Detailed Exploration around 4-Aminoquinolines Chemical Space to Navigate the Lysine Methyltransferase G9a and DNA Methyltransferase Biological Spaces

Article abstract of DOI:10.1021/acs.jmedchem.7b01925

Epigenetic regulators that exhibit aberrant enzymatic activities or expression profiles are potential therapeutic targets for cancers. Specifically, enzymes responsible for methylation at histone-3 lysine-9 (like G9a) and aberrant DNA hypermethylation (DNMTs) have been implicated in a number of cancers. Recently, molecules bearing a 4-aminoquinoline scaffold were reported as dual inhibitors of these targets and showed a significant in vivo efficacy in animal models of hematological malignancies. Here, we report a detailed exploration around three growing vectors born by this chemotype. Exploring this chemical space led to the identification of features to navigate G9a and DNMT1 biological spaces: not only their corresponding exclusive areas, selective compounds, but also common spaces. Thus, we identified from selective G9a and first-in-class DNMT1 inhibitors, >1 log unit between their IC₅₀ values, with IC₅₀ < 25 nM (e.g., 43 and 26, respectively) to equipotent inhibitors with IC₅₀ < 50 nM for both targets (e.g., 13). Their ADME/Tox profiling and antiproliferative efficacies, versus some cancer cell lines, are also reported.

Full text of DOI:10.1021/acs.jmedchem.7b01925

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Article
Detailed Exploration around 4-Aminoquinolines Chemical
Space to Navigate the Lysine Methyltransferase
G9a and DNA Methyltransferase Biological Spaces.
Obdulia Rabal, Juan A Sánchez-Arias, Edurne San José - Eneriz, Xabier Agirre, Irene De
Miguel, Leire Garate, Estibaliz Miranda, Elena Sáez, Sergio Roa, Jose Angel Martinez-
Climent, Yingying Liu, Wei Wu, Musheng Xu, Felipe Prosper, and Julen Oyarzabal
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01925 • Publication Date (Web): 11 Jun 2018
Downloaded from http://pubs.acs.org on June 11, 2018
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Detailed Exploration around 4-Aminoquinolines Chemical Space to
Navigate the
Lysine Methyltransferase
G9a
and
DNA
Methyltransferase Biological Spaces.
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Obdulia Rabal, Juan Antonio SánchezꢀArias, Edurne San JoséꢀEnériz, Xabier
2
1
2
2
1
2
Agirre, Irene de Miguel, Leire Garate, Estibaliz Miranda, Elena Sáez, Sergio Roa,
2
3
3
3
José Angel MartínezꢀCliment, Yingying Liu, Wei Wu, Musheng Xu, Felipe
2
,4
1,*
Prosper, and Julen Oyarzabal
1
2
Small Molecule Discovery Platform, Molecular Therapeutics Program, Area de
HematoꢀOncología, IDISNA, Ciberonc, Center for Applied Medical Research (CIMA),
University of Navarra, Avenida Pio XII 55, Eꢀ31008 Pamplona, Spain
3
WuXi Apptec (Tianjin) Co. Ltd., TEDA, No. 111 HuangHai Road, 4th Avenue, Tianjin
300456, PR China
4
Departmento de Hematología, Clinica Universidad de Navarra, University of Navarra,
Avenida Pio XII 36, Eꢀ31008 Pamplona, Spain
ABSTRACT
Epigenetic regulators that exhibit aberrant enzymatic activities or expression profiles are
potential therapeutic targets for cancers. Specifically, enzymes responsible for
methylation at histoneꢀ3 lysineꢀ9 (like G9a) and aberrant DNA hypermethylation
(DNMTs) have been implicated in a number of cancers. Recently, molecules bearing a
4ꢀaminoquinoline scaffold were reported as dual inhibitors of these targets and showed
a significant in-vivo efficacy in animal models of hematological malignancies. Here, we
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6
report a detailed exploration around three growing vectors born by this chemotype.
Exploring this chemical space led to the identification of features to navigate G9a and
DNMT1 biological spaces; not only their corresponding exclusive areas, selective
compounds, but also common spaces. Thus, we identified from selective G9a and firstꢀ
inꢀclass DNMT1 inhibitors, > 1 log unit between their IC50 values, with IC50 < 25nM
0
1
2
3
4
5
6
7
8
9
0
1
2
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0
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0
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6
7
8
9
0
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2
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7
8
9
0
(e.g. 43 and 26, respectively) to equipotent inhibitors with IC < 50nM for both targets
50
(e.g. 13). Their ADME/Tox profiling and antiproliferative efficacies, versus some
cancer cell lines, are also reported.
INTRODUCTION
Methyltransferases are responsible for the methylation of their corresponding substrates
(histones, proteins for protein methyltransferases (PMTs); or DNA, for DNA
methyltransferases (DNMTs)) using SꢀadenosylꢀLꢀmethionine (SAM or AdoMet) as
cofactor; thus acting as epigenetic writers that control epigenetic gene regulation and
1
,2
carcinogenesis. G9a (also known as KMT1C or EHMT2) catalyzes monoꢀ or
3
dimethylation of histone H3 lysine 9 (H3K9) and other protein targets such as p53. G9a
4
upregulation and overexpression is present in a variety of cancers. DNMT1, a protein
catalyzing methylation of C5ꢀcytosine of DNA, binds G9a and both proteins cooperate
5
in promoting transcriptional silencing of target genes. Moreover, pharmacologic or
siRNAꢀmediated inhibition of G9a synergises with DNMT1 inhibition in reducing cell
6
,7
proliferation. All these observations strongly suggested that dual inhibitors of both
targets could be valuable anticancer agents and we sought to identify such compounds.
8
A number of selective G9a inhibitors have been reported, with substrateꢀcompetitive
9
10
inhibitors 1 (UNCꢀ0638) and 2 (Aꢀ366) being extensively used as chemical probes to
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investigate the biological role of G9a in multiple diseases beyond cancer (Chart 1). The
development of potent reversible DNMT inhibitors is more appealing as most advanced
compounds include nucleoside mimetics such as 3 (Azacytidine) and 4 (Decitabine) that
incorporate into DNA and irreversible bind to DNMT1. As an alternative, current
reversible nonꢀnucleoside DNMT1 inhibitors lack potency, with IC50 values in the low
1
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11,12
micromolar range.
We recently reported the discovery of the quinolineꢀbased lead compound 5 (CMꢀ272)
as an inhibitor of G9a (IC50 = 8 nM) and DNMT1 (IC50 = 382 nM) with in vivo efficacy
in Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL) and
6
Diffuse Large Bꢀcell Lymphomaꢀ(DLBCL) xenogeneic models. Competition assays
and microscale thermophoresis demonstrated that inhibitors of this chemical series bind
6,13
to the substrate binding site of both targets.
Our efforts to identify this lead
compound utilized a combination of knowledgeꢀ and structureꢀbased approaches,
starting from the chemical structure of compound 1. Initial structureꢀactivity
relationship (SAR) studies were focused on determining the optimal substitution pattern
at the 2ꢀposition of the quinoline not only to achieve G9a and DNMT1 enzymatic
inhibition but also a cellular response and a suitable pharmacokinetic (PK) profile for in
1
3
vivo proofꢀofꢀconcept. Additional SAR exploration around the 4ꢀ, 6ꢀ and 7ꢀpositions
of the quinoline scaffold, aimed at further DNMT1 potency optimization and target
selectivity modulation, identified key chemical features that confer selectivity towards
G9a or DNMT1 (where selectivity involves >1 log unit difference between
corresponding IC values). In fact, firstꢀinꢀclass DNMT1 potent (low nanomolar, IC <
5
0
50
25 nM), selective and reversible inhibitors are achieved. In addition, antiproliferative
activities in cancer cells and preliminary ADME/Tox of this exploration are discussed.
Furthermore, novel Rꢀgroups mimicking the lysine side chain of histone substrate (G9a)
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or the cytosine of DNA (DNMT1) are introduced. Our results and compounds not only
represent interesting, and firstꢀinꢀclass, tools for targeting relevant epigenetic enzymes
but also constitute valuable alternatives for the design of epigenetic inhibitors targeting
the substrateꢀbinding site of other methyltransferases.
0
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UNC-0638
A-366
IC50 G9a = 55 nM
IC50 G9a = 3.3 nM
IC50 DNMT1 = 2010 nM
IC50 DNMT1 > 50000 nM
3
4
5
5-Azacytidine
Decitabine
CM-272
IC50 G9a > 10000 nM
DNMT1 = 300 nM
IC50 G9a > 10000 nM
DNMT1 = 30 nM
IC50 G9a = 8 nM
IC50 DNMT1 = 382 nM
Chart 1. Known G9a inhibitors (1, 2), nucleoside DNMT1 inhibitors (3, 4) and inhibitor (5).
G9a IC50 values of 1, 3, 4 and 5 and DNMT1 IC50 values for 1 and 5 as determined internally
(
see footnote in Table 1). Alternatively, the reported IC50 value for compound 1 (UNCꢀ0638)
was < 15 nM (G9a, using a SAHHꢀcoupled assay) and 1287 nM (DNMT1, using a radioactive
1
4
methyl transfer assay). Discrepancies with initially reported DNMT1 IC50 values for 1 (IC50
=
9
10
1
07,000 nM) have been attributed to a different assay format. IC values for compound 2
5
0
1
5
15
(
G9a, DNMT1) and 3, 4 (DNMT1, corresponding to the lower concentration at which DNA
hypomethylation is observed) have been previously reported.
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RESULTS
SAR in Biochemical G9a and DNMT1 Assays
SAR examination of the 2ꢀposition of the quinoline scaffold revealed 5ꢀalkylꢀ2ꢀfuryl
groups (5ꢀmethyl in 5 and 5ꢀethyl in 6, Table 1) as the optimal groups for yielding
1
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functional cellular activity. Thus, we decided to alternatively use these substituents as
initial reference points for subsequent SAR exploration of the 4ꢀ, 6ꢀ and 7ꢀpositions of
the quinoline scaffold, using a sequential approach (Chart 2).
Chart 2. Sequential SAR exploration strategy for positions 4, 6 and 7 of the quinoline scaffold;
2
position 2 is fixed with a 2ꢀfuryl ring substituted at position 5 (where R is methyl or ethyl)
The 4ꢀamino moiety was first explored (Table 1). According to the predicted binding
6
mode of 5 and 6 (Figure 1AꢀB) into both methyltransferases and Xꢀray structure of 1
9
and related quinazoline analogues into G9a, this position extends out toward the so
called solvent accessible area, suggesting that chemically diverse groups, exploring the
1
6
biological space, could be wellꢀtolerated. Removal of the basic nitrogen of 5
compounds 7-11, 16) caused a significant drop in activity against both targets, and only
(
the piperidone derivative 11 retained potent DNMT1 activity (IC50 < 500 nM). This
result is consistent with the predicted binding mode of 6 with G9a, as the positively
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charged nitrogen of the piperidine ring interacts with Asp1078 (Figure 1A). With
respect to DNMT1, no explicit contacts were predicted by docking between the
piperidine ring and the protein (Figure 1B), although a negative electrostatic potential is
observed in this area (Figure 1C). Homologation of the piperidine ring of 5 with a
0
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methylene linker (compounds 12 -13) greatly improved DNMT1 activity (IC50 < 50
nM) while causing minor potency loss against G9a (IC50
∼ 15 nM). Given the poor
6
pharmacokinetic profile of 12, the gemꢀdimethyl analogue 15 was synthesized as the
methylene linker was viewed as a potential metabolic liability. As 15 was significantly
less potent against both targets, we concentrated on alternatives to the piperidine ring by
17
limiting molecular flexibility with spiropiperidines (17, 18, 20, 21), spiropyrrolidines
(
19, 22) and bridged piperazines (23-26). In general, these conformationally constrained
analogs share a biochemical profile similar to that of 5, especially in terms of selectivity
towards G9a over DNMT1. Exceptionally, racemic analogs 20-22, whose basic nitrogen
predictably positions more distant from that of the piperidine (in green in Figure 1D
compared to the other docked spiro derivatives in orange and 5 in violet, that overlap
well), were less potent against G9a (IC50 > 50 nM). DNMT1 SAR was flat, with no
general improvements in activity (200ꢀ300 nM, pIC50 ranging between 6.5 and 6.7, with
less than 0.3 log units compared to 5 and 6) except for some remarkable analogs such as
18
the homologated 3ꢀazabicyclo[3.2.1]octane of 25 (IC50 = 80 nM). As expected, loss of
the hydrogen bond donor of the 4ꢀamino group of 5 by methylation (27) or replacement
6
by an oxygen atom (28) was detrimental for G9a activity. Interestingly, similar results
were observed against DNMT1 and are in agreement with a predicted contact between
this 4ꢀamino group and Ser1233 of DNMT1 (Ser1230 in human DNMT1, hDNMT1,
Figure 1B), what might explain the decreased DNMT1 activity of 27 and 28. In
summary, this exploration of the 4ꢀposition either mainly retained the selectivity profile
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of lead compound 5 (e.g. with constrained rings) or equally potent compounds were
obtained at the cost of losing G9a activity (e.g. 11). Only the homologated compounds
12 and 13 exhibit both G9a and DNMT1 activities in the low nanomolar range (< 50
nM) and can be regarded as nonꢀselective, especially in comparison with their
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
counterparts 5 and 6 (absolute pIC50 differences between both targets of 0.3 for 12 and
13 versus 1.7 (5) and 2.1 (6)).
Table 1. Exploration of the 4-position
Cpd
5
R1
R2
Me
G9a IC50 (nM) DNMT1 IC50 (nM)
6,13
8
382
1
3
6
Et
2
234
7
8
Me
Me
436
735
1030
1790
9
1
Me
Me
244
105
781
0
1080
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1
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2
2
2
2
2
2
2
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cpd
11
R1
R2
Me
G9a IC50 (nM) DNMT1 IC50 (nM)
146
297
6
1
1
1
2
3
4
Me
Et
16
32
0
1
2
3
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5
6
7
8
9
0
1
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6
7
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9
0
1
2
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
18
40
Me
Me
Me
Me
13
205
722
624
140
15
115
699
15
1
1
6
7
18
Me
7
211
1
2
9
0
Me
Me
11
63
201
123
2
1
Et
94
220
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Cpd
22
R1
R2
Me
G9a IC50 (nM) DNMT1 IC50 (nM)
76
290
23
Me
41
235
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
4
5
6
Me
Et
21
5
261
80
Me
13
234
2
7
8
Me
Me
935
1320
1870
6
2
2870
For data in Tables 1ꢀ4 and Chart 1, all biochemical results are the average of at least two
independent replicates performed at different days. If absolute pIC50 difference was higher than
0
.3 log units, additional replicates were performed until satisfying the experimental error (by
discarding individual results with values outside 2 MADs of the mean value).
Exploration of the 6ꢀposition with small groups (29-33, Table 2), designed to modulate
ADME properties, produced a notable decrease in activity, especially against G9a,
affording equally potent compounds against both targets in the midꢀnanomolar or lowꢀ
micromolar range. Only replacement of the methoxy group by chlorine (31) had
negligible impact on DNMT1 activity (IC50 = 298 nM) compared to 5, so no further
exploration around this position was continued. Inspection of the binding cavity for this
derivatization position did not reveal any specific contacts between this substitution
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2
2
2
2
2
2
2
3
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3
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3
3
3
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4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pattern and both proteins as to rationalize the weaker activities observed against both of
them.
Table 2. Exploration of the 6-position
0
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cpd
R3
G9a IC50 (nM)
DNMT1 IC50 (nM)
29
30
31
32
532
950
859
645
342
298
6110
2080
3
3
1450
588
Finally, we modulated the 7ꢀ(3ꢀpyrrolidinꢀ1ꢀylpropoxy) moiety that predictably mimics
the lysine side chain (G9a) or the cytosine of DNA (DNMT1) (Table 3). The 7ꢀmethoxy
analogue 34, lacking the basic nitrogen of 5 at the 7ꢀposition, was considerably less
potent against G9a (IC50 = 2060 nM, absolute pIC50 difference of 2.4 log units), likely
as a result of missing interactions with Leu1086 (hydrogenꢀbond) and Tyr1154 (cationꢀ
π interaction) (Figure 1A). The impact of this moiety on DNMT1 was less notorious
(
IC50 raising from 382 nM for 5 to 750 nM for 34, < 0.3 log units), suggesting that the
predicted interaction between the basic nitrogen of pyrrolidine and catalytic Glu1269
Glu1266 in hDNMT1, Figure 1B) was not so important for the inhibitory activity of
this chemical series. Thus, we speculated that selective DNMT1 inhibitors could be
(
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developed by exploring amine analogs of pyrrolidine with different basicity and
distance extension from the quinoline core. Compounds in Table 3 were designed and
synthesized. G9a potency was attenuated in all cases, with piperidine 37 being the only
replacement retaining G9a IC50 value below 100 nM. Interestingly, the 4ꢀpiperidyl (40),
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1ꢀmethylꢀ4ꢀpiperidyl (41), 2ꢀmethylꢀ2ꢀazaspiro[3.3]heptanꢀ6ꢀyl (43) and 2ꢀisopropylꢀ2ꢀ
azaspiro[3.3]heptanꢀ6ꢀyl (44) groups conferred high DNMT1 potency (< 100 nM),
yielding compounds selective for DNMT1 over G9a (> 1 log units). The reduced
19
basicity of all these groups (estimated pK
position 7 of 6.33, 8.75, 7.26 for 40, 41 and 43, respectively), compared to that of the
pyrrolidin ring of 5 (pK of 12.1) and 37 (pK of 11.8) seems to play a major role on
a
with Pipeline Pilot for the amine nitrogen at
a
a
target selectivity, as on the other hand the cationic environment overlaps well among all
these derivatives (Figure 1E). While some of these amines in Table 3 have already been
10,18,20
examined as lysine mimetics
that reduce the lipophilicity of the parent compound
5 (acyclic amine 35, piperidine 37, morpholine 38, 3ꢀ(3ꢀfluoropyrrolidinꢀ1ꢀyl)propyl
39), the DNMT1 potency of the spirocyclic derivative 43 (IC50 = 21 nM) was
considered as a key SAR finding and a novelty in epigenetic drug design. Decreasing its
basicity with Nꢀisopropyl (44) and Nꢀcyclopropyl (45) capping groups correlated with
loss of potency. Compounds 46-50 were obtained by combining the identified optimal
groups at positions 4 and 7 (Table 4). Unfortunately, the excellent DNMT1 potency of
43 was not retained when this group was combined with the most potent 4ꢀsubstituents
21
in Table 1 to afford analogues 47-50, suggesting nonꢀadditive SAR effects. Despite
lower DNMT1 inhibitory activity (between 187 and 501 nM for 47-50), all these
compounds displayed DNMT1 selectivity over G9a comparable to that of 43.
Table 3. Exploration of the 7-position
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cpd
R4
R2
Me
G9a IC50 (nM)
2060
DNMT1 IC50 (nM)
750
34
3
5
Et
Et
123
599
425
212
36
37
Et
21
247
446
833
3
3
8
9
Et
3910
1470
Me
4
4
4
0
1
2
Me
Me
838
73
83
1150
Me
Me
Me
7550
351
414
21
43
44
378
83
45
Me
3260
494
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Table 4. Combined exploration of positions 4 and 7
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1
1
2
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cpd R4
R1
R2 G9a IC50 (nM) DNMT1 IC50 (nM)
46
Me 3080
200
47
48
Me 3220
Me 3450
475
250
4
5
9
0
Me 5130
Me 5410
187
501
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2
2
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
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5
5
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5
5
5
5
5
5
5
6
0
1
2
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4
5
6
7
8
9
0
1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Predicted complex of 6 with G9a (A, PDB entry 3RJW) and DNMT1 (B, PDB entry
4DA4). (C) Electrostatic potential of DNMT1 (only negative, in red), especially at the regions
accommodating both basic centers of compound 6. (D) Overlaid conformations of docked
spirocyclic and bridged derivatives in Table 1 to compound 5 (violet), showing that the basic
nitrogen of compounds 20-22 (green, all stereoisomers considered) lies distal to the basic
nitrogen of the rest of spirocycles and bridged structures (17-19, 23-26, orange). (E) Overlaid
conformations of docked compounds 5, 41, 43 and 44 showing that the basic nitrogen overlays
well among them.
Having identified interesting compounds with different selectivity profiles towards G9a
and/or DNMT1 (13, 43), we analyzed their selectivity at 10 µM against a panel of
epigenetic targets comprising 14 lysine and arginine methyltransferases, DNMTs
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(
DNMT3A, DNMT3B), bromodomains (BRD2, CREBBP and BAZ2B) and histone
demethylases (JMJD2C, JMJD3, JMJD1A) (Supplementary Table 1). Despite we
6
initially anticipated potent GLP inhibition (as for 12 ), given the close similarity among
G9a and GLP, compounds 13 and 43 displayed high selectivity towards G9a (GLP IC50
values higher than 20 µM). Reasons for this selectivity are not clear considering the
high structural similarity between 12 and 13. Oppositely, in agreement with initial
prediction, all three compounds are potent inhibitors of DNMT3A and DNMT3B
(>95% inhibition at 10 µM). For the remaining targets, inhibition values were < 50%,
with the exception of PRMT1 for 13 (84%) and JMJD3 for 43 (71%).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Antiproliferative effect and preliminary ADME
Following our screening funnel, the antiproliferative response of compounds with
potent inhibitory activity (G9a < 100 nM and DNMT1 < 500 nM) was tested against
ALL cell lines (CEMOꢀ1) and DLBCL (OCIꢀLy3 and OCIꢀLy10) cell lines (Table 5).
All selected compounds displayed potent antiproliferative effects, with GI50 values
below 1 µM against at least one cell line. However, no evident correlation was found
between G9a/DNMT1 activity/selectivity, passive membrane permeability of
compounds as measured using the parallel artificial membrane permeation assay
(PAMPA), and phenotypic response, this last being greatly dependent on the cell type.
For example, compounds 12 and 43, two of the most interesting compounds from the
viewpoint of G9a/DNMT1 selectivity, mainly differing in their G9a activity (16 nM
versus 351 nM) and being equally very potent against DNMT1 (32 versus 21 nM), have
the same growth inhibitory activity against CEMOꢀ1 (GI50
∼ 75 nM) but diverse
response against DLBCL cell lines: OCIꢀLy10 cell line (< 31 nM versus 253 nM for 12
and 43, respectively) and the opposite response against OCIꢀLy3 (202 versus 74 nM for
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2
2
2
2
2
2
2
3
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3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2 and 43, respectively). Low toxicity of compounds, assayed against the nonꢀtumoural
hepatic cell line THLEꢀ2 (Table 5), was regarded as a requisite for further compound
progression into in vitro preliminary ADME, ideally affording a therapeutic window
(absolute difference between pGI50 and pLC50) of a least one log unit against one out of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the four tumoural cell lines (mostly CEMOꢀ1, Table 6). Selected compounds in Table 6
showed low cytochrome P450 inhibition of five major isoforms (< 25% at 10 µM) and
were inactive against hERG (pIC50 < 4), thus limiting the potential for cardiotoxicity.
As no compound with a novel selectivity profile (e.g. 43) improved the metabolic
stability of lead compound 5, especially in human liver microsomes, compounds were
not further progressed into PK studies.
Table 5. Antiproliferative response of selected compounds against cancer cell lines
and non-tumoural hepatic cell line THLE-2
Cpd
CEMO-1 OCI-Ly3 OCI-
THLE-2
LC50
PAMPA G9a
GI50
GI50
(nM)
Ly10
GI50
Pe
pIC50 –
[a]
[a]
[c]
(nM)
(nM)
(nm/s)
DNMT1
pIC50
[a]
[b]
(nM)
72 h
6
5
6
218
56
409
95
455
64
1780
2320
12.9
23.9
1.7
2.1
1
3
1
1
172
75
93
215
202
277
N.D.
243
170
<31
133
N.D.
100
7950
1300
954
777
992
841
8380
731
517
1980
527
3.4
0.3
0.3
0.3
1.2
1.0
1.5
1.3
0.3
0.4
0.6
0.8
1.2
1.3
6
12
13
14
11.0
16.2
19.2
10.5
19.3
3.3
15.9
37.7
7.6
659
235
426
768
424
186
252
156
115
671
17
18
19
20
21
22
23
25
26
374
246
237
205
557
N.D.
N.D.
257
112
638
131
313
N.D.
N.D.
33.7
36.1
3.6
<164
4150
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782
N.D.
N.D.
2310
24.5
ꢀ0.1
35
36
370
184
196
1070
272
73
86
1100
596
576
1380
N.D.
253
3520
2040
1380
24800
1030
698
14.9
36.6
8.3
2.9
12
7.1
14.9
11.1
0.5
ꢀ0.5
1.1
ꢀ1.1
ꢀ1.1
ꢀ1.2
ꢀ1.2
ꢀ1.4
230
271
<63
N.D.
74
3
4
4
4
4
4
7
0
1
3
6
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
504
452
12
186
N.D.
2530
4650
Na] .D.
[
N.D. = Not Determined. Proliferation assays are the average of three replicates at different
[
b]
days. THLEꢀ2 cytotoxicity results after 72 hours of incubation are the average of at least two
independent experiments performed at different days. If absolute pLC50 difference was higher
than 1 log unit, additional replicates were performed until satisfying the experimental error (by
[c]
discarding individual results with values outside 3 MADs of the mean value). The PAMPA
assay was performed in triplicate. Depending on permeability values (Pe, nm/s), compounds can
be regarded as poor (Pe < 10 nm/s); moderate (10 < Pe < 30 nm/s) or good (> 30 nm/s)
22
permeators.
Table 6. ADME profile of selected compounds
Cpd 1A2 2C9 2C19 2D6 3A4 HLM MLM hERG
(
%) (%) (%)
(%) (%) (%)
(%)
IC50
ꢁM)
[a]
[a]
[a]
[a]
[a]
[b]
[b]
(
6,13
5
1
1
1
2.8 0.56 2.5
9.3 3.2 6.5
4.9 0.0 99.7 70.9
22.4 1.2 82.7 71.9
>100
>100
N.D.
>100
1
3
7
7.1 16.8 23.1 19.3 2.2 69.0 59.1
0
0
0
0
0
68.6 40.6
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
0
6
0
0
0
0
2.5
6.3
5.3
0
0
0
80.4 53.3
60.3 43.9
N.D.
N.D.
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
1
7.2 3.8
0
0
0
81.7 81.7
N.D.
N.D.
4
3
6.1 6.3
0
21.5 6.9 49.8 43.3
[a]
[b]
N.D. = Not Determined. % inhibition at 10 ꢁM. % compound remaining after a 20ꢀmin
incubation in human or mouse liver microsomes (HLM and MLM respectively). All assays
were performed in duplicate, with the exception of hERG binding data (n=1).
Finally, the functional cellular potency of selected compounds was assessed by
quantifying the global levels of H3K9me2 (Western Blot) and 5ꢀmethylcytosine (5mC)
(Dot Blot) in the CEMOꢀ1 cell line following 96 h of exposure. As an example, the
selected reference compounds 13 and 43 were tested. Both compounds reduced the
H3K9me2 and 5mC levels in a concentrationꢀdependent manner (Figure 2); Western
Blots are quantized and reported in the Supplementary Information (Figure S1). In
summary, these results demonstrate the functional dual effect of compounds 13 and 43
against the methyltranferase activity of G9a and DNMTs.
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4
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5
5
5
5
5
5
5
6
0
1
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5
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9
0
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0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. H3K9me2 and 5mC hallmarks in the CEMOꢀ1 cell line after treatment with
compounds 13 and 43. A) H3K9me2 levels after 96 hours of treatment with different doses of
compounds 13 and 43 in CEMOꢀ1 cell line. H3 total was used as loading control. B) 5mC levels
after 96 hours of treatment with different doses of compounds 13 and 43.
Chemistry
Preparation of compounds 7ꢀ11, 13 and 16 with different substituents at 4ꢀposition of
the quinoline were prepared as outlined in Scheme 1 from commercially available 2ꢀ
methoxyꢀ5ꢀnitroꢀphenol (51). This alcohol was first converted into compound 52
through Mitsunobu reaction and then intermediate 54 was achieved after hydrogenation
and reaction with POCl
3
in malonic acid. Then, Suzuki coupling with boronic esters 2ꢀ
(5ꢀethylꢀ2ꢀfuryl)ꢀ4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolane or 4,4,5,5ꢀtetramethylꢀ2ꢀ(5ꢀ
methylꢀ2ꢀfuryl)ꢀ1,3,2ꢀdioxaborolane afforded compounds 55a and 55b which were
finally converted into desired 4ꢀaminoquinolines 7ꢀ11, 13 and 16 by Pdꢀcoupling
reaction using different amines.
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 1. Synthesis of compounds 7ꢀ11, 13 and 16.
i)
iii)
iv)
51
54
5
2: R = NO2
ii)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
53: R = NH2
7
: R1 = CH , R
=
=
=
3
2
8
: R1 = CH , R
v) or vi)
3
2
2
9: R1 = CH , R
3
55a: R1 = CH3
55b: R1 = CH CH3
2
10: R1 = CH , R
=
3
2
2
11: R1 = CH , R
=
3
13: R1 = CH CH , R =
2
2
3
1
6: R1 = CH , R =
3 2
Conditions: i) 3ꢀpyrrolidinꢀ1ꢀylꢀpropanꢀ1ꢀol, PPh , DEAD, THF, rt, 5 h; ii) Pd/C, H (1 atm), MeOH, rt, 3
3
2
h; iii) POCl
3
, malonic acid, rt, 4 h, then, 90 ºC, overnight; iv) 2ꢀ(5ꢀethylꢀ2ꢀfuryl)ꢀ4,4,5,5ꢀtetramethylꢀ
1,3,2ꢀdioxaborolane or 4,4,5,5ꢀtetramethylꢀ2ꢀ(5ꢀmethylꢀ2ꢀfuryl)ꢀ1,3,2ꢀdioxaborolane, Na CO or K CO ,
2
3
2
3
3 4 2
Pd(PPh ) , 1,4ꢀdioxane or 1,4ꢀdioxane /H O (15:1), 110 ºC, MW or conventional heating, 4ꢀ12 h; v)
corresponding amine, Cs CO , BINAP, Pd (dba) , 1,4ꢀdioxane, 120ꢀ130 ºC, MW or conventional heating
2
3
2
3
2 3
, 3ꢀ12 h; vi) corresponding amine, tꢀBuONa, xantphos, Pd (dba) , toluene, 100 ºC, MW, 2 h.
Synthesis of isopropyl analogue 14, gemꢀdimethyl analogue 15, spiropiperidines 17, 18,
0 and 21, spiropyrrolidines 19 and 22, bridged piperazines 23ꢀ26 and methylated
2
analogue 27 is described in Scheme 2. Starting from previously described 4ꢀ
chloroquinolines 55a or 55b, BOCꢀprotected amines 56a-k were prepared by
BuchwaldꢀHartwig amination and tertiary amine 56l using tertꢀbutyl 4ꢀ
(methylamino)piperidineꢀ1ꢀcarboxylate and PTSA in
tꢀBuOH. Then, the BOC
protecting group was removed under acidic conditions and desired methyl or isopropyl
substituent was installed by reductive amination. All analogues with chiral centers and
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8
9
pseudostereocenters in this manuscript (11, 19-26, 39, 50) were prepared as racemic
mixtures.
Scheme 2. Synthesis of compounds 14, 15 and 17ꢀ27.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
n
n
n
Cy N-BOC
Cy N-H
Cy N-R
5
i) or ii)
iii)
55a-b
iv) or v)
5
6a: R1 = CH ; R , R , R = H; n = 1; Cy1
57a: R1 = CH ; R , R , R = H; n = 1; Cy1
3
2
3
4
14: R1 = CH ; R , R , R = H; R5 = CH(CH ) ; n = 1; Cy1
3 2 3 4 3 2
3
2
3
4
5
6b: R , R , R = CH ; R = H; n = 1; Cy1
57b: R , R , R = CH ; R = H; n = 1; Cy1
1
3
4
3
2
15: R , R , R , R = CH ; R = H; n = 1; Cy1
1 3 4 5 3 2
1
3
4
3
2
5
6c: R 1 = CH ; R = H; n = 0; Cy2
57c: R 1 = CH ; R = H; n = 0; Cy2
3
2
17: R , R = CH ; R = H; n = 0; Cy2
3
2
1
5
3
2
5
6d: R
= CH
; R
= H; n = 0; Cy3
57d: R
1
= CH
3
; R
2
= H; n = 0; Cy3
18: R
= CH
; R
= H; R = CH(CH ) ; n = 0; Cy2
5 3 2
1
3
2
1
3
2
56e: R1 = CH ; R = H; n = 0; Cy4
57e: R1 = CH ; R = H; n = 0; Cy4
3
2
19: R , R = CH ; R = H; n = 0; Cy3
3
2
1
5
3
2
5
6f: R
= CH
CH
; R
= H; n = 0; Cy4
57f: R1 = CH CH ; R = H; n = 0; Cy4
2
3
2
20: R
, R
= CH
; R
= H; n = 0; Cy4
1
2
3
2
1
5
3
2
5
6g: R1 = CH ; R = H; n = 0; Cy5
57g: R1 = CH ; R = H; n = 0; Cy5
3
2
21: R1 = CH CH ; R = H; R5 = CH ; n = 0; Cy4
2 3 2 3
3
2
56h: R1 = CH ; R = H; n = 0; Cy6
57h: R1 = CH ; R = H; n = 0; Cy6
3
2
22: R , R = CH ; R = H; n = 0; Cy5
1 5 3 2
3
2
5
6i: R1 = CH ; R , R , R = H; n = 1; Cy6
57i: R1 = CH ; R , R , R = H; n = 1; Cy6
3
2
3
4
23: R , R = CH ; R = H; n = 0; Cy6
1 5 3 2
3
2
3
4
5
6j: R1 = CH CH ; R , R , R = H; n = 1; Cy6
57j: R1 = CH CH ; R , R , R = H; n = 1; Cy6
2
3
2
3
4
24: R , R = CH ; R , R , R = H; n = 1; Cy6
1 5 3 2 3 4
2
3
2
3
4
5
6k: R1 = CH ; R2 = H; n = 0; Cy7
57k: R
1
= CH
3
; R
2
= H; n = 0; Cy7
25: R1 = CH CH ; R , R , R = H; R5 = CH ; n = 1; Cy6
2 3 2 3 4 3
3
5
6l: R , R2 = CH ; n = 0; Cy1
57l: R , R = CH ; n = 0; Cy1
1
2
3
26: R , R = CH ; R = H; n = 0; Cy7
1
3
1
1
5
2
3
2
27: R
, R
, R
= CH
; n = 0; Cy1
5
3
Cy1 =
Cy2 =
Cy3 =
Cy4 =
Cy5 =
Cy6 =
Cy7 =
Conditions: i) corresponding amine, Cs
2
CO
3
, BINAP, Pd
2
3
(dba) , 1,4ꢀdioxane, 115ꢀ130 ºC, MW or
conventional heating , 5ꢀ48 h; ii) tertꢀbutyl 4ꢀ(methylamino)piperidineꢀ1ꢀcarboxylate, PTSA, tꢀBuOH,
20 ºC, 48 h; iii) HCl/EtOAc (1.0 or 2.0 M) or HCl/MeOH (2.0 or 4.0 M), 16ꢀ25 ºC, 1ꢀ16 h; iv) acetone,
1
NaBH CN, AcOH, iꢀPrOH, 50ꢀ60 ºC, 15ꢀ16 h; v) (HCHO) , HCOOH, NaBH(OAc) , MeOH, rt or 60 ºC,
3
n
3
12 h or overnight.
Compounds with a hydrogen atom, a chlorine atom and a ꢀOCF
3
group at position 6 of
the quinoline (29, 31 and 32 respectively) were synthesized from intermediates 60a-c
through Mitsunobu reaction and reduction of nitro group (Scheme 3). Then, key
intermediates 63a-b were achieved after heating with POCl and malonic acid and
3
compound 63c by a three step protocol (reaction with ethyl 3ꢀchloroꢀ3ꢀoxoꢀpropanoate,
3
ester hydrolysis and reaction with POCl ). Subsequent reaction with 4,4,5,5ꢀ
tetramethylꢀ2ꢀ(5ꢀmethylꢀ2ꢀfuryl)ꢀ1,3,2ꢀdioxaborolane and BuchwaldꢀHartwig amination
led us to desired compounds 29, 31 and 32.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 3. Synthesis of compounds 29, 31 and 32.
i)
ii)
iii)
vi) or vii) viii) and ix)
6
1a: R1 = H, R2 = NO2
61b: R = Cl, R = NO
61c: R = OCF , R = NO
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
58
59
6
0a: R1 = H
0b: R1 = Cl
1
2
2
6
1
3
2
vi ) or v)
60c: R1 = OCF3
6
2a: R1 = H, R2 = NH2
2b: R1 = Cl, R2 = NH2
6
62c: R1 = OCF , R2 = NH2
3
x)
xi) or xii)
6
6
6
3a: R1 = H
64a: R1 = H
29: R1 = H
3b: R1 = Cl
3c: R1 = OCF3
64b: R1 = Cl
64c: R1 = OCF3
31: R1 = Cl
32: R1 = OCF3
Conditions: i) 4,4,5,5ꢀtetramethylꢀ2ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ1,3,2ꢀdioxaborolane,
KOAc, Pd(dppf)Cl
2
, 1,4ꢀdioxane, 90 ºC, 10 h; ii) NaOH, H
2
O
2
, THF, 30 minutes, 22 ºC; iii) 3ꢀpyrrolidinꢀ
1
ꢀylpropanꢀ1ꢀol, PPh , DEAD or DIAD, THF, 0 ºC or rt, 5ꢀ10 h; iv) Pd/C, H (40 Psi), MeOH, rt, 2ꢀ15 h;
3
2
4 2 3
v) Fe, NH Cl, EtOH/H O (4:1), rt, 3 h; vi) POCl , malonic acid, rt, 4 h, then 90 ºC, overnight; vii),
DMAP, pyridine, CH Cl , ꢀ70 ºC, 1 h, then ethyl 3ꢀchloroꢀ3ꢀoxoꢀpropanoate, 20 ºC, 15 h; viii)
2
2
LiOH·H
methylꢀ2ꢀfuryl)ꢀ1,3,2ꢀdioxaborolane, Na
0ꢀ110 ºC, MW or conventional heating, 2ꢀ15 h; xi) 1ꢀmethylpiperidinꢀ4ꢀamine, xꢀPhos, Pd
BuOK, 1,4ꢀdioxane, 130 ºC, MW, 4 h; xii) 1ꢀmethylpiperidinꢀ4ꢀamine, BINAP, Pd (dba) , Cs CO , 1,4ꢀ
2
O, THF/MeOH/H
2
O (4:2:3), 20 ºC, 15 h; ix) POCl
3
, 100 ºC, 3 h; x) 4,4,5,5ꢀtetramethylꢀ2ꢀ(5ꢀ
, 1,4ꢀdioxane/H O (5:1, 15:1 or 10:1),
(dba) , tꢀ
2
CO or K CO , Pd(PPh
3
2
3
3
)
4
2
8
2
3
2
3
2
3
dioxane, 80ꢀ110 ºC, 12ꢀ15 h.
Compounds with a hydroxy and a cyano substituent at position 6 of the quinoline
compounds 30 and 33) were also prepared. As illustrated in Scheme 4, from previously
described 5, compound 30 was afforded using BBr in CH Cl Then,
trifluoromethanesulfonate intermediate 65, obtained by reaction with PhN(OTf) , was
(
3
2
2
.
2
2 3 4
converted into desired compound 33 using Zn(CN) and Pd(PPh ) in DMF.
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Scheme 4. Synthesis of compounds 30 and 33.
i)
ii)
5
30
65: R = OTf
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
iii)
3
3: R = CN
Conditions: i) BBr
3
, CH
2
Cl
2
2
, 0 ºC, 2 h; ii) PhN(OTf) , DIEA, DMF, 0 ºC, 2 h, then 25 ºC, 12h; iii)
Zn(CN) , Pd(PPh ) , DMF, 110 ºC, 12 h.
2
3 4
To continue with exploration at position 7 of the quinoline, compounds 35, 36 and 39 were
synthesized (Scheme 5). Starting from commercially available 5ꢀaminoꢀ2ꢀmethoxyꢀphenol (66),
intermediate 67 was prepared by reaction with POCl . Substitution at position 7 was then
3
installed using 1,3ꢀdibromopropane and Nꢀmethylmethanamine or 5ꢀazaspiro[2.4]heptane to
obtain compounds 68a-b. On the other hand, intermediate 68c was achieved in only one step by
Mitsunobu reaction with 3ꢀ(3ꢀfluoropyrrolidinꢀ1ꢀyl)propanꢀ1ꢀol. Then, dichloroquinolines 68a-c
were converted into desired compounds 35, 36 and 39 by the two step protocol previously
described: Suzuki coupling and BuchwladꢀHartwig amination.
Scheme 5. Synthesis of compounds 35, 36 and 39.
i)
ii) and iii)
or iv)
v) and vi)
66
67
6
8a: R1
6
8b: R1
6
8c: R1
=
=
=
3
5: R1
=
=
=
, R2 = CH CH
2
3
3
6: R1
, R2 = CH CH
2
3
3
9: R1
, R2 = CH3
Conditions: i) POCl
corresponding amine, K
DEAD, THF, 20 ºC 16 h; v) 2ꢀ(5ꢀethylꢀ2ꢀfuryl)ꢀ4,4,5,5ꢀtetramethylꢀ 1,3,2ꢀdioxaborolane or 4,4,5,5ꢀ
3
, malonic acid, 95 ºC, 12 h; ii) 1,3ꢀdibromopropane, K
2
CO
3
, CH
3
CN, 60 ºC, 88 h; iii)
2
CO , CH
3
3 3
CN, 60 ºC, 16 h; iv) 3ꢀ(3ꢀfluoropyrrolidinꢀ1ꢀyl)propanꢀ1ꢀol, PPh ,
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tetramethylꢀ2ꢀ(5ꢀmethylꢀ2ꢀfuryl)ꢀ1,3,2ꢀdioxaborolane, K
2
CO
3
, Pd(PPh
3 4 2
) , 1,4ꢀdioxane/H O (1:1 or 10:1),
1
10 ºC, 16 h; vi) 1ꢀmethylpiperidinꢀ4ꢀamine, Cs CO , Pd
2
3
2
(dba)
3
, BINAP, 1,4ꢀdioxane, 120 ºC, 12ꢀ16 h.
Compounds with a piperidine or morpholine at position 7 were also prepared as outlined
in Scheme 6. Conversion of commercially available 2ꢀmethoxyꢀ5ꢀnitroꢀphenol (51) to
intermediate 69 was first achieved by reaction with 1,3ꢀdibromopropane. Then,
substitution by piperidine or morpholine and subsequent hydrogenation led us to
compounds 70a-b which were converted into corresponding 2,4ꢀdichloroanilines 71a-b
after reaction with ethyl 3ꢀchloroꢀ3ꢀoxoꢀpropanoate, ester hydrolysis and reaction with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
POCl . Finally, desired 4ꢀaminoquinolines 37 and 38 were isolated through Suzuki
coupling and amination under BuchwaldꢀHartwig conditions.
Scheme 6. Synthesis of compounds 37 and 38.
O
O
O
O
i)
ii) and iii)
vi), v) and vi)
HO
NO2
Br
NO2
N
O
NH2
X
51
69
70a: X = CH2
7
0b: X = O
N
Cl
HN
N
O
O
O
vii) and viii)
O
N
N
Cl
N
O
X
X
7
1a: X = CH2
37: X = CH2
38: X = O
7
1b: X = O
Conditions: i) 1,3ꢀdibromopropane, Cs
CH CN, 90 ºC, 16 h; iii) Pd/C, MeOH, H
ethyl 3ꢀchloroꢀ3ꢀoxoꢀpropanoate, 15 ºC, 16 h; v) LiOH·H
6 h; vi) POCl , 100 ºC, 2 h; vii) 2ꢀ(5ꢀethylꢀ2ꢀfuryl)ꢀ4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolane, K
Pd(PPh , 1,4ꢀdioxane or 1,4ꢀdioxane/H O (1:1), 110 ºC, 16 h; viii) 1ꢀmethylpiperidinꢀ4ꢀamine, Cs
Pd (dba) , BINAP, 1,4ꢀdioxane, 120 ºC, 16 h.
2
CO
(30 Psi), 15 ºC, 2 h; iv) DMAP, pyridine, CH
O, THF/MeOH/H O (20:20:13 or 3:3:2), 15 ºC,
3
, DMF, 20 ºC, 16 h; ii) piperidine or morpholine, Cs
2 3
CO ,
3
2
2
2
Cl , ꢀ78 ºC, then
2
2
1
3
2
2
CO
CO
3
3
,
,
3
)
4
2
2
3
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Preparation of compounds 34, 40-45 is shown in Scheme 7. In this case the synthesis
started with commercially available anilines 72a-b and 66 which were transformed into
2
,4ꢀdichloroanilines 73a-b and 67 following methods described above. Then, 5ꢀmethylꢀ
ꢀfuryl at position 2 was installed through Suzuki coupling to afford intermediates 74a-
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c. Compound 34 with a methoxy group at position 7 of the quinoline, was then prepared
from intermediate 74a by BuchwaldꢀHartwig amination using 1ꢀmethylpiperidinꢀ4ꢀ
amine as in other cases. 4ꢀPiperidyl derivatives 40 and 41 were synthesized from
intermediate 74b through Mitsunobu reaction with tertꢀbutyl 4ꢀ(hydroxymethyl)
piperidineꢀ1ꢀcarboxylate, coupling with 1ꢀmethylpiperidinꢀ4ꢀamine, removal of BOC
protecting group under acidic conditions and reductive amination in the case of
methylated derivative 41. Compound 42 was prepared from intermediate 75 (obtained
from 74c by Suzuki coupling and hydrogenation), after reaction with tertꢀbutyl 4ꢀ
methylsulfonyloxypiperidineꢀ1ꢀcarboxylate and acidic removal of BOC protecting
group. Finally, 2ꢀazaspiro[3.3]heptanꢀ6ꢀyl derivatives 43ꢀ45 were isolated after reaction
of intermediate 75 with tertꢀbutyl 6ꢀmethylsulfonyloxyꢀ2ꢀazaspiro[3.3]heptaneꢀ2ꢀ
carboxylate and methylation using LiAlH for compound 43 or deprotection with TFA
4
and reductive amination or cyclopropanation for compounds 44 and 45 respectively.
Scheme 7. Synthesis of compounds 34, 40-45.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
i) or
v)
vi) or
ii), iii) and iv)
vi) and vii)
6
6: R1 = H
67: R
73a: R
73b: R
1
= H
= CH
= Bn
74a: R1 = CH3
74b: R1 = H
74c: R1 = Bn
34: R1
75: R1
= CH3
= H
7
2a: R1 = CH3
1
3
72b: R1 = Bn
1
xi) and ix) or
xi) and xii) or
xi), xiii) and xiv)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
viii), ix) and vi) or
viii), ix), vi) and x)
40: n = 1, Cy =
41: n = 1, Cy =
, R2 = H
n
, R2 = CH3
, R2 = H
Cy
4
4
4
4
2: n = 0, Cy =
3: n = 0, Cy =
4: n = 0, Cy =
5: n = 0, Cy =
, R2 = CH3
, R2 = isopropyl
, R2 = cyclopropyl
Conditions: i) POCl , malonic acid, rt, 4 h, then 90 ºC, overnight or 95 ºC, 12 h; ii) Et N, ethyl 3ꢀchloroꢀ
3
3
3
ꢀoxoꢀpropanoate, CH
2
Cl
2
, 25 ºC, 12 h; iii) LiOH·H
2
O, THF/MeOH/H
2
3
O (3:3:2), 25 ºC, 16 h; iv) POCl ,
9
0 ºC, 2 h; v) 4,4,5,5ꢀtetramethylꢀ2ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ1,3,2ꢀdioxaborolane,
CO or K CO , Pd(PPh , 1,4ꢀdioxane or 1,4ꢀdioxane/H O (15:1), 100ꢀ120 ºC, MW or conventional
Na
2
3
2
3
3
)
4
2
heating, 2ꢀ16 h; vi) 1ꢀmethylpiperidinꢀ4ꢀamine, Cs CO , BINAP, Pd (dba) , 1,4ꢀdioxane, 110ꢀ120 ºC,
2
3
2
3
MW or conventional heating, 3ꢀ16h; vii) Pd/C, H
2
(50 Psi), MeOH, 25 ºC, 16 h; viii) tertꢀbutyl 4ꢀ
(
hydroxymethyl) piperidineꢀ1ꢀcarboxylate, PPh , DIAD, THF, 0 ºC, 8 h; ix) HCl/EtOAc (1.0 or 2.0 M),
3
2
5 ºC, 2ꢀ3 h; x) (HCHO)
methylsulfonyloxypiperidineꢀ1ꢀcarboxylate or tertꢀbutyl 6ꢀmethylsulfonyloxyꢀ2ꢀazaspiro[3.3]heptaneꢀ2ꢀ
carboxylate, Cs CO , DMF, 100 ºC, 16 h; xii) LiAlH , THF, 70 ºC, 16 h; xiii) TFA, CH Cl , 18 ºC, 2 h;
xiv) acetone or (1ꢀethoxycyclopropoxy)ꢀtrimethylꢀsilane, NaBH CN, AcOH, iꢀPrOH or tꢀBuOH, 60 ºC,
n 3
, NaBH(OAc) , HCOOH, 1,4ꢀdioxane, 100 ºC, 2 h; xi) tertꢀbutyl 4ꢀ
2
3
4
2
2
3
1
6 h.
Synthesis of analogues 46ꢀ48 and 50 was performed as described in Scheme 8. From
intermediate 74b, compound 46 was prepared through Mitsunobu reaction, Pd mediated
coupling with tertꢀbutyl 2ꢀaminoꢀ7ꢀazaspiro[3.5]nonaneꢀ7ꢀcarboxylate, deprotection in
2
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acidic media and reductive amination. On the other hand, compounds 47, 48 and 50
were prepared from 74c. In this case, this intermediate was transformed into compounds
7
9a-c after amination with different BOCꢀ protected amines and hydrogenation. Then,
substituent at position 7 was installed by reaction with tertꢀbutyl 6ꢀmethylsulfonyloxyꢀ
ꢀazaspiro[3.3]heptaneꢀ2ꢀcarboxylate. Finally, BOC protecting groups were removed
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
using TFA and free secondary amines were methylated through reductive amination.
Scheme 8. Synthesis of compounds 46ꢀ48 and 50.
Cy1
Cy1
m
m
iii)
78a-c
79a-c
ii)
Cy1
iv)
m
i)
ii)
74b: R1 = H
n
76
77a-d
7
4c: R1 = Bn
Cy2
v) or vi)
Cy1
Cy1
m
m
4
6: n = 1, m = 0, Cy1
4
7: n = 0, m = 1, Cy1
4
8: n = 0, m = 0, Cy1
5
0: n = 0, m = 0, Cy1
=
=
=
=
, Cy2
=
vii)
, Cy2
=
n
n
80a-d
Cy2
Cy
2
, Cy2
=
, Cy2
=
Conditions: i) tertꢀbutyl 4ꢀ(hydroxymethyl) piperidineꢀ1ꢀcarboxylate, PPh
corresponding amine, Cs CO , BINAP, Pd (dba) , or Pd(dba) , 1,4ꢀdioxane, 110ꢀ130 ºC, 12ꢀ16 h; iii)
Pd/C, MeOH, H (50 Psi), 25 ºC, 16 h; iv) tertꢀbutyl 6ꢀmethylsulfonyloxyꢀ2ꢀazaspiro[3.3]heptaneꢀ2ꢀ
carboxylate, Cs CO , DMF, 100 ºC, 16 h; v) HCl/EtOAc (2.0 M), 25 ºC, 2 h; vi) TFA, CH Cl , 18 ºC, 2
h; vii) (HCHO) , NaBH CN or NaBH(OAc) , HCOOH, MeOH, 60ꢀ70 ºC, 12ꢀ16 h.
3
, DIAD, THF, 0 ºC, 8 h; ii)
2
3
2
3
2
2
2
3
2
2
n
3
3
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Finally, derivative 49 was synthesized as outlined in Scheme 9. To prepare this
compound, BOC protecting group of intermediate 78b was removed and then an
isopropyl group was installed by reductive amination. Compound 83 was then achieved
after hydrogenation. Subsequent Mitsunobu reaction using tertꢀbutyl 6ꢀ
methylsulfonyloxyꢀ2ꢀazaspiro[3.3]heptaneꢀ2ꢀcarboxylate led us to intermediate 84
which was finally converted into desired analogue 49 by acidic deprotection and
reductive amination.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 9. Synthesis of compound 49.
i)
ii)
78b
81
82
iii)
iv)
v)
83
84
8
5: R = H
vi)
4
9: R = CH3
Conditions: i) HCl/EtOAc (2.0 M), 18 ºC, 2 h; ii) acetone, NaBH
Pd/C, MeOH, H (50 Psi), 20 ºC, 8 h; iv) tertꢀbutyl 6ꢀmethylsulfonyloxyꢀ2ꢀazaspiro[3.3]heptaneꢀ2ꢀ
3
carboxylate, Cs CO , DMF, 100 ºC, 16 h; v) TFA, DCM, 18 ºC, 2 h; vi) (HCHO) , NaBH(OAc) ,
3
CN, AcOH, iꢀPrOH, 60 ºC, 16 h; iii)
2
2
3
n
HCOOH, MeOH, 60 ºC, 16 h.
DISCUSSION AND CONCLUSIONS
In summary, we present a detailed exploration around three main growing vectors born
by the 2ꢀ(2ꢀfuryl)ꢀ4ꢀaminoquinoline scaffold. Exploring this chemical space led to the
identification of key chemical features (substructures) that guide our navigation to wellꢀ
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defined biological spaces covering the above mentioned epigenetic targets; thus, we
discovered selective G9a inhibitors (5, 6 and 26), firstꢀinꢀclass potent and selective
DNMT1 inhibitors (43) as well as equipotent dual inhibitors targetting G9a and
DNMT1 (12 and 13). These molecules are valuable tools for comparative cellular
studies and to investigate the mechanistic role and function of these targets in different
diseases. In this regard, all the above mentioned compounds showed remarkable
antiproliferative effects against different hematological cancer cell lines and an adequate
therapeutic window. Thus, these firstꢀinꢀclass substrate competitive and reversible
inhibitors, covering different activity profiles, may serve as valuable starting points for
drug discovery programs.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
Chemistry. General Procedure.
Unless otherwise noted, all reagents and solvents were of the highest commercial
quality and used without further purification. All experiments dealing with moisture
sensitive compounds were conducted under N . Flash column chromatography was
2
performed on silica gel, particle size 60 Å, mesh = 230ꢀ400 (Merck) under standard
techniques. Automated flash column chromatography was performed using readyꢀtoꢀ
connect cartridges from Varian, on irregular silica gel, particle size 15ꢀ40 µm (normal
phase disposable flash columns) on a Biotage SPX flash purification system.
Microwaveꢀassisted reactions were performed in a Biotage Smith Synthesis microwave
reactor. The NMR spectroscopic data were recorded on a Bruker AV400 or VARIAN
4
00MR spectrometer with standard pulse sequences, operating at 400 MHz. Chemical
shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane
TMS), which was used as internal standard. The abbreviations used to explain
(
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