Discovery of uracil-bearing DAPYs derivatives as novel HIV-1 NNRTIs via crystallographic overlay-based molecular hybridization

Article abstract of DOI:10.1016/j.ejmech.2017.02.047

A novel series of uracil-bearing DAPYs derivatives were designed and synthesized via structure-based molecular hybridization to discover compounds with improved anti-resistance profiles. Anti-HIV activity of the designed compounds was tested in MT-4 cell

Full text of DOI:10.1016/j.ejmech.2017.02.047

Accepted Manuscript
Discovery of uracil-bearing DAPYs derivatives as novel HIV-1 NNRTIs via
crystallographic overlay-based molecular hybridization
Heng Zhang, Ye Tian, Dongwei Kang, Zhipeng Huo, Zhongxia Zhou, Huiqing Liu, Erik
De Clercq, Christophe Pannecouque, Peng Zhan, Xinyong Liu
PII:
S0223-5234(17)30120-4
10.1016/j.ejmech.2017.02.047
EJMECH 9243
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 January 2017
Revised Date: 13 February 2017
Accepted Date: 17 February 2017
Please cite this article as: H. Zhang, Y. Tian, D. Kang, Z. Huo, Z. Zhou, H. Liu, E. De Clercq, C.
Pannecouque, P. Zhan, X. Liu, Discovery of uracil-bearing DAPYs derivatives as novel HIV-1 NNRTIs
via crystallographic overlay-based molecular hybridization, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.02.047.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Discovery of uracil-bearing DAPYs derivatives as novel HIV-1 NNRTIs via
crystallographic overlay-based molecular hybridization
By structure-based molecular hybridization, a new HIV-1 NNRTI 16d was discovered
with excellent activity and high selectivity.
CN
O
N
CN
O
N
N
N
NH
NH
O
O
Molecular
Hybridization
+
Cl
O
O
N
NH
O
N
NH
O
N
N
Cl
Br
NH2
Cl
JLJ506 (6)
16d
1
EC50 = 5.6 nM (WT)
SI > 50000 (WT)
EC50 = 34.2 nM (E138K)

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Discovery of uracil-bearing DAPYs derivatives as novel HIV-1
NNRTIs via crystallographic overlay-based molecular hybridization
a
a
a
c
Heng Zhang a, Ye Tian a, Dongwei Kang , Zhipeng Huo , Zhongxia Zhou , Huiqing Liu , Erik
De Clercq b, Christophe Pannecouque b, Peng Zhan a,*, Xinyong Liu a,*.
a
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of
Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road,
250012 Ji’nan, Shandong, PR China
b
Rega Institute for Medical Research, KU Leuven Minderbroedersstraat 10, B-3000 Leuven,
Belgium
c
Institute of Pharmacology, School of Medicine, Shandong University, 44 West Culture Road,
250012 Ji’nan, Shandong, PR China
Abstract: A novel series of uracil-bearing DAPYs derivatives were designed and
synthesized via structure-based molecular hybridization to discover compounds with
improved anti-resistance profiles. Anti-HIV activity of the designed compounds was
tested in MT-4 cell cultures. The most promising compound 16d showed excellent
activity with EC50 value of 5.6 nM against wide-type HIV-1 and low cytotoxicity (SI >
50000). Activity against the clinic prevalent mutant strains was also tested, suggesting
that 16d was sensitive to E138K (EC50 = 34.2 nM). Primary drug-like properties, such
as water solubility and logP, were evaluated by experiment or calculation, which
indicated that introducing an uracil can improve solubility. The molecular modeling
accompanied with the preliminary SAR correlations paved the way for the next round
of rational design of potent anti-HIV agents.
Key words: NNRTIs, DAPYs, uracil, molecular hybridization, physicochemical
properties, drug design
1. Introduction
HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) are widely
investigated and constituted essential components of high active antiretroviral therapy
(HAART) broadly used in the clinic for the advantages of high potency, low toxicity
and high specificity[1-4]. However, for the replication infinity and genetic variability
Corresponding authors. Tel: +86 531 88380270; fax: +86 531 88382731; E-mail address:
zhanpeng1982@sdu.edu.cn (Zhan P.); xinyongl@sdu.edu.cn (Liu X.Y.)

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of HIV-1, drug resistance of first generation NNRTIs (Nevirapine, Delavirdine and
Efavirenz) arises rapidly. Considerable efforts towards seeking novel NNRTIs that are
effective against wild-type (WT) and resistant viruses are applied, which culminated
in the approval of second generation NNRTIs Etravirine (ETV) and Rilpivirine (RPV)
(Fig. 1)[5-7]. Both ETV and RPV belong to diarylpyrimidine derivatives (DAPYs)
and exhibit good anti-resistance profiles[8]. However, they have low solubility and
poor oral bioavailability for their special structures, thus limiting their clinical use [5,
9]. What’s more, some mutant strains that resistant to DAPYs have also been reported
including K103N, E138K, L100I/K103N and Y181C/K103N. Therefore, searching
for novel chemical structures derived from DAPY scaffold, with acceptable resistance
profiles against clinically relevant mutations and favorable pharmacokinetic profiles,
is being actively pursed[10, 11].
Fig 1. Structures and binding model of DAPYs.
Based on an established common “four-point pharmacophore model” of DAPYs
(Fig. 1): the hydrophobic domain, the hydrogen bond interaction domain, tolerant
region I and tolerant region II (entrance channel)[12], modifications were applied on
all four domains. Potent compounds (as shown in Fig 2) with comparable or better
potency than ETV and RPV were reported [13-15].

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Fig 2. Highly potent DAPY derivatives reported in recent years.
Among all these four regions, tolerant region I was opening toward a
solvent/protein interface, thus it was the most flexible domain and could
accommodate diverse moieties[15-19]. Extension of smaller NNRTIs into tolerant
region I has led to the discovery of potent and structurally novel derivatives with
improved activities and optimized physicochemical properties[16]. Recently, our
group disclosed a series of thiophene[3,2-d]pyrimidine-based NNRTIs (exemplified
by 5) with significantly improved drug resistance profiles against a comprehensive
panel of prevalent RT mutant viruses[15]. Molecular docking indicated that the newly
introduced 1-(4-sulfonamidebenzyl)piperidine in compound 5 (Fig 2) can form extra
hydrogen bonds with the backbone of K104 and V106, explaining the maintenance of
activity against the mutant HIV-1 strains.

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Fig 3. Rational design of target compounds.
In continuation of our research in this field, we wondered whether the introduction
of alternative substitutions in this region provided compounds with interesting
anti-resistance features. Recently, catechol diethers (exemplified by JLJ506 in Fig 3)
were disclosed as novel NNRTIs with distinguishing anti-resistance profiles and
favorable solubility [20, 21]. Crystallographic overlay-based molecular hybridization
represented a rational approach of molecular hybridization compared to other
ligand-based methodologies, and it was widely used in current drug design[22-24]
.
Overlapping the crystal structures of JLJ506 and ETV indicated that they had similar
shape and binding mode in RT (Fig 4)[21, 25]. The flexible and polar uracil group
pointed toward the tolerant region I, and was suitable for the solvent/protein interface.
To discover novel NNRTIs with high anti-resistance profile and good water solubility,
a series of compounds (general formula I) were designed by introducing of ethyluracil
into DAPYs via crystallographic overlay-based molecular hybridization (Fig 3)[26,
27]. Besides, the substitution position of ethyluracil on the pyrimidine ring, and
different substituents on the pyrimidine ring were also investigated (general formula
II and III).

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Fig 4. Overlay of crystal structures of ETV (green, PDB code: 3MEC) and JLJ506 (cyan, PDB
code: 4H4M).
Herein we report the synthesis, antiviral activity, some measured and calculated
physicochemical properties like logP and water solubility of these novel DAPYs
derivatives bearing an uracil moiety. Preliminary structure-activity relationships
(SARs) are also discussed in detail to gain further insights into this series of
analogues.
2. Chemistry
All target compounds were synthesized via several expeditious and straightforward
synthetic routes shown in Scheme 1 and Scheme 2. In Scheme 1, the key
intermediate 1-(2-aminoethyl)uracil hydrochloride (12) was prepared from uracil
following a previously described protocol[28]. Then, treatment of commercially
available 2,4-dichloropyridines (13a-d) with corresponding substituted phenols and
K2CO3 in DMF or acetone provided 2-chloro-4-phenoxypyrimidine intermediates
14a-l, or react with 2,4,6-trimethylaniline and Et3N in THF at 0˚C to produce
2-chloro-N-mesityl-5-nitropyrimidin-4-amine (15) [26, 29, 30]. Subsequently,
intermediates 14a-l were reacted with 12 in N-Methyl pyrrolidone (NMP) in the
present of N,N-Diisopropylethylamine (DIPEA) by heating at 100˚C for 6-12h to
afford corresponding compounds 16a-d, 17a-d, 18a-d, 21a-d, 22 and 23[17, 31].

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Compounds 17a-d can be aminated in 1,4-dioxane with NH3·H2O to give compounds
19a-d, and further bromination of 19b with bromine in DMF at low temperature
afforded compounds 20[32]. In Scheme 2, treatment of 15 with 12 and Et3N in THF
at 60˚C yielded compound 23 and then 23 was further reduced by SnCl2·2H2O in
ethanol under reflux to provide compound 24[29]. Furthermore, for convenience,
compound 24 was converted to its hydrochloride salt.
Scheme 1. Reagents and conditions: (a) i) benzoyl chloride, pyridine, CH3CN, rt; ii) K2CO3, H2O,
1,4-dioxane (1:2 v/v), rt, 30 min; (b) Di-t-butylcarbonate, Et3N, 1,4-dioxane; (c) TBAI, K2CO3,
DMF, 70 ˚C; (d) i) trifluoroacetic acid, DCM, rt; ii) HCl/EtOH.
Scheme 2. Reagents and conditions: (a) K2CO3, DMF or acetone, rt; (b) 2,4,6-trimethylaniline,

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NEt3, THF, 0˚C; (c) 12, DIPEA, NMP, 100˚C; (d) 12, Et3N, THF, 60˚C; (e) NH3·H2O, 1,4-dioxane,
120˚C; (f) Br2, DMF, rt; (g) SnCl2·H2O, EtOH, reflux.
In addition, 3-(4-hydroxy-3,5-dimethylphenyl)acrylonitrile (28) was prepared from
commercial reagents 25 and 26 in two steps (Scheme 3): a Heck reaction of
4-bromo-3,5-dimethylphenol with acrylamide and then dehydration with POCl3[33].
Scheme 3. Reagents and conditions: (a) Tri(o-tolyl)phosphine, Pd(CH3COO)2, Et3N, DMA, 120˚C;
(b) POCl3.
The synthesized compounds were characterized by physicochemical and spectral
1
means. The MS and H NMR, 13C NMR spectral data was in agreement with the
assigned molecular structures. Compounds 16 and 21 or 22 can be synthesized and
separated in one step, when analyzing the NMR spectrum, an interesting phenomenon
was found that, two hydrogens of the ethyl linker were splited and had a different
chemical shift, as well as the polar hydrogen. Furthermore, introducing of electron
withdrawing group aggravated this situation compared with electron donating group.
By comparing these two structures, we speculated that a tautomeric isomer was
existed as showed in Scheme 4 (more spectrum information in Supplementary Data).
Scheme 4. Proposed explanation for the spectrum and structure identification.

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3. Results and discussion
3.1. Anti-HIV activity in MT-4 cells
Anti-HIV activity and cytotoxicity of the newly synthesized compounds were
evaluated in vitro in MT-4 cells infected with the WT HIV-1 (strain IIIB) and the
common clinical mutant Y181C/K103N (also known as RES056), as well as HIV-2
(strain ROD). The biological results, interpreted as EC50 values, CC50 values and
selectivity index (SI) values, are summarized in Table 1. Zidovudine (AZT),
Nevirapine (NVP), Delavirdine mesylate (DLV), Efavirenz (EFV) and Etravirine
(ETV) were used as reference drugs. In addition, in order to investigate the potency of
designed compounds against the prevalent resistance HIV-1 strains, antiviral activity
against mutant strains including L100I, K103N, Y181C, Y188L, E138K and
F227L/V106A of 16d and 19d was also tested, and results were listed in Table 2.
Table 1．Activity against HIV-1 (IIIB and RES056) and HIV-2 (ROD) strains and cytotoxicity in
MT-4 cells.
O
O
O
O
NH
NH
NH
NH
N
O
N
O
N
O
N
O
R3
R3
5
3
4 N
O
4
2
6
O
NH
O
NH
NH
HN
R1
N
NH
N
5
R1
1
N
3 N
N
1
N
Cl
N
6
2
R2
Cl
22
21
23-24
16-20
EC50(µM)a
RES056
Compds
R1
R2
R3
CC50(µM)b
SI(IIIB)c
HIV-1IIIB
ROD
>2.49
Cl
Cl
Cl
Cl
H
H
H
2,4,6-Me
0.0078±0.0028
0.046±0.0072
0.88±0.43
>2.49
>11.65
>322.3
2.49±1.15
>11.65
317
≥255
>367
>50454
85
16a
16b
16c
16d
17a
17b
17c
17d
2,6-Me-4-CN
3,5-Me
>11.65
>322.3
H
>322.3
H
2,6-Me-4-CV 0.0056±0.0018 23.29±14.04 >284.8
>284.8
Cl
Cl
Cl
Cl
2,4,6-Me
2,6-Me-4-CN
3,5-Me
0.52±0.31
7.73±0.92
>56.47
>44.13
>20.64
ND
>44.13 44.13±28.53
>20.64 20.64±6.75
H
3
H
>56.47
>284.8
>56.47
>284.8
X1
H
2,6-Me-4-CV
0.12±0.064
>284.8
>2273

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H
H
H
H
H
H
H
H
Br
H
H
2,4,6-Me
2,6-Me-4-CN
3,5-Me
0.53±0.27
6.66±4.76
48.81±11.01
0.034±0.0098
2.70±0.56
18.90±6.27
>45.82
>271.7
>35.84
>74.82
>309.1
>119.3
>203.5
ND
>271.7 271.7±109.8
>35.84 35.84±21.80
>74.82 74.82±33.97
515
5
18a
18b
18c
18d
19a
19b
19c
19d
20
H
2
H
2,6-Me-4-CV
2,4,6-Me
>309.1
>309.1
>9102
44
NH2
>119.3 119.3±24.5
>203.5 203.5±76.7
>127.3 127.3±47.71
NH2 2,6-Me-4-CN
NH2 3,5-Me
11
<1
NH2 2,6-Me-4-CV
NH2 2,6-Me-4-CN
2,4,6-Me
0.17±0.021
0.034±0.0059
>42.30
>298.0
>264.7
ND
>298.0
>264.7
>298.0
>264.7
>1737
>7684
≤2
>76.40 76.40±42.20
>27.88 27.88±5.81
>99.53 99.53±49.48
>26.25 26.25±6.69
21a
21b
21c
21d
22
2,6-Me-4-CN
3,5-Me
9.38±1.77
>99.53
>27.88
ND
3
--
<1
2,6-Me-4-CVd
--
5.81±0.85
>2.89
>26.25
>2.89
>3.33
>299.1
5
>2.89
>3.33
2.89±1.99
≥3.33
<1
NO2
NH2
0.083±0.038
14.93±3.20
0.26
≥40
>20
>58
>1212
733
>1053
733
23
--
>299.1
>199.1
>15.02
>6.34
24
NVP
EFV
ETV
AZT
DLV
0.0052
--
0.0034
NDe
2.46
0.0071
>7.48
0.032
30.40
a
Concentration required to achieve 50% protection of MT-4 cells against HIV-1-induced
cytopathic effect, as determined by the MTT method; values are the mean ± SD of at least two
parallel tests.
b Concentration required to reduce the viability of uninfected cells by 50%, as determined by the
MTT method; values were averaged from at least four independent experiments.
c Selectivity index (SI): CC50/EC50.
d CV = cyanovinyl.
e ND: not determined.
As we can see from Table 1, most compounds exhibited high potency against WT
HIV-1 with EC50 value in micromolar or submicromolar. The most potent compound

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16d had an EC50 value of 5.6 nM that was comparable with EFV (5.2 nM) and ETV
(3.4 nM) and 16d also showed low cytotoxicity with SI > 50000. Compound 16a also
displayed excellent potency (EC50 = 7.8 nM) that was better than first generation
NNRTIs DLV (32 nM) and NVP (260 nM) and in the same order of magnitudes with
EFV and ETV. Unfortunately, all compounds except 16d lost activity against the
K103N/Y181C mutant strain. All compounds were inactive to HIV-2, indicating a
high selectivity to HIV-1. These promising results proved that introducing of the
ethyluracil into DAPYs was practicable and provided alternative NNRTIs with novel
scaffold.
Based on the anti-HIV-1(IIIB) assay results, preliminary SARs analysis were
summarized as follows:
First of all, the substitution pattern in the pyrimidine ring played an essential role in
maintaining antiviral activity, ortho phenoxy-substituted compound 22 (EC50 > 2.89
µM) lost its activity while the meta alternatives 16a (EC50 = 7.8 nM) performed much
better. Meanwhile, meta substituents in different position also demonstrated different
potency. Comparing the two different meta pattern, 2,4-disubstituted pyrimidine
analogues were more potent than 2,6-disubstituted derivatives (17a-d vs 21a-d).
Next, variations of substituents on the benzene ring were considered, except three
privilege fragments in DAPYs (2,4,6-Me, 2,4-Me-4-CN and 2,6-Me-4-CV), a new
3,5-Me disubstituent was introduced which was common in indolylarylsulfones
HIV-1 inhibitors[34]. The results demonstrated that 2,4,6-trisubstitution was more
active than 3,5-disubstitution, and activity was in the following sequence regardless of
substituents on pyrimidine ring: 2,6-Me-4-CV > 2,4,6-Me > 2,4-Me-4-CN > 3,5-Me
as indicated from all 16-21 series compounds. Among all designed compounds, 16d
(EC50 = 5.6 nM) exhibited the best activity probably because the cyanovinyl group
can form π-π stacking with W229 residues, and this can also explained why 16d
showed weak activity against the K103N/Y181C double mutant strain[25].
Subsequently, modifications were emphasized on pyrimidine, a hydrophobic
chlorine group was first introduced into the pyrimidine ring. Incorporation of a
chlorine to C5 of pyrimidine increased the activity for more than 10 folds (16a-d vs

ACCEPTED MANUSCRIPT
18a-d). In addition, replacing of the C5 hydrogen of 19b with a bromo atom increased
the anti-HIV-1 activity for more than 550 folds (20 vs 19b). However, changing the
position of chlorine to C6 of pyrimidine gave opposite result (17a-d vs 18a-d)
indicating the importance of substitution position.
Finally, the link atom between benzene ring and pyrimidine was studied. Changing
from oxygen to nitrogen gave compound 23 (EC50 = 0.083 µM) which was as potent
as 16b (EC50 = 0.046 µM). However, reduction of the nitro group to amino nearly lose
the antiviral activity, suggesting the sensitivity of the entrance channel as well as the
preference for hydrophobic moieties.
In addition, cytotoxicity of all compounds was lower than ETV as we can see from
Table 1. Substituents in both pyrimidine and benzene ring influenced the cytotoxicity,
for example C5 substituted compound 16a (CC50 = 2.49 µM) was more toxic than
unsubstituted counterpart 18a (CC50 = 271.7 µM) while the 3,5-dimethyl compound
16c (CC50 > 322.3 µM) demonstrated lower cytotoxicity than 18c (CC50 = 74.8 µM),
at the same time, it almost didn’t show any influence on the 2,6-dimethyl-4-cynovinyl
compounds (16d and 18d). But it was clear that 2,6-dimethyl-4-cynovinyl compounds
not only had higher activity but also showed the lower toxicity.
Furthermore, two compounds, 16d and 19d, were selected to test their activity
against the prevalent HIV-1 mutant strains, and the data was listed in Table 2.
Compounds 16d showed better performance than first generation compounds DLV
and NVP except Y188L and F227L/V106A strains, and activity of 16d was higher
than EFV against K103N. Generally, 16d and 19d were tolerant to E138K mutant
strain and showed moderate change (5 folds for 16d and 6 folds for 19d) of potency;
when it came to K103N, L100I and Y181C, both were modest with an increase for
more than 10 folds; as for double mutant strain F227L/V106A and K103/Y181C
strains both lose their activity.
Table 2. Activity against common clinical resistance HIV-1 strains in MT-4 cells.
EC50(µM)(fold resistance)
Compds
L100I
K103N
Y181C
Y188L
E138K
F227L+V106A

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16d
19d
0.43±0.0032(77) 0.086±0.0098(15) 0.15±0.024(27) 51.95±20.62(9277) 0.034±0.014(6)
≥96.61
>298.0
21.92±4.74(129)
0.83±0.32
0.53±0.11(3)
6.78±0.080
4.82±1.21(28)
6.22±0.080
>298.0(>1753)
9.61±1.86
0.83±0.29(5)
0.23±0.056
NVP
EFV
ETV
AZT
DLV
7.17±1.86
0.10±0.011
0.12±0.005
0.0068±0.0006
0.016±0.0003
0.0071±0.001
2.04±0.45
0.34±0.016
0.022±0.0004
0.011±0.002
0.69±0.011
0.0051±0.0007
0.014±0.002
0.013±0.004
0.043±0.005
0.21±0.0076
0.017±0.0004
0.0050±0.0002
1.87±0.12
0.0066±0.002
0.0052±0.0001
2.10±0.26
0.0032±0.0002
0.0083±0.0002
2.48±0.00
Taken together, incorporating of a flexible ethylurical group to the DAPYs
provided a novel series of HIV-1 inhibitors with moderate to excellent activity. SARs
of the newly designed compounds indicated that they probably bound RT with similar
conformation with DAPYs, and provided direction for further modifications.
3.2. HIV-1 RT inhibition assay
A RT inhibition assay was performed to confirm our design of the NNRTIs, and
compound 16d was chosen as represent. Anti-HIV-1 RT assay was performed in
recombinant HIV-1 RT (RT kit, Roche), and the result was provided in Table 3. As
indicated, 16d (IC50 = 1.58 µM) showed comparable activity with ETV (IC50 = 1.00
µM) and NVP (IC50 = 1.39 µM) in low micromolar range. However, for the poor
solubility of ETV and 16d, compounds were crystalized when diluting, leading to the
poor result of both compounds. Even so, the results proved that the newly synthesized
compounds could effectively inhibit the activity of HIV-1 RT, thus acting as typical
HIV-1 NNRTIs.
Table 3. Inhibitory activity against HIV-1 RT (WT).
compds
IC50 (µM)
NVP
1.39
ETV
1.00
16d
1.58
3.3. Molecular modeling analysis
Molecular modeling (docking) was performed using the Sybyl-X1.1 software to get
deeper insight into the molecular bases of the inhibitory potency and to rationalize
SARs of these analogues. Compound 20 and 16d were chosen as representative
compounds and compared with the lead compounds ETV and RPV to verify our
design.

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As illustrated in Fig. 5A-B, binding model of 20 was similar to that of ETV with
the classical “horseshoe shape” of DAPYs. Benzene ring of 20 oriented to the
hydrophilic domain and formed π-π stacking interaction with RT, but it was shifted
toward Y181 and Y188; the N1 of pyrimidine ring, 2-NH and the 6-NH2 formed triple
hydrogen bond with K101 and E138. The newly introduced urical group was located
in the tolerant region I and formed extra hydrogen bond with H235 as expected.
Binding of 16d was showed as Fig. 5C-D, it was different from 20 although both of
them demonstrated “horseshoe shape”. Even though the benzene ring of 16d was
surrounded by hydrophilic amino acid, it was shift “down” explaining the lower
activity comparing with RPV. Besides, one reason why RPV was insensitive to mutant
strains was due to the cyanovinyl group that can form π-π interaction with W229, shift
of benzene ring may lead to the loss of activity against mutant strains, indeed the
antiviral results proved this as 16d nearly lose activity against Y188L and double
mutant strains. The hydrogen bonds between RPV and backbone of K101 was
conserved in 16d and not affected by introducing of chloro atom. The right ring of
16d and RPV matched well even though they were quite different in structure, but the
difference between 16d and 20 was that the urical group was in opposite direction,
leading to the loss of hydrogen bond with H235.

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Fig 5. Predicted binding models of 20 (PDB ID: 3MEC) and 16d (PDB ID: 2ZD1). (a) binding of
20 (magentas) with RT wt; (b) superimposition of 20 and ETV (yellow); (c) binding of 16d (green)
with RT wt; (d) superimposition of 16d and RPV (orange). Hydrogen bonds are indicated by red
dotted lines to key amino acids.
3.4. In silico calculation of physicochemical properties and measured log P and water
solubility
Water solubility and hydrophilicity/lipophilicity are important drug-like properties
to evaluate compounds as they need to be absorbed and distributed in the body. As we
all know that poor solubility of DAPYs limited their clinical use even though they had
good activity and anti-resistance profiles, so attentions were emphasized on
developing novel DAPYs derivatives with improved solubility[35]. The solubility and
logP of designed compounds were tested by using HPLC, and some other
physicochemical properties such as molecular weight and melting point were also
included. According to Lipinski's rule of five, main parameters of compound 16d, 18b,

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18d, 19d and ETV were evaluated (Table 4).
Table 4. Main physicochemical propertiesa of 16d, 18b, 18d, 19d and ETV.
Parameters Rule of five
EC50 (nM)
ETV
3.4
16d
5.6
18b
6660
378.38
9
18d
34
19d
170
419.44
10
MW
nON
< 500
< 10
< 5
438.87
9
404.42
9
435.28
7
nOHNH
nrotb
2
2
2
4
3
≤ 10
< 140
< 5
7
6
7
7
4
tPSA
125.70
3.33a
125.70
-0.11
125.70
2.51a
151.73
1.21
120.65
> 5b
logP
Solubility
(pH = 7.4)d
Solubility
(pH = 2.0)d
< 0.1
ND
62.91
> 100
< 1
ND
0.43
<< 1b
<< 1b
91.69
mp
273-275˚C 192-195˚C 268-270˚C 229-231˚C 255-257˚Cc
a
Using free on-line software (http://www.molinspiration.com/); MW = molecular weight; nON =
no. of hydrogen bond acceptors; nOHNH = no. of hydrogen bond donors; nrotb = no. of rotatable
bonds; log P = logarithm of partition coefficient.
b See Ref.[36].
c See Ref.[32].
d Measured in phosphate buffer by HPLC method in duplicate.
Introducing of an uracil into DAPYs increased the solubility (18b vs ETV), and the
acid solvent was more suitable. The amino substituent 19d increased the solubility,
while incorporation of hydrophilic group decreased the solubility which was contrast
to the SARs summarized above, so a balance should be found to discover compounds
with good antiviral activity and better solubility that can be administrated orally. The
high melting point and the low solubility indicated that the low solubility of designed
compounds was probably due to the intermolecular hydrophobic interaction and
hydrogen bond, so introducing of steric group or reducing the number of hydrogen
bond donor or accepter that decreased the intermolecular interaction would improve
the DAPYs’ solubility.

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4. Conclusion
In conclusion, based on the new binding model of DAPYs, a novel series of
compounds were designed and synthesized by crystallographic based molecular
hybridization. All the designed compounds were evaluated for their bioactivities
against HIV-1 (IIIB strain and K103N/Y181C double-mutated strains) and HIV-2
(ROD) in MT-4 cells, as well as against HIV-1 RT activity. Activity of compounds
16d and 19d against the prevalent mutant strains were also tested to investigated their
anti-resistance profiles. As expected, most of designed compounds showed moderate
to excellent anti-HIV-1 potency and low toxicity. These efforts resulted in the
discovery of compound 16d (EC50 = 5.6 nM, SI > 50000), which showed excellent
activity against WT HIV-1 and EC50 values at double digital nanomolar level against
K103N and E138K mutant strains, much better than clinically-used drugs NVP and
DLV, and 16d also showed moderate activity to L100I and Y181C strains. Preliminary
SARs were summarized based on the anti-HIV-1 assay and it was found that
incorporating a hydrophobic group of the C5 of pyrimidine was favored for the
activity. Activity against RT was performed, and confirmed that the antiviral potency
of designed compounds was due to the inhibition of HIV-1 RT. Furthermore,
molecular modeling studies were carried out to predict the interactions between these
inhibitors and HIV-1 RT. Some physicochemical properties like log P and water
solubility were also measured. This valuable information provided deeper insight into
these promising structures, and further efforts to improve the antiviral activity of these
uracil bearing derivatives will be reported in due course.
5. Experimental section
5.1. Chemistry
All melting points were measured with a micromelting point apparatus and are
1
uncorrected. H NMR and 13C NMR spectra were obtained on a Bruker AV-400
spectrometer (Bruker BioSpin, Switzerland), or Bruker AV-300 spectrometer (Bruker
BioSpin, Switzerland), using tetramethylsilane (TMS) as internal reference. The
solvent used was DMSO-d6 unless otherwise indicated. Chemical shifts were
expressed in δ units (ppm) and J values were presented in hertz (Hz). Mass

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spectrometry was performed on an LC Autosampler Standard G1313A instrument
(Applied Biosystems/MDS Sciex, Concord, ON, Canada). TLC was performed on
pre-coated HUANGHAI HSGF254, 0.15-0.20 mm TLC-plates (Yantai Jiangyou
Silica Gel Development Co., Ltd, Yantai, Shandong, PR China), and spots were
visualized by irradiation with UV light (λ 254 nm). Flash column chromatography
was performed on columns packed with silica gel 60 (200-300 mesh) (Qingdao
haiyang silica gel desiccant co., Ltd, Qingdao, PR China). Solvents were of reagent
grade, and if necessary, were purified and dried by standard methods. The key
reagents were purchased from commercial suppliers and without further purification
when used. Rotary evaporators served in concentration of the reaction solutions at
reduced pressure.
5.1.1 Synthesis of 3-benzoylpyrimidine-2,4(1H,3H)-dione (8)
A solution of uracil (7) (1.12 g, 10.0 mmol), benzoyl chloride (2.60 mL, 22.4
mmol), and pyridine (4 mL) in dry acetonitrile (20 mL) were stirred at room
temperature for 24h. The mixture was concentrated under reduced pressure and the
residue was partitioned between dichloromethane (50 mL) and water (50 mL). The
organic layer was separated and then evaporated under reduced pressure. The residue
was dissolved in a mixture of aqueous potassium carbonate (0.5 mol/L, 10 mL) and
dioxane (20 mL) and stirred for 30 min at room temperature. The pH value was
adjusted to 5 with glacial acetic acid and the residue was then concentrated under
reduced pressure, then saturated aqueous sodium hydrogen carbonate (100 mL) was
added and mixture was stirred for 1h. The products were filtered and the residue was
washed with cold water (3 × 10 mL). Crystallization of this material from aqueous
acetone gave the title compound 8 as a white crystal (2.35 g, 54.0%); mp: 175-177˚C.
1H NMR (300MHz, DMSO-d6) δ: 11.60 (s, 1H, NH), 7.95 (d, J=7.8 Hz, 2H, Ph-H),
7.78 (t, J=7.5 Hz, 1H, =CH) 7.58-7.69 (m, 3H, Ph-H), 5.74 (d, J=7.5, 1H, =CH).
ESI-MS: m/z 217.4 (M+1)+, 239.4 (M+23)+. C11H8N2O3 (216.05).
5.1.2 Synthesis of tert-butyl (2-bromoethyl)carbamate (10)
Di-t-butylcarbonate (4.00 g, 18.3 mmol) and triethylamine (2.35 mL, 16.7 mmol)
were added dropwise to a solution of 2-aminoethylbromide hydrogenbromide (3.42 g,

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16.7 mmol) in 1,4-dioxane (30 mL) at 0˚C. The mixture was stirred for 30 min at 0˚C
and then for 48h at room temperature. Residue was filtered and the filtrate
concentrated under reduced pressure. The resultant was dissolved in dichloromethane
(100 mL), and the solution was washed with water (3 × 30 mL). The organic layer
was dried over anhydrous Na2SO4 and evaporated to dryness to give 10 as an oily
residue (3.65 g, 98.0%); This intermediate was used for the next step without further
purification.
5.1.3
Synthesis
of
tert-butyl
(2-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethyl)carbamate (11)
A mixture of intermediate 8 (0.54 g, 2.5 mmol), K2CO3 (0.35 g, 2.5 mmol),
intermediate 10 (0.45 g, 2.0 mmol) and tetrabutylammonium iodide (TBAI) (0.37 g,
0.25 mmol) were dissolved in DMF (20 mL), and heated at 70˚C for 24h. After
cooling to room temperature, residue was filtered and the filtrate concentrated under
reduced pressure. The resultant was washed with ethyl acetate until no impurity was
detected and filtered, solid was collected to afford pale yellow powder (0.55 g,
1
61.3%); mp: 162-165˚C. H NMR (400 MHz, CDCl3) δ: 8.05 (d, J = 7.6 Hz, 2H,
Ph-H), 7.65 (t, J = 7.4 Hz, 1H, Ph-H), 7.50 (t, J = 7.8 Hz, 2H, Ph-H), 7.23 (d, J = 7.9
Hz, 1H, =CH), 5.75 (d, J = 7.9 Hz, 1H, =CH), 4.87 (s, 1H, NH), 3.86 (s, 2H, CH2),
3.43 (s, 2H, CH2), 1.45 (s, 9H, t-butyl-H). ESI-MS: m/z 360.4 (M+1)+, 382.5 (M+23)+.
C18H21N3O5 (359.15).
5.1.4 Synthesis of 1-(2-aminoethyl)pyrimidine-2,4(1H,3H)-dione hydrogen
chloride(12)
Intermediate 11 (0.51 g, 1.4 mmol) was dissolved in trifluoroacetic
acid/dichloromethane (1:1) (2.28 g, 20.0 mmol) and stirred for 4h at room
temperature. After the reaction, residue was concentrated under reduced pressure and
the traced amount of trifluoroacetic acid was removed by formation of azeotropic
mixture with toluene. The resultant was dilute with ethanol and the hydrochloric acid
in ethanol was added till no solid was produced, residue was filtered to afford white
1
solid (0.15 g, 68.1% calculated as uricalyl-1-ethyl amine). mp: 160-162˚C. H NMR
(400 MHz, DMSO-d6) δ: 11.31 (br, 1H, NH), 7.87 (br, 2H, NH2), 7.48 (d, J = 7.6 Hz,

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1H, =CH), 5.63 (d, J = 7.6 Hz, 1H, =CH), 4.01 (t, J = 6.0 Hz, 2H, CH2), 3.01 (t, J =
6.0 Hz, 2H, CH2). ESI-MS: m/z 156.3 (M+1)+. C6H9N3O2 (155.07).
5.1.5 Synthesis of (2E)-3-(4-hydroxy-3,5-dimethylphenyl)-2-propenenitrile (28)
A mixture of 4-bromo-2,6-dimethylphenol (5.00 g, 25.0 mmol), acrylamide (2.70 g,
37.5 mmol), tri(o-tolyl)phosphine (2.30 g, 7.5 mmol), palladium acetate (0.56 g, 2.5
mmol) were added to a two-neck bottle and triethylamine (5.5 mL) and
N,N-dimethylacetamide (50 mL) in a dropping funnel (pressure equalization) and
bubble for 10min under N2. The solvent was added in and mixture was heated at
130-140˚C for 24h. After cooling, the residue was filtered and concentrated under
reduced pressure. Resultant was dissolved in ethyl acetate (100 mL) and washed with
water (3 × 40 mL), then with brine and dried over anhydrous Na2SO4. Resultant was
evaporated to dryness and further purification was using silica-gel column
1
chromatography to give a pale gray solid (0.78 g, 48.4%). mp: 162-164˚C. H NMR
(400 MHz, DMSO-d6) δ: 8.65 (s, 1H, OH), 7.37 (s, 1H, NH), 7.25 (d, J = 15.8 Hz, 1H,
=CH), 7.13 (s, 2H, Ph-H), 6.95 (s, 1H, NH), 6.39 (d, J = 15.8 Hz, 1H, =CH), 2.17 (s,
6H, CH3 × 2). ESI-MS: m/z 192.5 (M+1)+. C11H13NO2 (191.09).
Intermediate 27 (1.00 g, 5.2 mmol) was dissolved in POCl3 (6 mL) in an ice bath
and reacted for 30min, then stirred overnight at room temperature. The mixture was
poured into ice water and extracted with ethyl acetate (50 mL), organic layer was
dried over anhydrous Na2SO4 and evaporated to dryness. The residue was purified by
silica-gel column chromatography to give a white solid (0.79 g, 87.2%). mp:
115-119˚C. ESI-MS: m/z 174.1 (M+1)+. C11H11NO (173.08).
5.1.6 Synthesis of 2-chloro-5-nitro-N-(2,4,6-trimethylphenyl)-4-Pyrimidinamine (15)
Triethylamine (1.53 mL, 11.0 mmol) and 2,4,6-trimethylaniline (1.40 mL, 10.0
mmol) were added dropwise to a solution of 2,4-dimethyl-5-nitropyrimidine (1.94 g,
10.0 mmol) in an ice bath, and stirred for 3h. The reaction mixture was concentrated
and water (50 mL) was added, then extracted with ethyl acetate (3 × 50 mL). The
organic layer was dried over anhydrous Na2SO4 and evaporated to dryness, the
resultant was recrystallized from ethyl acetate to give yellow crystal (2.50 g, 85.4%).
1
mp: 170-173˚C. H NMR (400 MHz, CDCl3): δ: 9.56 (s, 1H, NH), 9.15 (s, 1H,

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pyrimidine-H), 6.98 (s, 2H, Ph-H), 2.33 (s, 3H, CH3), 2.17 (s, 6H, CH3 × 2). ESI-MS:
m/z 293.3(M+1)+. C13H13ClN4O2 (292.07).
5.1.7 General procedure for the synthesis of intermediates (14a-l)
A mixture of 13a-l (1 equiv), substituted phenol (1.2 equiv) and K2CO3 (1.2 equiv)
in DMF (or acetone) was stirred overnight at room temperature. The residue was
filtered and concentrated under reduced pressure, ethyl acetate was added and washed
with water and brine. Organic layer was dried over anhydrous Na2SO4 and evaporated
to dryness, then recrystallized from ethyl acetate/petroleum ether to give the
corresponding intermediates.
5.1.7.1 2-chloro-4-(2,4,6-trimethylphenoxy)pyrimidine (14a).
White crystal, yield: 47.9%, mp: 68-70˚C, 1H NMR (400 MHz, CDCl3) δ: 8.40 (d, J
= 5.7 Hz, 1H, pyrimidine-H), 6.92 (s, 2H, Ph-H), 6.64 (d, J = 5.7 Hz, 1H,
pyrimidine-H), 2.31 (s, 3H, CH3), 2.07 (s, 6H, CH3 × 2). ESI-MS: m/z 249.4 (M+1)+.
C13H13ClN2O (248.07).
5.1.7.2 4-[(2-chloro-4-pyrimidinyl)oxy]-3,5-dimethylbenzonitrile (14b)
1
White powder, yield: 48.2%, mp: 191-193˚C, H NMR (400 MHz, CDCl3) δ: 8.51
(d, J = 5.7 Hz, 1H, pyrimidine-H), 7.44 (s, 2H, Ph-H), 6.90 (d, J = 5.7 Hz, 1H,
pyrimidine-H), 2.15 (s, 6H, CH3 × 2). ESI-MS: m/z 260.3 (M+1)+. C13H10ClN3O
(259.05).
5.1.7.3 2-chloro-4-(3,5-dimethylphenoxy)pyrimidine (14c)
White crystal, yield: 66.7%, mp: < 60˚C, 1H NMR (400 MHz, CDCl3) δ: 8.40 (d, J
= 5.7 Hz, 1H, pyrimidine-H), 6.93 (s, 1H, Ph-H), 6.75 (s, 2H, Ph-H), 6.72 (d, J = 5.7
Hz, 1H, pyrimidine-H), 2.34 (s, 6H, CH3 × 2). ESI-MS: m/z 235.3(M+1)+.
C12H11ClN2O (234.06).
5.1.7.4 E-3-[4-[(2-chloro-4-pyrimidinyl)oxy]-3,5-dimethylphenyl]-2-Propenenitrile
(14d)
1
Yellow crystal, yield: 79.0%, mp: 158-162˚C, H NMR (400 MHz, CDCl3) δ: 8.47
(d, J = 5.7 Hz, 1H, pyrimidine-H), 7.34 (d, J = 16.6 Hz, 1H, =CH), 7.21 (s, 2H, Ph-H),
6.83 (d, J = 5.7 Hz, 1H, pyrimidine-H), 5.84 (d, J = 16.6 Hz, 1H, =CH), 2.13 (s, 6H,
CH3 × 2). ESI-MS: m/z 286.3 (M+1)+. C15H12ClN3O (285.07).

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5.1.7.5 2,6-dichloro-4-(2,4,6-trimethylphenoxy)pyrimidine (14e)
White crystal, yield: 69.6%, mp: 124-126˚C, 1H NMR (400 MHz, CDCl3) δ: 6.93 (s,
2H, Ph-H), 6.65 (s, 1H, pyrimidine-H), 2.31 (s, 3H, CH3), 2.07 (s, 6H, CH3 × 2).
ESI-MS: m/z 283.3 (M+1)+. C13H12Cl2N2O (282.03).
5.1.7.6 4-[(2,6-dichloro-4-pyrimidinyl)oxy]-3,5-dimethylbenzonitrile (14f)
White solid, yield: 59.2%, mp: 197-198˚C, 1H NMR (400 MHz, CDCl3) δ: 7.45 (s,
2H, Ph-H), 6.93 (s, 1H, pyrimidine-H), 2.15 (s, 6H, CH3 × 2). ESI-MS: m/z
294.1(M+1)+, 311.1 (M+18)+. C13H9Cl2N3O (293.01).
5.1.7.7 2,6-dichloro-4-(3,5-dimethylphenoxy)pyrimidine (14g)
White solid, yield: 78.4%, mp: 58-60˚C, 1H NMR (400 MHz, CDCl3) δ: 6.95 (s, 1H,
pyrimidine-H), 6.74 (s, 2H, Ph-H), 6.71 (s, 1H, Ph-H), 2.35 (s, 6H, CH3 × 2). ESI-MS:
m/z 269.4 (M+1)+. C12H10Cl2N2O (268.02).
5.1.7.8
E-3-[4-[(2,6-dichloro-4-pyrimidinyl)oxy]-3,5-dimethylphenyl]-2-propenenitrile (14h)
White solid, yield: 57.8%, mp: 154-157˚C, 1H NMR (400 MHz, CDCl3) δ: 7.35 (d,
J = 16.6 Hz, 1H, =CH), 7.22 (s, 2H, Ph-H), 6.86 (s, 1H, pyrimidine-H), 5.85 (d, J =
16.6 Hz, 1H, =CH), 2.13 (s, 6H, CH3 × 2). ESI-MS: m/z 320.4 (M+1)+. C15H11Cl2N3O
(319.03).
5.1.7.9 2,5-dichloro-4-(2,4,6-trimethylphenoxy)pyrimidine (14i)
1
White solid, yield: 70.9%, mp: 99-101˚C, H NMR (400 MHz, CDCl3) δ: 8.46 (s,
1H, pyrimidine-H), 6.92 (s, 2H, Ph-H), 2.31 (s, 3H, CH3), 2.07 (s, 6H, CH3 × 2).
ESI-MS: m/z 283.3 (M+1)+. C13H12Cl2N2O (282.03).
5.1.7.10 4-[(2,5-dichloro-4-pyrimidinyl)oxy]-3,5-dimethylbenzonitrile (14j)
White crystal, yield: 75.0%, mp: 209-211˚C, 1H NMR (400 MHz, CDCl3) δ: 8.53 (s,
1H, pyrimidine-H), 7.46 (s, 2H, Ph-H), 2.16 (s, 6H, CH3 × 2). ESI-MS: m/z 294.3
(M+1)+. C13H9Cl2N3O (293.01).
5.1.7.11 2,5-dichloro-4-(3,5-dimethylphenoxy)pyrimidine (14k)
White crystal, yield: 71.6%, mp: 120-121˚C, 1H NMR (400 MHz, CDCl3) δ: 8.45 (s,
1H, pyrimidine-H), 6.93 (s, 1H, Ph-H), 6.78 (s, 2H, Ph-H), 2.35 (s, 6H, CH3 × 2).
ESI-MS: m/z 269.4 (M+1)+. C12H10Cl2N2O (268.02).

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5.1.7.12
E-3-[4-[(2,5-dichloro-4-pyrimidinyl)oxy]-3,5-dimethylphenyl]-2-propenenitrile (14l)
White crystal, yield: 75.4%, mp: 215-217˚C, 1H NMR (400 MHz, CDCl3) δ: 8.51 (s,
1H, pyrimidine-H), 7.35 (d, J = 16.6 Hz, 1H, =CH), 7.22 (s, 2H, Ph-H), 5.85 (d, J =
16.6 Hz, 1H, =CH), 2.14 (s, 6H, CH3 × 2). ESI-MS: m/z 320.3 (M+1)+. C15H11Cl2N3O
(319.03).
5.1.8 General procedure for the synthesis of target compounds 16a-d~18a-d
A mixture of intermediate 14a-l (1 equiv), intermediate 12 (1.2 equiv) and
N,N-diisopropylethylamine (2.4 equiv) in NMP was heated at 100˚C for 4-6h. After
cooling, ethyl acetate was added and washed with water for three times and then
washed with brine. Organic layer was dried over anhydrous Na2SO4 and evaporated to
dryness. The residue was purified by silica-gel column chromatography and then
recrystallized from methanol/chloroform to give the target compounds.
5.1.8.1
1-(2-((5-chloro-4-(2,4,6-trimethylphenoxy)pyrimidin-2-yl)amino)ethyl)
pyrimidine-2,4-(1H,3H)-dione (16a)
1
White powder, yield: 46.5%. mp: 245-248˚C. H NMR (400 MHz, DMSO-d6) δ:
11.14 (s, 1H, uracil-NH), 8.24 (s, 1H, pyrimidine-H), 7.57 (s, 0.4H, NH), 7.33 (s, 1H,
=CH), 6.94 (s, 2H, Ph-H), 6.73 (s, 0.4H, NH), 5.37 (s, 1H, =CH), 3.73 (s, 1H, CH2),
13
3.40 (s, 2H, CH2), 2.98 (s, 1H, CH2), 2.25 (s, 3H, CH3), 2.01 (s, 6H, CH3 × 2). C
NMR (100 MHz, DMSO) δ: 164.23, 163.28, 160.98, 157.70, 151.34, 147.40, 146.42,
135.09, 130.09, 129.56, 103.55, 100.65, 47.75, 46.35, 20.82, 16.32. ESI-MS: m/z
402.5 (M+1)+. C19H20ClN5O3 (401.13).
5.1.8.2
4-((5-chloro-2-((2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethyl)amino)
pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (16b)
1
White powder, yield: 36.7%. mp: 298-300˚C. H NMR (400 MHz, DMSO-d6) δ:
11.14 (s, 1H, uracil-NH), 8.30 (s, 1H, pyrimidine-H), 7.71 (s, 2H, Ph-H), 7.63 (s, 0.4H,
NH), 7.36 (s, 1H, =CH), 6.97 (s, 0.4H, NH), 5.38 (s, 1H =CH), 3.74 (s, 1H, CH2),
3.41 (s, 2H, CH2), 2.99 (s, 1H, CH2), 2.10 (s, 6H, CH3 × 2). 13C NMR (100 MHz,
DMSO) δ: 164.24, 162.57, 160.84, 158.28, 153.40, 151.31, 146.42, 133.01, 132.93,
118.98, 109.11, 103.12, 100.69, 47.69, 46.31, 16.10. ESI-MS: m/z 413.5 (M+1)+.

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C19H17ClN6O3 (412.11).
5.1.8.3 1-(2-((5-chloro-4-(3,5-dimethylphenoxy)pyrimidin-2-yl)amino)ethyl)
pyrimidine-2,4-(1H,3H)-dione (16c)
Pale yellow crystal, yield: 40.7%. mp: 236-238˚C. 1H NMR (400 MHz, DMSO-d6)
δ: 11.16 (s, 1H, uracil-NH), 8.24 (s, 1H, pyrimidine-H), 7.48 (br, 2H, =CH, NH), 6.90
(s, 2H, Ph-H), 6.82 (s, 1H, Ph-H), 5.42 (d, J = 7.4 Hz, 1H, =CH), 3.74 (s, 1H, CH2),
13
3.54 (s, 1H, CH2), 3.42 (s, 1H, CH2), 3.09 (s, 1H, CH2), 2.28 (s, 6H, CH3 × 2). C
NMR (100 MHz, DMSO) δ: 164.26, 164.17, 160.81, 157.88, 152.46, 151.40, 146.40,
145.90, 139.48, 127.45, 119.58, 100.98, 47.72, 46.28, 21.23. ESI-MS: m/z: 388.1165
(M+1)+. C18H18ClN5O3 (387.82).
5.1.8.4
(E)-3-(4-((5-chloro-2-((2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethyl)
amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (16d)
1
Brown powder, yield: 59.8%. mp: 273-275˚C. H NMR (400 MHz, DMSO-d6) δ:
11.14 (s, 1H, uracil-NH), 8.27 (s, 1H, pyrimidine-H), 7.59 (d, J = 16.5 Hz, 2H, =CH),
7.48 (s, 2H, Ph-H), 7.35 (s, 1H, =CH), 6.42 (d, J = 16.6 Hz, 1H, =CH), 5.38 (s, 1H,
=CH), 3.74 (s, 1H, CH2), 3.40 (s, 2H, CH2), 2.98 (s, 1H, CH2), 2.08 (s, 6H, CH3 × 2).
13C NMR (100 MHz, DMSO) δ: 164.24, 162.92, 160.89, 158.06, 151.80, 151.34,
150.36, 146.42, 131.87, 131.53, 128.59, 119.33, 103.26, 100.62, 97.00, 47.67, 46.08,
16.37. ESI-MS: m/z 439.1295 (M+1)+. C21H19ClN6O3 (438.87).
5.1.8.5
1-(2-((4-chloro-6-(2,4,6-trimethylphenoxy)pyrimidin-2-yl)amino)ethyl)
pyrimidine-2,4-(1H,3H)-dione (17a)
1
White solid, yield: 33.1%. mp: 204-205˚C. H NMR (400 MHz, DMSO-d6) δ:
11.17 (d, 1H, uracil-NH), 8.32 (s, 0.3H, pyrimidine-H), 7.85 (s, 0.4H, NH), 7.62 (s,
0.6H, pyrimidine-H), 7.38 (s, 0.6H, NH), 6.94 (d, 2H, Ph-H), 6.34-6.05 (m, 1H, =CH),
5.40 (d, 1H, =CH), 3.75 (s, 1H), 3.52 (s, 1H, CH2), 3.42 (s, 1H, CH2), 3.24-3.06 (m,
1H, CH2), 2.24 (s, 3H, CH3), 2.01 (s, 6H, CH3 × 2). 13C NMR (100 MHz, DMSO) δ:
170.09, 164.25, 162.27, 161.36, 151.35, 147.33, 146.33, 135.11, 129.66, 100.69,
94.79, 79.65, 48.00, 46.61, 20.79, 16.40. ESI-MS: m/z 402.5 (M+1)+. C19H20ClN5O3
(401.13).
5.1.8.6
4-((6-chloro-2-((2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethyl)amino)

ACCEPTED MANUSCRIPT
pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (17b)
1
White solid, yield: 41.3%. mp: 228-230˚C. H NMR (400 MHz, DMSO-d6) δ:
10.97 (d, 1H, uracil-NH), 7.75 (s, 0.5H, NH), 7.66 (s, 2H, Ph-H), 7.55 (s, 0.5H, NH),
7.37 (s, 1H, pyrimidine-H), 6.37 (d, J = 17.4 Hz, 1H, =CH), 5.52-5.46 (m, 1H, =CH),
3.91 (s, 1H, CH2), 3.62 (s, 1H, CH2), 3.39 (s, 1H, CH2), 3.06 (s, 1H, CH2), 2.11 (d, 6H,
13
CH3 × 2). C NMR (100 MHz, DMSO) δ: 168.95, 163.73, 162.15, 161.48, 152.01,
140.89, 133.01, 118.99, 108.89, 100.18, 94.56, 93.62, 79.65, 38.95, 38.18, 16.22.
ESI-MS: m/z 413.5 (M+1)+, 435.4 (M+23)+. C19H17ClN6O3 (412.11).
5.1.8.7
1-(2-((4-chloro-6-(3,5-dimethylphenoxy)pyrimidin-2-yl)amino)ethyl)
pyrimidine-2,4-(1H,3H)-dione (17c)
1
White powder, yield: 37.0%. mp: 248-251˚C. H NMR (400 MHz, DMSO-d6) δ:
11.18 (d, 1H, uracil-NH), 7.84 (s, 0.5H, NH), 7.68 (s, 0.5H, NH), 7.43 (s, 0.5H,
pyrimidine-H), 7.11 (s, 0.5H, pyrimidine-H), 6.91 (s, 1H, =CH), 6.81 (s, 2H, Ph-H),
6.20 (d, 1H, Ph-H), 5.44 (d, J = 7.5 Hz, 1H, =CH), 3.76 (s, 1H, CH2), 3.64 (s, 1H,
CH2), 3.44 (s, 1H, CH2), 3.26 (s, 1H, CH2), 2.28 (s, 6H, CH3 × 2). 13C NMR (100
MHz, DMSO) δ: 171.13, 170.83, 164.25, 162.15, 161.28, 152.50, 151.44, 146.36,
145.94, 139.65, 127.55, 119.44, 101.07, 100.92, 95.72, 94.62, 47.98, 46.96, 21.23.
ESI-MS: m/z 388.4 (M+1)+, 410.4 (M+23)+. C18H18ClN5O3 (387.82).
5.1.8.8
(E)-3-(4-((6-chloro-2-((2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethyl)
amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (17d)
Pale yellow powder, yield: 60.8%, mp: 290-292˚C. 1H NMR (400 MHz, DMSO-d6)
δ: 11.16 (d, 1H, uracil-NH), 7.95-7.52 (m, 2H, pyrimidine-H, =CH), 7.47 (d, 2H,
Ph-H), 7.38 (s, 0.5H, =CH), 6.94 (s, 0.5H, =CH), 6.39 (dd, J = 22.2, 18.9 Hz, 2H,
=CH), 5.39 (dd, J = 22.9, 7.6 Hz, 1H, =CH), 3.74 (d, 1H, CH2), 3.53-3.36 (m, 2H,
CH2), 3.09 (d, 1H, CH2), 2.12-2.02 (m, 6H, CH3 × 2). 13C NMR (100 MHz, DMSO) δ:
169.91, 169.50, 164.25, 164.14, 162.12, 161.54, 151.72, 151.44, 151.25, 150.37,
146.32, 145.56, 131.80, 131.61, 128.67, 128.55, 119.33, 100.70, 96.94, 94.97, 47.94,
46.45, 31.15, 16.49. ESI-HRMS: m/z 439.1272 (M+1)+. C21H19ClN6O3 (438.87).
5.1.8.9
1-(2-((4-(2,4,6-trimethylphenoxy)pyrimidin-2-yl)amino)ethyl)pyrimidine-2,4-(1H,3H)-

ACCEPTED MANUSCRIPT
dione (18a)
1
White powder, yield: 66.3%. mp: 216-218˚C. H NMR (400 MHz, DMSO-d6) δ:
11.16 (s, 1H, uracil-NH), 8.10 (d, J = 4.9 Hz, 1H, pyrimidine-H), 7.33 (s, 1H,
pyrimidine-H), 7.13 (s, 1H, NH), 6.93 (s, 2H, Ph-H), 6.09 (s, 1H, =CH), 5.37 (s, 1H,
=CH), 3.74 (s, 1H, CH2), 3.45 (s, 2H, CH2), 3.18 (s, 1H, CH2), 2.25 (s, 3H, CH3), 2.00
(s, 6H, CH3 × 2). 13C NMR (100 MHz, DMSO) δ: 169.15, 164.23, 162.96, 160.00,
151.38, 147.50, 146.17, 134.77, 130.32, 129.57, 100.66, 96.08, 48.00, 39.70, 20.82,
16.45. ESI-MS: m/z 368.3 (M+1)+. C19H21N5O3 (367.40).
5.1.8.10
4-((2-((2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethyl)amino)pyrimidin-4-yl)oxy)-
3,5-dimethylbenzonitrile (18b)
1
White solid, yield: 49.4%. mp: 192-195˚C. H NMR (400 MHz, DMSO-d6) δ:
10.96 (s, 1H, uracil-NH), 8.15 (d, J = 8.6 Hz, 1H, pyrimidine-H), 7.65 (s, 2H, Ph-H),
7.36 (s, 1H, pyrimidine-H), 7.03 (s, 1H, =CH), 6.22 (s, 1H, NH), 5.49 (d, J = 7.1 Hz,
1H, =CH), 3.91 (s, 1H, CH2), 3.66 (s, 1H, CH2), 3.41 (s, 1H, CH2), 3.08 (s, 1H, CH2),
2.09 (s, 6H, CH3 × 2). 13C NMR (100 MHz, DMSO) δ: 168.23, 163.68, 162.83,
160.43, 153.80, 152.01, 140.83, 133.17, 132.87, 119.14, 108.58, 100.25, 95.90, 79.65,
38.97, 16.27. ESI-MS: m/z 379.5 (M+1)+. C19H18N6O3 (378.14).
5.1.8.11
1-(2-((4-(3,5-dimethylphenoxy)pyrimidin-2-yl)amino)ethyl)pyrimidine-2,4-(1H,3H)-di
one (18c)
1
White powder, yield: 55.8%. mp: 215-217˚C. H NMR (400 MHz, DMSO-d6) δ:
11.17 (s, 1H, uracil-NH), 8.12 (d, J = 5.2 Hz, 1H, pyrimidine-H), 7.35 (s, 1H,
pyrimidine-H), 7.22 (s, 1H, Ph-H), 6.89 (s, 1H, =CH), 6.78 (s, 2H, Ph-H), 6.07 (s, 1H,
NH), 5.42 (d, J = 7.1 Hz, 1H, =CH), 3.73 (s, 2H, CH2), 3.45 (s, 1H, CH2), 3.28 (s, 1H,
CH2), 2.27 (s, 6H, CH3 × 2). 13C NMR (100 MHz, DMSO) δ: 170.00, 164.29, 162.86,
160.33, 152.84, 151.44, 146.27, 139.54, 127.20, 119.56, 100.87, 97.00, 47.88, 21.25.
ESI-MS: m/z 354.4 (M+1)+. C18H19N5O3 (353.38).
5.1.8.12
(E)-3-(4-((2-((2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethyl)amino)
pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (18d)

ACCEPTED MANUSCRIPT
1
White solid, yield: 74.8%. mp: 268-270˚C. H NMR (400 MHz, DMSO-d6) δ:
11.13 (s, 1H, uracil-NH), 8.15 (d, J = 4.0 Hz, 1H, pyrimidine-H), 7.59 (d, J = 16.2 Hz,
1H, =CH), 7.46 (s, 2H, Ph-H), 7.33 (s, 1H, pyrimidine-H), 7.15 (s, 1H, =CH), 6.40 (d,
J = 16.3 Hz, 1H, =CH), 6.20 (s, 1H, NH), 5.37 (d, J = 4.0 Hz, 1H, =CH), 3.74 (s, 1H,
CH2), 3.45 (s, 2H, CH2), 3.12 (s, 1H, CH2), 2.06 (s, 6H, CH3 × 2). 13C NMR (100
MHz, DMSO) δ: 168.74, 164.23, 162.84, 160.27, 152.00, 151.35, 150.47, 146.38,
131.76, 131.55, 129.41, 128.58, 119.38, 100.65, 96.73, 48.03, 46.87, 16.53. ESI-MS:
m/z 405.6 (M+1)+. C21H20N6O3 (404.42).
5.1.8.13
1-(2-((2-chloro-6-(2,4,6-trimethylphenoxy)pyrimidin-4-yl)amino)ethyl)
pyrimidine-2,4-(1H,3H)-dione (21a)
1
White powder, yield: 23.3%. mp: 200-202˚C. H NMR (400 MHz, DMSO-d6) δ:
11.19 (s, 1H, uracil-NH), 7.81 (s, 1H, pyrimidine-H), 7.45 (d, J = 7.6 Hz, 1H, =CH),
6.95 (s, 2H, Ph-H), 5.40-5.48 (m, 2H, =CH, NH), 3.76 (t, J = 5.3 Hz, 2H, CH2),
3.59-3.35 (m, 2H, CH2), 2.25 (s, 3H, CH3), 2.01 (s, 6H). 13C NMR (100 MHz, DMSO)
δ: 169.15, 166.30, 164.20, 159.69, 151.49, 147.20, 146.25, 135.32, 130.18, 129.92,
100.96, 84.37, 60.21, 48.13, 20.79, 16.26. ESI-MS: m/z 402.5 (M+1)+. C19H20ClN5O3
(401.13).
5.1.8.14
4-((2-chloro-6-((2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethyl)amino)
pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (21b)
1
White solid, yield: 28.5%. mp: 239-242˚C. H NMR (400 MHz, DMSO-d6) δ:
11.39-11.04 (m, 1H, uracil-NH), 7.99 (d, 1H, pyrimidine-H), 7.69 (s, 2H, Ph-H),
7.29-7.60 (m, 1H, =CH), 5.73 (s, 1H, NH), 5.43 (d, J = 7.6 Hz, 1H, =CH), 3.79 (s, 2H,
CH2), 3.56 (s, 1H, CH2), 3.44 (s, 1H, CH2), 2.09 (s, 6H, CH3 × 2). 13C NMR (100
MHz, DMSO) δ: 166.32, 164.21, 161.70, 159.53, 153.29, 151.53, 146.27, 133.06,
118.94, 108.99, 100.99, 85.20, 79.65, 48.17, 47.30, 16.17. ESI-MS: m/z 413.5 (M+1)+,
435.4 (M+23)+. C19H17ClN6O3 (412.11).
5.1.8.15
1-(2-((2-chloro-6-(3,5-dimethylphenoxy)pyrimidin-4-yl)amino)ethyl)
pyrimidine-2,4-(1H,3H)-dione (21c)
1
White solid, yield: 25.0%. mp: 210-212˚C. H NMR (400 MHz, DMSO-d6) δ:
11.21 (d, 1H, uracil-NH), 7.93 (s, 1H, pyrimidine-H), 7.46 (d, J = 7.8 Hz, 1H, =CH),

ACCEPTED MANUSCRIPT
6.90 (s, 1H, Ph-H), 6.77 (s, 2H, Ph-H), 5.71-5.29 (m, 2H, =CH2, NH), 3.89-3.64 (m,
2H, CH2), 3.52 (s, 1H, CH2), 3.42 (s, 1H, CH2), 2.28 (s, 6H, CH3 × 2). 13C NMR (100
MHz, DMSO) δ: 166.15, 165.48, 164.23, 152.86, 151.51, 146.26, 139.13, 126.99,
119.19, 101.15, 99.32, 86.20, 47.27, 39.23, 21.25. ESI-MS: m/z 388.4 (M+1)+, 410.4
(m+23)+. C18H18ClN5O3 (387.82).
5.1.8.16
(E)-3-(4-((2-chloro-6-((2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethyl)amino)
pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (21d)
1
White powder, yield: 18.2%. mp: 160-165˚C. H NMR (400 MHz, DMSO-d6) δ:
11.20 (s, 1H, uracil-NH), 7.94 (s, 1H, pyrimidine-H), 7.59 (d, J = 16.8 Hz, 1H, =CH),
7.47 (s, 3H, Ph-H, =CH), 6.42 (d, J = 16.8 Hz, 1H, =CH), 5.62 (s, 1H, =CH), 5.43 (s,
1H, NH), 3.78 (s, 2H, CH2), 3.42 (s, 2H, CH2), 2.07 (s, 6H, CH3 × 2). 13C NMR (100
MHz, DMSO) δ: 168.51, 166.31, 164.21, 159.63, 151.51, 150.32, 146.27, 131.88,
131.63, 128.86, 119.31, 100.96, 97.05, 84.85, 79.65, 48.16, 39.18, 16.39. ESI-HRMS:
m/z 439.1284 (M+1)+. C21H19ClN6O3 (438.87).
5.1.8.17
1-(2-((2-chloro-4-(mesityloxy)pyrimidin-5-yl)amino)ethyl)pyrimidine-2,4-(1H,3H)
-dione (22)
1
White powder, yield: 24.0%. mp: 254-256˚C. H NMR (400 MHz, DMSO-d6) δ:
10.94 (s, 1H, uracil-NH), 8.21 (s, 1H, pyrimidine-H), 7.35 (d, J = 7.4 Hz, 1H, =CH),
7.22 (s, 1H), 6.91 (s, 2H, Ph-H), 5.48 (d, J = 7.5 Hz, 1H, =CH), 3.90 (s, 1H, NH),
3.59 (s, 1H, CH2), 3.37 (s, 2H, CH2), 2.98 (s, 1H, CH2), 2.24 (s, 3H, CH3), 2.01 (s, 6H,
13
CH3 × 2). C NMR (100 MHz, DMSO) δ: 163.69, 163.09, 161.13, 158.12, 157.54,
152.02, 147.46, 140.81, 134.94, 130.11, 129.53, 103.07, 100.25, 39.17, 20.81, 16.33.
ESI-MS: m/z 402.5 (M+1)+. C19H20ClN5O3 (401.13).
5.1.9 General procedure for the synthesis of target compounds 19a-d
Ammonium hydroxide was added to a solution of 17a-d in 1,4-dioxane and heated
in an autoclave at 120˚C for 12 h. After cooling to 50-60˚C, water was added slowly
to the reaction mixture, then cooled to 5-10˚C. The resultant precipitate was filtered
and washed with chilled 1,4-dioxane and water, and then recrystallized from

ACCEPTED MANUSCRIPT
methanol/chloroform to give the target compounds.
5.1.9.1 1-(2-((4-amino-6-(2,4,6-trimethylphenoxy)pyrimidin-2-yl)amino)ethyl)
pyrimidine-2,4-(1H,3H)-dione (19a)
1
White powder, yield: 70.1%. mp: 214-217˚C. H NMR (400 MHz, D MSO-d6) δ:
11.18 (s, 1H, uracil-NH), 7.44 (d, J = 7.2 Hz, 1H, =CH), 6.89 (s, 2H, Ph-H), 6.58 (s,
1H, NH), 6.23 (s, 1H, NH2), 6.00 (s, 1H, NH2), 5.57-5.33 (m, 1H, =CH), 4.92 (s, 1H,
NH), 3.72 (d, J = 24.0 Hz, 2H, CH2), 3.37 (d, J = 24.5 Hz, 2H, CH2), 2.23 (s, 3H,
CH3), 2.01 (s, 6H, CH3 × 2). 13C NMR (100 MHz, DMSO) δ: 169.92, 166.81, 164.27,
162.79, 151.44, 147.95, 146.46, 134.29, 130.56, 130.52, 129.50, 100.96, 60.23, 48.09,
20.81, 16.44. ESI-MS: m/z 383.4 (M+1)+. C19H22N6O3 (382.42).
5.1.9.2
4-((6-amino-2-((2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethyl)amino)
pyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (19b)
1
White powder, yield: 60.3%. mp: 220-222˚C. H NMR (400 MHz, DMSO-d6) δ:
11.21 (s, 1H, uracil-NH), 7.62 (s, 2H, Ph-H), 7.47 (d, J = 7.6 Hz, 1H, =CH), 6.94 (s,
1H, pyrimidine-H), 6.04 (s, 2H, NH2), 5.48 (d, J = 7.5 Hz, 1H, =CH), 5.11 (s, 1H,
NH), 3.77 (s, 2H, CH2), 3.43 (s, 2H, CH2), 2.09 (s, 6H, CH3 × 2). 13C NMR (100 MHz,
DMSO) δ: 168.90, 165.96, 164.27, 163.49, 154.42, 151.53, 146.47, 133.35, 132.81,
119.20, 108.09, 100.99, 76.27, 55.38, 48.13, 16.34. ESI-MS: m/z 394.3 (M+1)+.
C19H19N7O3 (393.40).
5.1.9.3
1-(2-((4-amino-6-(3,5-dimethylphenoxy)pyrimidin-2-yl)amino)ethyl)
pyrimidine-2,4-(1H,3H)-dione (19c)
1
White powder, yield: 66.7%. mp: 280-282˚C. H NMR (400 MHz, DMSO-d6) δ:
11.17 (s, 1H, uracil-NH), 7.42 (s, 1H, =CH), 6.84 (s, 1H, pyrimidine-H), 6.72 (s, 2H,
Ph-H), 6.62 (s, 1H, Ph-H), 6.28 (s, 2H, NH2), 5.45 (s, 1H, =CH), 4.99 (s, 1H, NH),
3.72 (s, 2H, CH2), 3.33 (s, 2H, CH2), 2.26 (s, 6H, CH3 × 2). 13C NMR (100 MHz,
DMSO) δ: 170.79, 166.73, 164.33, 162.68, 158.96, 158.49, 153.71, 151.48, 146.35,
139.35, 126.62, 119.47, 100.84, 77.78, 47.91, 21.26. ESI-HRMS: m/z 369.1680
(M+1)+. C18H20N6O3 (368.39).
5.1.9.4
(E)-3-(4-((6-amino-2-((2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethyl)
amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (19d)