Design and Synthesis of Novel Epigenetic Inhibitors Targeting Histone Deacetylases, DNA Methyltransferase 1, and Lysine Methyltransferase G9a with in Vivo Efficacy in Multiple Myeloma

Article abstract of DOI:10.1021/acs.jmedchem.0c02255

Concomitant inhibition of key epigenetic pathways involved in silencing tumor suppressor genes has been recognized as a promising strategy for cancer therapy. Herein, we report a first-in-class series of quinoline-based analogues that simultaneously inhibit histone deacetylases (from a low nanomolar range) and DNA methyltransferase-1 (from a mid-nanomolar range, IC50 < 200 nM). Additionally, lysine methyltransferase G9a inhibitory activity is achieved (from a low nanomolar range) by introduction of a key lysine mimic group at the 7-position of the quinoline ring. The corresponding epigenetic functional cellular responses are observed: Histone-3 acetylation, DNA hypomethylation, and decreased histone-3 methylation at lysine-9. These chemical probes, multitarget epigenetic inhibitors, were validated against the multiple myeloma cell line MM1.S, demonstrating promising in vitro activity of 12a (CM-444) with GI50 of 32 nM, an adequate therapeutic window (>1 log unit), and a suitable pharmacokinetic profile. In vivo, 12a achieved significant antitumor efficacy in a xenograft mouse model of human multiple myeloma.

Full text of DOI:10.1021/acs.jmedchem.0c02255

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Article
Design and Synthesis of Novel Epigenetic Inhibitors Targeting
Histone Deacetylases, DNA Methyltransferase 1, and Lysine
Methyltransferase G9a with In Vivo Efficacy in Multiple Myeloma
Obdulia Rabal, Edurne San José-Enériz, Xabier Agirre, Juan Antonio Sánchez-Arias, Irene de Miguel,
Raquel Ordonez, Leire Garate, Estíbaliz Miranda, Elena Sáez, Amaia Vilas-Zornoza,
̃
Antonio Pineda-Lucena, Ander Estella, Feifei Zhang, Wei Wu, Musheng Xu, Felipe Prosper,*
and Julen Oyarzabal*
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ABSTRACT: Concomitant inhibition of key epigenetic pathways involved in silencing tumor suppressor genes has been recognized
as a promising strategy for cancer therapy. Herein, we report a first-in-class series of quinoline-based analogues that simultaneously
inhibit histone deacetylases (from a low nanomolar range) and DNA methyltransferase-1 (from a mid-nanomolar range, IC50 < 200
nM). Additionally, lysine methyltransferase G9a inhibitory activity is achieved (from a low nanomolar range) by introduction of a
key lysine mimic group at the 7-position of the quinoline ring. The corresponding epigenetic functional cellular responses are
observed: histone-3 acetylation, DNA hypomethylation, and decreased histone-3 methylation at lysine-9. These chemical probes,
multitarget epigenetic inhibitors, were validated against the multiple myeloma cell line MM1.S, demonstrating promising in vitro
activity of 12a (CM-444) with GI50 of 32 nM, an adequate therapeutic window (>1 log unit), and a suitable pharmacokinetic profile.
In vivo, 12a achieved significant antitumor efficacy in a xenograft mouse model of human multiple myeloma.
analogues that, following conversion to the triphosphate,
incorporate into the DNA and covalently bind to DNMTs.
Such non-specific mechanism of action translates into
significant toxicity limiting the dose and efficacy. Despite
recent progress in the development of non-nucleoside-based
DNMTi binding to the catalytic site of DNMTs, none has
entered into clinical phases.2 For HDACs, the pipeline is more
advanced: a considerable number of potent HDAC inhibitors
(HDACi) have been discovered; the pharmacophore pattern
to inhibit them is well-established and five HDACi have been
INTRODUCTION
■
Epigenetic modifications play an important role in the
regulation of gene expression and transcription and are
implicated in cancer and many other diseases. Due to the
reversibility of epigenetic alterations, there has consequently
been a focus upon directing probe and drug discovery efforts
toward the identification of novel antitumor targets. Among
the many epigenetic protein families, DNA methyltransferases
(DNMTs) and histone deacetylases (HDACs), enzymes that
respectively add methylation marks to DNA and erase
acetylation marks from histones, were strongly implicated in
cancer through extensive studies of their biological mecha-
nisms and target validation via epigenetic medicinal chemistry.
As a result, both are molecular targets of FDA-approved drugs
for the treatment of hematologic malignancies, with a
considerable number of active clinical trials and many
inhibitors under development.1 Approved DNMT inhibitors
(DNMTi), Azacitidine and Decitabine, are nucleoside
Received: December 29, 2020
Published: March 4, 2021
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Chart 1. pan-HDACi (1,30 2,31 332), Dual DNMT, and HDACi (4),19 Dual G9a and DNMT1 Inhibitors (5, 6),21 G9a Inhibitor
(7)24 and Dual G9a and HDACi (8);23 IC50 Values for 4 Were Determined Internally (See Footnote in Table 1) and the Rest
of the Biochemical IC50 Values Extracted from the Corresponding References; for 8, the Enzymatic Activity of HDAC Was
Measured in Intact Cells by the Homogeneous Cellular HDAC Assay Method Using the K562 Cell Line and the Ability to
Block G9a by H3K9me2 Cell Immunofluorescence In-Cell Western (ICW) Assays Using the MDA-MB-231 Cell Line23
approved: SAHA (Vorinostat, 1), Belinostat, Panobinostat
(LBH-589, 2), Romidepsin (Depsipeptide), and Chidamide,3,4
the latter in China only. The clinical development of HDACi is
hampered by dose-limiting toxicity, which can lead to poor
single agent efficacy. Given that combination regimens are the
mainstay of modern cancer therapy to achieve optimal clinical
efficacy and the interplay between DNA methylation and
histone deacetylation,5,6 combination studies of HDACi and
DNMTi have been pursued, which demonstrate a synergistic in
vitro and in vivo antitumor activity and the induction of tumor
suppressor genes (TSGs).6−11 As additive toxicity is often
observed with combination therapy,12 dual DNMT and
HDACi may overcome this hurdle.13
Only a few compounds have been reported having this dual
DNMT/HDAC inhibitory profile: psammaplins14 (although
its DNMT inhibitory activity is controversial15) and related
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Figure 1. Focused exploration around three diversity points (R1, R2, and R3) of the quinoline ring to achieve first-in-class multitarget epigenetic
inhibitors targeting DNMT1, HDAC, and, optionally, G9asome examples are illustrated.
analogues,16 (−)-epigallocatechin-3-gallate,17 DC-517,18 and
NSC-319745-based hydroxamic acid derivatives,19 these last
being claimed as the first de novo-designed dual DNMT−
HDAC molecules. However, as exemplified with the advanced
compound 4 in Chart 1, the NSC-319745-based chemical
series still lacks potent DNMT1 inhibitory activity (e.g., <70%
inhibition at 100 μM). In the same line, DC-517-based
analogues (e.g., C02S) have IC50 values in the low micromolar
range against DNMTs and HDAC1.18
Based on the well-established pharmacophore of HDACi
and structural information available, incorporation of HDAC
inhibitory activity to our chemical series seemed initially
achievable considering the predicted binding mode of
compounds 5 and 6 at the substrate-binding site of both
methyltransferases; then, to achieve this goal, we explored
around R2 (Figure 1). Moreover, owing to the structural
knowledge gained from the SAR exploration of our lead
compounds 5 and 6 and the optimization of 7 toward more
potent G9a inhibitors,24,25 we hypothesized that the inhibitory
activity against G9a might be compatible with DNMT1 and
HDAC inhibition to yield a triple epigenetic inhibitor;
therefore, an exploration around R3 was also performed
(Figure 1).
Thus, using knowledge- and structure-based approaches, we
designed a synthetically feasible focused exploration around
the quinoline scaffold; the synthetic approach enables to
explore three diversity points (Figure 1) that are key for
primary activities. This exploration led to first-in-class
multitarget epigenetic inhibitors targeting G9a, DNMT1, and
HDAC. Herein, we report the discovery of chemical probes
(e.g., 9a and 12a) that confirm the feasibility of the proposed
multitarget epigenetic inhibition, which was not only validated
by biochemical assays but also by their corresponding
functional cellular responses: activities versus these epigenetic
targets, in both validation scenarios, range from nM to low μM
(up to ∼1−2 μM). In addition, this initial exploration led to
basic SAR guidelines to achieve these two target compound
profiles, (a) G9a, DNMT1, and HDAC inhibition as well as
(b) DNMT1 and HDAC inhibition, and to identify absorption,
distribution, metabolism, and excretion (ADME) properties
that may require an optimization (e.g., permeability and
solubility). From a drug discovery perspective, these chemical
probes are key starting points to become optimized lead
compounds according to project requirements (e.g., specific
inhibition profile for selected targets, therapeutic window,
ADME properties, etc.), fulfilling the corresponding target
product profile.
Here, considering polypharmacology as an attractive strategy
to overcome the main limitations of the single target therapy
leading to a superior therapeutic effect and a reduction of
potential mechanism(s) of drug resistance,20 we set out with
the goal of designing novel molecules able to inhibit DNMT
and HDAC simultaneously. The chemical structures of our
proprietary non-nucleoside epigenetic inhibitors CM-272 (5)
and CM-579 (6) served as starting points because of their
good potency against DNMT1 (IC50 of 382 nM and 32 nM,
respectively, Chart 1) and because they reversibly bind at the
substrate-binding site.21 Of note, these compounds display also
nanomolar inhibitory activity against G9a (EHMT2), a histone
methyltransferase responsible for the mono- and di-methyl-
ation of the lysine 9 of the histone 3 (H3K9).21,22 We
postulated that combined H3K9 hypomethylation and hyper-
acetylation as a result of concurrent inhibition of G9a and
HDAC should additionally contribute to relieve transcriptional
repression of TSG in cancer. In fact, the anticancer response of
dual G9a/HDAC inhibitory profile has also been examined for
a series of quinazoline derivatives of BIX-01294 (7),
exemplified by compound 8 in Chart 1.23 Therefore, achieving
first-in-class molecules as DNMT1, HDAC and, optionally,
G9a inhibitors targeting two or three different epigenetic marks
simultaneously should expectedly result in chemical probes to
validate the feasibility of the proposed multitarget epigenetic
inhibition (dual and triple inhibition, IC50 < 10 μM against
each target of interest) as well as to study epigenetic targets
from a mechanistic perspective (e.g., role in gene transcription)
and to analyze their phenotypic and antiproliferative responses.
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Table 1. Hybrid Compounds with Triple HDAC, DNMT1, and G9a Inhibitory Activity: Exploring Around R2 and R3
a
Positions
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Table 1. continued
a
For data in Tables 1−3 and Chart 1, all IC50 values are the average of at least two independent replicates performed at different days. If the
absolute pIC50 difference was higher than 0.3 log units, additional measurements were performed until satisfying the experimental error (by
discarding individual results with values outside 2 MADs of the mean value). Standard Deviations are reported together with the corresponding
b
IC50 values in brackets and in italics. N.D. = not determined. 1 (reference pan-HDACi),30 5 (dual G9a and DNMT1 inhibitor, potent G9a
inhibition),21 and 6 (dual G9a and DNMT1 inhibitor, potent DNMT1 inhibition)21 are utilized as positive controls for all biochemical assays
reported in Tables 1−3. Synthetic approaches for all new molecules are shown in Scheme 1 (compounds 9a−k), Scheme 2 (10a−b) and Scheme 3
(11).
Figure 2. Predicted complex of compound 9a with G9a [(A) PDB accession code: 3RJW],25 DNMT1 [(B) PDB accession code: 4DA4],36
HDAC1 [(C) PDB accession code: 4BKX]37 and HDAC6 [(D) PDB accession code: 5EDU]38experimental validation of proposed binding
modes will be experimentally performed by biophysical methods in due course.
As mentioned above, DNMT1 and HDAC are molecular
targets of FDA-approved drugs for the treatment of
hematologic malignancies; however, multiple myeloma
(MM) still remains incurable.26 Despite approval by the
FDA and the EMA of the pan-HDACi 2 in combination with
bortezomib and deixamethasone for the treatment of relapsed
or refractory MM,27 this is still an unmet medical need. Taking
into account that not only histone 3 (H3) acetylation is altered
their corresponding functional cellular responses: histone-3
acetylation, DNA hypomethylation, and decreased histone-3
methylation at lysine-9. This exploration also led to the
identification of compound 12a (CM-444) as a pharmaco-
logical tool compound: suboptimal but adequate ADME and
pharmacokinetic (PK) profiles as well as therapeutic window
to perform an in vivo proof of concept. In vivo efficacy of 12a
was assessed in a xenograft mouse model of human MM.
in MM patients but also the DNA methylation pattern,28,29
a
RESULTS
combination of HDAC and DNMT1 inhibition may be an
adequate therapeutic strategy. In fact, very recently it was
shown that a combination of azacitine and BG45 (HDACi)
exhibited synergistic cell growth inhibition of MM tumor cells
and induced cell growth inhibition in in vivo models.6
Thus, the antiproliferative response of these chemical probes
was tested against MM tumor cells MM1.S. Those molecules
with a potent antiproliferative response (GI50), 9a as a triple
inhibitor and 12a as a dual inhibitor, were selected to monitor
■
Rational Design of Hybrid Compounds with HDAC,
DNMT1, and G9a Inhibitory Activity. The classical
pharmacophore for HDAC inhibition consists of a (i) a zinc-
binding group (ZBG) that chelates the zinc ion in the active
site, (ii) a recognition capping group that interacts with the rim
of the catalytic tunnel, and (iii) a hydrophobic linker
connecting the ZBG and the cap group. This pharmacophore
pattern has been successfully exploited to derive a plethora of
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a
Table 2. Hybrid Compounds with Dual DNMT1 and HDAC Activity: Exploration of the 4 Position
a
N.D. = not determined. A corresponding synthetic approach is shown in Scheme 4 (compounds 12a−k).
novel multifunctional HDACi compounds, most of them
bearing a hydroxamic group as the ZBG.10,33 Compounds 5
and 6 are substrate competitive inhibitors against G9a and
DNMTs, with predicted binding modes established by docking
and consistent with SAR.21,34,35 Examination of these protein−
ligand complex models suggested that linking a hydroxamic
moiety to the methylpiperidine (position 4 of the quinoline
ring; R2 in Figure 1) would project this ZBG substituent
toward the solvent exposed area of both, G9a and DNMT, and
be well tolerated from a potency perspective. From the
viewpoint of HDAC inhibitory activity, the quinoline core of 5
and 6 would serve as a cap group. Following this rationale, and
owing to its chemical similarity, the pyrimidylhydroxamic acid
of quisinostat (3) was incorporated into 6 to yield compound
9a (Table 1 and synthesis in the Chemistry Section). As shown
in Figure 2, this compound retains the binding mode of the
parent compound into G9a and DNMT1 and fits predictably
well into the binding site cavities of HDAC1 and HDAC6.
These two HDAC isoforms were chosen for routinely
biochemical screening as representative of class I (HDAC1)
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and class IIb (HDAC6) HDAC isoforms. In fact, compound
9a is a potent HDACi, with a preference for class I (HDAC1,
HDAC2, and HDAC3 IC50 of 3, 21, and 5 nM) over class IIb
isoform (HDAC6 IC50 of 152 nM). For the G9a and DNMT1
profiles, a significant drop in activity was observed compared to
the parent compound 6 although 9a satisfies our initial
requirement of targeting the three epigenetic families
(respective G9a and DNMT1 IC50 values of 269 and 1160
nM, compared to 16 and 32 nM for 6). On this basis, various
analogues with different linker rings with varying lengths,
hydrophobicities, and flexibilities were synthesized (com-
pounds 9b−9k, Table 1). In general, flexible alkyl rings (9c)
and methylene-homologated rings (9d, 9e) yielded com-
pounds with improved G9a (IC50 < 100 nM), DNMT1 (IC50
< 500 nM) and HDAC6 (IC50 < 100 nM) potencies compared
to that of compound 9a, although at the cost of losing at least
one log unit of HDAC1 activity. Conserving the basic nitrogen
of the piperidine ring confers the highest potency against G9a
(9d, 9e, and 9g),34,35 with the striking exception of the
cyclohexyl moiety of 9c (G9a IC50 of 55 nM), the triple
inhibitor compound in Table 1 with the most promising well-
balanced biochemical profile against all targets. Substitution of
the 5-methyl-2-furyl group of the initial hit 9a by methyl to
yield 10a was beneficial for G9a and DNMT activities while
retaining a similar HDAC inhibitory profile to that of 9a.
Finally, compound 11 was designed in an attempt to increase
the DNMT1 potency of 9a by incorporation of a 4-piperidyl
moiety at the 7 position.35 As this replacement of the 7-(3-
pyrrolidin-1-ylpropoxy) group did not meet the expected
improvement, SAR exploration of the 7 position (R3, Figure 1)
with alternative groups with basic nitrogens was no longer
continued.
group of 12a, its azabicyclo analogue 12c, and the cyclohexyl
linker (12e) were among the best compounds in Table 2 (their
IC50 vs DNMT1 are ∼1−3 μM).
Finally, a small exploration of the 2-position of the quinoline
(R1, Figure 1) was carried out by keeping constant the initial
pyrimidylhydroxamic group of 12a and replacing its heteroaryl
ring, 5-methyl-2-furyl, by a methyl (13a), cyclohexyl (13b),
phenyl (13c), N-linked piperidine (13d), 2,5-dimethyl-3-furyl
(13e), and 5-methylthiophen-2-yl (13f) (Table 3). However,
Table 3. Hybrid Compounds: Exploration of the 2 Position
a
(R1)
Hybrid Compounds with Dual HDAC and DNMT1
Inhibitory Activity. During the course of our SAR
exploration around compounds 5 and 6, it was noticed that
removal of the basic nitrogen at position 7 of the quinoline
scaffold (R3, Figure 1) was detrimental for G9a activity.35 This
is consistent with the predicted role of the 7-(3-pyrrolidin-1-
ylpropoxy) group mimicking the lysine side chain (Figure 2A).
Similar conclusions were drawn during the optimization of 725
that can be explained on the basis of missing key interactions
with Leu1086 (hydrogen-bond) and Tyr1154 (cation−π
interaction) (Figure 2A). The impact of replacing this lysine
mimic group by a methoxy group was initially less impacting
on DNMT1 activity for the quinoline analogues.35 With the
goal of blocking G9a inhibitory activity, a handful of 7-
methoxyquinolines with the optimal linkers from previous SAR
explorations were prepared (12a, 12d, 12e, 12g, Table 2), as
well as some additional linkers designed to constrain the
conformation of the hydroxamic moiety (12b and 12j, Table
a
Corresponding synthetic approach is shown in Scheme 5
(compounds 13a−f).
compared to 12a, this exploration had a minor impact on the
HDAC profile (HDAC1 IC50 values are ∼10 nM); all these
substitutions were detrimental to DNMT1 inhibition: IC50
values > 10 μM for all these new molecules are reported in
Table 3, resulting in HDAC inhibition alone.
These initial explorations around these three diversity points
(R1, R2, and R3) borne by the quinoline scaffold, represented
in Figure 1 and exemplified in Tables 1−3, led to some key
general conclusions; a preliminary SAR guideline:
2). As expected, all of them were inactive against G9a (IC50
>
10 μM). DNMT1 activity was less affected, spanning mid-
nanomolar (again, for the flexible methylene-homologated
rings of 12g and 12h) to low-micromolar ranges. For HDACs,
inhibitory profiles between corresponding matched pairs in
Table 1 are in general consistent (e.g., 12a vs 9a, 12d vs 9b
and 12e vs 9c), highlighting the minimal impact of the
methoxy group moiety at the rim surface of the HDACs.
Considering a well-balanced contribution of the primary
targets, compounds 12g and 12h had an adequate profile (IC50
∼ 300 nM vs DNMT1 and HDAC1; and, ∼100 nM against
HDAC6). On the other hand, giving special emphasis to
HDAC1 inhibition (IC50 < 50 nM), the pyrimidylhydroxamic
• Substituents at R1, position 2 of the quinoline ring, have
a huge impact on DNMT1 activity; in fact, among all
explored analogues (Table 3), only the 5-methy-2-furyl
(12a) was able to maintain a capacity of inhibition (IC50
< 10 μM).
• The classical pharmacophore for HDAC inhibition
(containing a ZBG, e.g., hydroxamic acid33,39 or ortho-
aminoanilides40,41) should be located at R2, position 4
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molecules the IC50 values of which are >1 μM for more than
one target of interest (compounds 12j-trans and 12j-cis).
Exploration around the R1 position (Figure 1) only led to
HDACi lack of DNMT1 inhibition (IC50 values > 10 μM).
Then, for comparison purposes, only the most potent
compound from Table 3 progressed to the cellular assay
against MM1.S: 13b.
of the quinoline ring, thus causing minimal impact on
G9a and DNMT1 inhibition (solvent exposed according
to the proposed binding modes) and, on the other hand,
quinoline serves as the cap group for binding to
HDACas described in Figure 2. This substitution
pattern led to potent inhibition of HDAC enzymatic
activity (Tables 1−3)
As seen in Table 4, triple inhibitors exhibited GI50 values in
the low micromolar range (9a−i) comparable to that achieved
by G9a-DNMT1 inhibitors 5 and 6 or were inactive below 10
μM (10a−b,11) possibly due to their poor PAMPA
permeability (Pe ∼ <1 nm/s). Dual HDAC-DNMT1
compounds (12a−12g) with reduced molecular weight and
slightly higher permeability (although still poor permeators,
with Pe < 10 nm/s) mostly tended to show GI50 < 1 μM.
Among them, compound 12a achieved the most potent
antiproliferative response (GI50 = 32 nM) close to the
reference compound 2 (panobinostat, GI50 = ∼ 9 nM), ∼0.5
log units difference, and with decreased toxicity against the
healthy hepatic cell line THLE-2 (LC50 794 vs 22 nM); in fact,
the therapeutic window of compound 12a (1.4 log units) is 3
times larger than the corresponding therapeutic window of 2
(0.4 log units)Table 4. Furthermore, a pairwise comparison
between 12a and the most potent HDAC selective inhibitor of
this chemical series (13b, Table 3) shows that dual inhibition
leads to a more potent antiproliferative activity against MM1.S
(>0.75 log units difference) and a better therapeutic window
(Table 4). On the other hand, we should also highlight that
12a is >1.5 log units more potent against MM1.S than
reference dual G9a and DNMT1 inhibitors (compounds 5 and
6).
• The 7-(3-pyrrolidin-1-ylpropoxy) group mimics the
lysine side chain; then, as previously reported by our
group,35 removal of the basic nitrogen at position 7 of
the quinoline scaffold (located at R3) was detrimental
for G9a activity. Thus, as illustrated in Table 1 versus
Table 2, we can easily modulate the G9a inhibitory
activity.
Finally, we also explored an alternative scaffold to the
quinoline ring: quinazoline. Interestingly, the corresponding
quinazoline-based pair of the selected compound 12a (details
below), compound 14 (Chart 2 and synthesis in the Scheme
Chart 2. Selected compound 12a and its corresponding
quinazoline-matched pair 14.
When tested against other MM cell lines (JJN3, KMS28BM,
and H929), compound 12a also showed good antiproliferative
activity with GI50 values between 100 and 550 nM. 9a, as
representative of the triple inhibitors (described below), which
was also tested versus these cell lines and showed more modest
activities; in fact, GI50 values are between 1 and 6 μM (Table
5).
Finally, in order to assess the functional cellular response of
inhibiting these epigenetic targets (Tables 1 and 2), two
chemical probes were selected as representatives: 9a, as G9a,
DNMT1 and HDACi; and, 12a, as DNMT1 and HDACi. In
fact, 12a is the most potent molecule among dual inhibitors
versus MM1.S. On the other hand, 9a and 9i are the most
potent chemical probes among triple inhibitors; but, because
9a is >0.9 log units more potent versus G9a than 9i, 9a was
selected as the representative triple inhibitor to monitor its
corresponding functional hallmarks. Further biochemical
profiling versus two DNMT and seven HDAC additional
isoforms was also performed (described in Table S1,
Supporting Information) for these two selected chemical
probes, 9a and 12a. These results suggest that inhibition of
these DNMT and HDAC additional isoforms may contribute
to antiproliferative efficacy against MM1.S cell line as well as to
monitor cellular functional responses.
The global levels of histone-3 lysine-9 acetylation (H3K9Ac)
and H3K9me2 hallmarks were monitored by Western Blot in
MM1.S cells after 48 h of exposure of the selected compounds
in a concentration-dependent manner (Figure 3A) as well as in
comparison with the reference compounds for each target:
decitabine as DNMTi,2 panobinostat as HDACi3,4 and A-366
as G9a inhibitor42 (Figure S1 in the Supporting Information).
As shown in Figure 3A, both compounds led to an increase in
6), does not exhibit DNMT1 activity (IC50 > 10 μM), thereby
indicating the impact of the quinoline scaffold on the DNMT1
activityas previously reported for G9a and DNMT1
inhibitors.34
Cellular Response: Antiproliferative Effect and Func-
tional Hallmarks. New molecules fulfilling target compound
profiles, (a) G9a, DNMT1, and HDAC inhibition or (b)
DNMT1 and HDAC inhibition (Tables 1 and 2), and covering
diversity in terms of chemical space and biological responses
were selected to test their antiproliferative activity using human
cancer cells of MM, cell line MM1.S. From Table 1, only four
triple inhibitors did not progress to the cellular assay against
MM1.S: those chemical probes which IC50 values are >1 μM
for more than one target of interest (compounds 9g, 9h, 9j-
trans and 9j-cis); 9i and 11 were exceptions, diversity in
chemical space, and were tested. From Table 2, we also
discarded four double inhibitors: compounds the IC50 values of
which are >8 μM versus DNMT1 (12f and 12k) and those
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a
Scheme 1. Synthesis of Compounds 9a−k
a
Conditions: (i) 4,4,5,5-tetramethyl-2-(5-methyl-2-furyl)-1,3,2-dioxaborolane, Na2CO3, Pd(PPh3)4, 1,4-dioxane/H2O (15:1), 110 °C, MW, 4 h;
(ii) corresponding amine, Pd2(dba)3, BINAP, Cs2CO3, 1,4-dioxane, 110−120 °C, 16 h; (iii) HCl/EtOAc(2.0 M) or HCl/MeOH (2.0 M), 20 °C,
30 min to 16 h; (iv) ethyl 4-oxocyclohexanecarboxylate, ZnCl2/diethyl ether, NaBH3CN, MeOH, 40 °C, 15.5 h; (v) LiOH·H2O, EtOH/H2O (2:1)
or THF/MeOH/H2O (5:1:3 or 3:1:1), 20−25 °C, 1−16 h; (vi) THPONH2, HOBt, DIEA, EDCI, DMF, 20 °C, 2−16 h.
H3K9Ac at concentrations above 500 nM (12a) and 1 μM
(9a), such as the control compound panobinostat (Figure S1
in the Supporting Information). The higher dose required for
compound 9a might be due to its poorer permeability, given
that both compounds are equally active against HDAC1,
HDAC2, and HDAC3. In line with the lack of biochemical
activity of 12a against G9a (as reported in Table 2), no
alteration of H3K9me2 mark was detected, while this mark was
significantly reduced after treatment with doses of compound
9a above 1 μM (Figure 3A), such as the reference compound
A-366 (Figure S1 in the Supporting Information).
On the other hand, DNA methylation alteration was
monitored in MM1.S cells by pyrosequencing analysis of
several CpGs located in the promoter region of the POU4F2
gene, which is differentially methylated in MM and involved in
the TP53 pathway deregulation. As previously reported,34
doses below their IC50 values and long incubation times of
treating every day were used to avoid cell killing and thus to be
able to monitor their impact on DNA methylation; in this case,
9a was tested at 2 μM and 12a at 10 nM were used for 5 days.
Pyrosequencing analysis was focused on the promoter area
POU4F2 (Figure 3B) where treatment with compound 9a
clearly leads to hypomethylation, ∼15%, of the CpG position
“3” and incubation with 12a hypomethylates CpG positions
“4” and “5” in ∼10% each. This DNA hypomethylating activity,
monitored by POU4F2 pyrosequencing, was also observed in
the JJN.3 cell line treated with 9a (at 1 μM) and 12a (at 100
nM) for 5 days (Figure S2 in the Supporting Information).
In Vivo Antitumoral Efficacy in MM. As shown in Table
4, compound 12a exhibited a potent antiproliferative response
against the MM1.S cell line (GI50 = 32 nM) and an acceptable
therapeutic window (>1 log unit); thus, this molecule was
selected to test the efficacy in an in vivo xenogeneic mouse
model. Compound 9a, the selected triple inhibitor, exhibited a
moderate antiproliferative response (GI50 ∼ 2 μM) as well as
therapeutic window (<1 log unit), an identical profile to 9i. A
preliminary PK study was performed for 9a, but the mice (n =
5) died 10 min after its administration at 10 mg/kg (i.p.).
Then, the dose was reduced to 5 mg/kg (i.p.); but, 15 min
after administration of 9a, during the blood extraction process,
the mice (n = 5) also died (data not shown). Thus, any efforts
for in vivo testing of 9a were discontinued.
Before the in vivo efficacy test, the corresponding PK study
of the compound 12a was done; in addition, a preliminary
ADME profile was also performed (Table 6). This ADME data
show that 12a inhibits two P450 isoforms (1A2 and 3A4) by
more than 50% at 10 μM; then, P450 inhibition should be
taken into account during the optimization process. Moreover,
the reported ADME profiling highlights two key critical
aspects: (i) 12a metabolic stability, in human and mouse
cryopreserved hepatocytes (≤50% remaining after 60 min,
respectively), is not optimal and (ii) its solubility is poor
(below the limit of quantification), <0.987 μg/mL.
These two factors led to suboptimal PKs. The low metabolic
stability in hepatocytes translated in a high clearance and a
reduced half-life when administered to BALB/c-RAG2‑/‑γc‑/‑
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a
Scheme 2. Synthesis of Compounds 10a−b
a
Conditions: (i) methyl boronic acid, K2CO3, Pd(PPh3)4, 1,4-dioxane/H2O (10:1), 110 °C, MW, 3 h; (ii) corresponding amine, t-BuONa,
xantphos, Pd2(dba)3, toluene, 110 °C, MW, 1−2 h; (iii) HCl/1,4-dioxane (4.0 M), rt, 1−3 h; (iv) ethyl 2-chloropyrimidine-5-carboxylate, K2CO3,
CH3CN, rt, 3 h; (v) LiOH·H2O, THF/MeOH/H2O (10:1:3), rt, overnight; (vi) THPONH2, HOBt, NMM, EDC·HCl, DMF, rt, overnight; (vii)
HCl/1,4-dioxane (4.0 M), rt, 1 h.
mice for PK profiling (Table 7; details are described in the
Supporting Information, Tables S2 and S3); in addition, the
poor solubility of compound 12a also precludes its use in an in
vivo efficacy assay when resuspended in a 100% saline solution.
However, if the vehicle is not only a saline solution but also
DMSO and Tween 20 (10% each) then, 12a revealed an
acceptable profile with an adequate half-life (∼8.5 h) and an
acceptable exposure (AUC0−24h = 916.15) to achieve in vivo
efficacy versus the MM1.S cell line with GI50 value of 32 nM.
Compared with the PK profile of 12a using the 100% saline
solution, the formulation containing DMSO and Tween 20
leads to a half-life that is twice longer as well as to an exposure
1.5 times higher. Additional medicinal chemistry efforts are
required to optimize this molecule and should focus, among
others, on three critical factors: metabolic stability, solubility,
and permeability (its PAMPA, Table 4, is also poor).
compound was utilized as a pharmacological tool compound
to test the in vivo efficacy and its mechanism of action (dual
DNMT and HDAC inhibition) in terms of tumor growth in a
mouse model using human MM xenografts, one of the most
widely used models for the evaluation of in vivo efficacy of
anticancer drugs.43
The mouse model showed that tumor growth is prevented
using this inhibitor 12a in tumors induced by subcutaneously
injecting 10 × 106 MM1.S cells in BALB/c-RAG2‑/‑γc‑/‑ mice,
a model previously used in several studies.44 The treatment
with compound 12a started when all mice presented tumors 12
days after myeloma cell inoculation. These mice were then
treated with 10 mg/kg (i.p.) of compound 12a administered
daily during 5 consecutive days followed by 2 rest days and
sacrificed at day 35 after cell inoculation. The mice were
controlled for signs of morbidity (behavior and body weight
loss), not observing differences in the body weight of the
animals (Figure S3 in the Supporting Information), and the
Given the acceptable PK profile of 12a, using saline together
with 10% DMSO and 10% Tween 20 as vehicle, this
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a
Scheme 3. Synthesis of Compound 11
a
Conditions: (i) POCl3, malonic acid, 95 °C, 12 h; (ii) tert-butyl 4-(bromomethyl)piperidine-1-carboxylate, Cs2CO3, DMF, 80 °C, 16 h; (iii)
4,4,5,5-tetramethyl-2-(5-methyl-2-furyl)-1,3,2-dioxaborolane, Pd(PPh3)4, K2CO3, 1,4-dioxane/H2O (4:1), 90 °C, 16 h; (iv) ethyl 2-[4-
(aminomethyl)-1-piperidyl]pyrimidine-5-carboxylate, Pd2(dba)3, BINAP, Cs2CO3, 1,4-dioxane, 110 °C, 16 h; (v) LiOH·H2O, THF/H2O (2:1),
15 °C, 16 h; (vi) THPONH2, HOBt, DIEA, EDCI, DMF, 15 °C, 16 h; (vii) HCl/EtOAc (1.0 M), 15 °C, 16 h.
tumor volume was monitored every 3−5 days. As shown in
Figure 4, treatment with 12a produced a significant (p value <
0.05) overall tumor growth inhibition, average tumor volumes
at day 35 of 1788 1164 and 3836 1696 mm3 for treated
and control groups, respectively.
as well as DNA methylation levels; assay concentrations, for
both chemical probes, ranged from nanomolars to low
micromolars (up to ∼1−2 μM).
Taking into account that MM remains still incurable26 and
that different epigenetic layers are altered in MM patients,28,29
a combination of HDAC and DNMT1 inhibition may be an
adequate therapeutic strategy. Thus, those novel multitarget
inhibitors showing potent double and triple inhibitory activities
were tested versus the MM1.S cell line and 12a showed a
potent antiproliferative activity (GI50 is 32 nM). Furthermore,
pairwise comparisons between molecules of this chemical
series, for example, reference G9a and DNMT1 inhibitors (5
and 6) as well as the most potent HDAC selective inhibitor of
this chemical series (13b) versus 12a, show that dual DNMT
and HDAC inhibition leads to more potent antiproliferative
activity against MM1.S.
DISCUSSION AND CONCLUSIONS
■
We presented a detailed account of knowledge- and structure-
based design of first-in-class multitarget epigenetic inhibitors
targeting DNMT1, HDAC and, optionally, G9a. Synthetic
approaches, schemes described below, enabled to explore three
diversity points around the quinoline scaffold (Figure 1).
These explorations not only identified the impact of each
substitution pattern but also the role of each of the growing
vectors (R1, R2, and R3) on different epigenetic targets
(preliminary SAR described above). In addition, these focused
explorations led to chemical probes fulfilling our initial
objective (dual and triple inhibition, IC50 < 10 μM against
each target of interest) as well as to other compounds that only
inhibit HDACs (IC50 values for DNMT1 and G9a are >10
μM). Furthermore, replacement of the quinoline scaffold by
quinazoline eliminates DNMT1 inhibitory activity (IC50 > 10
μM), thereby validating the quinoline ring as the central core
for the achievement of this multitarget epigenetic inhibition.
Two chemical probes fulfilling the established target
compound profiles, (i) 9a as triple inhibitor (G9a, DNMT1
and HDAC) and (ii) 12a as double inhibitor (DNMT1 and
HDAC), were selected to validate their functional cellular
responses and clearly showed the impact on their correspond-
ing epigenetic marks: global levels of H3K9Ac and H3K9me2
Compound 12a shows a potent antiproliferative activity,
exhibits an adequate therapeutic window (>1 log unit) and,
based on a special vehicle, a suitable PK was achieved. Thus,
12a was assayed in vivo as a pharmacological tool compound to
test its antitumor efficacy in a xenograft mouse model of
human MM; a significant efficacy was achieved. However, 12a
is a chemical probe and requires an optimization process to
evolve and become a new molecule with optimized drug-like
properties: a lead compound. In fact, to overcome its poor
ADME profile (special stress on solubility, permeability, and
metabolic stability) and achieve an optimal PK, a focused
exploration around R2 and R3 positions is currently on-going.
The reported chemical probes are key tool compounds (i) to
validate the feasibility of this proposal: multitarget epigenetic
inhibition, (ii) to define a preliminary SAR guideline (focused
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a
Scheme 4. Synthesis of Compounds 12a−k
a
Conditions: (i) POCl3, malonic acid, 100 °C, 16 h; (ii) 4,4,5,5-tetramethyl-2-(5-methyl-2-furyl)-1,3,2-dioxaborolane, Pd(PPh3)4, K2CO3, 1,4-
dioxane/H2O (10:1), 90 °C, 16 h; (iii) corresponding amine, Pd2(dba)3, BINAP, Cs2CO3, 1,4-dioxane, 120−140 °C, 12−16 h; (iv) HCl/MeOH
(2.0 M) or HCl/EtOAc (1.0 or 2.0 M), 20−25 °C, 20 min −16 h; (v) ethyl 2-chloropyrimidine-5-carboxylate, K2CO3, DMF or CH3CN, 40−50
°C, 12−16 h; (vi) corresponding aldehyde or ketone, NaBH3CN, CH3COOH or ZnCl2, MeOH, 40−70 °C, 2−15.5 h; (vii) LiOH·H2O, THF/
H2O (2:1) or THF/MeOH/H2O (6:1:1 or 3:1:1), 15−25 °C, 2−16 h; (viii) THPONH2, HOBt, DIEA, EDCI, DMF, 15−25 °C, 2−16 h.
on a double and triple inhibition: DNMT1, HDAC and,
optionally, G9a), as well as to identify critical ADME issues
(e.g., permeability and solubility). and (iii) to initiate a new
drug discovery project that requires a multitarget epigenetic
inhibitor as starting points (e.g., as 12a for MM) to progress to
an optimized lead molecule fulfilling the corresponding target
product profile. Thus, these first-in-class molecules are paving
the way for achieving multitarget epigenetic inhibition
(DNMT1, HDAC, and, optionally, G9a) according to
requirements that every drug discovery project may need
(e.g., inhibition profile of selected targets, ADME properties,
etc.)
Synthesis of compounds 10a and 10b is shown in Scheme 2.
In this case, compound 15 was reacted with methyl boronic
acid and intermediate 2034,35 was isolated. Then, amines 22a
and 22b35 were prepared by reaction with tert-butyl 4-
aminopiperidine-1-carboxylate or tert-butyl 4-(aminomethyl)-
piperidine-1-carboxylate respectively using Pd2(dba)3 as the
catalyst and subsequent deprotection of corresponding
intermediates 21a35 and 21b35 in acidic media. These amines
were then substituted with a pyrimidyl ester to obtain
compounds 23a and 23b. Finally, the desired hydroxamates
were obtained by ester hydrolysis, synthesis of THP-protected
intermediates 25a and 25b, and acidic deprotection.
Next, compound 11 with a 4-piperidyl moiety at the 7-
position was prepared as outlined in Scheme 3. In this case, the
synthesis started from commercially available aniline 26, which
was converted into 2,4-dichloroquinoline 2735 by reaction with
POCl3. Then, the piperidyl group at position 7 and 5-methyl-2-
furyl group at position 2 of the quinoline were installed under
standard conditions to obtain compound 29.35 Conversion of
this intermediate into desired hydroxamate 11 was achieved
using the similar synthetic procedure as described above
(Buchwald−Hartwig amination, ester hydrolysis, synthesis of
protected hydroxamic acid and removal of THP protecting
group).
CHEMISTRY
■
The preparation of target compounds is summarized in the
following schemes. Compounds 9a−k (Scheme 1) were
synthesized from previously described 2,4-dichloroquinoline
15,21 which was converted into the desired key intermediate
1634,35 through Suzuki coupling. Then, different substitutions
at the 4-position of the quinoline were installed using
Buchwald−Hartwig amination conditions and esters 17a−k
were isolated. Hydrolysis of these esters led us to
corresponding carboxylic acids 18a−k. Finally, the desired
hydroxamates 9a−k were obtained by reaction with
THPONH2 and acidic cleavage of the protecting group.
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a
Scheme 5. Synthesis of Compounds 13a−f
a
Conditions: (i) corresponding boronic ester, Pd(PPh3)4 or Pd(dppf)Cl2, K2CO3, 1,4-dioxane/H2O (10:1 or 5:1), 90−100 °C, 6−16 h; (ii)
piperidine or ethyl 2-[4-(aminomethyl)-1-piperidyl]pyrimidine-5-carboxylate, Pd2(dba)3, BINAP, Cs2CO3, 1,4-dioxane,100−140 °C, 12−48 h; (iii)
Pd/C, H2 (15 Psi), MeOH, 20 °C, 16 h; (iv) LiOH·H2O, THF/MeOH/H2O (3:1:1) or THF/H2O (2:1), 15−25 °C, 16 h; (v) THPONH2,
HOBt, DIEA, EDCI, DMF, 15−20 °C, 12−16 h; (vi) HCl/EtOAc (1.0 or 2.0 M), 15−25 °C, 4−16 h.
a
Scheme 6. Synthesis of Compound 14
a
Conditions: (i) ethyl 2-[4-(aminomethyl)-1-piperidyl]pyrimidine-5-carboxylate, K2CO3, DMF, 80 °C, 12 h; (ii) 4,4,5,5-tetramethyl-2-(5-methyl-
2-furyl)-1,3,2-dioxaborolane, Pd(PPh3)4, K2CO3, 1,4-dioxane, 100 °C, 12 h; (iii) LiOH·H2O, THF/H2O (2:1), 25 °C, 12 h; (iv) THPONH2,
HOBt, DIEA, EDCI, DMF, 25 °C, 12 h; (v) TFA, CH3CN/H2O (1:1), 60 °C, 5 min.
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Table 4. Antiproliferative Response of Selected Hybrid Compounds against the MM1.S MM Cancer Cell Line and the Healthy
Hepatic Cell Line THLE-2
b
c
d
e
f
g
Cpd
MM1.S GI50 nM
MM1.S pGI50
THLE-2 LC50 nM 72 h
THLE-2 pLC50
PAMPA Pe (nm/s)
therapeutic window
2
8.9 (0.1)
8.05
5.97
5.42
5.68
5.35
<5
5.45
5.38
5.43
5.74
<5
22 (14.5)
7.66
5.75
5.89
4.96
4.95
4.58
5.41
4.66
5.01
4.87
5.18
<4
4.48
4.71
6.10
5.51
5.87
5.63
5.02
6.69
5.65
4.98
5.64
14.2 (0.7)
12.9 (1.4)
11.0 (0.7)
0 (0)
1.0 (0.2)
0 (0)
0 (0)
0 (0)
0.5 (0.7)
1.1 (0.9)
0.14 (0.2)
0 (0)
0.75 (0.6)
0.25 (0.3)
5.04 (0.5)
5.96 (0.58)
9.59 (0.66)
5.34 (0.5)
3.93 (0.76)
5.56 (0.32)
4.01 (0.1)
0.68 (0.5)
0.39
0.22
toxic
0.72
0.40
N.A.
0.04
0.72
0.42
0.87
toxic
N.A.
521,34
621,35
9a
1041 (62.1)
3842 (92.1)
2077 (409.5)
4505 (2181)
>10,000
3571 (1417)
4207 (97)
3763 (42.5)
1811 (199)
>10,000
>10,000
>10,000
>10,000
32 (27.3)
1025 (73.2)
459 (19.9)
520 (29.6)
5826 (920)
217 (32.3)
1938 (272)
1985 (458)
1780 (596)
1300 (590)
10,900 (1980)
11,300 (2496)
26,100 (5940)
3890 (516.2)
22,050 (9970)
9700 (367.7)
13,500 (0.5)
6656 (82)
h
9b
9c
9d
9e
9f
9i
9k
10a
10b
11
12a
12b
12c
12d
12e
12g
12h
12i
13b
i
h
i
<5
<5
<5
>100,000
i
33,250 (7778)
19,700 (282)
794 (17.0)
N.A.
N.A.
i
7.50
5.99
6.34
6.28
5.24
6.66
5.71
5.70
6.71
1.40
0.48
0.47
0.65
0.22
3090 (438.4)
1350 (21.2)
2330 (155.6)
9600 (1590)
204 (110.3)
2227 (129)
10,410 (1797)
2298 (277)
h
toxic
0.06
0.72
1.07
195 (53.2)
2.1 (0.7)
a
b
c
N.D. = not determined. MM1.S proliferation assays are the average of three replicates on different days. Antiproliferative efficacy vs MM1.S
d
(GI50 values) are also reported in log units, as pGI50. THLE-2 cytotoxicity results after 72 h of incubation are the average of at least two
independent experiments performed on different days. If absolute pLC50 difference was higher than 1 log unit, additional replicates were performed
e
until satisfying the experimental error (by discarding individual results with values outside 3 MADs of the mean value). Cytotoxicity vs THLE-2
f
(LC50 values) are also reported in log units, as pLC50. The PAMPA assay was performed in triplicate. Depending on permeability values (Pe, nm/
g
s), compounds can be regarded as poor (Pe < 10 nm/s); moderate (10 < Pe < 30 nm/s), or good (>30 nm/s) permeators.33 The therapeutic
h
Window describes the difference between efficacy (pGI50 values vs MM1.S) and toxicity (pLC50 values vs THLE-2). Antiproliferative effects
i
against the healthy cell line (THLE-2) are more potent than, or equal to, the tumor cell line (MM1.S): Toxicity. N.A. = not applicable (GI50 or/
and LC50 are undetermined). Standard deviations are reported together with the corresponding experimental values (GI50, LC50 and Pe) in brackets
and in italics.
1
Synthesis microwave reactor. The H NMR spectroscopic data were
Table 5. GI50 Values of Compounds 9a and 12a vs
Additional Cell Lines of MM
a
recorded on a Bruker AV400 or VARIAN 400MR spectrometer with
standard pulse sequences and 13C NMR on a Bruker AVII-600
compound JJN3, GI50 (nM) KMS28BM, GI50 (nM) H929, GI50 (nM)
equipped with a 5 mm TCI cryoprobe and processed using
MestreNova. H NMR chemical shifts (δ) are reported in parts per
1
9a
12a
6035 (856)
482 (230)
1299 (151)
547 (30.5)
981 (189)
103 (19.5)
million (ppm) and relative to the residual protons of deuterated
reagents, which are corrected by tetramethylsilane (TMS). The
abbreviations used to explain multiplicities are s = singlet, d = doublet,
t = triplet, m = multiplet, br s = broad singlet. Coupling constants (J)
are in hertz. HPLC-analysis was performed using a Shimadzu LC-
20AB with a Luna-C18(2), 5 μm, 2.0 × 50 mm column at 40 °C and
UV detection. Flow from the column was split to the MS detector
(Agilent 1200, 6110MS or Agilent 1200, 6120MS Quadropole),
configured with an electrospray source or API/APCI (N2 as the
nebulizer gas, with the source temperature at 50 °C, ChemStation
LC/MSD quad software). UHPLC-analysis was carried out using a
BEH C18, 1.7 mm, 2.1 × 50 column at 40 °C and UV detection. The
HPLC or UHPLC purity of all reported compounds, which were
subjected to pharmacological evaluation, is ≥95% except for
compound 42, the purity of which is 94.53. No effort was put on
yield optimization.
2-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]-1-piperidyl]pyrimidine-5-carbohy-
droxamic Acid (9a). A solution of compound 19a (80 mg, 0.114
mmol) in HCl/MeOH (10 mL, 2.0 M) was degassed and purged with
N2 3 times and the mixture was stirred at 20 °C for 16 h. Then, the
mixture was concentrated in reduced pressure at 40 °C and the
residue was purified by prep-HPLC (method 10 described in the
Supporting Information) to afford pure compound 9a (29.6 mg, 42%)
a
These results are the average of three replicates on different days.
Standard deviations are reported together with the corresponding
experimental GI50 values in brackets and in italics.
Finally, preparation of compounds 12a−k and 13a−f with a
methoxy group at position 7 and following the same synthetic
strategy as described for compounds above is outlined in
Schemes 4 and 5. Synthetic strategy for intermediates 51, 54,
57, 60, 64, 68, 73 and 75 is described in Scheme 7.
EXPERIMENTAL SECTION
■
Chemistry. General Procedure. Unless otherwise noted, all
starting materials, reagents, and solvents were purchased from
commercial suppliers and used without further purification. Air-
sensitive reactions were conducted under N2. Flash column
chromatography was performed on silica gel (230−400 mesh particle
size) under standard techniques. Automated flash column chromatog-
raphy was performed using ready-to-connect cartridges from Varian
on irregular silica gel, particle size 15−40 μm (normal phase
disposable flash columns) on a Biotage SPX flash purification system.
Microwave-assisted reactions were obtained in a Biotage Smith
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Figure 3. Changes in the epigenetic hallmarks following treatment of MM1.S cells with compounds 12a and 9a. (A) H3K9Ac and H3K9me2
dose−response western blots after incubation for 48 h with the indicated dose range of 12a and 9a. (B) POU4F2 gene pyrosequencing after
treatment with 12a at 10 nM and 9a at 2 μM for 5 days. All assays were performed in duplicate.
Table 6. ADME Profile of Compound 12a
1A2
2C9
2C19
a
2D6
3A4
HH
HH
MH
MH
kinetic solubility
a
a
a
a
b
c
d
b
c
d
e
Cpd
(%)
(%)
(%)
(%)
(%)
HH (%)
50.8 (10)
(t1/2
)
(CLint)
MH (%)
14.3 (0.9)
(t1/2
)
(CLint)
(μg/mL)
12a
58.8
0.0
5.6
10.8
50.8
71.1
54.2
22.8
410.7
<0.987
a
b
% inhibition at 10 μM. % compound remaining after a 60 min incubation in pooled human or C57 mouse hepatocytes (HH and MH
c
d
respectively); standard deviations are reported in brackets and in italics. Half-life (min) in HH and MH. In vivo CLint (mL/min/kg) in HH and
MH. Below the limit of quantification (BLQ), which is 0.987 μg/mL. All assays were performed in duplicate.
e
a
Table 7. PK Profile of Compound 12a
Cpd
route
vehicle
dose (mg/kg)
AUC0−24h (nM h)
t1/2 (h)
Cl/F (L/h)
Vz/F (L)
12a
12a
i.p.
i.p.
saline
80% saline, 10% Tween20 and 10% DMSO
10
10
591.1
916.15
3.8
8.46
0.8
0.53
4.6
6.56
a
Species: BALB/c-RAG2‑/‑γc‑/‑ mice; i.p. means intraperitoneal administration, saline: NaCl 0.9%; n = 5 and time points: 0.25, 1, 2, 4, 8, and 24 h.
as a yellow solid; mp 126−127 °C. 1H NMR (CD3OD, 400 MHz): δ
8.64 (s, 2H), 7.74 (s, 1H), 7.53 (d, J = 3.6 Hz, 1H), 7.48 (s, 1H), 7.00
(s, 1H), 6.42 (t, J = 3.2 Hz, 1H), 4.35 (t, J = 5.6 Hz, 2H), 4.05 (s,
3H), 3.85−3.78 (m, 2H), 3.56−3.48 (m, 4H), 3.19−3.10 (m, 3H),
3.02−2.92 (m, 3H), 2.53 (s, 3H), 2.40−2.38 (m, 2H), 2.36−2.23 (m,
3H), 2.09−2.07 (m, 2H), 1.99−1.96 (m, 2H), 1.37−1.31 (m, 2H);
three exchangeable protons (NH from 4-aminoquinoline and NH−
OH from hydroxamic group) were not observed. 13C NMR (151
MHz, MeOD): δ 164.04, 161.50, 157.63, 156.94 (2C), 154.55,
153.66, 149.46, 144.11, 140.00, 134.50, 115.66, 113.43, 110.62,
109.59, 101.55, 100.51, 91.81, 66.67, 55.73, 54.08 (2C), 53.06, 48.38,
43.61 (2C), 35.78, 29.59 (2C), 25.22, 22.64 (2C), 12.41. ESI-MS m/
z: calcd for C33H41N7O5, 615.3; m/z: found, 616.3 [M + H]+. HPLC
(method 1): Rt is 2.01 min and purity is 98.97%. HRMS [M + H]+:
calcd, 616.3242; found, 616.3235, Δ = 1.1 ppm.
4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]benzenecarbohydroxamic Acid
(9b). A mixture of compound 18b (80 mg, 0.155 mmol),
THPONH2(27 mg, 0.232 mmol), HOBt (25 mg, 0.186 mmol),
EDCI (36 mg, 0.186 mmol), and DIEA (40 mg, 0.310 mmol) in
DMF (3 mL) was degassed and purged with N2 3 times and the
mixture was stirred at 20 °C for 5 h. Then, the mixture was
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Scheme 7. (a−h) Synthesis of Intermediates: 51, 54, 57, 60,
a
64, 68, 73, and 75
Figure 4. Compound 12a shows in vivo efficacy in an MM1.S tumor
model. The tumor volume of MM1.S cells subcutaneously injected
and treated with the vehicle (80% saline, 10% Tween 20, and 10%
DMSO) or compound 12a (10 mg/Kg for 5 consecutive days
followed by 2 rest days) (n = 9). Statistical significance was calculated
by a two-tailed Student’s t-test. *p-value ≤ 0.05.
concentrated in reduced pressure at 40 °C and the residue was
purified by prep-HPLC (method 30 described in the Supporting
Information) to afford pure compound 9b (22.4 mg, 27%) as a yellow
solid; mp 100−101 °C. 1H NMR (DMSO-d6, 400 MHz): δ 13.36 (s,
1H), 11.22 (s, 1H), 9.43 (s, 1H), 7.89 (s, 1H), 7.77 (d, J = 8.4 Hz,
2H), 7.63 (s, 2H), 7.53 (d, J = 8.4 Hz, 2H), 6.99 (s, 1H), 6.52 (s,
1H), 4.93 (d, J = 6.0 Hz, 2H), 4.26−4.24 (m, 2H), 3.98 (s, 3H),
3.67−6.64 (m, 2H), 3.37−3.35 (m, 2H), 3.10−3.07 (m, 2H), 2.47 (s,
3H), 2.28−2.26 (m, 2H), 2.08−2.06 (m, 2H), 1.90−1.89 (m, 2H).
ESI-MS m/z: calcd for C30H34N4O5, 530.2; m/z: found, 531.3 [M +
H]+. HPLC (method 1): Rt is 1.75 min and purity is 96.17%. HRMS
[M + H]+: calcd, 531.2602; found, 531.2643, Δ = 7.7 ppm.
4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]cyclohexanecarbohydroxamic
Acid (9c). A solution of compound 19c (60 mg, 0.097 mmol) in HCl/
EtOAc (10 mL, 2.0 M) was stirred at 20 °C for 3 h. Then, the mixture
was concentrated in reduced pressure at 40 °C and the residue was
purified by prep-HPLC (method 25 described in the Supporting
Information) to afford pure compound 9c (9.6 mg, 18%) as a yellow
solid; mp 127−128 °C. 1H NMR (CD3OD, 400 MHz): δ 7.73 (d, J =
4.4 Hz, 1H), 7.51 (d, J = 3.6 Hz, 1H), 7.46 (s, 1H), 6.93 (s, 1H), 6.43
(d, J = 3.2 Hz, 1H), 4.36−4.33 (m, 2H), 4.04 (s, 3H), 3.83 (m, 2H),
3.51−3.46 (m, 4H), 3.17 (m, 2H), 2.50 (s, 3H), 2.39−2.36 (m, 2H),
2.21 (m, 2H), 2.09−2.06 (m, 3H), 2.01−1.99 (m, 2H), 1.86−1.83
(m, 3H), 1.62−1.56 (m, 2H), 1.22−1.13 (m, 2H); three exchangeable
protons (NH from 4-aminoquinoline and NH−OH from hydroxamic
group) were not observed. ESI-MS m/z:calcd for C30H40N4O5, 536.3;
m/z: found, 537.5 [M + H]+. HPLC (method 2): Rt is 3.02 min and
purity is 97.84%. HRMS [M + H]+: calcd, 537.3071; found, 537.3126,
Δ = 10.2 ppm.
4-[[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]-1-piperidyl]methyl]-
benzenecarbohydroxamic Acid (9d). A solution of compound 19d
(65 mg, 0.091 mmol) in HCl/EtOAc (5 mL, 2.0 M) was stirred at 20
°C for 0.5 h. Then, the solution was concentrated to give the residue,
which was purified by prep-HPLC (method 31 described in the
Supporting Information) to afford pure compound 9d (15.4 mg,
1
27%) as a yellow solid; mp 135−136 °C. H NMR (CD3OD, 400
MHz): δ 7.84 (d, J = 8.0 Hz, 2H), 7.71 (s, 1H), 7.60 (d, J = 8.0 Hz,
2H), 7.53 (d, J = 3.6 Hz, 1H), 7.47 (s, 1H), 6.98 (s, 1H), 6.42 (d, J =
3.6 Hz, 1H), 4.41−4.28 (m, 4H), 4.02 (s, 3H), 3.81 (s, 2H), 3.66−
3.44 (m, 6H), 3.23−2.91 (m, 4H), 2.49 (s, 3H), 2.43−2.31 (m, 2H),
2.29−1.98 (m, 7H), 1.65 (d, J = 12.4 Hz, 2H); three exchangeable
protons (NH from 4-aminoquinoline and NH−OH from hydroxamic
group) were not observed. ESI-MS m/z: calcd for C36H45N5O5,
627.3; m/z: found, 628.3 [M + H]+. HPLC (method 1): Rt is 1.443
a
Specific reaction conditions for each synthetic step are explicitly
detailed in each scheme (a−h).
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min and purity is 97.63%. HRMS [M + H]+: calcd, 628.3493; found,
628.3558, Δ = 10.3 ppm.
(m, 3H), 2.40−2.37 (m, 2H), 2.22 (s, 3H), 2.09−2.07 (m, 2H),
1.94−1.92 (m, 2H), 1.84−1.82 (m, 4H), 1.59−1.57 (m, 1H), 1.36−
1.33 (m, 1H); three exchangeable protons (NH from 4-aminoquino-
line and NH−OH from hydroxamic group) were not observed. ESI-
MS m/z:calcd for C36H44N4O5, 612.3; m/z: found, 613.4 [M + H]+.
HPLC (method 1): Rt is 2.127 min and purity is 99.19%. HRMS [M +
H]+: calcd, 613.3384; found, 613.3408, Δ = 3.9 ppm.
4-[[4-[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
p o x y ) - 4 - q u i n o l y l ] a m i n o ] - 1 - p i p e r i d y l ] m e t h y l ] -
benzenecarbohydroxamic Acid (9e). A solution of compound 19e
(80 mg, 0.115 mmol) in HCl/EtOAc (10 mL, 2.0 M) was stirred at
20 °C for 2 h. Then, the mixture was concentrated in reduced
pressure at 40 °C and the residue was purified by prep-HPLC
(method 32 described in the Supporting Information) to afford pure
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]phenyl]benzenecarbohydroxamic
Acid (9i). A solution of compound 19i (80 mg, 0.116 mmol) in HCl/
EtOAc (5 mL, 2.0 M) was stirred at 20 °C for 2 h. Then, the mixture
was concentrated in reduced pressure at 40 °C and the residue was
purified by prep-HPLC (method 10 described in the Supporting
Information) to afford pure compound 9i (22.9 mg, 32%) as a yellow
1
compound 9e (9.3 mg, 13%) as a yellow solid; mp 137−138 °C. H
NMR (CD3OD, 400 MHz): δ 7.90 (d, J = 8.4 Hz, 2H), 7.81 (s, 1H),
7.67 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 3.2 Hz, 1H), 7.50 (s, 1H), 7.07
(s, 1H), 6.44 (d, J = 2.8 Hz, 1H), 4.47 (s, 2H), 4.36−4.29 (m, 3H),
4.03 (s, 3H), 3.83 (s, 2H), 3.66 (s, 2H), 3.51−3.47 (m, 2H), 3.32−
3.31 (m, 2H), 3.17−3.14 (m, 2H), 2.51 (s, 3H), 2.40−2.37 (m, 4H),
2.21−2.06 (m, 6H); three exchangeable protons (NH from 4-
aminoquinoline and NH−OH from hydroxamic group) were not
observed. ESI-MS m/z:calcd for C35H43N5O5, 613.3; m/z: found,
614.4 [M + H]+. HPLC (method 1): Rt is 1.428 min and purity is
98.36%. HRMS [M + H]+: calcd, 614.3337; found, 614.3387, Δ = 8.1
ppm.
1
solid; mp 119−120 °C. H NMR (CD3OD, 400 MHz): δ 7.77 (s,
3H), 7.67−7.65 (m, 4H), 7.55−7.53 (m, 2H), 7.46−7.38 (m, 2H),
6.91 (d, J = 3.6 Hz, 1H), 6.39 (s, 1H), 4.36 (s, 2H), 4.06 (s, 3H), 3.87
(s, 2H), 3.53−3.50 (m, 2H), 3.17 (s, 2H), 2.47−2.39 (m, 6H), 2.23−
2.09 (m, 5H); three exchangeable protons (NH from 4-aminoquino-
line and NH−OH from hydroxamic group) were not observed. ESI-
MS m/z:calcd for C36H38N4O5, 606.3; m/z: found, 607.3 [M + H]+.
HPLC (method 1): Rt is 2.009 min and purity is 100%. HRMS [M +
H]+: calcd, 607.2915; found, 607.2961, Δ = 7.6 ppm.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
p o x y ) - 4 - q u i n o l y l ] a m i n o ] m e t h y l ] - 1 - p i p e r i d y l ] -
benzenecarbohydroxamic Acid (9f). A solution of compound 19f
(85 mg, 0.122 mmol) in HCl/EtOAc (5 mL, 2.0 M) was stirred at 20
°C for 0.5 h. Then, the mixture was concentrated to give a residue,
which was purified by prep-HPLC (method 25 described in the
Supporting Information) to afford pure compound 9f (25.1 mg, 33%)
as a yellow solid; mp 125−126 °C. 1H NMR (CD3OD, 400 MHz): δ
7.73 (s, 1H), 7.63 (d, J = 9.0 Hz, 2H), 7.53 (s, 1H), 7.47 (s, 1H),
7.02−6.95 (m, 3H), 6.42 (d, J = 2.0 Hz, 1H), 4.35 (s, 2H), 4.04 (s,
3H), 3.92 (d, J = 13.0 Hz, 2H), 3.85 (m, 2H), 3.58 (d, J = 6.4 Hz,
2H), 3.50 (t, J = 7.0 Hz, 2H), 3.18 (m, 2H), 2.88 (t, J = 12.0 Hz, 2H),
2.50 (s, 3H), 2.39 (d, J = 5.6 Hz, 2H), 2.22 (m, 2H), 2.09 (m, 3H),
1.98 (d, J = 12.0 Hz, 2H), 1.60−1.44 (m, 2H); three exchangeable
protons (NH from 4-aminoquinoline and NH−OH from hydroxamic
group) were not observed. ESI-MS m/z: calcd for C35H43N5O5,
613.3; m/z: found, 614.4 [M + H]+. HPLC (method 1): Rt is 1.797
min and purity is 98.70%. HRMS [M + H]+: calcd, 614.3337; found,
614.3369, Δ = 5.2 ppm.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
p o x y ) - 4 - q u i n o l y l ] a m i n o ] m e t h y l ] - 1 - p i p e r i d y l ] -
cyclohexanecarbohydroxamic Acid (9g). A solution of compound
19g (50 mg, 0.071 mmol) in HCl/EtOAc (10 mL, 2.0 M) was stirred
at 20 °C for 0.5 h. Then, the solution was concentrated to give a
residue, which was purified by prep-HPLC (method 33 described in
the Supporting Information) to afford pure compound 9g (5.3 mg,
12%) as a yellow oil. 1H NMR (CD3OD, 400 MHz): δ 7.77 (s, 1H),
7.59 (d, J = 3.2 Hz, 1H), 7.51 (s, 1H), 6.99 (s, 1H), 6.43 (d, J = 3.2
Hz, 1H), 4.36 (t, J = 5.2 Hz, 2H), 4.04 (s, 3H), 3.83 (s, 2H), 3.64−
3.60 (m, 5H), 3.58−3.49 (m, 2H), 3.18−3.09 (m, 5H), 2.50 (s, 3H),
2.40 (d, J = 6.0 Hz, 2H), 2.21−2.17 (m, 6H), 2.08 (d, J = 8.8 Hz,
5H), 1.95 (d, J = 8.8 Hz, 2H), 1.73−1.63 (m, 4H); three
exchangeable protons (NH from 4-aminoquinoline and NH−OH
from hydroxamic group) were not observed. ESI-MS m/z:calcd for
C35H49N5O5, 619.4; m/z: found, 620.4 [M + H]+. HPLC (method 1):
Rt is 1.464 min and purity is 98.13%. HRMS [M + H]+: calcd,
620.3806; found, 620.3818, Δ = 1.9 ppm.
rac-trans 4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-
1 - y l p r o p o x y ) - 4 - q u i n o l y l ] a m i n o ] m e t h y l ] p h e n y l ] -
cyclohexanecarbohydroxamic Acid (9j-trans) and rac-cis 4-[4-[[[6-
Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpropoxy)-4-
quinolyl]amino]methyl]phenyl]cyclohexanecarbohydroxamic Acid
(9j-cis). A solution of compound 19j (105 mg, 0.150 mmol) in HCl/
EtOAc (10 mL, 2.0 M) was stirred at 20 °C for 0.5 h. Then, the
solution was concentrated and the residue was purified by prep-HPLC
(method 10 described in the Supporting Information) to afford pure
compound 9j-cis (11.9 mg, 13%) as a yellow solid and pure
compound 9j-trans (3.3 mg, 4%) as a yellow solid. 9j-trans: mp 93−
1
94 °C. H NMR (CD3OD, 400 MHz): δ 7.78 (s, 1H), 7.46 (s, 1H),
7.40−7.34 (m, 3H), 7.31−7.29 (m, 2H), 6.95 (s, 1H), 6.40 (d, J = 3.4
Hz, 1H), 4.82 (s, 2H), 4.36 (t, J = 5.4 Hz, 2H), 4.04 (s, 3H), 3.83 (m,
2H), 3.52−3.49 (m, 2H), 3.19−3.14 (m, 2H), 2.67 (m, 1H), 2.47 (s,
3H), 2.46−2.37 (m, 3H), 2.23 (m, 3H), 2.10−1.85 (m, 6H), 1.75−
1.69 (m, 4H); two exchangeable protons were not observed. ESI-MS
m/z:calcd for C36H44N4O5, 612.3; m/z: found, 613.4 [M + H]+.
HPLC (method 1): Rt is 2.100 min and purity is 100%. HRMS [M +
H]+: calcd, 613.3384; found, 613.3451. 9j-cis: mp 134−135 °C.1H
NMR (CD3OD, 400 MHz): δ 7.77 (s, 1H), 7.46 (s, 1H), 7.39−7.37
(m, 3H), 7.27−7.25 (m, 2H), 6.93 (s, 1H), 6.39 (d, J = 3.4 Hz, 1H),
4.81 (s, 2H), 4.35 (m, 2H), 4.03 (s, 3H), 3.83 (s, 2H), 3.52−3.48 (m,
2H), 3.21−3.10 (m, 2H), 2.55 (m, 1H), 2.47 (s, 3H), 2.38 (m, 3H),
2.22 (s, 2H), 2.07 (m, 3H), 1.80−1.93 (m, 4H), 1.68 (m, 2H), 1.55−
1.49 (m, 2H); two exchangeable protons were not observed. ESI-MS
m/z:calcd for C36H44N4O5, 612.3; m/z: found, 613.3 [M + H]+.
HPLC (method 1): Rt is 2.040 min and purity is 100%. HRMS [M +
H]+: calcd, 613.3384; found, 613.3407, Δ = 3.8 ppm.
4-[4-[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]-1-piperidyl]benzenecarbohydroxamic
Acid (9k). A solution of compound 19k (100 mg, 0.146 mmol) in
HCl/EtOAc (5 mL, 2.0 M) was stirred at 20 °C for 3 h. Then, the
solution was concentrated in reduced pressure at 40 °C and the
residue was purified by prep-HPLC (method 5 described in the
Supporting Information) to afford pure compound 9k (23.4 mg, 27%)
as a yellow solid; mp 138−139 °C. 1H NMR (CD3OD, 400 MHz): δ
7.79 (s, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.56 (s, 1H), 7.48 (br, 1H),
7.10 (s, 1H), 7.05 (d, J = 9.2 Hz, 2H), 6.43 (s, 1H), 4.35 (s, 2H), 4.24
(m, 1H), 4.07−4.02 (m, 5H), 3.83 (m, 2H), 3.51−3.48 (m, 2H),
3.18−3.12 (m, 4H), 2.51 (s, 3H), 2.38 (m, 2H), 2.21−2.18 (m, 4H),
2.07 (m, 2H), 1.95−1.92 (m, 2H); three exchangeable protons (NH
from 4-aminoquinoline and NH−OH from hydroxamic group) were
not observed. ESI-MS m/z:calcd for C34H41N5O5, 599.3; m/z: found,
600.4 [M + H]+. HPLC (method 1): Rt is 1.862 min and purity is
98.07%. HRMS [M + H]+: calcd, 600.3180; found, 600.3245, Δ =
10.8 ppm.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
p o x y ) - 4 - q u i n o l y l ] a m i n o ] m e t h y l ] c y c l o h e x y l ] -
benzenecarbohydroxamic Acid (9h). A solution of compound 19h
(15 mg, 0.022 mmol) in HCl/EtOAc (3 mL, 2.0 M) was stirred at 20
°C for 1 h. Then, the mixture was concentrated in reduced pressure at
40 °C and the residue was purified by prep-HPLC (method 34
described in the Supporting Information) to afford pure compound
1
9h (8.5 mg, 64%) as a yellow oil. H NMR (CD3OD, 400 MHz): δ
7.75 (d, J = 6.4 Hz, 1H), 7.70−7.68 (m, 2H), 7.51 (d, J = 3.6 Hz,
1H), 7.47−7.46 (m, 2H), 7.43−7.33 (m, 1H), 7.00 (d, J = 8.0 Hz,
1H), 6.43 (d, J = 3.2 Hz, 1H), 4.37−4.34 (m, 2H), 4.05−4.04 (m,
3H), 3.84 (s, 2H), 3.76−3.74 (m, 1H), 3.56−3.54 (m, 1H), 3.50−
3.48 (m, 2H), 3.31−3.18 (m, 2H), 2.77−2.62 (m, 1H), 2.51−2.48
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2-[4-[[[6-Methoxy-2-methyl-7-(3-pyrrolidin-1-ylpropoxy)-4-
quinolyl]amino]methyl]-1-piperidyl]pyrimidine-5-carbohydroxa-
mic Acid (10a). A solution of compound 25a (50 mg, 0.080 mmol) in
HCl/1,4-dioxane (10 mL, 4.0 M) was stirred at room temperature for
1 h. Then, the reaction mixture was concentrated to give compound
hydroxylamine hydrochloride (44 mg, 0.286 mmol), EDCI (41 mg,
0.214 mmol), DIEA (37 mg, 0.286 mmol), and HOBt (29 mg, 0.214
mmol) in DMF (5.00 mL) was degassed and purged with N2 3 times,
and then the mixture was stirred at 25 °C for 12 h. Then, the reaction
mixture was concentrated in vacuum to give a residue, which was
purified by prep-HPLC (method 15 described in the Supporting
Information) to afford pure compound 12b (40.6 mg, 56%) as a
yellow solid; mp 181−182 °C. 1H NMR (CD3OD, 400 MHz): δ 8.70
(s, 2H), 7.72 (s, 1H), 7.56−7.54 (m, 2H), 7.43 (s, 1H), 7.12 (s, 1H),
6.44 (d, J = 2.88 Hz, 1H), 5.07−4.97 (m, 2H), 4.38−4.29 (m, 1H),
4.02 (s, 3H), 3.99 (s, 3H), 3.27−3.20 (m, 2H), 2.52 (s, 3H), 2.25−
2.16 (m, 2H), 1.85−1.72 (m, 2H); two exchangeable protons were
not observed. ESI-MS m/z:calcd for C26H28N6O5, 504.2; m/z: found,
505.3 [M + H]+. HPLC (method 1): Rt is 2.12 min and purity is
96.03%. HRMS [M + H]+: calcd, 505.2194; found, 505.2179, Δ = 3.0
ppm.
1
10a (20 mg, 46%) as a yellow oil. H NMR (CD3OD, 400 MHz): δ
8.67 (s, 2H), 7.69 (s, 1H), 7.19 (s, 1H), 6.66 (s, 1H), 4.30 (m, 2H),
4.04 (s, 3H), 3.47 (m, 2H), 3.09 (m, 8H), 2.65 (s, 3H), 2.26 (m, 3H),
1.96 (m, 8H), 1.34 (m, 2H); three exchangeable protons (NH from
4-aminoquinoline and NH−OH from hydroxamic group) were not
observed. ESI-MS m/z: calcd for C29H39N7O4, 549.3; m/z: found,
550.3 [M + H]+. HPLC (method 1): Rt is 2.85 min and purity is
95.59%. HRMS [M + H]+: calcd, 550.3136; found, 550.3161, Δ = 4.5
ppm.
2-[4-[[6-Methoxy-2-methyl-7-(3-pyrrolidin-1-ylpropoxy)-4-
quinolyl]amino]-1-piperidyl]pyrimidine-5-carbohydroxamic Acid
(10b). A solution of compound 25b (60 mg, 0.097 mmol) in HCl/
1,4-dioxane (10 mL, 4.0 M) was stirred at room temperature for 1 h.
Then, the reaction mixture was concentrated to give the desired
2-[8-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-3-azabicyclo[3.2.1]octan-3-yl]pyrimidine-5-carbohydroxa-
mic Acid (12c). A solution of compound 38c (0.1 g, 0.159 mmol) in
HCl/EtOAc (10 mL, 1.0 M) was stirred at 15 °C for 2 h. Then, the
reaction mixture was concentrated to give a residue, which was
purified by prep-HPLC (method 16 described in the Supporting
Information) to afford pure compound 12c (15 mg, 17%) as a white
1
compound 10b (31 mg, 60%) as a white solid; mp 192−193 °C. H
NMR (CD3OD, 400 MHz): δ 8.73 (s, 2H), 7.83 (s, 1H), 7.23 (s,
1H), 6.89 (s, 1H), 4.37−4.28 (m, 3H), 4.04 (s, 3H), 3.86 (m, 2H),
3.52 (m, 2H), 3.33 (m, 4H), 3.19 (m, 2H), 2.71 (s, 3H), 2.41 (m,
2H), 2.23 (m, 4H), 2.11 (m, 2H), 1.89 (m, 2H); three exchangeable
protons (NH from 4-aminoquinoline and NH−OH from hydroxamic
group) were not observed. ESI-MS m/z: calcd for C28H37N7O4,
535.3; m/z: found, 536.3 [M + H]+. HPLC (method 2): Rt is 2.80 min
and purity is 95.69%. HRMS [M + H]+: calcd, 536.2980; found,
536.3024, Δ = 8.2 ppm.
1
solid; mp 180−181 °C. H NMR (CD3OD, 400 MHz): δ 8.69−8.65
(m, 2H), 7.76−7.74 (d, J = 6 Hz, 1H), 7.56−7.50 (m, 1H), 7.45 (s,
1H), 7.06−7.03 (m, 1H), 6.44−6.41 (m, 1H), 4.64−4.62 (m, 1H),
4.37−4.34 (m, 1H), 4.04−4.01 (m, 7H), 3.53−3.44 (m, 2H), 3.08−
3.05 (m, 1H), 2.51−2.50 (d, J = 5.2 Hz, 3H), 2.46 (br s, 1H), 2.42−
2.39 (m, 2H), 1.97−1.90 (m, 2H), 1.66−1.58 (m, 2H); three
exchangeable protons (NH from 4-aminoquinoline and NH−OH
from hydroxamic group) were not observed. ESI-MS m/z:calcd for
C29H32N6O5, 544.2; m/z: found, 545.3 [M + H]+. HPLC (method 1):
Rt is 2.357 min and purity is 99.30%. HRMS [M + H]+: calcd,
545.2507; found, 545.2495, Δ = 2.2 ppm.
2-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(4-piperidylmethoxy)-
4-quinolyl]amino]methyl]-1-piperidyl]pyrimidine-5-carbohydroxa-
mic Acid (11). A solution of compound 32 (300 mg, 0.381 mmol) in
HCl/EtOAc (1.0 M, 10 mL) was stirred at 15 °C for 16 h. Then, the
reaction mixture was concentrated to give a residue, which was
purified by prep-HPLC (method 35 described in the Supporting
Information) to afford pure compound 11 (13 mg, 5%) as a yellow
4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]benzenecarbohydroxamic Acid (12d). A mixture of
compound 37d (60 mg, 0.143 mmol), THPONH2 (20 mg, 0.172
mmol), HOBt (23 mg, 0.172 mmol), EDCI (33 mg, 0.172 mmol),
and DIEA (37 mg, 0.287 mmol) in DMF (3 mL) was degassed and
purged with N2 3 times, and then the mixture was stirred at 20 °C for
3 h. Then, the mixture was concentrated in reduced pressure at 40 °C
and the residue was purified by prep-HPLC (method 10 described in
the Supporting Information) to afford pure compound 12d (27.1 mg,
1
solid; mp 155−156 °C. H NMR (CD3OD, 400 MHz): δ 8.65 (s,
2H), 7.71 (s, 1H), 7.52 (d, J = 3.6 Hz, 1H), 7.45 (s, 1H), 6.99 (s,
1H), 6.42 (s, 1H), 4.95−4.93 (m, 1H), 4.13 (d, J = 5.6 Hz, 2H), 4.02
(s, 3H), 3.58−3.56 (m, 2H), 3.52−3.49 (m, 2H), 3.13−3.01 (m, 4H),
2.50 (s, 3H), 2.31−2.23 (br s, 2H), 2.18−2.14 (m, 2H), 1.99−1.96
(m, 2H), 1.79−1.73 (m, 2H), 1.40−1.30 (m, 3H); four exchangeable
protons were not observed. ESI-MS m/z: calcd for C32H39N7O5,
601.3; m/z: found, 602.4 [M + H]+. UHPLC (method 3): Rt is 2.36
min and purity is 96.28%. HRMS [M + H]+: calcd, 602.3085; found,
602.3063, Δ = 3.6 ppm.
1
44%) as a yellow solid; mp 180−181 °C. H NMR (DMSO-d6, 400
MHz): δ 13.26 (s, 1H), 11.22 (s, 1H), 9.36 (s, 1H), 7.86 (s, 1H), 7.77
(d, J = 8.4 Hz, 2H), 7.59 (s, 2H), 7.53 (d, J = 8.0 Hz, 2H), 6.98 (s,
1H), 6.51 (d, J = 2.4 Hz, 1H), 4.92 (d, J = 5.2 Hz, 2H), 3.97 (s, 6H),
2.47 (s, 3H); one exchangeable proton was not observed. ESI-MS m/
z: calcd for C24H23N3O5, 433.2; m/z: found, 434.3 [M + H]+. HPLC
(method 1): Rt is 2.01 min and purity is 97.66%. HRMS [M + H]+:
calcd, 434.1710; found, 434.1746, Δ = 8.3 ppm.
2-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]pyrimidine-5-carbohydroxamic Acid (12a). A
mixture of compound 38a (80 mg, 0.133 mmol, 1.00 eq) in HCl/
EtOAc (5.00 mL, 2M) was degassed and purged with N2 3 times, and
then the mixture was stirred at 25 °C for 3 h under an N2 atmosphere.
The reaction mixture was concentrated in vacuo to give a residue. The
residue was purified by prep-HPLC (method 5 described in the
Supporting Information) to afford pure compound 12a (39.10 mg,
0.061 mmol, 45.91% yield, 98.74% purity) as a white solid; mp 136−
137 °C. 1H NMR (CD3OD, 400 MHz): δ 8.65 (s, 2H), 7.69 (s, 1H),
7.54 (d, J = 3.54 Hz, 1H), 7.43 (s, 1H), 6.99 (s, 1H), 6.42 (dd, J =
3.54 Hz, 0.88 Hz, 1H), 4.95−4.89 (m, 2H), 4.03 (s, 3H), 4.02 (s,
3H), 3.56 (d, J = 7.06 Hz, 2H), 3.05−2.96 (m, 2H), 2.85−2.71 (m,
1H), 2.50 (s, 3H), 2.29−2.18 (m, 1H), 2.02−1.94 (m, 2H), 1.43−
1.29 (m, 2H); two exchangeable protons were not observed. 13C
NMR (151 MHz, MeOD): δ 163.59, 161.07, 157.03, 156.49 (2C),
154.59, 153.89, 149.08, 143.55, 139.27, 134.07, 115.01, 113.04,
109.71, 109.03, 100.80, 98.99, 91.14, 55.08, 54.93, 47.93, 43.05 (2C),
35.14, 29.10 (2C), 11.88. ESI-MS m/z: calcd for C27H30N6O5, 518.2;
m/z: found, 519.3 [M + H]+. HPLC (method 1): Rt is 2.37 min and
purity is 98.74%. HRMS [M + H]+: calcd, 519.2361; found, 519.2335,
Δ = 5.0 ppm.
4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]cyclohexanecarbohydroxamic Acid (12e). A mixture of
compound 37e (50 mg, 0.118 mmol), THPONH2 (28 mg, 0.235
mmol), HOBt (19 mg, 0.141 mmol), EDCI (27 mg, 0.141 mmol),
and DIEA (30 mg, 0.235 mmol) in DMF (20 mL) was degassed and
purged with N2 3 times and the mixture was stirred at 20 °C for 3 h.
Then, the mixture was concentrated in reduced pressure at 40 °C to
give a residue, which was purified by prep-HPLC (method 10
described in the Supporting Information) to afford compound 12e
(17.2 mg, 33%) as a yellow solid; mp 148−149 °C. 1H NMR
(DMSO-d6, 400 MHz): δ 13.20 (s, 1H), 10.39 (s, 1H), 8.75 (d, J =
5.6 Hz, 1H), 7.75 (s, 1H), 7.69 (d, J = 3.2 Hz, 1H), 7.55 (s, 1H), 6.86
(s, 1H), 6.50 (d, J = 3.2 Hz, 1H), 3.94 (s, 6H), 3.45−3.40 (m, 2H),
2.50−2.47 (m, 3H), 2.01−1.95 (m, 1H), 1.89−1.86 (m, 2H), 1.72−
1.68 (m, 3H), 1.42−1.39 (m, 2H), 1.10−1.05 (m, 2H). ESI-MS m/z:
calcd for C24H29N3O5, 439.2; m/z: found, 440.3 [M + H]+. HPLC
(method 1): Rt is 2.13 min and purity is 96.86%. HRMS [M + H]+:
calcd, 440.2180; found, 440.2204, Δ = 5.5 ppm.
2-[4-[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-1-
piperidyl]pyrimidine-5-carbohydroxamic Acid (12b). A mixture of
compound 37b (70 mg, 0.143 mmol), O-(tetrahydro-2H-pyran-2-yl)
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6-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]pyridine-3-carbohydroxamic Acid (12f). A mixture of
compound 37f (50 mg, 0.119 mmol), THPONH2(28 mg, 0.238
mmol), EDCI (46 mg, 0.238 mmol), HOBt (32 mg, 0.238 mmol),
and DIEA (46 mg, 0.357 mmol) in DMF (10 mL) was degassed and
purged with N2 3 times, and then the mixture was stirred at 20 °C for
16 h. Then, the reaction mixture was quenched with water (2 mL)
and concentrated in vacuum to give a residue. The residue was
dissolved in 0.5 M HCl aqueous solution and stirred for 30 min.
Then, the reaction mixture was concentrated in vacuum to give a
residue, which was purified by prep-HPLC (method 42 described in
the Supporting Information) to afford pure compound 12f (3.5 mg,
6%) as a white solid.mp 138−139 °C. 1H NMR (CD3OD, 400 MHz):
δ 8.92 (s, 1H), 8.17 (d, J = 8 Hz, 1H), 7.72 (s, 1H), 7.63 (d, J = 8.4
Hz, 1H), 7.45 (s, 1H), 7.38 (d, J = 3.6 Hz, 1H), 6.94 (s, 1H), 6.39 (d,
J = 3.2 Hz, 1H), 5.02 (s, 2H), 4.04 (s, 6H), 2.46 (s, 3H); three
exchangeable protons (NH from 4-aminoquinoline and NH−OH
from hydroxamic group) were not observed. ESI-MS m/z: calcd for
C23H22N4O5, 434.2; m/z: found, 435.2 [M + H]+. UHPLC (method
3): Rt is 2.35 min and purity is 96.76%. HRMS [M + H]+: calcd,
435.1663; found, 435.1639, Δ = 5.5 ppm.
4-[[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]methyl]benzenecarbohydroxamic Acid (12g).
A mixture of compound 37g (50 mg, 0.097 mmol), THPONH2 (13
mg, 0.116 mmol), HOBt (15 mg, 0.116 mmol), EDCI (22 mg, 0.116
mmol), and DIEA (25 mg, 0.194 mmol) in DMF (3 mL) was
degassed and purged with N2 3 times and the mixture was stirred at
20 °C for 2 h. Then, the mixture was concentrated in reduced
pressure at 40 °C and the residue was purified by prep-HPLC
(method 18 described in the Supporting Information) to afford pure
compound 12g (21.5 mg, 41%) as a yellow solid; mp 162−163 °C.
1H NMR (CD3OD, 400 MHz): δ 7.85 (d, J = 7.6 Hz, 2H), 7.66 (s,
1H), 6.42 (d, J = 3.6 Hz, 1H), 4.01 (d, J = 5.2 Hz, 6H), 3.61−3.55
(m, 4H), 2.86−2.77 (m, 3H), 2.59−2.42 (m, 8H), 2.21−2.18 (m,
3H), 1.66−1.55 (m, 2H); three exchangeable protons (NH from 4-
aminoquinoline and NH−OH from hydroxamic group) were not
observed. ESI-MS m/z: calcd for C27H34N4O5, 494.3; m/z: found,
495.4 [M + H]+. HPLC (method 1): Rt is 1.644 min and purity is
100%. HRMS [M + H]+: calcd, 495.2602; found, 495.2636, Δ = 6.9
ppm.
rac-cis 4-[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]cyclohexanecarbohydroxamic Acid (12j-cis). A solution of
compound 38j-cis (30 mg, 0.059 mmol) in HCl/EtOAc (10 mL, 2.0
M) was stirred at 20 °C for 3 h. Then, the mixture was concentrated
in reduced pressure at 40 °C and the residue was purified by prep-
HPLC (method 20 described in the Supporting Information) to
afford pure compound 12j-cis (5.4 mg, 21%) as a yellow solid; mp
173−174 °C. 1H NMR (CD3OD, 400 MHz): δ 7.74 (s, 1H), 7.53 (d,
J = 3.2 Hz, 1H), 7.42 (s, 1H), 7.01 (s, 1H), 6.42 (d, J = 2.8 Hz, 1H),
4.02 (d, J = 3.6 Hz, 6H), 3.97−3.94 (m, 1H), 2.51 (s, 3H), 2.23−2.16
(m, 3H), 1.96−1.80 (m, 4H), 1.68−1.63 (m, 2H); three exchangeable
protons (NH from 4-aminoquinoline and NH−OH from hydroxamic
group) were not observed. ESI-MS m/z:calcd for C23H27N3O5, 425.2;
m/z: found, 426.2 [M + H]+. HPLC (method 1): Rt is 2.069 min and
purity is 100%. HRMS [M + H]+: calcd, 426.2023; found, 426.2070,
Δ = 11.0 ppm.
rac-trans 4-[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]cyclohexanecarbohydroxamic Acid (12j-trans). A solution
of compound 38j-trans (20 mg, 0.039 mmol) in HCl/EtOAc (10 mL,
2.0 M) was stirred at 20 °C for 3 h. Then, the mixture was
concentrated in reduced pressure at 40 °C and the residue was
purified by prep-HPLC (method 21 described in the Supporting
Information) to afford pure compound 12j-trans (3.6 mg, 21%) as a
yellow oil. 1H NMR (CD3OD, 400 MHz): δ 7.80 (s, 1H), 7.49 (d, J =
3.2 Hz, 1H), 7.42 (s, 1H), 6.96 (s, 1H), 6.41 (d, J = 2.4 Hz, 1H), 4.04
(s, 1H), 4.03 (s, 6H), 2.50 (s, 3H), 2.42 (s, 1H), 2.15−2.06 (m, 4H),
1.95−1.93 (m, 2H), 1.80−1.79 (m, 2H); three exchangeable protons
(NH from 4-aminoquinoline and NH−OH from hydroxamic group)
were not observed. ESI-MS m/z: calcd for C23H27N3O5, 425.2; m/z:
found, 426.2 [M + H]+. HPLC (method 1): Rt is 2.131 min and purity
is 100%. HRMS [M + H]+: calcd, 426.2023; found, 426.2052, Δ = 6.8
ppm.
6-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]pyridine-3-carbohydroxamic Acid (12k). A
solution of compound 37k (95 mg, 0.189 mmol, 1 equiv), O-
tetrahydropyran-2-ylhydroxylamine (44.29 mg, 0.378 mmol, 2 equiv),
HOBt (51.08 mg, 0.378 mmol, 2 equiv), EDCI (72.47 mg, 0.378
mmol, 2 equiv), and DIEA (73.29 mg, 0.567 mmol, 98.78 μL, 3
equiv) in DMF (5 mL) was degassed and purged with N2 three times,
and then the mixture was stirred at 25 °C for 12 h under an N2
atmosphere. The reaction mixture was adjusted to pH ∼ 5 with aq
HCl (1 M) and concentrated to give a residue, which was purified by
prep-HPLC (method 17 described in the Supporting Information).
Compound 12k was obtained as a light yellow solid: 19.4 mg, 0.038
mmol, 19.8% yield, 99.94% purity; mp 183−184 °C. 1H NMR
(CD3OD, 400 MHz): δ 8.40 (s, 1H), 8.05 (d, J = 9.5 Hz, 1H), 7.71
(s, 1H), 7.52 (d, J = 3.4 Hz, 1H), 7.45 (s, 1H), 7.15 (d, J = 9.4 Hz,
1H), 6.99 (s, 1H), 6.42 (d, J = 2.6 Hz, 1H), 4.42 (d, J = 13.7 Hz, 2H),
4.03 (s, 3H), 4.02 (s, 3H), 3.59 (d, J = 7.0 Hz, 2H), 3.18 (t, J = 12.1
Hz, 2H), 2.50 (s, 3H), 2.28 (m, 1H), 2.12−2.01 (m, 2H), 1.54−1.42
(m, 2H); three exchangeable protons were not observed. ESI-MS m/
z:calcd for C28H31N5O5, 517.2; m/z: found, 518.3 [M + H]+. HPLC
(method 1): Rt is 1.553 min and purity is 99.94%. HRMS [M + H]+:
calcd, 518.2398; found, 518.2409, Δ = 2.1 ppm.
1H), 7.61 (d, J = 7.6 Hz, 2H), 7.52 (d, J = 3.6 Hz, 1H), 7.44 (s, 1H),
6.97 (s, 1H), 6.43 (d, J = 3.6 Hz, 1H), 4.37 (s, 2H), 4.03−4.02 (s,
3H), 4.02−4.01 (s, 3H), 3.63−3.50 (m, 4H), 3.09−3.03 (m, 2H),
2.50 (s, 3H), 2.18−2.14 (m, 3H), 1.66−1.63 (m, 2H); three
exchangeable protons (NH from 4-aminoquinoline and NH−OH
from hydroxamic group) were not observed. ESI-MS m/z: calcd for
C30H34N4O5, 530.3; m/z: found, 531.4 [M + H]+. UHPLC (method
3): Rt is 2.04 min and purity is 100%. HRMS [M + H]+: calcd,
531.2602; found, 531.2620, Δ = 3.4 ppm.
5-[[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]methyl]thiophene-2-carbohydroxamic Acid
(12h). A mixture of compound 37h (60 mg, 0.115 mmol),
THPONH2 (16 mg, 0.138 mmol), HOBt (19 mg, 0.138 mmol),
EDCI (26 mg, 0.138 mmol), and DIEA (18 mg, 0.138 mmol) in
DMF (3 mL) was degassed and purged with N2 3 times and the
mixture was stirred at 20 °C for 16 h. Then, the mixture was
concentrated in reduced pressure at 40 °C and the residue was
purified by prep-HPLC (method 19 described in the Supporting
Information) to afford pure compound 12h (5.0 mg, 8%) as a yellow
1
solid; mp 165−166 °C. H NMR (CD3OD, 400 MHz): δ 7.68 (s,
1H), 7.55 (s, 1H), 7.52 (d, J = 3.2 Hz, 1H), 7.45 (s, 1H), 7.32 (d, J =
2.8 Hz, 1H), 6.97 (s, 1H), 6.42 (d, J = 3.2 Hz, 1H), 4.58 (s, 2H),
4.03−4.01 (m, 6H), 3.60 (m, 4H), 3.13−3.06 (m, 2H), 2.50 (s, 3H),
2.20−2.16 (m, 3H), 1.68 (s, 2H); three exchangeable protons (NH
from 4-aminoquinoline and NH−OH from hydroxamic group) were
not observed. ESI-MS m/z: calcd for C28H32N4O5S, 536.2; m/z:
found, 537.2 [M + H]+. HPLC (method 1): Rt is 1.76 min and purity
is 95.42%. HRMS [M + H]+: calcd, 537.2166; found, 537.2132, Δ =
6.3 ppm.
3-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]cyclobutanecarbohydroxamic Acid (12i). A
solution of compound 38i (50 mg, 0.086 mmol) in HCl/EtOAc
(10 mL, 2.0 M) was stirred at 20 °C for 20 min. Then, the mixture
was concentrated in reduced pressure at 40 °C and the residue was
purified by prep-HPLC (method 17 described in the Supporting
Information) to afford pure compound 12i (14.7 mg, 34%) as a
yellow solid; mp 150−151 °C. 1H NMR (CD3OD, 400 MHz): δ 7.67
(d, J = 3.6 Hz, 1H), 7.52 (d, J = 3.6 Hz, 1H), 7.44 (s, 1H), 6.97 (s,
2-[4-[[(6,7-Dimethoxy-2-methyl-4-quinolyl)amino]methyl]-1-
piperidyl]pyrimidine-5-carbohydroxamic Acid (13a). A solution of
compound 42a (300 mg, 0.559 mmol) in HCl/EtOAc (10 mL, 1.0
M) was stirred at 20 °C for 16 h. Then, the reaction mixture was
concentrated in vacuum and the residue was purified by prep-HPLC
(method 8 described in the Supporting Information) to afford pure
1
compound 13a (4 mg, 2%) as a yellow oil. H NMR (CD3OD, 400
MHz): δ 8.65 (s, 2H), 7.68 (s, 1H), 7.13 (s, 1H), 6.68 (s, 1H), 4.95−
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4.93 (m, 2H), 4.01−4.00 (m, 6H), 3.49−3.47 (m, 2H), 3.02−2.96
(m, 2H), 2.64 (s, 3H), 2.21−2.17 (m, 1H), 1.96−1.93 (m, 2H),
1.37−1.30 (m, 2H); three exchangeable protons (NH from 4-
aminoquinoline and NH−OH from hydroxamic group) were not
observed. ESI-MS m/z: calcd for C23H28N6O4, 452.2; m/z: found,
453.3 [M + H]+. HPLC (method 1): Rt is 1.528 min and purity is
98.28%. HRMS [M + H]+: calcd, 453.2245; found, 453.2209, Δ = 7.9
ppm.
(m, 2H); three exchangeable protons were not observed. ESI-MS m/
z: calcd for C28H32N6O5, 532.2; m/z: found, 533.3 [M + H]+. HPLC
(method 1): Rt is 2.288 min and purity is 100%. HRMS [M + H]+:
calcd, 533.2518; found, 533.2549, Δ = 5.8 ppm.
2-(4-(((6,7-Dimethoxy-2-(5-methylthiophen-2-yl)quinolin-4-yl)-
amino)methyl)piperidin-1-yl)-N-hydroxypyrimidine-5-carboxa-
mide (13f). To a solution of compound 42f (40 mg, 0.065 mmol, 1
equiv) in MeCN (5 mL) and water (5 mL) was added TFA (73.71
mg, 0.647 mmol, 47.86 μL, 10 equiv). The mixture was stirred at 60
°C for 5 min. HPLC showed that the starting material was consumed
completely and one main peak was detected. The solvent was
removed. The residue was purified by prep-HPLC (method 45
described in the Supporting Information) to afford pure compound
13f (24.8 mg, 0.046 mmol, 71.06% yield and 98.54% purity) as an off-
yellow solid; its mp is 177−178 °C. 1H NMR (CD3OD, 400 MHz): δ
8.65 (s, 2H), 7.80 (d, J = 3.8 Hz, 1H), 7.70 (s, 1H), 7.39 (s, 1H), 7.03
(d, J = 2.8 Hz, 1H), 6.83 (s, 1H), 4.94 (br s, 2H), 4.04 (s, 3H), 4.03
(s, 3H), 3.56 (d, J = 6.9 Hz, 2H), 3.01 (t, J = 12.2 Hz, 2H), 2.62 (s,
3H), 2.22 (br s, 1H), 1.97 (d, J = 12.7 Hz, 2H), 1.37−1.34 (m, 2H);
three exchangeable protons were not observed. ESI-MS m/z: calcd for
C27H30N6O4S, 534.2; m/z: found, 535.3 [M + H]+. HPLC (method
1): Rt is 2.186 min and purity is 98.543%. HRMS [M + H]+: calcd,
535.2122; found, 535.2138, Δ = 3.0 ppm.
2-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)quinazolin-4-yl]-
amino]methyl]-1-piperidyl]pyrimidine-5-carbohydroxamic Acid
(14). To a solution of 47 (130 mg, 0.215 mmol) in CH3CN/H2O
(1:1, 10 mL) was added TFA (1.54 g, 13.51 mmol, 1 mL) and the
mixture was stirred at 60 °C for 5 min. Then, the residue was purified
by prep-HPLC (method 14 described below) to afford pure
compound 42 (64.3 mg, 56%) as an off-white solid; mp 160−161
°C. 1H NMR (MeOD, 400 MHz): δ 8.64 (s, 2H), 7.71 (s, 1H), 7.58
(d, J = 3.5 Hz, 1H), 7.35 (s, 1H), 6.47 (d, J = 2.5 Hz, 1H), 4.90 (br s,
2H), 4.04 (s, 3H), 4.01 (s, 3H), 3.78 (d, J = 6.8 Hz, 2H), 3.01 (br t, J
= 11.5 Hz, 2H), 2.53 (s, 3H), 2.22 (br s, 1H), 1.94 (br d, J = 11.7 Hz,
2H), 1.39−1.31 (m, 2H); three exchangeable protons (NH from 4-
aminoquinoline and NH−OH from hydroxamic group) were not
observed. ESI-MS m/z: calcd for C26H29N7O5, 519.2; m/z: found,
520.3 [M + H]+. HPLC (method 1): Rt is 1.897 min and purity is
94.528%. HRMS [M + H]+: calcd, 520.2303; found, 520.2309, Δ =
1.2 ppm.
2-[4-[[(2-Cyclohexyl-6,7-dimethoxy-4-quinolyl)amino]methyl]-1-
piperidyl]pyrimidine-5-carbohydroxamic Acid (13b). A solution of
compound 42b (50 mg, 0.083 mmol) in HCl/EtOAc (10 mL, 2.0 M)
was stirred at 20 °C for 4 h. Then, the mixture was concentrated in
reduced pressure at 40 °C and the residue was purified by prep-HPLC
(method 6 described in the Supporting Information) to afford pure
1
compound 13b (9 mg, 21%) as a yellow solid; mp 155−156 °C. H
NMR (CD3OD, 400 MHz): δ 8.65 (s, 2H), 7.67 (s, 1H), 7.24 (s,
1H), 6.62 (s, 1H), 4.91 (d, J = 13.2 Hz, 2H), 4.01 (d, J = 2.4 Hz, 6H),
3.51 (d, J = 6.8 Hz, 2H), 3.03−2.97 (m, 2H), 2.87−2.84 (m, 1H),
2.19 (m, 1H), 2.03−1.94 (m, 6H), 1.72−1.50 (m, 3H), 1.49−1.32
(m, 5H); three exchangeable protons (NH from 4-aminoquinoline
and NH−OH from hydroxamic group) were not observed. ESI-MS
m/z:calcd for C28H36N6O4, 520.3; m/z: found, 521.3 [M + H]+.
HPLC (method 1): Rt is 2.351 min and purity is 100%. HRMS [M +
H]+: calcd, 521.2871; found, 521.2837, Δ = 6.5 ppm.
2-[4-[[(6,7-Dimethoxy-2-phenyl-4-quinolyl)amino]methyl]-1-
piperidyl]pyrimidine-5-carbohydroxamic Acid (13c). A solution of
compound 42c (60 mg, 0.100 mmol) in HCl/EtOAc (10 mL, 2.0 M)
was stirred at 20 °C for 4 h. Then, the mixture was concentrated in
reduced pressure at 40 °C and the residue was purified by prep-HPLC
(method 7 described in the Supporting Information) to afford pure
compound 13c (23.3 mg, 45%) as a yellow solid; mp 165−166 °C. 1H
NMR (CD3OD, 400 MHz): δ 8.64 (s, 2H), 7.91 (t, J = 5.2 Hz, 2H),
7.75 (s, 1H), 7.66−7.65 (m, 3H), 7.39 (s, 1H), 6.96 (s, 1H), 4.92−
4.89 (m, 2H), 4.04 (s, 6H), 3.59 (d, J = 6.8 Hz, 2H), 3.00 (d, J = 12.0
Hz, 2H), 2.24 (s, 1H), 1.97 (d, J = 12.0 Hz, 2H), 1.39−1.31 (s, 2H);
three exchangeable protons (NH from 4-aminoquinoline and NH−
OH from hydroxamic group) were not observed. ESI-MS m/z: calcd
for C28H30N6O4, 514.3; m/z: found, 515.2 [M + H]+. HPLC (method
1): Rt is 2.157 min and purity is 100%. HRMS [M + H]+: calcd,
515.2401; found, 515.2363, Δ = 7.4 ppm.
Ethyl 2-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-yl-
propoxy)-4-quinolyl]amino]methyl]-1-piperidyl]pyrimidine-5-car-
boxylate (17a). A mixture of compound 1634,35 (200 mg, 0.499
mmol), ethyl 2-[4-(aminomethyl)-1-piperidyl]pyrimidine-5-carboxy-
late (51, 198 mg, 0.748 mmol), Pd2(dba)3 (46 mg, 0.050 mmol),
BINAP (31 mg, 0.050 mmol), and Cs2CO3 (325 mg, 0.998 mmol) in
1,4-dioxane (10 mL) was degassed and purged with N2 3 times and
the mixture was stirred at 120 °C for 16 h. Then, the mixture was
concentrated in reduced pressure at 40 °C to give a residue and this
residue was poured into water (10 mL) and extracted with EtOAc (10
mL × 3). The combined organic phase was washed with brine (10 mL
× 2), dried with anhydrous Na2SO4, filtered, and concentrated in
vacuum to give a residue. The residue was purified by prep-HPLC
(method 23 described in the Supporting Information) to afford pure
compound 17a (80 mg, 25%) as a yellow solid. ESI-MS m/z: calcd for
C35H44N6O5, 628.3; m/z: found, 629.4 [M + H]+. This compound
was used in the next step without further characterization.
Ethyl 4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-yl-
propoxy)-4-quinolyl]amino]methyl]benzoate (17b). A mixture of
compound 1634,35 (100 mg, 0.249 mmol), ethyl 4-(aminomethyl)-
benzoate (58 mg, 0.324 mmol), Cs2CO3 (162 mg, 0.499 mmol),
BINAP (15 mg, 0.025 mmol), and Pd2(dba)3 (23 mg, 0.025 mmol) in
1,4-dioxane (3 mL) was degassed and purged with N2 3 times and the
mixture was stirred at 120 °C for 16 h. Then, the mixture was filtrated
and the filtrate was concentrated in reduced pressure at 40 °C to give
compound 17b (100 mg, 74%) as a yellow solid. ESI-MS m/z: calcd
for C32H37N3O5, 543.3; m/z: found, 544.3 [M + H]+. This
intermediate was used in the next step without further purification
or characterization.
2-[4-[[[6,7-Dimethoxy-2-(1-piperidyl)-4-quinolyl]amino]methyl]-
1-piperidyl]pyrimidine-5-carbohydroxamic Acid (13d). A solution
of compound 42d (0.3 g, 0.495 mmol) in HCl/EtOAc (10 mL, 1.0
M) was stirred at 15 °C for 16 h. Then, the reaction mixture was
concentrated in vacuum to give a residue, which was purified by prep-
HPLC (method 8 described in the Supporting Information) to afford
pure compound 13d (65.6 mg, 25%) as a white solid; mp 129−130
1
°C. H NMR (CD3OD, 400 MHz): δ 8.65 (m, 2H), 7.56 (s, 1H),
7.25 (s, 1H), 5.91 (s, 1H), 4.92 (s, 2H), 3.96 (s, 6H), 3.67 (s, 4H),
3.41−3.39 (m, 2H), 3.03−2.97 (m, 2H), 2.21−2.19 (br s, 1H), 1.96−
1.94 (m, 2H), 1.77 (s, 6H), 1.36−1.30 (m, 2H); three exchangeable
protons (NH from 4-aminoquinoline and NH−OH from hydroxamic
group) were not observed. ESI-MS m/z: calcd for C27H35N7O4,
521.3; m/z: found, 522.4 [M + H]+. HPLC (method 1): Rt is 2.233
min and purity is 98.77%. HRMS [M + H]+: calcd, 522.2823; found,
522.2834, Δ = 2.1 ppm.
2-[4-[[[2-(2,5-dimethyl-3-furyl)-6,7-dimethoxy-4-quinolyl]-
amino]methyl]-1-piperidyl]pyrimidine-5-carbohydroxamic Acid
(13e). To a solution of compound 42e (0.6 g, 0.973 mmol, 1
equiv) in MeCN (5 mL) and water (10 mL) was added TFA (770.00
mg, 6.75 mmol, 0.5 mL, 6.94 equiv).The mixture was stirred at 60 °C
for 1 h. LCMS showed that the reaction was completed and one main
peak with the desired m/z was detected. MeCN was removed in
vacuum, the residue was freeze-dried by the lyophilizer. Compound
13e was obtained as a light yellow solid: 0.42 g, 0.789 mmol, 81.1%
1
yield and 100% purity; its mp is 176−177 °C. H NMR (CD3OD,
400 MHz): δ 8.65 (s, 2H), 7.71 (s, 1H), 7.32 (s, 1H), 6.68 (s, 1H),
6.47 (s, 1H), 4.92 (br d, J = 13.2 Hz, 2H), 4.03 (s, 6H), 3.53 (br d, J =
7.1 Hz, 2H), 2.99 (br t, J = 11.9 Hz, 2H), 2.56 (s, 3H), 2.35 (s, 3H),
2.21 (br d, J = 4.2 Hz, 1H), 1.96 (br d, J = 11.0 Hz, 2H), 1.38−1.30
Ethyl 4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-yl-
propoxy)-4-quinolyl]amino]methyl]cyclohexanecarboxylate (17c).
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A mixture of compound 1634,35 (200 mg, 0.499 mmol), ethyl 4-
(aminomethyl)cyclohexanecarboxylate (139 mg, 0.748 mmol),
BINAP (31 mg, 0.050 mmol), Pd2(dba)3 (46 mg, 0.050 mmol),
and Cs2CO3 (325 mg, 0.998 mmol) in 1,4-dioxane (20 mL) was
degassed and purged with N2 3 times and the mixture was stirred at
120 °C for 16 h. The mixture was cooled to 20 °C and concentrated
in reduced pressure at 40 °C. The residue was poured into water (20
mL) and extracted with CH2Cl2 (20 mL × 3). The combined organic
phase was washed with brine (20 mL × 2), dried with anhydrous
Na2SO4, filtered, and concentrated in vacuum to afford compound
17c (200 mg, 73%) as a yellow solid. ESI-MS m/z: calcd for
C32H43N3O5, 549.3; m/z: found, 550.3 [M + H]+. This intermediate
was used in the next step without further purification or character-
ization.
Methyl 4-[[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-
ylpropoxy)-4-quinolyl]amino]methyl]-1-piperidyl]methyl]benzoate
(17d). A mixture of compound 1634,35 (140 mg, 0.349 mmol), methyl
4-[[4-(aminomethyl)-1-piperidyl]methyl]benzoate (54, 92 mg, 0.349
mmol), Pd2(dba)3 (32 mg, 0.035 mmol), BINAP (22 mg, 0.035
mmol), and Cs2CO3 (228 mg, 0.698 mmol) in 1,4-dioxane (20 mL)
was degassed and purged with N2 3 times and the mixture was stirred
at 120 °C for 16 h. Then, the mixture was filtered and the filtrate was
concentrated. The residue was diluted with water (10 mL) and
extracted with EtOAc (15 mL). The organic phase was acidized with
aqueous HCl (1.0 N) to pH = 3 and the organic phase was separated.
The aqueous phase was alkalized by saturated NaHCO3 solution to
pH = 8 and extracted with EtOAc (15 mL × 3). The combined
organic layers were washed with brine (5 mL), dried over Na2SO4,
filtered, and concentrated under reduced pressure to afford compound
17d (220 mg, crude) as a yellow solid. ESI-MS m/z: calcd for
C37H46N4O5, 626.4; m/z: found, 627.4 [M + H]+. This intermediate
was used in the next step without further purification or character-
ization.
Methyl 4-[[4-[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-
ylpropoxy)-4-quinolyl]amino]-1-piperidyl]methyl]benzoate (17e).
A mixture of compound 1634,35 (200 mg, 0.499 mmol), methyl 4-
[(4-amino-1-piperidyl)methyl]benzoate (57, 248 mg, 0.998 mmol),
BINAP (31 mg, 0.050 mmol), Pd2(dba)3 (46 mg, 0.050 mmol), and
Cs2CO3 (325 mg, 0.998 mmol) in 1,4-dioxane (20 mL) was degassed
and purged with N2 3 times and the mixture was stirred at 110 °C for
16 h. The mixture was cooled to 20 °C and concentrated in reduced
pressure at 40 °C. The residue was poured into water (20 mL) and
extracted with EtOAc (10 mL × 3). The combined organic phase was
washed with brine (10 mL × 2), dried with anhydrous Na2SO4,
filtered, and concentrated in vacuum to give a residue, which was
purified by prep-TLC (SiO2, CH2Cl2/MeOH = 10:1) to afford pure
compound 17e (150 mg, 49%) as a yellow solid. ESI-MS m/z: calcd
for C36H44N4O5, 612.3; m/z: found, 613.3 [M + H]+. This compound
was used in the next step without further characterization.
Methyl 4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-
ylpropoxy)-4-quinolyl]amino]methyl]-1-piperidyl]benzoate (17f). A
mixture of compound 1634,35 (300 mg, 0.748 mmol), methyl 4-[4-
(aminomethyl)-1-piperidyl]benzoate (60, 204 mg, 0.823 mmol),
Pd2(dba)3 (69 mg, 0.075 mmol), BINAP (47 mg, 0.075 mmol), and
Cs2CO3 (488 mg, 1.50 mmol) in 1,4-dioxane (30 mL) was degassed
and purged with N2 3 times and the mixture was stirred at 120 °C for
16 h. Then, the mixture was filtered and the filtrate was concentrated
to give a residue, which was washed with a solution of PE/EtOAc =
10/1 to afford compound 17f (220 mg, 48%) as a yellow solid. ESI-
MS m/z: calcd for C36H44N4O5, 612.3; m/z: found, 613.3 [M + H]+.
This intermediate was used in the next step without further
purification or characterization.
filtrate was concentrated to give a residue. The residue was washed
with a solution of PE/EtOAc = 15/1 to afford intermediate tert-butyl
4-[[[6-methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpropoxy)-4-
quinolyl]amino]methyl]piperidine-1-carboxylate (250 mg, 34%) as a
yellow solid. ESI-MS m/z: calcd for C33H46N4O5, 578.4; m/z: found,
579.4 [M + H]+. Then, a solution of this intermediate (250 mg, 0.432
mmol) in HCl/EtOAc (5 mL, 2.0 M) was stirred at 20 °C for 1 h.
The mixture was concentrated to give the crude product. The crude
product was diluted with MeOH (15 mL) and NaHCO3 solid was
added into the mixture to pH = 8. The mixture was filtered and the
filtrate was concentrated to give intermediate 6-methoxy-2-(5-methyl-
2-furyl)-N-(4-piperidylmethyl)-7-(3-pyrrolidin-1-ylpropoxy)quinolin-
4-amine(180 mg, 87%) as a yellow solid. ESI-MS m/z: calcd for
C28H38N4O3, 478.3; m/z: found, 479.3 [M + H]+. Finally, to a
solution of ZnCl2/diethyl ether (1.0 M, 20 μL) was added NaBH3CN
(52 mg, 0.827 mmol) in MeOH (10 mL) and the mixture was stirred
at 20 °C for 30 min. Then, a mixture of ethyl 4-oxocyclohex-
anecarboxylate (70 mg, 0.414 mmol) and intermediate 6-methoxy-2-
(5-methyl-2-furyl)-N-(4-piperidylmethyl)-7-(3-pyrrolidin-1-
ylpropoxy)quinolin-4-amine (180 mg, 0.376 mmol) in MeOH (40
mL) was added into the reaction solution. The mixture was heated at
40 °C for 15.5 h. Then, the reaction was quenched with water (10
mL) and the mixture was filtered. The filtrate was concentrated and
extracted with a solution of CH2Cl2/MeOH = 3:1. The combined
organic layers were washed with brine (10 mL), dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure to afford
compound 17g (155 mg, 65%) as a white solid. ESI-MS m/z: calcd
for C37H52N4O5, 632.4; m/z: found, 633.3 [M + H]+. This
intermediate was used in the next step without further purification
or characterization.
Methyl 4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-
ylpropoxy)-4-quinolyl]amino]methyl]cyclohexyl]benzoate (17h). A
mixture of compound 1634,35 (200 mg, 0.499 mmol), methyl 4-[4-
(aminomethyl)cyclohexyl]benzoate (64, 185 mg, 0.748 mmol),
BINAP (31 mg, 0.050 mmol), Pd2(dba)3 (46 mg, 0.050 mmol),
and Cs2CO3 (325 mg, 0.998 mmol) in 1,4-dioxane (10 mL) was
degassed and purged with N2 3 times and the mixture was stirred at
120 °C for 16 h. Then, the mixture was concentrated in reduced
pressure at 40 °C; the residue was poured into water (10 mL) and
extracted with EtOAc (10 mL × 3). The combined organic phase was
washed with brine (10 mL × 2), dried with anhydrous Na2SO4,
filtered, and concentrated in vacuum to give a residue, which was
purified by prep-TLC (SiO2, CH2Cl2/MeOH = 5:1) to afford pure
compound 17h (150 mg, 49%) as a yellow solid. ESI-MS m/z: calcd
for C37H45N3O5, 609.3; m/z: found, 610.3 [M + H]+. This compound
was used in the next step without further characterization.
Methyl 4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-
ylpropoxy)-4-quinolyl]amino]methyl]phenyl]benzoate (17i). A mix-
ture of compound 1634,35 (200 mg, 0.499 mmol), methyl 4-[4-
(aminomethyl)phenyl]benzoate (68, 180 mg, 0.748 mmol), BINAP
(31 mg, 0.050 mmol), Pd2(dba)3 (46 mg, 0.050 mmol), and Cs2CO3
(325 mg, 0.998 mmol) in 1,4-dioxane (15 mL) was degassed and
purged with N2 3 times and the mixture was stirred at 110 °C for 16
h. Then, the mixture was concentrated in reduced pressure at 40 °C;
the residue was poured into water (10 mL) and extracted with EtOAc
(10 mL × 3). The combined organic phase was washed with brine
(10 mL × 2), dried with anhydrous Na2SO4, filtered, and
concentrated in vacuum to give a residue, which was purified by
prep-TLC (SiO2, CH2Cl2/MeOH = 10:1) to afford pure compound
17i (200 mg, 66%) as a yellow solid. ESI-MS m/z: calcd for
C37H39N3O5, 605.3; m/z: found, 606.2 [M + H]+. This compound
was used in the next step without further characterization.
Ethyl 4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-yl-
p r o p o x y ) - 4 - q u i n o l y l ] a m i n o ] m e t h y l ] - 1 - p i p e r i d y l ] -
cyclohexanecarboxylate (17g). A mixture of compound 1634,35 (500
mg, 1.25 mmol), tert-butyl 4-(aminomethyl)piperidine-1-carboxylate
(267 mg, 1.25 mmol), Pd2(dba)3 (114 mg, 0.125 mmol), BINAP (78
mg, 0.125 mmol), and Cs2CO3 (813 mg, 2.49 mmol) in 1,4-dioxane
(10 mL) was degassed and purged with N2 3 times and the mixture
was stirred at 120 °C for 16 h. Then, the mixture was filtered and the
Ethyl 4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-yl-
p r o p o x y ) - 4 - q u i n o l y l ] a m i n o ] m e t h y l ] p h e n y l ] -
cyclohexanecarboxylate (17j). A mixture of compound 1634,35 (400
mg, 0.998 mmol), ethyl 4-[4-(aminomethyl)phenyl]-
cyclohexanecarboxylate (73, 261 mg, 0.998 mmol), Pd2(dba)3 (92
mg, 0.100 mmol), BINAP (62 mg, 0.100 mmol), and Cs2CO3 (650
mg, 2.00 mmol) in 1,4-dioxane (50 mL) was degassed and purged
with N2 3 times and the mixture was stirred at 120 °C for 16 h. Then,
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the mixture was filtered and the filtrate was concentrated to give a
residue, which was washed with a solution of PE/EtOAc = 10/1 to
give compound 17j (162 mg, 26%) as a yellow solid. ESI-MS m/z:
calcd for C38H47N3O5, 625.3; m/z: found, 626.2 [M + H]+. This
compound was used in the next step without further purification or
characterization.
mg, 0.735 mmol) in THF/MeOH/H2O (3:1:1, 5.0 mL) was stirred at
20 °C for 16 h. Then, the mixture was concentrated and the residue
was diluted with water (0.5 mL) and extracted with EtOAc (3 mL).
The aqueous layer was acidified to pH = 3−4 with 1 N HCl and
concentrated to dryness to afford compound 18e (150 mg, crude) as a
yellow solid. ESI-MS m/z: calcd for C35H42N4O5, 598.3; m/z: found,
599.3 [M + H]+. This intermediate was used in the next step without
further purification or characterization.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]-1-piperidyl]benzoic Acid (18f). To
a solution of compound 17f (220 mg, 0.359 mmol) in EtOH/H2O
(2:1, 6.0 mL) was added LiOH·H2O (31 mg, 0.718 mmol) and the
mixture was stirred at 20 °C for 3 h. Then, the solution was
concentrated to give a residue. The residue was acidized with 1.0 M
aqueous HCl to pH = 3 and the mixture was concentrated to give
compound 18f (155 mg, 72%) as a yellow solid. ESI-MS m/z: calcd
for C35H42N4O5, 598.3; m/z: found, 599.3 [M + H]+. This
intermediate was used in the next step without further purification
or characterization.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]-1-piperidyl]cyclohexanecarboxylic
Acid (18g). To a solution of compound 17g (155 mg, 0.245 mmol) in
EtOH/H2O (2:1, 12.0 mL) was added LiOH·H2O (21 mg, 0.490
mmol) and the mixture was stirred at 20 °C for 2 h. Then, the
solution was concentrated to give a residue. The residue was acidized
with 1.0 M aqueous HCl to pH = 3 and the mixture was concentrated
to give compound 18g (105 mg, 71%) as a yellow solid. ESI-MS m/z:
calcd for C35H48N4O5, 604.4; m/z: found, 605.4 [M + H]+. This
intermediate was used in the next step without further purification or
characterization.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]cyclohexyl]benzoic Acid (18h). A
mixture of compound 17h (150 mg, 0.245 mmol) and LiOH·H2O
(31 mg, 0.735 mmol) in THF/MeOH/H2O (3:1:1, 5.0 mL) was
stirred at 20 °C for 16 h. Then, the mixture was concentrated and the
residue was diluted with water (2 mL) and extracted with EtOAc (3
mL). The aqueous layer was acidified to pH = 3−4 with 1.0 N HCl.
The filter was concentrated to dryness to give a residue, which was
purified by prep-HPLC (method 24 described in the Supporting
Information) to afford pure compound 18h (60 mg, 41%) as a yellow
solid. ESI-MS m/z: calcd for C36H43N3O5, 597.3; m/z: found, 598.3
[M + H]+. This compound was used in the next step without further
characterization.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]phenyl]benzoic Acid (18i). A mix-
ture of compound 17i (200 mg, 0.330 mmol) and LiOH·H2O (41
mg, 0.990 mmol) in THF/MeOH/H2O (3:1:1, 5.0 mL) was stirred at
20 °C for 5 h. Then, the mixture was concentrated and the residue
was diluted with water (0.5 mL) and extracted with EtOAc (3 mL).
The aqueous phase was acidified to pH = 3−4 with 1.0 N HCl and
concentrated to dryness to give compound 18i (150 mg, 77%) as a
yellow solid. ESI-MS m/z: calcd for C36H37N3O5, 591.3; m/z: found,
592.3 [M + H]+. This intermediate was used in the next step without
further purification or characterization.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]phenyl]cyclohexanecarboxylic
Acid (18j). To a solution of compound 17j (160 mg, 0.256 mmol) in
EtOH/H2O (2:1, 6.0 mL) was added LiOH·H2O (21 mg, 0.511
mmol), which was stirred at 20 °C for 2 h. Then, the mixture was
acidized with 1.0 M HCl to pH = 3 and concentrated to give a
residue. The residue was diluted with MeOH (5 mL), filtered, and the
filtrate was concentrated to give compound 18j (135 mg, 88%) as a
yellow solid. ESI-MS m/z: calcd for C36H43N3O5, 597.3; m/z: found,
598.3 [M + H]+. This compound was used in the next step without
further purification or characterization.
Methyl 4-[4-[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-
ylpropoxy)-4-quinolyl]amino]-1-piperidyl]benzoate (17k). A mix-
ture of compound 1634,35 (300 mg, 0.748 mmol), methyl 4-(4-amino-
1-piperidyl)benzoate (75, 210 mg, 0.898 mmol), BINAP (93 mg,
0.150 mmol), Pd2(dba)3 (137 mg, 0.150 mmol), and Cs2CO3 (488
mg, 1.50 mmol) in 1,4-dioxane (20 mL) was degassed and purged
with N2 3 times and the mixture was stirred at 110 °C for 16 h. Then,
the mixture was concentrated in reduced pressure at 40 °C; the
residue was poured into water (20 mL) and extracted with EtOAc (20
mL × 3). The combined organic phase was washed with brine (20 mL
× 2), dried with anhydrous Na2SO4, filtered, and concentrated in
vacuum to give a residue, which was purified by prep-TLC (SiO2,
CH2Cl2/MeOH = 10:1) to afford pure compound 17k (200 mg,
44%) as a yellow solid. ESI-MS m/z: calcd for C35H42N4O5, 598.3; m/
z: found, 599.3 [M + H]+. This compound was used in the next step
without further characterization.
2-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]-1-piperidyl]pyrimidine-5-carbox-
ylic Acid (18a). To a solution of compound 17a (100 mg, 0.155
mmol) in THF/MeOH/H2O (5:1:3, 9.0 mL) was added LiOH·H2O
(10 mg, 0.233 mmol) and the mixture was stirred at 25 °C for 12 h.
Then, the reaction mixture was concentrated and the pH was adjusted
pH to 3−4 with 3.0 M aqueous HCl and the solid was precipitated.
The solid was collected to give compound 18a (90 mg, 94%) as a
yellow solid. ESI-MS m/z: calcd for C33H40N6O5, 600.3; m/z: found,
601.3 [M + H]+. This intermediate was used in the next step without
further purification or characterization.
4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]benzoic Acid (18b). A mixture of
compound 17b (100 mg, 0.184 mmol) and LiOH·H2O (15 mg, 0.368
mmol) in THF/MeOH/H2O (3:1:1, 5.0 mL) was degassed and
purged with N2 3 times and the mixture was stirred at 20 °C for 16 h.
Then, the mixture was concentrated and the residue was poured into
water (2 mL) and extracted with EtOAc (3 mL). The mixture was
adjusted to pH 3 with 2.0 M HCl. Then, the solution was filtered and
the filter cake was concentrated to dryness to give compound 18b (80
mg, 84%) as a yellow solid. ESI-MS m/z: calcd for C30H33N3O5,
515.2; m/z: found, 516.3 [M + H]+. This intermediate was used in
the next step without further purification or characterization.
4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]cyclohexanecarboxylic Acid (18c).
A mixture of compound 17c (200 mg, 0.364 mmol) and LiOH·H2O
(45 mg, 1.09 mmol) in THF/MeOH/H2O (3:1:1, 5.0 mL) was
degassed and purged with N2 3 times and the mixture was stirred at
20 °C for 16 h. Then, the mixture was concentrated and the residue
was poured into water (3 mL) and extracted with EtOAc (5 mL). The
mixture was acidified to pH = 3−4 with 1 N HCl and filtered. The
filter cake was concentrated to dryness to give compound 18c (100
mg, 52%) as a yellow solid. ESI-MS m/z: calcd for C30H39N3O5,
521.3; m/z: found, 522.3 [M + H]+. This intermediate was used in
the next step without further purification or characterization.
4-[[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]-1-piperidyl]methyl]benzoic Acid
(18d). To a mixture of compound 17d (220 mg, 0.351 mmol) in
EtOH/H2O (2:1, 6.0 mL) was added LiOH·H2O (29 mg, 0.702
mmol) and the mixture was stirred at 20 °C for 1 h. Then, the
solution was concentrated to give a residue. The residue was acidized
with 1.0 M aqueous HCl to pH = 3. Then, the mixture was
concentrated to give compound 18d (140 mg, 65%) as a white solid.
ESI-MS m/z: calcd for C36H44N4O5, 612.3; m/z: found, 613.3 [M +
H]+. This intermediate was used in the next step without further
purification or characterization.
4-[4-[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]-1-piperidyl]benzoic Acid (18k). A mixture
of compound 17k (200 mg, 0.334 mmol) and LiOH·H2O (42 mg,
1.00 mmol) in THF/MeOH/H2O (3:1:1, 5.0 mL) was stirred at 20
°C for 16 h. Then, the mixture was concentrated and the residue was
diluted with water (0.5 mL) and extracted with EtOAc (3 mL). The
4-[[4-[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]-1-piperidyl]methyl]benzoic Acid (18e). A
mixture of compound 17e (150 mg, 0.245 mmol) and LiOH·H2O (31
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aqueous phase was acidified to pH = 3−4 with 1.0 N HCl and
concentrated to dryness to afford compound 18k (150 mg, 76%) as a
yellow solid. ESI-MS m/z: calcd for C34H40N4O5, 584.3; m/z: found,
585.3 [M + H]+. This intermediate was used in the next step without
further purification or characterization.
with water (10 mL) and extracted with EtOAc (15 mL). The organic
phase was separated and the aqueous phase was concentrated to give
the residue, which was purified by prep-HPLC (method 10 described
in the Supporting Information) to afford pure compound 19f (85 mg,
61%) as a yellow solid. ESI-MS m/z: calcd for C40H51N5O6, 697.4; m/
z: found, 698.4 [M + H]+. This compound was used in the next step
without further characterization.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]-1-piperidyl]-N-tetrahydropyran-2-
yloxy-cyclohexanecarboxamide (19g). A mixture of compound 18g
(105 mg, 0.174 mmol), THPONH2 (22 mg, 0.191 mmol), EDCI (37
mg, 0.191 mmol), HOBt (35 mg, 0.260 mmol), and DIEA (45 mg,
0.347 mmol) in DMF (10 mL) was degassed and purged with N2 3
times and the mixture was stirred at 20 °C for 2 h. Then, the mixture
was concentrated and the residue was purified by prep-HPLC
(method 18 described in the Supporting Information) to afford pure
compound 19g (50 mg, 41%) as a yellow solid. ESI-MS m/z: calcd for
C40H57N5O6, 703.4; m/z: found, 704.5 [M + H]+. This compound
was used in the next step without further characterization.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]cyclohexyl]-N-tetrahydropyran-2-
yloxy-benzamide (19h). A mixture of compound 18h (60 mg, 0.1
mmol), THPONH2 (20 mg, 0.168 mmol), HOBt (14 mg, 0.101
mmol), EDCI (19 mg, 0.101 mmol), and DIEA (22 mg, 0.101 mmol)
in DMF (5 mL) was degassed and purged with N2 3 times and then
the mixture was stirred at 20 °C for 2 h. Then, the mixture was
concentrated in reduced pressure at 40 °C and the residue was
purified by prep-HPLC (method 28 described in the Supporting
Information) to afford pure compound 19h (15 mg, 21%) as a yellow
solid. ESI-MS m/z: calcd for C41H52N4O6, 696.4; m/z: found, 697.4
[M + H]+. This compound was used in the next step without further
characterization.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]phenyl]-N-tetrahydropyran-2-
yloxy-benzamide (19i). A mixture of compound 18i (150 mg, 0.253
mmol), THPONH2 (56 mg, 0.477 mmol), HOBt (39 mg, 0.286
mmol), EDCI (55 mg, 0.286 mmol), and DIEA (37 mg, 0.286 mmol)
in DMF (10 mL) was degassed and purged with N2 3 times and the
mixture was stirred at 20 °C for 3 h. Then, the mixture was
concentrated in reduced pressure at 40 °C and the residue was
purified by prep-HPLC (method 5 described in the Supporting
Information) to afford pure compound 19i (80 mg, 46%) as a yellow
solid. ESI-MS m/z: calcd for C41H46N4O6, 690.4; m/z: found, 691.4
[M + H]+. This compound was used in the next step without further
characterization.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]phenyl]-N-tetrahydropyran-2-
yloxy-cyclohexanecarboxamide (19j). A mixture of compound 18j
(135 mg, 0.226 mmol), THPONH2 (27 mg, 0.234 mmol), EDCI (45
mg, 0.234 mmol), HOBt (43 mg, 0.319 mmol), and DIEA (55 mg,
0.425 mmol) in DMF (10 mL) was degassed and purged with N2 3
times and the mixture was stirred at 20 °C for 2 h. Then, the mixture
was concentrated to give a residue, which was purified by prep-HPLC
(method 29 described in the Supporting Information) to afford pure
compound 19j (105 mg, 67%) as a yellow solid. ESI-MS m/z: calcd
for C41H52N4O6, 696.4; m/z: found, 697.4 [M + H]+. This compound
was used in the next step without further characterization.
4-[4-[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]-1-piperidyl]-N-tetrahydropyran-2-yloxy-
benzamide (19k). A mixture of compound 18k (200 mg, 0.342
mmol), THPONH2(75 mg, 0.644 mmol), HOBt (52 mg, 0.387
mmol), EDCI (74 mg, 0.387 mmol), and DIEA (50 mg, 0.387 mmol)
in DMF (10 mL) was degassed and purged with N2 3 times and the
mixture was stirred at 20 °C for 2 h. Then, the mixture was
concentrated in reduced pressure at 40 °C and the residue was
purified by prep-HPLC (method 24 described in the Supporting
Information) to afford pure compound 19k (100 mg, 43%) as a
yellow solid. ESI-MS m/z: calcd for C39H49N5O6, 683.4; m/z: found,
684.4 [M + H]+. This compound was used in the next step without
further characterization.
2-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]-1-piperidyl]-N-tetrahydropyran-2-
yloxy-pyrimidine-5-carboxamide (19a). A mixture of compound 18a
(80 mg, 0.133 mmol), DIEA (34 mg, 0.266 mmol), HOBt (21 mg,
0.160 mmol), THPONH2 (23 mg, 0.200 mmol), and EDCI (30 mg,
0.160 mmol) in DMF (5.00 mL) was degassed and purged with N2 3
times and the mixture was stirred at 20 °C for 16 h. Then, the mixture
was concentrated in reduced pressure at 40 °C to give a residue that
was poured into water (10 mL) and extracted with EtOAc (10 mL ×
3). The combined organic phase was washed with brine (10 mL × 2),
dried with anhydrous Na2SO4, filtered, and concentrated in vacuum to
give a residue. The residue was purified by prep-HPLC (method 25
described in the Supporting Information) to afford pure compound
19a (60 mg, 64%) as a yellow solid. ESI-MS m/z: calcd for
C38H49N7O6, 699.4; m/z: found, 700.4 [M + H]+.This compound was
used in the next step without further characterization.
4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]-N-tetrahydropyran-2-yloxy-cyclo-
hexanecarboxamide (19c). A mixture of compound 18c (100 mg,
0.192 mmol), THPONH2 (45 mg, 0.383 mmol), HOBt (31 mg,
0.230 mmol), EDCI (44 mg, 0.230 mmol), and DIEA (50 mg, 0.383
mmol) in DMF (3 mL) was degassed and purged with N2 3 times and
the mixture was stirred at 20 °C for 2 h. Then, the mixture was
concentrated in reduced pressure at 40 °C. The residue was poured
into water (10 mL) and extracted with EtOAc (10 mL × 3). The
combined organic phase was washed with brine (10 mL × 2), dried
with anhydrous Na2SO4, filtered, and concentrated in vacuum. The
residue was purified by prep-HPLC (method 26 described in the
Supporting Information) to afford pure compound 19c (60 mg, 50%)
as a yellow solid. ESI-MS m/z: calcd for C35H48N4O6, 620.4; m/z:
found, 621.4 [M + H]+. This compound was used in the next step
without further characterization.
4-[[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]-1-piperidyl]methyl]-N-tetrahydro-
pyran-2-yloxy-benzamide (19d). A mixture of compound 18d (140
mg, 0.229 mmol), THPONH2 (30 mg, 0.251 mmol), EDCI (45 mg,
0.251 mmol), HOBt (44 mg, 0.321 mmol), and DIEA (56 mg, 0.457
mmol) in DMF (10 mL) was degassed and purged with N2 3 times
and the mixture was stirred at 20 °C for 2 h. Then, the mixture was
concentrated and the residue was diluted with water (10 mL) and
extracted with EtOAc (15 mL). The organic phase was concentrated
to give a residue, which was purified by prep-HPLC (method 27
described in the Supporting Information) to afford pure compound
19d (65 mg, 40%) as a yellow solid. ESI-MS m/z: calcd for
C41H53N5O6, 711.4; m/z: found, 712.3 [M + H]+. This compound
was used in the next step without further characterization.
4-[[4-[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]-1-piperidyl]methyl]-N-tetrahydropyran-2-
yloxy-benzamide (19e). A mixture of compound 18e (150 mg, 0.250
mmol), THPONH2 (55 mg, 0.472 mmol), HOBt (38 mg, 0.283
mmol), EDCI (54 mg, 0.283 mmol), and DIEA (37 mg, 0.283 mmol)
in DMF (10 mL) was degassed and purged with N2 3 times and the
mixture was stirred at 20 °C for 5 h. Then, the mixture was
concentrated in reduced pressure at 40 °C and the residue was
purified by prep-HPLC (method 19 described in the Supporting
Information) to afford pure compound 19e (80 mg, 46%) as a yellow
solid. ESI-MS m/z: calcd for C40H51N5O6, 697.4; m/z: found, 698.3
[M + H]+. This compound was used in the next step without further
characterization.
4-[4-[[[6-Methoxy-2-(5-methyl-2-furyl)-7-(3-pyrrolidin-1-ylpro-
poxy)-4-quinolyl]amino]methyl]-1-piperidyl]-N-tetrahydropyran-2-
yloxy-benzamide (19f). A mixture of compound 18f (120 mg, 0.200
mmol), THPONH2 (26 mg, 0.220 mmol), EDCI (40 mg, 0.220
mmol), HOBt (38 mg, 0.300 mmol), and DIEA (49 mg, 0.400 mmol)
in DMF (10 mL) was degassed and purged with N2 3 times and the
mixture was stirred at 20 °C for 2 h. Then, the mixture was diluted
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6-Methoxy-2-methyl-N-(4-piperidylmethyl)-7-(3-pyrrolidin-1-
ylpropoxy)quinolin-4-amine (22a). A solution of compound 21a35
(250 mg, 0.49 mmol) in HCl/1,4-dioxane (10 mL, 4.0 M) was stirred
at room temperature for 1 h. Then, the reaction mixture was
concentrated to give compound 22a (200 mg, 99%) as a white solid.
ESI-MS m/z: calcd for C24H36N4O2, 412.3; m/z: found, 413 [M +
H]+. This intermediate was used in the next step without further
purification or characterization.
Ethyl 2-[4-[[[6-Methoxy-2-methyl-7-(3-pyrrolidin-1-ylpropoxy)-4-
quinolyl]amino]methyl]-1-piperidyl]pyrimidine-5-carboxylate
(23a). To a solution of compound 22a (41 mg, 0.1 mmol) in
acetonitrile (10 mL) were added K2CO3 (69 mg, 0.5 mmol) and ethyl
2-chloropyrimidine-5-carboxylate(37 mg, 0.2 mmol) and the solution
was stirred at room temperature for 3 h. Then, the mixture was
concentrated to give compound 23a (50 mg, 89%) as a yellow solid.
ESI-MS m/z: calcd for C31H42N6O4, 562.3; m/z: found, 563 [M +
H]+. This intermediate was used in the next step without further
purification or characterization.
Ethyl 2-[4-[[6-Methoxy-2-methyl-7-(3-pyrrolidin-1-ylpropoxy)-4-
quinolyl]amino]-1-piperidyl]pyrimidine-5-carboxylate (23b). To a
solution of compound 22b35 (120 mg, 0.3 mmol) in acetonitrile (15
mL) were added K2CO3 (138 mg, 1 mmol) and ethyl 2-
chloropyrimidine-5-carboxylate (88 mg, 0.45 mmol) and the mixture
was stirred at room temperature for 3 h. Then, the mixture was
concentrated to give compound 23b (0.15 g, 91%) as a yellow solid.
ESI-MS m/z: calcd for C30H40N6O4, 548.3; m/z: found, 549 [M +
H]+. This intermediate was used in the next step without further
purification or characterization.
2-[4-[[[6-Methoxy-2-methyl-7-(3-pyrrolidin-1-ylpropoxy)-4-
quinolyl]amino]methyl]-1-piperidyl]pyrimidine-5-carboxylic Acid
(24a). To a solution of compound 23a (56 mg, 0.1 mmol) in
THF/MeOH/H2O (10:1:3, 10 mL) was added LiOH·H2O (21 mg,
0.5 mmol) and the resulting mixture was stirred at room temperature
overnight. Then, the mixture was diluted with water and the pH was
adjusted to 2−3 with 1.0 N aqueous HCl. Then, the mixture was
extracted with EtOAc and the combined organic layers were
concentrated to give compound 24a (45 mg, 83%). ESI-MS m/z:
calcd for C29H38N6O4, 534.3; m/z: found, 535 [M + H]+. This
intermediate was used in the next step without further purification or
characterization.
2-[4-[[6-Methoxy-2-methyl-7-(3-pyrrolidin-1-ylpropoxy)-4-
quinolyl]amino]-1-piperidyl]pyrimidine-5-carboxylic Acid (24b).
To a solution of compound 23b (0.2 g, 0.365 mmol) in THF/
MeOH/H2O (10:1:3, 10 mL) was added LiOH·H2O (78 mg, 1.82
mmol) and the resulting mixture was stirred at room temperature
overnight. Then, the mixture was diluted with water and the pH was
adjusted to 2−3 with 1.0 N aqueous HCl. Then, the mixture was
extracted with EtOAc and the combined organic phase was
concentrated to give compound 24b (0.15 g, 79%). ESI-MS m/z:
calcd for C28H36N6O4, 520.3; m/z: found, 521 [M + H]+. This
intermediate was used in the next step without further purification or
characterization.
2-[4-[[[6-Methoxy-2-methyl-7-(3-pyrrolidin-1-ylpropoxy)-4-
quinolyl]amino]methyl]-1-piperidyl]-N-tetrahydropyran-2-yloxy-
pyrimidine-5-carboxamide (25a). To a solution of compound 24a
(45 mg, 0.084 mmol) in DMF (10 mL) were added EDC·HCl (29
mg, 0.17 mmol), HOBt (23 mg, 0.17 mmol), THPONH2 (20 mg,
0.17 mmol), and NMM (34 mg, 0.34 mmol) and the mixture was
stirred at room temperature overnight. Then, the mixture was
extracted with EtOAc and the combined organic layers were washed
with brine, dried over anhydrous Na2SO4, filtered, and concentrated
to give a residue, which was purified by prep-HPLC (method 41
described in the Supporting Information) to afford pure compound
25a (25 mg, 48%). ESI-MS m/z: calcd for C34H47N7O5, 633.4; m/z:
found, 634 [M + H]+. This compound was used in the next step
without further characterization.
and NMM (61 mg, 0.6 mmol) and the mixture was stirred at room
temperature overnight. Then, the mixture was extracted with EtOAc
and the combined organic layers were washed with brine, dried over
anhydrous Na2SO4, filtered, and concentrated to give a residue, which
was purified by prep-HPLC (method 39 described in the Supporting
Information) to afford pure compound 25b (60 mg, 48%). ESI-MS
m/z: calcd for C33H45N7O5, 619.3; m/z: found, 620 [M + H]+. This
compound was used in the next step without further characterization.
Ethyl 2-[4-[[[7-[(1-tert-Butoxycarbonyl-4-piperidyl)methoxy]-6-
methoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]methyl]-1-
piperidyl]pyrimidine-5-carboxylate (30). A mixture of compound
2935 (270 mg, 0.554 mmol), ethyl 2-[4-(aminomethyl)-1-piperidyl]-
pyrimidine-5-carboxylate (51, 293 mg, 1.11 mmol), Pd2(dba)3 (51
mg, 0.055 mmol), BINAP (34 mg, 0.055 mmol), and Cs2CO3 (451
mg, 1.39 mmol) in 1,4-dioxane (10 mL) was degassed and purged
with N2 3 times and the mixture was stirred at 110 °C for 16 h. Then,
the reaction mixture was concentrated in vacuum and the residue was
dissolved in EtOAc (50 mL) and filtered. The filtrate was
concentrated in vacuum to give a residue, which was purified by
prep-TLC (CH2Cl2/MeOH = 10:1) to afford pure compound 30
(200 mg, 50%) as a yellow solid. ESI-MS m/z: calcd for C39H50N6O7,
714.4; m/z: found, 715.4 [M + H]+. This compound was used in the
next step without further characterization.
2-[4-[[[7-[(1-tert-Butoxycarbonyl-4-piperidyl)methoxy]-6-me-
thoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]methyl]-1-piperidyl]-
pyrimidine-5-carboxylic Acid (31). To a solution of compound 30
(200 mg, 0.280 mmol) in THF/H2O (2:1, 30.0 mL) was added
LiOH·H2O (23 mg, 0.559 mmol) and the mixture was stirred at 15
°C for 16 h. Then, the reaction mixture was adjusted to pH 6 with 0.1
M HCl aqueous solution and concentrated in vacuum to obtain
compound 31 (200 mg, crude) as a yellow solid. ESI-MS m/z: calcd
for C37H46N6O7, 686.3; m/z: found, 687.4 [M + H]+.This
intermediate was used in the next step without further purification
or characterization.
tert-Butyl 4-[[6-Methoxy-2-(5-methyl-2-furyl)-4-[[1-[5-(tetrahy-
dropyran-2-yloxycarbamoyl)pyrimidin-2-yl]-4-piperidyl]-
methylamino]-7-quinolyl]oxymethyl]piperidine-1-carboxylate (32).
A mixture of compound 31 (200 mg, 0.291 mmol), THPONH2(68
mg, 0.582 mmol), EDCI (112 mg, 0.582 mmol), HOBt (79 mg, 0.582
mmol), and DIEA (113 mg, 0.873 mmol) in DMF (10 mL) was
degassed and purged with N2 3 times and the mixture was stirred at
15 °C for 16 h. Then, the reaction mixture was quenched with water
(10 mL) and concentrated in vacuum to give compound 32 (300 mg,
crude) as a yellow oil. ESI-MS m/z: calcd for C42H55N7O8, 785.5; m/
z: found, 786.5 [M + H]+. This intermediate was used in the next step
without further purification or characterization.
Ethyl 2-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]methyl]-1-piperidyl]pyrimidine-5-carboxylate (36a). A mix-
ture of compound 3535 (100 mg, 0.329 mmol), ethyl 2-[4-
(aminomethyl)-1-piperidyl]pyrimidine-5-carboxylate (51, 104.43
mg, 0.395 mmol, 1.20 equiv), Pd2(dba)3 (30.15 mg, 0.033 mmol,
0.10 equiv), BINAP (41.00 mg, 0.066 mmol, 0.20 equiv), and Cs2CO3
(214.54 mg, 0.658 mmol, 2.00 equiv) in 1,4-dioxane (3.00 mL) was
degassed and purged with N2 3 times, and then the mixture was
stirred at 140 °C for 12 h under an N2 atmosphere. The reaction
mixture was filtrated and the filtrate was concentrated in vacuo to give
the compound 36a (120.00 mg, 0.225 mmol, 68.6% yield) as a yellow
solid. ESI-MS m/z: calcd for C29H33N5O5, 531.3; m/z: found, 532.3
[M + H]+. This intermediate was used in the next step without further
purification or characterization.
Ethyl 2-[4-[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]-1-piperidyl]pyrimidine-5-carboxylate (36b). A mixture of
compound 3535 (250 mg, 0.823 mmol), tert-butyl 4-aminopiperidine-
1-carboxylate(247 mg, 1.23 mmol), Pd2(dba)3 (75 mg, 0.082 mmol),
BINAP (102 mg, 0.164 mmol), and Cs2CO3 (536 mg, 1.65 mmol) in
1,4-dioxane (10.0 mL) was degassed and purged with N2 3 times, and
then the mixture was stirred at 120 °C for 12 h. Then, the reaction
mixture was filtrated and the filtrate was concentrated under vacuum
to give intermediate tert-butyl 4-[[6,7-dimethoxy-2-(5-methyl-2-
furyl)-4-quinolyl]amino]piperidine-1-carboxylate (350 mg, 91%) as
a yellow solid. ESI-MS m/z: calcd for C26H33N3O5, 467.2; m/z:
2-[4-[[6-Methoxy-2-methyl-7-(3-pyrrolidin-1-ylpropoxy)-4-
quinolyl]amino]-1-piperidyl]-N-tetrahydropyran-2-yloxy-pyrimi-
dine-5-carboxamide (25b). To a solution of compound 24b (104
mg, 0.2 mmol) in DMF (10 mL) were added EDC·HCl (69 mg, 0.4
mmol), HOBt (54 mg, 0.4 mmol), THPONH2 (35 mg, 0.3 mmol),
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found, 468.3 [M + H]+. Then, a mixture of this intermediate (350 mg,
0.748 mmol) in HCl/MeOH (10.0 mL, 2.0 M) was degassed and
purged with N2 3 times and the mixture was stirred at 25 °C for 5 h.
The reaction mixture was concentrated under vacuum and the residue
was extracted with water (5 mL × 4) and washed with EtOAc (5 mL
× 3). The combined aqueous phase was concentrated under reduced
pressure to give intermediate 6,7-dimethoxy-2-(5-methyl-2-furyl)-N-
(4-piperidyl)quinolin-4-amine (220 mg, 80%) as a yellow solid. ESI-
MS m/z: calcd for C21H25N3O3, 367.2; m/z: found, 368.3 [M + H]+.
Finally, a mixture of this intermediate (100 mg, 0.272 mmol), ethyl 2-
chloropyrimidine-5-carboxylate (66 mg, 0.354 mmol), and K2CO3
(113 mg, 0.816 mmol) in DMF (10.0 mL) was degassed and purged
with N2 3 times, and then the mixture was stirred at 50 °C for 12 h.
Then, the reaction mixture was concentrated under vacuum and the
residue was extracted with EtOAc (10 mL × 3). The combined
organic layer was washed with water (10 mL × 1), dried over Na2SO4,
filtered, and concentrated to afford compound 36b (100 mg, 71%) as
a yellow solid. ESI-MS m/z: calcd for C28H31N5O5, 517.2; m/z:
found, 518.3 [M + H]+. This intermediate was used in the next step
without further purification or characterization.
Ethyl 2-[8-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]methyl]-3-azabicyclo[3.2.1]octan-3-yl]pyrimidine-5-carbox-
ylate (36c). A mixture of compound 3535 (200 mg, 0.658 mmol), tert-
butyl 8-(aminomethyl)-3-azabicyclo[3.2.1]octane-3-carboxylate (190
mg, 0.790 mmol), Cs2CO3 (429 mg, 1.32 mmol), BINAP (41 mg,
0.066 mmol), and Pd2(dba)3 (60 mg, 0.066 mmol) in 1,4-dioxane (20
mL) was degassed and purged with N2 3 times, and then the mixture
was stirred at 120 °C for 16 h. Then, the mixture was concentrated in
reduced pressure at 40 °C; the residue was poured into water (100
mL) and extracted with EtOAc (30 mL × 3). The combined organic
phase was washed with brine (10 mL × 2), dried with anhydrous
Na2SO4, filtered, and concentrated in vacuum. The residue was
purified by prep-TLC (SiO2, CH2Cl2/MeOH = 10:1) to afford
intermediate tert-butyl 8-[[[6,7-dimethoxy-2-(5-methyl-2-furyl)-4-
quinolyl]amino]methyl]-3-azabicyclo[3.2.1]octane-3-carboxylate
(250 mg, 75%) as a yellow solid. ESI-MS m/z: calcd for C29H37N3O5,
507.3; m/z: found, 508.3 [M + H]+. Then, a solution of this
intermediate (250 mg, 0.492 mmol) in HCl/EtOAc (20 mL, 2.0 M)
was stirred at 20 °C for 16 h and then, the mixture was concentrated
in reduced pressure at 40 °C to afford N-(3-azabicyclo[3.2.1]octan-8-
ylmethyl)-6,7-dimethoxy-2-(5-methyl-2-furyl)quinolin-4-amine (200
mg, 99%) as a yellow solid. ESI-MS m/z: calcd for C24H29N3O3,
407.3; m/z: found, 408.3 [M + H]+. Finally, to a solution of this
amine (500 mg, 1.23 mmol) in CH3CN (20 mL) were added K2CO3
(509 mg, 3.68 mmol) and ethyl 2-chloropyrimidine-5-carboxylate
(343 mg, 1.84 mmol) and the mixture was stirred at 40 °C for 16 h.
Then, the reaction mixture was filtered and concentrated in vacuum
to give compound 36c (0.4 g, 58%) as a yellow oil. ESI-MS m/z: calcd
for C31H35N5O5, 557.3; m/z: found, 558.2 [M + H]+.This
intermediate was used in the next step without further purification
or characterization.
compound 36e (100 mg, 67%) as a yellow solid. ESI-MS m/z: calcd
for C26H32N2O5, 452.2; m/z: found, 453.3 [M + H]+. This
intermediate was used in the next step without further purification
or characterization.
Methyl 6-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]methyl]pyridine-3-carboxylate (36f). A mixture of com-
pound 3535 (50 mg, 0.164 mmol), commercially available methyl 6-
(aminomethyl)pyridine-3-carboxylate (55 mg, 0.329 mmol),
Pd2(dba)3 (30 mg, 0.033 mmol), BINAP (31 mg, 0.049 mmol),
and Cs2CO3 (134 mg, 0.411 mmol) in 1,4-dioxane (10 mL) was
degassed and purged with N2 3 times, and then the mixture was
stirred at 120 °C for 16 h. Then, the reaction mixture was filtered and
concentrated in vacuum to give a residue. The residue was purified by
prep-TLC (CH2Cl2/MeOH = 10:1) to afford pure compound 36f
(40 mg, 56%) as a yellow solid. ESI-MS m/z: calcd for C24H23N3O5,
433.2; m/z: found, 434.2 [M + H]+. This compound was used in the
next step without further characterization.
Ethyl 4-[[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]methyl]-1-piperidyl]methyl]benzoate (36g). A mixture of
compound 3535 (500 mg, 1.65 mmol), tert-butyl 4-(aminomethyl)-
piperidine-1-carboxylate (530 mg, 2.47 mmol), Cs2CO3 (1.08 g, 3.30
mmol), BINAP (102 mg, 0.165 mmol), and Pd2(dba)3 (151 mg,
0.165 mmol) in 1,4-dioxane (10 mL) was degassed and purged with
N2 3 times and the mixture was stirred at 120 °C for 16 h. Then, the
mixture was concentrated in reduced pressure at 40 °C and the
residue was poured into water (10 mL) and extracted with EtOAc (10
mL × 3). The combined organic phase was washed with brine (10 mL
× 2), dried with anhydrous Na2SO4, filtered, and concentrated in
vacuum to give the crude product, which was purified by silica gel
column chromatography (PE/EtOAc = 1:0 to 0:1, CH2Cl2/MeOH =
1:0 to 1:1) to afford pure intermediate tert-butyl 4-[[[6,7-dimethoxy-
2-(5-methyl-2-furyl)-4-quinolyl]amino]methyl]piperidine-1-carboxy-
late (500 mg, 63%) as a yellow solid. ESI-MS m/z: calcd for
C27H35N3O5, 481.3; m/z: found, 482.3 [M + H]+. Then, a solution of
this intermediate (500 mg, 1.04 mmol) in HCl/MeOH (20 mL, 2.0
M) was degassed and purged with N2 3 times and then stirred at 20
°C for 5 h. Then, the mixture was concentrated in reduced pressure at
40 °C to give intermediate 6,7-dimethoxy-2-(5-methyl-2-furyl)-N-(4-
piperidylmethyl)quinolin-4-amine (400 mg, crude) as a yellow solid.
ESI-MS m/z: calcd for C22H27N3O3, 381.2; m/z: found, 382.2 [M +
H]+. Finally, a mixture of this amine (100 mg, 0.262 mmol), ethyl 4-
formylbenzoate (93 mg, 0.524 mmol), CH3COOH (16 mg, 0.262
mmol), and NaBH3CN (49 mg, 0.786 mmol) in MeOH (3 mL) was
degassed and purged with N2 3 times and the mixture was stirred at
70 °C for 2 h. Then, the solution was concentrated in reduced
pressure at 40 °C to give a residue, which was purified by prep-HPLC
(method 10 described in the Supporting Information) to afford pure
compound 36g (70 mg, 49%) as a yellow solid. ESI-MS m/z: calcd for
C32H37N3O5, 543.3; m/z: found, 544.2 [M + H]+. This compound
was used in the next step without further characterization.
Ethyl 5-[[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]methyl]-1-piperidyl]methyl]thiophene-2-carboxylate (36h).
Ethyl 4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]methyl]benzoate (36d). A mixture of compound 3535 (100
mg, 0.329 mmol), ethyl 4-(aminomethyl)benzoate (77 mg, 0.428
mmol), Cs2CO3 (214 mg, 0.658 mmol), Pd2(dba)3 (30 mg, 0.033
mmol), and BINAP (20 mg, 0.033 mmol) in 1,4-dioxane (3 mL) was
degassed and purged with N2 3 times, and then the mixture was
stirred at 120 °C for 16 h. Then, the mixture was concentrated in
reduced pressure at 40 °C to afford compound 36d (100 mg, 68%) as
a yellow solid. ESI-MS m/z: calcd for C26H26N2O5, 446.2; m/z:
found, 447.2 [M + H]+. This intermediate was used in the next step
without further purification or characterization.
Ethyl 4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]methyl]cyclohexanecarboxylate (36e). A mixture of com-
pound 3535 (100 mg, 0.329 mmol), ethyl 4-(aminomethyl)-
cyclohexanecarboxylate (122 mg, 0.659 mmol), BINAP (20 mg,
0.033 mmol), Pd2(dba)3 (30 mg, 0.033 mmol), and Cs2CO3 (214 mg,
0.658 mmol) in 1,4-dioxane (5 mL) was degassed and purged with N2
3 times and the mixture was stirred at 120 °C for 16 h. Then, the
mixture was concentrated in reduced pressure at 40 °C to give
A
mixture of 6,7-dimethoxy-2-(5-methyl-2-furyl)-N-(4-
piperidylmethyl)quinolin-4-amine (62 mg, 0.163 mmol, intermediate
described in the synthesis of compound 36g), ethyl 5-formylth-
iophene-2-carboxylate (60 mg, 0.325 mmol), CH3COOH (9 mg,
0.163 mmol), and NaBH3CN (30 mg, 0.488 mmol) in MeOH (3
mL) was degassed and purged with N2 3 times and the mixture was
stirred at 70 °C for 2 h. Then, the solution was concentrated in
reduced pressure at 40 °C to afford compound 36h (60 mg, 76%) as a
yellow solid. ESI-MS m/z: calcd for C30H35N3O5S, 549.2; m/z: found,
550.2 [M + H]+. This intermediate was used in the next step without
further purification or characterization.
Methyl 3-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]methyl]-1-piperidyl]cyclobutanecarboxylate (36i). To a
solution of NaBH3CN (72 mg, 1.15 mmol) was added ZnCl2 (78
mg, 0.576 mmol) in MeOH (5 mL) and the mixture was stirred at 20
°C for 30 min. Then, a mixture of 6,7-dimethoxy-2-(5-methyl-2-
furyl)-N-(4-piperidylmethyl)quinolin-4-amine(200 mg, 0.524 mmol,
intermediate described in the synthesis of compound 36g) and methyl
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3-oxocyclobutanecarboxylate (73 mg, 0.576 mmol) in MeOH (10
mL) was added and the solution was stirred at 40 °C for 15.5 h. Then,
the mixture was filtered and concentrated in vacuum to give
compound 36i (200 mg, 77%) as a yellow solid. ESI-MS m/z:
calcd for C28H35N3O5, 493.3; m/z: found, 494.3 [M + H]+. This
intermediate was used in the next step without further purification or
characterization.
0.9, 3.5 Hz, 1H), 4.54 (d, J = 13.1 Hz, 2H), 4.04 (s, 3H), 4.03 (s, 3H),
3.86 (s, 3H), 3.57 (d, J = 7.2 Hz, 2H), 3.08−2.97 (m, 2H), 2.50 (s,
3H), 2.23 (m, 1H), 1.99 (br d, J = 10.9 Hz, 2H), 1.47−1.34 (m, 2H);
one exchangeable proton was not observed. ESI-MS m/z: calcd for
C29H32N4O5, 516.2; m/z: found, 517.3 [M + H]+.
2-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]pyrimidine-5-carboxylic Acid (37a). A mixture
of compound 36a (120.00 mg, 0.226 mmol, 1.00 equiv) and LiOH·
H2O (18.94 mg, 0.452 mmol, 2.00 equiv) in THF/MeOH/H2O
(10.00/1.00/1.00 mL) was degassed and purged with N2 3 times, and
then the mixture was stirred at 25 °C for 12 h under an N2
atmosphere. The reaction mixture was concentrated under reduced
pressure to remove THF and MeOH to give a residue. The residue
was diluted with water 3 mL and quenched by 2 M HCl to adjust the
pH to 4. Then, the yellow solid was precipitated and collected. The
yellow solid was concentrated in vacuo to give the compound 37a
(90.00 mg, 0.179 mmol, 79.2% yield) as a yellow solid. ESI-MS m/z:
calcd for C27H29N5O5, 503.2; m/z: found, 504.3 [M + H]+. This
intermediate was used in the next step without further purification or
characterization.
2-[4-[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-1-
piperidyl]pyrimidine-5-carboxylic Acid (37b). A mixture of com-
pound 36b (100 mg, 0.193 mmol) and LiOH·H2O (24 mg, 0.579
mmol) in THF/MeOH/H2O (6:1:1, 4.00 mL) was degassed and
purged with N2 for 3 times, and then the mixture was stirred at 25 °C
for 12 h. Then, the reaction mixture was concentrated in vacuum and
the residue was adjusted to pH 4 with 4.0 M HCl. The obtained solid
was filtrated and collected to afford compound 37b (70 mg, 74%) as a
yellow solid. ESI-MS m/z: calcd for C26H27N5O5, 489.2; m/z: found,
490.3 [M + H]+. This intermediate was used in the next step without
further purification or characterization.
2-[8-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-3-azabicyclo[3.2.1]octan-3-yl]pyrimidine-5-carboxylic
Acid (37c). To a solution of compound 36c (0.4 g, 0.717 mmol) in
THF/H2O (2:1, 30 mL) was added LiOH·H2O (45 mg, 1.08 mmol)
and the mixture was stirred at 15 °C for 2 h. Then, the pH was
adjusted to 6 with 1.0 M HCl aqueous solution and the mixture was
concentrated in vacuum to afford compound 37c (0.4 g, crude) as a
brown solid. ESI-MS m/z: calcd for C29H31N5O5, 529.2; m/z: found,
530.2 [M + H]+. This intermediate was used in the next step without
further purification or characterization.
4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]benzoic Acid (37d). A mixture of compound 36d (100 mg,
0.224 mmol) and LiOH·H2O (19 mg, 0.448 mmol) in THF/MeOH/
H2O (3:1:1, 2.5 mL) was degassed and purged with N2 for 3 times,
and then the mixture was stirred at 20 °C for 5 h. Then, the mixture
was concentrated, the residue was diluted with water (2 mL) and the
residue was extracted with EtOAc (3 mL). The aqueous phase was
adjusted pH to 3−4 with 2.0 M HCl and the obtained yellow solid
was precipitated and filtrated. The filter cake was concentrated to
dryness to give compound 37d (60 mg, 64%) as a yellow solid. ESI-
MS m/z: calcd for C24H22N2O5, 418.2; m/z: found, 419.2 [M + H]+.
This intermediate was used in the next step without further
purification or characterization.
rac-cis Ethyl 4-[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]cyclohexanecarboxylate (36j-cis) and rac-trans Ethyl 4-
[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
cyclohexanecarboxylate (36j-trans). A mixture of compound 3535
(200 mg, 0.658 mmol), ethyl 4-aminocyclohexanecarboxylate (169
mg, 0.987 mmol), Cs2CO3 (429 mg, 1.32 mmol), BINAP (41 mg,
0.066 mmol), and Pd2(dba)3 (60 mg, 0.066 mmol) in 1,4-dioxane (20
mL) was degassed and purged with N2 3 times and the mixture was
stirred at 120 °C for 16 h. Then, the mixture was filtered and
concentrated in vacuum. The residue was purified by prep-HPLC
(method 11 described in the Supporting Information) to afford pure
compound 36j-cis (100 mg, 34%) as a yellow solid and 36j-trans (50
mg, 17%) as a yellow solid. 36j-cis: 1H NMR (CD3OD, 400 MHz): δ
7.74 (s, 1H), 7.52 (d, J = 3.2 Hz, 1H), 7.42 (s, 1H), 6.99 (s, 1H), 6.41
(d, J = 2.4 Hz, 1H), 4.18−4.13 (m, 2H), 4.01 (d, J = 2.0 Hz, 6H),
2.50 (s, 3H), 2.41−2.38 (m, 1H), 2.22−2.13 (m, 4H), 1.78−1.62 (m,
5H), 1.29−1.25 (m, 3H); one exchangeable proton was not observed.
ESI-MS m/z: calcd for C25H30N2O5, 438.2; m/z: found, 439.2 [M +
H]+. 36j-trans: 1H NMR (CD3OD, 400 MHz): δ 7.77−7.76 (m, 1H),
7.52 (d, J = 3.2 Hz, 1H), 7.44−7.43 (m, 1H), 7.02−7.00 (m, 1H),
6.43 (d, J = 2.8 Hz, 1H), 4.24−4.19 (m, 2H), 4.03 (d, J = 5.6 Hz,
6H), 2.76 (s, 1H), 2.52 (s, 3H), 2.32 (s, 2H), 2.29−2.24 (m, 1H),
2.05−2.00 (m, 2H), 1.85−1.74 (m, 4H), 1.33−1.29 (m, 3H); one
exchangeable proton was not observed. ESI-MS m/z: calcd for
C25H30N2O5, 438.2; m/z: found, 439.2 [M + H]+.
Methyl 6-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]methyl]-1-piperidyl]pyridine-3-carboxylate (36k). A mixture
of compound 3535 (0.2 g, 0.659 mmol, 1 equiv), tertbutyl 4-
(aminomethyl)piperidine-1-carboxylate (282.22 mg, 1.32 mmol, 2
equiv), Pd2(dba)3 (120.59 mg, 0.132 mmol, 0.2 equiv), BINAP
(123.00 mg, 0.198 mmol, 0.3 equiv), and Cs2CO3 (536.35 mg, 1.65
mmol, 2.5 equiv) in dioxane (10 mL) was degassed and purged with
N2 3 times, and then the mixture was stirred at 120 °C for 16 h under
an N2 atmosphere. LC−MS showed that the reaction was completed
and one main peak with the desired m/z was detected. The mixture
was filtered and the filtrate was concentrated. The residue was purified
by flash silica gel chromatography (ISCO, 20 g SepaFlash Silica Flash
Column) using as eluent a 0−70% ethylacetate/petroleum ether
gradient at 100 mL/min. Compound tert-butyl 4-[[[6,7-dimethoxy 2-
(5-methyl-2-furyl)-4-quinolyl]amino]methyl]piperidine-1-carboxylate
(0.2 g, 0.415 mmol, 63.07% yield) was obtained as a pure light yellow
solid. ESI-MS m/z: calcd for C27H35N3O5, 481.3; m/z: found, 482.3
[M + H]+. Then, a solution of this intermediate (0.2 g, 0.415 mmol, 1
eq) in HCl/EtOAc (4.0 M, 10 mL) was degassed and purged with N2
3 times, and then the mixture was stirred at 25 °C for1 h under an N2
atmosphere. LC−MS showed that the reaction was completed and
one main peak with the desired m/z was detected. The solvent was
removed and the pure intermediate, 6,7-dimethoxy-2-(5-methyl-2-
furyl)-N-(4-piperidylmethyl)quinolin-4-amine (140 mg, 0.335 mmol,
80.66% yield), was obtained as a light yellow solid. ESI-MS m/z: calcd
for C22H27N3O3, 381.2; m/z: found, 382.0 [M + H]+. Finally, to a
mixture of this amine (140 mg, 0.335 mmol, 1 equiv) in DMF (10
mL) were added K2CO3 (138.89 mg, 1.00 mmol, 3 equiv) and methyl
6-chloropyridine-3-carboxylate (68.97 mg, 0.402 mmol, 1.2 equiv).
The mixture was stirred at 50 °C for 12 h. HPLC and LC−MS
showed that the reaction was completed and one main peak with the
desired m/z was detected. The mixture was filtered and the filtrate
was concentrated to give a residue, which was purified by prep-HPLC
(method 43 described in the Supporting Information) to afford the
pure compound 36k (0.1 g, 0.194 mmol, 57.8% yield) as a light
yellow solid.1H NMR (CD3OD, 400 MHz): δ 8.66 (d, J = 1.9 Hz,
1H), 8.03 (dd, J = 2.4, 9.2 Hz, 1H), 7.69 (s, 1H), 7.51 (d, J = 3.3 Hz,
1H), 7.44 (s, 1H), 6.99 (s, 1H), 6.87 (d, J = 9.3 Hz, 1H), 6.42 (dd, J =
4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]cyclohexanecarboxylic Acid (37e). A mixture of compound
36e (100 mg, 0.221 mmol) and LiOH·H2O (18 mg, 0.442 mmol) in
THF/MeOH/H2O (3:1:1, 5.0 mL) was degassed and purged with N2
for 3 times and the mixture was stirred at 20 °C for 16 h. Then, the
mixture was concentrated and the residue was diluted with H2O (2
mL) and extracted with EtOAc (5 mL). Then pH of the aqueous
phase was adjusted to 3−4 with 2.0 M HCl. The obtained solid was
filtered and concentrated to dryness to give compound 37e (50 mg,
53%) as a yellow solid. ESI-MS m/z: calcd for C24H28N2O5, 424.2; m/
z: found, 425.2 [M + H]+. This intermediate was used in the next step
without further purification or characterization.
6-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]pyridine-3-carboxylic Acid (37f). To a solution of compound
36f (40 mg, 0.092 mmol) in THF/H2O (2:1, 15 mL) was added
LiOH·H2O (8 mg, 0.184 mmol) and the mixture was stirred at 20 °C
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for 16 h. Then, the reaction mixture was adjust to pH 6 and
concentrated in vacuum to give compound 37f (50 mg, crude) as a
yellow solid. ESI-MS m/z: calcd for C23H21N3O5, 419.2; m/z: found,
420.2 [M + H]+.This intermediate was used in the next step without
further purification or characterization.
equiv). The mixture was stirred at 20 °C for 12 h. The mixture was
adjusted pH ∼ 6 with HCl (2 M) and extracted with ethyl acetate (10
mL, three times). The combined organic phase was washed with brine
(10 mL), dried with anhydrous Na2SO4, filtered and concentrated in
vacuum. Compound 37k (95 mg, 0.189 mmol, 97.7% yield) was
obtained as a light yellow solid. ESI-MS m/z: calcd for C28H30N4O5,
502.2; m/z: found, 503.3 [M + H]+.This intermediate was used in the
next step without further purification or characterization.
2-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]-N-tetrahydropyran-2-yloxy-pyrimidine-5-car-
boxamide (38a). A mixture of compound 37a (120.00 mg, 0.238
mmol, 1.00 equiv), O-tetrahydropyran-2-ylhydroxylamine (54.91 mg,
0.358 mmol, 1.50 equivas hydrochloride), HOBt (48.30 mg, 0.358
mmol, 1.50 equiv), EDCI (68.53 mg, 0.358 mmol, 1.50 equiv) and
DIEA (61.60 mg, 0.477 mmol, 83.24 μL, 2.00 equiv) in DMF (10.00
mL) was degassed and purged with N2 for 3 times, and then the
mixture was stirred at 25 °C for 12 h under N2 atmosphere. LC−MS
showed 70% of desired compound was detected. The reaction mixture
was filtrated and the filtrate was concentrated in vacuo to give a
residue. The residue was extracted with EtOAc (20 mL, 3 times) and
washed with water (10 mL, twice). The combined organic layers were
washed with brine (10 mL), dried over Na2SO4, filtered, and
concentrated under reduced pressure to give a residue. The residue
was purified by HPLC (method 2 described in the Supporting
Information) to afford compound 38a (80.00 mg, 0.133 mmol, 55.7%
yield) as a yellow solid. ESI-MS m/z: calcd for C32H38N6O6, 602.3;
m/z: found, 603.3 [M + H]+.
2-[8-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-3-azabicyclo[3.2.1]octan-3-yl]-N-tetrahydropyran-2-yloxy-
pyrimidine-5-carboxamide (38c). A mixture of compound 37c (200
mg, 0.378 mmol), THPONH2 (88 mg, 0.755 mmol), EDCI (109 mg,
0.566 mmol), HOBt (76 mg, 0.566 mmol), and DIEA (146 mg, 1.13
mmol) in DMF (10 mL) was degassed and purged with N2 3 times,
and then the mixture was stirred at 15 °C for 16 h. Then, the reaction
mixture was poured into H2O (100 mL) and extracted with EtOAc
(20 mL × 3). The organic layers were dried over Na2SO4, filtered, and
concentrated in vacuum to obtain compound 38c (0.1 g, 42%) as a
yellow solid. ESI-MS m/z: calcd for C34H40N6O6, 628.3; m/z: found,
629.3 [M + H]+.This intermediate was used in the next step without
further purification or characterization.
3-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]-N-tetrahydropyran-2-yloxy-cyclobutanecar-
boxamide (38i). A mixture of compound 37i (90 mg, 0.188 mmol),
THPONH2 (44 mg, 0.375 mmol), DIEA (48 mg, 0.375 mmol),
HOBt (30 mg, 0.225 mmol), and EDCI (43 mg, 0.225 mmol) in
DMF (5 mL) was degassed and purged with N2 3 times and the
mixture was stirred at 20 °C for 16 h. Then, the mixture was
concentrated in reduced pressure at 40 °C, the residue was poured
into water (10 mL), and it was extracted with EtOAc (10 mL × 3).
The combined organic phase was washed with brine (10 mL × 2),
dried with anhydrous Na2SO4, filtered, and concentrated in vacuum.
The residue was purified by prep-HPLC (method 12 described in the
Supporting Information) to afford pure compound 38i (50 mg, 46%)
as a yellow solid. ESI-MS m/z: calcd for C32H42N4O6, 578.3; m/z:
found, 579.3 [M + H]+. This intermediate was used in the next step
without further characterization.
4-[[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]methyl]benzoic Acid (37g). A mixture of
compound 36g (70 mg, 0.129 mmol) and LiOH·H2O (11 mg,
0.257 mmol) in THF/MeOH/H2O (3:1:1, 2.5 mL) was degassed and
purged with N2 for 3 times, and then the mixture was stirred at 20 °C
for 16 h. Then, the mixture was concentrated and the residue was
diluted with water (0.5 mL) and extracted with EtOAc (3 mL). Then
aqueous phase was acidified with 2.0 M HCl to adjust pH to 3−4.
The solution was filtered and the filter cake was concentrated to
dryness to give compound 37g (50 mg, 75%) as a yellow solid. ESI-
MS m/z: calcd for C30H33N3O5, 515.3; m/z: found, 516.3 [M + H]+.
This intermediate was used in the next step without further
purification or characterization.
5-[[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]methyl]thiophene-2-carboxylic Acid (37h). A
mixture of compound 36h (100 mg, 0.182 mmol) and LiOH·H2O
(23 mg, 0.545 mmol) in THF/MeOH/H2O (3:1:1, 2.5 mL) was
stirred at 20 °C for 16 h. Then, the mixture was concentrated and the
residue was diluted with water (2 mL) and extracted with EtOAc (3
mL). The aqueous layer was acidified to pH = 3−4 with 1 N HCl and
filtered. The filter cake was concentrated to dryness to give compound
37h (60 mg, 63%) as a yellow solid. ESI-MS m/z: calcd for
C28H31N3O5S, 521.2; m/z: found, 522.2 [M + H]+. This intermediate
was used in the next step without further purification or character-
ization.
3-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]cyclobutanecarboxylic Acid (37i). A mixture of
compound 36i (200 mg, 0.405 mmol) and LiOH·H2O (51 mg, 1.22
mmol) in MeOH/THF/H2O (1:3:1, 15.0 mL) was stirred at 20 °C
for 16 h. Then, the mixture was concentrated and the residue was
diluted with H2O (2 mL) and extracted with EtOAc (3 mL). Then
the aqueous layer was acidified to pH = 3−4 with 1 N HCl and
filtered. The filter cake was concentrated to dryness to give compound
37i (100 mg, 51%) as a yellow solid. ESI-MS m/z: calcd for
C27H33N3O5, 479.2; m/z: found, 480.3 [M + H]+. This intermediate
was used in the next step without further purification or character-
ization.
rac-cis 4-[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]cyclohexanecarboxylic Acid (37j-cis). A mixture of com-
pound 36j-cis (70 mg, 0.160 mmol) and LiOH·H2O (20 mg, 0.479
mmol) in MeOH/THF/H2O (1:3.1, 10.0 mL) was stirred at 20 °C
for 3 h. Then, the mixture was concentrated and the residue was
diluted with water (3 mL) and extracted with EtOAc (5 mL). Then
the aqueous layer was acidified to pH = 3−4 with 1 N HCl and
filtered. The filter was concentrated to dryness to afford compound
37j-cis (60 mg, 91%) as a yellow solid. ESI-MS m/z: calcd for
C23H26N2O5, 410.2; m/z: found, 411.2 [M + H]+. This intermediate
was used in the next step without further purification or character-
ization.
rac-trans 4-[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]cyclohexanecarboxylic Acid (37j-trans). A mixture of
compound 36j-trans (50 mg, 0.114 mmol) and LiOH·H2O (14 mg,
0.342 mmol) in MeOH/THF/H2O (1:3:1, 8.0 mL) was stirred at 20
°C for 16 h. Then, the mixture was concentrated and the residue was
diluted with water (3 mL) and extracted with EtOAc (5 mL). Then
the aqueous layer was acidified to pH = 3−4 with 1 N HCl and
filtered. The filter was concentrated to dryness to give compound 37j-
trans (50 mg, crude) as a yellow solid. ESI-MS m/z: calcd for
C23H26N2O5, 410.2; m/z: found, 411.2 [M + H]+. This intermediate
was used in the next step without further purification or character-
ization.
rac-cis 4-[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]-N-tetrahydropyran-2-yloxy-cyclohexanecarboxamide
(38j-cis). A mixture of compound 37j-cis (60 mg, 0.146 mmol),
THPONH2(34 mg, 0.292 mmol), DIEA (38 mg, 0.292 mmol), HOBt
(24 mg, 0.175 mmol), and EDCI (34 mg, 0.175 mmol) in DMF (10
mL) was degassed and purged with N2 3 times and the mixture was
stirred at 20 °C for 16 h. Then, the mixture was concentrated in
reduced pressure at 40 °C and the residue was purified by prep-HPLC
(method 13 described in the Supporting Information) to afford
compound 38j-cis (30 mg, 40%) as a yellow solid. ESI-MS m/z: calcd
for C28H35N3O6, 509.3; m/z: found, 510.3 [M + H]+. This
intermediate was used in the next step without further character-
ization.
6-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]pyridine-3-carboxylic Acid (37k). To a solution
of compound 36k (0.1 g, 0.194 mmol, 1 equiv) in THF (5 mL) and
water (5 mL) was added LiOH·H2O (24.37 mg, 0.581 mmol, 3
rac-trans 4-[[6,7-Dimethoxy-2-(5-methyl-2-furyl)-4-quinolyl]-
amino]-N-tetrahydropyran-2-yloxy-cyclohexanecarboxamide
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(38j-trans). A mixture of compound 37j-trans (50 mg, 0.122 mmol),
THPONH2(29 mg, 0.244 mmol), DIEA (31 mg, 0.244 mmol), HOBt
(20 mg, 0.146 mmol), and EDCI (28 mg, 0.146 mmol) in DMF (10
mL) was degassed and purged with N2 3 times and the mixture was
stirred at 20 °C for 16 h. Then, the mixture was concentrated in
reduced pressure at 40 °C and the residue was purified by prep-HPLC
(method 3 described in the Supporting Information) to afford pure
compound 38j-trans (20 mg, 32%) as a yellow solid. ESI-MS m/z:
calcd for C28H35N3O6, 509.3; m/z: found, 510.3 [M + H]+. This
intermediate was used in the next step without further character-
ization.
times. The mixture was stirred under H2 (15 Psi) at 20 °C for 16 h.
Then, the mixture was filtered and concentrated in vacuum to afford
compound 40b (200 mg, 66%) as a yellow solid. ESI-MS m/z: calcd
for C30H39N5O4, 533.3; m/z: found, 534.3 [M + H]+. This
intermediate was used in the next step without further purification
or characterization.
Ethyl 2-[4-[[(6,7-Dimethoxy-2-phenyl-4-quinolyl)amino]methyl]-
1-piperidyl]pyrimidine-5-carboxylate (40c). A mixture of compound
39c46 (150 mg, 0.500 mmol), ethyl 2-[4-(aminomethyl)-1-piperidyl]-
pyrimidine-5-carboxylate (51, 145 mg, 0.550 mmol), Cs2CO3 (326
mg, 1.00 mmol), BINAP (31 mg, 0.050 mmol), and Pd2(dba)3 (45
mg, 0.050 mmol) in 1,4-dioxane (15 mL) was degassed and purged
with N2 3 times, and then the mixture was stirred at 100 °C for 16 h.
Then, the mixture was filtered and concentrated in vacuum. The
residue was purified by prep-TLC (SiO2, CH2Cl2/MeOH = 10:1) to
afford pure compound 40c (200 mg, 76%) as a yellow solid. ESI-MS
m/z: calcd for C30H33N5O4, 527.3; m/z: found, 528.3 [M + H]+. This
compound was used in the next step without further characterization.
Ethyl 2-[4-[[[6,7-Dimethoxy-2-(1-piperidyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]pyrimidine-5-carboxylate (40d). A mixture of
compound 39d (250 mg, 0.815 mmol), ethyl 2-[4-(aminomethyl)-1-
piperidyl]pyrimidine-5-carboxylate (51, 323 mg, 1.22 mmol),
Pd2(dba)3 (75 mg, 0.081 mmol), BINAP (51 mg, 0.081 mmol),
and Cs2CO3 (531 mg, 1.63 mmol) in 1,4-dioxane (10 mL) was
degassed and purged with N2 3 times, and then the mixture was
stirred at 120 °C for 16 h. Then, the reaction mixture was
concentrated in vacuum to give a residue. The residue was purified
by prep-TLC (CH2Cl2/MeOH = 10:1) to afford pure compound 40d
4-Chloro-6,7-dimethoxy-2-(1-piperidyl)quinoline (39d). A mix-
ture of compound 3435 (1.00 g, 3.87 mmol), piperidine (330 mg, 3.87
mmol), Cs2CO3 (2.52 g, 7.75 mmol), Pd2(dba)3 (355 mg, 0.387
mmol), and BINAP (241 mg, 0.387 mmol) in 1,4-dioxane (100 mL)
was degassed and purged with N2 3 times, and then the mixture was
stirred at 120 °C for 16 h. Then, the mixture was concentrated in
reduced pressure at 48 °C and the residue was poured into water (200
mL). The aqueous phase was extracted with EtOAc (100 mL × 3)
and the combined organic phase was concentrated in reduced
pressure at 48 °C to afford compound 39d (300 mg, 25%) as a yellow
solid, which was used in the next step without further purification. 1H
NMR (CD3OD, 400 MHz): δ 7.56 (s, 1H), 7.47 (s, 1H), 7.42 (s,
1H), 4.02 (s, 3H), 3.98 (s, 3H), 3.84 (s, 4H), 1.82 (s, 6H). ESI-MS
m/z: calcd for C16H19ClN2O2, 306.1; m/z: found, 307.1 [M + H]+.
4-Chloro-2-(2,5-dimethyl-3-furyl)-6,7-dimethoxy-quinoline
(39e). A mixture of compound 3435 (2 g, 7.75 mmol, 1 equiv), 2-(2,5-
dimethyl-3-furyl)-4,4,5,5 tetramethyl-1,3,2-dioxaborolane (1.72 g,
7.75 mmol, 1 equiv), K2CO3 (2.14 g, 15.50 mmol, 2 equiv),
Pd(PPh3)4 (895.44 mg, 0.775 mmol, 0.1 equiv) in dioxane (20 mL),
and water (5 mL) was degassed and purged with N2 3 times, and then
the mixture was stirred at 80 °C for 2 h under an N2 atmosphere.
LC−MS showed that the reaction was completed and one main peak
with the desired m/z was detected. The mixture was filtered and the
filtrate was concentrated in vacuum. The residue was purified by flash
silica gel chromatography (ISCO, 40 g SepaFlash Silica Flash
Column) using as eluent a 0−15% ethylacetate/petroleum ether
gradient at 100 mL/min. Compound 39e was obtained as a light
yellow solid: 1 g, 3.15 mmol, 40.61% yield. ESI-MS m/z: calcd for
C17H16ClNO3, 317.1; m/z: found, 318.1 [M + H]+. This compound
was used in the next step without further characterization.
Ethyl 2-[4-[[(6,7-Dimethoxy-2-methyl-4-quinolyl)amino]methyl]-
1-piperidyl]pyrimidine-5-carboxylate (40a). A mixture of compound
39a45,46 (200 mg, 0.841 mmol), ethyl 2-[4-(aminomethyl)-1-
piperidyl]pyrimidine-5-carboxylate (51, 334 mg, 1.26 mmol),
Pd2(dba)3 (154 mg, 0.168 mmol), BINAP (157 mg, 0.252 mmol),
and Cs2CO3 (548 mg, 1.68 mmol) in 1,4-dioxane (10 mL) was
degassed and purged with N2 3 times, and then the mixture was
stirred at 120 °C for 48 h. Then, the reaction mixture was filtered and
concentrated in vacuum to give a residue. The residue was purified by
prep-TLC (SiO2, CH2Cl2/MeOH = 10:1) to afford pure compound
40a (230 mg, 59%) as a yellow oil. ESI-MS m/z: calcd for
C25H31N5O4, 465.2; m/z: found, 466.3 [M + H]+. This compound
was used in the next step without further characterization.
1
(270 mg, 62%) as a yellow solid. H NMR (CD3OD, 400 MHz): δ
8.78 (s, 2H), 7.37 (s, 1H), 7.07 (s, 1H), 6.92 (s, 1H), 4.35−4.29 (m,
2H), 3.92 (s, 6H), 3.72 (s, 1H), 3.58 (br s, 4H), 3.30−3.27 (m, 2H),
3.04−2.98 (m, 2H), 2.22 (s, 2H), 1.98−1.95 (m, 2H), 1.87−1.86 (m,
1H), 1.70 (s, 6H), 1.36 (s, 3H), 1.31−1.27 (m, 2H). ESI-MS m/z:
calcd for C29H38N6O4, 534.3; m/z: found, 535.3 [M + H]+.
Ethyl 2-[4-[[[2-(2,5-Dimethyl-3-furyl)-6,7-dimethoxy-4-quinolyl]-
amino]methyl]-1-piperidyl]pyrimidine-5-carboxylate (40e). A mix-
ture of compound 39e (1 g, 3.15 mmol, 1 equiv), ethyl 2-[4-
(aminomethyl)-1-piperidyl]pyrimidine-5-carboxylate (51, 1.23 g, 4.09
mmol, 1.3 equiv), Pd2(dba)3 (576.35 mg, 0.629 mmol, 0.2 equiv),
BINAP (587.86 mg, 0.944 mmol, 0.3 equiv), and Cs2CO3 (3.08 g,
9.44 mmol, 3 equiv) in 1,4-dioxane (20 mL) was degassed and purged
with N2 3 times, and then the mixture was stirred at 120 °C for 12 h
under an N2 atmosphere. LC−MS showed that the reaction was
completed and one main peak with the desired m/z was detected. The
mixture was filtered and the filtrate was concentrated in vacuum. The
residue was purified by flash silica gel chromatography (ISCO, 40 g
SepaFlash Silica Flash Column) using as eluent a 0−100%
ethylacetate/petroleum ether gradient at 100 mL/min. Compound
40e was obtained as a light yellow solid: 0.8 g, 1.47 mmol, 46.6%
yield. ESI-MS m/z: calcd for C30H35N5O5, 545.3; m/z: found, 546.2
[M + H]+. This compound was used in the next step without further
characterization.
Ethyl 2-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-thienyl)-4-quinolyl]-
amino]methyl]-1-piperidyl]pyrimidine-5-carboxylate (40f). A mix-
ture of compound 39f46 (0.17 g, 0.532 mmol, 1 equiv), ethyl 2-[4-
(aminomethyl)-1-piperidyl]pyrimidine-5-carboxylate (51, 207.86 mg,
0.691 mmol, 1.3 equiv), Pd2(dba)3 (97.35 mg, 0.106 mmol, 0.2
equiv), BINAP (99.3 mg, 0.160 mmol, 0.3 equiv), and Cs2CO3 (519.6
mg, 1.59 mmol, 3 equiv) indioxane (10 mL) was degassed and purged
with N2 3 times, and then the mixture was stirred at 120 °C for 12 h
under an N2 atmosphere. LC−MS showed that the reaction was
completed and one main peak with the desired m/z was detected. The
mixture was filtered and the filtrate was concentrated. The residue was
purified by flash silica gel chromatography (ISCO, 12 g SepaFlash
Silica Flash Column) using as eluent a 0−80% ethylacetate/petroleum
ether gradient at 50 mL/min. Compound 40f was obtained as a light
yellow solid (0.15 g, 0.274 mmol, 51.5% yield), which was used in the
next step without further purification. 1H NMR (CD3OD, 400 MHz):
δ 8.85 (s, 2H), 7.44 (br s, 1H), 7.37 (s, 1H), 6.86 (s, 1H), 6.79−6.78
(m, 1H), 6.75 (s, 1H), 4.99 (d, J = 13.2 Hz, 2H), 4.75 (br s, 1H), 4.35
Ethyl 2-[4-[[(2-Cyclohexyl-6,7-dimethoxy-4-quinolyl)amino]-
methyl]-1-piperidyl]pyrimidine-5-carboxylate (40b). A mixture of
compound 39g46 (300 mg, 0.987 mmol), ethyl 2-[4-(aminomethyl)-
1-piperidyl]pyrimidine-5-carboxylate (51, 313 mg, 1.19 mmol),
Cs2CO3 (643 mg, 1.98 mmol), BINAP (61 mg, 0.099 mmol), and
Pd2(dba)3 (90 mg, 0.099 mmol) in 1,4-dioxane (20 mL) was degassed
and purged with N2 3 times, and then the mixture was stirred at 120
°C for 16 h. Then, the mixture was filtered and concentrated in
vacuum. The residue was purified by prep-TLC (SiO2, CH2Cl2/
MeOH = 10:1) to afford pure intermediate ethyl 2-[4-[[[2-
(cyclohexen-1-yl)-6,7-dimethoxy-4-quinolyl]amino]methyl]-1-
piperidyl]pyrimidine-5-carboxylate (300 mg, 57%) as a yellow solid.
ESI-MS m/z: calcd for C30H37N5O4, 531.3; m/z: found, 532.4 [M +
H]+. To a solution of this intermediate (300 mg, 0.564 mmol) in
MeOH (20 mL) was added Pd/C (10%, 30 mg) under an H2
atmosphere. The suspension was degassed and purged with H2
3
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(q, J = 7.2 Hz, 2H), 4.02 (s, 6H), 3.34 (t, J = 6.2 Hz, 2H), 3.03−2.97
(m, 2H), 2.55 (s, 3H), 2.15−2.05 (m, 1H), 2.01 (d, J = 11.9 Hz, 2H),
1.38 (t, J = 7.2 Hz, 5H). ESI-MS m/z: calcd for C29H33N5O4S, 547.2;
m/z: found, 548.3 [M + H]+.
was obtained as a yellow solid: 120 mg, 0.231 mmol, 84.3% yield. ESI-
MS m/z: calcd for C27H29N5O4S, 519.2; m/z: found, 520.2 [M + H]+.
This intermediate was used in the next step without further
purification or characterization.
2-[4-[[(6,7-Dimethoxy-2-methyl-4-quinolyl)amino]methyl]-1-pi-
peridyl]-N-tetrahydropyran-2-yloxy-pyrimidine-5-carboxamide
(42a). A mixture of compound 41a (300 mg, 0.685 mmol),
THPONH2 (161 mg, 1.37 mmol), EDCI (263 mg, 1.37 mmol),
HOBt (185 mg, 1.37 mmol), and DIEA (265 mg, 2.06 mmol) in
DMF (30 mL) was degassed and purged with N2 3 times, and then
the mixture was stirred at 20 °C for 16 h. Then, the reaction mixture
was quenched with water (10 mL) and concentrated in vacuum to
give a residue. The residue was extracted with EtOAc (3 mL × 3) and
the organic layers were dried over Na2SO4 and concentrated in
vacuum to give compound 42a (300 mg, 82%) as a yellow oil. ESI-MS
m/z: calcd for C28H36N6O5, 536.3; m/z: found, 537.3 [M + H]+. This
intermediate was used in the next step without further purification or
characterization.
2-[4-[[(6,7-Dimethoxy-2-methyl-4-quinolyl)amino]methyl]-1-
piperidyl]pyrimidine-5-carboxylic Acid (41a). To a solution of
compound 40a (230 mg, 0.494 mmol) in THF/H2O (2:1, 30 mL)
was added LiOH·H2O (41 mg, 0.988 mmol) and the mixture was
stirred at 20 °C for 16 h. Then, the reaction mixture was adjusted to
pH 6 and concentrated in vacuum to give compound 41a (300 mg,
crude) as a yellow solid that was used in the next step without further
1
purification. H NMR (CD3OD, 400 MHz): δ 8.91 (s, 2H), 7.76 (s,
1H), 7.20 (s, 1H), 6.69 (s, 1H), 4.82−4.80 (m, 2H), 4.02−4.00 (m,
6H), 3.53−3.51 (m, 2H), 3.28−3.21 (m, 2H), 2.66 (s, 3H), 2.35 (s,
1H), 2.08−2.05 (m, 2H), 1.50−1.48 (m, 2H); two exchangeable
protons were not observed. ESI-MS m/z: calcd for C23H27N5O4,
437.2; m/z: found, 438.3 [M + H]+.
2-[4-[[(2-Cyclohexyl-6,7-dimethoxy-4-quinolyl)amino]methyl]-1-
piperidyl]pyrimidine-5-carboxylic Acid (41b). A mixture of com-
pound 40b (200 mg, 0.375 mmol) and LiOH·H2O (47 mg, 1.12
mmol) in MeOH/THF/H2O (1:3:1, 5 mL) was stirred at 20 °C for
16 h. Then, the mixture was concentrated and the residue was diluted
with water (2 mL) and extracted with EtOAc (3 mL). Then, the
aqueous phase was acidified to pH = 3−4 with 1 N HCl and filtered.
The filter cake was concentrated to dryness to give compound 41b
(120 mg, 63%) as a yellow solid. ESI-MS m/z: calcd for C28H35N5O4,
505.3; m/z: found, 506.3 [M + H]+. This intermediate was used in
the next step without further purification or characterization.
2-[4-[[(6,7-Dimethoxy-2-phenyl-4-quinolyl)amino]methyl]-1-
piperidyl]pyrimidine-5-carboxylic Acid (41c). A mixture of com-
pound 40c (200 mg, 0.379 mmol) and LiOH·H2O (47 mg, 1.14
mmol) in MeOH/THF/H2O (1:3:1, 8 mL) was stirred at 20 °C for
16 h. Then, the mixture was concentrated and the residue was diluted
with water (5 mL) and extracted with EtOAc (10 mL). The aqueous
phase was acidified to pH = 3−4 with 1 N HCl and filtered. The filter
cake was concentrated to dryness to afford compound 41c (150 mg,
79%) as a yellow solid. ESI-MS m/z: calcd for C28H29N5O4, 499.2; m/
z: found, 500.3 [M + H]+. This intermediate was used in the next step
without further purification or characterization.
2-[4-[[[6,7-Dimethoxy-2-(1-piperidyl)-4-quinolyl]amino]methyl]-
1-piperidyl]pyrimidine-5-carboxylic Acid (41d). To a solution of
compound 40d (270 mg, 0.505 mmol) in THF/H2O (2:1, 15 mL)
was added LiOH·H2O (32 mg, 0.757 mmol) and the mixture was
stirred at 15 °C for 16 h. Then, the pH was adjusted to 5 with 2.0 M
HCl aqueous solution and the mixture was concentrated in vacuum to
obtain crude compound 41d (300 mg, crude) as a yellow oil. ESI-MS
m/z: calcd for C27H34N6O4, 506.3; m/z: found, 507.3 [M + H]+.This
intermediate was used in the next step without further purification or
characterization.
2-[4-[[[2-(2,5-Dimethyl-3-furyl)-6,7-dimethoxy-4-quinolyl]-
amino]methyl]-1-piperidyl]pyrimidine-5-carboxylic Acid (41e). To
a solution of 40e (0.8 g, 1.47 mmol, 1 equiv) in THF (10 mL)and
water (5 mL) was added LiOH·H2O (307.61 mg, 7.33 mmol, 5
equiv).The mixture was stirred at 25 °C for 12 h. LC−MS showed
that the reaction was completed and one main peak with the desired
m/z was detected. The mixture was adjusted to pH ∼ 5 with aqueous
HCl (2 M) at room temperature. Some precipitate was formed and,
after filtration, the solid was collected to afford compound 41e as a
light yellow solid: 0.6 g, 1.16 mmol, 79.1% yield. ESI-MS m/z: calcd
for C28H31N5O5, 517.2; m/z: found, 518.2 [M + H]+. This
intermediate was used in the next step without further purification
or characterization.
2-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-thienyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]pyrimidine-5-carboxylic Acid (41f). To a
solution of 40f (150 mg, 0.274 mmol, 1 equiv) in THF (8 mL)
and water (4 mL) was added LiOH·H2O (57.46 mg, 1.37 mmol, 5
equiv). The mixture was stirred at 25 °C for 12 h. LC−MS showed
that the reaction was completed and one main peak with the desired
m/z was detected. The mixture was adjusted to pH ∼ 5 with aqueous
HCl (2 M) at room temperature and then filtered. Compound 41f
2-[4-[[(2-Cyclohexyl-6,7-dimethoxy-4-quinolyl)amino]methyl]-1-
piperidyl]-N-tetrahydropyran-2-yloxy-pyrimidine-5-carboxamide
(42b). A mixture of compound 41b (120 mg, 0.237 mmol),
THPONH2 (56 mg, 0.475 mmol), DIEA (61 mg, 0.475 mmol),
HOBt (38 mg, 0.285 mmol), and EDCI (55 mg, 0.295 mmol) in
DMF (10 mL) was degassed and purged with N2 3 times, and then
the mixture was stirred at 20 °C for 16 h. Then, the mixture was
concentrated in reduced pressure at 40 °C and the residue was
purified by prep-HPLC (method 3 described in the Supporting
Information) to afford pure compound 42b (50 mg, 35%) as a yellow
solid. ESI-MS m/z: calcd for C33H44N6O5, 604.3; m/z: found, 605.4
[M + H]+. This compound was used in the next step without further
characterization.
2-[4-[[(6,7-Dimethoxy-2-phenyl-4-quinolyl)amino]methyl]-1-pi-
peridyl]-N-tetrahydropyran-2-yloxy-pyrimidine-5-carboxamide
(42c). A mixture of compound 41c (150 mg, 0.300 mmol),
THPONH2 (70 mg, 0.600 mmol), DIEA (77 mg, 0.600 mmol),
HOBt (48 mg, 0.360 mmol), and EDCI (69 mg, 0.360 mmol) in
DMF (15 mL) was degassed and purged with N2 3 times, and then
the mixture was stirred at 20 °C for 16 h. Then, the mixture was
concentrated in reduced pressure at 40 °C and the residue was
purified by prep-HPLC (method 4 described in the Supporting
Information) to afford pure compound 42c (60 mg, 33%) as a yellow
solid. ESI-MS m/z: calcd for C33H38N6O5, 598.3; m/z: found, 599.3
[M + H]+. This compound was used in the next step without further
characterization.
2-[4-[[[6,7-Dimethoxy-2-(1-piperidyl)-4-quinolyl]amino]methyl]-
1-piperidyl]-N-tetrahydropyran-2-yloxy-pyrimidine-5-carboxamide
(42d). A mixture of compound 41d (300 mg, 0.592 mmol),
THPONH2 (139 mg, 1.18 mmol), EDCI (227 mg, 1.18 mmol),
HOBt (160 mg, 1.18 mmol), and DIEA (229 mg, 1.78 mmol) in
DMF (10 mL) was degassed and purged with N2 3 times, and then
the mixture was stirred at 15 °C for 16 h. Then, the reaction mixture
was quenched with water (5 mL) and concentrated in vacuum to give
a residue. The residue was dissolved in 20 mL of CH3CN (20 mL)
and filtered. The filtrate was concentrated in vacuum to afford
compound 42d (500 mg, crude) as a brown oil. ESI-MS m/z: calcd
for C32H43N7O5, 605.3; m/z: found, 606.4 [M + H]+.This
intermediate was used in the next step without further purification
or characterization.
2-[4-[[[2-(2,5-Dimethyl-3-furyl)-6,7-dimethoxy-4-quinolyl]-
amino]methyl]-1-piperidyl]-N-tetrahydropyran-2-yloxy-pyrimi-
dine-5-carboxamide (42e). A mixture of compound 41e (0.6 g, 1.16
mmol, 1 equiv), O-tetrahydropyran-2-ylhydroxylamine (271.60 mg,
2.32 mmol, 2 equiv), HOBt (313.28 mg, 2.32 mmol, 2 equiv), EDCI
(444.46 mg, 2.32 mmol, 2 equiv), and DIEA (749.11 mg, 5.80 mmol,
1.01 mL, 5 equiv) in DMF (8 mL) was degassed and purged with N2
3 times, and then the mixture was stirred at 25 °C for 12 h under an
N2 atmosphere. LC−MS showed that the reaction was completed and
one main peak with the desired m/z was detected. The residue was
poured into ice-water (w/w = 1/1) (30 mL). The aqueous phase was
extracted with ethyl acetate (20 mL, three times). The combined
organic phase was washed with brine (30 mL), dried with anhydrous
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J. Med. Chem. 2021, 64, 3392−3426

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Na2SO4, concentrated in vacuum, and purified by prep-HPLC
(method 44 described in the Supporting Information) to afford the
pure compound 42e, as a light yellow solid: 0.6 g, 0.973 mmol, 83.9%
yield. ESI-MS m/z calcd for C33H40N6O6, 616.3; m/z: found, 617.3
[M + H]+.
2-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)quinazolin-4-yl]-
amino]methyl]-1-piperidyl]-N-tetrahydropyran-2-yloxy-pyrimi-
dine-5-carboxamide (47). A mixture of 46 (150 mg, 0.297 mmol),
THPONH2 (70 mg, 0.594 mmol), HOBt (80 mg, 0.594 mmol),
EDCI (114 mg, 0.594 mmol), and DIEA (192 mg, 1.49 mmol) in
DMF (10 mL) was degassed and purged with N2 3 times, and then
the mixture was stirred at 25 °C for 12 h under an N2 atmosphere.
Then, the residue was poured into ice-water (w/w = 1/1) (10 mL)
and the aqueous phase was extracted with EtOAc (10 mL × 3). The
combined organic phase was washed with brine (10 mL), dried with
anhydrous Na2SO4, filtered, and concentrated in vacuum to afford 47
(130 mg, crude) as a light yellow solid. ESI-MS m/z: calcd for
C31H37N7O6, 603.3; m/z: found, 604.3 [M + H]+. This intermediate
was used in the next step without further purification or character-
ization.
Synthesis of Intermediates. Ethyl 2-[4-[(tert-
Butoxycarbonylamino)methyl]-1-piperidyl]pyrimidine-5-carboxy-
late (50). A mixture of tert-butyl N-(4-piperidylmethyl)carbamate
(48, 1.00 g, 4.67 mmol), ethyl 2-chloropyrimidine-5-carboxylate (49,
870 mg, 4.67 mmol), BINAP (290 mg, 0.467 mmol), Cs2CO3 (3.04 g,
9.34 mmol), and Pd2(dba)3 (427 mg, 0.467 mmol) in 1,4-dioxane
(100 mL) was degassed and purged with N2 3 times, and then the
mixture was stirred at 110 °C for 16 h. Then, the mixture was
concentrated in reduced pressure at 40 °C, the residue was poured
into water (50 mL), and it was extracted with EtOAc (30 mL × 3).
The combined organic phase was washed with brine (30 mL × 2),
dried with anhydrous Na2SO4, filtered, and concentrated in vacuum.
The residue was purified by silica gel column chromatography (PE/
EtOAc = 1:0 to 2:1) to afford pure 50 (1.20 g, 70%) as a yellow solid.
ESI-MS m/z: calcd for C18H28N4O4, 364.2; m/z: found, 365.2 [M +
H]+.
Ethyl 2-[4-(Aminomethyl)-1-piperidyl]pyrimidine-5-carboxylate
(51). A solution of 50 (1.20 g, 3.29 mmol) in HCl/EtOAc (30 mL,
2.0 M) was stirred at 20 °C for 16 h. Then, the mixture was
concentrated in reduced pressure at 40 °C, the residue was poured
into water (20 mL), and it was extracted with EtOAc (10 mL × 3).
The aqueous phase was concentrated in vacuum and then dissolved in
MeOH (20 mL). NaHCO3 (200 mg) was added and the mixture was
stirred at 25 °C for 1 h. The mixture was filtered and concentrated in
vacuum to afford 51 (700 mg, 80%) as a white solid, which was used
for the synthesis of target compounds without further purification. 1H
NMR (CD3OD, 400 MHz): δ 8.77 (d, J = 8.0 Hz, 2H), 4.95−4.91
(m, 2H), 4.35−4.29 (m, 2H), 3.04−2.97 (m, 2H), 2.78−2.77 (m,
2H), 1.95−1.86 (m, 3H), 1.37−1.34 (m, 3H), 1.24−1.20 (m, 2H).
ESI-MS m/z: calcd for C13H20N4O2, 264.2; m/z: found, 265.2 [M +
H]+.
Methyl 4-[[4-[(tert-Butoxycarbonylamino)methyl]-1-piperidyl]-
methyl]benzoate (53). To a solution of methyl 4-formylbenzoate
(52, 200 mg, 1.22 mmol) in MeOH (20 mL) were added NaBH3CN
(230 mg, 3.66 mmol), AcOH (77 mg, 1.28 mmol), and tert-butyl N-
(4-piperidylmethyl)carbamate (48, 275 mg, 1.28 mmol) and the
mixture was stirred at 80 °C for 3 h. Then, the reaction was quenched
with water (5 mL) and MeOH was removed. The residue was
extracted with EtOAc (30 mL × 3). The combined organic layers
were washed with brine (10 mL), dried over anhydrous Na2SO4,
filtered, and concentrated under reduced pressure to give a residue,
which was purified by prep-TLC (SiO2, PE/EtOAc = 1/1) to afford
pure 53 (220 mg, 50%) as a white solid. ESI-MS m/z: calcd for
C20H30N2O4, 362.2; m/z: found, 363.1 [M + H]+.
2-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-thienyl)-4-quinolyl]amino]-
methyl]-1-piperidyl]-N-tetrahydropyran-2-yloxy-pyrimidine-5-car-
boxamide (42f). A mixture of compound 41f (50 mg, 0.096 mmol, 1
equiv), O-tetrahydropyran-2-ylhydroxylamine (22.54 mg, 0.192
mmol, 2 equiv), HOBt (26.0 mg, 0.192 mmol, 2 equiv), EDCI
(36.89 mg, 0.192 mmol, 2 equiv), and DIEA (62.18 mg, 0.481 mmol,
83.8 μL, 5 equiv) in DMF (5 mL) was degassed and purged with N2 3
times, and then the mixture was stirred at 25 °C for 12 h under an N2
atmosphere. LC−MS showed that the reaction was completed and
one main peak with the desired m/z was detected. The residue was
poured into ice-water (w/w = 1/1) (6 mL). The aqueous phase was
extracted with ethyl acetate (5 mL, 3 times). The combined organic
phase was washed with brine (10 mL), dried with anhydrous Na2SO4,
filtered, and concentrated in vacuum. Compound 42f was obtained as
a light yellow solid (40mg). ESI-MS m/z: calcd for C32H38N6O5S,
618.3; m/z: found, 619.3 [M + H]+. This intermediate was used in
the next step without further purification or characterization.
Ethyl 2-[4-[[(2-Chloro-6,7-dimethoxy-quinazolin-4-yl)amino]-
methyl]-1-piperidyl]pyrimidine-5-carboxylate (44). A mixture of
commercially available 2,4-dichloro-6,7-dimethoxy-quinazoline (43)
(0.2 g, 0.772 mmol), ethyl 2-[4-(aminomethyl)-1-piperidyl]-
pyrimidine-5-carboxylate (51, 232 mg, 0.772 mmol, HCl), and
K2CO3(320 mg, 2.32 mmol) in DMF (10 mL) was degassed and
purged with N2 3 times, and then the mixture was stirred at 80 °C for
12 h under an N2 atmosphere. Then, the mixture was filtered and the
filtrate was concentrated to give a residue. The residue was purified by
flash silica gel chromatography (ISCO; 12 g SepaFlash Silica
FlashColumn, Eluent of 0−40% EtOAc/PE gradient at 100 mL/
1
min) to obtain pure 44 (200 mg, 53%) as a light yellow solid. H
NMR (CDCl3, 400 MHz): δ 8.83 (s, 2H), 7.15 (s, 1H), 6.85 (s, 1H),
5.73 (br s, 1H), 4.94 (br d, J = 13.2 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H),
4.00 (s, 3H), 3.98 (s, 3H), 3.63 (t, J = 6.4 Hz, 2H), 3.02−2.95 (m,
2H), 2.13−2.12 (m, 1H), 1.93 (br d, J = 11.2 Hz, 2H), 1.39−1.32 (m,
5H). ESI-MS m/z: calcd for C23H27ClN6O4, 486.2; m/z: found, 487.2
[M + H]+.
Ethyl 2-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)quinazolin-4-yl]-
amino]methyl]-1-piperidyl]pyrimidine-5-carboxylate (45). A mix-
ture of 44 (150 mg, 0.308 mmol),4,4,5,5-tetramethyl-2-(5-methyl-2-
furyl)-1,3,2-dioxaborolane (64 mg, 0.308), K2CO3 (128 mg, 0.924
mmol) and Pd(PPh3)4 (36 mg, 0.031 mmol) in 1,4-dioxane (10 mL)
was degassed and purged with N2 3 times, and then the mixture was
stirred at 100 °C for 12 h under an N2 atmosphere. Then, the mixture
was filtered and the filtrate was concentrated. The residue was purified
by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica
FlashColumn, eluent of 0−40% EtOAc/PE gradient at 75 mL/min)
1
to obtain pure 45 (140 mg, crude) as a light yellow solid. H NMR
(CDCl3, 400 MHz): δ 8.83 (s, 2H), 7.36 (s, 1H), 7.16 (d, J = 3.3 Hz,
1H), 6.88 (s, 1H), 6.15 (dd, J = 0.9, 3.1 Hz, 1H), 5.58 (br s, 1H), 4.94
(br d, J = 13.5 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 4.00 (d, J = 0.9 Hz,
6H), 3.68 (t, J = 6.4 Hz, 2H), 3.02−2.95 (m, 2H), 2.46 (s, 3H),
2.20−2.17 (m, 1H), 1.96 (br d, J = 10.8 Hz, 2H), 1.41−1.33 (m, 5H).
ESI-MS m/z: calcd for C28H32N6O5, 532.2; m/z: found, 533.3 [M +
H]+.
2-[4-[[[6,7-Dimethoxy-2-(5-methyl-2-furyl)quinazolin-4-yl]-
amino]methyl]-1-piperidyl]pyrimidine-5-carboxylic Acid (46). To a
solution of 45 (190 mg, 0.356 mmol) in THF/H2O (2:1, 12 mL) was
added LiOH·H2O (45 mg, 1.07 mmol) and the mixture was stirred at
25 °C for 12 h. Then, the mixture was adjusted to pH ∼ 5 with
aqueous HCl (2 M) and extracted with EtOAc (5 mL × 3). The
combined organic phase was washed with brine (10 mL), dried with
anhydrous Na2SO4, filtered, and concentrated in vacuum to afford 46
(150 mg, 83%) as a light yellow solid. ESI-MS m/z: calcd for
C26H28N6O5, 504.2; m/z: found, 505.3 [M + H]+. This intermediate
was used in the next step without further purification or character-
ization.
Methyl 4-[[4-(Aminomethyl)-1-piperidyl]methyl]benzoate (54).
A solution of 53 (220 mg, 0.607 mmol) in HCl/EtOAc (10 mL, 2.0
M) was stirred at 20 °C for 1 h. Then, the mixture was concentrated
to give a residue. The residue was diluted with MeOH and the pH
was adjusted to 7−8 with NaHCO3. Then, the mixture was filtered
and the filtrate was concentrated to give 54 (140 mg, 88%) as a white
solid, which was used for the synthesis of compound 17d without
further purification. 1H NMR (CD3OD, 400 MHz): δ 7.98 (d, J = 8.0
Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 3.91 (s, 3H), 3.58 (d, J = 6.4 Hz,
2H), 2.94−2.90 (m, 2H), 2.56−2.53 (m, 2H), 2.08−2.02 (m, 2H),
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J. Med. Chem. 2021, 64, 3392−3426