Discovery and Characterization of a Highly Potent and Selective Aminopyrazoline-Based in Vivo Probe (BAY-598) for the Protein Lysine Methyltransferase SMYD2

Article abstract of DOI:10.1021/acs.jmedchem.5b01890

Protein lysine methyltransferases have recently emerged as a new target class for the development of inhibitors that modulate gene transcription or signaling pathways. SET and MYND domain containing protein 2 (SMYD2) is a catalytic SET domain containing methyltransferase reported to monomethylate lysine residues on histone and nonhistone proteins. Although several studies have uncovered an important role of SMYD2 in promoting cancer by protein methylation, the biology of SMYD2 is far from being fully understood. Utilization of highly potent and selective chemical probes for target validation has emerged as a concept which circumvents possible limitations of knockdown experiments and, in particular, could result in an improved exploration of drug targets with a complex underlying biology. Here, we report the development of a potent, selective, and cell-active, substrate-competitive inhibitor of SMYD2, which is the first reported inhibitor suitable for in vivo target validation studies in rodents.

Full text of DOI:10.1021/acs.jmedchem.5b01890

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Article
Discovery and Characterization of a Highly Potent
and Selective Aminopyrazoline-Based in vivo Probe
(BAY-598) for the Protein Lysine Methyltransferase SMYD2
Erik Eggert, Roman C. Hillig, Silke Köhr, Detlef Stöckigt, Jörg Weiske, Naomi Barak, Jeffrey
Mowat, Thomas Brumby, Clara D. Christ, Antonius ter Laak, Tina Lang, Amaury E. Fernández-
Montalván, Volker Badock, Hilmar Weinmann, Ingo V. Hartung, Dalia Barsyte-Lovejoy,
Magdalena Szewczyk, Steven Kennedy, Fengling Li, Masoud Vedadi, Peter J. Brown,
Vijayaratnam Santhakumar, Cheryl H. Arrowsmith, Timo Stellfeld, and Carlo Stresemann
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01890 • Publication Date (Web): 13 Apr 2016
Downloaded from http://pubs.acs.org on April 15, 2016
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Discovery and Characterization of a Highly Potent
and Selective AminopyrazolineꢀBased in vivo Probe
(BAYꢀ598) for the Protein Lysine Methyltransferase
SMYD2
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†
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Erik Eggert, Roman C. Hillig, Silke Koehr, Detlef Stöckigt, Jörg Weiske, Naomi Barak,
†
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†
†
Jeffrey Mowat, Thomas Brumby, Clara D. Christ, Antonius ter Laak, Tina Lang, Amaury E.
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FernandezꢀMontalvan, Volker Badock, Hilmar Weinmann, Ingo V. Hartung, Dalia Barsyteꢀ
‡
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‡,#
Lovejoy, Magdalena Szewczyk, Steven Kennedy, Fengling Li, Masoud Vedadi, Peter J.
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‡,§
,†
Brown, Vijayaratnam Santhakumar, Cheryl H. Arrowsmith,
Timo Stellfeld,* and Carlo
†
,
Stresemann *
†
Drug Discovery, Bayer AG, 13353 Berlin, Germany
‡
Structural Genomics Consortium, University of Toronto, Toronto, ON, M5G 1L7
#
§
Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, M5S 1A8.
Princess Margaret Cancer Centre and Department of Medical Biophysics, University of
Toronto, Toronto, ON, M5G 2M9.
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ABSTRACT: Protein lysine methyltransferases have recently emerged as a new target class for
the development of inhibitors that modulate gene transcription or signaling pathways. SET and
MYND domain containing protein 2 (SMYD2) is a catalytic SET domain containing
methyltransferase reported to monomethylate lysine residues on histone and nonꢀhistone
proteins. Although several studies have uncovered an important role of SMYD2 in promoting
cancer by protein methylation, the biology of SMYD2 is far from being fully understood.
Utilization of highly potent and selective chemical probes for target validation has emerged as a
concept which circumvents possible limitations of knockdown experiments and, in particular,
could result in an improved exploration of drug targets with a complex underlying biology. Here,
we report the development of a potent, selective and cellꢀactive, substrateꢀcompetitive inhibitor
of SMYD2, which is the first reported inhibitor suitable for in vivo target validation studies in
rodents.
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INTRODUCTION
SMYD2 is a catalytic SET domain containing protein methyltransferase reported to
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monomethylate lysine residues on histone and nonꢀhistone proteins. SMYD2 has been proposed
as a potential therapeutic target in cancer. Its overexpression has been reported in cancer cell
lines as well as in esophageal squamous carcinoma (ESCC), bladder carcinoma, gastric cancer
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and pediatric acute lymphoblastic leukemia patients. In these studies, SMYD2 overexpression
often correlated with lower survival rate and was suggested to be a clinically relevant prognostic
marker. Knockdown of SMYD2 in overexpressing ESCC, bladder and gastric cancer cell line
2
models significantly reduced cell proliferation. Initially, SMYD2 was characterized as
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methylating H3 lysine 36 and lysine 4 when interacting with HSP90a. Methylation of histones
by SMYD2 has been connected to increased transcription of genes involved in cellꢀcycle
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regulation, chromatin remodeling and transcriptional regulation. In addition, several studies
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have uncovered an important role of SMYD2 methylation activity toward nonꢀhistone proteins
closely connected to cancer. This is in line with the emerging concept that posttranslational
methylation of nonꢀhistone proteins (e.g., of transcription factors) by protein methyltransferases
can also substantially alter protein function. Thereby, a regulatory role of lysine methylation can
probably be extended to multiple cellular pathways, besides transcriptional regulation and
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histones.
So far, the bestꢀcharacterized example of SMYD2 methylation of a nonꢀhistone
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protein is the tumor suppressor transcription factor p53.
Transcriptional activity of p53 is
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inhibited by SMYD2ꢀmediated posttranslational methylation at lysine 370 (K370).
The
structural basis of p53 methylation by SMYD2 has been characterized by solving the crystal
structure of a ternary complex with the cofactor product Sꢀadenosylhomocysteine and a p53ꢀ
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derived substrate peptide. It has been proposed that methylation at K370 reduces the DNAꢀ
binding efficiency of p53 and subsequently prevents the transcriptional activation of p53 target
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genes. In the same study, a knockdown of SMYD2 and treatment with doxorubicin led to an
increase in p53ꢀmediated cellꢀcycle arrest and apoptosis. In line with these observations, low
SMYD2 gene expression was suggested as a predictive marker for an improved response to
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neoadjuvant chemotherapy in breast cancer patients. Besides p53, several other proteins have
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been identified as SMYD2 substrates, including the estrogen receptor (ER),
PARP1,
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retinoblastoma protein (Rb) and HSP90.
Mechanistically, methylation of HSP90 has been
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connected to the normal physiological role of SMYD2 in muscle biology,
as well as in
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cancer. These studies indicate that SMYD2 has many substrates and various potentially tissueꢀ
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specific physiological and pathogenic functions. SMYD2 therefore represents a very attractive
target for further exploration in different diseaseꢀrelevant models. Nevertheless, the biology of
SMYD2 is still poorly understood, and the molecular contribution of individual substrates to
specific knockdown phenotypes remains largely unknown. For a more unbiased interpretation of
biological experiments, fully profiled chemical probes can substantially contribute to preclinical
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target validation.
Although first cellularꢀactive probe inhibitors of SMYD2 have been
described (Figure 1), there is a need for structurally orthogonal chemical probes to enable crossꢀ
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validation studies and thereby rule out unspecific effects. The publication of 1 (AZ505) was
the first disclosure of a coꢀcrystal structure of an inhibitor bound to SMYD2 and paved the way
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for further studies, leading to the discovery of 2 (LLYꢀ507), and 3 (Aꢀ893), which have
reported significantly improved potency. Analysis of the respective coꢀcrystal structures reveals
that all three inhibitors bind in a similar fashion, occupying the same binding pockets.
Furthermore, the cellular activity of the known inhibitors is limited, and no data about in vivo
applicability have been published. Here we report the discovery of a potent and selective
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aminopyrazolineꢀbased small molecule inhibitor (S)ꢀ4 (BAYꢀ598).
We show that (S)ꢀ4 has a
distinctly different binding mode compared to previous inhibitors, utilizing a dichlorophenyl
moiety as so far unprecedented chemical motif for addressing the methylꢀlysine binding pocket
of SMYD2. For the first time we are presenting in vivo xenograft and DMPK data for a SMYD2
inhibitor. In addition to previously described inhibitors (S)-4 shows very potent cellular activity
combined with reasonable DMPK properties (Figure 1). Furthermore, we are indicating the
potential that this inhibitor might offer to the field of protein methyltransferases in the quest to
fully explore the underlying complex biology and therapeutic potential of SMYD2 by validating
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AHNAK protein as a new cellular substrate.
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RESULTS AND DISCUSSION
The potential link between SMYD2 and cancer motivated us to screen the Bayer compound
collection, with the aim of identifying smallꢀmolecule inhibitors of the enzyme. To this end, a
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scintillation proximity assay (SPA) was set up using recombinant Hisꢀtagged SMYD2, a
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biotinylated p53ꢀderived peptide substrate and tritiated Sꢀadenosylꢀ
L
ꢀmethionine ( HꢀSAM)
(Figure 2A). Of the three million compounds tested in a primary HTS, we identified more than
2300 confirmed hits which inhibited SMYD2 with IC50 values below 15 µM. Among the
multiple structural clusters and singletons in the hit list, several offered starting points with low
micromolar potency and tractable chemical matter, and our attention was drawn to pyrazolines
such as compound 5. Initial hits and later derivatives of the pyrazoline series showed stabilizing
effects in an SMYD2 thermal shift assay (TSA) (Figure 2B). In addition, binding of compound 6
to SMYD2 was validated by isothermal titration calorimetry (ITC), which indicated a
submicromolar binding constant (K = 540 nM, Figure 2C) and a high enthalpic contribution to
d
the binding energy. As the latter reduces the likelihood of nonspecific offꢀtarget activities, the
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ITC data underscored the attractiveness of pyrazoline hit 5.
It transpired that compounds of
this structural series had been prepared during the course of an inꢀhouse program as antagonists
for proteaseꢀactivated receptor 1 (PAR1), a Gꢀproteinꢀcoupled receptor which is highly
expressed in platelets and plays an important role in thrombin signaling and platelet
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aggregation.
Chemistry. The pyrazoline compounds of interest were synthesized according to previously
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described procedures. Synthesis of the required intermediates 10a–f started from the
commercially available 2ꢀaminoꢀ1ꢀphenylethanones 7 (Scheme 1). Protection of the amines as
the allyl carbamates 8 was followed by Mannich reaction with formaldehyde and piperidine, and
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the resulting product mixture was treated with hydrazine monohydrate to install the pyrazoline
moiety (compounds 9). Subsequent reaction with diphenyl Nꢀcyanocarbonimidate resulted in
intermediates 10a–f.
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The synthesis of compounds 5, 6 and 12–24 began with 10a (Scheme 2). Treatment of 10a with
the respective aniline or amine provided intermediates 11a–i. Deprotection to the 4ꢀ
aminopyrazolines and further elaboration led to the desired amides 5, 6 and 12–23 by a sequence
of optional reductive Nꢀalkylation, and amide formation. For the hydroxyacetyl derivatives 5, 6,
and 12–21, the amide was formed by using acetoxyacetyl chloride, followed by treatment of the
crude amide with methanolic potassium carbonate to remove the acetyl moiety. The amino acid
amide 22 was prepared by a coupling with 9ꢀfluorenylmethoxycarbonyl (Fmoc) protected
glycine (HATU, NMM, DMF), followed by removal of the Fmoc group with piperidine. Amide
formation with methoxyacetylchloride gave compound 23. Furthermore, oxoimidazolidine 24
was prepared by reaction of the Nꢀunsubstituted 4ꢀaminopyrazoline derivative with chloroacetyl
chloride and treatment of the resulting chloroacetamide with urotropine.
Compounds 4 and 25–29 were prepared from intermediates 10b–f, by the addition of 3ꢀ
(difluoromethoxy)aniline, followed by installation of the Nꢀethylated hydroxyacetamide as
described above (Scheme 3).
The synthesis of compounds 30–34 started from intermediate 10d by addition of the respective
aniline derivatives, followed by introduction of the Nꢀethylated hydroxyacetamide (Scheme 4).
Compounds 4, 28 and 30–34 were separated into their enantiomers by preparative chiral HPLC
or chiral Supercritical Fluid Chromatography. For compounds 6 and 4, the integrity of the
pyrazoline stereocenter was tested. The compounds were stable to racemization in aqueous
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solution at pH 7, as well as in mouse and human plasma at 37 °C, for at least 48 hours. However,
clean racemization was obtained under basic conditions and microwave irradiation (DBU, THF,
90 °C).
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Structure–Activity Relationships. Our initial structure–activity relationship (SAR) studies of 3ꢀ
(4ꢀchlorophenyl)pyrazoline derivatives with respect to SMYD2 inhibition are summarized in
3
Table 1. For the carboximidamide Nꢀsubstituent (R ), a metaꢀsubstituted phenyl seemed optimal,
with the 3ꢀ(difluoromethoxy)phenyl derivative 6 displaying highest potency and binding
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efficiency (IC = 0.8 µM, BEI= 12.5). Small para substituents are tolerated, such as with the 3ꢀ
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chloroꢀ4ꢀfluorophenyl, 3,4ꢀdichlorophenyl and 2,2ꢀdifluoroꢀ1,3ꢀbenzodioxolꢀ5ꢀyl derivatives 5,
2 and 13 (IC = 1.7, 3.3 and 2.2 µM, respectively). In contrast, the 4ꢀ(difluoromethoxy)phenyl
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derivative 14 is more than 15ꢀfold less active than the corresponding metaꢀderivative 6. The
extended 3ꢀchlorophenethyl derivative 15 showed no measurable activity (IC >20 µM),
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implying that the space within the relevant binding pocket is limited. Nonꢀaromatic derivatives,
such as 4,4,4ꢀtrifluorobutylꢀsubstituted 16 or the cyclopropylethyl derivative 18, are less potent
than 6 (IC = 6.9 and 8.3 µM for 16 and 18, respectively). Surprisingly, the corresponding nꢀ
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butyl derivative 17 is inactive (IC >20 µM). The SAR for PAR1 antagonism at the
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carboximidamide Nꢀsubstituent appears to be rather flat: all compounds 5, 6 and 12–18 are
potent PAR1 antagonists, with IC values below 100 nM.
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With respect to the amide moiety, it became clear that the presence and orientation of a
hydrogenꢀbond donor has a large impact on potency towards SMYD2 (Table 2). Variation of the
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amide Nꢀalkyl substituents (R ) revealed that the Nꢀethyl derivative is preferred: secondary
amide 19 (IC = 10.9 µM) is about 10ꢀfold less active, and the Nꢀpropyl derivative 20 is also
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less potent, than the corresponding Nꢀethyl derivative 6 (IC = 0.8 µM), whereas the larger
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cyclopropylmethyl substituent in 21 resulted in a loss of potency (IC >20 µM). Replacement of
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the hydroxyacetyl in compound 12 (IC = 3.3 µM) by an aminoacetyl group gave the equipotent
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derivative 22 (IC = 2.8 µM). In contrast, the methoxyacetyl derivative 23, lacking the
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hydrogenꢀbond donor, is inactive (IC >20 µM). In compound 24, where the hydrogenꢀbond
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donor is fixed in an oxoimidazolidine ring, there is significantly reduced potency (IC = 6 µM)
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relative to the Nꢀethyl derivative 6. Based on the available amide derivatives, with BEI values in
the same range (10 to 11), an improvement in the binding efficiency was not envisaged.
Furthermore, alterations of the amide moiety did not offer a path forward to selectivity against
PAR1. Compounds 22, 23 and 24 are in a similar potency range for PAR1 antagonism, and
greater than 10ꢀfold more potent against PAR1 (IC = 130, 30 and 100 nM, respectively) than
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SMYD2.
At this stage, we selected compound 6 for coꢀcrystal structure determination with SMYD2,
based on its potency and promising biophysical properties. Compound 6 was soaked into crystals
of SMYD2 grown in the presence of SAM. The crystal structure revealed that compound 6 binds
into the substrate peptide binding pocket of SMYD2; the observed binding mode is consistent
with the previously established SAR. There is a very good steric and electrostatic fit of 6 to the
substrate binding site of SMYD2 (Figure 3A and 3B). The pyrazoline and the NH of the
carboximidamide form hydrogen bonds to Gly183. The 4ꢀchlorophenyl substituent inserts into
the lysine binding channel and is engaged in πꢀstacking interactions with Phe184 and Tyr240.
There is a good fit of the 3ꢀ(difluoromethoxy)phenyl substituent into the adjacent hydrophobic
pocketꢀ1, with the difluoromethoxy group pointing into a hydrophobic subpocket. Although
racemic 6 was used for soaking, the density maps reveal that only the Sꢀenantiomer is bound.
The S configuration at the pyrazoline provides an optimal exit vector for the hydroxyacetamide
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substituent, which occupies pocketꢀ2 and forms two hydrogen bonds with Thr185 (Figure 3A).
The N'ꢀnitrile of the carboximidamide contributes to a waterꢀbridged hydrogen bond with
Ser196. Based on the crystal structure of compound 6, we envisioned that further exploration of
the aniline and amide moieties would not lead to a significant improvement of potency and
binding efficiency. Therefore, we elected to focus on derivatives of the 4ꢀchlorophenyl
substituent, which were underrepresented in our compound library.
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With the coꢀcrystal structure in hand, we employed molecular modeling for the prioritization
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of derivatives. In particular, WaterMap calculations,
which estimate the position and the
thermodynamic properties of water molecules in the ligandꢀfree structure, suggested the
introduction of a second substituent at the 3ꢀposition of the 4ꢀchlorophenyl group. To test this
hypothesis, a few derivatives were synthesized (see Table 3). The unsubstituted phenyl
derivative 25 is inactive, while the 4ꢀbromo derivative 26 (IC = 1.1 µM) is as potent as the 4ꢀ
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chloro derivative 6, indicating the importance of a hydrophobic substituent at the 4ꢀposition. As
predicted by WaterMap calculations, introduction of a second substituent at the meta position, as
exemplified by 3,4ꢀdichloro derivative 4 (IC = 0.08 µM, BEI = 13.5) and 4ꢀchloroꢀ3ꢀmethyl
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derivative 28 (IC = 0.08 µM, BEI = 14), resulted in significantly improved potency (ca. 10ꢀfold
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greater than 6) and binding efficiency. As highlighted in Figure 3C, the two chloro substituents
are coꢀlocated with two calculated water sites that have high free energy, suggesting an optimal
water displacement by 3,4ꢀdichlorophenyl and thus a lower binding free energy than for the 4ꢀ
chlorophenyl or the unsubstituted phenyl derivative, consistent with the observed potency
difference for these derivatives. However, introduction of a third substituent leads to a decrease
in potency compared to the 3,4ꢀdisubstituted derivatives, as exemplified by 4ꢀchloroꢀ3,5ꢀ
dimethyl derivative 27 (IC = 0.57 µM). Compound 29, with the bulkier 1,3ꢀbenzodioxole
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moiety, is also less potent (IC = 4.2 µM), suggesting that the SAR at this position is rather
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steep.
Compound 4 was selected for further biological and crystallographic studies. The coꢀcrystal
was obtained by soaking (S)ꢀ4 into crystals of SAMꢀbound SMYD2. Structure determination
revealed that (S)ꢀ4 features an almost identical binding mode as compound 6, as evidenced in the
overlay of both structures (Figure 3D). In line with the observed S configuration of compound 6
and (S)ꢀ4, we observed a greater than 50ꢀfold difference in the IC50 values of the two
enantiomers of 4 [27 nM for (S)ꢀ4 vs 1.7 µM for (R)ꢀ4, see Table 4]. Introduction of the
additional 3ꢀchloro substituent also resulted in a dramatic decrease in the antagonistic effect on
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PAR1 [IC = 1.7 µM for (S)ꢀ4 and >30 µM for (R)ꢀ4]. Although (S)ꢀ4 proved to be the active
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isomer for both SMYD2 inhibition and PAR1 antagonism, there is a greater than 50ꢀfold
selectivity for SMYD2 relative to PAR1.
The coꢀcrystal structures of compound 6 and (S)ꢀ4 revealed that the pyrazolineꢀbased SMYD2
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inhibitors feature a different binding mode to other recently reported inhibitors. Thus, 1 and 2
(
Figure 4A) have published IC values of 0.12 µM and <15 nM, respectively, and like (S)ꢀ4 they
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bind to the substrate binding site of SMYD2. The structures of the three inhibitors are
superimposed in Figure 4B. All ligands occupy the lysine binding channel and the adjacent
hydrophobic pocketꢀ1; however, (S)ꢀ4 employs pocketꢀ2 via hydrogenꢀbond interactions with its
hydroxyacetyl moiety (cf. Figure 2A), which are not present in 1 or 2. 1 and 2, on the other hand,
occupy a remote hydrophobic pocketꢀ3 which is not addressed by (S)ꢀ4. Figure 4C provides a
view into the lysine binding channel (the respective motifs are highlighted in color on the
structures in Figure 4A). 2 binds with its Nꢀalkylpyrrolidine moiety at this position, which is
structurally closely related to the lysine side chain of a methylated substrate peptide. 1 and (S)ꢀ4
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address the lysine channel with aromatic substituents. Noteworthy, the 3,4ꢀdichlorophenyl group
of 1 and of (S)ꢀ4 bind to different positions.
Based on 3,4ꢀdichlorophenyl as a novel lysine channel binding motif, we identified several
pyrazoline derivatives as potent SMYD2 inhibitors (see Table 4). Aiming to identify a candidate
for in vivo experiments, we focused on the potent Sꢀenantiomers of these derivatives, profiling
them in pharmacokinetic assays in vitro. (S)ꢀ4 showed moderate stability upon incubation with
rat hepatocytes (CLblood = 2.5 L/h/kg), as well as moderate apparent permeability (34 nm/s) and a
hint of active transport in the Caco2 assay (efflux ratio = 5). In comparison, the 4ꢀchloroꢀ3ꢀ
methylphenyl derivative (S)ꢀ28 had similar permeability and efflux (33 nm/s, efflux ratio = 7),
and slightly lower metabolic stability in rat hepatocytes (CLblood = 2.8 L/h/kg). Compounds (S)ꢀ
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30 and (S)ꢀ32–34 exhibited high metabolic stability (CLblood = 1.8–0.3 L/h/kg); however, these
derivatives have very low aqueous solubility (<5 mg/L), thereby limiting their suitability for in
vivo experiments. On the other hand, the methoxyphenyl derivative (S)ꢀ31 has high aqueous
solubility (163 mg/L) and moderate permeability with a hint of active transport (62 nm/s, efflux
ratio = 3) in the Caco2 assay; nevertheless, (S)ꢀ31 displayed low stability upon incubation with
rat hepatocytes (CLblood = 3.2 L/h/kg), and thus bioavailability is expected to be low. Based on
these data, (S)ꢀ4 was selected for further in vitro and in vivo studies. The in vivo
pharmacokinetic properties of (S)ꢀ4 were first evaluated by a singleꢀdose administration of (S)ꢀ4
at 0.4 mg/kg by i.v. bolus or 0.8 mg/kg p.o., respectively, to rats: there was moderate blood
clearance (1.6 L/h/kg) and a low bioavailability of 24% (see Supporting Information Figure
S1A). These data prompted us to assess the exposure of (S)ꢀ4 following oral administration.
Hence, we treated mice with 10–100 mg/kg po q.d. which are well tolerated doses of (S)ꢀ4. As a
result, unbound IC [cellular methylation InꢀCell Western (ICW) assay – see Figure 7AꢀC] is
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covered for ~9 to ~12 hours at steady state when 10 and 100 mg/kg, respectively, are
administered (see Supporting Information Figure S1B).
Potency, Selectivity and Inhibition Mode. (S)ꢀ4 showed potent in vitro inhibition of SMYD2
with an IC of 27 ±7 nM in the biochemical SPA assay (Figure 5A). To analyze the mode of
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inhibition and affinity of (S)ꢀ4, our SMYD2 protein preparation was characterized with respect to
its apparent Michaelis–Menten constants for SAM and the p53 peptide substrate (data not
shown). The Km(app) values of 60 nM and 1 µM, respectively, were in excellent agreement with
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the constants previously reported using a similar assay. Then, we performed IC50
determinations at increasing concentrations of one substrate and a fixed amount of the other ([S]
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=
Km(app)), and applied the Cheng–Prusoff relationship as described elsewhere. Increasing the
concentration of peptide substrate resulted in a linear increase in IC (Figure 4B, upper panel),
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as would be expected for a competitive mode of inhibition. Fitting these data to the
corresponding Cheng–Prusoff competitive inhibition model revealed a Ki(app) of 8 ± 1 nM (SD).
On the other hand, when SAM concentrations were titrated to saturation, we observed a decrease
in IC which converged to a constant value (Figure 4B, lower panel). This type of behavior
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toward SAM corresponds to an uncompetitive mode of inhibition. Consequently, the IC50 vs
S]/Km(app) plot fits best to the Cheng–Prusoff model for uncompetitive inhibition, yielding an
[
inhibitor constant αKi(app) of 28 ± 3 nM (SD). Our data suggest that (S)ꢀ4 is a peptideꢀ
competitive, SAMꢀuncompetitive inhibitor of SMYD2 methyltransferase activity, which
preferably binds to the SMYD2–SAM substrate complex. Interestingly, SMYD2 has been
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reported to follow a random sequential Bi Bi mechanism of substrate binding,
but the
uncompetitive mode of inhibition of (S)ꢀ4 regarding the SAM cofactor suggests an ordered
sequential Bi Bi mode of substrate binding, where SAM would be required to bind before the
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peptide substrate. Similar results have recently been described for inhibitors of the SAMꢀ
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dependent arginine methyltransferase PRMT5.
For further evaluation of selectivity, (S)ꢀ4 was tested on a panel of 32 additional
methyltransferases, including closely related family members SMYD3, SUV420H1 and
SUV420H2. As a result, (S)ꢀ4 displayed >100ꢀfold selectivity for SMYD2, with very weak
residual activity toward the closest related methyltransferase SMYD3 (IC ~3 µM) (Figure 5C).
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In addition to the methyltransferases, (S)ꢀ4 was also profiled in the commercially available
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KINOMEscan (DiscoveRx) and LeadProfilingScreen (Eurofins Panlabs) assay panels to fully
determine relative selectivity and specificity for kinases and other primary molecular targets,
including several CNS targets. Overall, we were able to confirm the high selectivity and
specificity of (S)ꢀ4 for SMYD2 inhibition (see Supporting Information Tables S1 and S2).
Cellular Methylation Activity on p53.
The ability of (S)ꢀ4 to inhibit SMYD2 was tested by monitoring its effects on cellular p53
methylation using different cellular mechanistic assays. First, we generated a polyclonal
antibody (SY46) for the specific detection of p53 monomethylation at lysine 370, as described
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elsewhere. This antibody was then tested on recombinant p53 protein which had been in vitro
methylated by SMYD2 in a western blot. Specificity for methylated p53 was confirmed by the
exclusive detection of the in vitro methylated p53 protein isoforms, whereas the nonꢀmethylated
p53 was not detected (Figure 6A). Endogenous methylation of p53 protein was characterized by
treatment of KYSEꢀ150 esophageal cancer cells with increasing concentrations of (S)ꢀ4 for 5
days. The KYSEꢀ150 cell line model was selected based on a described SMYD2 gene
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amplification and a heterozygous R248Q mutation in p53 (COSMIC) leading to p53 protein
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accumulation without a stress stimulus. After treatment with (S)ꢀ4, a significant reduction of
methylation was detected confirming that p53 is a cellular target of SMYD2ꢀdependent
methylation (Figure 6B). Nevertheless overall endogenous detection of p53 protein methylation
led to weak signals, hence this method was not useful for the determination of a cellular IC .
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Therefore, we additionally employed an established system with a transient FLAGꢀtagged
SMYD2 and FLAGꢀtagged p53 overexpression in HEK293T cells as benchmark assay. This
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assay has been used previously to characterize the structurally unrelated SMYD2 inhibitor 2.
As shown in Figure 6C, (S)ꢀ4 showed a concentrationꢀdependent decrease in p53 methylation
without affecting p53 total protein levels. A cellular IC of 58 nM was determined (Figure 6D),
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which confirms that (S)ꢀ4 is the most potent cellularꢀactive SMYD2 inhibitor known to date
(Figure 1).
Characterization and Inhibition of SMYD2 Mediated AHNAK methylation
To further characterize the effects of our aminopyrazolineꢀbased inhibitors on the cellular
methylation activity of SMYD2, we generated a polyclonal cell line derived from KYSEꢀ150
with stable Nꢀterminal 2xcꢀmycꢀtagged SMYD2 overexpression to maximize cellular
methylation activity. In an immunofluorescence analysis, SMYD2 was localized mainly in the
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cytosol (Supplementary Figure S2A), as reported by others. Surprisingly, the antibody SY46
directed against methylated p53 showed a very strong signal specifically in the clones with
SMYD2 overexpression in the immunochemistry staining not derived from p53 protein
(Supplementary Figure S2BꢀC). We validated the novel SMYD2 substrate giving rise to the
strong immunochemistry staining signal by knockdown and overexpression experiments in
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additional cell lines to be derived from AHNAK protein (Figure 7B and Suplementary Figure
2DꢀF). AHNAK methylation has been very recently reported in a proteomics study by other as
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an additional methylation substrate of SMYD2 further confirming our results. Importantly, we
used the strong AHNAK methylation signal to set up an In Cell Western (ICW) assay for cellular
optimization of our lead series (Figure 7A). The methylation signal was reduced by treatment
with increasing concentrations of (S)ꢀ4, derivatives of the aminopyrazoline series or 1 (Figure
7A). In addition (S)ꢀ4 specifically reduced methylation of AHNAK without altering AHNAK
protein expression in a western blot (Figure 7B). Importantly IC values in the cellular ICW
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assay for aminopyrazolines correlated with potency in the scintillation proximity assay (Figure
7C), and were also comparable to the p53 methylation assay (see Figure 6D). Our results of the
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methylation of AHNAK and the recent identification of many additional methylation targets
clearly point towards additional roles beyond p53 regulation of SMYD2, and further studies
supported by potent and selective inhibitors as (S)ꢀ4 are needed to fully elucidate the underlying
biology.
Characterization of (S)-4 in Proliferation and Apoptosis Assays.
To explore the potential effects of (S)ꢀ4 on proliferation, we tested a panel of 240 different
cancer cell lines (OncoPanel 240/Eurofins Panlabs). Cell lines were longꢀterm cultured with (S)ꢀ
4 for 10 days to allow for a sufficient translation of demethylation of SMYD2 substrates to
potential antiproliferative effects, which takes 48–72 hours (Supplementary Figure S2C). (S)ꢀ4
exposure resulted in only limited responses in a subset of cell lines: an antiproliferative response
with IC <10 µM was seen in 21 cell lines (~9%); however, most cell lines (83%) did not reach
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0% proliferation inhibition with 20 µM of (S)ꢀ4 (Figure 8A). In addition, there was no clear
preference for a specific tissue origin of the responding cancer cell line. Thus, SMYD2 inhibition
by (S)ꢀ4 has only limited proliferation effects in a small subset of cancer cell lines under the
employed conditions. Based on the observation that p53 protein is methylated by SMYD2, which
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should lead to suppression of apoptosis, we were additionally interested in the effects of (S)ꢀ4 in
combination with an apoptotic stimulus. KYSEꢀ150, U2OS and A2780 cell lines were preꢀ
treated with (S)ꢀ4 or inactive derivative 25 (see Table 3) for 2 days (demethylation phase),
followed by treatment with doxorubicin (apoptotic trigger). (S)ꢀ4, but not 25, significantly
improved caspase 3/7 activation in all three tested cell lines without inducing apoptosis alone
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(Figure 7B). Thus, SMYD2 inhibition can enhance apoptotic responses.
The First Chemical Probe Suitable for In Vivo Characterization of SMYD2 Inhibition.
Functional validation of novel potential cancer targets such as the protein methyltransferase
SMYD2 relies on appropriate model systems in vitro as well as in vivo. Additionally, chemical
probe inhibitors also suitable for in vivo applications are highly desirable. Most reported
activities of SMYD2 are not directly involved in survival signaling of cancer cells. Hence, in
vitro proliferation assays may not adequately cover the full phenotype of SMYD2 inhibition, and
more complex (in vivo) assays are thus required. The research work characterizing SMYD2 in
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heart and skeletal muscle cells by knockdown experiments in vivo
clearly illustrates the
complex underlying biology of SMYD2 and the necessity for chemical probes suitable for in
vivo applications. Therefore, we were interested in establishing if SMYD2 methylation activity
in tumor cells can be inhibited by (S)ꢀ4 in vivo. To this end, mice bearing subcutaneous tumor
xenografts (tumor tissue derived from the SMYD2ꢀoverexpressing KYSEꢀ150 cell line) were
treated orally with 10, 30, 70 or 100 mg/kg (S)ꢀ4, or vehicle (PEG 400/water 8:2), once daily for
3
days. After the treatment period, tumors were harvested and analyzed ex vivo for methylation
of AHNAK by dotꢀblotting. For detection of the methylation signals, SY46 methylation antibody
was used (see Figure 7A and Supplementary Figure S2AꢀF). (S)ꢀ4 significantly reduced the
methylation with doses starting from 30 mg/kg, with most significant effects in the 100 mg/kg
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treated group (P <0.001, Student’s t test) (Figure 9A). Treatment with 10 mg/kg (S)ꢀ4 resulted in
no significant effect on the methylation level. Exposure at 10 mg/kg is close to the level of the
cellular IC for ~9 hours, which may indicate a need for an even higher exposure as the IC to
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achieve in vivo effects on demethylation.
Then the KYSEꢀ150 esophageal xenograft model was used to evaluate if the observed
improved apoptosis induction in the in vitro setting after treatment with doxorubicin (Figure 8B)
could translate to antitumor efficacy in vivo. Four groups of tumorꢀbearing mice were treated as
follows: Group 1 (control group) was only treated with vehicle (Solutol/ethanol/water 1:1:8) iv
q.d. and once at day 4 with the vehicle used for doxorubicin (saline) iv; Group 2 was treated with
(S)ꢀ4 at 500 mg/kg po q.d.; Group 3 was treated with 10 mg/kg doxorubicin iv once at day 4;
Group 4 was treated with a combination of (S)ꢀ4 and doxorubicin. There was a slight reduction
in area (Figure 9B) and weight (Figure 9C) of tumors from mice treated with the combination of
(S)ꢀ4 and doxorubicin relative to tumors from the control group. Combination treatment only
resulted in a minor increase in treatmentꢀrelated body weight loss (Figure 9D). The combination
treatment reached a T/C level (based on tumor weight) of 0.46, which is significant (Figure 9C).
Therefore combination of a SMYD2 inhibitor with a chemotherapeutic agent resulted in reduced
cancer cell growth in vivo. In comparison, the monotherapy groups treated with only doxorubicin
or (S)ꢀ4 showed no significant antitumor efficacy relative to the vehicle control group. These
data are consistent with the observed limited cellular proliferation effects of (S)ꢀ4 in the cell line
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panel (Figure 8A) and indicate that, in contrast to SMYD2 knockdown, a catalytic inhibition
may be insufficient to induce cell death in the KYSEꢀ150 esophageal model. Furthermore, the
monotherapeutic approach with doxorubicin did not result in any antitumor efficacy in the
KYSEꢀ150 xenograft model.
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CONCLUSION
In summary, we have identified (S)ꢀ4 as a potent, selective and cellꢀactive, substrateꢀ
competitive inhibitor of SMYD2. Our data show that SMYD2 inhibition can enhance efficacy of
doxorubicin in vivo, which confirms our in vitro observation of higher caspase 3/7 activation
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(Figure 8B). Our results (S)ꢀ4 are also in agreement with an earlier study where an increased
apoptosis induction in cells with an SMYD2 knockdown was observed. Nonetheless, in our
initial explorative in vivo study with (S)ꢀ4, effects on xenografted tumors were only moderate. In
addition, high doses of (S)ꢀ4 were needed in vitro as well as in vivo relative to the concentration
needed to achieve effects on methylation. Therefore, we cannot exclude the possibility that
additional, soꢀfar unexplored activities of SMYD2 might be responsible for the observed effects.
This again underlines the necessity to identify suitable chemical probes for more extensive target
validation campaigns to fully explore the complex biology of SMYD2 and other targets. In this
regard, (S)ꢀ4 will be a highly valuable tool for the further exploration of SMYD2 biology, not
only for in vitro but also for in vivo studies.
EXPERIMENTAL SECTION
Chemistry
General Procedures. All reagents and solvents were used as purchased, unless otherwise
specified. All final products were at least 95% pure, as determined by analytical HPLC.
Materials. Intermediate 10a and compounds 5, 6 and 12–24 were synthesized according to the
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methods described previously. 1H NMR and determined to be >95%. 1H NMR spectra were
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recorded on Bruker Avance III HD spectrometers operating at 300, 400, or 500 mHz. The
chemical shifts (δ) reported are given in parts per million (ppm), and the coupling constants (J)
are in hertz (Hz). The spin multiplicities are reported as s = singlet, br s = broad singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, and br = broad. The LC/MS analysis was
performed on Waters Acquity UPLCMS SingleQuad with a Acquity UPLC BEH C18 column
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(1.7 µm, 50x2.1mm) at 60 °C, using water + 0.1 vol % formic acid (99%) and acetonitrile as
mobile phase at a flow rate of 0.8 ml/min and a DAD detector (210ꢀ400 nm). LC/MS/MS was
performed on a CTC PAL autosampler, an Agilent 1200 HPLC, and a ABSciex 4000 mass
spectrometer.
Assignment of stereochemistry. For all separated enentiomers, it was assumed based on the
coꢀcrystal structure of 4, that the active enantiomer (SMYD2 inhibition) has Sꢀconfiguration.
Phenyl
4-{[(Allyloxy)carbonyl]amino}-3-(4-bromophenyl)-N-cyano-4,5-dihydro-1H-
pyrazole-1-carboximidate (10b). Compound 10b was prepared as described for 10d. Starting
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from 7b (37.7 g, 0.18 mol), 10b was obtained as white solid (40.1 g, 49% over 4 steps). H NMR
(
400 MHz, CDCl ): δ = 4.26–4.42 (m, 3H), 4.66 (dd, J = 13.3, 6.0 Hz, 1H), 4.74 (dd, J = 13.1,
3
5.8 Hz, 1H), 5.30 (d, J = 10.3 Hz, 1H), 5.40 (d, J = 16.8 Hz, 1H), 5.67 (td, J = 8.8, 4.5 Hz, 1H),
6.00 (ddt, J = 16.8, 11.0, 5.6 Hz, 1H), 7.02–7.11 (m, 2H), 7.19–7.34 (m, 5H), 7.58 (d, J = 8.5 Hz,
+
2
H). LCꢀMS (ESI): m/z = 468.1/470.1 [M + H] .
Phenyl
4-{[(Allyloxy)carbonyl]amino}-3-(4-chloro-3,5-dimethylphenyl)-N-cyano-4,5-
dihydro-1H-pyrazole-1-carboximidate (10c). Compound 10c was prepared as described for
10d. Starting from 7c (35 g, 0.15 mol) to obtain 10c (29.3 g, 43% over 4 steps) as white solid.
1
H NMR (400 MHz, [D] DMSO): δ = 2.32 (s, 6H), 4.03–4.18 (m, 1H), 4.39–4.61 (m, 2H), 5.06–
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.27 (m, 2H), 5.49–5.66 (m, 1H), 5.79–5.94 (m, 1H), 5.75–5.95 (m, 1H), 7.26 (d, J = 7.8 Hz,
2
H), 7.30–7.37 (m, 1H), 7.41–7.55 (m, 2H), 7.56–7.73 (m, 2H), 8.17 (d, J = 8.7 Hz, 1H). LCꢀMS
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Phenyl
4-{[(Allyloxy)carbonyl]amino}-N-cyano-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-
(10d). To stirred solution of 2ꢀaminoꢀ1ꢀ(3,4ꢀ
pyrazole-1-carboximidate
a
dichlorophenyl)ethanone hydrochloride (1:1) (7d; 116 g, 0.480 mol) in water (500 mL) was
added allyl chloroformate (56.5 mL, 0.530 mol) in DCM (800 mL). The reaction mixture was
cooled to 0 °C and K CO (207 g, 1.49 mol) in water (1 L) was added dropwise over 1 h. The
2
3
mixture was allowed to warm to rt, stirred overnight, then diluted with DCM (500 mL). The
organic phase was washed with saturated aqueous NH Cl solution (400 mL) followed by brine
4
(500 mL), then dried over MgSO , filtered and concentrated in vacuo. The crude mixture was
4
purified by dry flash column chromatography (eluent: DCM/heptane 2:1, 3:1, 4:1; DCM; EtOAc)
to yield allyl [2ꢀ(3,4ꢀdichlorophenyl)ꢀ2ꢀoxoethyl]carbamate (8d; 120 g, 87%) as a white
1
crystalline solid. H NMR (400 MHz, CDCl ): δ = 4.62 (d, J = 5.5 Hz, 2H), 4.68 (d, J = 4.6 Hz,
3
2
H), 5.25 (dd, J = 1.4, 10.5 Hz, 1H), 5.35 (dd, J = 1.4, 16.9 Hz, 1H), 5.72 (br s, 1H), 5.94 (m,
1H), 7.60 (d, J = 8.2 Hz, 1H), 7.79 (dd, J = 2.1, 8.5 Hz, 1H), 8.06 (d, J = 1.8 Hz, 1H). LCꢀMS
+
(ESI): m/z = 288.1 [M + H] . To a stirred suspension of 8d (50.0 g, 0.174 mol) in EtOH (390
mL) was added formaldehyde solution (20 mL, 0.261 mol; 37 wt% in water) followed by the
dropwise addition of piperidine (26 mL, 0.261 mol) in EtOH (130 mL) over 30 min. The reaction
mixture was stirred overnight; TLC indicated consumption of 8d. The volatiles were removed by
evaporation to yield an orange oil which was not further purified. To a solution of this crude
material in EtOH (480 mL) was added hydrazine monohydrate (29.6 mL, 0.609 mol) and the
reaction mixture was heated to reflux for 2.5 h. Then, the mixture was allowed to cool to rt,
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concentrated and poured over iceꢀcooled saturated NH Cl solution (300 mL). The crude product
4
was extracted with EtOAc (1.5 L) and the combined organic layers were washed with brine (300
mL), then dried over MgSO , filtered and concentrated to yield allyl [3ꢀ(3,4ꢀdichlorophenyl)ꢀ4,5ꢀ
4
10
11
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60
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dihydroꢀ1Hꢀpyrazolꢀ4ꢀyl]carbamate (9d; 50.0 g, 91%) as a pale yellow solid. H NMR (400
MHz, [D] DMSO): δ = 3.24 (m, 1H), 3.59 (m, 1H), 4.39–4.54 (m, 2H), 5.08–5.25 (m, 3H), 5.79–
6
5
.90 (m, 1H), 7.52 (dd, J = 1.8, 8.3 Hz, 1H), 7.57 (br s, 1H), 7.59 (m, 1H), 7.68 (d, J = 1.8 Hz,
+
1
H), 7.84 (d, J = 8.7 Hz, 1H). LCꢀMS (ESI): m/z = 314.1 [M + H] . To a stirred suspension of 9d
(50.0 g, 0.159 mol) in iꢀPrOH (860 mL) was added diphenyl Nꢀcyanocarbonimidate (38.0 g,
.159 mol). The reaction mixture was heated to reflux, at which point the suspension dissolved
0
into solution; after a further 10 min at reflux, a white precipitate formed. The mixture was stirred
at reflux for a further 1 h, then allowed to slowly cool to rt overnight. The precipitate was
collected by filtration and washed with Et O (2 × 250 mL). The resulting white solid was
2
1
allowed to dry to yield 10d (48.6 g, 67%) as a white solid. H NMR (400 MHz, [D] DMSO): δ =
6
4
.13 (apparent d, J = 8.2 Hz, 1H), 4.47 (m, 3H), 5.14 (m, 2H), 5.51–5.63 (m, 1H), 5.79–5.90 (m,
1
H), 7.23 (d, J = 7.3 Hz, 2H), 7.30 (t, J = 7.8 Hz, 1H), 7.45 (m, 2H), 7.79 (br m, 2H), 7.97 (br s,
+
1H), 8.19 (d, J = 8.7 Hz, 1H). LCꢀMS (ESI): m/z = 458.0 [M + H] .
Phenyl 4-{[(Allyloxy)carbonyl]amino}-3-(4-chloro-3-methylphenyl)-N-cyano-4,5-dihydro-
1H-pyrazole-1-carboximidate (10e). Compound 10e was prepared as described for 10d.
1
Starting from 7e (3.1 g, 13.9 mmol) to obtain 10e (2.12 g, 34.4% over 4 steps) as white solid. H
NMR (400 MHz, CDCl ): δ = 2.18 (s, 3H), 4.30 (d, J = 6.4 Hz, 2H), 4.64 (d, J = 6.0 Hz, 2H),
3
5
.24 (d, J = 10.5 Hz, 1H), 5.34 (d, J = 16.9 Hz, 1H), 5.60–5.70 (m, 1H), 5.94 (ddt, J = 5.6, 10.9,
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6.8 Hz, 1H), 6.89–7.35 (m, 7H), 7.48 (m, 1H), 7.54 (m, 1H). LCꢀMS (ESI): m/z = 438.2 [M +
+
H] .
Phenyl 4-{[(Allyloxy)carbonyl]amino}-3-(1,3-benzodioxol-5-yl)-N-cyano-4,5-dihydro-1H-
pyrazole-1-carboximidate (10f). Compound 10f was prepared as described for 10d, starting
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from 7f (12.0 g, 55.7 mmol) to obtain 10f (9.1 g, 38% over 4 steps) as white solid. H NMR (400
MHz, [D] DMSO): δ = 4.05–4.08 (m, 1H), 4.43–4.47 (m, 3H), 5.1–5.2 (m, 2H), 5.50–5.55 (m,
6
1
H), 5.80–5.90 (m, 1H), 6.08 (s, 2H), 7.05 (d, J = 7.8 Hz, 1H), 7.21–7.27 (m, 2H), 7.27–7.41 (m,
+
3H), 7.43–7.50 (m, 2H), 8.16 (d, J = 8.7 Hz, 1H). LCꢀMS (ESI): m/z = 434.2 [M + H] .
General Procedure for the Synthesis of Allyl [1-(N'-Cyano-N-arylcarbamimidoyl)-3-
phenyl-4,5-dihydro-1H-pyrazol-4-yl]carbamates 11j–s. To a stirred solution of the respective
aniline derivative (3 equiv) in anhydrous THF (2 mL/mmol) at –78 °C was added nꢀBuLi (3
equiv, 2 M in hexane) dropwise, while maintaining the reaction temperature below –65 °C. The
reaction mixture was stirred at –78 °C for 1 h, then the respective phenyl 4ꢀ
{
[(allyloxy)carbonyl]amino}ꢀNꢀcyanoꢀ3ꢀphenylꢀ4,5ꢀdihydroꢀ1Hꢀpyrazoleꢀ1ꢀcarboximidate 10 (1
equiv) in anhydrous THF (30 mL/mmol) was added dropwise, while maintaining the reaction
temperature below –65 °C. The reaction mixture was stirred at –78 °C for 2 h, then slowly
poured over saturated NH Cl solution (30 mL/mmol based on 10). The crude product was
4
extracted into EtOAc (30 mL/mmol); the combined organic layers were washed with brine (15
mL/mmol), dried over MgSO , filtered and concentrated. The crude solid was precipitated from a
4
minimum volume of EtOAc, collected by filtration and washed with Et O to give the
2
corresponding intermediate 11.
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Allyl [3-(4-Bromophenyl)-1-{N'-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-
dihydro-1H-pyrazol-4-yl]carbamate (11j). Compound 11j was prepared from 10b (5.0 g, 10.7
mmol) and 3ꢀ(difluoromethoxy)aniline. 11j was not pure after chromatography, and was used as
10
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such in further steps (3.8 g, purity 77 %ꢀUV, 51 %). LCꢀMS (ESI): m/z = 533.1 [M + H] .
Allyl
[3-(4-Chloro-3,5-dimethylphenyl)-1-{N'-cyano-N-[3-(difluoromethoxy)phenyl]-
carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]carbamate (11k). Compound 11k was prepared
from 10c (10.0 g, 22.1 mmol) and 3ꢀ(difluoromethoxy)aniline. 11k was obtained as white solid
1
(9.1 g, 80 %). H NMR (400 MHz, CDCl ): δ = 2.32 (s, 6H), 4.3–4.6 (m, 4H), 5.13 (dd, J = 1.1,
3
1
0.3 Hz, 1H), 5.19 (dd, J = 1.4, 17.4 Hz, 1H), 5.5–5.6 (m, 1H), 5.7–5.9 (m, 1H), 6.53 (t,
J = 73.8 Hz, 1H), 6.87 (br d, J = 8.7 Hz, 1H), 7.1–7.3 (m, 4H), 7.50 (s, 2H), 8.35 (s, 1H). LCꢀMS
+
(
ESI): m/z = 517.2 [M + H] .
Allyl [1-{N'-Cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-
4,5-dihydro-1H-pyrazol-4-yl]carbamate (11l). Compound 11l was prepared from 10d (20.0 g,
1
4
3.6 mmol) and 3ꢀ(difluoromethoxy)aniline. 11l was obtained as white solid (21.2 g, 93 %). H
NMR (400 MHz, [D] DMSO): δ = 4.08 (dd, J = 4.6, 11.9 Hz, 1H), 4.36–4.53 (m, 3H), 5.11 (dd,
6
J = 1.4, 10.5 Hz, 1H), 5.17 (dd, J = 1.4, 17.4 Hz, 1H), 5.50–5.59 (m, 1H), 5.77–5.90 (m, 1H),
6
.99 (dd, J = 2.3, 8.2 Hz, 1H), 7.16 (m, 1H), 7.21 (t, J = 73.7 Hz, 1H), 7.22 (m, 1H), 7.39 (t,
J = 8.3 Hz, 1H), 7.73–7.81 (m, 2H), 8.15 (d, J = 1.4 Hz, 1H), 8.17 (d, J = 8.7 Hz, 1H), 9.79 (br s,
+
1H). LCꢀMS (ESI): m/z = 523.2 [M + H] .
Allyl
[3-(4-Chloro-3-methylphenyl)-1-{N'-cyano-N-[3-(difluoromethoxy)phenyl]-
carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]carbamate (11m). Compound 11m was
prepared from 10e (10.0 g, 22.8 mmol) and 3ꢀ(difluoromethoxy)aniline. 11m was obtained as
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white solid (9.6 g, 84 %). H NMR (400 MHz, [D] DMSO): δ = 2.33–2.40 (m, 3H), 4.03–4.15
6
(
m, 1H), 4.41–4.56 (m, 3H), 5.13–5.36 (m, 2H), 5.53–5.63 (m, 1H), 5.82–5.94 (m, 1H), 7.00–
7
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.08 (m, 1H), 7.19–7.29 (m, 2H), 7.25 (t, J = 74 Hz, 1H, partially masked by other peaks), 7.40–
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.49 (m, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.73 (dd, J = 1.7, 8.2 Hz, 1H), 7.84–7.90 (m, 1H), 8.2 (br
+
d, J = 8.6 Hz, 1H), 9.75 (br s, 1H). LCꢀMS (ESI): m/z = 503.2 [M + H] .
Allyl
[3-(1,3-Benzodioxol-5-yl)-1-{N'-cyano-N-[3-(difluoromethoxy)phenyl]-
carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]carbamate (11n). Compound 11n was prepared
from 10f (2.0 g, 4.6 mmol) and 3ꢀ(difluoromethoxy)aniline. 11n was obtained as white solid (1.7
1
g, 74 %). H NMR (400 MHz, [D] DMSO): δ = 4.09 (dd, J = 4.6, 11.9 Hz, 1H), 4.43 (t, J = 11.2
6
Hz, 1H), 4.50 (d, J = 4.1 Hz, 2H), 5.16 (dd, J = 1.4, 10.5 Hz, 1H), 5.22 (dd, J = 1.6, 17.2 Hz,
1
H), 5.49–5.60 (m, 1H), 5.82–5.93 (m, 1H), 6.11 (s, 2H), 6.95–7.09 (m, 2H), 7.16–7.32 (m, 4H),
7
.39 (m, 1H), 7.61 (d, J = 1.4 Hz, 1H), 8.21 (d, J = 8.7 Hz, 1H), 9.63 (s, 1H). LCꢀMS (ESI): m/z
+
=
499.2 [M + H] .
Allyl [1-{N'-Cyano-N-[3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-
4
,5-dihydro-1H-pyrazol-4-yl]carbamate (11o). Compound 11o was prepared from 10d (10 g,
1
21.8 mmol) and 3ꢀ(trifluoromethoxy)aniline. 11o was obtained as beige solid (7.4 g, 63 %). H
NMR (400 MHz, [D] DMSO): δ = 4.13 (dd, J = 4.8, 11.9 Hz, 1H), 4.42–4.56 (m, 3H), 5.12–5.25
6
(
m, 2H), 5.50–5.63 (m, 1H), 5.83–5.94 (m, 1H), 7.19 (br d, J = 8.1 Hz, 1H), 7.35–7.44 (m, 2H),
7.46–7.56 (m, 1H), 7.77–7.85 (m, 2H), 8.13–8.24 (m, 2H), 9.89 (br s, 1H). LCꢀMS (ESI): m/z =
+
5
40.8 [M + H] .
Allyl
{1-[N'-Cyano-N-(3-methoxyphenyl)carbamimidoyl]-3-(3,4-dichlorophenyl)-4,5-
dihydro-1H-pyrazol-4-yl}carbamate (11p). Compound 11p was prepared from 10d (2.0 g, 4.4
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mmol) and 3ꢀmethoxyaniline. 11p was obtained as white solid (1.5 g, 71 %). H NMR (400
MHz, [D] DMSO): δ = 3.3 (s, 3H, masked by water signal), 3.97–4.07 (m, 1H), 4.33–4.48 (m,
6
1
H), 5.40 (s, 1H), 5.74–5.82 (m, 1H), 6.95 (br d, J = 7.1 Hz, 1H), 7.14–7.26 (m, 3H), 7.34–7.43
10
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(
m, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.81 (dd, J = 1.9, 8.5 Hz, 1H), 8.08 (s, 1H), 8.76 (br d, J = 8.8
+
Hz, 1H), 9.51 (br s, 1H). LCꢀMS (ESI): m/z = 487.1 [M + H] .
Allyl
[1-{N'-Cyano-N-[6-(difluoromethoxy)pyridin-2-yl]carbamimidoyl}-3-(3,4-
dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate (11q). Compound 11q was prepared
from 10d (5.0 g, 10.9 mmol) and 6ꢀ(difluoromethoxy)pyridinꢀ2ꢀamine. 11q was obtained as offꢀ
1
white solid (2.9 g, 43 %). H NMR (400 MHz, [D] DMSO): δ = 3.95 (dd, J = 5.0, 12.4 Hz, 1H),
6
4
.32 (t, J = 11.7 Hz, 1H), 4.4–4.5 (m, 2H), 5.13 (m, 2H), 5.51 (m, 1H), 5.84 (m, 1H), 6.82 (d,
J = 8.2 Hz, 1H), 7.13 (d, J = 7.8 Hz, 1H), 7.58 (t, J = 72.8 Hz, 1H), 7.7–7.8 (m, 2H), 7.91 (t,
J = 7.8 Hz, 1H), 8.05 (d, 1H), 8.18 (d, J = 1.8 Hz, 1H), 10.45 (s, 1H). LCꢀMS (ESI): m/z = 524.1
+
[
M + H] .
Allyl
[1-{N-[4-Chloro-3-(difluoromethoxy)phenyl]-N'-cyanocarbamimidoyl}-3-(3,4-
dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate (11r). Compound 11r was prepared
from 10d (2.0 g, 4.4 mmol) and 4ꢀchloroꢀ3ꢀ(difluoromethoxy)aniline. 11r was obtained as white
1
solid (1.5 g, 62 %). H NMR (400 MHz, [D] DMSO): δ = 4.01–4.17 (m, 1H), 4.42–4.56 (m,
6
3
H), 5.12–5.25 (m, 2H), 5.53–5.64 (m, 1H), 5.82–5.94 (m, 1H), 7.28 (t, J = 73 Hz, 1H, partially
masked by other peaks), 7.34 (dd, J = 2.4, 8.7 Hz, 1H), 7.42 (m, 1H), 7.61 (d, J = 8.6 Hz, 1H),
+
7
.81 (s, 2H), 8.13–8.25 (m, 2H), 9.91 (br s, 1H). LCꢀMS (ESI): m/z = 557.1 [M + H] .
Allyl
[1-{N'-Cyano-N-[3-(difluoromethoxy)-5-fluorophenyl]carbamimidoyl}-3-(3,4-
dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate (11s). Compound 11s was prepared
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from 10d (1.6 g, 3.4 mmol) and 3ꢀ(difluoromethoxy)ꢀ5ꢀfluoroaniline. 11s was obtained as white
1
solid (0.8 g, 45 %). H NMR (400 MHz, [D] DMSO): δ = 4.13 (dd, J = 4.6, 11.9 Hz, 1H), 4.41–
6
4.53 (m, 3H), 5.10–5.25 (m, 2H), 5.51–5.63 (m, 1H), 5.78–5.94 (m, 1H), 6.96 (dd, J = 2.2, 9.7
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Hz, 1H), 7.10 (d, J = 5.1 Hz, 1H), 7.18 (br d, J = 10.9 Hz, 1H), 7.29 (t, J = 73 Hz, 1H, partially
masked by other peaks), 7.74–7.85 (m, 2H), 8.10–8.30 (m, 2H), 9.88 (br s, 1H). LCꢀMS (ESI):
+
m/z = 541.2 [M + H] .
N-(1-{N'-Cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-phenyl-4,5-dihydro-1H-
pyrazol-4-yl)-N-ethyl-2-hydroxyacetamide (25) and N-[3-(4-Bromophenyl)-1-{N'-cyano-N-
[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-
hydroxyacetamide (26). To a stirred solution of 11j (3.8 g, 77 %ꢀUV, 5.5 mmol) in degassed
THF (150 mL) was added 1,3ꢀdimethylbarbituric acid (1.8 g, 11.0 mmol), followed by
tetrakis(triphenylphosphine)palladium(0) [Pd(PPh ) ; 0.25 g, 0.22 mmol]. The reaction mixture
3
4
was stirred under argon for 12 h, then cautiously quenched with saturated NaHCO solution (150
3
mL) and extracted into EtOAc (150 mL). The organic layer was washed with brine (100 mL),
then dried over Na SO , filtered and concentrated. The crude material was purified by dry flash
2
4
column chromatography (eluent: EtOAc; MeOH) to yield 4ꢀaminoꢀ3ꢀ(4ꢀbromophenyl)ꢀN'ꢀcyanoꢀ
Nꢀ[3ꢀ(difluoromethoxy)phenyl]ꢀ4,5ꢀdihydroꢀ1Hꢀpyrazoleꢀ1ꢀcarboximidamide (2.4 g, 95 %ꢀUV,
1
9
2%). H NMR (400 MHz, [D] DMSO): δ = 2.38 (br s, 2H), 4.02 (dd, J = 4.4, 11.5 Hz, 1H),
6
4.37 (dd, J = 9.9, 11.4 Hz, 1H), 4.81 (dd, J = 4.5, 9.9 Hz, 1H), 6.98 (dd, J = 2.0, 8.1 Hz, 1H),
.22 (t, J = 74.0 Hz, 1H), 7.20–7.23 (m, 1H), 7.27 (ddd, J = 0.8, 2.0, 8.1 Hz, 1H), 7.40 (t, J = 8.1
7
Hz, 1H), 7.64–7.70 (m, 2H), 7.98–8.03 (m, 2H), 9.62 (br s, 1H). LCꢀMS (ESI): m/z = 449.3
+
[
M + H] . To the amine product (2.30 g, 5.1 mmol) in MeOH (70 mL) at rt was added
acetaldehyde (0.27 g, 6.1 mmol). The reaction mixture was stirred at rt for 1 h, and at 40 °C for 1
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h, then it was cooled to 0 °C and NaBH (0.22 g, 5.7 mmol) was added in small portions. The
4
mixture was stirred at rt for 1 h, then poured over saturated NaHCO solution (10 mL). The
3
volatiles were removed by evaporation and the resulting aqueous slurry was extracted with
EtOAc (2 × 10 mL). The combined organic layers were washed with brine (100 mL), then dried
over Na SO , filtered and concentrated to yield a crude black oil. The crude material was
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purified by dry flash column chromatography (eluent: DCM; MeOH) to yield 3ꢀ(4ꢀ
bromophenyl)ꢀN'ꢀcyanoꢀNꢀ[3ꢀ(difluoromethoxy)phenyl]ꢀ4ꢀ(ethylamino)ꢀ4,5ꢀdihydroꢀ1Hꢀ
pyrazoleꢀ1ꢀcarboximidamide (1.11 g, 45%) as the main Nꢀethylation product. LCꢀMS (ESI): m/z
+
=
477.1 [M + H] . The corresponding debrominated derivative N'ꢀcyanoꢀNꢀ[3ꢀ
(difluoromethoxy)phenyl]ꢀ4ꢀ(ethylamino)ꢀ3ꢀphenylꢀ4,5ꢀdihydroꢀ1Hꢀpyrazoleꢀ1ꢀ
carboximidamide (0.41 g, 19%) was obtained as a byproduct. LCꢀMS (ESI): m/z = 398.3
+
[
(
M + H] . To the debrominated byproduct (410 mg, 1 mmol) in DCM (15 mL) were added Et N
3
0.15 mL, 1.1 mmol), acetoxyacetyl chloride (0.12 mL, 1.1 mmol) and DMAP (126 mg, 1 mmol)
and the mixture was stirred at rt for 1 h. EtOAc (15 mL) was added, and the mixture was washed
with saturated aqueous NH Cl solution (20 mL) and brine (20 mL). The organic phase was dried
4
over Na SO and concentrated. The crude product was purified by dry flash column
2
4
chromatography (eluent: DCM; MeOH) to yield 504 mg of an impure intermediate, which was
dissolved in MeOH (12 ml), K CO (140 mg, 1.01 mmol) was added, and the mixture was heated
2
3
to reflux for 30 min. After cooling, to the reaction mixture was added saturated aqueous NH Cl,
4
and the mixture was extracted with EtOAc. The organic phase was dried over Na SO ,
2
4
concentrated and the residue was purified by flash column chromatography (eluent: DCM,
MeOH) and subsequent preparative HPLC to yield Nꢀ(1ꢀ{N'ꢀcyanoꢀNꢀ[3ꢀ
difluoromethoxy)phenyl]carbamimidoyl}ꢀ3ꢀphenylꢀ4,5ꢀdihydroꢀ1Hꢀpyrazolꢀ4ꢀyl)ꢀNꢀethylꢀ2ꢀ
(
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hydroxyacetamide (25; 50 mg, 10%). H NMR (300 MHz, [D] DMSO): δ = 1.03 (br s, 3H),
6
3
.16–3.33 (m, 2H), 3.92–4.25 (m, 3H), 4.47 (br t, J = 11.7 Hz, 1H), 4.75 (br t, J = 5.5 Hz, 1H),
6
.94–7.03 (m, 1H), 7.19–7.30 (m, 3H), 7.36–7.52 (m, 4H), 7.75–7.84 (m, 2H), 9.77 (br s, 1H).
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+
LCꢀMS (ESI): m/z = 457.2 [M + H] .
To the main product from the Nꢀethylation step (1.1 g, 2.3 mmol) in DCM (30 mL) were added
Et N (0.34 mL, 2.4 mmol), acetoxyacetyl chloride (0.27 mL, 2.4 mmol) and DMAP (282 mg, 2.3
3
mmol) and the mixture was stirred at rt for 1 h. EtOAc (30 mL) was added, and the mixture was
washed with saturated aqueous NH Cl solution (40 mL) and brine (40 mL). The organic phase
4
was dried over Na SO and concentrated. The crude product was purified by dry flash column
2
4
chromatography (eluent: DCM; MeOH) to yield 1.2 g of an impure intermediate. 200 mg of this
intermediate were dissolved in MeOH (5 ml), K CO (48 mg, 0.35 mmol) was added, and the
2
3
mixture was heated to reflux for 30 min. After cooling, the reaction mixture was concentrated
and the residue was purified by HPLC to yield (Nꢀ[3ꢀ(4ꢀbromophenyl)ꢀ1ꢀ{N'ꢀcyanoꢀNꢀ[3ꢀ
(
difluoromethoxy)phenyl]carbamimidoyl}ꢀ4,5ꢀdihydroꢀ1Hꢀpyrazolꢀ4ꢀyl]ꢀNꢀethylꢀ2ꢀ
1
hydroxyacetamide (26; 50 mg, 24%). H NMR (400 MHz, [D] DMSO): δ = 1.05 (br s, 3H),
6
3.27–3.36 (m, 2H), 3.98–4.18 (m, 3H), 4.48 (t, J = 11.7 Hz, 1H), 4.72 (t, J = 4.9 Hz, 1H), 7.00
(
d, J = 7.8 Hz, 1H), 7.21 (s, 1H), 7.23 (t, J = 74.0 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 7.41 (t,
J = 8.2 Hz, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.3 Hz, 2H), 9.78 (br s, 1H). LCꢀMS (ESI):
+
m/z = 535.1 [M + H] .
N-[3-(4-Chloro-3,5-dimethylphenyl)-1-{N'-cyano-N-[3-(difluoromethoxy)phenyl]-
carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide
(27).
Compound 27 was prepared as described for 4, starting from 11k (4.9 g, 9.6 mmol). 27 was
1
obtained as grey solid (1.6 g, 32 % over 3 steps). H NMR (300 MHz, [D] DMSO): δ = 1.05 (br
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s, 3H), 2.32 (s, 6H), 3.23–3.44 (m, 2H), 3.94–4.15 (m, 3H), 4.44 (br t, J = 11.7 Hz, 1H), 4.69 (br
s, 1H), 6.92–7.02 (m, 1H), 7.14–7.27 (m, 2H), 7.33–7.43 (m, 1H), 7.56 (s, 2H), 9.73 (br s, 1H).
+
LCꢀMS (ESI): m/z = 518 [M + H] .
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N-[1-{N'-Cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-
dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide (4). To a stirred solution of 11l (14.2
g, 27.0 mmol) in degassed THF (370 mL) was added 1,3ꢀdimethylbarbituric acid (17.0 g, 108
mmol), followed by Pd(PPh ) (2.40 g, 2.16 mmol). The reaction mixture was stirred under
3
4
argon for 15 min, then cautiously quenched with saturated NaHCO solution (400 mL) and
3
extracted into EtOAc (400 mL). The organic layer was washed with brine (200 mL), then dried
over MgSO , filtered and concentrated. The crude material was purified by dry flash column
4
chromatography (eluent: EtOAc/heptane 1:1, 2:1; EtOAc; MeOH/EtOAc 1:100) to yield 4ꢀ
aminoꢀN'ꢀcyanoꢀ3ꢀ(3,4ꢀdichlorophenyl)ꢀNꢀ[3ꢀ(difluoromethoxy)phenyl]ꢀ4,5ꢀdihydroꢀ1Hꢀ
1
pyrazoleꢀ1ꢀcarboximidamide (9.3 g, 78%) as an orange oil. H NMR (400 MHz, [D] DMSO): δ
6
=
3.95–4.02 (m, 1H, under EtOAc signal), 4.35 (m, 1H), 4.83 (dd, J = 4.6, 9.6 Hz, 1H), 7.01 (dd,
J = 2.1, 8.0 Hz, 1H), 7.22 (t, J = 2.1 Hz, 1H), 7.24 (t, J = 73.7 Hz, 1H), 7.27 (m, 1H), 7.38 (t,
J = 7.8 Hz, 1H), 7.48–7.61 (m, 1H), 7.72 (d, J = 8.2 Hz, 1H), 8.00 (dd, J = 2.1, 8.5 Hz, 1H), 8.31
+
(
d, J = 1.8 Hz, 1H), 9.67 (br s, 1H). LCꢀMS (ESI): m/z = 439.1 [M + H] . To a stirred solution of
the amine product (9.30 g, 21.2 mmol) in MeOH (170 mL) at 0 °C was added acetaldehyde (1.12
g, 25.4 mmol), followed by the portionwise addition of NaBH (0.96 g, 25.4 mmol) over 20 min.
4
The mixture was stirred for 30 min, then poured over saturated NaHCO solution (100 mL). The
3
volatiles were removed by evaporation and the resulting aqueous slurry was extracted with
EtOAc (2 × 100 mL). The combined organic layers were washed with brine (100 mL), then dried
over MgSO , filtered and concentrated to yield a crude black oil. The crude material was purified
4
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