European Journal of Medicinal Chemistry 115 (2016) 463e483

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Design, synthesis and biological evaluation of pyrido[2,3-d]pyrimidin-

7-(8H)-ones as HCV inhibitors


Marta Camarasa a, Raimon Puig de la Bellacasa a, Alex L. Gonzalez a, Raül Ondon~ o a,
Roger Estrada a, Sandra Franco b, Roger Badia b, Jose Este 
 b, Miguel Angel Martínez b,
a b  I. Borrell
 , Bonaventura Clotet , Jose a, *
Jordi Teixido
a
Grup d’Enginyeria Molecular, Institut Químic de Sarria
, Universitat Ramon Llull, Via Augusta 390, E-08017 Barcelona, Spain
b
noma de Barcelona, 08916 Badalona, Spain
Retrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Auto

a r t i c l e i n f o a b s t r a c t

Article history: The design and selection of a combinatorial library of pyrido[2,3-d]pyrimidin-7(8H)-ones (4) has allowed
Received 28 July 2015 the synthesis of 121 compounds, using known and new synthetic methodologies, and the evaluation of
Received in revised form the inhibitory activity against hepatitis C virus (HCV) genotype 1b replicon. Among these compounds, 21
3 March 2016
{4,10} and 24{2,10} presented very high activities [EC50 ¼ 0.027 mM (CC50 ¼ 5.3 mM) and EC50 ¼ 0.034 mM
Accepted 18 March 2016
Available online 21 March 2016
(CC50 ¼ 13.5 mM), respectively] and high selectivity indexes, 196 and 397. These values are similar to the
EC50 reported for sofosbuvir (2) (0.048 mM) using a similar methodological approach and the same virus
subtype. 21{4,10} and 24{2,10} are obtained through shorter synthetic itineraries than sofosbuvir and 24
Keywords:
Hepatitis C
{2,10} is achiral contrary to sofosbuvir which presents 4 stereogenic centers. In silico studies suggest that
HCV genotype 1b replicon 21{4,10} and 24{2,10} inhibits NS5B polymerase through allosteric site binding.
Small molecule inhibitor © 2016 Elsevier Masson SAS. All rights reserved.
Pyrido[2,3-d]pyrimidin-7-(8H)-ones

1. Introduction
Hepatitis C is an infectious disease caused by the hepatitis C
virus (HCV), which constitutes the leading cause of chronic liver
diseases. HCV has infected an estimated 150e200 million people
being an important health care problem worldwide [1]. The virus
persists in the liver in about 85% of those infected.
HCV belongs to the genus Hepacivirus, a member of the family
Flaviviridae. HCV is a positive-sense single-stranded RNA virus of
approximately 9.6 Kb. Based on genetic differences between HCV
isolates, the hepatitis C virus species is classified into seven
Abbreviations: BOP, (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate; m-CPBA, 3-Chloroperbenzoic acid; DBU, 1,8-Diazabicyclo
[5.4.0]undec-7-ene; DDQ, 2,3-Dichloro-5,6-dicyano-p-benzoquinone; PSII, Palm
site II of NS5B polymerase; PyBOP, (Benzotriazol-1-yloxy)tripyrrolidinophospho-
nium hexafluorophosphate.
* Corresponding author.
E-mail addresses: martacamarasan@gmail.com (M. Camarasa), raimonpc@gmail.
com (R. Puig de la Bellacasa), alejandro.lopez@iqs.url.edu (A.L.
Gonza lez),
raulondonom@iqs.url.edu (R. Ondon ~ o), roger.estrada@iqs.url.edu (R. Estrada),
sfranco@irsicaixa.es (S. Franco), rbadia@irsicaixa.es (R. Badia), jaeste@irsicaixa.es
(J. Este ), MMartinez@irsicaixa.es (M.A.  Martínez), jordi.teixido@iqs.url.edu
), bclotet@irsicaixa.es (B. Clotet), j.i.borrell@iqs.url.edu (J.I. Borrell).
(J. Teixido
genotypes (1e7) with several subtypes within each genotype [2],
which differ by 30e35% of the nucleotide sites over the complete
genome. For instance, subtypes 1a and 1b are found worldwide and
cause 60% of hepatitis C cases.
HCV encodes structural (E1, E2, C and p7) and nonstructural
proteins (NS2, NS3/4A, NS4B, NS5A, NS5B) [3]. Nonstructural pro-
teins represent the main targets for intensive anti-HCV drug dis-
covery programs.
Before the development of direct acting antivirals (DAAs),
treatment for hepatitis C was based on a combination of (pegy-
lated) interferon-alpha (PEG-IFN-a) plus ribavirin (1) for 24 or 48
weeks, depending on the HCV genotype [4]. This combination
therapy yields a sustained virologic response (SVR) in more than
40% of patients infected by genotype 1 and 4 viruses and about
80% of those infected by genotypes 2 and 3. Besides the limited
efficacy on HCV type 1, this combination therapy has significant
side effects and is poorly tolerated in many patients. The most
common side effects that may occur after treatment with PEG-
IFN-a are: nausea, diarrhea, fever, chills, muscle and joint pain,
difficulty concentrating, thyroid disorders, hair loss, insomnia, ir-
ritability, mild to severe depression, and rarely, suicidal thoughts.
Other serious side effects that may occur are bone marrow
toxicity, cardiovascular disorders, hypersensitivity disorders,

http://dx.doi.org/10.1016/j.ejmech.2016.03.055
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
464 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
Fig. 1. Ribavirin (1), sofosbuvir (2), pyrido[3,2-d]pyrimidines active against HCV (3),
and pyrido[2,3-d]pyrimidin-7-(8H)-ones (4).
endocrine disorders, pulmonary disorders, colitis, pancreatitis and
ophthalmologic disorders. PEG-IFN-a can cause neuropsychiatric,
autoimmune, ischemic and infectious fatal. With respect to riba-
virin, anemia is often the most frequent side effect during the
treatment of hepatitis C. Ribavirin can also cause decreased white
blood cells and platelets [5].
In 2011, the first two DAAs (boceprevir and telaprevir) that
targeted NS3/4A protease viral protein were introduced to the
market. Added to PEG-IFN-a and ribavirin increased the SVR in
genotype infected patients. However, several disadvantages were
reported [6]. Thus, further pursuit of this strategy as a potential
treatment was warranted. More recently, sofosbuvir (2, Sovaldi™)
[7], a nucleotide analog, has been approved for the treatment of
HCV infection. Treatment with this new drug without PEG-IFN-a
provides higher cure rates, less side effects, and a reduction of the
duration of the therapy. Sofosbuvir inhibits the NS5B polymerase,
one of the most attractive targets of HCV because it presents a
highly conserved region in the genome with respect to the different
genotypes [8]. Sofosbuvir has changed the prognosis of this severe
disease, however the price associated with the treatment (in the
range 20,000e40,000 V per one year) has engendered considerable
controversy including patent invalidity claims (Fig. 1).
We consider that this scenario shows that there is still a need for
clinically effective agents for the treatment of infections caused the
hepatitis C virus (HCV), which may improve current therapeutic
strategies, in particular, which may reduce unwanted side effects of
current drugs.
We decided to focus on the NS5B polymerase in order to develop
Scheme 1. Synthesis of 7-oxopyrido[2,3-d]pyrimidines 9, 10, and 11 from a,b-unsaturated esters 5 via pyridones 7.
a non-nucleotide inhibitor. As starting point we consider a series of
patents of Gilead Sciences on the use of pyrido[3,2-d]pyrimidines
(3) as HCV inhibitors (see for instance [9]) due to the structural
similarity between such compounds and the 7-oxopyrido[2,3-d]
pyrimidines (4) developed by our group.
In this context, our group has a broad experience in the syn-
thesis of 4-amino-5,6-dihydropyrido[2,3-d]pyrimidin-7-(8H)-ones
(9) (Scheme 1) from a,b-unsaturated esters (5). Thus, 2-methoxy-6-
oxo-1,4,5,6-tetrahydropyridin-3-carbonitriles (7) are obtained by
reaction of an a,b-unsaturated ester (5) and malononitrile (6) in
NaOMe/MeOH [10]. Treatment of pyridones 7 with guanidine sys-
tems (8, R4 ¼ H, alkyl, aryl) affords 4-amino-pyrido[2,3-d]pyrimi-
dines (9, R4 ¼ H, alkyl, aryl) [11].
More recently, we have developed protocols for the dehydro-
genation of 5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-ones 12 to
pyrido[2,3-d]pyrimidin-7(8H)-ones 11 [12] and a methodology for
the synthesis of 2-arylamino substituted 4-amino-5,6-
dihydropyrido[2,3-d]pyrimidin-7(8H)-ones (9, R4 ¼ aryl) via the
corresponding 3-aryl substituted pyridopyrimidines 10, formed
upon treatment of pyridones 7 with an arylsubstituted guanidine 8
in dioxane, which underwent the Dimroth rearrangement to the
desired 4-aminopyridopyrimidines (9, R4 ¼ aryl) with NaOMe/
MeOH. The overall yields of such two-step protocol are in general
higher than the reaction between pyridones 7 and an arylsub-
stituted guanidine 8 (Scheme 1) [13].
The present paper deals with the use of such methodology for
the synthesis of pyrido[2,3-d]pyrimidin-7-(8H)-ones as a potential
treatment of HCV.
2. Results and discussion
2.1. Virtual combinatorial library of pyrido[2,3-d]pyrimidin-7-
(8H)-ones
Inhibitory activity of several privileged scaffolds has been
intensively researched as potential anti-HCV candidates. Among
them, Gilead Sciences reported a large series of pyrido[3,2-d]py-
rimidines with promising activities but the main HCV target of
these molecules remains unclear [9]. A chemical and structural
similarity search of the crystallized targets, using a subset of pyrido
[3,2-d]pyrimidines as query, yielded similarity scores higher than
0.7 for molecules targeting NS5B polymerase (see Methods for
details), thus we hypothesize that this scaffold could potentially
target this HCV enzyme. Nevertheless, in view of the potency of
pyrido[3,2-d]pyrimidines and own expertise complemented by
computer-assisted rational design, we chose to evaluate the activity
 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483 465

of a combinatorial library of pyrido[2,3-d]pyrimidin-7-(8H)-ones

for optimization.
In order to construct our virtual combinatorial library of pyrido
[2,3-d]pyrimidin-7-(8H)-ones (4), we decided to take into account
the structural information contained in a series of patents of Gilead
Sciences on the use of pyrido[3,2-d]pyrimidines (3) as HCV in-
hibitors (WO2008/077651, WO2008/077650, WO2010/002998,
WO2008/077649, and WO2006/120252). The large number of
compounds included in such patents (800) led us to reduce them
on the basis of the values of the EC50 (effective concentration
required to inhibit 50% of replicating cells containing the HCV
replicon) and cytotoxic CC50 (concentration needed to induce cell
death in 50% of treated cells). It should be noted that these patents
do not give the exact value of the EC50 or the CC50 but only classified
compounds in three different categories for each parameter: EC50
(<0.1 mM; between 0.1 and 0.25 mM; and between 0.25 and 2 mM)
and CC50 (>25 mM; between 5 and 25 mM; and <5 mM). Conse-
quently, we decided to select molecules with EC50 < 0.1 mM and,
simultaneously, CC50 > 25 mM. This assumption drastically reduced
the number of molecules to be considered from the initial 800 to
236 from which we studied the nature of the substituents R1, R2 and
R3 present in structures 3. Thus, we analyzed the most repeated
substituents present at positions R1, R2 and R3 of these compounds
and we classified them according to frequency of occurrence from
major to minor (see supplementary information).
This protocol allowed us to select 31 potential substituents at R1,
32 at R2 and 89 at R3. The use of this set of substituents in our pyrido
[2,3-d]pyrimidin-7-(8H)-ones (4) afforded a potential virtual
combinatorial library of 31  32  89 ¼ 88,288 compounds, which Fig. 2. a,b-Unsaturated esters 5{x} used for library production.
could be even higher if we considered different substituents at R4
(H, Me, Et, CH2C6H5, ..).
Given the overall size of the potential library, a diverse selection complete in the case of PyBOP. In that way we obtained 14{2}, 14
of 200 compounds 4 was carried out based on the R1, R2 and R3 {4}, and 14{5} in 74e93% yield.
residues with higher frequencies of occurrence (including substi- Once obtained the intermediates 14{x}, we started to study the
tution at R4) and the commercial availability of the necessary substitution by different amines and alcohols of general structure
building blocks. Similarity scores of these molecules ranged be- R2-H and we realized that the direct substitution of the benzo-
tween 0.8 and 1.2 in a scale of 0e2, which implies high similarity of triazol-1-yloxy group was only possible with secondary amines
pyrido[2,3-d]pyrimidin-7-(8H)-ones compared to those forming a (such as pyrrolidine) or alcohols (in the presence of Cs2CO3). Using
complex with NS5B (see Supporting Information). The resulting this later protocol we obtained the corresponding 4-methoxy
sublibrary was synthesized and submitted to biological evaluation substituted (15{2,1} and 15{5,1}), 4-ethoxy substituted (15{2,2}),
(see Fig. 1). 4-(2-methoxyethoxy) substituted (15{5,3}), and 4-(2-
propoxyethoxy) substituted (15{5,4}) pyridopyrimidines.
2.2. Chemistry Consequently, for the synthesis of the 4-amino substituted
pyridopyrimidines 15{x,y} it was necessary to modify our approach
In order to obtain the selected sublibrary, including those pyrido and to react the starting 4-oxopyrido[2,3-d]pyrimidine 13{x} with
[2,3-d]pyrimidin-7-(8H)-ones (4) with complex disubstitution on the corresponding amine, BOP and DBU in anhydrous acetonitrile
positions C2 and C4 of the pyrimidine ring, we developed a new heated at 90  C for 2 days in a one-pot process. In that way we
synthetic strategy for the construction of the pyridopyrimidine obtained the corresponding 4-pentylamino substituted (15{2,5}
nucleus [14]. Thus, the treatment of 2-aryl-substituted acrylates 5 and 15{5,5}), 4-(2-propoxyethylamino) substituted (15{2,6} and 15
{1e10} (Fig. 2) with 2,6-diaminopyrimidine-4(3H)-one (12) with 1 {5,6}), and 4-pyrrolidino substituted (15{2,7} and 15{5,7}) pyr-
equivalent of sodium methoxide in ethylene glycol at 180  C for 3 h idopyrimidines in 40e78% yield. Nevertheless, in some cases it was
under microwave irradiation yielded the corresponding 2-amino- necessary to carry out the substitution on the corresponding ben-
6-aryl-5,6-dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-diones (13 zotriazol-1-yloxy substituted intermediate 14{x}. Thus we obtained
{1e10}) in 68e94% yield (Scheme 2). the 4-(2-propoxyethylamino) derivative 15{4,6} and the 4-
For the introduction of an amino or alkoxy group at position benzylamino substituted compound 15{5,8}. Although we tried to
C4 of 4-oxopyrido [2,3-d]pyrimidines 13{1e10}, we activated the use these protocols for the substitution of the benzotriazol-1-yloxy
C4 carbonyl group for nucleophilic displacement upon treatment group by ammonia to introduce a 4-amino group, the reaction did
with BOP or PyBOP [15e17]. This strategy involves the synthesis not proceed in any case. Consequently, the synthesis of 2,4-
of a benzotriazol-1-yloxy substituted intermediate 14{x}, which diaminopyrido[2,3-d]pyrimidines must be carried out using the
is a good leaving group for a nucleophilic substitution with an procedures previously described by our group (see Scheme 1).
amine or alcohol to yield the corresponding 4-amino or 4-alkoxy However, we explored the reaction between the methyl 2-
derivative 15{x,y} (Scheme 2). After several trials we found that arylacrylate 5{2}, sodium methoxide and 2,6-diaminopyrimidin-
the treatment of the corresponding 13{x} with BOP and DBU in 4(3H)-one in anhydrous ethylene glycol under microwave irradia-
anhydrous acetonitrile at rt for 2 days under nitrogen atmo- tion and we obtained the 2,4-diaminopyridopyrimidine 15{2,4} in
sphere afforded the corresponding 14{x}, the reaction not being
466 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
Scheme 2. Synthetic methodologies used for the preparation of the pyrido[2,3-d]pyrimidin-7-(8H)-ones included in the tested sublibrary tested against HCV.
26% yield.
We studied several methodologies for the dehydrogenation of
5,6-dihydropyrido[2,3-d]pyrimidine-7(8H)-ones to the corre-
sponding aromatic compounds [12], however we have not found a
general procedure and the reaction conditions and the oxidizing
agent strongly depend on the structure of the starting pyr-
idopyrimidine. In this work we used three different reaction
conditions: (a) NaH in DMSO at 100  C [12]; (b) DDQ in 1,2-
dichloroethane at 100  C; (c) activated MnO2 in AcOH at reflux.
Using the adequate protocol we obtained compounds 16{2}, 16
{2,5}, 16{2,6}, 16{2,7}, 16{4,6}, 16{5,9}, 16{5,5}, and 16{5,6} in
50e90% yield.
The aromatic compounds 16{x,y} were finally methylated on the
lactam nitrogen using MeI in NaH/DMSO at rt to afford the methyl
derivatives 17{x,y}. Thus we obtained compounds 17{2,1}, 17{2,5},
17{2,6}, 17{2,7}, and 17{5,3} in 26e89% yield.
On the other hand, the synthesis of pyridopyrimidines 23{x,y,z}
with a double complex substitution at C2 and C4 of the pyrimidine
ring also needed the development of a new synthetic strategy
(Scheme 2). Thus, the reaction between 2-aryl-substituted acry-
lates 5{1e10} (Fig. 2) and 6-amino-2-(methylthio)pyrimidin-4(3H)-
one (18) with K2CO3 as base in 2-propanol at 170  C for 3 h under
microwave irradiation yielded the corresponding 2-(methylthio)-
6-aryl-5,6-dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-diones (19
{1e5} and 19{7e11}) in 56e88% yield (Scheme 2) [14].
The direct substitution of the methylthio group of compounds
19{x} was possible with nucleophilic amines, such as benzylamine
which afforded in 2-propanol under microwave irradiation the
corresponding 2-benzylamino derivatives 21{2,8} and 21{5,8} in
53% and 36% respectively from 19{2} and 19{8}, respectively.
Similarly, the 2-(4-fluorobenzyl) amino derivative 21{2,9} in 40%
yield.
However, our main interest was the introduction of arylamino
substituents at position C2 of the pyrimidine ring. Consequently,
we decided to convert the methylthio group present in compounds
19{x} into a methylsulfonyl group to favor the nucleophilic
displacement. According to the literature we tested several
oxidizing agents (H2O2/AcOH, H2O2/NbC/EtOH [18], H2O2/EtOH,
carbamide peroxide/MeOH, oxone® (potassium perox-
ymonosulfate)/MeOH/H2O) but the conversion was never com-
plete, affording mixtures of the desired sulfone 20{x} and the
corresponding sulfoxide.
Finally, the treatment of compounds 19{x} with m-CPBA in DMF
at 0  C afforded the corresponding methylsulfonyl substituted
derivatives 20{x} in 60e74% yield. Using such protocol we obtained
compounds 20{1}, 20{2}, 20{4}, 20{5}, 20{8}, and 20{11}.
The subsequent treatment of compounds 20{x} with the corre-
sponding anilines afforded compounds 21{1,10}, 21{2,10}-21{2,13},
21{4,10}, and 21{8,10} in 67e94% yield.
Similarly, the treatment of 20{2} with sodium phenoxide in 2-
propanol afforded the 2-phenoxy substituted pyridopyrimidine
21{2,14} in 72% yield.
Once obtained the 2-substituted pyridopyrimidines 21{x,y}, we
used the same protocol described above for the introduction of a
second substituent at C4. Thus, we treated compounds 21{x,y} with
BOP and DBU in anhydrous acetonitrile to afford the corresponding
4-benzotriazol-1-yloxy substituted intermediate 22{x,y}. In that
way we obtained 22{2,10}, 22{2,14}, and 22{11,10} in 66e88% yield.
Subsequently, we substituted the 4-benzotriazol-1-yloxy group
of compounds 22{x,y} by amines also following the protocol pre-
viously described to afford pyridopyrimidines 23{x,y,z} bearing
substituents at C2 and C4 (23{2,10,6} and 23{2,14,6} were obtained
in 64% and 54% yield, respectively).
Then, compounds 23{x,y,z} were aromatized to the corre-
sponding 24{x,y,z} using the dehydrogenation protocols described
above. Thus, 24{2,10,6} and 24{2,14,6} were obtained in 78% and
75% yield, respectively.
To complete the sublibrary, we used the protocol for the syn-
thesis of 4-amino-2-arylamino substituted pyridopyrimidines 9
described in Scheme 1 [13] to obtain compounds 9{1}, 9{4}, 9{5}, 9
{6}, and 9{7} in 32e74% yield from pyridines 7{x} via intermediates
10{x} [13,19], which were subsequently dehydrogenated to 11{1}, 11
{4}, 11{5}, 11{6}, and 11{7} in 59e95% yield [19].
These later compounds were alkylated on the lactam nitrogen
with different C1eC6 linear and branched alkyl chains to afford
compounds 25{x,y,z,k}. In that way we obtained compounds 25
{4,10,4,1} [19], 25{5,10,4,5}, 25{5,12,4,1}, 25{5,10,4,1}, 25{6,10,4,1}
[19], 25{1,10,4,1} [19], 25{5,10,4,6}, 25{5,10,4,3}, 25{5,10,4,7}, 25
{7,10,4,1} [19], 25{5,10,4,2}, and 25{5,10,4,4} included in Table 1,
among others.
Finally, taking into account that all the members of this sub-
library present a substituent at position C4 of the pyrimidine ring
(oxo, amino, alkylamino, alkoxy), we decided to include three
unsubstituted compounds for comparison purposes. Thus, starting
from compound 26 [20] we obtained 27 and 28 in 87 and 96% yield,
respectively (Fig. 3).
 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483 467

Table 1
EC50 and CC50 of pyrido[2,3-d]pyrimidines against LucUbiNeo-ET cell line and Huh-7/Lunet cell line, respectively.

Entry Code Structure EC50 (mg/ml) EC50 (mM) CC50 (mM) CC50 (mg/ml)

1 21{4,10} 0.010 0.027 5.3 1.95

2 24{2,10} 0.012 0.034 13.5 4.71

3 25{4,10,4,1} [19] 0.06 0.16 10.4 3.94

4 25{5,10,4,5} 0.1 0.22 184.9 84

5 25{5,12,4,1} [25] 0.11 0.22 3.2 1.58

6 28 0.09 0.23 0.9 0.34

7 25{5,10,4,1} [25] 0.1 0.24 0.5 0.22

8 25{6,10,4,1} [19] 0.15 0.30 0.8 0.42

9 11{1} [19] 0.16 0.49 13.8 4.54

10 25{1,10,4,1} [19] 0.30 0.90 23.5 8.07

11 29 [25] 0.46 0.90 2.6 1.32

12 25{5,10,4,6} 0.50 0.99 240.9 109.9

13 22{2,10} 0.60 1.28 12.2 5.68

14 22{11,10} 0.70 1.35 5.5 2.83

15 25{5,10,4,3} 0.62 1.4 181.1 79.8

16 16{2,6} 0.74 2.1 129.9 46.44

(continued on next page)

468 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483

Table 1 (continued )

Entry Code Structure EC50 (mg/ml) EC50 (mM) CC50 (mM) CC50 (mg/ml)

17 25{5,10,4,7} 1.00 2.3 29.3 13

18 25{7,10,4,1} [19] 1.22 2.8 6.3 2.7

19 16{5,5} 1.24 3.2 11.0 4.32

20 24{2,14,6} 1.43 3.3 79.8 24.3

21 17{2,6} 1.75 4.7 54.3 20.16

22 25{5,10,6,1} 2.54 5.1 84.7 42.22

23 25{5,10,4,2} 2.70 6.3 >125

24 11{6} [19] 3.77 7.7 166 81.02

25 24{2,13} 3.55 9.4 46 17.24

26 21{5,8} 3.98 9.6 69 28.5

27 17{5,9} 4.39 10.4 50.1 21.16

28 17{2,7} 3.57 10.5 111.4 37.81

29 30 [14] 3.38 11.2 38.3 11.54

30 25{5,10,4,4} 5.41 11.9 e >125

31 23{5,10,6} 6.01 12.4 50.5 24.55

32 17{5,4} 5.6 16.7 120.2 40.4

33 11{4} [19] 6.39 17.5 2.4 0.89

 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
Table 1 (continued )
Entry Code Structure
34 22{2,14}
35 17{2,5}
36 15{5,3}
Fig. 3. 4-Unsubstituted pyrido-[2,3-d]pyrimidines included in the sublibrary tested
against HCV.
2.3. Biological evaluation of the HCV antiviral activity
Once the library of pyrido[2,3-d]pyrimidines was synthesized,
we evaluated the HCV antiviral activity and the toxicity of such
compounds. Consequently, we determined the EC50 and the CC50.
For testing the EC50 (mg/mL), we used a stable cell line,
LucUbiNeo-ET, that contains a subgenomic HCV genotype 1b
replicon, I389/NS3-3’/LucUbiNeo-ET, that express luciferase
constitutively [21,22].
To evaluate the cellular toxicity (CC50, mg/mL), we used another
cell line, Huh-7/Lunet [23], that originally carried a selectable
luciferase HCV 1b replicon and was cured by treatment with an
HCV-specific inhibitor.
As positive controls for these assays we used two commercially
available HCV NS3 protease inhibitors Inh I and Inh II (Fig. 4) with
EC50 ¼ 0.0608 mM (CC50 > 2.0 mM) and EC50 ¼ 0.0032 mM
(CC50 > 2.0 mM), respectively [24].
A total of 121 compounds were evaluated, including those
described in the present paper and pyrido[2,3-d]pyrimidines
coming from other research programs within our group (such as 29
Fig. 4. Commercially available HCV NS3 protease inhibitors Inh I and Inh II used as
positive controls for the assays.
469
EC50 (mg/ml) EC50 (mM) CC50 (mM) CC50 (mg/ml)
8.72 18.6 62.7 3.40
7.44 20.9 79.8 28.37
8.45 22.0 65.0 24.91
[25] and 30 [14]). The 36 compounds with the lower EC50 are
included in Table 1.
The results contained in Table 1 show that there are 12 com-
pounds with an EC50 < 1 mM, 9 compounds in the range
1 < EC50 < 5 mM, and 15 compounds in the range 5 < EC50 < 22 mM.
If we focus on the first group of compounds, there are two
compounds with very low EC50 and good CC50: 21{4,10} with
EC50 ¼ 0.027 mM (CC50 ¼ 5.3 mM) and 24{2,10} with
EC50 ¼ 0.034 mM (CC50 ¼ 13.5 mM), respectively. Moreover, it is
interesting to note what their selectivity index or therapeutic index,
which is the ratio CC50/EC50 and gives an idea safety of these po-
tential drugs, are 196 and 397.
Furthermore, the EC50 of 21{4,10} and 24{2,10} are in the same
range of the EC50 of the HCV NS3 protease inhibitors Inh I and Inh II
used as internal controls and, more importantly, are of the same
order of magnitude of the EC50 of sofosbuvir in a similar HCV 1b
replicon test (0.048 mM).
In order to try to locate the target of our antiviral compounds,
we carried out an HCV NS5B drug resistance assay without
concluding results. This experiment consisted of trying to isolate
replicons resistant to our most potent compounds propagating the
replicon at concentrations equivalent to the EC90. We obtained
resistant populations (EC50 between 32.7 and 63.7 mg/mL) but
when we sequenced them, we did not found mutations clearly
associated with non-nucleotide inhibitors of the NS5B polymerase.
2.4. Molecular docking study of 21{4,10} and 24{2,10} on NS5B
polymerase
As mentioned above, we hypothesized that NS5B polymerase
should be a feasible target for substituted pyrido[2,3-d]pyrimidin-
7-(8H)-ones due to the high similarity scores of these molecules
with the available NS5B-ligand X-ray complexes. NS5B is one of the
main targets for HCV replication inhibition, and several clinical
reports increased the attractiveness of using this enzyme as a
therapeutic target [26e39]. NS5B inhibitors can be classified in two
major groups: active site nucleotide/nucleoside analogues (Nucs)
and nonnucleoside inhibitors (NNIs) that bind to specific allosteric
sites on the enzyme. The latter, achieve inhibition by inducing
conformational changes into the protein. Several NS5B e inhibitor
complex structures have been characterized, which leads to an
attractive NNIs database for screening and comparison
[29e31,36,37]. These crystal structures revealed at least four
pockets as potential allosteric inhibitory sites, each of which is
characterized by specific contacts and hydrogen bonding patterns
that depend on the scaffold derivatization. Sites I and II occur in the
thumb domain (TSI and TSII respectively), while sites III and IV can
be found in the palm domain (also known as PSI and PSII
470 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
respectively).
In view of the promising anti-HCV activity of our molecules, we
rationalized its potency through in silico approaches. Despite a
wealth of structural information of NS5B complexes we could not
assure the binding domain of pyrido[2,3-d]pyrimidin-7-(8H)-ones
by the previous similarity strategy. Thus, we proceeded with a
cross-docking protocol consisting in the systematical docking of
ligands into the different allosteric binding sites of NS5B. The ob-
tained conformations were then refined allowing the enzyme to
accommodate the small molecules through an induced-fit method.
Our findings suggest that compounds 21{4,10} and 24{2,10} bind
to NS5B PSII with affinities close to the reference compounds.
Indeed, a high structural similarity should imply a similar binding
mode to the active site. For instance, 24{2,10} binds to PSII repro-
ducing a similar pose to (4-((N-(5-cyclopropyl-2-(4-fluorophenyl)-
3-(methylcarbamoyl)benzofuran-6-yl)methylsulfonamido)
methyl)-2-fluorophenyl)boronic acid (PDB ID 4KE5) [37]. The py-
rimidine core forms a cation-p stacking interaction with the
conserved R200 residue. At the same time, the fluorophenyl ring
occupies a hydrophobic pocket of the palm cleft with an excellent
superposition to the reference (Fig. 5). However, the lack of water
bridges and the lower number of hydrogen-bond donor and ac-
ceptors provided by 24{2,10} should reduce the potency of this
compound. Previous reports hypothesized that potent PSII in-
hibitors blocked polymerization by inactivating the b-hairpin flap
though hydrogen bond binding to Y448. This tyrosine is conserved
through all the genotypes, located at the tip of the b-flap comprised
of F193, P197 and M414, which can form a hydrogen-bond either
with K200 or potential PSII inhibitors. Unfortunately, our com-
pound is not able to form hydrogen bonding with Y448 due to
unfavorable positioning of the NHeC6H5 group. These observations
are consistent with the 20-fold increase relative to EC50 of our
compound for the 1b replicon, if compared to the crystal structure
ligand.
More interestingly, we found that compound 21{4,10} yields a
highest affinity complex which is in accordance with the biological
activity observed. Comparing both structures it becomes clear that
Fig. 5. Representation of 24{2,10} binding to the PSII of NS5B. (A) Superposition of compound with the reference crystal structure (PDB ID 4KE5). 24{2,10} and the crystal structure
ligand are depicted as orange and green sticks respectively. (B) Positioning of 24{2,10} into the PSII pocket. (C) Relative positioning of the compound into the b-flap pocket. Non-
polar hydrogens were removed for clarity purposes in all the representations.
21{4,10} optimize the intermolecular contacts and reduces the
sterical hindrance of the pyrido[2,3-d]pyrimidin-7-(8H)-one sub-
stituents (Fig. 6). However, the core positioning is inverted so the
difluoro phenyl ring points towards the NS5B active site and locates
at the initiation of the primer strand. A recent structure of NS5B
cocrystallized with sofosbuvir diphosphate and viral RNA demon-
strates the conformational adaptions of the enzyme during RNA
replication [40]. Intriguingly, superposition of the molecular
docking complexes and NS5B-RNA structure suggests that both 21
{4,10} and 24{2,10} can potentially interact with riboses of the
primer strand to disrupt polymerization (Fig. 6).
3. Conclusions
Using the structural information contained in a series of patents
of Gilead Sciences on the use of pyrido[3,2-d]pyrimidines (3) as
HCV inhibitors, we designed a library of pyrido[2,3-d]pyrimidin-
7(8H)-ones (4) including more than 89,000 compounds. After a
diversity selection, we synthesized 121 compounds using the
synthetic methodologies contained in Scheme 2. In addition, we
determined the EC50 (mg/mL) using a stable cell line containing a
subgenomic HCV subtype 1b replicon. Moreover, we also deter-
mined the cellular toxicity (CC50, mg/mL) using a cell line that
originally carried a selectable luciferase HCV 1b replicon and was
cured by treatment with an HCV-specific inhibitor.
Among these compounds, 21{4,10} and 24{2,10} presented very
high activities [EC50 ¼ 0.027 mM (CC50 ¼ 5.3 mM) and
EC50 ¼ 0.034 mM (CC50 ¼ 13.5 mM), respectively] and high selec-
tivity indexes, 196 and 397.
The EC50 of 21{4,10} and 24{2,10} are of the same order of
magnitude of the EC50 of sofosbuvir in a similar HCV 1b replicon
test (0.048 mM) [41]. It is interesting to note that sofosbuvir, a
compound containing 4 stereogenic centers, is synthesized through
a long multistep synthesis [42] while 21{4,10} presents only one
stereogenic center and is obtained in three steps from commer-
cially available starting materials. Similarly, the achiral 24{2,10} is
obtained in four steps also from commercially available starting
 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
Fig. 6. (A) Superposition of 24{2,10} (orange) and 21{4,10} (green) in the PSII binding
site. (B) Representation of a potential interaction of 21{4,10} with RNA and a cocrys-
tallized sofosbuvir diphosphate unit.
materials.
Although it has not been possible yet to stablish the molecular
target for these compounds, chemoinformatic studies suggest a
high degree of similarity between 21{4,10} and 24{2,10} and PSII
inhibitors of NS5B polymerase. Further cross docking analysis
shows an excellent binding of our compounds with the hydro-
phobic pocket of the palm cleft, as it has been described in other
structural studies with known PSII inhibitors. An optimal cation-p
stacking of the pyrido[2,3-d]pyrimidine core of 21{4,10} and 24
{2,10}, combined with the optimal positioning of the 2,6-
difluorophenyl and the p-fluorophenyl substituents respectively,
seems to be compatible with the interaction of these compounds
with RNA riboses of the primer strand which could disrupt poly-
merization, in good agreement with the observed biological
activity.
Taken together, these results point out to achiral compound 24
{2,10} as a promising candidate to be further developed as drug for
the treatment of hepatitis C. Further studies should be conducted to
validate such hypothesis.
471
4. Experimental
4.1. Synthesis and characterization of compounds
4.1.1. General
All solvents and chemicals were reagent grade. Unless otherwise
mentioned, all solvents and chemicals were purchased from com-
mercial vendors (Fluka, Aldrich, ABCR and ACROS Organics) and
used without purification. Compounds 13{1e10}, 19{1e5}, and 19
{7e10} were prepared as previously described [14].1H and 13C NMR
spectra were recorded on a Varian 400-MR spectrometer that was
operating at a field strength of 400 and 100.6 MHz, respectively.
Chemical shifts were reported in parts per million (d) and coupling
constants (J) were in Hz by using, in the case of 1H NMR spectros-
copy, tetramethylsilane (TMS) as an internal standard, and in the
case of 13C NMR spectroscopy, solvent residual peak was taken as
reference: CDCl3 at 77.0 ppm, DMSO-d6 at 39.5 ppm. Standard and
peak multiplicities are designed as follows: s, singlet; d, doublet;
dd, doublet of doublets; dt, doublet of triplets t, triplet; br, broad
signal. IR spectra were recorded in a Thermo Scientific Nicolet iS10
FTIR spectrophotometer with Smart iTr. Wavenumbers (n) are
expressed in cm1. MS data (m/z (%), EI, 70 eV) were obtained by
using an Agilent Technologies 5975 spectrometer and a Hewlett
Packard HP5988A quadrupole mass spectrometer operating in
electronic ionization (EI) mode at 70 eV and at 4 kV accelerating
potential, or a Bruker Biotoff II spectrometer operating in electro-
spray ionization (ESI) mode with a Time of Flight (TOF) detector.
HRMS data were obtained by using a VG AutoSpec (Micromass
Instruments) Trisector EBE high resolution spectrometer (EI or FAB
mode), a Bruker Biotof II mass spectrometer (ESI TOF mode).
Elemental microanalyses were obtained on a EuroVector In-
struments Euro EA elemental analyzer. The melting points were
determined with a Büchi-Tottoli 530 capillary apparatus and are
uncorrected. Automatic flash chromatography was performed in an
Isco Combiflash medium pressure liquid chromatograph with
RediSep® silica gel columns (35e70 mm) using a suitable mixture of
solvents as eluent. Microwave irradiation experiments were carried
out in an Initiator™ (Biotage) microwave apparatus, operating at a
frequency of 2.45 GHz with continuous irradiation power from 0 to
400 W. Reactions were carried out in 0.5, 2.5, 5, and 20 mL glass
tubes, sealed with aluminum/Teflon crimp tops, which can be
exposed up to 250  C and 20 bar internal pressure. Temperature
was measured with an IR sensor on the outer surface of the process
vial. After the irradiation period, the reaction vessel was cooled
rapidly to 50  C by air jet cooling.
4.1.2. General procedure for the synthesis of 4-((1H-benzo[d][1,2,3]
triazol-1-yl)oxy)-6-aryl-3,4,5,6-tetrahydropyrido[2,3-d]pyrimidine-
7(8H)-ones (14{x})
The corresponding 2-amino-6-aryl-5,6-dihydropyrido[2,3-d]
pyrimidine-4,7(3H,8H)-diones (13{x}) (0.50 mmol) [14] was sus-
pended in 13 mL of anhydrous acetonitrile. Then, BOP (0.75 mmol)
and DBU (1.25 mmol) were added into the solution. The system was
stirred at room temperature for 2 days under nitrogen atmosphere.
After this period, 200 mL of water were added and the resulting
precipitate was filtered, washed with water and dried in vacuo over
phosphorus pentoxide to afford the corresponding 14{x} that can
be used without further purification.
4.1.2.1. 4-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-2-amino-6-(4-
fluorophenyl)-3,4,5,6-tetrahydropyrido[2,3-d]pyrimidine-7(8H)-one
(14{2}). Starting from compound 13{2} to afford 14{2} in 74% yield,
white solid. IR (KBr), nmax (cm1): 3425, 3337, 3223, 1637, 1575,
1510, 1356, 1237, 1160, 1088, 847, 738. 1H NMR (400 MHz, DMSO-
d6): d 10.95 (s, 1H), 8.13 (dt, J ¼ 8.4, 0.9 Hz, 1H), 7.70e7.61 (m, 2H),
472 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
7.51 (ddd, J ¼ 8.1, 6.4, 1.6 Hz), 7.39 (dd, J ¼ 8.7, 5.6 Hz, 2H), 7.21 (t,
J ¼ 8.9 Hz, 1H), 6.59 (s, 2H, NH2), 4.11 (dd, J ¼ 10.7, 6.9 Hz, 1H),
3.29e3.12 (m, 2H). 13C NMR (100.6 MHz, DMSO-d6): d 172.4, 166.0,
161.8, 161.0, 143.2, 135.4, 130.8 (d, J ¼ 7.9 Hz, C12), 129.5 (C17), 128.9,
125.6 (C18), 120.3 (C19), 115.6 (d, J ¼ 21.1 Hz, C13), 110.0, 109.8 (C16),
86.0 (C5), 45.4 (C7), 24.5 (C6).
4.1.2.2. 4-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-2-amino-6-(2,6-
difluorophenyl)-5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-one (14
{4}). Starting from compound 13{4} to afford 14{4} in 93% yield,
white solid. IR (KBr), nmax (cm1): 3374, 3207, 2895, 1698, 1651,
1571, 1470, 1371, 1273, 1247, 1008, 785. 1H NMR (400 MHz, DMSO-
d6): d 11.03 (s, 1H, NH), 8.13 (dt, J ¼ 8.4, 0.9 Hz, 1H, C15eCH), 7.80
(dt, J ¼ 8.3, 0.9 Hz, 1H, C18eCH), 7.64 (ddd, J ¼ 8.3, 6.9, 0.9 Hz, 1H,
C16eCH), 7.51 (ddd, J ¼ 8.4, 6.9, 1.0 Hz, 1H, C17eCH), 7.49e7.42 (m,
1H, C14eCH), 7.16 (t, J ¼ 8.7 Hz, 2H, C13-CHx2), 6.63 (s, 2H, NH2),
4.51 (dd, J ¼ 13.9, 7.5 Hz, 1H, C7eCH), 3.31e3.25 (m, 1H, C6eCH),
3.10e3.03 (m, 1H, C6eCH). 13C NMR (100.6 MHz, DMSO-d6): d 170.0,
165.5, 161.4, 160.9 (d, J ¼ 247.2 Hz, C12), 160.8 (d, J ¼ 246.8 Hz, C12),
160.4, 142.7, 129.9 (t, J ¼ 10.6 Hz, C14), 128.9 (C16), 128.5, 125.1
(C17), 119.8 (C15), 114.7 (t, J ¼ 18.5 Hz, C11), 111.8 (dd, J ¼ 22.3,
2.5 Hz, C13), 109.6 (C18), 85.1 (C5), 36.1 (C7), 22.4 (C6). Anal. Calcd.
(%) for C19H13F2N7O2: C, 55.75; H, 3.20; N, 23.95. Found: C, 56.06; H,
3.22; N, 23.78.
4.1.2.3. 4-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-2-amino-6-(2,6-
dichloro phenyl)-3,4,5,6-tetrahydropyrido[2,3-d]pyrimidine-7(8H)-
one (14{5}). Starting from compound 13{5} to afford 14{5} in 78%
yield, white solid. IR (KBr): n (cm1): 3382, 3324, 3215, 2898, 1670,
1648, 1571, 1367, 1241, 1089, 1037, 774, 734. 1H NMR (400 MHz,
DMSO-d6): d 11.00 (s, 1H), 8.13 (dt, J ¼ 8.4, 0.9 Hz, 1H), 7.81 (dt,
J ¼ 8.4, 1.0 Hz, 1H), 7.68e7.61 (m, 1H), 7.60e7.46 (m, 3H), 7.39 (t,
J ¼ 8.1 Hz, 1H), 6.63 (s, 2H, NH2), 4.91 (dd, J ¼ 12.6, 9.3 Hz, 1H),
3.31e3.16 (m, 2H). 13C NMR (100.6 MHz, DMSO-d6): 170.14, 166.17,
161.79, 160.48, 143.17, 135.79, 135.38, 135.23, 130.39, 130.20, 129.42,
128.94, 128.86, 125.58, 120.22, 110.05, 84.62, 43.20, 21.98. HRMS
(70 eV, EI): m/z calculated for C19H13N7O2Cl2: 441.0508, [M]þ,
found: 441.0507.
4.1.3. General procedure for the synthesis of 4-alkoxy-substituted
pyrido[2,3-d]pyrimidines 15{x,y}
A mixture of the corresponding 4-((1H-benzo[d][1,2,3]triazol-1-
yl)oxy)-6-aryl-3,4,5,6-tetrahydropyrido[2,3-d]pyrimidine-7(8H)-
one (14{x}) (0.42 mmol), Cs2CO3 (0.54 mmol) and the corre-
sponding alcohol (9 mL) was heated a reflux overnight. The
resulting solution was cooled down, water (100 mL) was added and
the reaction crude was neutralized with 2 M HCl. The resulting solid
was collected by filtration and washed with water, and EtOEt to
afford the corresponding 15{x,y}.
4.1.3.1. 2-Amino-6-(4-fluorophenyl)-4-methoxy-5,6-dihydropyrido
[2,3-d]pyrimidin-7(8H)-one (15{2,1}). Starting from compound 14
{2} and methanol to afford 15{2,1} in 39% yield, white solid. IR (KBr),
nmax (cm1): 3381, 3227, 1686, 1658, 1633, 1575, 1511, 1464, 1384,
1247. 1H NMR (400 MHz, DMSO-d6): d 10.49 (s, 1H, NH), 7.26 (dd,
J ¼ 8.7, 5.7 Hz, 2H, C12-CHx2), 7.14 (t, J ¼ 8.9 Hz, 2H, C13-CHx2), 6.40
(s, 2H, NH2), 3.86 (dd, J ¼ 9.9, 6.9 Hz, 1H, C7eCH), 3.80 (s, 3H; OCH3),
2.93e2.72 (m, 2H, C6eCH2). 13C NMR (100.6 MHz, DMSO-d6):
d 172.0, 166.7, 161.7, 158.3, 153.0, 135.4 (d, J ¼ 2.9 Hz, C11), 130.1 (d,
J ¼ 8.1 Hz, C12), 115.0 (d, J ¼ 21.3 Hz, C13), 86.5 (C5), 53.2 (OCH3),
45.3 (7), 24.6 (6). MS (70 eV, EI): m/z (%) ¼ 288.2 (100) [M]þ, 193.0
(25) [M-C6H4F]þ. HRMS (70 eV, EI): m/z calculated for C14H13N4O2F:
288.1028, [M]þ, found: 288.1023.
4 .1. 3 . 2 . 2 - A m i n o - 6 - ( 2 , 6 - d i ch l o ro p h e nyl ) - 4 - m e t h o x y- 5 , 6 -
dihydropyrido[2,3-d]pyrimidine-7(8H)-one (15{5,1}). Starting from
compound 14{5} and methanol to afford 15{5,1} in 96% yield, white
solid. IR (KBr), nmax (cm1): 3438, 3330, 3222, 2951, 2891, 1688,
1634, 1577, 1488, 1466, 1371, 1257, 1196, 774. 1H NMR (400 MHz,
TFA-d): d 7.37 (dd, J ¼ 15.0, 8.1 Hz, 2H), 7.24 (t, J ¼ 8.2 Hz, 1H), 5.08
(dd, J ¼ 13.3, 9.4 Hz, 1H), 4.11 (s, 3H), 3.30e3.08 (m, 2H). 13C NMR
(100.6 MHz, TFA-d): d 173.9, 170.8, 154.3, 146.6, 136.0, 134.7, 131.7,
130.2, 129.4, 128.4, 90.0, 55.7, 42.6, 21.0. MS (70 eV, EI): m/z
(%) ¼ 338.1 (60) [M]þ, 303.1 (100) [M-Cl]þ, 153.0 (67) [M-
C6H9N4O]þ. HRMS (70 eV, EI): m/z calculated for C14H12N4O2Cl2:
338.0340, [M]þ, found: 338.0337. Anal. Calcd. (%) for
C14H12Cl2N4O2: C, 49.58; H, 3.57; N, 16.52. Found: C, 49.41; H, 3.76;
N, 16.89.
4.1.3.3. 2-Amino-4-ethoxy-6-(4-fluorophenyl)-5,6-dihydropyrido
[2,3-d]pyrimidin-7(8H)-one (15{2,2}). Starting from compound 14
{2} and ethanol to afford 15{2,2} in 20% yield, white solid. IR (KBr),
nmax (cm1): 3408, 3338, 3220, 1686, 1632, 1575, 1511, 1434, 1227. 1H
NMR (400 MHz, DMSO-d6): d 10.45 (s, 1H, NH), 7.27 (dd, J ¼ 8.8,
5.6 Hz, 2H, C12-CHx2), 7.14 (t, J ¼ 8.8 Hz, 2H, C13-CHx2), 6.35 (s, 2H,
NH2), 4.26 (qd, J ¼ 7.2, 1.3 Hz, 2H, C15eCH2), 3.86 (dd, J ¼ 10.4,
7.2 Hz, 1H, C7eCH), 2.93e2.71 (m, 2H, C6eCH2), 1.25 (t, J ¼ 7.0 Hz,
3H, C16eCH3). 13C NMR (100.6 MHz, DMSO-d6): d 172.1, 166.4,
162.4, 161.8, 160.0, 158.4, 135.5 (C11), 130.2 (d, J ¼ 8,5 Hz, C12), 115.1
(d, J ¼ 21.0 Hz, C12), 86.7 (C5), 61.5 (C15), 45.4 (C7), 24.8 (C6), 14.6
(C16). MS (70 eV, EI): m/z (%) ¼ 302.1 (100) [M]þ, 287.1 (25) [M-
CH3]þ, 273.1 (42) [M-C2H5]þ, 258.1 (19) [M-OC2H5]þ. HRMS (70 eV,
EI): m/z calculated for C15H15N4O2F: 302.1183, [M]þ, found:
302.1179.
4.1.3.4. 2-Amino-6-(2,6-dichlorophenyl)-4-ethoxy-5,6-dihydropyrido
[2,3-d]pyrimidin-7(8H)-one (15{5,2}). Starting from compound 14
{5} and ethanol to afford 15{5,2} in 31% yield, white solid. IR (KBr),
nmax (cm1): 3409, 3329, 3220, 1686, 1633, 1577, 1435, 1373, 778. 1H
NMR (400 MHz, DMSO-d6): d 10.47 (s, 1H, NH), 7.54e7.48 (m, 2H,
C13-CHx2), 7.36 (t, J ¼ 8.1 Hz, 1H, C13eCH), 6.38 (s, 2H, NH2), 4.62
(dd, J ¼ 12.8, 9.3 Hz, 1H, C7eCH), 4.26 (qd, J ¼ 7.1, 2.1 Hz, 2H,
C15eCH2), 2.88e2.79 (m, 2H, C6eCH2), 1.25 (t, J ¼ 7.0 Hz, 3H,
C16eCH3). 13C NMR (100.6 MHz, DMSO-d6): d 169.5, 166.4,
161.83,157.9, 135.3, 134.7, 129.7, 128.3, 85.1 (C5), 61.4 (C15), 43.1
(C7), 22.2 (C6), 14.5 (C16). HRMS (70 eV, EI): m/z calculated for
C15H14N4O2Cl2: 352.0494, [M]þ, found: 352.0494.
4.1.3.5. 2-Amino-6-(2,6-dichlorophenyl)-4-(2-methoxyethoxy)-5,6-
dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{5,3}). Starting from
compound 14{5} and 2-methoxyethanol to afford 15{5,3} in 76%
yield, white solid. IR (KBr), nmax (cm1): 3404, 3218, 2894, 1688,
1633, 1576, 1434, 1372, 775. 1H NMR (400 MHz, DMSO-d6): d 10.54
(s, 1H, NH), 7.55e7.48 (m, 2H, C-13-CHx2), 7.37 (t, J ¼ 8.1 Hz, 1H,
C14eCH), 6.44 (s, 2H, NH2), 4.64 (dd, J ¼ 12.8, 9.4 Hz, 1H, C7eCH),
4.37e4.32 (m, 2H, C15eCH2), 3.60 (t, J ¼ 4.8 Hz, 2H, C16eCH2), 3.26
(s, 3H, OCH3), 2.93e2.79 (m, 2H, C6eCH2). 13C NMR (100.6 MHz,
DMSO-d6): d 169.8, 166.4, 161.6, 157.9, 135.3, 135.2, 134.7, 129.8
(C14), 129.7 (C13), 128.3 (C13), 85.2 (C5), 70.1 (C16), 64.8 (C15), 58.2
(OCH3), 43.1 (C7), 22.2 (C6). MS (70 eV, EI): m/z (%) ¼ 382.0 (15)
[M]þ, 351.0 (23) [M-CH3O]þ, 322.9 (33) [M-C3H6O]þ, 289.0 (100)
[M-C3H6OCl]þ, 205.9 (26) [C9H10N4O2]þ. HRMS (70 eV, EI): m/z
calculated for C16H16N4O3F4Cl2: 382.0598, [M]þ, found: 382.0599.
4.1.3.6. 2-Amino-6-(2,6-dichlorophenyl)-4-(2-propoxyethoxy)-5,6-
dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{5,9}). Starting from
compound 14{5} and 2-propoxyethanol to afford 15{5,9} in 38%
yield, white solid. IR (KBr), nmax (cm1): 3479, 3332, 3220, 2773,
1692, 1631, 1575, 1458, 1435, 1373, 777. 1H NMR (400 MHz, DMSO-
 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
d6): d 10.51 (s, 1H, NH), 7.58e7.47 (m, 2H, C13eCH), 7.37 (t,
J ¼ 8.1 Hz, 1H, C14eCH), 6.42 (s, 2H, NH2), 4.63 (dd, J ¼ 12.9, 9.2 Hz,
1H, C7eCH), 4.35e4.32 (m, 2H, C15eCH2), 3.63 (t, J ¼ 4.9 Hz, 2H,
C16eCH2), 3.35 (t, J ¼ 6.6 Hz, 2H, C17eCH2), 2.93e2.79 (m, 2H,
C6eCH2), 1.46 (h, J ¼ 7.3 Hz, 2H, C18eCH2), 0.80 (t, J ¼ 7.4 Hz, 3H,
CH3). 13C NMR (100.6 MHz, DMSO-d6): d 169.6, 166.4, 161.8, 158.0,
135.3, 135.1, 129.8, 128.3, 85.2 (C5), 71.9 (C17), 68.2 (C16), 65.0 (C15),
43.1 (C7), 22.4 (C18), 22.2 (C16), 10.4 (C19). Anal. Calcd. (%) for
C18H20Cl2N4O2: C, 52.57; H, 4.90; N, 13.62. Found: C, 52.76; H, 5.12;
N, 13.98.
4.1.4. General procedure for the synthesis of 4-amino-substituted
pyrido [2,3-d]pyrimidines 15{x,y}
Procedure A: 4.88 mmol of the corresponding 4-oxopyrido[2,3-
d]pyrimidine (13{x}) was suspended in acetonitrile (350 mL), then
BOP (2.805 g, 6.34 mmol) and DBU (1.1 mL, 7.32 mmol) were added
to the solution. The mixture was stirred at room temperature for
2 h. The corresponding amine (14.6 mmol) was added and the
mixture was heated at 90  C for 2 days. The solvent was concen-
trated under reduced pressure and water was added to the residue.
The solid was collected by filtration and washed with water and
EtOEt.
Procedure B: 0.419 mmol of the corresponding benzotriazol-1-
yloxy substituted intermediate 14{x} was suspended in acetoni-
trile (20 mL) and 3 equivalents of the corresponding amine
(1.257 mmol) was added to the suspension and the mixture was
heated at 140  C by microwave irradiation for 5 h. Then, water was
added to the residue and the solid was collected by filtration and
washed with water and EtOEt.
4.1.4.1. 2-Amino-6-(4-fluorophenyl)-4-(pentylamino)-5,6-
dihydropyrido [2,3-d]pyrimidin-7(8H)-one (15{2,5}). Starting from
compound 13{2} and pentylamine and following procedure A to
afford 15{2,5} in 40% yield, white solid. IR (KBr), nmax (cm1): 3395,
3333, 3219, 2930, 2869, 1694, 1630, 1578, 1510, 1469, 1376, 1214,
841. 1H NMR (400 MHz, acetone): d 7.38 (m, 2H, 2xCH), 7.11 (t,
J ¼ 8.8 Hz, 2H, 2xCH), 3.94 (dd, J ¼ 10.3, 7.2 Hz, 1H, CHeC7), 3.42
(dd, J ¼ 12.9, 6.6 Hz, 2H, CH2eC15), 2.87 (m, 2H, CH2eC6), 1.67e1.51
(m, 2H, CH2eC16), 1.41e1.25 (m, 4H, 2xCH2eC17, C18), 0.88 (t,
J ¼ 6.8 Hz, 3H, CH3). 13C NMR (100.6 MHz, acetone) d 174.4 (CO),
163.3, 162.9 (d, J ¼ 69.1 Hz, C14), 161.6, 156.6 (C10), 136.6 (d,
J ¼ 3.3 Hz, C11), 131.2 (d, J ¼ 8.1 Hz, C12), 115.8 (d, J ¼ 21.3 Hz, C31),
85.1 (C5), 46.7 (C7), 41.5 (C15), 29.2 (C16), 29.0 (C17), 26.6 (C6), 23.2
(C18), 14.4 (C19). MS (70 eV, EI): m/z (%) ¼ 343.3 [M]þ, 314.2 [M-
C2H5]þ, 300.1 [M-C3H9]þ, 286.1 [M-C4H9]þ, 272.1 [M-C5H11]þ.
4.1.4.2. 2-Amino-6-(4-fluorophenyl)-4-((2-propoxyethyl)amino)-5,6-
dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{2,6}). Starting from
compound 13{2} and 2-propoxyethylamine and following proce-
dure A to afford 15{2,6} in 63% yield, white solid. IR (KBr), nmax
(cm1): 3391, 3226, 2932, 2874, 1689, 1629, 1575, 1511, 1477, 1226.
1
H NMR (400 MHz, CDCl3): d 11.99 (s, 1H, NH), 7.26e7.20 (m, 2H,
C12-CHx2), 7.04 (t, J ¼ 8.7 Hz, 2H, C13-CHx2), 4.81e4.75 (m, 1H,
NH), 3.84 (dd, J ¼ 10.2, 7.3 Hz, 1H, C7eCH), 3.64e3.61 (m, 2H,
C15eCH2), 3.58e3.52 (m, 2H, C16eCH2), 3.39 (t, J ¼ 6.7 Hz, 2H,
C17eCH2), 2.85e2.67 (m, 2H, C16eCH2), 1.63e1.51 (m, 2H,
C18eCH2), 0.88 (t, J ¼ 7.4 Hz, 3H, CH3). 13C NMR (100.6 MHz, CDCl3):
d 173.8 (CO), 161.5 (d, J ¼ 161.0 Hz, C14), 161.1, 155.9, 134.4 (d,
J ¼ 3.4 Hz, C11), 129.9 (d, J ¼ 8.1 Hz, C12), 115.9 (d, J ¼ 21.2 Hz, C13),
84.5 (C5), 72.9 (C17), 69.3 (C16), 46.2 (C7), 40.8 (C15), 26.3 (C6), 22.9
(C18), 10.7 (C19). MS (70 eV, EI): m/z (%) ¼ 359.1 (24) [M]þ, 316.1
(11) [M-C3H5]þ, 301.1 (17) [M-C3H8O]þ, 286.1 (100) [M-C4H9O]þ,
273.1 (80) [M-C5H12O]þ. HRMS (70 eV, EI): m/z calculated for
C18H22N5O2F: 359.1758, [M]þ, found: 359.1742. Anal. Calcd. (%) for
C18H22FN5O2: C, 60.15; H, 6.17; F, 5.29; N, 19.49; O, 8.90. Found: C,
473
59.80; H, 6.04; N, 19.82.
4.1.4.3. 2-Amino-6-(4-fluorophenyl)-4-(pyrrolidin-1-yl)-5,6-
dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{2,7}). Starting from
compound 13{2} and pyrrolidine and following procedure A to
afford 15{2,7} in 44% yield, white solid. IR (KBr), nmax (cm1): 3441,
3329, 3227, 2963, 2873, 1695, 1639, 1608, 1540, 1510, 1448, 1340,
1232, 840. 1H NMR (400 MHz, DMSO-d6): d 10.31 (s, 1H, NH),
7.30e7.24 (m, 2H, C12-2xCH), 7.14 (t, J ¼ 8.9 Hz, 2H, C13-2xCH), 5.89
(s, 2H, NH2), 3.78 (dd, J ¼ 10.4, 6.6 Hz, 1H, C7eCH), 3.50e3.41 (m,
4H, 2xCH2), 3.16e3.02 (m,2H, C6eCH2) 1.85e1.67 (m, 4H, 2xCH2).
13
C NMR (100.6 MHz, DMSO-d6): d 171.6 (CO), 161.2, 161.1 (d,
J ¼ 241.2 Hz, C14) 160.7, 157.5, 135.5 (d, J ¼ 3.0 Hz, C11), 130.3 (d,
J ¼ 8.1 Hz, C12), 114.9 (d, J ¼ 21.0 Hz, C13), 86.3 (C5), 48.9 (C15), 45.9
(C7), 28.5 (C6), 25.0 (C16). HRMS (70 eV, EI): m/z calculated for
C17H18N5OF: 327.1495, [M]þ, found: 327.1496.
4.1.4.4. 2-Amino-6-(2,6-difluorophenyl)-4-((2-propoxyethyl)amino)-
5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{4,6}).
Starting from compound 14{4} and 2-propoxyethylamine and
following procedure B to afford 15{4,6} in 60% yield, white solid. IR
(KBr), nmax (cm1): 3416, 3332, 3212, 2929, 2871, 1630, 1577, 1470,
1388, 1007, 783. 1H NMR (400 MHz, DMSO-d6): d 10.25 (s, 1H, NH),
7.46e7.37 (m, 1H, C14eCH), 7.13 (t, J ¼ 8.8 Hz, 2H, C13-CHx2), 6.34
(s, 1H, NH), 5.91 (s, 2H, NH2), 4.16 (dd, J ¼ 14.0, 7.6 Hz, 1H, C7eCH),
3.44e3.43 (m, 4H, C15eCH2, C16eCH2), 3.34e3.31 (m, 2H,
C17eCH2), 2.97e2.78 (m, 2H, C6eCH2), 1.48 (h, J ¼ 7.4 Hz, 2H,
C18eCH2), 0.83 (t, J ¼ 7.4 Hz, 3H, C19eCH3). 13C NMR (100.6 MHz,
DMSO-d6): d 170.3, 160.8 (d, J ¼ 246.8 Hz, C12), 160.7 (d,
J ¼ 246.6 Hz, C12), 161.8, 160.2, 155.6, 129.7 (t, J ¼ 10.2 Hz, C14),
115.4 (t. J ¼ 18.7 Hz, C11), 111.7 (dd, J ¼ 1.9, 22.3 Hz, C13), 83.6 (C5),
71.7 (C17), 68.7 (C16), 40.0 (C15), 36.4 (C7), 24.0 (C6), 22.5 (C18),
10.5 (C19). Anal. Calcd. (%) for C18H21F2N5O2: C, 57.29; H, 5.61; N,
18.56. Found: C, 57.25; H, 3.41; N, 18.74.
4.1.4.5. 2-Amino-6-(2,6-dichlorophenyl)-4-(pentylamino)-5,6-
dihydropyrido [2,3-d]pyrimidin-7(8H)-one (15{5,5}). Starting from
compound 13{5} and pyrrolidine and following procedure A to
afford 15{5,5} in 62% yield, white solid. IR (KBr), nmax (cm1): 3505,
3392, 3323, 3206, 2929, 2870, 1675, 1629, 1577, 1472, 1436, 1385,
1287, 776. 1H NMR (400 MHz, DMSO-d6): d 10.57 (s, 1H, CONH), 7.53
(m, 2H, C12-CHx2), 7.37 (t, J ¼ 8.1 Hz, 1H, C13eCH), 6.30 (s, 1H, NH),
6.06 (s, 2H, NH2), 4.64 (dd, J ¼ 13.1, 8.9 Hz, 1H, C7eCH), 3.31e3.20
(m, 2H, C14eCH2), 2.94e2.65 (m, 2H, CH2), 1.49 (p, J ¼ 7.1 Hz, 2H,
C15eCH2), 1.27 (m, 4H, C15,C16eCH2x2), 0.85 (t, J ¼ 6.9 Hz, 3H,
CH3). 13C NMR (100.6 MHz, DMSO-d6): d 169.8, 161.8, 160.5, 154.9,
135.7, 135.2, 134.8, 129.7, 128.3, 82.5 (C5), 43.1 (C7), 40.3 (C14) 28.8
(C15), 28.7 (C16), 23.2 (C6), 22.0 (C17), 14.0 (C18). MS (70 eV, EI): m/
z (%) ¼ 393.1 (69) [M]þ,351.0 (78) [M-C3H7]þ,336.0 (91) [M-
C4H9]þ,322.9 (54) [M-C5H11]þ, 248.1 (43) [M-C6H3Cl2]þ, 206.1 (83)
[C9H12N5O]þ, 192.0 (69) [C8H8N5O]þ. HRMS (70 eV, EI): m/z calcu-
lated for C18H21N5OCl2: 393.1123, [M]þ, found: 393.1122.
4.1.4.6. 2-Amino-6-(2,6-dichlorophenyl)-4-((2-propoxyethyl)
amino)-5,6-dihydropyrido [2,3-d]pyrimidin-7(8H)-one (15{5,6}).
Starting from compound 13{5} and 2-propoxyethylamine and
following procedure A to afford 15{5,6} in 70% yield, white solid. IR
(KBr), nmax (cm1): 3440, 3333, 3217, 2928, 2869, 1631, 1576, 1478,
776. 1H NMR (400 MHz, DMSO-d6): d 10.28 (s, 1H, CONH), 7.53 (ddd,
J ¼ 14.8, 8.3, 1.3 Hz, 2H, C13-2xCH), 7.37 (t, J ¼ 8.1 Hz, 1H, C14eCH),
6.33 (s, 1H, NH), 5.95 (s, 2H, NH2), 4.60 (dd, J ¼ 13.1, 8.9 Hz, 1H,
C7eCH), 3.44 (m, 4H, C15eCH2, C16eCH2), 3.31e3.34 (m, 2H,
C17eCH2) 2.92e2.64 (m, 2H, C6eCH2), 1.48 (h, J ¼ 7.2 Hz, 2H,
C18eCH2), 0.84 (t, J ¼ 7.4 Hz, 3H, C19eCH3). 13C NMR (100.6 MHz,
DMSO-d6): d 169.5, 161.9, 155.1, 135.6, 135.1, 134.8, 129.7, 128.3,
474 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
82.62 (C5), 71.7 (C17), 68.6 (C16), 43.2 (C7), 39.5 (C15) 23.2 (C6),
22.5 (C18), 10.5 (C19). HRMS (70 eV, EI): m/z calculated for
C18H21N5O2Cl2: 409.1077, [M]þ, found: 409.1072. Anal. Calcd. (%) for
C18H21Cl2N5O2: C, 52.69; H, 5.16; N, 17.07. Found: C, 52.60; H, 65.36;
N, 17.24.
4.1.4.7. 2-Amino-6-(2,6-dichlorophenyl)-4-(pyrrolidin-1-yl)-5,6-
dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{5,7}). Starting from
compound 13{5} and pyrrolidine and following procedure A to
afford 15{5,7} in 78% yield, white solid. IR (KBr), nmax (cm1): 3468,
3309, 3203, 2971, 2874, 1686, 1614, 1540, 1457, 1441, 1386, 1340,
1286, 772. 1H NMR (400 MHz, DMSO-d6): d 10.11 (s, 1H, NH), 7.50
(dd, J ¼ 15.6, 8.4 Hz, 4H, C13-CHx2), 7.36 (t, J ¼ 8.0 Hz, 1H, C14eCH),
5.85 (s, 2H, NH2), 4.48 (dd, J ¼ 13.6, 8.0 Hz, 1H, C7eCH), 3.46 (m, 4H,
C15eCH2x2), 2.85e2.65 (m, 2H, C6eCH2), 1.76 (m, 4H, C16eCH2x2).
13
C NMR (100.6 MHz, TFA-d): d 173.7, 151.8, 151.6, 134.8, 130.7, 130.6,
129.6, 128.5, 86.8 (C5), 51.9 (C15), 42.5 (C7), 24.4 (C16, C7). MS
(70 eV, EI): m/z (%) ¼ 377.1 (100) [M]þ, 232.1 (73) [M-C6H3Cl2]þ,
218.0 (42) [M-C7H5Cl2]þ, 204.0 (59) [M-C8H7Cl2]þ, 176 (13) [M-
C10H11Cl2]þ. HRMS (70 eV, EI): m/z calculated for C17H17N5OCl2:
377.0801, [M]þ, found: 377.0810.
4.1.4.8. 2-Amino-4-(benzylamino)-6-(2,6-dichlorophenyl)-5,6-
dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{5,8}). Starting from
compound 14{5} and benzylamine and following procedure B to
afford 15{5,8} in 60% yield, white solid. 1H NMR (400 MHz, DMSO-
d6): d 10.18 (s, 1H, NH), 7.56e7.47 (m, 3H), 7.39e7.34 (m, 2H),
7.30e7.28 (m, 2H), 7.22e7.17 (m, 1H), 6.88 (t, J ¼ 6.0 Hz, 1H, NH),
5.90 (s, 2H, NH2), 4.62 (dd, J ¼ 13.1, 8.9 Hz, 1H, C7eCH), 4.52 (td,
J ¼ 9.2, 3.5 Hz, 2H, C15eCH2), 2.97e2.66 (m, 2H, C6eCH2). HRMS
(70 eV, EI): m/z calculated for C20H17N5OCl2: 413.0810, [M]þ, found:
413.0809.
4.1.5. 2,4-Diamino-6-(4-fluorophenyl)-5,6-dihydropyrido[2,3-d]
pyrimidin-7(8H)-one (15{2,4})
Methyl 2-(4-fluorophenyl)acrylate (5{2}) (4.00 mmol) was dis-
solved in anhydrous ethylene glycol (20 mL). 2,6-
diaminopyrimidin-4(3H)-one (4.00 mmol) and sodium methoxide
(4.00 mmol) were added into the solution. The mixture was heated
under microwave irradiation at 180  C for 3 h. Water was added and
the reaction crude was neutralized with a 2 M solution of HCl. The
solid was collected by filtration and washed with water, MeOH and
Et2O to afford 15{2,4} as a white solid in 26% yield. IR (KBr), nmax
(cm1): 3495, 3334, 3219, 3104, 2888, 1698, 1659, 1638, 1570, 1510,
1460, 1210, 837. 1H NMR (400 MHz, DMSO-d6): d 10.72 (s, 1H, NH),
7.31 (dd, J ¼ 8.8, 5.6 Hz, 2H, C12-CHx2), 7.15 (t, J ¼ 8.9 Hz, 2H, C13-
CHx2), 6.26 (s, 2H, C2eNH2), 6.01 (s, 2H, C4eNH2), 3.88 (dd,
J ¼ 10.4, 6.9 Hz, 1H, C7eCH), 2.90e2.69 (m, 2H, C6eCH2). 13C NMR
(100.6 MHz, DMSO-d6): d 172.6 (CO), 161.8, 161.7, 161.2 (d,
J ¼ 241 Hz, C14) 156.3, 135.7 (d, J ¼ 3.1 Hz, C11), 130.2 (d, J ¼ 7.9 Hz,
C12), 115.0 (d, J ¼ 20.9 Hz, C13), 83.9 (C5), 45.5 (C7), 25.6 (C6). MS
(70 eV, EI): m/z (%) ¼ 273.1 (100) [M]þ. HRMS (70 eV, EI): m/z
calculated for C13H12N5OF: 273.1026, [M]þ, found: 273.1026.
4.1.6. General procedure for aromatization of pyrido[2,3-d]
pyrimidines 13{x} or 15{x,y}
Procedure A: A mixture of 0.5 mmol of the corresponding pyr-
idopyrimidine 13{x} or 15{x,y} and 60.0 mg (1.5 mmol) of sodium
hydride (60% dispersion in mineral oil) in 5 mL of anhydrous DMSO
was heated for 4 h at 100  C protected from moisture. The resulting
solution was cooled down, water (300 mL) was added and it was
neutralized with AcOH. The resulting precipitate was filtered,
washed with MeOH and EtOEt and dried in vacuo over phosphorus
pentoxide to afford the corresponding 16{x,y}.
Procedure B: A mixture of 2.7 mmol of the corresponding
pyridopyrimidine 13{x} or 15{x,y} and 3.5 mmol of DDQ in 30 mL of
1,2-dichloroethane was heated at 100  C overnight protected from
moisture. The resulting solution was cooled down, water (300 mL)
was added and the resulting precipitate was filtered, washed with
EtOH and EtOEt and dried in vacuo over phosphorus pentoxide to
afford the corresponding 16{x,y}.
Procedure C: A mixture of 0.5 mmol of the corresponding pyr-
idopyrimidine 13{x} or 15{x,y} and 114 mg (1.0 mmol) of activated
MnO2 in 8.5 mL of acetic acid was refluxed for 3 h. The resulting hot
suspension was filtered and the solvent was removed in vacuo. The
resulting solid was refluxed with water in order to eliminate MnO2
traces, then filtrated and dried in vacuo over phosphorus pentoxide
to afford the corresponding 16{x,y}.
4.1.6.1. 2-Amino-6-(4-fluorophenyl)pyrido[2,3-d]pyrimidine-
4,7(3H,8H)-dione (16{2}). Starting from 13{2} and following pro-
cedure B to afford 16{2} in 90% yield, yellow solid. IR (KBr), nmax
(cm1): 3323, 3096, 2922, 1633, 1505, 1393, 1231, 1159, 582. 1H NMR
(400 MHz, DMSO-d6): d 11.92 (s, 1H, NHeCO), 7.84 (s, 1H, C6eCH),
7.71 (dd, J ¼ 8.9, 5.6 Hz, 2H, C12-2xCH), 7.19 (t, J ¼ 9.0 Hz, 2H, C13-
2xCH), 6.08 (s, 2H, NH2). 13C NMR (100.6 MHz, DMSO-d6) d 162.7
(CO), 162.4, 160.0, 156.2, 155.7, 134.0 (C7), 132.8 (d, J ¼ 3.0 Hz), 130.0
(d, J ¼ 31.8 Hz, C12), 121.9 (C6) 114.7 (d, J ¼ 84.1 Hz C13), 95.9 (C5).
HRMS (ESIeTOF): m/z calculated for C13H9FN4O2: 273.0775,
[MþH]þ, found: 273.0782.
4.1.6.2. 2-Amino-6-(4-fluorophenyl)-4-(pentylamino)pyrido[2,3-d]
pyrimidin-7(8H)-one (16{2,5}). Starting from 15{2,5} and following
procedure C to afford 16{2,5} in 73% yield, white solid. IR (KBr), nmax
(cm1): 3380, 3171, 2930, 2859, 1619, 1564, 1509, 1475, 1227, 837,
796. 1H NMR (400 MHz, DMSO-d6): d 12.11 (s, 1H, NHCO), 8.27 (s,
1H, C6eCH), 7.76 (m, 2H, C12-CHx2), 7.24 (m, 2H, C13-CHx2),
3.47e3.30 (m, 2H, C15eCH2) 1.65e1.54 (m, 2H, C16eCH2),
1.37e1.29 (m, 4H, C17eCH2, C18eCH2), 0.89 (t, J ¼ 6.8 Hz, 3H, CH3).
13
C NMR (100.6 MHz, DMSO-d6): d 162.8, 162.5, 160.0, 159.6, 155.5,
130.6, 133.1 (d, J ¼ 6.6 Hz, C11), 130.3 (d, J ¼ 7.7 Hz, C12), 121.0 (C7),
114.7 (d, J ¼ 21.0 Hz, C13), 91.7 (C5), 40.4 (C15), 28.8 (C17), 28.6
(C16), 22.1 (C18), 14.0 (C19). HRMS (70 eV, EI): m/z calculated for
C18H20N5OF: 341.1653, [M]þ, found: 341.1652.
4.1.6.3. 2-Amino-6-(4-fluorophenyl)-4-((2-propoxyethyl)amino)pyr-
ido[2,3-d]pyrimidin-7(8H)-one (16{2,6}). Starting from 15{2,6} and
following procedure C to afford 16{2,6} in 72% yield, white solid. IR
(KBr), nmax (cm1): 3380, 2177, 2961, 2873, 1621, 1566, 1509, 1481,
1228, 838,796. 1H NMR (400 MHz, DMSO-d6): d 12.26 (s, 1H, NH),
8.30 (s, 1H, C6eCH), 7.90 (s, 1H, NH), 7.83e7.73 (m, 2H, C12eCH2),
7.23 (t, J ¼ 8.7 Hz, 3H, C13eCH2), 6.98 (s, 2H, NH2), 3.60e3.55 (m,
4H, C15eCH2, C16eCH2), 3.40e3.35 (m, 2H, C17eCH2), 1.57e1.45
(m, 2H, C18eCH2), 0.85 (t, J ¼ 7.4 Hz, 3H, C19eCH3). 13C NMR
(100.6 MHz, DMSO-d6): d 162.6, 161.1 (d, J ¼ 245.0 Hz, C14), 159.6,
155.6, 133.1 (d, J ¼ 1.9 Hz, C11), 132.8, 130.1 (d, J ¼ 7.9 Hz, C12), 121.1,
121.0, 114.5 (d, J ¼ 21.1 Hz, C13), 91.5 (C5), 71.8 (C17), 68.5 (C16),
39.5 (C15), 22.4 (C18), 10.5 (C19). HRMS (ESIeTOF): m/z calculated
for C18H21FN5O2, 358.1678, [M-H]þ, found: 358.1674.
4.1.6.4. 2-Amino-6-(4-fluorophenyl)-4-(pyrrolidin-1-yl)pyrido[2,3-d]
pyrimidin-7(8H)-one (16{2,7}). Starting from 15{2,7} and following
procedure C to afford 16{2,7} in 82% yield, light brown solid. IR
(KBr), nmax (cm1):3459, 3311, 3181, 2970, 2874, 1635, 1606, 1530,
1511, 1452, 1452, 1224, 839. 1H NMR (400 MHz, DMSO-d6): d 11.77
(s, 1H, NH), 8.10 (s, 1H, C6eCH), 7.73 (m, 2H, C12-CHx2), 7.19 (t,
J ¼ 9.0 Hz, 2H, C13-CHx2), 6.65 (s, 2H, NH2), 3.76 (t, J ¼ 6.4 Hz, 4H,
C15eCH2x2), 1.91 (t, J ¼ 6.5 Hz, 4H, C16eCH2x2).
 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
4.1.6.5. 2-Amino-6-(2,6-difluorophenyl)-4-((2-propoxyethyl)amino)
pyrido[2,3-d]pyrimidin-7(8H)-one (16{4,6}). Starting from 15{4,6}
and following procedure A to afford 16{4,6} in 60% yield, light
brown solid. IR (KBr), nmax (cm1): 3387, 3188, 2931, 2858, 1627,
1568, 1466, 1004, 788. 1H NMR (400 MHz, DMSO-d6): d 11.93 (s, 1H,
NH), 8.16 (s, 1H, C6eCH), 7.70 (s, 1H, NH), 7.49e7.41 (m, 1H,
C14eCH), 7.20e7.12 (m, 2H, C13-CHx2), 6.81 (s, 2H, NH2), 3.50e3.57
(m, 4H, C15eCH2, C16eCH2), 3.35e3.40 (m, 2H, C17eCH2), 1.49 (h,
J ¼ 7.4 Hz, 2H, C18eCH2), 0.84 (t, J ¼ 7.4 Hz, 3H, C19eCH3). 13C NMR
(100.6 MHz, DMSO-d6): d 163.0, 161.9, 160.4 (d, J ¼ 246.6 Hz, C12),
160.3 (d, J ¼ 246.7 Hz, C12), 159.6, 156.4, 136.7, 129.9 (t, J ¼ 10.3 Hz,
C14), 113.8 (t, J ¼ 20.1 Hz, C11), 111.6e111.3 (m, C13), 111.2, 91.0 (C5),
71.8 (C17), 68.3 (C16), 40.2 (C15), 22.4 (C18), 10.5 (C19). HRMS
(70 eV, EI): m/z calculated for C18H19N5O2F2: 375.1507, [M]þ, found:
375.1509.
4.1.6.6. 2-Amino-6-(2,6-dichlorophenyl)-4-(2-propoxyethoxy)pyrido
[2,3-d]pyrimidin-7(8H)-one (16{5,9}). Starting from 15{5,9} and
following procedure B to afford 16{5,9} in 82% yield, white solid. IR
(KBr), nmax (cm1): 3392, 3183, 2962, 2868, 1626, 1553, 1509, 1432,
1336, 1252, 802. 1H NMR (400 MHz, DMSO-d6): d 12.00 (s, 1H, NH),
7.58e7.51 (m, 3H, C13-CHx2, C7eCH), 7.42 (dd, J ¼ 8.7, 7.4 Hz, 1H,
C14eCH), 7.21 (s, 2H, NH2), 4.51e4.45 (m, 2H, C15eCH2), 3.74e3.68
(m, 2H, C16eCH2), 3.38 (t, J ¼ 6.6 Hz, 2H, C17eCH2), 1.47 (h,
J ¼ 7.3 Hz, 2H, C18eCH2), 0.80 (t, J ¼ 7.4 Hz, 3H, CH3). 13C NMR
(100.6 MHz, DMSO-d6): d 166.1, 162.7, 161.4, 157.4, 150.8, 135.2,
134.5, 134.2 (C6), 130.3 (C14), 129.2, 128.0 (C13), 122.1, 91.6 (C5),
71.9 (C17), 67.9 (C16), 65.7 (C15), 22.4 (C18), 10.4 (C19). HRMS
(70 eV, EI): m/z calculated for C18H18N4O3Cl2: 408.0750, [M]þ,
found: 408.0756.
4.1.6.7. 2-Amino-6-(2,6-dichlorophenyl)-4-(pentylamino)pyrido[2,3-
d]pyrimidin-7(8H)-one (16{5,5}). Starting from 15{5,5} and
following procedure B to afford 16{5,5} in 51% yield, white solid. IR
(KBr), nmax (cm1): 3126, 1856, 1640, 1465, 1430, 169, 780. 1H NMR
(400 MHz, DMSO-d6): d 10.27 (s, 1H, NH), 8.89 (s, 1H, NH), 8.36 (s,
1H, C6eCH), 7.98 (s, 2H, NH2), 7.60 (d, J ¼ 7.8 Hz, 2H, C13-CHx2),
7.47 (dd, J ¼ 8.8, 7.4 Hz, 1H, C14eCH), 3.49 (q, J ¼ 6.8 Hz, 2H,
C15eCH2), 1.60 (m, 2H, C16eCH2), 1.37e1.25 (m, 4H, C17eCH2,
C18eCH2), 0.91e0.83 (m, 3H, C19eCH3). 13C NMR (100.6 MHz,
DMSO-d6): d 169.9, 161.3, 155.7, 154.9, 135.4 (C6), 135.0, 134.7, 133.9,
130.7 (C14), 129.7, 128.1 (C13), 86.8 (C5), 43.4 (C15), 28.5 (C17), 27.9
(C16), 21.8 (C18), 13.9 (C19). MS (70 eV, EI): m/z (%) ¼ 391.2 (20.6)
[M]þ, 356.3 (100) [M-Cl]þ, 326.1 (19.0) [M-C2H5Cl]þ, 300.1 (7.5) [M-
C4H9Cl]þ. HRMS (70 eV, EI): m/z calculated for C18H19N5OCl2:
391.0967, [M]þ, found: 391.0967.
4.1.6.8. 2-Amino-6-(2,6-dichlorophenyl)-4-((2-propoxyethyl)amino)
pyrido[2,3-d]pyrimidin-7(8H)-one (16{5,6}). Starting from 15{5,6}
and following procedure B to afford 16{5,6} in 52% yield, light
brown solid. IR (KBr), nmax (cm1): 3128, 2870, 1640, 1453, 1430,
1284, 796. 1H NMR (400 MHz, DMSO-d6): d 8.94 (s, 1H, NH), 8.36 (s,
1H, C6eCH), 7.62e7.55 (m, 2H, C13-CHx2), 7.47 (t, J ¼ 8.7 Hz, 1H,
C14eCH), 3.66 (t, J ¼ 4.8 Hz, 2H, C15eCH2), 3.58 (t, J ¼ 5.3 Hz, 2H,
C16eCH2), 3.36 (t, J ¼ 6.6 Hz, 2H, C17eCH2), 1.48 (h, J ¼ 7.4, 6.4 Hz,
2H, C18eCH2), 0.82 (t, J ¼ 7.4 Hz, 3H, C19eCH3). 13C NMR
(100.6 MHz, DMSO-d6): d 156.42, 155.79, 150.86, 135.99 (C6), 135.00
(CeCl), 133.81, 130.83 (C14), 129.23, 128.15 (C13), 113.74, 101.71 (C5),
71.80 (C17), 67.68 (C16), 41.25 (C15), 22.38 (C18), 10.46 (C19). HRMS
(70 eV, EI): m/z calculated for C18H19N5O2Cl2: 407.0916, [M]þ,
found: 407.0911.
4.1.7. General procedure for the methylation at N8 of pyrido[2,3-d]
pyrimidines 16{x,y}
To a solution of 0.7 mmol of the corresponding 16{x,y} in 20 mL
475
of DMSO, 28.0 mg (0.7 mmol) of sodium hydride (60% dispersion in
mineral oil) were added and the mixture was stirred for 1 h at room
temperature under nitrogen atmosphere. After this period,
0.7 mmol of methyl iodide were added dropwise and then stirred
overnight at rt. The reaction was quenched by addition of 300 mL of
water and the resulting precipitate was filtered, washed with water
and dried in vacuo over phosphorus pentoxide to afford the cor-
responding 17{x,y}.
4.1.7.1. 2-Amino-6-(4-fluorophenyl)-4-methoxy-8-methylpyrido[2,3-
d]pyrimidin-7(8H)-one (17{2,1}). Starting from 16{2,1} to afford 17
{2,1} in 71% yield, white solid. IR (KBr), nmax (cm1): 3494, 3338,
2926, 1608, 1587, 1510, 1468, 1390, 834, 796. 1H NMR (400 MHz,
CDCl3): d 7.88 (s, 1H, C7eCH), 7.65 (dd, J ¼ 8.9, 5.5 Hz, 2H, C12-
CHx2), 7.08 (t, J ¼ 8.8 Hz, 2H, C13-CHx2), 4.03 (s, 3H, OCH3), 3.71
(s, 3H, NCH3). 13C NMR (100.6 MHz, CDCl3) d 167.85, 163.72, 163.13,
161.27, 156.81, 132.95 (C7), 130.61 (d, J ¼ 7.9 Hz,C12-CHx2), 130.45
(C6), 125.64 (C11), 115.12 (d, J ¼ 21.3 Hz,C13-CHx2), 94.75 (C5),
54.42 (OCH3), 28.89 (NCH3). HRMS (70 eV, EI): m/z calculated for
C15H13N4O2F: 300.1025, [M]þ, found: 300.1023.
4.1.7.2. 2-Amino-6-(4-fluorophenyl)-8-methyl-4-(pentylamino)pyr-
ido[2,3-d]pyrimidin-7(8H)-one (17{2,5}). Starting from 16{2,5} to
afford 17{2,5} in 89% yield, white solid. IR (KBr), nmax (cm1): 3345,
2930, 2858, 1603, 1567, 1508, 1466, 835, 799. 1H NMR (400 MHz,
CDCl3): d 7.91 (s, 1H, C6eCH), 7.52e7.46 (m, 2H, C12eCHx2), 6.94 (t,
J ¼ 8.4 Hz, 2H, C13eCHx2), 5.64 (t, J ¼ 5.4 Hz, 1H, NH), 5.09 (s, 2H,
NH2), 3.63 (s, 3H, CH3), 3.53e3.41 (m, 2H, C15eCH2), 1.65e1.58 (m,
2H, C16eCH2), 1.40e1.29 (m, 4H, C17eCH2, C18eCH2), 0.90 (t,
J ¼ 7.2 Hz, 3H, CH3). 13C NMR (100.6 MHz, CDCl3): d 163.4, 162.9,
161.3 (d, J ¼ 166.4 Hz, C14), 161.0, 155.8, 133.2 (d, J ¼ 3.2 Hz, C11),
130.4 (d, J ¼ 7.8 Hz, C12), 129.4 (C6), 123.4 (C7), 114.9 (d, J ¼ 21.3 Hz,
C13), 92.8 (C5), 41.4 (C15), 29.3, 29.3, 28.78 (NeCH3), 22.5 (C18),
14.1 (C19). MS (70 eV, EI): m/z (%) ¼ 355.2 (100) [M]þ, 312.1 (42) [M-
C3H7]þ, 298.1 (61) [M-C4H9]þ, 285.1 (62) [M-C5H12]þ, 158.0 (40) [M-
C12H20FN]þ.
4.1.7.3. 2-Amino-6-(4-fluorophenyl)-8-methyl-4-((2-propoxyethyl)
amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17{2,6}). Starting from 16
{2,6} to afford 17{2,6} in 87% yield, white solid. IR (KBr), nmax
(cm1): 3341, 3199, 2932, 2872, 1603, 1570, 1509, 1468, 1227, 1160,
837, 799. 1H NMR (400 MHz, CDCl3): d 7.58 (dd, J ¼ 8.7, 5.6 Hz, 2H,
C12eCHx2), 7.47 (s, 1H, C6eCH), 7.05 (t, J ¼ 8.8 Hz, 2H, C13eCHx2),
5.81 (s, 1H, NH), 5.03 (s, 2H, NH2), 3.72 (q, J ¼ 5.1 Hz, 2H, C15eCH2),
3.68 (s, 3H, NCH3), 3.63 (t, J ¼ 5.0 Hz, 2H, C16eCH2), 3.44 (t,
J ¼ 6.7 Hz, 2H, C17eCH2), 1.61 (dq, J ¼ 14.2, 7.2 Hz, 2H, C18eCH2),
0.93 (t, J ¼ 7.4 Hz, 3H, C19eCH3). 13C NMR (100.6 MHz, CDCl3):
d 163.59, 162.00 (d, J ¼ 162.0 Hz), 160.32, 155.98, 155.52, 133.21,
130.50 (d, J ¼ 7.9 Hz, C12), 128.97 (C6), 124.01, 115.11 (d, J ¼ 21.0 Hz,
C13), 92.73 (C5), 73.04 (C17), 69.05 (C16), 41.06 (C15), 28.81 (NCH3),
22.91 (C18), 10.71 (C19). MS (70 eV, EI): m/z (%) ¼ 371.2 (73) [M]þ,
312.1 (15) [M-C3H7O]þ, 298.1 (86) [M-C4H9O]þ, 285.1 (100) [M-
C5H12O]þ, 158.0 (44) [M-C12H20NFO]þ. HRMS (70 eV, EI): m/z
calculated for C19H22N5O2F: 371.1758, [M]þ, found: 371.1755.
4.1.7.4. 2-Amino-6-(4-fluorophenyl)-8-methyl-4-(pyrrolidin-1-yl)
pyrido[2,3-d]pyrimidin-7(8H)-one (17{2,7}). Starting from 16{2,7}
to afford 17{2,7} in 59% yield, white solid. IR (KBr), nmax (cm1):
3508, 3345, 3233, 2964, 2868, 1638, 1590, 1564, 1524, 1504, 1443,
833, 795. 1H NMR (400 MHz, CDCl3): d 8.01 (s, 1H, C7eCH), 7.61 (dd,
J ¼ 8.8, 5.4 Hz, 2H, C12eCHx2), 7.08 (t, J ¼ 8.8 Hz, 2H, C13eCHx2),
4.89 (s, 2H, NH2), 3.82e3.75 (m, 4H, C15eCH2), 2.03e1.96 (m, 4H,
C16eCH2). 13C NMR (100.6 MHz, CDCl3): d 162.4, 162.1 (d,
J ¼ 265.8 Hz, C14), 161.0, 160.9, 157.8, 133.8 (C11), 133.3 (C6), 130.4
(d, J ¼ 7.8 Hz, C12), 122.2 (C7), 115.1 (d, J ¼ 21.2 Hz, C13), 94.6 (C5),
476 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
51.0 (C15), 29.2 (CH3), 25.8 (C16). HRMS (70 eV, EI): m/z calculated
for C18H18N5OF: 339.1493, [M]þ, found: 339.1495.
4 .1. 7 . 5 . 2 - A m i n o - 6 - ( 2 , 6 - d i ch l o r o p h e n yl ) - 8 - m e t h yl - 4 - ( 2 -
propoxyethoxy)pyrido[2,3-d]pyrimidin-7(8H)-one (17{5,9}).
Starting from 16{5,9} to afford 17{5,9} in 26% yield, white solid. IR
(KBr), nmax (cm1): 3336, 2960, 2872, 1658, 1585, 1464, 1429, 1338,
801. 1H NMR (400 MHz, DMSO-d6): d 7.62 (s, 1H, C6eCH), 7.54 (d,
J ¼ 8.0 Hz, 2H, CHeC13x2), 7.42 (dd, J ¼ 8.8, 7.3 Hz, 1H, CHeC14),
7.38 (s, 2H, NH2), 4.53e4.45 (m, 2H, C15eCH2), 3.77e3.68 (m, 2H,
C16eCH2), 3.59 (m, 2H, C17e CH2), 3.55 (s, 3H, NCH3), 1.46 (h,
J ¼ 7.1 Hz, 2H, C18eCH2), 0.79 (t, J ¼ 7.4 Hz, 3H, C19eCH3). 13C NMR
(100.6 MHz, DMSO-d6): d 166.6, 162.4, 160.6, 157.2, 135.2, 134.8,
132.8 (C6), 130.4 (C14), 128.0 (C13), 120.6, 109.6, 92.0 (C5), 71.9
(C17), 67.9 (C16), 65.9 (C15), 28.2 (NCH3), 22.4 (C18), 10.4 (C19).
HRMS (ESIeTOF): m/z calculated for C19H21Cl2N4O3: 223.0983,
[MþH]þ, found: 423.0985. Anal. Calcd. (%) for C19H20Cl2N4O3: C,
53.91; H, 4.76; N, 13.24. Found: C, 53.97; H, 4.94; N, 12.93.
4.1.7.6. 4-Amino-6-(2,6-dichlorophenyl)-8-methylpyrido[2,3-d]py-
rimidine-7(8H)-one (17{5,4}). Starting from 9{5,4} [25] and
following procedure A described in 4.1.6. to afford 16{5,4} in 97%
yield, white solid. IR (KBr), nmax (cm1): 3457, 3329, 3182, 2791,
1615, 1545, 1494, 1462, 1429, 1400, 1327, 1290, 1191, 936, 802, 775.
1
H NMR (400 MHz, DMSO-d6): d 12.17 (br s, 1H), 7.98 (s, 1H), 7.54 (d,
J ¼ 8.0 Hz, 2H), 7.40 (dd, J ¼ 8.6, 7.6 Hz, 1H), 7.17 (br s, 2H), 6.81 (br s,
2H). 13C NMR (100.6 MHz, DMSO-d6): d 163.1, 161.9, 161.5, 156.6,
136.3, 135.5, 134.8, 130.2, 128.0, 119.6, 90.5. MS (ESI-TOF): m/z
(%) ¼ 322.0 (100) [MþH]þ, 305.0 (9) [M-NH2]þ, 286.0 (9) [M-Cl]þ,
252.1 (7) [MþHeCl2]þ. HRMS (ESI-TOF): m/z calculated for
C13H10N5OCl2: 322.0257 [MþH]þ, found: 322.0256. 16{5,4} was
converted to 17{5,4} in 70% yield, white solid. IR (KBr), nmax (cm1):
3347, 3212, 2925, 1627, 1607, 1568, 1546, 1462, 1430, 1394, 1360,
1328, 1202 802. 1H NMR (400 MHz, DMSO-d6): d 7.99 (s, 1H), 7.55 (d,
J ¼ 7.9 Hz, 2H), 7.41 (dd, J ¼ 8.6, 7.5 Hz, 1H), 7.15 (br s, 2H), 6.67 (br s,
2H), 3.50 (s, 3H). 13C NMR (100.6 MHz, DMSO-d6): d 162.8, 162.1,
160.6, 156.7, 135.6, 135.5, 134.1, 131.0, 128.0, 118.4, 90.5, 28.0. MS
(ESI-TOF): m/z (%) ¼ 336.0 (100) [MþH]þ, 286.0 (3) [MþH-CH3Cl]þ,
266.1 (5) [MþH-Cl2]þ. HRMS (ESIeTOF): m/z calculated for
C14H12Cl2N5O: 336.0413, [MþH]þ, found: 336.0424.
4.1.8. 6-(2,4-dichlorophenyl)-2-(methylthio)-5,6-dihydropyrido
[2,3-d]pyrimidine-4,7(3H,8H)-dione (19{11})
2-(2,4-dichlorophenyl)acrylate (5{11}) (4.00 mmol) was dis-
solved in 2-propanol (20 mL), 6-amino-2-(methylthio)pyrimidin-
4(3H)-one (52) (5.60 mmol) and potassium carbonate (4.00 mmol)
were added into the solution. The mixture was heated under mi-
crowave irradiation at 170  C for 3 h. Then, the solvent was
removed under reduced pressure, water was added to the crude
and this was neutralized with a 2 M solution of HCl. The solid was
collected by filtration and washed with water, and Et2O to afford 19
{11} in 80% yield, white solid. IR (KBr): n (cm1): 3404, 3131, 2868,
1694, 1633, 1557, 1505, 1479, 1459, 1374, 1285, 1241, 1151, 1103, 964,
823, 576. 1H NMR (400 MHz, DMSO-d6): d 10.73 (s, 1H), 7.63 (d,
J ¼ 1.9 Hz, 1H), 7.43 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.40 (d, J ¼ 8.3 Hz, 1H),
4.19 (dd, J ¼ 12.8, 7.7 Hz, 1H, C7eCH), 2.89 (dd, J ¼ 16.2, 7.7 Hz, 1H,
C6eCH2), 2.75 (dd, J ¼ 16.1, 12.8 Hz, 1H, C6eCH2), 2.50 (s, 3H, SCH3).
13
C NMR (100.6 MHz, DMSO-d6): d 170.25, 161.8, 154.4, 135.9 (C12),
134.3 (C14), 132.6(C16), 132.0 (C12), 129.0 (C13), 127.6 (C15), 95.0
(C5), 43.3 (C7), 23.7 (C6), 12.8 (SCH3). Anal. Calcd. (%) for
C14H11Cl2N3O2S: C, 47.20; H, 3.11; N, 11.80; S, 9.00. Found: C, 47.07;
H, 2.97; N, 11.96; S, 9.01.
4.1.9. General procedure for the substitution of the methylthio
group of compounds 19{x} by benzylamines
The corresponding benzylamine (20 mmol) was added to the
solution of the 2-methylthio-substituted pyridopyrimidine (19{x})
(2 mmol) in 14 mL of 2-propanol and the mixture was heated under
microwave irradiation at 170  C for 5 h. Cyclohexane was added to
the solution and the solid was collected by filtration and washed
with cyclohexane, and Et2O to afford the corresponding (21{x,y}).
4.1.9.1. 2-(benzylamino)-6-(4-fluorophenyl)-5,6-dihydropyrido[2,3-
d]pyrimidine-4,7(3H,8H)-dione (21{2,8}). Starting from 19{2} and
benzylamine to afford 21{2,8} in 53% yield, white solid. IR (KBr),
nmax (cm1): 3423, 3180, 3068, 2958, 1689, 1644, 1612, 1513, 1229,
836, 582. 1H NMR (400 MHz, TFA-d): d 7.42e7.30 (m, 5H, CHArx5),
7.22 (dd, J ¼ 7.8, 5.2 Hz, 2H, CHeC12x2), 7.05 (t, J ¼ 8.5 Hz, 2H,
CHeC13x2), 4.75 (s, 2H, CH2eC15), 4.05 (dd, J ¼ 11.3, 7.3 Hz, 1H,
CHeC7), 3.24 (dd, J ¼ 16.8, 7.4 Hz, 1H, C6eCH2), 3.03 (dd, J ¼ 16.8,
11.4 Hz, 1H, C6eCH2). 13C NMR (100.6 MHz, TFA-d): d 179.9, 168.9,
166.5 (d, J ¼ 235.5 Hz, C14) 153.9, 150.6, 136.2, 134.7 (d, J ¼ 3.4 Hz,
C11), 134.0 (d, J ¼ 8.5 Hz, C12) 133.7 (CAr), 133.7 (CAr), 131.6 (CAr),
120.3 (d, J ¼ 22.7 Hz, C13) 96.2 (C5), 50.7 (C15), 49.7 (C7), 27.8 (C6).
HRMS (70 eV, EI): m/z calculated for C20H17N4O2F: 324.0183, [M]þ,
found: 324.0181. Anal. Calcd. (%) for C20H17FN4O2: C, 65.93; H, 4.70;
N, 15.38. Found: C, 65.53; H, 4.47; N, 15.49.
4.1.9.2. 2-(benzylamino)-6-(2,6-dichlorophenyl)-5,6-dihydropyrido
[2,3-d]pyrimidine-4,7(3H,8H)-dione (21{5,8}). Starting from 19{5}
and benzylamine to afford 21{5,8} in 36% yield, white solid. IR (KBr),
nmax (cm1): 3263, 3181, 3066, 2934, 1686, 1639, 1613, 1597, 1545,
1520, 1374, 1289, 770. 1H NMR (400 MHz, DMSO-d6): d 10.57 (s, 1H,
NH), 10.27 (s, 1H, NH), 7.50 (ddd, J ¼ 16.6, 8.1, 1.3 Hz, 2H,
CHeC13x2), 7.40e7.31 (m, 5H, CH-Ar), 7.27 (d, J ¼ 7.0 Hz, 1H, CH-
Ar), 6.99 (s, 1H, NH-Bz), 4.53 (dd, J ¼ 13.4, 8.9 Hz, 1H, CHeC7),
4.47 (d, J ¼ 6.1 Hz, 2H), 2.84e2.64 (m, 2H, CH2eC6). 13C NMR
(100.6 MHz, DMSO-d6): d 169.7, 161.6, 155.5, 153.7, 139.1, 135.4,
135.2, 134.7, 129.7, 129.7, 128.3, 128.3, 127.6, 127.0, 87.1 (C5), 43.4
(C15), 43.4 (C7), 22.4 (C6). MS (70 eV, EI): m/z (%) ¼ 414.0 (100),
[M]þ, 379.1 [M-Cl]þ, 228.9 (34.0) [C12H12N4O]þ, 105.8 (62.6)
[C7H8N]þ, 90.9 (100) [C7H7]þ. HRMS (70 eV, EI): m/z calculated for
C20H16N4O2Cl2: 414.0650, [M]þ, found: 414.0650. Anal. Calcd. (%)
for C20H16Cl2N4O2: C, 57.84; H, 3.88; N, 13.49. Found: C, 57.71; H,
3.78; N, 13.47.
4.1.9.3. 2-((4-fluorobenzyl)amino)-6-(4-fluorophenyl)-5,6-
dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (21{2,9}).
Starting from 19{2} and 4-flurobenzylamine to afford 21{2,9} in 40%
yield, white solid. IR (KBr), nmax (cm1): 3424, 3248, 3181, 2931,
1637, 1611, 1513, 1230, 834. 1H NMR (400 MHz, TFA-d): d 7.31 (dd,
J ¼ 8.6, 5.0 Hz, 2H, C17-CHx2), 7.19 (dd, J ¼ 8.7, 5.0 Hz, 2H, C12-
CHx2), 7.06e7.00 (m, 4H, C18-CHx2, C13-CHx2), 4.70 (s, 2H,
C15eCH2), 4.02 (dd, J ¼ 11.2, 7.2 Hz, 1H, C7eCH), 3.22 (dd, J ¼ 16.8,
7.4 Hz, 1H, C6eCH2), 3.00 (dd, J ¼ 16.8, 11.4 Hz, 1H, C6eCH2). 13C
NMR (100.6 MHz, DMSO-d6): d 179.9, 168.0 (d, J ¼ 248.8 Hz, C14),
167.6 (d, J ¼ 247.7 Hz), 165.3, 154.0, 150.7, 134.7 (d, J ¼ 3.5 Hz, C11),
134.0 (d, J ¼ 8.5 Hz, C12), 133.7 (C17), 132.2 (C16), 120.5 (d,
J ¼ 24.5 Hz), 120.4 (d, J ¼ 22.3 Hz), 96.1 (C5), 50.0 (C15), 49.7 (C7),
27.8 (C6). HRMS (70 eV, EI): m/z calculated for C20H16N4O2F2:
382.1241, [M]þ, found: 382.1241.
4.1.10. General procedure for oxidation of 2-(methylthio)-5,6-
dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-diones (19{x}) to
methylsulfones 20{x}
m-CPBA (18 mmol) was suspended in 20 mL of DMF and was
added dropwise into a solution of the 2-methylthio-substituted
pyridopyrimidine (19{x}) (6 mmol) in 20 mL of DMF a 0  C. The
 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
mixture was stirred overnight at room temperature. Then the sol-
vent was removed under reduced pressure and the residue was
suspended in EtOEt and the solid was collected by filtration and
washed with water, and EtOEt to afford the corresponding 2-
methylsulfonyl-substituted pyrido[2,3-d]pyrimidine (20{x}).
4.1.10.1. 2-(methylsulfonyl)-6-phenyl-5,6-dihydropyrido[2,3-d]py-
rimidine-4,7(3H,8H)-dione (20{1}). Starting from 19{1} to afford 20
{1} in 74% yield, white solid. IR (KBr), nmax (cm1): 3429, 3200,
3136, 3022, 2919, 2866, 1691, 1643, 1612, 1486, 1341, 1235, 1165,
1134, 699. 1H NMR (400 MHz, DMSO-d6): d 13.29 (s, 1H, NH), 11.32
(s, 1H, NH), 7.36e7.31 (m, 2H, C13-CHx2), 7.30e7.22 (m, 3H, C12-
CHx2, C14eCH), 4.00 (dd, J ¼ 9.4, 7.3 Hz, 1H, C7eCH), 3.31 (s, 3H,
SCH3), 3.14e3.00 (m, 2H, C6eCH2). 13C NMR (100.6 MHz, DMSO-
d6): d 171.4, 167.1, 162.6, 158.6, 138.4, 128.5, 128.0, 127.1 (C12), 100.4
(C5), 44.8 (C7), 39.1 (SCH3), 24.7 (C6). Anal. Calcd. (%) for
C14H13N3O4S: C, 52.66; H, 4.10; N, 13.16; S, 10.04. Found: C, 52.32; H,
4.16 N, 13.02; S, 10.34.
4.1.10.2. 6-(4-fluorophenyl)-2-(methylsulfonyl)-5,6-dihydropyrido
[2,3-d]pyrimidine-4,7(3H,8H)-dione (20{2}). Starting from 19{2} to
afford 20{2} in 60% yield, white solid. IR (KBr): n (cm1): 3393,
3036, 2867, 1635, 1607, 1513, 1490, 1337, 1233, 1160, 839, 767, 541. 1H
NMR (400 MHz, DMSO-d6): d 13.28 (s, 1H, NH), 11.33 (s, 1H, NH),
7.30 (dd, J ¼ 8.7, 5.8 Hz, 2H, C12-CHx2), 7.17 (t, J ¼ 8.9 Hz, 2H, C13-
CHx2), 4.03 (dd, J ¼ 10.2, 7.4 Hz, 1H, C7eCH), 3.31 (s, 3H, SCH3),
3.14e2.94 (m, 2H, C6eCH2). 13C NMR (100.6 MHz, DMSO-d6):
d 171.4, 167.0, 162.7, 161.3 (d, J ¼ 243.2 Hz, C14), 158.7, 134.6 (d,
J ¼ 3.1 Hz, C11), 130.2 (d, J ¼ 8.1 Hz, C12), 115.2 (d, J ¼ 21.3 Hz, C13),
100.4 (C5), 44.1 (C7), 29.5 (SCH3), 24.8 (C6). HRMS (70 eV, EI): m/z
calculated for C14H12N3O4FS: 337.0533, [M]þ, found: 337.0533.
Anal. Calcd. (%) for C14H12FN3O4S: C, 49.85; H, 3.59; N, 12.46; S, 9.51.
Found: C, 49.68; H, 3.84; N, 12.70; S, 9.18.
4 .1.10 . 3 . 6 - ( 2 , 6 - d i fl u o r o p h e n yl ) - 2 - ( m e t h yl s u l f o n yl ) - 5 , 6 -
dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (20{4}).
Starting from 19{4} to afford 20{4} in 60% yield, white solid. IR
(KBr): n (cm1): 3432, 3219, 3027, 2923, 1704, 1649, 1612, 1474,
1459, 1339, 1235, 1164, 1134, 1010, 783, 542. 1H NMR (400 MHz,
DMSO-d6): d 13.33 (s, 1H), 11.41 (s, 1H), 7.44 (tt, J ¼ 8.4, 6.6 Hz, 1H,
C14eCH), 7.14 (t, J ¼ 8.8 Hz, 2H, C13-CHx2), 4.46 (dd, J ¼ 14.0, 7.6 Hz,
1H, C7eCH), 3.31 (s, 3H, SCH3), 3.10 (dd, J ¼ 16.5, 7.7 Hz, 1H,
C6eCH2), 2.94e2.82 (m, 1H, C6eCH2). 13C NMR (100.6 MHz, DMSO-
d6): d 169.6, 167.0, 162.7, 162.0 (d, J ¼ 8.3 Hz, C12), 159.6 (d,
J ¼ 8.2 Hz, C12), 158.7, 130.0 (t, J ¼ 10.6 Hz, C11), 114.5 (t, J ¼ 18.6 Hz,
C14), 111.8 (dd, J ¼ 22.3, 3.1 Hz, C13), 100.0 (C5), 39.2 (SCH3), 35.3
(C7), 23.4 (C6). Anal. Calcd. (%) for C14H11F2N3O4S: C, 47.32; H, 3.12;
N, 11.83; S, 9.02. Found: C, 47.14; H, 3.19 N, 11.72; S, 9.28.
4 .1.10 . 4 . 6 - ( 2 , 6 - d i ch l o r o p h e nyl ) - 2 - ( m e t hyl s u l fo nyl ) - 5 , 6 -
dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (20{5}).
Starting from 19{5} to afford 20{5} in 98% yield, white solid. 1H NMR
(400 MHz, DMSO-d6): d 11.35 (s, 1H, NH), 7.53 (dd, J ¼ 12.3, 8.1 Hz,
2H, C13-CHx2), 7.39 (t, J ¼ 8.1 Hz, 1H, C14eCH), 4.87 (dd, J ¼ 12.9,
9.2 Hz, 1H, C7eCH), 3.31 (s, 3H, SCH3), 3.06 (dd, J ¼ 17.1, 9.4 Hz, 1H,
C6eCH2), 2.99 (dd, J ¼ 17.0, 13.5 Hz, 1H, C6eCH2).
4.1.10.5. 2-(methylsulfonyl)-6-(naphthalen-1-yl)-5,6-dihydropyrido
[2,3-d]pyrimidine-4,7(3H,8H)-dione (20{8}). Starting from 19{8} to
afford 20{8} in 68% yield, white solid. IR (KBr), nmax(cm1): 3432,
3211, 3022, 1690, 1641, 1614, 1487, 1345, 1225, 1167, 1136, 775. 1H
NMR (400 MHz, DMSO-d6): d 13.24 (s, 1H, NH), 11.44 (s, 1H, NH),
8.13e8.07 (m, 1H, C19eCH), 8.00e7.94 (m, 1H, C16eCH), 7.87 (d,
J ¼ 8.2 Hz, 1H, C12eCH), 7.58e7.51 (m, 2H, C17eCH, C18eCH), 7.48
(t, J ¼ 7.2 Hz, 1H, C13eCH), 7.40 (dd, J ¼ 7.2, 1.2 Hz, 1H, C14eCH),
477
4.85 (dd, J ¼ 10.2, 7.9 Hz, 1H, C7eH), 3.33 (s, 3H, SCH3), 3.23e3.13
(m, 2H, C6eCH2). 13C NMR (100.6 MHz, DMSO-d6): d 171.5, 167.1,
162.7, 158.8, 135.0, 133.6, 131.1, 129.0 (C16), 127.8 (C12), 126.2 (C17),
125.7 (C18), 125.5 (C14, C13), 124.0 (C19), 100.6 (C5), 41.5 (C7), 39.1
(SCH3), 25.0 (C6). Anal. Calcd. (%) for C18H15N3O4S: C, 58.53; H, 4.09;
N, 11.38; S, 8.68. Found: C, 58.47; H, 4.18 N, 11.57; S, 8.69.
4 .1.10 . 6 . 6 - ( 2 , 4 - d i c h l o r o p h e n yl ) - 2 - ( m e t hyl s u l f o n yl ) - 5 , 6 -
dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (20{11}).
Starting from 19{11} to afford 20{11} in 70% yield, white solid. IR
(KBr), nmax(cm1): 3432, 3279, 3040, 2925, 2868, 1651, 1492, 1329,
1276, 1231, 1156, 1132, 944, 766, 572, 529. 1H NMR (400 MHz,
DMSO-d6): d 13.34 (s, 1H, NH), 11.42 (s, 1H, NH), 7.65 (dd, J ¼ 1.5,
0.9 Hz, 1H), 7.46 (s, 1H, C13eCH), 7.46 (d, J ¼ 0.9 Hz, 1H), 4.38 (dd,
J ¼ 11.6, 9.3 Hz, 1H, C7eCH), 3.31 (s, 3H, SCH3), 3.07e2.81 (s, 2H,
C6eCH2). 13C NMR (100.6 MHz, DMSO-d6): d 169.9, 167.0, 162.7,
158.6, 135.3, 134.4, 132.7, 132.1, 129.0 (C13), 127.6, 100.2 (C5), 42.6
(C7), 28.5 (SCH3), 23.8 (C6). Anal. Calcd. (%) for C14H11Cl2N3O4S: C,
43.31; H, 2.86; N, 10.82; S, 8.26. Found: C, 43.48; H, 2.66; N, 10.91; S,
8.11.
4.1.11. General procedure for the substitution of the methylsulfonyl
group of pyridopyrimidines 20{x} by arylamines
The corresponding arylamine (10 mmol) was added to a solution
of the 2-methylsulfonyl-substituted pyridopyrimidine (20{x})
(2 mmol) in 14 mL 2-propanol and the mixture was heated under
microwave irradiation at 170  C for 5 h. Cyclohexane was added to
the solution and the solid obtained was collected by filtration and
washed with cyclohexane and Et2O to afford the corresponding 21
{x,y}
4.1.11.1. 6-Phenyl-2-(phenylamino)-5,6-dihydropyrido[2,3-d]pyrimi-
dine-4,7(3H,8H)-dione (21{1,10}). Starting from 20{1} and aniline to
afford 21{1,10} in 82% yield, white solid. IR (KBr), nmax(cm1): 3401,
3058, 1615, 1654, 1462, 750, 395. 1H NMR (400 MHz, DMSO-d6):
d 10.48 (s, 2H, NHx2), 8.80 (s, 1H, NH), 7.73e7.66 (m, 2H, C16-CHx2),
7.34e7.22 (m, 7H, C17-CHx2, C12-CHx2, C13-CHx2, C14eCH), 7.02
(tt, J ¼ 7.3, 1.2 Hz, 1H, C8eCH), 3.82 (dd, J ¼ 8.5, 7.1 Hz, 1H, C7eCH),
2.92e2.72 (m, 2H, C6eCH2). 13C NMR (100.6 MHz, DMSO-d6):
d 172.4, 139.7, 139.1, 129.2, 128.8, 128.8, 128.5, 128.4, 127.3, 123.0
(C18), 119.8 (C16), 114.3, 90.7 (C5), 46.2 (C7), 25.0 (C6). HRMS
(70 eV, EI): m/z calculated for C19H16N4O2: 332.1273, [M]þ, found:
332.1275.
4.1.11.2. 6-(4-fluorophenyl)-2-(phenylamino)-5,6-dihydropyrido[2,3-
d]pyrimidine-4,7(3H,8H)-dione (21{2,10}). Starting from 20{2} and
aniline to afford 21{2,10} in 94% yield, white solid. IR (KBr), nmax
(cm1): 3400, 3186, 3049, 1655, 1614, 1511, 1461, 1232. 1H NMR
(400 MHz, DMSO-d6): d 10.47 (s, 2H, 2xNH), 9.25 (s, 1H, NH-Ph),
7.79e7.67 (m, 2H, 2xCHPh), 7.33e7.24 (m, 4H, 2xCHPh þ C12-CHx2),
7.15 (t, J ¼ 8.9 Hz, 2H, C13-CHx2), 7.04e6.98 (m, 1H), 3.85 (dd,
J ¼ 9.6, 7.0 Hz, 1H), 2.92e2.70 (m, 2H, C6eCH2). 13C NMR
(100.6 MHz, DMSO-d6): d 172.0, 161.8, 161.3 (d, J ¼ 242.8 Hz, C14),
155.6, 151.1, 138.8, 135.5 (d, J ¼ 3.1 Hz, C11), 130.1 (d, J ¼ 8.1 Hz, C12),
128.8, 122.6, 119.5, 115.2 (d, J ¼ 21.1 Hz, C13) 90.4 (C5), 45.1 (C7),
24.7 (C6). HRMS (70 eV, EI): m/z calculated for C19H15N4O2F:
350.1179, [M]þ, found: 350.1179.
4.1.11.3. 6-(4-fluorophenyl)-2-((4-fluorophenyl)amino)-5,6-
dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (21{2,11}).
Starting from 20{2} and 4-fluoroaniline to afford 21{2,11} in 67%
yield, white solid. IR (KBr), nmax (cm1): 3409, 3171, 2958, 1645,
1603, 1512, 1242, 836. 1H NMR (400 MHz, DMSO-d6): d 10.58 (s, 1H,
NH), 10.48 (s, 1H, NH), 8.88 (s, 1H, NH), 7.71 (dd, J ¼ 9.1, 4.9 Hz, 2H,
C16-CHx2), 7.28 (dd, J ¼ 8.7, 5.6 Hz, 2H, C12-CHx2), 7.13 (m, 4H,
478 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
C13-CHx2, C17-CHx2), 3.84 (dd, J ¼ 9.5, 7.1 Hz, 1H, C7eCH), 2.86
(dd, J ¼ 16.0, 7.1 Hz, 1H, C6eCH), 2.73 (dd, J ¼ 16.0, 9.6 Hz, 1H,
C6eCH). 13C NMR (100.6 MHz, DMSO-d6): d 171.9, 161.2 (d,
J ¼ 242.6 Hz, C14), 157.8 (d, J ¼ 239.4 Hz, C18), 156.2, 155.4, 154.8,
135.4 (d, J ¼ 3.1 Hz, C11), 135.0 (C15), 130.0 (d, J ¼ 8.1 Hz, C12), 121.3
(d, J ¼ 7.6 Hz, C16), 115.2 (d, J ¼ 17.7 Hz, C13), 115.0 (d, J ¼ 16.7 Hz,
C17), 90.3 (C5), 45.0 (C7), 24.6 (C6). HRMS (70 eV, EI): m/z calcu-
lated for C19H14N4O2F2: 368.1085, [M]þ, found: 368.1081.
4.1.11.4. 2-((4-bromophenyl)amino)-6-(4-fluorophenyl)-5,6-
dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (21{2,12}).
Starting from 20{2} and 4-bromoaniline to afford 21{2,12} in 74%
yield, white solid. IR (KBr), nmax (cm1): 3400, 3163, 2963, 1643,
1600, 1511, 1488, 1236, 835, 795, 771. 1H NMR (400 MHz, DMSO-d6):
d 10.53 (s, 1H, NHx2), 8.93 (s, 1H, NH), 7.69 (d, J ¼ 8.9 Hz, 2H,
C17eCHx2), 7.43 (d, J ¼ 8.8 Hz, 2H, C16eCHx2), 7.28 (dd, J ¼ 8.7,
5.6 Hz, 2H, C12eCHx2), 7.15 (t, J ¼ 8.9 Hz, 2H, C13eCHx2), 3.85 (dd,
J ¼ 9.6, 7.1 Hz, 1H, C7eCH), 2.91e2.69 (m, 2H, C6eCH2). 13C NMR
(100.6 MHz, DMSO-d6): d 171.9, 161.2 (d, J ¼ 242.8 Hz, C14), 158.5,
158.2, 155.4, 138.2 (C15), 135.4 (d, J ¼ 3.1 Hz, C11), 131.4 (C17), 130.0
(d, J ¼ 8.1 Hz, C12), 121.4 (C16), 115.1 (d, J ¼ 21.3 Hz, C13), 114.0
(C18), 90.6 (C5), 45.0 (C7), 24.6 (C6). HRMS (70 eV, EI): m/z calcu-
lated for C19H14N4O2FBr: 428.0284, [M]þ, found: 428.0287.
4.1.11.5. 6-(4-fluorophenyl)-2-((4-methoxyphenyl)amino)-5,6-
dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (21{2,13}).
Starting from 20{2} and 1.2 equivalents of 4-methoxyaniline to
afford 21{2,13} in 79% yield, white solid. IR (KBr), nmax (cm1): 3404,
3148, 2813, 1656, 1615, 1509, 1457, 1228, 844. 1H NMR (400 MHz,
DMSO-d6): d 10.40 (s, 1H, NH), 8.62 (s, 1H, NH), 7.56 (d, J ¼ 9.0 Hz,
2H, C17-CHx2), 7.28 (dd, J ¼ 8.7, 5.6 Hz, 2H, C12eCHx2), 7.15 (t,
J ¼ 8.9 Hz, 2H, C13eCHx2), 6.87 (d, J ¼ 9.0 Hz, 2H, C16eCHx2), 3.83
(dd, J ¼ 9.5, 7.0 Hz, 1H, C7eCH), 3.73 (s, 3H, OCH3), 2.89e2.69 (m,
2H, C6eCH2). 13C NMR (100.6 MHz, DMSO-d6): d 171.9, 161.4, 161.1
(d, J ¼ 242.8 Hz, C14), 156.2, 155.1, 154.8, 135.4 (d, J ¼ 3.1 Hz, C11),
131.55, 130.0 (d, J ¼ 8.1 Hz, C12), 121.5 (C17), 115.0 (d, J ¼ 21.3 Hz,
C13), 113.9 (C16), 89.8 (C5) 55.2 (OMe), 45.1 (C7), 24.6 (C6). HRMS
(70 eV, EI): m/z calculated for C20H17N4O3F: 380.1285, [M]þ, found:
380.1286.
4.1.11.6. 6-(2,6-difluorophenyl)-2-(phenylamino)-5,6-dihydropyrido
[2,3-d]pyrimidine-4,7(3H,8H)-dione (21{4,10}). Starting from 20{4}
and aniline to afford 21{4,10} in 83% yield, white solid. IR (KBr),
nmax(cm1): 3423, 3231, 3060, 2931, 1701, 1643, 1627, 1593, 1469,
1235, 1009, 786. 1H NMR (400 MHz, DMSO-d6): d 10.58 (s, 2H,
2xNH), 8.83 (s, 1H, NH), 7.74e7.68 (m, 2H, C15eCHx2), 7.46e7.40
(m, 1H, C14eCH), 7.34e7.27 (m, 2H, C16eCHx2), 7.13 (t, J ¼ 8.8 Hz,
2H, C13eCHx2), 7.03 (t, J ¼ 7.4 Hz, 1H, C17eCH), 4.19 (dd, J ¼ 14.1,
7.7 Hz, 1H, C7eCH), 2.90 (dd, J ¼ 15.7, 7.8 Hz, 1H, C6eCH2),
2.53e2.55 (m, 1H, C6eCH2). 13C NMR (100.6 MHz, DMSO-d6):
d 170.0, 162.6, 160.8 (d, J ¼ 246.7 Hz, C12), 160.7 (d, J ¼ 246.8 Hz,
C12), 155.5, 138.6, 129.7 (t, J ¼ 10.3 Hz, C14), 128.8 (C15), 122.7 (C17),
119.4 (C16), 115.2 (t, J ¼ 18.6 Hz, C11), 113.9, 111.7 (d, J ¼ 22.4, 3.2 Hz,
C13), 90.1 (C5), 36.4 (C7), 23.3 (C6). HRMS (70 eV, EI): m/z calcu-
lated for C19H14N4O2F2: 368.1085, [M]þ, found: 368.1087.
4.1.11.7. 6-(Naphthalen-1-yl)-2-(phenylamino)-5,6-dihydropyrido
[2,3-d]pyrimidine-4,7(3H,8H)-dione (21{8,10}). Starting from 20{8}
and aniline to afford 21{8,10} in 67% yield, white solid. IR (KBr),
nmax(cm1): 3430, 3167, 3054, 1644, 1595, 1579, 1540, 1495, 1235,
777. 1H NMR (400 MHz, DMSO-d6): d 10.64 (s, 1H, NH), 8.86 (s, 1H,
NH), 8.09 (d, J ¼ 7.7 Hz, 1H, C19eCH), 7.98e7.93 (m, 1H, C16eCH),
7.85 (d, J ¼ 8.1 Hz, 1H, C14eCH), 7.74 (d, J ¼ 7.7 Hz, 2H, C21eCHx2),
7.60e7.50 (m, 3H, C17eCH, C18eCH), 7.46 (dd, J ¼ 8.2, 7.1 Hz, 1H,
C13eCH), 7.41e7.29 (m, 3H, C12eCH, C22eCHx2), 7.04 (t, J ¼ 7.3 Hz,
1H, C23eCH), 4.64 (dd, J ¼ 9.1, 7.6 Hz, 1H, C7eCH), 3.00 (dd, J ¼ 16.1,
7.7 Hz, 1H, C6eCH2), 2.85 (dd, J ¼ 16.1, 9.1 Hz, 1H, C6eCH2). 13C NMR
(100.6 MHz, DMSO-d6): d 172.0, 171.0, 162.9, 155.4, 138.7, 135.9,
133.7, 131.0, 128.8 (C22,C16), 127.5 (C14), 126.1, 125.6, 125.4 (C13),
125.1 (C12), 123.9 (C19), 122.6 (C23), 119.4 (C21), 90.3 (C5), 42.5
(C7), 24.8 (C6). HRMS (70 eV, EI): m/z calculated for C23H18N4O2:
382.1430, [M]þ, found: 382.1429.
4.1.12. 6-(4-fluorophenyl)-2-phenoxy-5,6-dihydropyrido[2,3-d]
pyrimidine-4,7(3H,8H)-dione (21{2,14})
Phenol (4 mmol) was heated at 60  C in a mixture of sodium
methoxyde (4 mmol) in 2-propanol (20 mL) for 20 min. 20{2}
(2 mmol) was added to the mixture and it was heated under mi-
crowave irradiation at 170  C for 3 h. The solvent was eliminated
under reduced pressure, water was added to the residue and the
mixture was neutralized with HCl 2 M. The solid was collected by
filtration, washed with cyclohexane and Et2O to afford 21{2,14} in
72% as a white solid. IR (KBr), nmax (cm1): 3408, 3129, 2902, 1692,
1632, 1577, 1513, 1469, 1337, 1220. 1H NMR (400 MHz, DMSO-d6):
d 12.58 (s, 1H, NH), 10.48 (s, 1H, NH), 7.44 (dd, J ¼ 8.6, 7.3 Hz, 2H,
C12eCHx2), 7.28e7.25 (m, 5H, C15-CHx2, C16eCHx2, C17eCH),
7.15 (t, J ¼ 8.9 Hz, 2H, C13eCHx2), 3.84 (dd, J ¼ 10.0, 7.1 Hz, 1H, C7),
2.88 (dd, J ¼ 16.2, 7.1 Hz, 1H, C6eCH), 2.76 (dd, J ¼ 16.2, 10.2 Hz, 1H,
C6eCH). 13C NMR (100.6 MHz, DMSO-d6): d 171.5, 163.2, 161.2 (d,
J ¼ 242.7 Hz, C14), 157.6, 155.6, 151.6, 135.1 (d, J ¼ 3.1 Hz, C11), 130.1
(d, J ¼ 8.9 Hz, C12), 129.8 (C16), 125.8 (C17), 121.6 (C15), 115.1 (d,
J ¼ 21.3 Hz, C13), 93.7 (C5), 44.7 (C7), 24.5 (C6). HRMS (70 eV, EI): m/
z calculated for C19H14N3O3F: 351.1019, [M]þ, found: 351.1033.
4.1.13. 4-((1H-benzo[d] [1,2,3]triazol-1-yl)oxy)-6-(4-fluorophenyl)-
2-(phenylamino)-5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-one
(22{2,10})
Starting from compound 21{2,10} and using procedure
described in 4.1.2. to afford 22{2,10} in 88% yield, white solid. 1H
NMR (400 MHz, DMSO-d6): d 11.19 (s, 1H, NH), 9.46 (s, 1H, NH), 8.23
(d, J ¼ 8.5 Hz, 1H), 7.76 (dt, J ¼ 8.4, 0.9 Hz, 1H), 7.69e7.64 (m, 1H),
7.56 (ddd, J ¼ 8.2, 6.9, 1.1 Hz, 1H), 7.43 (dd, J ¼ 8.7, 5.5 Hz, 2H,
C12eCHx2), 7.23 (t, J ¼ 8.9 Hz, 2H, C13eCHx2), 6.95e6.90 (m, 2H),
6.85e6.79 (m, 2H), 6.75e6.77 (m, 1H, C18eCH), 4.19 (dd, J ¼ 11.0,
7.0 Hz, 1H, C7eCH), 3.41e3.29 (m, 2H, C6eCH2). HRMS (70 eV, EI):
m/z calculated for C25H18N7O2F: 467.1506, [M]þ, found: 467.1508.
4.1.14. 4-((1H-benzo[d] [1,2,3]triazol-1-yl)oxy)-6-(4-fluorophenyl)-
2-phenoxy-5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-one (22
{2,14})
Starting from compound 21{2,14} and using procedure
described in 4.1.2. to afford 22{2,14} in 72% yield, white solid. IR
(KBr), nmax (cm1): 3061, 2977, 2863, 1714, 1635, 1581, 1512, 1412,
1364, 1219, 1099, 746. 1H NMR (400 MHz, DMSO-d6): d 11.50 (s, 1H,
NH), 8.07 (dt, J ¼ 8.4, 0.9 Hz, 1H, C22), 7.70e7.60 (m, 2H, C19eCH,
C20eCH), 7.53e7.49 (m, 1H, C21eCH), 7.41 (dd, J ¼ 8.7, 5.5 Hz, 2H,
C12eCHx2), 7.22 (t, J ¼ 8.9 Hz, 2H, C13eCHx2), 7.11e7.00 (m, 3H,
C18eCH, C17eCHx2), 6.84e6.78 (m, 2H, C16eCHx2), 4.20 (dd,
J ¼ 11.5, 7.1 Hz, 1H, C7eCH), 3.44e3.34 (m, 2H, C6eCH2). 13C NMR
(100.6 MHz, DMSO-d6): d 171.5, 165.9, 162.2, 162.1, 161.4 (d,
J ¼ 243.5 Hz, C14), 151.6, 142.5, 134.3 (d, J ¼ 3.1 Hz, C11), 130.5 (d,
J ¼ 8.2 Hz, C12), 129.1 (C20), 129.1 (C17), 128.3, 125.1 (C21), 125.0
(C18), 120.8 (C16), 119.9 (C22), 115.2 (d, J ¼ 21.3 Hz, C13), 109.2
(C19), 92.9 (C5), 44.3 (C7), 24.0 (C6). HRMS (70 eV, EI): m/z calcu-
lated for C25H17N6O3F: 468.1346, [M]þ, found: 468.1337. Anal. Calcd.
(%) for C25H17FN6O3: C, 64.10; H, 3.66; N, 17.94. Found: C, 64.06; H,
3.62; N, 18.04.
 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
4.1.15. 4-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-6-(2,4-
dichlorophenyl)-2-(phenylamino)-5,6-dihydropyrido[2,3-d]
pyrimidin-7(8H)-one (22{11,10})
Starting from compound 21{11,10} and using procedure
described in 4.1.2. to afford 22{11,10} in 66% yield, white solid. IR
(KBr): n (cm1): 3418, 3274, 3143, 2983, 1687, 1652, 1581, 1555,
1461, 1327, 1252, 1088, 1064, 1043, 901, 814, 741, 603, 507. 1H NMR
(400 MHz, DMSO-d6): d 11.27 (s, 1H,), 9.48 (s, 1H), 8.22 (d, J ¼ 8.4 Hz,
1H), 7.81 (dt, J ¼ 8.4, 0.9 Hz, 1H), 7.70 (d, J ¼ 2.2 Hz, 1H, C13eCH),
7.66 (ddd, J ¼ 8.3, 6.9, 0.9 Hz, 1H), 7.58 (d, J ¼ 8.4 Hz, 1H), 7.56 (ddd,
J ¼ 8.1, 6.9, 1.0 Hz, 1H), 7.51 (dd, J ¼ 8.4, 2.2 Hz, 1H), 7.03e6.78 (m,
4H), 6.78e6.72 (m, 1H, C18eCH), 4.55 (dd, J ¼ 11.6, 9.0 Hz, 1H,
C7eCH), 3.35 (d, J ¼ 8.9 Hz, 2H, C6eCH2). 13C NMR (100.6 MHz,
DMSO-d6): d 170.4, 165.0, 160.3, 157.2, 142.9, 139.3, 135.4, 134.6,
132.8, 132.2, 129.2, 129.0, 128.6, 127.9, 127.6, 125.2, 121.6, 119.9,
118.5, 109.5, 87.9, 43.3.
4.1.16. 6-(4-fluorophenyl)-2-(phenylamino)-4-((2-propoxyethyl)
amino)-5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-one (23{2,10,6})
Starting from 22{2,10} using procedure B described in 4.1.4. and
2-propoxyethylamine to afford 23{2,10,6} in 64% yield, white solid.
1
H NMR (400 MHz, DMSO-d6): d 10.28 (s, 1H, CONH), 8.82 (s, 1H,
NHPh), 7.81 (d, J ¼ 7.7 Hz, 2H, C16eCHx2), 7.32 (dd, J ¼ 8.7, 5.6 Hz,
2H, C12eCHx2), 7.22e7.12 (m, 4H, C17eCHx2, C13eCHx2), 6.85 (t,
J ¼ 7.3 Hz, 1H, C18eCH), 6.70 (s, 1H, C4eNH), 3.92 (dd, J ¼ 10.5,
6.9 Hz, 1H, C7eCH), 3.54e3.51 (m, 4H, C19eCH2, C20eCH2),
3.37e3.29 (m, 2H, C21eCH2), 2.99e2.71 (m, 2H, C6eCH2), 1.48 (p,
J ¼ 7.0 Hz, 2H, C22eCH2), 0.84 (t, J ¼ 7.4 Hz, 3H, CH3). 13C NMR
(100.6 MHz, DMSO-d6): d 171.7, 161.2 (d, J ¼ 242.8 Hz, C14), 160.0,
158.0, 155.5, 141.4, 135.7 (d, J ¼ 3.1 Hz, C11), 130.3 (d, J ¼ 7.9 Hz, C12),
128.2 (C17), 120.3 (C18), 118.4 (C16), 115.0 (d, J ¼ 21.3 Hz, C13), 86.1
(C5), 71.8 (C21), 68.7 (C20), 45.3 (C7), 41.4 (19), 25.6 (C6), 22.5 (C22),
10.5(C23). HRMS (70 eV, EI): m/z calculated for C24H26N5O2F:
435.2071, [M]þ, found: 435.2070.
4.1.17. 6-(4-fluorophenyl)-2-phenoxy-4-((2-propoxyethyl)amino)-
5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-one (23{2,14,6})
Starting from 22{2,14} using procedure B described in 4.1.4. and
2-propoxyethylamine to afford 23{2,14,6} in 54% yield, white solid.
IR (KBr), nmax (cm1): 3466, 3412, 2961, 2333, 2856, 1700, 1623,
1587, 1510, 1407, 1348, 1214. 1H NMR (400 MHz, DMSO-d6): d 8.01 (s,
1H, NH), 7.35 (t, J ¼ 7.7 Hz, 2H, C12eCHx2), 7.23e7.12 (m, 5H,
C16eCHx2, C17eCHx2, C18eCH), 7.01 (t, J ¼ 8.6 Hz, 2H, C13-CHx2),
5.14 (s, 1H, NH), 3.83 (dd, J ¼ 9.7, 7.2 Hz, 1H, C7eCH), 3.61e3.44 (m,
4H, C19eCH2, C20eCH2), 3.35 (t, J ¼ 6.7 Hz, 2H, C21eCH2),
2.94e2.68 (m, 2H, C6eCH2), 1.54 (h, J ¼ 7.1 Hz, 2H, C22eCH2), 0.87
(t, J ¼ 7.4 Hz, 3H, C23eCH2). 13C NMR (100.6 MHz, DMSO-d6):
d 170.8, 163.9, 162.3 (d, J ¼ 246.6 Hz, C14), 161.4, 156.2, 153.1, 133.8
(d, J ¼ 3.3 Hz, C11), 129.8 (d, J ¼ 8.1 Hz, C12) 129.2 (C17), 125.0 (C18),
122.0 (C16), 115.9 (d, J ¼ 21.5 Hz, C13), 88.8 (C5), 72.8 (C21), 68.9
(C20), 45.9 (C7), 41.1 (C19), 26.0 (C6), 22.8 (C22), 10.6 (C23). HRMS
(70 eV, EI): m/z calculated for C24H25N4O3F: 436.1911, [M]þ, found:
436.1913. Anal. Calcd. (%) for C24H25N4O3F: C, 66.04; H, 5.77; N,
12.84. Found: C, 66.09; H, 6.08; N, 12.56.
4.1.18. 6-(2,6-dichlorophenyl)-2-(phenylamino)-4-((2-
propoxyethyl)amino)-5,6-dihydropyrido[2,3-d]pyrimidine-7(8H)-
one (23{5,10,6})
A dispersion of 9{5,10} [13] (200.1 mg, 0.5 mmol) in acetic acid
(10 mL) is treated with sodium nitrite (173 mg, 2.51 mmol) for 2 h at
room temperature. Then, the mixture is diluted with water
(100 mL), filtered and the resulting cake intensively washed with
water. This cake is recovered by solubilization with ethanol, which
can be removed by vacuum distillation. The resulting slurry is
dispersed in 1 M aqueous hydrochloric acid 1 M (30 mL) by
479
ultrasounds and mechanical stirring for 1 h at room temperature
and then heated at 80  C for 1 h. The resulting dispersion is diluted
with water (300 mL), filtered and washed with water, ethanol and
diethyl ether to afford 144 mg (0.36 mmol, yield 72%) of 6-(2,6-
dichlorophenyl)-2-(phenylamino)-5,8-dihydropyrido[2,3-d]pyrim-
idine-4,7(3H,6H)-dione 21{5,10} as a white solid. IR (KBr), nmax
(cm1): 3395, 3222, 3106, 2898, 1732, 1645, 1621, 1598, 1563, 1510,
1497, 1455, 1436, 1367, 1330, 1234, 758. 1H NMR (400 MHz, DMSO-
d6): d 10.55 (br s, 1H), 10.52 (br s, 1H), 8.80 (br s, 1H), 7.72 (dd, J ¼ 8.7,
1.0 Hz, 2H), 7.58e7.46 (m, 2H), 7.37 (t, J ¼ 8.1 Hz, 1H), 7.33e7.23 (m,
2H), 7.03 (tt, J ¼ 7.4, 1.0 Hz, 1H), 4.61 (dd, J ¼ 13.4, 9.0 Hz, 1H), 2.87
(dd, J ¼ 15.9, 9.0 Hz, 1H), 2.74 (dd, J ¼ 15.9, 13.5 Hz, 1H). 13C NMR
(100.6 MHz, DMSO-d6): d 169.7, 161.1, 155.0, 151.0, 138.7, 135.5,
135.2, 134.7, 129.8, 129.8, 128.8, 128.3, 122.5, 119.2, 89.1, 43.2, 22.5.
MS (ESI-TOF): m/z (%) ¼ 401.1 (100) [MþH]þ, 365.1 (3) [M-Cl]þ.
HRMS (ESI-TOF): m/z calculated for C19H15N4O2Cl2: 401.0567
[MþH]þ, found: 401.0563. 401.2 mg (1.0 mmol) of 6-(2,6-
dichlorophenyl)-2-(phenylamino)-5,8-dihydropyrido[2,3-d]pyrim-
idine-4,7(3H,6H)-dione 21{5,10} were suspended in acetonitrile
(75 mL), then BOP (574.6 g, 1.30 mmol) and DBU (225 mL,
1.50 mmol) were added to the solution. The mixture was stirred at
room temperature for 2 days. The solvent was removed in vacuo
and water was added. The resulting solid was filtered and dried
under reduced pressure over phosphorus pentoxide. Without
further purification, 259.2 mg (0.5 mmol) of 22{5,10} and 182 mL
(1.5 mmol) of 2-propoxyethylamine were solved in 10 mL of
acetonitrile and heated at 140  C for 5 h. The solvent was concen-
trated under reduced pressure and water was added to the residue.
The solid was collected by filtration and washed with water and
EtOEt to afford 6-(2,6-dichlorophenyl)-2-(phenylamino)-4-((2-
propoxyethyl)amino)-5,6-dihydropyrido[2,3-d]pyrimidine-7(8H)-
one (23{5,10,6}) in 78% yield as a yellow solid. IR (KBr), nmax (cm1):
3441, 3286, 3207, 3132, 2960, 2923, 2871, 1683, 1637, 1600, 1581,
1553, 1523, 1499, 1441, 1375, 1349, 1289, 1248, 1110, 1087, 985, 829,
781, 754, 693. 1H NMR (400 MHz, DMSO-d6) d 10.31 (br s, 1H), 8.82
(br s, 1H), 7.83 (dd, J ¼ 8.7, 1.0 Hz, 2H), 7.54 (m, 2H), 7.39 (t,
J ¼ 8.1 Hz, 1H), 7.19 (dd, J ¼ 8.5, 7.4 Hz, 2H), 6.85 (br t, J ¼ 7.3 Hz, 1H),
6.60 (s, 1H), 4.68 (dd, J ¼ 13.1, 8.9 Hz, 1H), 3.58e3.48 (m, 4H), 3.35 (t,
J ¼ 6.6 Hz, 2H), 2.99e2.90 (m, 1H), 2.77 (dd, J ¼ 15.7, 13.2 Hz, 1H),
1.49 (hex, J ¼ 7.3 Hz, 2H), 0.84 (t, J ¼ 7.4 Hz, 3H). 13C NMR
(100.6 MHz, DMSO-d6): d 169.2, 160.1, 158.1, 155.1, 141.34, 135.6,
135.2, 134.8, 129.8, 129.7, 128.3, 128.2, 120.3, 118.4, 84.6, 71.7, 68.6,
43.1, 40.3, 23.4, 22.5, 10.5. MS (ESI-TOF): m/z (%) ¼ 486.1 (100)
[MþH]þ, 426.1 (11) [M-OPr]þ HRMS (ESI-TOF): m/z calculated for
C24H26N5O2Cl2: 486.1458, [MþH]þ, found: 486.1456. Anal. Calcd.
(%) for C24H25Cl2N5O2: C, 59.26; H, 5.18; N, 14.40. Found: C, 59.20; H,
5.29; N, 14.48.
4.1.19. Aromatization of pyrido[2,3-d]pyrimidines 21{x,y} or 23
{x,y,z}
4.1.19.1. 6-(4-fluorophenyl)-2-(phenylamino)-4-((2-propoxyethyl)
amino)pyrido[2,3-d]pyrimidin-7(8H)-one (24{2,10,6}). Starting from
23{2,10,6} and following procedure A described in 4.1.6. to afford 24
{2,10,6} in 78% yield, white solid. IR (KBr), nmax (cm1): 3445, 3059,
2928, 1636, 1603, 1570, 1499, 1451, 1310, 1228. 1H NMR (400 MHz,
DMSO-d6): d 11.80 (s, 1H, NH), 9.31 (s, 1H, NH), 8.32 (s, 1H, C6),
7.90e7.85 (m, 2H, C16eCHx2), 7.80 (dd, J ¼ 8.8, 5.7 Hz, 2H,
C12eCHx2), 7.27e7.22 (m, 4H, C13eCHx2, C17-CHx2), 6.96e6.91
(m, 1H, C18), 3.67e3.57 (m, 4H, C19eCH2, C20eCH2), 3.38 (t,
J ¼ 6.7 Hz, 2H, C21eCH2), 1.51 (h, J ¼ 7.2 Hz, 2H, C22eCH2), 0.84 (t,
J ¼ 7.4 Hz, 3H, C23eCH3). 13C NMR (100.6 MHz, DMSO-d6): d 162.4,
161.3 (d, J ¼ 244.6 Hz, C14), 158.9, 157.5, 155.0, 140.6 (C15), 133.0 (d,
J ¼ 7.9 Hz, C11), 132.2 (C6), 130.2 (d, J ¼ 7.7 Hx, C12), 128.3 (C17),
122.8 (C7), 121.4 (C18), 119.4 (C16), 114.6 (d, J ¼ 21.4 Hz, C13), 92.4
(C5), 71.9 (C21), 68.4 (C20), 41.4 (C19), 22.4 (C22), 10.5 (C23). HRMS
480 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
(70 eV, EI): m/z calculated for C24H24N5O2F: 433.1914, [M]þ, found:
433.1927. Anal. Calcd. (%) for C24H24FN5O2: C, 66.50; H, 5.58; N,
16.16. Found: C, 66.53; H, 5.86; N, 16.25.
4.1.19.2. 6-(4-fluorophenyl)-2-phenoxy-4-((2-propoxyethyl)amino)
pyrido[2,3-d]pyrimidin-7(8H)-one (24{2,14,6}). Starting from 23
{2,14,6} and following procedure A described in 4.1.6. to afford 24
{2,14,6} in 75% yield, white solid. IR (KBr), nmax (cm1): 3313, 2933,
2873, 1648, 1612, 1591, 1571, 1510, 1419, 1401, 1264, 1207. 1H NMR
(400 MHz, DMSO-d6): d 9.08 (s, 1H, NH), 7.64e7.58 (m, 3H,
C12eCHx2, C6eCH), 7.43e7.37 (m, 2H, C17eCHx2), 7.25e7.17 (m,
3H, C18eCHx2, C16eCH), 7.07 (t, J ¼ 8.7 Hz, 2H, C13-CHx2), 6.08 (s,
1H, NH), 3.69 (q, J ¼ 5.1 Hz, 2H, C19eCH2), 3.58 (t, J ¼ 5.0 Hz, 2H,
C20eCH2), 3.42 (t, J ¼ 6.7 Hz, 2H, C21eCH2), 1.64e1.58 (m, 2H,
C22.CH2), 0.93 (t, J ¼ 7.4 Hz, 3H, C23eCH3). 13C NMR (100.6 MHz,
DMSO-d6): d 164.8, 162.7 (d, J ¼ 248.1 Hz, C14), 162.4, 160.7, 155.2,
152.8, 131.7 (d, J ¼ 3.3 Hz, C11), 130.6, 130.4 (d, J ¼ 8.1 Hz, C12), 129.3
(C17), 127.9, 125.4 (C18), 122.1 (C16), 115.3 (d, J ¼ 21.4 Hz, C13), 94.5
(C5), 72.9 (C21), 68.7 (C20), 41.3 (C19), 22.7 (C22), 10.6 (C23). HRMS
(70 eV, EI): m/z calculated for C24H23N4O3F: 434.1754, [M]þ, found:
434.1758. Anal. Calcd. (%) for C24H23N4O3F: C, 66.35; H, 5.34; N,
12.90. Found: C, 66.57; H, 5.31; N, 12.51.
4.1.19.3. 6-(4-fluorophenyl)-2-(phenylamino)pyrido[2,3-d]pyrimi-
dine-4,7(3H,8H)-dione (24{2,10}). Starting from 21{2,10} and
following procedure A described in 4.1.6. to afford 24{2,10} in 78%
yield, white solid. IR (KBr), nmax (cm1): 3391, 2924, 2850, 1611,
1554, 1497, 1442, 1226. 1H NMR (400 MHz, DMSO-d6): d 12.18 (s, 1H,
NH), 10.81 (s, 1H, NH), 9.12 (s, 1H, NH), 7.89 (s, 1H, C6eCH),
7.78e7.72 (m, 4H, C12eCHx2, C16eCHx2), 7.35 (dd, J ¼ 8.6, 7.3 Hz,
2H, C17eCHx2), 7.21 (t, J ¼ 8.9 Hz, 2H, C13eCHx2), 7.14e7.08 (m,
1H, C18eCH). 13C NMR (100.6 MHz, DMSO-d6): d 162.4, 161.4 (d,
J ¼ 244.6 Hz, C14), 159.7, 155.1, 151.0, 137.9, 133.5 (C7), 132.6 (d,
J ¼ 3.1 Hz, C11), 130.1 (d, J ¼ 8.1 Hz, C12), 128.9 (C17), 123.7 (C18),
123.5, 120.1 (C16), 114.8 (d, J ¼ 21.2 Hz, C13), 97.2 (C5). HRMS (70 eV,
EI): m/z calculated for C19H13N4O2F: 348.1023, [M]þ, found:
348.1022.
4.1.19.4. 6-(4-fluorophenyl)-2-((4-methoxyphenyl)amino)pyrido[2,3-
d]pyrimidine-4,7(3H,8H)-dione (24{2,13}). Starting from 21{2,13}
and following procedure A described in 4.1.6. to afford 24{2,13} in
70% yield as a white solid.1H NMR (400 MHz, DMSO-d6): d 12.07 (s,
1H, NH), 10.74 (s, 1H, NH), 8.96 (s, 1H, NH), 7.87 (s, 1H, C6eCH), 7.74
(dd, J ¼ 8.9, 5.6 Hz, 2H, C12eCHx2), 7.62 (d, J ¼ 9.0 Hz, 2H,
C16eCHx2), 7.20 (t, J ¼ 8.9 Hz, 2H, C13eCHx2), 6.92 (d, J ¼ 9.0 Hz,
2H, C15eCHx2), 3.76 (s, 3H, OCH3). 13C NMR (100.6 MHz, DMSO-
d6): d 162.5, 160.1, 159.8, 155.8, 155.3, 151.3, 133.9, 133.6 (C6), 132.7
(d, J ¼ 2.9 Hz, C11), 130.7, 130.1 (d, J ¼ 8.0 Hz, C12), 123.3, 122.4
(C16), 114.8 (d, J ¼ 21.2 Hz, C13), 114.1 (C17), 97.0 (C5), 55.3 (C6).
4 .1.19 . 5 . 6 - ( 2 , 6 - d i ch l o ro p h e n yl ) - 2 - ( p h e n yl a m i n o ) - 4 - ( ( 2 -
propoxyethyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one (24{5,10,6}).
Starting from 23{5,10,6} and following procedure A described in
4.1.6. to afford 24{5,10,6} in 65% yield, brownish solid. IR (KBr), nmax
(cm1): 3209, 3060, 2929, 1614, 1596, 1573, 1499, 1450, 1304, 1263,
1191, 1108, 989, 903, 798. 1H NMR (400 MHz, DMSO-d6) d 11.85 (br s,
1H), 9.34 (br s, 1H), 8.09 (s, 1H), 7.91e7.82 (m, 3H), 7.60e7.53 (m,
2H), 7.44 (dd, J ¼ 8.6, 7.6 Hz, 1H), 7.26 (t, J ¼ 7.4 Hz, 2H), 6.95 (t,
J ¼ 7.3 Hz, 1H), 3.67e3.54 (m, 4H), 3.35 (t, J ¼ 6.7 Hz, 2H), 1.49 (h,
J ¼ 7.3 Hz, 2H), 0.83 (t, J ¼ 7.4 Hz, 3H). 13C NMR (100.6 MHz, DMSO-
d6): d 161.1, 159.4, 159.2, 155.9, 149.4, 140.5, 135.4, 135.0, 130.3,
128.3, 128.0, 121.5, 119.5, 109.6, 91.6, 71.8, 68.2, 40.5, 22.4, 10.5. MS
(ESIeTOF): m/z (%) ¼ 484.1 (75) [MþH]þ, 448.1 (100) [M-Cl]þ, 426.1
(54) [MþH-OPr]þ. HRMS (ESI-TOF): m/z calculated for
C24H24N5O2Cl2: 484.1302, [MþH]þ, found: 484.1293. Anal. Calcd.
(%) for C24H23Cl2N5O2: C, 59.51; H, 4.79; N, 14.46. Found: C, 59.85;
H, 5.63; N, 14.26.
4.1.20. General procedure for the alkylation at N8 of pyrido[2,3-d]
pyrimidines 24{x,y,z}
To a solution of (0.7 mmol) of the corresponding pyrido[2,3-d]
pyrimidine 24{x,y,z} in 10 mL of anhydrous DMSO, 28.0 mg
(0.7 mmol) of sodium hydride (NaH) (60% dispersion in mineral oil)
were added and the mixture was stirred for 1 h at room tempera-
ture under nitrogen atmosphere. After this period, (0.7 mmol) of
the corresponding alkyl halide were added dropwise and then
stirred overnight at room temperature. The reaction was quenched
by addition of 300 mL of water and the resulting precipitate was
filtered, washed with water and dried in vacuo over phosphorus
pentoxide to afford the corresponding pyrido[2,3-d]pyrimidine 25
{x,y,z,k}.
4.1.20.1. 4-Amino-6-(2,6-dichlorophenyl)-8-ethyl-2-(phenylamino)
pyrido[2,3-d]pyrimidine-7(8H)-one (25{5,10,4,2}). Starting from 24
{5,10,4} and following the general methodology described above to
afford 25{5,10,4,2} in 88% yield, brownish solid. IR (KBr), nmax
(cm1): 3386, 2974, 2929, 1670, 1614, 1570, 1517, 1497, 1472, 1446,
1401, 1337, 1279, 1238, 1185, 1030, 832, 803, 772, 753. 1H NMR
(400 MHz, DMSO-d6) d 9.41 (br s, 1H), 8.09 (s, 1H), 7.83 (dd, J ¼ 8.7,
1.0 Hz, 2H), 7.60e7.55 (m, 2H), 7.44 (dd, J ¼ 8.7, 7.5 Hz, 1H), 7.38 (br
s, 2H), 7.29 (dd, J ¼ 8.6, 7.4 Hz, 2H), 6.97 (tt, J ¼ 7.3, 1.1 Hz, 1H), 4.35
(q, J ¼ 6.9 Hz, 2H), 1.24 (t, J ¼ 7.0 Hz, 3H). 13C NMR (100.6 MHz,
DMSO-d6) d 161.7, 160.0, 159.2, 155.5, 140.4, 135.3, 135.3, 134.0,
130.2, 128.4, 128.0, 121.6, 120.4, 119.5, 91.6, 36.0, 13.0. MS (70 eV, EI):
m/z (%) ¼ 425.1 (1) [M]þ, 396.1 (1) [M-NEt]þ, 390.1 (2) [M-Cl]þ, 43.1
(100) [NEt]þ. HRMS (70 eV, EI): m/z calculated for C21H17N5OCl2:
425.0810, [M]þ, found: 425.0806.
4.1.20.2. 4-Amino-6-(2,6-dichlorophenyl)-2-(phenylamino)-8-
propylpyrido[2,3-d]pyrimidine-7(8H)-one (25{5,10,4,3}).
Starting from 24{5,10,4} and following the general methodology
described above to afford 25{5,10,4,3} in 86% yield, brownish solid.
IR (KBr), nmax (cm1): 3336, 3141, 2958, 2927, 2872, 1677, 1625,
1574, 1518, 1473, 1447, 1339, 1236, 1185, 1031, 802, 754, 694. 1H NMR
(400 MHz, DMSO-d6) d 9.39 (br s, 1H), 8.09 (s, 1H), 7.82 (dd, J ¼ 8.7,
1.0 Hz, 2H), 7.61e7.54 (m, 2H), 7.43 (dd, J ¼ 8.7, 7.5 Hz, 1H), 7.35 (br s,
2H), 7.31e7.23 (m, 2H), 6.97 (tt, J ¼ 7.3, 1.0 Hz, 1H), 4.31e4.21 (m,
2H), 1.76e1.64 (hex, J ¼ 7.4 Hz, 2H), 0.94 (t, J ¼ 7.5 Hz, 3H). 13C NMR
(100.6 MHz, DMSO-d6) d 161.6, 160.2, 159.1, 155.6, 140.4, 135.4,
135.3, 133.9, 130.2, 128.3, 128.0, 121.7, 120.4, 119.5, 91.6, 42.4, 20.8,
11.3. HRMS (ESI-TOF): m/z calculated for C22H20N5OCl2: 440.1039,
[MþH]þ, found: 440.1046.
4.1.20.3. 4-Amino-8-butyl-6-(2,6-dichlorophenyl)-2-(phenylamino)
pyrido[2,3-d]pyrimidine-7(8H)-one (25{5,10,4,4}). Starting from 24
{5,10,4} and following the general methodology described above to
afford 25{5,10,4,4} in 78% yield, brownish solid. IR (KBr), nmax
(cm1): 3346, 3150, 2959, 2927, 2859, 1626, 1573, 1518, 1497, 1471,
1445, 1339, 1234, 1202, 1031, 803, 772, 755, 692. 1H NMR (400 MHz,
DMSO-d6) d 9.39 (br s, 1H), 8.09 (s, 1H), 7.82 (dd, J ¼ 8.7, 1.1 Hz, 2H),
7.57 (m, 2H), 7.43 (dd, J ¼ 8.7, 7.4 Hz, 1H), 7.34 (br s, 2H), 7.30e7.24
(m, 2H), 6.97 (tt, J ¼ 7.3, 1.1 Hz, 1H), 4.33e4.23 (m, 2H), 1.66 (quint,
J ¼ 8.0 Hz, 2H), 1.39 (hex, J ¼ 7.5 Hz, 2H), 0.93 (t, J ¼ 7.4 Hz, 3H). 13C
NMR (100.6 MHz, DMSO-d6) d 161.7, 160.3, 159.2, 155.7, 140.4, 135.4,
135.4, 134.0, 130.4, 128.5, 128.1, 121.9, 120.6, 119.6, 91.8, 40.9, 29.7,
20.0, 13.9. MS (70 eV, EI): m/z (%) ¼ 453.1 (1) [M]þ, 418.1 (1) [M-Cl]þ,
396.1 (2) [M-But]þ, 57.0 (100) [But]þ. HRMS (70 eV, EI): m/z
calculated for C23H21N5OCl2: 453.1123, [M]þ, found: 453.1123.
 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
4.1.20.4. 4-Amino-6-(2,6-dichlorophenyl)-8-isobutyl-2-(phenyl-
amino)pyrido[2,3-d]pyrimidine-7(8H)-one (25{5,10,4,5}).
Starting from 24{5,10,4} and following the general methodology
described above to afford 25{5,10,4,5} in 83% yield, brownish solid.
IR (KBr), nmax (cm1): 3333, 3199, 2958, 2926, 2869, 1630, 1571,
1522, 1497, 1468, 1446, 1340, 1307, 1234, 1186, 1091, 1027, 799, 778,
753, 691. 1H NMR (400 MHz, DMSO-d6) d 9.38 (br s, 1H), 8.09 (s, 1H),
7.82 (dd, J ¼ 8.7, 1.1 Hz, 2H), 7.60e7.55 (m, 2H), 7.44 (dd, J ¼ 8.7,
7.5 Hz, 1H), 7.34 (br s, 2H), 7.32e7.23 (m, 2H), 6.98 (tt, J ¼ 7.3, 1.1 Hz,
1H), 4.18 (d, J ¼ 7.4 Hz, 2H), 2.29 (hept, J ¼ 7.0 Hz, 1H), 0.90 (d,
J ¼ 6.7 Hz, 6H). 13C NMR (100.6 MHz, DMSO-d6) d 161.7, 160.6, 159.0,
155.9, 140.4, 135.5, 135.4, 133.9, 130.3, 128.4, 128.1, 121.8, 120.6,
119.6, 91.7, 47.4, 26.9, 20.0. MS (70 eV, EI): m/z (%) ¼ 453.1 (1) [M]þ,
418.3 (1) [M-Cl]þ, 396.1 (4) [M-isoBut]þ, 57.0 (100) [isoBut]þ. HRMS
(70 eV, EI): m/z calculated for C23H21N5OCl2: 453.1123, [M]þ, found:
453.1128.
4.1.20.5. 4-Amino-6-(2,6-dichlorophenyl)-8-(2-methoxyethyl)-2-
(phenylamino)pyrido[2,3-d]pyrimidine-7(8H)-one (25{5,10,4,6}).
Starting from 24{5,10,4} and following the general methodology
described above to afford 25{5,10,4,6} in 83% yield, brownish solid.
IR (KBr), nmax (cm1): 3340, 2925, 1632, 1574, 1523, 1497, 1468, 1447,
1341, 1306, 1235, 1196, 1117, 1028, 801, 771, 754, 692. 1H NMR
(400 MHz, DMSO-d6) d 9.42 (br s, 1H), 8.10 (s, 1H), 7.82 (dd, J ¼ 8.7,
1.0 Hz, 2H), 7.61e7.55 (m, 2H), 7.44 (dd, J ¼ 8.7, 7.5 Hz, 1H), 7.40 (br
s, 2H), 7.31e7.25 (m, 2H), 6.97 (tt, J ¼ 7.3, 1.1 Hz, 1H), 4.50 (t,
J ¼ 6.6 Hz, 2H), 3.61 (t, J ¼ 6.6 Hz, 2H), 3.27 (s, 3H). 13C NMR
(100.6 MHz, DMSO-d6) d 161.8, 160.3, 159.1, 155.9, 140.3, 135.4,
135.3, 134.3, 130.4, 128.5, 128.1, 121.9, 120.3, 119.7, 91.7, 68.5, 58.2,
40.2. MS (ESI-TOF): m/z (%) ¼ 456.1 (51) [MþH]þ, 424.1 (100) [M-
OMe]þ. HRMS (ESI-TOF): m/z calculated for C22H20N5O2Cl2:
456.0989, [MþH]þ, found: 456.0988.
4.1.20.6. 4-Amino-6-(2,6-dichlorophenyl)-8-(2-hydroxyethyl)-2-
(phenylamino)pyrido[2,3-d]pyrimidine-7(8H)-one (25{5,10,4,7}).
Starting from 24{5,10,4} and following the general methodology
described above with an extra purification consisting of a chro-
matographic separation in silica and CH2Cl2/MeOH as solvent to
afford 25{5,10,4,7} in 34% yield, brownish solid. IR (KBr), nmax
(cm1): 3418, 2924, 1634, 1599, 1568, 1525, 1497, 1469, 1447, 1341,
1306, 1235, 1201, 1057, 1015, 802, 772, 691. 1H NMR (400 MHz,
DMSO-d6) d 9.40 (br s, 1H), 8.09 (s, 1H), 7.84 (dd, J ¼ 8.7, 1.0 Hz, 2H),
7.60e7.55 (m, 2H), 7.44 (dd, J ¼ 8.7, 7.5 Hz, 1H), 7.38 (br s, 2H), 7.28
(m, 2H), 6.96 (tt, J ¼ 7.3, 1.1 Hz, 1H), 4.82 (br t, J ¼ 5.3 Hz, 1H), 4.42 (t,
J ¼ 7.1 Hz, 2H), 3.64 (q, J ¼ 6.5 Hz, 2H). 13C NMR (100.6 MHz, DMSO-
d6) d 161.8, 160.4, 159.2, 156.0, 140.4, 135.4, 135.3, 134.2, 130.4, 128.6,
128.1, 121.8, 120.4, 119.6, 91.7, 58.0, 42.8. MS (ESI-TOF): m/z
(%) ¼ 442.1 (100) [MþH]þ, 424.1 (81) [M-OH]þ. HRMS (ESI-TOF): m/
z calculated for C21H18N5O2Cl2: 442.0832, [MþH]þ, found:
442.0839.
4.1.20.7. 6-(2,6-dichlorophenyl)-8-methyl-2-(phenylamino)-4-((2-
propoxyethyl)amino)pyrido[2,3-d]pyrimidine-7(8H)-one (25
{5,10,6,1}). Starting from 24{5,10,6} and following the general
methodology described above to afford 25{5,10,6,1} in 96% yield,
brownish solid. IR (KBr), nmax (cm1): 3338, 2930, 2872, 1642, 1577,
1519, 1498, 1474, 1446, 1341, 1316, 1229, 1184, 1103, 799, 753, 692. 1H
NMR (400 MHz, DMSO-d6) d 9.52 (br s, 1H), 8.14 (s, 1H), 7.93 (br s,
1H), 7.83 (d, J ¼ 7.6 Hz, 2H), 7.58 (m, 2H), 7.44 (dd, J ¼ 8.7, 7.5 Hz,
1H), 7.30 (t, J ¼ 7.4 Hz, 2H), 6.98 (t, J ¼ 7.3 Hz, 1H), 3.70e3.54 (m,
7H), 3.37 (t, J ¼ 6.6 Hz, 2H), 1.49 (hex, J ¼ 7.4 Hz, 2H), 0.83 (t,
J ¼ 7.4 Hz, 3H). 13C NMR (100.6 MHz, DMSO-d6) d 160.8, 160.3, 159.2,
156.2, 140.7, 135.8, 135.8, 133.8, 130.7, 128.9, 128.5, 122.2, 120.4,
120.0, 92.5, 72.2, 68.6, 41.1, 28.9, 22.9, 10.9. Anal. Calcd. (%) for
C25H25Cl2N5O2: C, 60.25; H, 5.06; N, 14.05. Found: C, 60.50; H, 4.84;
481
N, 13.79. MS (ESI-TOF): m/z (%) ¼ 498.1 (100) [MþH]þ, 438.1 (13)
[M-OC3H7]þ. HRMS (ESI-TOF): m/z calculated for C25H26N5O2Cl2:
498.1458, [MþH]þ, found: 498.1460.
4.2. Biology
4.2.1. Cell lines and cell culture
To analyze the antiviral activity of the compounds, we used a
stable cell line, LucUbiNeo-ET, that contains a subgenomic HCV
genotype 1b replicon, I389/NS3-3’/LucUbiNeo-ET, that express
luciferase constitutively [21,22]. To evaluate the cellular toxicity, we
use other cell line, Huh-7/Lunet [23], that originally carried a
selectable luciferase HCV 1b replicon and was cured by treatment
with an HCV-specific inhibitor. These cells are characterized by
high permissiveness for HCV RNA replication. LucUbiNeo-ET cell
line was cultured at 37  C in a humidified atmosphere with 5% CO2
in Dulbecco's modified Eagle medium (D-MEM) supplemented
with high glucose concentration (4.5 g/l) (Invitrogen), 10% (vol/vol)
heat-inactivated fetal bovine serum (Hyclone, Cultek), 2 mM L-
Glutamine (SigmaeAldrich), 1 mM sodium pyruvate (Sigma-
aldrich), 1% MEM NEEA (SigmaeAldrich), and 0.5 mg/mL of
Geneticin (G418) (Gibco, Invitrogen). Cured or Huh-7/Lunet cell line
was maintained at the same conditions as described above without
the addition of Geneticin.
4.2.2. Evaluation of the in vitro antiviral activity (EC50) and
cytotoxicity assays (CC50)
Antiviral activity was carried out in white-clear bottom 96-well
plates, and cytotoxicity assays were performed in clear 96-well
plates. Cells were seeded at a density of 104/well in 100ul D-MEM
without selection of antibiotics. After 24 h, nine fivefold serial
dilution of compounds, with 3 replicates at each dilution, were
prepared in D-MEM and added to the appropriate wells, yielding
concentrations of 125 to 0,00032 ug/ml in a final volume of 100 ul
of D-MEM. Following 2 days of incubation, we determined the
antiviral activity (EC50) by quantifying the luciferase with the
Luciferase Bright Glo assay (Promega). Briefly, LucUbiNeo-ET cells
were harvested with the addition of 100 ul of luciferase per well,
and after 2 min of shaking, the light was read in a Luminometer
Fluoroskan Ascent FL. Toxicity (CC50) was analyzed in the Lunet/
HuH7 cells by the addition of 10 ul of MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) per well. After 4 h of in-
cubation at 37  C in a humidified atmosphere with 5% CO2, the
amount of formazan produced was quantified spectrophotometri-
cally at 550/620 nm [43]. The EC50 and CC50 values were calculated
for each compound. These assays were done in duplicate and the
results shown in Table 1 represent the mean value of these
duplicates.
4.3. Computational studies
Chemical Similarity Searching. Chemical similarity was per-
formed using Omega 2.3.2 [44] and ROCS 3.0 (Rapid Overlay of
Chemical Structures) [45] software from the OpenEye Scientific
Software. Using the RCSB Protein Data Bank, NS5B X-ray structures
cocrystallized with a ligand were retrieved and classified according
to the binding site (sites I to IV). For each ligand, 100 conformations
were generated using Omega and compared to our in-house com-
pound database. Three-dimensional shape comparisons (both
shape Tanimoto coefficients and color scores) were performed with
ROCS. The alignment overlap was calculated using the color force
field implicit Mills-Dean [46]. The Tanimoto Combo score corre-
sponds to the sum of the shape Tanimoto coefficient and the color
score. Molecules with a Tanimoto Combo score higher than 0.7
were considered as similar while scores higher than 0.95 were
482 M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483
considered as highly similar.
Cross docking methodology. Complexes PDB ID 2WCX [31],
2WHO [30], 3BSC [36], 3H98 [29], 4KE5 [37] and 4NLD [32] were
selected based on the previous chemical similarity results and used
as target structures for our modeling studies. Water molecules
were deleted and the protein structures were prepared with
Schro€dinger Protein Preparation Wizard [47]. This workflow en-
sures chemical corrected and optimized protein structures for
further analysis. Hydrogen atom were added and bond order and
charges were assigned. A small number of minimization steps were
applied in order to relieve steric clashes using the OPLS 2005 force
field.
The ligand database used during the cross docking protocol
consist of our in house database as well as the ligands cocrystallized
in the aforementioned NS5B complexes. Compounds were sub-
mitted to the LigPrep utility which takes into account ionization
states, tautomers, stereochemistries and ring conformations at a pH
range close to 7.
Cross docking methodology was performed using Glide [48]
from the Schro €dinger Suite. All docking calculations were run in
extra precision mode and the grid was centered on the crystallo-
graphic position of the ligand. Further docking refinement was
achieved by an induced fit (IF) protocol and ligands were ranked
according to the IF docking score. All figures were prepared using
PyMOL [49].
Acknowledgment
Financial support by the Spanish Ministerio de Economia y
Competividad project SAF2010-C21617-C02 is greatly acknowl-
edged. M. Camarasa wants to thank IQS for a scholarship. R. Puig de
la Bellacasa wants to thank Generalitat de Catalunya for a grant
within the Talent empresa (TEM) 2009 program (2009 TEM 00128).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.03.055.
Taken together, comparison of crystallographic data with our
best candidates 21{4,10} and 24{2,10} suggest that substituted
pyrido[2,3-d]pyrimidin-7-(8H)-ones are a feasible target for NS5B
and can potentially disrupt the RNA processing channel.
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