Targeting Myeloid Differentiation Using Potent 2-Hydroxypyrazolo[1,5- a]pyridine Scaffold-Based Human Dihydroorotate Dehydrogenase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b00373

Human dihydroorotate dehydrogenase (hDHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis, the conversion of dihydroorotate to orotate. hDHODH has recently been found to be associated with acute myelogenous leukemia, a disease for wh

Full text of DOI:10.1021/acs.jmedchem.8b00373

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Article
Targeting myeloid differentiation using potent 2-
hydroxypyrazolo[1,5-a]pyridine scaffold-based human
dihydroorotate dehydrogenase (hDHODH) inhibitors
Stefano Sainas, Agnese Chiara Pippione, Elisa Lupino, Marta Giorgis, paola circosta, Valentina
Gaidano, parveen goyal, davide bonanni, Barbara Rolando, Alessandro Cignetti, Alex Ducime,
Mikael Andersson, Michael Järvå, Rosmarie Friemann, Marco Piccinini, Cristina Ramondetti,
Barbara Buccinnà, Salam Al-Karadaghi, Donatella Boschi, Giuseppe Saglio, and Marco L Lolli
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00373 • Publication Date (Web): 25 Jun 2018
Downloaded from http://pubs.acs.org on June 25, 2018
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Targeting Myeloid Differentiation Using Potent
2ꢀHydroxypyrazolo[1,5ꢀa]pyridine ScaffoldꢀBased
Human Dihydroorotate Dehydrogenase
(hDHODH) Inhibitors
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a
a
b
a
Stefano Sainas, Agnese C. Pippione, Elisa Lupino, Marta Giorgis, Paola
c,d
e,f
g
a
Circosta, Valentina Gaidano, Parveen Goyal, Davide Bonanni, Barbara
a
e,f
a
g
Rolando, Alessandro Cignetti, Alex Ducime, Mikael Andersson, Michael
h
g
b
b
Järvå, Rosmarie Friemann, Marco Piccinini, Cristina Ramondetti, Barbara
b
i
a
e,f
Buccinnà, Salam AlꢀKaradaghi, Donatella Boschi, Giuseppe Saglio and
a
Marco L. Lolli*
a
Department of Drug Science and Technology, University of Turin, 10125 Turin (Italy);
b
Department of Oncology, University of Turin, 10125 Turin (Italy);
c
Department of Molecular Biotechnology and Health Sciences, University of Turin, 10126 Turin (Italy);
d
Molecular Biotechnology Center, 10126 Turin (Italy);
e
Department of Clinical and Biological Sciences, University of Turin, 10043 Turin (Italy);
f
Mauriziano Hospital S.C.D.U. Hematology – 10128 Turin (Italy);
g
Department of Chemistry and Molecular Biology, University of Gothenburg, SE 405 Gothenburg (Sweden);
h
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, VIC
3086 Melbourne, Australia;
i
Department of Biochemistry and Structural Biology, Lund University, 221 00 Lund (Sweden);
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Keywords
Bioisosterism, dihydroorotate dehydrogenase (DHODH) inhibitors, leflunomide, brequinar,
autoimmune diseases, hydroxyazoles, pyrazolo[1,5ꢀa]pyridine, acute myelogenous leukemia
(AML), myeloid differentiation.
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Abstract
Human dihydroorotate dehydrogenase (hDHODH) catalyzes the rateꢀlimiting step in de
novo pyrimidine biosynthesis, the conversion of dihydroorotate to orotate. hDHODH has
recently been found to be associated with acute myelogenous leukemia, a disease for which
the standard of intensive care has not changed over decades. This work presents a novel class
of hDHODH inhibitors, which are based on an unusual carboxylic group bioisostere 2ꢀ
hydroxypyrazolo[1,5ꢀa]pyridine, that has been designed starting from brequinar, one of the
most potent hDHODH inhibitors. A combination of structureꢀbased and ligandꢀbased
strategies produced compound 4, which shows brequinarꢀlike hDHODH potency in vitro and
is superior in terms of cytotoxicity and immunosuppression. Compound 4 also restores
myeloid differentiation in leukemia cell lines at concentrations that are one log digit lower
than those achieved in experiments with brequinar. This paper reports the design, synthesis,
SAR, Xꢀray crystallography, biological assays and physicochemical characterization of the
new class of hDHODH inhibitors.
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1
.
INTRODUCTION
Human dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11) is a flavinꢀdependent
enzyme located in the inner mitochondrial membrane and involved in the de novo pyrimidine
biosynthesis. hDHODH is a therapeutic target that has been validated for the treatment of
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1ꢀ3
autoimmune diseases and cancer. While a variety of hDHODH inhibitors have been
studied over the years, leflunomide and its metabolite teriflunomide are still the only
4
hDHODHꢀtargeting drugs (Figure 1), approved for the treatment of rheumatoid arthritis and
5, 6
other autoimmune diseases. Brequinar, one of the most potent hDHODH inhibitors known
to date, has been identified during the search for new compounds that would display clinical
7
benefits similar to those of leflunomide, but without the associated side effects.
Unfortunately, brequinar was discarded as a therapeutic agent when submitted to clinical
8
9
trials for cancer, and for the prevention of organ transplant rejection, due to its severe side
1, 9, 10
effects, a narrow therapeutic window and inconsistent pharmacokinetics.
1
1, 12
In the fall of 2016, two publications,
demonstrated the central role that hDHODH plays
in acute myelogenous leukemia (AML), a disease for which standard intensive care has
13
undergone little change over the last four decades. In AML, the most common acute
leukemia in adults that affects the myeloid lineage of white blood cells, leukemic cells lose
their ability to differentiate into adult white blood cells. This leads to the accumulation in the
bone marrow of immature cells, which are characterized by high proliferation potential and
known to interfere with the production of normal blood cells. The disease progresses rapidly
11
and is typically fatal within weeks or months if left untreated. A study by Sykes et al.,
suggested that hDHODH plays a central role in the regulation of myeloid differentiation in
both in vitro and in vivo models, and thus opens up totally new perspectives for AML
treatment. Using brequinar, the authors showed that hDHODH inhibition has a profound
effect on the induction of myeloid differentiation, delay of disease development and
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reduction of the burden of leukemiaꢀinitiating cells in various AML mouse models, human
cell line xenografts, patientꢀderived xenografts and syngeneic mouse models.
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Figure 1. Leflunomide, brequinar and hydroxyazole analogues 1 – 3.
The discovery of the central role of hDHODH in AML immediately attracted the interest of
the pharmaceutical industry. Two newlyꢀpatented hDHODH inhibitors are currently being
investigated for the treatment of AML. The first one, ASLAN003 (by ASLAN
Pharmaceuticals), entered Phase II clinical trials in November 2017 (NCT03451084), while
the second compound by Bayer (BAY2402234), entered Phase I clinical trials in January
2018 (NCT03404726). In May 2018, the patent containing the BAY2402234 structure and a
15
description of the activity profile has been published.
2, 4 16ꢀ
The large number of new hDHODH inhibitors that have been reported in recent years,
19
and the rising interest from the industry suggest that there is a wide interest towards the
development of new hDHODH inhibitors.
1
4, 20
We have recently introduced a new generation of hDHODH inhibitors
that were designed
via scaffoldꢀhopping replacement of brequinar's acidic moiety with a variety of acidic
14, 20ꢀ24
hydroxylated azoles.
Three of these compounds (1, 2 and 3), which are based on
hydroxythiadiazole, hydroxytriazole and hydroxypyrazole respectively (Figure 1), showed
high hDHODH inhibitory activity in vitro, with compound 1 being the best in the series with
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an IC value of 16 nM. The Xꢀray crystallographic structures of 1, 2 and 3 in complex with
50
hDHODH showed that the acidic scaffolds of the compounds interact with Arg136 at subsite
14
2
of hDHODH ubiquinone binding site, in a way reminiscent of the interactions formed by
25
the carboxylic group of brequinar. In addition, each scaffold was able to establish
interactions with the small lipophilic pocket created by Val143 and Val134, known as subsite
4 (the location of the subsites is shown by numbers in Figure 3). When assessed for
antiproliferative activity, the compounds were found to be effective in the same concentration
range as brequinar, although with much lower cytotoxicity, showing cytotoxic effects first at
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70ꢀtimes the concentrations required to inhibit cell proliferation.
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Figure 2. Structures of compounds 4 – 10, which are based on the 2ꢀhydroxypyrazolo[1,5ꢀ
a]pyridine scaffold and characterized by the presence of either a biphenylic (a), or biphenyl
ether substituent (b).
This work reports the design of a new series of potent hDHODH inhibitors based on 2ꢀ
hydroxypyrazolo[1,5ꢀa]pyridine as an acidic scaffold, a system that is still relatively
unexplored in the literature. Here we report the first scaffoldꢀhopping study of this structure
aimed at generating a bioisostere with a carboxylic function. In the first series, besides
investigating the moiety itself (compound 4 and 7, Figure 2a), we have also investigated the
effect of introducing a methyl group into the pyridine ring in order to improve its lipophilic
interaction with subsite 4 (compounds 5 and 6, Figure 2a). In the second series (compounds 8
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ꢀ
10, Figure 2b), we replaced the biphenyl substituent with a more flexible and polar diphenyl
26
ether in order to improve pharmacokinetics and provide more drugꢀlike properties. This
paper reports and discusses the design principles, modeling, synthesis, SAR and Xꢀray
crystallographic analysis of this new generation of hDHODH inhibitors. In addition,
biological assays (including cell viability, proliferation, cytotoxicity, immunosuppression and
myeloid differentiation), physicoꢀchemical characterization and preliminary drugꢀlike
properties are also presented.
2. RESULTS AND DISCUSSION
2.1 Chemistry: synthesis of target compounds 4 – 10.
The chemical strategies used to produce 17a and the regiosubstituted, protected 2ꢀ
hydroxypyrazolo[1,5ꢀa]pyridine building blocks 20aꢀc, which are useful in the syntheses of
target compounds 4 ꢀ 10, are shown in scheme 1.
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Scheme 1. Synthetic strategies for the preparation of substituted hydroxylated pyrazolo[1,5ꢀ
a]pyridine intermediates. i) HOSA, H
2
O, 90 °C; ii) K
ethanol, 90 °C; iv) tꢀBuO K , dry THF v) 0.1 N HCl; vi) Cs
Cs CO , BnBr, dry DMF, viii) 5 M NaOH, ethanol, 70°C.
2
CO
3
, EtOH; iii) diethylmalonate,
ꢀ
+
2
CO
3
, MeI, dry THF, 40 °C; vii)
2
3
Compounds 15a,b,c were prepared via a slight modification to a known procedure (Scheme
27
1), starting from either pyridine or the corresponding substituted pyridines. Compounds 12a
ꢀ c were obtained by aminating the substituted pyridines 11a ꢀ c, using hydroxylamineꢀOꢀ
2 3
sulfonic acid (HOSA) as the aminating reagent. The products were treated with K CO to
give the yields of 13 that were reacted with diethyl malonate in EtOH to give intermediate
types 14, which were, in turn, converted into the desired compounds 15a ꢀ c, in the presence
ꢀ
+
of a strong base (tꢀBuO K ), with 18 – 21 % overall yields.
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Although some examples have been reported in the literature, the reactivity pattern that 2ꢀ
hydroxypyrazolo[1,5ꢀa]pyridines show towards alkylating agents has never been fully
14, 29
investigated. Both Oꢀ and Nꢀ alkylation patterns must always be considered,
when
considering the reactivity of substituted hydroxylated azoles. The type of the heteroatom
within the heterocyclic system and the choice of the alkylating agent used usually control the
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14, 23, 30
alkylation pattern.
Moving onwards in Scheme 1, the alkylation of 15a ꢀ c with benzyl
bromide also gave the corresponding Nꢀalkylated derivatives 18a ꢀ c (ratio 5 – 29 %), besides
desired Oꢀalkylated compounds 19a ꢀ c, in each case. A similar result was obtained when
methyl iodide was used as an alkylating agent on 15a, producing the methylated isomers 16a
and 17a in 35 % and 59 % yields, respectively. 2DꢀNMR spectroscopy was used to
univocally attribute the relative isomeric structures (see Supplementary material). Esters 19a
ꢀ c were then hydrolyzed under basic conditions to obtain the corresponding acids 20a ꢀ c in
good yields, which were then used for the preparation of targets 4 – 6, 8 ꢀ 10, as described in
scheme 2.
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Scheme 2. Synthesis of targets 4 ꢀ 10: i) oxalyl chloride, dry DMF, dry THF; ii) AlMe , dry
2 2
toluene, reflux; iii) H , Pd/C, 37 % HCl, ethanol; iv) dry toluene, reflux; v) H , Pd/C, dry
THF, vi) 5M NaOH, ethanol, r.t.
Starting from acids 20a ꢀ c (see Scheme 1), the corresponding acyl chlorides were obtained
via treatment with oxalyl chloride in dichloromethane and used directly after drying without
any further purification. In order to improve their reactivity with acyl chlorides, 2,3,5,6ꢀ
tetrafluoroꢀ4ꢀphenylaniline (21), was converted into its corresponding dimethylaluminum
amide. The desired amides 22a ꢀ c were obtained in the 38 ꢀ 45 % yield range. Interestingly,
the benzylic protection transposed from the exocyclic oxygen to the endocyclic N1 nitrogen
1
3
during coupling (see Supplementary for the characterization details. The CꢀNMR chemical
shifts of the CH benzylic nucleus were diagnostic for the structural attribution of Nꢀbenzyl
2
or Oꢀbenzyl derivatives of 22a –c and 26 – 28 respectively as for compounds 18a and 19a).
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On the other hand, the coupling of the acyl chloride, derived from 20a, with phenoxy anilines
23 ꢀ 25 produced the expected Oꢀbenzylꢀprotected amides 26 ꢀ 28, as desired. This difference
in reactivity led us to correlate the migration of the benzyl group with the presence of a
Lewis acid in the reaction mixture. Compounds 22a ꢀ c and 26 ꢀ 28 were then converted to
the desired target compounds 4 ꢀ 6, 8 ꢀ 10 by applying roomꢀpressure catalytic hydrogenation
conditions. The same approach was applied to the preparation of 7. In this case, the acidic
compound obtained from the hydrolysis of 17b was quite unstable, meaning that any
isolation attempt resulted in decarboxylation. We avoided this decomposition by isolating the
intermediate as a sodium salt and transforming it into the corresponding acyl chloride. The
acyl chloride was stable enough to react with the dimethylaluminum amide of 2,3,5,6ꢀ
tetrafluoroꢀ4ꢀphenylaniline producing compound 7 with a 27 % yield.
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2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.2 Inhibition of
hDHODH and structureꢀactivity relationships (SAR).
Initially, we evaluated recombinant hDHODH inhibition activity of compounds 4 ꢀ 10 using
14
brequinar, teriflunomide and the hydroxytriazole analogues 2, as reference compounds
(Table 1). Among the firstꢀgeneration compounds 1 – 3, compound 2 showed the best
balance between hDHODH potency and cell cytotoxicity and, for this reason, it was
considered to be the most promising compound and included in this study as reference.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Immunoꢀ
b
b
c
Proliferation
Proliferation
Cytotoxicity
Immunoꢀ
a
d
hDHODH
suppression
d
Compound
IC50 ± SE
IC50 ± SE
(effect ≥ 30%)
(µM)
suppression
IC50 ± SE (µM)
IC50 ± SE (µM) +
Uridine
(
µM)
(µM) + Uridine
IC50 ± SE (µM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Brequinar
0.0018±0.0003
0.388±0.064
0.045±0.013
0.0012 ± 0.0002
0.0043±0.0005
0.035 ± 0.003
> 5
0.91±0.07
94.17±2.08
nd
48.2±0.8
53±3
3.74±0.06
59.64±2.18
nd
1
4
Teriflunomide
43.22±1.24
1.88±0.06
0.75±0.04
0.82±0.03
1.56±0.08
nd
54.3±3.1
1
4
2
4
nd
>100
8.9±0.7
nd
68.69±2.35
35.62±0.98
88.45±1.48
nd
60.4±1.2
41.3±1.5
48.6±2.3
nd
0.78±0.06
0.77±0.08
1.08±0.10
nd
57.15±2.06
46.84±1.27
52.39±1.46
nd
5
6
7
8
0.760 ± 0.136
0.480 ± 0.031
0.043 ± 0.005
89.66±1.64
67.55±1.21
1.47±0.06
95.63±2.11
>100
>100
69.25±2.47
35.26±2.34
0.84±0.16
>100
9
>100
>100
10
55.13±2.05
>100
74.69±1.63
Table 1: Biological effects of compounds 2, 4 – 10, brequinar and teriflunomide. The effect
a)
of the compounds (expressed as IC value, except for cytotoxicity), on hDHODH in vitro
50
b)
c)
assay;
cell proliferation inhibition (Jurkat T cells);
cytotoxicity, concentration of
d)
compounds causing a significant (≥ 30%) cytotoxic effect (Jurkat T cells); PHAꢀstimulated
PBMC proliferation inhibition. The “nd” notation indicates that the compound was not tested
in that specific assay.
2
ꢀHydroxypyrazolo[1,5ꢀa]pyridine analogue 4 (IC50 = 1.2 nM), was found to be the most
potent inhibitor in the series, as it had 320 times higher activity when compared to
teriflunomide (IC50 = 388 nM), and comparable activity to brequinar (IC50 = 1.8 nM) in the
enzymatic assay. A crystallographic study (see section 2.3) was carried out to experimentally
determine the binding pose of compound 4 in the ubiquinone binding pocket of hDHODH. It
is interesting to note here how brequinarꢀlike potency was obtained even when a weaker
a
acidic moiety (a table containing the pK values of hydroxylꢀpyrazolo[1,5ꢀa]pyridine
1
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analogues is included in the Supplementary section, see Table S2), was involved in the key
interaction with Arg136 at subsite 2. Moreover, this interaction is essential for activity, as the
neutral compound 7 is totally inactive.
Further, we focused on the possibility of decorating the “pyridine” part of compound 4, to
add an interaction with the small lipophilic pocket created by Val134 and Val143 at subsite 4.
Here, we carried out relative binding freeꢀenergy calculations (i.e., the relative difference in
binding energy between two compounds) using a nonꢀphysical thermodynamic cycle. The
differences in binding affinities for the proteinꢀligand complexes were calculated with the aid
of freeꢀenergy perturbation (FEP) approach based on Molecular Dynamics (MD) simulations,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
31
the methodological detail are given in the supplementary information. The four available
positions of 4, indicated in Table 2, were explored using MD/FEP methyl and chlorine scans
32, 33
to identify the most promising sites for a beneficial hydrogen substitution.
H to Cl ꢁꢁG
3
H to CH ꢁꢁG
(
kcal/mol)
(kcal/mol)
C4
C5
C6
C7
ꢀ0.35 ± 0.02
ꢀ0.48 ± 0.02
0.87 ± 0.02
ꢀ1.43 ± 0.02
1.49 ± 0.07
1.19 ± 0.07
2.59 ± 0.07
0.15 ± 0.09
Table 2. MD/FEP results of the change in calculated free energy of binding (in kcal/mol),
and the computed uncertainty, for the introduction of chlorine and methyl substituents on the
2ꢀhydroxypyrazolo[1,5ꢀa]pyridine motif of compound 4.
The calculated ꢁꢁG values indicate that the chlorine group is generally preferred over the
methyl in all the positions, since negative values represent higher binding affinity. Among
the four sites, position 7 is the most energetically profitable for a substitution as it is
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
associated with the lower energy value of ꢀ1.43 for the chlorine and has only a marginal
effect on the energy in the case of the methyl group (0.15). Replacement of the hydrogen in
position 6 is less favorable as it is evident from the higher positive energy values for the
chlorine (0.87) and the methyl (2.59). Positions 4 and 5 show comparable behavior, with
substitution in 5 only slightly better tolerated than in 4 for both groups.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A probable reason of a better tolerability of the chlorine substitution as compared to the
methyl group could be a result of the interaction of the 2ꢀhydroxypyrazolo[1,5ꢀa]pyridine
moiety with the side chain of His56 (see section 2.3). Here, the πꢀstaking interaction energy
of the aromatic rings could be complemented by the electronꢀwithdrawing effect of the
34
additional chlorine in compound 4. The FEP results also show that methyl modulations did
not lead to an improvement in ꢁꢁG, suggesting that additional lipophilic interactions did not
contribute to the binding affinity. This also supports the importance of the electrostatic
effects of the substituents on the interactions. Remarkably, similar variations of ꢁꢁG were
found among the four positions for both methyl and chlorine scans. It was also noted that the
most tolerated position for substituents was at C7 (Table 2) ꢀ it appears that small groups at
this position could make use of the space created around Val143, Val134 and Arg136. On the
other hand, the predicted changes in the free energy revealed that replacements at C6 do not
provide any advantages, presumably due to the steric affects associated with the side chains
of Val134 and Val143. FEP analysis on C4 and C5 showed comparable results, suggesting
that the placement of additional lipophilic groups at subsite 3 of the enzyme did not
contribute to any new favorable interactions.
Moving to experimental assessment and taking into account the MD/FEP results, the two
most tolerated positions 5 and 7 were considered in the next investigations. Since the chlorine
derivatives of 4 are not currently synthetically accessible, compounds with methyl
substituents in positions 7 and 5 were synthetized (Scheme 1), leading to compounds 5 and 6,
respectively. While the substitution at position 5 decreased the activity 29 fold (6, IC50 = 35
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7
8
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nM), as compared to compound 4, the substitution at position 7 yielded a similar profile (5,
IC50 = 4.3 nM).
In the second series (compounds 8 ꢀ 10, Figure 2), the replacement of the biphenylic
substituent was investigated in an attempt to improve pharmacokinetics and obtain more
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
26
14, 20
drugꢀlike compounds. In our earlier studies,
optimal interactions with the lipophilic
subsite 1 were only guaranteed with a tetrafluoro substitution on the first ring.
3
5
Conformational analyses, underlined the role of incremental fluorine substitution on the
first ring in stabilizing the brequinarꢀlike binding mode, which has previously been found to
25
be connected with higher inhibitory potency. For example, the removal of two or three
fluorine atoms from the biphenylic scaffold of triazole analogue 2 resulted in a dramatic drop
1
4
in inhibitory activity. However, the presence of the tetrafluorobiphenylic substituent is
26
detrimental for the solubility of the derivatives. We therefore decided to design analogues
that lacked a biphenylic scaffold in order to explore novel possibilities. Inspiration was taken
36
from a recent study by Das et al, in which brequinar–like activity was replicated by a
brequinarꢀrelated compound that included a substituted diphenylether; compounds 8 – 10,
which contain a variety of diphenylether substituents, were thus designed. Although a
dramatic drop in activity was observed in moving from 4 (IC50 = 1.2 nM) to 8 (IC50 = 760
nM), compound 8 firstly proved the possibility to design inhibitors where the biphenylic is
replaced by the diphenylether scaffold. The addition of a methyl group at position 3 of the
first ring (compound 9), led to activity moving closer to the teriflunomide range (IC50 = 480
nM). Adding a second methyl at position 6 (compound 10), finally allowed a nM range to be
reached (IC50 = 43 nM). On the basis of these results, we envision that the increased rigidity
granted by the double methyl substitution and the increase in hydrophobicity might be crucial
for activity.
2.3 Binding mode analysis: crystallographic and molecular modeling studies.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In order to achieve further insights into the binding mode of this new generation of 2ꢀ
hydroxypyrazolo[1,5ꢀa]pyridine moietyꢀbased inhibitors, we determined the crystal structure
of the complex between 4, as it is one of the most potent hDHODH inhibitors yet described,
with the protein. The crystals diffracted to 1.58 Å (PDB id: 6FMD) and the structure was
determined by molecular replacement. Xꢀray data and refinement statistics are summarized in
Table S1 (Supplementary). The inhibitor was clearly bound in the ubiquinone binding site
with high quality electron density (see Supplementary. Figure S2). It shows a binding mode
that can be perfectly superimposed onto the previously reported crystal structure of the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
14
complex with brequinar (Figure. 3). As can be seen in Figure 3, which shows the entrance
of the binding pocket, the tetrafluorobiphenilic moiety of compound 4 is positioned in subsite
1, a lipophilic cavity that is built up by Met43, Leu42, Leu46, Ala59, Phe62, Phe98 Leu68,
Leu359 and Pro364. The hydroxylꢀpyrazolo[1,5ꢀa]pyridine moiety is extended over subsites
2
and 3, occupying the innermost part of the pocket. In addition, there is an ion bridge
extending to the side chain of Arg136 and a hydrogen bond with the side chain of Gln47. The
pyridine moiety is seen to be able to fit perfectly within the lipophilic subsite 4 (Val134 and
Val143). As this interaction is the only difference between this and the previous generation of
inhibitors, it appears to be the reason for compound 4’s high potency, which is more than 10
1
4
times that of our best earlier generation compound, the thiadiazole 1 (IC 16 nM).
5
0
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Ubiquinone binding sites of hDHODH coꢀcrystallized with compound 4 (blue)
(PDB id: 6FMD), superimposed on the complex with brequinar analog, (PDB id: 1D3G);
only the ligand is shown in pink. Hydrogen bonds are shown as dashed lines. Flavin
mononucleotide and orotate are represented in grey and yellow, respectively.
Compound 10 represents a good starting point for future modulation of the small
diphenylether series (8 – 10). Its activity towards hDHODH is just comparable to that
observed for triazole 2, which is one of the best compounds in the first series. In order to
better understand the role played by substitutions at the first diphenylether ring in influencing
activity, a MD study was performed on compounds 4, 8, 9 and 10. Starting from the docked
conformation of the four compounds in the target binding site, 50 ns long MD simulation for
each compound was carried out. The root mean square deviations (RMSD) for the ligands
and the protein α carbon chain were calculated with respect to the starting structures. In each
system analyzed, the protein structures reached stability after a few ns of simulation and then
remained stable with an RMSD of around 0.95 Å (Figure S1). More interesting still is the
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
change in RMSD of the atomic coordinates of the ligands in the binding site. Figure 4 shows
the average conformations of compounds 4, 8, 9, 10 assumed during the MD simulations and
the relative RMSD plots.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4: The graphs show the evolution of the RMSD values (calculated for ligand heavy
atoms as compared with starting structures) of the hDHODH complex analyzed by MD
simulation. A, B, C and D in the figure correspond to compounds 4, 8, 9, 10, respectively.
The compounds and surrounding amino acids are in the average conformations assumed
during MD simulations.
While compound 4 shows a stable conformation, with an average RMSD of around 0.6 Å
(Figure 4A), in all the other simulations considerably higher RMSD values were obtained for
compound 8 (Figure 4B). The unsubstituted first ring in compound 8 allows free rotation of
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the phenylꢀOꢀphenyl dihedral angle inside subsite 1, leading to the conformational variability
seen along the MD trajectories. During the simulation, as seen in Figure 4B, compound 8
appears to have two relatively stable conformations in which one is similar to the starting
docking pose, identified by an RMSD of around 0.6 Å. In the second, the distal phenyl of the
diphenylether points towards Tyr38, leading to an RMSD of around 2.0 Å. As long as
activity values are concerned, the alternating behavior observed for compound 8 results in a
loss of an interaction, which is reflected in reduced activity. Substitution on the first ring in
compounds 9 and 10 (Figure 4C and 4D), led to a more stable profile. In fact, after an initial
increase in RMSD, the compounds reached a steady conformation with average RMSDs of
around 1.6 and 1.2 Å, respectively. Activity values appear to indicate that the additional
degree of freedom associated with the diphenylether is strictly correlated with the
compounds’ binding affinity. Indeed, the introduction of methyl substituents in compounds 9
and 10 seems to stabilize the favored binding conformations leading to a more optimal
interaction with the protein. It is worth noting that the activity of compound 9, which is only
slightly higher than that of 8, clearly indicates how a single substitution is not sufficient to
achieve the rigidity required for optimal binding affinity. On the other hand, the double
methyl substitution in compound 10 is able to recover the activity, which reaches a nM range,
suggesting that molecule rigidity is one of the key attributes for hDHODH structureꢀactivity
relationship (SAR).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Although MD analysis gives important insights into the role of the diphenylether moiety in
the binding to hDHODH, the free energy of binding (FEB) was still required for the
understanding of the structureꢀactivity relationships. MM/GBSA calculations were used to
provide a quantitative way to evaluate the different components of interaction energy that
contributes to binding of compounds 4, 8, 9 and 10. All the free energies were computed on
the last 40 ns of the simulated trajectory. Detailed results are summarized in Table 3.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
b
c
d
e
f
Ligand
IC (nM) ꢁTotal
ꢁEvdW
ꢁEelec
ꢁEelec.solv
78.91
86.46
84.09
86.55
ꢁGgas
ꢁGsolv
5
0
4
8
9
1.2
ꢀ40.87
ꢀ36.08
ꢀ37.18
ꢀ40.32
ꢀ55.44
ꢀ53.80
ꢀ53.80
ꢀ56.68
ꢀ57.57
ꢀ62.28
ꢀ60.92
ꢀ63.42
ꢀ113.02
ꢀ116.08
ꢀ114.72
ꢀ120.09
72.14
80.00
77.54
79.77
760
480
43
10
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a)
Table 3. Free energy analysis for the binding of compounds 4, 8, 9 and 10 to hDHODH.
b)
Final estimated binding free energy calculated from the terms below. Nonbonded van der
c)
d)
e)
Waals. Nonbonded electrostatics. Electrostatics contribution to solvation. Total gas
f)
phase energy. Sum of nonpolar and polar contributions to solvation.
The MMꢀGBSA calculations show a good agreement with the experimental activity data.
According to the experiments, the difference in FEB of compound 4 (ꢁTotal = ꢀ40.87
kcal/mol) and 8 (ꢁTotal = ꢀ36.08 kcal/mol) is found more than 4 kcal/mol, suggesting that 4
is the most active in the series. Moving to compounds 9 (ꢁTotal = ꢀ37.18 kcal/mol) and 10
(ꢁTotal = ꢀ40.32 kcal/mol), obtained by modulation of the first ring of compound 8, an
improvement of the ꢁTotal can be observed. While the computed free binding energy for
compound 9 is still close to 8, compound 10 shows a free energy value close to 4. Detailing
the binding free energy composition, it can be noticed how electrostatic components
contribute more in 8, 9 and 10, if compared with 4. On the other hand, electrostatic solvation
(
ꢁEele,solv) disfavors the binding of the diphenylether series because of a greater deꢀsolvation
penalty respect the tetrafluorinated biphenyl scaffold. Remarkably, considering the van der
Waals contributes (ꢁEvdW), only 10 reaches a value that overcome compound 4. A perꢀ
residue energy decomposition (PRED) analysis was also performed in order to identify the
key residues that contribute to binding affinity at the binding site.
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Figure 5. The perꢀresidue free energy contribution to binding for compounds 4, 8, 9, 10.
As shown in Figure 5, Arg136 and Glu47 mainly contribute to binding, pointing to the
significance of the interactions with these residues. PRED results are consistent with the
binding mode proposed for compound 9 (data not shown), and 10 (Figure 6). For Met43,
Leu46 and Leu58, the energy contributions suggest a higher interaction with 9 and 10 respect
compound 4. This result is in agreement with the observed slightly different position assumed
by 10 at binding side if compared with 4. Moreover, perꢀresidue free energy analysis also
suggest that Phe62 is a key residue for binding. On the basis of Xꢀray structure (Figure 6), the
side chain of Phe62 is involved in a πꢀπ interaction with the distal phenyl of 4. The binding
contribution of Phe62 for 9 and 10 (ꢀ1.05 kcal/mol and ꢀ1.09 kcal/mol, respectively) is less
than about 1 kcal/mol compared to 4 (ꢀ1.97 kcal/mol). This is according to binding mode of
10, where the staking interaction with Phe62 is partially lost because the shifting of the
second ring of the diphenylether toward the cavity composed by Leu42, Leu46 and Tyr38.
Taken together, MD and FBE analysis are coherently with bioassay results and explain the
SAR of replacement on diphenylether scaffold, highlighting how methyl groups on the first
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ring gave an important contribute to stabilize the bioactive conformation by decreasing ligand
flexibility and optimizing the interactions with subsite 1 and 2.
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Figure 6. Ubiquinone binding sites of hDHODH with bound compound 4 (PDB id: 6FMD,
protein in green and ligand in blue), superimposed on the average structure of the hDHODHꢀ
compound 10 complex produced in MD simulation (protein and ligand in tan). Hydrogen
bonds are shown as dashed line. Flavin mononucleotide and orotate are represented in grey
and yellow, respectively.
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.4 Cell based assays.
.4.1 Proliferation, cytotoxicity and immunosuppression on Jurkat cells.
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After evaluating compounds 4 ꢀ 10 for their ability to inhibit recombinant hDHODH in vitro,
active compounds 4 ꢀ 6 and 8 ꢀ 10 were tested for their effects on cell proliferation in Jurkat
T cells (Table 1). The stability of the compounds under the applied experimental conditions
was also checked and they were all found to be stable (See Supplementary). The potent
hDHODH in vitro activity observed for compounds 4 and 5 was translated into a potent
antiproliferative effect, which was slightly superior to that of brequinar itself in both cases.
Compounds 6 and 8 ꢀ 10 displayed similar profiles, although weaker hDHODH potency was
reflected in weaker antiproliferative effects. Besides 4 and 5, 6 and 10 also outperformed
teriflunomide, both showing antiproliferative effects that were 30 times more potent. The
DHODHꢀdependence of the antiproliferative effects of compounds 4 ꢀ 6 and 8 ꢀ 10 were also
3
7
tested by assaying their activity in the presence of 100 µM uridine. As shown in Table 1,
the antiproliferative effects were reverted by the addition of exogenous uridine, which
strongly indicates that the compounds act as pyrimidine biosynthesis inhibitors, and thus
inhibit Jurkat cell proliferation via this mechanism. The exception to this is 8, which is
probably too weak as hDHODH inhibitor to produce a reverse uridineꢀmediated effect. In
order to evaluate whether the antiproliferative effects resulted from cell death, cytotoxicity
was evaluated on Jurkat T cells using the CellTox green assay and the concentration of
compounds that was able to cause 30 % cell death were detected. Compound 4 had no
negative effect on cell viability up to 60 µM, while 5 and 6 were found to be cytotoxic in a
concentration similar to that of brequinar. Intriguingly, no negative effect on cell viability
was observed for compounds 8 ꢀ 10, for which a diphenylether was used to introduce subsite
1
interactions, even when they were tested at a concentration of 100 µM. This result is quite
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interesting as it shows how promising drugꢀlike profiles can be obtained using this moiety for
targeting subsite 1 interactions.
In order to investigate whether the immunosuppressive activity of the compounds, their effect
on the proliferation of phytohaemagglutininꢀactivated peripheral blood mononuclear cells
(PBMCs) was evaluated and compared with that of brequinar. As shown in Table 1, the
antiproliferative effect of brequinar is 10 times greater than that of teriflunomide (3.74 and
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54.3 ꢂM, respectively), which confirms earlier research. It was observed that potent activity
against hDHODH correlates with the potent inhibition of activated PBMC proliferation for
all tested compounds. This inhibition, however, can be reversed by the addition of exogenous
uridine, suggesting that the immunosuppressive activity of the compounds may be due to the
inhibition of de novo pyrimidine nucleotide synthesis.
2.4.2 Proliferation, cytotoxicity and myeloid differentiation in Leukemia cells.
In the last phase of our study, we evaluated the effects of our hDHODH inhibitors on two
AML cell lines (U937 and THP1). We decided to compare compound 4, judged as the best
compromise between potency and cytotoxicity, triazole 2, the top compound from the first
series, which displays very low cytotoxicity, and brequinar, which was used as a positive
control. In the initial experiments, we evaluated cell viability using CFSEꢀbased assays. As
shown in Figure 7A, both compound 4 and brequinar show strong and concentrationꢀ
dependent cytotoxicity, while compound 2 is only able to induce cell death at high doses.
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Figure 7. A) Cytotoxicity induced by different concentrations of brequinar, compound 2 and
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on U937 and THP1. Statistical significance: *p < 0.05; ** p < 0.01 between our compounds
and brequinar. B) Cytotoxicity is totally reversed when uridine is added to compound 4, but
not to compound 2, both in U937 and THP1. Statistical significance: *p < 0.05; ** p < 0.01
between each compound ± uridine. Uridine was added at 100 µM concentration. C)
Proliferation inhibition exerted by different concentrations of brequinar, compound 2 and 4
on U937 and THP1. Statistical significance: *p < 0.05; ** p < 0.01; ***p < 0.001 between
treated and untreated cells (DMSO only). # p < 0.05 between our compounds and brequinar.
DMSO = dimethyl sulfoxide, i.e., the solvent of all tested compounds.
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The cytotoxicity of compound 4 was totally reversed when uridine was added, but this was
not the case for compound 2 (Figure 7B). This suggests that, in the case of compound 4
cytotoxicity may only be ascribed to hDHODH inhibition, while cytotoxicity of compound 2
at high doses is probably associated with offꢀtarget effects. Moreover, cytotoxicity against
U937 was slightly more evident than that against THP1, which probably reflects the
heterogeneity of AML. CFSEꢀbased proliferation assays were also performed and, as
expected, compound 4, brequinar and, to a lesser extent, compound 2, all greatly reduced cell
proliferation, as shown in Figure 7C. Interestingly, compound 4 seemed to be more effective
than brequinar at lower concentrations both in proliferation and cytotoxicity experiments.
While results from the proliferation assays were expected, cytotoxicity data, at first sight,
contradicted the JurkatꢀT cell experiments, which had shown our compounds’ very low
toxicity. However, as mature cells have a much shorter halfꢀlife than immature ones, we
hypothesized that the considerable cytotoxicity observed in AML cell lines, but not in Jurkatꢀ
T cells, had to be ascribed to the differentiation induced in leukemic cells by hDHODH
inhibitors. We therefore investigated the differentiation effect induced by compounds 2, 4
and brequinar on our AML cell lines at several concentrations. The differentiation process
was tracked by analyzing CD11b and CD14 expression, as these antigens are typically
present in mature myeloid cells. In particular, cell differentiation could be best evaluated with
CD11b on U937 and with CD14 on THP1; similar, but less prominent results were obtained
with CD11b on THP1 (see supplementary Figure S3). Our experiments clearly demonstrated
that both compound 4 and brequinar induced a strong differentiation in U937 and THP1 cells,
as shown in Figure 8A, 8B and supplementary Figure S3. After the treatment with these
compounds, in fact, the expression of CD11b and CD14 increased significantly day by day,
depending on compound concentrations.
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Figure 8. A) Kinetic of differentiation induced by various concentrations of brequinar,
compound 4 and 2, on U937, expressed as the proportion of CD11b positive cells. B) Kinetic
of differentiation induced by various concentrations of brequinar and compound 4 on THP1,
expressed as the proportion of CD14 positive cells. C) The differentiation induced on U937
by brequinar (left panel) and compound 4 (right panel) is reversed when uridine is added. The
differentiation analysis is performed at day 3. D) The differentiation induced on THP1 by
brequinar (left panel) and compound 4 (right panel) is reversed when uridine is added. The
differentiation analysis is performed at day 3. DMSO indicates cells treated with dimethyl
sulfoxide only. #4 = compound 4; #2 = compound 2; BRQ = brequinar; ur = uridine. Uridine
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was added at 100 ꢂM concentration in all experiments. Statistical significance: *p < 0.05; **
p < 0.01; ***p < 0.001.
Notably, compound 4 induced a differentiation effect that was comparable to that of
brequinar at a 1ꢀlog inferior concentration. Compound 2, on the other hand, only induced a
mild CD11b increase in U937 cells that only occurred at high doses (10 ꢂM), where it was
associated with significant cell death. These data, together with the cytotoxicity results,
indicated that compound 2 was not able to induce myeloid differentiation and caused offꢀ
target toxicity at high doses. For this reason, compound 2 was excluded from further
experiments with THP1 and uridine.
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In order to further demonstrate the connection between differentiation and hDHODH
inhibition, the differentiation experiments were repeated in presence of uridine, and the
complete rescue of the phenomenon was observed (Figure 8C, D). Differentiation
experiments had to be stopped after 4 days as differentiated cells progressively died. With
this in mind, we can see how compound 4 and brequinar alone caused the death of the vast
majority of leukemic cells in vitro, even though the proportion of daily differentiating cells
reached a 40 % maximum (Figure 9 and Figure S4 in supplementary). Again, compound 4
was able to induce a massive death of leukemic cells already at 0.1 ꢂM, i.e., at a 1ꢀlog
inferior concentration compared to brequinar.
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Figure 9. Parallelism between the daily proportion of differentiating cells and cells viability.
The proportion of differentiating cells is expressed with bars, and the reference axis is on the
left; cells viability is expressed with lines, and the reference axis is on the right. The bottom
arrow indicates the concentration of drugs. Experiments were performed both on U937 and
THP1 and differentiation was evaluated, respectively, with CD11b and CD14 expression. See
supplementary Figure S4 for experiments with 10 µM concentrations. Statistical significance:
*p < 0.05; ** p < 0.01; ***p < 0.001 between treated and untreated cells. DMSO = dimethyl
sulfoxide.
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