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Design, Synthesis and Biological Evaluation of Pyrido (2,3-d) Pyrimidin-7 - (8H) - Ones PDF
Design, Synthesis and Biological Evaluation of Pyrido (2,3-d) Pyrimidin-7 - (8H) - Ones PDF
Design, Synthesis and Biological Evaluation of Pyrido (2,3-d) Pyrimidin-7 - (8H) - Ones PDF
Research paper
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 genotypes (1e7) with several subtypes within each genotype [2],
which differ by 30e35% of the nucleotide sites over the complete
Hepatitis C is an infectious disease caused by the hepatitis C genome. For instance, subtypes 1a and 1b are found worldwide and
virus (HCV), which constitutes the leading cause of chronic liver cause 60% of hepatitis C cases.
diseases. HCV has infected an estimated 150e200 million people HCV encodes structural (E1, E2, C and p7) and nonstructural
being an important health care problem worldwide [1]. The virus proteins (NS2, NS3/4A, NS4B, NS5A, NS5B) [3]. Nonstructural pro-
persists in the liver in about 85% of those infected. teins represent the main targets for intensive anti-HCV drug dis-
HCV belongs to the genus Hepacivirus, a member of the family covery programs.
Flaviviridae. HCV is a positive-sense single-stranded RNA virus of Before the development of direct acting antivirals (DAAs),
approximately 9.6 Kb. Based on genetic differences between HCV treatment for hepatitis C was based on a combination of (pegy-
isolates, the hepatitis C virus species is classified into seven 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
Abbreviations: BOP, (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate; m-CPBA, 3-Chloroperbenzoic acid; DBU, 1,8-Diazabicyclo 80% of those infected by genotypes 2 and 3. Besides the limited
[5.4.0]undec-7-ene; DDQ, 2,3-Dichloro-5,6-dicyano-p-benzoquinone; PSII, Palm efficacy on HCV type 1, this combination therapy has significant
site II of NS5B polymerase; PyBOP, (Benzotriazol-1-yloxy)tripyrrolidinophospho- side effects and is poorly tolerated in many patients. The most
nium hexafluorophosphate.
common side effects that may occur after treatment with PEG-
* Corresponding author.
E-mail addresses: martacamarasan@gmail.com (M. Camarasa), raimonpc@gmail.
IFN-a are: nausea, diarrhea, fever, chills, muscle and joint pain,
com (R. Puig de la Bellacasa), alejandro.lopez@iqs.url.edu (A.L. Gonza lez), difficulty concentrating, thyroid disorders, hair loss, insomnia, ir-
raulondonom@iqs.url.edu (R. Ondon ~ o), roger.estrada@iqs.url.edu (R. Estrada), ritability, mild to severe depression, and rarely, suicidal thoughts.
sfranco@irsicaixa.es (S. Franco), rbadia@irsicaixa.es (R. Badia), jaeste@irsicaixa.es Other serious side effects that may occur are bone marrow
(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).
toxicity, cardiovascular disorders, hypersensitivity disorders,
(J. Teixido
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
Scheme 1. Synthesis of 7-oxopyrido[2,3-d]pyrimidines 9, 10, and 11 from a,b-unsaturated esters 5 via pyridones 7.
M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483 465
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. derivatives 20{x} in 60e74% yield. Using such protocol we obtained
We studied several methodologies for the dehydrogenation of compounds 20{1}, 20{2}, 20{4}, 20{5}, 20{8}, and 20{11}.
5,6-dihydropyrido[2,3-d]pyrimidine-7(8H)-ones to the corre- The subsequent treatment of compounds 20{x} with the corre-
sponding aromatic compounds [12], however we have not found a sponding anilines afforded compounds 21{1,10}, 21{2,10}-21{2,13},
general procedure and the reaction conditions and the oxidizing 21{4,10}, and 21{8,10} in 67e94% yield.
agent strongly depend on the structure of the starting pyr- Similarly, the treatment of 20{2} with sodium phenoxide in 2-
idopyrimidine. In this work we used three different reaction propanol afforded the 2-phenoxy substituted pyridopyrimidine
conditions: (a) NaH in DMSO at 100 C [12]; (b) DDQ in 1,2- 21{2,14} in 72% yield.
dichloroethane at 100 C; (c) activated MnO2 in AcOH at reflux. Once obtained the 2-substituted pyridopyrimidines 21{x,y}, we
Using the adequate protocol we obtained compounds 16{2}, 16 used the same protocol described above for the introduction of a
{2,5}, 16{2,6}, 16{2,7}, 16{4,6}, 16{5,9}, 16{5,5}, and 16{5,6} in second substituent at C4. Thus, we treated compounds 21{x,y} with
50e90% yield. BOP and DBU in anhydrous acetonitrile to afford the corresponding
The aromatic compounds 16{x,y} were finally methylated on the 4-benzotriazol-1-yloxy substituted intermediate 22{x,y}. In that
lactam nitrogen using MeI in NaH/DMSO at rt to afford the methyl way we obtained 22{2,10}, 22{2,14}, and 22{11,10} in 66e88% yield.
derivatives 17{x,y}. Thus we obtained compounds 17{2,1}, 17{2,5}, Subsequently, we substituted the 4-benzotriazol-1-yloxy group
17{2,6}, 17{2,7}, and 17{5,3} in 26e89% yield. of compounds 22{x,y} by amines also following the protocol pre-
On the other hand, the synthesis of pyridopyrimidines 23{x,y,z} viously described to afford pyridopyrimidines 23{x,y,z} bearing
with a double complex substitution at C2 and C4 of the pyrimidine substituents at C2 and C4 (23{2,10,6} and 23{2,14,6} were obtained
ring also needed the development of a new synthetic strategy in 64% and 54% yield, respectively).
(Scheme 2). Thus, the reaction between 2-aryl-substituted acry- Then, compounds 23{x,y,z} were aromatized to the corre-
lates 5{1e10} (Fig. 2) and 6-amino-2-(methylthio)pyrimidin-4(3H)- sponding 24{x,y,z} using the dehydrogenation protocols described
one (18) with K2CO3 as base in 2-propanol at 170 C for 3 h under above. Thus, 24{2,10,6} and 24{2,14,6} were obtained in 78% and
microwave irradiation yielded the corresponding 2-(methylthio)- 75% yield, respectively.
6-aryl-5,6-dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-diones (19 To complete the sublibrary, we used the protocol for the syn-
{1e5} and 19{7e11}) in 56e88% yield (Scheme 2) [14]. thesis of 4-amino-2-arylamino substituted pyridopyrimidines 9
The direct substitution of the methylthio group of compounds described in Scheme 1 [13] to obtain compounds 9{1}, 9{4}, 9{5}, 9
19{x} was possible with nucleophilic amines, such as benzylamine {6}, and 9{7} in 32e74% yield from pyridines 7{x} via intermediates
which afforded in 2-propanol under microwave irradiation the 10{x} [13,19], which were subsequently dehydrogenated to 11{1}, 11
corresponding 2-benzylamino derivatives 21{2,8} and 21{5,8} in {4}, 11{5}, 11{6}, and 11{7} in 59e95% yield [19].
53% and 36% respectively from 19{2} and 19{8}, respectively. These later compounds were alkylated on the lactam nitrogen
Similarly, the 2-(4-fluorobenzyl) amino derivative 21{2,9} in 40% with different C1eC6 linear and branched alkyl chains to afford
yield. compounds 25{x,y,z,k}. In that way we obtained compounds 25
However, our main interest was the introduction of arylamino {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}
substituents at position C2 of the pyrimidine ring. Consequently, [19], 25{1,10,4,1} [19], 25{5,10,4,6}, 25{5,10,4,3}, 25{5,10,4,7}, 25
we decided to convert the methylthio group present in compounds {7,10,4,1} [19], 25{5,10,4,2}, and 25{5,10,4,4} included in Table 1,
19{x} into a methylsulfonyl group to favor the nucleophilic among others.
displacement. According to the literature we tested several Finally, taking into account that all the members of this sub-
oxidizing agents (H2O2/AcOH, H2O2/NbC/EtOH [18], H2O2/EtOH, library present a substituent at position C4 of the pyrimidine ring
carbamide peroxide/MeOH, oxone® (potassium perox- (oxo, amino, alkylamino, alkoxy), we decided to include three
ymonosulfate)/MeOH/H2O) but the conversion was never com- unsubstituted compounds for comparison purposes. Thus, starting
plete, affording mixtures of the desired sulfone 20{x} and the from compound 26 [20] we obtained 27 and 28 in 87 and 96% yield,
corresponding sulfoxide. respectively (Fig. 3).
Finally, the treatment of compounds 19{x} with m-CPBA in DMF
at 0 C afforded the corresponding methylsulfonyl substituted
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)
Table 1 (continued )
Entry Code Structure EC50 (mg/ml) EC50 (mM) CC50 (mM) CC50 (mg/ml)
Table 1 (continued )
Entry Code Structure EC50 (mg/ml) EC50 (mM) CC50 (mM) CC50 (mg/ml)
[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-
Fig. 3. 4-Unsubstituted pyrido-[2,3-d]pyrimidines included in the sublibrary tested tential drugs, are 196 and 397.
against HCV.
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
2.3. Biological evaluation of the HCV antiviral activity used as internal controls and, more importantly, are of the same
order of magnitude of the EC50 of sofosbuvir in a similar HCV 1b
Once the library of pyrido[2,3-d]pyrimidines was synthesized, replicon test (0.048 mM).
we evaluated the HCV antiviral activity and the toxicity of such In order to try to locate the target of our antiviral compounds,
compounds. Consequently, we determined the EC50 and the CC50. we carried out an HCV NS5B drug resistance assay without
For testing the EC50 (mg/mL), we used a stable cell line, concluding results. This experiment consisted of trying to isolate
LucUbiNeo-ET, that contains a subgenomic HCV genotype 1b replicons resistant to our most potent compounds propagating the
replicon, I389/NS3-3’/LucUbiNeo-ET, that express luciferase replicon at concentrations equivalent to the EC90. We obtained
constitutively [21,22]. resistant populations (EC50 between 32.7 and 63.7 mg/mL) but
To evaluate the cellular toxicity (CC50, mg/mL), we used another when we sequenced them, we did not found mutations clearly
cell line, Huh-7/Lunet [23], that originally carried a selectable associated with non-nucleotide inhibitors of the NS5B polymerase.
luciferase HCV 1b replicon and was cured by treatment with an
HCV-specific inhibitor.
2.4. Molecular docking study of 21{4,10} and 24{2,10} on NS5B
As positive controls for these assays we used two commercially
polymerase
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
As mentioned above, we hypothesized that NS5B polymerase
(CC50 > 2.0 mM), respectively [24].
should be a feasible target for substituted pyrido[2,3-d]pyrimidin-
A total of 121 compounds were evaluated, including those
7-(8H)-ones due to the high similarity scores of these molecules
described in the present paper and pyrido[2,3-d]pyrimidines
with the available NS5B-ligand X-ray complexes. NS5B is one of the
coming from other research programs within our group (such as 29
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
Fig. 4. Commercially available HCV NS3 protease inhibitors Inh I and Inh II used as thumb domain (TSI and TSII respectively), while sites III and IV can
positive controls for the assays. 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
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.
M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483 471
4. Experimental
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
Fig. 6. (A) Superposition of 24{2,10} (orange) and 21{4,10} (green) in the PSII binding
400 W. Reactions were carried out in 0.5, 2.5, 5, and 20 mL glass
site. (B) Representation of a potential interaction of 21{4,10} with RNA and a cocrys-
tallized sofosbuvir diphosphate unit. 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
materials.
rapidly to 50 C by air jet cooling.
Although it has not been possible yet to stablish the molecular
target for these compounds, chemoinformatic studies suggest a
4.1.2. General procedure for the synthesis of 4-((1H-benzo[d][1,2,3]
high degree of similarity between 21{4,10} and 24{2,10} and PSII
triazol-1-yl)oxy)-6-aryl-3,4,5,6-tetrahydropyrido[2,3-d]pyrimidine-
inhibitors of NS5B polymerase. Further cross docking analysis
7(8H)-ones (14{x})
shows an excellent binding of our compounds with the hydro-
The corresponding 2-amino-6-aryl-5,6-dihydropyrido[2,3-d]
phobic pocket of the palm cleft, as it has been described in other
pyrimidine-4,7(3H,8H)-diones (13{x}) (0.50 mmol) [14] was sus-
structural studies with known PSII inhibitors. An optimal cation-p
pended in 13 mL of anhydrous acetonitrile. Then, BOP (0.75 mmol)
stacking of the pyrido[2,3-d]pyrimidine core of 21{4,10} and 24
and DBU (1.25 mmol) were added into the solution. The system was
{2,10}, combined with the optimal positioning of the 2,6-
stirred at room temperature for 2 days under nitrogen atmosphere.
difluorophenyl and the p-fluorophenyl substituents respectively,
After this period, 200 mL of water were added and the resulting
seems to be compatible with the interaction of these compounds
precipitate was filtered, washed with water and dried in vacuo over
with RNA riboses of the primer strand which could disrupt poly-
phosphorus pentoxide to afford the corresponding 14{x} that can
merization, in good agreement with the observed biological
be used without further purification.
activity.
Taken together, these results point out to achiral compound 24
4.1.2.1. 4-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-2-amino-6-(4-
{2,10} as a promising candidate to be further developed as drug for
fluorophenyl)-3,4,5,6-tetrahydropyrido[2,3-d]pyrimidine-7(8H)-one
the treatment of hepatitis C. Further studies should be conducted to
(14{2}). Starting from compound 13{2} to afford 14{2} in 74% yield,
validate such hypothesis.
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, 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 -
J ¼ 8.9 Hz, 1H), 6.59 (s, 2H, NH2), 4.11 (dd, J ¼ 10.7, 6.9 Hz, 1H), dihydropyrido[2,3-d]pyrimidine-7(8H)-one (15{5,1}). Starting from
3.29e3.12 (m, 2H). 13C NMR (100.6 MHz, DMSO-d6): d 172.4, 166.0, compound 14{5} and methanol to afford 15{5,1} in 96% yield, white
161.8, 161.0, 143.2, 135.4, 130.8 (d, J ¼ 7.9 Hz, C12), 129.5 (C17), 128.9, solid. IR (KBr), nmax (cm1): 3438, 3330, 3222, 2951, 2891, 1688,
125.6 (C18), 120.3 (C19), 115.6 (d, J ¼ 21.1 Hz, C13), 110.0, 109.8 (C16), 1634, 1577, 1488, 1466, 1371, 1257, 1196, 774. 1H NMR (400 MHz,
86.0 (C5), 45.4 (C7), 24.5 (C6). 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,
4.1.2.2. 4-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-2-amino-6-(2,6-
130.2, 129.4, 128.4, 90.0, 55.7, 42.6, 21.0. MS (70 eV, EI): m/z
difluorophenyl)-5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-one (14
(%) ¼ 338.1 (60) [M]þ, 303.1 (100) [M-Cl]þ, 153.0 (67) [M-
{4}). Starting from compound 13{4} to afford 14{4} in 93% yield,
C6H9N4O]þ. HRMS (70 eV, EI): m/z calculated for C14H12N4O2Cl2:
white solid. IR (KBr), nmax (cm1): 3374, 3207, 2895, 1698, 1651,
338.0340, [M]þ, found: 338.0337. Anal. Calcd. (%) for
1571, 1470, 1371, 1273, 1247, 1008, 785. 1H NMR (400 MHz, DMSO-
C14H12Cl2N4O2: C, 49.58; H, 3.57; N, 16.52. Found: C, 49.41; H, 3.76;
d6): d 11.03 (s, 1H, NH), 8.13 (dt, J ¼ 8.4, 0.9 Hz, 1H, C15eCH), 7.80
N, 16.89.
(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,
4.1.3.3. 2-Amino-4-ethoxy-6-(4-fluorophenyl)-5,6-dihydropyrido
1H, C14eCH), 7.16 (t, J ¼ 8.7 Hz, 2H, C13-CHx2), 6.63 (s, 2H, NH2),
[2,3-d]pyrimidin-7(8H)-one (15{2,2}). Starting from compound 14
4.51 (dd, J ¼ 13.9, 7.5 Hz, 1H, C7eCH), 3.31e3.25 (m, 1H, C6eCH),
{2} and ethanol to afford 15{2,2} in 20% yield, white solid. IR (KBr),
3.10e3.03 (m, 1H, C6eCH). 13C NMR (100.6 MHz, DMSO-d6): d 170.0,
nmax (cm1): 3408, 3338, 3220, 1686, 1632, 1575, 1511, 1434, 1227. 1H
165.5, 161.4, 160.9 (d, J ¼ 247.2 Hz, C12), 160.8 (d, J ¼ 246.8 Hz, C12),
NMR (400 MHz, DMSO-d6): d 10.45 (s, 1H, NH), 7.27 (dd, J ¼ 8.8,
160.4, 142.7, 129.9 (t, J ¼ 10.6 Hz, C14), 128.9 (C16), 128.5, 125.1
5.6 Hz, 2H, C12-CHx2), 7.14 (t, J ¼ 8.8 Hz, 2H, C13-CHx2), 6.35 (s, 2H,
(C17), 119.8 (C15), 114.7 (t, J ¼ 18.5 Hz, C11), 111.8 (dd, J ¼ 22.3,
NH2), 4.26 (qd, J ¼ 7.2, 1.3 Hz, 2H, C15eCH2), 3.86 (dd, J ¼ 10.4,
2.5 Hz, C13), 109.6 (C18), 85.1 (C5), 36.1 (C7), 22.4 (C6). Anal. Calcd.
7.2 Hz, 1H, C7eCH), 2.93e2.71 (m, 2H, C6eCH2), 1.25 (t, J ¼ 7.0 Hz,
(%) for C19H13F2N7O2: C, 55.75; H, 3.20; N, 23.95. Found: C, 56.06; H,
3H, C16eCH3). 13C NMR (100.6 MHz, DMSO-d6): d 172.1, 166.4,
3.22; N, 23.78.
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
4.1.2.3. 4-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-2-amino-6-(2,6- (C16). MS (70 eV, EI): m/z (%) ¼ 302.1 (100) [M]þ, 287.1 (25) [M-
dichloro phenyl)-3,4,5,6-tetrahydropyrido[2,3-d]pyrimidine-7(8H)- CH3]þ, 273.1 (42) [M-C2H5]þ, 258.1 (19) [M-OC2H5]þ. HRMS (70 eV,
one (14{5}). Starting from compound 13{5} to afford 14{5} in 78% EI): m/z calculated for C15H15N4O2F: 302.1183, [M]þ, found:
yield, white solid. IR (KBr): n (cm1): 3382, 3324, 3215, 2898, 1670, 302.1179.
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, 4.1.3.4. 2-Amino-6-(2,6-dichlorophenyl)-4-ethoxy-5,6-dihydropyrido
J ¼ 8.4, 1.0 Hz, 1H), 7.68e7.61 (m, 1H), 7.60e7.46 (m, 3H), 7.39 (t, [2,3-d]pyrimidin-7(8H)-one (15{5,2}). Starting from compound 14
J ¼ 8.1 Hz, 1H), 6.63 (s, 2H, NH2), 4.91 (dd, J ¼ 12.6, 9.3 Hz, 1H), {5} and ethanol to afford 15{5,2} in 31% yield, white solid. IR (KBr),
3.31e3.16 (m, 2H). 13C NMR (100.6 MHz, DMSO-d6): 170.14, 166.17, nmax (cm1): 3409, 3329, 3220, 1686, 1633, 1577, 1435, 1373, 778. 1H
161.79, 160.48, 143.17, 135.79, 135.38, 135.23, 130.39, 130.20, 129.42, NMR (400 MHz, DMSO-d6): d 10.47 (s, 1H, NH), 7.54e7.48 (m, 2H,
128.94, 128.86, 125.58, 120.22, 110.05, 84.62, 43.20, 21.98. HRMS C13-CHx2), 7.36 (t, J ¼ 8.1 Hz, 1H, C13eCH), 6.38 (s, 2H, NH2), 4.62
(70 eV, EI): m/z calculated for C19H13N7O2Cl2: 441.0508, [M]þ, (dd, J ¼ 12.8, 9.3 Hz, 1H, C7eCH), 4.26 (qd, J ¼ 7.1, 2.1 Hz, 2H,
found: 441.0507. 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
4.1.3. General procedure for the synthesis of 4-alkoxy-substituted
(C7), 22.2 (C6), 14.5 (C16). HRMS (70 eV, EI): m/z calculated for
pyrido[2,3-d]pyrimidines 15{x,y}
C15H14N4O2Cl2: 352.0494, [M]þ, found: 352.0494.
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)-
4.1.3.5. 2-Amino-6-(2,6-dichlorophenyl)-4-(2-methoxyethoxy)-5,6-
one (14{x}) (0.42 mmol), Cs2CO3 (0.54 mmol) and the corre-
dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{5,3}). Starting from
sponding alcohol (9 mL) was heated a reflux overnight. The
compound 14{5} and 2-methoxyethanol to afford 15{5,3} in 76%
resulting solution was cooled down, water (100 mL) was added and
yield, white solid. IR (KBr), nmax (cm1): 3404, 3218, 2894, 1688,
the reaction crude was neutralized with 2 M HCl. The resulting solid
1633, 1576, 1434, 1372, 775. 1H NMR (400 MHz, DMSO-d6): d 10.54
was collected by filtration and washed with water, and EtOEt to
(s, 1H, NH), 7.55e7.48 (m, 2H, C-13-CHx2), 7.37 (t, J ¼ 8.1 Hz, 1H,
afford the corresponding 15{x,y}.
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
4.1.3.1. 2-Amino-6-(4-fluorophenyl)-4-methoxy-5,6-dihydropyrido (s, 3H, OCH3), 2.93e2.79 (m, 2H, C6eCH2). 13C NMR (100.6 MHz,
[2,3-d]pyrimidin-7(8H)-one (15{2,1}). Starting from compound 14 DMSO-d6): d 169.8, 166.4, 161.6, 157.9, 135.3, 135.2, 134.7, 129.8
{2} and methanol to afford 15{2,1} in 39% yield, white solid. IR (KBr), (C14), 129.7 (C13), 128.3 (C13), 85.2 (C5), 70.1 (C16), 64.8 (C15), 58.2
nmax (cm1): 3381, 3227, 1686, 1658, 1633, 1575, 1511, 1464, 1384, (OCH3), 43.1 (C7), 22.2 (C6). MS (70 eV, EI): m/z (%) ¼ 382.0 (15)
1247. 1H NMR (400 MHz, DMSO-d6): d 10.49 (s, 1H, NH), 7.26 (dd, [M]þ, 351.0 (23) [M-CH3O]þ, 322.9 (33) [M-C3H6O]þ, 289.0 (100)
J ¼ 8.7, 5.7 Hz, 2H, C12-CHx2), 7.14 (t, J ¼ 8.9 Hz, 2H, C13-CHx2), 6.40 [M-C3H6OCl]þ, 205.9 (26) [C9H10N4O2]þ. HRMS (70 eV, EI): m/z
(s, 2H, NH2), 3.86 (dd, J ¼ 9.9, 6.9 Hz, 1H, C7eCH), 3.80 (s, 3H; OCH3), calculated for C16H16N4O3F4Cl2: 382.0598, [M]þ, found: 382.0599.
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, 4.1.3.6. 2-Amino-6-(2,6-dichlorophenyl)-4-(2-propoxyethoxy)-5,6-
J ¼ 8.1 Hz, C12), 115.0 (d, J ¼ 21.3 Hz, C13), 86.5 (C5), 53.2 (OCH3), dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{5,9}). Starting from
45.3 (7), 24.6 (6). MS (70 eV, EI): m/z (%) ¼ 288.2 (100) [M]þ, 193.0 compound 14{5} and 2-propoxyethanol to afford 15{5,9} in 38%
(25) [M-C6H4F]þ. HRMS (70 eV, EI): m/z calculated for C14H13N4O2F: yield, white solid. IR (KBr), nmax (cm1): 3479, 3332, 3220, 2773,
288.1028, [M]þ, found: 288.1023. 1692, 1631, 1575, 1458, 1435, 1373, 777. 1H NMR (400 MHz, DMSO-
M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483 473
d6): d 10.51 (s, 1H, NH), 7.58e7.47 (m, 2H, C13eCH), 7.37 (t, 59.80; H, 6.04; N, 19.82.
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, 4.1.4.3. 2-Amino-6-(4-fluorophenyl)-4-(pyrrolidin-1-yl)-5,6-
C16eCH2), 3.35 (t, J ¼ 6.6 Hz, 2H, C17eCH2), 2.93e2.79 (m, 2H, dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{2,7}). Starting from
C6eCH2), 1.46 (h, J ¼ 7.3 Hz, 2H, C18eCH2), 0.80 (t, J ¼ 7.4 Hz, 3H, compound 13{2} and pyrrolidine and following procedure A to
CH3). 13C NMR (100.6 MHz, DMSO-d6): d 169.6, 166.4, 161.8, 158.0, afford 15{2,7} in 44% yield, white solid. IR (KBr), nmax (cm1): 3441,
135.3, 135.1, 129.8, 128.3, 85.2 (C5), 71.9 (C17), 68.2 (C16), 65.0 (C15), 3329, 3227, 2963, 2873, 1695, 1639, 1608, 1540, 1510, 1448, 1340,
43.1 (C7), 22.4 (C18), 22.2 (C16), 10.4 (C19). Anal. Calcd. (%) for 1232, 840. 1H NMR (400 MHz, DMSO-d6): d 10.31 (s, 1H, NH),
C18H20Cl2N4O2: C, 52.57; H, 4.90; N, 13.62. Found: C, 52.76; H, 5.12; 7.30e7.24 (m, 2H, C12-2xCH), 7.14 (t, J ¼ 8.9 Hz, 2H, C13-2xCH), 5.89
N, 13.98. (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
4.1.4. General procedure for the synthesis of 4-amino-substituted C NMR (100.6 MHz, DMSO-d6): d 171.6 (CO), 161.2, 161.1 (d,
pyrido [2,3-d]pyrimidines 15{x,y} J ¼ 241.2 Hz, C14) 160.7, 157.5, 135.5 (d, J ¼ 3.0 Hz, C11), 130.3 (d,
Procedure A: 4.88 mmol of the corresponding 4-oxopyrido[2,3- J ¼ 8.1 Hz, C12), 114.9 (d, J ¼ 21.0 Hz, C13), 86.3 (C5), 48.9 (C15), 45.9
d]pyrimidine (13{x}) was suspended in acetonitrile (350 mL), then (C7), 28.5 (C6), 25.0 (C16). HRMS (70 eV, EI): m/z calculated for
BOP (2.805 g, 6.34 mmol) and DBU (1.1 mL, 7.32 mmol) were added C17H18N5OF: 327.1495, [M]þ, found: 327.1496.
to the solution. The mixture was stirred at room temperature for
2 h. The corresponding amine (14.6 mmol) was added and the 4.1.4.4. 2-Amino-6-(2,6-difluorophenyl)-4-((2-propoxyethyl)amino)-
mixture was heated at 90 C for 2 days. The solvent was concen- 5,6-dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{4,6}).
trated under reduced pressure and water was added to the residue. Starting from compound 14{4} and 2-propoxyethylamine and
The solid was collected by filtration and washed with water and following procedure B to afford 15{4,6} in 60% yield, white solid. IR
EtOEt. (KBr), nmax (cm1): 3416, 3332, 3212, 2929, 2871, 1630, 1577, 1470,
Procedure B: 0.419 mmol of the corresponding benzotriazol-1- 1388, 1007, 783. 1H NMR (400 MHz, DMSO-d6): d 10.25 (s, 1H, NH),
yloxy substituted intermediate 14{x} was suspended in acetoni- 7.46e7.37 (m, 1H, C14eCH), 7.13 (t, J ¼ 8.8 Hz, 2H, C13-CHx2), 6.34
trile (20 mL) and 3 equivalents of the corresponding amine (s, 1H, NH), 5.91 (s, 2H, NH2), 4.16 (dd, J ¼ 14.0, 7.6 Hz, 1H, C7eCH),
(1.257 mmol) was added to the suspension and the mixture was 3.44e3.43 (m, 4H, C15eCH2, C16eCH2), 3.34e3.31 (m, 2H,
heated at 140 C by microwave irradiation for 5 h. Then, water was C17eCH2), 2.97e2.78 (m, 2H, C6eCH2), 1.48 (h, J ¼ 7.4 Hz, 2H,
added to the residue and the solid was collected by filtration and C18eCH2), 0.83 (t, J ¼ 7.4 Hz, 3H, C19eCH3). 13C NMR (100.6 MHz,
washed with water and EtOEt. 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),
4.1.4.1. 2-Amino-6-(4-fluorophenyl)-4-(pentylamino)-5,6- 115.4 (t. J ¼ 18.7 Hz, C11), 111.7 (dd, J ¼ 1.9, 22.3 Hz, C13), 83.6 (C5),
dihydropyrido [2,3-d]pyrimidin-7(8H)-one (15{2,5}). Starting from 71.7 (C17), 68.7 (C16), 40.0 (C15), 36.4 (C7), 24.0 (C6), 22.5 (C18),
compound 13{2} and pentylamine and following procedure A to 10.5 (C19). Anal. Calcd. (%) for C18H21F2N5O2: C, 57.29; H, 5.61; N,
afford 15{2,5} in 40% yield, white solid. IR (KBr), nmax (cm1): 3395, 18.56. Found: C, 57.25; H, 3.41; N, 18.74.
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, 4.1.4.5. 2-Amino-6-(2,6-dichlorophenyl)-4-(pentylamino)-5,6-
J ¼ 8.8 Hz, 2H, 2xCH), 3.94 (dd, J ¼ 10.3, 7.2 Hz, 1H, CHeC7), 3.42 dihydropyrido [2,3-d]pyrimidin-7(8H)-one (15{5,5}). Starting from
(dd, J ¼ 12.9, 6.6 Hz, 2H, CH2eC15), 2.87 (m, 2H, CH2eC6), 1.67e1.51 compound 13{5} and pyrrolidine and following procedure A to
(m, 2H, CH2eC16), 1.41e1.25 (m, 4H, 2xCH2eC17, C18), 0.88 (t, afford 15{5,5} in 62% yield, white solid. IR (KBr), nmax (cm1): 3505,
J ¼ 6.8 Hz, 3H, CH3). 13C NMR (100.6 MHz, acetone) d 174.4 (CO), 3392, 3323, 3206, 2929, 2870, 1675, 1629, 1577, 1472, 1436, 1385,
163.3, 162.9 (d, J ¼ 69.1 Hz, C14), 161.6, 156.6 (C10), 136.6 (d, 1287, 776. 1H NMR (400 MHz, DMSO-d6): d 10.57 (s, 1H, CONH), 7.53
J ¼ 3.3 Hz, C11), 131.2 (d, J ¼ 8.1 Hz, C12), 115.8 (d, J ¼ 21.3 Hz, C31), (m, 2H, C12-CHx2), 7.37 (t, J ¼ 8.1 Hz, 1H, C13eCH), 6.30 (s, 1H, NH),
85.1 (C5), 46.7 (C7), 41.5 (C15), 29.2 (C16), 29.0 (C17), 26.6 (C6), 23.2 6.06 (s, 2H, NH2), 4.64 (dd, J ¼ 13.1, 8.9 Hz, 1H, C7eCH), 3.31e3.20
(C18), 14.4 (C19). MS (70 eV, EI): m/z (%) ¼ 343.3 [M]þ, 314.2 [M- (m, 2H, C14eCH2), 2.94e2.65 (m, 2H, CH2), 1.49 (p, J ¼ 7.1 Hz, 2H,
C2H5]þ, 300.1 [M-C3H9]þ, 286.1 [M-C4H9]þ, 272.1 [M-C5H11]þ. 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,
4.1.4.2. 2-Amino-6-(4-fluorophenyl)-4-((2-propoxyethyl)amino)-5,6- 135.7, 135.2, 134.8, 129.7, 128.3, 82.5 (C5), 43.1 (C7), 40.3 (C14) 28.8
dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{2,6}). Starting from (C15), 28.7 (C16), 23.2 (C6), 22.0 (C17), 14.0 (C18). MS (70 eV, EI): m/
compound 13{2} and 2-propoxyethylamine and following proce- z (%) ¼ 393.1 (69) [M]þ,351.0 (78) [M-C3H7]þ,336.0 (91) [M-
dure A to afford 15{2,6} in 63% yield, white solid. IR (KBr), nmax C4H9]þ,322.9 (54) [M-C5H11]þ, 248.1 (43) [M-C6H3Cl2]þ, 206.1 (83)
(cm1): 3391, 3226, 2932, 2874, 1689, 1629, 1575, 1511, 1477, 1226. [C9H12N5O]þ, 192.0 (69) [C8H8N5O]þ. HRMS (70 eV, EI): m/z calcu-
1
H NMR (400 MHz, CDCl3): d 11.99 (s, 1H, NH), 7.26e7.20 (m, 2H, lated for C18H21N5OCl2: 393.1123, [M]þ, found: 393.1122.
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, 4.1.4.6. 2-Amino-6-(2,6-dichlorophenyl)-4-((2-propoxyethyl)
C15eCH2), 3.58e3.52 (m, 2H, C16eCH2), 3.39 (t, J ¼ 6.7 Hz, 2H, amino)-5,6-dihydropyrido [2,3-d]pyrimidin-7(8H)-one (15{5,6}).
C17eCH2), 2.85e2.67 (m, 2H, C16eCH2), 1.63e1.51 (m, 2H, Starting from compound 13{5} and 2-propoxyethylamine and
C18eCH2), 0.88 (t, J ¼ 7.4 Hz, 3H, CH3). 13C NMR (100.6 MHz, CDCl3): following procedure A to afford 15{5,6} in 70% yield, white solid. IR
d 173.8 (CO), 161.5 (d, J ¼ 161.0 Hz, C14), 161.1, 155.9, 134.4 (d, (KBr), nmax (cm1): 3440, 3333, 3217, 2928, 2869, 1631, 1576, 1478,
J ¼ 3.4 Hz, C11), 129.9 (d, J ¼ 8.1 Hz, C12), 115.9 (d, J ¼ 21.2 Hz, C13), 776. 1H NMR (400 MHz, DMSO-d6): d 10.28 (s, 1H, CONH), 7.53 (ddd,
84.5 (C5), 72.9 (C17), 69.3 (C16), 46.2 (C7), 40.8 (C15), 26.3 (C6), 22.9 J ¼ 14.8, 8.3, 1.3 Hz, 2H, C13-2xCH), 7.37 (t, J ¼ 8.1 Hz, 1H, C14eCH),
(C18), 10.7 (C19). MS (70 eV, EI): m/z (%) ¼ 359.1 (24) [M]þ, 316.1 6.33 (s, 1H, NH), 5.95 (s, 2H, NH2), 4.60 (dd, J ¼ 13.1, 8.9 Hz, 1H,
(11) [M-C3H5]þ, 301.1 (17) [M-C3H8O]þ, 286.1 (100) [M-C4H9O]þ, C7eCH), 3.44 (m, 4H, C15eCH2, C16eCH2), 3.31e3.34 (m, 2H,
273.1 (80) [M-C5H12O]þ. HRMS (70 eV, EI): m/z calculated for C17eCH2) 2.92e2.64 (m, 2H, C6eCH2), 1.48 (h, J ¼ 7.2 Hz, 2H,
C18H22N5O2F: 359.1758, [M]þ, found: 359.1742. Anal. Calcd. (%) for C18eCH2), 0.84 (t, J ¼ 7.4 Hz, 3H, C19eCH3). 13C NMR (100.6 MHz,
C18H22FN5O2: C, 60.15; H, 6.17; F, 5.29; N, 19.49; O, 8.90. Found: C, 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), pyridopyrimidine 13{x} or 15{x,y} and 3.5 mmol of DDQ in 30 mL of
22.5 (C18), 10.5 (C19). HRMS (70 eV, EI): m/z calculated for 1,2-dichloroethane was heated at 100 C overnight protected from
C18H21N5O2Cl2: 409.1077, [M]þ, found: 409.1072. Anal. Calcd. (%) for moisture. The resulting solution was cooled down, water (300 mL)
C18H21Cl2N5O2: C, 52.69; H, 5.16; N, 17.07. Found: C, 52.60; H, 65.36; was added and the resulting precipitate was filtered, washed with
N, 17.24. EtOH and EtOEt and dried in vacuo over phosphorus pentoxide to
afford the corresponding 16{x,y}.
4.1.4.7. 2-Amino-6-(2,6-dichlorophenyl)-4-(pyrrolidin-1-yl)-5,6- Procedure C: A mixture of 0.5 mmol of the corresponding pyr-
dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{5,7}). Starting from idopyrimidine 13{x} or 15{x,y} and 114 mg (1.0 mmol) of activated
compound 13{5} and pyrrolidine and following procedure A to MnO2 in 8.5 mL of acetic acid was refluxed for 3 h. The resulting hot
afford 15{5,7} in 78% yield, white solid. IR (KBr), nmax (cm1): 3468, suspension was filtered and the solvent was removed in vacuo. The
3309, 3203, 2971, 2874, 1686, 1614, 1540, 1457, 1441, 1386, 1340, resulting solid was refluxed with water in order to eliminate MnO2
1286, 772. 1H NMR (400 MHz, DMSO-d6): d 10.11 (s, 1H, NH), 7.50 traces, then filtrated and dried in vacuo over phosphorus pentoxide
(dd, J ¼ 15.6, 8.4 Hz, 4H, C13-CHx2), 7.36 (t, J ¼ 8.0 Hz, 1H, C14eCH), to afford the corresponding 16{x,y}.
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 4.1.6.1. 2-Amino-6-(4-fluorophenyl)pyrido[2,3-d]pyrimidine-
C NMR (100.6 MHz, TFA-d): d 173.7, 151.8, 151.6, 134.8, 130.7, 130.6,
4,7(3H,8H)-dione (16{2}). Starting from 13{2} and following pro-
129.6, 128.5, 86.8 (C5), 51.9 (C15), 42.5 (C7), 24.4 (C16, C7). MS
cedure B to afford 16{2} in 90% yield, yellow solid. IR (KBr), nmax
(70 eV, EI): m/z (%) ¼ 377.1 (100) [M]þ, 232.1 (73) [M-C6H3Cl2]þ,
(cm1): 3323, 3096, 2922, 1633, 1505, 1393, 1231, 1159, 582. 1H NMR
218.0 (42) [M-C7H5Cl2]þ, 204.0 (59) [M-C8H7Cl2]þ, 176 (13) [M-
(400 MHz, DMSO-d6): d 11.92 (s, 1H, NHeCO), 7.84 (s, 1H, C6eCH),
C10H11Cl2]þ. HRMS (70 eV, EI): m/z calculated for C17H17N5OCl2:
7.71 (dd, J ¼ 8.9, 5.6 Hz, 2H, C12-2xCH), 7.19 (t, J ¼ 9.0 Hz, 2H, C13-
377.0801, [M]þ, found: 377.0810.
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
4.1.4.8. 2-Amino-4-(benzylamino)-6-(2,6-dichlorophenyl)-5,6-
(d, J ¼ 31.8 Hz, C12), 121.9 (C6) 114.7 (d, J ¼ 84.1 Hz C13), 95.9 (C5).
dihydropyrido[2,3-d]pyrimidin-7(8H)-one (15{5,8}). Starting from
HRMS (ESIeTOF): m/z calculated for C13H9FN4O2: 273.0775,
compound 14{5} and benzylamine and following procedure B to
[MþH]þ, found: 273.0782.
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), 4.1.6.2. 2-Amino-6-(4-fluorophenyl)-4-(pentylamino)pyrido[2,3-d]
5.90 (s, 2H, NH2), 4.62 (dd, J ¼ 13.1, 8.9 Hz, 1H, C7eCH), 4.52 (td, pyrimidin-7(8H)-one (16{2,5}). Starting from 15{2,5} and following
J ¼ 9.2, 3.5 Hz, 2H, C15eCH2), 2.97e2.66 (m, 2H, C6eCH2). HRMS procedure C to afford 16{2,5} in 73% yield, white solid. IR (KBr), nmax
(70 eV, EI): m/z calculated for C20H17N5OCl2: 413.0810, [M]þ, found: (cm1): 3380, 3171, 2930, 2859, 1619, 1564, 1509, 1475, 1227, 837,
413.0809. 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),
4.1.5. 2,4-Diamino-6-(4-fluorophenyl)-5,6-dihydropyrido[2,3-d] 3.47e3.30 (m, 2H, C15eCH2) 1.65e1.54 (m, 2H, C16eCH2),
pyrimidin-7(8H)-one (15{2,4}) 1.37e1.29 (m, 4H, C17eCH2, C18eCH2), 0.89 (t, J ¼ 6.8 Hz, 3H, CH3).
13
Methyl 2-(4-fluorophenyl)acrylate (5{2}) (4.00 mmol) was dis- C NMR (100.6 MHz, DMSO-d6): d 162.8, 162.5, 160.0, 159.6, 155.5,
solved in anhydrous ethylene glycol (20 mL). 2,6- 130.6, 133.1 (d, J ¼ 6.6 Hz, C11), 130.3 (d, J ¼ 7.7 Hz, C12), 121.0 (C7),
diaminopyrimidin-4(3H)-one (4.00 mmol) and sodium methoxide 114.7 (d, J ¼ 21.0 Hz, C13), 91.7 (C5), 40.4 (C15), 28.8 (C17), 28.6
(4.00 mmol) were added into the solution. The mixture was heated (C16), 22.1 (C18), 14.0 (C19). HRMS (70 eV, EI): m/z calculated for
under microwave irradiation at 180 C for 3 h. Water was added and C18H20N5OF: 341.1653, [M]þ, found: 341.1652.
the reaction crude was neutralized with a 2 M solution of HCl. The
solid was collected by filtration and washed with water, MeOH and
4.1.6.3. 2-Amino-6-(4-fluorophenyl)-4-((2-propoxyethyl)amino)pyr-
Et2O to afford 15{2,4} as a white solid in 26% yield. IR (KBr), nmax
ido[2,3-d]pyrimidin-7(8H)-one (16{2,6}). Starting from 15{2,6} and
(cm1): 3495, 3334, 3219, 3104, 2888, 1698, 1659, 1638, 1570, 1510,
following procedure C to afford 16{2,6} in 72% yield, white solid. IR
1460, 1210, 837. 1H NMR (400 MHz, DMSO-d6): d 10.72 (s, 1H, NH),
(KBr), nmax (cm1): 3380, 2177, 2961, 2873, 1621, 1566, 1509, 1481,
7.31 (dd, J ¼ 8.8, 5.6 Hz, 2H, C12-CHx2), 7.15 (t, J ¼ 8.9 Hz, 2H, C13-
1228, 838,796. 1H NMR (400 MHz, DMSO-d6): d 12.26 (s, 1H, NH),
CHx2), 6.26 (s, 2H, C2eNH2), 6.01 (s, 2H, C4eNH2), 3.88 (dd,
8.30 (s, 1H, C6eCH), 7.90 (s, 1H, NH), 7.83e7.73 (m, 2H, C12eCH2),
J ¼ 10.4, 6.9 Hz, 1H, C7eCH), 2.90e2.69 (m, 2H, C6eCH2). 13C NMR
7.23 (t, J ¼ 8.7 Hz, 3H, C13eCH2), 6.98 (s, 2H, NH2), 3.60e3.55 (m,
(100.6 MHz, DMSO-d6): d 172.6 (CO), 161.8, 161.7, 161.2 (d,
4H, C15eCH2, C16eCH2), 3.40e3.35 (m, 2H, C17eCH2), 1.57e1.45
J ¼ 241 Hz, C14) 156.3, 135.7 (d, J ¼ 3.1 Hz, C11), 130.2 (d, J ¼ 7.9 Hz,
(m, 2H, C18eCH2), 0.85 (t, J ¼ 7.4 Hz, 3H, C19eCH3). 13C NMR
C12), 115.0 (d, J ¼ 20.9 Hz, C13), 83.9 (C5), 45.5 (C7), 25.6 (C6). MS
(100.6 MHz, DMSO-d6): d 162.6, 161.1 (d, J ¼ 245.0 Hz, C14), 159.6,
(70 eV, EI): m/z (%) ¼ 273.1 (100) [M]þ. HRMS (70 eV, EI): m/z
155.6, 133.1 (d, J ¼ 1.9 Hz, C11), 132.8, 130.1 (d, J ¼ 7.9 Hz, C12), 121.1,
calculated for C13H12N5OF: 273.1026, [M]þ, found: 273.1026.
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
4.1.6. General procedure for aromatization of pyrido[2,3-d]
for C18H21FN5O2, 358.1678, [M-H]þ, found: 358.1674.
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 4.1.6.4. 2-Amino-6-(4-fluorophenyl)-4-(pyrrolidin-1-yl)pyrido[2,3-d]
hydride (60% dispersion in mineral oil) in 5 mL of anhydrous DMSO pyrimidin-7(8H)-one (16{2,7}). Starting from 15{2,7} and following
was heated for 4 h at 100 C protected from moisture. The resulting procedure C to afford 16{2,7} in 82% yield, light brown solid. IR
solution was cooled down, water (300 mL) was added and it was (KBr), nmax (cm1):3459, 3311, 3181, 2970, 2874, 1635, 1606, 1530,
neutralized with AcOH. The resulting precipitate was filtered, 1511, 1452, 1452, 1224, 839. 1H NMR (400 MHz, DMSO-d6): d 11.77
washed with MeOH and EtOEt and dried in vacuo over phosphorus (s, 1H, NH), 8.10 (s, 1H, C6eCH), 7.73 (m, 2H, C12-CHx2), 7.19 (t,
pentoxide to afford the corresponding 16{x,y}. J ¼ 9.0 Hz, 2H, C13-CHx2), 6.65 (s, 2H, NH2), 3.76 (t, J ¼ 6.4 Hz, 4H,
Procedure B: A mixture of 2.7 mmol of the corresponding C15eCH2x2), 1.91 (t, J ¼ 6.5 Hz, 4H, C16eCH2x2).
M. Camarasa et al. / European Journal of Medicinal Chemistry 115 (2016) 463e483 475
4.1.6.5. 2-Amino-6-(2,6-difluorophenyl)-4-((2-propoxyethyl)amino) of DMSO, 28.0 mg (0.7 mmol) of sodium hydride (60% dispersion in
pyrido[2,3-d]pyrimidin-7(8H)-one (16{4,6}). Starting from 15{4,6} mineral oil) were added and the mixture was stirred for 1 h at room
and following procedure A to afford 16{4,6} in 60% yield, light temperature under nitrogen atmosphere. After this period,
brown solid. IR (KBr), nmax (cm1): 3387, 3188, 2931, 2858, 1627, 0.7 mmol of methyl iodide were added dropwise and then stirred
1568, 1466, 1004, 788. 1H NMR (400 MHz, DMSO-d6): d 11.93 (s, 1H, overnight at rt. The reaction was quenched by addition of 300 mL of
NH), 8.16 (s, 1H, C6eCH), 7.70 (s, 1H, NH), 7.49e7.41 (m, 1H, water and the resulting precipitate was filtered, washed with water
C14eCH), 7.20e7.12 (m, 2H, C13-CHx2), 6.81 (s, 2H, NH2), 3.50e3.57 and dried in vacuo over phosphorus pentoxide to afford the cor-
(m, 4H, C15eCH2, C16eCH2), 3.35e3.40 (m, 2H, C17eCH2), 1.49 (h, responding 17{x,y}.
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), 4.1.7.1. 2-Amino-6-(4-fluorophenyl)-4-methoxy-8-methylpyrido[2,3-
160.3 (d, J ¼ 246.7 Hz, C12), 159.6, 156.4, 136.7, 129.9 (t, J ¼ 10.3 Hz, d]pyrimidin-7(8H)-one (17{2,1}). Starting from 16{2,1} to afford 17
C14), 113.8 (t, J ¼ 20.1 Hz, C11), 111.6e111.3 (m, C13), 111.2, 91.0 (C5), {2,1} in 71% yield, white solid. IR (KBr), nmax (cm1): 3494, 3338,
71.8 (C17), 68.3 (C16), 40.2 (C15), 22.4 (C18), 10.5 (C19). HRMS 2926, 1608, 1587, 1510, 1468, 1390, 834, 796. 1H NMR (400 MHz,
(70 eV, EI): m/z calculated for C18H19N5O2F2: 375.1507, [M]þ, found: CDCl3): d 7.88 (s, 1H, C7eCH), 7.65 (dd, J ¼ 8.9, 5.5 Hz, 2H, C12-
375.1509. 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,
4.1.6.6. 2-Amino-6-(2,6-dichlorophenyl)-4-(2-propoxyethoxy)pyrido 161.27, 156.81, 132.95 (C7), 130.61 (d, J ¼ 7.9 Hz,C12-CHx2), 130.45
[2,3-d]pyrimidin-7(8H)-one (16{5,9}). Starting from 15{5,9} and (C6), 125.64 (C11), 115.12 (d, J ¼ 21.3 Hz,C13-CHx2), 94.75 (C5),
following procedure B to afford 16{5,9} in 82% yield, white solid. IR 54.42 (OCH3), 28.89 (NCH3). HRMS (70 eV, EI): m/z calculated for
(KBr), nmax (cm1): 3392, 3183, 2962, 2868, 1626, 1553, 1509, 1432, C15H13N4O2F: 300.1025, [M]þ, found: 300.1023.
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, 4.1.7.2. 2-Amino-6-(4-fluorophenyl)-8-methyl-4-(pentylamino)pyr-
C14eCH), 7.21 (s, 2H, NH2), 4.51e4.45 (m, 2H, C15eCH2), 3.74e3.68 ido[2,3-d]pyrimidin-7(8H)-one (17{2,5}). Starting from 16{2,5} to
(m, 2H, C16eCH2), 3.38 (t, J ¼ 6.6 Hz, 2H, C17eCH2), 1.47 (h, afford 17{2,5} in 89% yield, white solid. IR (KBr), nmax (cm1): 3345,
J ¼ 7.3 Hz, 2H, C18eCH2), 0.80 (t, J ¼ 7.4 Hz, 3H, CH3). 13C NMR 2930, 2858, 1603, 1567, 1508, 1466, 835, 799. 1H NMR (400 MHz,
(100.6 MHz, DMSO-d6): d 166.1, 162.7, 161.4, 157.4, 150.8, 135.2, CDCl3): d 7.91 (s, 1H, C6eCH), 7.52e7.46 (m, 2H, C12eCHx2), 6.94 (t,
134.5, 134.2 (C6), 130.3 (C14), 129.2, 128.0 (C13), 122.1, 91.6 (C5), J ¼ 8.4 Hz, 2H, C13eCHx2), 5.64 (t, J ¼ 5.4 Hz, 1H, NH), 5.09 (s, 2H,
71.9 (C17), 67.9 (C16), 65.7 (C15), 22.4 (C18), 10.4 (C19). HRMS NH2), 3.63 (s, 3H, CH3), 3.53e3.41 (m, 2H, C15eCH2), 1.65e1.58 (m,
(70 eV, EI): m/z calculated for C18H18N4O3Cl2: 408.0750, [M]þ, 2H, C16eCH2), 1.40e1.29 (m, 4H, C17eCH2, C18eCH2), 0.90 (t,
found: 408.0756. 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),
4.1.6.7. 2-Amino-6-(2,6-dichlorophenyl)-4-(pentylamino)pyrido[2,3- 130.4 (d, J ¼ 7.8 Hz, C12), 129.4 (C6), 123.4 (C7), 114.9 (d, J ¼ 21.3 Hz,
d]pyrimidin-7(8H)-one (16{5,5}). Starting from 15{5,5} and C13), 92.8 (C5), 41.4 (C15), 29.3, 29.3, 28.78 (NeCH3), 22.5 (C18),
following procedure B to afford 16{5,5} in 51% yield, white solid. IR 14.1 (C19). MS (70 eV, EI): m/z (%) ¼ 355.2 (100) [M]þ, 312.1 (42) [M-
(KBr), nmax (cm1): 3126, 1856, 1640, 1465, 1430, 169, 780. 1H NMR C3H7]þ, 298.1 (61) [M-C4H9]þ, 285.1 (62) [M-C5H12]þ, 158.0 (40) [M-
(400 MHz, DMSO-d6): d 10.27 (s, 1H, NH), 8.89 (s, 1H, NH), 8.36 (s, C12H20FN]þ.
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, 4.1.7.3. 2-Amino-6-(4-fluorophenyl)-8-methyl-4-((2-propoxyethyl)
C15eCH2), 1.60 (m, 2H, C16eCH2), 1.37e1.25 (m, 4H, C17eCH2, amino)pyrido[2,3-d]pyrimidin-7(8H)-one (17{2,6}). Starting from 16
C18eCH2), 0.91e0.83 (m, 3H, C19eCH3). 13C NMR (100.6 MHz, {2,6} to afford 17{2,6} in 87% yield, white solid. IR (KBr), nmax
DMSO-d6): d 169.9, 161.3, 155.7, 154.9, 135.4 (C6), 135.0, 134.7, 133.9, (cm1): 3341, 3199, 2932, 2872, 1603, 1570, 1509, 1468, 1227, 1160,
130.7 (C14), 129.7, 128.1 (C13), 86.8 (C5), 43.4 (C15), 28.5 (C17), 27.9 837, 799. 1H NMR (400 MHz, CDCl3): d 7.58 (dd, J ¼ 8.7, 5.6 Hz, 2H,
(C16), 21.8 (C18), 13.9 (C19). MS (70 eV, EI): m/z (%) ¼ 391.2 (20.6) C12eCHx2), 7.47 (s, 1H, C6eCH), 7.05 (t, J ¼ 8.8 Hz, 2H, C13eCHx2),
[M]þ, 356.3 (100) [M-Cl]þ, 326.1 (19.0) [M-C2H5Cl]þ, 300.1 (7.5) [M- 5.81 (s, 1H, NH), 5.03 (s, 2H, NH2), 3.72 (q, J ¼ 5.1 Hz, 2H, C15eCH2),
C4H9Cl]þ. HRMS (70 eV, EI): m/z calculated for C18H19N5OCl2: 3.68 (s, 3H, NCH3), 3.63 (t, J ¼ 5.0 Hz, 2H, C16eCH2), 3.44 (t,
391.0967, [M]þ, found: 391.0967. 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):
4.1.6.8. 2-Amino-6-(2,6-dichlorophenyl)-4-((2-propoxyethyl)amino) d 163.59, 162.00 (d, J ¼ 162.0 Hz), 160.32, 155.98, 155.52, 133.21,
pyrido[2,3-d]pyrimidin-7(8H)-one (16{5,6}). Starting from 15{5,6} 130.50 (d, J ¼ 7.9 Hz, C12), 128.97 (C6), 124.01, 115.11 (d, J ¼ 21.0 Hz,
and following procedure B to afford 16{5,6} in 52% yield, light C13), 92.73 (C5), 73.04 (C17), 69.05 (C16), 41.06 (C15), 28.81 (NCH3),
brown solid. IR (KBr), nmax (cm1): 3128, 2870, 1640, 1453, 1430, 22.91 (C18), 10.71 (C19). MS (70 eV, EI): m/z (%) ¼ 371.2 (73) [M]þ,
1284, 796. 1H NMR (400 MHz, DMSO-d6): d 8.94 (s, 1H, NH), 8.36 (s, 312.1 (15) [M-C3H7O]þ, 298.1 (86) [M-C4H9O]þ, 285.1 (100) [M-
1H, C6eCH), 7.62e7.55 (m, 2H, C13-CHx2), 7.47 (t, J ¼ 8.7 Hz, 1H, C5H12O]þ, 158.0 (44) [M-C12H20NFO]þ. HRMS (70 eV, EI): m/z
C14eCH), 3.66 (t, J ¼ 4.8 Hz, 2H, C15eCH2), 3.58 (t, J ¼ 5.3 Hz, 2H, calculated for C19H22N5O2F: 371.1758, [M]þ, found: 371.1755.
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 4.1.7.4. 2-Amino-6-(4-fluorophenyl)-8-methyl-4-(pyrrolidin-1-yl)
(100.6 MHz, DMSO-d6): d 156.42, 155.79, 150.86, 135.99 (C6), 135.00 pyrido[2,3-d]pyrimidin-7(8H)-one (17{2,7}). Starting from 16{2,7}
(CeCl), 133.81, 130.83 (C14), 129.23, 128.15 (C13), 113.74, 101.71 (C5), to afford 17{2,7} in 59% yield, white solid. IR (KBr), nmax (cm1):
71.80 (C17), 67.68 (C16), 41.25 (C15), 22.38 (C18), 10.46 (C19). HRMS 3508, 3345, 3233, 2964, 2868, 1638, 1590, 1564, 1524, 1504, 1443,
(70 eV, EI): m/z calculated for C18H19N5O2Cl2: 407.0916, [M]þ, 833, 795. 1H NMR (400 MHz, CDCl3): d 8.01 (s, 1H, C7eCH), 7.61 (dd,
found: 407.0911. 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,
4.1.7. General procedure for the methylation at N8 of pyrido[2,3-d] C16eCH2). 13C NMR (100.6 MHz, CDCl3): d 162.4, 162.1 (d,
pyrimidines 16{x,y} J ¼ 265.8 Hz, C14), 161.0, 160.9, 157.8, 133.8 (C11), 133.3 (C6), 130.4
To a solution of 0.7 mmol of the corresponding 16{x,y} in 20 mL (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 4.1.9. General procedure for the substitution of the methylthio
for C18H18N5OF: 339.1493, [M]þ, found: 339.1495. 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})
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 - (2 mmol) in 14 mL of 2-propanol and the mixture was heated under
propoxyethoxy)pyrido[2,3-d]pyrimidin-7(8H)-one (17{5,9}). microwave irradiation at 170 C for 5 h. Cyclohexane was added to
Starting from 16{5,9} to afford 17{5,9} in 26% yield, white solid. IR the solution and the solid was collected by filtration and washed
(KBr), nmax (cm1): 3336, 2960, 2872, 1658, 1585, 1464, 1429, 1338, with cyclohexane, and Et2O to afford the corresponding (21{x,y}).
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), 4.1.9.1. 2-(benzylamino)-6-(4-fluorophenyl)-5,6-dihydropyrido[2,3-
7.38 (s, 2H, NH2), 4.53e4.45 (m, 2H, C15eCH2), 3.77e3.68 (m, 2H, d]pyrimidine-4,7(3H,8H)-dione (21{2,8}). Starting from 19{2} and
C16eCH2), 3.59 (m, 2H, C17e CH2), 3.55 (s, 3H, NCH3), 1.46 (h, benzylamine to afford 21{2,8} in 53% yield, white solid. IR (KBr),
J ¼ 7.1 Hz, 2H, C18eCH2), 0.79 (t, J ¼ 7.4 Hz, 3H, C19eCH3). 13C NMR nmax (cm1): 3423, 3180, 3068, 2958, 1689, 1644, 1612, 1513, 1229,
(100.6 MHz, DMSO-d6): d 166.6, 162.4, 160.6, 157.2, 135.2, 134.8, 836, 582. 1H NMR (400 MHz, TFA-d): d 7.42e7.30 (m, 5H, CHArx5),
132.8 (C6), 130.4 (C14), 128.0 (C13), 120.6, 109.6, 92.0 (C5), 71.9 7.22 (dd, J ¼ 7.8, 5.2 Hz, 2H, CHeC12x2), 7.05 (t, J ¼ 8.5 Hz, 2H,
(C17), 67.9 (C16), 65.9 (C15), 28.2 (NCH3), 22.4 (C18), 10.4 (C19). CHeC13x2), 4.75 (s, 2H, CH2eC15), 4.05 (dd, J ¼ 11.3, 7.3 Hz, 1H,
HRMS (ESIeTOF): m/z calculated for C19H21Cl2N4O3: 223.0983, CHeC7), 3.24 (dd, J ¼ 16.8, 7.4 Hz, 1H, C6eCH2), 3.03 (dd, J ¼ 16.8,
[MþH]þ, found: 423.0985. Anal. Calcd. (%) for C19H20Cl2N4O3: C, 11.4 Hz, 1H, C6eCH2). 13C NMR (100.6 MHz, TFA-d): d 179.9, 168.9,
53.91; H, 4.76; N, 13.24. Found: C, 53.97; H, 4.94; N, 12.93. 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).
4.1.7.6. 4-Amino-6-(2,6-dichlorophenyl)-8-methylpyrido[2,3-d]py- HRMS (70 eV, EI): m/z calculated for C20H17N4O2F: 324.0183, [M]þ,
rimidine-7(8H)-one (17{5,4}). Starting from 9{5,4} [25] and found: 324.0181. Anal. Calcd. (%) for C20H17FN4O2: C, 65.93; H, 4.70;
following procedure A described in 4.1.6. to afford 16{5,4} in 97% N, 15.38. Found: C, 65.53; H, 4.47; N, 15.49.
yield, white solid. IR (KBr), nmax (cm1): 3457, 3329, 3182, 2791,
1615, 1545, 1494, 1462, 1429, 1400, 1327, 1290, 1191, 936, 802, 775. 4.1.9.2. 2-(benzylamino)-6-(2,6-dichlorophenyl)-5,6-dihydropyrido
1
H NMR (400 MHz, DMSO-d6): d 12.17 (br s, 1H), 7.98 (s, 1H), 7.54 (d, [2,3-d]pyrimidine-4,7(3H,8H)-dione (21{5,8}). Starting from 19{5}
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, and benzylamine to afford 21{5,8} in 36% yield, white solid. IR (KBr),
2H). 13C NMR (100.6 MHz, DMSO-d6): d 163.1, 161.9, 161.5, 156.6, nmax (cm1): 3263, 3181, 3066, 2934, 1686, 1639, 1613, 1597, 1545,
136.3, 135.5, 134.8, 130.2, 128.0, 119.6, 90.5. MS (ESI-TOF): m/z 1520, 1374, 1289, 770. 1H NMR (400 MHz, DMSO-d6): d 10.57 (s, 1H,
(%) ¼ 322.0 (100) [MþH]þ, 305.0 (9) [M-NH2]þ, 286.0 (9) [M-Cl]þ, NH), 10.27 (s, 1H, NH), 7.50 (ddd, J ¼ 16.6, 8.1, 1.3 Hz, 2H,
252.1 (7) [MþHeCl2]þ. HRMS (ESI-TOF): m/z calculated for CHeC13x2), 7.40e7.31 (m, 5H, CH-Ar), 7.27 (d, J ¼ 7.0 Hz, 1H, CH-
C13H10N5OCl2: 322.0257 [MþH]þ, found: 322.0256. 16{5,4} was Ar), 6.99 (s, 1H, NH-Bz), 4.53 (dd, J ¼ 13.4, 8.9 Hz, 1H, CHeC7),
converted to 17{5,4} in 70% yield, white solid. IR (KBr), nmax (cm1): 4.47 (d, J ¼ 6.1 Hz, 2H), 2.84e2.64 (m, 2H, CH2eC6). 13C NMR
3347, 3212, 2925, 1627, 1607, 1568, 1546, 1462, 1430, 1394, 1360, (100.6 MHz, DMSO-d6): d 169.7, 161.6, 155.5, 153.7, 139.1, 135.4,
1328, 1202 802. 1H NMR (400 MHz, DMSO-d6): d 7.99 (s, 1H), 7.55 (d, 135.2, 134.7, 129.7, 129.7, 128.3, 128.3, 127.6, 127.0, 87.1 (C5), 43.4
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, (C15), 43.4 (C7), 22.4 (C6). MS (70 eV, EI): m/z (%) ¼ 414.0 (100),
2H), 3.50 (s, 3H). 13C NMR (100.6 MHz, DMSO-d6): d 162.8, 162.1, [M]þ, 379.1 [M-Cl]þ, 228.9 (34.0) [C12H12N4O]þ, 105.8 (62.6)
160.6, 156.7, 135.6, 135.5, 134.1, 131.0, 128.0, 118.4, 90.5, 28.0. MS [C7H8N]þ, 90.9 (100) [C7H7]þ. HRMS (70 eV, EI): m/z calculated for
(ESI-TOF): m/z (%) ¼ 336.0 (100) [MþH]þ, 286.0 (3) [MþH-CH3Cl]þ, C20H16N4O2Cl2: 414.0650, [M]þ, found: 414.0650. Anal. Calcd. (%)
266.1 (5) [MþH-Cl2]þ. HRMS (ESIeTOF): m/z calculated for for C20H16Cl2N4O2: C, 57.84; H, 3.88; N, 13.49. Found: C, 57.71; H,
C14H12Cl2N5O: 336.0413, [MþH]þ, found: 336.0424. 3.78; N, 13.47.
4.1.9.3. 2-((4-fluorobenzyl)amino)-6-(4-fluorophenyl)-5,6-
4.1.8. 6-(2,4-dichlorophenyl)-2-(methylthio)-5,6-dihydropyrido dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (21{2,9}).
[2,3-d]pyrimidine-4,7(3H,8H)-dione (19{11}) Starting from 19{2} and 4-flurobenzylamine to afford 21{2,9} in 40%
2-(2,4-dichlorophenyl)acrylate (5{11}) (4.00 mmol) was dis- yield, white solid. IR (KBr), nmax (cm1): 3424, 3248, 3181, 2931,
solved in 2-propanol (20 mL), 6-amino-2-(methylthio)pyrimidin- 1637, 1611, 1513, 1230, 834. 1H NMR (400 MHz, TFA-d): d 7.31 (dd,
4(3H)-one (52) (5.60 mmol) and potassium carbonate (4.00 mmol) J ¼ 8.6, 5.0 Hz, 2H, C17-CHx2), 7.19 (dd, J ¼ 8.7, 5.0 Hz, 2H, C12-
were added into the solution. The mixture was heated under mi- CHx2), 7.06e7.00 (m, 4H, C18-CHx2, C13-CHx2), 4.70 (s, 2H,
crowave irradiation at 170 C for 3 h. Then, the solvent was C15eCH2), 4.02 (dd, J ¼ 11.2, 7.2 Hz, 1H, C7eCH), 3.22 (dd, J ¼ 16.8,
removed under reduced pressure, water was added to the crude 7.4 Hz, 1H, C6eCH2), 3.00 (dd, J ¼ 16.8, 11.4 Hz, 1H, C6eCH2). 13C
and this was neutralized with a 2 M solution of HCl. The solid was NMR (100.6 MHz, DMSO-d6): d 179.9, 168.0 (d, J ¼ 248.8 Hz, C14),
collected by filtration and washed with water, and Et2O to afford 19 167.6 (d, J ¼ 247.7 Hz), 165.3, 154.0, 150.7, 134.7 (d, J ¼ 3.5 Hz, C11),
{11} in 80% yield, white solid. IR (KBr): n (cm1): 3404, 3131, 2868, 134.0 (d, J ¼ 8.5 Hz, C12), 133.7 (C17), 132.2 (C16), 120.5 (d,
1694, 1633, 1557, 1505, 1479, 1459, 1374, 1285, 1241, 1151, 1103, 964, J ¼ 24.5 Hz), 120.4 (d, J ¼ 22.3 Hz), 96.1 (C5), 50.0 (C15), 49.7 (C7),
823, 576. 1H NMR (400 MHz, DMSO-d6): d 10.73 (s, 1H), 7.63 (d, 27.8 (C6). HRMS (70 eV, EI): m/z calculated for C20H16N4O2F2:
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), 382.1241, [M]þ, found: 382.1241.
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). 4.1.10. General procedure for oxidation of 2-(methylthio)-5,6-
13
C NMR (100.6 MHz, DMSO-d6): d 170.25, 161.8, 154.4, 135.9 (C12), dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-diones (19{x}) to
134.3 (C14), 132.6(C16), 132.0 (C12), 129.0 (C13), 127.6 (C15), 95.0 methylsulfones 20{x}
(C5), 43.3 (C7), 23.7 (C6), 12.8 (SCH3). Anal. Calcd. (%) for m-CPBA (18 mmol) was suspended in 20 mL of DMF and was
C14H11Cl2N3O2S: C, 47.20; H, 3.11; N, 11.80; S, 9.00. Found: C, 47.07; added dropwise into a solution of the 2-methylthio-substituted
H, 2.97; N, 11.96; S, 9.01. 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 477
mixture was stirred overnight at room temperature. Then the sol- 4.85 (dd, J ¼ 10.2, 7.9 Hz, 1H, C7eH), 3.33 (s, 3H, SCH3), 3.23e3.13
vent was removed under reduced pressure and the residue was (m, 2H, C6eCH2). 13C NMR (100.6 MHz, DMSO-d6): d 171.5, 167.1,
suspended in EtOEt and the solid was collected by filtration and 162.7, 158.8, 135.0, 133.6, 131.1, 129.0 (C16), 127.8 (C12), 126.2 (C17),
washed with water, and EtOEt to afford the corresponding 2- 125.7 (C18), 125.5 (C14, C13), 124.0 (C19), 100.6 (C5), 41.5 (C7), 39.1
methylsulfonyl-substituted pyrido[2,3-d]pyrimidine (20{x}). (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.
4.1.10.1. 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 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 -
{1} in 74% yield, white solid. IR (KBr), nmax (cm1): 3429, 3200, dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (20{11}).
3136, 3022, 2919, 2866, 1691, 1643, 1612, 1486, 1341, 1235, 1165, Starting from 19{11} to afford 20{11} in 70% yield, white solid. IR
1134, 699. 1H NMR (400 MHz, DMSO-d6): d 13.29 (s, 1H, NH), 11.32 (KBr), nmax(cm1): 3432, 3279, 3040, 2925, 2868, 1651, 1492, 1329,
(s, 1H, NH), 7.36e7.31 (m, 2H, C13-CHx2), 7.30e7.22 (m, 3H, C12- 1276, 1231, 1156, 1132, 944, 766, 572, 529. 1H NMR (400 MHz,
CHx2, C14eCH), 4.00 (dd, J ¼ 9.4, 7.3 Hz, 1H, C7eCH), 3.31 (s, 3H, DMSO-d6): d 13.34 (s, 1H, NH), 11.42 (s, 1H, NH), 7.65 (dd, J ¼ 1.5,
SCH3), 3.14e3.00 (m, 2H, C6eCH2). 13C NMR (100.6 MHz, DMSO- 0.9 Hz, 1H), 7.46 (s, 1H, C13eCH), 7.46 (d, J ¼ 0.9 Hz, 1H), 4.38 (dd,
d6): d 171.4, 167.1, 162.6, 158.6, 138.4, 128.5, 128.0, 127.1 (C12), 100.4 J ¼ 11.6, 9.3 Hz, 1H, C7eCH), 3.31 (s, 3H, SCH3), 3.07e2.81 (s, 2H,
(C5), 44.8 (C7), 39.1 (SCH3), 24.7 (C6). Anal. Calcd. (%) for C6eCH2). 13C NMR (100.6 MHz, DMSO-d6): d 169.9, 167.0, 162.7,
C14H13N3O4S: C, 52.66; H, 4.10; N, 13.16; S, 10.04. Found: C, 52.32; H, 158.6, 135.3, 134.4, 132.7, 132.1, 129.0 (C13), 127.6, 100.2 (C5), 42.6
4.16 N, 13.02; S, 10.34. (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,
4.1.10.2. 6-(4-fluorophenyl)-2-(methylsulfonyl)-5,6-dihydropyrido 8.11.
[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, 4.1.11. General procedure for the substitution of the methylsulfonyl
3036, 2867, 1635, 1607, 1513, 1490, 1337, 1233, 1160, 839, 767, 541. 1H group of pyridopyrimidines 20{x} by arylamines
NMR (400 MHz, DMSO-d6): d 13.28 (s, 1H, NH), 11.33 (s, 1H, NH), The corresponding arylamine (10 mmol) was added to a solution
7.30 (dd, J ¼ 8.7, 5.8 Hz, 2H, C12-CHx2), 7.17 (t, J ¼ 8.9 Hz, 2H, C13- of the 2-methylsulfonyl-substituted pyridopyrimidine (20{x})
CHx2), 4.03 (dd, J ¼ 10.2, 7.4 Hz, 1H, C7eCH), 3.31 (s, 3H, SCH3), (2 mmol) in 14 mL 2-propanol and the mixture was heated under
3.14e2.94 (m, 2H, C6eCH2). 13C NMR (100.6 MHz, DMSO-d6): microwave irradiation at 170 C for 5 h. Cyclohexane was added to
d 171.4, 167.0, 162.7, 161.3 (d, J ¼ 243.2 Hz, C14), 158.7, 134.6 (d, the solution and the solid obtained was collected by filtration and
J ¼ 3.1 Hz, C11), 130.2 (d, J ¼ 8.1 Hz, C12), 115.2 (d, J ¼ 21.3 Hz, C13), washed with cyclohexane and Et2O to afford the corresponding 21
100.4 (C5), 44.1 (C7), 29.5 (SCH3), 24.8 (C6). HRMS (70 eV, EI): m/z {x,y}
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. 4.1.11.1. 6-Phenyl-2-(phenylamino)-5,6-dihydropyrido[2,3-d]pyrimi-
Found: C, 49.68; H, 3.84; N, 12.70; S, 9.18. 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,
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 - 3058, 1615, 1654, 1462, 750, 395. 1H NMR (400 MHz, DMSO-d6):
dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (20{4}). d 10.48 (s, 2H, NHx2), 8.80 (s, 1H, NH), 7.73e7.66 (m, 2H, C16-CHx2),
Starting from 19{4} to afford 20{4} in 60% yield, white solid. IR 7.34e7.22 (m, 7H, C17-CHx2, C12-CHx2, C13-CHx2, C14eCH), 7.02
(KBr): n (cm1): 3432, 3219, 3027, 2923, 1704, 1649, 1612, 1474, (tt, J ¼ 7.3, 1.2 Hz, 1H, C8eCH), 3.82 (dd, J ¼ 8.5, 7.1 Hz, 1H, C7eCH),
1459, 1339, 1235, 1164, 1134, 1010, 783, 542. 1H NMR (400 MHz, 2.92e2.72 (m, 2H, C6eCH2). 13C NMR (100.6 MHz, DMSO-d6):
DMSO-d6): d 13.33 (s, 1H), 11.41 (s, 1H), 7.44 (tt, J ¼ 8.4, 6.6 Hz, 1H, d 172.4, 139.7, 139.1, 129.2, 128.8, 128.8, 128.5, 128.4, 127.3, 123.0
C14eCH), 7.14 (t, J ¼ 8.8 Hz, 2H, C13-CHx2), 4.46 (dd, J ¼ 14.0, 7.6 Hz, (C18), 119.8 (C16), 114.3, 90.7 (C5), 46.2 (C7), 25.0 (C6). HRMS
1H, C7eCH), 3.31 (s, 3H, SCH3), 3.10 (dd, J ¼ 16.5, 7.7 Hz, 1H, (70 eV, EI): m/z calculated for C19H16N4O2: 332.1273, [M]þ, found:
C6eCH2), 2.94e2.82 (m, 1H, C6eCH2). 13C NMR (100.6 MHz, DMSO- 332.1275.
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, 4.1.11.2. 6-(4-fluorophenyl)-2-(phenylamino)-5,6-dihydropyrido[2,3-
C14), 111.8 (dd, J ¼ 22.3, 3.1 Hz, C13), 100.0 (C5), 39.2 (SCH3), 35.3 d]pyrimidine-4,7(3H,8H)-dione (21{2,10}). Starting from 20{2} and
(C7), 23.4 (C6). Anal. Calcd. (%) for C14H11F2N3O4S: C, 47.32; H, 3.12; aniline to afford 21{2,10} in 94% yield, white solid. IR (KBr), nmax
N, 11.83; S, 9.02. Found: C, 47.14; H, 3.19 N, 11.72; S, 9.28. (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),
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 - 7.79e7.67 (m, 2H, 2xCHPh), 7.33e7.24 (m, 4H, 2xCHPh þ C12-CHx2),
dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (20{5}). 7.15 (t, J ¼ 8.9 Hz, 2H, C13-CHx2), 7.04e6.98 (m, 1H), 3.85 (dd,
Starting from 19{5} to afford 20{5} in 98% yield, white solid. 1H NMR J ¼ 9.6, 7.0 Hz, 1H), 2.92e2.70 (m, 2H, C6eCH2). 13C NMR
(400 MHz, DMSO-d6): d 11.35 (s, 1H, NH), 7.53 (dd, J ¼ 12.3, 8.1 Hz, (100.6 MHz, DMSO-d6): d 172.0, 161.8, 161.3 (d, J ¼ 242.8 Hz, C14),
2H, C13-CHx2), 7.39 (t, J ¼ 8.1 Hz, 1H, C14eCH), 4.87 (dd, J ¼ 12.9, 155.6, 151.1, 138.8, 135.5 (d, J ¼ 3.1 Hz, C11), 130.1 (d, J ¼ 8.1 Hz, C12),
9.2 Hz, 1H, C7eCH), 3.31 (s, 3H, SCH3), 3.06 (dd, J ¼ 17.1, 9.4 Hz, 1H, 128.8, 122.6, 119.5, 115.2 (d, J ¼ 21.1 Hz, C13) 90.4 (C5), 45.1 (C7),
C6eCH2), 2.99 (dd, J ¼ 17.0, 13.5 Hz, 1H, C6eCH2). 24.7 (C6). HRMS (70 eV, EI): m/z calculated for C19H15N4O2F:
350.1179, [M]þ, found: 350.1179.
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 4.1.11.3. 6-(4-fluorophenyl)-2-((4-fluorophenyl)amino)-5,6-
afford 20{8} in 68% yield, white solid. IR (KBr), nmax(cm1): 3432, dihydropyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (21{2,11}).
3211, 3022, 1690, 1641, 1614, 1487, 1345, 1225, 1167, 1136, 775. 1H Starting from 20{2} and 4-fluoroaniline to afford 21{2,11} in 67%
NMR (400 MHz, DMSO-d6): d 13.24 (s, 1H, NH), 11.44 (s, 1H, NH), yield, white solid. IR (KBr), nmax (cm1): 3409, 3171, 2958, 1645,
8.13e8.07 (m, 1H, C19eCH), 8.00e7.94 (m, 1H, C16eCH), 7.87 (d, 1603, 1512, 1242, 836. 1H NMR (400 MHz, DMSO-d6): d 10.58 (s, 1H,
J ¼ 8.2 Hz, 1H, C12eCH), 7.58e7.51 (m, 2H, C17eCH, C18eCH), 7.48 NH), 10.48 (s, 1H, NH), 8.88 (s, 1H, NH), 7.71 (dd, J ¼ 9.1, 4.9 Hz, 2H,
(t, J ¼ 7.2 Hz, 1H, C13eCH), 7.40 (dd, J ¼ 7.2, 1.2 Hz, 1H, C14eCH), 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 1H, C23eCH), 4.64 (dd, J ¼ 9.1, 7.6 Hz, 1H, C7eCH), 3.00 (dd, J ¼ 16.1,
(dd, J ¼ 16.0, 7.1 Hz, 1H, C6eCH), 2.73 (dd, J ¼ 16.0, 9.6 Hz, 1H, 7.7 Hz, 1H, C6eCH2), 2.85 (dd, J ¼ 16.1, 9.1 Hz, 1H, C6eCH2). 13C NMR
C6eCH). 13C NMR (100.6 MHz, DMSO-d6): d 171.9, 161.2 (d, (100.6 MHz, DMSO-d6): d 172.0, 171.0, 162.9, 155.4, 138.7, 135.9,
J ¼ 242.6 Hz, C14), 157.8 (d, J ¼ 239.4 Hz, C18), 156.2, 155.4, 154.8, 133.7, 131.0, 128.8 (C22,C16), 127.5 (C14), 126.1, 125.6, 125.4 (C13),
135.4 (d, J ¼ 3.1 Hz, C11), 135.0 (C15), 130.0 (d, J ¼ 8.1 Hz, C12), 121.3 125.1 (C12), 123.9 (C19), 122.6 (C23), 119.4 (C21), 90.3 (C5), 42.5
(d, J ¼ 7.6 Hz, C16), 115.2 (d, J ¼ 17.7 Hz, C13), 115.0 (d, J ¼ 16.7 Hz, (C7), 24.8 (C6). HRMS (70 eV, EI): m/z calculated for C23H18N4O2:
C17), 90.3 (C5), 45.0 (C7), 24.6 (C6). HRMS (70 eV, EI): m/z calcu- 382.1430, [M]þ, found: 382.1429.
lated for C19H14N4O2F2: 368.1085, [M]þ, found: 368.1081.
(70 eV, EI): m/z calculated for C24H24N5O2F: 433.1914, [M]þ, found: (%) for C24H23Cl2N5O2: C, 59.51; H, 4.79; N, 14.46. Found: C, 59.85;
433.1927. Anal. Calcd. (%) for C24H24FN5O2: C, 66.50; H, 5.58; N, H, 5.63; N, 14.26.
16.16. Found: C, 66.53; H, 5.86; N, 16.25.
4.1.20. General procedure for the alkylation at N8 of pyrido[2,3-d]
4.1.19.2. 6-(4-fluorophenyl)-2-phenoxy-4-((2-propoxyethyl)amino)
pyrimidines 24{x,y,z}
pyrido[2,3-d]pyrimidin-7(8H)-one (24{2,14,6}). Starting from 23
To a solution of (0.7 mmol) of the corresponding pyrido[2,3-d]
{2,14,6} and following procedure A described in 4.1.6. to afford 24
pyrimidine 24{x,y,z} in 10 mL of anhydrous DMSO, 28.0 mg
{2,14,6} in 75% yield, white solid. IR (KBr), nmax (cm1): 3313, 2933,
(0.7 mmol) of sodium hydride (NaH) (60% dispersion in mineral oil)
2873, 1648, 1612, 1591, 1571, 1510, 1419, 1401, 1264, 1207. 1H NMR
were added and the mixture was stirred for 1 h at room tempera-
(400 MHz, DMSO-d6): d 9.08 (s, 1H, NH), 7.64e7.58 (m, 3H,
ture under nitrogen atmosphere. After this period, (0.7 mmol) of
C12eCHx2, C6eCH), 7.43e7.37 (m, 2H, C17eCHx2), 7.25e7.17 (m,
the corresponding alkyl halide were added dropwise and then
3H, C18eCHx2, C16eCH), 7.07 (t, J ¼ 8.7 Hz, 2H, C13-CHx2), 6.08 (s,
stirred overnight at room temperature. The reaction was quenched
1H, NH), 3.69 (q, J ¼ 5.1 Hz, 2H, C19eCH2), 3.58 (t, J ¼ 5.0 Hz, 2H,
by addition of 300 mL of water and the resulting precipitate was
C20eCH2), 3.42 (t, J ¼ 6.7 Hz, 2H, C21eCH2), 1.64e1.58 (m, 2H,
filtered, washed with water and dried in vacuo over phosphorus
C22.CH2), 0.93 (t, J ¼ 7.4 Hz, 3H, C23eCH3). 13C NMR (100.6 MHz,
pentoxide to afford the corresponding pyrido[2,3-d]pyrimidine 25
DMSO-d6): d 164.8, 162.7 (d, J ¼ 248.1 Hz, C14), 162.4, 160.7, 155.2,
{x,y,z,k}.
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 4.1.20.1. 4-Amino-6-(2,6-dichlorophenyl)-8-ethyl-2-(phenylamino)
(70 eV, EI): m/z calculated for C24H23N4O3F: 434.1754, [M]þ, found: pyrido[2,3-d]pyrimidine-7(8H)-one (25{5,10,4,2}). Starting from 24
434.1758. Anal. Calcd. (%) for C24H23N4O3F: C, 66.35; H, 5.34; N, {5,10,4} and following the general methodology described above to
12.90. Found: C, 66.57; H, 5.31; N, 12.51. 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,
4.1.19.3. 6-(4-fluorophenyl)-2-(phenylamino)pyrido[2,3-d]pyrimi- 1401, 1337, 1279, 1238, 1185, 1030, 832, 803, 772, 753. 1H NMR
dine-4,7(3H,8H)-dione (24{2,10}). Starting from 21{2,10} and (400 MHz, DMSO-d6) d 9.41 (br s, 1H), 8.09 (s, 1H), 7.83 (dd, J ¼ 8.7,
following procedure A described in 4.1.6. to afford 24{2,10} in 78% 1.0 Hz, 2H), 7.60e7.55 (m, 2H), 7.44 (dd, J ¼ 8.7, 7.5 Hz, 1H), 7.38 (br
yield, white solid. IR (KBr), nmax (cm1): 3391, 2924, 2850, 1611, 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
1554, 1497, 1442, 1226. 1H NMR (400 MHz, DMSO-d6): d 12.18 (s, 1H, (q, J ¼ 6.9 Hz, 2H), 1.24 (t, J ¼ 7.0 Hz, 3H). 13C NMR (100.6 MHz,
NH), 10.81 (s, 1H, NH), 9.12 (s, 1H, NH), 7.89 (s, 1H, C6eCH), DMSO-d6) d 161.7, 160.0, 159.2, 155.5, 140.4, 135.3, 135.3, 134.0,
7.78e7.72 (m, 4H, C12eCHx2, C16eCHx2), 7.35 (dd, J ¼ 8.6, 7.3 Hz, 130.2, 128.4, 128.0, 121.6, 120.4, 119.5, 91.6, 36.0, 13.0. MS (70 eV, EI):
2H, C17eCHx2), 7.21 (t, J ¼ 8.9 Hz, 2H, C13eCHx2), 7.14e7.08 (m, m/z (%) ¼ 425.1 (1) [M]þ, 396.1 (1) [M-NEt]þ, 390.1 (2) [M-Cl]þ, 43.1
1H, C18eCH). 13C NMR (100.6 MHz, DMSO-d6): d 162.4, 161.4 (d, (100) [NEt]þ. HRMS (70 eV, EI): m/z calculated for C21H17N5OCl2:
J ¼ 244.6 Hz, C14), 159.7, 155.1, 151.0, 137.9, 133.5 (C7), 132.6 (d, 425.0810, [M]þ, found: 425.0806.
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,
4.1.20.2. 4-Amino-6-(2,6-dichlorophenyl)-2-(phenylamino)-8-
EI): m/z calculated for C19H13N4O2F: 348.1023, [M]þ, found:
propylpyrido[2,3-d]pyrimidine-7(8H)-one (25{5,10,4,3}).
348.1022.
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.
4.1.19.4. 6-(4-fluorophenyl)-2-((4-methoxyphenyl)amino)pyrido[2,3-
IR (KBr), nmax (cm1): 3336, 3141, 2958, 2927, 2872, 1677, 1625,
d]pyrimidine-4,7(3H,8H)-dione (24{2,13}). Starting from 21{2,13}
1574, 1518, 1473, 1447, 1339, 1236, 1185, 1031, 802, 754, 694. 1H NMR
and following procedure A described in 4.1.6. to afford 24{2,13} in
(400 MHz, DMSO-d6) d 9.39 (br s, 1H), 8.09 (s, 1H), 7.82 (dd, J ¼ 8.7,
70% yield as a white solid.1H NMR (400 MHz, DMSO-d6): d 12.07 (s,
1.0 Hz, 2H), 7.61e7.54 (m, 2H), 7.43 (dd, J ¼ 8.7, 7.5 Hz, 1H), 7.35 (br s,
1H, NH), 10.74 (s, 1H, NH), 8.96 (s, 1H, NH), 7.87 (s, 1H, C6eCH), 7.74
2H), 7.31e7.23 (m, 2H), 6.97 (tt, J ¼ 7.3, 1.0 Hz, 1H), 4.31e4.21 (m,
(dd, J ¼ 8.9, 5.6 Hz, 2H, C12eCHx2), 7.62 (d, J ¼ 9.0 Hz, 2H,
2H), 1.76e1.64 (hex, J ¼ 7.4 Hz, 2H), 0.94 (t, J ¼ 7.5 Hz, 3H). 13C NMR
C16eCHx2), 7.20 (t, J ¼ 8.9 Hz, 2H, C13eCHx2), 6.92 (d, J ¼ 9.0 Hz,
(100.6 MHz, DMSO-d6) d 161.6, 160.2, 159.1, 155.6, 140.4, 135.4,
2H, C15eCHx2), 3.76 (s, 3H, OCH3). 13C NMR (100.6 MHz, DMSO-
135.3, 133.9, 130.2, 128.3, 128.0, 121.7, 120.4, 119.5, 91.6, 42.4, 20.8,
d6): d 162.5, 160.1, 159.8, 155.8, 155.3, 151.3, 133.9, 133.6 (C6), 132.7
11.3. HRMS (ESI-TOF): m/z calculated for C22H20N5OCl2: 440.1039,
(d, J ¼ 2.9 Hz, C11), 130.7, 130.1 (d, J ¼ 8.0 Hz, C12), 123.3, 122.4
[MþH]þ, found: 440.1046.
(C16), 114.8 (d, J ¼ 21.2 Hz, C13), 114.1 (C17), 97.0 (C5), 55.3 (C6).
4.1.20.4. 4-Amino-6-(2,6-dichlorophenyl)-8-isobutyl-2-(phenyl- N, 13.79. MS (ESI-TOF): m/z (%) ¼ 498.1 (100) [MþH]þ, 438.1 (13)
amino)pyrido[2,3-d]pyrimidine-7(8H)-one (25{5,10,4,5}). [M-OC3H7]þ. HRMS (ESI-TOF): m/z calculated for C25H26N5O2Cl2:
Starting from 24{5,10,4} and following the general methodology 498.1458, [MþH]þ, found: 498.1460.
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, 4.2. Biology
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), 4.2.1. Cell lines and cell culture
7.82 (dd, J ¼ 8.7, 1.1 Hz, 2H), 7.60e7.55 (m, 2H), 7.44 (dd, J ¼ 8.7, To analyze the antiviral activity of the compounds, we used a
7.5 Hz, 1H), 7.34 (br s, 2H), 7.32e7.23 (m, 2H), 6.98 (tt, J ¼ 7.3, 1.1 Hz, stable cell line, LucUbiNeo-ET, that contains a subgenomic HCV
1H), 4.18 (d, J ¼ 7.4 Hz, 2H), 2.29 (hept, J ¼ 7.0 Hz, 1H), 0.90 (d, genotype 1b replicon, I389/NS3-3’/LucUbiNeo-ET, that express
J ¼ 6.7 Hz, 6H). 13C NMR (100.6 MHz, DMSO-d6) d 161.7, 160.6, 159.0, luciferase constitutively [21,22]. To evaluate the cellular toxicity, we
155.9, 140.4, 135.5, 135.4, 133.9, 130.3, 128.4, 128.1, 121.8, 120.6, use other cell line, Huh-7/Lunet [23], that originally carried a
119.6, 91.7, 47.4, 26.9, 20.0. MS (70 eV, EI): m/z (%) ¼ 453.1 (1) [M]þ, selectable luciferase HCV 1b replicon and was cured by treatment
418.3 (1) [M-Cl]þ, 396.1 (4) [M-isoBut]þ, 57.0 (100) [isoBut]þ. HRMS with an HCV-specific inhibitor. These cells are characterized by
(70 eV, EI): m/z calculated for C23H21N5OCl2: 453.1123, [M]þ, found: high permissiveness for HCV RNA replication. LucUbiNeo-ET cell
453.1128. line was cultured at 37 C in a humidified atmosphere with 5% CO2
in Dulbecco's modified Eagle medium (D-MEM) supplemented
4.1.20.5. 4-Amino-6-(2,6-dichlorophenyl)-8-(2-methoxyethyl)-2- with high glucose concentration (4.5 g/l) (Invitrogen), 10% (vol/vol)
(phenylamino)pyrido[2,3-d]pyrimidine-7(8H)-one (25{5,10,4,6}). heat-inactivated fetal bovine serum (Hyclone, Cultek), 2 mM L-
Starting from 24{5,10,4} and following the general methodology Glutamine (SigmaeAldrich), 1 mM sodium pyruvate (Sigma-
described above to afford 25{5,10,4,6} in 83% yield, brownish solid. aldrich), 1% MEM NEEA (SigmaeAldrich), and 0.5 mg/mL of
IR (KBr), nmax (cm1): 3340, 2925, 1632, 1574, 1523, 1497, 1468, 1447, Geneticin (G418) (Gibco, Invitrogen). Cured or Huh-7/Lunet cell line
1341, 1306, 1235, 1196, 1117, 1028, 801, 771, 754, 692. 1H NMR was maintained at the same conditions as described above without
(400 MHz, DMSO-d6) d 9.42 (br s, 1H), 8.10 (s, 1H), 7.82 (dd, J ¼ 8.7, the addition of Geneticin.
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, 4.2.2. Evaluation of the in vitro antiviral activity (EC50) and
J ¼ 6.6 Hz, 2H), 3.61 (t, J ¼ 6.6 Hz, 2H), 3.27 (s, 3H). 13C NMR cytotoxicity assays (CC50)
(100.6 MHz, DMSO-d6) d 161.8, 160.3, 159.1, 155.9, 140.3, 135.4, Antiviral activity was carried out in white-clear bottom 96-well
135.3, 134.3, 130.4, 128.5, 128.1, 121.9, 120.3, 119.7, 91.7, 68.5, 58.2, plates, and cytotoxicity assays were performed in clear 96-well
40.2. MS (ESI-TOF): m/z (%) ¼ 456.1 (51) [MþH]þ, 424.1 (100) [M- plates. Cells were seeded at a density of 104/well in 100ul D-MEM
OMe]þ. HRMS (ESI-TOF): m/z calculated for C22H20N5O2Cl2: without selection of antibiotics. After 24 h, nine fivefold serial
456.0989, [MþH]þ, found: 456.0988. dilution of compounds, with 3 replicates at each dilution, were
prepared in D-MEM and added to the appropriate wells, yielding
4.1.20.6. 4-Amino-6-(2,6-dichlorophenyl)-8-(2-hydroxyethyl)-2- concentrations of 125 to 0,00032 ug/ml in a final volume of 100 ul
(phenylamino)pyrido[2,3-d]pyrimidine-7(8H)-one (25{5,10,4,7}). of D-MEM. Following 2 days of incubation, we determined the
Starting from 24{5,10,4} and following the general methodology antiviral activity (EC50) by quantifying the luciferase with the
described above with an extra purification consisting of a chro- Luciferase Bright Glo assay (Promega). Briefly, LucUbiNeo-ET cells
matographic separation in silica and CH2Cl2/MeOH as solvent to were harvested with the addition of 100 ul of luciferase per well,
afford 25{5,10,4,7} in 34% yield, brownish solid. IR (KBr), nmax and after 2 min of shaking, the light was read in a Luminometer
(cm1): 3418, 2924, 1634, 1599, 1568, 1525, 1497, 1469, 1447, 1341, Fluoroskan Ascent FL. Toxicity (CC50) was analyzed in the Lunet/
1306, 1235, 1201, 1057, 1015, 802, 772, 691. 1H NMR (400 MHz, HuH7 cells by the addition of 10 ul of MTT (3-(4,5-dimethylthiazol-
DMSO-d6) d 9.40 (br s, 1H), 8.09 (s, 1H), 7.84 (dd, J ¼ 8.7, 1.0 Hz, 2H), 2-yl)-2,5-diphenyltetrazolium bromide) per well. After 4 h of in-
7.60e7.55 (m, 2H), 7.44 (dd, J ¼ 8.7, 7.5 Hz, 1H), 7.38 (br s, 2H), 7.28 cubation at 37 C in a humidified atmosphere with 5% CO2, the
(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, amount of formazan produced was quantified spectrophotometri-
J ¼ 7.1 Hz, 2H), 3.64 (q, J ¼ 6.5 Hz, 2H). 13C NMR (100.6 MHz, DMSO- cally at 550/620 nm [43]. The EC50 and CC50 values were calculated
d6) d 161.8, 160.4, 159.2, 156.0, 140.4, 135.4, 135.3, 134.2, 130.4, 128.6, for each compound. These assays were done in duplicate and the
128.1, 121.8, 120.4, 119.6, 91.7, 58.0, 42.8. MS (ESI-TOF): m/z results shown in Table 1 represent the mean value of these
(%) ¼ 442.1 (100) [MþH]þ, 424.1 (81) [M-OH]þ. HRMS (ESI-TOF): m/ duplicates.
z calculated for C21H18N5O2Cl2: 442.0832, [MþH]þ, found:
442.0839. 4.3. Computational studies
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