47–7.44 (m, 2H), 6.77 (s, 1H),
3.20 ppm (s, 2H); 13C NMR (100 MHz, CD3OD, 258C): d=175.8,
148.5, 133.3, 131.7, 131.5, 129.5, 127.2, 111.9, 33.5 ppm; HRMS (ESI):
m/z calcd for C11H12N3O2+: 218.0924 [M+H]+, found: 218.0924; IR
(neat): n˜ =3337, 2360, 2340, 1734, 1684, 1653, 1559, 1539, 1506,
1
TFA over 40 min): H NMR (400 MHz, CD3OD, 258C): d=9.21 (d, J=
2.8 Hz, 1H), 8.86 (dd, J=8.8, 2.8 Hz, 1H), 8.18 (d, J=8.8 Hz, 1H),
8.12 (d, J=6.4 Hz, 2H), 7.34 (s, 1H), 7.23 ppm (d, J=6.4 Hz, 2H);
HRMS (ESI) m/z calcd for C16H9Br2N7O4+: 521.9156 [M+H]+, found:
521.9185.
In vitro preparation of neural stem cells: Neural stem cells (NSCs)
were obtained as described in ref. [40] and [41] with slight modifi-
cations. The J1 mouse ESC line was obtained from the American
Type Culture Collection (ATCC) and maintained on 0.1% gelatin-
coated dishes with mitomycin C-treated mouse embryonic fibro-
blasts (MEF, Kitayama Labes). The medium for culturing of the ESCs
consisted of Dulbecco’s modified Eagle medium (DMEM, Wako)
supplemented with 15% FBS (fetal bovine serum, Hyclone), 1% l-
glutamine (Gibco), 1% penicillin-streptomycin (P/S, Gibco), 1%
nonessential amino acids (Gibco), 0.18% 2-mercaptoethanol
(Gibco), and 1000 UmLꢀ1 leukemia inhibitory factor (LIF, Millipore).
The ES cells were differentiated into embryoid bodies (EBs) by the
hanging drop method (7500 cells per droplet of 20 mL) for 3 days.
The EBs were then transferred to nonadhesive bacterial dishes
(Thermo) with astrocyte-conditioned medium (ACM, Sumitomo Ba-
kelite) supplemented with 20 ngmLꢀ1 rhFGF-2 (recombinant
human basic fibroblast growth factor) and 20 ngmLꢀ1 rhEGF (re-
combinant human epidermal growth factor, R&D) for 4 days to
give rise to NSCs in the EBs. After that, the EBs were transferred to
matrigel (BD)-coated dishes in neurobasal medium (Gibco) supple-
mented with 2% B-27 supplement (Gibco), 1% P/S, 20 ngmLꢀ1
rhFGF-2, and 20 ngmLꢀ1 rhEGF. Ten days later, the NSCs that mi-
grated out of the EBs were collected and cryopreserved until use
for the neural differentiation assay.
1085 cmꢀ1
.
General procedure for the one-pot synthesis of the N1-sustitut-
ed ageladine A derivatives (3a–o): Exemplary shown for the prep-
aration of N1-(4-methoxyphenyl)-4-(pyridine-2-yl)-imidazo[4,5-c]pyr-
idin-2-amine (3a). To
a
solution of cyanamide (37.8 mg,
0.900 mmol) and 4-methoxyaniline (25a, 45.5 mg, 0.300 mmol) in
MeOH (0.30 mL), conc. HCl (75 mL) was added. After stirring under
reflux for 12 h, the solution was cooled down to room temperature
and neutralized with NEt3 (125 mL, 0.900 mmol). After adding the
dialdehyde 9 (50.0 mg, 0.600 mmol), the resulting mixture was
stirred at room temperature for 1 h and the pyridine 2-carboalde-
hyde 13a (28.3 mmol, 0.300 mmol) and NH4Cl (79.5 mg,
1.49 mmol) were sequentially added. After stirring under reflux for
additional 2 h, the resulting solution was concentrated in vacuo.
The residue was purified by the reversed-phase HPLC (20–60% of
MeCN containing 0.1% TFA over 40 min) to give 3a as a yellow
solid. Yield: 21 mg (22%); 1H NMR (500 MHz, CD3OD, 258C): d=
9.25 (d, J=8.0 Hz, 1H), 8.85 (d, J=4.5 Hz, 1H), 8.27 (d, J=6.0 Hz,
1H), 8.08 (dd, J=8.0, 8.0 Hz, 1H), 7.58 (dd, J=8.0, 4.5 Hz, 1H), 7.52
(d, J=9.0 Hz, 2H), 7.27–7.24 (m, 3H), 3.93 ppm (s, 3H); 13C NMR
(125 MHz, CD3OD, 258C): d=163.0, 162.8, 160.7, 150.8, 148.8,
139.0, 134.6, 134.2, 129.7, 126.8, 126.6, 125.6, 119.3, 117.0, 105.7,
Differentiation of neural stem cells: The cryopreserved neural
stem cells were seeded on a matrigel-coated 96 well plate or
a 6 cm dish with neurobasal medium supplemented with 2% B-
27 supplement, 1% P/S, 20 ngmLꢀ1 rhFGF-2, and 20 ngmLꢀ1 rhEGF.
After culturing for three days, fresh medium without rhFGF-2 and
rhEGF was added to induce the selective neural differentiation
(neurobasal medium with 2% B-27 supplement and 1% P/S) or to
Chem. Eur. J. 2016, 22, 1 – 11
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