Structure-Guided Development of Covalent and Mutant-Selective Pyrazolopyrimidines to Target T790M Drug Resistance in Epidermal Growth Factor Receptor

Article abstract of DOI:10.1021/acs.jmedchem.7b00515

Reversible epidermal growth factor receptor (EGFR) inhibitors prompt a beneficial clinical response in non-small cell lung cancer patients who harbor activating mutations in EGFR. However, resistance mutations, particularly the gatekeeper mutation T790M, limit this efficacy. Here, we describe a structure-guided development of a series of covalent and mutant-selective EGFR inhibitors that effectively target the T790M mutant. The pyrazolopyrimidine-based core differs structurally from that of aminopyrimidine-based third-generation EGFR inhibitors and therefore constitutes a new set of inhibitors that target this mechanism of drug resistance. These inhibitors exhibited strong inhibitory effects toward EGFR kinase activity and excellent inhibition of cell growth in the drug-resistant cell line H1975, without significantly affecting EGFR wild-type cell lines. Additionally, we present the in vitro ADME/DMPK parameters for a subset of the inhibitors as well as in vivo pharmacokinetics in mice for a candidate with promising activity profile.

Full text of DOI:10.1021/acs.jmedchem.7b00515

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Article
Structure-Guided Development of Covalent and Mutant-
Selective Pyrazolopyrimidines to Target T790M Drug
Resistance in Epidermal Growth Factor Receptor
Julian Engel, Steven Smith, Jonas Lategahn, Hannah L. Tumbrink, Lisa Goebel, Christian
Becker, Elisabeth Hennes, Marina Keul, Anke Unger, Heiko Müller, Matthias Baumann,
Carsten Schultz-Fademrecht, Georgia Günther, Jan G. Hengstler, and Daniel Rauh
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00515 • Publication Date (Web): 30 Aug 2017
Downloaded from http://pubs.acs.org on August 30, 2017
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Structure-Guided Development of Covalent and
Mutant-Selective Pyrazolopyrimidines to
Target T790M Drug Resistance in Epidermal
Growth Factor Receptor
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1,‡
1,‡
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Julian Engel , Steven Smith , Jonas Lategahn , Hannah L. Tumbrink , Lisa Goebel ,
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Christian Becker , Elisabeth Hennes , Marina Keul , Anke Unger , Heiko Müller ,
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Matthias Baumann , Carsten SchultzꢀFademrecht , Georgia Günther , Jan G. Hengstler ,
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Daniel Rauh *
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Faculty of Chemistry and Chemical Biology, TU Dortmund University, OttoꢀHahnꢀStraße
4a, Dꢀ44227 Dortmund, Germany
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Lead Discovery Center GmbH, OttoꢀHahnꢀStraße 15, Dꢀ44227 Dortmund, Germany
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Leibniz Research Centre for Working Environment and Human Factors (IfADo), TU
Dortmund University, Dꢀ44139 Dortmund, Germany
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ABSTRACT
Reversible Epidermal Growth Factor Receptor (EGFR) inhibitors prompt a beneficial
clinical response in nonꢀsmall cell lung cancer patients who harbor activating mutations in
EGFR. However, resistance mutations, particularly the gatekeeper mutation T790M, limit
this efficacy. Here, we describe a structureꢀguided development of a series of covalent and
mutantꢀselective EGFR inhibitors that effectively target the T790M mutant. The
pyrazolopyrimidineꢀbased core differs structurally from that of aminopyrimidine based thirdꢀ
generation EGFR inhibitors and therefore constitutes a new set of inhibitors that target this
mechanism of drug resistance. These inhibitors exhibited strong inhibitory effects toward
EGFR kinase activity and excellent inhibition of cell growth in the drugꢀresistant cell line
H1975, without significantly affecting EGFR wildꢀtype cell lines. Additionally, we present
the in vitro ADME/DMPK parameters for a subset of the inhibitors as well as in vivo
pharmacokinetics in mice for a candidate with promising activity profile.
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INTRODUCTION
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Firstꢀgeneration Epidermal Growth Factor Receptor (EGFR) inhibitors gefitinib and
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erlotinib provided clinical benefit in patients harboring somatic activating mutations in the
gene encoding EGFR such as the L858R point mutation and an exon 19 deletion that drive
oncogenesis in nonꢀsmall cell lung cancer (NSCLC) (Figure 1). Treatment with tyrosine
kinase inhibitors (TKIs) achieved impressive objective response rates of 50ꢀ80 % and a
doubled median progressionꢀfree survival, therefore representing a prime example of targeted
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ꢀ7
cancer therapy that opened up a new era in cancer treatment. However, patients acquired
resistances and suffered a relapse within 18 months of treatment. Biopsies of resistant tumors
revealed a single point mutation of the gatekeeper residue (T790M) as the major resistance
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ꢀ10
mechanism.
Replacement of the threonine with methionine leads to steric repulsion of the
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ꢀaminoquinazolineꢀbased firstꢀgeneration EGFR inhibitors and results in a slightly different
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binding mode that accounts for a dramatic loss of inhibitory activity.
In addition to the
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steric repulsion, the change in ATP affinity is well documented.
While the L858R point
mutation leads to a distinct loss of ATP affinity to EGFR, the T790M mutation restores the
ATP affinity to almost the same level as in wildꢀtype EGFR, rendering ATPꢀcompetitive
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4, 17
inhibitors less effective in the presence of cellular ATP concentrations.
Secondꢀ
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generation EGFR inhibitors that also contain a 4ꢀaminoquinazoline core (afatinib and
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dacomitinib ) were developed and thought to overcome T790M drug resistance, as they
incorporate a Michael acceptor that covalently modified a unique cysteine (Cys797) at the lip
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of the EGFR ATPꢀbinding site (Figure 1).
These covalent inhibitors, characterized by
marginal offꢀrates as well as maximal drug–target residence time, were expected to inhibit
T790M drugꢀresistant EGFR efficiently, but at clinically achievable concentrations they
displayed insufficient efficacy in patients due to severe side effects. The clinical data revealed
that 4ꢀaminoquinazoline inhibitors are inappropriate to target the gatekeeper mutant variant
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of EGFR, as they likewise inhibit wildꢀtype EGFR, accounting for the observed side effects
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and represent a doseꢀlimiting factor.
Hence, a thirdꢀgeneration of EGFR inhibitors
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(
COꢀ1686 (rociletinib ) and AZD9291 (osimertinib ), structurally based on an
aminopyrimidine, were developed and seemed to avoid a steric clash with the mutant
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methionine gatekeeper (Figure 1). Both covalent inhibitors showed excellent in vivo potency
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as well as reduced onꢀtarget toxicity and could progress into human clinical trials.
Rociletinib has recently been updated to an objective response rate of 45 % to T790Mꢀ
positive patients. Furthermore, elevated levels of glucose in the blood were observed which
could be explained by a noncovalent metabolite that inhibits the insulinꢀlike growth factor 1
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and the insulin receptor and, thus, its clinical development was stopped.
In fact, only one
thirdꢀgeneration covalent inhibitor has yet achieved FDA approval. Osimertinib showed
excellent results in clinical studies as well as reduced onꢀtarget toxicity and was approved by
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the FDA as Tagrisso in 2015.
Here, we present the expanded structureꢀguided development and the biochemical as well
as cellular evaluation of a pyrazolopyrimidineꢀbased series of covalent inhibitors that target
the drugꢀresistant EGFR mutant T790M in a highly mutantꢀselective manner. In a previous
publication, we presented complex crystal structures of mutant EGFR, which revealed the
binding mode of this class of pyrazolopyrimidines and gave a first explanation for the
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potency and selectivity toward the gatekeeper mutated variant. Based on these preliminary
results, we now further investigated the structureꢀactivity relationship (SAR) and examined
the in vitro ADME/DMPK parameters for a representative set of the pyrazolopyrimidines as
well as in vivo pharmacokinetics for one candidate that displayed the most promising overall
profile.
RESULTS
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Structure-Guided Development of Pyrazolopyrimidine-Based Inhibitors
Prompted by the failure of secondꢀgeneration EGFR inhibitors in clinical studies and
further stimulated by the significant impact of the first covalent T790M inhibitors beyond 4ꢀ
aminoquinazolines osimertinib and rociletinib, we sought to identify a new set of covalent
inhibitors that specifically target the gatekeeper mutant. Considering the knowledge we
gained from firstꢀ and secondꢀgeneration inhibitors as well as from our previous work, our
structureꢀguided design was focused on the following three main criteria, which we expected
to be crucial for the development of an effective inhibitor that targets the drugꢀresistant
gatekeeper mutant form of EGFR: (i) a distinct scaffold to specifically target the Metꢀ
gatekeeper mutant avoiding steric interference; (ii) mutantꢀselective inhibition, while sparing
the wildꢀtype form as an essential requirement to reduce onꢀtarget toxicity that is associated
with severe side effects; and (iii) an electrophile optimized with respect to geometry and
orientation for the efficient alkylation of Cys797 as a critical element to overcome T790M
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drug resistance.
We focused our design efforts on the wellꢀstudied 4ꢀamino
pyrazolopyrimidine as a core element as this moiety supplies an appropriate hingeꢀbinding
element and allows for the rapid and straightforward synthesis of a focused collection of
small molecules for exploring the efficiency of potent inhibition of the kinase activity of
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EGFRꢀT790M, the selectivity profiles, and covalent binding characteristics.
We
identified N1 of the pyrazolopyrimidine as a suitable position for the attachment of a Michael
acceptor linked to a 3ꢀpiperidine as the electrophilic system that has been shown to be
appropriate for alkylation of an analogous cysteine to EGFR (Cys481 in Bruton’s tyrosine
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kinase, BTK) (Figure 2).
Furthermore, we have learned from previous studies on EGFR
inhibitors that a restricted rotational flexibility of the electrophile’s linker, which enables a
preferred orientation toward Cys797, has a major effect on the kinetics of the covalent bond
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formation that may drive the inhibitory potency. As 4ꢀamino pyrazolopyrimidines, with a
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PP1 derived substitution pattern, provide this potential, we focused our design on the SARs
of various substitutions at the 3ꢀposition to investigate in what way a change in size,
orientation, and electronic properties will affect the activity and selectivity on the different
EGFR mutant variants (Figure 2 and 3). We initially designed derivatives that contained
rather simple moieties at 3ꢀposition that differed in their electronic properties (e.g.,
containing a halogen, ethyne, or cyclopropyl). To further investigate the tolerable size of Cꢀ3
substitutions, we included benzyl and phenyl moieties with different types of halogen
substitutions in the meta and para positions. Ultimately, we designed derivatives with
sterically advanced bicyclic moieties at Cꢀ3 to exploit the maximum tolerable size of
lipophilic extensions into the pocket adjacent to the gatekeeper residue Met790.
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Synthesis of a Focused Library of Covalent Pyrazolopyrimidines
We synthesized a small, focused library of covalent pyrazolopyrimidines in which we paid
particular attention to the derivatization of the 3ꢀposition of the pyrazolopyrimidine core to
elucidate on the maximal tolerable size for the pocket near the methionine gatekeeper
(
Met790) and the potential for beneficial interactions while retaining the crucial direct hinge
contact. Further efforts were directed to exploring covalent bonding to Cys797 by altering the
type of Michael acceptors and to producing reversible counterparts for comparative
evaluation. We employed a general synthetic route for synthesizing pyrazolopyrimidines 1dꢀz
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(
Scheme 1). Iodination of the commercially available 1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀ
amine (2) at the 3ꢀposition resulted in the formation of compound 3. Subsequent Mitsunobu
coupling was performed with NꢀBocꢀprotected (S)ꢀ3ꢀhydroxypiperidine to generate chiral
compound 4, which served as a generic intermediate for rapid library generation. Suzuki
coupling with various aryl, benzyl, and heteroaryl boronic acids or esters as well as
Sonogashira coupling with cyclopentaneꢀequipped alkynes led to intermediate 5. Subsequent
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Boc deprotection with TFA resulted in secondary amine 6, which was decorated with a
Michael acceptor using acryloyl chloride, resulting in structures 1d, 1g, 1i, 1k, 1n, and 1p-t,
1v, and 1y-z, or with 4ꢀ(dimethylamino)ꢀ2ꢀbutenoic acid to generate amides 1f and 1m via
standard amide coupling. Reversible counterparts (1e, 1h, 1j, 1l, and 1o) were synthesized
with propionyl chloride. Compounds 1a-c were synthesized through a slightly altered
synthetic approach presented in the Supporting Information (Schemes S1 ꢀ 3). The entire
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chemical data of compounds 1u, 1x and 1w have been previously described.
Covalent Pyrazolopyrimidines Effectively Inhibit the Kinase Activity of the Drug-
Resistant EGFR Mutant
We used an activityꢀbased assay to evaluate the inhibitory activity of pyrazolopyrimidines
1aꢀz on wildꢀtype, activating (L858R), and drugꢀresistant (L858R/T790M) EGFR (Table 1).
Notably, structure 1a inhibited the wildꢀtype, L858R, and L858R/T790M variants of EGFR
(IC = 6.9 µM, 2.5 µM, and 1.6 µM, respectively) and shows a reasonable selectivity toward
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the drugꢀresistant mutant of EGFR (ratio WT:T790M = 4.3). Since compound 1a possesses a
hydrogen atom at Cꢀ3 that is not suitable for beneficial interaction with Met790, the observed
activity is directly linked to the inhibitory effect of the acrylamide piperidineꢀdecorated
4ꢀamino pyrazolopyrimidine core, rendering it potentially useful for covalently targeting
EGFRꢀT790M. The introduction of small lipophilic moieties showed an interesting effect on
all variants of EGFR. While an ethyne at the 3ꢀposition (compound 1c) led to an increased
activity on all EGFR variants, but a loss of selectivity (ratio WT:T790M = 2) the introduction
of iodine (compound 1b) resulted in an even lower kinase activity, but a very good selectivity
toward the double mutant of EGFR (EGFRꢀWT IC50 = 0.3 µM, EGFRꢀL858R/T790M
IC = 0.02 µM and ratio WT:T790M = 14, respectively). For structure 1d, which is equipped
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compared to 1b, indicating an insufficient interaction within the lipophilic subpocket that is
in line with the Xꢀray based modeling studies (Figure 4A). Phenyl derivatives with an
additional fluorine or trifluoromethyl group in the meta or para position (1g, 1i, 1l, and 1n,
respectively) or the introduction of the bioisosteric cyclopropylꢀmoiety (1p) tended to follow
the same inhibitory profile. Notably, introduction of a 4ꢀphenol moiety increased the
inhibition of L858R (IC50 = 0.003 µM) and L858R/T790M (IC50 = 0.04 µM), but led to a
complete loss of T790Mꢀselectivity (ratio WT:T790M = 0.5). The phenol moiety seemed to
have a major influence on the activity, most probably due to a contact with an adjacent
glutamate residue (Glu762) of the helix C (Figure 4B).
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To further investigate the potential benefits of the penetration in the subpocket near the
gatekeeper residue, we attached ethynylꢀ and propꢀ1ꢀynyl bridged cyclopentane moieties (1s
and 1t, respectively) to restrict the substituents in an orientation facing the gatekeeper
residue. In contrast to compound 1c (terminal alkyne), compounds 1s (IC50 = 1.4 µM) and
1t (IC = 0.9 µM) displayed less inhibitory activity against L858R/T790M and more
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strongly inhibited activating mutant L858R (IC = 0.06 µM and 0.1 µM, respectively),
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sustainably indicating that the extended substitutions underwent steric repulsion with
Met790. Notably, the benzylꢀequipped compound 1r demonstrated potent kinase inhibition of
the relevant mutant variants of EGFR displaying a 24ꢀfold increased inhibition against
EGFRꢀL8585/T790M compared to the phenylꢀderivative (1d). The more flexible benzyl
group probably adopts an orientation toward Met790 and particularly Lys745 that allows a
more beneficial interaction (Figure 4C). Triggered by this observation, we sought to increase
the size of the substituents and attached bicyclic aromatic moieties (compounds 1u-z).
Therefore, we incorporated 1ꢀnaphthyl (compound 1x) and 2ꢀnaphthyl (compound 1u)
moieties at the 3ꢀposition of the pyrazolopyrimidine scaffold. These alterations increased the
inhibition of the activating mutation (3ꢀfold increase on L858R compared to 1r) and
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particularly of the drugꢀresistant (L858R/T790M; 1x IC50 = 0.002 µM; 1u IC50 < 0.001 µM)
mutants of EGFR, leading to a superior selectivity over EGFRꢀWT (ratio WT:T790M;
1x = 30; 1u > 20). Conversion of naphthyl into an azaindole (compound 1v) or a
methylindole (compound 1w) yielded comparable trends with IC values in the singleꢀdigit
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nanomolar range. As these inhibitors showed preferential inhibition of the gatekeeper
mutated form of EGFR, the highly lipophilic and sterically demanding moieties seemed to
induce crucial interactions, while avoiding adverse interference with the gatekeeper residue
(Figure S1). In addition to their high potency, the bicyclic aromatic substitutions reduced the
inhibition of wildꢀtype EGFR, with up to 30ꢀfold mutant selectivity for the T790M mutant.
As an exception, the 7ꢀazaindole containing compound 1v displayed 10ꢀfold reduced mutant
selectivity, which is possibly caused by an additional interaction with the threonine
gatekeeper residue, leading to increased inhibition of wildꢀtype.
While the selectivity for the T790M mutant for 1y and 1z remains virtually the same
(around 3.5) a tenꢀfold decrease in activity for all EGFR variants could be observed by the
introduction of the bulky dimethylamino group at the 4ꢀposition of the naphthyl compared to
the fluorine group. According to the model of 1y in EGFRꢀT790M, the decrease in activity
stems supposedly from steric properties (Figure 4D). The introduction of a more sterically
demanding Michael acceptor (dimethylamino butenamide) led to compounds 1f and 1m and
resulted in a roughly 30ꢀfold less activity against the EGFR variant L858R/T790M
(compared to acrylamideꢀcontaining counterparts 1d and 1k, respectively, suggesting that
acrylamide linked to a piperidine is the more favored electrophile for these specific
pyrazolopyrimidines. The observed difference in activity between these two Michael
acceptors again illustrates the relevance of the development and optimization of an
electrophilic system (Michel acceptor and linker) for each covalent inhibitor scaffold.
Remarkably, the reversible counterparts of our pyrazolopyrimidine inhibitors (compounds 1e,
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1h, 1j, 1l, and 1o) did not significantly inhibit mutant L858R/T790M. Mass spectrometry of
EGFRꢀT790M treated with compounds 1u, 1x or 1w confirmed covalent modification
consistent with a complete labeling of the targeted protein. NanoꢀLCꢀMS/MS analysis
35
precisely verified a single alkylation of Cys797.
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Mutant-Selective Inhibitory Effects on Drug-Resistant NSCLC Cell Line H1975
We evaluated the inhibitory efficacy of pyrazolopyrimidineꢀbased compounds 1a-z on a
cell line with an EGFR activating mutation (HCC827, EGFRꢀdelE746_A750) and on the
drugꢀresistant NSCLC cell line H1975, which harbors EGFRꢀL858R/T790M. To investigate
mutantꢀselective characteristics, we also tested the compounds’ effect on different cancer cell
lines harboring wildꢀtype EGFR (A431, Table 2). Additionally, we tested on KRASꢀmutated
cell lines (A549, and H358; Table 2, and Table S1, respectively) to exclude the possibility of
offꢀtarget toxicity. Cells were treated with compounds 1a-z (0ꢀ30 µM) for 96 h. Gefitinib,
afatinib, Nꢀ(3ꢀ((5ꢀChloroꢀ2ꢀ((2ꢀmethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)pyrimidinꢀ
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ꢀyl)oxy)phenyl)acrylamide(7, WZ4002 ), osimertinib, rociletinib, and ibrutinib served as
references. Compounds 1u-x demonstrated excellent inhibitory effects on HCC827
EC50 = <0.01ꢀ0.50 µM) and on H1975 cells (EC50 = 0.1ꢀ0.5 µM) without significantly
(
impacting EGFR wildꢀtype cell lines (A431, EC50 = 1.4ꢀ29 µM; A549, EC50 = 3.5ꢀ23 µM;
H358, EC = 2.3ꢀ20 µM or no inhibition). Remarkably, the 2ꢀnaphthylꢀcontaining compound
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1
u was the most active inhibitor (EC = 0.1 µM against H1975 cells), while very high
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selectivity for the drugꢀresistant over the WT cell line was achieved with compounds 1w and
x (30ꢀfold and 60ꢀfold, respectively). Compound 1v evoked an inhibitory effect in EGFR
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wildꢀtype cells (EC50 = 1.4, 3.5, and 2.3 µM in A431, A549, and H358, respectively),
consistent with its strong in vitro inhibition of wildꢀtype EGFR. Compound 1y, which
contains a dimethylamine at the 4 position of the naphthyl moiety, exhibited less activity in
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H1975 cells (EC50 = 2.7 µM), in line with our activityꢀbased data. Also consistent with our
activityꢀbased assay, compounds with lessꢀlipophilic extensions at the pyrazolopyrimidine Cꢀ
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(compounds 1c, 1d, 1g, 1i, 1k, and 1p) had less effect on H1975 cells (EC50 = 1.8ꢀ16 µM),
while the unsubstituted derivative 1a did not display inhibitory activity. Notably, compounds
1q (4ꢀphenol substitution at Cꢀ3) and 1b (iodine substitution at Cꢀ3) strongly inhibited the
T790M mutant in the biochemical setting (IC50 = 0.04 µM and 0.02 µM, respectively,
Table 1), but were less potent against H1975 cells (EC50 = 1.84 µM and 3.9 µM,
respectively). In accordance with our activityꢀbased data, reversible inhibitors 1e, 1h, 1j, 1l,
and 1o did not affect any of the tested cancer cell lines, in contrast to their covalent
counterparts (compounds 1d, 1g, 1i, 1k, and 1n), highlighting how crucial a maximized drugꢀ
target residence time (characteristics of covalent inhibition) is to target T790M drug
resistance. Further, the dimethylamino butenamide Michael acceptor equipped inhibitors 1f
and 1m demonstrated significantly less inhibitory potency against H1975 cells
(EC = 30 µM for compound 1f and EC = 21 µM for compound 1m) than their respective
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acrylamide analogues (compound 1d, EC = 3.7 µM; compound 1k, EC = 4.2 µM), also
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consistent with our activityꢀbased assay data.
ADME/DMPK parameters of a subset of pyrazolopyrimidines
For a representative subset of pyrazolopyrimidines, the physicochemical and in vitro
ADME parameters such as kinetic solubility and permeability as well as plasma and
metabolic stability were examined (Table 3). Despite the increased lipophilicity of the most
active derivatives reflected by their higher clogP and clogD7.4 values, the compounds
displayed high aqueous kinetic solubility (SolRank) with values of 248ꢀ461 µM in buffer at
pH 7.4 with the exception of 1x (kinetic solubility concentration = 38 µM) that contains a 1ꢀ
naphthyl moiety.
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As the lipophilicity of the most promising pyrazolopyrimidines was significantly raised in
the course of the optimization cycles, the determination of the ligandꢀlipophilicity efficiency
(LLE; defined as pIC50 – clogP/D and describing how efficient a ligand exploits its
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lipophilicity) became of interest to substantiate a specific target interaction. The most
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promising compounds being effective on H1975 drugꢀresistant cell lines (1u, 1v, 1w, and 1x)
displayed LLE values regarding H1975 between 3.2 ꢀ 5.2 that are in an acceptable range
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(Table 3, and Figure S2).
Moreover, all compounds tested showed high penetration in the artificial membrane
permeation assay (PAMPA) (59ꢀ82 % flux), except for the more polar azaindole derivative
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(1v) (27 % flux). 1u and 1w were further measured in the Cacoꢀ2 cell permeability assay as
a cellular model to examine intestinal drug absorption. Both compounds demonstrated a high
cellular apical (A) to basolateral (B) permeability with low efflux ratios of 0.3 (1u) and 1.0
(1w) predicting good oral absorption.
All tested covalent pyrazolopyrimidines seemed to be extremely stable in both mouse and
human plasma (> 86 %). With respect to plasma protein binding, the 2ꢀnaphthyl containing
derivative was the only compound that showed a somewhat lower free fraction
(fu100% = 0.02) compared to the other derivatives of the series that revealed satisfactory fu100%
values ranging from 0.09 to 0.37 which are significantly higher than the fraction unbound
levels determined for osimertinib, rociletinib and ibrutinib (Table 3). As expected, the
intrinsic clearance (Cl ) of the compounds was very high in both mouse and human liver
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microsomes, thus being similarly unstable as the structureꢀrelated clinical reference ibrutinib.
Nevertheless, the overall profile encouraged us to investigate the pharmacokinetics of this
compound class in vivo.
We selected 1w for a first in vivo experiment and administered the compound by the
intravenous (IV), intraperitoneal (IP), and oral (PO) route to CDꢀ1 mice (Table 4) at doses of
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2 mg/kg (IV), 20 mg/kg (IP), and 20 mg/kg (PO). We observed no adverse effects indicating
that the compound was generally well tolerated. In line with the in vitro ADME data, we
observed relatively low exposure (AUC) combined with a high volume of distribution and a
high clearance of the compound after intravenous administration. This resulted in a short
halfꢀlife of only 0.6 hours. For the intraperitoneal route, however, we measured a
disproportionately high exposure of 1888 h·ng/mL indicating a good IP bioavailability. The
highest concentration measured in plasma (Cmax, total) of 4539 ng/mL (= 11.32 µM) represents
a nearly 50ꢀfold cover of the cellular EC50 of 0.23 µM obtained in H1975 cells while the
highest free plasma concentration was still nearly sixꢀfold above this value (Cmax,
free = 1.36 µM). Thus, despite the relatively short halfꢀlife of 0.2 hours, the total plasma
concentration was above the H1975 EC50 for approximately 75 min, and the free plasma
concentration was above the EC50 for about 30 min which could be sufficient to see efficacy
in a proofꢀofꢀconcept study in vivo (Figure S3). In contrast, administration by oral gavage
resulted in a very low exposure of 28.6 h·ng/mL indicative of a poor oral bioavailability,
despite the promising permeability values obtained in the Cacoꢀ2 assay. It remains to be
determined, whether this could be a consequence of metabolism during intestinal absorption
or whether the Cacoꢀ2 assay did not correctly reflect the in vivo situation. Nevertheless, the
intraperitoneal route seems to be the most appropriate route of administration to further
profile 1w in preclinical in vivo studies.
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DISCUSSION
Patients that suffer from advanced NSCLC are treated with cytotoxic chemotherapy, which
dramatically decreases the patient healthꢀrelated quality of life and fails to exert sustainable
therapeutic effects. For this reason, selective drugs must be developed, for example, TKIs
may achieve impressive effects in NSCLC patients that harbor specific activating mutations
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in EGFR. Although therapy is beneficial in these patients, treatment is limited due to the
acquisition of drugꢀresistance mutations. Thirdꢀgeneration EGFR inhibitors irreversibly target
the oncogenic and drugꢀresistant T790M variant by alkylating a rare cysteine (Cys797).
Rociletinib and osimertinib were the most promising clinical candidates for the treatment of
advanced NSCLC, of which only osimertinib (Tagrisso) has been FDA approved, whereas
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the clinical development of rociletinib has recently been stopped by Clovis Oncology. The
latest progress and the fact that there are only very few approved drugs for this indication to
date shows the persistent interest for the development of new T790Mꢀtargeting drugs.
Here, we reported the structureꢀguided development of a series of covalent
pyrazolopyrimidines that selectively target T790M drugꢀresistant EGFR without affecting the
wildꢀtype protein. Our design relied on the classical hingeꢀbinding motif 4ꢀamino
pyrazolopyrimidine as the starting point, to which we introduced a 3ꢀpiperidine linker for
attaching a Michael acceptor for covalent modification of Cys797. Based on modeling
studies, we focused our chemical efforts on derivatization of Cꢀ3 of the pyrazolopyrimidine
core, which we considered as the hot spot for beneficial interactions with the methionine
gatekeeper. Recently, we studied the interactions of substituted indazoles with the back
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pocket of the active site. To investigate the alkylation reaction, we varied the electrophilic
system. A focused smallꢀmolecule library contained a series of EGFR inhibitors that
displayed excellent (subnanomolar) inhibitory effects against the drugꢀresistant variant
L858R/T790M and reduced inhibition of wildꢀtype EGFR in activityꢀbased studies. The most
promising compounds 1u, 1w, 1x, and 1z, equipped with aromatic bicyclic moieties
demonstrated a mutantꢀselective profile during biochemical characterization, with excellent
inhibitory effects on H1975 cells (EC50 up to 0.1 µM) but not on cancer cells harboring wildꢀ
type EGFR.
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In our collection of compounds, the Cꢀ3 substitution of the pyrazolopyrimidine was the hot
spot for increased inhibitory potency and selectivity toward the T790M mutant. Of note,
bicyclic moieties with a strong lipophilic character were highly active against H1975 cells, as
these motifs avoid steric interference, but rather directly interact with the Met790 gatekeeper
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residue as well as the alkyl side chain of Lys745. In contrast, compounds with less
lipophilic substituents at 3ꢀposition were less effective as inhibitors due to the weakening of
favorable interactions. Variation of the Michael acceptor demonstrated that an acrylamide is
the preferred electrophile for these types of pyrazolopyrimidines. Reversible counterparts that
cannot undergo Michael addition to form a covalent bond with Cys797 were inactive in both
biochemical and cellular studies, substantiating the need of covalent inhibition of drugꢀ
resistant EGFR. It is crucial to differentiate between the two distinct binding events that
occur with covalent ligands. The initial binding of the inhibitor is almost completely based on
the intrinsic affinity of the inhibitor to the target and reversible. After the reversible binding,
the electrophilic warhead can form a covalent bond with the target kinase if it is properly
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positioned to a suitable nucleophile. For this reason it is important to differentiate these two
binding steps in a series of kinetic measurements, which has been done for a selected set of
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the herein presented compounds in Engel and Becker et al. The structureꢀactivity
relationship of the entire series of pyrazolopyrimidines observed in the activityꢀbased assay
translated nicely into a cellular setting for mutant as well as wildꢀtype EGFR.
The in vitro ADME profile of 1w represented the most promising overall profile,
displaying a high aqueous kinetic solubility as well as an excellent cellular permeability with
a low efflux ratio. The high stability in both mouse and human plasma together with a
satisfactory free fraction in mouse plasma stimulated an in vivo pharmacokinetic study in
mice that revealed a high exposure by the intraperitoneal route. The total plasma
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concentration was above the H1975 EC50 for approximately 75 min that could be sufficient to
see efficacy in a proofꢀofꢀconcept study in vivo.
In summary, we identified a novel class of covalent and mutantꢀselective thirdꢀgeneration
EGFR inhibitors that exhibited strong effects on the drugꢀresistant H1975 cell line while
displaying marginal inhibitory activity on EGFR wildꢀtype cell lines, a characteristic that
may ultimately overcome issues of onꢀtarget toxicity. The first in vivo experiment of 1w
revealed good exposure by the intraperitoneal route that seems to be the most appropriate
route of administration to further profile 1w in in vivo studies. The discovery of this class of
inhibitors may stimulate further development of therapeutics to overcome drug resistance in
NSCLC.
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Experimental Section
Activity-Based Assay for IC50 Determination
IC50 determinations for EGFR and its mutants (Carna Biosciences, lot13CBSꢀ0005K for
EGFRꢀWT; Invitrogen, lot279551C for EGFRꢀL858R and Invitrogen, lot350247C for EGFRꢀ
L858R/T790M) were performed with the HTRF KinEASEꢀTK assay from Cisbio according
to the manufacturer’s instructions. Briefly, the amount of EGFR in each reaction well was set
to 0.60 ng EGFRꢀWT (0.67 nM), 0.10 ng EGFRꢀL858R (0.11 nM) or 0.07 ng EGFRꢀ
T790M/L858R (0.08 nM). An artificial substrate peptide (TKꢀsubstrate from Cisbio) was
phosphorylated by EGFR. After completion of the reaction (reaction times: 25 min for WT,
15 min for L858R and 20 min for T790M/L858R), the reaction was stopped by addition of
buffer containing EDTA as well as, an antiꢀphosphotyrosine antibody labeled with europium
cryptate and streptavidin labeled with the fluorophore XL665. FRET between europium
cryptate and XL665 was measured after an additional hour of incubation to quantify the
phosphorylation of the substrate peptide. ATP concentrations were set at their respective Kmꢀ
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values (9.5 µM for EGFRꢀWT, 25 µM for EGFRꢀL858R, and 20 µM for EGFRꢀ
L858R/T790M) in conjunction with substrate concentrations of 1 µM, 225 nM, and 275 nM,
respectively. Kinase and inhibitor were preincubated for 30 min before the reaction was
started by addition of ATP and substrate peptide. An EnVision multimode plate reader
(Perkin Elmer) was used to measure the fluorescence of the samples at 620 nm (Euꢀlabeled
antibody) and 665 nm (XL665 labeled streptavidin) 50 µs after excitation at 320 nm. The
quotient of both intensities for reactions made with eight different inhibitor concentrations
was then analyzed using the Quattro Software Suite for IC50ꢀdetermination. Each reaction
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was performed in duplicate, and at least three independent determinations of each IC were
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Cell Culture
A431, HCC827, H1975, A549, and H358 cells were obtained from the American Type
Culture Collection (ATCC). A431 cells were cultured in DMEM high glucose media (Life
Technologies, Germany), and HCC827, H1975, A549, as well as H358 cells were cultured in
RPMI media (Life Technologies, Germany), both containing
Lꢀglutamine and supplemented
with 10% FBS (PANꢀBiotech, Germany) and 1% PenStrep (Life Technologies, Germany) in
a humidified incubator at 37 °C and 5% CO
October 2014 by STR profiling of 16 alleles.
Viability Assay
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. Cell line authentication was performed last in
Cells were seeded on day 1 at cell numbers that ensure linearity and optimal signal
intensity (100−300 cells/well, 25 ꢁL). After culturing for 24 h in serumꢀ and antibioticsꢀ
containing media in humidified chambers at 37 °C/5% CO , the cells were incubated for 96 h
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with EGFR inhibitors in serial dilutions (14 nM to 30 µM) with DMSO and staurosporine as
control. Compound dilutions were generated using the acoustic dispensing system “ECHO
520 Liquid Handler” from Labcyte (Sunnyvale, CA) and the according dose−response
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software “Echo DoseꢀResponse v1.5.4”. Viability studies were carried out on day 5 using
CellTiterGlo Assay (Promega, U.S.) which is a homogeneous method of determining the
number of viable cells in culture. It is based on quantification of ATP, indicating the presence
of metabolically active cells. For these studies, CellTiterGlo Reagent was prepared according
to the instructions of the kit and diluted 1:1 with the complete growth medium suitable for the
corresponding cell line. Then, reagent and assay plates were equilibrated at room temperature
for 20 min. Equal volumes of the reagent were added to the volume of culture medium
present in each well (25 µL). The plates were shaken for 2 min on an orbital shaker to induce
cell lysis. The microplates were then incubated at room temperature for 20 min for
stabilization of the luminescent signal. Following incubation, the luminescence was recorded
on an EnVision microplate reader (PerkinElmer) using 500 ms integration time. The data
were then analyzed using the Quattro Software Suite for EC50ꢀdetermination. As quality
control, the Z′ꢀfactor was calculated from 16 positive and negative control values. Only assay
results showing a Z′ꢀfactor ≥ 0.5 were used for further analysis. All experimental points were
measured in duplicates for each plate and were replicated in at least two plates.
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For determining kinetic solubility, the compound was diluted from a 10 mM stock in
DMSO to a final concentration of 500 ꢁM in 50 mM Hepes buffer, pH 7.4. Following an
incubation of 90 min at room temperature on a shaker, the aqueous dilution was filtrated
through a 0.2 ꢁm PVDF filter, and the optical density between 250 and 500 nm was measured
at intervals of 10 nm. The kinetic solubility was calculated from the area under the curve
(AUC) between 250 and 500 nm and normalized to absorption of a dilution of the compound
in acetonitrile.
Absorption was assessed using PAMPA (Parallel artificial membrane permeability assay)
and Cacoꢀ2 cell culture. For PAMPA, the compound was diluted from a 10 mM stock in
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DMSO to a final concentration of 500 ꢁM in 50 mM Hepes buffer pH 7.4 and transferred
onto a transwell membrane covered with a membraneꢀforming solution of 10 % 1.2ꢀDioleylꢀ
snꢀglycerꢀ3ꢀphosphocholine (Sigma Aldrich) and 0.5 % (w/v) cholesterol (Sigma Aldrich) in
dodecane. Following an incubation of 20 h at room temperature in a wet chamber, the optical
density of the solution in the receiver well was measured between 250 and 500 nm in
intervals of 10 nm. The percent flux was calculated from the AUC between 250 and 500 nm
and normalized to the absorption of the compound following a 20 h incubation in a parallel
transwell containing a membrane covered with 50 % methanol in 50 mM Hepes buffer
pH 7.4.
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For Cacoꢀ2 cell culture, a 10 mM DMSO stock of the compound was diluted to a final
concentration of 5 ꢁM in HBSS buffer pH 7.4 and incubated for 2 h at 37 °C and 5 % CO
a monolayer of Cacoꢀ2 cells (ATCC) that had been grown on a transwell membrane
Millipore, Schwalbach, Germany) for 21 d. The compound concentration was measured in
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(
the receiver as well as the donor well. Apparent permeability (Papp) from either the apical to
basolateral direction or vice versa was calculated by the equation: Papp = 1/AC0 (dQ/dt),
where A is the membrane surface area, C0 is the donor drug concentration at t = 0, and dQ/dt
is the amount of drug transported within the given time period of 2 h.
Plasma stability was measured by LCꢀMSꢀbased determination of % remaining of selected
compounds at a concentration of 5 µM after incubation in 100 % plasma obtained from
different species for 1 h at 37 °C.
Assessment of plasma protein binding was measured by equilibrium dialysis by incubating
the compound of interest at a concentration of 5 µM for 6 h at 37 °C in 50 % plasma in buffer
(v/v) followed by LCꢀMSꢀbased determination of final compound concentrations. The
resulting fraction unbound at 50 % plasma (fu %) is extrapolated to the fraction unbound at
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100 % plasma (fu100 %) using the following equation: fu100 % = fu50 %/(2ꢀfu50 %).
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5
5
6
Metabolic stability under oxidative conditions was measured in liver microsomes from
different species by LCꢀMSꢀbased measuring of depletion of compound at a concentration of
3
µM over time up to 50 min at 37 °C. Based on compound halfꢀlife t1/2, in vitro intrinsic
clearance CL was calculated.
int
0
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2
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9
0
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8
9
0
To determine the pharmacokinetics of 1w totally 24 male RjOrl:SWISS (CD1) mice, age 8ꢀ
1
0 weeks, body weight (bw) between 29 and 31 g, were purchased from Janvier, Saintꢀ
Berthevin Cedex, France. Mice were fed ad libitum with “AlleinꢀFutter für Rattenꢀ
Mäusehaltung” from ssniff special diets GmbH, Germany. They had free access to water and
/
were kept in a 12 h day/night rhythm. All experiments were approved by the local authorities.
Pharmacokinetics were studied for the oral, intraperitoneal (ip), and intravenous (iv) route of
administration. For the oral and intraperitoneal routes a stock solution of 8 mg 1w in 4 mL of
a solution consisting to 10 % of DMSO and to 90 % of an aqueous solution of 30 % 2ꢀ
hydroxypropylꢀβꢀcyclodextrin was prepared. To reach doses of 20 mg/kg bw, 300 µl were
administered per 30 g of mouse bw. For the intravenous route of administration a 2 mg/ml
DMSO solution was prepared and 30 µl were applied to a 30 g mouse to reach a
concentration of 2 mg/kg.bw. Mice were sacrificed 5, 15, 45 and 135 min after administration
of the test compound and approximately 100 µl blood was taken from the left heart chamber.
Three mice were analyzed per time point and route of administration (oral, ip, and iv),
respectively. Immediately after sampling blood was centrifuged at 16,200 ×g for 10 min at
4 °C and plasma was stored at ꢀ80 °C until LC/MS analysis of 1w. Prior to LCꢀMS/MS
analysis, plasma proteins were precipitated with acetonitrile containing an internal standard
and samples were filtered. A calibration curve was obtained from spiked blank plasma
samples. 1w was measured using a Shimadzu UPLC system connected to a QTrap 4000
hybrid triple quadrupole/linear ion trap mass spectrometer (AB Sciex). The regression
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equation of the calibration curve was used to calculate plasma concentrations.
Pharmacokinetic parameters were calculated using the PKSolver software2.
Chemistry
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Unless otherwise noted, all reagents and solvents were purchased from Acros, Fluka,
SigmaꢀAldrich or Merck and used without further purification. Dry solvents were purchased
1
13
as anhydrous reagents from commercial suppliers. H and C NMR spectra were recorded on
a Bruker Avance DRX 400 or Bruker Avance DRX 500 spectrometer at 400, 500, 600 or 700
1
and 101, 126 or 176 MHz, respectively. H chemical shifts are reported in δ (ppm) as s
(singlet), d (doublet), dd (doublet of doublet), t (triplet), q (quartet), m (multiplet), and bs
6
(broad singlet) and are referenced to the residual solvent signal. DMSOꢀd (2.50). Coupling
13
constants (J) are expressed in hertz (Hz). C spectra are referenced to the residual solvent
6
signal DMSOꢀd (39.52). Highꢀresolution electrospray ionization mass spectra (ESIꢀFTMS)
were recorded on a Thermo LTQ Orbitrap (highꢀresolution mass spectrometer from Thermo
Electron) coupled to an “Accela” HPLC system supplied with a “Hypersil GOLD” column
(
Thermo Eletron). Analytical TLC was carried out on Merck 60 F245 aluminumꢀbacked
silica gel plates. Compounds were purified by column chromatography using Baker silica gel
40ꢀ70 µm particle size) or Flash Chromatography on a Biotage Isolera One using Biotage
(
SNAP, SNAP Ultra, ZIP Sphere or ZIP KPꢀSil columns (25 g, 10 g, 5 g or 120 g,
respectively) monitored by UV at λ = 254 nm and 280 nm. Preparative HPLC was conducted
on an Agilent HPLC system (1200 series) with a VP 125/21 Nucleodur C18 column from
MachereyꢀNagel and monitored by UV at λ = 210 n, 254 nm. All final compounds were
purified to >95 % purity as determined by highꢀperformance liquid chromatography (HPLC).
Purity was measured using Agilent 1200 series HPLC systems with UV detection at 210 nm
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3 2
(system: Agilent Eclipse XDBꢀC18 4.6 mm × 150 mm, 5 ꢁM, 10ꢀ100 % CH CN in H O,
with 0.1 % TFA, for 15 min at 1.0 mL/min).
General Procedure A: SuzukiꢀCrossꢀCoupling of iodinated pyrazolopyrimidines (3) with
various boronic acids.
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tertꢀButylꢀ(R)ꢀ3ꢀ(4ꢀaminoꢀ3ꢀiodoꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ1ꢀyl)piperidineꢀ1ꢀ
carboxylate (3) and the corresponding boronic acid were dissolved in a mixture of DME and
Ethanol (4 mL, 3/1, v/v) and treated with saturated sodium carbonate solution. The reaction
mixture was flushed with argon for 5 min After that tetrakis(triphenylphosphine)palladium(0)
was added. The mixture was heated to 90 °C and stirred for 12 h under argon atmosphere.
The reaction mixture was filtered through a plug of Celite and after that washed using
saturated sodium hydrogen carbonate solution and extracted three times with ethyl acetate,
washed with water and saturated sodium chloride solution. The combined organic layers were
dried over anhydrous sodium sulfate and evaporated to dryness. The resulting crude product
was purified via column chromatography or flash chromatography (normal and reversed
phase).
General Procedure B: Deprotection of the secondary amine.
The protected pyrazolopyrimidines (5a-o) were dissolved in 3 mL DCM and cooled to
0
°C. To this stirred solution, 3 mL of a solution of TFA in DCM (25/75, v/v) was added
dropwise. The reaction mixture was warmed to room temperature and stirred for 1 h. The
reaction mixture was diluted with DCM and the pH adjusted to 8 with sat. sodium hydrogen
carbonate solution and extracted five times with a solution of 10 % MeOH in DCM. The
combined organic layers were dried over anhydrous sodium sulfate and evaporated to
dryness. The deprotected amines were taken up in THF and cooled to 0 °C. The solution was
treated with DIPEA and stirred for 15 min at 0 °C under argon atmosphere. A solution of
acryolyl chloride or propionyl chloride in THF was added dropwise at this temperature and
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stirred until the reaction was complete (15 – 45 min) at 0 °C. The reaction mixture was
quenched with sat. sodium hydrogen carbonate solution and extracted three times with ethyl
acetate, washed with water and saturated sodium chloride solution. The combined organic
layers were dried over anhydrous sodium sulfate and evaporated to dryness. The resulting
crude product was purified by column chromatography, flash chromatography (normal and
reversed phase) or preparative HPLC.
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(R)-1-(3-(4-Amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one
(
1a)
Compound 1a was prepared according to General Procedure B using 5a (50 mg, 0.2 mmol,
.0 eq.), DIPEA (269 µL, 1.6 mmol, 10.0 eq.) and acryolyl chloride (14 µL, 0.2 mmol,
1
0
1
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7
8
9
0
1.1 eq.). The crude product was purified by column chromatography (MeOH/DCM (1/99, v/v
ꢀ
3/97, v/v)). Yield: 15 mg (0.1 mmol, 38 % over two steps).
1
6
H NMR (500 MHz, DMSOꢀd ): δ8.33 (s, 1H), 8.26 (d, J = 14.5 Hz, 1H), 6.89 – 6.64 (m,
1
H), 6.15 – 6.04 (m, 1H), 5.73 – 5.59 (m, 1H), 4.70 – 4.59 (m, 1H), 4.52 (d, J = 12.4 Hz,
0
.5H), 4.27 – 4.21 (m, 0.5H), 4.19 – 4.13 (m, 0.5H), 4.11 – 4.05 (m, 0.5H), 3.63 (m, 0.5H),
3.20 – 3.11 (m, 1H), 2.98 – 2.92 (m, 0.5H), 2.25 – 2.16 (m, 1H), 2.12 – 2.05 (m, 1H), 1.94 –
1
.87 (m, 1H), 1.65 – 1.53 (m, 1H).
+
HRMS Berechnet: 273.15 for C13
18 6
H N O [M+H] . Found: 273.15.
(R)-1-(3-(4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-
1-one (1b)
Compound 1b was prepared according to General Procedure B using 4 (80 mg, 0.2 mmol,
.0 eq.), DIPEA (204 µL, 1.2 mmol, 1.2 eq.) and acryolyl chloride (28 µL, 0.3 mmol,
.9 eq.). The crude product was purified by column chromatography (MeOH/DCM (1/99, v/v
1
1
ꢀ
2/98, v/v)). Yield: 12 mg (0.03 mmol, 17 % over two steps).
1
H NMR (600 MHz, DMSOꢀd ) δ 8.21 (s, 1H), 7.90 (bs, 1H), 6.89 – 6.69 (m, 1H), 6.63
6
(
bs, 1H), 6.15 – 6.05 (m, 1H), 5.73 – 5.59 (m, 1H), 4.65 – 4.54 (m, 1H), 4.54 – 4.48 (m,
0
.5H), 4.30 – 4.24 (m, 0.5H), 4.20 – 4.14 (m, 0.5H), 4.09 – 4.03 (m, 0.5H), 3.62 – 3.56 (m,
.5H), 3.19 – 3.07 (m, 1H), 2.92 – 2.86 (m, 0.5H), 2.22 – 2.15 (m, 1H), 2.08 – 2.02 (m, 1H),
0
1.91 – 1.83 (m, 1H), 1.61 – 1.47 (m, 1H).
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C NMR (151 MHz, DMSOꢀd ) δ 165.4, 164.9, 158.2, 156.5, 153.7, 153.6, 128.8, 128.7,
1
28.0, 127.9, 103.7, 103.6, 89.8, 89.7, 53.9, 53.1, 49.6, 46.2, 45.5, 41.9, 30.1, 30.0, 25.4,
3.9.
2
+
LCꢀMS (ESIꢀMS): calcd: 399.04346 for C H IN O [M+H] . Found: 399.04248.
13
16
6
10
11
12
13
14
15
16
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19
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23
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40
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43
44
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59
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(R)-1-(3-(4-Amino-3-ethynyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-
en-1-one (1c)
Compound 1c was prepared according to General Procedure B using 11 (400 mg,
1
.2 mmol, 1.0 eq.), DIPEA (995 µL, 5.9 mmol, 5.0 eq.) and acryolyl chloride (129 µL,
1
.6 mmol, 1.4 eq.). The crude product was purified by column chromatography
(MeOH/DCM (1/99, v/v ꢀ 3/97, v/v). Yield: 147 mg (0.5 mmol, 43 % over two steps).
1
6
H NMR (500 MHz, DMSOꢀd ) δ 8.24 (s, 1H), 8.05 (bs, 1H), 6.88 – 6.68 (m, 1H), 6.54
(
bs, 1H), 6.54 (bs, 1H), 6.15 – 6.05 (m, 1H), 4.66 (s, 1H), 4.65 – 4.59 (m, 1H), 4.51 (d,
J = 11.3 Hz, 0.5H), 4.29 – 4.16 (m, 1H), 4.07 (d, J = 13.4 Hz, 0.5H), 3.64 – 3.58 (m, 0.5H),
3.19 – 3.09 (m, 1H), 2.95 – 2.89 (m, 0.5H), 2.22 – 2.15 (m, 1H), 2.10 – 2.05 (m, 1H), 1.93 –
1
.85 (m, 1H), 1.60 – 1.52 (m, 1H).
13
6
C NMR (126 MHz, DMSOꢀd ) δ 164.6, 164.5, 157.7, 156.5, 152.8, 152.7, 128.3, 128.2,
1
27.6, 127.5, 125.1, 125.0, 101.0, 100.9, 86.2, 75.2, 53.4, 52.5, 49.1, 45.6, 45.1, 41.5, 29.5,
9.4, 24.9, 23.4.
2
+
HRMS (ESIꢀMS): calcd: 297.14647 for C H N O [M+H] . Found: 297.14584.
15
17
6
(R)-1-(3-(4-Amino-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-
en-1-one (1d)
Compound 1d was prepared according to General Procedure B using 5b (286 mg,
0.7 mmol, 1.0 eq.), 100 mg (0.3 mmol, 1.0 eq) of the deprotected compound were then used
according to General Procedure B with DIPEA (340 µL, 2.0 mmol, 5.9 eq.) and acryolyl
chloride (30 µL, 0.4 mmol, 1.1 eq.). The crude product was purified by column
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chromatography (MeOH/DCM (1/99, v/v ꢀ 4/96, v/v)) and subsequent preparative HPLC
2
(H O/ACN (20/80, v/v) + 0.2 % TFA as additive). Yield: 18 mg (0.05 mmol, 15 %).
1
H NMR (500 MHz, DMSOꢀd
6
) δ 8.31 (s, 1H), 7.68 (d, J = 7.2 Hz, 2H), 7.59 – 7.53 (m,
2H), 7.52 – 7.47 (m, 1H), 7.04 (bs, 2H), 6.94 – 6.63 (m, 1H), 6.23 – 6.02 (m, 1H), 5.73 – 5.56
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
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1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(m, 1H), 4.81 – 4.66 (m, 1H), 4.56 (d, J = 10.9 Hz, 0.5H), 4.20 (d, J = 11.2 Hz, 1H), 4.07 (d,
J = 12.3 Hz, 0.5H), 3.77 – 3.67 (m, 0.5H), 3.29 – 3.16 (m, 1H), 3.07 – 2.99 (m, 0.5H), 2.34 –
2.22 (m, 1H), 2.17 – 2.10 (m, 1H)), 1.98 – 1.90 (m, 1H), 1.67 – 1.52 (m, 1H).
1
3
6
C NMR (126 MHz, DMSOꢀd ) δ 165.4, 158.4, 154.6, 155.3, 144.7, 133.6, 130.0, 129.6,
129.3, 129.1, 128.3, 128.0, 98.2, 53.6, 52.9, 49.8, 46.5, 47.0, 42.5, 30.2, 30.5, 25.7, 24.0.
+
LCꢀMS (ESIꢀMS): calcd: 349.17687 for C H N O [M+H] . Found: 349.17714.
19
21
6
(R)-1-(3-(4-Amino-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)propan-
1-one (1e)
Compound 1e was prepared according to General Procedure B using 5b (286 mg,
0.7 mmol, 1.0 eq.), 70 mg (0.2 mmol, 1.0 eq.) of the deprotected compound were then used
according to General Procedure B with DIPEA (243 µL, 1.4 mmol, 6.0 eq.) and propionyl
chloride (23 µL, 0.3 mmol, 1.1 eq.). The crude product was purified by column
chromatography (MeOH/DCM (1/99, v/v ꢀ 4/96, v/v) and subsequent preparative HPLC
2
(H O/ACN + 0.1 % TFA as additive). Yield: 43 mg (0.1 mmol, 50 % over two steps).
1
H NMR (500 MHz, DMSOꢀd ) δ 8.27 (d, J = 11.6 Hz, 1H), 7.67 (d, J = 7.3 Hz, 2H), 7.55
6
(dd, J = 7.4 Hz, 2H), 7.52 – 7.47 (m, 1H), 8.85 (bs, 2H), 4.79 – 4.60 (m, 1H), 4.57 – 4.51 (m,
0.5H) 4.22 (d, J = 13.0 Hz, 0.5H), 4.06 – 4.00 (m, 0.5H), 3.87 (d, J = 13.3 Hz, 0.5H), 3.62 –
3.55 (m, 0.5H), 3.16 – 3.07 (m, 1H), 2.90 – 2.82 (m, 0.5H), 2.42 – 2.19 (m, 3H), 2.16 – 2.07
(m, 1H), 1.94 – 1.84 (m, 1H), 1.69 – 1.47 (m, 1H), 1.04 – 0.94 (m, 3H).
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C NMR (126 MHz, DMSOꢀd ) δ 171.4, 171.3, 158.1, 155.6, 155.5, 154.0, 153.9, 143.7,
1
43.6, 132.9, 129.0, 128.6, 128.2, 97.4, 97.4, 52.7, 52.1, 49.1, 45.3, 44.8, 41.1, 29.6, 29.3,
5.6, 24.6, 23.4, 9.4.
2
+
HRMS (ESIꢀMS): caclcd: 351.19349 for C H N O [M+H] . Found: 351.19279.
1
9
23
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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(R,E)-1-(3-(4-Amino-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-
(
dimethylamino)but-2-en-1-one (1f)
Compound 1f was prepared according to General Procedure B using 5b (286 mg,
0
.7 mmol, 1.0 eq.), 50 mg (0.2 mmol, 1.0 eq) of the deprotected compound were then solved
in 2 ml DCM and treated with (E)ꢀ4ꢀ(dimethylamino)butꢀ2ꢀenoic acid hydrochloride (42 mg,
0.3 mmol, 1.5 eq.), 3ꢀ(ethyliminomethyleneamino)ꢀN,Nꢀdimethylpropanꢀ1ꢀamine
hydrochlorid (49 mg, 0.3 mmol, 1.5 eq.), benzotriazolꢀ1ꢀol (34 mg, 0.3 mmol, 1.5 eq.) and
DIPEA (173 µL, 1.0 mmol, 6.0 eq.). The reaction mixture was stirred for 12 h at room
temperature. The reaction mixture was diluted with DCM and the pH adjusted to 8 with sat.
sodium hydrogen carbonate solution and extracted four times with a solution of 10 % MeOH
in DCM. The combined organic layers were dried over anhydrous sodium sulfate and
evaporated to dryness. The crude product was purified by column chromatography
(
2
MeOH/DCM (2/98, v/v ꢀ 10/90, v/v and subsequent preparative HPLC (H O/ACN + 0.1 %
TFA as additive). Yield: 19 mg (0.05 mmol, 29 %).
1
H NMR (500 MHz, DMSOꢀd ) δ 8.27 (d, J = 3.8 Hz, 1H), 7.70 – 7.64 (m, 2H), 7.62 (bs,
6
2
–
3
H), 7.58 – 7.53 (m, 2H), 7.52 – 7.48 (m, 1H), 7.05 – 6.85 (m, 1H), 6.71 – 6.58 (m, 1H), 4.79
4.67 (m, 1H), 4.54 (d, J = 12.0 Hz, 0.5H), 4.28 – 4.17 (m, 1H), 4.08 (d, J = 13.4 Hz, 0.5H),
.87 (d, J = 6.9 Hz, 1H), 3.74 (d, J = 6.9 Hz, 1H), 3.77 – 3.70 (m, J = 8.9 Hz, 0.5H), 3.28 –
3.20 (m, 1H), 3.03 – 2.96 (m, 0.5), 2.72 (s, 3H), 2.65 (s, 3H), 2.31 – 2.21 (m, 1H), 2.16 – 2.09
m, 1H), 1.98 – 1.87 (m, 1H), 1.69 – 1.53 (m, 1H).
(
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6
C NMR (126 MHz, DMSOꢀd ) δ 163.5, 158.2, 155.8, 155.7, 154.1, 154.0, 143.9, 143.8,
1
32.9, 132.9, 132.4, 132.1, 129.2, 129.2, 129.0, 128.8, 128.3, 128.3, 97.4, 56.7, 52.8, 52.0,
9.4, 45.9, 45.3, 41.8, 41.6, 29.7, 29.6, 24.9, 23.4.
4
+
HRMS (ESIꢀMS): calcd: 406.23599 for C H N O [M+H] . Found: 406.23499.
22
28
7
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
(R)-1-(3-(4-Amino-3-(3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-
yl)prop-2-en-1-one (1g)
Compound 1g was prepared according to General Procedure B using 5c (239 mg,
0
.6 mmol, 1.0 eq.), 44 mg (0.1 mmol, 1.0 eq) of the deprotected compound were then used
according to General Procedure B with DIPEA (246 µL, 1.4 mmol, 10.2 eq.) and acryolyl
chloride (13.0 µL, 0.2 mmol, 1.1 eq.). The crude product was purified by column
chromatography (MeOH/DCM (1/99, v/v ꢀ 4/96, v/v). Yield: 15.0 mg (0.04 mmol, 29 %).
1
6
H NMR (500 MHz, DMSOꢀd ) δ 8.29 (s, 1H), 7.60 – 7.41 (m, 3H), 7.36 – 7.27 (m, 1H),
6
.90 – 6.61 (m, 1H), 6.09 (dd, J = 35.8, 16.9 Hz, 1H), 5.74 – 5.53 (m, 1H), 4.73 (s, 1H), 4.17
(s, 1H), 3.77 – 3.66 (m, 1H), 3.24 (dd, J = 11.7 Hz, 1H), 2.04 – 1.88 (m, 2H), 1.59 (s, 1H),
1
.32 – 1.19 (m, 2H).
19
6
F NMR (565 MHz, DMSOꢀd ) δ ꢀ74.34 (s, J = 293.7 Hz).
+
HRMS (ESIꢀMS): calcd: 367.16771 for C19
R)-1-(3-(4-Amino-3-(3-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-
yl)propan-1-one (1h)
6
H19ON F [M+H] . Found: 367.16812.
(
Compound 1h was prepared according to General Procedure B using 5c (239 mg,
0
.6 mmol, 1.0 eq.), 181 mg (0.4 mmol, 1.0 eq.) of the deprotected compound were then used
according to General Procedure B with DIPEA (249 µL, 1.4 mmol, 3.3 eq.) and propionyl
chloride (56 µL, 0.6 mmol, 1.5 eq.). The crude product was purified by column
chromatography (MeOH/DCM (1/99, v/v ꢀ 4/96, v/v). Yield: 36 mg (0.1 mmol, 23 %).
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Journal of Medicinal Chemistry
1
2
3
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8
9
1
H NMR (500 MHz, DMSOꢀd
J = 7.6 Hz, 1H), 7.47 – 7.41 (m, 1H), 7.35 – 7.30 (m, 1H), 7.17 (bs, 2H), 4.80 – 4.61 (m,
H), 4.54 – 4.48 (m, 0.5H), 4.20 (d, J = 13.0 Hz, 0.5H), 4.05 – 3.99 (m, 0.5H), 3.87 (d,
6
) δ 8.30 (d, J = 8.4 Hz, 1H), 7.62 – 7.56 (m, 1H), 7.50 (d,
1
J = 13.5 Hz, 0.5H), 3.61 – 3.55 (m, 0.5H), 3.17 – 3.09 (m, 1H), 2.93 – 2.82 (m, 0.5H), 2.42 –
10
11
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15
16
17
18
19
20
21
22
23
24
25
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27
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29
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31
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35
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37
38
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43
44
45
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47
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49
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52
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56
57
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59
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2.18 (m, 3H), 2.15 – 2.07 (m, 1H), 1.94 – 1.83 (m, 1H), 1.69 – 1.46 (m, 1H), 1.03 – 0.94 (m,
3H).
13
6
C NMR (126 MHz, DMSOꢀd ) δ 171.4, 163.5, 161.5, 157.6, 155.1, 153.8, 142.7, 134.9,
1
31.1, 124.4, 115.6, 115.4, 97.3, 52.8, 52.3, 49.0, 45.3, 44.7, 41.1, 29.5, 29.3, 25.6, 24.5,
3.5, 9.4.
2
1
9
F NMR (565 MHz, DMSOꢀd ) δ ꢀ73.70 (s, J = 299.4 Hz).
6
+
HRMS (ESIꢀMS): calcd: 369.18336 for C19
R)-1-(3-(4-Amino-3-(4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-
yl)prop-2-en-1-one (1i)
6
H21ON F [M+H] . Found: 369.18390
(
Compound 1i was prepared according to General Procedure B using 5d (78 mg, 0.2 mmol,
.0 eq.), DIPEA (326 µL, 1.9 mmol, 9.8 eq.) and acryolyl chloride (16.8 µL, 0.2 mmol,
.1 eq.). The crude product was purified by column chromatography (MeOH/DCM (1/99, v/v
1
1
ꢀ
3/97, v/v)). Yield: 44 mg (0.1 mmol, 63 % over two steps).
1
3
H NMR (500 MHz, CDCl ): δ 8,39 (s, 1H), 7.67 (m, 2H), 7.23 (t, 2H), 6.58 (m, 1H), 6.29
(d, 1H), 5.68 (d, 1H), 5.40 (s, 2H), 2.36 (m, 1H), 2.24 (m, 1H), 2.02 (m, 1H), 1.74 (m, 1H),
1.27 (m, 5H).
+
6
HRMS calcd: 367.16 for C19H20FN O [M+H] . Found: 367.17.
(R)-1-(3-(4-Amino-3-(4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-
yl)propan-1-one (1j)
Compound 1j was prepared according to General Procedure B using 5d (389 mg,
.9 mmol, 1.0 eq.), DIPEA (1.87 mL, 11.0 mmol, 11.7 eq.) and propionyl chloride (101 µL,
0
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