Design, synthesis, and biological evaluation of pyrimidine-2-carboxamide analogs: Investigation for novel RAGE inhibitors with reduced hydrophobicity and toxicity

Article abstract of DOI:10.1007/s12272-015-0596-5

This paper describes an investigation of novel RAGE inhibitors with improved drug-like properties. To identify the improved drug-like RAGE inhibitor, we designed and synthesized pyrimidine-2-carboxamide analogs based on our previous work. Several potent a

Full text of DOI:10.1007/s12272-015-0596-5

Arch. Pharm. Res.
DOI 10.1007/s12272-015-0596-5
RESEARCH ARTICLE
Design, synthesis, and biological evaluation of pyrimidine-2-
carboxamide analogs: investigation for novel RAGE inhibitors
with reduced hydrophobicity and toxicity
Seok-Ho Kim1 Young Taek Han2
•
Received: 13 January 2015 / Accepted: 1 April 2015
ꢀ The Pharmaceutical Society of Korea 2015
Abstract This paper describes an investigation of novel
RAGE inhibitors with improved drug-like properties. To
identify theimproved drug-like RAGE inhibitor, we designed
and synthesized pyrimidine-2-carboxamide analogs based on
our previous work. Several potent analogs with improved
hydrophilicity were identified by evaluation of RAGE in-
hibitory activity. In particular, one of the potent (diethy-
lamino)ethoxymethoxy analogs did not exhibit undesired
cytotoxicity in contrast with the parent RAGE inhibitors.
inflammatory diseases (Alexiou et al. 2010; Barlovic et al.
2011). In the brain of AD patients, RAGE interacts with
cytotoxic amyloid-b (Ab), a major component of patho-
logical plaques. It is a prime suspect of AD, and subsequently
induces the inflammatory events. In addition, RAGE at the
blood–brain barrier (BBB) is predominantly responsible for
the plasma-derived Ab influx into the brain (Schmidt et al.
2009). In this context, inhibition of the RAGE-Ab interac-
tion can reduce the accumulation rate of Ab into the brain,
neurotoxic inflammation, and consequent neuronal cell
death. For this reason, RAGE inhibitor has been regarded as
one of promising ‘‘disease-modifying agents’’ for AD
treatment (Deane et al. 2012).
Keywords RAGE Á RAGE inhibitor Á Pyrimidine-2-
carboxamide Á Drug-like property
Recently, we worked on the development of small-mole-
cule RAGE inhibitors, and reported a novel series of RAGE
inhibitors. A series of 2-aminopyrimidines including bis(4-
chlorophenyl)pyrimidine 1, which exhibited significant
therapeutic effects in an AD model study, as well as potent
in vitro RAGE inhibitory activity (IC50 = 16.5 lM), were
designed on the structural basis of one of the monomeric
AGEs (Han et al. 2012). Based on this previous study, we
identified a series of pyrazole-5-carboxamide analogs
through heterocycle modification and a subsequent structure–
activity relationship (SAR) study. Moreover, pyrazole-5-
carboxamide 2 exhibited more potent RAGE inhibitory ac-
tivity (IC50 = 1.9 lM), with improved aqueous solubility,
than 2-aminopyrimidine 1 (Han et al. 2014). Although such
significant anti-AD activities were observed, the hit com-
pounds 1 and 2 seemed to have considerable drawbacks as a
drug candidate, such as unsatisfactory pharmacokinetic pro-
files and undesired cytotoxicity (cell survival: 37.3 and
10.3 %, respectively, HT22 cells). We supposed that their
excessive hydrophobicities (ClogP = 9.21 and 7.95, re-
spectively. Calculated by ChemBioDraw Ultra 12.0) were
strongly related to the problems by inducing poor aqueous
Introduction
Receptor for advanced glycation end product (RAGE) is a
multi-ligand binding transmembrane receptor of the im-
munoglobulin superfamily. In chronic inflammatory disease
conditions, such as diabetes, atherosclerosis and Alzhei-
mer’s disease (AD), RAGE interactswith various pathogenic
ligands and activates nuclear factor-jB (NF-jB), a tran-
scription factor that plays a crucial role in various inflam-
matory responses (Bierhaus et al. 2005). Usually, RAGE is
over-expressed in chronic inflammatory diseases through
positive feedback loops (Li and Schmidt 1997). Therefore,
RAGE has been regarded as a therapeutic target for chronic
& Young Taek Han
hanyoungtaek@gmail.com
1
College of Pharmacy, CHA University, Pochen-Si,
Gyeonggi-do 487-010, Korea
2
College of Pharmacy, Dankook University,
Cheonan 330-714, Korea
123

S.-H. Kim et al.
solubility and immoderate non-specific protein binding
(Kerns and Di 2008). For these reasons, we turned our at-
tention to more hydrophilic analogs to identify novel RAGE
inhibitors with improved drug-like properties.
purification. Tetrahydrofuran was distilled from sodium
benzophenone ketyl. Dichloromethane was freshly distilled
from calcium hydride. All solvents used for routine product
isolation and chromatography were of reagent grade and
glass distilled. Reaction flasks were dried at 100 ꢁC before
use, and air and moisture sensitive reactions were per-
formed under argon. Flash column chromatography was
performed using silica gel 60 (230–400 mesh, Merck) with
the indicated solvents. Thin-layer chromatography was
performed using 0.25 mm silica gel plates (Merck). Mass
spectra were obtained using a VG Trio-2 GC–MS instru-
ment, and high resolution mass spectra were obtained using
To design the more hydrophilic analogs, we planned to
make use of our previous studies on 2-aminopyrimidines
and pyrazole-5-carboxamides, including information from
SAR analysis and molecular docking studies (Han et al.
2012, 2014). As shown in Fig. 1, we designed pyrimidine-
2-carboxamide analogs 3 by replacing the chloride and
2-amino moieties of the 2-aminopyrimidine 1 with the
more polar fluoride and carboxamide moieties of the 2,
respectively. By considering a molecular docking study of
the more active analog 2, we anticipated that these two
moieties would provide an improvement in RAGE in-
hibitory activities as well as hydrophilic properties. We
introduced a polar methoxy moiety to the pyrimidine-2-
carboxamide analogs, with the expectation of additional
interaction with RAGE and increasing hydrophilicity. In
addition, the positional effect of the (diethylamino)ethoxy
side chain and methoxy moiety was intensively examined
based on the previous observation that RAGE inhibitory
activities of the 2-aminopyrimidine analogs were strongly
influenced by the position of the alkoxy side chain. Herein,
we report design, synthesis and biological evaluation of the
pyrimidine-2-carboxamide analogs, an exploration for
novel RAGE inhibitors with improved drug-like properties.
1
a JEOL JMS-AX 505WA unit. H and 13C spectra were
recorded on a JEOL JNM-LA 300, Brucker Analytik
ADVANCE digital 400, ADVANCE digital 500 or JEOL
ECA-600 spectrometer in deuteriochloroform (CDCl3),
deuterioacetone (aetone-d6), deuteriodimethylsulfoxide
(DMSO-d6) or deuteriomethanol (CD3OD). Chemical
shifts are expressed in parts per million (ppm, d) downfield
from tetramethylsilane and are referenced to the deuterated
1
solvent. H-NMR data are reported in the order; chemical
shift, multiplicity (s, singlet; bs, broad singlet; d, doublet; t,
triplet; q, quartet; m, multiplet, and/or multiple resonance,
numbers of protons, and coupling constants in hertz (Hz).
General procedure A: synthesis of the carboxamide
analogs
To a solution of carboxylic acid (1 equiv) in CH2Cl2 were
added catalytic amount of DMF and oxalyl chloride (5 equiv)
at 0 ꢁC. After being stirred for 1 h at same temperature, the
reaction mixture was concentrated in vacuo and diluted with
THF. To the solution of the acid chloride were added cor-
responding aniline (1 equiv) and Et3N (5 equiv), and stirred
for 1 h at ambient temperature. The reaction mixture was
Material and method
General procedure
Unless noted otherwise, all starting materials and reagents
were obtained commercially and were used without further
Fig. 1 Design strategy for
pyrimidine-2-carboxamide
analogs
123

Design, synthesis, and biological evaluation of pyrimidine-2-carboxamide analogs…
quenched with H2O, and diluted with EtOAc. The organic
phase was washed with water and brine, dried over MgSO4,
and concentrated in vacuo. Purification of the residue via
flash column chromatography on silica gel (MeOH/CH2-
Cl2 = 1:10–20) afforded the titled analog.
was added 1,4-diazabicyclo[2.2.2]octane (DABCO,
446 mg, 9.68 mmol) at ambient temperature. After being
stirred for 5 h at same temperature, the reaction mixture was
diluted with EtOAc and water. The organic phase was
washed with water and brine, dried over MgSO4, and con-
centrated in vacuo. Purification of the residue via flash col-
umn chromatography on silica gel (EtOAc/n-hexane = 1:3)
afforded 1.67 g (88 %) of the carbonitrile 6: 1H-NMR
(300 MHz, CD3OD) d 8.61 (s, 1H), 8.40-8.37 (m, 4H),
7.34-7.30 (m, 4H); LRMS (FAB) m/z 294 (M ? H?).
General procedure B: Mitsunobu reaction of the phenols
To a solution of phenol (1 equiv), triphenylphosphine (1.3
equiv) and 2-(diethylamino)ethanol (1.3 equiv) in THF was
added diisopropyl azodicarboxylate (DIAD, 1.3 equiv) at
ambient temperature. After being stirred at ambient tem-
perature until no starting material could be observed by
TLC, the reaction mixture was quenched with H2O and
diluted with EtOAc. The organic layer was washed with
water and brine, dried over MgSO4, and concentrated in
vacuo. Purification of the residue via flash column chro-
matography on silica gel afforded the titled compound.
4,6-bis(4-fluorophenyl)pyrimidine-2-carboxylic acid (7)
To a solution of the pyrimidine-2-carbonitrile 6 (1.67 g,
5.69 mmol) in 50 % aq. EtOH (80 mL) was added KOH
(3.19 g, 56.9 mmol) at ambient temperature. After being
stirred under reflux condition for 8 h, the reaction mixture
was concentrated in vacuo and diluted with EtOAc. The
organic phase was acidified with 1 N HCl, and washed
with water and brine. The organic phase was dried over
MgSO4, and concentrated in vacuo. The crude pyrimidine-
2-carboxylic acid 7 (1.56 g, 88 %) was used for further
reaction without purification: 1H-NMR (500 MHz, DMSO-
d6) d 8.75 (s, 1H), 8.52-8.49 (m, 4H), 7.47-7.43 (m, 4H);
LRMS (FAB) m/z 313 (M ? H?).
General procedure C: reduction of nitrobenzenes
To a solution of nitrobenzene (1 equiv) in EtOH was added
SnCl2Á2H2O (5 equiv) at ambient temperature. The reaction
mixture was refluxed for 3 h and concentrated in vacuo. The
residue was diluted with EtOAc and saturated NaHCO3 so-
lution was added. The mixture was filteredusinga Celite pad.
The organic phase was washed with water and brine, dried
over MgSO4 and concentrated in vacuo. Purification of the
residue via flash column chromatography on silica gel af-
forded titled aniline as a brown oil.
N-(2-(2-(Diethylamino)ethoxy)phenyl)-4,6-bis(4-
fluorophenyl)pyrimidine-2-carboxamide (9a)
The acid 7 (17 mg, 0.054 mmol) afforded the carboxamide
9a (6.8 mg, 25 %) via general procedure A: 1H-NMR
(400 MHz, CD3OD) d 8.46 (d, 1H, J = 7.8 Hz), 8.38-
8.35 (m, 5H), 7.25 (t, 4H, J = 8.6 Hz), 7.10 (t, 1H,
J = 7.7 Hz), 7.02 (d, 1H, J = 8.0 Hz), 6.96 (t, 1H,
J = 7.6 Hz), 4.19 (t, 2H, J = 6.3 Hz), 2.98 (t, 2H,
J = 6.3 Hz), 2.58 (q, 4H, J = 7.1 Hz), 0.95 (t, 6H,
J = 7.1 Hz); 13C-NMR (CDCl3, 75 MHz) d 166.7, 164.5,
164.5, 163.4, 159.8, 158.4, 147.7, 132.4, 132.3, 129.7,
129.7, 129.6, 129.6, 127.6, 124.3, 121.5, 119.9, 116.3,
116.3, 116.1, 116.1, 112.7, 111.2, 67.5, 52.2, 48.1, 48.1,
12.1, 12.1; LRMS (FAB) m/z 503 (M ? H?); HRMS
(FAB) calcd for C29H29F2N4O2 (M ? H?): 503.2259;
found 503.2266.
2-Chloro-4,6-bis(4-fluorophenyl)pyrimidine (5)
To a solution of 2,4,6-trichloropyrimidine 4 (2.09 g,
11.4 mmol), 4-fluorophenylboronic acid (3.82 g, 27.3 m-
mol) and Na2CO3 (4.79 g, 57 mmol) in 50 % aq. THF
(110 mL) were added Pd(OAc)2 (128 mg, 0.57 mmol) and
PPh3 (598 mg, 2.28 mmol) at ambient temperature. After
being stirred under reflux condition until no starting ma-
terial could be observed by TLC, the reaction mixture was
cooled to ambient temperature and diluted with EtOAc.
The organic phase was washed with water and brine, dried
over MgSO4 and concentrated in vacuo. Purification of the
residue via flash column chromatography on silica gel
(EtOAc/n-hexane/CH2Cl2 = 1:40:5)
afforded
1.95 g
N-(3-(2-(Diethylamino)ethoxy)phenyl)-4,6-bis(4-
(57 %) of the chloropyrimidine 5: 1H-NMR (300 MHz,
CDCl3) d 8.17 - 8.12 (m, 4H), 7.91 (s, 1H), 7.20-7.18
(m, 4H); LRMS (FAB) m/z 303 (M ? H?).
fluorophenyl)pyrimidine-2-carboxamide (9b)
The acid 7 (15 mg, 0.047 mmol) afforded the carboxamide
9b (12 mg, 51 %) via general procedure A: 1H-NMR
(300 MHz, CD3OD) d 8.37-8.33 (m, 4H), 8.31 (s, 1H),
7.53 (t, 1H, J = 2.1 Hz), 7.33 (m, 1H), 7.26-7.16 (m, 5H),
6.72 (ddd, J = 1.1, 2.6, 8.3 Hz), 4.09 (t, 2H, J = 5.7 Hz),
2.92 (t, 2H, J = 5.7 Hz), 2.70 (q, 4H, J = 7.2 Hz), 1.11 (t,
4,6-bis(4-fluorophenyl)pyrimidine-2-carbonitrile (6)
To a solution of the 2-chloropyrimidine 5 (1.95 g, 6.45 m-
mol) and NaCN (464 mg, 9.68 mmol) in DMSO (45 mL)
123

S.-H. Kim et al.
6H, J = 7.2 Hz); 13C-NMR (CDCl3, 75 MHz) d 166.7,
164.5, 164.5, 163.3, 159.9, 159.5, 157.9, 138.6, 132.1,
132.1, 129.8, 129.7, 129.7, 129.6, 129.6, 116.4, 116.4,
116.1, 116.1, 113.0, 112.0, 111.3, 106.0, 66.4, 51.6, 47.8,
47.8, 11.8, 11.8; LRMS (FAB) m/z 503 (M ? H?); HRMS
(FAB) calcd for C29H29F2N4O2 (M ? H?): 503.2259;
found 503.2266.
2.92 (t, 2H, J = 6.1 Hz), 2.63 (q, 4H, J = 7.1 Hz), 1.05 (t,
6H, J = 7.1 Hz); LRMS (FAB) m/z 269 (M ? H?).
N,N-Diethyl-2-(2-methoxy-6-nitrophenoxy)ethanamine
(11d)
Nitrophenol 10d (254 mg, 1.50 mmol) afforded the etha-
1
namine 11d (282 mg, 70 %) via general procedure B: H-
N-(4-(2-(Diethylamino)ethoxy)phenyl)-4,6-bis(4-
NMR (300 MHz, CDCl3) d 7.86 (dd, 1H, J = 2.8, 9.0 Hz),
7.70 (d, 1H, J = 2.6 Hz), 6.89 (d, 1H, J = 8.8 Hz), 4.14 (t,
2H, J = 6.6 Hz), 3.90 (s, 3H), 2.92 (t, 2H, J = 6.6 Hz),
2.61 (q, 4H, J = 7.1 Hz), 1.04 (t, 6H, J = 7.1 Hz); LRMS
(FAB) m/z 269 (M ? H?).
fluorophenyl)pyrimidine-2-carboxamide (9c)
The acid 7 (10 mg, 0.032 mmol) afforded the carboxamide
9c (8 mg, 47 %) via general procedure A: 1H-NMR
(300 MHz, CD3OD) d 8.43-8.38 (m, 4H), 8.37 (s, 1H),
7.72 (d, 2H, J = 9.2 Hz), 7.26-7.20 (m, 4H), 6.93 (d, 2H,
J = 9.2 Hz), 4.09 (t, 2H, J = 5.7 Hz), 2.94 (q, 4H,
J = 7.1 Hz), 2.72 (q, 4H, J = 7.1 Hz), 1.12 (t, 6H,
J = 7.1 Hz); 13C-NMR (CDCl3, 100 MHz) d 166.3, 164.5,
164.5, 163.8, 159.7, 158.1, 155.9, 132.2, 132.2, 130.7,
129.7, 129.7, 129.6, 129.6, 121.3, 121.3, 116.4, 116.4,
116.2., 116.2, 115.0, 115.0, 112.9, 66.6, 51.7, 47.8, 47.8,
11.7, 11.7; LRMS (FAB) m/z 503 (M ? H?); HRMS
(FAB) calcd for C29H29F2N4O2 (M ? H?): 503.2259;
found 503.2266.
2-(2-(Diethylamino)ethoxy)-6-methoxyaniline (12a)
Ethanamine 11a (262 mg, 0.98 mmol) afforded the aniline
12a (189, 87 %) via general procedure C: 1H-NMR
(300 MHz, CDCl3) d 6.66-6.61 (m, 1H), 6.53-6.52 (m,
1H), 6.50-6.49 (m, 1H), 4.07 (t, 2H, J = 6.1 Hz), 3.83 (s,
3H), 2.89 (t, 2H, J = 6.1 Hz), 2.65 (q, 4H, J = 7.1 Hz),
1.07 (t, 6H, J = 7.1 Hz); LRMS (FAB) m/z 239
(M ? H?).
2-(2-(Diethylamino)ethoxy)-5-methoxyaniline (12b)
N,N-Diethyl-2-(3-methoxy-2-nitrophenoxy)ethanamine
(11a)
Ethanamine 11b (298 mg, 1.11 mmol) afforded the aniline
1
12b (172 mg, 65 %) via general procedure C: H-NMR
Nitrophenol 10a (254 mg, 1.50 mmol) afforded the etha-
1
namine 11a (262 mg, 65 %) via general procedure B: H-
(300 MHz, CDCl3) d 6.71 (d, 1H, J = 8.6 Hz), 6.29 (d,
1H, J = 2.9 Hz), 6.19 (dd, 1H, J = 2.9, 8.6 Hz), 3.97 (t,
2H, J = 6.0 Hz), 3.70 (s, 3H), 2.81 (t, 2H, J = 6.0 Hz),
2.61 (q, 4H, J = 7.1 Hz), 1.04 (t, 6H, J = 7.1 Hz); LRMS
(FAB) m/z 239 (M ? H?).
NMR (300 MHz, CDCl3) d 7.28 (t, 1H, J = 8.5 Hz), 6.61
(d, 1H, J = 3.5 Hz), 6.58 (d, 1H, J = 3.3 Hz), 4.09 (t, 2H,
J = 6.2 Hz), 3.85 (s, 3H), 2.83 (t, 2H, J = 6.3 Hz), 2.57
(q, 4H, J = 7.1 Hz), 1.01 (t, 6H, J = 7.1 Hz); LRMS
(FAB) m/z 269 (M ? H?).
2-(2-(Diethylamino)ethoxy)-4-methoxyaniline (12c)
N,N-Diethyl-2-(4-methoxy-2-nitrophenoxy)ethanamine
Ethanamine 11c (310 mg, 1.16 mmol) afforded the aniline
1
12c (171 mg, 62 %) via general procedure C: H-NMR
(11b)
(300 MHz, CDCl3) d 6.63 (d, 1H, J = 8.4 Hz), 6.44 (d,
1H, J = 2.6 Hz), 6.34 (dd, 1H, J = 2.7, 8.4 Hz), 4.03 (t,
2H, J = 6.4 Hz), 3.72 (s, 3H), 2.88 (t, 2H, J = 6.4 Hz),
2.63 (q, 4H, J = 7.1 Hz), 1.06 (t, 6H, J = 7.1 Hz); LRMS
(FAB) m/z 239 (M ? H?).
Nitrophenol 10b (254 mg, 1.50 mmol) afforded the etha-
1
namine 11b (298 mg, 74 %) via general procedure B: H-
NMR (300 MHz, CDCl3) d 7.35 (d, 1 h, J = 2.8 Hz),
7.09-7.00 (m, 2H), 4.10 (t, 2H, J = 6.1 Hz), 2.88 (t, 2H,
J = 6.1 Hz), 2.61 (q, 4H, J = 7.1 Hz), 1.04 (t, 6H,
J = 7.1 Hz); LRMS (FAB) m/z 269 (M ? H?).
2-(2-(Diethylamino)ethoxy)-3-methoxyaniline (12d)
N,N-Diethyl-2-(5-methoxy-2-nitrophenoxy)ethanamine
Ethanamine 11d (282 mg, 1.05 mmol) afforded the aniline
1
12d (225 mg, 90 %) via general procedure C: H-NMR
(11c)
(300 MHz, CDCl3) d 6.72 (d, 1H, J = 8.2 Hz), 6.27 (d,
1H, J = 2.6 Hz), 6.18 (dd, 1H, J = 2.6, 8.3 Hz), 4.00 (t,
2H, J = 6.8 Hz), 3.78 (s, 3H), 2.87 (t, 2H, J = 6.8 Hz),
2.62 (q, 4H, J = 7.1 Hz), 1.04 (t, 6H, J = 7.1 Hz); LRMS
(FAB) m/z 239 (M ? H?).
Nitrophenol 10c (255 mg, 1.50 mmol) afforded the etha-
1
namine 11c (310 mg, 77 %) via general procedure B: H-
NMR (300 MHz, CDCl3) d 7.95 (d, 1H, J = 9.2 Hz),
6.52-6.45 (m, 2H), 4.11 (t, 2H, J = 6.2 Hz), 3.85 (s, 3H),
123

Design, synthesis, and biological evaluation of pyrimidine-2-carboxamide analogs…
N,N-Diethyl-2-(2-methoxy-3-nitrophenoxy)ethanamine
3-(2-(Diethylamino)ethoxy)-2-methoxyaniline (15a)
(14a)
Ethanamine 14a (89 mg, 0.33 mmol) afforded the aniline
15a (36 mg, 52 %) via general procedure C: 1H-NMR
(300 MHz, CDCl3) d 6.80 (t, 1H, J = 8.1 Hz), 6.34-6.31
(m, 1H), 6.28-6.25 (m, 1H), 3.99 (t, 2H, J = 5.3 Hz), 3.80
(s, 3H), 2.74-2.72 (m, 2H), 2.61 (q, 4H, J = 7.1 Hz), 1.04
(t, 6H, J = 7.1 Hz); LRMS (FAB) m/z 239 (M ? H?).
To a solution of NaH (105 mg, 2.63 mmol) in DMF
(5 mL) was added 2-(diethylamino)ethanol (0.35 mL,
2.63 mmol) at 0 ꢁC for 10 min, and the reaction mixture
was stirred at same temperature for 30 min. To the reaction
mixture was added solution of 1-fluoro-2-methoxy-3-ni-
trobenzene 13a (317 mg, 1.75 mmol) in DMF (2 mL) at
ambient temperature. After being stirred at ambient tem-
perature until no starting material could be observed by
TLC, the reaction mixture was quenched with H2O and
diluted with EtOAc. The organic layer was washed with
water and brine, dried over MgSO4, and concentrated in
vacuo. Purification of the residue via flash column chro-
matography on silica gel (MeOH/CH2Cl2 = 1:50) afforded
5-(2-(Diethylamino)ethoxy)-2-methoxyaniline (15b)
Ethanamine 14b (314 mg, 1.17 mmol) afforded the aniline
1
15b (184 mg, 66 %) via general procedure C: H-NMR
(300 MHz, CDCl3) d 6.66 (d, 1H, J = 8.6 Hz), 6.31 (d,
1H, J = 2.9 Hz), 6.22 (dd, 1H, J = 2.8, 8.6 Hz), 3.95 (t,
2H, J = 6.4 Hz), 3.78 (s, 3H), 2.82 (t, 2H, J = 6.4 Hz),
2.61 (q, 4H, J = 7.1 Hz), 1.04 (t, 6H, J = 7.1 Hz); LRMS
(FAB) m/z 239 (M ? H?).
1
the ethanamine 14a (89 mg, 19 %). H-NMR (300 MHz,
CDCl3) d 7.31-7.28 (m, 1H), 7.09-7.07 (m, 2H), 4.21 (t,
2H, J = 6.4 Hz), 3.88 (s, 3H), 2.88 (t, 2H, J = 6.6 Hz),
2.62 (q, 4H, J = 7.2 Hz), 1.04 (t, 6H, J = 7.1 Hz); LRMS
(FAB) m/z 269 (M ? H?).
3-(2-(Diethylamino)ethoxy)-5-methoxyaniline (15c)
Ethanamine 14c (122 mg, 0.45 mmol) afforded the aniline
15c (73 mg, 78 %) via general procedure C: 1H-NMR
(300 MHz, CDCl3) d 5.91 (t, 1H, J = 2.1 Hz), 5.86-5.84
(m, 2H), 3.97 (t, 2H, J = 6.4 Hz), 3.71 (s, 3H), 3.62 (bs, 2H),
2.83 (t, 2H, J = 6.3 Hz), 2.61 (q, 4H, J = 7.1 Hz), 1.05 (t,
6H, J = 7.1 Hz); LRMS (FAB) m/z 239 (M ? H?).
N,N-Diethyl-2-(4-methoxy-3-nitrophenoxy)ethanamine
(14b)
Nitrophenol 13c (257 mg, 1.52 mmol) afforded the etha-
1
namine 14b (314 mg, 77 %) via general procedure B: H-
NMR (300 MHz, CDCl3) d 7.40 (d, 1H, J = 3.1 Hz), 7.10
(dd, 1H, J = 3.1, 9.2 Hz), 6.99 (d, 1H, J = 9.2 Hz), 4.00
(t, 2H, J = 6.1 Hz), 3.90 (s, 3H), 2.84 (t, 2H, J = 6.0 Hz),
2.61 (q, 4H, J = 7.1 Hz), 1.04 (t, 6H, J = 7.1 Hz); LRMS
(FAB) m/z 269 (M ? H?).
3-(2-(Diethylamino)ethoxy)-4-methoxyaniline (15d)
Ethanamine 14d (362 mg, 1.35 mmol) afforded the aniline
1
15d (244 mg, 76 %) via general procedure C: H-NMR
(300 MHz, CDCl3) d 6.68 (d, 1H, J = 8.4 Hz), 6.31 (d,
1H, J = 2.6 Hz), 6.21 (dd, 1H, J = 2.6, 8.4 Hz), 4.02 (t,
2H, J = 6.9 Hz), 3.76 (s, 3H), 3.41 (bs, 2H), 2.90 (t, 2H,
J = 6.9 Hz), 2.61 (q, 4H, J = 7.1 Hz), 1.04 (t, 6H,
J = 7.1 Hz); LRMS (FAB) m/z 239 (M ? H?).
N,N-Diethyl-2-(3-methoxy-5-nitrophenoxy)ethanamine
(14c)
Ethanamine 14c was prepared by the procedure for 14a,
using 13b (300 mg, 1.75 mmol) instead of 13a (122 mg,
26 %). 1H-NMR (300 MHz, CDCl3) d 7.36-7.33 (m, 2H),
6.74 (t, 1H, J = 2.2 Hz), 4.07 (t, 2H, J = 6.0 Hz), 3.83 (s,
3H), 2.87 (t, 2H, J = 6.0 Hz), 2.63 (q, 4H, J = 7.1 Hz), 1.06
(t, 6H, J = 7.1 Hz); LRMS (FAB) m/z 269 (M ? H?).
N,N-Diethyl-2-(2-methoxy-4-nitrophenoxy)ethanamine (17a)
4-Nitroguaiacol 16a (257 mg, 1.52 mmol) afforded the
ethanamine 17a (285 mg, 70 %) via general procedure B:
1H-NMR (300 MHz, CDCl3) d 7.88 (dd, 1H, J = 2.6,
8.8 Hz), 7.72 (d, 1H, J = 2.4 Hz), 6.91 (d, 1H,
J = 8.8 Hz), 4.16 (t, 2H, J = 6.6 Hz), 3.92 (s, 3H), 2.93 (t,
2H, J = 6.6 Hz), 2.63 (q, 4H, J = 7.1 Hz), 1.05 (t, 6H,
J = 7.1 Hz); LRMS (FAB) m/z 269 (M ? H?).
N,N-Diethyl-2-(2-methoxy-5-nitrophenoxy)ethanamine
(14d)
5-Nitroguaiacol 13d (256 mg, 1.51 mmol) afforded the
ethanamine 14d (361 mg, 89 %) via general procedure B:
1H-NMR (300 MHz, CDCl3) d 7.88 (dd, 1H, J = 2.6,
9.0 Hz), 7.77 (d, 1H, J = 2.6 Hz), 6.87 (d, 1H,
J = 9.0 Hz), 4.13 (t, 2H, J = 6.5 Hz), 3.93 (s, 3H), 2.93 (t,
2H, J = 6.5 Hz), 2.63 (q, 4H, J = 7.1 Hz), 1.06 (t, 6H,
J = 7.1 Hz); LRMS (FAB) m/z 269 (M ? H?).
N,N-Diethyl-2-(3-methoxy-4-nitrophenoxy)ethanamine
(17b)
Nitrophenol 16b (257 mg, 1.52 mmol) afforded the etha-
1
namine 17b (330 mg, 81 %) via general procedure B: H-
123

S.-H. Kim et al.
NMR (300 MHz, CDCl3) d 7.97 (d, 1H, J = 9.0 Hz), 6.54
(d, 1H, J = 2.4 Hz), 6.48 (dd, 1H, J = 2.6, 9.1 Hz), 4.08
(t, 2H, J = 6.1 Hz), 3.92 (s, 3H), 2.86 (t, 2H, J = 6.2 Hz),
2.62 (q, 4H, J = 7.1 Hz), 1.05 (t, 6H, J = 7.1 Hz); LRMS
(FAB) m/z 269 (M ? H?).
(CDCl3, 75 MHz) d 166.7, 164.4, 164.4, 163.3, 159.4,
158.6, 156.8, 148.9, 132.4, 132.4, 129.7, 129.7, 129.6,
129.6, 121.3, 120.6, 116.3, 116.3, 116.0, 116.0, 112.6,
104.4, 99.6, 67.4, 55.6, 52.1, 48.1, 48.1, 12.0, 12.0; LRMS
(FAB) m/z 533 (M ? H?); HRMS (FAB) calcd for C30-
H31F2N4O3 (M ? H?): 533.2364; found 533.2361.
4-(2-(Diethylamino)ethoxy)-3-methoxyaniline (18a)
N-(2-(2-(Diethylamino)ethoxy)-5-methoxyphenyl)-4,6-
Ethanamine 17a (285 mg, 1.06 mmol) afforded the aniline
1
18a (166 mg, 66 %) via general procedure C: H-NMR
bis(4-fluorophenyl)pyrimidine-2-carboxamide (21)
(300 MHz, CDCl3) d 6.72 (d, 1H, J = 8.4 Hz), 6.27 (d,
1H, J = 2.6 Hz), 6.18 (dd, 1H, J = 2.6, 8.4 Hz), 3.99 (t,
2H, J = 6.9 Hz), 3,78 (s, 3H), 3.41 (bs, 2H), 2.86 (t, 2H,
J = 6.9 Hz), 2.61 (q, 4H, J = 7.1 Hz), 1.04 (t, 6H,
J = 7.1 Hz); LRMS (FAB) m/z 239 (M ? H?).
The acid 7 (8 mg, 0.026 mmol) and aniline 12b (6.2 mg,
0.026 mmol) afforded the carboxamide 21 (13 mg, 58 %)
1
via general procedure A: H-NMR (300 MHz, CD3OD) d
8.52 (s, 1H), 8.48-8.43 (m, 4H), 8.22 (d, 1H, J = 2.9 Hz),
7.36-7.30 (m, 4H), 7.02 (d, 1H, J = 9.2 Hz), 6.70 (dd,
1H, J = 2.9, 9.0 Hz), 4.20 (t, 2H, J = 6.2 Hz), 3.79 (s,
3H), 2.99 (t, 2H, J = 6.3 Hz), 2.58 (q, 4H, J = 7.2 Hz),
0.94 (t, 6H, J = 7.1 Hz); 13C-NMR (CDCl3, 75 MHz) d
166.7, 164.4, 164.4, 163.3, 159.9, 158.2, 154.1, 141.8,
132.2, 132.2, 129.7, 129.7, 129.6, 129.6, 128.2, 116.3,
116.3, 116.0, 116.0, 112.7, 112.3, 109.8, 105.5, 68.0, 55.8,
52.2, 48.0, 48.0, 12.0, 12.0; LRMS (FAB) m/z 533
(M ? H?); HRMS (FAB) calcd for C30H31F2N4O3
(M ? H?): 533.2364; found 533.2376.
4-(2-(Diethylamino)ethoxy)-2-methoxyaniline (18b)
Ethanamine 17b (330 mg, 1.23 mmol) afforded the aniline
1
18b (240 mg, 73 %) via general procedure C: H-NMR
(300 MHz, CDCl3) d 6.61 (d, 1H, J = 8.4 Hz), 6.45 (d,
1H, J = 2.6 Hz), 6.33 (dd, 1H, J = 2.6, 8.4 Hz), 3.97 (t,
2H, J = 6.5 Hz), 3.80 (s, 3H), 2.83 (t, 2H, J = 6.4 Hz),
2.62 (q, 4H, J = 7.1 Hz), 1.05 (t, 6H, J = 7.1 Hz); LRMS
(FAB) m/z 239 (M ? H?).
N-(2-(2-(Diethylamino)ethoxy)-6-methoxyphenyl)-4,6-
N-(2-(2-(Diethylamino)ethoxy)-3-methoxyphenyl)-4,6-
bis(4-fluorophenyl)pyrimidine-2-carboxamide (22)
bis(4-fluorophenyl)pyramidine-2-carboxamide (19)
The acid 7 (8 mg, 0.026 mmol) and aniline 12a (6.2 mg,
0.026 mmol) afforded the carboxamide 22 (16 mg, 66 %)
The acid 7 (8 mg, 0.026 mmol) and aniline 12d (6.2 mg,
0.026 mmol) afforded the carboxamide 19 (12 mg, 47 %)
1
via general procedure A: H-NMR (300 MHz, CD3OD) d
1
via general procedure A: H-NMR (300 MHz, CD3OD) d
8.58 (s, 1H), 8.54-8.50 (m, 4H), 7.36-7.28 (m, 5H),
6.82-6.78 (m, 2H), 4.27 (t, 2H, J = 4.9 Hz), 3.89 (s, 3H),
3.13-3.11 (m, 2H), 2.84-2.80 (m, 4H), 1.01 (t, 6H,
J = 7.2 Hz); 13C-NMR (acetone-d6, 150 MHz) d 166.9,
165.4, 165.4, 165.3, 161.6, 159.8, 157.4, 156.4, 134.0,
134.0, 131.4, 131.4, 131.4, 131.4, 128.9, 117.1, 117.1,
116.9, 116.9, 116.3, 114.1, 106.7, 105.8, 68.5, 56.7, 52.7,
48.7, 48.7, 12.4, 12.4; LRMS (FAB) m/z 533 (M ? H?);
HRMS (FAB) calcd for C30H31F2N4O3 (M ? H?):
533.2364; found 533.2371.
8.42-8.37 (m, 4H), 8.36 (s, 1H), 7.59 (d, 1H, J = 2.4 Hz),
7.31 (dd, 1H, J = 2.4, 8.6 Hz), 7.26-7.20 (m, 4H), 6.93
(d, 1H, J = 8.8 Hz), 4.11 (t, 2H, J = 5.9 Hz), 2.77 (q, 4H,
J = 7.2 Hz), 1.13 (t, 6H, J = 7.1 Hz); 13C-NMR (CDCl3,
75 MHz) d 166.7, 164.6, 164.6, 163.4, 159.8, 158.1, 149.8,
145.4, 132.2, 132.2, 131.7, 129.7, 129.7, 129.6, 129.6,
116.4, 116.4, 116.2, 116.2, 113.7, 113.0, 111.7, 105.0,
67.6, 56.0, 51.7, 47.8, 47.8, 11.6, 11.6; LRMS (FAB) m/z
533 (M ? H?); HRMS (FAB) calcd for C30H31F2N4O3
(M ? H?): 533.2364; found 533.2376.
N-(3-(2-(Diethylamino)ethoxy)-2-methoxyphenyl)-4,6-
N-(2-(2-(Diethylamino)ethoxy)-4-methoxyphenyl)-4,6-
bis(4-fluorophenyl)pyrimidine-2-carboxamide (23)
bis(4-fluorophenyl)pyrimidine-2-carboxamide (20)
The acid 7 (8 mg, 0.026 mmol) and aniline 15a (6.2 mg,
0.026 mmol) afforded the carboxamide 23 (15 mg, 76 %)
The acid 7 (8 mg, 0.026 mmol) and aniline 12c (6.2 mg,
0.026 mmol) afforded the carboxamide 20 (9 mg, 41 %)
1
via general procedure A: H-NMR (400 MHz, CDCl3) d
1
via general procedure A: H-NMR (300 MHz, CD3OD) d
10.87 (s, 1H), 8.31-8.25 (m, 5H), 8.10 (s, 1H), 7.25-7.21
(m, 4H), 7.11 (t, 1H, J = 8.3 Hz), 6.73 (d, 1H,
J = 8.0 Hz), 4.19 (t, 2H, J = 6.7 Hz), 3.88 (s, 3H), 2.84 (t,
2H, J = 6.6 Hz), 2.44 (q, 4H, J = 6.9 Hz), 0.82 (t, 6H,
J = 7.1 Hz); 13C-NMR (acetone-d6, 125 MHz) d 167.2,
8.41-8.33 (m, 6H), 7.30-7.24 (m, 4H), 6.59 (d, 1H,
J = 2.6 Hz), 6.52 (dd, 1H, J = 2.6, 8.8 Hz), 4.18 (t, 2H,
J = 6.2 Hz), 3.78 (s, 3H), 3.01 (t, 2H, J = 6.2 Hz), 2.61
(q, 4H, J = 7.1 Hz), 0.97 (t, 6H, J = 7.1 Hz); 13C-NMR
123

Design, synthesis, and biological evaluation of pyrimidine-2-carboxamide analogs…
165.6, 165.6, 165.2, 161.4, 161.3, 153.6, 138.2, 134.0,
134.0, 133.8, 131.5, 131.5, 131.4, 131.4, 125.1, 117.3,
117.3, 117.1, 117.1, 114.5, 113.3, 109.5, 72.2, 56.7, 53.7,
48.1, 48.1, 12.0, 12.0; LRMS (FAB) m/z 533 (M ? H?);
HRMS (FAB) calcd for C30H31F2N4O3 (M ? H?):
533.2364; found 533.2367.
1.13 (t, 6H, J = 7.1 Hz); 13C-NMR (CDCl3, 75 MHz) d
166.7, 164.3, 164.3, 163.4, 159.8, 158.1, 153.2, 142.8,
132.2, 132.1, 129.6, 129.6, 129.5, 129.5, 128.1, 116.4,
116.4, 116.1, 116.1, 112.4, 111.0, 110.0, 106.6, 66.5, 56.6,
51.5, 47.7, 47.7, 11.5, 11.5; LRMS (FAB) m/z 533
(M ? H?); HRMS (FAB) calcd for C30H31F2N4O3
(M ? H?): 533.2364; found 533.2359.
N-(3-(2-(Diethylamino)ethoxy)-4-methoxyphenyl)-4,6-
bis(4-fluorophenyl)pyrimidine-2-carboxamide (24)
N-(4-(2-(Diethylamino)ethoxy)-2-methoxyphenyl)-4,6-
bis(4-fluorophenyl)pyramidine-2-carboxamide (27)
The acid 7 (8 mg, 0.026 mmol) and aniline 15b (6.2 mg,
0.026 mmol) afforded the carboxamide 24 (17 mg, 79 %)
The acid 7 (8 mg, 0.026 mmol) and aniline 18a (6.2 mg,
0.026 mmol) afforded the carboxamide 27 (18 mg, 75 %)
1
via general procedure A: H-NMR (300 MHz, CDCl3) d
1
9.91 (s, 1H), 8.25-8.19 (m, 4H), 8.08 (s, 1H), 7.65-7.64
(m, 1H), 7.25-7.19 (m, 5H), 6.86 (d, 1H, J = 8.6 Hz),
4.15 (t, 2H, J = 6.7 Hz), 3.84 (s, 3H), 2.97 (t, 2H,
J = 6.6 Hz), 2.66 (q, 4H, J = 7.1 Hz), 1.08 (t, 6H,
J = 7.1 Hz); 13C-NMR (CDCl3, 75 MHz) d 166.7, 164.5,
164.5, 163.3, 159.7, 158.0, 148.4, 146.4, 132.1, 132.1,
131.1, 129.7, 129.7, 129.6, 129.6, 116.4, 116.4, 116.1,
116.1, 112.9, 112.1, 111.9, 106.0, 67.0, 56.2, 51.4, 47.7,
47.7, 11.7, 11.7; LRMS (FAB) m/z 533 (M ? H?); HRMS
(FAB) calcd for C30H31F2N4O3 (M ? H?): 533.2364;
found 533.2367.
via general procedure A: H-NMR (300 MHz, CDCl3) d
8.56 (d, 1H, J = 8.3 Hz), 8.28-8.24 (m, 4H), 8.08 (s, 1H),
7.24-7.19 (m, 4H), 6.56-6.53 (m, 2H), 4.07 (t, 2H,
J = 6.1 Hz), 3.95 (s, 3H), 2.89 (t, 2H, J = 6.1 Hz), 2.66
(q, 4H, J = 7.1 Hz), 1.08 (t, 6H, J = 7.1 Hz); 13C-NMR
(CDCl3, 75 MHz) d 166.6, 164.2, 164.2, 163.3, 159.3,
158.3, 156.0, 149.6, 132.2, 132.2, 129.6, 129.6, 129.5,
129.5, 121.1, 120.3, 116.3, 116.3, 116.0, 116.0, 112.3,
104.8, 99.2, 66.6, 56.0, 51.7, 47.8, 47.8, 11.8, 11.8; LRMS
(FAB) m/z 533 (M ? H?); HRMS (FAB) calcd for C30-
H31F2N4O3 (M ? H?): 533.2364; found 533.2361.
N-(3-(2-(Diethylamino)ethoxy)-5-methoxyphenyl)-4,6-
N-(4-(2-(Diethylamino)ethoxy)-3-methoxyphenyl)-4,6-
bis(4-fluorophenyl)pyrimidine-2-carboxamide (25)
bis(4-fluorophenyl)pyrimi-dine-2-carboxamide (28)
The acid 7 (8 mg, 0.026 mmol) and aniline 15c (6.2 mg,
0.026 mmol) afforded the carboxamide 25 (18 mg, 80 %)
The acid 7 (8 mg, 0.026 mmol) and aniline 18b (6.2 mg,
0.026 mmol) afforded the carboxamide 28 (10 mg, 46 %)
1
1
via general procedure A: H-NMR (300 MHz, CDCl3) d
via general procedure A: H-NMR (300 MHz, CDCl3) d
9.97 (s, 1H), 8.25-8.20 (m, 4H), 8.09 (s, 1H), 7.26-7.20
(m, 4H), 7.13-7.11 (m, 1H), 7.03-7.01 (m, 1H), 6.30 (t,
1H, J = 2.0 Hz), 4.08 (t, 2H, J = 6.2 Hz), 3.80 (s, 3H),
2.89 (t, 2H, J = 6.1 Hz), 2.66 (q, 4H, J = 7.1 Hz), 1.08 (t,
6H, J = 7.1 Hz); 13C-NMR (CDCl3, 75 MHz) d 166.7,
164.6, 164.6, 163.4, 161.2, 160.2, 160.0, 157.8, 139.2,
132.1, 132.1, 129.7, 129.7, 129.6, 129.6, 116.4, 116.4,
116.2, 116.2, 113.0, 98.7, 98.3, 97.3, 66.2, 55.5, 51.5, 47.7,
47.7, 11.5, 11.5; LRMS (FAB) m/z 533 (M ? H?); HRMS
(FAB) calcd for C30H31F2N4O3 (M ? H?): 533.2364;
found 533.2357.
9.94 (s, 1H), 8.26-8.21 (m, 4H), 8.09 (s, 1H), 7.82 (d, 1H,
J = 2.4 Hz), 7.26-7.21 (m, 4H), 7.06 (dd, 1H, J = 2.4,
8.6 Hz), 6.90 (d, 1H, J = 8.6 Hz), 4.09 (t, 2H,
J = 6.9 Hz), 3.91 (s, 3H), 2.92 (t, 2H, J = 6.8 Hz), 2.64
(q, 4H, J = 7.1 Hz), 1.06 (t, 6H, J = 7.1 Hz); 13C-NMR
(CDCl3, 75 MHz) d 166.7, 164.5, 164.5, 163.3, 159.8,
157.8, 149.6, 145.3, 132.2, 132.2, 131.5, 129.7, 129.7,
129.6, 129.6, 116.4, 116.4, 116.1, 116.1, 113.3, 113.0,
111.6, 104.8, 67.5, 56.0, 51.6, 47.8, 47.8, 11.7, 11.7; LRMS
(FAB) m/z 533 (M ? H?); HRMS (FAB) calcd for C30-
H31F2N4O3 (M ? H?): 533.2364; found 533.2376.
N-(5-(2-(Diethylamino)ethoxy)-2-methoxyphenyl)-4,6-
ELISA test
bis(4-fluorophenyl)pyrimidine-2-carboxamide (26)
One microgram of purified biotinylated-human-RAGE,
1 lL of 10 lM Ab solution and 20 lM of the compound in
100 lL of TBS-T with 2.5 % BSA were incubated on a
streptavidin-coated plate for 60 min at ambient tem-
perature. After washing the plate with TBS-T, the horse-
radish-peroxidase conjugated 4G8 antibody (4G8-HRP,
1:1000 dilution) in 100 lL of TBS-T with 2.5 % BSA was
added into each well to detect the bound Ab. The plate was
The acid 7 (8 mg, 0.026 mmol) and aniline 15d (6.2 mg,
0.026 mmol) afforded the carboxamide 26 (9 mg, 41 %)
1
via general procedure A: H-NMR (300 MHz, CD3OD) d
8.12-8.08 (m, 4H), 8.03 (s, 1H), 7.85 (d, 1H, J = 2.9 Hz),
7.07-7.01 (m, 4H), 6.67 (d, 1H, J = 9.0 Hz), 6.45 (dd,
1H, J = 2.9, 8.8 Hz), 3.91 (t, 2H, J = 5.7 Hz), 3.73 (s,
3H), 2.91 (t, 2H, J = 5.6 Hz), 2.74 (q, 4H, J = 7.1 Hz),
123

S.-H. Kim et al.
incubated for 60 min at ambient temperature. After wash-
ing with TBS-T, the plate was developed with TMB sub-
strate: the reaction was stopped with sulfuric acid. The
absorbance was read on a Sunrise plate reader (TECHAN)
at 450 nm.
Mitsunobu reaction, respectively. Nitrobenzyl ethers 14a,
b were synthesized from 3-fluoro-nitrobenzenes 13a, b by
the aromatic substitution reaction, respectively, because the
corresponding methoxy-3-nitrophenols were not commer-
cially available. The meta-(diethylamino)ethoxy anilines
15a–d were also readily prepared by nitro-reduction of
nitrobenzenes 14a–d, respectively. As shown in Scheme 4,
the sequential reaction of the Mitsunobu reaction and nitro-
reduction afforded para-(diethylamino)ethoxy anilines
18a, b with moderate yields from 4-nitrophenols 16a, b,
respectively.
Cell viability test
For determining cell viability, the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) assay was
used. Hippocampal HT22 cells were seeded in 96-well
plates with a density of 5000 cells per well, and cultured in
DMEM with 10 % FBS for 24 h. The medium was re-
placed with serum free DMEM before treatment. Com-
pound or DMSO were diluted in DMEM and added in each
wells. After 18 h of treatment, the cells were incubated
with MTT at a final concentration of 500 lg/mL for 4 h.
After incubation, the medium was removed and MTT
formazan was dissolved in 100 lL of DMSO. The ab-
sorbance was read with a microplate reader (Sunrise,
TECHAN, Australia) at 570 nm. Cell viability was ex-
pressed as a percentage of the control.
In the same manner as analogs 9a–c, methoxy-substi-
tuted analogs 19–28 were prepared by amidation reaction
between the acid intermediate 7 and the corresponding
anilines, as shown in Scheme 5.
Biological study
RAGE inhibitory activities of the synthesized analogs were
evaluated through ELISAs (enzyme-linked immunosorbent
assays) using 20 lM of each analog (Han et al. 2012, 2014).
The assay results are summarized in Fig. 2. Compared to the
positive control 1, analogs 9a–9c (ClogP = 7.04) were re-
garded as more hydrophilic analogs. However, these ana-
logs, except meta analog 9b (29 %), did not exhibit any
RAGE inhibitory activity. Considering methoxy derivatives
(23–26) of the meta analog 9b, introduction of a methoxy
moiety seemed to contribute to the improvement of hy-
drophilicity, as well as RAGE inhibitory activity to some
extent. For example, compared with the 2-aminopyrimidine
1 and meta analog 9b, 5-methoxy analog 25 and 6-methoxy
analog 26 exhibited significant improvement in not only
RAGE inhibitory activity (159 % and 167 %, respectively),
but also hydrophilicity (ClogP = 7.02 and 6.43, respec-
tively). Introduction of the methoxy moiety at the 2-position
and the 4-position also improved the meta analog 9b,
although the activities of 23 (76 %) and 24 (64 %) were not
greater than 2-aminopyrimidine 1. We could not observe any
RAGE inhibitory activity in the ortho analog 9a and para
analog 9c, but their methoxy derivatives, 21 and 27, exhib-
ited potent RAGE inhibitory activity (151 and 152 %, re-
spectively) with improved hydrophilic property
(ClogP = 6.43).
Results and discussion
Chemistry
The pyrimidine-2-carboxamide analogs were readily pre-
pared from commercially available 2,4,6-trichloro-pyrim-
idine 4 as shown in Scheme 1. Regioselective Suzuki
reaction (Delia et al. 2006) between trichloropyrimidine 4
and 4-fluorophenylboronic acid provided 4,6-bis(4-
fluorophenyl)pyrimidine 5, which was treated with NaCN
and DABCO to afford carbonitrile 6 (Irie et al. 2008).
Hydrolysis of the carbonitrile 6 yielded an acid interme-
´
diate 7 (Suarez-Varela et al. 2008). And the carboxamide
analogs 9a–c were synthesized by an amidation reaction
between the acid intermediate 7 and known aniline coun-
terparts 8a–c (Han et al. 2012), respectively.
To synthesize various methoxy-substituted pyrimidine-
2-carboxamide analogs, methoxy-substituted aniline
counterparts were prepared from commercially available
nitrobenzenes as described in Schemes 2, 3 and 4. Aniline
counterparts possessing a (diethylamino)ethoxy moiety at
ortho-position 12a–d were afforded from methoxy-2-ni-
trophenols as shown in Scheme 2. The Mitsunobu reaction
of nitrophenols 10a–d with 2-(diethylamino)ethanol gave
phenyl ethers 11a–d, which were subjected to nitro-re-
duction to afford ortho-(diethylamino)ethoxy anilines 12a–
d, respectively.
Through this investigation, we identified several more
active and hydrophilic pyrimidine-2-carboxamide analogs,
such as 21, 25, 26, and 27. With the expectation that im-
proved hydrophilicity may reduce cytotoxicity, we
evaluated cytotoxicities of the identified pyrimidine-2-
carboxamide analogs by MTT assay, using hippocampal
HT22 cells. As shown in Table 1, analog 21 did not exhibit
any cytotoxic effects as we had expected. Analogs 25–27
displayed severe cytotoxicity, which was even higher than
the parent analogs, 1 and 2.
As shown in Scheme 3, nitrobenzyl ethers 14c, d were
also afforded from methoxy-3-nitrophenols 13c,d by the
123

Design, synthesis, and biological evaluation of pyrimidine-2-carboxamide analogs…
Scheme 1 Reagents and conditions: a 4-fluorophenylboronic acid,
Pd(OAc)2, PPh3, Na2CO3, THF/H2O (1:1), reflux, 57 %, b NaCN,
DABCO, DMSO, rt, 88 %, c aq. KOH, EtOH/H2O (1:1), reflux,
88 %, d oxalyl chloride, DMF, CH2Cl2, 0 ꢁC, e anilines (8a–c), Et3N,
THF, rt, 25 % (9a), 51 % (9b), 47 % (9c)
Scheme 2 Reagents and
conditions: a 2-
(diethylamino)ethanol, DIAD,
PPh3, THF, rt, 65–77 %
b SnCl2Á2H2O, EtOH, reflux,
62–90 %
Scheme 3 Reagents and
conditions: a 2-
(diethylamino)ethanol, NaH,
DMF, rt, 19 % (14a), 26 %
(14c), b 2-
(diethylamino)ethanol, DIAD,
PPh3, THF, rt, 77 % (14b),
89 % (14d), c SnCl2Á2H2O,
EtOH, reflux, 52–78 %
123

S.-H. Kim et al.
Scheme 4 Reagents and conditions: a 2-(diethylamino)ethanol, DIAD, PPh3, THF, rt, 70 % (17a), 81 % (17b), b SnCl2Á2H2O, EtOH, reflux,
66 % (18a), 73 % (18b)
Scheme 5 Reagents and
conditions: a oxalyl chloride,
DMF, CH2Cl2, 0 ꢁC, b anilines
(12a–d, 15a–d, and 18a, b),
Et3N, THF, rt, 41–80 % (in 2
steps)
Fig. 2 Relative RAGE
inhibitory activity of the
analogs. Inhibitory activities
were determined by duplicate
experiments using 20 lM of the
synthesized analogs and
expressed in comparison with
the activity of 1
Table 1 Evaluation of
cytotoxicity of the analogs
Analog (10 lM)
1
2
21
25
26
27
Cell viability (%)
37.3 1.7
10.3 7.5
100.4 3.2
5.4 0.2
4.5 0.2
5.3 0.3
Cell viabilities were determined by duplicate MTT assay in hippocampal HT22 cells using 10 lM of the
synthesized analogs
In summary, to identify novel RAGE inhibitors that
have favorable drug-like properties, we designed and syn-
thesized a series of pyrimidine-2-carboxamides based on
our preceding studies. Evaluation of their RAGE inhibitory
activities led us to identify several potent pyrimidine-2-
carboxamide analogs, with improved hydrophilicity. In
particular, by contrast with the parent analogs 1 and 2, the
2-(2-(diethylamino)ethoxy)-5-methoxyphenyl analog 21
123

Design, synthesis, and biological evaluation of pyrimidine-2-carboxamide analogs…
Han, Y.T., G.-I. Choi, D. Son, N.-J. Kim, H. Yun, S. Lee, D.-J.
did not exhibit undesired cytotoxic effect. Further research
on the identified pyrimidine-2-carboxamide RAGE in-
hibitor is currently in progress.
Chang, H.-S. Hong, H. Kim, H.-J. Ha, Y.-H. Kim, H.-J. Park, J.
Lee, and Y.-G. Suh. 2012. Ligand-based design, synthesis and
biological evaluation of 2-aminopyrimidines, a novel series of
RAGE (Receptor for Advanced Glycation End Products)
inhibitors. Journal of Medicinal Chemistry 55: 9120–9135.
Han, Y.T., K. Kim, G.-I. Choi, H. An, D. Son, H. Kim, H.-J. Ha, J.-H.
Son, S.-J. Chung, H.-J. Park, J. Lee, and Y.-G. Suh. 2014.
Pyrazole-5-carboxamides, novel inhibitors of receptor for ad-
vanced glycation end products (RAGE). European Journal of
Medicinal Chemistry 79: 128–142.
Acknowledgments This work was supported by National Research
Foundation grant funded by the Korea government (MEST) as a part
of Global Drug Candidate Development Program for Neurodegen-
erative Diseases (2011-0018334).
Irie, O., T. Ehara, A. Iwasaki, F. Yokokawa, J. Sakaki, H. Hirao, T.
Kanazawa, N. Teno, M. Horiuchi, I. Umemura, H. Gunji, K.
Masuya, Y. Hitomi, G. Iwasaki, K. Nonomura, K. Tanabe, H.
Fukaya, T. Kosaka, C.R. Snell, and A. Hallett. 2008. Discovery
of selective and nonpeptidic cathepsin S inhibitors. Bioorganic
and Medicinal Chemistry Letters 18: 3959–3962.
Kerns, E.H., and L. Di. 2008. Drug-like properties: Concepts,
structure design and methods from ADME to toxicity optimiza-
tion. London: Academic Press.
Li, J., and A.M. Schmidt. 1997. Characterization and functional
analysis of the promoter of RAGE, the receptor for advanced
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