Naphthyridines as novel bet family bromodomain inhibitors

Article abstract of DOI:10.1002/cmdc.201300259

Bromodomains (BRDs) are small protein domains found in a variety of proteins that recognize and bind to acetylated histone tails. This binding affects chromatin structure and facilitates the localisation of transcriptional complexes to specific genes, thereby regulating epigenetically controlled processes including gene transcription and mRNA elongation. Inhibitors of the bromodomain and extra-terminal (BET) proteins BRD2-4 and T, which prevent bromodomain binding to acetyl-modified histone tails, have shown therapeutic promise in several diseases. We report here the discovery of 1,5-naphthyridine derivatives as potent inhibitors of the BET bromodomain family with good cell activity and oral pharmacokinetic parameters. X-ray crystal structures of naphthyridine isomers have been solved and quantum mechanical calculations have been used to explain the higher affinity of the 1,5-isomer over the others. The best compounds were progressed in a mouse model of inflammation and exhibited dose-dependent anti-inflammatory pharmacology.

Full text of DOI:10.1002/cmdc.201300259

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DOI: 10.1002/cmdc.201300259
Naphthyridines as Novel BET Family Bromodomain
Inhibitors
Olivier Mirguet,*[a, b] Yann Lamotte,[a] Chun-wa Chung,[c] Paul Bamborough,[c]
Delphine Delannꢀe,[a] Anne Bouillot,[a] FranÅoise Gellibert,[a, b] Gael Krysa,[a] Antonia Lewis,[d]
Jason Witherington,[e] Pascal Huet,[a] Yann Dudit,[a] Lionel Trottet,[a] and Edwige Nicodeme[a]
Bromodomains (BRDs) are small protein domains found in a va-
riety of proteins that recognize and bind to acetylated histone
tails. This binding affects chromatin structure and facilitates
the localisation of transcriptional complexes to specific genes,
thereby regulating epigenetically controlled processes includ-
ing gene transcription and mRNA elongation. Inhibitors of the
bromodomain and extra-terminal (BET) proteins BRD2–4 and T,
which prevent bromodomain binding to acetyl-modified his-
tone tails, have shown therapeutic promise in several diseases.
We report here the discovery of 1,5-naphthyridine derivatives
as potent inhibitors of the BET bromodomain family with good
cell activity and oral pharmacokinetic parameters. X-ray crystal
structures of naphthyridine isomers have been solved and
quantum mechanical calculations have been used to explain
the higher affinity of the 1,5-isomer over the others. The best
compounds were progressed in a mouse model of inflamma-
tion and exhibited dose-dependent anti-inflammatory pharma-
cology.
Introduction
The bromodomain and extra C-terminal (BET) domain family of
bromodomains (BRDs) consists of four proteins, each contain-
ing two discrete bromodomain ‘reader’ modules which recog-
nize the e-N-acetylation state of specific lysine residues found
within histone tails and other proteins.[1] Whereas three mem-
bers of this family (BRD2, BRD3 and BRD4) are ubiquitously ex-
pressed, the fourth member, BRDT, is a tissue-restricted, chro-
matin-associated protein expressed in pachytene spermato-
cytes, diplotene spermatocytes, and round spermatids.[2] On
recognition of certain acetyl lysine marks, the bromodomain
displays a key role in regulating further protein–protein inter-
actions as part of chromatin remodelling, and despite not
modifying post-translational marks through acetylation or iter-
ative methylation directly, the function of the bromodomain
ultimately manifests itself in transcriptional activity control.[3]
The recent disclosure of potent, selective small-molecule inhib-
itors by members of this group[4,5] and others[6,7] of the BET
family of bromodomains demonstrates that epigenetic reader
proteins could be as tractable to small-molecule drug discov-
ery as their epigenetic enzyme counterparts. The structurally
related triazolodiazepine compounds I-BET762 (1) and (+)-JQ1
(2; Figure 1) are potent (nanomolar), BET-family-selective, and
cell-active BET family inhibitors. I-BET762 (1) has recently en-
tered clinical trials for NUT midline carcinoma.[8] Isoxazoloqui-
noline compounds such as I-BET151 (3) were also described as
potent BET protein inhibitors,[9] and more recently, Pfizer/SGC
published 3-methyl-1,2,3,4-tetrahydroquinazolin-2-one (4) as
new chemical probe for BET protein inhibition.[7a]
[a] Dr. O. Mirguet, Y. Lamotte, D. Delannꢀe, Dr. A. Bouillot, Dr. F. Gellibert,
G. Krysa, P. Huet, Y. Dudit, Dr. L. Trottet, Dr. E. Nicodeme
Centre de Recherches FranÅois Hyafil, GlaxoSmithKline R&D
25 Avenue du Quꢀbec, 91140 Villebon-sur-Yvette (France)
E-mail: oliviermirguet@yahoo.fr
[b] Dr. O. Mirguet, Dr. F. Gellibert
Current address: Institut de Recherches Servier
11 Rue des Moulineaux, 92150 Suresnes (France)
We have previously reported the BRD affinities of a series of
potent and cell-active isoxazoloquinolines (e.g., 3, 5, 6).[10] In
the present study, new naphthyridine analogues were synthe-
sized and tested on BET family bromodomains to determine
their affinities towards all BRD subtypes. The main objective of
this work was to expand the chemical diversity of BET inhibi-
tors and enhance solubility of the parent chemical series,
namely the isoxazoloquinoline derivatives. Adding one extra
nitrogen atom to the quinoline template would allow us to
prepare salts and thus increase water solubility of the com-
pounds. At the beginning of this work, the best position for
the extra nitrogen atom was first investigated.
[c] C.-w. Chung, Dr. P. Bamborough
Computational & Structural Chemistry, Molecular Discovery Research
GlaxoSmithKline R&D
Medicines Research Centre, Gunnels Wood Road
Stevenage, Hertfordshire SG1 2NY (UK)
[d] A. Lewis
Screening & Compound Profiling, Molecular Discovery Research
GlaxoSmithKline R&D
Medicines Research Centre, Gunnels Wood Road
Stevenage, Hertfordshire SG1 2NY (UK)
[e] Dr. J. Witherington
Epinova, GlaxoSmithKline R&D
Medicines Research Centre, Gunnels Wood Road
Stevenage, Hertfordshire SG1 2NY (UK)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300259.
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yields. Final saponification of
the ethyl ester with sodium hy-
droxide gave the target com-
pounds.
Scheme 2 depicts the synthe-
sis of 1,5-naphthyridine deriva-
tives. Suzuki coupling reaction
between bromopyridine 17 and
(3,5-dimethyl-4-isoxazoyl)boron-
ic acid in the presence of
barium hydroxide and a catalytic
amount of (tetrakistriphenyl-
phosphine)palladium(0) afford-
ed 18. The latter was directly
engaged in the naphthyridine
ring synthesis to give 1,5-naph-
thyridin-4-one 19 in good yield.
Chlorination followed by ther-
Figure 1. Structurally diverse reported BET inhibitors.
mal condensation with 2-(tert-
butyl)aniline afforded ester 20
in good yield. Finally, desired compound 25 was obtained fol-
lowing treatment with sodium hydroxide.
Results and Discussion
The synthetic routes of target compounds, namely 1,5-, 1,6-
and 1,8-naphthyridines, are depicted in Scheme 1 and 2. As re-
ported in Scheme 1, the treatment of the appropriate bromo-
pyridine 7 and 8 with (3,5-dimethyl-4-isoxazoyl)boronic acid in
the presence of barium hydroxide and a catalytic amount of
(tetrakistriphenylphosphine)palladium(0) afforded 9 and 10, re-
spectively, in good yields. Gould–Jacobs conditions, that is,
condensation of the aromatic amine with diethyl 2-(ethoxyme-
thylene)malonate and thermal cyclisation, led to the formation
of 1,8-naphthyridin-4-one 11 and 1,6-naphtyridin-4-one 12 in
55% and 76% yield, respectively over two steps. Phosphoryl
chloride-mediated chlorination followed by thermal condensa-
tion with 2-(tert-butyl)aniline led to esters 13 and 14 in good
Compounds were tested in BRD2, BRD3 and BRD4 fluores-
cence polarization binding assays. Unfortunately, both 1,8- and
1,6- naphthyridine derivatives 15 and 16 were less potent
against the BET bromodomains than quinoline 5 (Table 1).
Docking studies gave identical binding modes for 5, 15 and
16, and did not offer an obvious explanation for this observa-
tion. Therefore, crystal structures of the N-terminal bromodo-
main of BRD4 were solved in complex with representative
compounds 5 and 15.
Compound 5 binds to BRD4 in a similar way as it does to
BRD2 in our previously published complex,[10b] with the di-
methyl isoxazole filling the acetyl-lysine pocket close to
Asn140 (Figure 2a,b). The isoxazole heteroatoms
participate in hydrogen-bonding interactions with
a network of water molecules, the closest of which
also bridges to the side chain hydroxy of Tyr97. The
quinoline ring occupies a narrow lipophilic channel
between Leu92 and Pro82. The nitrogen of the quin-
oline forms a hydrogen bond to another water mol-
ecule, and the terminal carboxylate is exposed to
solvent at the mouth of the pocket.
One interesting difference between the BRD4 and
BRD2 structures is in the positioning of the tert-butyl
phenyl substituent. In our BRD2 complex,[10b] the
phenyl ring of 5 could be resolved unambiguously
and was found to sit upon the hydrophobic WPF
shelf, near Trp97, Pro98, and Ile162. Other members
of this series with different phenyl substituents had
similar BRD2 binding modes. However, in the BRD4
complex, the electron density around the tert-butyl
phenyl group is more ambiguous. A better fit to this
Scheme 1. Representative synthesis of 1,8-naphthyridine 15 and 1,6-naphthyridine 16.
Reagents and conditions: a) (3,5-dimethyl-4-isoxazoyl)boronic acid (1 equiv), cat.
Pd(PPh3)4, Ba(OH)2·8H2O (2.5 equiv), dimethoxyethane (DME)/H2O (5:1 v/v), 908C; b) di-
ethyl 2-(ethoxymethylene)malonate (1.05 equiv), 1208C; c) diphenyl ether, 2608C;
d) POCl3, 1208C; e) 2-(tert-butyl)aniline, solvent or neat, reflux; f) 1n NaOH, EtOH, reflux.
is a flipped ring orientation, in which the tert-butyl
group interacts with the analogous BRD4 residues
Trp81, Pro82 and Ile146 (Figure 2c). This result was
unexpected, although we believe that both orienta-
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tions are energetically similar
because the binding affinities of
analogues that lack the tert-
butyl substituent described in
this manuscript and elsewher-
e[10b] have comparable affinity.
When bound to BRD4, com-
pound 15 adopts a very similar
binding mode and geometry to
5 (Figure 2d). In each complex,
the torsion angle between the
dimethyl isoxazole and the
quinoline ring of 5, and that be-
tween the dimethyl isoxazole
and the naphthyridine ring of
15, is about 508. The additional
ring nitrogen has very little ap-
parent consequence, and the
crystal structure does not ex-
plain the lower potency of 15
and 16. To try to understand
this, we carried out in vacuo
Scheme 2. Representative synthesis of 1,5-naphthyridines 25–29. Reagents and conditions: a) (3,5-dimethyl-4-isox-
azoyl)boronic acid (3 equiv), cat. Pd(PPh3)4, Ba(OH)2·8H2O (2.0 equiv), DME, H2O, 908C; b) diethyl 2-(ethoxymethyl-
ene)malonate (1.3 equiv), 1308C, 93% over two steps; c) diphenylether, 2608C, 58%; d) POCl3, 1208C, 63%;
e) R2NH2, solvent, reflux; f) 1n NaOH, EtOH, reflux.
Table 1. BET and cellular activity for naphthyridine isomers.[a]
quantum mechanical calculations. The bound conformation of
5 in its complex with BRD2,[10b] in which the quinoline–isoxa-
zole torsion angle was approximately 578, was used as the
starting point for optimisation (6-31G**-B3LYP). This was re-
peated for modelled compounds 15, 16 and 25. The naphthyr-
idine isomers 15 and 16 both relax to a minimum-energy con-
formation, in which the naphthyridine–isoxazole torsion angle
is flatter than that of quinoline 5 (Table 1), and of the 6-me-
thoxy quinoline analogue 6, which was also modelled. We
therefore rationalize the lower activity of 15 and 16 relative to
Compd
pIC50
Calcd
BRD2 (FP)[b] BRD3 (FP)[b] BRD4 (FP)[b] PBMC (IL-6)[c] MET [8][d]
5
6.1
5.2
5.8
6.2
6.3
5.3
6.0
6.5
5.9
5.2
5.9
6.3
5.5
4.2
4.7
4.9
47
25
25
51
15
16
25
[a] All values are means of nꢀ2. [b] Measured using fluorescence polari-
zation. [c] Measured using PBMCs after an LPS challenge. [d] MET=Mini-
mum-energy torsion.
Figure 2. a) The complex between BRD4 and 5 showing water-mediated hydrogen bonds. b) Surface representation of BRD4 in complex with 5. c) The com-
plex between BRD4 (green) and 5 (orange), overlaid on the complex of BRD2 (blue) and 5 (cyan), showing the alternative aniline position. Residue numbers
shown are for BRD4. d) The complex between BRD4 and 15. e) The complex between BRD4 and 25. f) The complex between BRD4 and 36.
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5 according to their internal geometric energy in the bound
state: incorporation of a nitrogen atom next to the inter-ring
bond disfavours the relatively nonplanar conformation that the
compounds are forced to adopt by the binding site.
Table 2. BET and cellular activity for 1,5-naphthyridine analogues.[a]
Compd
pIC50
BRD4 (FP)[b]
BRD2 (FP)[b]
BRD3 (FP)[b]
PBMC (IL-6)[c]
Because of the relatively low activity of these compounds,
we turned to a third naphthyridine template, the 1,5-isomer.
This offered the additional advantage of the facile incorpora-
tion of the methoxy substituent at the 6-position, which as re-
ported previously afforded an increase in potency in the quin-
oline series. The 1,5-naphthyridine 25 showed improved bind-
ing affinity relative to the 1,8- and 1,6-regioisomers 15 and 16
(Table 1). The calculated minimum naphthyridine–isoxazole tor-
sion for 25 in the gas phase is 518; so, this result is consistent
with the geometric argument outlined above for the different
activities of 5, 15, and 16. A crystal structure of 25 bound to
BRD4 was solved, which showed a very similar binding mode
to that of 5 (Figure 2e). The small increase in binding potency
of 25 over 5 is comparable to that observed when adding
a methoxy group to the quinoline series[10b] and probably
arises from favourable contacts made with the side chain of
Ile146.
6
6.3
6.2
5.8
5.2
5.8
5.2
5.4
6.5
6.5
6.1
5.6
6.0
5.7
5.9
6.2
6.3
5.9
5.3
5.8
5.4
5.6
6.3
4.9
5.9
25
30
31
32
33
34
5.3
NT[d]
5.8
5.6
[a] All values are means of nꢀ2. [b] Measured using fluorescence polari-
zation. [c] Measured using PBMCs after an LPS challenge. [d] NT=Not
tested.
nolines (compare the activity of 32, BRD4 pIC50 =5.8, with its
direct analogue 6, BRD4 pIC50 =6.2),[10b] we were sufficiently
encouraged to persist with this series. Cyclisation of the quino-
line series to give imidazolones had been shown to be benefi-
cial.[10a,b] We thus decided to explore the cyclic analogues of
1,5-naphthyridines as shown in Scheme 3. Desired compounds
35–39 were synthesized from the corresponding carboxylic
acids (compounds 25–29), which underwent a one-step Cur-
tius rearrangement using diphenylphosphoryl azide (DPPA) in
the presence of triethylamine in refluxing DMF. The key SAR
findings are summarized in Table 3. The cyclic naphthyridines
were more or less equipotent to 25, with 37 (R=2-fluorophen-
yl) being the least potent.
These encouraging results led us to use the synthetic route
depicted in Scheme 3 to prepare analogues of 25. To attempt
to improve the cellular activity of the series, all carboxylic acids
(compounds 25–29) were then transformed into their corre-
sponding neutral primary amides (compounds 30–34) using
standard peptide-coupling chemistry.
Profiling was carried out against the three BET subtypes, as
well as in peripheral blood mononuclear cells
(PBMCs), where inhibition of cytokine IL-6 was
measured after a lipopolysaccharide (LPS) challenge
(Table 2).
Table 3. BET and cellular activity for 1,5-naphthyridine analogues.[a]
[d]
As observed in the quinoline series,[10b] potency on
the BET proteins was in general lower for amides
(compounds 30–34) than for the acids (e.g., com-
pound 25) but cellular activity increased given the
true neutral nature of those compounds (Table 2). In
all cases, no drop-off in cellular activity was observed
for amides (compounds 30–34). Unfortunately, struc-
ture–activity relationships (SAR) for substituents at
position 4 of the 1,5-naphthyridine series proved to
be flat, with little improvement seen for a range of
substituents. Although not quite as potent as the qui-
Compd
pIC50
Solubility
FaSSIF
CLint
HSA
[%]
BRD2
(FP)[b]
BRD3
(FP)[b]
BRD4
(FP)[b]
PBMC
(IL-6)[c]
[mLminꢁ1 gꢁ1
]
RLM
[mgmLꢁ1
]
HLM
25
35
36
37
38
39
6.2
6.1
6.0
5.3
6.3
6.3
6.5
6.4
6.3
6.0
6.6
6.6
6.3
6.3
6.2
5.6
6.5
6.5
4.9
6.4
6.5
5.5
6.6
6.8
61
116
107
NT[e]
105
106
0.3
3.3
0.6
NT[e]
2.2
<0.2
0.6
99.1
98.2
97.9
NT[e]
97.3
98.4
0.3
NT[e]
0.2
3.4
1.3
[a] All values are means of nꢀ2. [b] Measured using fluorescence polarization.
[c] Measured using PBMCs, after an LPS challenge. [d] CLint =intrinsic clearance; rat
(RLM) and human (HLM) liver microsomes. [e] NT=Not tested.
Scheme 3. Syntheses of 1,5-naphthyridine analogues 30–39. Reagents and conditions: a) HATU (1.2 equiv), Et3N (1.2 equiv), NH3 (0.5m in 1,4-dioxane), DMF/
CH2Cl2 (1:1 v/v), RT; b) DPPA (1.1 equiv), Et3N (1.1 equiv), DMF, reflux.
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A further crystal structure was solved of the complex be-
tween BRD4 and 36 (Figure 2 f). It showed an overall similar
binding mode to that of 5. However, the additional cyclic con-
straints remove any possibility for the pendant phenyl ring to
interact directly with the WPF shelf residues Trp81, Ile146 and
Met149. This interaction is made instead by the 2-OCF3 sub-
stituent. This observation provides a rationale for the reduced
potency of compounds lacking this group, for example, 2-fluo-
rophenyl analogue 37 compared to 35 and 36.
Cellular activity was greatly improved for compounds of this
subseries, which warranted further profiling. As shown in
Table 3, all cyclized analogues were found to be more soluble
in fasted-state simulated intestinal fluid (FaSSIF) than the car-
boxylic acid starting point 25, and all compounds were found
to be metabolically stable in human liver microsomes (HLM).
The results on rat liver microsomes (RLM) were more divergent,
with compound 36 being the more metabolically stable,
whereas compound 39 was the least stable. Regarding binding
to human serum albumin (HSA), all cyclized compounds were
found to be less bound than the carboxylate 25.
Figure 3. In vivo anti-inflammatory pharmacology in BALB/c mice for com-
pounds 36 and 38.
Conclusions
From that set of cyclized 1,5-naphthyridines, we decided to
select both compounds 36 and 38 for further in vivo profiling
due to their lower clearance in RLM. Those two compounds
were thus administered to rats by oral and intravenous routes
to determine their pharmacokinetic profile (Table 4). Both com-
In this report, we have summarized our efforts to optimize
a series of naphthyridine derivatives as bromodomain and
extra-terminal (BET) domain inhibitors. Assessment of the best
position for the nitrogen atom resulted in the synthesis of 1,5-
naphthyridine compounds, which show efficacy in a mouse
model of inflammation. Cyclised analogues in particular have
good cell activity, good in vivo pharmacokinetics in the rat as-
sociated with good solubility, making a valuable scaffold with
which to probe BET family of bromodomain epigenetic readers
and their functions.
Table 4. In vivo pharmacokinetic data in rats for compounds 36 and
38.[a]
1
Compd
AUC0–inf
[ng hꢁ1 mLꢁ1
CLb
Vd
[Lkgꢁ1
t = [h]
i.v.
F [%]
p.o.
2
]
[mL minꢁ1 kgꢁ1
]
]
36
38
9062
5700
7.7
11.3
0.9
5.9
1.4
2.6
83
76
Experimental Section
[a] Parameters obtained after single p.o. at 5 mgkgꢁ1 of compound in
HPMC 0.5%/Tween 80 1%/H2O formulation and i.v. at 1 mgkgꢁ1 in
DMSO/PEG/cyclodextrine solution dosing to CD rat (n=2). Abbreviations:
area under the curve (AUC); blood clearance (CLb); volume of distribution
Chemistry
General: All commercial chemicals and solvents are reagent grade
and were used without further purification unless otherwise speci-
fied. All reactions except those in aq media were carried out with
the use of standard techniques for the exclusion of moisture. Reac-
tions performed under microwave irradiation utilized either a Biot-
age Initiator or CEM Discover Microwave. Reactions were moni-
tored by TLC on 0.2 mm silica gel plates (ALUGRAM SIL G/UV254,
Macherey–Nagel) and visualized with UV light. Final compounds
were typically purified either by flash chromatography on silica gel
(40–63 mm, Merck) or by recrystallization. All final compounds ana-
1
(Vd); elimination half-life (t = ); bioavailability (F).
2
pounds were well absorbed and showed good oral availability.
In vivo clearance was found to be moderate for both com-
pounds, resulting in limited systemic exposure after oral
dosing. Nevertheless, the pharmacokinetic profile showed that
both compounds were suitable for chronic oral administration
and thus progressed into an acute inflammation mouse model.
BALB/c mice were dosed orally with 10 or 30 mgkgꢁ1 of the
compound or vehicle, and were challenged one hour later
with 30 mgkgꢁ1 LPS i.p. Ninety minutes after LPS challenge,
mice treated with both compounds displayed reduced levels
of liver plasminogen activator inhibitor-1 (PAI-1) mRNA
(Figure 3). Compound 36 showed a dose–response profile with
a reduction of liver PAI-1 mRNA of 41% and 80% at
10 mgkgꢁ1 and 30 mgkgꢁ1, respectively. Compound 38 exhib-
ited a 60% reduction of the acute phase protein PAI-1 mRNA
at 10 mgkgꢁ1.
1
lysed were >95% pure unless otherwise indicated. H and 13C NMR
spectra were recorded on a Brucker 300 MHz Avance DPX. Chemi-
cal shifts are reported in parts per million (ppm, d units). Splitting
patterns are designed as s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet; br s, broad singlet. HRMS was performed on a Micro-
mass LCT (TOF) spectrometer coupled to an analytical HPLC,
XTERRA-MS C18 column (30ꢂ3 mm id, 2.5 mm), eluting with 0.01m
NH4OAc in H2O and 100% CH3CN, using the following elution gra-
dient: 0!100% CH3CN over 4 min and 100% CH3CN over 1 min at
1.1 mLminꢁ1 at 408C. Mass spectra were acquired in either posi-
tive- or negative-ion mode using electrospray ionisation (ESI). Melt-
ing points were measured with a Kofler bench and were uncorrect-
ed.
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6-(3,5-Dimethylisoxazol-4-yl)pyridin-2-amine (9): Pd(PPh3)4 (1.0 g,
0.9 mmol, 0.03 equiv) was added to a solution of 6-bromopyridin-
2-amine (4.98 g, 28.8 mmol), (3,5-dimethylisoxazol-4-yl)boronic acid
(4.0 g, 28.8 mmol, 1 equiv) and Ba(OH)2·8H2O (22.7 g, 71.9 mmol,
2.5 equiv) in dimethoxyethane (DME)/H2O (5:1 v/v, 120 mL). The re-
action mixture was stirred at reflux overnight before being concen-
trated to dryness in vacuo. The residue was taken up in CH2Cl2 and
washed with H2O. The organic layer was dried over Na2SO4, filtered
and concentrated. The crude product was purified by flash chro-
matography on silica gel using CH2Cl2/CH3OH as eluent (100:0!
98:2 v/v) to give compound 9 as a white solid (4.9 g, 90%):
1H NMR (300 MHz, CDCl3): d=7.48 (m, 1H), 6.67 (dd, J=7.4, 1.0 Hz,
1H), 6.44 (dd, J=8.2, 1.0 Hz, 1H), 4.53 (br s, 2H), 2.54 (s, 3H),
2.40 ppm (s, 3H); LC–MS (ESI): m/z: 190 [M+H]+; tR =2.02 min.
using CH2Cl2/CH3OH as eluent (98:2 v/v) to give diethyl 2-(((2-(3,5-
dimethylisoxazol-4-yl)pyridin-4-yl)amino)methylene)malonate
as
a white solid (6.6 g, 92%): mp: 109–1108C; 1H NMR (300 MHz,
CDCl3): d=10.98 (d, J=13.2 Hz, 1H), 8.58 (d, J=6.0 Hz, 1H), 8.53
(d, J=13.2 Hz, 1H), 7.08–6.86 (m, 2H), 4.32 (q, J=7.2 Hz, 2H), 4.27
(q, J=7.1 Hz, 2H), 2.58 (s, 3H), 2.42 (s, 3H), 1.38 (t, J=7.1 Hz, 3H),
1.34ppm (t, J=7.2 Hz, 3H); LC–MS (ESI): m/z: 360 [M+H]+; tR =
2.92 min.
Step 2: Diphenyl ether (40 mL) was heated to 2608C and diethyl 2-
(((2-(3,5-dimethylisoxazol-4-yl)pyridin-4-yl)amino)methylene)malo-
nate (1.79 g, 5 mmol) was added portion by portion over 2 h. The
solution was cooled down and concentrated to half-volume. Cyclo-
hexane was added to induce precipitation and the resulting solid
was filtered, dried in vacuo and recrystallized in N,N-dimethylfor-
mamide (DMF) to give 12 as an off-white solid (1.3 g, 83%):
1H NMR (300 MHz, CDCl3): d=10.72 (br s, 1H), 9.27 (s, 1H), 7.88 (s,
1H), 7.51- 7.42 (m, 2H), 7.11 (m, 1H), 7.00 (m, 1H), 6.93 (m, 1H),
4.47 (q, J=7.0 Hz, 2H), 2.47 (s, 3H), 2.33 (s, 3H), 1.48 ppm (t, J=
7.0 Hz, 3H); LC–MS (ESI): m/z: 314 [M+H]+; tR =2.02 min.
2-(3,5-Dimethylisoxazol-4-yl)pyridin-4-amine
(3.0 g, 2.6 mmol, 0.03 equiv) was added to amine 8 (14.9 g,
86.2 mmol), (3,5-dimethylisoxazol-4-yl)boronic acid (12.1 g,
(10):
Pd(PPh3)4
86.2 mmol, 1 equiv) and Ba(OH)2·8H2O (68.0 g, 215.5 mmol,
2.5 equiv) in DME/H2O (5:1 v/v, 120 mL). The reaction mixture was
stirred at reflux for 3 days before being concentrated to dryness.
The residue was taken up in CH2Cl2 and washed with H2O. The or-
ganic layer was dried over Na2SO4, filtered and concentrated in
vacuo. The crude product was purified by flash chromatography
on silica gel using CH2Cl2/CH3OH as eluent (100:0!90:10 v/v) to
Ethyl 4-((2-(tert-butyl)phenyl)amino)-7-(3,5-dimethylisoxazol-4-
yl)-1,8-naphthyridine-3-carboxylate (13): Step 1: A solution of
ethyl 7-(3,5-dimethylisoxazol-4-yl)-4-oxo-1,4-dihydro-1,8-naphthyri-
dine-3-carboxylate (1.1 g, 3.5 mmol) in POCl3 (15 mL) was stirred at
reflux overnight. The solvent was then removed in vacuo. Toluene
was added and the solvents were removed in vacuo. This proce-
dure was repeated twice and the resulting compound, ethyl 4-
chloro-7-(3,5-dimethylisoxazol-4-yl)-1,8-naphthyridine-3-carboxylate,
was used without further purification.
1
give compound 10 as a white solid (8.2 g, 50%): H NMR (300 MHz,
CDCl3): d=8.28 (d, J=5.5 Hz, 1H), 6.53 (d, J=2.3 Hz, 1H), 6.48 (dd,
J=5.5, 2.3 Hz, 1H), 4.24 (br s, 2H), 2.53 (s, 3H), 2.39 ppm (s, 3H);
13C NMR (75 MHz, CDCl3): d=167.0 (s), 158.7 (s), 153.1 (s), 151.1 (s),
150.3 (d), 116.4 (s), 108.9 (d), 108.0 (d), 12.1 (q), 11.2 ppm (q);
LC–MS (ESI): m/z: 190 [M+H]+; tR =1.29 min.
Step 2: Crude ethyl 4-chloro-7-(3,5-dimethylisoxazol-4-yl)-1,8-naph-
thyridine-3-carboxylate (assumed 300 mg, 0.74 mmol) and 2-(tert-
butyl)aniline (222 mg, 1.5 mmol, 2 equiv) in 1,4-dioxane (10 mL)
were heated to reflux for 3 h. After cooling, the residue was taken
up in CH2Cl2 and washed with saturated NaHCO3. The organic layer
was dried over Na2SO4, filtered and concentrated in vacuo. The
crude product was purified by flash chromatography on silica gel
using CH2Cl2/CH3OH as eluent (100:0!99:1 v/v). The resulting
solid was then triturated in pentane to give compound 13 as
Ethyl 7-(3,5-dimethylisoxazol-4-yl)-4-oxo-1,4-dihydro-1,8-naph-
thyridine-3-carboxylate (11): Step 1: A mixture of amine 9 (4.7 g,
24.9 mmol) and diethyl 2-(ethoxymethylene)malonate (5.65 g,
26.1 mmol, 1.05 equiv) was stirred at 1208C for 20 min. The mix-
ture was cooled down and diluted with CH2Cl2 and washed with
H2O. The organic layer was dried over Na2SO4, filtered and concen-
trated in vacuo. The crude product was triturated in isopropyl
ether to give diethyl 2-(((6-(3,5-dimethylisoxazol-4-yl)pyridin-2-yl)a-
mino)methylene)malonate as a white solid (8.0 g, 90%): 1H NMR
(300 MHz, CDCl3): d=11.09 (d, J=12.6 Hz, 1H), 9.26 (d, J=12.6 Hz,
1H), 7.72 (m, 1H), 7.10 (d, J=7.5 Hz, 1H), 6.77 (d, J=8.1 Hz, 1H),
4.32 (q, J=7.1 Hz, 2H), 4.23 (q, J=7.2 Hz, 2H), 2.65 (s, 3H), 2.50 (s,
3H), 1.38 (t, J=7.2 Hz, 3H), 1.31 ppm (t, J=7.1 Hz, 3H); LC–MS
(ESI): m/z: 360 [M+H]+; tR =3.25 min.
1
a yellow solid (100 mg, 6% over 2 steps): mp: 165–1678C; H NMR
(300 MHz, CDCl3): d=11.02 (br s, 1H), 9.44 (s, 1H), 7.68 (d, J=
8.9 Hz, 1H), 7.60 (d, J=7.9 Hz, 1H), 7.29 (t, J=7.5 Hz, 1H), 7.13 (t,
J=7.5 Hz, 1H), 7.06 (d, J=8.9 Hz, 1H), 6.91 (d, J=7.5 Hz, 1H), 4.48
(q, J=7.1 Hz, 2H), 2.70 (s, 3H), 2.53 (s, 3H), 1.56 (s, 9H), 1.48 ppm
(t, J=7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=169.5 (s), 168.6 (s),
158.9 (s), 158.8 (s), 155.3 (d), 154.9 (s), 153.8 (s), 144.4 (s), 140.3 (s),
136.4 (d), 127.8 (d), 127.5 (d), 127.2 (d),127.1 (d), 118.1 (d), 115.4 (s),
111.5 (s), 105.1 (s), 61.4 (t), 35.3 (s), 30.4 (3q), 14.3 (q), 13.2 (q),
12.1 ppm (q); HRMS: m/z [M+H]+ calcd for C26H29N4O3: 445.2240,
found: 445.2200; LC–MS (ESI): m/z: 445 [M+H]+; tR =3.92 min.
Step 2: Diphenyl ether (140 mL) was heated to 2408C and diethyl
2-(((6-(3,5-dimethylisoxazol-4-yl)pyridin-2-yl)amino)methylene)malo-
nate (7.95 g, 22.1 mmol) was added portion by portion over 1 h.
The solution was cooled down and concentrated to dryness. The
resulting solid was triturated in isopropyl ether to give compound
Ethyl 4-((2-(tert-butyl)phenyl)amino)-7-(3,5-dimethylisoxazol-4-
yl)-1,6-naphthyridine-3-carboxylate (14): Step 1: A solution of 12
(1.26 g, 4.0 mmol) in POCl3 (20 mL) was stirred at 908C for 48 h.
The solvent was then removed in vacuo. Toluene was added and
the solvents were removed in vacuo. This procedure was repeated
twice. After trituration in di-isopropyl ether, the resulting com-
pound, ethyl 4-chloro-7-(3,5-dimethylisoxazol-4-yl)-1,6-naphthyri-
dine-3-carboxylate, was obtained as a white solid (900 mg, 68%):
mp: 2608C; LC–MS (ESI): m/z: 332 [M+H]+; tR =3.10 min.
1
11 as a brown solid (4.2 g, 61%): mp: >2608C; H NMR (300 MHz,
[D6]DMSO): d=12.73 (br s, 1H), 8.54 (d, J=8.3 Hz, 1H), 8.53 (s, 1H),
7.65 (d, J=8.3 Hz, 1H), 4.22 (q, J=7.1 Hz, 2H), 2.69 (s, 3H), 2.49 (s,
3H), 1.28 ppm (t, J=7.1 Hz, 3H); LC–MS (ESI): m/z: 314 [M+H]+;
tR =2.18 min.
Ethyl 7-(3,5-dimethylisoxazol-4-yl)-4-oxo-1,4-dihydro-1,6-naph-
thyridine-3-carboxylate (12): Step 1: A mixture of amine 10 (3.8 g,
20.0 mmol) and diethyl 2-(ethoxymethylene)malonate (4.54 g,
21 mmol, 1.05 equiv) was stirred at 1208C for 1 h. The mixture was
then diluted with CH2Cl2 and washed with H2O. The organic layer
was dried over Na2SO4, filtered and concentrated in vacuo. The
crude product was purified by flash chromatography on silica gel
Step 2: Ethyl 4-chloro-7-(3,5-dimethylisoxazol-4-yl)-1,6-naphthyri-
dine-3-carboxylate (206 mg, 0.62 mmol) and 2-(tert-butyl)aniline
(93 mg, 0.62 mmol, 1 equiv) were heated to fusion in a sealed tube
for 1 min. After cooling, the residue was taken up in CH2Cl2 and
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washed with H2O. The organic layer was dried over Na2SO4, filtered
and concentrated in vacuo. The crude product was purified by
flash chromatography on silica gel using CH2Cl2/CH3OH as eluent
(100:0!99:1 v/v). The resulting solid was triturated in pentane to
give 14 as an off-white solid (140 mg, 51%): mp: 166–1688C;
1H NMR (300 MHz, CDCl3): d=11.21 (s, 1H), 9.33 (s, 1H), 8.66 (s,
1H), 7.71 (s, 1H), 7.62 (dd, J=8.0, 1.4 Hz, 1H), 7.32 (m, 1H), 7.18
(m, 1H), 7.01 (d, J=7.9 Hz, 1H), 4.48 (q, J=7.0 Hz, 2H), 2.58 (s, 3H),
2.44 (s, 3H), 1.57 (s, 9H), 1.49 ppm (t, J=7.0 Hz, 3H); 13C NMR
(75 MHz, CDCl3): d=168.5 (s), 168.2 (s), 158.7 (s), 155.8 (d), 154.7
(s), 153.6 (s), 151.1 (d), 149.5 (s), 144.7 (s), 140.2 (s), 128.1 (d), 127.8
(d), 127.7 (d), 127.0 (d), 120.5 (d), 115.3 (s), 113.1 (s), 105.0 (s), 61.5
(t), 35.3 (s), 30.4 (3q), 14.3 (q), 12.7 (q), 11.7 ppm (q); HRMS: m/z
[M+H]+ calcd for C26H29N4O3: 445.2240, found: 445.2207; LC–MS
(ESI): m/z: 445 [M+H]+; tR =4.01 min.
(100:0!98:2 v/v) to give the resulting compound, diethyl 2-(((5-
(3,5-dimethylisoxazol-4-yl)-6-methoxypyridin-3-yl)amino)methyle-
ne)malonate, as a brown oil (18 g, 93% over 2 steps): 1H NMR
(300 MHz, CDCl3): d=10.62 (d, J=13.5 Hz, 1H), 8.04 (d, J=13.5 Hz,
1H), 7.76 (d, J=2.8 Hz, 1H), 6.94 (d, J=2.8 Hz, 1H), 4.00 (q, J=
7.2 Hz, 2H), 3.93 (q, J=7.0 Hz, 2H), 3.63 (s, 3H), 2.02 (s, 3H), 1.87
(s, 3H), 1.07 (t, J=7.2 Hz, 3H), 1.00 ppm (t, J=7.0 Hz, 3H); LC–MS
(ESI): m/z: 388 [MꢁH]ꢁ; tR =3.15 min.
Step 2: Diphenyl ether (100 mL) was heated to 2608C and diethyl
2-(((6-(3,5-dimethylisoxazol-4-yl)pyridin-2-yl)amino)methylene)malo-
nate (18.0 g, 46.0 mmol) was added portion by portion over 3 h.
Cyclohexane/isopropyl ether (1:1 v/v) was added to induce precipi-
tation. The resulting solid was triturated in isopropyl ether to give
compound as a beige solid 19 (9.1 g, 58%): 1H NMR (300 MHz,
[D6]DMSO): d=8.55 (s, 1H), 7.96 (s, 1H), 4.23 (q, J=7.1 Hz, 2H),
3.98 (s, 3H), 2.35 (s, 3H), 2.15 (s, 3H), 1.29 ppm (t, J=7.1 Hz, 3H);
LC–MS (ESI): m/z: 344 [M+H]+; tR =2.06 min.
4-((2-(tert-Butyl)phenyl)amino)-7-(3,5-dimethylisoxazol-4-yl)-1,8-
naphthyridine-3-carboxylic acid di-hydrochloride (15): NaOH
(0.25 mL, 1n in H2O, 0.25 mmol, 2.5 equiv) was added to a solution
of 13 (44 mg, 0.1 mmol) in EtOH (5 mL). The reaction mixture was
stirred at reflux overnight before being cooled down and
quenched with 1n HCl. The resulting solid was filtered and dried
to give compound 15 as a yellow solid (25 mg, 51%): mp: 225–
2278C; 1H NMR (300 MHz, CDCl3): d=13.12 (s, 1H), 9.52 (s, 1H),
7.74–7.62 (m, 2H), 7.48 (d, J=8.1 Hz, 1H), 7.53 (d, J=9.1 Hz, 1H),
7.34 (t, J=8.1 Hz, 1H), 7.19 (d, J=9.1 Hz, 2H), 2.64 (s, 3H), 2.42 (s,
3H), 1.43 ppm (s, 9H); 13C NMR (75 MHz, [D6]DMSO): d=170.7 (s),
167.7 (s), 158.7 (s), 156.5 (s), 155.1 (s), 150.1 (s), 146.1 (d), 144.0 (s),
139.2 (s), 137.4 (s), 136.9 (d), 128.2 (d), 128.1 (d), 128.0 (d), 126.4
(d), 119.4 (d), 113.9 (s), 110.7 (s), 35.0 (s), 30.1 (3q), 13.1 (q),
11.8 ppm (q); HRMS: m/z [M+H]+ calcd for C24H25N4O3: 417.1927,
found: 417.1942; LC–MS (ESI): m/z: 417 [M+H]+; tR =2.76 min.
Procedure A for synthesis of compounds 20–24: Step 1: A solu-
tion of 19 (6.5 g, 18.9 mmol) in POCl3 (25 mL) was stirred at 1008C
for 10 min under microwave irradiation. The reaction mixture was
evaporated to dryness. Toluene was added, and the solvents were
removed in vacuo. This procedure was repeated twice. After cool-
ing, the residue was taken up in CH2Cl2 and washed with 1n
NaOH. The organic layer was dried over Na2SO4, filtered and con-
centrated in vacuo. The crude product was purified by flash chro-
matography on silica gel using CH2Cl2/CH3OH (98:2 v/v) as eluent
to give ethyl 4-chloro-7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-1,5-
naphthyridine-3-carboxylate as an off-white solid (4.4 g, 63%):
1H NMR (300 MHz, [D6]DMSO): d=9.08 (s, 1H), 8.44 (s, 1H), 4.45 (q,
J=7.1 Hz, 2H), 4.13 (s, 3H), 2.39 (s, 3H), 2.20 (s, 3H), 1.40 ppm (t,
J=7.1 Hz, 3H); 13C NMR (75 MHz, [D6]DMSO): d=167.6 (s), 163.7
(s), 160.5 (s), 159.0 (s), 147.6 (d), 143.0 (s), 140.7 (d), 139.7 (s), 137.4
(s), 126.3 (s), 121.2 (s), 110.2 (s), 62.1 (t), 54.4 (q), 14.0 (q), 11.5 (q),
10.3 ppm (q); HRMS: m/z [M+H]+ calcd for C17H17ClN3O4:
362.0908, found: 362.0916; LC–MS (ESI): m/z: 362 [M+H]+; tR =
3.43 min.
4-((2-(tert-Butyl)phenyl)amino)-7-(3,5-dimethylisoxazol-4-yl)-1,6-
naphthyridine-3-carboxylic acid di-hydrochloride (16): NaOH
(0.45 mL, 1N in H2O, 0.45 mmol) was added to a solution of 14
(80 mg, 0.18 mmol) in EtOH (10 mL). The reaction mixture was
stirred at 808C overnight before being cooled down and concen-
trated to dryness. The crude product was acidified with HCl in
ether and the resulting solid was filtered and dried to give 16 as
a brown solid (53 mg, 60%): mp: 211–2138C; 1H NMR (300 MHz,
[D6]DMSO): d=13.01 (br s, 1H), 9.26 (s, 1H), 8.36 (d, J=7.7 Hz, 2H),
7.72 (d, J=7.9 Hz, 1H), 7.52 (m, 1H), 7.44–7.24 (m, 2H), 2.60 (s,
3H), 2.39 (s, 3H), 1.46 ppm (s, 9H); HRMS: m/z [M+H]+ calcd for
C24H25N4O3: 417.1927, found: 417.1919; LC–MS (ESI): m/z: 417
[M+H]+; tR =2.59 min.
Step 2: The appropriate amine (1–2 equiv) was added to a solution
of ethyl 4-chloro-7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-1,5-naph-
thyridine-3-carboxylate (1 equiv) in 1,4-dioxane. The reaction mix-
ture was stirred at 1008C overnight, before it cooled down and
concentrated to dryness. The crude material was purified by flash
chromatography on silica gel using CH2Cl2/CH3OH (98:2 v/v) as
eluent to give compound 20–24 as solids.
Ethyl 4-((2-(tert-butyl)phenyl)amino)-7-(3,5-dimethylisoxazol-4-
yl)-6-methoxy-1,5-naphthyridine-3-carboxylate (20): 2-tert-Butyla-
niline (2.7 mL, 17.3 mmol, 2 equiv) was used in step 2 of procedur-
e A: 1H NMR (300 MHz, CDCl3): d=11.85 (br s, 1H), 9.16 (s, 1H),
8.75 (s, 1H), 7.56 (m, 1H), 7.34–7.19 (m, 2H), 7.05 (m, 1H), 4.53 (m,
2H), 3.04 (s, 3H), 2.37 (s, 3H), 2.18 (s, 3H), 1.48 (s, 9H), 1.22 ppm (t,
J=7.0 Hz, 3H); HRMS: m/z [M+H]+ calcd for C27H31N4O4: 475.2345,
found: 475.2338; LC–MS (ESI): m/z: 475 [M+H]+; tR =4.11 min.
5-(3,5-Dimethylisoxazol-4-yl)-6-methoxypyridin-3-amine
(18):
Pd(PPh3)4 (2.85 g, 0.0025 mmol, 0.05 equiv) was added to a solution
of amine 17 (10.0 g, 50.0 mmol), (3,5-dimethylisoxazol-4-yl)boronic
acid (21.1 g, 150.0 mmol, 3 equiv) and Ba(OH)2·8H2O (31.0 g,
100.0 mmol, 2.0 equiv) in DME/H2O (6:1 v/v; 290 mL). The reaction
mixture was stirred at reflux for 4 h before being concentrated to
dryness. The crude brown oil 18 was used without further purifica-
1
tion in the next step (15 g): H NMR (300 MHz, CDCl3): d=7.70 (d,
J=2.8, 1H), 6.88 (d, J=2.8, 1H), 3.87 (s, 3H), 2.31 (s, 3H), 2.17 ppm
(s, 3H).
Ethyl 7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-4-((2-(trifluorome-
thoxy)phenyl)amino)-1,5-naphthyridine-3-carboxylate (21): 2-(Tri-
fluoromethoxy)aniline (0.27 mL, 2.0 mmol, 1 equiv) was used in
step 2 of procedure A: 1H NMR (300 MHz, [D6]DMSO): d=9.11 (s,
1H), 8.29 (s, 1H), 7.64–7.28 (m, 4H), 4.27 (q, J=7.1 Hz, 2H), 3.37 (s,
3H), 2.35 (s, 3H), 2.13 (s, 3H), 1.30 ppm (t, J=7.1 Hz, 3H); HRMS:
m/z [M+H]+ calcd for C24H22F3N4O5: 503.1542, found: 503.1501.
Ethyl 7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-4-oxo-1,4-dihydro-
1,5-naphthyridine-3-carboxylate (19): Step 1: A mixture of 18 (as-
sumed 12 g, 55.0 mmol) and diethyl 2-(ethoxymethylene)malonate
(14.4 mL, 71.5 mmol, 1.3 equiv) was stirred at reflux for 4 h. The
mixture was cooled down, and the resulting solid was filtered and
washed with isopropyl ether. The crude product was purified by
flash chromatography on silica gel using CH2Cl2/CH3OH as eluent
Ethyl 7-(3,5-dimethylisoxazol-4-yl)-4-((2-fluorophenyl)amino)-6-
methoxy-1,5-naphthyridine-3-carboxylate (22): 2-Fluoroaniline
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(0.19 mL, 2.0 mmol, 1 equiv) was used in step 2 of procedure A:
1H NMR (300 MHz, [D6]DMSO): d=9.05 (s, 1H), 8.34 (s, 1H), 7.47 (m,
1H), 7.43–7.23 (m, 3H), 4.09 (q, J=7.0 Hz, 2H), 3.62 (s, 3H), 2.37 (s,
3H), 2.16 (s, 3H), 1.24 ppm (t, J=7.0 Hz, 3H); HRMS: m/z [M+H]+
calcd for C23H22FN4O4; 437.1625, found: 437.1586; LC–MS (ESI): m/z:
437 [M+H]+; tR =3.54 min.
2.18 (s, 3H), 1.87–1.60 (m, 7H), 1.34–1.01 ppm (m, 5H); HRMS: m/z
[M+H]+ calcd for C22H27N4O4: 411.2032, found: 411.2036.
Procedure C for synthesis of compounds 30–34: O-(7-Azabenzo-
triazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate
(HATU; 1.2 equiv) and Et3N (1.2 equiv) was added to a solution of
carboxylic acid (1 equiv) in DMF/CH2Cl2 (1:1 v/v). The reaction mix-
ture was stirred at RT for 1 h, and NH3(0.5m in 1,4-dioxane, 2–
4 equiv) was added. After stirring overnight at RT, the mixture was
concentrated to dryness, and the crude material was taken up in
CH2Cl2 and washed with H2O. The organic layer was dried over
Na2SO4, filtered and concentrated in vacuo. The crude product was
purified by flash chromatography on silica gel using CH2Cl2/CH3OH
(98:2 v/v) as eluent to give the desired compound as a solid.
Ethyl 4-(benzylamino)-7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-
1,5-naphthyridine-3-carboxylate (23): Benzylamine (0.21 mL,
2.0 mmol, 1 equiv) was used in step 2 of procedure A: 1H NMR
(300 MHz, [D6]DMSO): d=9.29 (br s, 1H), 8.87 (s, 1H), 8.11 (s, 1H),
7.43–7.22 (m, 5H), 5.55 (m, 2H), 4.31 (q, J=7.0 Hz, 2H), 3.87 (s,
3H), 2.35 (s, 3H), 2.15 (s, 3H), 1.32 ppm (q, J=7.0 Hz, 3H); LC–MS
(ESI): m/z: 433 [M+H]+; tR =3.66 min.
Ethyl 4-((cyclohexylmethyl)amino)-7-(3,5-dimethylisoxazol-4-yl)-
6-methoxy-1,5-naphthyridine-3-carboxylate (24): 1-Cyclohexyl-
methanamine (0.26 mL, 2.0 mmol, 1 equiv) was used in step 2 of
4-((2-(tert-Butyl)phenyl)amino)-7-(3,5-dimethylisoxazol-4-yl)-6-
methoxy-1,5-naphthyridine-3-carboxamide (30): Prepared as de-
scribed in procedure C: H NMR (300 MHz, [D6]DMSO): d=11.40 (s,
1H), 8.95 (s, 1H), 8.38 (m, 1H), 8.07 (s, 1H), 7.69 (m, 1H), 7.42 (m,
1H), 7.10 (m, 2H), 6.89 (m, 1H), 3.04 (s, 3H), 2.30 (s, 3H), 2.09 (s,
3H), 1.46 ppm (s, 9H); HRMS: m/z [M+H]+ calcd for C25H28N5O3:
446.2192, found: 446.2180.
1
1
procedure A: H NMR (300 MHz, [D6]DMSO): d=8.84 (s, 1H), 8.08 (s,
1H), 4.35 (q, J=7.2 Hz, 2H), 4.00 (s, 3H), 3.31 (m, 2H), 2.36 (s, 3H),
2.17 (s, 3H), 1.87–1.57 (m, 7H), 1.36 (t, J=7.2 Hz, 3H), 1.30–
0.97 ppm (m, 5H); HRMS: m/z [M+H]+ calcd for C24H31N4O4:
439.2343, found: 439.2308; LC–MS (ESI): m/z: 439 [M+H]+; tR =
4.14 min.
7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-4-((2-(trifluoromethoxy)-
phenyl)amino)-1,5-naphthyridine-3-carboxamide (31): Prepared
as described in procedure C: mp: 1338C; 1H NMR (300 MHz,
[D6]DMSO): d=11.4 (br s, 1H), 9.02 (s, 1H), 8.49 (br s, 1H), 8.17 (s,
1H), 7.92 (br s, 1H), 7.44 (m, 1H), 7.43–7.32 (m, 2H), 7.26 (m, 1H),
3.19 (s, 3H), 2.33 (s, 3H), 2.12 ppm (s, 3H); HRMS: m/z [M+H]+
calcd for C22H19F3N5O4: 474.1389, found: 474.1347; LC–MS (ESI):
m/z: 474 [M+H]+; tR =3.09 min.
Procedure B for synthesis of compounds 25–29: 1n NaOH
(2 equiv) was added to a solution of ethyl ester (1 equiv) in EtOH.
The reaction mixture was stirred at reflux overnight before being
concentrated to dryness. The residue was taken up in H2O and
quenched with 1n HCl until precipitation. The resulting solid was
filtered and dried to give the corresponding carboxylic acid as
a solid.
7-(3,5-Dimethylisoxazol-4-yl)-4-((2-fluorophenyl)amino)-6-me-
4-((2-(tert-Butyl)phenyl)amino)-7-(3,5-dimethylisoxazol-4-yl)-6-
methoxy-1,5-naphthyridine-3-carboxylic acid (25): Prepared as
described in procedure B: H NMR (300 MHz, CDCl3): d=13.05 (br s,
1H), 9.38 (s, 1H), 8.73 (s, 1H), 7.52 (m, 1H), 7.26–7.15 (m, 2H), 7.06
(m, 1H), 3.04 (s, 3H), 2.35 (s, 3H), 2.18 (s, 3H), 1.46 ppm (s, 9H);
HRMS: m/z [M+H]+ calcd for C25H27N4O4: 447.2032, found:
447.1993; LC–MS (ESI): m/z: 447 [M+H]+; tR =2.56 min.
thoxy-1,5-naphthyridine-3-carboxamide (32): Prepared as de-
scribed in procedure C: mp: 2828C; H NMR (300 MHz, [D6]DMSO):
d=8.93 (s, 1H), 8.14 (s, 1H), 7.38–7.05 (m, 4H), 3.25 (s, 3H), 2.32 (s,
3H), 2.11 ppm (s, 3H); HRMS: m/z [M+H]+ calcd for C21H19FN5O3:
408.1472, found: 408.1436.
1
1
4-(Benzylamino)-7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-1,5-
naphthyridine-3-carboxamide (33): Prepared as described in pro-
cedure C: mp: 1678C; 1H NMR (300 MHz, [D6]DMSO): d=8.76 (s,
1H), 8.08 (s, 1H), 7.43–7.24 (m, 5H), 5.48 (s, 2H), 3.88 (s, 3H), 2.35
(s, 3H), 2.16 ppm (s, 3H); HRMS: m/z [M+H]+ calcd for C22H22N5O3:
404.1722, found: 404.1707; LC–MS (ESI): m/z: 404 [M+H]+; tR =
2.11 min.
7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-4-((2-(trifluoromethoxy)-
phenyl)amino)-1,5-naphthyridine-3-carboxylic acid (26): Prepared
as described in procedure B: mp: 2308C; 1H NMR (300 MHz,
[D6]DMSO): d=9.20 (s, 1H), 8.29 (s, 1H), 7.57–7.35 (m, 4H), 3.17 (s,
3H), 2.33 (s, 3H), 2.12 ppm (s, 3H); HRMS: m/z [M+H]+ calcd for
C22H18F3N4O5: 475.1229, found: 475.1200.
4-((Cyclohexylmethyl)amino)-7-(3,5-dimethylisoxazol-4-yl)-6-me-
7-(3,5-Dimethylisoxazol-4-yl)-4-((2-fluorophenyl)amino)-6-me-
thoxy-1,5-naphthyridine-3-carboxamide (34): Prepared as de-
scribed in procedure C: mp: 2748C; H NMR (300 MHz, [D6]DMSO):
1
thoxy-1,5-naphthyridine-3-carboxylic acid (27): Prepared as de-
1
scribed in procedure B: mp: 2868C; H NMR (300 MHz, [D6]DMSO):
d=8.79 (s, 1H), 8.42 (br s, 1H), 8.07 (s, 1H), 7.96 (br s, 1H), 4.20 (br
s, 1H), 4.06 (s, 3H), 2.39 (s, 3H), 2.19 (s, 3H), 1.87–1.59 (m, 6H),
1.34–0.97 ppm (m, 6H); HRMS: m/z [M+H]+ calcd for C22H28N5O3:
410.2191, found: 410.2168; LC–MS (ESI): m/z: 410 [M+H]+; tR =
4.23 min.
d=9.10 (s, 1H), 8.22 (s, 1H), 7.47 (m, 1H), 7.37–7.17 (m, 3H), 3.24
(s, 3H), 2.33 (s, 3H), 2.12 ppm (s, 3H); HRMS: m/z [M+H]+ calcd
for C21H18FN4O4: 409.1312, found: 409.1321.
4-(Benzylamino)-7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-1,5-
naphthyridine-3-carboxylic acid (28): Prepared as described in
procedure B: mp: 2558C; H NMR (300 MHz, [D6]DMSO): d=8.93 (s,
1H), 8.12 (s, 1H), 7.44–7.26 (m, 5H), 5.64 (s, 2H), 3.84 (s, 3H), 2.36
(s, 3H), 2.16 ppm (s, 3H); HRMS: m/z [M+H]+ calcd for C22H21N4O4:
405.1563, found: 405.1554.
Procedure D for synthesis of compounds 35–39: Diphenylphos-
phoryl azide (DPPA; 1.1 equiv) and Et3N (1.1 equiv) was added to
a solution of carboxylic acid (1 equiv) in DMF. The reaction mixture
was stirred at reflux for 1–2 h before being cooled down and con-
centrated to dryness. The crude product was purified by flash chro-
matography on silica gel using CH2Cl2/CH3OH (98:2 v/v) as eluent
and recrystallized in isopropyl ether to give the desired compound
as a solid.
1
4-((Cyclohexylmethyl)amino)-7-(3,5-dimethylisoxazol-4-yl)-6-me-
thoxy-1,5-naphthyridine-3-carboxylic acid (29): Prepared as de-
1
scribed in procedure B: mp: 2608C; H NMR (300 MHz, [D6]DMSO):
d=8.93 (s, 1H), 8.15 (s, 1H), 4.30 (m, 2H), 4.03 (s, 3H), 2.38 (s, 3H),
1-(2-(tert-Butyl)phenyl)-7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-
1H-imidazo[4,5-c][1,5]naphthyridin-2(3H)-one (35): Prepared as
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described in procedure D: mp: 300–3108C; 1H NMR (300 MHz,
[D6]DMSO): d=8.68 (s, 1H), 8.17 (s, 1H), 7.69 (m, 1H), 7.46 (m, 1H),
7.33 (m, 1H), 7.23 (m, 1H), 3.17 (s, 3H), 2.28 (s, 3H), 2.07 (s, 3H),
1.21 ppm (m, 9H); 13C NMR (75 MHz, [D6]DMSO): d=166.7 (s), 158.9
(s), 158.0 (s), 154.4 (s), 147.9 (s), 140.9 (d), 136.7 (s), 134.0 (s), 132.0
(d), 131.9 (d), 129.4 (s), 129.0 (d), 128.3 (d), 128.2 (s), 126.6 (d),
125.2 (s), 114.9 (s), 110.7 (s), 52.7 (q), 35.6 (s), 31.3 (3q), 11.4 (q),
10.3 ppm (q); HRMS: m/z [M+H]+ calcd for C25H26N5O3: 444.2036,
found: 444.2003; LC–MS (ESI): m/z: 444 [M+H]+; tR =3.09 min.
is significantly (>50%) bound, and in the presence of a sufficient
concentration of a potent inhibitor, the anisotropy of the unbound
fluorescent ligand is measurably different from the bound value.
All data was normalized to the mean of 16 high and 16 low control
wells on each plate. A four-parameter curve fit of the following
form was then applied: y=a+((bꢁa)/(1+(10^x/10^c)^d), where
a is the minimum, b is the Hill slope, c is the pIC50 and d is the
maximum (for a complete protocol, see the Supporting Informa-
tion Section 1.1).
7-(3,5-Dimethylisoxazol-4-yl)-8-methoxy-1-(2-(trifluoromethoxy)-
Use of animals: All animal studies were ethically reviewed and car-
ried out in accordance with European Directive 2010/63/EU and
the GSK Policy on the Care, Welfare and Treatment of Animals.
phenyl)-1H-imidazo[4,5-c][1,5]naphthyridin-2(3H)-one (36): Pre-
1
pared as described in procedure D: mp=1388C; H NMR (300 MHz,
[D6]DMSO): d=11.90 (s, 1H), 8.70 (s, 1H), 8.22 (s, 1H), 7.81 (m, 1H),
7.72–7.55 (m, 3H), 3.26 (s, 3H), 2.29 (s, 3H), 2.08 ppm (s, 3H);
13C NMR (75 MHz, [D6]DMSO): d=166.8 (s), 158.9 (s), 158.5 (s), 153.0
(s), 145.5 (s), 141.0 (d), 136.5 (s), 132.0 (d), 131.9 (d), 130.6 (d), 128.3
(s), 128.1 (s), 127.9 (s), 127.7 (d), 125.2 (s), 121.4 (d), 119.9 (q, J=
258.0 Hz), 115.3 (s), 110.7 (s), 53.0 (q), 11.3 (q), 10.2 ppm (q);
19F NMR (300 MHz, [D6]DMSO): d=ꢁ56.6 ppm (s); HRMS: m/z
[M+H]+ calcd for C22H17F3N5O4: 472.1233, found: 472.1230; LC–MS
(ESI): m/z: 472 [M+H]+; tR =2.87 min.
Accession codes
Atomic coordinates of BRD4-BD1:compound 5 (PDB code: 4BW3),
BRD4-BD1:compound 15 (PDB code: 4BW2), BRD4-BD1:compound
25 (PDB code: 4BW3) and BRD4-BD1:compound 36 (PDB code:
4BW4) have been deposited with the Protein Data Bank (PDB).
Glossary
7-(3,5-dimethylisoxazol-4-yl)-1-(2-fluorophenyl)-8-methoxy-1H-
imidazo[4,5-c][1,5]naphthyridin-2(3H)-one (37): Prepared as de-
scribed in procedure D: mp: 290–3008C; 1H NMR (300 MHz,
[D6]DMSO): d=8.70 (s, 1H), 8.22 (s, 1H), 7.72 (m, 1H), 7.62–7.35 (m,
3H), 3.32 (s, 3H), 2.29 (s, 3H), 2.09 ppm (s, 3H); 13C NMR (75 MHz,
[D6]DMSO): d=166.8 (s), 159.0 (s), 158.6 (s), 158.5 (d, J=248.1 Hz),
153.1 (s), 141.0 (d), 136.6 (s), 132.1 (d), 131.5 (d), 130.7 (t, J=
7.7 Hz), 128.1 (s), 127.9 (s), 125.2 (s), 124.5 (t, J=3.3 Hz), 123.3 (d,
J=12.6 Hz), 116.0 (t, J=19.3 Hz), 115.4 (s), 110.8 (s), 53.2 (q), 11.3
(q), 10.3 ppm (q); 19F NMR (300 MHz, [D6]DMSO): d=ꢁ120.8 ppm
(s); HRMS: m/z [M+H]+ calcd for C21H17FN5O3: 406.1315, found:
406.1312; LC–MS (ESI): m/z: 406 [M+H]+; tR =2.68 min.
BET: bromodomain and extra-terminal; NUT: nuclear protein in
testis; BRD2/3/4, bromodomain-containing protein 2/3/4; BRDT:
bromodomain, testis-specific; FP: fluorescence polarization; IL-6, in-
terleukin-6; PBMC: peripheral blood mononuclear cell; RLM: rat
liver microsomes; HLM: human liver microsomes; DPPA: diphenyl-
phosphoryl azide; NT: not tested. HATU: O-(7-azabenzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate; PAI-1: plasmi-
nogen activator inhibitor-1; FaSSIF: fasted-state simulated intestinal
fluid; LPS: lipopolysaccharides.
Acknowledgements
1-Benzyl-7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-1H-imidazo-
[4,5-c][1,5]naphthyridin-2(3H)-one (38): Prepared as described in
procedure D: mp: 2748C; H NMR (300 MHz, [D6]DMSO): d=8.64 (s,
1H), 8.23 (s, 1H), 7.32–7.24 (m, 5H), 5.71 (s, 2H), 3.82 (s, 3H), 2.33
(s, 3H), 2.13 ppm (s, 3H); HRMS: m/z [M+H]+ calcd for C22H20N5O3:
402.1566, found: 402.1547; LC–MS (ESI): m/z: 402 [M+H]+; tR =
2.80 min.
1
The authors would like to thank Romain Gosmini and the many
colleagues that have contributed to the ApoA1 program and
those that have taken time to pass on useful comments in the
preparation of this manuscript.
1-(Cyclohexylmethyl)-7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-
1H-imidazo[4,5-c][1,5]naphthyridin-2(3H)-one (39): Prepared as
described in procedure D: mp: 2278C; 1H NMR (300 MHz,
[D6]DMSO): d=8.58 (s, 1H), 8.23 (s, 1H), 4.25 (d, J=7.7 Hz, 2H),
4.06 (s, 3H), 2.36 (s, 3H), 2.17 (s, 3H), 1.73–1.55 (m, 5H), 1.21–
1.02 ppm (m, 6H); 13C NMR (75 MHz, [D6]DMSO): d=166.8 (s), 159.0
(s), 158.6 (s), 154.2 (s), 141.3 (d), 136.5 (s), 131.7 (d), 128.4 (s), 128.2
(s), 124.8 (s), 115.1 (s), 110.9 (s), 53.9 (q), 47.8 (t), 37.7 (d), 29.9 (t),
26.0 (t), 25.2 (t), 11.4 (q), 10.4 ppm (q); HRMS: m/z [M+H]+ calcd
for C22H26N5O3: 408.2036, found: 408.2059; LC–MS (ESI): m/z: 408
[M+H]+; tR =3.13 min.
Keywords: BET family bromodomains inhibitors · epigenetic
readers · inflammation · naphthyridines
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Biological assays
Fluorescence polarization (FP) assays: The binding of compounds to
His6-tagged BRD2–4 was assessed using a fluorescence anisotropy
binding assay. Generally the method involves incubating the bro-
modomain protein (BRD2 (1--473), BRD3 (1--435) or BRD4 (1--477)),
fluorescent ligand and a variable concentration of test compound
together to reach thermodynamic equilibrium, under conditions
such that in the absence of test compound the fluorescent ligand
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Received: June 10, 2013
Published online on && &&, 0000
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Place your BET! Naphthyridine deriva-
tives were designed and synthesized as
potent inhibitors of the BET bromodo-
main family with good cell activity and
oral pharmacokinetic parameters. X-ray
crystal structures were solved and quan-
tum mechanical calculations were used
to explain the higher affinity of the 1,5-
isomer over the others. The best com-
pounds were progressed in a mice
model of inflammation and exhibited
dose-dependent anti-inflammatory
pharmacology.
O. Mirguet,* Y. Lamotte, C.-w. Chung,
P. Bamborough, D. Delannꢀe, A. Bouillot,
F. Gellibert, G. Krysa, A. Lewis,
J. Witherington, P. Huet, Y. Dudit,
L. Trottet, E. Nicodeme
&& – &&
Naphthyridines as Novel BET Family
Bromodomain Inhibitors
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