Design, synthesis and evaluation of 2,4-disubstituted pyrimidines as cholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.04.108

A group of 2,4-disubstituted pyrimidine derivatives (7a-e, 8a-e and 9a-d) that possess a variety of C-2 aliphatic five- and six-membered heterocycloalkyl ring in conjunction with a C-4 arylalkylamino substituent were designed, synthesized and evaluated as cholinesterase (ChE) inhibitors. The steric and electronic properties at C-2 and C-4 positions of the pyrimidine ring were varied to investigate their effect on ChE inhibitory potency and selectivity. The structure-activity relationship (SAR) studies identified N-benzyl-2-thiomorpholinopyrimidin-4-amine (7c) as the most potent cholinesterase inhibitor (ChEI) with an IC₅₀ = 0.33 μM (acetylcholinesterase, AChE) and 2.30 μM (butyrylcholinesterase, BuChE). The molecular modeling studies indicate that within the AChE active site, the C-2 thiomorpholine substituent was oriented toward the cationic active site region (Trp84 and Phe330) whereas within the BuChE active site, it was oriented toward a hydrophobic region closer to the active site gorge entrance (Ala277). Accordingly, steric and electronic properties at the C-2 position of the pyrimidine ring play a critical role in ChE inhibition.

Full text of DOI:10.1016/j.bmcl.2010.04.108

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3606–3609
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of 2,4-disubstituted pyrimidines as
cholinesterase inhibitors
Tarek Mohamed a,b, Praveen P. N. Rao a,
*
a School of Pharmacy, Health Sciences Campus, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
b Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of 2,4-disubstituted pyrimidine derivatives (7a–e, 8a–e and 9a–d) that possess a variety of C-2
aliphatic five- and six-membered heterocycloalkyl ring in conjunction with a C-4 arylalkylamino substi-
tuent were designed, synthesized and evaluated as cholinesterase (ChE) inhibitors. The steric and elec-
tronic properties at C-2 and C-4 positions of the pyrimidine ring were varied to investigate their effect
on ChE inhibitory potency and selectivity. The structure–activity relationship (SAR) studies identified
N-benzyl-2-thiomorpholinopyrimidin-4-amine (7c) as the most potent cholinesterase inhibitor (ChEI)
Received 9 April 2010
Revised 23 April 2010
Accepted 26 April 2010
Available online 28 April 2010
Keywords:
with an IC50 = 0.33 lM (acetylcholinesterase, AChE) and 2.30 lM (butyrylcholinesterase, BuChE). The
Acetylcholinesterase AChE
Butyrylcholinesterase BuChE
2,4-Disubstituted pyrimidines
Molecular modeling
molecular modeling studies indicate that within the AChE active site, the C-2 thiomorpholine substituent
was oriented toward the cationic active site region (Trp84 and Phe330) whereas within the BuChE active
site, it was oriented toward a hydrophobic region closer to the active site gorge entrance (Ala277).
Accordingly, steric and electronic properties at the C-2 position of the pyrimidine ring play a critical role
in ChE inhibition.
Ó 2010 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a progressive neuro degenerative
disorder that affects regions of the brain that control cognition,
memory, language, speech and awareness to one’s physical sur-
roundings.1 The characteristics of AD include the progressive loss
of cholinergic neural transmission, formation of abeta-amyloid pla-
ques (Ab-plaques) and neurofibrillary tangles (NFTs) of hyperphos-
phorylated tau protein, all of which support the cholinergic, b-
amyloid and tau hypotheses of AD.2 Although the cholinergic
hypothesis is one of the earliest and most studied pathways, recent
findings suggests a link between diminished cholinergic neural
transmission and Ab-plaque toxicity in AD pathogenesis generat-
ing a renewed interest in the cholinergic hypothesis.3
In this regard, several fused heterocyclic ring templates were
developed as ChE inhibitors (ChEIs) (Fig. 1). The acridine derivative
tacrine (1) was one of the earliest ChEI developed to treat AD.7 The
related derivative bis-7-tacrine (2), is a highly potent ChEI.8 Fur-
thermore, b-carbolines (3) and phenothiazines (4) were developed
as dual AChE and BuChE inhibitors.9–11 As part of our program to
develop novel ChEIs, we herein report the design, synthesis and
evaluation of a class of 2,4-disubstituted pyrimidine derivatives
(7a–e, 8a–e and 9a–d) based on a non-fused heterocyclic ring
According to the cholinergic hypothesis, the pathogenesis of AD
is a result of the progressive decline of cholinergic transmission
mediated via the neurotransmitter acetylcholine (ACh).2 The ChE
enzymes, AChE and BuChE are hydrolytic enzymes that act on
ACh to terminate its actions in the synaptic cleft by cleaving the
neurotransmitter to choline and acetate.4 Recent studies have
shown that AD pathogenesis is characterized by the rapid loss of
AChE activity in the early stages of the disease along with the
increasing ratio of BuChE to AChE as the disease progresses.5,6
These findings support the need to control the activity of the ChE
enzymes at different stages of AD progression.
NH2
N
N
N
H
N
H
7
N
1
2
( )
( )
t-BuO
O
OMe
N
N+
Me
N
S
(4)
Me
H
(3)
* Corresponding author. Tel.: +1 519 888 4567x21317; fax: +1 519 888 7910.
E-mail address: praopera@uwaterloo.ca (P.P.N. Rao).
Figure 1. Structures of some fused heterocyclic ring templates that exhibit ChE
inhibition (tacrine 1; bis-tacrine 2; b-carboline 3; phenothiazine 4).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.108

T. Mohamed, P. P. N. Rao / Bioorg. Med. Chem. Lett. 20 (2010) 3606–3609
3607
Table 1
template. Their synthetic protocol, AChE/BuChE inhibition, struc-
ture–activity relationship (SAR) studies and molecular modeling
investigations are described.
The N-benzyl, N-phenethyl and naphthyl methyl 2-chloropyr-
imidin-4-amine intermediates (6a–c) were synthesized from 2,4-
dichloropyrimidine starting material (5) by a nucleophilic aromatic
substitution reaction using a base such as N,N-diisopropylethyl-
amine (DIPEA) in moderate yield (60–65%) (Scheme 1).12 In the
next step, the C-2 chlorine is displaced by various cyclic secondary
amines (R1 = pyrrolidine, morpholine, thiomorpholine, 1-methylpi-
In vitro ChE (AChE and BuChE) inhibition assay data for compounds 7a–e, 8a–e and
9a–d
Compound
AChE
IC50
BuChE
IC50 (lM)
SIb
C log Pc
a
a
(
lM)
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9.80
0.50
0.33
0.56
0.39
12.00
4.80
25.00
14.40
7.90
26.40
68.30
2.30
>100
2.90
13.80
>100
>100
>100
17.70
8.90
28.00
34.70
2.60
0.37
0.007
0.14
<0.006
0.13
0.87
<0.05
<0.25
<0.14
0.45
0.65
0.56
2.96
2.14
2.97
2.70
4.04
3.61
2.79
3.62
3.35
4.69
4.14
3.31
4.15
3.87
3.27
10.09
perazine, 4-methylpiperidine) under rigorous conditions in
a
sealed pressure vessel using n-butanol as a solvent to afford the
target 2,4-disubstituted pyrimidine derivatives (7a–e, 8a–e and
9a–d) in good yield (75–85%) (Scheme 1).13,14
The potency and selectivity of the synthesized pyrimidine
derivatives (7a–e, 8a–e and 9a–d) were evaluated in vitro using
an inhibition assay with AChE (electric eel) and BuChE (equine ser-
um). The inhibition data (IC50 values) are reported in Table 1.15–17
Amongst the N-benzyl series of compounds (7a–e), varying the
C-2 substituents modulated the ChE inhibitory profile (Table 1).
The presence of a C-2 five-membered pyrrolidine substituent in
5.80
15.70
15.50
17.60
0.173
0.0018
0.45
6.77
9.11
0.17
Tacrine (1)
Bis-tacrine (2)
0.019
0.0105
a
The in vitro test compound concentration required to produce 50% inhibition of
AChE (electric eel) or BuChE (equine serum). The result (IC50 M) is the mean of
,
l
two separate determinations (n = 4) and the deviation from the mean is <10% of the
7a led to the inhibition of both AChE (IC50 = 9.80
(IC50 = 26.40 M), although 7a was not as potent as the reference
compound tacrine (1, AChE IC50 = 0.173 M; BuChE IC50
0.019 M; Table 1). In contrast, the presence of a six-membered
lM) and BuChE
mean value.
b
Selectivity index (SI) = AChE IC50/BuChE IC50
The C log P value was calculated using the ChemDraw Ultra program, Version
.
l
c
l
=
11.0, CambridgeSoft company.
l
C-2 substituent such as a morpholine (7b), a thiomorpholine (7c),
1-methylpiperazine (7d) and a 4-methylpiperidine (7e) afforded
compounds that exhibited potent AChE inhibition (IC50 val-
ues = 0.33–0.56
weak (7b, BuChE IC50 = 68.30
IC50 > 100 M) whereas, compounds 7c (AChE IC50 = 0.33
BuChE IC50 = 2.30 M) and 7e (AChE IC50 = 0.39 M; BuChE
IC50 = 2.90 M) exhibited dual inhibition of both AChE and BuChE.
l
M range). However, 7b and 7d exhibited either
M) or no inhibition of BuChE (7d,
M;
Cl
N
l
l
l
(i)
(i)
l
l
Cl
N
l
(5)
From the N-benzyl series (7a–e), 7c (N-benzyl-2-thiomorpholino-
pyrimid-4-amine) was identified as the most potent ChE inhibitor
(i)
(AChE IC50 = 0.33 lM; BuChE IC50 = 2.30 lM; Table 1) with supe-
rior AChE inhibitory potency and selectivity.
Amongst the N-phenethyl series (8a–e), 8a with a C-2 five-
NH
NH
NH
membered pyrrolidine substituent (AChE IC50 = 12.00
IC50 = 13.80 M) and 8e with a C-2 six-membered 4-methylpiper-
idine substituent (AChE IC50 = 7.90 M; BuChE IC50 = 17.70 M)
lM; BuChE
l
N
N
N
l
l
N
Cl
N
Cl
Cl
N
exhibited dual ChE inhibition with 8e exhibiting superior AChE
selectivity, although they were not as potent compared to the N-
benzyl counterpart 7e (Table 1). Interestingly, the presence of a
six-membered C-2 substituent such as a morpholine (8b), thi-
omorpholine (8c) and a 1-methylpiperazine (8d) led to a loss of
(6b)
(ii)
(6a)
(6c)
(ii)
(ii)
BuChE inhibition (BuChE IC50 > 100 lM, Table 1). In general, the
N-phenethyl derivatives (8a–e) exhibited decreased AChE inhibi-
tory potency compared to their N-benzyl counterparts (7a–e) and
the AChE inhibitory potency was dependent on the electronic
and steric parameters of C-2 substituents and the activity was of
the order: morpholine > 4-methylpiperidine > pyrrolidine > 1-
methylpiperazine > thiomorpholine.
NH
NH
NH
N
N
N
R1
R1
N
R1
N
N
Amongst the naphthyl methyl series (9a–d), 9a containing a C-2
(7a-e)
(9a-d)
(8a-e)
five-membered pyrrolidine substituent (AChE IC50 = 5.80
BuChE IC50 = 8.90 M; Table 1) exhibited dual ChE inhibition
whereas 9b (AChE IC50 = 15.70 M; BuChE IC50 = 28.0 M) and 9c
(AChE IC50 = 15.50 M; BuChE IC50 = 34.70 M) possessing a C-2
lM;
l
l
l
HN
HN
HN
l
l
HN
HN
R1 =
six-membered morpholine or a thiomorpholine substituent were
equipotent AChE inhibitors with weak BuChE inhibition. It was
interesting to note that compound 9d with a C-2 methylpiperazine
substituent exhibited 6.7-fold selectivity toward BuChE (AChE
N
S
O
(b)
Me
Me
(d)
(a)
(c)
(e)
IC50 = 17.60
not as potent relative to tacrine (AChE IC50 = 0.173
IC50 = 0.019 M; SI = 9.11; Table 1). These in vitro data indicate
that the ChE inhibition was also dependent on the volume of
l
M; BuChE IC50 = 2.60
l
M; SI = 6.77) although it was
Scheme 1. Reagents and conditions: (i) DIPEA, phenylmethanamine (6a) or
l
M; BuChE
2-phenylethanamine (6b) or (naphthalen-1-yl)methanamine (6c), EtOH,
0 to
70–80 °C, reflux 3 h; (ii) n-BuOH, R1 = pyrrolidine, morpholine, thiomorpholine,
1-methylpiperazine and 4-methylpiperidine, respectively, 145–150 °C, 30–40 min.
l

3608
T. Mohamed, P. P. N. Rao / Bioorg. Med. Chem. Lett. 20 (2010) 3606–3609
pyrimidine derivatives (7a–e, 8a–e and 9a–d) with the naphthyl
methyl derivatives (9a–d) exhibiting BuChE inhibition as well
due to their increased volume/size compared to N-phenethyl ser-
ies. Furthermore, theoretical log P values for the synthesized com-
pounds correlate well (C log P = 2.14–4.69 range; Table 1) with the
reference compound tacrine (C log P = 3.27) supporting their abil-
ity to reach the central nevous system (CNS).
The enzyme–ligand binding interactions of the most potent ChE
inhibitor, 7c (N-benzyl-2-thiomorpholinopyrimidin-4-amine, AChE
IC50 = 0.33 lM; BuChE IC50 = 2.30 lM; SI = 0.14), was investigated
by molecular modeling studies (Fig. 2).18 The docking study of 7c
within the active site of TcAChE (Fig. 2) indicates that the pyrimidine
ring was oriented closer to the catalytic triad (His440) at the bottom
of the active site and was stacked between Trp84 and Phe330 in the
cationic active site (CAS) (distance < 5 Å). This observation is consis-
tent with the binding pattern of the central pyridine ring in tacrine.7
The pyrimidine N-1 of 7c was about 2.68 Å away from NH of indole
(Trp84) and was about 2.88 Å away from backbone C@O (Gly441),
respectively. The C-2 thiomorpholine ring was oriented in a non-po-
lar region comprised of Gly80, Ser81, Trp84, Phe330, Leu333,
Tyr334, Trp432, Ile439, Met436 and Tyr442 (distance < 5 Å). The
nitrogen atom of thiomorpholine was undergoing a hydrogen-bond-
ing interaction with indole NH of Trp84 (distance = 3.05 Å) whereas
the distance between OH of Tyr442 and nitrogen atom of thi-
omorpholine was about 3.90 Å. The sulphur atom of thiomorpholine
was undergoing hydrophobic interactions with the side chains of
Met436 and Leu333 (distance < 5 Å). Interestingly, the sulphur atom
of thiomorpholine was also forming a weak hydrogen bond with OH
of Tyr334 (distance = 3.59 Å). The C-4 N-benzyl ring was oriented in
a hydrophobic region comprised of Gly117, Gly118, Gly123, Leu127
and Tyr130 closer to the ‘peripheral’ anionic site (PAS, Trp279)
(distance < 5 Å).
A similar docking experiment was conducted to investigate the
binding interactions of 7c within the active site of mammalian
BuChE (Fig. 3). The BuChE active site lacks the PAS present in AChE
and its gorge entry is lined by smaller aliphatic amino acid residues
whereas in AChE it is lined by bulkier aromatic amino acids. This
makes the volume of BuChE active site about 60% larger than that
of AChE.6,19 In contrast to TcAChE binding, the pyrimidine ring of 7c
binds in the centre of BuChE active site such that the C-4 N-benzyl
ring was oriented toward the catalytic triad (His438). The pyrimi-
dine was in a region comprised of polar amino acids such as Asp70,
Figure 3. Docking of N-benzyl-2-thiomorpholinopyrimidin-4-amine (7c) in the
active site of mammalian BuChE (PDB code: 1P0I). Hydrogen atoms are not shown
for clarity.
Ser79, Asn83 and Thr120 (distance < 5 Å). The N-1 of pyrimidine
was about 3.91 Å away from OH of Thr120. The C-2 thiomorpholine
ring was in a hydrophobic region closer to the active site gorge
comprised of Gly116, Gly117, Leu286 and Pro285 (distance < 5 Å).
The sulphur atom of the thiomorpholine ring was about 3.94 Å
away from backbone C@O of Pro285. The distance between
Ala277, which is present at the entrance of the active site, and C-
2 of the thiomorpholine ring was about 11.89 Å. In addition, The
C-4 N-benzyl substituent was in a hydrophobic region closer to
the CAS comprised of Trp82, Ala328, Phe329, Trp430, Met437,
His438 and Tyr440 (distance < 5 Å). These binding interaction
studies are consistent with the ChE inhibitory data obtained for
7c (Table 1).
In general, the presence of either a C-4 N-benzyl (7a–c and, 7e)
or naphthyl methyl (9a–d) substituent with varying C-2 groups
provided dual ChE (AChE/BuChE) inhibition. In contrast, the pres-
ence of a C-4 N-phenethyl substituent as in 8b–d led to a loss in
BuChE inhibition. These studies indicate that a non-fused pyrimi-
dine based ring template can be developed as a dual ChE inhibitor
by modulating the steric and electronic parameters at C-2 and C-4
positions.
In summary, the current study supports the hypothesis that (i) a
pyrimidine ring serves as a suitable template in the design of novel
ChE inhibitors; (ii) the ChE inhibition was sensitive to substituent
electronic and steric parameters at C-2 and C-4 position of the
pyrimidine ring and (iii) compound 7c (AChE IC50 = 0.33
lM;
BuChE IC50 = 2.30 M; SI = 0.14) with a C-4 N-benzyl substituent
l
and a C-2 six-membered thiomorpholine substituent was identi-
fied as a dual ChE inhibitor.
Acknowledgements
The authors thank the Department of Biology and the School of
Pharmacy at the University of Waterloo for supporting this re-
search project.
Figure 2. Docking of N-benzyl-2-thiomorpholinopyrimidin-4-amine (7c) in the
active site of TcAChE (PDB code: 1ACJ). Red lines represent hydrogen-bonding
interactions. Hydrogen atoms are not shown for clarity.

T. Mohamed, P. P. N. Rao / Bioorg. Med. Chem. Lett. 20 (2010) 3606–3609
3609
was placed in an oil bath at 145–150 °C and stirred for 30–40 min. The n-BuOH
was evaporated in vacuo and the residue was re-dissolved in a solvent mixture
of EtOAc and DCM in ꢀ3:1 ratio and washed successively with saturated
NaHCO3 and NaCl solution (1ꢁ 15 mL), respectively. The aqueous layer was
washed with EtOAc (3ꢁ 5 mL) and the organic layer was dried over anhydrous
MgSO4 then filtered. The solution was evaporated in vacuo and the residue
obtained was further purified as needed by a silica gel column chromatography
using EtOAc/hexanes (3:1) as eluent to afford the respective 2,4-disubstituted
pyrimidine derivatives (7a–e, 8a–e and 9a–d).
References and notes
1. (a) Farlow, M. R. Am. J. Health Syst. Pharm. 1998, 55, S5; (b) Holden, M.; Kelly, C.
Adv. Psychiatry Treat. 2002, 8, 89.
2. (a) Parihar, M. S.; Hemnani, T. J. Clin. Neurosci. 2004, 11, 456; (b) Klafki, H.;
Staufenbiel, M.; Kornhuber, J.; Wiltfang, J. Brain 2006, 129, 2840.
3. (a) Pakaski, M.; Kalman, J. Neurochem. Int. 2008, 53, 103; (b) Matharu, B.;
Gibson, G.; Parsons, R.; Huckerby, T. N.; Moore, S. A.; Cooper, L. J.; Millichamp,
R.; Allsop, D.; Austen, B. J. Neurol. Sci. 2009, 280, 49.
4. Shen, T.; Tai, K.; Henchman, R. H.; McCammon, J. A. Acc. Chem. Res. 2002, 35,
332.
5. Greig, N. H.; Utsuki, T.; Ingram, D. K.; Wang, Y.; Pepeu, G.; Scali, C.; Yu, Q.;
Mamczarz, J.; Holloway, H. W.; Giordano, T.; Chen, D.; Furukawa, K.;
Sambamurti, K.; Brossi, A.; Lahiri, D. K. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
17213.
6. Darvesh, S.; Hopkins, D. A.; Geula, C. Nat. Rev. Neurosci. 2003, 4, 131.
7. Harel, M.; Schalk, I.; Ehret-Sabatier, L.; Bouet, F.; Goeldner, M.; Hirth, C.;
Axelsen, P. H.; Silman, I.; Sussman, J. L. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9031.
8. Pang, P.; Quiram, P.; Jelacic, T.; Hong, F.; Brimijoin, S. J. Biol. Chem. 1996, 271,
23646.
9. Schott, Y.; Decker, M.; Rommelspacher, H.; Lehmann, J. Bioorg. Med. Chem. Lett.
2006, 16, 5840.
10. Darvesh, S.; Darvesh, K. V.; McDonald, R. S.; Mataija, D.; Walsh, R.; Mothana, S.;
Lockridge, O.; Martin, E. J. Med. Chem. 2008, 51, 4200.
Analytical data for N-benzyl-2-thiomorpholinopyrimidin-4-amine (7c): Yield,
77%; brown solid; mp 85–87 °C; IR (film): 3258 (NH) cmꢂ1
;
1H NMR
(300 MHz, CDCl3): d 2.54–2.58 (m, 4H, thiomorpholine S-CH2), 4.03–4.07 (m,
4H, thiomorpholine N-CH2), 4.47 (d, J = 5.6 Hz,
2H, CH2-NH), 5.03 (br s, 1H, CH2-NH), 5.66 (d, J = 5.7 Hz, 1H, pyrimidine H-5),
7.24–7.33 (m, 5H, benzyl H-2, H-3, H-4, H-5 and H-6), 7.84 (d, J = 5.7 Hz, 1H,
pyrimidine H-6); 13C NMR (75 MHz, CDCl3) d 26.76 (S-CH2), 45.11 (CH2-NH),
46.32 (N-CH2), 94.11 (pyrimidine C-5), 127.32, 127.37 and 128.60 (benzyl C-2,
C-3, C-4, C-5 and C-6), 138.76 (benzyl C-1), 155.94 (pyrimidine C-2), 160.90
(pyrimidine C-6), 162.97 (pyrimidine C-4); HREIMS Calcd for C15H18N4S (M+)
m/z 286.3952, found m/z 286.1872.
15. Adsersen, A.; Kjølbye, A.; Dall, O.; Jäger, A. K. J. Ethnopharmacol. 2007, 113, 179.
16. Giovanni, S. D.; Borloz, A.; Urbain, A.; Marston, A.; Hostettmann, K.; Carrupt, P.
A.; Reist, M. Eur. J. Pharm. Sci. 2008, 33, 109.
17. Cholinesterase inhibition assay: The ability of the test compounds (7a–e, 8a–e
and 9a–d) to inhibit electric eel AChE (product number C3389, Sigma, Ann
Arbor, MI) and equine serum BuChE (product number C1057, Sigma, St. Louis,
11. Darvesh, S.; Pottie, I. R.; Darvesh, K. V.; McDonald, R. S.; Walsh, R.; Conard, S.;
Penwell, A.; Mataija, D.; Martin, E. Bioorg. Med. Chem. 2010, 18, 2232.
12. Nugiel, D. A.; Cornelius, L. A. M.; Corbett, J. W. J. Org. Chem. 1997, 62, 201.
13. Fiorini, M. T.; Abell, C. Tetrahedron Lett. 1998, 39, 1827.
14. General procedure for the synthesis of 4-substituted-2-chloropyrimidin-4-amines
MO) was determined using Ellman’s method (IC50 values, lM). Stock solutions
of test compounds were dissolved in a minimum volume of DMSO (1%) and
were diluted using the buffer solution (50 mM Tris–HCl, pH 8.0, 0.1 M NaCl,
0.02 M MgCl2ꢃ6H2O). In 96-well plates, 160
acid) (1.5 mM DTNB), 50 l of AChE (0.22 U/mL prepared in 50 mM Tris–HCl,
pH 8.0, 0.1% w/v bovine serum albumin, BSA) or 50 l of BuChE (0.12 U/mL
prepared in 50 mM Tris–HCl, pH 8.0, 0.1% w/v BSA) were incubated with 10
of various concentrations of test compounds (0.001–100 M) at room
temperature for 5 min followed by the addition of the substrates (30 l)
l
l 5,50-dithiobis(2-nitrobenzoic
(6a–c): To
a mixture of 5 (2 g, 13.4 mmol) and the respective amine
l
[13.4 mmol, phenylmethanamine, 2-phenylethanamine or (naphthalen-1-
yl)methanamine, respectively] in 25 mL of EtOH, kept at 0 °C (ice-bath),
DIPEA (2.43 mL, 14.7 mmol) was added. The reaction was allowed to stir on the
ice-bath for 5 min and was refluxed at 75–80 °C for 3 h. After cooling to 25 °C,
the EtOH was evaporated in vacuo and the residue was re-dissolved in a
solvent mixture of EtOAc and dichloromethane (DCM) in ꢀ3:1 ratio and
washed successively with saturated NaHCO3 and NaCl solution (1ꢁ 15 mL),
respectively. Aqueous layer was washed with EtOAc (3ꢁ 15 mL) and the
combined organic layer was dried over anhydrous MgSO4 then filtered. The
organic layer was evaporated in vacuo and the resulting solid or oily residue
was further purified by silica gel column chromatography using EtOAc/hexanes
(3:1) as eluent to afford the respective intermediate (6a–c).
General procedure for the synthesis of 2,4-disubstituted-pyrimidin-4-amines
(7a–e, 8a–e and 9a–d): To a solution of 6a–c (0.9–1.0 mmol) in 3 mL of
n-BuOH kept in a pressure vessel (PV) with stirring, the respective secondary
amine (1.20 mmol, pyrrolidine, morpholine, thiomorpholine, 1-methyl
piperazine or 4-methylpiperidine, respectively) was added. The sealed PV
l
ll
l
l
acetylthiocholine iodide (15 mM ATCl) or S-butyrylthiocholine iodide (15 mM
BTCI) and the absorbance was measured at different time intervals (0, 60, 120
and 180 s) at a wavelength of 405 nm. Percent inhibition was calculated by the
comparison of compound-treated to various control incubations. The
concentration of the test compound causing 50% inhibition (IC50, lM) was
calculated from the concentration-inhibition response curve (duplicate to
quadruplicate determinations).
18. Yu, G.; Rao, P. N. P.; Chowdhury, M. A.; Abdellatif, K. R.; Dong, Y.; Das, D.;
Velázquez, C. A.; Suresh, M. R.; Knaus, E. E. Bioorg. Med. Chem. Lett. 2010, 20,
2168.
19. Saxena, A.; Redman, A. M. G.; Jiang, X.; Lockridge, O.; Doctor, B. P. Biochemistry
1997, 36, 14642.