Cell Penetrant Inhibitors of the KDM4 and KDM5 Families of Histone Lysine Demethylases. 2. Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives

Article abstract of DOI:10.1021/acs.jmedchem.5b01538

Following the discovery of cell penetrant pyridine-4-carboxylate inhibitors of the KDM4 (JMJD2) and KDM5 (JARID1) families of histone lysine demethylases (e.g., 1), further optimization led to the identification of non-carboxylate inhibitors derived from pyrido[3,4-d]pyrimidin-4(3H)-one. A number of exemplars such as compound 41 possess interesting activity profiles in KDM4C and KDM5C biochemical and target-specific, cellular mechanistic assays.

Full text of DOI:10.1021/acs.jmedchem.5b01538

Article
pubs.acs.org/jmc
Cell Penetrant Inhibitors of the KDM4 and KDM5 Families of Histone
Lysine Demethylases. 2. Pyrido[3,4‑d]pyrimidin-4(3H)‑one
Derivatives
Susan M. Westaway,*,† Alex G. S. Preston,*,† Michael D. Barker,† Fiona Brown,‡ Jack A. Brown,†
Matthew Campbell,† Chun-wa Chung,‡ Gerard Drewes,§ Robert Eagle,‡ Neil Garton,† Laurie Gordon,‡
Carl Haslam,‡ Thomas G. Hayhow,† Philip G. Humphreys,† Gerard Joberty,§ Roy Katso,‡
Laurens Kruidenier,†,∇ Melanie Leveridge,‡ Michelle Pemberton,‡ Inma Rioja,† Gail A. Seal,†
Tracy Shipley,† Onkar Singh,‡ Colin J. Suckling,∥ Joanna Taylor,‡ Pamela Thomas,‡ David M. Wilson,†,⊥
Kevin Lee,†,# and Rab K. Prinjha†
†Epinova Discovery Performance Unit, Medicines Research Centre, GlaxoSmithKline R&D, Stevenage SG1 2NY, U.K.
‡Platform Technology and Sciences, Medicines Research Centre, GlaxoSmithKline R&D, Stevenage SG1 2NY, U.K.
§Cellzome GmbH, a GSK Company, Meyerhofstrasse 1, 69117 Heidelberg, Germany
∥Department of Pure and Applied Chemistry, WestCHEM Research School, University of Strathclyde, Glasgow G1 1XL, U.K.
S
* Supporting Information
ABSTRACT: Following the discovery of cell penetrant pyridine-4-carboxylate inhibitors of the KDM4 (JMJD2) and KDM5
(JARID1) families of histone lysine demethylases (e.g., 1), further optimization led to the identification of non-carboxylate
inhibitors derived from pyrido[3,4-d]pyrimidin-4(3H)-one. A number of exemplars such as compound 41 possess interesting
activity profiles in KDM4C and KDM5C biochemical and target-specific, cellular mechanistic assays.
enzymes, we were keen to determine whether we could make
further improvements through the design of less acidic and thus
potentially more cell penetrant compounds, targeting cellular
activity pIC50 ≥ 6 (IC50 ≤ 1 μM). In a similar, recently reported
approach, optimization of the broad spectrum 2-oxoglutarate-
dependent dioxygenase inhibitor 8-hydroxyquinoline-5-carbox-
ylic acid, which included removal of the carboxylate, led to
increased cellular activity and a reduced differential between
cellular and biochemical activity against members of the KDM4
family.5
INTRODUCTION
■
There is currently rapidly expanding interest in how epigenetic
control of gene expression is implicated in various disease
states.1−3 As a consequence, “probe molecules” with proven
cellular activity against a specific epigenetic target or family or
targets, for example, the “writers”, “erasers”, or “readers” of
post-translational modifications of nuclear histone proteins, can
play a key role in understanding how modulation of such
targets could affect disease progression. In the preceding
article4 we reported on our initial efforts to identify inhibitors of
the KDM4 (JMJD2) family of histone lysine demethylases that
led to the discovery and optimization of a series of cell-active 3-
aminopyridine-4-carboxylate based inhibitors of both the
KDM4 and the KDM5 (JARID1) families, for example,
compounds 1 and 2 (Figure 1).
Special Issue: Epigenetics
Received: October 1, 2015
While these compounds may prove useful for further in vitro
phenotypic exploration of the roles of KDM4 and KDM5
© XXXX American Chemical Society
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Figure 1. Pyridine-4-carboxylic acid inhibitors of KDM4 and KDM5
families of histone lysine demethylases (KDMs).
RESULTS AND DISCUSSION
■
As described in the preceding article,4 we found that the
fragment core of KDM4C cell active molecules 1 and 2, 3-
amino-4-pyridinecarboxylic acid 3, was an inhibitor of the
KDM4 family with micromolar activity in our biochemical
RapidFire mass spectrometry (RFMS)-based assays.6 Selectivity
vs the related Jumonji enzyme KDM6B (JMJD3) and the prolyl
hydroxylase EGLN3 (also known as PHD3), a more distantly
related 2-oxoglutarate (2-OG) dependent dioxygenase enzyme,
was encouraging with activity against these targets assessed
using RFMS and homogeneous time-resolved fluorescence
(HTRF) assay formats, respectively4 (Table 1).
Figure 2. X-ray crystal structure of 3 bound to KDM4D active site
(PDB code 5FP9).
Scheme 1. Synthesis of Bicyclic Pyridine Derivatives 4, 6,
a
and 7
Table 1. Inhibition Profile of 3-Amino-4-pyridinecarboxylic
Acid 3 in KDM4 and KDM6B RFMS Assays and an EGLN3
HTRF Assay4,7
pIC50
KDM4A
KDM4C
KDM4D
KDM4E
KDM6B
EGLN3
6.3
6.2
5.8
5.9
4.2
a
<4.3
a
4.2, n = 12; <4.0, n = 12.
a
Conditions: (a) HC(NH)NH2.HOAc, N,N-dimethylacetamide, 150
°C, 16 h; (b) 1,1′-CDI, PhNH2, 2Me-THF, 50 °C, 20 h; (c) n-BuLi,
DMF, THF, −70 to 0 °C; (d) 35% N2H4 in water, reflux, 2 h; (e) conc
HCl, water, NaNO2, SO3, rt; (f) 2 M HClaq, reflux, 5 h.
With the aim of improving cell penetration, we targeted a
range of less acidic, bicyclic fragments based on 3 that
conserved the key sp2 nitrogen atom that interacts with the
catalytic metal within the active site and that also contained
functionality that could potentially form similar interactions
with K210 and Y136 as shown for 3 bound to KDM4D (Figure
2). Compounds 4, 6, and 7 were synthesized according to
Scheme 1. Thus, pyrido[3,4-d]pyrimidin-4(3H)-one 4 was
prepared utilizing a literature method involving treatment of 3-
amino-4-pyridinecarboxylic acid with formamidine acetate8 in a
moderate yield of 39%. Pyrido[3,4-d]pyridazin-1(2H)-one 6
was prepared via a three-step literature-based synthesis9
wherein 4-pyridinecarboxylic acid was derivatized as the N-
phenylamide 10 and then deprotonated with n-BuLi and
treated with N,N-dimethylformamide to give the crude
isoindolin-1-one intermediate 11.
Reaction with aqueous hydrazine then gave the target bicycle
6. 1H-Pyrazolo[3,4-c]pyridin-3(2H)-one 7 was prepared in two
steps from 3-amino-4-pyridinecarboxylic acid via conversion of
the amine to the hydrazine 12 by treatment with sodium nitrite
and sulfur dioxide followed by ring closure under acidic
conditions in 25% overall yield.10 Additionally, 2,6-naphthyr-
idin-1(2H)-one 5 was sourced commercially. The data from
screening compounds 4−7 in the KDM4C RFMS biochemical
assay are shown in Table 2. Compounds 5−7 did not show
detectable activity in this assay, indicating the preference for an
sp2 nitrogen atom at the 1-position. The pyrido[3,4-
d]pyrimidin-4(3H)-one 4 showed encouraging activity in the
KDM4C assay with pIC50 = 5.7 and was tested in the RFMS
assays for other KDM4 family members giving pIC50 of 5.9, 5.0,
and 5.1 for KDM4A, -D, and -E, respectively. Additionally, 4
gave no detectable activity in our KDM6B and EGLN3 assays
(pIC50 <4.0 and <4.3, respectively).
X-ray crystallography of 4 bound into KDM4D confirmed
that the compound, as designed, adopts a very similar binding
mode to the pyridine carboxylate 3 (Figure 3a). It was also
apparent from this structure that 4 possessed a high level of
shape complementarity in the region of the 5- and 6-positions
that offered little scope for further substitution (Figure 3b).
Indeed, the introduction of methyl substituents into either of
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Table 2. Activity of Bicyclic Analogues 4−9 vs KDM4C7
Scheme 2. Synthesis of 1-Benzyl and 1-Phenylpropyl
Analogues 8 and 9
a
compd
KDM4C RFMS pIC50
5.7
a
4
5
6
7
8
9
Conditions: (a) HCONH2, POCl3, 0 °C to rt, 16.5 h.
a
<4.0
<4.0
interactions with the active site iron atom. However, this
proved unsuccessful with all compounds prepared giving pIC50
≤ 4.6 (Supporting Information Table 1). We then turned our
attention to exploration of the 2-postion of compound 4, and
the effects of a directly attached aryl were investigated in the
first instance with the synthesis of key exemplars shown in
Scheme 3. The ethyl 3-amido-4-pyridine carboxylates were
reacted with methanolic ammonia12 followed by aqueous
sodium hydroxide to furnish compounds 15−17 in yields of 7−
60%.
<4.0
4.6
4.0, <4.0
a
See ref 11.
these positions resulted in IC50 values greater than 100 μM
(data not shown). The cLogP and ChromLogDpH7.4 of 4 are
−0.7 and −0.6, respectively, and these values are suboptimal for
passive permeability into cells. Thus, the 1-, 2-, and 8-positions
appeared the most viable for exploration of the SAR and
modulation of physicochemical properties.
a
Scheme 3. Synthesis of Aryl and Hetaryl Analogues 15−17
We hypothesized that substitution of the nitrogen atom at
the 1-position would give a tautomeric derivative of 4 that
could still potentially form suitable interactions with the key
lysine and tyrosine residues, even though this substitution
would result in the loss of the hydrogen bond donor (HBD) at
the 3-position; indeed 3-amino-4-pyridinecarboxylic acid 3 will
predominantly exist as the ionized form under physiological
conditions and thus will not have hydrogen bond donor
capability in the equivalent position. The 1-benzyl and 1-(3-
phenylpropyl) derivatives of 4, compounds 8 and 9, were
synthesized via cyclization of the corresponding 3-amino-4-
pyridinecarboxylic acids 13 and 14 with formamide in the
presence of phosphorus oxychloride, as shown in Scheme 2.
However, we were somewhat surprised to find that these
analogues 8 and 9 showed considerably reduced potency at
KDM4C when compared to core 4 (Table 2) and the parent
acids 13 and 14 (pIC50 = 7.0, 6.7, respectively).
a
Conditions: (a) NH3 (7 M in MeOH), rt, 4 h, or 0.88 NH3, MeOH,
rt, 21.5 h, then NaOH (10 Maq), EtOH.
The introduction of a phenyl ring into the 2-position, 15
resulted in 10-fold lower potency at KDM4C (pIC50 = 4.7)
when compared to 4 (Table 3). However, some of the activity
could be regained by moving to the 3-pyridyl analogue 16 with
pIC50 = 5.1 at KDM4C.
A variety of substituents was investigated to determine if any
further increases in potency could be achieved. Meta
substitution of a phenyl or 3-pyridyl ring relative to the
position of attachment to the core was most beneficial, with
compound 17 giving the best albeit a modest 4-fold increase in
potency to pIC50 = 5.7 (IC50 = 2 μM) (Table 3). To assess
These data indicated the importance of the HBD at the 3-
position. Therefore, keeping this in place, we next synthesized a
small set of compounds with substituents at the 8-position,
including some that could potentially combine with the
nitrogen atom in the 7-postion of the core to form bidentate
Figure 3. X-ray structure of 4 bound to KDM4D active site (PDB code 5FPA).
C
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Table 3. Activity of 2-Aryl Analogues 15−17 vs KDM4C7
The S- and O-linked analogues 20 and 21 gave the highest
level of activity in the KDM4C RFMS assay (pIC50 = 6.1 and
6.0, respectively), followed by the NH analogue 19 and then
the CH2 analogue 18, which was 12- to 16-fold less active
(Table 4). The increase in potency from CH2 through to O and
S corresponds to a decrease in the pKa value of the 3-NH, and
we hypothesize that the increased acidity contributes to a
stronger H-bonding interaction between this NH and the key
tyrosine residue described previously. Furthermore, the O-
linked analogue 21 was also starting to show detectable activity
in the KDM4C cell based assay on some test occasions, albeit at
a low level.
Ethoxy analogue 21 was used as a starting point to further
optimize biochemical and cellular potency in this series, and a
set of O-linked six-membered ring containing analogues was
synthesized as outlined in Scheme 5. The thioethyl analogue 20
was oxidized with either mCPBA or peracetic acid to give either
the sulfonyl intermediate 20a or mixtures of the sulfonyl and
sulfinyl intermediates 20b. The proportions of each of the
oxidized sulfur species present in the crude product mixture
20b were variable between reaction runs, and the final step,
treatment with an appropriate nucleophile, was carried out
directly on isolated crude material due to the relative instability
of these species (see Experimental Section for details).
Optimization of this route was required with some of the less
reactive nucleophiles. Thus, 20 was protected with the SEM
group to give 23, and this was then reacted with mCPBA. The
crude oxidized intermediate 23a was then reacted with the
required alcohol in the presence of cesium carbonate, and a
final Lewis acid-mediated deprotection step furnished the
required product. The data obtained from testing these
analogues in the KDM4C RFMS and cell imaging assays are
shown in Table 5.
compd
KDM4C RFMS pIC50
4
5.7
4.7
5.1
5.7
15
16
17
cellular activity, compound 17 was tested in the previously
described KDM4C mechanistic, high content imaging assay.4 In
this assay U2OS cells were subjected to a Bacmam-mediated
transfection with full-length HALO-tagged KDM4C in the
presence of test compound at 37 °C for 24 h, followed by
determination of the level of global H3K9Me3 demethylation
compared to positive and negative controls. Unfortunately the
level of biochemical activity observed for 17 (pIC50 = 5.7) did
not translate into measurable cellular activity in this assay
(pIC50 < 4.0).
As part of an investigation into a range of small aliphatic
substituents at the 2-postion of 4, a series of compounds was
prepared wherein an ethyl group was linked to the core through
CH2, NH, S, and O linker groups. The methylene linked
analogue 18 was prepared in a manner similar to that utilized
for the directly attached aryl analogues shown in Scheme 3,
whereas the heteroatom linked compounds 19−21 were
prepared as outlined in Scheme 4. Thus, 3-amino-4-
pyridinecarboxylic acid was cyclized with thiourea to give the
thione 22.13 Methylation with iodomethane and finally
treatment with ethylamine or sodium ethoxide under micro-
wave heating then furnished 19 and 21, respectively. Thioethyl
analogue 20 was prepared by treatment of 22 with iodoethane
under basic conditions.
The phenyl analogue 24 gave similar potency to the ethyl
compound 21 with pIC50 = 6.2 in the KDM4C RFMS
biochemical assay and encouragingly showed improved activity
in the cellular assay with pIC50 = 4.9. The benzyl analogue 25
was less potent in both assay formats. The cyclohexyl and
cyclohexylmethyl analogues 26 and 27 also showed decreased
a
Scheme 4. Synthesis of S-, N-, and O-Linked Analogues 19−21
a
Conditions: (a) H2NC(S)NH2, 160 °C, 16 h, 77%; (b) EtI, 1 M NaOHaq, MeOH, rt, 1 h, 88%; (c) MeI, 1 M NaOHaq, MeOH, rt, 16 h, 64%; (d)
EtNH2 (2 M in THF), microwave, 120 °C, 4 h, then 150 °C, 4 h, 63%; (e) NaOEt (21% w/w in EtOH), microwave, 140 °C, 4 h, 14%.
D
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Table 4. KDM4C Biochemical and Cell Activity of 18−217
compd
KDM4C RFMS pIC50
KDM4C cell pIC50
<4.0
pKa
4
5.7
4.9
5.5
6.1
6.0
8.3
8.8
8.6
6.8
7.5
a
18
19
20
21
nd
a
nd
<4.0
b
4.2
a
b
nd = not determined. 4.2, n = 3; <4.0, n = 3.
a
Scheme 5. Synthesis of Aryloxy, Hetaryloxy, and Alkoxy Analogues 24−43
a
Conditions: (a) (i) mCPBA, NMP, rt, 3 h or (ii) mCPBA, THF, rt, overnight or (iii) 39% MeCO3H in AcOH, THF, 3 Å molecular sieves, rt,
overnight; (b) ROH, NaH, DMF, 110 °C, overnight; (c) SEM-Cl, K2CO3, DMF, 50 °C, overnight; (d) mCPBA, THF, rt, 1 h; (e) ROH, CsCO3,
1,4-dioxane, 60 °C, 3 h; (f) MgBr2.Et2O, MeNO2, 95 °C, 1 h.
Table 5. KDM4C Biochemical and Cell Activity with Lipophilicity Parameters of Analogues 24-327
compd
R
KDM4C RFMS pIC50
KDM4C cell pIC50
cLogP
ChromLogDpH7.4
24
25
26
27
28
29
30
31
32
Ph
6.2
5.7
5.5
4.9
<4.0
4.6
1.4
1.6
1.2
2.8
CH2Ph
c-hexyl
2.3
3.7
a
CH2-c-hexyl
3-pyridyl
5.5
<4.0
4.9
2.9
4.5
6.3
6.3
6.7
6.4
6.0
−0.1
2.1
−0.2
2.0
4-ClPh
5.0
(3-OH)Ph
5.0
0.8
0.3
(4-Cl-3-OH)Ph
(6-iPr)-3-pyridyl
5.4
1.4
1.1
5.4
1.4
1.5
a
5.5, n = 2; <4, n = 1.
potency in the KDM4C RFMS assay compared to the phenyl
compound 24. Introduction of a nitrogen atom into the phenyl
ring was well tolerated with the 3-pyridyl compound 28
possessing a similar profile to 24 in terms of both biochemical
and cellular potency. Substitution of the phenyl ring also gave
analogues of interest with the 4-chlorophenyl analogue 29
being equipotent with the phenyl compound 24. Introduction
of the 3-hydroxy substituent 30 gave a 3-fold increase in
potency to pIC50 = 6.7. Combining these two substituents,
however, did not result in additive SAR with compound 31
possessing intermediate KDM4C RFMS activity, pIC50 = 6.4,
although this combination of substituents did result in a modest
improvement in cellular activity with 31 giving pIC50 = 5.4 in
the KDM4C cell imaging assay. A similar trend was noted for
compound 32 whereby addition of a 6-isopropyl substituent to
28 gave a compound with cell activity pIC50 = 5.4.
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Table 6. KDM4C Biochemical and Cell Activity with Lipophilicity Parameters of Analogues 33−457
compd
R
KDM4C RFMS pIC50
KDM4C cell pIC50
cLogP
ChromLogDpH7.4
33
34
35
36
37
38
39
40
41
42
43
44
45
H
6.4
6.4
6.3
5.1
5.1
5.3
5.7
5.4
5.6
5.3
5.4
5.3
5.2
4.9
<4.0
−0.5
−0.5
0.4
−0.6
−0.3
0.7
Me
iPr
a
c-pentyl
c-Hex
6.2
1.0
1.7
6.4
6.3
6.2
6.4
6.3
1.6
2.2
c-Hep
2.1
2.9
CH2(c-hexyl)
Ph
2.2
2.9
1.5
1.9
CH2Ph
1.5
1.8
b
CH2(3-OMe)Ph
CH2-3-Pyr
Me
6.2
1.4
2.0
6.2
5.6
0.0
0.3
−0.2
−0.2
0.0
c
Me
5.7
nd
−0.2
a
b
c
6.2, n = 8; <4.0, n = 1. 6.2, n = 9; <4.0 n = 1;. nd = not determined.
Figure 4. X-ray structure of 33 bound to KDM4D active site (PDB code 5FPB).
In a parallel investigation, a series of pyrazolyloxy derivatives
of 4 was also prepared (Scheme 5), and the data from testing
these compounds are shown in Table 6. Somewhat surprisingly
the parent pyrazole 33 showed a relatively high level of cellular
activity in the KDM4C cell imaging assay given its highly polar
nature (pIC50 = 5.1, cLogP = −0.5, ChromLogD = −0.6). A
crystal structure of 33 bound to KDM4D showed excellent
shape complementarity with the protein (Figure 4a). One
pyrazole nitrogen forms a water-bridged interaction to Q88
with limited potential for substitution. In contrast the other
nitrogen provides a vector for growth out toward solvent and
the opportunity to pick up additional interactions (Figure 4b).
As expected, N-substitution of the pyrazole ring was well
tolerated when this substitution gave the 4-pyrazolyl isomers;
for example, Me 34, i-Pr 35, c-pentyl 36, phenyl 40, and benzyl
41 substitution gave compounds with biochemical potency in
the range pIC50 = 6.2−6.4. The introduction of methyl groups
into the 3- and 5- positions of the pyrazole ring (44, 45) was
not beneficial to activity due to the restricted size of the pocket
in these regions as illustrated in Figure 4c. We observed that the
effects of increasing lipophilicity on cellular potency were small;
thus, by comparison of 36, 37, 38, and 39, there was a 16-fold
increase in ChromLogD but KDM4C cell imaging activity
remained broadly stable at pIC50 = 5.3−5.7 (IC50 = 2−5 μM).
This suggests these compounds may be actively transported
into cells and that increasing potency through modifications
immediately adjacent to the 2-OG binding pocket may be
challenging. These data also indicate a significant component of
activity for this series resides in the fragment-like core that
mimics the key interactions of 2-OG within the KDM4
enzymes.
Indeed, from examination of X-ray crystallography data of a
number of examples, including the 3-pyridine analogue 28,
phenol analogue 30, and the pyrazole 33, we found
heteroatoms strategically placed to form direct or water-
mediated hydrogen bonding interactions with the protein were
successful in making these contacts. However, at best, only
modest potency gains were observed when compared with the
core 4 and the phenoxy analogue 24. Therefore, substitution
into this region may produce sufficient entropic and enthalpic
penalties to attenuate the positive effects of forming a range of
positive interactions (H-bonding, van der Waals), giving rise to
relatively flat SAR. Nevertheless, the cell activity demonstrated
by a range of these compounds and their novelty as non-
carboxylate KDM4 inhibitors led us to further profile key
exemplars as shown in Table 7. Also included is the inactive,
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Table 7. Further Profiling of Cell Active Pyridopyrimidinone Inhibitors 31, 32, 36, 39, and 41 with “Negative Control”
Analogue 467
target
assay type
value
31
32
36
39
41
46
KDM4A
KDM4C
KDM4D
KDM4E
KDM6B
KDM5C
EGLN3
KDM4C
KDM5C
RFMS
pIC50
pIC50
pIC50
pIC50
pIC50
pIC50
pIC50
pIC50
pIC50
6.4
5.8
6.0
6.2
6.4
6.1
6.2
6.2
<4.0
<4.0
<4.0
<4.0
<4.0
a
b
RFMS
6.4
6.2
6.2
6.3
RFMS
6.5
6.3
6.4
6.4
RFMS
6.9
6.4
6.3
6.3
e
RFMS
<4.0
6.7
nd
<4.0
6.9
<4.0
7.1
<4.0
7.2
c
RFMS
6.9
4.5
e
HTRF
cell imaging
<4.0
5.4
nd
5.4
4.3
<4.0
5.7
<4.0
5.3
<4.0
5.3
<4.0
<4.0
<4.0
d
cell imaging
4.5
5.3
5.6
5.4
a
b
c
d
e
6.2, n = 8; <4.0, n = 1. <4.0, n = 5; 4.4, n = 1. 4.5, n = 3; <4.0, n = 7. 4.5, n = 2; <4.0 n = 4. nd = not determined.
“negative control” analogue 46 of compound 41 wherein the
nitrogen atom has been translocated from the 7- to the 8-
position of the core and is therefore no longer able to interact
with the active site iron.
2-OG dependent dioxygenases, including other KDM enzymes,
a chemical proteomics approach has been developed in our
laboratories, and the activity/selectivity profile determined for
the 1-benzylpyrazolyloxy derivative 41 using this approach will
be published in due course.15
Compound 41 and its analogues, for example, those shown
in Table 7, may have utility in probing the effects of inhibition
of KDM4 and KDM5 enzymes in a cellular phenotypic context
when used in combination with an inactive control molecule
such as 46. Additionally, these compounds may also serve as a
staging point in the design and synthesis of further nonacidic
inhibitors of these two families or indeed other members of the
broader 2-OG utilizing class of enzymes.
While these compounds are equipotent across the KDM4
family and have maintained high levels of selectivity vs KDM6B
and the more distantly related prolyl hydroxylase EGLN3, we
also found that like the 3-amino-4-pyridinecarboxylic acids
described previously,4 they are potent inhibitors of the H3K4
demethylase KDM5C (JARID1C). These molecules also show
activity in the KDM5C cell imaging assay4 but in general with a
larger differential between biochemical and cellular potency
values for this target than for KDM4C. Additionally, the six-
membered aryl analogues 31 and 32 appear ∼10-fold selective
for KDM4C over KDM5C in the cell assay, as they showed a
larger drop in potency from biochemical to cell assay for
KDM5C than the pyrazolyloxy analogues 36, 39, and 41 which
appear equipotent for both targets in this format. The reasons
for this observation are not clear, as both assays utilize the same
cell type, but as previously suggested in the accompanying
article,4 differences in affinity for the natural cofactor 2-OG
and/or differences in expression levels of the enzymes in the
cells could potentially play a role.
EXPERIMENTAL SECTION
■
Chemistry. All commercial chemicals and solvents are reagent
grade and were used without further purification unless otherwise
specified. Reactions were monitored by thin-layer chromatography on
0.2 mm silica gel plates (POLYGRAM SIL G/UV254, Macherey-
Nagel) and were visualized with UV light. Compounds were typically
purified by automated flash silica chromatography (Biotage SP4),
manual chromatography on prepacked cartridges (SPE), or mass
directed autopreparative chromatography (MDAP). Where specifically
indicated the following MDAP methods were used. For the formic
method, the HPLC analysis was conducted on a Sunfire C18 column
(150 mm × 30 mm i.d., 5 μm packing diameter) at ambient
temperature, eluting with 0.1% formic acid in water and 0.1% formic
acid in acetonitrile using an elution gradient. The UV detection was an
averaged signal from wavelength of 210−350 nm. The mass spectra
were recorded on a Waters ZQ mass spectrometer using alternate-scan
positive and negative electrospray. Ionization data were rounded to the
nearest integer. For the high pH method, the HPLC analysis was
conducted on a XBridge C18 column (100 mm × 30 mm i.d., 5 μm
packing diameter) at ambient temperature, eluting with 10 mM
ammonium bicarbonate in water adjusted to pH 10 with ammonia
solution, and acetonitrile using an elution gradient. The UV detection
was an averaged signal from wavelength of 210−350 nm. The mass
spectra were recorded on a Waters ZQ mass spectrometer using
alternate-scan positive and negative electrospray. Ionization data were
rounded to the nearest integer. 1H and 13C NMR spectra were
recorded on either a Bruker DPX-400 spectrometer at 400 and 126
MHz, respectively, or a Bruker AV-600 spectrometer at 600 and 150
MHz, respectively. Chemical shifts are reported in parts per million
(ppm, δ units). Splitting patterns are designated as s, singlet; d,
Compounds 31, 32, 36, 39, and 41 have been tested against
our in-house panel of nonrelated drug and liability targets
(Supporting Information Tables 2−6) and show a range of
broadly selective profiles.
CONCLUSION
■
We have designed and synthesized a range of non-carboxylate
inhibitors of the KDM4 (JMJD2) and KDM5 (JARID1)
families of histone lysine demethylases based on pyrido[3,4-
d]pyrimidin-4(3H)-one 4.14 A number of exemplars possess
interesting activity profiles in target-specific, cellular mecha-
nistic assays against overexpressed KDM4C (compounds 31,
32, 36, 39, and 41, IC50 = 2−5 μM) and against overexpressed
KDM5C (compounds 36, 39, and 41, IC50 = 2.5−5 μM). As
described above, a limited set of biochemical assays was used to
assess selectivity during this program of work with encourag-
ingly low levels of activity observed vs KDM6B and EGLN3.
To enable assessment of selectivity against the broader class of
G
DOI: 10.1021/acs.jmedchem.5b01538
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Journal of Medicinal Chemistry
Article
doublet; t, triplet; q, quartet; m, multiplet; br, broad; etc. LCMS
spectra were recorded on an Acquity UPLC BEH C18 column (50
mm × 2.1 mm i.d., 1.7 μm packing diameter) at 40 °C. The UV
detection was a summed signal from wavelength of 210−350 nm. The
mass spectra were recorded on a Waters ZQ mass spectrometer using
alternate-scan positive and negative electrospray. Ionization data were
rounded to the nearest integer. As specifically indicated, the
compounds were eluted by one of the following LCMS methods.
For the formic method, elution was with 0.1% v/v solution of formic
acid in water (solvent A) and 0.1% v/v solution of formic acid in
acetonitrile (solvent B) using the following elution gradient 0−1.5 min
3−100% B, 1.5−1.9 min 100% B, 1.9−2.1 min 3% B at a flow rate of 1
mL/min. For the high pH method, elution was with 10 mM
ammonium bicarbonate in water adjusted to pH 10 with ammonia
solution (solvent A) and acetonitrile (solvent B) using the following
elution gradient 0−1.5 min 1−97% B, 1.5−1.9 min 97% B, 1.9−2.1
min 100% B at a flow rate of 1 mL/min. For the TFA method, elution
was with 0.1% v/v solution of trifluoroacetic acid in water (solvent A)
and 0.1% v/v solution of trifluoroacetic acid in acetonitrile (solvent B)
using the following elution gradient: 0−1.5 min 3−100% B, 1.5−1.9
min 100% B, 1.9−2.0 min 100−3% B at a flow rate of 1 mL/min. High
resolution mass spectra (HRMS) were acquired as profile data using a
Thermo Scientific LTQ Orbitrap mass spectrometer, equipped with an
ESI interface, over a mass range of 120−1000 Da at a mass resolution
of 100 000. A commercial calibration solution (Pierce LTQ ESI
positive ion calibration solution) was used to externally calibrate the
instrument prior to analysis. Ionization was achieved with a spray
voltage of 4 kV, a capillary voltage of 45 V, tube lens voltage of 110 V,
and sheath and auxiliary gas flows of 40 and 20 (arbitrary units),
respectively. The capillary temperature was maintained at 300 °C. The
elemental composition was calculated using Xcalibur (version 2.0.7)
for the [M + H]+ and the mass error quoted as ppm; an error of <5
ppm indicates that the measured mass is consistent with the proposed
formula. Melting point analysis was carried out using a Stuart SMP40
melting point apparatus, and melting points are uncorrected. The
purity of all compounds screened in the biological assays was found to
be ≥95% by LCMS analysis unless otherwise specified.
Step 2. N-Phenylisonicotinamide 10 (1.14 g, 5.75 mmol) was
dissolved in THF (36 mL) under N2, and the mixture cooled to −70
°C. n-Butyllithium (7.91 mL, 12.65 mmol) was added cautiously
keeping the reaction temperature below −60 °C. The reaction was
stirred at −30 °C for 30 min and warmed to 0 °C for 5 min. The
reaction was cooled to −70 °C and DMF (0.891 mL, 11.50 mmol)
added cautiously. The reaction was allowed to warm to rt overnight,
acidified to pH 2 with 2 M HCl(aq) and the organic layer separated.
The aqueous layer was extracted with DCM, and the organic phases
were dried and concentrated. The residue was purified by silica gel
column chromatography, eluting with a gradient of 75−100% EtOAc/
cyclohexane followed by 0−10% DCM/MeOH to give crude 3-
hydroxy-2-phenylisoindolin-1-one 11 as a brown solid (84 mg, 21%).
LCMS (TFA method) retention time 0.52 min, [M + H]+ = 227.
Step 3. The crude 3-hydroxy-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-
1-one 11 (84 mg) was stirred in 35% hydrazine in water (2.4 g, 26.2
mmol) and the mixture heated to reflux under N2 for 2 h. The mixture
was azeotroped to dryness with toluene and the residue purified by
MDAP (high pH method) to give 6 as a pale brown solid (7.3 mg).
LCMS (high pH method) retention time 0.39 min, 94% pure by area,
[M + H]+ = 195. 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.06 (d, J =
5.3 Hz, 1H), 8.53 (s, 1H), 8.98 (d, J = 5.3 Hz, 1H), 9.33 (d, J = 0.8 Hz,
1H), 13.00 (br s, 1H).
1H-Indazol-3(2H)-one, 7. Step 1. Concentrated HCl (22 mL, 264
mmol) was added to a stirred suspension of 3-aminoisonicotinic acid
(2.8 g, 20.27 mmol) in water (20 mL) and the resulting suspension
cooled in an ice bath. Sodium nitrite (1.6 g, 23.19 mmol) was added
cautiously and the resulting solution stirred for 1 h. The solution was
added dropwise to a solution of water while sparging with SO2(g), and
the resulting suspension was stirred for 1 h and allowed to stand
overnight. The suspension was stirred for 5 min and filtered; the
resulting filter cake was washed with water, water/MeOH (1:1) and
dried in vacuo to give 3-hydrazinylisonicotinic acid dihydrochloride 12
1
as a yellow solid (3.02g, 56%). H NMR (400 MHz, DMSO-d6) δ
ppm: 8.02 (br s, 2H), 8.74 (s, 1H), 8.93 (br s, 1H).
Step 2. 3-Hydrazinylisonicotinic acid dihydrochloride 12 (1 g, 4.42
mmol) was added to a stirred solution of 2 M HCl (aq) (50 mL, 100
mmol) and the mixture heated to reflux for 5 h. The solution was
evaporated in vacuo to a yellow solid. This solid was suspended in
water (30 mL) and 50% NaOH (aq) added until the resulting
suspension was adjusted to pH 7. The precipitate was recrystallized
from the solution to give 7 as an orange solid (269 mg, 45%). LCMS
Pyrido[3,4-d]pyrimidin-4(3H)-one, 4. Formamidine acetate
(1.507 g, 14.48 mmol) was added in a single portion to a stirred
suspension of 3-aminoisonicotinic acid (1 g, 7.24 mmol) in N,N-
dimethylacetamide (8 mL) at rt under N2. The resultant suspension
was heated to 150 °C for 16 h. The resulting solution was then cooled
to rt which resulted in a suspension forming. Water was added,
followed by sat. NaHCO3 (aq), and the resulting suspension was
filtered. The collected solid was washed with sat. NaHCO3 (aq), H2O,
MeOH, and Et2O to give 4 as a pale brown solid (418 mg, 39%).
LCMS (formic method) retention time 0.33 min, [M + H]+ = 148. 1H
NMR (600 MHz, DMSO-d6) δ ppm: 7.96 (d, J = 5.1 Hz, 1H), 8.23 (s,
1H), 8.67 (d, J = 5.1 Hz, 1H), 9.06 (s, 1H), 12.60 (br s, 1H).
2,6-Naphthyridin-1(2H)-one, 5. Sourced from DL chiral
chemicals. LCMS (high pH method) retention time 0.44 min, [M +
1
(high pH method) retention time 0.19 min, [M + H]+ = 135. H
NMR (400 MHz, DMSO-d6) δ ppm: 7.61 (dd, J = 5.5, 1.3 Hz, 1H),
8.10 (d, J = 5.4 Hz, 1H), 8.81 (d, J = 1.2 Hz, 1H), 10.87 (br s, 1H),
12.14 (br s, 1H).
1-Benzylpyrido[3,4-d]pyrimidin-4(1H)-one, 8. Step 1. 3-Fluo-
roisonicotinic acid (250 mg, 1.772 mmol) was suspended in
benzylamine (0.5 mL, 4.57 mmol) and irradiated in a microwave at
150 °C for 2 h. The crude mixture was dissolved in MeOH and
applied to an aminopropyl column. The column was washed with
MeOH and then 10% 2 M HCl (aq) in MeOH. The appropriate
fractions were combined and evaporated to give a yellow oil, 3-
(benzylamino)isonicotinic acid (323 mg, 80% yield). LCMS (TFA
1
H]+ = 147. H NMR (600 MHz, DMSO-d6) δ ppm: 6.67 (d, J = 7.3
Hz, 1H), 7.33 (d, J = 7.3 Hz, 1H), 7.97 (d, J = 5.5 Hz, 1H), 8.62 (d, J
= 5.5 Hz, 1H), 9.06 (s, 1H).
1
Pyrido[3,4-d]pyridazin-1(2H)-one, 6. Step 1. Isonicotinic acid (5
g, 40.6 mmol) and 1,1′-carbonyldiimidazole (7.90 g, 48.7 mmol) were
stirred in 2-methyl-THF (50 mL), and the mixture was heated to 50
°C for 30 min. Aniline (4.82 mL, 52.8 mmol) was added, and the
mixture was stirred at 50 °C for 20 h. The mixture was cooled to rt,
and a solid precipitated. The organic mixture was washed with water
and brine and dried using MgSO4. The aqueous washings were
extracted with DCM, combined with the washed organic layer, and
evaporated to give a brown solid. The solid was triturated with EtOAc
and the resulting solid washed with EtOAc and dried via filtration to
give N-phenylisonicotinamide 10 as a white crystalline solid (5.96 g,
70%). LCMS (formic method) retention time 0.55 min, [M + H]+ =
199. 1H NMR (400 MHz, CDCl3) δ ppm: 7.19−7.25 (m, 3H), 7.41 (t,
J = 8.0 Hz, 2H), 7.66 (d, J = 8.1 Hz, 2H), 7.72 (dd, J = 4.6, 1.0 Hz,
2H), 7.93 (br s, 1H), 8.81 (dd, J = 4.8, 1.0 Hz, 1H).
method) retention time 0.56 min, [M + H]+ = 229. H NMR (400
MHz, DMSO-d6) δ ppm: 4.63 (s, 2H), 7.26−7.32 (m, 1H), 7.34−7.45
(m, 7 H), 7.47−7.52 (m, 2H), 7.96 (s, 2H), 8.23 (s, 1H), 8.34 (br s,
2H).
Step 2. 3-(Benzylamino)isonicotinic acid (76 mg, 0.333 mmol) was
suspended in formamide (0.5 mL, 12.54 mmol) under N2 and the
mixture cooled to ∼0 °C in an ice bath. Phosphorus oxychloride
(0.124 mL, 1.332 mmol) was added dropwise. The mixture was stirred
for 30 min at 0 °C followed by a further 16 h at rt. The reaction
mixture was concentrated, and the residue purified by MDAP (high
pH method) to give 8 (12.5 mg, 15% yield) as a cream solid. LCMS
1
(TFA method) retention time 0.59 min, [M + H]+ = 238. H NMR
(400 MHz, CDCl3) δ ppm: 5.38 (s, 2H), 7.28−7.23 (m, 3H), 7.43−
7.37 (m, 2H), 8.13 (d, J = 5.2 Hz, 1H), 8.41 (s, 1H), 8.71 (d, J = 5.2
Hz, 1H), 8.79 (s, 1H).
H
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Journal of Medicinal Chemistry
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1-(3-Phenylpropyl)pyrido[3,4-d]pyrimidin-4(1H)-one, 9. Step
1. A suspension of 3-fluoroisonicotinic acid (308 mg, 2.183 mmol) in
3-phenylpropan-1-amine (0.5 mL, 2.183 mmol) was heated by
microwave irradiation in a sealed vial to 120 °C for 8 h. The reaction
mixture was diluted with DCM and purified using silica gel column
chromatography, eluting with a gradient of 0−10% MeOH/DCM to
give 3-((3-phenylpropyl)amino)isonicotinic acid (510 mg, 91% yield)
as a yellow oil. LCMS (TFA method) retention time 0.1 min, [M +
H]+ = 257.
Step 2. Ammonium hydroxide (0.032 mL, 0.818 mmol) was added
to a solution of ethyl 3-(nicotinamido)isonicotinate (222 mg, 0.818
mmol) in MeOH and stirred at rt for 1.5 h. An additional amount of
ammonium hydroxide (1 mL) was added to the reaction mixture and
stirred at rt for 20 h. The solvent was removed under reduced pressure
after which the crude solid was dissolved in EtOH and stirred at 0 °C
for 5 min. Then NaOH(aq) (0.50 mL, 10 M in H2O, 4.09 mmol) was
added dropwise and stirred at 0 °C for 5 min after which the flask was
removed from the cooling bath and stirred at rt for 3 h. 2 M HCl (aq)
was added dropwise, and the resultant solution was loaded onto an
aminopropyl column. The column was eluted with MeOH, and the
appropriate fractions were combined and evaporated under reduced
pressure to give a white solid (201 mg). The crude solid was purified
by MDAP (high pH method) to give a white solid (104 mg). The
white solid was then dissolved in MeOH and loaded onto a SCX
cartridge that had been prewashed with MeOH. The column was
washed with MeOH and then 2 M NH3 in MeOH. The appropriate
fraction were combined and evaporated under reduced pressure to
give 16 as a white solid (25 mg, 14%). LCMS (high pH method)
retention time 0.43 min, [M + H]+ = 225. 1H NMR (400 MHz,
DMSO-d6) δ ppm: 7.59 (dd, J = 7.8, 4.8 Hz, 1H), 7.97 (d, J = 5.0 Hz,
1H), 8.51 (d, J = 7.8 Hz, 1H), 8.64 (d, J = 5.0 Hz, 1H), 8.76 (d, J = 3.8
Hz, 1H), 9.11 (s, 1H), 9.32 (s, 1H).
2-(5-Fluoropyridin-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-one,
17. Step 1. Ethyl 3-aminoisonicotinate (300 mg, 1.805 mmol), 5-
fluoronicotinic acid (289 mg, 1.986 mmol), and N-ethyl-N-
isopropylpropan-2-amine (0.946 mL, 5.42 mmol) were dissolved in
DCM (6 mL). 1-Propanephosphonic acid cyclic anhydride (1.505 mL,
2.53 mmol) was added, and the mixture was stirred at rt for 5 h.
Further 5-fluoronicotinic acid (130 mg), N-ethyl-N-isopropylpropan-2-
amine (0.315 mL), and 1-propanephosphonic acid cyclic anhydride
(0.5 mL) were added, and the mixture was stirred for 16 h. The
reaction was diluted with sat. NaHCO3 (aq) and extracted with DCM.
The organic phase was dried using a hydrophobic frit and
concentrated in vacuo. The crude was purified using silica gel column
chromatography, eluting with a gradient of 20−60% EtOAc/
cyclohexane to give ethyl 3-(5-fluoronicotinamido)isonicotinate (343
mg, 66%) as a yellow solid. LCMS (formic method) retention time
0.64 min, [M + H]+ = 290.
Step 2. Ethyl 3-(5-fluoronicotinamido)isonicotinate (343 mg, 0.711
mmol) was dissolved in 7 M NH3 in MeOH (6 mL, 42.0 mmol) and
allowed to stir at rt for 16 h. 10 M NaOH (0.711 mL, 7.11 mmol) was
added and the mixture stirred for 24 h. The basic mixture was
neutralized with 2 M HCl (aq) and filtered, washed with water
followed by MeOH to give the 17 as a white solid (64 mg, 37%).
LCMS (formic method) retention time 0.56 min, [M + H]+ = 243. 1H
NMR (400 MHz, DMSO-d6) δ ppm: 8.01 (dd, J = 5.3, 0.8 Hz, 1H),
8.42 (ddd, J = 9.8, 2.5, 1.8 Hz, 1H), 8.71 (d, J = 5.1 Hz, 1H), 8.82 (d, J
= 2.8 Hz, 1H), 9.16 (s, 1H), 9.21 (t, J = 1.5 Hz, 1H), 13.06−13.10 (m,
1H).
2-Propylpyrido[3,4-d]pyrimidin-4(3H)-one, 18. Step 1. To
ethyl 3-aminoisonicotinate (200 mg, 1.204 mmol) in EtOAc (15
mL) was added butyric acid (0.221 mL, 2.407 mmol), N-ethyl-N-
isopropylpropan-2-amine (0.841 mL, 4.81 mmol), and 2,4,6-tripropyl-
1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (2.87 mL, 4.81 mmol).
The mixture was heated to 60 °C under N2 for 72 h. The reaction
mixture was partitioned between NaHCO3 (aq) and EtOAc. The
aqueous phase was washed with EtOAc, and the combined organic
fractions were washed with water and evaporated to afford a dark
brown oil. The crude oil was purified by silica gel column
chromatography, eluting with a gradient of 0−80% EtOAc/cyclo-
hexane to give ethyl 3-butyramidoisonicotinate (109 mg, 38%) as a
yellow oil. LCMS (formic method) retention time 0.85 min, [M + H]+
= 237.
Step 2. 3-((3-Phenylpropyl)amino)isonicotinic acid (153 mg, 0.597
mmol) was dissolved in formamide (2 mL, 50.2 mmol) and cooled to
0−5 °C under N2. Phosphorus oxychloride (0.14 mL, 1.502 mmol)
was added dropwise, and the mixture was stirred at 0−5 °C for 30 min,
followed by a further 1 h at rt. A further portion of phosphorus
oxychloride (0.17 mL, 1.824 mmol) was added and the mixture stirred
at rt for 16 h. The reaction mixture was concentrated and purified by
MDAP (high pH method) to give 9 (26.7 mg, 17% yield). LCMS
(formic method) retention time 0.71 min, [M + H]+ = 266. 1H NMR
(400 MHz, CDCl3) δ ppm: 2.29 (dt, J = 7.3 Hz, 5.3 Hz, 2H), 2.79 (t, J
= 7.3 Hz, 2H), 4.16 (t, J = 5.3 Hz, 2H), 7.38−7.16 (m, 5 H), 8.10 (d, J
= 5.1 Hz, 1H), 8.15 (s, 1H), 8.72 (d, J = 5.1 Hz, 1H), 8.75 (s, 1H).
2-Phenylpyrido[3,4-d]pyrimidin-4(3H)-one, 15. Step 1. Benzo-
yl chloride (0.227 mL, 1.956 mmol) was added dropwise to a stirred
biphasic solution of ethyl 3-aminoisonicotinate (250 mg, 1.504 mmol)
in DCM (5 mL) and sat. K2CO3 (aq) (5 mL) at rt. After stirring
rapidly at rt for 1 h, H2O and DCM were added. The separated
aqueous phase was extracted with DCM, the combined organic phase
was passed through a hydrophobic frit and evaporated under reduced
pressure to give an orange oil. This oil was purified by silica gel column
chromatography using a gradient of 0−30% EtOAc/cyclohexane to
give ethyl 3-benzamidoisonicotinate (196 mg, 48%). LCMS (formic
1
method) retention time 1.01 min, [M + H]+ = 271. H NMR (400
MHz, CDCl3) δ ppm: 1.48 (t, J = 7.1 Hz, 3H), 4.50 (q, J = 7.1 Hz,
2H), 7.28 (s, 2H), 7.50 (t, J = 8.0 Hz, 1H), 7.53−7.67 (m, 4H), 7.88
(d, J = 5.1 Hz, 1H), 8.07 (dd, J = 7.8, 1.5 Hz, 2H), 8.12 (dd, J = 8.3,
1.5 Hz, 1H), 8.50 (d, J = 5.1 Hz, 1H), 10.27 (s, 1H), 11.67 (br s, 1H)
Step 2. 7 M NH3 in methanol (10 mL, 70.0 mmol) was added in a
single portion to a flask containing ethyl 3-benzamidoisonicotinate
(196 mg, 0.725 mmol) at rt. The resultant solution was stirred at rt for
4 h, and then the solvent was evaporated under reduced pressure to
give a white solid. The solid was dissolved in EtOH, and then NaOH
(aq) (0.73 mL, 10 M in H2O, 4.35 mmol) was added dropwise. The
resultant solution was stirred at rt for 1 h and then allowed to stand at
rt for 12 h. 2 M HCl (aq) (4 mL) was added, and the resultant
solution was evaporated under reduced pressure to give a yellow solid.
The solid was dissolved in EtOH and passed through an aminopropyl
column (20 g) that had been prewashed with EtOH. The column was
eluted with EtOH. The appropriate fractions were combined and
evaporated under reduced pressure to give a white solid (100 mg).
The solid was suspended in DMSO and filtered. The collected solid
was washed with DMSO, MeOH, and finally Et2O. The solid was then
air-dried under vacuo to give 15 as a white solid (11 mg, 7%). LCMS
(formic method) retention time 0.66 min, [M + H]+ = 224. 1H NMR
(400 MHz, DMSO-d6) δ ppm: 7.53−7.66 (m, 3H), 7.99 (dd, J = 5.3,
0.8 Hz, 1H), 8.20 (dd, J = 8.4, 1.5 Hz, 2H), 8.67 (d, J = 5.3 Hz, 1H),
9.13 (s, 1H), 12.85 (br s, 1H).
2-(Pyridin-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-one, 16. Step 1.
Nicotinoyl chloride hydrochloride (268 mg, 1.504 mmol) was added
to a stirred suspension of ethyl-3-amino-4-pyridine carboxylate (250
mg, 1.505 mmol) in DCM (10 mL) at 0 °C under N2. The solution
was then stirred for 5 min after which the flask was removed from the
cooling bath and stirring was continued at rt for 3 h. Saturated
NaHCO3 (aq) and DCM were added, and the separated aqueous
phase was extracted with DCM. The combined organic phases were
passed through a hydrophobic frit and evaporated under reduced
pressure to give a brown oil (508 mg). The oil was purified by silica gel
column chromatography, eluting with a gradient of 0−100% EtOAc/
cyclohexane to give ethyl 3-(nicotinamido)isonicotinate as a light
yellow solid (222 mg, 54%). LCMS (high pH method) retention time
0.79 min, [M + H]+ = 272.
Step 2. Ethyl 3-butyramidoisonicotinate (109 mg, 0.461 mmol) was
taken up in 7 M NH3 in MeOH (15 mL, 105 mmol) and stirred at 50
°C for 16 h. After this time the solvent was evaporated in vacuo to
afford a green/gray crystalline solid. The crude product was
recrystallized from MeOH and dried in vacuo to afford 18 (47 mg,
54%) as a green crystalline solid. LCMS (formic method) retention
I
DOI: 10.1021/acs.jmedchem.5b01538
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Journal of Medicinal Chemistry
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1
time 0.51 min, [M + H]+ = 190. H NMR (400 MHz, DMSO-d6) δ
4(3H)-one 20 (2 g, 80% w/w, 7.72 mmol) in THF (100 mL), and
mCPBA (77% in water) (4.33 g, 19.3 mmol) was added, and the slurry
was stirred at rt for 3 h. The slurry was diluted with an equal volume of
IPA (100 mL) and was concentrated under reduced pressure. The
slurry was rediluted with IPA (100 mL) and was concentrated in vacuo
again. The precipitated solid was isolated by filtration, washed with
IPA (20 mL) followed by Et2O (20 mL), and dried under suction to
give 20a as a white free-flowing powder (2.1 g, ∼80% w/w) . LCMS
(formate method) retention time 0.43 min, [M + H]+ = 240.
Representative Procedure for the Oxidation of 20 to a
Mixture of 2-(Ethylsulfonyl)pyrido[3,4-d]pyrimidin-4(3H)-one
and 2-(Ethylsulfinyl)pyrido[3,4-d]pyrimidin-4(3H)-one, 20b.
39% Peracetic acid in AcOH (2.054 mL, 12.06 mmol) was added to
a stirred suspension of 2-(ethylthio)pyrido[3,4-d]pyrimidin-4(3H)-
one 20 (1g, 4.83 mmol) in THF (50 mL) over 3 Å molecular sieves (1
g). The resulting suspension was stirred for 24 h. A further portion of
39% peracetic acid in AcOH (2.054 mL, 12.06 mmol) was added and
the mixture stirred for 16 h. The mixture was filtered and the resulting
solid washed with toluene, methyl tert-butyl ether and dried under
vacuo to give crude 20b as an almost white solid (1.84 g, ∼40% w/w)
which was used directly in the subsequent steps without further
purification. The crude mixture contained a 2:1 ratio of 2-
(ethylsulfonyl)pyrido[3,4-d]pyrimidin-4(3H)-one and 2-
(ethylsulfinyl)pyrido[3,4-d]pyrimidin-4(3H)-one. LCMS (TFA meth-
od) retention time 0.40 min, [M + H]+ = 224 (sulfoxide), and
retention time 0.42 min, [M + H]+ = 240 (sulfone).
ppm: 0.95 (t, J = 7.5 Hz, 3H), 1.72−1.82 (m, 2H), 2.62 (t, J = 7.6 Hz,
2H), 7.91 (d, J = 5.1 Hz, 1H), 8.61 (d, J = 5.1 Hz, 1H), 8.99 (s, 1H),
12.49 (br s, 1H).
2-Thioxo-2,3-dihydropyrido[3,4-d]pyrimidin-4(1H)-one, 22.
3-Aminoisonicotinic acid (5 g, 36.2 mmol) and thiourea (3.31 g,
43.4 mmol) were stirred at 160 °C for 16 h. The reaction was cooled
to 100 °C and was treated with water and then cooled to rt. The
resulting precipitate was removed by filtration and dried to give the 22
as a light brown solid (5.711 g, 88%). LCMS (formic method)
retention time 0.38 min, [M + H]+ = 180. H NMR (400 MHz,
1
DMSO-d6) δ ppm: 7.78 (dd, J = 5.1, 0.7 Hz, 1H), 8.48 (d, J = 5.1 Hz,
1H), 8.72 (s, 1H), 12.71 (br s, 1H), 12.90 (br s, 1H).
2-(Ethylamino)pyrido[3,4-d]pyrimidin-4(3H)-one, 19. Step 1.
To a suspension of 2-thioxo-2,3-dihydropyrido[3,4-d]pyrimidin-
4(1H)-one 22 (5.7 g, 31.8 mmol) in a mixture of 1 M NaOH (50
mL, 50.0 mmol) and MeOH (50 mL) was added methyl iodide (2.98
mL, 47.7 mmol) dropwise, and the reaction was stirred at rt under N2
for 16 h. The reaction was adjusted to pH 7 by the addition of 1 M
HCl (aq) and the MeOH removed under reduced pressure. The
resulting precipitate was removed by filtration, washed with water, and
then dried in a vacuum oven to give 2-(methylthio)pyrido[3,4-
d]pyrimidin-4(3H)-one as a white solid (3.91 g, 95%). LCMS (formic
method) retention time 0.51 min, [M + H]+ = 194. H NMR (400
MHz, DMSO-d6) δ ppm: 2.61 (s, 3H), 7.86 (d, J = 5.1 Hz, 1H), 8.57
(d, J = 5.1 Hz, 1H), 8.92 (s, 1H), 12.93 (br s, 1H).
1
Further Representative Procedure for the Oxidation of 20 to
a Mixture of 2-(Ethylsulfonyl)pyrido[3,4-d]pyrimidin-4(3H)-
one and 2-(Ethylsulfinyl)pyrido[3,4-d]pyrimidin-4(3H)-one,
20b. mCPBA (2 g, 11.59 mmol) was added to a stirred suspension
of 2-(ethylthio)pyrido[3,4-d]pyrimidin-4(3H)-one 20 (1g, 4.82
mmol) in THF (50 mL), and the mixture was stirred for 16 h. The
resulting fine suspension was filtered, and the filtrate was stirred with
solid sodium metabisulfite (2 g, 10.52 mmol) for 5 min, filtered, and
evaporated to a brown slurry. This slurry was triturated with IPA,
filtered, and washed with further IPA to give the crude 20b as a pale
yellow solid (674 mg, ∼85% w/w) which was used in subsequent
reactions without further purification. The crude mixture contained a
4:1 ratio of 2-(ethylsulfonyl)pyrido[3,4-d]pyrimidin-4(3H)-one and 2-
(ethylsulfinyl)pyrido[3,4-d]pyrimidin-4(3H)-one. LCMS (TFA meth-
od) retention time 0.41 min, [M + H]+ = 224 (sulfoxide), and
retention time 0.43 min, [M + H]+ = 240 (sulfone).
2-Phenoxypyrido[3,4-d]pyrimidin-4(3H)-one, 24. A reaction
tube was charged with phenol (71 mg, 0.754 mmol), DMF (5 mL),
and NaH (51 mg, 60% w/w in oil, 1.275 mmol). The reaction was
stirred at rt for 3 h. After this time a mixture of 2-(ethylsulfonyl)-
pyrido[3,4-d]pyrimidin-4(3H)-one and 2-(ethylsulfinyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one 20b (150 mg, 0.477 mmol) was added. The
reaction was stirred at 110 °C for 2 days. Further phenol (30 mg,
0.319 mmol) and NaH (51 mg, 60% w/w in oil, 1.275 mmol) were
added, and the reaction was stirred at 110 °C for 16 h. The reaction
was concentrated and slurried in hot DMSO before the precipitated
solid was removed by filtration through a plug of cotton wool. The
filtrate was purified by MDAP (formic method) to give 24 as a tan
solid (13 mg, 11%). LCMS (formic method) retention time 0.66 min,
[M + H]+ = 240. 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.16−7.24
(m, 3H), 7.41 (t, J = 8.0 Hz, 2H), 7.74 (d, J = 5.1 Hz, 1H), 8.29 (br s,
2H), 8.59 (s, 1H).
Step 2. 2-(Methylthio)pyrido[3,4-d]pyrimidin-4(3H)-one (100 mg,
0.518 mmol) was placed in a microwave vial, and ethylamine (2 M
solution in THF) (2 mL, 4.00 mmol) was added. The reaction was
irradiated at 120 °C for 4 h followed by 150 °C for 4 h. The reaction
was diluted with MeOH and eluted through an SCX column, eluting
with MeOH and 2 M NH3 in MeOH. The methanolic ammonia
fraction was evaporated to give an orange solid. This solid was purified
using silica gel column chromatography, eluting with a gradient of 0−
5% DCM/MeOH to give 19 as a white solid (82 mg, 83%) . LCMS
(formic method) retention time 0.37 min, [M + H]+ = 191. 1H NMR
(400 MHz, DMSO-d6) δ ppm: 1.16 (t, J = 7.1 Hz, 3H), 3.34−3.42 (m,
2H), 6.44 (br s, 1H), 7.68 (d, J = 5.1 Hz, 1H), 8.25 (d, J = 5.1 Hz,
1H), 8.64 (s, 1H), 11.17 (br s, 1H).
2-Ethoxypyrido[3,4-d]pyrimidin-4(3H)-one, 21. 2-
(Methylthio)pyrido[3,4-d]pyrimidin-4(3H)-one (100 mg, 0.518
mmol) (see compound 19, step 1, for preparation) and NaOEt
(1950 μL, 21% w/w in EtOH, 5.18 mmol) were placed in a
microwaveable vial and irradiated at 140 °C for 4 h. The reaction was
concentrated to an orange solid that was suspended in MeOH; the
resulting precipitate was removed by filtration and the filtrate
concentrated to give a crude solid. This solid was purified using
MDAP (formic method). Appropriate fractions were combined and
eluted through an NH2 SPE column with MeOH. The MeOH was
concentrated and dried to give 21 as a white solid (14 mg, 14%).
LCMS (formic method) retention time 0.49 min, [M + H]+ = 192. 1H
NMR (400 MHz, DMSO-d6) δ ppm: 1.36 (t, J = 7.1 Hz, 3H), 4.47 (q,
J = 7.1 Hz, 2H), 7.83 (d, J = 5.1 Hz, 1H), 8.47 (d, J = 5.1 Hz, 1H),
8.82 (s, 1H), 12.50−12.66 (m, 1H).
2-(Ethylthio)pyrido[3,4-d]pyrimidin-4(3H)-one, 20. 2-Thioxo-
2,3-dihydropyrido[3,4-d]pyrimidin-4(1H)-one 22 (1.355 g, 7.56
mmol) was taken up in MeOH (20 mL) and 1 M NaOH (20 mL,
20.00 mmol) and stirred at rt for 5 min. Ethyl iodide (0.733 mL, 9.07
mmol) was added dropwise, and the reaction was stirred at rt for 1 h.
The reaction was concentrated to remove the MeOH, and the
resulting yellow solution was adjusted to pH 6 with 2 M HCl (aq). A
yellow precipitate formed which was removed by filtration and dried to
give the 20 as a yellow solid (1.479 g, 94%). LCMS (formic method)
2-(Benzyloxy)pyrido[3,4-d]pyrimidin-4(3H)-one, 25. Phenyl-
methanol (0.048 mL, 0.460 mmol) and NaH (20.0 mg, 60% w/w in
oil, 0.500 mmol) were added to DMF (2.5 mL) at 0 °C and stirred for
5 min. A mixture of 2-(ethylsulfonyl)pyrido[3,4-d]pyrimidin-4(3H)-
one and 2-(ethylsulfinyl)pyrido[3,4-d]pyrimidin-4(3H)-one 20b (100
mg, 0.307 mmol) was added to DMF (2.5 mL), and this suspension
was added to the reaction mixture. The reaction was heated to 110 °C
for 1 h and then cooled to rt and stirred for 2.5 days. Further
phenylmethanol (0.048 mL, 0.460 mmol) and NaH (16.7 mg, 60% w/
w in oil) were added to DMF (1 mL), and this was added to the
reaction mixture. The reaction was heated to 110 °C and stirred for 2
h. The reaction was quenched with water and extracted with EtOAc.
1
retention time 0.62 min, [M + H]+ = 208. H NMR (400 MHz,
DMSO-d6) δ ppm: 1.36 (t, J = 7.5 Hz, 3H), 3.24 (q, J = 7.5 Hz, 2H),
7.87 (d, J = 5 Hz, 1H), 8.56 (d, J = 5 Hz, 1H), 8.91 (s, 1H), 12.80−
12.92 (m, 1H).
Representative Procedure for the Oxidation of 20 to 2-
(Ethylsulfonyl)pyrido[3,4-d]pyrimidin-4(3H)-one, 20a. A round-
bottom flask was charged with 2-(ethylthio)pyrido[3,4-d]pyrimidin-
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Journal of Medicinal Chemistry
Article
The organic phase was washed with 10% LiCl (aq), brine and dried
using a hydrophobic frit. The solvent was removed in vacuo and the
resulting solid triturated with MeOH to give 25 (21 mg, 27%) as a
white solid. LCMS (formic method) retention time 0.76 min, [M +
concentrated to a slurry in vacuo. The slurry was diluted with IPA and
concentrated in vacuo to a minimum volume. The precipitated solid
was isolated by filtration, washed with IPA followed by Et2O, and dried
under vacuo to give the crude sulfone/sulfoxide intermediate 20b as a
white powder. The 4-chlorophenol (0.095 mL, 0.965 mmol) was taken
up in DMF (5 mL), and NaH (77 mg, 60% w/w in oil, 1.930 mmol)
was added with continuous stirring at rt. The reaction was left stirring
for 5 min, and then the intermediate white powder 20b was added.
The reaction was heated to 110 °C and stirred for 16 h. The reaction
was allowed to cool to rt, and MeOH was added. The solvent was
removed in vacuo and the crude product was purified by silica gel
column chromatography, eluting with a gradient of 0−100% EtOAc/
cyclohexane followed by 0−5% MeOH/DCM to give 29 (17 mg, 6%)
as a white solid. LCMS (formic method) retention time 0.80 min, [M
+ H]+ = 274/276. 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.41 (d, J
= 8.8 Hz, 2H), 7.55 (d, J = 8.8 Hz, 2H), 7.89 (d, J = 5.1 Hz, 1H), 8.53
(d, J = 5.1 Hz, 1H), 8.70 (s, 1H), 13.15 (br s, 1H).
1
H]+ = 254. H NMR (400 MHz, DMSO-d6) δ ppm: 5.51 (s, 2H),
7.35−7.45 (m, 3H), 7.53 (d, J = 6.8 Hz, 2H), 7.86 (d, J = 5.1 Hz, 1H),
8.51 (d, J = 5.1 Hz, 1H), 8.88 (s, 1H).
2-(Cyclohexyloxy)pyrido[3,4-d]pyrimidin-4(3H)-one, 26. Cy-
clohexanol (62.8 mg, 0.627 mmol) was taken up in NMP (2 mL) and
treated with NaH (25.08 mg, 60% w/w in oil, 0.627 mmol). The
reaction was then treated with a mixture of 2-(ethylsulfonyl)pyrido-
[3,4-d]pyrimidin-4(3H)-one and 2-(ethylsulfinyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one 20b (50 mg, 0.166 mmol) and allowed to
stand at rt for 16 h. The reaction was diluted with water and was
extracted with EtOAc, and the organic layer was washed with brine,
dried using a hydrophobic frit, and concentrated to an oil. This oil was
purified using silica gel column chromatography, eluting with a
gradient of 0−50% EtOAc/cyclohexane to give 26 (3 mg, 7%) as a
white solid. LCMS (formic method) retention time 0.82 min, [M +
2-(3-Hydroxyphenoxy)pyrido[3,4-d]pyrimidin-4(3H)-one,
30. To NaH (80 mg, 60% w/w in oil, 1.998 mmol) at rt under N2 was
added resorcinol (55 mg, 0.499 mmol) in DMF (4 mL). After 20 min,
2-(ethylsulfonyl)pyrido[3,4-d]pyrimidin-4(3H)-one 20a (520 mg,
0.999 mmol) was added, and the reaction was heated to 110 °C for
16 h. The reaction was quenched with water and the solvent was
evaporated in vacuo affording a red/black solid. This was taken up in
MeOH and filtered. The filtrate was purified by silica gel column
chromatography, eluting with a gradient of 0−20% MeOH/DCM to
give crude 30. The crude was purified by MDAP (formic method) to
give 30 (8 mg, 6%) as a pink solid. LCMS (formic method) retention
1
H]+ = 246. H NMR (400 MHz, DMSO-d6) δ ppm: 1.23−1.35 (m,
1H), 1.36−1.47 (m, 2H), 1.50−1.60 (m, 3H), 1.70−1.80 (m, 2H),
1.99 (m, J = 3.5 Hz, 2H), 5.13−5.20 (m, 1H), 7.83 (d, J = 5.1 Hz, 1H),
8.47 (d, J = 5.1 Hz, 1H), 8.82 (s, 1H), 12.50 (br s, 1H).
2-(Cyclohexylmethoxy)pyrido[3,4-d]pyrimidin-4(3H)-one,
27. Cyclohexylmethanol (0.085 mL, 0.692 mmol) in anhydrous DMF
(2 mL) was added to NaH (50.2 mg, 60% w/w in oil, 1.255 mmol)
under N2. After 10 min of stirring a mixture of 2-(ethylsulfonyl)-
pyrido[3,4-d]pyrimidin-4(3H)-one and 2-(ethylsulfinyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one 20b (150 mg, 0.570 mmol) in DMF (2 mL) was
added and the reaction mixture heated at 110 °C under N2 for 7 h.
Further NaH (25 mg, 60% w/w in oil) was added and the reaction
mixture heated at 110 °C under N2 for 72 h. The reaction mixture was
quenched with water. Sat. NaHCO3(aq), 10% LiCl (aq), and brine
were added. The product was extracted with EtOAc and passed
through a hydrophobic frit and concentrated. The resulting crude was
purified by silica gel column chromatography, eluting with a gradient
of 0−75% EtOAc/cyclohexane to give 27 (8 mg, 5% yield) as a white
powder. LCMS (formic method) retention time 0.94 min, [M + H]+ =
1
time 0.55 min, [M + H]+ = 256. H NMR (400 MHz, DMSO-d6) δ
ppm: 6.69−6.76 (m, 3H), 7.25 (t, J = 8.0 Hz, 1H), 7.87 (d, J = 5.1 Hz,
1H), 8.50 (d, J = 5.1 Hz, 1H), 8.70 (s, 1H), 9.71−9.84 (m, 1H).
2-((1H-Pyrazol-4-yl)oxy)pyrido[3,4-d]pyrimidin-4(3H)-one,
33. Step 1. Tosic acid monohydrate (0.294 g, 1.546 mmol) and 3,4-
dihydro-2H-pyran (2.82 mL, 30.9 mmol) were added to a solution of
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (3 g,
15.46 mmol), and the resulting suspension was heated to 75 °C
with a drying tube attached for 3 h. The resulting solution was cooled,
washed with sat. NaHCO3 (aq) and brine, then dried (MgSO4) and
evaporated in vacuo to a pale brown oil. The residue was purified using
silica gel column chromatography, eluting with a gradient of 0−33%
EtOAc/cyclohexane to give crude 1-(tetrahydro-2H-pyran-2-yl)-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (2.85 g) as a
colorless oil. LCMS (high pH method) retention time1.03 min, [M +
H]+ = 279.
Step 2. A premixed solution of 2 M NaOH (5.98 mL, 11.96 mmol)
and 30% H2O2 (aq) (1.222 mL, 11.96 mmol) was added to a solution
of crude 1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-di-
oxaborolan-2-yl)-1H-pyrazole (2.85g) from step 1, in THF (50 mL).
The resulting suspension was stirred for 2 h. The reaction mixture was
adjusted to pH 9 with 2 M HCl (aq) and extracted with EtOAc. The
organic layer was washed with water and brine, dried (MgSO4), and
evaporated to a colorless oil. This oil was purified by silica gel column
chromatography, eluting with a gradient of 25−75% EtOAc/
cyclohexane to give 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-ol
(1.12g, 90%) as a white solid. LCMS (high pH method) retention
time 0.51 min, [M + H]+ = 169.
Step 3. NaH (182 mg, 60% w/w in oil, 4.55 mmol) was added
cautiously to a solution of 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-
ol (249 mg, 1.480 mmol) in DMF (5 mL) under N2, and the resulting
suspension was stirred for 5 min. A mixture of 2-(ethylsulfonyl)-
pyrido[3,4-d]pyrimidin-4(3H)-one and 2-(ethylsulfinyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one 20b (300 mg, 1.137 mmol) was added, and the
resulting suspension was heated to 110 °C for 1.5 h, cooled to rt,
acidified with 1 M HCl (5 mL), and neutralized to pH 7 with sat.
aqueous NaHCO3. The resulting suspension was evaporated in vacuo
to dryness and purified by silica gel column chromatography, eluting
with a gradient of 0−10% DCM/MeOH to give 2-((1-(tetrahydro-2H-
pyran-2-yl)-1H-pyrazol-4-yl)oxy)pyrido[3,4-d]pyrimidin-4(3H)-one
(93 mg, 23%) as an off white solid. LCMS (high pH method)
retention time 0.53 min, [M + H]+ = 314.
1
260. H NMR (400 MHz, DMSO-d6) δ ppm: 1.00−1.12 (m, 2H),
1.14−1.32 (m, 3H), 1.62−1.69 (m, 1H), 1.69−1.84 (m, 5 H), 4.24 (d,
J = 6.1 Hz, 2H), 7.83 (d, J = 4.8 Hz, 1H), 8.48 (d, J = 5.1 Hz, 1H),
8.83 (s, 1H).
2-(Pyridin-3-yloxy)pyrido[3,4-d]pyrimidin-4(3H)-one, 28.
Pyridin-3-ol (44 mg, 0.463 mmol) in DMF (1 mL) was added
dropwise over 0.5 min to a stirred suspension of NaH (34 mg, 60% w/
w in oil, 0.850 mmol) in DMF (2 mL) at rt under N2. After 30 min, a
mixture of 2-(ethylsulfonyl)pyrido[3,4-d]pyrimidin-4(3H)-one and 2-
(ethylsulfinyl)pyrido[3,4-d]pyrimidin-4(3H)-one 20b (100 mg, 0.380
mmol) in DMF (1 mL) was added. The reaction was heated to 110 °C
for 18 h under N2 then cooled to rt. Water, sat. NaHCO3 (aq), 10%
LiCl (aq), brine (aq), and EtOAc were added to the reaction mixture,
and the phases were separated. The aqueous portion was extracted
with EtOAc and then adjusted to pH 7 and further extracted with
EtOAc. All of the organic phases were combined, passed through a
hydrophobic frit, and the solvent was evaporated in vacuo to give a
cream solid. The solid was purified by silica gel column
chromatography, eluting with a gradient of 0−10% MeOH/DCM to
give 28 (30 mg, 29%) as a white solid. LCMS (formic method)
1
retention time 0.46 min, [M + H]+ = 241. H NMR (400 MHz,
DMSO-d6) δ ppm: 7.57 (dd, J = 8.3, 4.8 Hz, 1H), 7.86−7.92 (m, 2H),
8.53−8.58 (m, 2H), 8.65 (d, J = 2.8 Hz, 1H), 8.70 (s, 1H), 13.26 (br s,
1H).
2-(4-Chlorophenoxy)pyrido[3,4-d]pyrimidin-4(3H)-one, 29.
2-(Ethylthio)pyrido[3,4-d]pyrimidin-4(3H)-one 20 (200 mg, 0.965
mmol) was added to THF (10 mL), and mCPBA (77% in water) (416
mg, 2.413 mmol) was added with continuous stirring. The reaction
was stirred at rt under N2 for 4 h. Further mCPBA (77% in water)
(0.965 mmol) was added, and the reaction was stirred for 1.5 h. The
resultant slurry was filtered using a hydrophobic frit and washed with
THF. The filtrate was diluted with an equal volume of IPA and
K
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Journal of Medicinal Chemistry
Article
Step 4. 2 M aq HCl (0.064 mL, 0.128 mmol) was added to a stirred
suspension of 2-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)oxy)-
pyrido[3,4-d]pyrimidin-4(3H)-one (40 mg, 0.128 mmol) in MeOH (2
mL). The suspension was stirred for 16 h and the resulting solution
concentrated in vacuo. The residue was dry-loaded on to silica column
and then eluted with a gradient of 0−20% DCM/MeOH to a give 33
(16 mg, 49%) as a white solid. LCMS (TFA method) retention time
[3,4-d]pyrimidin-4(3H)-one and 2-(ethylsulfinyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one 20b (200 mg, 0.342 mmol) was added and
the reaction was stirred for 16 h under N2. Further NaH (16.7 mg,
60% w/w in oil, 0.417 mmol) was added, and the reaction was stirred
for a further 4 h. The reaction was cooled to rt, and MeOH was added.
The solvent was removed in vacuo and the crude solid was purified by
silica gel column chromatography, eluting with a gradient of 0−100%
EtOAc/cyclohexane to give 35 (22 mg, 24%) as a white solid. LCMS
(formic method) retention time 0.60 min, [M + H]+ = 272. 1H NMR
(400 MHz, DMSO-d6) δ ppm: 1.45 (d, J = 6.6 Hz, 6H), 4.50 (dt, J =
13.3, 6.6 Hz, 1H), 7.61 (s, 1H), 7.88 (d, J = 5.1 Hz, 1H), 8.08 (s, 1H),
8.54 (d, J = 5.3 Hz, 1H), 8.85 (s, 1H), 13.06 (br s, 1H).
2-((1-Cycloheptyl-1H-pyrazol-4-yl)oxy)pyrido[3,4-d]-
pyrimidin-4(3H)-one, 38. Step 1. To 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (1 g, 5.15 mmol) and cycloheptanol
(0.626 mL, 5.15 mmol) in THF (10 mL) were added triphenylphos-
phine (2.70 g, 10.31 mmol) and (E)-di-tert-butyldiazene-1,2-
dicarboxylate (1.187 g, 5.15 mmol). The reaction mixture was stirred
under N2 at rt for 48 h. The reaction mixture was concentrated under
reduced pressure to give a yellow gum which was purified by silica gel
column chromatography, eluting with a gradient of 0−100% EtOAc/
cyclohexane to give a mixture of 1-cycloheptyl-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-pyrazole and (1-cycloheptyl-1H-pyrazol-
4-yl)boronate (ratio 5:1) (717 mg, 28%) as a white solid. LCMS
(formic method) retention time 0.74 min, [M + H]+ = 209, retention
time 1.26 min, [M + H]+ = 291.
Step 2. To a solution of the mixture of 1-cycloheptyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole/(1-cycloheptyl-1H-
pyrazol-4-yl)boronate from step 1 (717 mg, 1.007 mmol) in THF (10
mL) was added 2 M NaOH (aq) (1.511 mL, 3.02 mmol) and 30%
H2O2 (aq) (0.206 mL, 2.015 mmol). The solution was stirred at rt for
2 h. The reaction was treated with sat. Na2SO3 (aq) and was then
acidified to pH 2 with 2 M HCl (aq). The reaction was extracted with
EtOAc. The organic phase was dried over MgSO4 and concentrated to
give a crude residue. This residue was purified by silica gel column
chromatography, eluting with a gradient of 0−100% EtOAc/
cyclohexane to give 1-cycloheptyl-1H-pyrazol-4-ol (422 mg) as a
colorless gum that was used in the next step without further
purification. LCMS (formic method) retention time 0.77 min, [M +
H]+ = 181.
1
0.36 min, [M + H]+ = 230. H NMR (400 MHz, DMSO-d6) δ ppm:
7.66 (br s, 1H), 7.88 (d, J = 5.1 Hz, 1H), 8.03 (br s, 1H), 8.54 (d, J =
5.1 Hz, 1H), 8.83 (s, 1H), 13.06−13.09 (m, 1H).
2-((1-Methyl-1H-pyrazol-4-yl)oxy)pyrido[3,4-d]pyrimidin-
4(3H)-one, 34. Step 1. 1-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)-1H-pyrazole (1.01g, 4.89 mmol) was dissolved in THF
(10 mL). 2 M NaOH (aq) (4.89 mL, 9.79 mmol) and 30% H2O2 (aq)
(1 mL, 9.79 mmol) were added, and the reaction was stirred for 2 h at
rt. The reaction was adjusted to pH 2 with 2 M HCl (aq) and then
partitioned between water and DCM. The aqueous layer was extracted
with DCM and the combined organics were washed with sat. Na2S2O5
(aq) and concentrated in vacuo to give a crude residue. This residue
was purified by silica gel column chromatography, eluting with a
gradient of 0−70% EtOAc/cyclohexane to give 1-methyl-1H-pyrazol-
1
4-ol (48 mg, 0.489 mmol) as a white solid. H NMR (400 MHz,
DMSO-d6) δ ppm: 3.31 (br s, 3H) 6.94 (s, 1H), 7.13 (s, 1H), 8.28 (br
s, 1H).
Step 2. 1-Methyl-1H-pyrazol-4-ol (48 mg, 0.489 mmol) and NaH
(33.4 mg, 60% w/w in oil, 0.835 mmol) were added to DMF (4 mL)
and stirred at rt for 5 min. 2-(Ethylsulfonyl)pyrido[3,4-d]pyrimidin-
4(3H)-one 20a (100 mg, 0.284 mmol) was taken up in DMF (1 mL)
and added to the reaction mixture which was heated to 110 °C under
N2 for 4 h. 1-Methyl-1H-pyrazol-4-ol (20 mg, 0.201 mmol) and NaH
(16.7 mg, 60% w/w in oil, 0.416 mmol) were added, and the reaction
was stirred at 110 °C under N2 for 16 h. The reaction was quenched
with water and extracted into EtOAc. The aqueous layer was adjusted
to pH 4 with 2 M HCl (aq) and extracted with EtOAc. The combined
organics were washed with 10% LiCl (aq) and brine and dried using a
hydrophobic frit. The solvent was removed in vacuo to give a solid
which was purified by silica gel column chromatography, eluting with a
gradient of 0−10% MeOH/DCM to yield crude product. This crude
product was further purified by MDAP (formic method) to give a
white solid. This solid was taken up in a minimum amount of MeOH
and DMSO and eluted through an NH2 SPE column with 2 M NH3 in
MeOH. All fractions were collected and the solvent was removed in
vacuo to give 34 as a white solid. (11 mg, 16%). LCMS (formic
Step 3. To a suspension of 1-cycloheptyl-1H-pyrazol-4-ol (69 mg,
0.306 mmol) in DMF (10 mL) was added NaH (61.2 mg, 60% w/w in
oil, 1.531 mmol). The slurry was stirred for 15 min at rt under N2.
Then 2-(ethylsulfonyl)pyrido[3,4-d]pyrimidin-4(3H)-one 20a (95 mg,
0.337 mmol) was added, and the reaction mixture was stirred for 2
days under N2 at 110 °C. The reaction was quenched by adding
MeOH, and the resultant solution was concentrated in vacuo to give a
black solid. This solid was purified by reverse phase column
chromatography, eluting with 0−50% MeCN/H2O to give crude
desired product. This crude product was further purified using a
MDAP (high pH method) to give 38 (14 mg, 14%) as a light brown
solid. LCMS (formic method) retention time 0.87 min, [M + H]+ =
1
method) retention time 0.45 min, [M + H]+ = 244. H NMR (400
MHz, DMSO-d6) δ ppm: 3.86 (s, 3H), 7.58 (s, 1H), 7.88 (d, J = 5.3
Hz, 1H), 8.03 (s, 1H), 8.55 (d, J = 5.1 Hz, 1H), 8.84 (s, 1H), 13.05 (br
s, 1H).
2-((1-Isopropyl-1H-pyrazol-4-yl)oxy)pyrido[3,4-d]pyrimidin-
4(3H)-one, 35. Step 1. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-
yl)-1H-pyrazole (1.5 g, 7.73 mmol) and Cs2CO3 (3.78 g, 11.6 mmol)
were suspended in MeCN (15 mL) and stirred at rt for 10 min. 2-
Bromopropane (1.097 mL, 11.6 mmol) was added and the reaction
was stirred at 60 °C for 16 h. Further 2-bromopropane (0.6 mL, 6.4
mmol) was added and the reaction stirred at 60 °C for 16 h. The
reaction was cooled and triturated with Et2O to give 1-isopropyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.31g,
62%) as a brown oil. LCMS (formic method) retention time 0.98
min, [M + H]+ = 237.
Step 2. A mixture of 1-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (1 g, 4.24 mmol), 30% H2O2 (aq)
(0.865 mL, 8.47 mmol), and 2 M NaOH (aq) (4.24 mL, 8.47 mmol)
in THF (5 mL) was stirred at rt for 5 h. The reaction was adjusted to
pH 2 with 2 M HCl (aq) and extracted with DCM and EtOAc. The
combined organics were washed with 10% Na2S2O5 (aq) and dried
using a hydrophobic frit. The solvent was removed in vacuo to give 1-
isopropyl-1H-pyrazol-4-ol (850 mg, 159%) as an orange oil. LCMS
(formic method) retention time 0.43 min, [M + H]+ = 127.
Step 3. 1-Isopropyl-1H-pyrazol-4-ol (116 mg, 0.919 mmol) and
NaH (33.4 mg, 60% w/w in oil, 0.835 mmol) were added to DMF (5
mL) at rt and stirred for 5 min. A mixture of 2-(ethylsulfonyl)pyrido-
1
326. H NMR (400 MHz, DMSO-d6) δ ppm: 1.47−1.57 (m, 2H),
1.57−1.67 (m, 3H), 1.70−1.80 (m, 2H), 1.89−1.99 (m, 2H), 2.00−
2.08 (m, 2H), 4.34 (tt, J = 9.5, 4.7 Hz, 1H), 7.58 (s, 1H), 7.87 (d, J =
5.1 Hz, 1H), 8.06 (s, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.83 (s, 1H).
2-((1-Cyclohexyl-1H-pyrazol-4-yl)oxy)pyrido[3,4-d]-
pyrimidin-4(3H)-one, 37. Step 1. 1-Cyclohexyl-1H-pyrazol-4-ol was
prepared using a similar method to that described for 1-cycloheptyl-
1H-pyrazol-4-ol described in the preparation of compound 38. LCMS
(formic method) retention time 0.69 min, [M + H]+ = 167.
Step 2. 2-(Ethylthio)pyrido[3,4-d]pyrimidin-4(3H)-one 20 (387
mg, 1.868 mmol) was taken up in THF (10 mL). mCPBA (77% in
water) (1172 mg, 5.23 mmol) was added, and the mixture was stirred
at rt for 4 h by which time LCMS showed conversion of 20 to the
sulfone intermediate 20a. The solvent was evaporated in vacuo, and
the residue was triturated with IPA and filtered. The collected solid
was washed with IPA and Et2O and air-dried affording a pale yellow
powder. To this powder was added 1-cyclohexyl-1H-pyrazol-4-ol (207
mg, 0.747 mmol) and NaH (59.8 mg, 60% w/w in oil, 1.494 mmol) in
L
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Journal of Medicinal Chemistry
Article
DMF (4 mL). The mixture was heated to 110 °C under N2 for 16 h.
The reaction was poured into 10% LiCl (aq) and extracted with
EtOAc. The organic fractions were dried using a hydrophobic frit and
concentrated to a brown oil. This oil was purified by MDAP (formic
method) to give 37 (10 mg, 4%) as a off-white solid. LCMS (formic
1.09 min, [M + H]+ = 314, retention time 0.63 min, [M + H]+ = 232,
retention time 0.83 min, [M + H]+ = 188.
Step 2. To the mixture of (1-(3-methoxybenzyl)-1H-pyrazol-4-
yl)boronic acid, 1-(3-methoxybenzyl)-1H-pyrazole, and 1-(3-methox-
ybenzyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole
(9:1:4) from step 1 (1.044 g, 3.32 mmol) in THF (10 mL) were added
2 M NaOH (aq) (4.98 mL, 9.97 mmol) and 30% H2O2 (aq) (0.679
mL, 6.65 mmol). The reaction mixture was stirred for 2 h at rt and was
quenched by adding sat. Na2SO3 (aq) and adjusted to pH 4 with 2 M
HCl (aq). The resultant solution was concentrated and partitioned
between water and EtOAc. The organics were dried over Na2SO4 and
concentrated to give crude residue. This residue was purified by silica
gel column chromatography, eluting with a gradient of 0−100%
EtOAc/cyclohexane to give 1-(3-methoxybenzyl)-1H-pyrazol-4-ol
(390 mg, 58%) as a white solid. LCMS (formic method) retention
time 0.66 min, [M + H]+ = 205.
Step 3. To a solution of 1-(3-methoxybenzyl)-1H-pyrazol-4-ol (93
mg, 0.455 mmol) in DMF (15 mL) was cautiously added NaH (48
mg, 60% w/w in oil, 1.200 mmol). The mixture was stirred at rt for 10
min. Then 2-(ethylsulfonyl)pyrido[3,4-d]pyrimidin-4(3H)-one 20a
(143 mg, 84% w/w, 0.501 mmol) was added and the reaction stirred
at 110 °C for 16 h. Further 2-(ethylsulfonyl)pyrido[3,4-d]pyrimidin-
4(3H)-one 20a (74 mg, 84% w/w, 0.455 mmol) was added and the
mixture heated at 110 °C for a further 16 h and then quenched with
MeOH and concentrated. The resulting residue was partitioned with
EtOAc and water. The organic layer was dried using a hydrophobic frit
and concentrated to give crude product. This was purified by MDAP
(formic method) to give 42 (38 mg, 24%) as a white solid. LCMS
(formic method) retention time 0.77 min, [M + H]+ = 350. 1H NMR
(400 MHz, DMSO-d6) δ ppm: 3.75 (s, 3H), 5.31 (s, 2H), 6.83−6.86
(m, 2H), 6.87−6.91 (m, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.65 (d, J = 0.9
Hz, 1H), 7.88 (dd, J = 5.1, 0.9 Hz, 1H), 8.19 (s, 1H), 8.54 (d, J = 5.1
Hz, 1H), 8.82 (s, 1H), 13.09 (br s, 1H).
2-((1-(Pyridin-3-ylmethyl)-1H-pyrazol-4-yl)oxy)pyrido[3,4-
d]pyrimidin-4(3H)-one, 43. Step 1. 4-(4,4,5,5-Tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (1 g, 5.15 mmol) was dissolved in
MeCN (20 mL) and was treated with Cs2CO3 (5.04 g, 15.46 mmol)
followed by 3-(bromomethyl)pyridine hydrobromide (2.61 g, 10.31
mmol). The mixture was stirred at rt for 16 h. The insoluble materials
were removed by filtration and the filtrate was concentrated in vacuo
to give an orange oil. The oil was purified by silica gel column
chromatography using a gradient of 0−100% EtOAc/cyclohexane to
give 3-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-
yl)methyl)pyridine (316 mg, 22% yield) as a colorless oil. LCMS
(formic method) retention time 0.65 min, [M + H]+ = 286.
Step 2. 3-((4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyr-
azol-1-yl)methyl)pyridine (316 mg, 0.831 mmol) was dissolved in
THF (10 mL) and was treated with 2 M NaOH (aq) (1.7 mL, 3.40
mmol) and 30% H2O2 (aq) (0.170 mL, 1.662 mmol) and the mixture
stirred at rt for 1 h. The reaction was neutralized with 2 M HCl (aq),
and the sample was loaded onto an SCX column and eluted with
MeOH then 2 M NH3 in MeOH. The appropriate fractions were
combined and evaporated to give 1-(pyridin-3-ylmethyl)-1H-pyrazol-
4-ol (178 mg, 122% yield) as a colorless oil which solidified. LCMS
(high pH method) retention time 0.42 min, [M + H]+ = 176.
Step 3. A round-bottom flask was charged with 2-(ethylthio)pyrido-
[3,4-d]pyrimidin-4(3H)-one 20 (210 mg, 1.013 mmol), THF (10
mL), and mCPBA (77% in water) (568 mg, 2.53 mmol). The slurry
was stirred at rt for 4 h. IPA (10 mL) was added, and the mixture was
concentrated to approximately 7 mL. Further IPA (10 mL) was added,
and the solution was again concentrated in vacuo to approximately 7
mL. The resultant slurry was filtered; the residue was washed with
Et2O and dried under vacuo to give sulfone/sulfoxide intermediate
20b. A round-bottom flask was charged with 1-(pyridin-3-ylmethyl)-
1H-pyrazol-4-ol (178 mg, 1.013 mmol), DMF (10 mL), and NaH (81
mg, 60% w/w in oil, 2.027 mmol). The mixture was stirred at rt for 20
min prior to the addition of the above prepared intermediate 20b. The
reaction was heated at 110 °C under N2 for 16 h. The reaction was
cooled to rt and was quenched with water. The volatiles were removed
in vacuo to leave a brown residue. The residue was triturated with hot
1
method) retention time 0.79 min, [M + H]+ = 312. H NMR (400
MHz, DMSO-d6) δ ppm: 1.22 (qt, J = 12.9, 3.3 Hz, 1H), 1.41 (qt, J =
12.9, 3.3 Hz, 2H), 1.63−1.71 (m, 2H), 1.75 (dd, J = 12.5, 3.3 Hz, 1H),
1.83 (dt, J = 12.9, 3.3 Hz, 2H), 2.04 (dd, J = 12.6, 2.6 Hz, 2H), 4.12
(tt, J = 11.6, 3.8 Hz, 1H), 7.59 (s, 1H), 7.86 (dd, J = 5.1, 0.7 Hz, 1H),
8.06 (s, 1H), 8.51 (d, J = 5.1 Hz, 1H), 8.83 (s, 1H).
2-((1-Benzyl-1H-pyrazol-4-yl)oxy)pyrido[3,4-d]pyrimidin-
4(3H)-one, 41. Step 1. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-
yl)-1H-pyrazole (500 mg, 2.58 mmol) was taken up in MeOH (5 mL)
and 1 M NaOH (aq) (5.15 mL, 5.15 mmol). The reaction mixture was
stirred for 5 min at rt. Then (bromomethyl)benzene (0.368 mL, 3.09
mmol) was added and the reaction stirred at rt for 16 h. Further 1 M
NaOH (aq) (2.58 mL, 2.58 mmol) and (bromomethyl)benzene
(0.307 mL, 2.58 mmol) were added, and the reaction was stirred at rt
for 2.5 days. The reaction was adjusted to pH 5 with 2 M HCl (aq)
and then extracted with EtOAc. The solvent was removed in vacuo
and the crude residue was purified by silica gel column
chromatography, eluting with a gradient of 0−25% EtOAc/cyclo-
hexane to give 1-benzyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1H-pyrazole (170 mg, 23%) as a white solid. LCMS (formic
method) retention time 1.09 min, [M + H]+ = 285.
Step 2. 1-Benzyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazole (170 mg, 0.598 mmol) was dissolved in THF (5 mL) and
cooled to 0 °C. 2 M NaOH (aq) (0.598 mL, 1.197 mmol) and 30%
H2O2 (aq) (0.122 mL, 1.197 mmol) were added, and the reaction was
stirred at rt for 40 min. The reaction was adjusted to pH 2 with 2 N
HCl (aq) and then partitioned with water and DCM. The organic
phase was dried using a hydrophobic frit and concentrated to give a
crude residue. This residue was purified using silica gel column
chromatography, eluting with a gradient of 0−50% EtOAc/cyclo-
hexane to give 1-benzyl-1H-pyrazol-4-ol as a white solid (80 mg, 77%).
LCMS (formic method) retention time 0.63 min, [M + H]+ = 175.
Step 3. 1-Benzyl-1H-pyrazol-4-ol (80 mg, 0.459 mmol) and NaH
(33.4 mg, 60% w/w in oil, 0.835 mmol) were added to DMF (4 mL)
and stirred at rt for 5 min. 2-(Ethylsulfonyl)pyrido[3,4-d]pyrimidin-
4(3H)-one 20a (100 mg, 0.305 mmol) dissolved in DMF (1 mL) was
added to the reaction mixture which was heated to 110 °C and stirred
for 5 h. Further NaH (17 mg, 60% w/w in oil, 0.417 mmol) was added
and the reaction stirred for a further 18 h. The reaction was quenched
with water and extracted into EtOAc. The organic phase was washed
with 10% LiCl (aq) and then washed with brine and concentrated to
give a yellow solid. This solid was triturated in MeOH to give 41 (33
mg, 34%) as a white solid. LCMS (formic method) retention time 0.74
min, [M + H]+ = 320. HRMS: C17H14N5O2 requires [M + H]+
1
320.1142, found 320.1142 (error −0.1 ppm). H NMR (400 MHz,
DMSO-d6) δ ppm: 5.34 (s, 2H), 7.27−7.34 (m, 3H), 7.35−7.41 (m,
2H), 7.65 (s, 1H), 7.88 (d, J = 5.1 Hz, 1H), 8.19 (s, 1H), 8.54 (d, J =
4.8 Hz, 1H), 8.82 (s, 1H), 13.1 (br s, 1H). 13C NMR (126 MHz,
DMSO-d6) δ ppm: 162.7, 154.5, 149.4, 144.9, 143.3, 137.8, 135.6,
131.4, 129.0, 128.2, 128.1, 125.3, 122.3, 118.9, 56.0. Mp 214−216 °C.
2-((1-(3-Methoxybenzyl)-1H-pyrazol-4-yl)oxy)pyrido[3,4-d]-
pyrimidin-4(3H)-one, 42. Step 1. To a solution of 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1 g, 5.15 mmol) in
MeCN (10 mL) were added Cs2CO3 (2.52 g, 7.73 mmol) and 1-
(bromomethyl)-3-methoxybenzene (0.794 mL, 5.41 mmol). The
reaction mixture was stirred for 24 h at 80 °C. The resultant slurry
was concentrated and partitioned between water and EtOAc. The
organic phase was concentrated to give crude residue which was
purified using silica gel column chromatography, eluting with a
gradient of 0−100% EtOAc/cyclohexane to give a three-component
mixture of (1-(3-methoxybenzyl)-1H-pyrazol-4-yl)boronic acid with 1-
(3-methoxybenzyl)-1H-pyrazole and 1-(3-methoxybenzyl)-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (9:1:4) (1.04g,
27%) as a colorless solid. LCMS (formic method) retention time
M
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Journal of Medicinal Chemistry
Article
EtOH, and the resulting slurry was filtered. The filtrate was
concentrated in vacuo to give a red residue which was purified by
MDAP (high pH method) to give 43 (59 mg, 18%) as a yellow solid.
LCMS (high pH method) retention time 0.46 min, purity 91% by area,
dissolved. The solution was cooled again and was saturated with CO2
until a cream colored precipitate had formed. The solid was isolated by
filtration and dried in vacuo to give pyrido[2,3-d]pyrimidine-
2,4(1H,3H)-dione (4.8 g, 82%) as a white solid. LCMS (formic
method) retention time 0.36 min, [M + H]+ = 164. H NMR (400
1
1
[M + H]+ = 321. H NMR (400 MHz, DMSO-d6) δ ppm: 5.40 (s,
2H), 7.41 (dd, J = 7.7, 4.5 Hz, 1H), 7.67 (s, 1H), 7.70 (dt, J = 8.0, 1.5
Hz, 1H), 7.88 (d, J = 5.1 Hz, 1H), 8.26 (s, 1H), 8.54 (d, J = 5.1 Hz,
2H), 8.56 (d, J = 1.7 Hz, 1H), 8.82 (s, 1H), 13.12 (br s, 1H).
2-((1,3-Dimethyl-1H-pyrazol-4-yl)oxy)pyrido[3,4-d]-
pyrimidin-4(3H)-one, 44. Step 1. A premixed solution of 30% H2O2
(aq) (0.270 mL, 2.64 mmol) and 2 M NaOH (aq) (1.322 mL, 2.64
mmol) was added to a solution of 1,3-dimethyl-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-1H-pyrazole (534 mg, 2.404 mmol) in THF
(10 mL). The resulting suspension was stirred for 2 h and evaporated
to dryness. The residue was purified by silica gel column
chromatography, eluting with a gradient of 50−100% EtOAc/
cyclohexane to give 1,3-dimethyl-1H-pyrazol-4-ol (205 mg, 72%) as
a white solid. LCMS (TFA method) retention time 0.23 min, [M +
H]+ = 112.
Step 2. NaH (115 mg, 60% w/w in oil, 2.88 mmol) was added to a
solution of 1,3-dimethyl-1H-pyrazol-4-ol (105 mg, 0.936 mmol) in
DMF (5 mL) under N2, and the resulting suspension was stirred for 5
min. A mixture of 2-(ethylsulfonyl)pyrido[3,4-d]pyrimidin-4(3H)-one
and 2-(ethylsulfinyl)pyrido[3,4-d]pyrimidin-4(3H)-one 20b (150 mg,
0.568 mmol) was added, and the resulting suspension was heated to
110 °C for 1.5 h. The mixture was cooled to rt, acidified to pH 4 with
2 M HCl (aq), and diluted with EtOAc. The aqueous layer was
separated, and the organic layer was washed with water and brine and
then dried (MgSO4) and evaporated to give a dark red solid. This solid
was purified using silica gel column chromatography, eluting with a
gradient of 0−6% DCM/MeOH to give an off white solid which was
triturated with tert-butylmethyl ether and dried to give 44 (9 mg, 5%)
as a white solid. LCMS (TFA method) retention time 0.45 min, [M +
H]+ = 258. 1H NMR (400 MHz, DMSO-d6) δ ppm: 2.07 (s, 3H), 3.78
(s, 3H), 7.88 (d, J = 4.6 Hz, 1H), 7.91 (s, 1H), 8.53 (d, J = 5.1 Hz,
1H), 8.81 (s, 1H), 13.06 (br s, 1H).
2-((1,5-Dimethyl-1H-pyrazol-4-yl)oxy)pyrido[3,4-d]-
pyrimidin-4(3H)-one, 45. Step 1. To a solution of 1,5-dimethyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (500 mg,
2.251 mmol) in THF (10 mL) were added 2 M NaOH (aq) (3.38
mL, 6.75 mmol) and 30% H2O2 (aq) (0.460 mL, 4.50 mmol). The
solution was stirred for 2 h at rt. The excess of H2O2 was quenched by
adding sat. Na2SO3 (aq), and the mixture was adjusted to pH 4 with 2
M HCl (aq). The mixture was extracted with EtOAc, dried over
MgSO4, and concentrated to a crude residue which was purified using
silica gel column chromatography, eluting with a gradient of 0−100%
EtOAc/cyclohexane to give 1,5-dimethyl-1H-pyrazol-4-ol (120 mg,
35%) as a white solid. 1H NMR (400 MHz, MeOH-d4) δ ppm: 2.18 (s,
3 H), 3.69 (s, 3 H), 7.01 (s, 1 H).
Step 2. To 1,5-dimethyl-1H-pyrazol-4-ol (88 mg, 0.785 mmol) in
DMF (10 mL) was added NaH (23.54 mg, 60% w/w in oil, 0.981
mmol). The suspension was stirred for 15 min under N2 at rt. 2-
(Ethylsulfonyl)pyrido[3,4-d]pyrimidin-4(3H)-one 20a (258 mg, 80%
w/w, 0.863 mmol) was added, and the reaction mixture was stirred
overnight at 110 °C. Further NaH (23.54 mg, 60% w/w in oil, 0.981
mmol) was added, and the reaction mixture was stirred for 20 h at 100
°C under N2. 1,5-Dimethyl-1H-pyrazol-4-ol (44 mg, 0392 mmol) was
added, and the reaction mixture was stirred overnight under N2 at 110
°C. MeOH and water were added to the reaction which was then
concentrated to give a crude solid. This solid was purified by MDAP
(formic method) to give 45 (6.3 mg, 3%) as a white solid. LCMS
(formic method) retention time 0.49 min, [M + H]+ = 258. 1H NMR
(400 MHz, DMSO-d6) δ ppm: 2.16 (s, 3H), 3.76 (s, 3H), 7.47 (s,
1H), 7.84 (d, J = 5.2 Hz, 1H), 8.46 (d, J = 5.2 Hz, 1H), 8.73 (s, 1H).
2-((1-Benzyl-1H-pyrazol-4-yl)oxy)pyrido[2,3-d]pyrimidin-
4(3H)-one, 46. Step 1. 2-Aminonicotinic acid (5g, 36.2 mmol) and
urea (12.70 g, 145 mmol) were stirred at 180 °C for 15 min, then at
200 °C for 15 min and then 210 °C for 1 h. The reaction was cooled
to rt. To the resulting solid was added 2 M NaOH (approximately 50
mL), and the mixture was stirred at 50 °C until all the solid had
MHz, DMSO-d6) δ ppm: 5.43 (br s, 1H), 7.01 (dd, J = 7.6, 4.7 Hz,
1H), 8.13 (dd, J = 7.6, 2.0 Hz, 1H), 8.48 (dd, J = 4.7, 1.96 Hz, 1H),
10.96 (br s, 1H).
Step 2. Pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (1g, 6.13 mmol)
was taken up in phosphorus oxychloride (20 mL, 215 mmol) and
stirred at 110 °C under N2 for 16 h. The reaction was azeotroped with
toluene. The residue was treated with 2 N NaOH (20 mL, 40.0 mmol)
and stirred at rt for 2 h. The mixture was adjusted to pH 5 with 2 M
HCl (aq) and extracted with EtOAc. The combined organic fractions
were dried using a hydrophobic frit and concentrated in vacuo to give
2-chloropyrido[2,3-d]pyrimidin-4(3H)-one as a pale yellow solid (723
mg, 65%). LCMS (formic method) retention time 0.39 min, [M + H]+
= 182. 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.58 (dd, J = 7.8, 4.7
Hz, 1H), 8.49 (dd, J = 7.8, 2.0 Hz, 1H), 8.94 (dd, J = 4.7, 2.0 Hz, 1H),
13.59 (br s, 1H).
Step 3. 1-Benzyl-1H-pyrazol-4-ol (767 mg, 2.203 mmol) (see
compound 41, steps 1 and 2, for preparation) was taken up in DMF (4
mL). NaH (44.1 mg, 60% w/w in oil, 1.101 mmol) was added, and
after 5 min 2-chloropyrido[2,3-d]pyrimidin-4(3H)-one (200 mg, 1.101
mmol) was added. The mixture was heated to 110 °C under N2 for 2
days. The reaction was filtered, and the solvent was evaporated in
vacuo. The residue was purified by MDAP (formic method) to give 46
(87 mg, 25%). LCMS (formic method) retention time 0.71 min, [M +
H]+ = 320. HRMS: C17H14N5O2 requires [M + H]+ 320.1142, found
1
320.1142 (error −0.1 ppm). H NMR (400 MHz, MeOH-d4) δ ppm:
5.35 (s, 2H), 7.30−7.34 (m, 3H), 7.35−7.40 (m, 2H), 7.45 (dd, J =
7.8, 4.7 Hz, 1H), 7.70 (s, 1H), 8.20 (s, 1H), 8.57 (dd, J = 7.8, 2.0 Hz,
1H), 8.78 (dd, J = 4.7, 2.0 Hz, 1H). 13C NMR (126 MHz, DMSO-d6)
δ ppm: 164.1, 158.5, 156.0, 155.9, 137.8, 136.3, 135.5, 131.6, 129.0,
128.2, 128.1, 122.3, 121.2, 115.1, 56.0. Mp 265−270 °C (dec).
2-(Ethylthio)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-
d]pyrimidin-4(3H)-one, 23. A mixture of 2-(ethylthio)pyrido[3,4-
d]pyrimidin-4(3H)-one 20 (5 g, 24.13 mmol), DMF (50 mL), K2CO3
(6.67 g, 48.3 mmol), and SEM chloride (4.22 g, 25.3 mmol) was
stirred at 50 °C for 3 h. The reaction temperature was increased to 80
°C, and heating continued for a further 4 h. The reaction was cooled
to rt prior to the addition of further SEM chloride (2 g, 12.00 mmol).
The mixture was stirred at 50 °C under N2 for 16 h. The reaction was
cooled to rt, and further SEM chloride (0.8 g, 4.80 mmol) was added
dropwise. The reaction was stirred at 50 °C for 4 h. The reaction was
cooled to rt, the insoluble material was removed by filtration through
Celite (EtOAc eluent), and the filtrate was concentrated in vacuo to
give an orange oil. This oil was purified using silica gel column
chromatography, eluting with a gradient of 0−20% EtOAc/cyclo-
hexane to give 23 (6.36 g, 78% yield) as a yellow oil which solidified
on standing. LCMS (formic method) retention time 1.37 min, [M +
H]+ = 338. 1H NMR (400 MHz, CDCl3) δ ppm: 0.01−0.04 (m, 10H),
1.01 (t, J = 8.5 Hz, 2H), 1.44−1.50 (m, 4 H), 3.35 (q, J = 7.5 Hz, 2H),
3.73 (t, J = 8.0 Hz, 2H), 5.63 (s, 2H), 7.98 (d, J = 5.1 Hz, 1H), 8.61 (d,
J = 5.1 Hz, 1H), 9.00 (s, 1H).
2-(4-Chloro-3-hydroxyphenoxy)pyrido[3,4-d]pyrimidin-
4(3H)-one, 31. Step 1. 5-Bromo-2-chlorophenol (2g, 9.64 mmol) was
dissolved in DMF (20 mL). (2-(Chloromethoxy)ethyl)trimethylsilane
(1.922 mL, 10.60 mmol) and K2CO3 (2.66 g, 19.28 mmol) were
added, and the mixture was stirred at rt for 20 h. 10% aq LiCl (40 mL)
was added, and the aqueous portion was extracted with EtOAc (40
mL). The organic layer was passed though a hydrophobic frit and was
evaporated in vacuo affording a colorless oil. This was purified by silica
gel column chromatography, eluting with 0−30% EtOAc/cyclohexane
to give (2-((5-bromo-2-chlorophenoxy)methoxy)ethyl)trimethylsilane
(3.23g) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ ppm: 0.00 (s,
9H), 0.96 (m, 2H), 3.79 (m, 2H), 5.26 (s, 2H), 7.06 (dd, J = 8.0, 2 Hz,
1H), 7.21 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H).
Step 2. (2-((5-Bromo-2-chlorophenoxy)methoxy)ethyl)-
trimethylsilane (1 g, 2.96 mmol) was dissolved in 2-methyl-THF
N
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Journal of Medicinal Chemistry
Article
(15 mL). Bis(pinocolato)diboron (0.759 g, 2.99 mmol), Pd(dppf)Cl2
(0.108 g, 0.148 mmol), and potassium acetate (0.436 g, 4.44 mmol)
were added, and the mixture was heated to 80 °C under nitrogen for
16 h. The reaction was filtered through Celite and the filtrate was
concentrated in vacuo affording a colorless oil which was purified by
silica gel column chromatography, eluting with 0−15% EtOAc/
cyclohexane. Relevant fractions were combined and evaporated in
vacuo to give (2-((2-chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenoxy)methoxy)ethyl)trimethylsilane as a colorless oil (409
mg). 1H NMR (400 MHz, CDCl3) δ ppm: 0.00 (s, 9H), 0.97 (m, 2H),
1.33 (s, 12H), 3.83 (m, 2H), 5.33 (s, 2H), 7.37 (m, 2H), 7.55 (s, 1H).
Step 3. (2-((2-Chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenoxy)methoxy)ethyl)trimethylsilane (409 mg, 0.776 mmol) was
dissolved in THF. 30% w/w H2O2 (159 μL, 1.552 mmol) and 2 M
NaOH (776 μL, 1.552 mmol) were added, and the mixture was stirred
at rt for 4 h and then left standing at rt for 2 days. Water (20 mL) was
added, and the mixture was extracted with DCM (30 mL). The
organic layer was passed through a hydrophobic frit and was
evaporated in vacuo affording a colorless oil which was purified by
silica gel column chromatography, eluting with a gradient of 0−20%
EtOAc/cyclohexane to give 4-chloro-3-((2-(trimethylsilyl)ethoxy)-
Step 2. A round-bottom flask was charged with 5-(benzyloxy)-2-
bromopyridine (1 g, 3.79 mmol), 4,4,5,5-tetramethyl-2-(prop-1-en-2-
yl)-1,3,2-dioxaborolane (950 mg, 5.65 mmol), 1,4-dioxane (20 mL),
tetrakis(triphenylphosphine)palladium(0) (0.088 g, 0.076 mmol), and
2 M Na2CO3 (5.68 mL, 11.36 mmol). The mixture was degassed and
was warmed to 90 °C under a blanket of N2 overnight. The mixture
was cooled to rt, diluted with EtOAc and water, and the layers were
mixed and separated. The organics were washed with brine, passed
through a hydrophobic frit, and concentrated in vacuo to give a yellow
oil. The oil was purified by silica gel column chromatography, eluting
with a gradient of 0−25% EtOAc/cyclohexane to give 5-(benzyloxy)-
2-(prop-1-en-2-yl)pyridine (768 mg, 63% yield) as a colorless oil. This
material contained unreacted starting material 5-(benzyloxy)-2-
bromopyridine (∼20% by LCMS) and was used in the next step
without further purification. LCMS (high pH method) retention time
1.18 min, [M + H]+ = 264/266, starting material; retention time 1.24
min [M + H]+ = 226, product.
Step 3. A hydrogenation flask was charged with 5-(benzyloxy)-2-
(prop-1-en-2-yl)pyridine (664 mg, 2.063 mmol) from step 2, EtOH
(10 mL), and palladium hydroxide on carbon (66 mg, 0.470 mmol). A
hydrogenation head was fitted, and the reaction was stirred at rt under
an atmosphere of H2 overnight. The mixture was passed through
Celite (EtOAc eluent), and the filtrate was concentrated in vacuo to
give an off-white solid. The sample was purified by silica gel column
chromatography, eluting with a gradient of 0−40% EtOAc/cyclo-
hexane to give 6-isopropylpyridin-3-ol (228 mg, 81% yield) as a white
solid. LCMS (formic method) retention time 0.34 min, [M + H]+ =
138.
Step 4. 2-(Ethylthio)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido-
[3,4-d]pyrimidin-4(3H)-one 23 (237 mg, 0.702 mmol), THF (10
mL), and mCPBA (77% weight in water) (393 mg, 1.755 mmol) were
stirred at rt for 1 h. The mixture was concentrated in vacuo to half
volume, then diluted with DCM. The organics were washed with sat.
NaHCO3 (aq), dried using a hydrophobic frit, and concentrated to
give a yellow oil. This oil was dissolved in 1,4-dioxane (10 mL). 6-
Isopropylpyridin-3-ol (96 mg, 0.702 mmol) and Cs2CO3 (686 mg,
2.107 mmol) were added, and the reaction was stirred at 60 °C under
N2 for 3 h. The reaction was diluted with EtOAc and the organics were
washed with sat. NaHCO3 (aq) and brine, dried using a hydrophobic
frit, and concentrated in vacuo to give a yellow solid. This solid was
purified using silica gel column chromatography, eluting with a
gradient of 0−20% EtOAc/cyclohexane to give 2-((6-isopropylpyridin-
3-yl)oxy)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one (122 mg, 0.180 mmol, 25.7% yield) as a
colorless oil. LCMS (formic method) retention time 1.31 min, [M +
H]+ = 413.
Step 5. To 2-((6-Isopropylpyridin-3-yl)oxy)-3-((2-(trimethylsilyl)-
ethoxy)methyl)pyrido[3,4-d]pyrimidin-4(3H)-one (122 mg, 0.180
mmol) in nitromethane (10 mL) was added magnesium bromide
diethyl etherate (140 mg, 0.541 mmol), and the reaction was stirred at
95 °C for 1 h. The mixture was cooled to rt and concentrated to give a
tan residue. The residue was slurried in MeOH and the solid was
isolated by filtration, washed with MeOH, and dried to give 32 (25
mg, 49%) as a buff solid. LCMS (formic method) retention time 0.63
min, [M + H]+ = 283. HRMS: C15H15N4O2 requires [M + H]+
283.1190, found 283.1191 (error 0.4 ppm). 1H NMR (400 MHz,
DMSO-d6) δ ppm: 1.28 (d, J = 6.9 Hz, 6 H), 3.10 (dt, J = 13.9, 6.9 Hz,
1H), 7.43 (d, J = 8.6 Hz, 1H), 7.78 (dd, J = 8.6, 2.9 Hz, 1H), 7.90 (d, J
= 5.9 Hz, 1H), 8.53 (d, J = 7.8 Hz, 2H), 8.71 (s, 2H), 13.23 (br s, 1H).
13C NMR (126 MHz, DMSO-d6) δ ppm: 164.5, 162.6, 155.3, 149.2,
1
methoxy)phenol as a colorless oil (135 mg, 63%). H NMR (400
MHz, CDCl3) δ ppm: 0.00 (s, 9H), 0.96 (m, 2H), 3.79 (m, 2H), 5.26
(s, 2H), 4.98 (s, 1H), 6.42 (dd, J = 8, 2.0 Hz, 1H), 6.74 (d, J = 2.0 Hz,
1H), 7.18 (d, J = 8.0 Hz, 1H).
Step 4. 2-(Ethylthio)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido-
[3,4-d]pyrimidin-4(3H)-one 23 (332 mg, 0.982 mmol) was taken up
in 2-methyl-THF (10 mL). mCPBA (77% in water) (339 mg, 1.965
mmol) was added, and the mixture was stirred at rt for 2 h. The
reaction mixture was washed with sat. NaHCO3 (aq), and the organic
layer was passed through a hydrophobic frit before being evaporated in
vacuo to give a yellow oil. To the yellow oil was added 4-chloro-3-((2-
(trimethylsilyl)ethoxy)methoxy)phenol (135 mg, 0.491 mmol) in 1,4-
dioxane (10 mL) and Cs2CO3 (320 mg, 0.982 mmol). The mixture
was heated to 60 °C under N2 for 16 h. The reaction was filtered, and
the solvent was evaporated in vacuo. The residue was purified by silica
gel column chromatography, eluting with a gradient of 0−30%
EtOAc/cyclohexane to give 2-(4-chloro-3-((2-(trimethylsilyl)ethoxy)-
methoxy)phenoxy)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-
d]pyrimidin-4(3H)-one (217 mg, 80%). LCMS (formic method)
retention time 1.64 min, [M + H]+ = 550.
Step 5. 2-(4-Chloro-3-((2-(trimethylsilyl)ethoxy)methoxy)-
phenoxy)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one (217 mg, 0.394 mmol) was taken up in
nitromethane (10 mL). Magnesium bromide diethyl etherate (306
mg, 1.183 mmol) was added, and the mixture was stirred at 95 °C
under nitrogen for 16 h. The solvent was evaporated in vacuo and the
residue was purified by silica gel column chromatography with a
gradient of 0−100% EtOAc/cyclohexane to give 31 as a pale brown
solid (45 mg, 39%). LCMS (formic method) retention time 0.67 min,
[M + H]+ = 290. HRMS: C13H9ClN3O3 requires [M + H]+ 290.0327,
1
found 290.0329 (error 0.6 ppm). H NMR (400 MHz, DMSO-d6) δ
ppm: 6.82 (dd, J = 8.7, 2.7 Hz, 1H), 6.94 (d, J = 2.8 Hz, 1H), 7.43 (d, J
= 8.6 Hz, 1H), 7.89 (d, J = 5.1 Hz, 1H), 8.53 (d, J = 5.1 Hz, 1H), 8.72
(s, 1H), 10.57 (s, 1H), 13.09 (br s, 1H). 13C NMR (126 MHz,
DMSO-d6) δ ppm: 162.4, 155.0, 154.2, 150.8, 149.4, 145.0, 143.4,
130.7, 125.3, 118.8, 117.7, 114.0, 110.8. Mp 268 °C (dec).
2-((6-Isopropylpyridin-3-yl)oxy)pyrido[3,4-d]pyrimidin-
4(3H)-one, 32. Step 1. A round-bottom flask was charged with 6-
bromopyridin-3-ol (2 g, 11.49 mmol), polymer-bound triphenylphos-
phine (3 mmol/g) (9.20 g, 27.6 mmol), DCM (70 mL), and benzyl
alcohol (1.45 mL, 13.95 mmol). To the mixture was added diisopropyl
azodicarboxylate (2.68 mL, 3.79 mmol) dropwise. The slurry was
stirred at rt under a blanket of N2 for 2 days. The insoluble materials
were removed by filtration through a hydrophobic frit, and the filtrate
was concentrated in vacuo to give a brown oil. The oil was purified by
silica gel column chromatography, eluting with a gradient of 0−40%
EtOAc/cyclohexane to give 5-(benzyloxy)-2-bromopyridine (1.98 g,
65% yield) as a colorless solid. LCMS (high pH method) retention
time 1.16 min, [M + H]+ = 264/266.
146.7, 145.0, 143.2, 142.7, 130.6, 125.3, 122.0, 118.9, 35.5, 23.0. Mp
248 °C (dec).
2-((1-Cyclopentyl-1H-pyrazol-4-yl)oxy)pyrido[3,4-d]-
pyrimidin-4(3H)-one, 36. Step 1. 1-Cyclopentyl-1H-pyrazol-4-ol was
prepared using a similar method to that described for 1-cycloheptyl-
1H-pyrazol-4-ol described in the preparation of compound 38. LCMS
(formic method) retention time 0.60 min, [M + H]+ = 153.
Step 2. To a solution of mCPBA (77% in water) (664 mg, 2.96
mmol) in 2-methyl-THF, (5 mL) was added 2-(ethylthio)-3-((2-
(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]pyrimidin-4(3H)-one 23
O
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Journal of Medicinal Chemistry
Article
(400 mg, 1.185 mmol). The resultant suspension was stirred under N2
for 1 h. The reaction was diluted with 2-methyl-THF (20 mL) and
washed with sat. NaHCO3 (aq); the organic layer was passed through
a hydrophobic frit and the solvent was evaporated to give a pale yellow
solid. This solid was redissolved in 1,4-dioxane (5 mL), and then
Cs2CO3 (1158 mg, 3.56 mmol) and 1-cyclopentyl-1H-pyrazol-4-ol
(180 mg, 1.185 mmol) were added. The resultant suspension was
stirred at 60 °C under N2 for 16 h. The reaction was concentrated and
purified by MDAP (formic method) to give 2-((1-cyclopentyl-1H-
pyrazol-4-yl)oxy)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one (87 mg, 0.203 mmol) as a clear oil. LCMS
(formic method) retention time 1.33 min, [M + H]+ = 428.
Step 3. To a solution of 2-((1-cyclopentyl-1H-pyrazol-4-yl)oxy)-3-
((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]pyrimidin-4(3H)-one
(87 mg, 0.203 mmol) in nitromethane (5 mL) at 0 °C was added
magnesium bromide diethyl etherate (112 mg, 0.610 mmol)
portionwise. The reaction was stirred at 95 °C for 2 h. The solvent
was removed and the crude purified by MDAP (formic method) to
give 36 (21 mg, 34%) as a cream solid. LCMS (formic method)
retention time 0.74 min, [M + H]+ = 298. HRMS: C15H16N5O1 2
requires [M + H]+ 298.1299, found 298.1298 (error −0.3 ppm). H
NMR (400 MHz, DMSO-d6) δ ppm: 1.62−1.68 (m, 2H), 1.76−1.83
(m, 2H), 1.90−2.00 (m, 2H), 2.04−2.14 (m, 2H), 4.69 (quin, J = 7.0
Hz, 1H), 7.60 (s, 1H), 7.87 (dd, J = 5.1, 0.7 Hz, 1H), 8.07 (s, 1H),
8.51 (d, J = 5.1 Hz, 1H), 8.82 (s, 1H), 13.04−13.11 (m, 1H). 13C
NMR (126 MHz, DMSO-d6) δ ppm: 163.7, 155.7, 149.4, 144.4, 143.9,
135.5, 130.7, 125.2, 120.6, 118.9, 63.1, 32.9, 24.2. Mp 224−228 °C
(decomp).
2-((1-(Cyclohexylmethyl)-1H-pyrazol-4-yl)oxy)pyrido[3,4-d]-
pyrimidin-4(3H)-one, 39. Step 1. 4-(4,4,5,5-Tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole (2 g, 10.31 mmol) was taken up in
DMF (30 mL) and cooled to 0 °C. NaH (0.824 g, 60% w/w in oil,
20.61 mmol) was added portionwise, and the reaction was stirred at rt
under N2 for 30 min until effervescence ceased. (Bromomethyl)-
cyclohexane (1.5 mL, 10.75 mmol) was added slowly, and the reaction
was stirred under the same conditions for 2.3 h. Further
(bromomethyl)cyclohexane (0.5 mL) was added and the reaction
stirred under the same conditions for 1 h. Water was added to the
reaction followed by extraction with EtOAc. The organics were washed
with 10% LiCl (aq) followed by brine and dried by passing through a
hydrophobic frit. The solvent was removed in vacuo to give a pale
yellow oil which was purified using silica gel column chromatography,
eluting with a gradient of 0−100% EtOAc/cyclohexane to give 1-
(cyclohexylmethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
1H-pyrazole as a colorless oil (1.41g). LCMS (formic method)
retention time 1.27 min, [M + H]+ = 291.
Step 2. 1-(Cyclohexylmethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)-1H-pyrazole (1.41 g, 4.86 mmol) was taken up in THF (10
mL), and 2 M NaOH (4.86 mL, 9.72 mmol) was added. H2O2 (30%
w/w, 0.993 mL, 9.72 mmol) was slowly added, and the reaction was
stirred at rt under N2for 1 h. The reaction was adjusted to pH 2 using
2 M HCl (aq) and was partitioned between water and DCM. The
aqueous layer was extracted into DCM (×3), and the combined
organics were passed through a hydrophobic frit. The solvent was
removed in vacuo to give a pale yellow oil which was purified using
silica gel column chromatography, eluting with a gradient of 0−40%
EtOAc/cyclohexane to give 1-(cyclohexylmethyl)-1H-pyrazol-4-ol as a
colorless oil (893 mg). LCMS (formic method) retention time 0.81
min, [M + H]+ = 181.
and was diluted with EtOAc. The organics were washed with sat.
NaHCO3 (aq), and the aqueous layer was extracted with further
EtOAc. The combined organic layers were washed with brine, dried
using a hydrophobic frit, and concentrated. The residue was purified
using silica gel column chromatography, eluting with a gradient of 0−
30% EtOAc/cyclohexane to give 2-((1-(cyclohexylmethyl)-1H-pyr-
azol-4-yl)oxy)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one (138 mg, 0.303 mmol). LCMS (formic method)
retention time 1.43 min, [M + H]+ = 456.
Step 4. 2-((1-(Cyclohexylmethyl)-1H-pyrazol-4-yl)oxy)-3-((2-
(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]pyrimidin-4(3H)-one
(138 mg, 0.303 mmol) was taken up in nitromethane (10 mL) and the
resulting solution cooled to 0 °C. Magnesium bromide diethyl etherate
(235 mg, 0.909 mmol) was added and the reaction stirred at rt for 1 h
under N2 and then at 95 °C for 1 h. The reaction was cooled to rt and
the solvent removed in vacuo to give a yellow solid. The solid was
triturated with MeOH to give 39 (53 mg, 54%) as a yellow solid.
LCMS (formic method) retention time 0.90 min, [M + H]+ = 326.
HRMS: C17H20N5O2 requires [M + H]+ 326.1612, found 326.1609
(error −0.2 ppm). 1H NMR (400 MHz, DMSO-d6) δ ppm: 0.90−1.03
(m, 2H), 1.12−1.27 (m, 3H), 1.54 (d, J = 11.3 Hz, 2H), 1.63 (d, J =
8.6 Hz, 1H), 1.69 (d, J = 12.2 Hz, 2H), 1.83 (dtt, J = 14.8, 7.4, 7.4, 3.6,
3.6 Hz, 1H), 3.95 (d, J = 7.3 Hz, 2H), 7.60 (s, 1H), 7.88 (d, J = 5.1 Hz,
1H), 8.04 (s, 1H), 8.54 (d, J = 5.1 Hz, 1H), 8.82 (s, 1H), 13.07 (br s,
1H). 13C NMR (126 MHz, DMSO-d6) δ ppm: 162.4, 154.3, 149.4,
145.0, 143.2, 135.1, 130.7, 125.3, 122.4, 118.9, 58.4, 38.7, 30.3, 26.4,
25.6. Mp 242−244 °C.
2-((1-Phenyl-1H-pyrazol-4-yl)oxy)pyrido[3,4-d]pyrimidin-
4(3H)-one, 40. Step 1. A round-bottom flask was charged with aniline
(0.840 mL, 9.2 mmol), water (4 mL), and HCl (37%) (2 mL, 65.8
mmol). The stirred solution was cooled to 0 °C in an ice bath. To the
cold solution was added sodium nitrite (0.635 g, 9.20 mmol) in water
(5 mL), dropwise over a period of 10 min, maintaining the
temperature at 0 °C. A solution of 4-chloro-3-oxobutanoic acid
(1.44 g, 10.55 mmol) in water (5 mL) was added dropwise, also at 0
°C. Finally, a solution of sodium acetate (1.51 g, 18.41 mmol) in water
(10 mL) was added dropwise over a period of 30 min. The mixture
was warmed to rt and stirred overnight. The mixture was partitioned
with EtOAc, the layers were mixed and separated, and the aqueous
portion was re-extracted with EtOAc. The organics were combined
and passed through a hydrophobic frit and were concentrated in vacuo
to give crude (E)-1-chloro-3-(2-phenylhydrazono)propan-2-one (1.6
g) as a red solid. LCMS (formic method) retention time 0.97 min, [M
+ H]+ = 197/199.
Step 2. A round-bottom flask was charged with ground NaOH (0.82
g, 20.50 mmol) and MeOH (10 mL). The slurry was stirred at rt for
30 min prior to the portionwise addition of (E)-1-chloro-3-(2-
phenylhydrazono)propan-2-one (1.6 g, 8.14 mmol) from step 1. The
vessel was sealed and stirred at rt over the weekend. The solvent was
removed in vacuo and the residue slurried with water. The insoluble
materials were removed by filtration, and the filtrate was neutralized
with concentrated HCl. The aqueous portion was extracted with
EtOAc (×2), and the organics were combined, washed with brine, and
passed through a hydrophobic frit. Concentration of the filtrate in
vacuo gave crude 1-phenyl-1H-pyrazol-4-ol as a red solid (866 mg).
LCMS (formic method) retention time 0.69 min, [M + H]+ = 161.
Step 3. A mixture of 2-(ethylthio)-3-((2-(trimethylsilyl)ethoxy)-
methyl)pyrido[3,4-d]pyrimidin-4(3H)-one 23 (200 mg, 0.593 mmol),
THF (15 mL), and mCPBA (77% weight in water) (332 mg, 1.481
mmol) was stirred at rt for 1 h. The mixture was concentrated to half
the volume and was diluted with DCM, washed with sat. NaHCO3
(aq), dried using a hydrophobic frit, and concentrated to give an
orange oil. The oil was redissolved in 1,4-dioxane (15 mL) and was
treated with 1-phenyl-1H-pyrazol-4-ol (95 mg, 0.593 mmol) and
Cs2CO3 (579 mg, 1.778 mmol). The reaction was stirred at 60 °C
under N2 for 16 h. The reaction was cooled to rt and diluted with
EtOAc and water; the organic phase was washed with brine, dried
using a hydrophobic frit, and concentrated in vacuo to give an orange
oil. This oil was purified using silica gel column chromatography,
eluting with a gradient of 0−20% EtOAc/cyclohexane to give 2-((1-
Step 3. 2-(Ethylthio)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido-
[3,4-d]pyrimidin-4(3H)-one 23 (500 mg, 1.481 mmol) was taken up
in THF (10 mL). mCPBA (77% in water) (830 mg, 3.70 mmol) was
added, and the reaction was stirred for 1 h under N2. The reaction was
concentrated to half volume and diluted with DCM. The organics
were washed with sat. NaHCO3 (aq), dried using a hydrophobic frit,
and concentrated to give a yellow residue. The yellow residue was
taken up in 1,4-dioxane (20 mL), and 1-(cyclohexylmethyl)-1H-
pyrazol-4-ol (401 mg, 2.222 mmol) was added to the resulting
solution, followed by Cs2CO3 (1448 mg, 4.44 mmol). The reaction
was stirred at 60 °C under N2 for 16 h. The reaction was cooled to rt
P
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phenyl-1H-pyrazol-4-yl)oxy)-3-((2-(trimethylsilyl)ethoxy)methyl)-
pyrido[3,4-d]pyrimidin-4(3H)-one (89 mg, 34.5% yield) as a colorless
oil. LCMS (formic method) retention time 1.36 min, [M + H]+ = 436.
Step 4. To 2-((1-phenyl-1H-pyrazol-4-yl)oxy)-3-((2-(trimethylsilyl)-
ethoxy)methyl)pyrido[3,4-d]pyrimidin-4(3H)-one (89 mg, 0.153
mmol) and nitromethane (10 mL) was added magnesium bromide
diethyl etherate (119 mg, 0.460 mmol), and the slurry was heated to
95 °C under N2 for 1 h. The reaction was cooled to rt and
concentrated to give an orange solid. The solid was triturated with
MeOH to give 40 (22 mg, 47%) as a buff solid. LCMS (formic
Anthony Shillings, Kate Simpson, Penny Smee, Rob Tanner,
Claire Tebbutt, Laura Williams, and Huizhen Zhao for assay
reagent production, development, management, and data
generation; Robert Sheppard for molecular modeling and
SAR discussions.
ABBREVIATIONS USED
■
KDM, lysine demethylase; JMJD, Jumonji domain-containing
protein; RFMS, RapidFire mass spectrometry; HTRF, homo-
geneous time-resolved fluorescence; JARID, Jumonji AT-rich
interactive domain; 2-OG, 2-oxoglutarate; EGLN, egg-laying
deficiency protein ninelike protein; PHD, prolyl hydroxylase
domain protein; SCX, strong cation exchange; SPE, solid phase
extraction; SEM, ((trimethylsilyl)ethoxy)methyl
1
method) retention time 0.79 min, [M + H]+ = 306. H NMR (400
MHz, DMSO-d6) δ ppm: 7.35 (t, J = 7.5 Hz, 1H), 7.54 (t, J = 8.1 Hz,
2H), 7.89 (dd, J = 7.8, 1.2 Hz, 2H), 7.91 (dd, J = 5.1, 0.7 Hz, 1H), 7.99
(s, 1H), 8.57 (d, J = 5.1 Hz, 1H), 8.83 (s, 1H), 8.90 (s, 1H), 13.23 (br
s, 1H).
ASSOCIATED CONTENT
* Supporting Information
■
REFERENCES
S
■
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01538.
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LCMS spectra and selectivity profiling data for 31, 32,
36, 39, and 41; KDM4C RFMS assay data for
compounds 47−49; X-ray crystallography methods and
data for compounds 3, 4, and 33 bound to KDM4D
(PDF)
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Molecular formula strings (CSV)
Accession Codes
X-ray crystal structures have been deposited in the Protein Data
Bank as follows: compound 3 bound to KDM4D, PDB code
5FP9; compound 4 bound to KDM4D, PDB code 5FPA;
compound 33 bound to KDM4D, PDB code 5FPB.
AUTHOR INFORMATION
Corresponding Authors
*S.M.W.: e-mail, sue.m.westaway@gsk.com; telephone, +44 (0)
1438763469.
*A.G.S.P.: e-mail, alex.gs.preston@gsk.com; telephone, +44 (0)
1438790152.
■
(6) Hutchinson, S. E.; Leveridge, M. V.; Heathcote, M.; Francis, P.;
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36, 39, 46. The pIC50 data reported from the earlier KDM4C RFMS
assay format (ref 11) for compounds 6 and 9 are the mean of at least
two values with SEM ≤ 0.3. In a similar early KDM4A RFMS format
compound 4 gave pIC50 = 5.7, n = 4, SEM < 0.1.
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Present Addresses
⊥D.M.W.: Oncology Innovative Medicines and Early Develop-
ment, AstraZeneca, Cambridge Science Park, Milton Road,
Cambridge CB4 OWG, U.K.
#K.L.: Bicycle Therapeutics Limited, Meditrina Building,
Babraham Research Campus, Cambridge CB22 3AT, UK.
∇L.K.: Takeda Pharmaceuticals, 10275 Science Center Drive,
San Diego, CA 92121, U.S.
Notes
The authors declare the following competing financial
interest(s): All authors except C.J.S. were GlaxoSmithKline
and Cellzome full-time employees at the time this work was
carried out.
ACKNOWLEDGMENTS
■
The authors thank Aymeric Bencib, Sarah Chambers, Louise
Coghill, Georgia Malcolm, Michael Popadynec, Pradip Songara,
and Yuteng Wu for synthetic chemistry contributions; Iain Reid
for pKa determination; Shenaz Bunally for ChromLogD
determination; Bill Leavens for HRMS analysis; Argyrides
Argyrou, Anshu Bhardwaja, Angela Bridges, Matthew Burns,
Rachel Grimley, Michelle Heathcote, Thau Ho, Jon Hutch-
inson, Sue Hutchinson, John Martin, Lisa Miller, Linda Myers,
Kelvin Nurse, Rebecca Randle, Steve Ratcliffe, Mike Rees,
(11) pIC50 data presented for compound 5 are from an earlier version
of the optimized KDM4C RFMS assay reported in refs 4 and 6, from
which all other data in this article are presented. Analogues 6, 8, and 9
were tested in both assay formats. In the early format pIC50 values
Q
DOI: 10.1021/acs.jmedchem.5b01538
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Journal of Medicinal Chemistry
Article
were <4.0, 4.8, and 4.3, respectively and these data are similar to those
reported in Table 2 for the optimized format where pIC50 values were
<4.0, 4.6, and 4.3, respectively.
(12) Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi,
T.; Okada, M.; Ohta, M.; Tsukamoto, S.; Parker, P.; Workman, P.;
Waterfield, M. Synthesis and biological evaluation of 4-morpholino-2-
phenylquinazolines and related derivatives as novel PI3 kinase p110α
inhibitors. Bioorg. Med. Chem. 2006, 14, 6847−6858.
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quinazolines as PDE10 inhibitors. U.S. Pat. Appl. Publ.
US20110319409, 2011.
(14) This work was originally disclosed as part of an oral
presentation: Westaway, S. M. Novel cell active inhibitors of histone
lysine demethylases (KDMs): Progress and challenges in the discovery
of tool inhibitors of JumonjiD2 (JmjD2) and JumonjiD3 (JmjD3).
Abstracts of Papers, 246th National Meeting and Exposition of the
American Chemical Society, Indianapolis, IN, U.S., September 8−12,
2013; American Chemical Society: Washington, DC, 2013; MEDI-26.
Compounds 21, 24, 25, 29, 35, 36, and 41 have also been disclosed as
KDM inhibitors in the following patent application: Kanouni, T.;
Stafford, J. A.; Veal, J. M.; Wallace, M. B. Histone demethylase
inhibitors. PCT Int. Appl. WO2014151106, 2014.
(15) Joberty, G.; Boesche, M.; Brown, J. A.; Eberhard, D.; Garton, N.
S.; Muelbaier, M.; Ramsden, N.; Reader, V.; Rueger, A.; Sheppard, R.
J.; Westaway, S. M.; Bantscheff, M.; Lee, K.; Wilson, D. M.; Prinjha, R.
K.; Drewes, G. Interrogating the druggability of the 2-oxoglutarate-
dependent dioxygenase target class by chemical proteomics.
Unpublished results, 2015.
R
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