Synthesis and biological evaluation of a triazole-based library of pyrido[2,3-d]pyrimidines as FGFR3 tyrosine kinase inhibitors

Article abstract of DOI:10.1039/b923882d

A library of pyrido[2,3-d]pyrimidines was designed as inhibitors of FGFR3 tyrosine kinase allowing possible interactions with an unexploited region of the ATP binding-site. This library was built-up with an efficient step of click-chemistry giving easy access to triazole-based compounds bearing a large panel of substituents. Among the 27 analogues synthesized, more than half exhibited 55-89% inhibition of in vitro FGFR3 kinase activity at 2 μM and one (19g) was able to inhibit auto-phosphorylation of mutant FGFR3-K650M in transfected HEK cells.

Full text of DOI:10.1039/b923882d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological evaluation of a triazole-based library of
pyrido[2,3-d]pyrimidines as FGFR3 tyrosine kinase inhibitors†
Laurent Le Corre,a Anne-Lise Girard,a Johannes Aubertin,b Franc¸ois Radvanyi,b Catherine Benoist-Lasselin,c
Aure´lie Jonquoy,c Emilie Mugniery,c Laurence Legeai-Mallet,c Patricia Busca*a and Yves Le Merrer*a
Received 13th November 2009, Accepted 17th February 2010
First published as an Advance Article on the web 11th March 2010
DOI: 10.1039/b923882d
A library of pyrido[2,3-d]pyrimidines was designed as inhibitors of FGFR3 tyrosine kinase allowing
possible interactions with an unexploited region of the ATP binding-site. This library was built-up with
an efficient step of click-chemistry giving easy access to triazole-based compounds bearing a large panel
of substituents. Among the 27 analogues synthesized, more than half exhibited 55–89% inhibition of
in vitro FGFR3 kinase activity at 2 mM and one (19g) was able to inhibit auto-phosphorylation of
mutant FGFR3-K650M in transfected HEK cells.
Introduction
Receptor tyrosine kinases (RTKs) play an important role in
proliferation, motility and differentiation, and can therefore
become potent oncoproteins when mutated or overexpressed.1 The
family of fibroblast growth factor receptor (FGFR), one of the
twenty sub-classes of RTKs, consists of four homologous receptors
numbered FGFR1-4.2 Initially identified at the germinal level,
activating mutations within FGFR3 are responsible for human
skeletal disorders3 ranging from hypochondroplasia (the mildest
form)4 to thanatophoric dysplasia (the neonatal lethal form)5
through achondroplasia (the most frequent form).6 Interestingly,
several mutations involved in chondrodysplasia were also identi-
fied somatically in human tumours.7 It is now well established that
FGFR3 can act as an oncogene in bladder cancers,8 as well as
in multiple myeloma,9 cervical10 or prostate11 cancers and benign
skin tumours.12
Over the years, extensive efforts have been made to developATP-
competitive inhibitors as target-directed therapeutics for RTK-
associated diseases, mostly in the field of cancer.13 Rational design
Fig. 1 Pharmacophore model of ATP binding site.
of such inhibitors usually refers to the general pharmacophore
FGFR1 and VEGFR2,16 and was more recently identified as an
model built up by Traxler and Furet14 showing that the highly
FGFR3 inhibitor.17 The crystal structure of unphosphorylated
conserved ATP binding site is divided into five subregions:
FGFR1 with PD17307418 (Fig. 2a) has been determined (2FGI)
hydrophobic regions I and II, the adenine, the ribose and the
and the electron density map shows good supporting density for
phosphate-binding regions (Fig. 1). We observed the same topol-
ogy in the 3D-structure of the kinase domain of FGFR3 according
to a homology-based model.15 Amongst the plethora of inhibitors
the pyrido[2,3-d]pyrimidine ring system, the dimethoxyphenyl (6-
position) and tert-butyl urea (7-position) groups, but shows weaker
density for the diethylaminobutylamino chain (2-position).
reported so far, PD173074, a compound from the pyrido[2,3-
Such electron density is consistent with a high contribution to
d]pyrimidine class, was found to exhibit high potency against
potency of aromatic moieties that occupy the adenine binding site
and hydrophobic pocket I with an excellent surface complemen-
tarity, and a neglectable contribution for the aliphatic butyl chain
that lies in hydrophobic pocket II.19
aUniversite´ Paris Descartes, UMR 8601-CNRS, 45 rue des Saints-Pe`res,
75006 Paris, France. E-mail: yves.le-merrer@parisdescartes.fr, patricia.
busca@parisdescartes.fr; Fax: + 33-142868387; Tel: + 33-142862176
In this context, we became interested in modifying this alkyl-
amino group at position 2 in order to establish new interactions
with the large cavity that encompasses hydrophobic and ribose
pockets. For example, when co-crystallized with FGFR1 (1FGI),20
SU5402, which is another FGFR3 inhibitor, exhibits a pyrrole ring
in hydrophobic pocket II with the methyl group in Van der Waals
contact with Gly 567 and a carboxylate group hydrogen bonded
with Asn 568, which is part of the ribose pocket (Fig. 2b). We
bCNRS UMR144, Institut Curie, 26 rue d’Ulm, 75005 Paris, France. E-mail:
francois.radvanyi@curie.fr; Fax: + 33-156246349; Tel: +33-156246339
cUniversite´ Paris Descartes, INSERM U781, Hoˆpital Necker-Enfants
Malades, 149 rue de Se`vres, 75015 Paris, France. E-mail: laurence.
legeai-mallet@inserm.fr; Fax: + 33-1473485 14; Tel: + 33-144494000 ext
97833/97830
† Electronic supplementary information (ESI) available: 1. Numbering
system, 2. Comprehensive experimental section, 3. 1H and 13C NMR
Spectra. 4. Biological data. See DOI: 10.1039/b923882d
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Scheme 1 Rationale for the synthesis of clicked analogs.
Based on the above considerations and our ongoing efforts
to develop FGFR3 inhibitors,27 we report herein the synthesis
of the first “click” library of RTK inhibitors based on the
pyrido[2,3-d]pyrimidine skeleton of the PD173074 structure and
their biological evaluations.
Results and discussion
Chemical Synthesis
Fig. 2 X-Ray analysis of FGFR1-inhibitors crystals.
Our aim was to build a library of triazole-based inhibitors through
efficient click-chemistry connection between the core structure of
PD173074 and various substituents. For this purpose, we prepared
a collection of azide building blocks according to different
literature protocols, depending on the commercial availability of
the starting material (Scheme 2).
Azides 228 and 429 were prepared by standard nucleophilic
substitution from their parent bromide 1 and chloride 3, re-
spectively, whereas alcohol 5 was converted to azide 6 using a
facile one-pot reaction.30 Guanidino derivative 9 was obtained
from azidoamine 8 treated with a guanylation agent.31 We first
attempted to synthesize 8 from ethane-1,2-diamine according to
a procedure previously described in the literature.32 However, we
found out that starting from the corresponding bromoamine 7
was a more efficient pathway. Of note, bromine displacement was
followed by N-Boc protection in order to facilitate the purification
step. Moreover, N-Boc was cleaved with TFA, which allowed
a better storage of azido-amine 8 as its ammonium salt. The
synthesis of azides 11 was achieved in two steps via standard
conversion of alcohols 10 to mesylate or tosylate intermediates,
therefore aimed at the synthesis of PD173074 analogs bearing
in position 2 a large panel of substituents, in terms of size and
polarity. Such compounds could lead to enhanced activity and/or
selectivity. A complementary study, focusing on heteroaromatic
substituents has been recently reported by Sanofi Aventis.21 In
order to quickly obtain a small library of inhibitors with a
large structural diversity, we decided to introduce modifications
through click reaction, as depicted in Scheme 1. As a matter of
fact, since Sharpless introduced the click chemistry concept,22 the
Huisgen [3+2] cycloaddition has proved to be a powerful tool
in drug discovery.23 Beside synthetic interest, the resulting 1,2,3-
triazole unit is not just a passive linker but a rather active phar-
macophore that may significantly contribute to protein binding.24
Moreover, 1,2,3-triazole moiety has been used with success to
optimize ZD4190, a VEGFR inhibitor from the quinazoline
family25 and very recently to discover a new PfPK7 protein kinase
inhibitor.26
Scheme 2 Reagents and conditions: (a) NaN3, DMF, 60 ◦C, 48 h, 51%. (b) NaN3, H2O, reflux, 24 h, 36%. (c) NaN3, PPh3, CCl4/DMF, 90 ◦C, 1 h, 56%.
(d) i. NaN3, H2O, 70 ◦C, 5 h; ii. KOH, Boc2O, tBuOH/H2O, r.t., 24 h, 95%. (e) TFA, DCM, r.t., 1 h, 95%. (f) DIPEA, BocNH-C(NBoc)-Im, r.t., 24 h,
64%. (g) i. BuLi, TsCl, THF, 70 ◦C, 15 h (11a) or MsCl, TEA, DCM (11b, c); ii. NaN3, DMF, 90 ◦C, 5 h. (h) MsCl, TEA, DCM, 0 ◦C, 1 h then r.t., 2 h,
76%. (i) NaN3, MeCN, reflux, 24 h, 38%. (j) R2NH, TEA, MeCN, reflux, 16–24 h.
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Scheme 3 Reagents and conditions: (a). 28% aq NH4OH, TEA, THF, r.t., 2 h 30, 76%. (b) LiAlH4, THF, 25 ◦C, 45 min, 78%. (c) MnO2, CHCl3, r.t., 16 h,
93%. (d) NaH, ArCH2CN, DMF, 5 ◦C, 5 h then r.t., 18 h, 91%. (e) NaH, tBuNCO, DMF, 0 ◦C, 8 h then r.t., 16 h, 90%. (f) mCPBA, CHCl3, 0 ◦C, 2h30,
80%. (g) propargylamine, dioxane, 50 ◦C, 24 h, 86%. (h) RN3, CuSO4·H2O, sodium ascorbate, tBuOH/H2O (2 : 1), r.t., 20 h-3 d. (i) 3M HCl, dioxane.
which were in turn reacted with sodium azide, affording 11a27 in
moderate yield compared to 11b, c. The last set of azides 14 was
prepared from ethylene glycol 12 via a route adapted from the
literature.33 Thus, after bis-mesylation of diol 12, one of the mesyl
groups was displaced by sodium azide to give intermediate 13,
and the other one was substituted with commercially available
amines providing 14a–e in good to excellent yields. We next
turned our attention to the synthesis of the PD173074 core
structure bearing an alkyne handle in 2-position. Interestingly, the
synthetic pathway previously reported for the pyridopyrimidine
scaffold allows the introduction of various primary amines in this
position,21 giving us the opportunity to use propargylamine as
alkyne carrier. Therefore, key derivative 17 was prepared following
a literature procedure, the main steps of which are summarized in
Scheme 3.
Starting from commercially available 4-chloro-5-carbethoxy-2-
methylthiopyrimidine 15, nucleophilic substitution of chlorine
with aqueous ammonia followed by reduction of the ester to
alcohol with lithium-aluminium hydride and oxidation with
manganese oxide afforded amino-aldehyde 16 in high yield.
Condensation of the aldehyde with dimethoxyphenyl-acetonitrile
and subsequent protection of the amine were achieved prior to
oxidation of methyl sulfide in sulfone, leading to pyridopyrimidine
range of azides reacted smoothly under these conditions with
good to excellent yields. In the particular case of 19c, a further
step of acidolysis allowed the removal of Boc protecting groups,
furnishing guanidino derivative 19d as an hydrochloride salt.
Next, with the aim of studying the influence of chain length, we
undertook the synthesis of various pyridopyrimidine derivatives
22a–c bearing an alkyne portion tethered by 2, 3 or 4 methylene
groups as shown in Scheme 4. Indeed, a flexible linker may allow
substituents to interact very nicely with surrounding residues in the
ATP-binding site. Thus, the homopropargyl alcohol and its higher
homologous 20 were converted to their corresponding mesylates
which were in turn reacted with sodium azide as described in
the literature.34 In order to facilitate the purification step, the
reduction of the azido group was achieved with ethylenebis
(diphenylphosphine) instead of triphenylphosphine. In addition,
difficulties to isolate free amines were circumvented via the
formation of their hydrochloride salts. This modified methodology
provided aminoalkyne 21b–c in excellent yields, except for 21a,
probably because of its low molecular weight. Subsequent SNAr
with sulfone 17 was carried out in presence of DIPEA and
afforded pyridopyrimidine 22a–c. Further 1,3-cycloadditions were
performed under usual conditions to afford new triazoles 23a–f.
Finally, we were interested in obtaining the azido-analog of
alkyne 18 in order to examine the impact of triazole orientation.
Unfortunately, the lack of synthetic method to prepare such
azido-methanamine derivative restricted us to the synthesis of
homologous 24 bearing a two methylene spacer as shown in
Scheme 5. For this purpose, sulfone 17 was reacted with azi-
doamine 8 in presence of DIPEA to afford pyridopyrimidine 24.
Click experiments were realized using a series of commercially
available alkynes 25a–d whereas 25e was prepared by guanylation
of propargylamine. Cycloadditions were performed under usual
conditions, as described above, and readily afforded triazoles
26a–f. In the particular case of 26e, a further acidolysis step
allowed the removal of Boc protecting groups, yielding the
guanidino derivative 26f as an hydrochloride salt.
R
17.Attempts to perform the oxidation step with oxoneꢀ instead
of mCPBA did not result in yield increase. The sulfone was in
turn reacted with propargylic amine to provide 2-alkynylamino
derivative 18. Having alkyne 18 and azide partners in hand,
we investigated the click assembling reaction. According to the
standard procedure, we performed 1,3-dipolar cycloaddition with
a slight excess of azido reactants, CuSO4 as copper source and
sodium ascorbate as reducing agent. Thus, as expected for the
Cu(I) catalyzed version of Huisgen reaction, these conditions
produced 1,4-disubstituted triazoles 19 as single products. We
also found out that tBuOH/H2O was the best solvent system
and most of the click reactions were completed within 20 h
at room temperature. As can be seen in Scheme 3, the overall
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Scheme 4 Reagents and conditions: (a) i. MsCl, TEA, DCM, 0 ◦C, 3 h; 55% (n = 2), 100% (n = 3), 91% (n = 4). (b) NaN3, DMF, 80 ◦C, 3 h 30; 61%
(n = 2), 100% (n = 3), 52% (n = 4). (c) i. Ph2P-(CH2)2-PPh2, THF, H2O, r.t., 16h; ii. 10% HCl; 44% (n = 2), 100% (n = 3), 61% (n = 4). (d) 17, DIPEA,
dioxane, 70 ◦C, 20 h. (e) RN3, CuSO4·H2O, sodium ascorbate, tBuOH/H2O (2 : 1), r.t., 20 h-3 d.
◦
≡
Scheme 5 Reagents and conditions: (a) DIPEA, 8, dioxane, 50 C, 24 h, 80%. (b) RC CH, CuSO4·H2O, sodium ascorbate, tBuOH/H2O (2 : 1), r.t., 20 h.
(c) 3 M HCl, dioxane.
Biological evaluations
Finally, focusing on the most potent derivatives (i.e. residual
kinase activity < 25% at 2 mM) highlights that the best triazole
substitution is either an aromatic ring (i.e. pyridine 19j or
phenol 19h) or an aza-containing heterocycle (i.e. pyrrolidine 19k,
piperidine 19g or piperazine 19f). Of note, any further substitution
of these moieties resulted in decreased inhibition potency (19l–m
versus 19f and 19i versus 19h).
Within this 1st series, pyridine 19j, pyrrolidine 19k and piperi-
dine 19g were chosen as points of reference for the synthesis of
the 2nd set of analogues. These compounds bear a spacer, the
length of which varies from two to four carbon atoms. All of the 9
analogues (22a–23f) were less active than their parent compounds,
respectively, indicating that increasing the chain length had a
negative effect on potency.
Finally, the 3rd group of derivatives, built up to examine the
impact of the triazole orientation, was examined. All of the 5
compounds 26a–f exhibited a complete loss of activity, indicating
that triazole geometry may be crucial for biological activity.
Amongst the most promising inhibitors, we next selected 19g
and 19h for further cellular assays. To examine the FGFR3
tyrosine phosphorylation level after inhibitor treatment, we used
HEK cells transfected with FGFR3-K650M construct,35 K650M
being an activating mutation responsible for SADDAN, a severe
achondroplasia. Immunoblot analysis reported in Fig. 4 shows
that after treatment at 2 mM for one night, the level of phos-
phorylated FGFR3 was still detected with 19h (lane 2) whereas
The inhibitory potency of all target compounds was examined
in vitro at 500 nM and 2 mM using the recombinant FGFR3
kinase domain. PD173074 was used as the reference inhibitor and
the amount of residual phosphotyrosine products was estimated
by measurement of absorbance at 450 nm (see experimental section
for details). Ranking of inhibitor activities was similar for 500 nM
and 2 mM inhibitor concentration (see ESI, section 4†). For the
sake of clarity, only results observed at 2 mM are shown in Fig. 3.
The general trend shows that most of the active compounds
belongs to the first series (19a–19m, Scheme 3) and the less active
compounds are mainly part of the last series (26a–f, Scheme 5).
All of the 13 analogues tested within the 1st series were able to
inhibit phosphorylation at 2 mM and most of them with similar
potency to the parent PD173074.
Therefore, introduction of 1,2,3-triazole moiety in position 2 of
the pyridopyrimidine scaffold seems to have no significant negative
effect on potency. However, alkyne 18 is unexpectedly one of the
most potent inhibitors, which is nevertheless consistent with the
hydrophobicity of region II. On the other hand, compound 19b
is the only one within this 1st series to display less than 50%
inhibition at 2 mM, indicating that the carboxylic acid function,
which is negatively charged at physiological pH, is less tolerated
than other functions (phenol, alkoxy, guanidine and amine) which
are neutral or positively charged under physiological conditions.
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Fig. 3 Percentage of in vitro activity of recombinant FGFR3 kinase domain in presence of synthesized compounds at 2 mM; values are presented as
means of triplicates, error bars indicate one standard deviation. Percentage was calculated relative to the DMSO control.
Experimental
Chemical Synthesis
All non aqueous reactions were carried out under an argon
atmosphere. All commercial reagents and anhydrous solvents were
purchased from Aldrich or Acros and were used without further
purification. Thin layer chromatography (TLC) was performed
with Merck 60F-254 precoated silica (0.2 mm) on glass. Flash
column chromatography was performed with Merck Kieselgel 60
(40–63 mm). NMR spectra were recorded on a Bruker AM250 or
a Bruker Avance II 500 instrument in CDCl3 at 298 K (unless
indicated) and chemical shifts (d) are reported in ppm. Mass
spectra, electrospray (ESI) and high resolution (HRMS) were
recorded by the Service de Spectrome´trie de Masse, ICSN, Gif
sur Yvette. Elemental analyses were performed by the Service de
Microanalyse, ICSN, Gif sur Yvette.
Fig. 4 Immunodetection of phosphorylated FGFR-3 in HEK transfected
cells without (lane 1) or with a tyrosine kinase inhibitor treatment (lanes 2,
3 and 4 with 19h, 19g and PD173074, respectively). Immunoprecipitation
(IP) and immunoblotting (IB) protocols are detailed in the experimental
section.
it was strongly reduced with 19g and PD173074 (lanes 3 and 4,
respectively) compared to the level of unphosphorylated FGFR3
which was the same in the four cellular extracts (data not shown).
Discrepancy of inhibition may reveal that 19h, bearing a phenol
substituent, is less able to pass through the cellular membrane than
PD173074 and 19g, that both bear an alkylamino substituent.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-(prop-2-ynylamino)-
pyrido[2,3-d]pyrimidin-7-yl)urea (18). A mixture of 1721 (3.81 g,
8.29 mmol) and propargylamine (1.42 mL, 20.73 mmol) in dry
Conclusions
◦
The present study identifies a new class of FGFR3 kinase
inhibitors, designed from a pyrido[2,3-d]pyrimidine central core
to examine possible interactions with an unexplored region of
the ATP binding-site. The key step of click- chemistry allowed
a rapid and efficient synthesis of various analogues bearing a
large panel of substituents in terms of size and polarity. Among
the 27 analogues synthesized, more than half exhibited 55–89%
inhibition of in vitro FGFR3 kinase activity at 2 mM and one was
able to inhibit auto-phosphorylation of mutant FGFR3-K650M
in transfected HEK cells. The present investigation highlights that
the targetted region of the ATP binding-site accepts 1,2,3-triazole
moiety, with a preference for positively charged substituents.
dioxane (65 mL) was heated at 50 C for 20 h. The solvent was
then concentrated in vacuo and the crude residue was purified
by flash column chromatography (CH2Cl2–MeOH 98 : 2) to give
alkyne 18 (3.1 g, 86%) as a white solid. Rf 0.26 (CH2Cl2–MeOH
1
98 : 2); H NMR: d 10.23 (br s, 1H, NHCO), 8.78 (s, 1H, H-4),
7.71 (s, 1H, H-5), 7.15 (br s, 1H, NHtBu), 6.56–6.44 (m, 3H, H-2¢,
H-4¢, H-6¢), 5.56 (br t, J = 5.6, 1H, NHCH2), 4.39 (dd, J = 5.6,
2.5, 2H, H-1¢¢), 3.83 (s, 6H, OCH3), 2.27 (t, J = 2.5, 1H, H-3¢¢),
1.53 (s, 9H, CMe3); 13C NMR: d 162.1, 161.9, 161.1, 158.6, 155.9,
152.8, 137.2, 136.7, 123.3, 110.3, 107.4, 101.0, 80.5, 71.3, 55.7,
51.1, 31.5, 29.1; HRMS (ESI): Calc. for C23H26N6O3Na [M+Na]+
457.1964, found 457.1945.
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General procedure for the cycloaddition reactions
(m, 8H, OCH3, H-b), 1.42 (s, 9H, CMe3); MS (ESI): m/z = 563
[M-HCl +H]+ 100%.
A mixture of alkyne (1 eq.), azide (1 eq.), sodium ascor-
bate (0.2 eq.), CuSO4·5H2O (0.1 eq.) in 2 : 1 tBuOH/H2O
(26 mL/1 mmol) was vigorously stirred at room temperature
for several h (TLC control). Typical reaction times are from
20 h to 3 d. After disappearance of alkyne, the mixture was
diluted with CH2Cl2 and washed with water, dried (MgSO4) then
concentrated in vacuo. Purification of the crude residue afforded
the corresponding triazoles.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-((1-(2-(piperidin-1-
yl)ethyl)-1H -1,2,3-triazol-4-yl)methylamino)pyrido[2,3-d]pyrim-
idin-7-yl)urea (19e). Rf 0.37 (CH2Cl2–MeOH/NEt3 90 : 10 : 0.5);
1H NMR: d 10.31 (br s, 1H, NHCO), 8.73 (br s, 1H, H-4), 7.75
(br s, 1H, Htri), 7.68 (s, 1H, H-5), 7.13 (br s, 1H, NHtBu), 6.55–6.40
(m, 3H, H-2¢, H-4¢, H-6¢), 5.96 (br t, J = 6.0, 1H, NHCH2), 4.84
(d, J = 6.0, 2H, H-1¢¢), 4.42 (t, J = 6.3, 2H, H-a), 3.80 (s, 6H,
OCH3), 2.73 (t, J = 6.3, 2H, H-b), 2.45–2.30 (m, 4H, Hpip), 1.53–
1.28 (m, 15H, CMe3, Hpip); 13C NMR: d 162.4, 161.8, 161.1, 158.6,
155.7, 152.8, 145.3, 137.3, 136.7, 123.1, 122.8, 110.0, 107.4, 100.9,
58.3, 55.7, 55.6, 54.6, 51.0, 47.9, 37.2, 29.1, 25.9, 24.2; Anal. Calc.
for C23H26N6O3·0.7H2O: C, 59.92; H, 6.94; N, 23.29; found: C,
59.90; H, 6.89; N, 23.24; HRMS (ESI): Calc. for C30H40N10O3Na
[M+Na]+ 611.3183, found 611.3181.
1-tert-Butyl-3-(2-((1-(2-(diethylamino)ethyl)-1H-1,2,3-triazol-
4 - yl)methylamino) - 6 - (3,5 - dimethoxyphenyl)pyrido[2,3 - d]pyri-
midin-7-yl)urea (19a). Rf 0.24 (CH2Cl2–MeOH 93 : 7); 1H NMR:
d 10.33 (br s, 1H, NHCO), 8.75 (s, 1H, H-4), 7.72 (br s, 1H, Htri),
7.68 (s, 1H, H-5), 7.15 (br s, 1H, NHtBu), 6.56–6.44 (m, 3H, H-2¢,
H-4¢, H-6¢), 5.94 (t, J = 5.9, 1H, NHCH2), 4.86 (d, J = 5.9, 2H,
H-1¢¢), 4.37 (t, J = 6.4, 2H, H-a), 3.89 (s, 6H, OCH3), 2.85 (t, J =
6.4, 2H, H-b), 2.51 (q, J = 7.1, 4H, CH2CH3), 1.49 (s, 9H, CMe3);
1.01 (t, J = 7.1, 6H, CH2CH3); 13C NMR: d 162.4, 161.8, 161.1,
158.5, 155.7, 152.8, 145.1, 137.2, 136.7, 123.1, 122.8, 110.0, 107.3,
100.8, 55.7, 55.6, 53.1, 51.0, 49.2, 47.4, 37.2, 29.1, 12.0; Anal. Calc.
for C29H40N10O3: C, 60.40; H, 6.99; N, 24.29; found: C, 59.98; H,
7.13; N, 24.16; HRMS (ESI): Calc. for C29H40N10O3Na [M+Na]+
599.3183, found 599.3144.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-((1-(2-(4-methylpip-
erazin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methylamino)pyrido[2,3-
d]pyrimidin-7-yl)urea (19f). Rf 0.12 (CH2Cl2–MeOH/NEt3
1
90 : 10 : 0.3); H NMR: d 10.31 (br s, 1H, NHCO), 8.79 (s, 1H,
H-4), 7.78 (s, 1H, Htri), 7.73 (s, 1H, H-5), 7.19 (s, 1H, NHtBu),
6.60–6.45 (m, 3H, H-2¢, H-4¢, H-6¢), 6.00 (br t, J = 5.5, 1H,
NHCH2), 4.86 (d, J = 5.5, 2H, H-1¢¢), 4.47 (t, J = 6.5, 2H, H-
a), 3.86 (s, 6H, OCH3), 2.84 (t, J = 6.5, 2H, H-b), 2.65–2.30 (m,
8H, Hpip), 2.24 (s, 3H, NCH3), 1.53 (s, 9H, CMe3); 13C NMR: d
162.4, 161.8, 161.2, 158.6, 155.7, 152.8, 145.4, 137.3, 136.7, 123.1,
122.8, 110.1, 107.3, 100.8, 57.6, 55.7, 55.6, 55.0, 53.1, 51.0, 47.9,
46.0, 37.2, 29.2; HRMS (ESI): Calc. for C30H41N11O3Na [M+Na]+
626.3292, found 626.3284.
3-(4-((7-(3-tert-Butylureido)-6-(3,5-dimethoxyphenyl)pyrido-
[2,3-d]pyrimidin-2-ylamino) methyl)-1H -1,2,3-triazol-1-yl)pro-
panoic acid (19b). Rf 0.17 (CH2Cl2–MeOH 90 : 10); 1H NMR: d
10.18 (br s, 1H, NHCO), 8.84 (br s, 1H, H-4), 7.95 (br s, 1H, Htri),
7.69 (br s, 1H, H-5), 6.55–6.38 (m, 3H, H-2¢, H-4¢, H-6¢), 4.93 (br s,
2H, H-1¢¢), 4.69 (t, J = 5.0, 2H, H-a), 3.80 (s, 6H, OCH3), 2.85 (m,
2H, H-b), 1.45 (s, 9H, CMe3); 13C NMR: d 175.3, 162.5, 161.9,
161.7, 156.2, 152.6, 145.2, 137.3, 136.4, 123.6, 123.2, 109.7, 107.4,
101.0, 55.7, 55.6, 51.5, 45.9, 37.9, 35.1, 29.2; MS (ESI): m/z = 550
[M+H]+ 100%; HRMS (ESI): Calc. for C26H31N9O5Na [M+Na]+
572.2346, found 572.2358.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-((1-((1-methylpiperi-
din-3-yl)methyl)-1H -1,2,3-triazol-4-yl)methylamino)pyrido[2,3-
d]pyrimidin-7-yl)urea (19g). Rf 0.17 (CH2Cl2–MeOH 90 : 10); 1H
NMR: d 10.30 (br s, 1H, NHCO), 8.75 (br s, 1H, H-4), 7.69 (br s,
1H, H-5), 7.61 (br s, 1H, Htri), 7.16 (br s, 1H, NHtBu), 6.51–6.47
(m, 3H, H-2¢, H-4¢, H-6¢), 6.25 (br s, 1H, NHCH2), 4.85 (d, J =
5.7, 2H, H-1¢¢), 4.24 (d, J = 7.1, 2H, H-a), 3.81 (s, 6H, OCH3),
2.19 (s, 3H, NCH3), 1.48 (s, 9H, CMe3), 2.68–1.00 (m, 8H, Hpip);
13C NMR: d 162.6, 162.1, 161.4, 158.8, 156.0, 153.0, 145.8, 137.4,
136.9, 123.2, 122.9, 110.4, 107.7, 101.2, 59.3, 56.2, 56.1, 55.9, 54.0,
51.2, 46.7, 37.5, 37.4, 29.4, 27.8, 24.5; HRMS (ESI): Calc. for
C30H40N10O3Na [M+Na]+ 611.3183, found 611.3165.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-((1-(2-(N,N¢-bis-
(tert-butoxycarbonyl)guanidino)ethyl)-1H-1,2,3-triazol-4-yl)meth-
ylamino)pyrido[2,3-d]pyrimidin-7-yl)urea hydrochloride (19c). Rf
0.23 (CH2Cl2–MeOH 96 : 4); 1H NMR: d 11.42 (s, 1H, NHBoc),
10.31 (br s, 1H, NHCO), 8.76 (s, 1H, H-4), 8.53 (t, J = 5.3, NHgua),
7.71 (s, 1H, Htri), 7.69 (s, 1H, H-5), 7.18 (s, 1H, NHtBu), 6.60–6.40
(m, 3H, H-2¢, H-4¢, H-6¢), 5.99 (br s, 1H, NHCH2), 4.89 (d, J =
5.5, 2H, H-1¢¢), 4.55 (t, J = 5.3, 2H, H-a), 3.91 (m, 2H, H-b), 3.82
(s, 6H, OCH3), 1.49 and 1.47 (s, 27H, CMe3); 13C NMR: d 163.3,
162.3, 161.7, 161.1, 158.4, 156.4, 155.7, 153.0, 152.7, 145.6, 137.2,
136.6, 122.8, 110.0, 107.3, 100.8, 83.5, 79.6, 55.6, 51.0, 49.3, 40.6,
37.1, 29.1, 28.3, 28.1; MS (ESI): m/z = 785 [M+Na]+ 100%.
1-(2-((1-(4-Hydroxybenzyl)- 1H -1,2,3-triazol-4-yl)methyl-
amino)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl)-3-
1
tert-butylurea (19h). Rf 0.17 (CH2Cl2–MeOH 95 : 5); H NMR
(500 MHz, CDCl3+CD3OD): d 8.97 (br s, 1H, H-4), 7.66 (s, 1H,
H-5), 7.50 (s, 1H, Htri), 7.01 (d, J = 7.0, 2H, H-b¢, H-f¢), 6.69 (d,
J = 7.0, 2H, H-c¢, H-e¢), 6.55–6.35 (m, 3H, H-2¢, H-4¢, H-6¢), 5.31
(s, 2H, H-a), 4.70 (br s, 2H, H-1¢¢), 3.77 (s, 6H, OCH3), 1.37 (s, 9H,
CMe3); 13C NMR (125 MHz, CDCl3+CD3OD): d 161.9, 161.8,
161.4, 157.5, 155.5, 153.1, 145.0, 137.4, 136.3, 129.7, 125.2, 123.1,
122.4, 115.9, 109.9, 107.3, 101.0, 55.5, 53.9, 51.1, 36.8, 28.8; MS
(ESI): m/z = 583 [M]+ 100%.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-((1-(2-guanidino-
ethyl)-1H-1,2,3-triazol-4-yl)methylamino)pyrido[2,3-d]pyrimidin-
7-yl)urea hydrochloride (19d). To a solution of triazole 19c
(30 mg, 0.039 mmol) in dioxane (3 mL) was added 3 N HCl
(1.3 mL) and the mixture was stirred at room temperature for 3 h
30 then concentrated in vacuo. The residue was triturated with
Et2O and the solid was filtered then dried under vacuum to afford
guanidine 19d (22 mg, 93%) as a yellow solid. 1H NMR (CD3OD):
d 9.09 (s, 1H, H-4), 8.41 (s, 1H, Htri), 8.13 (s, 1H, H-5), 6.80–6.60
(m, 3H, H-2¢, H-4¢, H-6¢), 4.90–4.60 (m, 4H, H-a, H-1¢¢), 3.95–3.75
1-(2-((1-(Benzo[d][1,3]dioxol-5-ylmethyl)-1H -1,2,3-triazol-4-
yl)methylamino)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-
1
7-yl)-3-tert-butylurea (19i). Rf 0.21 (CH2Cl2–MeOH 95 : 5); H
NMR (DMF): d 10.41 (br s, 1H, NHCO), 9.02 (br s, 1H, NHtBu),
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8.13 (br s, 1H, C-5¢), 8.08 (s, 1H, H-5), 8.02 (s, 1H, H-4), 7.29 (br s,
1H, NHCH2), 6.98–6.82 (m, 3H, H-b¢, H-e¢, H-f¢), 6.78–6.64 (m,
3H, H-2¢, H-4¢, H-6¢), 6.05 (s, 2H, OCH2O), 5.57 (s, 2H, H-a), 4.81
(m, 2H, H-1¢¢), 3.89 (s, 6H, OCH3), 1.43 (s, 9H, CMe3); 13C NMR
(DMF): d 163.6–162.9, 159.2, 156.2, 153.4, 149.0, 148.6, 147.8,
139.2, 138.5, 131.2, 123.8, 122.9, 122,7, 111.1, 109.5, 109.3, 108.3,
102.6, 101.3, 56.4, 56.3, 53.9, 51.3, 37.9, 29.5; HRMS (ESI): Calc.
for C31H33N9O5Na [M+Na]+ 634.2502, found 634.2488.
H-1¢¢), 4.45 (t, J = 6.0, 2H, H-a), 3.89 (m, 2H, CH2-pyrrole), 3.82
(s, 6 H, OCH3), 2.86 (t, J = 6.0, 2H, H-b), 2.63–2.40 (m, 10H, Hpip
,
CH2-piperazine), 2.17 (s, 6H, CH3-pyrrole), 1.49 (s, 9H, CMe3);
13C NMR: d 162.4, 161.8, 161.2, 158.6, 155.7, 152.8, 145.3, 137.2,
136.6, 127.4, 122.9, 122.7, 110.1, 107.4, 105.4, 100.8, 58.4, 57.6,
55.7, 55.6, 53.6, 53.1, 51.0, 47.8, 41.5, 37.3, 29.1, 12.6; MS (ESI):
m/z = 712 [M+H]+ 100%; HRMS (ESI): Calc. for C37H50N12O3Na
[M+Na]+ 733.4034, found 733.4027.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-((1-(pyridin-2-yl-
methyl)-1H -1,2,3-triazol-4-yl)methylamino)pyrido[2,3-d]pyrimi-
din-7-yl)urea (19j). 1H NMR: d 10.26 (br s, 1H, NHCO), 8.72
(s, 1H, H-4), 8.56 (d, J = 4.9, 1H, H-f¢), 7.76 (br s, 1H, Htri),
7.67 (s, 1H, H-5), 7.65 (td, J = 7.8, 1.7, 1H, H-d¢), 7.28–7.10 (m,
3H, H-e¢, H-c¢, NHtBu), 6.53–6.43 (m, 3H, H-2¢, H-4¢, H-6¢), 5.93
(t, J = 5.8, 1H, NHCH2), 5.61 (s, 2H, H-a), 4.85 (d, J = 5.8,
2H, H-1¢¢), 3.80 (s, 6H, OCH3), 1.43 (s, 9H, CMe3); 13C NMR: d
162.4, 161.8, 161.2, 158.6, 155.7, 154.6, 152.8, 149.9, 146.0, 137.4,
137.3, 136.7, 123.5, 123.1, 122.9, 122.4, 110.1, 107.5, 100.9, 55.8,
55.7, 55.6, 51.0, 37.3, 29.1; MS (ESI): m/z = 591 [M+Na]+ 100%;
HRMS (ESI): Calc. for C29H32N10O3Na [M+Na]+ 591.2557, found
591.2554.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-(but-3-ynylamino)-
pyrido[2,3-d]pyrimidin-7-yl)urea (22a). To a solution of 1721
(414 mg, 0.90 mmol) in dry dioxane (10 mL) was added 3-
butyn-1-amine hydrochloride 21a (95 mg, 0.90 mmol) and DIPEA
(0.31 mL, 1.81 mmol). The mixture was heated at 70 ◦C for 20 h,
and then concentrated in vacuo. The crude residue was diluted with
CH2Cl2, washed with a saturated solution of NaHCO3 (20 mL)
and water (20 mL), dried (MgSO4) then concentrated in vacuo.
Purification by column chromatography (CH2Cl2–MeOH 98 : 2)
gave alkyne 22a (251 mg, 60%) as a yellow solid. Rf 0.40 (CH2Cl2–
MeOH 95 : 5); 1H NMR: d 10.32 (br s, 1H, NHCO), 8.74 (s, 1H,
H-4), 7.68 (s, 1H, H-5), 7.16 (br s, 1H, NHtBu), 6.52–6.44 (m, 3H,
H-2¢, H-4¢, H-6¢), 5.96 (br s, 1H, NHCH2), 3.77 (s, 6H, OCH3),
3.73 (q, J = 6.8, 2H, H-1¢¢), 2.70–2.50 (m, 2H, H-2¢¢), 1.99 (t,
J = 2.6, H-4¢¢), 1.48 (s, 9H, CMe3); 13C NMR: d 162.4, 161.8,
161.2, 158.5, 155.8, 152.8, 137.1, 136.6, 122.9, 110.0, 107.4, 100.9,
81.8, 70.1, 55.7, 55.6, 51.1, 40.5, 29.0, 19.2; MS (ESI): m/z = 449
[M+H]+ 100%.
1-(2-((1-(1-Benzylpyrrolidin-3-yl)-1H-1,2,3-triazol-4-yl)methyl-
amino)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl)-3-
tert-butylurea (19k). Rf 0.70 (CH2Cl2–MeOH/NEt3 90 : 10 : 0.5);
1H NMR: d 10.35 (br s, 1H, NHCO), 8.77 (br s, 1H, H-4), 7.87
(br s, 1H, Htri), 7.70 (br s, 1H, H-5), 7.26–7.22 (m, 5H, HPh), 7.16
(br s, 1H, NHtBu), 6.53–6.46 (m, 3H, H-2¢, H-4¢, H-6¢), 6.13 (br s,
1H, NHCH2), 5.23-5.21 (m, 1H, H-c), 4.87 (d, J = 5.2, 2H, H-1¢¢),
3.82 (s, 6 H, OCH3), 3.70 (AB, J = 11.9, 2H, CH2Ph), 3.11–2.10 (m,
6H, H-b, H-d, H-e), 1.49 (s, 9H, CMe3); 13C NMR: d 162.7, 162.1,
161.4, 158.8, 156.0, 153.0, 146.0, 138.5, 137.4, 136.9, 128.9, 128.7,
127.7, 123.2, 120.8, 110.4, 107.6, 101.2, 60.3, 59.9, 59.7, 55.9, 55.8,
53.0, 51.2, 37.6, 33.0, 29.3; MS (ESI): m/z = 637 [M+H]+ 100%;
HRMS (ESI): Calc. for C34H40N10O3Na [M+Na]+ 659.3183, found
659.3192.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-(pent-4-ynylamino)-
pyrido[2,3-d]pyrimidin-7-yl)urea (22b). This compound was pre-
pared according the procedure outlined for compound 22a starting
from 17 (574 mg, 1.25 mmol), 4-pentyn-1-amine hydrochloride
21b (299 mg, 2.50 mmol) and DIPEA (0.86 mL, 5 mmol) in
dry dioxane (12 mL) at 50 ◦C for 20 h. Purification by flash
column chromatography (CH2Cl2–MeOH 98 : 2) furnish alkyne
22b (411 mg, 35%) as a yellow solid. Rf 0.27 (CH2Cl2–MeOH
1
95 : 5); H NMR: d 10.34 (br s, 1H, NHCO), 8.71 (s, 1H, H-4),
7.66 (s, 1H, H-5), 7.14 (br s, 1H, NHtBu), 6.50–6.46 (m, 3H, H-2¢,
H-4¢, H-6¢), 5.73 (m, 1H, NHCH2), 3.81 (s, 6H, OCH3), 3.70 (q,
J = 6.7, 2H, H-1¢¢), 2.34 (td, J = 6.7, 2.7, 2H, H-3¢¢), 1.99 (t, J =
2.7, 1H, H-5¢¢), 1.94 (qt, J = 6.7, 6.7, 2H, H-2¢¢), 1.49 (s, 9H, CMe3);
13C NMR: d 163.1, 162.0, 161.2, 158.6, 155.9, 153.0, 137.4, 137.0,
122.7, 110.1, 107.6, 101.1, 83.9, 69.3, 55.9, 55.8, 51.2, 41.0, 29.2,
28.2, 16.4; MS (ESI): m/z = 485 [M+Na]+ 100%; HRMS (ESI):
Calc. for C25H30N6O3Na [M+Na]+ 485.2277, found 485.2255.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-((1-(2-(4-(5-(trifluo-
romethyl)pyridin-2-yl)piperazin-1-yl)ethyl)-1H -1,2,3-triazol-4-
yl)methylamino)pyrido[2,3-d]pyrimidin-7-yl)urea (19l). Rf 0.21
1
(CH2Cl2–MeOH 95 : 5); H NMR: d 10.31 (br s, 1 H, NHCO),
8.71 (s, 1H, H-4), 8.33 (s, 1H, Hpyr), 7.78 (br s, 1H, Htri), 7.63
(s, 1H, H-5), 7.56 (d, J = 8.9, 1H, Hpyr), 7.17 (s, 1H, NHtBu),
6.64 (br s, 1H, NHCH2), 6.54 (d, J = 8.9, 1H, Hpyr), 6.50–6.39
(m, 3H, H-2¢, H-4¢, H-6¢), 4.84 (d, J = 5.5, 2H, H-1¢¢), 4.45 (t,
J = 5.8, 2H, H-a), 3.80 (s, 6H, OCH3), 3.49 (m, 4H, Hpip), 2.83
(t, J = 5.8, 2H, H-b), 2.51 (m, 4H, Hpip), 1.46 (s, 9H, CMe3);
13C NMR: d 162.4, 161.8, 161.2, 160.2, 158.5, 155.7, 152.8, 145.9,
145.5, 137.3, 134.6, 136.6, 134.6, 123.0, 122.5, 115.4, 110.1, 110.5,
105.6, 100.7, 57.6, 55.7, 55.6, 52.8, 51.0, 47.8, 44.6, 37.2, 29.2;
MS (ESI): m/z = 736 [M+H]+ 100%; HRMS (ESI): Calc. for
C35H41N12O3F3Na [M+Na]+ 757.3274, found 757.3278.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-(hex-5-ynylamino)-
pyrido[2,3-d]pyrimidin-7-yl)urea (22c). This compound was pre-
pared according the procedure outlined for compound 22b starting
from 17 (344 mg, 0.75 mmol), 5-hexyn-1-amine hydrochloride
21c (200 mg, 1.50 mmol) and DIPEA (0.50 mL, 2.9 mmol)
in dry dioxane (12 mL) at 70 ◦C for 17 h. Purification by
flash chromatography (CH2Cl2–MeOH 98 : 2) furnish alkyne 22c
(109 mg, 31%) as a yellow solid. Rf 0.28 (CH2Cl2–MeOH 99 : 1);
1H NMR (500 MHz) : d 10.35 (br s, 1H, NHCO), 8.75 (br s, 1H,
H-4), 7.69 (br s, 1H, H-5), 7.14 (br s, 1H, NHtBu), 6.58–6.48 (m,
3H, H-2¢, H-4¢, H-6¢), 5.49 (br s, 1H, NHCH2), 3.83 (s, 6H, OCH3),
3.63 (m, 2H, H-1¢¢), 2.28 (td, J = 7.0, 2.5, 2H, H-4¢¢), 1.96 (t, J =
2.5, 1H, H-6¢¢), 1.84 (qt, J = 7.0, 2H, H-2¢¢), 1.67 (qt, J = 7.0, 2H,
H-3¢¢); 1.46 (s, 9H, CMe3); 13C NMR (125 MHz): d 162.7, 161.7,
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-((1-(2-(4-(2-(2,5-
dimethyl-1H-pyrrol-1-yl)ethyl)piperazin-1-yl)ethyl)-1H-1,2,3-tria-
zol-4-yl)methylamino)pyrido[2,3-d]pyrimidin-7-yl)urea (19m). Rf
0.42 (CH2Cl2–MeOH 90 : 10); 1H NMR: d 10.29 (br s, 1H,
NHCO), 8.75 (s, 1H, H-4), 7.70 (s, 1H, Htri), 7.69 (s, 1H, H-5),
7.17 (s, 1H, NHtBu), 6.58–6.42 (m, 3H, H-2¢, H-4¢, H-6¢), 6.00 (t,
J = 5.8, 1H, NHCH2), 5.74 (s, 2H, Hpyr), 4.86 (d, J = 5.8, 2H,
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161.0, 157.0, 155.6, 152.8, 137.1, 136.7, 122.4, 109.7, 107.4, 100.8,
83.9, 68.7, 55.6, 51.0, 40.9, 29.0, 28.6, 25.9, 18.2; MS (ESI): m/z =
477 [M+H]+ 100%.
55.6, 51.0, 41.2, 29.0, 26.8, 25.4; MS (ESI): m/z = 633 [M+Na]+
100%; HRMS (ESI): Calc. for C32H38N10O3Na [M+Na]+ 633.3026,
found 633.3017.
1-(2-(2-(1-(1-Benzylpyrrolidin-3-yl)-1H -1,2,3-triazol-4-yl)-
ethylamino)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl)-
3-tert-butylurea (23a). Rf 0.43 (CH2Cl2–MeOH 90 : 10); 1H
NMR: d 10.35 (br s, 1H, NHCO), 8.70 (br s, 1H, H-4), 7.65
(br s, 1H, H-5), 7.63 (br s, 1H, Htri), 7.40–7.19 (m, 5H, HPh), 7.15
(br s, 1H, NHtBu), 6.50–6.45 (m, 3 H, H-2¢, H-4¢, H-6¢), 6.08 (t,
J = 5.5, 1H, NHCH2), 5.22–5.16 (m, 1H, H-c), 4.05–3.82 (m, 2H,
H-1¢¢), 3.80 (s, 6H, OCH3), 3.65 (s, 2H, CH2Ph), 3.25–2.75 (m,
5H, H-2¢¢, H-b, H-e), 2.65–2.35 (m, 2H, H-d, H-e), 2.15–1.95 (m,
1H, H-d), 1.48 (s, 9H, CMe3); 13C NMR: d 163.0, 162.0, 161.2,
158.9, 155.9, 153.0, 146.0, 138.5, 137.4, 136.9, 128.9, 128.7, 127.6,
122.8, 120.0, 110.1, 107.6, 101.1, 60.4, 59.9, 59.6, 55.9, 55.8, 52.9,
51.2, 41.2, 33.0, 29.3, 25.9; MS (ESI): m/z = 673 [M+Na]+ 100%;
HRMS (ESI): Calc. for C35H42N10O3Na [M+Na]+ 673.3339, found
673.3329.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-(3-(1-((1-methyl-
piperidin-3-yl)methyl)-1H-1,2,3-triazol-4-yl)propylamino)pyrido-
[2,3-d]pyrimidin-7-yl)urea (23e). Rf 0.11 (CH2Cl2–MeOH
1
90 : 10); H NMR (500 MHz): d 10.35 (br s, 1H, NHCO), 8.72
(br s, 1H, H-4), 7.66 (s, 1H, H-5), 7.32 (s, 1H, Htri), 7.13 (s, 1H,
NHtBu), 6,51–6.46 (m, 3H, H-2¢, H-4¢, H-6¢), 5.77 (br s, 1H,
NHCH2), 4.35–4.18 (m, 2H, H-a), 3.81 (s, 6H, OCH3), 3.71–3.56
(m, 2H, H-1¢¢), 2.85–2.68 (m, 4H, H-b¢, H-f¢, H-3¢¢), 2.29–2.20
(m, NCH3, H-c¢), 2.19–2.01 (m, 2H, H-f¢, H-2¢¢), 1.99–1.84 (m,
1H, H-b¢), 1.81–1.60 (m, 3H, H-d¢, H-e¢), 1.48 (s, 9H, CMe3),
1.15–0.98 (m, 1H, H-d¢); 13C NMR (125 MHz): d 162.9, 161.8,
161.0, 158.7, 155.7, 152.9, 147.6, 137.3, 136.8, 122.6, 121.2, 109.8,
107.5, 100.9, 58.9, 55.8, 55.6, 53.4, 51.0, 46.3, 41.1, 36.9, 29.1,
29.0, 27.3, 24.0, 23.2; MS (ESI): m/z = 639 [M+Na]+ 100%;
HRMS (ESI): Calc. for C32H44N10O3Na [M+Na]+ 639.3496,
found 639.3502.
1-(2-(3-(1-(1-Benzylpyrrolidin-3-yl)-1H-1,2,3-triazol-4-yl)prop-
ylamino)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl)-3-
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-(4-(1-((1-methyl-
piperidin-3-yl)methyl)-1H -1,2,3-triazol-4-yl)butylamino)pyrido-
[2,3-d]pyrimidin-7-yl)urea (23f). Rf 0.13 (CH2Cl2–MeOH
1
tert-butylurea (23b). Rf 0.29 (CH2Cl2–MeOH 95 : 5); H NMR:
d 10.33 (br s, 1H, NHCO), 8.68 (br s, 1H, H-4), 7.62 (br s, 1H,
H-5), 7.55 (br s, 1H, Htri), 7.40–7.26 (m, 5H, HPh), 7.14 (br s, 1H,
NHtBu), 6.47–6.44 (m, 3H, H-2¢, H-4¢, H-6¢), 5.99 (t, J = 5.2, 1H,
NHCH2), 5.19–5.12 (m, 1H, H-c), 3.78 (s, 6H, OCH3), 3.63–3.58
(m, 4H, H-1¢¢, CH2Ph), 3.10–2.70 (m, 5H, H-3¢¢, H-b, H-e), 2.65–
2.35 (m, 2H, H-d, H-e), 2.20–1.95 (m, 3H, H-2¢¢, H-d), 1.45 (s, 9H,
CMe3); 13C NMR: d 163.0, 162.0, 161.2, 158.9, 155.8, 153.0, 147.9,
138.6, 137.4, 137.0, 128.8, 128.7, 127.5, 122.7, 119.3, 110.0, 107.6,
101.0, 60.5, 59.9, 59.5, 55.8, 55.7, 52.9, 51.2, 41.3, 33.0, 29.2, 27.2,
23.5; MS (ESI): m/z = 687 [M+Na]+ 100%; HRMS (ESI): Calc.
for C36H44N10O3Na [M+Na]+ 687.3496, found 687.3472.
1
90 : 10); H NMR (500 MHz): d 10.34 (br s, 1H, NHCO), 8.72
(br s, 1H, H-4), 7.68 (s, 1H, H-5), 7.39 (s, 1H, Htri), 7.51 (s, 1H,
NHtBu), 6,70–6.35 (m, 3H, H-2¢, H-4¢, H-6¢), 5.72 (br s, 1H,
NHCH2), 4.45–4.15 (m, 2H, H-a), 3.83 (s, 6H, OCH3), 3.70–3.55
(m, 2H, H-1¢¢), 3.05–2.70 (m, 4H, H-b¢, H-f¢, H-4¢¢), 2.45–2.30
(m, 4H, NCH3, H-c¢), 2.28–2.12 (m, 1H, H-f¢), 2.10–1.99 (m,
1H, H-b¢), 1.97–1.64 (m, 7H, H-2¢¢, H-3¢¢, H-d¢, H-e¢), 1.49 (s,
9H, CMe3), 1.20–1.02 (m, 1H, H-d¢);13C NMR (125 MHz): d
162.9, 161.8, 161.0, 158.7, 155.7, 152.9, 148.1, 137.3, 136.8, 122.6,
121.4, 109.8, 107.6, 100.9, 58.9, 55.8, 55.6, 55.2, 53.4, 51.0, 46.2,
41.3, 36.7, 29.1, 29.0, 27.2, 26.9, 25.4, 24.0; MS (ESI): m/z = 631
[M+H]+ 100%; HRMS (ESI): Calc. for C33H46N10O3Na [M+Na]+
653.3652, found 653.3647.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-(3-(1-(pyridin-2-yl-
methyl)-1H -1,2,3-triazol-4-yl)propylamino)pyrido[2,3-d]pyrimi-
din-7-yl)urea (23c). Rf 0.28 (CH2Cl2–MeOH 95 : 5); 1H NMR: d
10.35 (br s, 1H, NHCO), 8.71 (br s, 1H, H-4), 8.64–8.55 (m, 1H,
H-f¢), 7.71–7.65 (m, 2H, H-5, H-d¢), 7.48 (br s, 1H, Htri), 7.25–
7.09 (m, 3H, NHtBu, H-c¢, H-e¢), 6.52–6.47 (m, 3H, H-2¢, H-4¢,
H-6¢), 5.86 (t, 1H, J = 7.0, NHCH2), 5.62 (s, 2H, CH2Pyr), 3.82
(s, 6H, OCH3), 3.64 (dd, 2H, J = 7.0, 7.0, H-1¢¢), 2.86 (t, 2H,
1 - (2 - (2 - Azidoethylamino) - 6 - (3,5 - dimethoxyphenyl)pyrido-
[2,3-d]pyrimidin-7-yl)-3-tert-butylurea (24). A mixture of 1721
(1.23 g, 2.68 mmol), 2-azidoethylamine salt 8 (1.02 g, 5.08 mmol)
and DIPEA (0.92 mL, 5.35 mmol) in dry dioxane (30 mL) was
warmed at 50 ◦C for 24 h; the solvent was removed in vacuo and
the crude residue was purified by flash column chromatography
(CH2Cl2–MeOH 99 : 1 to 98 : 2) to give 24 (987 mg, 80%) as a
J = 7.0, H-3¢¢), 2.17–2.07 (m, 2H, H-2¢¢), 1.48 (s, 9H, CMe3); 13
C
NMR: d 163.1, 162.0, 161.2, 158.9, 155.9, 155.0, 153.1, 150.0,
148.1, 137.6, 137.4, 137.0, 123.7, 122.7, 121.7, 110.0, 107.6, 101.0,
55.9, 55.8, 51.2, 41.3, 29.2, 23.4; MS (ESI): m/z = 597 [M+H]+
100%; HRMS (ESI): Calc. for C31H36N10O3Na [M+Na]+ 619.2870,
found 619.2881.
1
yellow solid. Rf 0.26 (CH2Cl2–MeOH 97 : 3); H NMR: d 10.26
(br s, 1H, NHCO), d 8.75 (br s, 1H, H-4), 7.69 (br s, 1H, H-5),
7.16 (br s, 1H, NHtBu), 6.55–6.41 (m, 3H, H-2¢, H-4¢, H-6¢), 5.71
(br s, 1H, NHCH2), 3.86–3.71 (m, 8H, OCH3, H-1¢¢), 3.67–3.57 (t,
J = 5.3, 2H, H-2¢¢), 1.48 (s, 9H, CMe3); 13C NMR: d 162.6, 161.9,
161.3, 158.7, 155.9, 152.9, 137.3, 136.7, 123.2, 110.3, 107.5, 101.0,
55.8, 55.7, 51.2, 50.6, 41.2, 29.1; MS (ESI): m/z = 488 [M+Na]+
100%; HRMS (ESI): Calc. for C22H27N9O3Na [M+Na]+ 488.2135,
found 488.2139.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-(4-(1-((pyridine-2-yl-
methyl)methyl)-1H -1,2,3-triazol-4-yl)butylamino)pyrido[2,3-d]-
pyrimidin-7-yl)urea (23d). Rf 0.27 (CH2Cl2–MeOH 95 : 5); 1H
NMR (500 MHz): d 10.34 (br s, 1H, NHCO), 8.79 (s, 1H, H-
4), 8.56 (d, J = 4.8, H-f¢), 7.81–7.61 (m, 2H, H-5, H-d), 7.52 (s,
1H, Htri), 7.35–7.10 (m, 3H, H-c¢, H-e¢, NHtBu), 6,60–6.40 (m, 3H,
H-2¢, H-4¢, H-6¢); 5.83 (br s, 1H, NHCH2), 5.64 (s, 2H, CH2pyr),
3.80 (s, 6H, OCH3), 3.66 (br s, 2H, H-1¢¢), 2.83 (t, 3H, J = 6.8, H-
4¢¢), 1.95–1.75 (m, 4H, H-2¢¢, H-3¢¢), 1.51 (s, 9H, CMe3); 13C NMR
(125 MHz): d 162.7, 161.8, 161.0, 158.6, 155.6, 154.9, 152.8, 149.8,
148.4, 137.3, 137.2, 136.8, 123.4, 122.4, 121.5, 109.7, 107.4, 100.8,
3-(1-(2-(7-(3-tert-Butylureido)-6-(3,5-dimethoxyphenyl)pyrido-
[2,3-d]pyrimidin-2-ylamino)ethyl)-1H-1,2,3-triazol-4-yl)propanoic
1
acid (26a). Rf 0.14 (CH2Cl2–MeOH 9 : 1); H NMR (DMF): d
12.43 (br s, 1H, CO2H), 10.47 (br s, 1H, NHCO), 8.99 (s, 1H, H-4),
8.09 (s, 1H, H-5), 7.92 (br s, 1H, Htri), 7.84 (br s, 1H, NHtBu), 6.80–
6.60 (m, 3H, H-2¢, H-4¢, H-6¢), 4.83–4.70 (m, 2H, H-2¢¢), 4.08–3.95
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(m, 2H, H-1¢¢), 3.88 (s, 6H, OCH3), 3.00–2.85 (m, 2H, H-a), 2.70–
2.55 (m, 2H, H-b), 1.48 (s, 9H, CMe3); 13C NMR (DMF): d 174.3,
164.0, 163.3, 162.9, 159.1, 156.2, 153.4, 147.1, 139.3, 138.5, 123.3,
122.9, 111.1, 108.3, 101.3, 56.4, 56.3, 51.4, 49.4, 42.8, 34.3, 29.1,
22.1; MS (ESI): m/z = 564 [M+H]+ 100%; HRMS (ESI): Calc.
for C27H33N9O3Na [M+Na]+ 586.2502, found 586.2509.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-(2-(4-(guanidino-
methyl)-1H-1,2,3-triazol-1-yl)ethylamino)pyrido[2,3-d]pyrimidin-
7-yl)urea hydrochloride (26f). 1H NMR (CD3OD): d 8.90 (s, 1H,
H-4), 8.04 (s, 1H, Htri), 7.86 (s, 1H, H-5), 6.65–6.50 (m, 3H, H-2¢,
H-4¢, H-6¢), 4.90–4.70 (m, 2H, H-2¢¢), 4.46 (s, 2H, CH2-guanidine),
4.05–3.95 (m, 2H, H-1¢¢), 3.84 (s, 6H, OCH3), 1.42 (s, 9H, CMe3);
MS (ESI): m/z = 563 [M-HCl +H]+ 100%; HRMS (ESI): Calc.
for C26H35N12O3 [M-HCl+H]+ 563.2955 found 563.2953.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-(2-(4-((dimethyl-
amino)methyl)-1H -1,2,3-triazol-1-yl)ethylamino)pyrido[2,3-d]-
1
Biological evaluations
pyrimidin-7-yl)urea (26b). Rf 0.30 (CH2Cl2–MeOH 80 : 20); H
NMR: d 10.28 (br s, 1H, NHCO), 8.74 (s, 1H, H-4), 7.93 (br s,
1H, H-5¢¢), 7.70 (s, 1H, H-5), 7.15 (br s, 1H, NHtBu), 6.56-6.44 (m,
3H, H-2¢, H-4¢, H-6¢), 5.83 (br s, 1H, NHCH2), 4.75 (t, J = 5.9, 2H,
H-2¢¢), 4.15–4.01 (m, 2H, H-1¢¢), 3.94 (br s, 2H, CH2NMe2), 3.81
(s, 6H, OCH3), 2.53 (s, 6H, CH3), 1.50 (s, 9H, CMe3); 13C NMR: d
162.5, 161.9, 161.2, 158.5, 155.9, 152.8, 146.1, 137.2, 136.6, 123.8,
123.3, 110.3, 107.5, 100.9, 55.8, 55.7, 54.3, 51.1, 49.2, 45.0, 42.0,
29.1; MS (ESI): m/z = 549 [M+H]+ 100%; HRMS (ESI): Calc.
for C27H36N10O3Na [M+Na]+ 571.2870, found 571.2875.
In vitro. Recombinant FGFR3 kinase domain (FGFR3 KD,
amino acid residues 439–806) was expressed in baculovirus-
infected Sf9 cells as hexa-histidine tagged protein. FGFR3 KD
was purified by affinity chromatography on Ni-NTA beads for
use in the assay. Nunc MaxiSorp 96-well plates were coated by
incubation with 100 mL/well of 10 mg mL-1 poly(Glu:Ala:Tyr,
6 : 3 : 1), (pGAT, Sigma Aldrich) in PBS overnight at 4 ◦C. Excess
pGAT was removed by aspiration and the plate was washed
2 times with PBS. The kinase reaction was performed in 100 mL
of 50 mM TRIS (pH 7.5) containing 0.01% BSA, 5 mM MgCl2,
5 mM MnCl2, 1 mM dithiothreitol, 5 mM ATP and ~8 ng of
FGFR3 KD. Inhibitors in DMSO were added; the final DMSO
concentration was 2% (v/v). After 15 min preincubation at
room temperature, phosphorylation was initiated by addition
of ATP and the assay was incubated for 1 h at 37 ◦C. The
kinase reaction was terminated by aspiration of the reaction
mixture and washing two times with PBST (0.05% Tween 20 in
PBS). Blocking was performed with 300 mL blocking buffer (3%
BSA in PBST) for 1 h at room temperature. For detection of
substrate phosphorylation, 100 mL PY99 anti-phosphotyrosine
antibody (Santa Cruz Biotechnology), diluted to 0.2 mg mL-1
with 1% BSA in PBST, were incubated for 1 h, removed by
aspiration and washed three times with PBST. 100 mL of HRP
conjugated anti-mouse antibody were added and incubated for
1 h at room temperature, removed by aspiration and washed
three times with PBST. The colorimetric signal was developed
by addition of 100 mL TMB substrate (TMB substrate kit,
Thermo Fisher Scientific) and stopped by the addition of 100 mL
2M sulfuric acid. The amount of phosphotyrosine product
in the wells was estimated by measurement of absorbance at
450 nm.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-(2-(4-(hydroxy-
methyl)-1H -1,2,3-triazol-1-yl)ethylamino)pyrido[2,3-d]pyrimi-
1
din-7-yl)urea (26c). Rf 0.42 (CH2Cl2–MeOH 80 : 20); H NMR
(500 MHz, 330 K): d 9.88 (br s, 1H, NHCO), 8.77 (s, 1H, H-4),
7.72 (br s, 1H, H-5), 7.59 (s, 1H, H-5¢¢), 6.57–6.46 (m, 3H, H-2¢,
H-4¢, H-6¢), 4.77 (s, 2H, CH2OH), 4.72 (t, J = 5.2, 2H, H-2¢¢), 4.17–
4.08 (m, 2H, H-1¢¢), 3.84 (s, 6H, OCH3), 1.52 (s, 9H, CMe3); 13C
NMR (500 MHz): d 162.2, 161.9, 161.3, 158.5, 156.0, 152.7, 137.3,
136.4, 123.3, 121.9, 110.0, 107.4, 100.9, 55.9, 55.6, 51.1, 48.9, 42.1,
29.0; MS (ESI): m/z = 522 [M+H]+ 100%; HRMS (ESI): Calc.
for C25H31N9O4Na [M+Na]+ 544.2397, found 544.2391.
1-tert-Butyl-3-(6-(3,5-dimethoxyphenyl)-2-(2-(4-(2-hydro-
xyethyl)-1H-1,2,3-triazol-1-yl)ethylamino)pyrido[2,3-d]pyrimidin-
7-yl)urea (26d). Rf 0.18 (CH2Cl2–MeOH 95 : 5); 1H NMR: d
10.29 (br s, 1H, NHCO), 8.70 (s, 1H, H-4), 7.70 (br s, 1H, H-
5), 7.41 (s, 1H, Htri), 7.20 (br s, 1H, NHtBu), 6.51–6.46 (m, 4H,
NHCH2, H-2¢, H-4¢, H-6¢), 4.78–4.62 (m, 2H, H-2¢¢), 4.05 (dd, J =
5.6, 5.6, 2H, H-1¢¢), 3.88 (t, J = 5.8, 2H, H-b), 3.81 (s, 6H, OCH3),
2.88 (t, J = 5.8, 2H, H-a), 1.48 (s, 9H, CMe3); 13C NMR: d 162.3,
161.8, 161.3, 158.4, 155.9, 152.7, 145.6, 137.2, 136.5, 123.3, 122.7,
110.2, 107.4, 100.9, 61.5, 55.9, 55.6, 51.1, 49.3, 42.1, 29.1, 28.8;
MS (ESI): m/z = 558 [M+Na]+ 100%; HRMS (ESI): Calc. for
C26H33N9O4Na [M+Na]+ 558.2553, found 558.2534.
In cellulo. Human Embryonic Kidney cells (HEK) stably
expressing the vitronectin receptor (293 VnR) were incubated in
DMEM supplemented with 10% FCS (Invitrogen), and antibi-
otics. Transfection of constructs (FGFR3-K650M) into cells at 50–
80% confluency using Fugene6 (Roche) was performed according
to the manufacturer’s instructions. The tyrosine kinase inhibitor
PD173074, 19g and 19h were dissolved in DMSO and used at
2 mM overnight.
Transfected cells were lysed in RIPA buffer (50 mM Tris-HCl
pH 7.6, 150 mM NaCl, 0.5% NP40, 0.25% Sodium deoxycholate,
supplemented with protease and phosphatase inhibitors). Im-
munoprecipitation (IP) was performed by incubating 3 mL rabbit
anti FGFR3 (Sigma)/500 mg protein with protein A-agarose
(Roche). Immunoblotting (IB): Rabbit- Anti-FGFR3 (Sigma) was
used at 1 : 1000 and the mouse anti-phosphotyrosine at 1 : 400
(Cell signaling). Anti-rabbit and anti-mouse HRP (Amersham)
secondary antibodies were used at a concentration of 1 : 10000
tert-Butyl(tert-butoxycarbonylamino)((1-(2-(7-(3-tert-butyl-
ureido) - 6 - (3,5 - dimethoxyphenyl)pyrido[2,3 - d]pyrimidin - 2 - yl-
amino)ethyl)-1H -1,2,3-triazol-4-yl)methylamino)methylenecar-
bamate (26e). Rf 0.19 (CH2Cl2–MeOH 96 : 4); 1H NMR
(500 MHz, 330 K): d 11.40 (s, 1H, NHBoc), 10.33 (br s, 1H,
NHCO), 8.73 (br s, 2H, H-4, NHgua), 7.70 (s, 1H, H-5), 7.55 (s,
1H, Htri), 7.19 (s, 1H, NHtBu), 6.60–6.40 (m, 3H, H-2¢, H-4¢, H-6¢),
6.32 (br s, 1H, NHCH2), 4.80–4.55 (m, 4H, H-2¢¢, CH2-guanidine),
4.05 (br s, 2H, H-1¢¢), 3.79 (s, 6H, OCH3), 1.46, 1.45 and 1.44 (s,
27 H, CMe3); 13C NMR: d 163.4, 162.4, 161.8, 161.1, 158.4, 156.0,
155.8, 153.0, 152.7, 144.0, 137.2, 136.5, 123.2, 110.3, 107.4, 100.9,
83.3, 79.4, 55.6, 51.0, 49.0, 41.8, 36.5, 29.0, 28.4, 28.1; MS (ESI):
m/z = 763 [M+H]+ 100%; HRMS (ESI): Calc. for C36H50N12O7Na
[M+Na]+ 785.3823, found 785.3836.
2172 | Org. Biomol. Chem., 2010, 8, 2164–2173
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