Accepted Manuscript Fragment-Based Hit Discovery and Structure-Based Optimization of Aminotriazoloquinazolines as Novel Hsp90 Inhibitors Elena Casale, Nadia Amboldi, Maria Gabriella Brasca, Dannica Caronni, Nicoletta Colombo, Claudio Dalvit, Eduard R. Felder, Gianpaolo Fogliatto, Arturo Galvani, Antonella Isacchi, Paolo Polucci, Laura Riceputi, Francesco Sola, Carlo Visco, Fabio Zuccotto, Francesco Casuscelli PII: DOI: Reference:
S0968-0896(14)00415-5 http://dx.doi.org/10.1016/j.bmc.2014.05.056 BMC 11617
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
9 April 2014 21 May 2014 23 May 2014
Please cite this article as: Casale, E., Amboldi, N., Brasca, M.G., Caronni, D., Colombo, N., Dalvit, C., Felder, E.R., Fogliatto, G., Galvani, A., Isacchi, A., Polucci, P., Riceputi, L., Sola, F., Visco, C., Zuccotto, F., Casuscelli, F., Fragment-Based Hit Discovery and Structure-Based Optimization of Aminotriazoloquinazolines as Novel Hsp90 Inhibitors, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.05.056
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Fragment-Based Hit Discovery and Structure-Based Optimization of Aminotriazoloquinazolines as Novel Hsp90 Inhibitors
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Elena Casale, Nadia Amboldi, Maria Gabriella Brasca, Dannica Caronni, Nicoletta Colombo, Claudio Dalvit, Eduard R. Felder, Gianpaolo Fogliatto, Arturo Galvani, Antonella Isacchi, Paolo Polucci, Laura Riceputi, Francesco Sola, Carlo Visco, Fabio Zuccotto, and Francesco Casuscelli FAXS-NMR Screening Hit
12b Ki (FP) = 0.012µM LE = 0.40
3 Ki (FP) = 37.2µM LE = 0.37
18a Ki (FP) = 0.179µM LE = 0.33
Bioorganic & Medicinal Chemistry j o ur n al h om e p a g e : w w w . e l s e v i er . c o m
Fragment-Based Hit Discovery and Structure-Based Optimization of Aminotriazoloquinazolines as Novel Hsp90 Inhibitors Elena Casalea,∗ Nadia Amboldia, Maria Gabriella Brascaa, Dannica Caronnia, Nicoletta Colomboa, Claudio Dalvita,†, Eduard R. Feldera, Gianpaolo Fogliattoa, Arturo Galvania, Antonella Isacchia, Paolo Poluccia, Laura Riceputia, Francesco Solaa, Carlo Viscoa, Fabio Zuccottoa,‡, and Francesco Casuscellia,∗ a
Oncology, Nerviano Medical Sciences, viale Pasteur 10, 20014, Nerviano (MI) I Current address: University of Neuchatel, Avenue de Bellevaux 51, 2000, Neuchatel CH ‡ Current address: University of Dundee, Dow street, Dundee DD1 5EH, Scotland, UK †
A RT IC L E I N F O
A BS T RA C T
Article history: Received Received in revised form Accepted Available online
In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.
Keywords: Keyword_1 Keyword_2 Keyword_3 Keyword_4 Keyword_5
Fragment based hit discovery FAXS-NMR screening Structure-based design Hsp90 inhibitors Anti-cancer agents
2014 Elsevier Ltd. All rights reserved.
∗ Corresponding author. Tel.: +39 0331-581041; fax: +39 0331-581347; e-mail:
[email protected] ∗ Corresponding author. Tel.: +39 0331-581326; fax: +39 0331-581347; e-mail:
[email protected]
a]quinazoline subtype was not described so far. We therefore
1. Introduction Heat shock protein (Hsp90) is a molecular chaperone that
prioritized the optimization of this fragment, whose structure-
plays a role in the conformational stability, maturation, and
guided evolution led us to obtain nanomolar inhibitors of Hsp90
function of several proteins, including many oncogenic kinases,
in just a few cycles of chemical modification and biological
whose dysfunctional activation has been collectively described as
testing. An alternative binding mode of optimized intermediates
constituting the hallmark traits of cancer. Hsp90 inhibitors cause
was revealed by crystallographic studies and offered an
the destabilization and degradation of more than a hundred
additional path for the medicinal chemistry expansion and further
1
2
Hsp90 client proteins, and showed promising antitumor activity
evolution of this attractive chemical class.
1c
in several preclinical model systems. For these reasons, Hsp90 emerged in the last decade as a major therapeutic target and a great deal of efforts have been dedicated to the discovery of new inhibitors3 and some of them are currently tested in clinical trials.4
2. Design and synthesis 2.1. NMR screening and Hit Identification In search of novel chemical entities as Hsp90 inhibitors we engaged in a fragment-based approach, which included the screening of an internal library of fragments followed by an
Fragment Based Drug Discovery (FBDD) is an established
extensive structure-guided design phase supporting inhibitor
approach for lead discovery and has been successfully applied in
evolution to improve potency and drug-like properties of the
drug research programs,5 with a number of examples concerning
initial hits.
the optimization of Hsp90 inhibitors.6 A high-quality fragment library together with valid biophysical techniques for screening, assisted by crystallography, provide remarkable scope in support of the medicinal chemistry elaboration of fragment hits into leads. Screening of a relatively small number of low molecular weight fragments can efficiently sample the relevant chemical space, even though fragments generally have low binding affinities for the intended target. Hence, the choice of sensitive and robust biophysical methods able to detect weak binders is crucial.7,8 Here, among the many methods for detecting the fragment binding, the FAXS (Fluorine chemical shift Anisotropy and eXchange for Screening) competition assay, which utilizes the favourable properties of Fluorine NMR spectroscopy,9 was chosen for Hsp90 fragment screening. In our experience this method has proven to be a powerful approach since it combines sensitivity,
efficiency,
reliability
and
throughput
while
minimizing protein consumption. Furthermore, it provides straightforward evidence of a molecule’s biophysical effects on the targeted binding site of a protein, helping to distinguish general unspecific binders. In the present work we report on the fragment-based approach for the identification of aminotriazoloquinazoline as an original chemotype in the context of Hsp90 inhibition efforts, attractive for its potential to introduce chemical novelty. Although the triazoloquinazoline scaffold has been previously utilised a.o. in adenosine receptor antagonists, 10 for the treatment of disorders mediated by purine receptors11, and also in phosphodiesterase PDE10A inhibitors,12 our 2-amino-5-alkyl-[1,2,4]triazolo[1,5-
A library of approximately 1200 fully characterized fragmentlike molecules including commercial compounds, proprietary building blocks and advanced intermediates from medicinal chemistry programs was assembled. Our definition of the fragments chemical space is based on the following parameters: MW (Molecular Weight) < 300 Da, (variable if halogen or sulphur atoms are present), PSA (Polar Surface Area) < 80 Å2, clogP < 3, number of rotatable bonds < 4, total number of Hbond acceptors/donors between 3 and 6.13 We also consider fragments with asymmetric centers and sp3-carbon rich molecules, which might be advantageous in exploring demanding biological targets.14 The resulting library of fragments was then subjected to chemoinformatic filters to ensure the absence of undesirable chemical functionalities, and screened with the SPAM (Solubility, Purity and Aggregation of the Molecule)15 filter approach for the exclusion of aggregation or low kinetic solubility as potential alert of PAINS16 (promiscuous or panassay interference compounds) frequently encountered during primary screening. A subset of this library including about 300 fluorinated fragments (CF or CF3 containing molecules),9c was first screened against the N-terminal domain of human Hsp90α for the identification of a binder to be used as a probe (“spy molecule”) in the FAXS NMR experiment. The two fluorinated binders 1 and 2, belonging to the frequently described aminopyrimidine chemical motif and structurally similar to the derivative found in the PDB structure 2XHR, were identified (Figure 1).17 These
inhibitors showed a competitive behavior with respect to ATP with a binding affinity constant (Ki) of 29.6 µM and 7.5 µM for 1
N
and 2 respectively, as measured in a fluorescence polarization
NH2 N
(FP) assay. Compound 2 having the benzodioxolane group was deemed to be a better probe for the identification of ATP binding
F
F
F
O 1 Ki (FP) = 29.6 µM
F N
O O
N
NH2
2 Ki (FP) = 7.5 µM
site ligands, as well as alternative site binders, and thus it was selected as the spy molecule. Despite its low solubility (~15 µM) was easily identified in the
19
N
F NMR direct binding assay and
performed well as spy molecule in the
19
H2N 2 1N
H2N N N N
F NMR competition
R
and synthesized molecules.
2
1
7 8
3 Ki (FP) = 37.2 µM Ki (FAXS-NMR) = 32.2 µM Ki (ITC) = 32.4 µM LE = 0.37a
Mixtures consisting of ten fragments were screened with the FAXS NMR displacement assay, using 6 µM of the spy molecule
Figure 1. Spy molecules 1 and 2, fragment hit 3 and the 2-
2 and 0.6 µM of the catalytic domain of Hsp90α, as described in
aminotriazoloquinazoline core structure
Figure SI 1).
R 5
N6
10 9
binding assay and in the Ki measurements of the identified hits
the Experimental Section (See also the supporting information
3 N N4
To facilitate the chemical expansion, the crystal structure of compound 3 bound to the ATPase domain (aa 9-236) of Hsp90α
A straightforward mixture deconvolution process identified 23
(Figure 2) was determined. Interestingly, compound 3 does not
ligands (hit rate of 1.9%). The hits progressed to crystallization
occupy the adenine binding site, rather it is located in an adjacent
trials and structural data were obtained for four compounds. The
induced-fit pocket by triggering a rearrangement of residues 103–
discovery and optimization of one of these fragments belonging
111, which in the presence of the ligand adopt a helical
to the resorcinol class of small molecule inhibitors of Hsp90, has
conformation. This second binding site has been documented
already been reported.18 In a complementary effort we pursued
previously and compounds with similar behavior have been
also the development of a novel ATP competitive inhibitor with a
reported.20 These conformational changes do not modify the
distinct chemotype. To this end the aminotriazoloquinazoline hit
adenine binding site, preserving the positions of the key residue
3 (Figure 1) was selected for full characterization and chemical
Asp93 and thus offering opportunities for the design of new
optimization. FAXS NMR titration measurement of 3 was
inhibitors taking advantage of additional interactions with the
performed and a Ki of 32.2 µM was calculated corresponding to a
enzyme.
ligand efficiency (LE) of 0.37 which was considered good19 and
Compound 3 binds deep inside the lipophilic pocket, with its
suited for the further exploration of the fragment class. The
propyl chain pointing out toward the ATP sugar region. The
binding affinity to Hsp90 was also confirmed by isothermal
interactions of the ligand with the protein are mainly
titration
supporting
hydrophobic. The triazoloquinazoline core is stacked between the
information Figure SI 2) and FP assays reporting a Ki of 32.4 µM
calorimetry
(ITC)
experiments
(See
side chain of Leu107 and Phe138 making a (face-to-face) π-π
and 37.2 µM respectively. The structures of the fluorinated
stacking with Phe138 and an additional face-to-edge π-stacking
binders 1 and 2 and fragment 3 with the corresponding Hsp90 Ki
contact with Trp162.20,21 Other favorable hydrophobic contacts
values and the ligand efficiency (LE) are reported in Figure 1.
occur with Met98, Leu103, Tyr139, Val150, whilst the polar interactions are only weak hydrogen bonds between the amino group and the backbone carbonyl group of Gly135 and a conserved water molecule. Additional hydrogen bonds are found between the nitrogen atoms of the scaffold (N1, N3 and N6) and three conserved water molecules (Figure 2).
a for 6a-d R
O
2
R OH
O
2
OH
b
NH
for 5e-v
NH 2
R 4
R1
Cpd
1
R
2
7a
n-Pr
7-OMe
7b
n-Pr
10-F
7c
n-Pr
7-Me
7d
n-Pr
7-Cl
R
O
c for 6e-v
N
O 5e-v R2
O
8
R1 *
*
OMe
H
NO 2
7f 7g
*
H
*
OMe
H
H
7h OMe MeO *
OMe
7i
H
OMe
*
H
1
R
H
O
H
O
7m
"
8-F
7n
"
8-SO2NH2
7o
"
10-F
7p
"
8,10-F
7q
"
10-Cl
7 7r
"
7-OPMB
7s
"
8-OPMB
7t
"
9-CN
7u
"
8-NO2
7v
"
8-COOH
MeO *
7j
N
OMe
OMe *
N 7
N
R2
7l
7e
NH2
N
7a-v
6a-v
7k
10
9
d 1
R
Cpd
2
OMe
Figure 2. X-ray structure of the initial fragment hit bound to the N-terminal domain of Hsp90. Compound 3 (yellow carbon atoms) and key residues of the active site (green carbon atoms) are shown. The grey surface represents the induced pocket, while ordered water molecules are shown as red spheres and hydrogen bonds as black dashed lines.
Amino-triazoloquinazoline 3 offered an attractive starting point for structure-based optimization mainly due to the good ligand efficiency, the chemical feasibility and the peculiar binding mode in the alternative binding site. Furthermore, the replacement of the propyl chain with well known structural motifs (i.e. resorcinol, aminopyrimidine)5 was expected to provide opportunities to explore the adenine-binding region. 2.2. Chemistry
Scheme 1. Synthesis of aminotriazoloquinazolines 7a-v. Reagents and conditions: (a) Butyric anhydride, 160 °C, microwave, 10 min. (b) Acyl chlorides R1 COCl, pyridine, DCM, 120 °C, microwave, 5 min; (c) Acetic anhydride, 130 °C, 10 min. (d) Aminoguanidine bicarbonate, pyridine, 180 °C, microwave, 20 min.
The acylated anthranilic acids 5e-v were precipitated with HCl, filtered, and used without further purification. Access to the key benzoxazinone derivatives 6e-v was achieved upon treatment of 5e-v with acetic anhydride at reflux for 5 minutes. The final products 7a-v were then obtained by reacting benzoxazinones 6av with aminoguanidine in the presence of pyridine. Compounds conveniently precipitated slowly in pure form from the reaction mixture. The overall yields ranged from 10 to 50 %, and the synthetic sequence tolerated a broad variety of substitutions.
Scheme 1 illustrates the aminotriazoloquinazoline synthesis 1
2
route with R diversification and variations of the R substituents on the benzene ring of the scaffold (7a-v).
22
Decoration and modification of the core structure were performed following suitable functional group transformations,
As previously
as reported in Scheme 2. Boron tribromide mediated deprotection
reported, the aminotriazoloquinazoline core structure can be
of methoxy compounds 7e, 7h, and 7i gave corresponding
obtained from a key benzoxazinone intermediate upon reaction
phenols 8a-c. Instead, the 8-hydroxyl derivative 9a and 9b were
with aminoguanidine bicarbonate, in pyridine, at reflux or by
obtained after cleavage of the PMB protecting group by treatment
microwave assisted heating. 12, 23
with HCl. The nitro group of 7f and 7u was transformed into the
A direct
conversion
of anthranilic acids 4a-d into
benzoxazinone 6a-d was performed upon treatment with neat butyric anhydride. Alternatively, starting from anthranilic acids 4e-v the access to the amide derivatives 5e-v was accomplished by reaction with the suitable acyl chlorides under microwave heating conditions.
amino group delivering compounds
10a-b
by standard
hydrogenation. Compound 11 was successfully prepared from the corresponding nitrile 7t upon treatment with borohydride/cobalt chloride. Halogenated derivatives such as 7- or 8-bromo intermediates were considered good synthons for expansion but unfortunately neither Suzuki nor Buchwald reactions worked.
Moreover, installation of a solubilizing tether via reductive
amination performed on compound 10b failed.
NH2
N NH2
N H2N
N
N
N
N
O
N R
8b
R1 =
N
-CH2-Ph-m,mdiOH
from 7e,h,i
from 7t
O O
N 9a R2 = 8-OH 9b R2 = 7-OH
HO a
11
N
N
8c R1 = -CH2-Ph-o,pdiOH
O
N
NH2
8a R1 = -(CH2)2-Ph-pOH
1
b
d from 7r,s NH 2
N N H2N
NH2
N
c
O
from 7u
O
N
R
N
2
R
R
1
2
N
O O
N 13a-b
13a R2 = -CONH(CH2)2OH c
NH2 N
N
from 7v
7
10b
N
f
N
N
NH2
N
N
from 7m
e from 7o
13b R2 = -CONH(CH2)3OH
e
from 7f
NH2
N
N NH2
N
HO
NH 2
NH
N
N N
2
N
O
O O
N 12a-g
O
N
10a
R
N
12a R2 = -NH(CH2)3OH
14
12b R2 = -NH(CH2)2OH 12c R2 = -NH(CH2)2NH2 12d R2 = -NH(CH2)2NMe2 12e R2 = -NH(CH2)2Npyrrolidine 12f R2 = -piperazineNMe 12g R2 = -NH(CH2)2OMe
Scheme 2. Synthesis of aminotriazoloquinazoline derivatives. Reagents and conditions: (a) 1 M BBr3 in DCM, rt, 120 h; (b) HCl 4 M dioxane, rt 18 h (c) Cyclohexene, 10% Pd/C, MeOH, 65 °C, 2 h; (d) NaBH4, CoCl2 x 12 H2 O, THF/MeOH, rt, 18 h; (e) Amines, 140 °C, 2-8 h; (f) Amines, TBTU, DiPEA, DCM, rt, 4 h
Conversely, introduction of such groups were successfully
As shown in Scheme 3, the intermediate 5p (prepared as
achieved by nucleophilic aromatic substitution24 on the 8- and
reported
10-fluoro derivatives 7m and 7o yielding the desired compounds
trimethylsilyldiazomethane to afford the 2-(2-benzo[1,3]dioxol-
with a nitrogen containing moiety 12a-g and. The amide
5-yl-acetylamino)-4,6-difluoro-benzoic acid methyl ester that
preparation following path f (Scheme 2) was performed by
upon treatment with a suitable amine such as N-methylpiperazine
activation of the carboxylic acid intermediate 7v with TBTU in
in
the presence of the selected amines delivering the desired
methylpiperazinyl)-6-F
DMSO
in
at
Scheme
80
°C
1)
was
afforded
derivative
reacted
selectively 15
via
the
the
with
4-(4-
4-fluorine
2
replacement, likely due to the bulky amine’s steric effect. After
substitents in 8 or 10 position afforded products with good
ester hydrolysis, the acid was condensed via benzoxazinone 16 to
potency and improved solubility, the syntheses of di-substituted
the aminotriazoloquinazoline 17 in a similar manner to that
derivatives with the best groups in position 8 and 10 were
previously
compound 13a-b in fair yields. Since the evaluation of R
attempted.
described.
F
F
O
O
OH F
F
O
O N
NH
a,b
O
NH
N
c, d
N
O 5p
N
O O
N
N
15
N
N
O
16
17
O
O
O
N
N
O
NH2
N
F e
O
O d,e NH 2
R N
F
2
N
F
N
N
O
f
O
NH 2
N N
F
N
N
18a R2 = -NH(CH2)2OH 18b R2 = -NH(CH2)3OH
O
18c R2 = -NH(CH2)2OMe
O
18d R2 = -NH(CH2)2NH2 18e R2 = -piperazineNMe
7p
Scheme 3. Synthesis of 8-10 di-substituted aminotriazoloquinazoline derivatives. Reagents and conditions: (a) TMS-CHN2 , MeOH, DCM, rt, 1h; (b) NMethylpiperazine, DMSO, 80 °C, 2 h; (c) LiOH, THF/MeO/H2O (7:2:1) rt, 18 h; (d) Acetic anhydride, 130 °C, 10 min; (e) Aminoguanidine bicarbonate, pyridine, 180 °C, microwave, 20 min; (f) Amines, 140 °C, 2-8 h.
Regrettably the attempts to expand the reaction with
hydrophilic head from the propyl group to target the above
additional amines proved to be limiting since the reaction with
mentioned interactions. Compound 8a and 10a were designed to
primary amines in similar conditions gave an unresolved mixture
assess this structure-based hypothesis (Table 1). While 10a
of regioisomers via the 4-fluoro or 6-fluoro replacement, also
provided a modest enhancement of the affinity, the phenolic
when a more steric hindered ester was evaluated.
derivative 8a was inactive, possibly because its solubility was
Alternatively, the introduction of solubilizing groups was successfully achieved by nucleophilic aromatic substitution on 8,10-difluoro-2-aminotriazoloquinazoline derivative 7p, obtained
very low
Table 1. Optimization iteration: biochemical potency, ligand efficiency and solubility NH2
from intermediate 5p as previously described, delivering
2 R 10 9
compounds 18a-e as reported in Scheme 3.24 However, the aromatic nucleophilic substitution did not derivatize the 8position, whereas
the 10-fluorine group resulted to be more
reactive and underwent the replacement, although preventing a bis substitution (Scheme 3).24 Results and Discussion The aminotriazoloquinazoline fragment 3 being commercially available, additional structural analogues were searched in databases from vendors. Twenty additional derivatives were then purchased and tested in the FAXS NMR binding assay, but only modest Hsp90 inhibition was measured. Initial medicinal chemistry efforts were focused on the Fragment growing approach.17a,25
To this
purpose,
a left-hand
substitution
exploration was performed with small R2 substituents on the
8
Cpd
R1
7
N N N
N R
1
R2
Ki (µM)a
LEb
Solc
3
n-Pr
H
32.2
0.37
70
7a
“
7-OMe
22.7
0.34
30
7b
“
10-F
28.6
0.35
86
7c
“
7-Me
16.7
0.37
10
7d
“
7-Cl
23.4
0.36
30
H
NEd
3
H
NEd
3
H
15.2
7e
* OMe
8a
* OH
10a
NH2 *
0.28
12
a Ki were obtained with FAXS NMR and values are an average of two or more determinations. bLigand Efficiency LE = -1.4log(Ki)/HAC; HAC = heavy atom count. 19 c Kinetic solubility PBS pH 7.4 µM. d No Effect .
phenyl ring, resulting in the structure–activity relationship (SAR) presented in Table 1. The analogues 7a-d containing minimal structural modifications gave some activity improvement.
Additionally, we developed a fragment linking strategy with the help of a large amount of structural information publicly
On the other hand, the co-crystal structure of 3 in Hsp90
available.17a,25 In particular, the overlay of the crystal structures of
(Figure 2) indicates that the propyl chain points to a solvent
radicicol analogues or resorcinol compounds (PDB 2IWU, 2BT0
exposed area towards a cluster of H bond accepting groups
and 2UWD) highlighted the possibility to extend the initial
(Asn51, Ser52, Asp54) interacting with a conserved water
fragment to access the adenine region. Therefore, linking of
molecule. Therefore, a possible development of 3 could extend a
aminotriazoloquinazoline with the well known resorcinol moiety
was considered an option to develop new derivatives likely to 17,25
establish interactions with the key residue Asp93.
suggestions (Figure 3A). As expected, the dihydroxyphenyl ring
Modeling
of 8b occupies the adenine site with a binding mode reminiscent
studies indicated the position 5 of the triazoloquinazoline core as
of other resorcinol containing compounds.26,27 The key hydrogen
the best one to connect our fragment to the resorcinol moiety
bond interaction between the carboxylate side chain of Asp93
using a flexible -CH2- linker. The in silico analysis also
and one of the two hydroxyl groups is conserved. In addition the
highlighted how either the ortho-para or the meta-meta resorcinol
two hydroxyl groups form a network of water mediated
regioisomers could reproduce the favourable network of
interactions with the neighboring residues Gly97, Ser52 and
interactions. Following this approach, compounds with an aryl
Leu48. The recognition of the resorcinol moiety seems to be the
group bearing two hydroxyls were synthesized according to
‘driving force’ of the binding and induces changes in the
Schemes 1 and 2. Incorporation of dihydroxyphenyl moieties
orientation of the aminotriazoloquinazoline moiety that adopts a
tethered to C-5 of the scaffold with a -CH2 - linker was performed
slightly different orientation compared to the initial hit 3 (Fig. 2),
leading to the compounds 8b and 8c (Table 2) that showed a
although it is still accommodated in the hydrophobic pocket.
fivefold improvement of affinity. The co-crystal structure of 8b in complex with Hsp90 confirmed molecular modeling
Figure 3. (A) View of compound 8b bound in the active site of Hsp90. 8b (yellow carbons atoms)) and key residues of the active site( green carbon atoms) are shown as sticks, ordered water molecules are shown as red spheres and hydrogen bonds as black dashed lines. (B) View of compound 7h bound in the active site of Hsp90 7h (yellow carbons atoms)) and key residues of the active site ( green carbon atoms) are shown as sticks, ordered water molecules are shown as red spheres and hydrogen bonds as black dashed lines.
Intriguingly, compounds 7h and 7i, synthetic intermediates
residues are water mediated: the three nitrogen atoms in
of 8b and 8c, resulted even more active. Taking into
position 2, 3, and 6 of the triazoloquinazoline core are bridged
consideration the binding mode of 8b it was not trivial to
by several water molecules with the side chain of Asp93,
explain the activity of 7h and its analogue as the two
Thr184 and Asn51, the backbone nitrogen of Gly97 and the
molecules lack the hydroxyl groups that are essential for the
backbone
interactions with Asp93 and the conserved water molecules.
dimethoxyphenyl moiety occupies the induced pocket and is
Thus, the structure of compound 7h in complex with Hsp90
stacked between the side chain of Leu107 and Phe138,
was solved (Figure 3B). It turns out that compound 7h binds in
overlapping well with the triazoloquinazoline core space of
a completely different manner compared to his hydroxyl
compound 3. This binding mode is reminiscent of the one
analogue 8b (Figure 3A). The molecule flips so that the
described for the purine based inhibitor PU3.20,28,29 The
aminotriazoloquinazoline group occupies the adenine pocket
phenomena
and its amino group forms the key hydrogen bond to the side
conformation, are well known during optimization of Hsp90
chain of Asp93. The additional interactions with the protein
inhibitors as previously shown by others.17a,25,30
oxygen
of
of
Leu48
fragment
and
adopting
Ile91.
different
The
3,5-
binding
This binding mode switch was unexpected and in light of
Activity data concerning analogues of 7h are summarized in
these results we could not exclude that the initial fragment 3
Table 2. The compounds with 4-methoxy and either a (2’-4’)
may bind to the protein in two different ways: either in the
or (3’-4’) or (2’-5’) di-methoxy benzyl group (7g, 7i-k) were
adenine pocket or in the hydrophobic site and that the
confirmed possessing good inhibitory activity comparable to
crystallographic structure just trapped only one of them. The
7h, these groups being buried in a very hydrophobic cavity and
identification of these two binding modes was a breakthrough
making (as observed for 7h in Figure 3B) predominantly face
that yielded the opportunity to develop two different series
to face π-stacking interactions with Phe138. Moreover, the
starting from the initial hit. The potency and ligand efficiency,
methoxy group in 3’ (as deduced from Figure 3B) is in place
as well as novelty and diversity, encouraged us to prioritize
and distance to make a weak interaction with the hydroxyl
compound 7h for further explorations.
group of Tyr139 that could explain the small difference in
Optimization of 7h initially focused on exploring the SAR of the substituent at position 5, taking inspiration from the already disclosed SAR developed for the optimization of the purine scaffold inhibitors.28,29 Substituents on the aryl ring of this set of analogues allowed probing of potential protein interactions with the helical-conformation side chains of Phe138, Tyr139 and Trp162.
potency of 7h, 7j and 7k compared to 7g and 7i. Additionally, the ortho substitution revealed a negligible effect on the binding (7g vs 7i or 7h vs 7j). Introduction of the five membered dioxolane ring in 2’,3’’ of the phenyl moiety led to 7l, resulting in the most potent compound of the series. It showed a Ki of 0.170 µM with high ligand efficiency (0.39) and a 200-fold improvement in activity with respect to the original hit, (inhibitory potency approaching the limit of
Table 2. Optimization iteration: biochemical potency,
detection in FAXS assay). All the other analogues bearing
ligand efficiency and solubility of compounds 3, 7f-k, and 8b-
either variously substituted phenyl groups or larger diphenyl
c
systems did not show significant improvement in binding NH2
affinity (data not shown). Further substitutions on the
N
benzodioxole moiety previously reported to be beneficial25 led
N N N
Cpd
R1
3
n-Pr
7g
* OMe
1
R
Ki (µM)a 32.2
0.37
Solc (µM) 70
3.5
0.33
3
LEb
to inactive compounds likely due to the poor solubility (data not shown). Structure studies of 7l confirmed the binding mode of the parent compound 7h. Interestingly the benzodioxole moiety occupies the pocket in a manner similar to the PU-DZ8 purines,28,29 perfectly “sandwiched” in the hydrophobic pocket
OMe
7h
2.6
*
0.31
4
Trp162 (See supporting information Figure SI 3).31
OMe
7i
MeO *
3.1
0.31
11
2.1
0.32
4
OMe
After the initial optimization process, the binding affinities of the new compounds reached the limits of sensitivity of the
MeO *
7j OMe
7k
with a water mediated interaction involving Tyr139 and
* OMe
assessed by Fluorescence Polarization (FP). Compound 7l was 2.4
0.31
4
O
tested in both FAXS NMR and Fluorescence Polarization (FP) assays and the Ki values observed were 0.170 µM and 0.035
OMe *
7l
FAXS assay, therefore activity evaluation and SAR were
d
0.170
0.39
15
µM respectively (Table 3).
O
With the benzodioxole moiety identified as the best
OH
8b
*
6.1
0.32
15
HO
8c a
* OH
substituent for position 5, the next step of the optimization process focused on the exploration of positions 7-10 by the
OH
8.4
0.31
30
Ki were obtained with FAXS NMR and values are an average of two or more determinations. bLigand Efficiency LE = -1.4log(Ki)/HAC; HAC = heavy atom count. c Kinetic solubility PBS pH 7.4 µM; d FAXS NMR assay detection limit
introduction of polar moieties aiming to improve both activity and solubility (Table 3), the latter representing a major concern and possibly a cause of the modest cellular activity measured. Towards this end, compound 11, which contains an amino-
methyl moiety attached on C-9, was prepared and tested.
pocket interacting with the side chains of Asp102 and Lys58.
Unfortunately we observed a decrease in the activity likely due
Surprisingly, the introduction of either an amino group or a
to steric repulsion. On the contrary, the insertion of a fluorine
hydroxyl group in position 8 (10b and 9a respectively) as well
atom in position 10 led to the most potent compound (7o) with
as a hydroxyl group on C-7 (9b) produced a marginally
the highest ligand efficiency (0.46 kcal per heavy atom), even
negative effect compared to 7l. However, these groups are
though position 10 is exposed to a severely hindered
better tolerated than halogen, such as for 7m, likely as a
environment by the presence of residues Ile96 and Gly97. A 3-
consequence of the electronic effect on the aromatic ring.
D structural insight of this compound (See supporting
A sulfonamide group was incorporated in position 8 in
information Figure SI 4) revealed that the fluorine atom
order to explore possible H-bonding opportunities with the side
occupies a small pocket defined by residues Ile96 and Gly97,
chains of Lys58 and Asp102 in this region. Compound 7n was
interacts with the carbonyl group of Gly97 and contributes to
obtained despite the synthetic difficulties, but no affinity
the anchorage with Asp93 through a water bridge network.
improvement was gained. The crystal structure of 7n indicated
Residues Ile96 and Gly97 restrain the shape and size of this
the presence of the predicted interactions: the sulfonamide
pocket, so that only very small C-10 groups could be tolerated,
group points toward the solvent and its nitrogen atom is
as indicated by the lack of activity observed with the 10-chloro
hydrogen bonded to Asp102, while one of the two oxygen
derivative 7q.
atom makes water mediated interactions with Lys58 and
Careful inspection of the crystal structure of 7l revealed that
His154 (see Supporting Information Figure SI 5). On the other
the 7 and 8-positions of the triazoloquinazoline core point
hand these interactions did not translate into an affinity
toward the solvent and are more suitable for the insertion of a
improvement. The impact of the type and number of polar
variety of substituents for the improvement of potency and
interactions introduced needs to be optimized experimentally,
adjustment of physicochemical properties. Modeling studies
as the competing effects of binding and solvation are hardly
suggested that we could gain additional potency by designing
predictable in quantitative terms.
analogues that extend into the solvent exposed ribose binding
Table 3. Inhibitor activity against Hsp90 and cellular antiproliferative activity. NH2 2 R 10 9
8
7
N N N
N
O O
0.035
A2780 IC50(µM)a 10
0.43
Sol c (µM) 6
8-F
0.690
5.047
0.35
76
8-SO2 NH2
0.088
8.823
0.35
13
d
Cpd
R2
Ki (FP) (µM)a
7l
H
7m 7n
LEb
7o
10-F
0.005
6.613
0.46
1
7p
8,10-diF
0.079
9.03
0.38
44
11
0.39
11
7q
10-Cl
>10
9a
8-OH
0.126
1
9b
7-OH
0.332
11.02
0.36
2
10b
8-NH2
0.081
5.961
0.40
76
11
9-CH2 NH2
>10
>10
12a
8-NH(CH2 )3OH
0.001d
2.119
0.43
117 20
12b
8-NH(CH2 )2OH
0.012
2.116
0.40
15
a
12c
8-NH(CH2)2 NH2
0.087
8.112
0.35
100
12d
8-NH(CH2 )2NMe2
0.03
9.189
0.35
205
12e
8-NH(CH2 )2(pyrrolidine)
0.084
8.303
0.31
160
12f
8-MePiperazine
0.045
9.01
0.33
52
12g
8-NH(CH2)2OMe
0.064
3.60
0.36
14
13a
8-CONH-(CH2)2-OH
0.056
9.5
0.34
192
13b
8-CONH-(CH2)3-OH
0.034
8.19
0.34
51
Ki (FP) values are an average of two or more determinations. b Ligand Efficiency LE = -1.4log(Ki)/HAC; HAC = heavy atom count. cThermodynamic solubility
pH 7 µM; dTight Binding.
Notwithstanding, molecular docking studies prompted us to
was hypothesized that incorporation of an amine into the alkyl
design chemistry allowing the straightforward introduction of
chain, which would be protonated at physiological pH, could
amines on C-8 to study the SAR of that position (Scheme 2). A
introduce an ionic interaction with Asp102, potentially
number of analogues with different alkyl chains with polar
providing increased affinity. To test these structure-based
ending, linked via an NH or a CO to position 8, were then
hypotheses, a series of aminoalkyl derivatives with a variety of
synthesized and representative examples are shown in Table 3
hydrophilic moieties was prepared (Scheme 2). As reported in
(12a-g and 13a-b). Ultimately, the hydroxyalkyl extension
Table 3, the amino alkyl derivatives 12c-e were less potent
proved potent and soluble, compounds 12a and 12b being at
compared to the hydroxyl analogues. These observations are in
least one order of magnitude more potent than the parent
contrast with the prediction, which did not take into account
compound 7l. We obtained an X-ray crystal structure of 12b
the energy balance of the solvation/desolvation step during the
that confirmed the predicted binding mode and suggested that
enzyme inhibitor binding process. Analogously, a larger
additional optimal interactions were driving the enhanced
substituent such as methyl piperazine in 12f was found almost
inhibitory affinity, since all other interactions appeared
equipotent to 7l.
conserved (Figure 4A). In fact the 3-D structure of 12b in complex with Hsp90 showed that position 8 is indeed the most solvent exposed of the scaffold. The hydrophilic OH group establishes a direct interactions with Asp102. This observation can be inferred to explain the improved potency of 12b (Ki = 0.012 µM) over compound 12g (Ki = 0.064 µM), which cannot make this contact. Similar considerations are also applicable for the C3 analog 12a; molecular docking studies suggest an
Amide links of the aminopropanol and aminoethanol chains (13a-b) did not match the level of improvement observed with the corresponding amine junctions (12a-b), thus revealing the significant role played by the mode of substituent projection into the target space of this area, and the resultant inappropriate distance for the OH group to establish a direct interaction with Asp102.
additional hydrophobic contact of the propyl chain with the
Although the introduction of such a tether with polar
Lys58 side chain that could explain the single digit nanomolar
endings improved the biochemical activity and solubility, an
biochemical activity. Based on the crystal structure of 12b it
antiproliferative activity increase was observed only in a few cases
(Table
3).
Figure 4: (A)Structure of 12b. (B) The ‘unexpected’ binding mode of 18a in HSP90.
Compound 7o described above showed the best ligand
unexpected binding mode different from that of 12b, where the
efficiency in this series, up to this point. Therefore, in order to
aminotriazoloquinazoline moiety is flipped due to steric
enlarge the SAR around triazoloquinazoline derivatives, we
clashes induced by the N-alkyl substituent. The benzodioxole
combined the best performing 8-substitutions with the 10-
moiety points toward the solvent and makes a hydrogen bond
fluoro core structure as reported in Table 4. Compound 17 was
with Lys58. The triazoloquinazoline ring fits into the induced
generally as effective as its parent counterparts (7o and 12f) in
hydrophobic pocket and, consequently, the aminotriazole NH2
inhibiting the Hsp90. Unfortunately, all the efforts to
loses the key interaction with Asp93 and entails a hydrogen
synthesize the desired compound with the best combination of
bond with the carbonyl group of Gly135, a specific feature in
8-hydroxypropylamine with 10-F were vain.
common with the bis-phenolic compound 8b in the original binding mode of this class (Figure 3A). The contribution of the
We nonetheless envisioned further investigation of the 8-10
fluorine atom seems to be essential for reaching activity (18a
disubstitution pattern by working on synthon 7p as reported in
vs 14) by occupying a small hydrophobic dimple defined by
Scheme 3. The 8-10-difluoro aminotriazoloquinazoline 7p,
residues Val150 and Trp162 (electronic effects). The
prepared from 3,5-difluoro anthranilic acid 4p, proved to be a
aminoethanol chain is located at the back of the induced pocket
valuable intermediate in our strategies (Scheme 3). Thus,
and extends into a narrow tunnel leading to the so called “north
introduction of the solubilizing group was successfully
opening”25 with the hydroxyl group, displacing a conserved
achieved by thermal nucleophilic aromatic substitution on the
water molecule and mimicking its interaction with the side
8,10-difluoro derivative 7p, to deliver compounds 18a-e. As
chain of Tyr139, Trp162 and the backbone carbonyl of
previously mentioned (Scheme 3), 1H NMR analysis indicated
Leu103. The structural information obtained for 18a showed
that they were the undesired regioisomers with the solubilizing
how this tunnel can be properly targeted by the ligand and
group in position 10 and the fluorine atom in 8. However, upon
offered another route for further compound optimization. A
testing, the compounds had residual Hsp90 inhibitory activity,
longer N-alkyl moiety or the methoxy capped tether abolished
although only 18a performed decently (Ki = 0.179 µM).
the activity (18b and 18c), likely due to entropic effects or
Figure 4B shows a view of 18a in the ligand binding pocket
because these modifications would not include the interactions
of Hsp90. Interestingly, the 3-D structure reveals once again an
of the replaced water molecule.
Table 4. Biochemical and cellular values for analogues combining optimal substituents at the 8- and 10-positions 2a
R
NH2 N N N
2b
R
N
O O
a
Ki (FP) (µM)a
A2780 IC50 (µM)a
LE
Sol c µM
0.40
15
R2a
R2b
12b
H
NH(CH2)2 OH
0.012
14
NH(CH2)2 OH
H
>5d
17
F
MePiperazine
0.009
1.55
0.33
38
18a
NH(CH2)2 OH
F
0.179
6.516
0.33
23
Cpd
2.116
1
18b
NH(CH2)3 OH
F
>5
>10
2
18c
NH(CH2 )2 OMe
F
>5
>10
1
18d
NH(CH2)2 NH2
F
0.656
6.948
0.31
171
18e
MePiperazine
F
0.257
9.103
0.30
205
Ki (FP) values are an average of two or more determinations. cThermodynamic solubility pH 7 µM. dNMR KI = 13.2 µM;
Selected compounds with potent inhibitory activity in the
Table 5. Correlation between antiproliferative activity and mechanism of action
Hsp90 biochemical assay were further characterized to establish that the antiproliferative activity measured so far was
17
A2780 IC50(µM)a 1.55
Her2 degrad IC50 (µM) 2.05
the depletion of the client protein Her2 (ErbB2) in BT474 cells
12a
2.119
2.56
supporting the premise that the antiproliferative activity was a
12b
2.11
2.88
consequence of chaperone inhibition (Table 5).
7o
6.313
6.706
10b
5.962
18.147
NVP-AUY922
0.001
0.010
Cpd
fully dependent on the inhibition of Hsp90. The Hsp90 dependent mechanism of action was monitored by following
No activity was observed when the selected compounds were tested against the biologically related ATPase Hsc70 as well as against a panel of 56 protein kinases (IC50 > 10 µM).32
a
Table 6 Physicochemical and ADME profile for compound 12a. PSAa
cLogPb
Solc
1.82
20
115.8 a
2 b
IC50 values are an average of two or more determinations.
33
Caco2_ABd
Caco2_ERe
PPBf
HLM g
RLMg
iv CLh
% Fosi
65.4
1
92.0
29.20
600
132
1.5
c
Polar surface Area, calculated in A . Calculated log P. Thermodynamic aqueous solubility, pH 7 (high throuput assay), in µg/mL. dApparent permeability
apical (A) to basolateral (B) in 10-6 cm/s. e Ratio of B
