Synthesis and evaluation of N-substituted 2-amino-4,5-diarylpyrimidines as selective adenosine A1receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2016.09.081

We report the synthesis and biological evaluation of new 2-amino-4,5-diarylpyrimidines as selective antagonists at the adenosine A₁receptor. The scaffold they are based upon is a deaza variation of a previously reported collection of 3-amino-5,6-diaryl-1,2,4-triazines, members of which had a subnanomolar affinity but limited selectivity over the A_2Asubtype. Initially, similar structure-affinity relationships at the 5-aryl ring were established, and then emphasis was put on increasing selectivity at the hA₁AR by introducing substituents on the N²-position, all the while maintaining a nanomolar affinity. Compound 3z, bearing a trans 4-hydroxycyclohexyl substituent, was identified as a potent (K_i(hA₁AR) = 7.7 nM) and selective (K_i(hA_2AAR) = 1389 nM) antagonist at the human adenosine A₁receptor. Computational docking was effected at the A₁and A_2Asubtypes, rationalizing the effect of the 4-hydroxycyclohexyl substituent on selectivity, in relation with the nature of the substituent on the 5-position of the pyrimidine.

Full text of DOI:10.1016/j.ejmech.2016.09.081

Accepted Manuscript
Synthesis and evaluation of N-substituted 2-amino-4,5-diarylpyrimidines as selective
adenosine A receptor antagonists
1
Georgios Alachouzos, Eelke B. Lenselink, Thea Mulder-Krieger, Henk de Vries,
Adriaan P. IJzerman, Julien Louvel
PII:
S0223-5234(16)30818-2
10.1016/j.ejmech.2016.09.081
EJMECH 8947
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 May 2016
Revised Date: 4 September 2016
Accepted Date: 25 September 2016
Please cite this article as: G. Alachouzos, E.B. Lenselink, T. Mulder-Krieger, H. de Vries, A.P. IJzerman,
J. Louvel, Synthesis and evaluation of N-substituted 2-amino-4,5-diarylpyrimidines as selective
adenosine A receptor antagonists, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
1
j.ejmech.2016.09.081.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and evaluation of N-substituted 2-amino-
4
,5-diarylpyrimidines as selective Adenosine A1
receptor antagonists
Georgios Alachouzos, Eelke B. Lenselink, Thea Mulder-Krieger, Henk de Vries, Adriaan P.
IJzerman and Julien Louvel*
Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden
University, P.O. Box 9502, 2300 RA Leiden (The Netherlands). *E-mail:
j.a.louvel@lacdr.leidenuniv.nl
6
ABBREVIATIONS: ADA, adenosine deaminase; Ac, acetyl; BCA, Bicinchoninic acid; BSA, bovine serum albumin; CPA, N -
cyclopentyladenosine; CHO, Chinese hamster ovary; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; DPCPX, 1,3-dipropyl-8-
cyclopentyl-xanthine; DTT, dithiothreitol; EC50, concentration of unlabelled ligand which elicits 50% of the maximum response (Emax) in a
functional assay at membranes of CHO cells stably expressing the adenosine A
maximum response elicited by an unlabelled ligand in a functional assay (relatively to CPA) at membranes of CHO cells stably expressing the
adenosine A receptor; GDP, guanosine diphosphate; GF, glass filter; GTP, guanosine triphosphate; GTPγS, guanosine 5'-O-[γ-thio]triphosphate;
HPLC, high-pressure liquid chromatography; IC50, concentration of unlabelled ligand which displaces 50% of [ H]DPCPX binding to membranes
of CHO cells stably expressing the adenosine A receptor; K , affinity of ligand; KRI, kinetic rate index; NMR, nuclear magnetic resonance; SAR,
structure-affinity relationships; THF, tetrahydrofuran; TLC, thin layer chromatography; Tris, tris(hydroxymethyl)aminomethane.
1
receptor; EDTA, ethylenediaminetetraacetic acid; Emax,
1
3
1
i
1

ACCEPTED MANUSCRIPT
ABSTRACT
We report the synthesis and biological evaluation of new 2-amino-4,5-diarylpyrimidines as
selective antagonists at the adenosine A receptor. The scaffold they are based upon is a deaza
1
variation of a previously reported collection of 3-amino-5,6-diaryl-1,2,4-triazines, members of
which had a subnanomolar affinity but limited selectivity over the A2A subtype. Initially, similar
structure-affinity relationships at the 5-aryl ring were established, and then emphasis was put on
2
increasing selectivity at the hA AR by introducing substituents on the N -position, all the while
1
maintaining a nanomolar affinity. Compound 3z, bearing a trans 4-hydroxycyclohexyl
substituent, was identified as a potent (K (hA AR) = 7.7 nM) and selective (K (hA AR) = 1389
i
1
i
2A
1
nM) antagonist at the human adenosine A receptor. Computational docking was effected at the
A
1
and A2A subtypes, rationalizing the effect of the 4-hydroxycyclohexyl substituent on
selectivity, in relation with the nature of the substituent on the 5-position of the pyrimidine.
1
Key words: Structure-affinity relationships; Adenosine A receptor; Adenosine A2A receptor;
selectivity; antagonists.
INTRODUCTION
The adenosine receptors are cell membrane-bound proteins belonging to the superfamily of G
protein-coupled receptors (GPCRs). Their physiological and clinical relevance has been
1 3
extensively studied, and all four subtypes (A , A2A, A2B and A ) have been suggested as targets
1
for the treatment of various diseases. In the past 35 years, a plethora of synthetic ligands that
selectively bind to a determined number of adenosine receptor subtypes have been developed,
which show a wide range of functional profiles of partial or full agonists and antagonists/inverse
agonists. Among those, the 6 examples depicted in Figure 1 are high affinity antagonists at the
2

ACCEPTED MANUSCRIPT
human adenosine A
1
1
receptor (hA AR), and display a selectivity of at least 100-fold against the
2
other subtypes.
N
N
O
N
O
N
N
H
N
N
H
N
H
N
O
N
O
Figure 1. Examples of previously reported selective hA
1
AR antagonists.
Recently, Heptares Therapeutics reported the synthesis and evaluation of 3-amino-5,6-diaryl-
1
,2,4-triazines, a new scaffold in the field of adenosine receptors, as small molecule antagonists
3
for the A AR. A few of these compounds had subnanomolar affinities at the A AR, but also
2
A
2A
at the A AR, the best selectivities ranging from 62-fold at the A AR to 91-fold at the A AR
1
2A
1
(
Figure 2). We decided to explore the possibility to increase the selectivity for the A
1
AR through
chemical modifications on a similar scaffold, mainly by introducing substituents on the exocyclic
amino group, an option which had not been explored on such a scaffold, but had borne its fruits
2
in other series of hA AR antagonists.
1
3

ACCEPTED MANUSCRIPT
Figure 2. 3-amino-5.6-diaryl-1,2,4-triazines reported by Heptares Therapeutics.
In the current study we report the synthesis and evaluation of 2-amino-4,5-diarylpyrimidines as
selective A AR antagonists. Some of these compounds are 4-deaza analogues of 3-amino-5,6-
1
diaryl-1,2,4-triazines reported by Heptares Therapeutics. They were evaluated in radioligand
displacement binding assays. Among the approximately 50 compounds synthesized partial
inverse agonist 3z stood out with high affinity and good selectivity for the human adenosine A1
receptor.
RESULTS AND DISCUSSION
Design and Synthesis.
The compounds were synthesized through a general approach which made use of the
corresponding diarylethanones 6a-u and 8, most of which have been described and were readily
4
available. For those previously unreported, phenylacetic acids 4o and 4p were converted to the
5
corresponding acyl chlorides in the presence of SOCl
2
, and in a subsequent step to the
6
corresponding Weinreb amides 5o and 5p, which were then treated with an excess of
phenylmagnesium bromide to afford 6o and 6p. Alternatively, 6t was obtained by deprotonation
4

ACCEPTED MANUSCRIPT
of 2-cyclopropyl-4-methylpyridine and subsequent treatment wih N-methoxy-N-
7
8
methylbenzamide. Diarylethanones 6a−6u and 8 were then reacted with DMF-dimethylacetal
9
to afford oxoenamines 7a−7u and 9, which were in turn cyclized into compounds 1a−1u and 2
1
0
in the presence of guanidine. Compounds 1v−1x bearing hydroxyl substituents were obtained
from the corresponding methoxy-bearing compounds after treatment with excess BBr3.
Compound 1y was obtained from corresponding benzyloxy compound 1p through catalytic
hydrogenolysis (Scheme 1).
Scheme 1. Synthesis of compounds 1a−y and 2. 6o, 6p and 6t are previously unreported.
OH
N
a)
O
O
O
RO
RO
4
4
o (R = Me)
p (R = Bn)
Cl
Cl 5o (R = Me)
5
p (R = Bn)
b)
NH2
N
N
N
Ph
Ph
c)
R1
d)
R1
e)
O
O
R1
N
6
a-6u
7a-u
1
a-u
(
see experimental section)
(
see Table 1)
NH2
N
NH2
NH2
NH2
N
N
N
N
N
N
N
f)
g)
(OMe)n
(OH)n
Cl
Cl
1j-l
1v-x
see Table 1)
OBn
OH
1y
(
1p
NH2
N
N
O
N
O
d)
e)
O
O
O
8
9
2
a
Reagents and conditions: a) i. SOCl
2 2 2
, DMF cat., CH Cl , rt, 16 h; ii. MeONHMe.HCl, pyridine,
CH Cl , 0 °C to rt, 16 h; b) PhMgBr, THF, −50 °C to −30 °C, 3 h; c) i. LDA, THF, −30 to −10
2
2
°
C, 1 h; ii. BzN(OMe)Me, THF, −50 °C to rt, 3 h; d) Me NCH(OMe) , 80 °C, 16 h; e)
2 2
5

ACCEPTED MANUSCRIPT
Guanidine.HCl, K CO , EtOH, 80 °C, 16 h; f) BBr , CH Cl , −78 °C to rt, 16 h; g) H , Pd/C,
2
3
3
2
2
2
MeOH:THF 3:4, rt, 3 h.
Compounds 3a−3u, bearing a substituted amine, were synthesized following two main strategies.
The amides were introduced by acylation of 1f, whereas the amines were obtained from enamine
2
7
f, except for 3f (N-Ph substituent). More precisely, compounds 3g, 3h and 3r (R = cPent, 2,2-
diphenylethyl and 4-hydroxybutyl respectively) were obtained through cyclization of 7f in the
presence of the corresponding N-alkyl guanidine. 7f was also transformed into sulfoxide 11 via a
1
1
sequence consisting of cyclization and methylation to sulfide 10, followed by oxidation with
Davis’ oxaziridine; treatment of 11 with an excess of the required primary amine afforded 3i−3q
1
2
and 3s-3v. 3f was obtained from 1f via a Buchwald−Hartwig coupling with bromobenzene
1
0
(Scheme 2).
Scheme 2. Synthesis of compounds 3a−v.
a
Reagents and conditions: a) RCOCl, pyridine, 200 °C (µW), 2 h; b) PhBr, Pd
2
(dba)
3
, XPhos,
2
Cs
2
CO
3
, dioxane, 150 °C (µW), 70 min; c) N-R guanidine.HCl, K
2
CO
3
, THF, 75 °C, 16 h; d)
6

ACCEPTED MANUSCRIPT
thiourea, KOt-Bu, EtOH, 80 °C, 1 h, then MeI, CH
CH Cl , 0 °C to rt, 16 h; f) R NH , THF, 60 °C, 16 h.
2 2
Cl , 0 °C, 30 min; e) Davis’ oxaziridine,
2
2
2
2
Biology. All the compounds were tested in a radioligand displacement assay on the adenosine
3
13
A receptor in the presence of 2.5 nM [ H]DPCPX. Compounds that displaced more than 80%
1
of the radioligand at a concentration of 1 µM (yielding an estimated Ki value of at least 300 nM)
were submitted to a full concentration range assay, through which their affinity was determined.
Subsequently, compounds with high A AR affinity (K < 100 nM) were also tested in a
1
i
3
14
displacement assay at hA AR using [ H]ZM241385 as the radioligand. Compound 3z was
2
A
also tested at the adenosine A2B and A receptor for a full selectivity profile, and further
3
3
5
15
evaluated in a [ S]GTPγS binding assay for its functional characteristics. Since binding
1
6
kinetics are getting more importance in early-stage drug discovery, compounds with a
3
displacement of [ H]DPCPX above 80% were also screened for their KRI value. This is a quick
and straightforward indication of the dissociation kinetics of a compound, in which values below
unity represent compounds that dissociate faster than the radioligand, while values above unity
1
7
represent compounds that dissociate slower. The most outspoken compounds in this study
displayed KRI values in the 1.2−1.4 range, corresponding to a residence time at the receptor in
1
7a
the 11−17 min range.
Structure−Affinity Relationships.
Compounds 1a, 1b, 1d−1j, 1n, 1q, 1t, 1u, 1w and 1y are deaza analogues of compounds
reported by Heptares Therapeutics, and 7 among the original 1,2,4-triazines have reported
1
2
affinities (R = Ph and R = Ph, 3-Cl-Ph, 3-CF -Ph, 2-naphthyl, 2-cPr-4-pyridyl, 4-OH-Ph and
3
3
-Cl-4-OH-Ph) (Table 1, last column). When comparing the affinity values, there is no
significant difference between the 1,2,4-triazine and the pyrimidine scaffolds, except in the case
7

ACCEPTED MANUSCRIPT
2
of 1w (R = 4-OH-Ph), for which the affinity is identical at both receptor subtypes, whereas the
1
,2,4-triazine analogue shows a greatly reduced affinity at hA AR (K = 214 nM vs 1.4 nM).
1
i
1) 2-amino unsubstituted derivatives (Table 1).
1
a. Aromatic ring at the 5-position (R = Ph).
The unsubstituted diphenyl derivative 1a only displaced 32% of the radioligand at hA AR;
1
adding a chloro substituent in the para position led to an even worse result (1g, 41%
displacement), whereas it gave a great increase at the meta position (1f, K = 55 nM) with
virtually no selectivity over hA2AAR. Replacing the chloro substituent with an isosteric methyl
group gave similar results (1c, 51% displacement; 1b, K = 205 nM). The same trend was
observed with trifluoromethyl substituents, with the para derivative displaying a low affinity (1r,
1% displacement), while the meta derivative shows high affinity (1r, K = 14 nM), albeit also at
i
i
5
i
the hA AR. Similar results were obtained with a methoxy substituent, with the meta-substituted
2
A
ligand outperforming the para-substituted one (1j, K = 54 nM vs 1k, 56% displacement); worthy
i
of note is that the ortho derivative yielded the lowest affinity (1i, 20% displacement). Changing
from chloro to fluoro gave opposite results, with the 4-fluoro derivative showing appreciable
i
affinity (1e, K = 160 nM) and the 3-fluoro derivative only partially displacing the radioligand at
a concentration of 1 µM (1d, 63% displacement). This seems to suggest that a polar substituent is
needed at the 4-position, whereas a more lipophilic substituent is favored at the 3-position.
Indeed, the hydroxyl derivatives confirmed this trend, with the 4-OH ligand displaying a
nanomolar affinity (1w, K = 1.4 nM) (and no selectivity over hA AR) while the 3-OH
i
2A
compound showed a 100-fold decrease (1v, K = 144 nM); the 4-pyridyl analogue behaved
i
i
similarly (1s, K = 17 nM).
8

ACCEPTED MANUSCRIPT
The addition of a second substituent was then studied. Combining chloro substituents at the 3
and 4 positions did not prove deleterious compared to the mono substitution at the 3 position (1h,
K = 38 nM vs 1f, K = 55 nM). The same effect was observed with methoxy susbtituents (1l, K =
i
i
i
1
9
04 nM vs 1j, K = 54 nM), and also with a meta chloro and a para methoxy substituent (1o, K =
i i
5 nM). This seems to indicate that 4-chloro and 4-methoxy substituents, which do not lead to a
change in affinity when compared to the unsubstituted 1a, are also innocuous on the effect of an
adjacent substituent. Replacing the methoxy group with a larger benzyloxy group led to a drop in
affinity (1p, 38% displacement vs 1o, K
i
= 95 nM), probably due to steric clashes with residues
of the binding pocket. The 3,4-dihydroxy substituted compound displayed an affinity that was
1
1
0-fold higher than the 3-OH compound, but 10-fold lower than its 4-OH counterpart (1x, K =
i
2 nM vs 1v and 1w). Disubstituting the same two positions with a cyclic methylenedioxy group,
on the other hand, led to a significant decrease in affinity (1n, 74% displacement). Changing the
2
methylenedioxy ring for a phenyl ring (R = naphthalen-2-yl) resulted in a good affinity (1u, K =
i
1
8 nM). Combining the 3-chloro substituent with the 4-hydroxy moiety gave the best affinity of
this series of compounds (1y, K = 0.46 nM), which was also observed in the 1,2,4-triazine series.
i
Finally, adding a third methoxy substituent on the 5-position canceled the beneficial effect due
to the methoxy group on the 3-position (1m, 56% displacement).
2
b. Aromatic ring at the 4-position (R = Ph).
Finally, replacing the phenyl ring at the 4-position with a 2-furyl ring, which had given good
1
8
results in a cyclized 2-amino-4,6-diarylpyrimidine series, led to a greatly decreased affinity (2,
2
6% displacement vs 1a, 56% displacement). This seems to indicate a significantly different
binding pose of the present 2-amino-4,5-diarylpyrimidines as compared to 2-amino-4,6-
diarylpyrimidines.
9

ACCEPTED MANUSCRIPT
1
Table 1. Affinity of compounds 1a-1y and 2 at the human A AR and A2AAR.
A
2AAR K
i
A AR Ki
nM) ± SEM
or % disp. at
K of aza
analogue at
1
i
KRI
at
A AR
1
(nM) ±
SEM or
(
1
2
R
R
b
A AR/A AR
1
2A
% disp.d
a
e
1
µM
2%
28; 36)
205 ± 21
1%
49; 53)
3%
62; 65)
(nM)
at 1 µM
3
c
c
1
a
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
3-Me-Ph
-
-
275/117
(
0.89
c
1
b
-
-
(
0.97; 0.81)
5
c
c
1
c
4-Me-Ph
-
-
-
(
(
6
c
c
1
d
3-F-Ph
-
-
-
0
.91
0.83; 0.99)
.96
c
1
e
4-F-Ph
160 ± 48
55 ± 19
-
-
(
(
0
1
f
3-Cl-Ph
120 ± 7.6
56/51
0.96; 0.96)
4
1%
c
c
1
g
4-Cl-Ph
-
-
-
(38; 44)
0.97
41%
(42 ; 39)
1
h
3,4-diCl-Ph
2-OMe-Ph
3-OMe-Ph
4-OMe-Ph
3,4-diOMe-Ph
3,4,5-triOMe-Ph
38 ± 9.5
0%
-
(
(
(
1.02; 0.93)
2
c
c
1
i
-
-
-
(
22; 18)
54 ± 3.6
6%
0.94
154 ±
10.5
1
j
-
0.94; 0.95)
5
c
c
1
k
-
-
-
(
56; 56)
104 ± 2.5
6%
0.93
c
1
l
-
-
1.05; 0.81)
5
c
c
1
m
-
-
-
(
55; 57)
4%
73; 74)
7
c
c
1
n
3,4-OCH
2
O-Ph
-
-
-
(
0.91
1
o
p
q
3-Cl-4-OMe-Ph
3-Cl-4-OBn-Ph
95 ± 3.0
251 ± 15
-
-
(
(
0.91; 0.91)
3
8%
c
c
1
-
-
(25; 50)
0.85
1
3-CF
3
-Ph
14 ± 0.90
98 ± 5.7
15/20
0.80; 0.90)
1
0

ACCEPTED MANUSCRIPT
5
1%
c
c
1
r
Ph
Ph
Ph
4-CF
3
-Ph
-
-
-
(
49; 54)
0
.89
0.85; 0.92)
.0
1.0; 1.0)
.1
1
s
4-pyridyl
17 ± 0.7
108 ± 4
36 ± 1.0
129 ± 9.3
-
(
1
1
t
2-cPr-4-pyridyl
1.76 ± 0.12
0.45/35
(
(
1
1
u
Ph
Ph
Ph
naphthalen-2-yl
3-OH-Ph
18 ± 1.1
144 ± 19
1.4 ± 0.07
3.0/102
-
1.0; 1.1)
c
c
1
v
-
-
0
.95
0.90; 1.0)
.90
1.0; 0.81)
.87
1
w
4-OH-Ph
1.3 ± 0.06
4.7 ± 1.17
1.3 ± 0.07
214/1.4
(
0
1
1
x
y
Ph
Ph
3,4-diOH-Ph
3-Cl-4-OH-Ph
Ph
12 ± 2.3
-
(
0
0.46 ± 0.08
0.14/1.4
-
(
0.83; 0.91)
2
6%
c
c
2
a
2-furyl
-
-
(
30; 22)
K ± SEM (n = 3) or % displacement (n ; n ) (n = 2), obtained from radioligand binding assays
i
1
2
3
b
with [ H]DPCPX on CHO cell membranes stably expressing human adenosine A receptors.
KRI (n ; n ) (n = 2), obtained from dual-point competition association assays with [ H]DPCPX
on CHO cell membranes stably expressing human adenosine A receptors. Not determined. %
displacement at 1 µM concentrations of specific [ H]ZM241385 binding on HEK 293 cell
1
3
1
2
c
d
1
3
e
membranes stably expressing human adenosine A2A receptors. Values from ref. 3.
2) 2-amino substituted derivatives (Table 2).
2
Next the introduction of a substituent at the N position was studied. The ligand bearing a 3-
chloro substituent was selected from the first series, because it showed a good affinity at hA AR
1
and allowed a straightforward, high-yielding synthesis of the required diarylethanone 6f through
a simple Friedel-Crafts acylation of 3-chlorophenylacetylchloride, which was readily available
from cheap 3-chlorophenylacetic acid (see Design and synthesis).
a. Amide substituents.
Five derivatives were synthesized, bearing substituents that had given good results in previous
1
5,19
studies on 2-amino-4,6-diarylpyrimidines and substituted azapurines.
Out of the linear alkyl
substituents, only the n-propyl substituted compound had an acceptable affinity of 106 nM (3a),
whereas the homologous n-butyl substituent led to a significant drop (3b, 52% displacement).
1
1

ACCEPTED MANUSCRIPT
All the branched and cyclic alkyl substituents failed to displace the radioligand more than 60% at
a 1 µM concentration (3c, 56%; 3d, 52%; 3e, 47%).
b. Amine substituents.
Various substituents were introduced on the nitrogen atom, most of which were inspired by
2
previous work carried out on A
1
AR ligands. Introducing an aromatic substituent led to a loss of
affinity (3f, R = Ph, 34% displacement), and adding methylene spacers did not bring any
improvement (3t, R = CH Ph, 16% displacement; 3u, R = CH CH Ph, 42% displacement; 3v, R
2
2
2
=
CH CH CH Ph, 33% displacement; 3h, R = CH CHPh , 25% displacement). Small linear
2 2 2 2 2
substituents led to a displacement of the radioligand of around 75% (3i, R = Me and 3k, R =
CH C≡C), and so did the smallest branched isopropyl 3j. Longer and more sterically hindered
2
carbon chains progressively decreased the affinity (3l, R = n-Bu, 56% displacement and 3m, R =
i-Pent, 39% displacement). Introducing a cyclopentyl substituent, known for bringing affinity
2
and selectivity in various series of A
1
AR antagonists, restored affinity (3g, K
i
= 94 nM) and
showed significant selectivity over the A2A receptor (23% displacement); however, expanding
the ring to a cyclohexane abolished that effect (3n, 58% displacement). Since only small
lipophilic substituents seemed to be the least deleterious to affinity, the decision was made to
introduce more hydrophilic groups. While an N,N-dimethyl-2-aminoethyl group led to a
displacement similar to the isosteric isopentyl (3s, 52% displacement), the introduction of
terminal alcohols greatly improved the affinity, with an optimal chain length of 3 methylene
units (3q, K
i
= 59 nM vs 3r, K
i
= 130 nM and 3p, K
i
= 180 nM); this modification also increased
3
the subtype selectivity, since 3q and 3r only displaced 9.5% and 13% of [ H]ZM241385
(
hA AR) at a concentration of 1 µM, respectively. Finally, introducing a trans 4-
2
2
0
hydroxycyclohexyl substituent, also present in compounds such as SLV320, gave a similar
1
2

ACCEPTED MANUSCRIPT
affinity (3o, K
i
= 30 nM) and an increase in selectivity, with 3o barely displacing the radioligand
from the A2A receptor. Taken together, the results obtained with these last 4 compounds suggest
2
that the maximal and optimal distance between the nitrogen atom at the N position and the
oxygen of the hydroxyl group is around 6 Å (dN-O(3q) = 4.9 Å < dN-O(3o) = 5.7 Å < dN-O(3r) =
2
1
6
.2 Å).
Table 2. Affinity of compounds 3a-3v at the human A AR and A AR.
1
2A
A AR K (nM) ±
KRI
at
A AR K (nM) ±
2A i
SEM or % disp.
1
i
R
SEM or % disp.
a
b
d
at 1 µM
A
1
AR
at 1 µM
c
c
3
a
C(O)nPr
C(O)nBu
C(O)iBu
C(O)cBu
C(O)cPent
Ph
106 ± 35
52% (54; 50)
56% (52; 59)
52% (50; 54)
47% (48; 46)
34% (37; 31)
94 ± 4.0
25% (24; 26)
75% (73; 76)
71% (71; 72)
76% (76; 75)
56% (55; 56)
39% (38; 41)
58% (56; 61)
30 ± 8.5
-
-
-
-
-
c
c
3b
-
c
c
3
c
-
c
c
3d
-
c
c
3
3
e
f
-c
-
c
-
-
3g
cPent
1.1 (1.1; 1.2)
23% (28; 19)
c
c
3h
CH
2
CHPh
2
-
-
c
c
3
i
Me
-c
-
c
3
j
iPr
-
-
-
-
-
c
c
3
k
CH C≡C
-
2
c
c
3
l
nBu
-
c
c
3
3
m
n
iPent
cHex
-
c
c
-
-
3
3
3
3
3
3
o
p
q
r
s
trans 4-OH-cHex
CH CH OH
CH CH CH OH
CH CH CH CH OH
CH CH NMe
CH Ph
CH CH
CH CH
1.0 (1.0; 1.0)
1.4% (−2.0; 4.9)
c
c
2
2
180 ± 19
-
-
59 ± 9.4
130 ± 3.5
52% (53; 52)
16% (16; 16)
42% (41; 43)
33% (32; 33)
0.88 (0.86; 0.90)
9.5% (11; 7.8)
13% (6.0; 20)
2
2
2
c
-
-
-
-
2
2
2
2
c
c
-
2
2
2
c
c
t
2
-
-
c
c
3u
2
2
Ph
2
c
c
3v
2
2
CH
Ph
-
-
a
K ± SEM (n = 3) or % displacement (n ; n ) (n = 2), obtained from radioligand binding assays
i
1
2
3
with [ H]DPCPX on CHO cell membranes stably expressing human adenosine A receptors.
1
1
3

ACCEPTED MANUSCRIPT
b
3
KRI (n
1 2
; n ) (n = 2), obtained from dual-point competition association assays with [ H]DPCPX
c
d
on CHO cell membranes stably expressing human adenosine A
displacement (n ; n ) (n = 2) at 1 µM concentrations of specific [ H]ZM241385 binding on HEK
1
receptors. Not determined. %
3
1
2
2
93 cell membranes stably expressing human adenosine A2A receptors.
It seemed logical to combine the best features – a 3-chloro-4-hydroxyphenyl, a 2-cyclopropyl-
2
4
-pyridyl or a 2-naphthalenyl at the 5 position and a trans 4-cyclohexanol at the N position −
into three final compounds, that would retain the high affinity of 1u, 1x and 1y all the while
displaying the same selectivity as 3o. 3x-3z were synthesized by cyclization of 7o, 7t and 7u
with in situ generated corresponding N-substituted guanidine hydroiodide 13, followed by
demethylation of the obtained 3w in the case of 3x (Scheme 3).
1
4

ACCEPTED MANUSCRIPT
Scheme 3. Synthesis of compounds 3x-3z.
N
OH
Cl
Ph
HN
O
O
N
N
b)
c)
7o
Cl
OH
3
x
OH
N
Ph
HN
OH
N
O
N
N
b)
S
a)
7t
HN
H2N
H2N
NH.HI
NH.HI
1
2
1
3
N
3
y
OH
HN
b)
N
N
N
Ph
O
7
u
3
z
a
Reagents and conditions: a) trans-4-aminocyclohexanol, THF, 78 °C, 3 h; b) K CO , EtOH,
2
3
8
0 °C, 16 h; c) BBr , CH Cl , −78 °C to r.t., 6 h.
3
2
2
1
3x-3z showed a high affinity at hA AR, similar or even better than the affinity of the
unsubstituted parent compounds. The selectivity over hA2AAR was low in the case of 3x (13-
fold), and improved in the case of 3y (61-fold); it was much better for 3z (180-fold). Worthy of
note was also the variation in selectivity as compared to the unsubstituted parent pyrimidine,
which was not identical for all three compounds: it went from barely 3-fold for 3y vs 1t up to 25-
fold for 3z vs 1u. 3z was then further tested in single-point displacements assays at the remaining
1
5

ACCEPTED MANUSCRIPT
two adenosine receptor subtypes: while it showed a significant displacement of 48% of
3
[
H]PSB603 from the hA AR at a concentration of 1 µM (thus giving it an IC of roughly 1
2B 50
3
µM), it showed virtually no affinity at the hA AR (Table 3).
Table 3. Affinity and functional data of compounds 3o, 3x−3z.
KRI
at
A AR
A AR
3
1
A AR
K (nM)
i
A AR
K (nM)
i
A AR
displ. (%)
A AR
Emax (%)
1
2A
2B
1
a
c
d
displ.
EC50g
h
b
f
A
1
AR
.0
1.0; 1.0)
.4
1.4; 1.4)
.2
1.2; 1.2)
.3
1.2; 1.3)
(%)
(nM)
1
1.4%
(−2.0; 4.9)
27
(23; 30)
3
3
3
3
o
x
y
30 ± 8.5
0.55 ± 0.02
1.06 ± 0.06
7.7 ± 0.10
278 ± 175
-11 ± 2
(
(
(
(
(2; 4)
1
e
e
e
e
7.2 ± 0.13
-
-
-
-
1
e
e
e
e
65 ± 5.1
-
-
-
-
1
48
(50; 46)
9
3
z
a
1389 ± 101
166 ± 92
−8 ± 0.8
(10; 8)
3
K ± SEM (n = 3) obtained from radioligand binding assays with [ H]DPCPX on CHO cell
membranes stably expressing human adenosine A receptors. KRI (n ; n ) (n = 2), obtained from
dual-point competition association assays with [ H]DPCPX on CHO cell membranes stably
expressing human adenosine A receptors. K ± SEM (n = 3) obtained from radioligand binding
assays with [ H]ZM241385 on HEK293 cell membranes stably expressing human adenosine A
receptors. % displacement (n ; n ) (n = 2) at 1 µM concentrations of specific [ H]PSB603
binding on CHO cell membranes stably expressing human adenosine A2B receptors. Not
determined. % displacement (n ; n ) (n = 2) at 1 µM concentrations of specific [ H]PSB11
i
b
1
1
2
3
c
1
i
3
2
A
d
3
1
2
e
f
3
1
2
binding on CHO cell membranes stably expressing human adenosine A receptors.
3
g
35
Concentration causing half-maximal stimulation of specific binding of [ S]GTPγS in CHO cell
membranes expressing human adenosine A receptors (n = 4). Maximum specific binding of
h
1
3
5
[
S]GTPγS in CHO cell membranes expressing human adenosine A receptors, with 0% being
1
the binding in the absence of any ligand (buffer) and 100% being the reference agonist CPA at a
−5.5
concentration of 10 M (n = 4).
1
6

ACCEPTED MANUSCRIPT
3
5
Compounds 3o and 3z were also tested in a [ S]GTPγS functional assay, and were
characterized as partial inverse agonists (relative to DPCPX, and with CPA being set at 100%)
with fair potency (Table 3 and Figure 3).
Figure 3. Functional profile of 3o, 3z, reference inverse agonist DPCPX and reference agonist
3
5
CPA, expressed as [ S]GTPγS binding in CHO cell membranes stably expressing human
3
5
adenosine A receptors (n=3). The curves were obtained by co-incubating [ S]GTPγS and
1
different concentrations of the ligands. The maximum response of CPA was set at 100%. Please,
note the different scales on the two halves of the Y-axis.
Computational study and discussion
In order to rationalize the observed differences in selectivity between 3x and 3z, a docking study
was performed in both the hA AR and hA AR. The 3UZC structure of the A receptor
1
2A
2A
published by Heptares Therapeutics was used as such and to build a homology model of the A1
3
a
receptor, which was subsequently optimized by induced-fit docking. In hA AR, the docking
1
pose of 3z clearly shows the usual and expected interaction of the exocyclic as well as one
endocyclic nitrogen atom with the Asn254 residue, while the naphthyl substituent is binding
deeper in the pocket, in a similar pose as reported for the 3-chloro-4-hydroxyphenyl group of the
3
a
original series of triazines. The hydroxyl group of the cyclohexanol moiety, on the other hand,
is involved in a hydrogen bond with Thr270, which is situated at the top of TM7. From this
1
7

ACCEPTED MANUSCRIPT
perspective, it seems clear that a polar functional group would be beneficial for affinity at
hA AR (Figure 4A). In hA AR, 3z shows a similar pose as in hA AR, but the residue at
1
2A
1
position 270 is a methionine, which is bigger than threonine, thus resulting in a steric clash and
the absence of a hydrogen bond. This seems to be a good explanation for the low affinity of 3z at
the hA AR. Conversely, when looking at the binding pose of 3x in hA AR, it sits deeper in the
2
A
2A
binding site; this was already observed in the 1,2,4-triazine series when comparing ligands that
were capable of forming hydrogen bonds with His278 at their 6-position (like 3x, via the OH
substituent) with ligands that were not (like 3z). More importantly in this binding pose, Met270
is turned away from 3x, thereby not leading to steric hindrance (Figure 4B).
A
B
Figure 4. Docking poses of ligands in hA AR and hA AR.
1
2A
A: Docking pose of 3z (LUF7279, black carbon atoms) in the homology model of hA AR,
1
showing hydrogen bonding interactions (yellow dashed lines) with the residues of the binding
site. B: Docking pose of 3z (LUF7279, black carbon atoms for the ligand, golden for the protein)
and 3x (LUF7558, green carbon atoms for ligand and protein) in hA AR, showing hydrogen
2
A
bonding interactions (yellow dashed lines) with the residues of the binding site. Non-carbon
atoms color code: white = hydrogen, blue = nitrogen, green = chlorine, red = oxygen, yellow =
sulfur.
CONCLUSION
1
8

ACCEPTED MANUSCRIPT
In conclusion, a substituent has been identified that led to a great increase in selectivity of 2-
2
amino-4,5-diarylpyrimidines as hA AR antagonists. This substituent, a N -trans-cyclohexanol
1
group, can be involved in favorable hydrogen bonding in hA AR, while this interaction is absent
1
in hA AR, as was established by computational studies. Eventually, the best ligand identified
2
A
was the partial inverse agonist 3z, bearing a 2-naphthyl substituent at the 5-position, which
showed a high affinity of 7.7 nM at hA AR, with a 180-fold selectivity over hA R, a lower but
1
2A
significant binding affinity at hA2BAR, and virtually no affinity at hA
3
AR. It was also discovered
that replacing the previously reported 1,2,4-triazine core with a pyrimidine moiety did not lead to
significant changes in the affinity at hA
1
AR.
EXPERIMENTAL SECTION
Chemistry. All solvents and reagents were purchased from commercial sources and were of
analytical grade. Demineralized water is simply referred to as H O, and was used in all cases
2
unless stated otherwise (i.e. brine). Melting points were measured using a Stuart Scientific
1
13
1
SMP3. H and C NMR spectra were recorded on a Bruker AV 400 liquid spectrometer ( H
1
3
NMR, 400 MHz; C NMR, 100 MHz) at ambient temperature. Chemical shifts are reported in
parts per million (ppm), are designated by δ and are downfield to the internal standard
3
tetramethylsilane (TMS) in CDCl . Coupling-constants are reported in Hz and are designated as
J. Analytical purity of the final compounds was determined by high pressure liquid
chromatography (HPLC) with a Phenomenex Gemini 3µ C18 110A column (50 x 4.6 mm, 3
µm), measuring UV absorbance at 254 nm. Sample preparation and HPLC method was − unless
stated otherwise − as follows: 0.3−0.8 mg of compound was dissolved in 1 mL of a 1:1:1
mixture of CH CN/H O/tBuOH and eluted from the column within 15 minutes, with a three
3
2
2 3 2
component system of H O/CH CN/1% TFA in H O, decreasing polarity of the solvent mixture in
1
9

ACCEPTED MANUSCRIPT
time from 80/10/10 to 0/90/10. All compounds showed a single peak at the designated retention
time and are at least 95% pure. Liquid chromatography–mass spectrometry (LC–MS) analyses
were performed using a Thermo Finnigan Surveyor − LCQ Advantage Max LC-MS system and
a Gemini C18 Phenomenex column (50 × 4.6 mm, 3 µm). The sample preparation was the same
as for HPLC analysis. The elution method was set up as follows: 1–4 min isocratic system of
th
H
2
O/CH
3
CN/1% TFA in H
2
O, 80:10:10, from the 4 min, a gradient was applied from 80:10:10
to 0:90:10 within 9 min, followed by 1 min of equilibration at 0:90:10 and 1 min at 80:10:10.
Thin-layer chromatography (TLC) was routinely performed to monitor the progress of reactions,
using aluminum coated Merck silica gel F254 plates. Purification by column chromatography
was achieved by use of Grace Davison Davisil silica column material (LC60A 30−200 µM).
Solutions were concentrated using a Heidolph laborota W8 2000 efficient rotary evaporation
apparatus and by a high vacuum on a Binder APT line Vacuum Drying Oven. The procedure for
a series of similar compounds is given as a general procedure for all within that series, annotated
by the numbers of the compounds. The procedure for the synthesis of similar compounds is
given as a general procedure; deviations therefrom are given below for each compound.
All reactions were carried out in inert atmosphere (under N
purged with N before use. Reflux was carried out using an oil bath as a heat source.
General procedure A: Synthesis of N-methoxy-N-Methyl-phenylacetamides (5o-5p) from
2
), and glassware was dried and
2
2
2
23
phenylacetic acids (4o -4p ): To a solution of phenylacetic acid (1 eq) in CH Cl (0.6 M) was
2
2
added thionyl chloride (2 eq), followed by a few drops of DMF. The reaction mixture was stirred
for 16 h at rt, upon which the reaction was concentrated in vacuo, to yield pure phenylacetyl
chloride, which was used in the next step without any purification or analysis. To a solution of
phenylacetyl chloride (1 eq) in CH Cl2 (0.1 M) was added N-methoxy-N-methylamine
2
2
0

ACCEPTED MANUSCRIPT
hydrochloride (1.1 eq). The reaction was cooled to 0 °C, upon which pyridine (1.1eq) was added.
The reaction mixture was stirred for 16 h at rt, upon which the reaction was concentrated in
vacuo, and the crude mixture partitioned between a mixture of brine: CH Cl :Et O (2:1:1 ratio)
2
2
2
equal to 0.5 x the original reaction volume. The organic layer was separated, dried over MgSO4
and concentrated in vacuo, to yield pure N-methoxy-N-methyl-phenylacetamide, which was used
in the next step without any purification.
General procedure B: Synthesis of 2-phenylacetophenones (6o-6p) from N-methoxy-N-
Methyl-phenylacetamides (5o-5p): A solution of Weinreb phenylacetamide (1 eq) in THF (0.5
2
M) was cooled to −50°C. To this solution was added 2.8 M PhMgBr solution in Et O (3.2 eq).
The reaction was stirred at −30 °C for 3 h, upon which a solution of sat. aq. NH Cl equal to 0.75
4
x the original reaction volume was added and the mixture extracted three times with Et O (total
2
volume equal to 1.5 x the original reaction volume). The combined organic layers were washed
with brine equal to 0.5 x the original reaction volume, and were subsequently dried over MgSO4
and concentrated in vacuo. The residue was purified by column flash chromatography, using
CH
engaged in the next step.
o: liquid, only available: H NMR (400 MHz, CDCl ) δ 8.01−7.98 (m, 2H), 7.57 (tt, J = 7.4,
2 2
Cl /PE as a solvent system, to give 2-phenylacetophenones 6o-6p, which were directly
1
6
3
1
.2 Hz, 1H), 7.49−7.45 (m, 2H), 7.28 (d, J = 2.2 Hz, 1H), 7.12 (dd, J = 8.4, 2.2 Hz, 1H), 6.88 (d,
J = 8.4 Hz, 1H), 4.20 (s, 2H), 3.87 (s, 3H).
1
6
p: liquid, only available: H NMR (400 MHz, CDCl ) δ 8.00−7.98 (m, 2H), 7.57 (tt, J = 7.4,
3
1
.9 Hz, 1H), 7.49−7.44 (m, 3H), 7.39−7.36 (m, 2H), 7.33−7.30 (m, 2H), 7.07 (dd, J = 8.4, 2.2
Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 5.13 (s, 2H), 4.19 (s, 2H).
2
1

ACCEPTED MANUSCRIPT
2-(2-Cyclopropylpyridin-4-yl)-1-phenylethan-1-one (6t). LDA (1M solution in
THF/hexanes, 3.5 mL, 3.5 mmol) was added to THF (2.25 mL) at −30 °C. A solution of 2-
cyclopropyl-4-methylpyridine (388 mg, 2.9 mmol) in THF (2.9 mL) was then added dropwise,
and the orange mixture was stirred at −10 °C for 1 h. It was then cooled to −50 °C and N-methyl-
N-methoxybenzamide (441 mL, 2.9 mmol) was added dropwise. The mixture was allowed to
warm to room temperature, at which it was stirred for 16 h. Water (20 mL) was added, and the
mixture was extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over
MgSO and concentrated in vacuo. The crude residue was purified by flash chromatography on
4
1
silica gel (50% Et
2
O in Pet Ether) to afford 6t as a yellow oil (366 mg, 53%). H NMR (400
MHz, CDCl ) δ 8.39 (d, J = 5.0 Hz, 1H), 8.02−8.00 (m, 2H), 7.63−7.59 (m, 1H), 7.52−7.48 (m,
3
2
4
1
H), 7.06 (s, 1H), 6.96 (dd, J = 5.0, 1.6 Hz, 1H), 4.25 (s, 2H), 2.04−1.98 (m, 1H), 1.05−0.96 (m,
1
3
3
H); C NMR (100 MHz, CDCl ) δ 196.3, 163.3, 149.5, 143.3, 136.4, 133.7, 128.9, 128.6,
22.6, 121.7, 44.8, 17.2, 10.0 ; LCMS: ESI [M + H]: 237.50.
General procedure C: Synthesis of 3-dimethylamino-1,2-diphenylprop-2-en-1-one (7a-7t)
2
4
from 2-arylacetophenones (6a-6u): To neat 2-arylacetophenone (1 eq) was added N,N-
dimethylformamide dimethyl acetal (3 eq). The reaction was stirred at 80 °C for 16 h, upon
which the reaction was concentrated in vacuo, to yield pure 3-dimethylamino-1,2-diphenylprop-
2
-en-1-one, which was used in the next step without any purification or analysis.
General procedure D: Synthesis of 2-amino-4,5-diarylpyrimidines (1a-t) from 3-
dimethylamino-1,2-diphenylprop-2-en-1-one (7a-u): To a solution of 3-dimethylamino-1,2-
diphenylprop-2-en-1-one (1 eq) in EtOH (0.3 M) was added K CO (3 eq). Subsequently,
2
3
guanidine-HCl (1 eq) was added. The reaction was stirred at reflux for 16 h, upon which H O
2
(0.5 x original reaction volume) was added. In the event that precipitation of the product
2
2

ACCEPTED MANUSCRIPT
2 2
occurred, the precipitate was filtered, and washed generously with CH Cl . In the event that
precipitation did not occur the mixture was extracted 3 times with CH Cl (3 x original reaction
2
2
volume). The combined organic layers were washed with brine (1 x original reaction volume),
dried over MgSO and concentrated in vacuo, to yield impure 2-amino-5,6-diphenylpyrimidine.
4
Impure 2-amino-5,6-diarylpyrimidines were purified by column flash chromatography, or by
PTLC, using MeOH/CH Cl as a solvent system, to give 2-amino-5,6-diarylpyrimidines in >95%
2
2
purity.
,5-diphenylpyrimidin-2-amine (1a): 1a was obtained from 7a (commercially available), and
purified by column flash chromatography (10% MeOH in CH Cl ), with a yield of 55% over two
4
2
2
steps, as a white solid (according to general procedures C and D). m.p.: 230.1 °C; Rf = 0.1
1
(
CHCl ); H NMR (400 MHz, CDCl ) δ 8.25 (s, 1H), 7.28−7.25 (m, 8H), 7.10-7.09 (m, 2H),
3
3
1
3
6
1
.83 (br s, 2H); C NMR (100 MHz, CDCl
3
) δ 163.7, 162.8, 159.5, 138.3, 137.2, 129.3, 129.2,
28.8, 128.4, 127.8, 126.7, 121.9; HPLC: 7.57 min; LCMS: ESI [M + H]: 248.20.
2
5
4
-phenyl-5-(m-tolyl)pyrimidin-2-amine (1b): 1b was obtained from 7b, and purified by
column flash chromatography (10% MeOH in CH Cl ), with a yield of 42% over two steps, as a
2
2
white solid (according to general procedures C and D) ). m.p.: 159.2 °C; Rf = 0.3 (1:9
1
MeOH/CH Cl ); H NMR (400 MHz, CDCl ) δ 8.32 (s, 1H), 7.40−7.36 (m, 2H), 7.34−7.27 (m,
2
2
3
1
3
2
H), 7.26−7.23 (m, 2H), 7.14 (t, J = 7.5 Hz, 1H), 5.29 (br s, 2H), 2.28 (s, 3H); C NMR (100
MHz, CDCl ) δ 165.0, 162.2, 159.7, 138.2, 138.1, 136.9, 130.1, 129.5, 129.2, 128.4, 128.1,
3
1
27.9, 126.8, 124.2, 21.5; HPLC: 8.30 min; LCMS: ESI [M + H]: 262.20.
-phenyl-5-(p-tolyl)pyrimidin-2-amine (1c): 1c was obtained from 7c, and purified by
column flash chromatography (10% MeOH in CH Cl ), with a yield of 40% over two steps, as a
white solid (according to general procedures C and D). m.p.: 232.7 °C; Rf = 0.3 (1:9
2
5
4
2
2
2
3

ACCEPTED MANUSCRIPT
1
MeOH/CH
.29−7.25 (m, 5H), 7.08 (d, J = 7.4 Hz, 2H), 6.77 (br s, 2H), 2.26 (s, 3H); C NMR (100 MHz,
DMSO-d ) δ 163.8, 162.7, 159.5, 148.4, 138.5, 135.9, 134.3, 129.3, 129.1, 128.8, 127.9, 121.9,
2 2 6
Cl ); H NMR (400 MHz, DMSO-d ) δ 8.22 (s, 1H), 7.98 (d, J = 7.4 Hz, 2H),
13
7
6
2
0.7; HPLC: 8.30 min; LCMS: ESI [M + H]: 262.20.
-(3-fluorophenyl)-4-phenylpyrimidin-2-amine (1d): 1d was obtained from 7d, and
purified by column flash chromatography (10% MeOH in CH Cl ), with a yield of 5% over two
2
5
5
2
2
steps, as a white solid (according to general procedures C and D). m.p.: 208.1 °C; Rf = 0.3 (1:9
1
MeOH/CH Cl ); H NMR (400 MHz, CDCl ) δ 8.32 (s, 1H), 7.38−7.22 (m, 6H), 6.95 (td, J = 8.5
2
2
3
Hz, 2.5 Hz, 1H), 6.98 (dd, J = 7.7 Hz, 0.7 Hz, 1H) 6.83 (dt, J = 9.7 Hz, 2.2 Hz, 1H), 5.32 (br s,
1
3
2
1
H); C NMR (100 MHz, CDCl
3
) δ 165.3, 164.0, 162.3, 161.6, 159.6, 139.3 (d), 137.7, 130.2,
30.1, 129.5 (d), 128.3, 125.4, 125.3, 123.1, 116.5, 116.3, 114.3, 114.1; HPLC: 8.29 min;
LCMS: ESI [M + H]: 266.13.
-(4-fluorophenyl)-4-phenylpyrimidin-2-amine (1e): 1e was obtained from 7e,
purified by column flash chromatography (10% MeOH in CH Cl ), with a yield of 34% over two
2
6
5
and
2
2
steps, as a white solid (according to general procedures C and D). m.p.: 244.1 °C; Rf = 0.3 (1:9
1
MeOH/CH
2
Cl
2
); H NMR (400 MHz, CDCl
3
+ CD
3
OD) δ 8.27 (s, 1H), 7.36−7.26 (m, 5H),
1
3
7
1
.10−6.94 (m, 4H); C NMR (100 MHz, CDCl ) δ 165.2, 163.1, 161.7, 160.7, 158.9, 137.3,
3
32.3, 132.2, 130.8, 130.7, 129.1, 129.0, 127.9, 122.6, 115.3, 115.1; HPLC: 8.00 min; LCMS:
ESI [M + H]: 266.13.
-(3-chlorophenyl)-4-phenylpyrimidin-2-amine (1f): 1f was obtained from 7f, and purified
by column flash chromatography (10% MeOH in CH Cl ), with a yield of 28% over two steps,
2
5
5
2
2
as a white solid (according to general procedures C and D). m.p.: 182.3 °C; Rf = 0.3 (1:9
1
MeOH/CH Cl ); H NMR (400 MHz, CDCl + CD OD) δ 8.28 (s, 1H), 7.43−7.14 (m, 8H),
2
2
3
3
2
4

ACCEPTED MANUSCRIPT
1
3
6
1
.96−6.93 (m, 1H); C NMR (100 MHz, CDCl ) δ 165.3, 161.9, 159.1, 138.3, 137.1, 134.2,
3
29.5, 129.1, 129.0, 128.0, 127.6, 127.1, 122.3; HPLC: 8.70 min; LCMS: ESI [M + H]: 282.13.
2
7
5
-(4-chlorophenyl)-4-phenylpyrimidin-2-amine (1g): 1g was obtained from 7g, and
purified by column flash chromatography (10% MeOH in CH Cl ), with a yield of 9% over two
2
2
steps, as a white solid (according to general procedures C and D). m.p.: 260.0 °C (decomp.); Rf =
1
0
8
1
.3 (1:9 MeOH/CH Cl ); H NMR (400 MHz, CDCl + CD OD) δ 8.27 (s, 1H), 7.38−7.31 (m,
2
2
3
3
1
3
H), 7.25 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.4 Hz, 2H); C NMR (100 MHz, CDCl ) δ 165.3,
3
59.2, 137.3, 135.0, 133.2, 130.6, 129.4, 129.2, 128.7, 128.2, 122.6 ; HPLC: 8.70 min; LCMS:
ESI [M + H]: 282.13.
-(3,4-dichlorophenyl)-4-phenylpyrimidin-2-amine (1h): 1h was obtained from 7h, and
purified by column flash chromatography (12% MeOH in CH Cl ), with a yield of 38% over two
2
6
5
2
2
steps, as a white solid (according to general procedures C and D). m.p.: 203.8 °C; Rf = 0.25 (1:9
1
MeOH/CH Cl ); H NMR (400 MHz, CDCl + CD OD) δ 8.23 (s, 1H), 7.32 (br s, 7H),
2
2
3
3
1
3
6
1
.87−6.86 (m, 1H) ; C NMR (100 MHz, CDCl ) δ 165.4, 159.0, 137.0, 136.7, 132.5, 131.3,
3
30.7, 130.3, 129.5, 129.1, 128.7, 128.2, 121.3 ; HPLC: 9.50 min; LCMS: ESI [M + H]: 316.00.
2
6
5
-(2-methoxyphenyl)-4-phenylpyrimidin-2-amine (1i): 1i was obtained from 7i, and
purified by column flash chromatography (1% MeOH in CH Cl ), with a yield of 28% over two
2
2
steps, as an off-white solid (according to general procedures C and D). m.p.: 215.6 °C; Rf = 0.2
1
(
1:19 MeOH/CH Cl ); H NMR (400 MHz, CDCl ) δ 8.29 (s, 1H), 7.35 (d, J = 1.6 Hz, 2H),
2
2
3
7
.29−7.20 (m, 3H), 7.14 (dd, J = 7.5 Hz, 1.7 Hz, 1H) 6.95 (dt, J = 7.4 Hz, 0.9 Hz, 1H), 6.76 (d, J
1
3
=
8.0 Hz, 1H), 5.15 (br s, 2H), 3.36 (s, 3H) ; C NMR (100 MHz, CDCl ) δ 162.3, 160.2, 156.6,
3
1
39.2, 131.3, 129.3, 128.9, 128.4, 127.9, 126.2, 121.0, 120.7, 111.2, 55.0 ; HPLC: 7.68 min;
LCMS: ESI [M + H]: 278.07.
2
5

ACCEPTED MANUSCRIPT
2
7
5
-(3-methoxyphenyl)-4-phenylpyrimidin-2-amine (1j): 1j was obtained from 7j, and
purified by column flash chromatography (8% MeOH in CH Cl ), with a yield of 65% over two
2
2
steps, as an off-yellow solid (according to general procedures C and D). m.p.: 172.3 °C; Rf = 0.4
1
(
1:9 MeOH/CH Cl ); H NMR (400 MHz, CDCl ) δ 8.32 (s, 1H), 7.37 (dd, J = 7.8 Hz, 1.4 Hz,
2
2
3
2
6
1
7
H), 7.30−7.23 (m, 3H), 7.16 (t, J = 8.0 Hz, 1H) 6.79−6.76 (m, 1H), 6.69 (d, J = 7.6 Hz, 1H),
13
3
.62 (t, J = 2.0 Hz, 1H), 5.46 (br s, 2H), 3.34 (s, 3H) ; C NMR (100 MHz, CDCl ) δ 165.1,
62.3, 159.7, 159.6, 138.4, 138.1, 129.5, 129.2, 128.2, 124.0, 121.9, 115.0, 112.9, 55.2; HPLC:
.90 min; LCMS: ESI [M + H]: 278.07.
2
8
5
-(4-methoxyphenyl)-4-phenylpyrimidin-2-amine (1k): 1k was obtained from 7k, and
purified by column flash chromatography (10% MeOH in CH Cl ), with a yield of 24% over two
2
2
steps, as an off-white solid (according to general procedures C and D). m.p.: 232.7 °C; Rf = 0.3
1
(
1:9 MeOH/CH
2
Cl
2
); H NMR (400 MHz, CDCl
3
) δ 8.31 (s, 1H), 7.40−7.34 (m, 2H), 7.32−7.24
13
(m, 3H), 7.03 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 5.12 (br s, 2H), 3.80 (s, 3H);
C
NMR (100 MHz, CDCl ) δ 162.1, 159.8, 158.9, 138.3, 135.1, 130.6, 129.5, 129.1, 128.2, 123.9,
3
1
21.1, 114.1, 55.4; HPLC: 7.90 min; LCMS: ESI [M + H]: 278.07.
-(3,4-dimethoxyphenyl)-4-phenylpyrimidin-2-amine (1l): 1l was obtained from 7l, and
purified by column flash chromatography (10% MeOH in CH Cl ), with a yield of 55% over two
2
9
5
2
2
steps, as a white solid (according to general procedures C and D). m.p.: 175.3 °C; Rf = 0.3 (1:9
1
MeOH/CH Cl ); H NMR (400 MHz, CDCl ) δ 8.35 (s, 1H), 7.39−7.37 (m, 2H), 7.29−7.27 (m,
2
2
3
1
3
3
H), 6.83−6.74 (m, 2H), 6.49 (s, 1H), 5.29 (br s, 2H), 3.88 (s, 3H), 3.58 (s, 3H); C NMR (100
MHz, CDCl ) δ 165.0, 162.1, 159.6, 148.7, 148.3, 138.3, 129.5, 129.4, 129., 128.2, 124.0, 121.5,
3
1
13.1, 111.3, 55.9, 55.7; HPLC: 7.20 min; LCMS: ESI [M + H]: 308.07.
2
6

ACCEPTED MANUSCRIPT
3
0
4-phenyl-5-(3,4,5-trimethoxyphenyl)pyrimidin-2-amine (1m): 1m was obtained from 7m,
and purified by column flash chromatography (10% MeOH in CH Cl ), with a yield of 17% over
2
2
two steps, as an orange solid (according to general procedures C and D). m.p.: 172.2 °C; Rf = 0.3
1
(
1:9 MeOH/CH Cl ); H NMR (400 MHz, CDCl ) δ 8.36 (s, 1H), 7.39−7.37 (m, 2H), 7.30−7.26
2
2
3
1
3
(
m, 3H), 6.28 (s, 1H), 5.38 (br s, 2H), 3.84 (s, 3H), 3.64 (s, 6H); C NMR (100 MHz, CDCl
3
) δ
62.3, 162.2, 159.3, 153.2, 138.2, 132.3, 129.3, 129.2, 128.2, 124.1, 106.9, 61.1, 56.1; HPLC:
.60 min; LCMS: ESI [M + H]: 338.07.
-(benzo[d][1,3]dioxol-5-yl)-4-phenylpyrimidin-2-amine (1n): 1n was obtained from 7n,
and purified by column flash chromatography (10% MeOH in CH Cl ), with a yield of 34% over
1
7
3
1
5
2
2
two steps, as a white solid (according to general procedures C and D). m.p.: 182.4 °C; Rf = 0.3
1
(
(
(
1:9 MeOH/CH
2
Cl
2
); H NMR (400 MHz, CDCl
3
) δ 8.21 (s, 1H), 7.40−7.38 (m, 2H), 7.31−7.26
m, 3H), 6.77 (d, J =7.8 Hz, 1H), 6.63 (dd, J = 7.8 Hz, 1.7 Hz, 1H), 6.59 (d, J = 1.7 Hz, 1H) 6.12
1
3
br s, 2H), 5.95 (s, 2H); C NMR (100 MHz, CDCl ) δ 164.9, 163.8, 160.4, 148.6, 147.6, 139.6,
3
1
32.3, 130.3, 129.6, 128.6, 123.7, 123.6, 110.7, 109.1, 102.0; HPLC: 7.80 min; LCMS: ESI [M +
H]: 292.07.
5-(3-chloro-4-methoxyphenyl)-4-phenylpyrimidin-2-amine (1o): 1o was obtained from 7o,
2 2
and purified by column flash chromatography (10% MeOH in CH Cl ), with a yield of 53% over
two steps, as a white solid (according to general procedures C and D). m.p.: 210.9 °C; Rf = 0.3
1
(
1:9 MeOH/CH Cl ); H NMR (400 MHz, CDCl ) δ 8.27 (s, 1H), 7.39−7.28 (m, 5H), 7.14 (d, J
2
2
3
1
3
=
2.0 Hz, 1H), 7.03 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 3.88 (s, 3H); C NMR
) δ 165.9, 163.6, 160.3, 155.2, 139.2, 131.7, 131.3, 130.4, 130.1, 130.0, 128.9,
23.0, 122.7, 113.2, 56.6; HPLC: 8.18 min; LCMS: ESI [M + H]: 312.00.
(100 MHz, CDCl
3
1
2
7

ACCEPTED MANUSCRIPT
5-(3-chloro-4-benzyloxyphenyl)-4-phenylpyrimidin-2-amine (1p): 1p was obtained from
7
p, and purified by column flash chromatography (5% MeOH in CH Cl ), with a yield of 25%
2 2
over two steps, as a white solid (according to general procedures C and D). m.p.: 143.7 °C; Rf =
1
0
.5 (1:9 MeOH/CH Cl ); H NMR (400 MHz, CDCl ) δ 8.27 (s, 1H), 7.45−7.25 (m, 9H), 7.18
2
2
3
13
(
d, J = 1.6 Hz, 1H), 6.87−6.82 (m, 2H), 5.38 (br s, 2H), 5.12 (s, 2H); C NMR (100 MHz,
CDCl ) δ 165.1, 162.3, 159.6, 153.4, 137.9, 136.4, 131.0, 130.7, 129.5, 129.3, 128.9, 128.7,
3
1
28.3, 128.2, 127.2, 122.6, 114.0, 70.9; HPLC: 9.90 min; LCMS: ESI [M + H]: 388.10.
-phenyl-5-(3-(trifluoromethyl)phenyl)pyrimidin-2-amine (1q): 1q was obtained from 7q,
and purified by column flash chromatography (1% MeOH in CH Cl ), with a yield of 40% over
2
5
4
2
2
two steps, as a pale yellow solid (according to general procedures C and D). m.p.: 208.4 °C; Rf =
1
0
7
1
3
.55 (1:19 MeOH/CH Cl ); H NMR (400 MHz, CDCl ) δ 8.34 (s, 1H), 7.51 (d, J = 7.6 Hz, 2H),
2
2
3
1
3
.39−7.26 (m, 8H), 5.38 (br s, 2H); C NMR (100 MHz, CDCl ) δ 165.0, 162.4, 159.6, 137.8,
3
37.4, 132.8, 131.1, 130.8, 129.4, 128.9, 128.3, 126.1 (q, J = 3.4Hz ), 125.3, 123.9 (q, J =
.9Hz), 122.7; HPLC: 8.89 min; LCMS: ESI [M + H]: 316.20.
2
8
4-phenyl-5-(4-(trifluoromethyl)phenyl)pyrimidin-2-amine (1r): 1r was obtained from 7r,
and purified by column flash chromatography (2% MeOH in CH Cl ), with a yield of 27% over
2
2
two steps, as a pale yellow solid (according to general procedures C and D). m.p.: 199.3 °C; Rf =
1
0
7
1
.45 (1:19 MeOH/CH Cl ); H NMR (400 MHz, CDCl ) δ 8.35 (s, 1H), 7.45 (dd, J = 15.6 Hz,
2
2
3
1
3
.9 Hz, 2H), 7.36−7.22 (m, 7H), 5.56 (s, 2H); C NMR (100 MHz, CDCl
3
) δ 165.4, 162.5,
59.8, 140.9, 137.6, 129.8, 129.6, 129.5, 128.4, 125.6 (q, J = 3.7 Hz); HPLC: 9.08 min; LCMS:
ESI [M + H]: 316.20.
-phenyl-5-(pyridin-4-yl)pyrimidin-2-amine (1s): 1s was obtained from 7s, and purified
by column flash chromatography (1% MeOH in CH Cl ), with a yield of 27% over two steps, as
3
2
32
4
2
2
2
8