Substituent Effects on Drug-Receptor H-bond Interactions: Correlations Useful for the Design of Kinase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b01342

Investigation of troponin I-interacting kinase (TNNI3K) as a potential target for the treatment of heart failure has produced a series of substituted N-methyl-3-(pyrimidin-4-ylamino)benzenesulfonamide inhibitors that display excellent potency and selectivity against a broad spectrum of protein kinases. Crystal structures of prototypical members bound to the ATP-binding site of TNNI3K reveal two anchoring hydrogen bond contacts: (1) from the hinge region amide N-H to the pyrimidine nitrogen and (2) from the sulfonamide N-H to the gatekeeper threonine. Evaluation of various para-substituted benzenesulfonamides defined a substituent effect on binding affinity resulting from modulation of the sulfonamide H-bond donor strength. An opposite electronic effect emerged for the hinge NH-pyrimidine H-bond interaction, which is further illuminated in the correlation of calculated H-bond acceptor strength and TNNI3K affinity for a variety of hinge binding heterocycles. These fundamental correlations on drug-receptor H-bond interactions may be generally useful tools for the optimization of potency and selectivity in the design of kinase inhibitors.

Full text of DOI:10.1021/acs.jmedchem.6b01342

Article
pubs.acs.org/jmc
Substituent Effects on Drug−Receptor H‑bond Interactions:
Correlations Useful for the Design of Kinase Inhibitors
Brian G. Lawhorn,*,† Joanne Philp,† Alan P. Graves,‡ Dennis A. Holt,† Gregory J. Gatto, Jr.,†
and Lara S. Kallander†
†Heart Failure Discovery Performance Unit and ‡Platform Technology and Sciences, GlaxoSmithKline, 709 Swedeland Road, King of
Prussia, Pennsylvania 19406, United States
S
* Supporting Information
ABSTRACT: Investigation of troponin I-interacting kinase
(TNNI3K) as a potential target for the treatment of heart
failure has produced a series of substituted N-methyl-3-
(pyrimidin-4-ylamino)benzenesulfonamide inhibitors that dis-
play excellent potency and selectivity against a broad spectrum
of protein kinases. Crystal structures of prototypical members
bound to the ATP-binding site of TNNI3K reveal two
anchoring hydrogen bond contacts: (1) from the hinge region
amide N−H to the pyrimidine nitrogen and (2) from the
sulfonamide N−H to the gatekeeper threonine. Evaluation of
various para-substituted benzenesulfonamides defined a
substituent effect on binding affinity resulting from modulation
of the sulfonamide H-bond donor strength. An opposite electronic effect emerged for the hinge NH-pyrimidine H-bond
interaction, which is further illuminated in the correlation of calculated H-bond acceptor strength and TNNI3K affinity for a
variety of hinge binding heterocycles. These fundamental correlations on drug−receptor H-bond interactions may be generally
useful tools for the optimization of potency and selectivity in the design of kinase inhibitors.
INTRODUCTION
■
Troponin I-interacting protein kinase (TNNI3K) is a tyrosine-
like kinase that is specifically expressed in cardiomyocytes and
is an emerging target for cardiac disease.1 TNNI3K may play a
central role in heart failure as it has been associated with
progression of dilated cardiomyopathy and pathological
remodeling in ischemic heart disease.2−5 Overexpression of
TNNI3K exacerbates disease progression in ischemia/reperfu-
sion models, while its deletion is cardioprotective, suggesting
TNNI3K may be a key mediator of cardiac injury in patients
who receive percutaneous coronary intervention (PCI)
following an acute myocardial infarction (MI).5 When
administered during reperfusion therapy following acute MI
in mice, TNNI3K inhibitors reduced infarct size, preserved
cardiac function, and limited the adverse ventricular remodeling
associated with heart failure, confirming the value of TNNI3K
as a target for cardiac disease.5
We recently developed three series of related 7-deazapurine
(1), quinazoline (2), and 4,6-diaminopyrimidine (3) containing
TNNI3K inhibitors that share a common 3-N-methylsulfona-
mide moiety linked to a pyrimidine core (Figure 1).6,7 X-ray
cocrystal structures of representative members of this class
bound to the TNNI3K active site revealed that one pyrimidine
nitrogen serves as a hydrogen bond acceptor for the amide N−
H of the hinge region Ile542 (Figure 2).8 The sulfonamide
group forms a full complement of hydrogen bonds as it binds
TNNI3K, including a key H-bond donor interaction from the
Figure 1. Benzenesulfonamide TNNI3K inhibitors.6,7
sulfonamide N−H to the side chain OH of Thr539, which is
the gatekeeper residue of TNNI3K. In order to simultaneously
accommodate the Ile542 hydrogen bond contact and the
Thr539−sulfonamide hydrogen bond interaction, the pyrimi-
dine and benzene rings adopt a coplanar binding conformation
(Figure 2). The necessity of this orientation for TNNI3K
recognition has been demonstrated by introducing structural
modifications that would disrupt this binding mode, leading to
analogs with severely compromised activity against TNNI3K.6
Received: September 9, 2016
© XXXX American Chemical Society
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RESULTS AND DISCUSSION
■
Protein Acceptor−Ligand Donor Hydrogen Bond
Interaction. A series of 4-substituted benzenesulfonamide
analogs of 1 and 3 were synthesized and assessed for their
ability to bind to TNNI3K (Figure 3).6 Among the set of small
Figure 2. Cocrystal structure of a benzenesulfonamide inhibitor bound
to TNNI3K.6,8
While optimizing the benzenesulfonamide inhibitors we
noted that their activity at TNNI3K was highly sensitive to
substituents located para to the sulfonamide group and
hypothesized that the initial SAR may arise, at least in part,
through electronic effects that modulate the key H-bond
interactions formed upon binding to TNNI3K.6 Separately, we
observed an increased affinity for TNNI3K in analogs bearing
electron donating groups on the quinazoline scaffold suggesting
a potential electronic effect on the hinge binding H-bond
protein donor interaction, though competing steric effects
prevented a clear delineation of this connection using the
analogs examined in the previous study.7
Herein, we report the systematic evaluation of an expanded
set of para-substituted benzenesulfonamides that define a
fundamental relationship between substituent electronic
properties (σp) and affinity for a protein acceptor−ligand
donor hydrogen bonding interaction. We also disclose a set of
monosubstituted quinazolines within the same class of
inhibitors that establish a complementary correlation between
the Hammett constant (σp) and affinity for a protein donor−
ligand acceptor hydrogen bonding interaction. The latter
electronic effect was expanded by demonstrating the association
of calculated H-bond acceptor strength and TNNI3K affinity
for a variety of hinge binding heterocycles, thereby producing a
quantitative relationship for kinase hinge recognition that may
be generally applicable for designing drugs that target protein
kinases.
The effect of substituent electronic properties on covalent
bonding interactions between proteins and their substrates have
been reported, including electronic effects on ADP-ribosyl
transferase activity with substituted (benzylidineamino)-
guanidines, which supported an SN2 enzymatic reaction
mechanism.9 Poulter reported substituent effects on the
allylation of tryptophans catalyzed by dimethyallyl tryptophan
synthase pointing to an electrophilic aromatic substitution
reaction mechanism.10 A correlation between the Hammett
constant (σp) and the energy of a charge-transfer interaction
between substituted phenols and the flavin cofactor of old
yellow enzyme has also been observed.11 Boger and colleagues
defined a substituent effect on the inhibition of fatty acid amide
hydrolase by α-ketoheterocycles, where substituents were found
to electronically modulate a covalent interaction between an
active site serine residue and the inhibitor ketone functional
group.12 In this report, we expand upon these previously
established correlations between the Hammett constant (σp)
and protein−ligand interactions by defining fundamental
relationships between substituent electronic properties and
inhibitor affinity for drug−receptor hydrogen bond inter-
actions.
Figure 3. Effect of C4-benzenesulfonamide substituents.
functional groups that project into a spacious area of the
TNNI3K ATP-binding site (Figure 2), a relationship between
the substituent electronic properties and TNNI3K binding
affinity is evident (cf. 4−12 and 13−21), with strongly electron
donating groups (e.g., NHMe) leading to weak activity, while
electron withdrawing groups (e.g., OCF3) enhance potency
relative to the unsubstituted parent compounds. A small set of
mildly electron donating 4-substituted benzenesulfonamides
(22−25, 26−29) diverge from the general trend showing
enhanced activity values. Their more productive binding to
TNNI3K likely arises from their unique ability to form an
intramolecular H-bond with the neighboring aniline N−H
(Figure 3), which would stabilize the coplanar orientation
required for TNNI3K recognition (Figure 2).
A Hammett plot (TNNI3K pIC50 vs Hammett σp, Figure 4)
revealed that the TNNI3K potency of each compound is
dependent on the electronic nature of the substituent located
para to the sulfonamide moiety (R2 ≈ 0.9).13 Importantly, the
substituents (22−25, 26−29) that engage in a preorganizing
intramolecular H-bond (Figure 4, red points) deviate
productively from the main trend forming a separate but
consistent structure−activity relationship that tracks with their
electronic properties, confirming the general correlation rather
than negating it. The steep and positive slopes (ρ = 1.6, 2.2)
associated with the correlations suggest a significant electronic
modulation in which negative charge is built on the ligand as it
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protein serves as a H-bond acceptor and the ligand acts as a H-
bond donor.
In a previous report, we noted that incorporating an N,N-
dimethylamino substituent at C4 (30, IC50 = 50 nM) produced
activity at TNNI3K that departed from the weak inhibition
demonstrated by the N-monomethylamino variant (4, IC50
=
6300 nM).6 The potent activity of 30 results from its twisted
conformation (Figure 5), a characteristic of ortho-substituted
Figure 5. Conformations of anilines 4 and 30.
N,N-dialkylanilines that arises due to the severe steric clash that
exists between one of their N-alkyl groups and the neighboring
ortho substituent in the alternative coplanar orientation. The
twisted conformation disrupts the resonance between the
aniline nitrogen lone pair and the adjacent aryl ring and thereby
abolishes the elecron donating effect of the N,N-dimethylamino
sustituent on the para-sulfonamide group.6,17 Thus, the
divergent TNNI3K activity of 4 and 30 provides additional
support for the importance of the observed electronic effect of
C4-substituents on the sulfonamide H-bond donor interaction.
To further probe this relationship, a set of cyclic and acyclic
disubstitued amino groups were installed on the 7-deazapurine
(1) and 4,6-diaminopyrimidine (3) templates and evaluated
against TNNI3K (Figure 6). Interestingly, the pyrrolidine-
bearing analogs (e.g., 31, IC50 = 320 nM) displayed reduced
potency against TNNI3K compared to 30 (IC50 = 50 nM) but
were more active than 4 (IC50 = 6300 nM), suggesting that the
pyrrolidine may adopt a partially twisted conformation as a
result of its reduced steric interaction with the neighboring
aniline NH. This orientation would restore in part the
resonance electron donating character of the pyrrolidine
nitrogen lone pair, leading to reduced hydrogen bond donor
strength in the para-sulfonamide and diminished TNNI3K
binding affinity. Consistent with this interpretation, increasing
the steric demand of the pyrrolidine via methylation (32, 33) or
its conversion to a piperidine (35) restored the TNNI3K
binding affinity. Incorporation of fluorine atoms into the N,N-
dialkyl substituents consistently increased their TNNI3K
activity (e.g., 34 vs 31, 36 vs 35, 38 vs 37) as would be
expected based on the benefit that inductive electron
withdrawal would impart on the para-sufonamide H-bond
donor capacity. Similar observations were found with the 4,6-
diaminopyrimidines (39−46) solidifying our understanding of
the electronic and stereoelectronic effects on the sulfonamide
NH hydrogen bond donor interaction with the TNNI3K
Thr539 residue.
Figure 4. Plots of TNNI3K pIC50 vs σp for para-substituted
benzenesulfonamides (1, 3, 4−29).
binds to TNNI3K. Presumably, the 4-substituent elicits an
electronic effect on the para-sulfonamide group, which forms
three hydrogen bonds with TNNI3K (Figure 2). Of these, the
sulfonamide-NH H-bond donor interaction with Thr539 would
be enhanced by electron withdrawing groups and destabilized
by electron donating groups, as a partial negative charge builds
on the ligand when it forms this contact (Figure 4). Thus, the
Hammett equation defines the sulfonamide H-bond interaction
with Thr539 as a key driver for TNNI3K recognition in the
series and illustrates the substantial energetic consequence (∼4
kcal/mol) of electronically tuning drug−receptor hydrogen
bonds.
The dominating effect of the H-bond donor interaction is
consistent with the large hydrogen bond donor capacity of
sulfonamides and their relatively weak hydrogen bond acceptor
strength.14 A theoretical study analyzing the effects of para-
substituents on aryl sulfonamides revealed a corrleation
between Hammett σp and sulfonamide gas-phase acidities.15
Computation of geometrical parameters for the same series of
para-substituted sulfonamides demonstrated that electron
donating groups significantly increase the sulfonamide S−N
bond length but have little impact on SO bond lengths,
suggesting that para-substituents may have a much larger
electronic effect on sulfonamide-NH versus sulfonamide SO
hydrogen-bonding.15 Further, a correlation of Hammett σp with
Bronsted acid strength (pKa) has been reported for a series of
substituted N-benzylbenzenesulfonamides that established
experimentally the ability of para-substituents to electronically
modulate sulfonamide acidity.16 Notably, the magnitude of this
effect (ρ = 1.5) is similar to that observed in the relationship
between TNNI3K affinity and substituent electronic properties
for para-substituted-benzenesulfonamides (Figure 4). There-
fore, we conclude that the electronic effect on TNNI3K binding
results from modifying the sulfonamide-NH hydrogen bond
donor strength, which establishes a new fundamental relation-
ship between Hammett σp and inhibitor affinity where the
Protein Donor−Ligand Acceptor Hydrogen Bond
Interaction. A series of 6-substituted quinazoline inhibitors
related to 2 were synthesized and assessed as TNNI3K
inhibitors (Figure 7). This set of analogs confirmed our
previous observation that electron donating groups (47−49)
enhance the TNNI3K affinity of the quinazoline inhibitors and
expanded the structure−function relationship by demonstrating
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Figure 6. Effect of N,N-dialkyl-4-benzenesulfonamide substituents.
negative sign of the ρ-value is opposite the trend observed for
the benzenesulfonamide substituent effect (Figure 4) and
signifies an increase in positive charge on the quinazoline as the
inhibitors bind to TNNI3K. X-ray structures of related
inhibitors imply that a key hydrogen bond exists between the
quinazoline N1 and the hinge region Ile542 (Figure 2), where
the quinazoline serves as the H-bond acceptor and the protein
residue fulfills the role of H-bond donor. In all probability, the
6-substituents modulate this hydrogen bond contact as both the
direction and magnitude (ρ = −1.2) of the electronic effect
suggest the potentiation of a critical kinase recognition element
where a partial positive charge builds on the ligand (Figure 8).
Thus, the Hammett analysis confirms the importance of the
quinazoline N1 hydrogen bond with Ile542 for TNNI3K
binding, demonstrates the extraordinary sensitivity of the hinge
H-bond interaction to electronic tuning (∼4 kcal/mol), and
defines a quantitative structure−activity relationship for kinase
hinge recognition.
Figure 7. 6-Substituted quinazolines.
that electron withdrawing groups (51, 52) diminish TNNI3K
inhibition.7 A Hammett plot (TNNI3K pIC50 vs Hammett σp,
Figure 8) revealed a strong correlation between the electronic
The electronic effect that governs the TNNI3K hinge region
H-bond interaction may be relevant for many kinase inhibitors
under development as drugs, since the vast majority engage in
an analogous H-bond interaction within their respective
targets.18 Although a clear correlation may not always be
evident due to conflicting binding effects, which obscure the
relationship, the electronic effect is anticipated to be an
energetically meaningful component of a broad range of kinase
hinge binding events. In fact, evidence of electronic modulation
in analogous H-bond interactions within other kinases is
apparent from a retrospective analysis of the published
literature.19−22 Among early EGFR inhibitors, it was noted
that incorporating electron donating groups (NH2 or OMe)
onto a quinazoline scaffold enhanced potency, with the 6,7-di-
MeO substitution pattern leading to substantial improvement
over the unsubstituted parent.19 Similarly, 6,7- or 5,7-di-MeO
quinoline inhibitors of PDGFR were 20-fold more active than
their unsubstituted counterparts.20 However, without the
benefit of protein crystal structures, the authors were unable
to rationalize this intriguing observation. A series of 6-methoxy,
7-alkoxy-quinazolines were reported as potent VEGFR
inhibitors, but models of these compounds bound to the
ATP-site revealed no specific interactions or steric limitations
Figure 8. Plot of TNNI3K pIC50 vs σp for 6-substituted quinazolines
(47−52).
properties of the 6-substituent and inhibitor affinity (R2 =
0.97), suggesting these functional groups serve as remote
modulators of ring electronics. Indeed, the 6-substituents are
expected to project into a vacant area near the opening of the
TNNI3K binding site (Figure 2) where they are free from
competing steric, hydrophobic, and conformational effects that
would interfere with the observed electronic correlation. The
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that could account for the SAR of those substiuents.21 In a
separate series of Type II inhibitors, a 6,7-di-MeO-quinoline
hinge-binding group displayed 10-fold greater affinity for
VEGFR compared to the unsubstituted quinoline, and the
authors speculated that the improved activity could arise from
potentiation of a putative aryl C8−H hydrogen bond donor
interaction to a backbone amide CO.22 However, in contrast
to the pyridyl C−H hydrogen bonds that have been observed in
kinase crystal structures, evidence that aryl C−H groups lacking
an adjacent heteroatom (such as the C8-quinazoline) serve as
H-bond donors is wanting.23
Consistent with these literature reports, we have observed
potency enhancements in the TNNI3K quinazoline template
when incorporating either the 6,7-di-MeO (10-fold) or 5,7-di-
MeO (100-fold) substituents, suggesting a similar phenomenon
is in play for multiple kinases.7 Based on the strong dependency
of TNNI3K affinity on the electronic properties of the
quinazoline substituents (Figure 8), we propose that the 6,7-
and 5,7-di-MeO substituent effect is largely due to electronic
modulation of the highly conserved hinge-binding protein
donor−ligand acceptor interaction. The dominant nature of
this hinge region amide NH hydrogen bond donor is not
surprising, since it serves as a key recognition element for ATP
binding, and its importance is evident from the fact that it is
satisfied with a hydrogen bond acceptor in nearly every
available kinase crystal structure.24 Importantly, the direction of
the electronic substituent effect (ρ = −1.2) on kinase hinge
binding is consistent with stabilizing a hydrogen bond acceptor
interaction and inconsistent with enhancing the hydrogen bond
donor strength of the ligand. Further, quantum level theory
confirms that the 5,7-di-MeO and 6,7-di-MeO substituent
patterns would increase the hydrogen bond accepting capability
of quinoline or quinazoline nitrogens to a degree that is
consistent with the enhancements they provide to kinase
affinity (vide infra). Thus, in the present study we present the
first systematic analysis of an often observed phenomenon,
which leads to the conclusion that electron donating groups
(e.g., MeO) on hinge binding heterocycles increase their kinase
affinity by augmenting the ligand hydrogen bond acceptor
strength.
Figure 9. 6-Substituted quinazolines.
Figure 10. Plot of B-RafV600E pIC50 (red) and TNNI3K pIC50 (black)
vs σp (47−52).
Figure 11. Overlay of 51 modeled in TNNI3K and B-Raf
structures.6−8
Divergent Electronic Effects Leading to Kinase
Selectivity. Quinazoline 50 and its analogues exhibit
significant activity against B-Raf, a serine/threonine kinase
that shares high ATP-binding site homology with TNNI3K
(82% similarity). We noted that the selectivity of each
compound for TNNI3K over B-Raf is sensitive to the
quinazoline substitution pattern and postulated a possible
electronic component as a driver for the TNNI3K specificity of
certain initial analogues.7 To clearly delineate this electronic
effect on kinase selectivity, quinazolines 47−52 were evaluated
for B-Raf inhibition (Figure 9) and a Hammet analysis revealed
a divergent substituent electronic effect on B-Raf potency
(Figure 10). Consistent with expected behavior based on their
weakening of the quinazoline H-bond acceptor strength,
electron withdrawing groups (51, 52) reduced activity at B-
Raf, similar to observations with TNNI3K (Figure 10).
However, electron donating groups (47−49) also diminished
activity against B-Raf leading to a biphasic substituent effect
that is indicative of divergent binding interactions for B-Raf vs
TNNI3K.
TNNI3K Leu595 residue that lies below the quinazoline is
replaced by Phe582 within the B-Raf structure. Consequently,
the quinazoline inhibitors enagage in π−π interactions in B-Raf
that are not available within TNNI3K (Figure 11). Analogous
π-stacking contacts have been observed between B-Raf and
earlier inhibitors, suggesting these interactions are a general
feature of B-Raf recognition.25 We anticipate the π-stacking
interactions between the quinazoline inhibitors and B-Raf
would be destabilized by electron-donating groups due to
enhanced repulsion between electron rich quinazolines and the
π-clouds of Phe582 and Trp530.26 Consistent with this
interpretation, substituent electronic effects on parallel and
offset π-stacking interactions have been reported that
demonstrate a correlation between π−π interaction energy
and aryl electron density.27 Thus, within B-Raf, the substituent
effect on π−π stacking opposes the electronic effect on the
hinge region H-bond interaction leading to the altered
electronic relationship for B-Raf hinge binding vs TNNI3K
(Figure 10). The lack of this competing electronic effect within
TNNI3K can be exploited to establish significant bias over B-
Raf as illustrated in 3-{[6,7-bis(methyloxy)-4-quinazolinyl]-
amino}-4-(dimethylamino)-N-methylbenzenesulfonamide hy-
drochloride (GSK114) and its relatives (e.g., 47−49),7 and
An overlay of 51 modeled in the TNNI3K and B-Raf crystal
structures (Figure 11) reveals two key residue differences.
Tyr541 of TNNI3K corresponds to Trp530 in B-Raf, and the
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Figure 12. TNNI3K activity for a variety of hinge binding heterocycles.
this serves as a well-defined example of introducing selectivity
through optimization of electrostatic complentarity.28
competing binding effects (e.g., single-point vs dual H-
bonding), which confirms the dominance of this hydrogen
bond acceptor interaction for TNNI3K recognition. In fact,
deviations from the correlation are indicative of a significant
opposing binding effect as illustrated by comparison of
pyrimidine 59 and triazine 60. While the pyrimidine has a
high HBA strength (17.6 kcal/mol) the modest TNNI3K
affinity of 59 (IC50 = 80 nM) is below expectations based on
the correlation and presumably results from the conformational
flexibility of 59 (versus fused bicycles 47−58) and the
preference of the pyrimidine for the inactive s-trans
orientation.31 By contrast, the triazine exhibits a more modest
HBA strength (16.5 kcal/mol) yet imparts a higher affinity for
TNNI3K (60, IC50 = 10 nM) as the anilino-triazine readily
adopts the s-cis conformation required for hinge binding.31
The strong relationship between TNNI3K affinity and
calculated HBA strength (Figure 13) supports the conclusion
that the electronic effect uncovered in the Hammett correlation
(Figure 8) arises from modulation of a key H-bond interaction
between the pyrimidine N and the Ile542 backbone NH. The
ability to calculate HBA strength for a broad range of
heterocycles significantly extends the scope of the analysis of
electronic effects associated with protein−ligand H-bond
interactions. There are few reports of quantitative assessments
of hydrogen bonding in structure−activity relationships for
protein−ligand interactions, and the reported correlations are
modest.32 Thus, the excellent correlation between TNNI3K
affinity and calculated HBA strength provides an instructive
example of how this computed property can be used as a tool
for optimization of ligand H-bond acceptor-protein H-bond
donor interactions.33
Calculated Hydrogen Bond Acceptor Strengths. To
expand the substituent electronic effect on TNNI3K affinity
established with 6-substituted quinazolines (Figure 8), we
synthesized and evaluated a set of TNNI3K inhibitors
containing a range of hinge-binding heterocycles with varying
electronic properites (Figure 12).6,7 The set included both
monodentate (53−57) and bidentate (58−63) hinge binders
with a variety of substituent patterns that resulted in a broad
array of TNNI3K activities. An initial inspection of the results
revealed an apparent relationship between the electronic
properties of each heterocycle and TNNI3K binding, with
electron rich heterocycles (55, 62, 63) showing exceptional
potency (<10 nM) while electron poor hinge binders (53, 56)
had weak affinity (>100 nM). The hydrogen bond acceptor
strength (HBA) of each of the examined heterocycles (30, 47−
63) was quantified using ab initio quantum chemical
calculations (see Supporting Information).29 A plot of
calculated HBA versus TNNI3K pIC50 (Figure 13) revealed a
strong correlation between the hydrogen bonding capacity of
each heterocycle and its associated TNNI3K affinity.30
Importantly, the correlation holds despite the potential for
CONCLUSIONS
■
A systematic evaluation of two key hydrogen bonds formed
between a series of TNNI3K inhibitors and their protein target
was conducted that defined fundamental relationships between
substituent electronic properties and affinity through modu-
lation of protein−ligand hydrogen bonding interactions. Two
Figure 13. Plot of TNNI3K pIC50 vs calcd HBA strength for hinge
binding heterocycles (30, 47−63).
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and the required aniline (1.5 equiv) in i-PrOH (2 mL) was stirred at
80 °C for 16 h or subjected to microwave irradiation (150 °C) for 30
min before being cooled to room temperature. Analytically pure solids
were obtained after purification by reverse-phase HPLC (49−53), by
collecting the precipitate by filtration (55), or after flash chromatog-
raphy (54).
separate and complementary correlations between Hammett σp
and TNNI3K affinity were delineated that establish the large
energetic consequence (∼4 kcal/mol) of electronically tuning
drug−receptor hydrogen bonds for both protein acceptor−
ligand donor and protein donor−ligand acceptor H-bond
contacts. The latter electronic effect was found to diverge for
the related kinase B-Raf as a result of its unique π−π
interactions with an opposing electronic preference. The
electronic effect on the TNNI3K hinge binding feature was
expanded by demonstrating the association of calculated H-
bond acceptor strength and TNNI3K affinity for a variety of
hinge binding heterocycles. The simple, quantitative relation-
ships for modulating H-bond strength defined in this study may
be generally applicable for designing drugs that target protein
kinases and for optimizing ligand−receptor H-bond inter-
actions and may also encourage further investigations of
quantitative aspects of hydrogen bonding in SAR studies.
3-{[6,7-Bis(methyloxy)-4-quinazolinyl]amino}-N-methylbenzene-
sulfonamide hydrochloride (2). Compound 2 was prepared from 4-
chloro-6,7-dimethoxyquinazoline and 3-amino-N-methylbenzenesulfo-
1
namide using method A as a yellow solid in 45% yield (42 mg): H
NMR (400 MHz, DMSO-d6) δ 10.98 (br. s., 1H), 8.85 (s, 1H), 8.05−
8.18 (m, 3H), 7.73 (t, J = 7.91 Hz, 1H), 7.67 (d, J = 7.78 Hz, 1H), 7.59
(q, J = 4.43 Hz, 1H), 7.29 (s, 1H), 4.02 (d, J = 3.76 Hz, 6H), 2.48 (s,
3H). MS (m/z) 375.1 (M + H+).
3-((6-((4-Chlorophenyl)amino)pyrimidin-4-yl)amino)-N-methyl-
benzenesulfonamide (3). Compound 3 was prepared from 3-((6-
chloropyrimidin-4-yl)amino)-N-methylbenzenesulfonamide and 4-
chloroaniline using method A as a white solid in 38% yield (60
mg): 1H NMR (400 MHz, DMSO-d6) δ 9.60 (br. s., 1H), 9.43 (br. S.,
1H), 8.36 (s, 1H), 8.10 (br. S., 1H), 7.91 (d, J = 7.78 Hz, 1H), 7.64 (d,
J = 8.28 Hz, 2H), 7.52 (t, J = 7.78 Hz, 1H), 7.44 (d, J = 4.52 Hz, 1H),
7.36 (d, J = 7.53 Hz, 3H), 6.19 (s, 1H), 2.45 (d, J = 4.52 Hz, 3H). MS
(m/z) 390.0 (M + H+).
EXPERIMENTAL SECTION
■
General Experimental. Compounds were evaluated for activity
against TNNI3K and B-Raf as previously described.6 For the TNNI3K
enzyme assay, compound IC50 values less than 10 nM could not be
accurately obtained due to titration of TNNI3K in the assay. The
purity of each inhibitor was determined to be >95% (except as noted)
on an Agilent 1100 HPLC equipped with a Sunfire C18 5.0 μm
column (3.0 mm × 50 mm) using a gradient of 10% to 100% MeCN/
H2O/0.05% TFA at 1 mL/min flow rate with detection at 220 and 254
nm. Mass determinations were conducted using an Agilent 6110
quadrupole MS with postive ESI. Preparative HPLC was conducted on
a Waters 2525 system at a flow rate of 50 mL/min with 254 nm
detection using either a Sunfire C18 OBD 5 μm column (30 mm ×
150 mm) with a gradient of MeCN/H2O/0.1% TFA or an Xbridge
C18 OBD 5 μm column (30 mm × 150 mm) with a gradient of
MeCN/aq NH4OH, pH 10. Flash column chromatography was
conducted on silica gel eluting with EtOAc−hexanes, EtOAc−
petroleum ether, or MeOH−CH2Cl2 mixtures. Compounds were
synthesized using procedures that followed reported protocols,6,7,34
and specific experimental details have been published previously for
compounds 1, 4, 7, 11, 30, and 62.6 Reagents and building blocks were
purchased and used as is or synthesized following reported
procedures.6,34
3-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-4-(ethylamino)-N-
methylbenzenesulfonamide (5). Compound 5 was prepared from 4-
chloro-7H-pyrrolo[2,3-d]pyrimidine and 3-amino-4-(ethylamino)-N-
methylbenzenesulfonamide using method B as a white solid in 12%
1
yield (28 mg): H NMR (400 MHz, DMSO-d6) δ ppm 12.52 (br. s.,
1H), 10.28 (br. S., 1H), 8.26 (s, 1H), 7.62 (dd, J = 8.78, 1.76 Hz, 1H),
7.54 (d, J = 2.01 Hz, 1H), 7.39 (br. s., 1H), 7.11 (q, J = 4.77 Hz, 1H),
6.90 (d, J = 8.78 Hz, 1H), 6.47 (br. S., 1H), 3.20 (q, J = 7.00 Hz, 2H),
2.38 (d, J = 5.02 Hz, 3H), 1.11 (t, J = 7.00 Hz, 3H). MS (m/z) 346.9
(M + H+).
N-Methyl-4-(propylamino)-3-(1H-pyrrolo[2,3-d]pyrimidin-4-
ylamino)benzenesulfonamide (6). Compound 6 was prepared from
4-chloro-7H-pyrrolo[2,3-d]pyrimidine and 3-amino-N-methyl-4-
(propylamino)benzenesulfonamide using method A as a white solid
1
in 12% yield (13 mg): H NMR (400 MHz, DMSO-d6) δ ppm 11.67
(br. s, 1H), 8.78 (s, 1H), 8.11 (s, 1H), 7.48 (s, 1H), 7.50 (d, J = 2.01
Hz, 1H), 7.12−7.19 (m, 1H), 7.05 (q, J = 4.77 Hz, 1H), 6.80 (d, J =
8.78 Hz, 1H), 6.29 (br. s, 1H), 5.79 (t, J = 5.65 Hz, 1H), 3.12 (q, J =
6.36 Hz, 2H), 2.37 (d, J = 4.77 Hz, 3H), 1.50−1.57 (m, 2H), 0.87 (t, J
= 7.40 Hz, 3H). MS (m/z) 361.1 (M + H+).
N-Methyl-2-(1H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-4-biphenyl-
sulfonamide trifluoroacetate (8). A mixture of 4-chloro-N-methyl-3-
(1H-pyrrolo[2,3-d]pyrimidin-4-ylamino)benzenesulfonamide (11) (70
mg, 0.207 mmol) and phenylboronic acid (37.9 mg, 0.311 mmol) in
1,4-dioxane (4 mL) and water (1 mL) was treated with K2CO3 (57.3
mg, 0.414 mmol) and PdCl2(dppf)·CH2Cl2 (16.92 mg, 0.021 mmol)
and heated at 140 °C for 4 h before being concentrated and subjected
to reverse phase HPLC to afford 8 as a white solid in 38% yield (41
Method A: General Coupling Method for Anilines and
Chloro-heterocycles. A mixture of a chloro-heterocycle and the
required aniline (1.3 equiv) in i-PrOH (2 mL) was treated with 1 M
aqueous HCl (1 equiv) and subjected to microwave irradiation (150
°C) for 15−60 min before being cooled to room temperature.
Analytically pure solids were obtained after purification by reverse-
phase HPLC (3, 6, 12, 16, 17, 20, 22, 24, 25, 28, 29, 34, 36, 38, 39,
40, 44, 46, 56, 57, 59) or flash column chromatography (41) or by
collecting the precipitate by filtration (2).
Method B: General Coupling Method for Anilines and
Chloro-heterocycles. A mixture of the indicated chloro-heterocycle,
the required aniline (1.3 equiv), and AgOTf (1 equiv) were dissolved
in either DMF (2 mL), NMP (2 mL), or i-PrOH (2 mL) and stirred at
80 °C overnight or subjected to microwave irradiation (150 °C) for 60
min before being diluted with MeOH and filtered. Analytically pure
solids were obtained after purification by reverse-phase HPLC (5, 10,
15, 18, 19, 21, 26, 27, 37, 43, 60, 61) or by flash column
chromatography (9, 13, 14, 42, 45, 58) or by collecting the precipitate
by filtration (23).
1
mg): H NMR (400 MHz, DMSO-d6) δ ppm 12.37 (br. s, 1H), 8.16
(s, 1H), 7.96−8.00 (m, 1H), 7.85 (m, 1H), 7.77−7.80 (m, 1H), 7.74
(d, J = 8.03 Hz, 1H), 7.65 (m, 1H), 7.46 (d, J = 7.03 Hz, 2H), 7.30−
7.38 (m, 4H), 6.41 (br. s, 1H), 2.53 (d, 3H (obscured by solvent)).
MS (m/z) 380.1 (M + H+).
N-Methyl-4-(methylthio)-3-(1H-pyrrolo[2,3-d]pyrimidin-4-
ylamino)benzenesulfonamide (9). Compound 9 was prepared from
4-chloro-1H-pyrrolo[2,3-d]pyrimidine and 3-amino-N-methyl-4-
(methylthio) benzenesulfonamide using method B as a white solid
1
in 13% yield (22 mg): H NMR (400 MHz, DMSO-d6) δ ppm 11.73
(br. s, 1H), 9.13 (s, 1H), 8.10 (s, 1H), 7.71−7.75 (m, 1H), 7.66 (dd, J
= 1.88, 8.41 Hz, 1H), 7.53 (d, J = 8.53 Hz, 1H), 7.46 (q, J = 4.68 Hz,
1H), 7.16−7.22 (m, 1H), 6.41 (br. s, 1H), 2.47 (s, 3H), 2.44 (d, J =
5.02 Hz, 3H). MS (m/z) 349.9 (M + H+).
Method C: General Method for Substitution of 19 with
Cyclic Amines. A mixture of 19 in DMSO (1 mL) was treated with
the indicated cyclic amine (11−32 equiv) and subjected to microwave
irradiation at 150 °C for 30 min before being concentrated and
filtered. Analytically pure solids were obtained after purification by
reverse-phase HPLC (32, 33, 35).
4-Fluoro-N-methyl-3-(1H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-
benzenesulfonamide (10). Compound 10 was prepared from 4-
chloro-1H-pyrrolo[2,3-d]pyrimidine and 3-amino-4-fluoro-N-methyl-
benzenesulfonamide using method B as a white solid in 42% yield (44
Method D: General Coupling Method for Anilines and 4-
Chloroquinazolines. A mixture of the required 4-chloroquinazoline
1
mg): H NMR (400 MHz, DMSO-d6) δ ppm 11.84 (br. s, 1H), 9.42
G
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Journal of Medicinal Chemistry
Article
(s, 1H), 8.28 (dd, J = 2.13, 7.40 Hz, 1H), 8.22 (s, 1H), 7.49−7.62 (m,
3H), 7.26−7.31 (m, 1H), 6.71−6.79 (m, 1H), 2.48 (d, J = 4.77 Hz,
3H). MS (m/z) 322.0 (M + H+).
(d, J = 8.78 Hz, 2H), 6.00 (s, 1H), 2.49 (d, J = 5.02 Hz, 3H), 2.35 (s,
3H. MS (m/z) 404.0 (M + H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-N-methyl-
4-(methylthio)benzenesulfonamide Trifluoroacetate (18). Com-
pound 18 was prepared from 6-chloro-N-(4-chlorophenyl)-4-pyrimi-
dinamine and 3-amino-N-methyl-4-(methylthio)benzenesulfonamide
using method B as a pale brown solid in 33% yield (92 mg): 1H NMR
(400 MHz, CD3OD) δ ppm 8.27−8.30 (m, 1H), 7.81−7.86 (m, 1H),
7.79 (d, J = 2.01 Hz, 1H), 7.56−7.61 (m, 1H), 7.39−7.45 (m, 4H),
5.85−5.88 (m, 1H), 2.53−2.59 (m, 6H). MS (m/z) 435.9 (M + H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-4-fluoro-N-
methylbenzenesulfonamide Trifluoroacetate (19). Compound 19
was prepared from 6-chloro-N-(4-chlorophenyl)-4-pyrimidinamine
and 3-amino-4-fluoro-N-methylbenzenesulfonamide using method B
as a brown solid in 15% yield (39 mg): 1H NMR (400 MHz, DMSO-
d6) δ ppm 9.53 (s, 1H), 9.39 (s, 1H), 8.44−8.53 (m, 1H), 8.34 (s,
1H), 7.62 (d, J = 8.78 Hz, 2H), 7.45−7.56 (m, 3H), 7.37 (d, J = 8.78
Hz, 2H), 6.30 (s, 1H), 2.45 (d, J = 4.77 Hz, 3H). MS (m/z) 407.9 (M
+ H+).
4-Chloro-3-({6-[(4-chlorophenyl)amino]-4-pyrimidinyl}amino)-N-
methylbenzenesulfonamide Trifluoroacetate (20). Compound 20
was prepared from 6-chloro-N-(4-chlorophenyl)-4-pyrimidinamine
and 3-amino-4-chloro-N-methylbenzenesulfonamide using method A
as a white solid in 7% yield (14 mg): 1H NMR (400 MHz, DMSO-d6)
δ ppm 9.52 (br. s., 1H), 9.19 (br. s., 1H), 8.30 (s, 1H), 8.21 (d, J =
2.01 Hz, 1H), 7.76 (d, J = 8.28 Hz, 1H), 7.58−7.65 (m, 3H), 7.48−
7.55 (m, 1H), 7.35−7.42 (m, 2H), 6.23 (s, 1H), 2.46 (d, J = 5.02 Hz,
3H). MS (m/z) 424.0 (M + H+).
N-Methyl-3-(1H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-4-
[(trifluoromethyl)oxy]benzenesulfonamide (12). Compound 12 was
prepared from 4-chloro-1H-pyrrolo[2,3-d]pyrimidine and 3-amino-N-
methyl-4-[(trifluoromethyl)oxy]benzenesulfonamide using method A
as a yellow solid in 7% yield (8 mg): 1H NMR (400 MHz, DMSO-d6)
δ ppm 11.85 (br. s., 1H), 9.43 (br. s., 1H), 8.30 (d, J = 1.76 Hz, 1H),
8.21 (s, 1H), 7.59−7.71 (m, 3H), 7.28 (d, J = 3.51 Hz, 1H), 6.71 (d, J
= 3.01 Hz, 1H), 2.49 (d, 3H, (obscured by solvent)). MS (m/z) 388.0
(M + H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-N-methyl-
4-(methylamino)benzenesulfonamide (13). Compound 13 was
prepared from 6-chloro-N-(4-chlorophenyl)-4-pyrimidinamine and 3-
amino-N-methyl-4-(methylamino)benzenesulfonamide using method
B as a purple solid in 6% yield (20 mg): 1H NMR (400 MHz, DMSO-
d6) δ ppm 9.24 (s, 1H), 8.37 (s, 1H), 8.21 (s, 1H), 7.62 (d, J = 9.03
Hz, 2H) 7.50 (dd, J = 8.53, 2.01 Hz, 1H), 7.44 (d, J = 2.01 Hz, 1H),
7.29 (d, J = 8.78 Hz, 2H), 7.08 (q, J = 5.10 Hz, 1H), 6.72 (d, J = 8.78
Hz, 1H), 5.94 (q, J = 4.60 Hz, 1H), 5.65 (s, 1H), 2.77 (d, J = 4.77 Hz,
3H), 2.36 (d, J = 5.02 Hz, 4H). MS (m/z) 419.1 (M + H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-N-methyl-
4-(ethylamino)benzenesulfonamide (14). Compound 14 was pre-
pared from 6-chloro-N-(4-chlorophenyl)-4-pyrimidinamine and 3-
amino-N-methyl-4-(ethylamino)benzenesulfonamide using method B
as a brown solid in 31% yield (95 mg): 1H NMR (400 MHz, DMSO-
d6) δ ppm 9.26 (s, 1H) 8.37 (s, 1H) 8.21 (s, 1H) 7.61 (m, J = 9.03 Hz,
2H) 7.41−7.52 (m, 2H) 7.30 (m, J = 9.04 Hz, 2H) 7.09 (q, J = 4.94
Hz, 1H) 6.79 (d, J = 9.29 Hz, 1H) 5.74 (t, J = 5.52 Hz, 1H) 5.70 (s,
1H) 3.13−3.24 (m, 2H) 2.36 (d, J = 5.02 Hz, 3H) 1.15 (t, J = 7.03 Hz,
3H). MS (m/z) 433.1 (M + H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-N-methyl-
4-[(trifluoromethyl)oxy]-benzenesulfonamide Trifluoroacetate (21).
Compound 21 was prepared from 6-chloro-N-(4-chlorophenyl)-4-
pyrimidinamine and 3-amino-N-methyl-4-[(trifluoromethyl)oxy]-
benzenesulfonamide using method B as beige solid in 9% yield (49
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-N-methyl-
4-(propylamino)benzenesulfonamide (15). Compound 15 was
prepared from 6-chloro-N-(4-chlorophenyl)-4-pyrimidinamine and 3-
amino-N-methyl-4-(propylamino)benzenesulfonamide using method
B as a gray solid in 15% yield (50 mg): 1H NMR (400 MHz, DMSO-
d6) δ ppm 9.78 (br. s., 1H), 9.05 (br. s., 1H), 8.35 (s, 1H), 7.49−7.59
(m, 3H), 7.45 (d, J = 2.01 Hz, 1H), 7.37 (d, J = 9.03 Hz, 2H), 7.10 (q,
J = 4.94 Hz, 1H), 6.84 (d, J = 8.78 Hz, 1H), 5.70 (s, 1H), 3.13 (t, J =
7.03 Hz, 2H), 2.37 (d, J = 4.77 Hz, 3H), 1.55 (sxt, J = 7.28 Hz, 2H),
0.89 (t, J = 7.40 Hz, 3H). MS (m/z) 447.1 (M + H+).
1
mg): H NMR (400 MHz, DMSO-d6) δ ppm 9.49 (br. s, 1H), 9.30−
9.34 (m, 1H), 8.49−8.51 (m, 1H), 8.32 (s, 1H), 7.61 (d, J = 9.03 Hz,
4H), 7.52−7.56 (m, 1H), 7.37 (d, J = 9.04 Hz, 2H), 6.33 (s, 1H), 2.48
(d, J = 5.02 Hz, 3H). MS (m/z) 474.0 (M + H+).
4-Hydroxy-N-methyl-3-(1H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-
benzenesulfonamide Trifluoroacetate (22). Compound 22 was
prepared from 6-chloro-7-deazapurine and 3-amino-4-hydroxy-N-
methylbenzenesulfonamide using method A as a light green solid in
38% yield (54 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.57 (br.
s, 1H), 10.46 (br. s, 1H), 8.31 (s, 1H), 7.88 (br. s, 1H), 7.62 (m, 1H),
7.46 (br. s, 1H), 7.35 (q, J = 5.27 Hz, 1H), 7.19 (d, J = 8.53 Hz, 1H),
6.75−6.82 (m, 1H), 2.43 (d, J = 5.02 Hz, 3H). MS (m/z) 320.1 (M +
H+).
4-Amino-3-({6-[(4-chlorophenyl)amino]-4-pyrimidinyl}amino)-N-
methylbenzenesulfonamide trifluoroacetate (16). Compound 16
was prepared from 6-chloro-N-(4-chlorophenyl)-4-pyrimidinamine
and 3,4-diamino-N-methylbenzenesulfonamide using method A as a
N-Methyl-4-(methyloxy)-3-(1H-pyrrolo[2,3-d]pyrimidin-4-
ylamino)benzenesulfonamide (23). Compound 23 was prepared
from 6-chloro-7-deazapurine and 3-amino-N-methyl-4-(methyloxy)-
benzenesulfonamide using method B as a white solid in 45% yield (74
1
brown solid in 2% yield (32 mg): H NMR (400 MHz, DMSO-d6) δ
ppm 10.02 (br. s., 1H, C4-aniline-NH), 9.38 (br. s., 1H, C6-aniline-
NH), 8.41 (s, 1H, pyridine C2-H), 7.37−7.58 (m, 6H, 4-chlorophenyl
C-H, benzenesulfonamide C2-H, benzenesulfonamide C6-H), 7.07−
7.15 (m, 1H, SO2NHCH3) 6.88 (d, J = 8.53 Hz, 1H,
benzenesulfonamide C5-H), 5.70 (br. s., 1H, pyridine C5-H), 2.37
(d, J = 4.02 Hz, 3H, SO2NHCH3). 13C NMR (500 MHz, DMSO-d6) δ
158.86 (pyrimidine C6), 158.58 (pyrimidine C4), 154.50 (pyrimidine
C2), 148.74 (benzenesulfonamide C4), 138.04 (4-chlorophenyl C1),
129.30 (4-chlorophenyl C4), 129.25 (4-chlorophenyl C3,C5), 127.53
(benzenesulfonamide C2,C6), 125.31 (benzenesulfonamide C1),
122.94 (4-chlorophenyl C4), 119.93 (benzenesulfonamide C3),
115.17 (benzenesulfonamide C5), 83.50 (pyrimidine C5), 29.04
(SO2NHCH3). For compound 16, the 1H NMR and 13C NMR
resonances were assigned on the basis of gCOSY45, gHMQC,
gHMBC, and 13C GASPE spectra, and all 2D correlations are in
agreement with the assigned structure. MS (m/z) 405.0 (M + H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-N,4-dime-
thylbenzenesulfonamide Trifluoroacetate (17). Compound 17 was
prepared from 3-[(6-chloro-4-pyrimidinyl)amino]-4-(diethylamino)-
N-methylbenzenesulfonamide and 4-chloroaniline using method A as a
white solid in 23% yield (160 mg): 1H NMR (400 MHz, DMSO-d6) δ
(ppm) 9.53 (br. s., 1H), 9.04 (br. s., 1H), 8.33 (s, 1H), 7.89 (s, 1H),
7.65 (d, J = 9.04 Hz, 2H), 7.57 (s, 2H), 7.48 (q, J = 5.10 Hz, 1H), 7.41
1
mg): H NMR (400 MHz, DMSO-d6) δ ppm 11.79 (br. s, 1H), 8.69
(s, 1H), 8.43 (d, J = 2.26 Hz, 1H), 8.22 (s, 1H), 7.54 (dd, J = 2.26,
8.78 Hz, 1H), 7.37 (br. s, 1H), 7.28 (d, J = 8.53 Hz, 1H), 7.25 (d, 1H),
6.69 (d, J = 3.51 Hz, 1H), 3.93 (s, 3H), 2.44 (s, 3H). MS (m/z) 333.9
(M + H+).
4-(Ethyloxy)-N-methyl-3-(1H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-
benzenesulfonamide (24). Compound 24 was prepared from 6-
chloro-7-deazapurine and 3-amino-4-(ethyloxy)-N-methylbenzenesul-
1
fonamide using method A as a white solid in 39% yield (45 mg): H
NMR (400 MHz, DMSO-d6) δ ppm 11.81 (br. s, 1H), 8.58 (s, 1H),
8.45 (d, J = 2.26 Hz, 1H), 8.24 (s, 1H), 7.51 (dd, J = 8.53, 2.26 Hz,
1H), 7.27 (d, J = 5.52 Hz, 1H), 7.25 (s, 1H), 6.62 (d, J = 3.51 Hz, 1H),
4.20 (q, J = 6.86 Hz, 2H), 2.44 (s, 3 H), 1.35 (t, J = 7.03 Hz, 3H). MS
(m/z) 348.0 (M + H+).
N-Methyl-3-(1H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-4-[(2,2,2-
trifluoroethyl)oxy]benzenesulfonamide Trifluoroacetate (25). Com-
pound 25 was prepared from 6-chloro-7-deazapurine and 3-amino-N-
methyl-4-[(2,2,2-trifluoroethyl)oxy]benzenesulfonamide using method
1
A as an orange solid in 47% yield (79 mg): H NMR (400 MHz,
DMSO-d6) δ ppm 12.15 (br. s, 1H), 8.25 (s, 1H), 8.14 (br. s, 1H),
7.68 (m, 1H), 7.44−7.52 (m, 2H), 7.34 (br. s, 1H), 6.64 (br. s, 1H),
H
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4.92 (q, J = 8.78 Hz, 2H), 2.46 (d, J = 5.02 Hz, 3H). MS (m/z) 402.0
(M + H+).
Compound 34 was prepared from 4-chloro-1H-pyrrolo[2,3-d]-
pyrimidine and 3-amino-4-(3,3-difluoro-1-pyrrolidinyl)-N-methylben-
zenesulfonamide using method A as a brown solid in 38% yield (98
mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 8.25 (s, 1H), 7.61−7.67
(m, 2H), 7.31−7.40 (m, 1H), 7.24−7.31 (m, 1H), 7.08 (d, J = 8.03
Hz, 1H), 3.77 (t, J = 13.30 Hz, 2H), 3.57 (t, J = 7.15 Hz, 2H), 2.32−
2.46 (m, 5H). MS (m/z) 409.0 (M + H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-4-hydroxy-
N-methylbenzenesulfonamide Trifluoroacetate (26). Compound 26
was prepared from 6-chloro-N-(4-chlorophenyl)-4-pyrimidinamine
and 3-amino-4-hydroxy-N-methylbenzenesulfonamide using method
B as a brown solid in 16% yield (56 mg): 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.71 (br. s., 1H), 9.18 (br. s., 1H), 8.35 (s, 1H),
8.04 (s, 1H), 7.57 (d, J = 9.03 Hz, 2H), 7.43 (dd, J = 2.26, 8.53 Hz,
1H), 7.39 (d, J = 8.78 Hz, 2H), 7.26 (q, J = 4.94 Hz, 1H), 7.07 (d, J =
8.53 Hz, 1H), 6.15 (s, 1H), 2.40 (d, J = 4.77 Hz, 3H). MS (m/z) 405.9
(M + H+).
N-Methyl-4-(1-piperidinyl)-3-(1H-pyrrolo[2,3-d]pyrimidin-4-
ylamino)benzenesulfonamide Trifluoroacetate (35). Compound 35
was prepared from 19 and piperidine using method C as a yellow solid
1
in 54% yield (43 mg): H NMR (400 MHz, CD3OD) δ ppm 8.25−
8.29 (m, 1H), 7.86−7.93 (m, 2H), 7.50 (d, J = 3.76 Hz, 1H), 7.38−
7.45 (m, 1H), 6.80 (br. s, 1H), 3.01−3.09 (m, 4H), 2.56−2.62 (m,
3H), 1.41−1.51 (m, 2H), 1.27−1.37 (m, 4H). MS (m/z) 387.1 (M +
H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-N-methyl-
4-(methyloxy)benzenesulfonamide Trifluoroacetate (27). Com-
pound 27 was prepared from 6-chloro-N-(4-chlorophenyl)-4-pyrimi-
dinamine and 3-amino-N-methyl-4-(methyloxy)benzenesulfonamide
4-(3,3-Difluoro-1-piperidinyl)-N-methyl-3-(1H-pyrrolo[2,3-d]-
pyrimidin-4-ylamino)benzenesulfonamide Trifluoroacetate (36).
Compound 36 was prepared from 4-chloro-1H-pyrrolo[2,3-d]-
pyrimidine and 4-(3,3-difluoro-1-piperidinyl)-N-methyl-3-
(methylamino)benzenesulfonamide using method A as a brown solid
in 42% yield (110 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 12.56
(br. S, 1H), 8.30 (s, 1H), 8.10 (br. S, 1H), 7.70 (br. s., 1H), 7.37−7.57
(m, 3H), 6.68 (br. s., 1H), 3.27 (t, J = 11.04 Hz, 2H), 3.02 (br. s., 2H),
2.47 (d, J = 5.02 Hz, 3H), 1.92 (br. s., 2H), 1.48 (br. s., 2H). MS (m/
z) 423.1 (M + H+).
1
using method B as a white solid in 63% yield (313 mg): H NMR
(400 MHz, DMSO-d6) δ ppm 9.62 (br. s., 1H), 9.10 (br. s., 1H), 8.34
(s, 1H), 8.25 (br. s., 1H), 7.50−7.61 (m, 3H), 7.39 (d, J = 8.78 Hz,
2H), 7.34 (q, J = 4.85 Hz, 1H), 7.29 (s, 1H), 6.21 (s, 1H), 3.93 (s,
3H), 2.42 (d, J = 5.02 Hz, 3H). MS (m/z) 420.9 (M + H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-4-(ethyl-
oxy)-N-methylbenzenesulfonamide Trifluoroacetate (28). Com-
pound 28 was prepared from 6-chloro-N-(4-chlorophenyl)-4-pyrimi-
dinamine and 3-amino-4-(ethyloxy)-N-methylbenzenesulfonamide
1
using method A as a white solid in 16% yield (100 mg): H NMR
4-[Ethyl(methyl)amino]-N-methyl-3-(1H-pyrrolo[2,3-d]pyrimidin-
4-ylamino)benzenesulfonamide Trifluoroacetate (37). Compound
37 was prepared from 4-chloro-1H-pyrrolo[2,3-d] pyrimidine and 3-
amino-4-[ethyl(methyl) amino]-N-methylbenzenesulfonamide using
(400 MHz, DMSO-d6) δ ppm 9.34 (s, 1H), 8.62 (br. s., 1H), 8.27 (s,
2H), 7.54−7.62 (m, 2H), 7.43 (dd, J = 8.49, 2.09 Hz, 1H), 7.30−7.35
(m, 2H), 7.28 (q, J = 5.07 Hz, 1H), 7.21 (d, J = 8.60 Hz, 1H), 6.19 (s,
1H), 4.18 (q, J = 6.98 Hz, 2H), 2.39 (d, J = 5.07 Hz, 3H), 1.34 (t, J =
6.95 Hz, 3H). MS (m/z) 434.2 (M + H+).
1
method B as a white solid in 11% yield (26 mg): H NMR (400
MHz, CD3OD) δ ppm 8.19−8.29 (m, 1H), 7.93 (d, J = 2.26 Hz, 1H),
7.84 (dd, J = 2.26, 8.78 Hz, 1H), 7.42−7.47 (m, 1H), 7.37−7.42 (m,
1H), 6.71 (br. s, 1H), 3.12−3.21 (m, 2H), 2.82−2.86 (m, 3H), 2.56−
2.60 (m, 3H), 0.96−1.05 (m, 3H). MS (m/z) 361.0 (M + H+).
N-Methyl-4-[methyl(2,2,2-trifluoroethyl)amino]-3-(1H-pyrrolo-
[2,3-d]pyrimidin-4-ylamino)benzenesulfonamide Trifluoroacetate
(38). Compound 38 was prepared from 4-chloro-1H-pyrrolo[2,3-
d]pyrimidine and 3-amino-N-methyl-4-[methyl(2,2,2-trifluoroethyl)-
amino]benzenesulfonamide using method A as a purple solid in 4%
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-N-methyl-
4-[(2,2,2-trifluoroethyl)oxy]-benzenesulfonamide Trifluoroacetate
(29). Compound 29 was prepared from 6-chloro-N-(4-chlorophen-
yl)-4-pyrimidinamine and 3-amino-N-methyl-4-[(2,2,2-trifluoroethyl)-
oxy]benzenesulfonamide using method A as a white solid in 39% yield
1
(100 mg): H NMR (400 MHz, DMSO-d6) δ ppm 9.58 (br. s., 1H),
9.07 (br. s., 1H), 8.29 (s, 1H), 8.07 (d, J = 2.21 Hz, 1H), 7.52−7.59
(m, 3H), 7.38−7.44 (m, 2H), 7.31−7.38 (m, 2H), 6.11 (s, 1H), 4.89
(q, J = 8.82 Hz, 2H), 2.41 (d, J = 4.85 Hz, 3H). MS (m/z) 488.0 (M +
H+).
N-Methyl-4-(1-pyrrolidinyl)-3-(1H-pyrrolo[2,3-d]pyrimidin-4-
ylamino)benzenesulfonamide (31). A mixture of 19 (45 mg, 0.140
mmol) and pyrrolidine (0.5 mL, 6.05 mmol) was stirred at 80 °C
overnight before being concentrated. The residue was triturated with
EtOAc and dried to afford 31 as a tan solid in 41% yield (24 mg): 1H
NMR (400 MHz, DMSO-d6) δ ppm 11.63 (br. s, 1H), 9.00 (s, 1H),
8.09 (s, 1H), 7.45−7.53 (m, 2H), 7.07−7.16 (m, 2H), 6.88 (d, J = 8.53
Hz, 1H), 6.24 (br. s, 1H), 2.87 (t, J = 6.65 Hz, 2H), 2.35−2.40 (m,
3H), 1.73−1.83 (m, 4H), 1.65−1.73 (m, 2H). MS (m/z) 372.9 (M +
H+).
1
yield (2 mg): H NMR (400 MHz, DMSO-d6) δ ppm 12.59 (br. s.,
1H), 8.60 (s, 1H), 8.28 (s, 1H), 7.69−7.74 (m, 1H), 7.49 (d, J = 8.53
Hz, 1H), 7.42 (q, J = 4.85 Hz, 1H), 7.30−7.38 (m, 1H), 6.62 (dd, J =
3.51, 1.76 Hz, 1H), 3.93−4.02 (m, 2H), 2.93 (s, 3H), 2.44 (d, J = 5.02
Hz, 3H). MS (m/z) 415.1 (M + H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-N-methyl-
4-(1-pyrrolidinyl)benzenesulfonamide (39). Compound 39 was
prepared from 3-[(6-chloro-4-pyrimidinyl)amino]-N-methyl-4-(1-
pyrrolidinyl)benzenesulfonamide and 4-chloroaniline using method
A as a white solid in 25% yield (73 mg): 1H NMR (400 MHz, DMSO-
d6) δ ppm 9.69 (br. s., 1H), 9.23 (br. s., 1H), 8.29 (s, 1H), 7.48−7.60
(m, 3H), 7.44 (d, J = 2.26 Hz, 1H), 7.36 (d, J = 8.78 Hz, 2H), 7.18 (q,
J = 4.85 Hz, 1H), 6.92 (d, J = 9.03 Hz, 1H), 5.62 (br. s., 1H), 3.33 (br.
s., 4H), 2.38 (d, J = 5.02 Hz, 3H), 1.84 (br. s., 4H). MS (m/z) 459.1
(M + H+).
N-Methyl-4-(2-methyl-1-pyrrolidinyl)-3-(1H-pyrrolo[2,3-d]-
pyrimidin-4-ylamino)benzenesulfonamide Trifluoroacetate (32).
Compound 32 was prepared from 19 and 2-methylpyrrolidine using
1
method C as a tan solid in 48% yield (34 mg): H NMR (400 MHz,
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-N-methyl-
4-(2-methyl-1-pyrrolidinyl)benzenesulfonamide Trifluoroacetate
(40). Compound 40 was prepared from 3-[(6-chloro-4-pyrimidinyl)-
amino]-N-methyl-4-(2-methyl-1-pyrrolidinyl)benzenesulfonamide and
4-chloroaniline using method A as a brown solid in 30% yield (206
CD3OD) δ ppm 8.21 (s, 1H), 7.79 (dd, J = 1.88, 8.91 Hz, 1H), 7.75
(d, J = 2.01 Hz, 1H), 7.42 (d, J = 3.01 Hz, 1H), 7.19 (d, J = 9.04 Hz,
1H), 6.58 (br. s, 1H), 3.95 (t, J = 6.42 Hz, 1H), 3.46−3.59 (m, 1H),
3.22−3.29 (m, 1H), 2.56 (s, 3H), 2.08−2.19 (m, 1H), 1.83−1.96 (m,
1H), 1.65−1.81 (m, 1H), 1.45−1.59 (m, 1H), 1.14 (d, 3H). MS (m/z)
387.1 (M + H+).
1
mg): H NMR (400 MHz, DMSO-d6) δ ppm 9.94 (br. s., 1H), 9.54
(br. s., 1H), 8.35 (s, 1H), 7.49−7.57 (m, 3H), 7.38 (d, J = 8.78 Hz,
2H), 7.20−7.26 (m, 1H), 7.00−7.05 (m, 2H), 5.67−5.74 (m, 1H),
3.85−3.95 (m, 1H), 3.48 (br. s., 1H), 3.17 (s, 1H), 2.40 (d, J = 4.77
Hz, 4H), 2.05−2.15 (m, 1H), 1.82−1.92 (m, 1H), 1.61−1.74 (m, 1H),
1.42−1.53 (m, 1H), 1.03 (d, J = 5.77 Hz, 3H). MS (m/z) 473.1 (M +
H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-4-(2,5-di-
methyl-1-pyrrolidinyl)-N-methylbenzenesulfonamide Trifluoroace-
tate (41). Compound 41 was prepared from 3-[(6-chloro-4-
pyrimidinyl)amino]-4-(2,5-dimethyl-1-pyrrolidinyl)-N-methylbenze-
nesulfonamide and 4-chloroaniline using method A in 32% yield (12
4-(2,5-Dimethyl-1-pyrrolidinyl)-N-methyl-3-(1H-pyrrolo[2,3-d]-
pyrimidin-4-ylamino)benzenesulfonamide Trifluoroacetate (33).
Compound 33 was prepared from 19 and 2,5-dimethylpyrrolidine
1
using method C as an off-white solid in 8% yield (10 mg): H NMR
(400 MHz, DMSO-d6) δ ppm 12.43 (br. s, 1H), 10.05 (br s., 1H), 8.37
(s, 1H), 7.55 (m., 2H), 7.43 (s, 1H), 7.31 (m, 2H), 6.52 (br. s, 1H),
3.65 (m, 2H), 2.43 (d, J = 5.04 Hz, 3H), 2.01 (m, 2H), 1.66 (m, 2H),
1.05 (d, J = 5.79 Hz, 6H). MS (m/z) 401.1 (M + H+).
4-(3,3-Difluoro-1-pyrrolidinyl)-N-methyl-3-(1H-pyrrolo[2,3-d]-
pyrimidin-4-ylamino)benzenesulfonamide Trifluoroacetate (34).
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mg, 85% purity): 1H NMR (400 MHz, DMSO-d6) δ ppm 9.25 (br. s.,
1H), 8.86 (br. s., 1H), 8.15 (s, 1H), 7.70 (s, 1H), 7.52 (s, 2H), 7.45 (q,
J = 5.15 Hz, 1H), 7.35 (d, J = 8.38 Hz, 2H), 7.14 (d, 2H), 5.89 (s, 1H),
2.85 (d, J = 6.62 Hz, 2H), 2.76−2.83 (m, 1H), 2.38 (d, J = 4.85 Hz,
3H), 1.79 (m, J = 6.62 Hz, 1H), 1.15 (d, J = 7.06 Hz, 6H), 0.95 (d, J =
6.62 Hz, 6H). MS (m/z) 486.0 (M + H+).
1H), 8.78−8.87 (m, 1H), 8.75 (s, 1H), 8.06 (dd, J = 8.91, 1.88 Hz,
1H), 7.87 (d, J = 9.04 Hz, 1H), 7.74 (d, J = 2.01 Hz, 1H), 7.63 (dd, J =
8.78, 2.26 Hz, 1H), 7.32 (q, J = 5.02 Hz, 1H), 7.23 (d, J = 8.78 Hz,
1H), 2.80 (s, 6H), 2.43 (d, J = 5.02 Hz, 3H). MS (m/z) 387.0 (M +
H+).
3-((6-Aminoquinazolin-4-yl)amino)-4-(dimethylamino)-N-meth-
ylbenzenesulfonamide (48). A mixture of 52 (715 mg, 1.78 mmol) in
MeOH (50 mL) was treated with 10% Pd/C (189 mg, 0.178 mmol)
and stirred under H2 (balloon) at 25 °C for 2.5 h before being filtered
through Celite and concentrated. The residue was dissolved in
CH2Cl2/MeOH, washed with saturated aqueous NaHCO3, dried
(Na2SO4), and concentrated to give 48 as a yellow solid in 59% yield
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-4-(3,3-di-
fluoro-1-pyrrolidinyl)-N-methylbenzenesulfonamide Trifluoroace-
tate (42). Compound 42 was prepared from 6-chloro-N-(4-
chlorophenyl)-4-pyrimidinamine and 3-amino-4-(3,3-difluoro-1-pyrro-
lidinyl)-N-methylbenzenesulfonamide using method B as a brown
1
solid in 1% yield (6 mg): H NMR (400 MHz, CD3OD) δ ppm 8.18
1
(d, J = 0.75 Hz, 1H), 7.61 (dd, J = 8.78, 2.26 Hz, 1H), 7.55 (d, J = 2.26
Hz, 1H), 7.26−7.33 (m, 4H), 6.98 (d, J = 8.78 Hz, 1H), 5.62 (s, 1H),
3.62 (t, J = 13.05 Hz, 2H), 3.49 (t, J = 7.03 Hz, 2H), 2.40 (s, 3H),
2.27−2.38 (m, 2H). MS (m/z) 495.1 (M + H+).
(433 mg, 90% purity): H NMR (400 MHz, DMSO-d6) δ ppm 9.05
(br. s., 1H), 8.31 (s, 1H), 8.03−8.11 (m, 2H), 7.50 (dd, J = 8.66, 1.88
Hz, 1H), 7.22 (d, J = 8.53 Hz, 1H), 6.94 (dd, J = 8.91, 1.88 Hz, 1H),
6.60−6.69 (m, 1H), 6.55 (d, J = 2.01 Hz, 1H), 2.80 (d, J = 4.77 Hz,
3H), 2.76 (s, 6H), 2.43 (s, 3H). MS (m/z) 372.9 (M + H+).
4-(Dimethylamino)-N-methyl-3-{[6-(methyloxy)-4-quinazolinyl]-
amino}benzenesulfonamide Trifluoroacetate (49). Compound 49
was prepared from 4-chloro-6-(methyloxy)quinazoline and 3-amino-4-
(dimethylamino)-N-methylbenzenesulfonamide using method D as a
yellow solid in 50% yield (143 mg, 90% purity): 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.41 (s, 1H), 8.36 (s, 1H), 7.97 (d, J = 2.26 Hz,
1H), 7.73 (s, 1H), 7.68 (dd, J = 8.78, 2.26 Hz, 1H), 7.40−7.50 (m,
2H), 7.20 (s, 1H), 4.88 (q, J = 8.78 Hz, 2H), 3.95 (s, 3H), 3.94 (s,
3H), 2.46 (d, J = 4.77 Hz, 3H). MS (m/z) 388.1 (M + H+).
4-(Dimethylamino)-N-methyl-3-(4-quinazolinylamino)-
benzenesulfonamide Trifluoroacetate (50). Compound 50 was
prepared from 4-chloroquinazoline and 3-amino-4-(dimethylamino)-
N-methylbenzenesulfonamide using method D as an orange solid in
12% yield (30 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 10.77 (br.
s., 1H), 8.78−8.87 (m, 1H), 8.75 (s, 1H), 8.06 (dd, J = 8.91, 1.88 Hz,
1H), 7.87 (d, J = 9.04 Hz, 1H), 7.74 (d, J = 2.01 Hz, 1H), 7.63 (dd, J =
8.78, 2.26 Hz, 1H), 7.32 (q, J = 5.02 Hz, 1H), 7.23 (d, J = 8.78 Hz,
1H), 2.80 (s, 6H), 2.43 (d, J = 5.02 Hz, 3H). MS (m/z) 358.1 (M +
H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-N-methyl-
4-[(2R)-2-(trifluoromethyl)-1-pyrrolidinyl]benzenesulfonamide Tri-
fluoroacetate (43). Compound 43 was prepared from 6-chloro-N-
(4-chlorophenyl)-4-pyrimidinamine and 3-amino-N-methyl-4-[(2R)-2-
(trifluoromethyl)-1-pyrrolidinyl]benzenesulfonamide using method B
as a white solid in 9% yield (38 mg): 1H NMR (400 MHz, DMSO-d6)
δ ppm 9.57 (br. s., 1 H), 9.09 (br. s., 1 H), 8.30 (s, 1 H), 7.69 (d, J =
2.26 Hz, 1 H), 7.56 (d, J = 9.03 Hz, 2 H), 7.52 (dd, J = 8.78, 2.26 Hz, 1
H), 7.42 (d, J = 8.78 Hz, 1 H), 7.35 (d, J = 9.03 Hz, 3 H), 5.78 (s, 1
H), 4.75−4.88 (m, 1 H), 3.56−3.66 (m, 1 H), 3.13−3.24 (m, 1 H),
2.42 (d, J = 5.02 Hz, 3 H), 2.12−2.22 (m, 1 H), 1.88−2.00 (m, 2 H),
1.75−1.87 (m, 1 H). MS (m/z) 527.1 (M + H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-4-(3,3-di-
fluoro-1-piperidinyl)-N-methylbenzenesulfonamide Trifluoracetate
(44). Compound 44 was prepared from 6-chloro-N-(4-chlorophen-
yl)-4-pyrimidinamine and 3-amino-4-(3,3-difluoro-1-piperidinyl)-N-
methylbenzenesulfonamide using method A as a white solid in 14%
1
yield (76 mg): H NMR (400 MHz, DMSO-d6) δ ppm 9.68 (br. s.,
1H), 8.93 (br. s., 1H), 8.34 (s, 1H), 7.94 (br. s., 1H), 7.59 (d, J = 8.78
Hz, 2H), 7.56 (d, J = 8.53 Hz, 1H), 7.43 (q, J = 4.94 Hz, 1H), 7.38 (d,
J = 8.78 Hz, 2H), 7.34 (d, J = 8.53 Hz, 1H), 5.99 (s, 1H), 3.27 (t, J =
11.54 Hz, 2H), 3.03 (d, J = 5.02 Hz, 2H), 2.43 (d, J = 5.02 Hz, 3H),
1.94−2.06 (m, 2H), 1.67−1.75 (m, 2H). MS (m/z) 509.1 (M + H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-4-[ethyl-
(methyl)amino]-N-methylbenzenesulfonamide Trifluoroacetate
(45). Compound 45 was prepared from 6-chloro-N-(4-chlorophen-
yl)-4-pyrimidinamine and 3-amino-4-[ethyl(methyl)amino]-N-methyl-
benzenesulfonamide using method B as a pale yellow solid in 14%
3-[(6-Chloro-4-quinazolinyl)amino]-4-(dimethylamino)-N-meth-
ylbenzenesulfonamide (51). Compound 51 was prepared from 4,6-
dichloroquinazoline and 3-amino-4-(dimethylamino)-N-methylbenze-
nesulfonamide using method D as yellow solid in 13% yield (25 mg):
1H NMR (400 MHz, DMSO-d6) δ ppm 11.32 (br. s., 1H), 8.85 (s,
1H), 8.19 (d, J = 2.51 Hz, 1H), 7.87 (d, J = 9.29 Hz, 1H), 7.77 (dd, J =
9.29, 2.51 Hz, 1H), 7.72 (d, J = 2.26 Hz, 1H), 7.67 (dd, J = 8.53, 2.26
Hz, 1H), 7.33 (q, J = 4.94 Hz, 1H), 7.25 (d, J = 8.78 Hz, 1H), 3.97 (s,
3H), 2.82 (s, 6H), 2.39−2.47 (m, 3H). MS (m/z) 392.0 (M + H+).
4-(Dimethylamino)-N-methyl-3-[(6-nitro-4-quinazolinyl)amino]-
benzenesulfonamide (52). Compound 52 was prepared from 4-
chloro-6-nitroquinazoline and 3-amino-4-(dimethylamino)-N-methyl-
benzenesulfonamide using method D as a yellow solid in 80% yield
(765 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 10.59 (br. s., 1H),
9.66 (br. s., 1H) 8.55−8.70 (m, 2H) 7.95 (d, J = 10.04 Hz, 1H) 7.70
(s, 1H) 7.60 (d, J = 7.53 Hz, 1H) 7.30 (q, J = 5.02 Hz, 1H) 7.20 (d, J =
8.78 Hz, 1H) 2.79 (s, 6H) 2.43 (d, J = 5.02 Hz, 3H). MS (m/z) 402.9
(M + H+).
4-(Dimethylamino)-N-methyl-3-(pyrido[2,3-d]pyrimidin-4-
ylamino)benzenesulfonamide (53). Compound 53 was prepared
from 4-chloropyrido[2,3-d]pyrimidine and 3-amino-4-(dimethylami-
no)-N-methylbenzenesulfonamide using method D as a yellow solid in
4% yield (26 mg): 1H NMR (400 MHz, DMSO-d6) δ ppm 11.25 (br.
s., 1H), 9.15 (d, J = 4.02 Hz, 1H), 9.08 (d, J = 8.28 Hz, 1H), 8.87 (br.
s., 1H), 7.87 (dd, J = 8.41, 4.64 Hz, 1H), 7.77 (s, 1H), 7.64 (dd, J =
8.53, 2.26 Hz, 1H), 7.33 (q, J = 4.85 Hz, 1H), 7.24 (d, J = 8.53 Hz,
1H), 2.82 (s, 6H), 2.43 (d, J = 5.02 Hz, 3H). MS (m/z) 359.0 (M +
H+).
1
yield (54 mg, 90% purity): H NMR (400 MHz, DMSO-d6) δ ppm
9.70 (br. s., 1H), 9.09 (br. s., 1H), 8.34 (s, 1H), 7.82 (br. s., 1H), 7.57
(d, J = 8.28 Hz, 2H), 7.52 (d, J = 8.28 Hz, 1H), 7.38 (d, J = 8.53 Hz,
2H), 7.32 (d, J = 4.77 Hz, 1H), 7.23 (d, J = 8.53 Hz, 1H), 5.99 (s, 1H),
2.98−3.13 (m, 2H), 2.75 (s, 3H), 2.41 (d, J = 4.27 Hz, 3H), 1.01 (t, J
= 6.78 Hz, 3H). MS (m/z) 446.9 (M + H+).
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-N-methyl-
4-[methyl(2,2,2-trifluoroethyl)amino]benzenesulfonamide Trifluor-
oacetate (46). Compound 46 was prepared from 6-chloro-N-(4-
chlorophenyl)-4-pyrimidinamine and 3-amino-N-methyl-4-[methyl-
(2,2,2-trifluoroethyl)amino]benzenesulfonamide using method A as a
1
white solid in 3% yield (8 mg): H NMR (400 MHz, DMSO-d6) δ
ppm 9.53 (br. s., 1H), 8.98 (br. s., 1H), 8.31 (s, 1H), 7.84 (d, J = 2.01
Hz, 1H), 7.59 (d, J = 8.78 Hz, 2H), 7.55 (dd, J = 8.53, 2.26 Hz, 1H),
7.33−7.42 (m, 4H), 5.90 (s, 1H), 4.00 (q, J = 9.79, 2H), 2.99 (s, 3H),
2.42 (d, J = 5.02 Hz, 3H). MS (m/z) 501.1 (M + H+).
4-(Dimethylamino)-N-methyl-3-((6-(methylamino)quinazolin-4-
yl)amino)benzenesulfonamide Bis(trifluoroacetate) (47). A mixture
of 48 (129 mg, 0.346 mmol) in CH2Cl2 (3 mL) was treated with
AcOH (0.022 mL, 0.381 mmol) and 37% aqueous formaldehyde
(0.028 mL, 0.381 mmol) and stirred at 25 °C. After 5 min, sodium
triacetoxyborohydride (110 mg, 0.520 mmol) was added and the
mixture was stirred for 2 h before being quenched with the addition of
saturated aqueous NaHCO3 and extracted with a CH2Cl2/MeOH
mixture. The organic extract was dried (Na2SO4), concentrated, and
subjected to reverse phase HPLC to give 47 as a yellow solid in 1%
3-{[6,7-Bis(methyloxy)-4-quinazolinyl]amino}-4-(dimethylami-
no)-N-methylbenzenesulfonamide Hydrochloride (54). Compound
54 was prepared from 3-amino-4-(dimethylamino)-N-methylbenzene-
sulfonamide and 4-chloro-6,7-bis(methyloxy)quinazoline using meth-
1
od D as an orange solid in 35% yield (54 mg): H NMR (400 MHz,
1
yield (3 mg): H NMR (400 MHz, DMSO-d6) δ ppm 10.77 (br. s.,
DMSO-d6) δ 11.38 (br. s., 1H, aniline-NH), 8.81 (s, 1H, quinazoline
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Journal of Medicinal Chemistry
Article
C2-H), 8.29 (s, 1H, quinazoline C5-H), 7.59−7.71 (m, 2H,
benzenesulfonamide C2-H, benzenesulfonamide C6-H), 7.31−7.39
(m, 2H, quinazoline C8-H, SO2NHCH3), 7.23 (d, J = 9.54 Hz, 1H,
benzenesulfonamide C5-H), 4.00 (s, 3H, OCH3), 4.01 (s, 3H, OCH3),
2.82 (s, 6H, N(CH3)2), 2.43 (d, J = 4.27 Hz, 3H, SO2NHCH3). 13C
NMR (500 MHz, DMSO-d6) δ 160.01 (quinazoline C4), 157.27
(quinazoline C7), 152.17 (benzenesulfonamide C4), 151.16 (quinazo-
line C6), 149.45 (quinazoline C2), 136.04 (quinazoline C8a), 131.00
(benzenesulfonamide C3), 129.49 (benzenesulfonamide C2), 127.78
(benzensulfonamide C6), 127.05 (benzenesulfonamide C1), 119.03
(benzenesulfonamide C5), 108.14 (quinazoline C4a), 105.07
(quinazoline C5), 100.39 (quinazoline C8), 57.84 (OCH3), 57.38
(OCH3), 43.34 (N(CH3)2), 29.60 (SO2NHCH3). For compound 54,
the 1H NMR and 13C NMR resonances were assigned on the basis of
gCOSY45, gHMQC, gHMBC, and 13C GASPE spectra, and all 2D
correlations are in agreement with the assigned structure. MS (m/z)
418.0 (M + H+).
3-((9H-Pyrimido[4,5-b]indol-4-yl)amino)-4-(dimethylamino)-N-
methylbenzenesulfonamide Trifluoroacetate (61). Compound 61
was prepared from 4-chloro-9H-pyrimido[4,5-b]indole and 3-amino-4-
(dimethylamino)-N-methylbenzenesulfonamide using method B as a
1
white solid in 20% yield (16 mg): H NMR (400 MHz, DMSO-d6) δ
ppm 12.35 (br. s., 1H), 9.06 (br. s., 1H), 8.63, (br. s., 1H), 8.53 (s,
1H), 8.18 (d, J = 6.59 Hz, 1H), 7.59 (d, J = 7.81 Hz, 1H), 7.47−7.57
(m, 2H), 7.34−7.45 (m, 3H), 2.82 (s, 6H), 2.46 (d, J = 4.88 Hz, 3H).
MS (m/z) 397.1 (M + H+).
4-(Dimethylamino)-3-{[5-(2-furanyl)-1H-pyrrolo[2,3-d]pyrimidin-
4-yl]amino}-N-methylbenzenesulfonamide (63). A mixture of 3-[(5-
bromo-1H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-4-(dimethylamino)-
N-methylbenzenesulfonamide6 (0.20 g, 0.47 mmol), 2-furanylboronic
acid (0.26 g, 2.35 mmol), Pd(t-Bu3P)2 (0.024 g, 0.047 mmol), and 2 M
aqueous K2CO3 (1.18 mL, 2.35 mmol) in 1,4-dioxane (10 mL) was
subjected to microwave irradiation (150 °C) for 40 min before being
concentrated and subjected to flash chromatography (1% NH4OH/9%
MeOH/90% CHCl3). The solid product was triturated with MeOH to
3-{[5,7-Bis(methyloxy)-4-quinazolinyl]amino}-4-(dimethylami-
no)-N-methylbenzenesulfonamide (55). Compound 55 was prepared
from 3-amino-4-(dimethylamino)-N-methylbenzenesulfonamide and
4-chloro-5,7-bis(methyloxy)quinazoline using method D as a yellow
1
give 63 as an off white solid in 39% yield (80 mg): H NMR (400
MHz, DMSO-d6) δ ppm 12.35 (s, 1H), 9.20 (s, 1H), 9.15 (s, 1H),
8.41 (s, 1H), 7.79 (s, 1H), 7.74 (s, 1H), 7.41 (m, 1H), 7.36 (m, 3H),
6.72 (m, 2H), 2.64 (s, 6H), 2.49 (m, 3H). MS (m/z) 428.0 (M + H+).
Modeling Compound 51 to TNNI3K and B-raf. The Molecular
Operating Environment (MOE) from Chemical Computing Group
Inc. was used to model compound 51 to TNNI3K (PDB code 4YFI)6
and B-raf structures (PDB code 4YHT).7 The crystal structures were
prepared using the Structure Preparation tool in MOE. Atomic charges
were assigned using the PFROSST force-field parameters. Hydrogens
were added and their positions were optimized using the Protonate 3D
tool. Compound 51 was modeled to TNNI3K and B-raf using the
bound benzenesulfonamide ligand in the respective structure as a
guide. The crystallographic water molecule observed in TNNI3K and
B-raf that forms an H-bond interaction to the sulfonamide oxygen was
retained in each model. LigX was used to minimize the ligand, water
molecule, and nearby protein residues with the PFROSST molecular
mechanics force field, Born solvation model, and default settings to
optimize favorable interactions and relieve remaining steric clashes.
Calculated Hydrogen Bond Acceptor Strength. The hydrogen
bond acceptor (HBA) strengths were appoximated by computing the
geometry optimized energy difference between each heterocycle from
Supplemental Table 1 complexed with a coordinated hydrogen
fluoride (HF) molecule and the isolated heterocycle and HF molecule
as depicted in the following reaction scheme:
1
solid in 43% yield (61 mg): H NMR (400 MHz, DMSO-d6) δ ppm
9.87 (br. s., 1H), 8.57 (s, 1H), 8.17 (s, 1H), 7.92 (d, J = 8.82 Hz, 1H),
7.57−7.64 (m, 1H), 7.48−7.56 (m, 2H), 7.34 (s, 1H), 4.01 (s, 3H),
3.85 (s, 3H), 2.45 (d, J = 4.85 Hz, 3H). MS (m/z) 418.1 (M + H+).
4-(Dimethylamino)-N-methyl-3-(thieno[3,2-d]pyrimidin-4-
ylamino)benzenesulfonamide (56). Compound 56 was prepared
from 3-amino-4-(dimethylamino)-N-methylbenzenesulfonamide and
4-chlorothieno[3,2-d]pyrimidine using method A as a yellow solid in
1
53% yield (80 mg): H NMR (400 MHz, DMSO-d6) δ 10.84 (br. s.,
1H), 8.77 (s, 1H), 8.34 (d, J = 5.51 Hz, 1H), 7.64−7.69 (m, 2H), 7.49
(d, J = 5.29 Hz, 1H), 7.30−7.37 (m, 1H), 7.19 (d, J = 9.48 Hz, 1H),
2.78 (s, 6H), 2.39 (d, J = 4.63 Hz, 3H). MS (m/z) 364.0 (M + H+).
3-((5H-Pyrrolo[3,2-d]pyrimidin-4-yl)amino)-4-(dimethylamino)-
N-methylbenzenesulfonamide (57). Compound 57 was prepared
from 3-amino-4-(dimethylamino)-N-methylbenzenesulfonamide and
4-chloro-5H-pyrrolo[3,2-d]pyrimidine using method A as a yellow
1
solid in 27% yield (58 mg): H NMR (400 MHz, DMSO-d6) δ 12.31
(br. s., 1H), 10.32 (s, 1H), 8.75 (s, 1H), 7.96−8.09 (m, 2H), 7.62 (dd,
J = 2.26, 8.53 Hz, 1H), 7.26−7.39 (m, 2H), 6.69 (dd, J = 1.88, 2.89 Hz,
1H), 2.79 (s, 6H), 2.45 (d, J = 5.02 Hz, 3H). MS (m/z) 347.0 (M +
H+).
4-(Dimethylamino)-N-methyl-3-(1H-purin-6-ylamino)-
benzenesulfonamide (58). Compound 58 was prepared from 3-
amino-4-(dimethylamino)-N-methylbenzenesulfonamide and 2-chlor-
1
opurine using method B as a white solid in 45% yield (350 mg): H
NMR (400 MHz, DMSO-d6) δ ppm 13.30 (br. s., 1H), 8.89 (br. s,
1H), 8.83 (s, 1H), 8.46 (s, 1H), 8.32 (s, 1H), 7.43−7.50 (m, 1H),
7.33−7.42 (m, 2H), 2.75 (s, 6H), 2.46 (d, J = 5.05 Hz, 3H). MS (m/z)
348.0 (M + H+).
The energy difference for this reaction (ΔE) has been shown to
correlate very well with the Abraham parameter β, which describes
hydrogen bond acceptor propensity.29 Hydrogen fluoride was used in
this model as it is the smallest hydrogen bond donor and thus reduces
the complexity of the calculations and the chance of secondary
interactions. The benzenesulfonamide portion of each TNNI3K
inhibitor considered was replaced by a methyl in the modeled hinge
binding heterocycles to further simplify the calculations. Hydrogen
bonded complexes were initially generated by placing a HF molecule
1.85 Å from the heterocycle acceptor indicated in red in Supplemental
Table 1. The geometries of the HF molecule and each heterocycle and
heterocycle−HF complex were first optimized using density functional
theory (DFT) with the 6-31G** basis set. Taking these geometries as
starting points, single point energies were then computed using the 6-
311G** basis set. ΔE was computed by subtracting the resultant
energy of the heterocycle and HF molecules from the heterocycle−HF
complex. All quantum mechanical calculations were performed with
Jaguar from Schrodinger Inc.35
3-({6-[(4-Chlorophenyl)amino]-4-pyrimidinyl}amino)-4-(dimethy-
lamino)-N-methylbenzenesulfonamide Trifluoroacetate (59). Com-
pound 59 was prepared 3-[(6-chloro-4-pyrimidinyl)amino]-4-(dime-
thylamino)-N-methylbenzene-sulfonamide and 4-chloroaniline using
1
method A as an off-white solid in 21% yield (14 mg): H NMR (400
MHz, DMSO-d6) δ ppm 9.73 (br. s., 1H), 9.21 (br. s., 1H), 8.33 (s,
1H), 7.78−7.83 (m, 1H), 7.55 (d, J = 8.81 Hz, 2H), 7.51 (dd, J = 2.27,
8.56 Hz, 1H), 7.38 (d, J = 8.81 Hz, 2H), 7.30 (q, J = 4.95 Hz, 1H),
7.20 (d, J = 8.56 Hz, 1H), 6.01 (s, 1H), 2.77 (s, 6H), 2.41 (d, J = 5.04
Hz, 3H). MS (m/z) 432.9 (M + H+).
3-((4-((4-Chlorophenyl)amino)-1,3,5-triazin-2-yl)amino)-4-(dime-
thylamino)-N-methylbenzenesulfonamide Trifluoroacetate (60).
Compound 60 was prepared from 4-chloro-N-(4-chlorophenyl)-
1,3,5-triazin-2-amine and 3-amino-4-(dimethylamino)-N-methylbenze-
nesulfonamide using method B as a brown solid in 20% yield (45 mg):
1H NMR (400 MHz, DMSO-d6) δ ppm 9.93 (br. s., 1H), 8.34 (s, 1H),
7.84 (br. s., 1H), 7.66 (br. S., 2H), 7.54 (d, J = 8.53 Hz, 1H), 7.23−
7.35 (m, 3H), 7.20 (d, J = 8.53 Hz, 2H), 2.77 (s, 6H), 2.38 (d, J = 4.77
Hz, 3H). MS (m/z) 453.9 (M + H+).
K
DOI: 10.1021/acs.jmedchem.6b01342
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(6) Lawhorn, B. G.; Philp, J.; Zhao, S.; Louer, C.; Hammond, M.;
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Sweitzer, S.; Holt, D. A.; Gatto, G. J., Jr.; Kallander, L. S. Identification
of purines and 7-deazapurines as potent and selective type I inhibitors
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01342.
■
S
Table of calculated hydrogen bond acceptor strengths
(PDF)
SMILES representation of compounds with key data
(CSV)
AUTHOR INFORMATION
Corresponding Author
*E-mail: brian.2.lawhorn@gsk.com. Phone: 610-270-5347.
ORCID
Brian G. Lawhorn: 0000-0003-4618-5314
Notes
The authors declare the following competing financial
interest(s): Authors affilliated with GlaxoSmithKline have
received compensation in the form of salary and stock.
■
(12) Romero, F. A.; Hwang, I.; Boger, D. L. Delineation of a
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benzoic acid (pKa = 4.03) and 4-trifluoroethoxybenzoic acid (pKa =
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ACKNOWLEDGMENTS
■
We gratefully acknowledge Claire Jarvis, Tavake Piriou, Steve
Zhao, Chris Louer, Martha Sarpong, and Mark Seefeld for
assistance in compound synthesis, Nathan Gaul, Peter Caprioli,
Mehul Patel, Brian Dombroski, Joi Brown, Michael Schaber,
Evan Agyeman, and Laurie Carson for conducting the TNNI3K
and B-Raf assays, and Michael Kranz for providing the
hydrogen bond acceptor strength calculator.
ABBREVIATIONS USED
■
TNNI3K, troponin I-interacting kinase; H-bond, hydrogen
bond; PCI, percutaneous coronary intervention; MI, myocar-
dial infarction; σp, Hammett substituent constant; ρ, Hammett
reaction constant; pKa, −log10 Ka; Ka, acid dissociation
constant; pIC50, −log10 IC50; PDGFR, platelet-derived growth
factor receptor; HBA, hydrogen bond acceptor strength; dppf,
1,1′-bis(diphenylphosphino)ferrocene; MOE, molecular oper-
ating environment; PFROSST, parm@Frosst force field; ΔE,
change in energy
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